Extended Abstracts for the 51st International Symposium of
the Princess Takamatsu Cancer Research Fund, 2023

ENVIRONMENTAL IMPACT ON
CLONAL EVOLUTION AND CANCERDEVELOPMENT
November 14-16, 2023
Tokyo, Japan

Edited by :Hiroyuki Tsuda, Allan Balmain, Hitoshi Nakagama,
Fuyuki Ishikawa, and Yukari Totsuka

Princess Takamatsu Cancer Research Fund

CONTENTS

KEYNOTE LECTURE
Precision Medicine: Environmental Cancer
Curtis C. Harris 

1

ENVIRONMENTAL CARCINOGENESIS/OCCUPATIONAL CARCINOGENESIS
Occupational Urinary Bladder Cancers: Insights Into Aromatic AmineInduced Carcinogenesis
Hideki Wanibuchi 

6Environmental Toxicants Target Prostate Stem-Progenitor Cells to Drive
Prostate Carcinogenesis
10
Gail S. Prins 
Mechanism of Action and an Inflammatory Axis For Air PollutionInduced Non-Small Cell Lung Cancer
Charles Swanton i17DNA ADDUCTS
High-Resolution Mass Spectrometry-Based Approaches to Improve theCharacterization of Chemical Carcinogenesis
20
Silvia Balbo 
DNA Adductomics in Human Tissues
Haruhiko Sugimura 

24

Elucidation of Driver Adducts of Cancer Development Using
Comprehensive Analysis of DNA Adducts
Yukari Totsuka 30

GENETIC MUTATIONAL SIGNATURE
An Anthology of Unusual Patterns of Somatic Mutations in Cancer
Genomes
Ludmil B. Alexandrov 

35

Trans-Ancestry Mutational and Structural Alteration Spectrum Landscapeof Gastric Cancer
39
Tatsuhiro Shibata 
Can We Identify New Causes of Cancer through Large Scale Genomic
Studies ?
Paul J. Brennan 

43

Mutational Signatures of Chemotherapeutic Agents and Environmental
Carcinogens
45
David H. Phillips 

iiNAKAHARA MEMORIAL LECTURE
Environmental Carcinogenesis, Mutational Signatures and Rational Drug
Design48
Arthur P. Grollman 

ANIMAL MODELSThe Interplay between Environmental Mutagens and Inflammatory
Tumour Promoting Factors in Determining Cancer Risk.
Allan Balmain 

50

Carbon Nanotubes, Innovative Materials of The 21st Century:
Carcinogenicity Evaluation of CNTs with Different Wall Structures
Hiroyuki Tsuda 

55Collaborative Cross Mice: A Population-Based Model for Optimally
Deciphering Gene-Environment-Microbiome Interactions in
Carcinogenesis
Jian-Hua Mao 

62

NON-MUTAGENIC MECHANISMS OF CANCERPROMOTION (1)The Human Microbiome and Cancer Prevention
Jiyoung Ahn 

iii

66

Histone H3.3-Specific Chaperone HIRA is Required for Acquired
Tolerance, a Form of Phenotypic Plasticity
Fuyuki Ishikawa 

70

NON-MUTAGENIC MECHANISMS OF CANCERPROMOTION (2)Epigenetics and Environmental Origins of Cancer
Zdenko Herceg 72

Epigenetic Field for Cancerization due to Chronic Inflammation
Toshikazu Ushijima 

77

CANCER PREVENTION EPIDEMIOLOGICAL STUDIES
Obesity, Metabolic Dysfunction and Cancer: Understanding Causal
Mechanisms to Guide Prevention
Marc J. Gunter 

82

Molecular Prevention of Cancer with Aspirin
Andrew T. Chan 

86

PRECISION PREVENTION
Environmental Impact on the Gut Microbiome: Multi-Omics to
Mechanisms of Dietary Spinach
Roderick H. Dashwood iv93Adductomic Strategies for Emerging Risk Factors in Liver Cancer
John D. Groopman 

96

Inhibition of Colorectal Carcinogenesis in Familial Adenomatous
Polyposis
Michihiro Mutoh 

104

MICROBIOME AND CANCER
Microbes and Colon Cancer: Where are we ?
Cynthia L. Sears 

108

Comprehensive Genomic Profiling and Clonal Evolution of
Neuroendocrine Carcinomas of the Gastrointestinal System
Shinichi Yachida 

117

The Role of BRCA1 Tumor Suppressor in Helicobacter Pylori-MediatedGastric Carcinogenesis
121
Masanori Hatakeyama 

CLONAL EVOLUTION (1)Somatic Evolution, Cancer, and Our Inevitable Decline with Age –Inextricably Linked
James DeGregori 

124

Using Ultra-Sensitive Sequencing to Study Clonal Evolution and Cancer
Risk
127
Rosana Risques v

Understanding of GI Cancer Initiation and Progression Using Organoids
Toshiro Sato 
130

CLONAL EVOLUTION (2)
Decoding Normal Breast Tissue Biology and the Evolution ofPremalignant Breast Cancer
Nicholas E. Navin 

132Life Histories of Breast Cancer and Related Clones
Seishi Ogawa 

134

CONCLUDING REMARKS
Hiroyuki Tsuda 136

List of Participants 142

List of Previous International Symposia Series 

160

vi

1

PRECISION MEDICINE: ENVIRONMENTAL CANCER

Curtis C. Harris
Laboratory of Human Carcinogenesis
The National Institutes of Health, National Cancer Institute37 Convent Drive, Room 3068A
Bethesda, MD 20898-4258, USA(curtis_harris@nih.gov)

The International Princess Takamatsu Symposium keynote address will be presented inthree very brief historical sections: first the status of the field in 1991, then examples of theremarkable progress made by cancer researchers and finally a description of our recent
precision medicine studies of lung cancer.

In 1991, it was generally accepted that mutagenesis was a major mechanism of cancer

caused by environmental chemicals. An important cellular target was the p53 gene that had
been previously discovered by David Lane and L. Crawford1. p53 has been found to be themost mutated gene in human cancer. One of the highlights at the 22nd International Princess

Takamatsu symposium was the different p53 mutagenesis signatures by aflatoxin B12 andother environmental carcinogens. Multiple more complex mutation signatures were initiallydefined in DNA by Alexandrov and Stratton3.

This 51 st International Princess Takamatsu Symposium further advances ourunderstanding of environmental causes and mechanisms of carcinogenesis.

The keynote address will focus on the major and most costly types of lethal cancers

including lung cancer. 25 trillion international dollars during the next 25 years4, that includes

both tobacco smokers and an increasing number of never smokers (Fig. 1 and 2) who were

exposed to environmental and passive tobacco carcinogens, air pollutant another causes. The
multiomic framework is now defined as precision medicine5, 6 (Fig. 3).

2

C. C. Harris

Fig. 1

Fig. 2

We will then discuss biomarkers in cancer

and liquid biopsy. For example, creatine

riboside (CR) is associated with increased
risk of multiple types of cancer including
lung cancer, brain glioblastoma, breast
cancer, prostate cancer and including othertypes of cancers. Prospective studies have
Fig. 3

identified individuals at risk of cancer, cancer
diagnosis and response to therapy including

liquid biopsy of other potential biomarkers in therapeutic pathways and drugs to treat
human cancer7.

The organizers have orchestrated a comprehensive program with themes focused on

advancements recently made in identifying cellular and molecular mechanisms of theenvironmental and occupational cancers. Presentations will update the long-standing themesin carcinogenesis such as tumor promotion, inflammation, DNA addicts, DNA signatures,and the multiomics of precision medicine. Including the microbiome, and the increasing

cancer incidence linked to decline of anticancer defenses with increasing age. We haverecently been investigating the mechanisms of carcinogenesis and aging-related disease, e.g.,
Alzheimer’s disease, p53 isoforms involvement in cellular senescence8.

Keynote Lecture

3

4

C. C. Harris

Laboratory of Human Carcinogenesis 2023
References
1. D.P. Lane and L.V. Crawford. T antigen is bound to a host protein in SV40-transformed
cells, Nature 1979. 278: 261-3.
2. C. Hsu, R.A. Metcalf, T. Sun, J.A. Welsh, N.J. Wang and C.C. Harris. Mutational hotspots
in the p53 gene in human hepatocellular carcinomas, Nature 1991. 350: 427-8.

3. L.B. Alexandrov, Y.S. Ju, K. Haase, P. Van Loo, I. Martincorena, S. Nik-Zainal, et al.
Mutational signatures associated with tobacco smoking in human cancer. Science 2016.

354: 618-622.
4. S. Chen, Z. Cao, K. Prettner, M. Kuhn, J. Yang, L. Jiao, et al. Estimates and Projections of

the Global Economic Cost of 29 Cancers in 204 Countries and Territories From 2020 to2050. JAMA Oncol 2023. 9: 465-472.
5. Modified: Toward Precision Medicine: Building a Knowledge Network for Biomedical
Research and a New Taxonomy of Disease. (National Research Council 2011).
6. A. Vargas and C.C. Harris. Biomarker Development in the Precision Medicine Era. Nature
Review Cancer 2016. 18: 525-33.

7. E.A. Mathé, A.D. Patterson, M. Haznadar, S.K. Manna, K.W. Krausz, E.D. Bowman, P.G.
Shields, J.R. Idle, P.B. Smith, K. Anami, D.G. Kazandjian, E. Hatzakis, Gonzalez FJ, andHarris CC. Noninvasive urinary metabolomic profiling identifies diagnostic andKeynote Lecture5

prognostic markers in lung cancer. Cancer Res. 2014. 74: 3259-70.
8. K. Ungerleider … C.C. Harris. Neuroscience 2022. 498:190-202; K Ungerleider … C.C
Harris. Cell Cycle 2021. 20: 752-764; C. Turnquist … C.C. Harris. Neuro Oncol 2019. 21: 474-

485; N. von Muhlinen … C.C. Harris. Oncogene 2018. 37: 2379-2393; S. Joruiz … C.C
Harris. Submitted; J. Beck … C.C. Harris. Carcinogenesis 2020 (Review). 41: 1017-1029;
and C. Amor … S. W. Lowe. Nature 2020. 583: 127-132.

Curtis C. Harris, MD
1969-1970
1970 - 1972
1972 - 1975
1973 - 1976
1975 - 1981
1979 - 1981
1981 - Pres.

2013 - Pres.

Intern, Dept of Medicine, U.C.L.A. Hospital, Los
Angeles, CA and Clinical Training, NCI, NIH, Bethesda,
MD
Research Associate, Lung Cancer Unit, OASDC, DCCP,
NCI, NIH, Bethesda, MD
Head, Ultrastructure Unit, Pathogenesis Section, Lung
Cancer Branch, DCCP, NCI, NIH, Bethesda, MD
Resident and Trainee in Clinical Oncology, NCI-VA
Oncology Branch, Dept of Medicine, VA Hospital,
Washington, DC (4 months/year)
Head, Human Tissue Studies Section, Laboratory ofExperimental Pathology, DCCP, NCI, NIH, Bethesda,
MD
Associate Chief, Laboratory of Experimental Pathology,
NCI, NIH, Bethesda, MD
Chief, Laboratory of Human Carcinogenesis, CCR, NCI,
NIH and Head, Molecular Genetics and Carcinogenesis
Section, Laboratory of Human Carcinogenesis, CCR,
NCI, NIH, Bethesda, MD
Adjunct Professor, Department of Oncology, Georgetown
University Medical Center, Washington, DC

6

OCCUPATIONAL URINARY BLADDER CANCERS: INSIGHTSINTO AROMATIC AMINE-INDUCED CARCINOGENESIS

Hideki Wanibuchi
Department of Molecular Pathology
Osaka Metropolitan University Graduate School of Medicine
1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan(wani@omu.ac.jp)

Occupational bladder cancer is a subtype of bladder cancer resulting from prolonged

exposure to carcinogenic agents in the workplace. Aromatic amines, such as o-toluidine(OTD), 2-naphthylamine, 4-aminobiphenyl, and benzidine, are prominent carcinogens linked
to this occupational risk. In this symposium, we offer an overview of recent cases ofoccupational bladder cancer in Japan associated with exposure to OTD and acetoaceto-otoluidide (AAOT). Furthermore, we share novel insights into the metabolism andcarcinogenicity of these aromatic amines, based on our findings from rat and humanizedmouse models.

AAOT is utilized as an industrial intermediate in the synthesis of organic pigments.

Recently, cases of occupational urinary bladder cancer have been reported among workers inJapanese plants producing AAOT, which utilizes OTD as a raw material. The chronic
exposure of these workers to AAOT raised the concerns about its potential link to bladdercancer. In our studies, AAOT induced preneopasitic lesion (simple hyperplasia) on the rat
urinary bladder in the short period study [1], and promoted urinary bladder carcinogenesis
in a rat two-stage urinary bladder carcinogenesis model (Figure 1) [2]. We also detected thatthe most abundant urinary metabolite of AAOT was OTD, which was at least one order ofmagnitude higher than AAOT [1, 2]. These data suggest that AAOT is carcinogenic to theurinary bladder in rats. We also suggest that OTD, which is derived from AAOT metabolism,

could be pivotal to AAOT's carcinogenic effects. Mutation analyses reveal that bladdercancers in individuals exposed to either OTD or AAOT have diverse mutations in cancer-

related genes, presenting a mutation signature distinct from sporadic bladder cancers. Thisemphasizes the potential of AAOT, like other carcinogenic aromatic amines, as a humanEnvironmental Carcinogenesis/Occupational Carcinogenesis

7

Figure 1

Promoting effects of AAOT on N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) -induced rat
bladder carcinogenesis.

Figure 2

Inhibitory effects of apocynin (APO) on the carcinogenic effects of OTD in the rat bladderepithelium.

bladder carcinogen.

In our study investigating the carcinogenic mechanisms of AAOT and OTD, we identified

oxidative DNA adducts, including 8-OHdG, in the urothelium of rats treated with thesecompounds, using comprehensive DNA adduct analysis. Furthermore, our also found thatapocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, significantly
inhibited cell proliferation, DNA damage, and oxidative stress induced by OTD in a 4-week

rat bladder carcinogenicity study (Figure 2) [3]. These findings imply that oxidative stress
may play a crucial role in the development of urinary cancer caused by OTD.

8

H. Wanibuchi

In additional studies focusing on human metabolites, OTD was administered to micewith humanized livers, established through human hepatocyte transplantation. This
exposure resulted in the induction of CYP3A4 expression in the human liver cells of thesemice [4], suggesting that CYP3A4 is an important enzyme involved in the toxicity andcarcinogenicity in humans exposed to OTD.

As the list of chemicals associated with workplace exposure continues to grow,identifying chemicals that may trigger occupational bladder cancer becomes increasingly
important. Employing experimental models remains an essential approach in determining
the causes of occupational cancer and understanding the carcinogenic mechanisms.
References

1. Okuno T, Gi M, Fujioka M, Yukimatu N, Kakehashi A, Takeuchi A, Endo G, Endo Y,
Wanibuchi H. Acetoaceto-o-toluidide enhances cellular proliferative activity in theurinary bladder of rats. Toxicol Sci. 2019, 169, 56-64.
2. Yukimatsu N, Gi M, Okuno T, Fujioka M, Suzuki S, Kakehashi A, Yanagiba Y, Suda M,
Koda S, Nakatani T, Wanibuchi H. Promotion effects of acetoaceto-o-toluidide on
N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats. Arch
Toxicol. 2019, 93, 3617-3631.
3. Suzuki S, Gi M, Koyama M, Obikane A, Vachiraarunwong A, Fujioka M, Kakehashi A,
Totsuka Y, Wanibuchi H. Evaluation of the mechanisms involved in the development of
bladder toxicity following exposure to occupational bladder cancer causative chemicals
using DNA adductome analysis. Biomolecules. 2024, 14, 36.
4. Yokota Y, Suzuki S, Gi M, Yanagiba Y, Yoneda N, Fujioka M, Kakehashi A, Koda S,
Suemizu H, Wanibuchi H. o-Toluidine metabolism and effects in the urinary bladder ofhumanized-liver mice. Toxicology. 2023, 488, 153483.

Environmental Carcinogenesis/Occupational Carcinogenesis

Hideki Wanibuchi, MD, DMSc
1978-1984
1985-1989
1989-1992
1991-1992
1993-2000
2001-2005
2006-2022
2022-present

Graduated with M.D. from Osaka City University
Medical School
Graduated with DMSc. from Osaka City University
Graduate School of Medicine
Assistant Professor, Osaka City University Graduate
School of Medicine
Osaka City Overseas Researcher, University of
Amsterdam, Netherlands
Senior Assistant Professor, Osaka City University
Graduate School of Medicine
Associate Professor, Osaka City University Graduate
School of Medicine
Professor, Osaka City University, Graduate School of
Medicin
Professor, Osak Metropolitan University, Graduate
School of Medicine

910

ENVIRONMENTAL TOXICANTS TARGET PROSTATE STEMPROGENITOR CELLS TO DRIVE PROSTATE CARCINOGENESIS

Gail S. Prins
Department of Urology, Pathology and Physiology
College of Medicine
The University of Illinois at Chicago
820 S Wood St, Chicago, IL, 60612, USA(gprins@uic.edu)

It is long recognized that environmental factors play a major role in cancer etiology, andemerging data suggest that specific toxicants may contribute to an increased cancer risk,

including prostate cancer (1). While differentiated prostate epithelial cells can be directly

influenced by chemical exposures, properties of stem-progenitor cells make them compelling
targets of tumorigenesis. Stem cells are fundamental components of biological organization,
responsible for development and maintenance of tissues and organ systems (2). Embryonicstem cells are pluripotent cells with a robust proliferative capacity and ability to differentiate
into the three embryonic germ cell layers. Once tissues and organs are formed following

morphogenesis, adult tissue-specific stem cells maintain homeostasis within that structure,providing cells for natural tissue turn over and regeneration as well as response to injury.

The highly plastic state of stem and daughter progenitor cells during development andtissue maintenance permits the needed flexibility for proper tissue formation and repair.
Regrettably, this plasticity also provides an opportunity for aberrant cellular reprogrammingdue to inappropriate signaling from chemical exposures that can lead to persistent life-longeffects and tissue perturbation. Further to this point, the cancer stem cell hypothesis
identifies normal tissue stem cells and their immediate progenitors as putative targets forcell transformation and tumor initiation (3, 4).

W h i l e s t e m a n d p ro g e n i t o r c e l l s i n a l l s y s t e m s a re t i g h t l y re g u l a t e d b y t h e i r

microenvironment or stem cell niche, hormonally sensitive tissues appear to have anadditional layer of hormonal regulation of the stem and progenitor cells. Our research has
shown that prostate stem and progenitor cells (SPCs) express nuclear receptors that cantightly regulate stem cell self-renewal and progenitor cell lineage commitment andEnvironmental Carcinogenesis/Occupational Carcinogenesis

11

differentiation. Although negative for androgen receptor (AR), the human prostate SPC

population expresses estrogen receptors (ERα, ERβ, GPER), retinoid receptors (RARs, RXRs),
vitamin D receptor (VDR), peroxisome proliferator receptors (PPARs), Aryl Hydrocarbon

receptor (ArR) among others (5, 6). Early evidence demonstrates that when activated bytheir cognate ligands, these receptors mediate diverse effects including stem cell selfrenewal, progenitor cell amplification and differentiated lineage commitment.

Unfortunately, exposures to endocrine disrupting chemicals and other environmental
compounds have the potential to disrupt prostate homeostasis through these same

pathways. As such, research in my laboratory focuses on these long-lived cells as directenvironmental toxicant targets. Our findings have determined that several compounds,
including Bisphenol A (BPA), Per-and polyfluorylalkyl substances (PFAS), and inorganic

arsenic, reprogram normal human prostate SPC populations, leading to increased cancer
susceptibility. Of particular note, the observed alterations in SPC homeostasis leading totransformation are mediated through chemical-specific mechanisms.

Direct Effects of Bisphenol A on Human Prostate Stem and Progenitor Cells that Increase
Carcinogenic Susceptibility in Human Prostate Epithelium

To directly assess the actions of endogenous and exogenous factors on prostate SPCs andto evaluate the relevance of these findings to the human prostate gland, we derived in vitro

and in vivo systems utilizing primary cells cultured from prostates of young, disease-freeorgan donors. Adult prostate stem cells were enriched by FACS or 3-D matrigel culture toform prostaspheres (PS) (7). Exposure of primary human prostate epithelial cell cultures to

estradiol-17β results in an in stem-like cell numbers using the side-population FACS assay
and increased PS numbers and size indicative of enhanced stem cell self-renewal andprogenitor cell proliferation, respectively (8). Similarly, exposure to BPA, a ubiquitous
estrogenic-like chemical found in 1000s of consumer products, enhanced SPC growth atenvironmentally relevant levels and augmented expression of stemness genes (NANOG,
TBX3).

To understand the molecular underpinnings of BPA effects, the transcriptome andepigenome of SPCs were interrogated in spheroids grown in the presence of vehicle,
estradiol or BPA (9). Using an Affymetrix human genome microarray, a unique block of 26

genes was consistently down regulated across all donors and treatment groups of which 15
were identified as small noncoding nucleolar RNA (SNORDs). While the SNORDS were notsilenced by DNA methylation modifications, they were repressed by specific histone

modifications at H3K4, H3K9 and H3K27 (9). Together, these findings show that estrogenic

chemicals modify the epigenome of normal adult prostate SPCs. Since epigenetic
mechanisms are crucial in maintaining stem cell pluripotency and controlling differentiation

12

G. S. Prins

into progenitor cells and epithelial cell lineages (10), alterations in epigenetic marks may set
the stage for aberrant differentiation programs within the prostate epithelium which may, inturn, contribute to increased human prostate cancer risk.

We next demonstrated increased carcinogenic risk in prostate epithelium following

progenitor cell exposure to BPA through creation of an in vivo model using the SPCs isolated

from normal human prostates. Prostaspheres of SPCs were dispersed, combined withinductive rat urogenital sinus mesenchyme and grafted under the renal capsule of adultmale nude mice (8), forming normal prostate-like structures with differentiated humanprostate epithelium that expressed prostate specific antigen (PSA). Once mature tissues
formed, host mice were treated with elevated estradiol (E) and testosterone (T)supplementation for 2-4 months to drive carcinogenesis. Pathologic lesions were observedin the human prostate epithelium over time, progressing from hyperplasia, squamous

metaplasia and high-grade PIN to adenocarcinoma at a low incidence. To test whether BPA
could influence this process, host mice were orally exposed to low doses of BPA for two
weeks following engraftment, followed by T+E exposure for 4 months. The incidence ofmalignancy in the human prostate epithelium significantly increased from 13% in oilexposed controls given T+E to 33-36% in tissues exposed in vivo to BPA followed T+E. Thisfurther increased to 45% cancerous lesions when the SPCs were exposed to BPA in vitro

followed by 2 weeks of in vivo exposure. Together, these results demonstrate that exposures
to low doses of BPA increase the susceptibility of the human prostate epithelium to estrogen-driven carcinogenesis and that prostate SPCs are direct targets that propel this increased
cancer risk.

Inorganic arsenic transforms human prostate progenitor cells through p62-KEAP-NRF2

Inorganic arsenic (iAs) is a ubiquitously distributed environmental and industrial

toxicant, classified as a class I carcinogen by the International Agency for Research on Cancer(11). More than 137 million people in over 70 countries are exposed to iAs at levels of greaterthan 10 ppb, the U.S. Environmental Protection Agency drinking water standard (12).

Epidemiologic studies and experimental evidence link chronic iAs exposure with increased
risk of certain cancers (11, 13) and while not conclusive, emerging epidemiological datasuggests a direct causal relationship between iAs exposure and prostate cancer incidence
and mortality (14). As some studies using human prostate cell lines identified prostate SPCs
as arsenic targets for transformation (15), we investigated whether prostate SPCs from
disease-free organ donors were direct iAs targets and sought to identify the underlying
mechanisms of transformation.

Using the model systems described above, we found that exposure to 1 µM iAs (~75 ppb)

significantly increased stem cell numbers, inhibited cell differentiation in organoid cultures

Environmental Carcinogenesis/Occupational Carcinogenesis

13

and induced progenitor cell transformation as assessed by soft-agar colony formation.

Transcriptomic analysis found that brief exposure of prostaspheres (PS) to 1 µM iAs
augmented prostate carcinogenic pathways (kRAS, PTEN, p53, MEK), including activationof the KEAP1-NRF2 pathway. NRF2 was deemed essential for iAs-induced transformationof progenitor cells which was suppressed by NRF2 knockdown. Detailed studies
determined that this was mediated by a non-canonical p-p62 dependent pathway, elevatedduring autophagosome formation which allowed for KEAP1-NRF2 dissociation andtranslocation of NRF2 to the nucleus. Further, we established that autophagy-flux blockade

underpinned the p-p62 accumulation. Mechanistically, iAs suppressed V-ATPase subunit
VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein

degradation including p62. In vivo, chronic iAs exposure activated NRF2 in both epithelialand stroma cells of chimeric human prostate grafts and induced premalignant events. In

summary, we outlined a novel mechanism involving NRF2 dysregulation by which iAsinterferes with homeostasis of prostate SPCs and predisposes to tumorigenesis.

Per- and Polyfluoroalkyl Substances Target and Alter Human Prostate Stem-Progenitor Cells

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals utilized in various

industrial settings, and include products such as flame retardants, artificial film-forming
foams, cosmetics, non-stick cookware among others. Epidemiological studies suggest a link

between increased blood PFAS levels and prostate cancer incidence and our in vitro and invivo studies with benign and cancerous prostate cell lines confirmed the enhancement of celland tumor growth when exposed to elevated levels of several PFAS compounds (16). PFAS

chemicals are known to bind to PPARs in other organ systems, and since prostate stemprogenitor cells express PPARs, we asked whether they are direct PFAS targets that may be
involved in PFAS-driven prostate cancer incidence and mortality. Exposure of normal

human prostate SPCs to 10 nM PFOA or PFOS for four weeks using serial passage of

prostasphere cultures, markedly increased spheroid numbers and size indicative of elevatedstem cell self-renewal and progenitor cell proliferation (17). Transcriptome analysis using
single-cell RNA sequencing (scRNA-seq) confirmed enrichment of stem cells and early-stage
luminal cells, decreased basal progenitors and the emergence of a new cell cluster ofaberrantly differentiated luminal progenitor cells upon PFOS/PFOA exposure indicating

altered lineage commitment. Pathway analysis found enrichment of cancer-associatedsignaling pathways and enhanced glycolosis pathways. Metabolomic analysis of PFASexposed prostaspheres revealed increased glycolytic pathways including the Warburg effectas well as strongly enrichment of serine and glycine metabolism which may promote a pre-malignant SPC fate. Together, these findings identify human prostate SPCs as direct PFAS
targets with resultant reprogrammed transcriptomes and an oncogenic metabolome that

14

G. S. Prins

Figure 1

Environmental toxicants modify prostate SPC homeostasis that underpins transformation
in a chemical-specific manner.

may contribute to an elevated prostate cancer risk with chronic exposures.

In summary, our findings document that prostate SPC populations are direct targets formany environmental toxicants that reprogram and transform the cells through chemical-

specific mechanisms (Fig 1). Due to the long-lived nature of prostate stem cells, theseprocesses can underpin increased prostate carcinogenic potential over extended time periods.
References

1. L X, GS P. Environmental Factors in Cancer Risk. In: D H, Z L, VLW G, eds. Nutritional

Oncology: Nutrition in Cancer Prevention, Treatment and Survivorship. Vol First
Edition: CRC Press; 2022:459-474.
2. Weissman IL. Stem cells: units of development, units of regeneration, and units inevolution. Cell. 2000;100(1):157-168.
3. Wicha MS. Targeting breast cancer stem cells. Breast. 2009;18 Suppl 3:S56-58.
4. Batlle E, Clevers H. Cancer stem cells revisited. Nature medicine. 2017;23(10):1124-1134.
5. Prins GS, Hu W. Prostate Stem Cells, Hormones and Development. In: SD C, ed. StemCells and Prostate Cancer. Vol VII: Springer LLC; 2013:1-20.
6. Hu WY, Shi GB, Hu DP, Nelles JL, Prins GS. Actions of estrogens and endocrine

disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk.

Environmental Carcinogenesis/Occupational Carcinogenesis

15

Molecular and cellular endocrinology. 2012;354(1-2):63-73.

7. Hu WY, Shi GB, Lam HM, Hu DP, Ho SM, Madueke IC, Kajdacsy-Balla A, Prins GS.
Estrogen-initiated transformation of prostate epithelium derived from normal humanprostate stem-progenitor cells. Endocrinology. 2011;152(6):2150-2163.
8. Prins GS, Hu WY, Shi GB, Hu DP, Majumdar S, Li G, Huang K, Nelles JL, Ho SM, Walker
CL, Kajdacsy-Balla A, van Breemen RB. Bisphenol A promotes human prostate stemprogenitor cell self-renewal and increases in vivo carcinogenesis in human prostateepithelium. Endocrinology. 2014;155(3):805-817.

9. Ho SM, Cheong A, Lam HM, Hu WY, Shi GB, Zhu X, Chen J, Zhang X, Medvedovic M,
Leung YK, Prins GS. Exposure of Human Prostaspheres to Bisphenol A Epigenetically
Regulates SNORD Family Noncoding RNAs via Histone Modification. Endocrinology.

2015;156(11):3984-3995.
10. Mikkelsen T, Ku M, Jaffe D, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W,
Kim T, Koche R, Lee W, Mendenhall E, O'Donovan A, Presser A, Russ C, Xie X, Meissner
A, Wernig M, Jaenisch R, Nusbaum C, Lander E, Bernstein B. Genome-wide maps ofchromatin state in pluripotent and lineage-committed cells. Nature. 2007;448:553-560.
11. IARC. A review of human carcinogens: Part C: metals, arsenic, dust and fibres. IARC
Monogram. 2009:453-454.

12. Carlin DJ, Naujokas MF, Bradham KD, Cowden J, Heacock M, Henry HF, Lee JS, Thomas
D J , T h o m p s o n C , To k a r E J , Wa a l k e s M P, B i r n b a u m L S , S u k WA . A r s e n i c a n dEnvironmental Health: State of the Science and Future Research Opportunities.
Environmental health perspectives. 2016;124(7):890-899.
13. Tokar EJ, Benbrahim-Tallaa L, Ward JM, Lunn R, Sams RL, Waalkes MP. Cancer inexperimental animals exposed to arsenic and arsenic compounds. Critical reviews in
toxicology. 2010;40(10):912-927.

14. Yang Y, McDonald AC, Wang X, Pan Y, Wang M. Arsenic exposures and prostate cancer
risk: A multilevel meta-analysis. Journal of trace elements in medicine and biology : organ of

the Society for Minerals and Trace Elements (GMS). 2022;72:126992.
15. Tokar EJ, Diwan BA, Waalkes MP. Arsenic exposure transforms human epithelial stem/p ro g e n i t o r c e l l s i n t o a c a n c e r s t e m - l i k e p h e n o t y p e . E n i v o n H e a l t h P e r s p e c t .

2010;118(1):108-115.
16. Imir OB, Kaminsky AZ, Zuo QY, Liu YJ, Singh R, Spinella MJ, Irudayaraj J, Hu WY, Prins

GS, Madak Erdogan Z. Per- and Polyfluoroalkyl Substance Exposure Combined with
High-Fat Diet Supports Prostate Cancer Progression. Nutrients. 2021;13(11).
17. Hu WY, Lu R, Hu DP, Imir OB, Zuo Q, Moline D, Afradiasbagharani P, Liu L, Lowe S,
Birch L, Griend DJV, Madak-Erdogan Z, Prins GS. Per- and polyfluoroalkyl substancestarget and alter human prostate stem-progenitor cells. Biochemical pharmacology.
2022;197:114902.

16

G. S. Prins

Gail S. Prins, PhD
1974-1979
1979-1983
1983-1990
1991-present
2017-present

Graduated from University of Illinois Medical Center(Physiology)
Postdoctoral Researcher, Northwestern University,
ChicagoAssistant Professor, University of Chicago
Michael Reese Distinguished Professor, University of
Illinois at Chicago
Co-Director, Chicago Center for Health and Environment,
University of Illinois/University of Chicago

17

MECHANISM OF ACTION AND AN INFLAMMATORY AXIS
FOR AIR POLLUTION-INDUCED NON-SMALL CELL LUNGCANCER

Charles Swanton
Cancer Evolution and Genome Instability Laboratory
Francis Crick Institute
1 Midland Road, London NW1 1AT, United Kingdom(charles.swanton@crick.ac.uk)

Lung cancer in never-smokers is distinct from smoking-associated lung cancer in that it

has a low mutational burden and over half of patients have tumours with an EGFR mutation.
Proposed risk factors have included germline susceptibility loci, radon gas, second-handsmoke, and environmental air pollution.

We have hypothesised that environmental carcinogens like environmental pollution orsecond-hand smoke might cause lung cancer by promoting the expansion of pre-existing
mutant clones.

Work from TRACERx has demonstrated that lung cancer in never-smokers is associatedwith a low mutational burden that has very few mutations. Those mutations tend to be in anageing context or APOBEC context. The Sherlock study has demonstrated that there is no

carcinogenic mutational signature in 97% of Sherlock study never-smoking lung cancers.
Approximately 70 years ago, Doll and Hill were amongst the first to propose a link between

atmospheric air pollution from exhaust fumes and lung cancer, but in their seminal paper in1950 proved the association of tobacco with lung cancer. Over the course of the last four

decades, there've been at least six meta-analyses demonstrating an association of air
pollution with both lung cancer in smokers and lung cancer in never-smokers. The relative

risks appear to be greater in lung adenocarcinoma. We hypothesised that if environmental

air pollution is causing lung cancer in never-smokers it would have to fulfil four criteria. It
would have to explain the geographic distribution of the disease, we would have to provethat pollution directly causes lung cancer, we would have to explain how pollution could

18

C. Swantoncause cancer without directly causing DNA mutations and explain why lung cancer in neversmokers is more common in females. We reasoned that the classic mutational model might

not apply to environmental air pollution-associated lung cancer or lung cancer caused
directly by air pollution for the reason that there is no clear environmental carcinogenic

signature in these tumours. Instead, a potential model similar to the tumour promotion
model as first proposed by Berenblum in 1947 might be operating. Berenblum, and latterly

Balmain, proposed that tumours are promoted from cells with pre-existing mutations. We
wondered whether air pollution might act as a tumour promoter rather than a mutagenic
initiator.

Firstly, we conducted a pan-cancer analysis of cancer incidence in PM2.5s in UK biobank

participants. In over 407,000 participants we demonstrated an increased risk of lung cancer

controlled for multiple testing of approximately 1.08 for each μg/m3 increment of PM2.5. We

went on to find that PM2.5s are associated with EGFR mutant lung cancer incidence inEngland, Taiwan, and South Korea. These data are consistent with a meta-analysis foradenocarcinoma diagnoses per 5 μg/m3 rise in PM2.5s of approximately 1.55. Next, we set

up three GEMM models of EGFR or KRAS-mutant lung cancer and instilled intratracheal
PM2.5s every two days for three weeks in these mice after oncogene induction. In all threemodels, we found an increased incidence of cancer lesions or adenoma lesions in each of themouse models in a dose-dependent manner. We could find no evidence of clear mutagenic
signatures in these tumours and no significant increase in the number of mutations whenthese air pollution-associated tumours were subjected to whole genome sequencing
compared to saline control. Therefore, it did appear that Berenblum’s model might be
operating in these tumours. Next, we demonstrated that there was no significant rise in air

pollution associated lung cancers in a RAG2 IL-2 receptor gamma null mouse model
deficient in T and B cells as well as NK cells. We saw no increase at 50 μg of PM 2.5 exposurein the number of lesions per millimetre squared in mouse lungs suggesting that a competentimmune system is required for PM-mediated tumorigenesis. Next, we subjected mouseepithelial cells to transcriptional analysis and found evidence of an AT2 cell state and amacrophage recruitment state. Subsequent experiments demonstrated that a progenitor cellstate was initiated following air pollution exposure of mouse epithelial cells but only in thepresence of an EGFR activating mutation. This appeared to be secondary to macrophage

infiltrates in epithelial tissue following PM 2.5 exposure. We found evidence of increased
macrophage infiltrates in both mouse and human lungs exposed to air pollution. Analysis of
published data from Christopher Carlston and colleagues demonstrated interleukin-1 beta

(IL-1β) upregulation in both mouse and human lung tissue after acute PM2.5 exposure.
These data were pertinent given the results of the Cantos trial which has shown a dose-Environmental Carcinogenesis/Occupational Carcinogenesis

19

dependent reduction in new lung cancer primaries following anti-IL-1β therapy. Treatmentof mice with an anti-IL-1β antibody appeared to attenuate new tumour formation in thepresence of air pollution and anti- IL-1β antibody. We found evidence that one of theinitiating cells is the AT2 cell. Therefore, the AT2 cell appears to be a PM2.5 vulnerable
tumour cell-of-origin. Follow up experiments demonstrated that pollution exposed
macrophages elevate progenitor capacity of EGFR mutant AT2 cells exposed to room air.

Finally, in order to demonstrate that the Berenblum model might be correct, we had todemonstrate whether we had to assess whether there was evidence of mutant clones innormal tissue. Analysis of normal tissues using digital PCR with SAGA technologies or
TwinStrand Duplexseq analysis demonstrated EGFR activating mutations in 15% of normallung tissues and KRAS activating mutations in 53% of normal lung samples. These mutations

appeared to expand in pollution exposed lung and smoking exposed lung respectively.
These mutations appeared to occur due to the ageing process with evidence of an increased
number of activating mutations found in normal lung tissue with age in never smokers. In

summary, our data suggest that air pollution acts as an inflammatory mediator driving the
expansion of pre-existing oncogenic mutations in normal tissue in vulnerable progenitor

cells such as the AT2 cell. These data are consistent with animal model systems published byboth Balmain and Berenblum suggesting a route into molecular cancer prevention byblocking inflammatory pathways that might sustain or initiate the expansion of pre-existing
mutant clones in progenitor cells.

Charles Swanton, MD, PhD
1995
1998
1999
2013-present

Graduated from University College
London (Cellular Pathology
MBPhD
MBBS
Principal Investigator, Francis Crick Institute,London & UCL Cancer Institute20

HIGH-RESOLUTION MASS SPECTROMETRY-BASEDAPPROACHES TO IMPROVE THE CHARACTERIZATION OF
CHEMICAL CARCINOGENESIS

Silvia Balbo
Division of Environmental Health Sciences
School of Public Health
Masonic Cancer Center
University of Minnesota
CCRB, 2231 6th St. SE – Room 2-145 Minneapolis, MN 55455, USA(balbo006@umn.edu)
Characterizing chemical exposures and their health effects in humans, remains asignificant challenge. This is due in part to the difficulties related to capturing the complexityof exposures and their interaction with biological systems, but also to the fact that markers

that can help disentangle this complexity, are often at trace levels. Improved tools providing

greater analytical sensitivity, combined with comprehensive screening capabilities, areneeded to move this field of research forward. Our laboratory is focusing on developing
state-of-the-art high-resolution mass spectrometry-based methods to characterize theexposome and determine how it may interact with cellular targets playing a key role incarcinogenesis. These methods are based on the use of high-field orbital trap instrumentation(Thermo Scientific Orbitrap Lumos) which is very well suited for the analysis of challenging

low-abundance, high-complexity samples. We are developing approaches based on neutral
loss screening techniques paired with relative quantitation strategies to perform metabolic
profiling focused on the characterization of reactive chemicals in biological fluids. In parallel,

we are working on establishing a DNA adductomic approach based on a data dependentconstant neutral loss-MS3 (DDA-CNL/MS3) methodology as a comprehensive screening

method to characterize all covalent DNA modifications (DNA adducts), induced by various
exposures.1 We are now applying this approach for a more comprehensive characterizationof the DNA damage resulting from genotoxic exposures, such as tobacco smoke, thecharacterization of unknown genotoxins produced by the gut microbiome and known to belinked to increased risk of colon cancer, and to the development of markers to support

stratification of patients undergoing chemotherapy based on alkylating agents. An overview
of these three lines of research is presented.

DNA Adducts

21

In the case of tobacco smoke, the DNA damage resulting from the interaction of thetobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is

analyzed in the presence of inflammation, to investigate the driver adducts that are enhancedby the inflammatory process and that may be responsible for the initiation of carcinogenesis.

Lung DNA samples from an animal model in which animals are exposed to NNK and the
combination of NNK plus the inflammation-inducing agent Lipopolysaccharide (LPS) are

analyzed and compared to tissue samples from animals exposed to LPS only and controls.
This model is used because animals develop lung tumors after several weeks and the effectis increased in severity and number of tumors when animals are co-exposed with NNK and

LPS. Therefore, this model allows to monitor and follow the development of the DNA
damage from its initial formation to the actual development of tumors, and allows to fully
characterize the overall carcinogenic process.2 The use of our DNA adductomic approachallowed to detect and measure a large number of DNA adducts generated by NNK,

including expected monoadducts, protein and DNA cross links and it also allowed to
discover some modifications previously unknown, namely DNA-RNA crosslinks. 3 The

method is now used to analyze the development of the profile of adducts over time in thesame animal model, by analyzing lung DNA samples from animals exposed to NNK or LPS

and NNK at 1, 5 and 15 weeks after initial exposure. Also, in this case the results arecompared with samples collected from animals only treated with LPS or with the vehicle ascontrols.

In the case of using our DNA adductomic approach to discover new genotoxins producedby the gut microbiome, we investigated the DNA damaging effects of the genotoxin
colibactin, produced by the E.coli carrying the pks+ island and known to be associated withcolorectal cancer risk. This genotoxin has never been characterized before and the elucidationof the modification it results in when binding DNA allowed us to understand the structuralfeatures of the original molecule.4 Structural elucidation was possible by taking advantage ofthe various levels of fragmentations at high resolution that our method features. The

identified adduct was then analyzed and found in tissue from animals exposed to E.coli pks+

and was further investigated in samples obtained from human colonoscopies. The humansamples included in this work were collected from a biorepository created at the Universityof Minnesota, and approved by the University IRB. which includes Cystic Fibrosis patients.

These patients are at higher risk of developing colon cancer and their disease disrupts thepermeability of their gut mucosa. Therefore, samples from these patients were expected topotentially be more susceptible to the presence of the colibactin-derived DNA adduct. The

DNA adducts analysis of 85 Cystic Fibrosis samples was compared to that of 59 controls
after assessing the infection to E.coli pks+ using qPCR. Around 30% of participants werepositive to the infection, however only 2 individuals, among the Cystic Fibrosis group, were22

S. Balbo

found to be positive to the presence of the colibactin-derived DNA adducts. Interestingly
these two patients had several characteristics in common: they were both obtained from lungtransplant patients who were not taking any medication for Cystic Fibrosis and who werediagnosed with colon cancer or a precancerous lesion during the colonoscopy. Furthermore,
the E.Coli pks+ infection did not seem to be enough for the detection of the adducts andother parameters seemed to be necessary for the adduct to be detected. These preliminary
findings need to be further confirmed on a larger sample size. However, this is the firstreport of the presence of this adduct in human samples obtained from normal tissue
collected during screening colonoscopies.

Finally, our method has been used to characterize the DNA damage deriving from DNA

alkylating drugs used in chemotherapy, to investigate the potential use of the corresponding
DNA adducts as markers to stratify patients and discriminate those who may experience

toxicity or less effective outcomes from those benefiting from the treatment.

Cyclophosphamide-derived DNA adducts were characterized in vitro by exposing calf

thymus DNA to the drug in the presence of S9 metabolism and comparing the DNA adductprofile present in the exposed DNA only and not in the control DNA. A list of 40 adducts
was compiled. These DNA modifications were then investigated in peripheral blood DNA ofpatients undergoing cyclophosphamide treatment as part of the preparative regimen forbone marrow transplant. Twenty cyclophosphamide-derived DNA adducts were detected inpatients’ DNA. These findings set the stage for the investigation of the relationship between

each of these DNA modifications and therapy outcome, to indentify any DNA adductassociated with severe toxicity or lower response to the treatment. These investigations areaiming at developing markers to be used to predict response to therapy and adapt theregimen type and dosage in a more precise and personalized fashion.5

The examples presented, show the power of the MS-based methods that we aredeveloping aimed at generating innovative comprehensive tools for the characterization of
exposures in humans and for the elucidation of their effects on DNA. These approaches haveimportant implications for the development of new genotoxicity testing methods, thedevelopment of new biomonitoring tools, and the development and support of improved
cancer prevention and treatment strategies.

References
1. Stornetta A, Villalta PW, Hecht SS, Sturla SJ, Balbo S. Screening for DNA Alkylation
Mono and Cross-Linked Adducts with a Comprehensive LC-MS(3) Adductomic

Approach. Anal Chem. 2015 Dec 1;87(23):11706-13. doi: 10.1021/acs.analchem.5b02759.
Epub 2015 Nov 10. PMID: 26509677; PMCID: PMC5126974.

DNA Adducts

23

2. Melkamu T, Qian X, Upadhyaya P, O'Sullivan MG, Kassie F. Lipopolysaccharide
enhances mouse lung tumorigenesis: a model for inflammation-driven lung cancer. Vet
Pathol. 2013 Sep;50(5):895-902. doi: 10.1177/0300985813476061. Epub 2013 Feb 4. PMID:
23381924; PMCID: PMC4667800.
3. Dator RP, Murray KJ, Luedtke MW, Jacobs FC, Kassie F, Nguyen HD, Villalta PW, Balbo S.

Identification of Formaldehyde-Induced DNA-RNA Cross-Links in the A/J Mouse Lung
Tumorigenesis Model. Chem Res Toxicol. 2022 Nov 21;35(11):2025-2036. doi: 10.1021/acs.
chemrestox.2c00206. Epub 2022 Nov 10. PMID: 36356054; PMCID: PMC10336729.
4. Wilson MR, Jiang Y, Villalta PW, Stornetta A, Boudreau PD, Carrá A, Brennan CA, Chun E,
Ngo L, Samson LD, Engelward BP, Garrett WS, Balbo S, Balskus EP. The human gut
bacterial genotoxin colibactin alkylates DNA. Science. 2019 Feb 15;363(6428):eaar7785.

doi: 10.1126/science.aar7785. PMID: 30765538; PMCID: PMC6407708.
5. Guidolin V, Jacobs FC, MacMillan ML, Villalta PW, Balbo S. Liquid ChromatographyMass Spectrometry Screening of Cyclophosphamide DNA Damage In Vitro and in
Patients Undergoing Chemotherapy Treatment. Chem Res Toxicol. 2023 Aug
21;36(8):1278-1289. doi: 10.1021/acs.chemrestox.3c00008. Epub 2023 Jul 25. PMID:
37490747.

Silvia Balbo, PhD
07/2002
02/2006
03/2006-03/2008
04/2008-04/2014
2014- 20192019_ present

Master in Medicinal Chemistry from the Universityof Torino, Italy
PhD in Medicinal Chemistry from the University ofTorino, Italy
Post-doctoral Fellow at the International Agency for
Research on Cancer, Lyon France
Post-doctoral Fellow/ Research Assistant at theMasonic Cancer Center, University of Minnesota
Assistant Professor in the Divison of Environmental
Health Sciences of the School of Public Heath,
University of Minnesota
Associate Professor in the Divison of Environmental
Health Sciences of the School of Public Heath,
University of Minnesota

24

DNA ADDUCTOMICS IN HUMAN TISSUESHaruhiko Sugimura
Sasaki Institute Sasaki Foundation
2-2 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan(hsugimur@po.kyoundo.jp)“Adduct” is a synthetic term of “addition” and “product”, and “adductomics” is meant

for an approach of comprehensive capture of numerous adducts generated bothendogenously and exogenously in various biological systems. In the context ofenvironmental and/or chemical carcinogenesis, DNA adducts formation, with deficient or

insufficient repair of them, is a pivotal step toward carcinogenic mutagenesis (Fig 1). Fromthe standpoint DNA lesions, DNA adducts including base modifications by endogenous

enzymes, theoretically, are the most proximal or ultimate indicators of exposures, aconsequence of “exposomics”.

DNA adducts in tissues: Since successful formation of artificial cancer in rabbits [1],animal models and in vitro models of chemical carcinogenesis have provided theoreticalbases for how cancer arises, that is, the basis of cancer science. They lead to the concept ofcancer hallmarks and precision therapy of cancers.

Fig.1

Adducts formation in the pathway from exposure to mutations

DNA Adducts

25

In animal models, most often overdosed by chemicals, detections of DNA adducts andinvestigation of subsequent mutation spectrum/signature further established the main

theorem of carcinogenesis down to the details. DNA adducts in these animal models havebeen extensively reviewed focusing several different pathways [2, 3], such as alkyl adducts
and polyaromatic hydrocarbon adducts, according to different target organs.

Extrapolation to human beings: As we know, understandings on human carcinogenesis

based on these basic and experimental findings are dramatically crystalized in recent
achievements, comprehensive cataloguing of somatic DNA changes of various humancancers and reasoning these somatic changes as a reflection of carcinogenesis processincluding environmental exposures and life styles. Large scale acquisition of DNA mutations

based on massive parallel sequencing technology have also paved the way to current
targeted therapy of individual cancers according to individual somatic changes. Recently

clonal and subclonal mutations in apparently normal human tissues became detectable anda possibility to use them as indicators of risk in individuals is argued.

On the other hand, an adductomics approach, detection and interpretation of DNA

adducts in daily medical practice, though we used to expect it help identifying specificexposure and specific high-risk populations or individuals progresses more slowly.

The challenges in detection, identification, and quantitation of DNA adducts in humantissues were and still are in methodology and accessibility. 32P post-labelling method was

widely applied to detect DNA adducts in various settings including human tissues, but
specific identification of DNA adducts, supposed to exist as several different modified bases,
was not available. For the last two decades combination of double mass spectrometry andliquid chromatography enabled us to identify dozens of specific DNA adducts at single
experiment [4]. Now we became able to compare the tissue originated from animals exposed

to experimental carcinogens and from human tissues having cancers and resected by surgery
or autopsy. The findings would provide us additional and applicable information on

straightforward interpretations or misinterpretations of environmental or chemical
carcinogenesis of human and other animals.

Theoretically, when we have chemical standards of DNA adducts (isotopic ones)

synthesized we would be able to identify and estimate as many as the numbers of thesestandard compounds. We called it “adductome map” [4] (Fig 2). The first adductome map

was drawn based on just two autopsy lung tissues from a smoker and a never-smoker. Circle
size in Cartesian plane represent quantities of DNA adducts or modification calculated fromthe LC-MSMS. Annotations of the circles are far from complete, and detectability and semi-quantitation are the greatest challenges, too. Some adducts, nevertheless, are repeatedly

26

H. Sugimura

Fig 2

Adductome map from two lungs, a smoker (dark circle) and a never-smoker (light circle). From
the reference 4. Only some of the circles have annotations.

detected in human tissues.
Lipid peroxidation derived DNA adducts from different geographical populations:

Lipid peroxidation derived DNA adducts are detected at considerable levels in variousorgans of human tissues [5]. Our currently used protocol to identify and quantitate isrelatively stable and established. Then adductomics approach focusing these 7 DNA adducts

was applied to gastric mucosa from two populations. 12 and 10 cases of gastric mucosa wererecruited from Lujiang Hospital (Lujiang County, Anhui Province, China, the 5th highest

county of all the counties in China, 2010) and Hamamatsu University Hospital (Hamamatsu,
Japan). 7 lipid peroxidation derived DNA adducts were identified semi-quantitatively. Seven

lipid peroxidation-related DNA adducts [1,N6-etheno-2'-deoxyadenosine(εdA), butanone-

etheno-2'-deoxycytidine (BεdC), butanone-etheno-2'-deoxy-5-methylcytidine, butanoneetheno-2'-deoxyadenosine (BεdA), heptanone-etheno-2'-deoxycytidine, heptanone-etheno-2'deoxyadenosine (HεdA) and heptanone-etheno- 2'-deoxyguanosine] were identified in a

total of 22 gastric mucosa samples. The levels of these adducts ranged from 0 to 30,000 per
109 bases. Although the presence of Helicobacter pylori DNA in the mucosa was not related

to these adducts level, the levels of BεdC, BεdA and HεdA were higher in the Japanese

gastric mucosa samples. The profiles of these 7 adduct levels among the 21 cases werecapable of discriminating between the possible origins (China or Japan) of the gastric mucosa

samples. One outlier case was revealed to be EBV associated cases, with massive lymphoid

DNA Adducts

27

cell infiltration. From this experience, we think we will be able to suspect of geographic
origin of cancers by revealing adductomics profile [6].

Geospatial distribution of DNA adducts in human stomach: Gastric cancer arises more

frequently in the lesser curvature than greater curvature, thus geospatial distribution of

DNA adducts, presumable factors for carcinogenesis may influence this preferred region ofcancer occurrence in the human stomach. We did take DNAs from up to 27 sites per casefrom individual resected stomach for gastric cancer. We compared the quantities of C5-

methyl-2′-deoxycytidine, 2′-deoxyinosine, C5- hydroxymethyl-2′-deoxycytidine, N6-methyl2′-deoxyadenosine, εdA, N6-hydroxymethyl- 2′-deoxyadenosine, and C8-oxo-2′-

deoxyguanosine among the spatial sites taken in the stomach. Among these 7 DNA
modifications (adducts), there are no adducts preferentially found in the spatial sites of thestomach, where cancer more often occurs. There are some differences of the quantities ofthese DNA modifications in different locations such as between lesser and greater curvature,proximal and distal, and anterior and posterior walls of the individual stomach, but interindividual differences were greater than intraindividual (geospatial difference in individual
stomach) difference [7].

Life style information and DNA adducts profile: Lifestyle information was taken fromthe clinical and nursing records of 59 subjects who undertook gastrectomy and 7 autopsy
cases who did not have gastric cancer. For smoking, we classified patients into neversmokers, current smokers, and former smokers. Estimation of alcohol equivalents was madewith reference to the past literature. We collected the frequency and amounts of various
alcohol beverages consumed by each individual according to self-reported records, and thetotal intake was calculated from ethanol equivalents (grams) per drink.

The amount of εdA was greater in smokers than never smokers; in drinkers than neverdrinkers; and in the subjects who smoke and drink than those who never smoke nor drink.

There are few reports on life style information and DNA adducts, then these findings should
be corroborated further and mutation signature of tumors in those having relatively large
amount of εdA would be intriguing [8].

DNA adductomics in different organs: Multiple DNA adducts profile are reported inseveral organs using autopsy cases, but the information is still sparse. We investigated DNA
adductomics in urothelial mucosa and kidney. The profile of urothelial mucosa was quite

different from that of gastric mucosa. For example, lipid peroxidation derived DNA adducts
were barely detectable in urothelial mucosa resected for urothelial carcinoma or renal cell
carcinoma [9].

28

H. Sugimura

Hydroxymethyl cytosine as an epigenetic clock: In both gastrointestinal and urothelial

mucosa, 5-hydroxymethyl cytosine in non-tumor mucosa of the stomach or upper urinary
tract without cancer are greater than that in non-tumor mucosa of the stomach or upper

urinary tract with cancer. Decrease of 5-hydroxymethl cytosine may indicate increased
cancer predisposition in these organs [8, 9].

Conclusion: The data on adductomics in human tissue is still scarce especially from thestandpoint of prevailing theories such as alkylation mediated carcinogenesis. Refining themethodology is still necessary to provide us definitive evidences toward our long-heldunderstanding of human carcinogenesis. It takes a combination of pathology, epidemiology,
and molecular biology in addition to population studies [10, 11].
References
1. Ya m a g i w a K , I c h i k a w a K . E x p r i m e n t e l e S t u d i e ü b e r d i e P a t h o g e n e s e d e r
Epithelialgeschwülste. Mitteilungen Med Fakultät Kaiserl Univ Tokyo 1915; 15:295-344.
2. Lawry PD. Historical Origins of Current Concepts of Carcinogenesis. Adv Cancer Res.
1994; 65:17-111.
3. H e c h t S S . B i o c h e m i s t r y, B i o l o g y, a n d C a rc i n o g e n i c i t y o f To b a c c o - S p e c i f i c
N-Nitrosamines. Chem res Toxicol. 1998; 11(6):559-603.
4. Kanaly RA, Hanaoka T, Sugimura H, Toda H, Matsui S, Matsuda T. Development of the
adductome approach to detect DNA damage in humans. Antioxid Redox Signal 2006;
8(5-6): 993-1001.
5. Chou PH, Kageyama S, Matsuda S, Kanemoto K, Sasada Y, Oka M, Shinmura K, Mori H,
Kawai K, Kasai H, Sugimura H, Matsuda T. Detection of lipid peroxidation-induced
DNA adducts caused by 4-oxo-2(E)-nonenal and 4-oxo-2(E)-hexenal in human autopsy
tissues. Chem Res Toxicol. 2010; 23(9):1442-8.
6. Matsuda T, Tao H, Goto M, Yamada H, Suzuki M, Wu Y, Xiao N, He Q, Guo W, Cai Z,

Kurabe N, Ishino K, Matsushima Y, Shinmura K, Konno H, Maekawa M, Wang Y,
Sugimura H. Lipid peroxidation-induced DNA adducts in human gastric mucosa.
Carcinogenesis. 2013;34(1):121-7.
7. Iwashita Y, Ohnishi I, Matsushita Y, Ohtsuka S, Yamashita T, Inaba K, Fukazawa A,
Ochiai H, Matsumoto K, Kurono N, Matsushima Y, Mori H, Suzuki S, Suzuki S, Tanioka

F, Sugimura H. Geospatial Assessments of DNA Adducts in the Human Stomach: A
Model of Field Cancerization. Cancers (Basel). 2021;13(15):3728.
8. Ohnishi I, Iwashita Y, Matsushita Y, Ohtsuka S, Yamashita T, Inaba K, Fukazawa A,
Ochiai H, Matsumoto K, Kurono N, Matsushima Y, Mori H, Suzuki S, Suzuki S, Tanioka
F, Sugimura H. Mass spectrometric profiling of DNA adducts in the human stomach

DNA Adducts

29

associated with damage from environmental factors. Genes Environ. 2021;43(1):12.
9. Matsushita Y, Iwashita Y, Ohtsuka S, Ohnishi I, Yamashita T, Miyake H, Sugimura H. A
DNA adductome analysis revealed a reduction in the global level of C5-hydroxymethyl2'-deoxycytidine in the non-tumoral upper urinary tract mucosa of urothelial carcinoma
patients. Genes Environ. 2021;43(1):52.
10. Sugimura H. Susceptibility to human cancer: From the perspective of a pathologist.
Pathol Int. 2016;66(7):359-68.
11. Natsume H, Szczepaniak K, Yamada H, Iwashita Y, Gędek M, Šuto J, Ishino K, Kasajima R,

Matsuda T, Manirakiza F, Nzitakera A, Wu Y, Xiao N, He Q, Guo W, Cai Z, Ohta T,
Szekely T, Kadar Z, Sekiyama A, Oshima T, Yoshikawa T, Tsuburaya A, Kurono N, Wang
Y, Miyagi Y, Gurzu S, Sugimura H. Non-CpG sites preference in G:C > A:T transition of
TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared toEast Asian countries (China and Japan). Genes Environ. 2023;45(1):1.

Haruhiko Sugimura, MD, PhD
1987

Research Resident, Epidemiology Division, National
Cancer Center Research. Resident, Tokyo
1988-1990
Visiting Fellow, National Cancer Institute, NIH,
Bethesda, USA
1988-1991
Assistant of Professor, Department of Pathology, theUniversity of Tokyo
1991-1995
Associate Professor, Department of the First Pathology,
Hamamatsu University School of Medicine, Hamamatsu
1995-2023
Professor, Department of Tumor Pathology, Hamamatsu
University School of Medicine
2021-2022
Vice Director, Hamamatsu University School of
Medicine
2023- present. Director, Sasaki Institute Sasaki Foundation, Tokyo

30

ELUCIDATION OF DRIVER ADDUCTS OF CANCERDEVELOPMENT USING COMPREHENSIVE ANALYSIS OF
DNA ADDUCTS

Yukari Totsuka
Laboratory of Environmental Toxicology and Carcinogenesis
Nihon University School of Pharmacy,
7-7-1 Narashinodai, Funabashi-shi, Chiba, 274-8555, Japan(totsuka.yukari@nihon-u.ac.jp)

Humans are exposed to many chemicals in daily life, and it can be assumed that many

DNA adducts of exogenous/endogenous origin are generated in our bodies. These adducts

are triggers that introduce mutations in the genome, and are therefore considered to be

involved in cancer development 1, 2) . However, not all adducts contribute to humancarcinogenesis to the same extent. Therefore, it is important to find adducts that contributeto carcinogenesis (driver adducts) to identify carcinogenic factors and risk assessment3). With

rapid advances in mass spectrometry, several research groups have developed DNA
adductome analysis - an emerging method that simultaneously screens for multiple DNA
adducts and provides relevant structural information4-6) . Using this method, we havereported that the carcinogenic mechanism of 1,4-dioxane, a non-genotoxic liver carcinogen,involves DNA adducts derived from inflammatory reactions, and that adducts derived frompiperidine, which is contained in black pepper, are a factor in the development of esophageal
cancer in China7, 8).

On the other hand, whole genome/exome sequencing analyses of human cancers haverevealed the presence of characteristic mutational profiles, called ‘mutational signatures’(https://cancer.sanger.ac.uk/signatures/)9). Mutational signatures have been clearly linked

with relevant environmental exposure. Furthermore, a strong strand bias observed in amutational signature suggests that DNA adducts may induce contextual mutations at therelevant regions. These DNA adducts are considered as ‘driver adducts’ that are significant
for human cancer development.

We have analyzed driver adducts of chemicals that have been suggested to be responsible

for human cancer development. Occupational exposure to aromatic amines (AAs), such as

DNA Adducts

31

o-toluidine (OTD) is an important risk factor for urinary bladder cancer10). Recently, ten

workers at a Japanese chemical plant handling AAs were diagnosed with bladder cancer11).

All patients were exposed primarily to OTD and co-exposed to p-toluidine, o-anisidine,aniline, 2,4-xylidine, acetoacet-o-toluidine (AAOT), and o-chloroaniline. It has been reported
that AAOT can promote urinary bladder carcinogenesis in rats12). Thus, OTD and AAOT

were considered as the causative agents of occupational bladder cancer in this plant. In thepresent study, we evaluated the toxicity of AAs and analyzed the carcinogenic mechanisms
in rat bladders by comprehensive analysis of DNA adducts (DNA adductome)13). DNA wasextracted from the bladder epithelia of rats treated with AAs, including AAOT and OTD,

and adductome analysis was performed. Principal component analysis–discriminant

analysis (PCA-DA) revealed that OTD and AAOT could be clearly separated from thecontrols and others, including aniline and p-toluidine (Figure 1). This finding correspondedwith the morphological changes in the rat urinary epithelium, suggesting that DNA adductformation might contribute to the development of pre-cancerous lesions in the rat urinaryepithelium. After confirming the intensity of each adduct, four adducts named adduct32,
adduct242, adduct489 and adduct557, were screened as having characteristics of the OTD/
AAOT treatment (Figure 2). Comparing with the in-house DNA adduct database, three offour candidates (adduct32, 242 and 489) were identified as oxidative DNA adducts, such as

8-OH-dG, 8-OH-dA and 5-OH-dC based on mass fragmentation together with highresolution accurate mass (HRAM) spectrometry data (Figure 3)13) . Therefore, findings

suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium
exposed to OTD/AAOT. Consequently, another animal experiment determining the effect of

Figure 1 DNA adductome analysis of urinary bladder of rats exposed to AAs.
2D PCA–DA scores of DNA adducts obtained from the adductome analysis. Blue, control; red, OTD;
yellow, AAOT; orange, PT; and green, ANL. The clusters are indicated by ellipses: red for OTD,
yellow-green for AAOT, and blue for others.

32

Y. Totsuka

the antioxidant apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate
oxidase, on urinary epithelial hyperplasia induced by OTD was conducted to confirm thishypothesis. Apocynin treatment reduced moderate simple hyperplasia. In addition, labelingindices of Ki67, γ-H2AX, and 8-OHdG in the urinary bladders of rats treated with OTD/

apocynin were decreased in a dose-dependent manner compared to that of OTD-treated rats.
These findings indicate that oxidative stress may have contributed to the development ofurinary cancer induced by OTD.

Figure 2 he intensity of four adducts screened as characteristics of the OTD/AAOT treatment.
T
Legends are the same as Figure 1

Figure 3 P roduct ion scan and MS/MS fragmentation data of characteristic DNA adducts for OTD/AAOT.

DNA Adducts

33

References
1. Poirier, M.C. (2004) Chemical-induced DNA damage and human cancer risk. Nature
Reviews Cancer, 4, 630-637.

2. Poirier, M.C. (1997) DNA adducts as exposure biomarkers and indicators of cancer risk.
Environmental Health Perspectives, 105 (suppl 4), 907–912.

3. Totsuka, Y., Watanabe, M., Lin, Y. New horizons of DNA adductome for exploring
environmental causes of cancer. Cancer Sci. 2021, 112, 7–15.
4. Balbo, S., Turesky, R.J. and Villalta, P.W. (2014) DNA adductomics. Chemical Research in
Toxicology, 27, 356–366.

5. Kanaly, R.A., Hanaoka, T., Sugimura, H., Toda, H., Matsui, S. and Matsuda, T. (2006)
Development of the adductome approach to detect DNA damage in humans.
Antioxidants & Redox Signaling, 8, 993–1001.
6. Ishino, K., Kato, T., Kato, M., Shibata, T., Watanabe, M., Wakabayashi, K., Nakagama, H.
and Totsuka, Y. (2015) Comprehensive DNA adduct analysis reveals pulmonary
inflammatory response contributes to genotoxic action of magnetite nanoparticles.
International Journal of Molecular Sciences, 16, 3474–3492.
7. Totsuka, Y., Maesako, Y., Ono, H., Nagai, M., Kato, M., Gi, M., Wanibuchi, H., Fukushima,S., Shiizaki, K., Nakagama, H. Comprehensive analysis of DNA adducts (DNAadductome analysis) in the liver of rats treated with 1,4-dioxane. Proc. Jpn. Acad. Ser. B
Phys. Biol. Sci. 2020, 96, 180–187.

8. Totsuka Y, Lin Y, He Y, Ishino K, Sato H, Kato M, Nagai M, Elzawahry A, Totoki Y,
Nakamura H, Hosoda F, Shibata T, Matsuda T, Matsushima Y, Song G, Meng F, Li D, Liu

J, Qiao Y, Wei W, Inoue M, Kikuchi S, Nakagama H, Shan B. DNA adductome analysis
identifies N-nitrosopiperidine involved in the etiology of esophageal cancer in Cixian,
China. 2019, Chem Res Toxicol. 32, 1515-1527.
9. Alexandrov LB, Kim J, Haradhvala NJ, et al. The repertoire of mutational signatures inhuman cancer. Nature. 2020;578:94-101.

10. Cumberbatch, M.G.K.; Noon, A.P. Epidemiology, aetiology and screening of bladdercancer. Transl. Androl. Urol. 2019, 8, 5–11.
11. Nakano, M.; Omae, K.; Takebayashi, T.; Tanaka, S.; Koda, S. An epidemic of bladdercancer: Ten cases of bladder cancer in male Japanese workers exposed to ortho-toluidine.
J. Occup. Health 2018, 60, 307–311.
12. Yukimatsu, N.; Gi, M.; Okuno, T.; Fujioka, M.; Suzuki, S.; Kakehashi, A.; Yanagiba, Y.;
Suda, M.; Koda, S.; Nakatani, T.; et al. Promotion effects of acetoaceto-o-toluidide on
N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder carcinogenesis in rats. Arch.
Toxicol. 2019, 93, 3617–3631.

13. Suzuki S., Gi M., Komiya M., Obikane A., Vachiraarunwong A., Fujioka M., Kakehashi A.,

34

Y. Totsuka

Totsuka Y., Wanibuchi H. Evaluation of the Mechanisms Involved in the Development ofBladder Toxicity following Exposure to Occupational Bladder Cancer Causative
Chemicals Using DNA Adductome Analysis, Biomolecules, 2024, 14, 36.

Yukari Totsuka, PhD
1991-1993
1993-1997
2008-20202021-present

Graduated from Graduate School of Meiji Pharmaceutical
University
Research Staff, Carcinogenesis Division, National
Cancer Center Research InstituteLaboratory Head, Division of Cancer Development
Systems, National Cancer Center Research Institute
Professor, Laboratory of Environmental Toxicology andCarcinogenesis, Nihon University School of Pharmacy

35

AN ANTHOLOGY OF UNUSUAL PATTERNS OF SOMATIC
MUTATIONS IN CANCER GENOMES

Ludmil B. Alexandrov
Department of Cellular and Molecular Medicine
Shu Chien-Gene Lay Department of Bioengineering
University of California, San Diego
9500 Gilman Dr, La Jolla, CA 92093, USA(L2alexandrov@health.ucsd.edu)

Cancer is the most common human genetic disease with all cancers harboring larger

numbers of somatic mutations within their genomes [1]. These mutations are theconsequence of the activities of a multitude of mutational processes, including, the intrinsic

slight infidelity of the DNA replication machinery, exogenous or endogenous mutagen

exposures, enzymatic modifications of DNA, defective DNA repair machinery, and manyothers [1-3]. In some cancer types, a substantial proportion of somatic mutations are known
to be generated by exogenous mutagenic carcinogens, for example, tobacco smoking in lungcancers and ultraviolet light in skin cancers, or by abnormalities of DNA maintenance, forexample, defective DNA mismatch repair in certain colorectal and uterine cancers. In othercancer types, such as cancers of the central nervous system, most somatic mutations are due

to endogenous mutagenic processes with a significant contribution of the clock-likemutagenic processes that are attributed to normal ageing.

Each mutational process leaves an unusual mutagenic pattern on the genomes of cancer

cells, termed, mutational signature [1]. Prior computational developments have demonstrated

that bioinformatics tools can separate distinct mutational signatures when applied tonext-generation sequencing data from cancer samples. We previously developed
SigProfilerExtractor, an automated tool for accurate de novo extraction of mutationalsignatures for all types of somatic mutations, that allows a highly sensitive and specificdetection of mutational signatures in whole-genome and whole-exome sequenced cancers [4].

Applying SigProfilerExtractor to almost five thousand whole-genome and twenty thousand
whole-exome sequenced cancers, encompassing most types of human cancers, has revealed

more than 100 distinct mutational signatures with each signature providing significant novel

36

L. B. Alexandrov

capabilities that can be leveraged to both better prevent and better treat human cancer [1, 4].
In this extended abstract, by using exemplars, I will briefly present the utility of mutationalsignatures related to detecting environmental mutagens and to better selecting cancer
treatment approaches.

Analysis of cancer genomes has allowed identifying known and unknown mutagens thatare linked to environmental exposures and lifestyle choice [1-4]. Notable examples include
single base substitution (SBS) signatures SBS4, SBS7, SBS22, and SBS24. Specifically, signature

SBS4 i s di st i ng u i she d b y C: G >A: T a n d T: A> A: T mut a t i on s, di sp l a y i n g a st ron gtranscriptional strand bias indicative of damage to purine and suggesting a potential
interplay with transcription-coupled nucleotide excision repair (TC-NER). This signature is

exclusive to cancer types epidemiologically linked to tobacco smoking. Moreover, SBS4 ishighly enriched in the genomes of lung cancers from tobacco smokers compared to lifelong
non-smokers. SBS7 is identified in cancer types associated with ultraviolet light exposure,

such as melanomas and skin squamous cell carcinomas, and it is characterized by C:G>T:A
and CC:GG>TT:AA mutations at dipyrimidines. The signature exhibits a pronounced

transcriptional strand bias, suggesting the repair of pyrimidine photodimers by TC-NER.

SBS22 is primarily marked by T:A>A:T substitutions occurring at NpTpG and NpTpA

trinucleotides (mutated base is underlined), displaying a transcriptional strand bias

indicative of adenine damage. This mutational signature is exclusively found in cancer
samples exposed to aristolochic acid. Signature SBS24 also exhibits a C:G>A:T substitution

pattern with a transcriptional strand bias indicative of TC-NER. The mutation patterns of
SBS4 and SBS24 differ from each other with signature SBS24 exclusively found in livercancers from individuals exposed to aflatoxin. Importantly, all four hitherto describedmutational signatures have been experimentally validated by exposing cell lines to therespective mutagens. Thus, the characteristic signatures exhibited by different environmental

exposures and lifestyle choice allows unambiguously detecting mutagenic exposures. For
example, when sequencing liver cancers, one can easily determine whether a specificindividual has been consuming products with aflatoxin by the presence or absence of
signature SBS24. This type of analysis provides an unprecedented opportunity for detecting

previously known and unknown environmental mutagens and for providing mechanistic
understanding of epidemiological results related to change in cancer incidence rates acrossthe world.

In addition to detecting environmental mutagens, analysis of unusual patterns of somatic

mutations allows a better selection of cancer treatment approaches [3-6]. Failure of DNA
repair mechanisms results in the accumulation of specific mutational signatures. Importantly,

phenotypically leveraging these mutational footprints of deficiency allows identifying them

independent of the mechanism causing the actual DNA repair deficiency. A notable example

Genetic Mutational Signature37

for this is signature SBS3. Specifically, SBS3 displays a relatively uniform mutational patternand has been commonly detected in breast, ovary, pancreas, stomach, and esophageal

cancers [1-3]. This signature is linked to inactivating mutations in BRCA1 and BRCA2, crucialcomponents of error-free double-strand break (DSB) repair through homologous
recombination. Interestingly, signature SBS3 is also present in numerous samples without

mutations in BRCA1 and BRCA2, suggesting that the impairment of DSB repair via

homologous recombination might result from BRCA promoter methylation or theinactivation of another gene(s) within the homologous recombination pathway. In line withthis observation, SBS3 has been used as a more accurate predictor of the inability to repair

DSBs through homologous recombination [5-6], making it a valuable biomarker forpredicting response to platinum therapy and PARP inhibitor in breast and ovarian cancer.
This example illustrates the utility of mutational signatures as biomarkers for optimallytargeting cancer treatment. There are currently at least 11 mutational signatures that reflect

failure of DNA repair mechanisms, each of which may provide a potential opportunity forbetter management of cancer patients in the future.

Overall, the presented anthology of unusual patterns of somatic mutations uncovers thevariety of mutational processes that contribute to cancer development, offering insights into

the utility of mutational signatures for understanding cancer causes and informing on cancer
prevention and better cancer treatment.

References
1. Alexandrov LB, Kim J, Haradhvala NJ, Huang MN, Ng AWT, Wu Y, Boot A, Covington
KR, Gordenin DA, Bergstrom EN, Islam SMA, Lopez-Bigas N, Klimczak LJ, McPherson
JR, Morganella S, Sabarinathan R, Wheeler DA, Mustonen V, PCAWG Mutational
Signatures Working Group, Getz G, Rozen SG, Stratton MR, and PCAWG Consortium(2020) The repertoire of mutational signatures in human cancer. Nature 578:94–101.
2. Bergstrom EN, Luebeck J, Petljak M, Khandekar A, Barnes M, Zhang T, Steele CD, Pillay
N, Landi MT, Bafna V, Mischel PS, Harris RS, and Alexandrov LB (2022) Mapping
clustered mutations in cancer reveals APOBEC3 mutagenesis of ecDNA. Nature 602:510–
517.
3. Steele CD, Abbasi A, Islam SMA, Bowes A, Khandekar A, Haase K, Hames-Fathi S, Ajayi
D, Verfaillie A, Dhami P, McLatchie A, Lechner M, Light N, Shlien A, Malkin D, Feber A,
Proszek P, Lesluyes T, Mertens F, Flanagan AM, Tarabichi M, Van Loo P, Alexandrov LB,
and Pillay N (2022) Signatures of copy number alterations in human cancer. Nature,

606:984–991.
4. Islam SMA, Díaz-Gay M, Wu Y, Barnes M, Vangara R, Bergstrom EN, He Y, Vella M,

38

L. B. Alexandrov

Wang J, Teague JW, Clapham P, Moody S, Senkin S, Li YR, Riva L, Zhang T, Gruber AJ,
Steele CD, Otlu B, Khandekar A, Abbasi A, Humphreys L, Syulyukina N, Brady SW,

Alexandrov BS, Pillay N, Zhang J, Adams DJ, Martincorena I, Wedge DC, Landi MT,
Brennan P, Stratton MR, Rozen SG, and Alexandrov LB (2022) Uncovering novel
mutational signatures by de novo extraction with SigProfilerExtractor. Cell Genomics 2(11), 100179.
5. Alexandrov LB, Nik-Zainal S, Siu JC, Leung SY, and Stratton MR (2015) A mutationalsignature in gastric cancer suggests therapeutic strategies. Nature Communications, 6(article# 8683).
6. Tymon-Rosario JR, Manara P, Manavella DD, Bellone S, Hartwich TMP, Harold J, YangHartwich Y, Zipponi M, Choi J, Jeong K, Mutlu L, Yang K, Altwerger G, Menderes G,

Ratner E, Huang GS, Clark M, Andikyan V, Azodi M, Schwartz PE, Alexandrov LB, andSantin AD (2022) Homologous recombination deficiency (HRD) signature-3 in ovarianand uterine carcinosarcomas correlates with preclinical sensitivity to Olaparib, a poly(adenosine diphosphate [ADP]- ribose) polymerase (PARP) inhibitor. Gynecologic
Oncology, 166 (1), 117-125.

Ludmil B. Alexandrov, PhD
2010-20142014-20172018-2021
2021-present

PhD student, University of Cambridge and Wellcome
Sanger Institute
Oppenheimer Fellow, Theoretical Biology andBiophysics, Los Alamos National Lab
Assistant Professor, University of California San Diego
Associate Professor, University of California San Diego

39

TRANS-ANCESTRY MUTATIONAL AND STRUCTURALALTERATION SPECTRUM LANDSCAPE OF GASTRIC CANCER

Tatsuhiro Shibata
Laboratory of Molecular Medicine
The Institute of Medical Science, The University of Tokyo
Division of Cancer Genomics, National Cancer Research Institute4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan(tshibat@ims.u-tokyo.ac.jp)

Gastric cancer (GC) is the third most common cancer killer worldwide. Its incidence

varies from region to region. The highest rates are found in East Asia, Eastern Europe andSouth America, and the lowest in North America. In addition to germline variants, a numberof environmental risk factors have been reported, including chronic infection with

Helicobacter pylori (HP) or Epstein-Barr virus (EBV), and lifestyle factors such as salt intake,alcohol consumption and smoking. However, no comprehensive molecular study has been

conducted to uncover the somatic drivers associated with these geographic andepidemiological differences. Diffuse gastric cancer, a subtype of GC with a poor prognosis,
still has low five-year survival rates and the development of more effective treatment andprevention methods is expected. The diffuse type accounts for about 30% of cases, in which

eradication of H. pylori has little preventive effect. Carcinogenic exposures may differ
between races, and international collaborations can facilitate the discovery and study of
ethnic-specific carcinogens by comparing somatic signature profiles.
1. Trans-ethnic mutational landscape of gastric cancer

We have reported a comprehensive, multi-ancestral landscape of driver events in thelargest (more than 1400 cases) gastric cancer (GC) cohort, including 697 Japanese cases, 328

Caucasian cases, 391 cases from East Asian regions (China, Korea, Singapore and Vietnam)
and 41 cases from other areas1. Our mutational signature analysis revealed that different

mutational processes were represented by mutational signatures between subtypes, EBV
infection and ancestry groups. In particular, COSMIC-Sig16 contributed more frequently todiffuse type cases (P=0.0010), East Asian ancestry (P=2.9e-5) and alcohol consumption

40

T. Shibata(P=4.8e-6). Germline variations in alcohol dehydrogenase 1 B (ADH1B, rs1229984 AA/AG)

and aldehyde dehydrogenase 2 (ALDH2, rs671 AA/AG) were also significantly associatedwith higher COSMIC-Sig16 contribution (P=8.8e-5 and 2.2e-16, respectively).

We compared the sequence contexts of recurrent identical mutations with those of

COSMIC signatures. Characteristically, the hot-spot mutation context of RHOA, a hallmark
diffuse-type driver gene, was similar to that of COSMIC-Sig16. RHOA Y42C hot-spot
mutations were significantly enriched in the COSMIC-Sig16 high cases (P=3.0e-5) andalcohol drinking with ADH1B/ALDH2 germline variants (P=0.011, OR=5.4, P=0.0021,

OR=6.9, respectively) (Figure 1). These results demonstrate that germline variants (ADH1B/
ALDH2) and lifestyle (alcohol consumption) synergistically contribute to the acquisition of

specific driver mutations (RHOA Y42C) and may partly explain the higher incidence ofdiffuse-type GCs in the East Asian population.

2. Structural alteration spectrum of gastric cancer genomes

The assessment of non-coding region driver aberrations, such as extrachromosomal DNA(ecDNA) and complex chromosomal structural aberrations, is still inadequate and theirpatterns and processes remain poorly understood. To explore the molecular andepidemiological background of structural variation (SV) accumulation processes, we
examined 170 GC whole genomes and extracted SV signatures representing SV propensity2.

We identified six rearrangement signatures (RS). Unsupervised hierarchical clustering

Figure 1

RHOA Y32C mutation was enriched with East-Asian ancestry, diffuse-type, drinkers with
ALDH2 rs671 AA/GG allele cases with high COSMIC Sig16.

Genetic Mutational Signature

41

classified 170 cases into seven RS subtypes (subtype RS1-6 and subtype RS2/6); non-random

combinations of RSs revealed unique GC subtypes with one or a few dominant RSs
associated with unique driver events (BRCA1/2 defects, mismatch repair deficiency and TP53
mutation) and epidemiological backgrounds.

SV clusters (SVCs) were frequently detected in cancer genomes and are associated with

oncogene amplification. 36.6% of the total SVs were clustered, forming 3,457 SVCs in GC

g e n o m e s . We t h e n a n n o t a t e d t h e p r o p o s e d m o l e c u l a r m e c h a n i s m s : C F S , L 1

retrotransposition, non-allelic homologous recombination-mediated duplication, andbreakage-fusion-bridge cycles. Our analysis also identified other complex types of SVCs thatwere not classified as described above. They exhibited high SVAF breakpoints at theamplicon edges and contained a group of lower SVAFs densely located within the amplicon.

In some cases, these amplified segments were joined over distances > 2 Mb. We termed theformer a self-joining amplicon and the latter an assembled amplicon, both of which sharedwith similarities to ecDNA (Figure 2).

The chromosomal distribution of these SVCs with amplicon types revealed that they wereenriched in specific genomic regions containing GC oncogenes. In addition, previously

reported ecDNA-associated amplicon loci overlapped with self-joining/assembled amplicon

SVCs. In addition to GC oncogene loci, we discovered SVC-enriched amplified loci, such as
1q42.3, 13q34 and 18q12-13, where the target genes remain to be determined. Among these,the 1q42.3 genomic fragment has been reported to induce cellular senescence, suggesting the

Figure 2

Unique SV patterns are associated with extra-chromosomal DNA (ecDNA)

42

T. Shibata

existence of an unexplored oncogene.
References
1. Totoki Y, Saito-Adachi M, Shiraishi Y, Komura D, Nakamura H, Suzuki A, Tatsuno K,
Rokutan H, Hama N, Yamamoto S, Ono H, Arai Y, Hosoda F, Katoh H, Chiba K, Iida N,
Nagae G, Ueda H, Shihang C, Sekine S, Abe H, Nomura S, Matsuura T, Sakai E, Ohshima

T, Rino Y, Yeoh KG, So J, Sanghvi K, Soong R, Fukagawa A, Yachida S, Kato M, Seto Y,
Ushiku T, Nakajima A, Katai H, Tan P, Ishikawa S, Aburatani H, Shibata T. Multiancestry
genomic and transcriptomic analysis of gastric cancer. Nat Genet. 2023, 55, 581-594.
2. Saito-Adachi M, Hama N, Totoki Y, Nakamura H, Arai Y, Hosoda F, Rokutan H, Yachida S,
Kato M, Fukagawa A, Shibata T. Oncogenic structural aberration landscape in gastriccancer genomes. Nat Commun. 2023, 14, 3688.

Tatsuhiro Shibata, MD, PhD
1990-1994
1995-1997
2010-present
2014-present

Graduated from University of Tokyo, Graduate School of
Medicine University of Tokyo (Pathology)
Postdoctoral researcher, Developmental Biology Center,
University of California, Irvine
Chief, Division of Cancer Genomics, National Cancer
CenterProfessor, Laboratory of Molecular Medicine, The
Institute of Medical Science, The University of Tokyo

43

CAN WE IDENTIFY NEW CAUSES OF CANCER THROUGHLARGE SCALE GENOMIC STUDIES ?

Paul J. Brennan
Head of Genomic Epidemiology
International Agency for Research on Cancer (IARC)
25 avenue Tony Garnier CS 90627 - 69366 LYON CEDEX 07, France(brennanp@iarc.who.int)

International differences in the incidence of many cancer types indicate the existence of

carcinogen exposures that have not been identified by conventional epidemiology yet
potentially make a substantial contribution to cancer burden1. Some carcinogens generate

somatic mutations and, therefore, a complementary strategy for detecting past exposures is

to sequence the genomes of cancers from populations with different incidence rates inferring

underlying causes from differences in patterns of somatic mutations. As an example of thisapproach, we sequenced 962 renal cancers from 11 countries of varying incidence. Somatic
mutation profiles differed between countries. In Romania, Serbia and Thailand, mutationalsignatures likely caused by extracts of Aristolochia plants were present in most cases andrare elsewhere. In Japan, a mutational signature of unknown cause was found in >70% casesand <2% elsewhere. A further mutational signature of unknown cause was ubiquitous but

exhibited higher mutation loads in countries with higher kidney cancer incidence rates(p-value <6 × 10 −18 ). Known signatures of tobacco smoking correlated with tobacco
consumption, but no signature was associated with other known causes of renal cancer

including obesity or hypertension, suggesting non-mutagenic mechanisms of action
underlying these risk factors. The results indicate the existence of multiple, geographicallyvariable, mutagenic exposures potentially affecting 10s of millions of people and illustrate
the opportunities for new insights into cancer causation through large-scale global cancer
genomics.

44

P. J. Brennan

References
1. Brennan, P. & Davey-Smith, G. Identifying Novel Causes of Cancers to Enhance Cancer
Prevention: New Strategies Are Needed. JNCI: Journal of the National Cancer Institute 114,
353–360 (2022).

Paul J. Brennan, PhD
1990-1995
1989-1996
1997-2004
2005-present
2015-present
2021-present

PhD, Graduated from the Faculty of Medicine,
University of Manchester, Genetic Epidemiology
Lecturer in Epidemiology and Medical Statistics, ARC
Epidemiology Research Unit, University of Manchester
Medical School, UK
Scientist in the Unit of Environmental Cancer
Epidemiology and in the Gene-Environment
Epidemiology Group, IARC, France
Head, Section of Genetics, IARC, France
Honorary Professor, University of Bristol, UK
Head, Genomic Epidemiology Branch, IARC, France.

45

MUTATIONAL SIGNATURES OF CHEMOTHERAPEUTICAGENTS AND ENVIRONMENTAL CARCINOGENSDavid H. Phillips
Department of Analytical, Environmental and Forensic Sciences
School of Cancer and Pharmaceutical Sciences, Faculty of Life Sciences and Medicine
King’s College London
Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom(david.phillips@kcl.ac.uk)

Reporter gene assays, in which a single mutation from each experiment can contribute tot h e a s s e m b l y o f a m u t a t i o n s p e c t r u m f o r a n a g e n t , h a v e p ro v i d e d t h e b a s i s f o r

understanding the mutational processes induced by mutagenic agents and for providing
clues to the origins of mutations in human tumours1 . More recently exome and wholegenome sequencing of human tumours has revealed distinct patterns of mutation thatprovide additional clues for the causative origins of cancer. These can be tested by examining
the mutational signatures induced in experimental systems by putative cancer-causing

agents. The generation of multiple mutations per experiment represents a distinct advantage
over single gene assays, which generally produce one mutation per experiment.

We have investigated mutational signatures in transformed mouse embryo fibroblast

cells2, in human induced pluripotent stem cells3 (hIPSCs) and in human tissue organoidcultures. Proof of principle has established that mutational signatures generated in mouse
cells by simulated sunlight and aristolochic acid matched those signatures found in humanmelanomas and urothelial cancers, respectively2. Benzo[a]pyrene produced a signature thatis a close match to the smoking-related signature detected in the respiratory tract tumours of

tobacco smokers2. Each DNA sample was capable of generating up to tens of thousands ofmutations with which to construct the mutational signatures. In hIPSCs, of 79 agents tested

with or without metabolic activation at concentrations that produced measurable
cytotoxicity, 41 yielded characteristic single-base substitution (SBS) mutational signatures3.

Several exhibit similarity with signatures found in human tumours, including those
associated with simulated sunlight, aristolochic acid and benz0[a]pyrene already observedin mouse cells. Additionally, 6 agents produced double-base substitution (DBS) signatures

46

D. H. Phillips

and 8 produced indel (ID) signatures. Investigating mutation asymmetries across genome
topography revealed fully functional mismatch and transcription-coupled repair pathways
in iPSCs, the latter evident as strand bias in mutations.

In human tissue organoids there was a lesser requirement for an exogenous metabolizing

system, as the cultures were, in most cases, capable of activating test agents4, 5 . The

application of duplex sequencing has removed the need for cloning of mutated cells, as

sequencing both DNA strands enables errors in PCR amplification are eliminated from thesignatures. The signatures for benzo[a]pyrene (BaP), aflatoxin B1 (AFB1), aristolochic acid(AA1) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were qualitatively
similar in five different human tissue organoids – gastric, colon, liver, pancreas and kidney –but there were quantitative differences. The mutational signatures of a further 16
environmental carcinogens and 30 cancer chemotherapy agents have been investigated inhuman gastric organoids, providing additional insights into processes underlying the causesof human cancer.

References
1. Phillips, D.H. Mutational spectra and mutational signatures: insights into cancer
aetiology and mechanisms of DNA damage and repair. DNA Repair, 71, 6-11 (2018).
2. Nik-Zainal, S., Kucab, J.E., Morganella, S., Glodzik, D., Alexandrov, L.B., Arlt, V.M.,
Weningen, A., Hollstein, M., Stratton, M.R. and Phillips, D.H. The genome as a record ofenvironmental exposure. Mutagenesis, 30, 763-770 (2015).
3. Kucab, J.E., Zou, X., Morganella, S., Joel, M., Nanda, A.S., Nagy, E., Gomez, C.,

Degasperi, A., Harris, R., Jackson, S.P., Arlt, V.M., Phillips, D.H. and Nik-Zainal, S. A
compendium of mutational signatures of environmental and chemotherapeutic agents.
Cell, 177, 821-836 (2019).
4. Caipa Garcia, A.L., Kucab, J.E., Al-Serori, H., Beck, R.S.S., Fischer, F., Hufnagel, M.,
Hartwig, A., Floeder, A., Balbo, S., Francies, H., Garnett, M., Huch, M., Drost, J., Zilbauer,

M., Arlt, V.M. and Phillips, D.H. Metabolic activation of benzo[a]pyrene by human tissueorganoid cultures. Int. J. Mol. Sci., 24, e606 (2023).
5. Caipa Garcia, A.L., Kucab, J.E., Al-Serori, H., Beck, R.S.S., Bellamri, M., Turesky, R.,
Groopman, J., Francies, H., Garnett, M.J., Huch, M., Drost, J., Zilbauer, M., Arlt, V.M. andPhillips, D.H. The use of human tissue organoid cultures to study the metabolic
activation of dietary carcinogens. Chem. Res. Toxicol., in press (2023).

Genetic Mutational Signature

47

David H. Phillips, BA, PhD, DSc, FRCPath
1971-1975
1975-1978
1978-1980
1980-1981
1981-20112011-present

Magdalen College, Oxford University. BA in Chemistry
Institute of Cancer Research, London. PhD in
BiochemistryPost-doc, University of Wisconsin, USA
Post-doc, Stanford University, USA
Institute of Cancer Research, London
King’s College London

48

ENVIRONMENTAL CARCINOGENESIS, MUTATIONALSIGNATURES AND RATIONAL DRUG DESIGN

Arthur P. Grollman
Laboratory of Chemical Biology
Department of Pharmacological Sciences
Department of Medicine
Renaissance School of Medicine, Health Science Center
Stony Brook University, Stony Brook NY 11794, USA(arthur.grollman@stonybrook.edu)
The Aristolochiaceae family of herbaceous plants has been utilized for medicinal purposes

for more than 2500 years. Toxicologists have long been aware of its toxic properties. The

dramatic revelation that aristolochic acid (AA) is a powerful nephrotoxin and humancarcinogen drew attention to the extensive use of Aristolochia sp. worldwide. Twenty years ormore may elapse before AA-induced renal dysfunction becomes apparent or AA-induced
tumors develop. Some AA-DNA adducts are remarkably stable, having been detected
decades after exposure and lead to A:T → T:A mutations. Mutational analysis of the p53

tumor suppressor gene in upper urothelial cancers from Balkan Endemic Nephropathy
regions and from Taiwan, two area with high exposure to Aristolochia, revealed that thefrequency of A:T → T:A mutations was 78%. The genome-wide mutational signature for AA
has become a powerful tool for dissecting the epidemiology of AA-related pathologies. Thissignature has been detected in healthy tissues and in tumors from patients with many cancer
types including upper tract cancer, bladder cancer, renal cell cancer, liver cancer, and intrahepatic cholangiocarcinoma.

We, in collaboration with Dr. Robert Turesky of University of Minnesota and Dr. Chung-

Hsin Chen of National Taiwan University, are developing “adductomic” techniques to both

simultaneously identify the totality of long-lived bulky DNA-adducts in a patient DNA
sample and to determine the relationship of those adducts to mutational signatures in the

DNA sample. Aristolochic acid derived adducts serve as a valuable “internal control”
expected to be found in patient samples from Taiwan. To date, only aristolochic acid I

derived adducts of dA have been definitively linked to human disease. But several other AA
species exist in most Aristolochia. Dr. Turesky’s adductomic techniques has detected the dG

Nakahara Memorial Lecture49

and dC aristolactam-adducts derived from AAI. Also, for the first time, in rat DNA, he has
detected the dA adducts derived from AAIII, a common AA in Aristolochia. In the rat kidney,
AAIII forms adducts 30-fold more efficiently than AAI.

In the endemic regions of the Balkans contamination of feedstocks by Aristolochia is theroute of exposure to AA. Modern technology can be used to confirm the elimination ofAristolochia from agricultural fields. We are using remote sensing with high-resolution
multispectral cameras mounted on robotic drones. The images obtained are utilized for thedetection and precise localization of Aristolochia plants within wheat fields wherever homemade bread is a principal source of food.

Following bioactivation in the liver, unmodified AAs and their active species aretransported via circulation and elicit carcinogenic effects by binding to DNA of various

tissues. We apply x-ray crystallography, DNA adduct analysis, fluorescence quenching andisothermal titration calorimetry to elucidate the role of human serum albumin (HSA) in AA’s
carcinogenicity. These structural and thermodynamic analyses of AA-HSA interactions yieldsignificant insight into the binding, transport, toxicity and potential allostery of AAs. The
crystallographic data obtained for binding to HSA at 1.9A resolution is amongst the highest
resolution structures available for such ligand protein complexes.

In a separate venture we are repurposing classic medicines as antivirals to face modern

challenges such as the recent COVID-19 pandemic. Emetine has been used in the clinic forthe past century to treat amoebiasis and as a broad-spectrum antiviral agent. However, at thedoses required as an anti-amoebic, emetine has significant side-effects that lead to its

replacement over time. But emetine is effective against coronaviruses at 100-fold lower doses
than required for treating amoebic dysentery. Hence, we are initiating a clinical trial to test
the effectiveness of emetine as an anti-CoV-2 agent.

Arthur P. Grollman, MD
1955-1961
1961-1963
1963-1965
1965-1974
1975-2000
1975-present

Graduate School and Medical Residency, Johns Hopkins
School of Medicine
Research Associate, National Institutes of Health
Postdoctoral Fellow, Molecular Biology, Albert Einstein
College of Medicine
Professor, Pharmacology and Medicine, Albert Einstein
College of Medicine
Chair, Dept Pharmacological Sciences Stony Brook
University
Director, Laboratory of Chemical Biology, Professor of
Medicine, Stony Brook University

50

THE INTERPLAY BETWEEN ENVIRONMENTAL MUTAGENSAND INFLAMMATORY TUMOUR PROMOTING FACTORS IN
DETERMINING CANCER RISK.

Allan Balmain
University of California, San Francisco
Helen Diller Family Comprehensive Cancer Center
1450 3rd Street, San Francisco CA94158, USA(Allan.balmain@ucsf.edu)

Somatic point mutations in cancer driver genes are essential for the development of mostcancers, but deep sequencing studies have demonstrated that similar mutations canaccumulate in normal tissues during the ageing process. These observations have raised
many questions regarding the mechanisms that allow normal tissues to tolerate the presenceof cells carrying potent mutations, and the factors that stimulate the emergence of neoplastic
clones. In particular, the relative contributions of mutations and exogenous environmental

tumour promoters or endogenous processes related to chronic tissue damage, inflammation,and obesity, to cancer risk are presently unclear and highly controversial. We have shown,using mouse tumour models (1) that many known or suspected human carcinogens do not
induce a high mutational burden, or novel mutational signatures, thus raising the possibility
that the role of promoting factors in cancer risk has been underestimated. Moreover, recent

work from the Swanton laboratory has also demonstrated that environmental pollutants
such as air particulates may promote “initiated”, dormant pre-cancer cells to develop intolung cancers, for the first time explaining why non-smokers may develop EGFR mutatedlung adenocarcinomas (2).

In order to quantify the relative contributions of mutations and promoting factors tocancer risk, we have replicated the seminal studies of mouse multistage skin cancer
development carried out in the 1940s (3) suggesting that “initiated cells” are capable ofremaining dormant in normal mouse tissue for most of the mouse lifespan, but can bepromoted to form visible tumours at any time by repeated exposure to an irritant, tumour
promoting plant extract. In spite of the importance of these historic studies in formulatingpresent models of multistage carcinogenesis, and in demonstrating the critical role of theAnimal Models

51

promotion stage in cancer risk, the primary observations have never been replicated in themodern era and examined using next generation sequencing to quantify the numbers of
initiating mutations induced by carcinogens, to demonstrate their persistence in normal

tissue for long periods of time, and their continued responsiveness to tumour promoters.
Similarly, although classical tumour promoters are widely assumed to be non-mutagenic asthey fail to induce point mutations in bacterial assays such as the Ames test, the possibility

that irritant and promoting factors may contribute to increased mutation burden indirectly,

through activation of oxidative stress, DNA damage, or gross chromosomal alterations has
never been adequately investigated.

To address these unanswered questions, we carried out two series of experiments to a)test the hypothesis that chemically-induced tumours arise from long lived stem cells in themouse skin, and b) determine how many mutations are induced by a single initiating dose of
a mutagen (DMBA), and demonstrate that cells carrying these mutations lie dormant in theskin for most of the mouse lifespan without causing any obvious pathology. To address thefirst question, we carried out studies using reporter mice that enabled us to track the stem

cell population(s) that were capable of initiation in vivo using exogenous mutagens andtumour promoters (4). These studies led to the conclusion that chemically initiated tumours

arise from long-lived stem cells in the upper hair follicle rather than from the classical bulge

stem cell region, and furthermore confirmed the prediction by Berenblum and Shubik (3) thatthe most important and rate-limiting step in carcinogenesis was exposure to a promoting

factor rather than initiation. To address the second question, we carried out whole genome

sequencing (WGS) of over 100 mouse skin tumours induced by a range of mouse models
involving initiation by exposure to potent mutagens or genetic induction of Hras or Kras

mutations, followed by repeated exposure to strongly inflammatory processes such as tissue

damaging agents, wounding, and obesity. We also examined the permanence of mutations
induced by carcinogen exposure either during fetal development or in adult mice, followed
by a long latency period of 6-12 months before treatment with a tumour promoter (5).

Taken together, our results suggest that a single mutagenic exposure at any point in an

individual’s lifespan can result in sufficient mutations, in the correct combinations, for

initiation of carcinogenesis, and that these cells are not removed by the immune system orcompetition with normal cells during embryonic development, nor during an extended
period of adult life. We also conclude that the rate-limiting determinant of early tumour
growth is exposure to a tumour promoter or tissue injury, rather than the number of genomic

mutations. Clearly, mutations in cancer driver genes are essential for tumours to develop,
but the process of promotion is also essential to stimulate the outgrowth and clonal selectionof cells carrying these driver mutations. The constant stimulation of wound healing

52

A. Balmain

responses by tumour promoters leads to chromosome changes that have been noted
previously by cytological and genetic analysis of skin tumours (6, 7), and further selection of

specific chromosome changes may help to fix certain cell states essential for advancement
through the pre-malignant stage and progression to malignancy (8).

These considerations help us to reconcile various models of cancer initiation andprogression as being primarily due to (A) sequential accumulation of the point mutations

necessary to activate the various cancer “hallmark” properties, or (B) induction or

spontaneous occurrence of one or very few cancer driver mutations that are adequate forgenetic initiation, but require repeated exposure to agents that cause tissue damage in order toundergo clonal expansion and ultimately progression to malignancy (9). It is likely that large

scale chromosomal events (aneuploidy, loss of heterozygosity or deletions) can help to enable
progression through the biological stages of pre-malignancy until cells arise that have become

independent from continued promoter treatment, and emerge as fully malignant clones (Fig.1)

Figure 1 A (9): he sequential mutation model for cancer development compatible with the originalT
age-incidence data from Armitage and Doll suggesting around 6 “stages”, eachassociated with a mutational event, and acquisition of cancer hallmarks such as self
sufficiency in growth factors, resistance to cell death, recruitment of new bloodvessels (angiogenesis), and ability to invade and metastasize.
B: T he two stage clonal selection model, originally developed using mouse models (3),by which initiation is accomplished by a single treatment with an initiator, generatingdormant mutated cells that are dependent on exposure to a tumour promoting agent
or wounding in order to clonally expand. Tissue regeneration caused by TPA orwounding induces many of the cancer hallmark properties including increased growth
factor production, angiogenesis, invasive behaviour, and immuno-evasion. Chronic
treatment with a promoter is necessary to ensure clonal expansion, and initiated cellclones lose their competitive advantage if promotion is stopped. Progression throughthese stages of pre-neoplasia and malignant development involves acquisition of
independence from continued promoter treatment, through genomic alterations thatlead to genetic instability and loss of tumour suppressor gene activity.

Animal Models

53

Further studies of the exact mechanisms of tumour promotion can have major

implications for development of new strategies for cancer prevention.
References

1. Riva, L. et al. The mutational signature profile of known and suspected humancarcinogens in mice. Nat Genet 52, (11):1189-97 (2020).

2. Lung adenocarcinoma promotion by air pollutants.

Hill W, et al. Nature. 2023. PMID: 37020004
3. Berenblum I, Shubik P. The persistence of latent tumour cells induced in the mouse's skinby a single application of 9:10-dimethyl-1:2-benzanthracene (1949). Br J Cancer 3(3):3846.
4. Kandyba, E. et al. Chemically-induced skin tumours arise from long-lived stem cells of

the upper hair follicle. Submitted.
5. Yun Rose Li et al, The interplay between dormant mutated cells and tumor promotion bychronic tissue damage in determining cancer risk. Submitted
6. Aldaz CM, Trono D, Larcher F, Slaga TJ, Conti CJ. Sequential trisomization ofchromosomes 6 and 7 in mouse skin premalignant lesions.
Mol Carcinog. 1989;2(1):22-6. doi: 10.1002/mc.2940020104.PMID: 2499343
7. Bremner R, Kemp CJ, Balmain A. Induction of different genetic changes by different
classes of chemical carcinogens during progression of mouse skin tumors. Mol Carcinog.
1994 Oct;11(2):90-7. PMID: 7916997
8. Taylor, M. et al Gene networks reveal stem-cell state convergence during preneoplasia
and progression to malignancy in multistage skin carcinogenesis. Submitted.
9. Balmain A. Peto’s paradox revisited: black Box vs mechanistic approaches tounderstanding the roles of mutations and promoting factors in cancer. Eur J Epidemiol.
2022 Dec 13. doi: 10.1007/s10654-022-00933.PMID: 36512199

54

A. Balmain

Allan Balmain, PhD
1962-1966
1966-1969
1969-1971
1971-1972
1999-Present
2001-Present
2004-20192004-Present

University of Glasgow, Scotland (BSc, Hons. Chemistry)
University of Glasgow, Scotland (Ph.D. Organic
Chemistry)
University of Strasbourg, France (Royal Society ofEuropean Programme Fellow)
German Cancer Research Centre, Heidelberg (Alexander
Von Humboldt Stiftung Post Doctoral Fellow)
UCSF, Cancer Research Institute and Dept. ofBiochemistry and Biophysics (Professor in Residence)
UCSF Cancer Center (Barbara Bass Bakar Distinguished
Professor in Cancer Genetics)
UCSF Cancer Center (Director, Genome Analysis Core)UCSF Cancer Center (Co-Director, Cancer Genetics
Program)

55

CARBON NANOTUBES, INNOVATIVE MATERIALS OF THE
21st CENTURY: CARCINOGENICITY EVALUATION OF CNTs
WITH DIFFERENT WALL STRUCTURES

Hiroyuki Tsuda
Nanotoxicology Project
Nagoya City University Graduate School of Medical Sciences
3-1 Tanabedohri, Mizuho-ku, Nagoya 467-8603, Japan(htsuda@phar.nagoya-cu.ac.jp)

Carbon nanotubes (CNT) are composed of concentric one-atom thick graphene cylinders.

The carbon-carbon bonds of graphene are exclusively sp2, giving CNTs remarkable chemicaland physical properties. Consequently, CNTs have numerous industrial applications and theelimination of currently manufactured CNTs would likely have a significant negative impact.
However, the light weight and stability of CNTs also give rise to concern regarding potential
human toxicity. Therefore, hazardous CNTs need to be identified so that appropriate safety

precautions can be put into place that ensure the safe manufacture and use of these
extremely valuable materials.

In spite of the potential hazard to human health presented by respirable CNTs, very few

long term studies of CNTs administered into the lung have been carried out. In October,
2014, IARC assessed the carcinogenicity of CNTs. They concluded that there was sufficientevidence in experiment animals to classify multi-walled CNT-7 (MWCNT-7; also known as

Mitsui-7, MWNT-7) as possibly carcinogenic to humans (group 2B), but that there wasinadequate evidence in experimental animals to classify the carcinogenicity of single-walled

CNTs (SWCNTs) and MWCNTs other than MWCNT-7, placing all CNTs except for
MWNCT-7 as not classifiable as to their carcinogenicity to humans (Group 3) [1]: the final

report was published in 2017 [2]. Notably, at the time of the IARC assessment of CNTs, not asingle 2-year carcinogenic study of CNTs administered into the lung had been published.

Whole-body inhalation studies are extremely complex [3], and there are very few facilities

equipped to carry out such studies. At the time of this writing, only a single 2-year study of

CNTs administered by inhalation has been carried out [4]. Therefore, a reliable method thatadministers respirable CNTs to the lungs that reflects potential human exposure using

56

H. Tsuda

techniques and equipment that are more commonly accessible to research groups needs to
be used for the testing of the many different types of CNTs being produced. We haveestablished “intra-tracheal, intra-pulmonary spraying (TIPS)” as a method for theadministration of CNTs evenly throughout lung using readily available techniques andequipment [5].

Summary of 2-Year Carcinogenicity Studies of CNTs Administered to the Lung

In our studies MWCNTs were administered to rats eight times (total 0.5~1.5 mg/rat) overa 2-week period. The rats are then observed without any further treatment for up to 2 years.
We initially assessed the carcinogenicity of MWCNT-N, a thick straight MWCNT with 40

wall layers [6]. We found that these fibers induced lung tumors and pleural malignant
mesotheliomas. This was the first study to show the carcinogenicity of an MWCNT afteradministration into the lung. Next, a very thick, straight MWCNT, MWCNT-A (150 layers),

and a thin tangled MWCNT, MWCNT-B (15 layers), were tested [7]. Contrary to expectation,
we found that MWCNT-B was more carcinogenic to the lung than MWCNT-A. In this study,

3 rats administered 1.0 mg MWCNT-A developed lung carcinomas while no rats
administered vehicle developed lung carcinomas and no rats administered crocidolite

asbestos (a known human lung carcinogen) developed lung carcinomas, suggesting thatwhile the induction of lung carcinomas in the rats administered MWCNT-A was notstatistically significant, MWCNT-A likely has carcinogenic potential. We then tested a far

thinner CNT, double-walled CNT (DWCNT), and found that DWCNT was also carcinogenic

to the lung [8]. These studies indicate that CNT carcinogenicity in the lung is not restricted tolong, straight CNTs.

The Japan Bioassay Research Center (JBRC) published the results of its 2-year inhalation

exposure to MWCNT-7 in 2016 [4]. This study found that inhalation of MWCNT-7 induced

lung tumors, but unlike MWCNT-N, inhalation of MWCNT-7 did not induce mesothelioma

development. However, in a study by Numano et al., instillation of MWCNT-7 at 1.5 µg/rat
resulted in development of mesothelioma in 18 of 19 rats, the other rat died from a pituitary

tumor. The amount of MWCNT-7 fibers in the lungs of the male rats exposed to 2 mg/m3

MWCNT-7 at the end of 2 years in the JBRC study was 1.8 mg and the instilled dose of

MWCNT-7 in the Numano et al. study was 1.5 mg, suggesting that if the rats in the JBRC
study had survived for an addition 1 to 2 years, they would have developed mesotheliomas,
while in the Numano et al. study, if the rats had not developed malignant mesothelioma,

they would have developed lung tumors. A study by Hojo et al, supports these arguments.
Hojo et al, administered MWCNT-7 by instillation into the lung once every 4 weeks for 2
years [9]. They found that MWCNT-7 induced both lung tumors and mesothelioma.

Overall, administration of thick straight MWCNT-N and MWCNT-7 resulted in lung

Animal Models

57

tumor and mesothelioma development, while administration of thin tangled MWCNT-B andDWCNT resulted in lung tumor development but not mesothelioma development.
A Brief Discussion of Mechanism

Inflammation was assessed in four of the two year studies cited above. Exposure to

MWCNT-7, MWCNT-B, and DWCNT caused chronic inflammation [4, 7, 8, 9]. In a recent
study, rats were administered MWCNT-7, MWCNT-N, MWCNT-B, and DWCNT, and lungtissue was collected 6 weeks after administration and RNA sequences were analyzed.
Analysis of the up and down-regulated RNAs was consistent with inflammatory signaling

in the lung tissues of the CNT treated rats [Data presented at the PTCRF International
Symposium, manuscript in preparation]. These findings are in agreement with those of astudy by Hojo et al. that found that MWCNT-7 induced tumorigenesis in the lung wasconsistent with inflammation-induced carcinogenesis [10].

Importantly, macrophages were able to completely engulf the MWCNT-N, MWCNT-7,

MWCNT-A, MWNCT-B, and DWCNT fibers that we tested (Fig. 1). However, all these fiber
types persisted in the lung. In agreement with these findings, the inhalation studies

conducted by JBRC using MWCNT-7 also demonstrated that fibers that were completely
engulfed by macrophages persisted in the lung, resulting in chronic inflammation and lungtumor development. Harmful fibrous particles like asbestos are known to induce
inflammation via their interaction with macrophages. Fibers that cannot be removed fromthe lung cause repeated cycles of inflammation-induced tissue damage which is followed bytissue repair. These cycles of tissue damage and repair can cause DNA mutations that can be
fixed in daughter cells. One of the primary mechanisms by which non-degradable fibers are

retained in the lung is thought to be the inability of macrophages to completely engulf longfibers resulting in frustrated phagocytosis. This led to the assumption that short fibers may

Figure 1(A) MWCNT-A > 100 layers (Ø150 nm). (B) MWCNT-B 15 layers (Ø7.4 nm).

58

H. Tsuda

not be toxic. However, it is now clear that removal from the lung of MWCNTs that are shortand flexible enough to be completely engulfed by macrophages can also impeded, possibly
by generation of a “STOP” signal [3]. Consequently, short CNTs can also be carcinogenic.

Overall, carcinogenic CNTs have two effects on alveolar macrophages. First, phagocytosisof the CNTs by macrophages results in inflammatory signaling. Second, the phagocytosed
CNTs impede the ability of the macrophages to remove the CNT from the alveoli: CNTs

impede the ability of alveolar macrophages to sequester the CNTs in granulation tissue andthey also impede the macrophage from physically removing the CNTs out of the alveoli into

the ciliated airways. These two effects result in continuous inflammatory signaling andproduction of cytotoxic molecules that will result in the death of the macrophage and releaseof the CNTs, which are then phagocytosed by other macrophages attracted to the site of

inflammation. This results in the chronic inflammation, tissue damage and tissue repair,
DNA mutations, and the eventual development of tumors reported by the studies cited
above.

Importantly, the induction of tissue damage and DNA mutations in the lung differs fromthat in the pleural cavity. In the lung, interaction of biopersistent CNTs with macrophages

leads to chronic inflammation and tissue damage and DNA mutations. In contrast,

intraperitoneal injection studies identified CNTs that induced the development ofmesotheliomas as being straight (acicular) with low levels of agglomerate formation [11, 12,13]. These CNTs were cytotoxic to mesothelial cells and it was concluded that direct injury tomesothelial cells was responsible for the induction of inflammation and the subsequentdevelopment of mesotheliomas. In contrast, thin tangled CNTs were not cytotoxic tomesothelial cells and did not induce mesothelioma development. Mesothelial cell death
results in the release of HMGB1. In humans, HMGB1 release induced by asbestos drives

mesothelial cell transformation and the development of mesothelioma [14]. Thus, CNTs thatare not cytotoxic to mesothelial cells are not carcinogenic in the pleural cavity, but these

CNTs will interact with alveolar macrophages and consequently can be carcinogenic in thelung.

Two Non-carcinogenic CNTs

In another recent study we are investigating two types of CNTs that did not induce

tumorigenesis after 2 years, NC-1 and NC-2. RNA sequence analysis of lung tissues collected
6 weeks after administration of NC-1 indicted that there was a foreign-body reaction in thelung, however, the overall changes in RNA expression induced by NC-1 was very different
from that induced by carcinogenic-positive CNTs. Analysis of RNA expression indicted that
inflammatory signaling induced by NC-1 was was much lower than that induced bycarcinogenic-positive CNTs [Data presented at the PTCRF International Symposium,

Animal Models

Figure 259

S EM examination of CNTs in the lung at 6 weeks. (A) NC-1. (B) NC-2. (C) MWCNT-7.
Arrows indicate the engulfed CNTs.

manuscript in preparation]. The low inflammatory signaling was confirmed by quantitative
real time PCR. In addition, at 6 weeks there were no free fibers in the alveoli, and at the endof the 2 year study period no fibers outside of granulation tissue were present in the lung.
Thus, NC-1 and NC-1 interaction with macrophages induced little or no inflammatory

signaling, and NC-1 and NC-2 did not completely impede the ability of macrophages tosequester these CNTs in granulation tissue or to physically transport them out of the alveoli
and into the ciliated airways.

A notable observation was the difference in the appearance of macrophages interactingwith NT-1 and NT-2 compared to macrophages interacting with MWCNT-7 at 6 weeks (Fig.
2). This is in agreement with the premise that interaction of CNTs with alveolar macrophages
is a key component of the carcinogenic potential of CNTs in the lung.
References

1. Grosse Y, Loomis D, Guyton KZ, Lauby-Secretan B, El Ghissassi F, Bouvard V, et al.
Carcinogenicity of fluoro-edenite, silicon carbide fibres and whiskers, and carbon
nanotubes. Lancet Oncol. 2014;15 13:1427-8; doi: 10.1016/S1470-2045(14)71109-X.
https://www.ncbi.nlm.nih.gov/pubmed/25499275.
2. Group IW. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans;

volume 111-Some Nanomaterials and some Fibres. 2017.
3. Kasai T, Fukushima S. Exposure of Rats to Multi-Walled Carbon Nanotubes: Correlationof Inhalation Exposure to Lung Burden, Bronchoalveolar Lavage Fluid Findings, andLung Morphology. Nanomaterials (Basel). 2023;13 18; doi: 10.3390/nano13182598.
https://www.ncbi.nlm.nih.gov/pubmed/37764628.
4. Kasai T, Umeda Y, Ohnishi M, Mine T, Kondo H, Takeuchi T, et al. Lung carcinogenicity
of inhaled multi-walled carbon nanotube in rats. Part Fibre Toxicol. 2016;13 1:53; doi:

60

H. Tsuda

10.1186/s12989-016-0164-2. https://www.ncbi.nlm.nih.gov/pubmed/27737701.
5. Tsuda H, Alexander DB. Development of Intratracheal Intrapulmonary Spraying (TIPS)

administration as a feasible assay method for testing the toxicity and carcinogenic
potential of multiwall carbon nanotubes. In Vivo Inhalation Toxicity Screening Methods
for Manufactured Nanomaterials. 2019:145-63.
6. Suzui M, Futakuchi M, Fukamachi K, Numano T, Abdelgied M, Takahashi S, et al.
Multiwalled carbon nanotubes intratracheally instilled into the rat lung induce
development of pleural malignant mesothelioma and lung tumors. Cancer Sci. 2016;107

7:924-35; doi: 10.1111/cas.12954. https://www.ncbi.nlm.nih.gov/pubmed/27098557.
7. Saleh DM, Alexander WT, Numano T, Ahmed OHM, Gunasekaran S, Alexander DB, etal. Comparative carcinogenicity study of a thick, straight-type and a thin, tangled-type

multi-walled carbon nanotube administered by intra-tracheal instillation in the rat. Part
Fibre Toxicol. 2020;17 1:48; doi: 10.1186/s12989-020-00382-y. https://www.ncbi.nlm.nih.
gov/pubmed/33054855.
8. Saleh DM, Luo S, Ahmed OHM, Alexander DB, Alexander WT, Gunasekaran S, et al.
Assessment of the toxicity and carcinogenicity of double-walled carbon nanotubes in the
rat lung after intratracheal instillation: a two-year study. Part Fibre Toxicol. 2022;19 1:30;
doi: 10.1186/s12989-022-00469-8. https://www.ncbi.nlm.nih.gov/pubmed/35449069.
9. Hojo M, Maeno A, Sakamoto Y, Ohnuki A, Tada Y, Yamamoto Y, et al. Two-year
intermittent exposure of a multiwalled carbon nanotube by intratracheal instillationinduces lung tumors and pleural mesotheliomas in F344 rats. Part Fibre Toxicol. 2022;19
1 : 3 8 ; d o i : 1 0 . 11 8 6 / s 1 2 9 8 9 - 0 2 2 - 0 0 4 7 8 - 7 . h t t p s : / / w w w. n c b i . n l m . n i h . g o v /pubmed/35590372.
10. Hojo M, Maeno A, Sakamoto Y, Yamamoto Y, Taquahashi Y, Hirose A, et al. Time-Courseof Transcriptomic Change in the Lungs of F344 Rats Repeatedly Exposed to a
Multiwalled Carbon Nanotube in a 2-Year Test. Nanomaterials (Basel). 2023;13 14; doi:
10.3390/nano13142105. https://www.ncbi.nlm.nih.gov/pubmed/37513116.
11. Nagai H, Okazaki Y, Chew SH, Misawa N, Yamashita Y, Akatsuka S, et al. Diameter andrigidity of multiwalled carbon nanotubes are critical factors in mesothelial injury andcarcinogenesis. Proc Natl Acad Sci U S A. 2011;108 49:E1330-8; doi: 10.1073/
pnas.1110013108. https://www.ncbi.nlm.nih.gov/pubmed/22084097.
12. Rittinghausen S, Hackbarth A, Creutzenberg O, Ernst H, Heinrich U, Leonhardt A,
Schaudien D. The carcinogenic effect of various multi-walled carbon nanotubes(MWCNTs) after intraperitoneal injection in rats. Part Fibre Toxicol. 2014;11:59; doi:
10.1186/s12989-014-0059-z. https://www.ncbi.nlm.nih.gov/pubmed/25410479.
13. Sakamoto Y, Hojo M, Kosugi Y, Watanabe K, Hirose A, Inomata A, et al. Comparative

study for carcinogenicity of 7 different multi-wall carbon nanotubes with different
physicochemical characteristics by a single intraperitoneal injection in male Fischer 344

Animal Models

61

rats. J Toxicol Sci. 2018;43 10:587-600; doi: 10.2131/jts.43.587. https://www.ncbi.nlm.nih.
gov/pubmed/30298847.
14. Xue J, Patergnani S, Giorgi C, Suarez J, Goto K, Bononi A, et al. Asbestos induces

mesothelial cell transformation via HMGB1-driven autophagy. Proc Natl Acad Sci U S A.
2020;117 41:25543-52; doi: 10.1073/pnas.2007622117. https://www.ncbi.nlm.nih.gov/
pubmed/32999071.

Hiroyuki Tsuda, MD, PhD
1969
1975
1977-1979
1984
1989-1993
1993-2003
2003-2008
2008~

Graduated from Nagoya City University Medical School
Ph.D. (Pathology)
Post-doctoral researcher, Univ. of Toronto, Canada
Visiting Scientist, German Center for Cancer Research(DKFZ)
Associate Professor, Department of Pathology, Fujita
Health University, School of Medicine
Chief, Experimental Pathology and Chemotherapy
Division, National Cancer Center Research Institute,Tokyo
Professor, Molecular Toxicology, Nagoya City Univ.
Graduate School of Medial Sciences
Professor, Nanotoxicology Project, Nagoya City Univ.

62

COLLABORATIVE CROSS MICE: A POPULATION-BASED MODELFOR OPTIMALLY DECIPHERING GENE-ENVIRONMENTMICROBIOME INTERACTIONS IN CARCINOGENESIS

Jian-Hua Mao
Biological Systems and Engineering Division
Lawrence Berkeley National Laboratory
One Cyclotron Road, Berkeley, CA 94720, USA(JHMao@lbl.gov)

The development of most cancers is caused by the complex interplay between genetic

and environmental factors. The advent of biotechnologies, especially next generation

sequencing technologies, has allowed the rapid discovery of genetic variants that contributeto cancer risk. Hundreds of cancer risk genes have been identified, but only a small
proportion of cancer risk is explained by the genetic variants identified so far, known as“missing heritability”. It has been shown that gene-environment interactions can provide

valuable insights into missing heritability. Indeed, recent studies provide evidence that
additional genetic and environmental factors can be identified in studies that examine geneenvironment interactions.

We have recently realized that microbiomes play important roles in cancer development

and progression. It has been reported that the microbiome is involved in the vast majority ofcancer hallmarks such as genomics instability and mutation, deregulating cellular energetics,
avoiding immune destruction, inducing angiogenesis, sustaining proliferative signal, tumorpromoting inflammation, and activating invasion and metastasis. The microbiome has now

been suggested as a third factor that can affect cancer risk via its interaction with genetics
and environment. Incorporating the contribution of the microbiome to cancer risk will leadto a more complete understanding of cancer and may shed light on strategies to halt or

mitigate those contributions. Moreover, we have increased awareness of the impact ofbidirectional interactions between genetics and microbiota and between environmental

factors and microbiota on cancer risk. Understanding the causal role of the microbiome inthis complex interaction is essential for the development of microbiome-targeted therapy forthe prevention and treatment of cancer.

Animal Models

63

Gene-environment-microbiome interactions in cancer risk: It is widely accepted thatgene-environment interactions influence cancer risk. Environmental exposures can cause
genetic mutations, alter gene expression, etc., whereas interindividual variation in genetic

makeup strongly determines the effect of environmental exposure on cancer risk.
Consequently, investigations on gene–environment interactions are crucial in understanding

how genetic heterogeneity is influenced by various environmental factors, getting moreinsights into the biological nature of cancer.

In recent years, attention has been raised to bidirectional interactions between hostgenetics and microbiome. Increasing number of studies have shown that host genetics is oneof factors that determine the human microbiome composition and diversity. The genome

wide association studies (GWAS) have identified hundreds of genetic variants significantly
correlated to the abundance of distinct gut microbes. On the other hand, the microbiome

strongly influences the host genome. Mounting evidence has shown that gut microbiota
modulates host gene expression through dysregulation of the epigenetic landscape, directinterruption of host signaling cascades, chromatin remodeling, splicing alteration, andmiRNAs. In addition, gut microbiota can produce genotoxic metabolites that cause DNA
damage, subsequently leading to genomics instability, chromosomal aberrations, and genemutations. Therefore, the bidirectional interactions between genetics and microbiome play avital role in cancer risk.

Scientific research has begun to elucidate the various ways in which the humanmicrobiome interacts with environmental exposures. The gut microbiota is a pivotal player

in the toxicity of environmental pollutants, whereas environmental exposures are a crucial
factor for human microbial composition and function. The human microbiome canmetabolize environmental chemicals, either increasing or decreasing their toxicity to themammalian host. Although the roles of microbiomes in the metabolism of environmental
pollutants are established, there are substantial gaps in our understanding of the breadth ofpotential metabolic pathways of environmental pollutants in each microbiome. Alternately,
environmental pollutants can, directly and indirectly, alter the microbiome. These changes in
the microbiome by environmental pollutants can be detected by multi-omics. However, howenvironmental pollutants induce gut microbiota dysbiosis and its influence on cancer riskneed to be investigated in future studies. The collective findings indicate that thebidirectional interactions between environmental factors and microbiome play an important

contribution to cancer risk. Overall, we should consider genetic variants, environmental
factors, and microbiome as multiplicative/additive factors in the prediction of cancer risk.

Collaborative Cross is an optimal mouse model for deciphering gene-environment-

microbiome interactions in carcinogenesis: Many challenges have been faced in performing

64

Jian-Hua Maohuman studies to decipher gene-environment-microbiome interactions in carcinogenesis,

which partly originate from the complex, evolving, and expanding nature of genetic,
environmental, and microbial data collection. Mouse models have contributed greatly to ourcurrent understanding of gene-environment interactions in cancer development andprogression. More importantly, the mouse research community has established new
population-based resources, such as the Collaborative Cross (CC) mouse resource.

The CC is a large panel of new inbred mouse strains derived from a genetically diverse

set of eight inbred founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/LtJ, NZO/HlLtJ,
CAST/EiJ, PWK/PhJ, and WSB/EiJ). A targeted funneling breeding approach was used torandomize the genetic makeup of each inbred line. The CC collection allows studies tomimic the genetic diversity found in the human population and captures nearly 90% of the
known variation of laboratory mice [1]. This system is a new innovative resource forstudying systems genetics, allowing studies to rapidly map genetic loci at high resolution

and identify individual genes involved in complex diseases at unprecedented speed. An

advantage to using this system is that the genotype of each individual strain is alreadyknown, and since each CC line has been inbred, one can make repeated measurements in thesame genetically diverse series of mice. Our studies in CC mice have reported the diversity
in gut microbiome composition and metabolome [2, 3]; spontaneous tumor development [4];cancer susceptibility to thirdhand smoking exposure [5]; blood immune response toradiation exposure [6]; and azoxymethane-induced acute toxicity [7]. In our recent study, we

identified genetic and microbial determinants of azoxymethane-induced colorectal tumor
susceptibility with human translational value. Moreover, we showed critical importance ofinteraction between genetics and microbiome in colorectal tumor susceptibility, which
provides potential novel targets for personalized colorectal cancer prevention and treatment.
Overall, all these studies demonstrated large variation in tumor susceptibility and tumor-

related phenotypes across CC mouse strains, which suggests that CC mice phenotypically
mimic the human populations. Therefore, CC mice are an optimal model for decipheringgene-environment-microbiome interactions in carcinogenesis, in combination with othermouse model such as knockout and gnotobiotic mice, which will provide the information
about the causative mechanisms.

In conclusion, understanding gene-environment-microbiome interactions provides

crucial insights into biological mechanisms and strategies for cancer prevention and control.
References

1. Collaborative Cross C: The genome architecture of the Collaborative Cross mouse genetic
reference population. Genetics 2012, 190:389-401.

Animal Models

65

2. Snijders AM, Langley SA, Kim YM, Brislawn CJ, Noecker C, Zink EM, Fansler SJ, Casey
CP, Miller DR, Huang H, Karpen GH, Celniker SE, Brown JB, Borenstein E, Jansson JK,

Metz TO, Mao JH. Influence of early life exposure, host genetics and diet on the mouse
gut microbiome and metabolome. Nat Microbiol, 2: 16221 (2017).
3. Mao JH, Kim YM, Zhou YX, Hu D, Zhong C, Chang H, Brislawn C, Langley S, Wang Y,

Loulou Peisl BY, Celniker SE, Threadgill DW, Wilmes P, Orr G, Metz TO, Jansson JK,
Snijders AM. Genetic and metabolic links between the murine microbiome and memory.
Microbiome, 8: 53 (2020).
4. Wang P, Wang Y, Langley SA, Zhou YX, Jen KY, Sun Q, Brislawn CJ, Rojas CM, Wahl KL,
Wang T, Fan X, Jansson JK, Celniker SE, Zou X, Threadgill DW, Snijders AM, Mao JH.
Diverse tumor susceptibility in Collaborative Cross mice: Identification of a new mousemodel for human gastric tumorigenesis. Gut, 68: 1942-1952 (2019).

5. Yang H, Wang X, Wang P, He L, Schick SF, Jacob P, Benowitz N, Gundel LA, Zhu C, Xia Y,
Inman JL, Chang H, Snijders AM, Mao JH, Hang B. Thirdhand tobacco smoke exposure
increases the genetic background-dependent risk of pan tumor development in
Collaborative Cross mice. Environ Int, 174: 107876 (2023).
6. He L, Zhong C, Chang H, Inman JL, Celniker SE, Ioakeim-Ioannidou M, Liu KX, HaasKogan D, MacDonald SM, Threadgill DW, Kogan SC, Mao JH, Snijders AM. Geneticarchitecture of the acute and persistent immune cell response after radiation exposure.
Cell Genomics, 3: 100422 (2023).

7. Zhong C, He L, Lee SY, Chang H, Zhang Y, Threadgill DW, Yuan Y, Zhou F, Celniker SE,
Xia Y, Snijders AM, Mao JH. Host genetics and gut microbiota cooperatively contributeto azoxymethane-induced acute toxicity in Collaborative Cross mice. Arch Toxicol, 95:
949-958 (2021).

Jian-Hua Mao, PhD
1993-1997
1997-1999
2016-now
2017-now
2018-now

Graduated from University of Glasgow Department ofRadiation Oncology, University of Glasgow, Glasgow
Postdoctoral researcher, Department of Medical
Oncology, University of Glasgow, Glasgow
co-Director, Berkeley Biomedical Data Science Center(BBDS), Lawrence Berkeley National Laboratory
Senior Scientist, Biological Systems and Engineering
Division, Lawrence Berkeley National Laboratory
Adjunct Professor, Comparative Biochemistry Program,
University of California Berkeley

66

THE HUMAN MICROBIOME AND CANCER PREVENTION

Jiyoung Ahn
NYU Perlmutter Cancer Center
New York University Grossman School of Medicine
522 First Avenue, Smilow Research Building, New York, NY 10016, USA(Jiyoung.Ahn@nyulangone.org)

My research focuses on how the social environment influences the microbiome andcancer prevention. My recent contributions to cancer prevention fall into three maincategories:

1. Microbiome and cancer incidence: In our seminal papers relating periodontal disease to

the risk of orodigestive track cancer 1 , we tested the hypothesis that oral microbiome

dysbiosis is related to orodigestive track cancer. In a large cohort study, we demonstrated

that the oral carriage of commensal beta-proteobacteria predicts the subsequent developmentof head and neck cancer2 and esophageal cancer3 . Subsequently, we found that several

pathogenic periodontal bacteria are associated with an increased risk of pancreatic cancer4.

Mechanistic experiments revealed that cancer-associated oral microbiota play a pivotal rolein metabolizing carcinogens in the oral cavity, and their abundance is significantly altered inheavy smokers and alcohol drinkers5 . These studies were the first to establish the link

between oral microbiota and orodigestive track cancer development. The impact on the field

is further demonstrated by our recent invitation to contribute to the 2022 US Surgeon
General's Report on Oral Health.

2. Microbiome and cancer treatment response: We also demonstrated that the microbiome isa predictor of therapy response in cancer patients. My team also showed, in non-small celllung cancer patients, that microbiome and peripheral blood gene expression biomarkers may

improve risk prediction of recurrence in early-stage non-small cell lung cancer patients6. We
demonstrated, in melanoma patients, that the gut microbiome also is a potential predictor

for recurrence7 and toxicity8 after immune checkpoint inhibitor treatment. Bringing theNon-mutagenic Mechanisms of Cancer Promotion (1)67

microbiome discovery to clinical utilization, we recently developed risk prediction models
for immunotherapy decision-making in a large global immunotherapy trial (NYU Melanoma
SPORE project).

3. Sociobiome and cancer health disparities: As the microbiome is influenced by thephysical and socioeconomic environment9, I developed FAMiLI, the only Asian American-

enriched multi-ethnic cohort of 14,000 individuals, uniquely capturing microbiome,

environmental, acculturation, and sociocultural factors in pre- and post-immigration status.

This is particularly significant, as over 90% of Asian Americans in the U.S. are firstgeneration or second-generation immigrants. In this cohort, we found that the socioeconomic

neighborhood environment10 and dietary acculturation11 influence gut microbiota associatedwith colon carcinogenesis. This cohort also forms the foundation of the NYU Cancer Health
Disparities SPORE, which she serves as PI. As part of the SPORE initiative, our team istesting the novel hypothesis that the human microbiome contributes to racial and social
disparities in oro-digestive track cancers.

Figure 1

The effect of the environment on cancer is a complex set of interactions between multiple
exposures that, alone or more commonly interdependently, affect various structures andfunctions of the microbiome. This figure illustrates how environmental exposures have a
direct impact on the human microbiome, implicated in cancer. These environmental
exposures are influenced by and interrelated with the macroenvironment, including built
environment, and social environment, as well as the microenvironment, including smoking,alcohol, and dietary factors. Although external environmental impacts are illustrated,individual factors, such as age, sex, and genes, also interact with and eventually determine
exposure, dose, and any subsequent response and effect. Ahn et al, Annual Review of
Public Health, 2021

68

J. AhnReferences
1. Ahn J, Segars S, Hayes RB. Periodontal Disease, Porphyromonas Gingivalis (P. gingivalis)
Serum Antibody Levels and Orodigestive Track Cancer Mortality. Carcinogenesis.

2012;33(5):1055-8.
2. Hayes RB, Ahn J, Fan X, Peters BA, Wu J, Yang L, Purdue MP, Freedman ND, Gapstur
SM, Pei Z. Oral microbiome and prospective risk for head and neck cancer. JAMA
oncology. 2018;doi: 10.1001/jamaoncol.2017.4777. [Epub ahead of print].
3. Peters BA, Wu J, Pei Z, Yang L, Purdue MP, Freedman ND, Jacobs EJ, Gapstur SM, Hayes
RB, Ahn J. Oral Microbiome Composition Reflects Prospective Risk for Esophageal
Cancers. Cancer Research. 2017;77(23):6777-6787. doi: 10.1158/0008-5472.CAN-17-1296.
4. Fan X, Alekseyenko V, Jacobs E, Wu J, Peters B, Purdue MP, Miller G, Abnet CC, Hayes
RB, Ahn J. Human oral microbiota and prospective risk for pancreatic cancer: apopulation-based nested case-control study. Gut 2018;67(1):120-127. doi: 10.1136/
gutjnl-2016-312580. Epub 2019 Oct 14.
5. Wu J, Peters B, Dominianni C, Zhang Y, Pei Z, Yang L, Ma Y, Purdue MP, Jacobs EJ,
Gapstur SM, Li H, Alekseyenko AV, Hayes RB, Ahn J. Cigarette smoking and the oral
microbiome in a large study of American adults. ISME Journal. 2016(10):2432-46.
6. Peters BA, Pass HI, Burk RD, Xue X, Goparaju C, Sollecito CC, Grassi E, Segal LN, Tsay
JJ, Hayes RB, Ahn J. The lung microbiome, peripheral gene expression, and recurrencefree survival after resection of stage II non-small cell lung cancer. Genome medicine.
2022;14(1):121. Epub 20221027. doi: 10.1186/s13073-022-01126-7. PubMed PMID:
36303210; PMCID: PMC9609265.
7. Peters BA, Wilson M, Moran U, Pavlick A, Izsak A, Wechter T, Weber JS, Osman I, Ahn J.
Relating the gut metagenome and metatranscriptome to immunotherapy responses inmelanoma patients. Genome medicine. 2019;11(1):61. Epub 20191009. doi: 10.1186/

s13073-019-0672-4. PubMed PMID: 31597568; PMCID: PMC6785875.
8. Usyk M, Pandey A, Hayes RB, Moran U, Pavlick A, Osman I, Weber JS, Ahn J.
Bacteroides vulgatus and Bacteroides dorei predict immune-related adverse events inimmune checkpoint blockade treatment of metastatic melanoma. Genome medicine.
2021;13(1):160. Epub 20211013. doi: 10.1186/s13073-021-00974-z. PubMed PMID:
34641962; PMCID: PMC8513370.
9. Ahn J, Hayes RB. Environmental Influences on the Human Microbiome and Implications

for Noncommunicable Disease. Annual review of public health. 2021;42:277-92. doi: doi:
10.1146/annurev-publhealth-012420-105020; PMCID: PMC8641399.
10. Ahn J, Kwak S, Usyk M, Beggs D, Choi H, Ahdoot D, Wu F, Maceda L, Li H, Im EO, Han
HR, Lee E, Wu A, Hayes R. Sociobiome - Individual and neighborhood socioeconomic
status influence the gut microbiome in a multi-ethnic population in the US. Res Sq. 2023.

Non-mutagenic Mechanisms of Cancer Promotion (1)69Epub 20230419. doi: 10.21203/rs.3.rs-2733916/v1. PubMed PMID: 37131763; PMCID:
PMC10153375.
11. Peters BA, Yi SS, Beasley JM, Cobbs EN, Choi HS, Beggs DB, Hayes RB, Ahn J. US

nativity and dietary acculturation impact the gut microbiome in a diverse US population.
ISME J. 2020;14(7):1639-50. Epub 20200324. doi: 10.1038/s41396-020-0630-6. PubMed
PMID: 32210364; PMCID: PMC7305110.

Jiyoung Ahn, PhD
2000-2005
2006-20082009-present
2016-present

Graduated from Cornell University, Ithaca, NY
Postdoctoral researcher, National Cancer Institute/NIH,
Bethesda, MD
Professor, Department of Population Health, New York
University (NYU) Grossman School of Medicine, New
York, NY
Director of Epidemiology; Associate Center Director for
Population Science, NYU Perlmutter Cancer Center,
New York, NY

70

HISTONE H3.3-SPECIFIC CHAPERONE HIRA IS REQUIREDFOR ACQUIRED TOLERANCE, A FORM OF PHENOTYPIC
PLASTICITY

Fuyuki Ishikawa
Graduate School of Biostudies and Kyoto University Research Administration Center (KURA),
Kyoto University
Yoshida-Honmachi, Sakyo-ku, Kyoto, Kyoto 606-8501, Japan(ishikawa.fuyuki.7u@kyoto-u.ac.jp)

It remains an enigma as to why cancer cells, once they reach an advanced stage, continue

to exhibit robust growth and metastasis despite repeated treatments. Recent studies focus on
the potential implication of non-genetic changes of phenotypes, specifically the concept of“phenotypic plasticity”, in the context of cancer progression. Hormesis collectively
designates phenomena wherein low-dose stresses bestow pro-survival effects on cells andorganisms, as opposed to anti-survival effects induced by high-dose stresses. Acquired
tolerance (AT) is a form of hormesis, wherein pretreatment of cells with low-dose stressconfers resistance to subsequent lethal stresses. It has long been recognized that mild
ischemia induces resistance to subsequent profound ischemia in the heart and brain (alsoknown as post-conditioning tolerance). The anti-aging effect of caloric restriction is arguably
a hormetic effect brought about by mild starvation. They are possibly examples of AT.

Although AT has generated substantial attention to explain various physiological andpathological conditions, its molecular mechanisms remained largely unknown. To address

the question, we first devised experimental systems to study AT in fission yeast. Classicalforward genetics identified histone chaperone HIRA is required for AT in fission yeast1.

In this study, we have extended our analyses to humans and elucidated the molecularmechanism in detail2. Using normal human fibroblast WI-38 cells and cancer cell lines, wehave established low-dose heat stress (referred to as priming, P) elicited AT towards

subsequent lethal heat stress (L). When we knock-downed the HIRA gene encoding a

subunit of the mammalian HIRA trimeric complex, we found that AT was significantly

inactivated. We therefore concluded that HIRA plays a pivotal role in AT across species, fromfission yeast to humans.

Non-mutagenic Mechanisms of Cancer Promotion (1)71HIRA functions as a histone H3.3-specific chaperone. In contrast to the conventional

histone H3, H3.1 and H3.2, H3.3 is synthesized throughout the cell cycle and in nonproliferating cells. It is substituted for H3.1 and H3.3, and activate genes. Employing ChiPseq, we demonstrated that priming heat stress loaded histone H3.3 to stress-responsive

genes, including those encoding heat shock proteins (HSPs). RNA-seq experiments
consistently revealed that HSPs are profoundly up-regulated by priming + lethal (PL)conditions compared to P alone or L alone.

We furthermore conducted the DMBA-TPA-induced skin carcinogenesis in HIRA-/- andcontrol mice. Interestingly, tumors in KO mice showed significantly slow progression andfrequent spontaneous regression of tumors relative to control mice. We conclude that
HIRA-H3.3 axis constitutes a promising target to prevent cancer progression.

This research was conducted through a collaborative effort with Drs. Ryoji Yao (JapaneseFoundation for Cancer Research), Yuichi Wakabayashi (Chiba Cancer Center ResearchInstitute), Ryuichiro Nakato, and Katsuhiko Shirahige (Institute for Quantitative Biosciences,

The University of Tokyo). It was supported by Grants from The Japan Agency for Medical
Research and Development (AMED), MEXT KAKENHI Grant and Princess Takamatsu
Cancer Research Fund.

References
1. Chujo, M., Tarumoto, Y., Miyatake, K., Nishida, E., and Ishikawa, F. (2012). HIRA, aconserved histone chaperone plays an essential role in low-dose stress response via
transcriptional stimulation in fission yeast. J Biol Chem 287(28), 23440-23450. doi:
10.1074/jbc.M112.349944.
2. submitted.

Fuyuki Ishikawa, MD, PhD
1982
1984-1987
1987-1990
1990-1992
1992-2002
2002-2023
2023-

Graduated from the University of Tokyo Medical School
Postdoctoral Researcher, National Cancer Center
Research Institute, Tokyo
Postdoctoral Researcher, Department of Hematology, theUniversity of Tokyo Medical School
Postdoctoral Researcher, Colorado University(Supervisor: TR Cech)
Professor, Tokyo Institute of Technology
Professor, Grad School Biostudies. Kyoto University
Vice President, Kyoto University, and Director KURA

72

EPIGENETICS AND ENVIRONMENTAL ORIGINS OF CANCER

Zdenko Herceg
Epigenomics and Mechanisms BranchInternational Agency for Research on Cancer (IARC)
25 avenue Tony Garnier, CS 90627-69366 LYON CEDEX 07, France(HercegZ@iarc.who.int)

Epigenetics, traditionally defined as stable and heritable changes impacting gene

expression that are independent of changes on DNA sequence, has become a mainstrem incancer research owing to the recognition that epigenetic mechanisms are the key contributors
to cancer development and progression. Epigenome changes, with their ubiquitous presence

and great potential for modulation, have become a major focus in mechanistic studies andbiomarker discovery, and in epigenetics-based cancer therapy and prevention. A plethora ofrecent studies has also highlighted the important role of epigenome deregulation in mediating
gene–environment interactions and their effects throughout the tumorigenesis process. Thisis supported by growing evidence showing that epigenetic changes may be risk factor-specific(“fingerprints”), which should prove instrumental in the discovery of new biomarkers incancer [1]. This progress has been catalysed by advances in epigenomics, including the

emergence of powerful technologies and state-of-the-art in vitro as well as in silico

computational approaches. It is noteworthy that well-established risk factors of cancer, suchas smoking, alcohol, fungal toxins, biological agents, diet and age, as well as air pollution

and endocrine disruptors, have been shown to act though epigenome deregulation [2]. In

recent years, many international cohorts and consortia have been established, which together
with sequencing technologies have galvanized investigations of epigenetic precursors ofcancer and biomarkers of risk in the context of large-scale molecular epidemiology studies,including those aimed at functional characterization of the mechanistically important(driver) genes [3]. The intrinsic reversibility of epigenetic alterations raises the prospect ofthe development of novel therapeutic and preventive strategies.

Non-mutagenic Mechanisms of Cancer Promotion (2)73

Epigenetic signatures of early-life factors and childhood cancer risk

Growing evidence points to an origin in utero of pediatric cancers, when global

redistribution of the epigenome modifications occurs driving tissue differentiation [1].
Recent international efforts are aimed testing the hypothesis that molecular causes of cancers

in children and adolescents innvolve epigenetic changes driven by risk factors exposures.
We and other put forward the hypothesis that epigenetic changes associated with exposure
to environmental/lifestyle factors during pregnancy and disparity drivers can be identified

in blood cells at birth, and that these changes can serve as sensitive biomarkers in primaryand secondary prevention of pediatric cancer. Such studies build on recent advances in
identifying epigenetic markers of early-life factors and deciphering their precursor roles inpediatric cancer and its predisposing (intermediate) phenotypes at critical ages ofdevelopment, exploiting large international networks of prospective and retrospective
consortia some of which are coordinated by the International Agency for Research on Cancer(IARC, epichildcan.iarc.who.int, [4-6]). These include: (i) extending the exposure period fromfetal life to childhood and adolescence by bringing together new consortia, lager sample

sizes, and previously uncharacterized geographical regions, notably different ethnicities anddisparity populations (including low- and middle-income countries, LMICs), and (ii)innovative study designs and approaches (particularly those that can overcome statisticalpower limitations associated with rare diseases such as pediatric cancer). These international
efforts should improve our knowledge of the etiology of pediatric cancer and identify bothnovel biomarkers and clues to causation, thus providing an evidence base for cancer

prevention and strategies aimed at reducing disparities. The ability to monitor exposure tocancer-promoting factors and screen for early changes that suggest increased cancer risk will
have an important impact on the design and follow-up of preventive measures.

Identifying epigenetic driver genes (‘epidrivers’) in cancer and their link to environmental
carcinogens

Recent large-scale international sequencing studies of the human cancer genome,

spearheaded by The Cancer Genome Atlas (TCGA), revealed the high frequency ofmutations in epigenetic regulator genes (ERGs) in common human cancers. We hypothesizedthat these genes may act as “drivers” (“epidrivers”) of tumorigenesis and that they constitutecritical mechanisms promoting epigenome changes that are evident in human malignancies
and potentially synergize with environmental exposure.

We conducted a pan-cancer and comprehensive characterization of genomic andtranscriptomic alterations of ERGs by applying a battery of bioinformatics, biostatistics, andexperimental tools [3, 7]. Our analysis integrated (epi)genome, transcriptome, and DNA
methylome alterations in a comprehensive compendium of 426 ERGs across 33 cancer types,

74

Z. Herceg

comprising >11,000 tumor and normal tissues. We also developed and applied a conceptual

framework (including an orthogonal in vitro screen CRISPR-Cas9 screen targeting all ERGs)
for experimental identification and characterization of the mechanistically important
epidrivers that reshape the epigenome associated with environmental exposure.

We found that, in addition to mutations, copy number alterations (CNAs) in ERGs weremore frequent than previously anticipated. A subset of ERGs was frequently deregulated at

the genetic and/or RNA expression level across many malignancies, consistent with thenotion that disruption of some ERGs represents a shared driver mechanism operating acrossmultiple cancer types. By applying novel bioinformatics and statistical approaches,

integrating the strengths of various driver prediction algorithms, we identified ERGs withpotential driver roles within and across malignancies. We further orthogonally validated incellular models functionally important epidriver genes through CRISPR/Cas9 screen by
identifying a panel of ERGs with a potential epidriver role conferring on cancer cells the trait
associated with specific hallmarks of cancer [3].

Our ongoing studies are aimed at investigating whether specific epidrivers have a central

role in associating environmental exposures with cancer, including multiple phenotypic

traits (and “epigenetic signatures”) across the carcinogenesis process and using different

biological systems. The observed deregulations in ERGs represent snapshot portraits ofaccumulated molecular events (mutations and non-mutational alterations) captured at agiven (often advanced) time point of a multistep process; thus, the timing and role of critical

epidriver events at critical stages in cancer are poorly understood [8]. Therefore, epidriverbased disruption of cellular processes may not only assume a primary role at different stagesof tumorigenesis but also dictate cancer outcome and phenotype. Interestingly, epigenomic(chromatin) organization has been proposed as a major determinant not only of gene
expression programmes but also of the cancer mutational landscape. In line with this,chromatin accessibility and modification patterns were shown to explain a vast majority ofthe variance in mutation rates across cancer genomes, and that epigenomic features are best

predictors of local somatic mutation density. Our ongoing studies are aimed at testing the

hypothesis that changes in the epigenomic patterns as a consequence of epidriverderegulation may not only contribute to changes in the transcriptional programme but also

acting as an important non-mutagenic mechanism of cancer promotion (Figure 1). Thesestudies should make a contribution to understanding the causes of cancer by providing

insights into the mechanisms by which environmental factors promote carcinogenesis, aswell as powerful targets for personalized approaches in early cancer ‘interception’ strategies.

Non-mutagenic Mechanisms of Cancer Promotion (2)Figure 175

H ypothetical model of “epidrivers” enhancing/promoting potential of carcinogens.
Epidriver hits occurring in cells (as results of environmental exposure or stochastic events,
such as those associated with ageing) might trigger or promote the carcinogenic effect of
exposure to environmental carcinogens, leading to the development and progression ofcancer.

References
1. Herceg Z, Ghantous A, Wild CP, Sklias A, Casati L, Duthie SJ, Fry R, Issa JP, Kellermayer R,
Koturbash I, et al: Roadmap for investigating epigenome deregulation andenvironmental origins of cancer. Int J Cancer 2018, 142:874-882.
2. C h u n g F, H e r c e g Z : T h e p r o m i s e s a n d c h a l l e n g e s o f t o x i c o - e p i g e n o m i c s :Environmental chemicals and their impacts on the epigenome. Env Health Perspect
2019, in revision.
3. Halaburkova A, Cahais V, Novoloaca A, Araujo M, Khoueiry R, Ghantous A, Herceg Z:
Pan-cancer multi-omics analysis and orthogonal experimental assessment of
epigenetic driver genes. Genome Res 2020, 30:1517-1532.
4. Joubert BR, Felix JF, Yousefi P, Bakulski KM, Just AC, Breton C, Reese SE, Markunas CA,
Richmond RC, Xu CJ, et al: DNA Methylation in Newborns and Maternal Smoking in
Pregnancy: Genome-wide Consortium Meta-analysis. Am J Hum Genet 2016, 98:680-696.

5. Kupers LK, Monnereau C, Sharp GC, Yousefi P, Salas LA, Ghantous A, Page CM, Reese
SE, Wilcox AJ, Czamara D, et al: Meta-analysis of epigenome-wide association studies
in neonates reveals widespread differential DNA methylation associated withbirthweight. Nat Commun 2019, 10:1893.

6. Merid SK, Novoloaca A, Sharp GC, Kupers LK, Kho AT, Roy R, Gao L, Annesi-Maesano I,

76

Z. Herceg

Jain P, Plusquin M, et al: Epigenome-wide meta-analysis of blood DNA methylation in
newborns and children identifies numerous loci related to gestational age. Genome
Med 2020, 12:25.

7. Vicente A, Novoloaca A, Cahais V, Awada Z, Cuenin C, Spitz N, Carvalho AL,
Evangelista AF, Crovador CS, Reis RM, et al: Cutaneous and acral melanoma crossOMICs reveals prognostic cancer drivers associated with pathobiology and ultraviolet
exposure. Nat Commun 2022, 13:4115.
8. Feinberg AP, Koldobskiy MA, Gondor A: Epigenetic modulators, modifiers andmediators in cancer aetiology and progression. Nat Rev Genet 2016, 17:284-299.

Zdenko Herceg, MSc, PhD
1992-1995
1996-1997
1997-1999
1999-2006
2007-20202011 - now

PhD, University of St Andrews, Scotland, UK
Postdoc scientist, Institute of Molecular Pathology (IMP),
Vienna, Austria
Postdoc, International Agency for Res. on Cancer (IARC)
Lyon France
Scientist, International Agency for Res. on Cancer(IARC) Lyon FranceGroup Head, International Agency for Res. on Cancer(IARC) Lyon France
Section/Branch Head, IARC, Lyon France

77

EPIGENETIC FIELD FOR CANCERIZATION DUE TO CHRONICINFLAMMATION

Toshikazu Ushijima
Hoshi University
2-4-41 Ebara, Shinagawa-ku, Tokyo, 142-8501, Japan(tushijima142@hoshi.ac.jp)

Impact of chronic inflammation on normal-appearing epithelial cells

Chronic inflammation is induced by multiple environmental factors, including bacterial

and viral infection, mechanical irritation, and chemical irritation, and can cause cancers. It is

known that inflammation-associated cancers are heavily influenced by aberrant DNA
methylation, such as gastric cancer induced by Helicobacter pylori (HP) infection-triggered

inflammation [1]. Aberrant DNA methylation is not only present in cancer cells but also innormal-appearing tissues, long before cancer development, involving numerous genes [2].
Total deviation from a normal cell can be defined as an epigenetic burden, which can be
represented by the methylation level of a single marker gene [3]. Importantly, themethylation level of such a marker gene is correlated with gastric cancer risk, reflecting thepresence of an epigenetic field for cancerization (Fig. 1) [1]. The impact of epigenetic
alterations on gastric cancer risk was stronger than point mutations [4].
Induction mechanism of aberrant DNA methylation

Aberrant DNA methylation is induced by chronic inflammation, not by HP itself [5].

Comparison of multiple types of persistent inflammation, that caused by HP infection, highsalt, and high concentration of alcohol, showed that only HP infection-triggered

inflammation can induce severe aberrant DNA methylation, and indicated that IL-1β/TNF-αsignaling and increased Nos2 expression were important [6]. At the molecular level,simultaneous suppression of TET genes, due to activation of NF-κB signalling via IL-1β/

TNF-α signaling, and exposure to nitric oxide, due to increased NOS2 expression, wasresponsible for methylation induction (Fig. 2) [7]. It is noteworthy that many types of chronic

78

T. Ushijima

Fig. 1 Methylation burden in normal-appearing gastric mucosa.
Three representative samples were analyzed for their methylation deviation from normal stomach,and methylation burden was calculated as an inverse of a correlation coefficient. The methylation
burden clearly increased according to risk levels, and was closely correlated with methylation level ofa marker gene (modified from reference #3).

Fig. 2 Methylation induction by synergy of NF-κB activation and NO production.
Treatment of cells with TET3-targeting shRNA only induced mild aberrant DNA methylation (scatter
plot, upper left), and that of cells with NO donor only induced minimal aberrant DNA methylation(scatter plot, bottom left). In contrast, simultaneous treatment of cells with TET3-targeting shRNA
and NO donor induced extensive aberrant DNA methylation (scatter plot, right) (modified fromreference #7).

Non-mutagenic Mechanisms of Cancer Promotion (2)79inflammation associated with cancer are known to have high IL-1β/TNF-α and NOS2
expression, and the same mechanism is likely to be involved.

Clinical application of aberrant DNA methylation in normal tissues

Based the fact that the accumulation level of aberrant DNA methylation is correlated withcancer risk and that chronic inflammation is mechanistically involved in its induction,clinical application of the epigenetic field as a gastric cancer risk marker is on-going. A
multicenter prospective cohort study involving 826 gastric cancer patients cured byendoscopic treatment showed that risk of metachronous gastric cancer can be precisely
predicted by the methylation level of a single marker gene, miR-124a, in gastric mucosa (Fig.

3) [8, 9]. Now, another multicenter prospective study involving 1,880 healthy people withsevere atrophy after HP eradication has reached its target event number with a medianfollow-up period of 4 years, and its results will be disclosed soon.
Conclusion

Environmental exposure, especially chronic inflammation, induces aberrant DNA

Protocol and result of a multicenter prospective study to predict risk of metachronous gastriccancer after endoscopic treatment of initial gastric cancer.
A total of 850 gastric cancer patients cured for their initial gastric cancer were tested for HP infection,
and, if positive, received eradication. Finally, 826 patients were enrolled, and one piece of biopsy was
taken from their antrum. Methylation levels of three marker genes were measured, and they were
followed up by annual endoscopy for five years. When patients were stratified by the methylationlevel of miR-124a-3, the highest quartile (Q4) had much higher risk of metachronous gastric cancer
than the lowest quartile (Q1) with a hazard ratio of 3.0 (95%CI=1.58-5.72) (modified from references#8 and #9).
Fig. 3

80

T. Ushijima

methylation of many genes in normal tissues, and this process is likely to be involved in avariety of human disorders.

References
1. Maekita, T, Nakazawa, K, Mihara, M, Nakajima, T, Yanaoka, K, Iguchi, M, Arii, K,
Kaneda, A, Tsukamoto, T, Tatematsu, M, Tamura, G, Saito, D, Sugimura, T, Ichinose, M
and Ushijima, T. High levels of aberrant DNA methylation in Helicobacter pylori-infected
gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res,

12: 989-995, 2006.
2. Yamashita, S, Nanjo, S, Rehnberg, E, Iida, N, Takeshima, H, Ando, T, Maekita, T,
Sugiyama, T and Ushijima, T. Distinct DNA methylation targets by aging and chronic
inflammation: a pilot study using gastric mucosa infected with Helicobacter pylori. Clin
Epigenetics, 11: 191, 2019.

3. Irie, T, Yamada, H, Takeuchi, C, Liu, YY, Charvat, H, Shimazu, T, Ando, T, Maekita, T,
Abe, S, Takamaru, H, Kodama, M, Murakami, K, Sugimoto, K, Sakamoto, K and
Ushijima, T. The methylation level of a single cancer risk marker gene reflects
methylation burden in gastric mucosa. Gastric Cancer, 26: 667-676, 2023.
4. Yamashita, S, Kishino, T, Takahashi, T, Shimazu, T, Charvat, H, Kakugawa, Y, Nakajima, T,
Lee, YC, Iida, N, Maeda, M, Hattori, N, Takeshima, H, Nagano, R, Oda, I, Tsugane, S,
Wu, MS and Ushijima, T. Genetic and epigenetic alterations in normal tissues havedifferential impacts on cancer risk among tissues. Proc Natl Acad Sci U S A, 115: 13281333, 2018.
5. Niwa, T, Tsukamoto, T, Toyoda, T, Mori, A, Tanaka, H, Maekita, T, Ichinose, M,
Tatematsu, M and Ushijima, T. Inflammatory processes triggered by Helicobacter pylori
infection cause aberrant DNA methylation in gastric epithelial cells. Cancer Res, 70:

1430-1440, 2010.
6. Hur, K, Niwa, T, Toyoda, T, Tsukamoto, T, Tatematsu, M, Yang, HK and Ushijima, T.

Insufficient role of cell proliferation in aberrant DNA methylation induction andinvolvement of specific types of inflammation. Carcinogenesis, 32: 35-41, 2011.
7. Takeshima, H, Niwa, T, Yamashita, S, Takamura-Enya, T, Iida, N, Wakabayashi, M,
Nanjo, S, Abe, M, Sugiyama, T, Kim, YJ and Ushijima, T. TET repression and increased
DNMT activity synergistically induce aberrant DNA methylation. J Clin Invest, 130:

5370-5379, 2020.
8. Asada, K, Nakajima, T, Shimazu, T, Yamamichi, N, Maekita, T, Yokoi, C, Oda, I, Ando, T,
Yo s h i d a , T, N a n j o , S , F u j i s h i ro , M , G o t o d a , T, I c h i n o s e , M a n d U s h i j i m a , T.
Demonstration of the usefulness of epigenetic cancer risk prediction by a multicentre
prospective cohort study. Gut, 64: 388-396, 2015.

Non-mutagenic Mechanisms of Cancer Promotion (2)81

9. Maeda, M, Nakajima, T, Oda, I, Shimazu, T, Yamamichi, N, Maekita, T, Asada, K, Yokoi, C,
Ando, T, Yoshida, T, Nanjo, S, Fujishiro, M, Gotoda, T, Ichinose, M and Ushijima, T. Highimpact of methylation accumulation on metachronous gastric cancer: 5-year follow-up ofa multicentre prospective cohort study. Gut, 66: 1721-1723, 2017.

Toshikazu Ushijima, MD, PhD
1986
1991-1994
1994-1999
1999-2022
2022-present

Graduated from University of Tokyo School of Medicine
Staff Scientist, Carcinogenesis Division, National Cancer
Center Research InstituteSection Head, Carcinogenesis Division, National Cancer
Center Research InstituteChief, Division of Epigenomics (Carcinogenesis
Division), National Cancer Center Research Institute
President, Hoshi University

82

OBESITY, METABOLIC DYSFUNCTION AND CANCER:
UNDERSTANDING CAUSAL MECHANISMS TO GUIDEPREVENTION

Marc J. Gunter
Cancer Epidemiology and Prevention Research Unit
School of Public HealthImperial College London
London, United Kingdom(m.gunter@imperial.ac.uk)

Excess adiposity is a recognized risk factor for at least 13 cancer types including cancersof the colorectum, pancreas, liver, endometrium, ovary, kidney, gastric cardia, thyroid,

gallbladder, breast (post-menopausal), bladder as well as oesophageal adenocarcinoma andleukaemia1. Global obesity rates continue to increase, and therefore the incidence of cancers
attributable to excess adiposity are also likely to rise. In fact, it has been estimated that in the

coming decades, obesity will surpass smoking as the leading cause of cancer in somecountries2. Currently, 12% of adult males and 16% of adult females worldwide are classified

as obese3. Among women, the highest prevalence of obesity is in Central Asia, the Middle

East, and North Africa (age-standardised prevalence 35%). Among men, prevalence ishighest in high income Western countries (30%)3. South Asia is experiencing the fastest
relative increase in obesity rates, with prevalence increasing from 0.4% to 4.6% in recent
decades. Obesity is also increasing in children having risen from 0.7% to 5.6% in girls and
0.9% to 7.8% in boys over the past 30-40 years3.

Approximately 3.6% of all new cancer cases are estimated to be directly attributable tohigh body mass index (BMI) and this proportion is greater for women than for men (5.4%
and 1.9%, respectively)4 . High BMI is the third leading risk factor for the global cancer

burden, behind tobacco and alcohol, accounting for 4.7 and 4.3% of disability-adjusted life
years from cancer, for women and men, respectively5 . This burden is highest in North

America and has increased over time, but the fastest increases are occurring in low- andmiddle-income countries. Greater obesity prevalence in younger populations is also
hypothesized to be contributing to rising cancer diagnoses at earlier ages6.

Population-level strategies for reversing the obesity epidemic, even if successful, may beCancer Prevention Epidemiological Studies

83

insufficient for reducing cancer risk in many people with obesity, and effective, targeted
prevention strategies are urgently needed. Critical questions, however, include whom totarget and how? Tremendous metabolic and physiological heterogeneity occurs amongst
people with obesity and only some develop cancer. Metabolic sub-states exist within thestandard body mass index (BMI) sub-classes (e.g. defined by insulin levels)7,8, and BMI does

not capture variations in body composition, including fat and muscle distribution, metabolic

dysregulation and inflammation/immune function, which may be highly relevant to cancer
development. Biomarkers or well-characterised phenotypes that identify individuals at

higher risk of cancer could guide precision interventions that intercept pro-cancer
mechanisms.

Adipose tissue is a complex endocrine system that interacts with numerous organs tomaintain metabolic homeostasis. Body fat levels correlate with circulating levels of hundreds
to thousands of different metabolites, proteins, and other biomarkers9-12. It is possible that

only a small fraction of these, e.g. 5% or fewer, are relevant to cancer risk. Moreover, only

some individuals with obesity may have elevated levels of the cancer-pertinent factors.

Approximately 10-30% of individuals with obesity maintain metabolic health characterisedby an absence of conditions such as gallstones, diabetes, hyperinsulinemia, dyslipidaemia,

or fatty liver 7,8 . These individuals may have lower cancer risks compared with their

metabolically unhealthy counterparts, though risk remains higher than those who aremetabolically healthy and lean.

Metabolic phenotypes, not always well-captured by BMI, are hypothesized to underlie

the obesity-cancer link and promote the proliferation and survival of cells bearing oncogenic

mutations. These ‘metabotypes’ likely have their origins in dysregulated body composition
and adipose tissue; however, their underlying pro-cancer mechanisms are not understood.
Knowledge of how obesity, body composition and weight change impact the biology ofnormal tissues at anatomical sites where obesity is associated with tumour development, iscritical for understanding causal pathways and developing precise prevention interventions,targeted to mechanistic pathways. Human weight loss intervention studies using bariatricsurgery, diet or pharmacological methods, with collection of tissues and biospecimens pre-and post-weight loss can provide novel insights into how weight loss in obesity can modify
the molecular architecture of normal tissues 13,14 . In parallel, recent developments inepidemiological research, particularly the growing volume of health data and molecularmeasurements (e.g metabolomics, proteomics, imaging), has the potential to enhance ourunderstanding of the biological pathways underlying the obesity-cancer link. Triangulationof such data with those from experimental models could uncover novel mechanisms linking

obesity with cancer and ultimately may inform preventative strategies and formulation ofeffective public health policies.

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M. J. Gunter

References
1. Lauby-Secretan, B. et al. Body fatness and cancer. N. Engl. J. Med. 375, 794–798 (2016)
2. https://news.cancerresearchuk.org/2018/09/24/obesity-could-overtake-smoking-asbiggest-preventable-cause-of-cancer-in-women/
3. Abarca-Gómez, L. et al. Worldwide trends in body-mass index, underweight, overweight,
and obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement
studies in 128·9 million children, adolescents, and adults. The Lancet 390, 2627–2642(2017).
4. Arnold, M. et al. Global burden of cancer attributable to high body-mass index in 2012: a
population-based study. Lancet Oncol. 16, 36–46 (2015).

5. Tran, K. B. et al. The global burden of cancer attributable to risk factors, 2010–19: asystematic analysis for the Global Burden of Disease Study 2019. The Lancet 400, 563–591(2022).
6. Sung, H., Siegel, R. L., Rosenberg, P. S. & Jemal, A. Emerging cancer trends among young
adults in the USA: analysis of a population-based cancer registry. Lancet Public Health 4,

e137–e147 (2019).
7. Murphy, N. et al. Nested case-control study of metabolically defined body size
phenotypes and risk of colorectal cancer in the European Prospective Investigation into
Cancer and Nutrition (EPIC). PLOS Med. 13, e1001988 (2016)
8. Gunter MJ, et al. Breast cancer risk in metabolically healthy but overweight
postmenopausal women. Cancer Res. 2015 Jan 15;75(2):270-4.
9. Moore, S. C. et al. A Metabolomics Analysis of Body Mass Index and Postmenopausal
Breast Cancer Risk. JNCI J. Natl. Cancer Inst. (2018) doi:10.1093/jnci/djx244.
10. Cirulli, E. T. et al. Profound Perturbation of the Metabolome in Obesity Is Associatedwith Health Risk. Cell Metab. 29, 488-500.e2 (2019).
11. Zaghlool, S. B. et al. Revealing the role of the human blood plasma proteome in obesity
using genetic drivers. Nat. Commun. 12, 1279 (2021).

12. Goudswaard, L. J. et al. Effects of adiposity on the human plasma proteome:observational and Mendelian randomisation estimates. Int. J. Obes. 2005 45, 2221–2229(2021).
13. Beeken RJ, et al. The Impact of Diet-Induced Weight Loss on Biomarkers for Colorectal

Cancer: An Exploratory Study (INTERCEPT). Obesity 2017 Nov;25 Suppl 2:S95-S101
14. N a q v i A , e t a l . T h e i m p a c t o f o b e s i t y a n d b a r i a t r i c s u r g e r y o n t h e i m m u n e
microenvironment of the endometrium. Int J Obes (Lond). 2022 Mar;46(3):605-612.

Cancer Prevention Epidemiological Studies

85

Marc J. Gunter, PhD
1994-1998
1998-2002
2003-2006
2007-20112011-20162016-2023
2023-

Oxford University; B.A (hons) Biochemistry
Cambridge University; Ph.D. Biology
National Cancer Institute, NIH; Post-doctoral researcher
Albert Einstein College of Medicine, New York.
Assistant Professor
Imperial College London; Reader
International Agency for Research on Cancer, Head,
Nutrition and Metabolism Branch
Imperial College London; Chair in Cancer Epidemiology
and Prevention

86

MOLECULAR PREVENTION OF CANCER WITH ASPIRIN

Andrew T. Chan
Chief, Clinical and Translational Epidemiology Unit
Director of Epidemiology, MGH Cancer Center
Daniel K. Podolsky Professor of Medicine, Harvard Medical School
Harvard T.H. Chan School of Public Health
American Cancer Society Clinical Research Professor
Massachusetts General Hospital
100 Cambridge Street, 16th Floor
Boston, MA 02114, USA(achan@mgh.harvard.edu)
Over the last several decades, research into cancer prevention has focused on early

detection (e.g. screening) or unselected chemoprevention, defined as the use of agents (e.g.
medications, vitamins, or supplements) for the inhibition, delay, or reversal ofcarcinogenesis. Although screening has had many notable successes, there are barriers to

implementation and inherent limitations to its efficacy. For chemoprevention, the field haslargely tested putative agents through expensive Phase III clinical trials in large populationsof generally unselected individuals which has yielded few successes. A notable exception is
the remarkably consistent evidence demonstrating that aspirin is associated with a lower

risk of colorectal cancer (CRC), which led to a first-if-its-kind U.S. Preventive Services Task

Force (USPSTF) recommendation for population use of aspirin for cancer prevention in 2016.
The 2016 USPSTF recommendations were a milestone, endorsing for the first time low-dose

aspirin for prevention of colorectal cancer in addition to cardiovascular disease (CVD) for
individuals aged 50–59 years with a 10% ten-year cardiovascular risk [1].

A shift in guidelines: In 2022, the USPSTF released new guidelines reversing thisrecommendation based on an evidence synthesis that prioritized the results of new primary

CVD prevention aspirin RCTs completed after 2016: ASCEND (A Study of Cardiovascular
Events in Diabetes); ARRIVE (Aspirin to Reduce Risk of Initial Vascular Events); and

ASPREE (Aspirin to Prevent Events in the Elderly) [2-4]. These trials did not show a lower

risk of CRC. However, a crucial limitation of each of these trials is the short duration offollow-up (median 4.7 to 7.4 years), undoubtedly too brief for a protective benefit of aspirinCancer Prevention Epidemiological Studies

87

to emerge. Prior trials considered for the 2016 recommendations clearly demonstrated that

reduction in CRC and CRC mortality is not evident until at least 10 years, consistent withwhat might be expected for a slowly evolving process such as cancer.

Limitations of ASPREE’s duration of follow-up: The 2022 USPSTF also heavily weighted

findings from ASPREE, which enrolled U.S minority participants of at least 65 years of age
or Australian/U.S. White participants of at least 70 years of age. ASPREE showed low-dose

aspirin increased CRC mortality (as well as mortality from cancer arising from otheranatomic sites) after a median of 4.7 years, a sharp contrast with prior trial data [5].

However, ASPREE did not prespecify CRC mortality as an endpoint, included only a small
number of events, and did not adjust its analysis for multiple comparisons (18 different
cancer endpoints). The trial was also stopped prematurely due to futility for the primary

endpoint (disability and dementia-free survival), increasing the likelihood of artefactual

results. Thus, the USPSTF’s focus on the outlier finding of increased CRC mortality in
ASPREE as a potential signal of broader harm seems an overreach. In a subsequent study

examining incident cancer events rather than cancer deaths, we worked with the ASPREE

team to show that aspirin did not increase the incidence of overall cancer or CRC in ASPREE[6]. Taken together, an increase in cancer death after just 4.7 years of exposure without
anatomic specificity or a corresponding increase in cancer incidence weakens the case thatthe association of aspirin with cancer mortality in ASPREE was causal.

Limitations of ASPREE as a cohort of older adults: In addition, the 2022 USPSTF did notconsider the highly selected nature of the ASPREE cohort before weighing its results against
prior RCTs that have shown a cancer benefit. Not only were ASPREE participants older (atleast age 65-70 years), but the vast majority (85%) had never used aspirin. It should not be

surprising that starting aspirin late in life might be too late to influence cancer initiation

events that occur in mid-adulthood. Indeed, prior primary prevention trials suggest that thebenefits of aspirin for CRC are confined to individuals who start aspirin at a younger age.
For example, JPAD (Japanese Primary Prevention of Atherosclerosis with Aspirin for

Diabetes) showed aspirin resulted in a hazard ratio (HR) for developing CRC of 0.41 (95%CI, 0.15-0.97) for those age less than 65 years [7]. A key study informing the 2016 USPSTF
was long-term follow-up of the WHS (Women’s Health Study), the largest primaryprevention RCT of aspirin among women, which showed that CRC was reduced in thoserandomized to aspirin with a hazard ratio (HR) of 0.80 (95% CI, 0.67-0.97) after 10 years,
with a posttrial HR of 0.58 (95% CI, 0.42-0.80). Importantly, even in WHS, there appeared to
be potential modification of the effect by age: the aspirin group had a HR for CRC of 0.71

(95% CI, 0.52-0.98) among those age 45-54 years, with an attenuation of benefit for those who

were older [8]. In our recent study of nearly 95,000 participants in the Nurses’ Health Studyand Health Professionals Follow-up Study, we prospectively examined aspirin use over88

A. T. Chanmore than four decades and found that aspirin was associated with lower risk of CRC only if
initiated before age 70 [9].

Need for USPSTF to consider prior data: Finally, the 2022 USPSTF did not weigh several

well-conducted trials of aspirin showing a reduced risk of colorectal adenomas amongindividuals with a prior history of CRC or adenoma. These trials are compelling evidence of

causality as adenomas are the precursors to the vast majority of CRC and an establishedsurrogate endpoint for cancer. The 2022 USPSTF excluded these studies, reasoning that they

applied only to “high-risk” populations. However, since adenomas are present in 20-30% of

U.S. adults, many of whom are otherwise healthy, these trials should be considered as

relevant as the “primary prevention” trials relied on by the 2022 USPSTF which included
individuals selected for diabetes (ASCEND), moderate vascular risk (ARRIVE), or older age(ASPREE). The 2022 USPSTF also did not consider the recent results of CAPP2 (Colorectal
Adenoma/carcinoma Prevention Program) which convincingly showed aspirin reduced
CRC among participants Lynch syndrome after 10 years [10]. Although a “high-risk”

population, these are the only trial data with CRC as a prespecified primary endpoint andprovide compelling evidence of causality in the association of aspirin with CRC.

The need for precision prevention approaches: Thus, the evidence supporting that

aspirin reduces risk of CRC does not appear to have substantially changed since the 2016

USPSTF recommendation. The 2022 USPSTF’s reframing of the evidence on aspirin and CRC
as “inadequate” is largely based on an unnuanced assessment of primary prevention studies.

Notably, other expert bodies, including the American College of Gastroenterology and theAmerican Gastroenterological Association [11, 12], considered the same available evidence

as the USPSTF did, and they did not change their opinion about the value of aspirin for CRC
prevention in 2021.

The shifting USPSTF recommendations underscore the pitfalls of interpreting imperfectdata for cancer prevention in the interest of formulating broad recommendations based only

on blunt categories of cardiovascular risk and age. It was perhaps unrealistic to expect thatany cancer preventive agent, including aspirin, would make sense for all, or even most,individuals, particularly since resourcing and executing the requisite “one-size-fits-all” largescale trials with long-term follow-up for cancer events are a major challenge. This
underscores the importance of focusing future studies on “precision prevention” approaches
that incorporate risk-stratifying individuals by either clinical or molecular factors to sharpen

clinical recommendations to optimize risk-benefit. For example, starting aspirin at a younger

age during an optimal biological window when cancers initiate yet bleeding risk is low may
be a more impactful option than continuing the push to lower the age for screening eligibility

to stem the rise in early onset CRC. In the meantime, the 2022 USPSTF guidelines
unfortunately may serve to confuse patients and providers, potentially narrowing the optionCancer Prevention Epidemiological Studies

89

for an individualized discussion of the risks and benefits. If so, this would be a disservice toadvancing the field of cancer prevention for which there remains a high unmet need for lowcost, widely available agents with an established safety profile that can be used for the right
person, at the right time, in the right way.

Aspirin and liver cancer: Additional opportunities: Metabolic dysfunction-associatedsteatotic liver disease (MASLD) represents the most common cause of chronic liver diseasein Western countries, affecting over 30% of U.S. adults [13]. Up to one-third of patients with
MASLD develop progressive steatohepatitis and fibrosis, which can lead to cirrhosis,

hepatocellular carcinoma (HCC), and death [14, 15]. Despite significant investments in thedevelopment of therapies to treat steatotic liver disease (SLD), approved medications that

effectively and safely reverse steatosis, Aspirin may represent a promising and low-cost

strategy for treating MASLD and preventing progression to fibrosis, cirrhosis, and HCC. In

preclinical studies, aspirin prevented intrahepatic platelet activation and Kupffer cellinteractions, which are necessary for the development of steatohepatitis [16], and exhibited
anti-inflammatory and anti-tumor effects, by inhibiting pro-inflammatory cyclooxygenase-2(COX-2) and platelet-derived growth factor signaling [17], and by modulating bioactive

lipids [18]. Consistent with those findings, observational studies associated aspirin use witha lower prevalence of hepatic steatosis [19], and with reduced rates of disease progression toadvanced fibrosis [20]. We have also shown that aspirin is associated with lower risk of livercancer in two separate cohorts [21, 22]. Thus, we recently completed a Phase II randomized

clinical trial in adults with MASLD, to test the efficacy of low-dose aspirin compared toplacebo, for reducing liver fat and markers of inflammation and fibrosis among adults aged
18-70 years with established MASLD without cirrhosis for 6 months. At the 2023 American

Association for the Study of Liver Diseases, we reported that compared to placebo, aspirin
significantly reduced absolute hepatic fat content, the primary outcome. Of four secondary
outcomes, all were statistically significant, favoring aspirin. Nevertheless, given the current

lack of approved treatments for MASLD, aspirin represents an attractive, accessible, andlow-cost option, that now warrants further study. These data are currently submitted for

publication. Taken together with the observational data that aspirin is associated with alower risk of HCC, this trial provides causal data that aspirin can reduce hepatic fat, anestablished surrogate biomarker for fibrosis and eventual development of liver-related

mortality, including liver cancer. These data support the need for further studies examining
aspirin for HCC prevention, for which there is a high unmet need.
References
1. Drew DA, Chan AT. Aspirin in the prevention of colorectal neoplasia. Annu Rev Med.

90

A. T. Chan

2021;72(1):415-430. doi:10.1146/annurev-med-060319-120913
2. US Preventive Services Task Force, Davidson KW, Barry MJ, et al. Aspirin use to prevent

cardiovascular disease: US Preventive Services Task Force recommendation statement.
JAMA. 2022;327(16):1577. doi:10.1001/jama.2022.4983
3. Guirguis-Blake JM, Evans CV, Perdue LA, et al. Aspirin use to prevent cardiovasculardisease and colorectal cancer: updated evidence report and systematic review for the US
Preventive Services Task Force. JAMA. 2022;327(16):1585. doi:10.1001/jama.2022.3337
4. Chubak J, Kamineni A, Buist DS, et al. Aspirin Use for the Prevention of Colorectal Cancer:
An Updated Systematic Evidence Review for the US Preventive Services Task Force. Agency for
Healthcare Research and Quality (US); 2015. Accessed May 9, 2022. http://www.ncbi.
nlm.nih.gov/books/NBK321661/
5. McNeil JJ, Nelson MR, Woods RL, et al. Effect of aspirin on all-cause mortality in thehealthy elderly. N Engl J Med. 2018;379(16):1519-1528. doi:10.1056/NEJMoa1803955

6. McNeil JJ, Gibbs P, Orchard SG, et al. Effect of aspirin on cancer incidence and mortality
in older adults. JNCI: Journal of the National Cancer Institute. 2021;113(3):258-265.
doi:10.1093/jnci/djaa114
7. Ogawa H. Nakayama M, Morimoto, T, et al. Low-dose aspirin for primary prevention ofatherosclerotic events in patients with type 2 diabetes: a randomized controlled trial.
JAMA. 2008;300(18):2134. doi:10.1001/jama.2008.623
8. Cook NR, Lee IM, Zhang SM, et al. Alternate-day, low-dose aspirin and cancer risk: longterm observational follow-up of a randomized trial. Ann Intern Med. 2013;159(2):77.
doi:10.7326/0003-4819-159-2-201307160-00002
9. Guo CG, Ma W, Drew DA, et al. Aspirin use and risk of colorectal cancer among older
adults. JAMA Oncol. 2021;7(3):428. doi:10.1001/jamaoncol.2020.7338

10. Burn J, Sheth H, Elliott F, et al. Cancer prevention with aspirin in hereditary colorectal
cancer (Lynch syndrome), 10-year follow-up and registry-based 20-year data in the
CAPP2 study: a double-blind, randomised, placebo-controlled trial. The Lancet.

2020;395(10240):1855-1863. doi:10.1016/S0140-6736(20)30366-4
11. Shaukat A, Kahi CJ, Burke CA, Rabeneck L, et al. ACG clinical guidelines: colorectal
cancer screening 2021. Am J Gastroenterol. 2021;116(3):458-479. doi:10.14309/
ajg.0000000000001122
12. Liang PS, Shaukat A, Crockett SD. AGA clinical practice update on chemoprevention forcolorectal neoplasia: expert review. Clinical Gastroenterology and Hepatology .

2021;19(7):1327-1336. doi:10.1016/j.cgh.2021.02.014
13. Riazi K, Azhari H, Charette JH, et al. The prevalence and incidence of NAFLD
worldwide: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol
2022;7(9):851-61. doi: 10.1016/S2468-1253(22)00165-0 [published Online First:
2022/07/08]

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14. Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural
history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults.
Aliment Pharmacol Ther 2011;34(3):274-85. doi: 10.1111/j.1365-2036.2011.04724.x [published
Online First: 2011/06/01]
15. Simon TG, Roelstraete B, Khalili H, et al. Mortality in biopsy-confirmed nonalcoholic
fatty liver disease: results from a nationwide cohort. Gut 2020 doi: 10.1136/
gutjnl-2020-322786 [published Online First: 2020/10/11]
16. Malehmir M, Pfister D, Gallage S, et al. Platelet GPIbalpha is a mediator and potential
interventional target for NASH and subsequent liver cancer. Nat Med 2019;25(4):641-55.

doi: 10.1038/s41591-019-0379-5 [published Online First: 2019/04/03]
17. Paik YH, Kim JK, Lee JI, et al. Celecoxib induces hepatic stellate cell apoptosis throughinhibition of Akt activation and suppresses hepatic fibrosis in rats. Gut 2009;58(11):1517-

27. doi: 10.1136/gut.2008.157420 [published Online First: 2009/02/10]
18. Gilligan MM, Gartung A, Sulciner ML, et al. Aspirin-triggered proresolving mediators
stimulate resolution in cancer. Proc Natl Acad Sci U S A 2019;116(13):6292-97. doi:
10.1073/pnas.1804000116 [published Online First: 2019/03/14]
19. Shen H, Shahzad G, Jawairia M, et al. Association between aspirin use and theprevalence of nonalcoholic fatty liver disease: a cross-sectional study from the Third
National Health and Nutrition Examination Survey. Aliment Pharmacol Ther
2014;40(9):1066-73. doi: 10.1111/apt.12944 [published Online First: 2014/09/02]
20. Simon TG, Henson J, Osganian S, et al. Daily Aspirin Use Associated With Reduced Risk
For Fibrosis Progression In Patients With Nonalcoholic Fatty Liver Disease. Clin
Gastroenterol Hepatol 2019;17(13):2776-84 e4. doi: 10.1016/j.cgh.2019.04.061 [published
Online First: 2019/05/12]

21. Simon TG, Duberg AS, Aleman S, et al. Association of Aspirin with Hepatocellular
Carcinoma and Liver-Related Mortality. N Engl J Med 2020;382(11):1018-28. doi: 10.1056/
NEJMoa1912035 [published Online First: 2020/03/12]
22. Simon TG, Ma Y, Ludvigsson JF, Chong DQ, Giovannucci EL, Fuchs CS, Meyerhardt JA,
Corey KE, Chung RT, Zhang X, Chan AT.＊ Association between aspirin use and risk ofhepatocellular carcinoma. JAMA Oncol. 2018 Oct 4. PMCID: PMC6440745.

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A. T. Chan

Andrew T. Chan, MD, MPH
1997
1997-2004
2004
2015-present
2017-present
2017-present
2019-present
2021-present

MD, cum laude, Harvard Medical School
Internal medicine residency, chief resident in medicine,fellow in gastroenterology, Massachusetts General
Hospital
Masters in Public Health, Harvard T.H. Chan School of
Public Health
Chief, Clinical and Translational Epidemiology Unit,
Massachusetts General Hospital
Professor of Medicine, Harvard Medical School
Professor of Immunology and Infectious Diseases,
Harvard T.H. Chan School of Public HealthDirector of Epidemiology, Massachusetts General
Hospital Cancer Center
Daniel K. Podolsky Professor of Medicine, Harvard
Medical School

93

ENVIRONMENTAL IMPACT ON THE GUT MICROBIOME:
MULTI-OMICS TO MECHANISMS OF DIETARY SPINACH

Roderick H. Dashwood
Center for Epigenetics & Disease Prevention
John S. Dunn Chair in Disease Prevention
Texas A&M HEALTH and School of Medicine
2121 W. Holcombe Blvd., Houston, Texas 77030, USA(rdashwood@tamu.edu)

Complex interrelationships govern the dynamic interactions between gut microbes, thehost, and exogenous drivers of disease outcome. A multi-omics approach to cancer

interception by dietary spinach (SPI) was pursued [1, 2] via integrated metagenomic,transcriptomic and metabolomic studies in the polyposis in rat colon (Pirc) model [3]. SPI

fed for 26 weeks (10% wt/wt, freeze-dried in the diet) exhibited significant antitumor
efficacy in the colon and duodenum. Notably, in the Apc-mutant genetic background,
β-catenin remained over-expressed in adenomatous polyps. In both wild type and Apc-

mutant rats, increased gut microbiome diversity after 26-week dietary SPI consumption

coincided with reversal of taxonomic composition. Reshaping of the gut microbiome was notevident after 3-day SPI intake, requiring >1-2 weeks to approximate the increased α-diversity

observed at 26 weeks. By design, the fiber content was kept identical among the AIN-93
custom diets, and alternative mechanisms of action were sought.

Untargeted metabolomics corroborated metagenome prediction following chronic SPI

intake: Metagenomic data implicated linoleate and butanoate metabolism, tricarboxylic acidcycle, and pathways in cancer, which was supported by transcriptomic and metabolomic
analyses. Thus, tumor suppression by SPI involved marked reshaping of the gut microbiome

and changes in host RNA-miRNA networks. When colon polyps were compared withmatched normal-looking tissues via metabolomics, anticancer outcomes were linked to SPI-

derived linoleate bioactives with known proapoptotic/anti-inflammatory mechanisms [4], as
well as N-aceto-2-hydroxybutanoate, consistent with altered butanoate metabolism arisingfrom increased α-diversity of the gut microbiome.

Mechanistic studies after chronic SPI consumption supported HDAC inhibition and94

R. H. Dashwood

Fig. 1

HDAC inhibition and altered interferon-γ signaling by dietary spinach.

altered interferon-γ signaling: Follow-up mechanistic studies were undertaken in humanand murine colon cancer cells. In addition to corroborating the HDAC inhibitory activity ofsingle and combined metabolites, 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) and (S)-2-hydroxybutanoate elevated β2m protein levels, increased cell surface β2m expression, andinduced IL-2 secretion from T-cell hybridomas co-cultured with colon cancer cells, consistent

with enhanced MHC-I cell surface presentation. A working model proposes downregulationof MHC class I-dependent antigen presentation via epigenetic silencing mechanisms thatwere reversed by SPI-derived linoleate and gut microbiome-associated hydroxylated
butanoate metabolites (Fig. 1). Immunoepigenetic regulation at the adenoma stage warrants

further investigation with a view to delaying colectomy and drug intervention in at-riskpatients [5, 6].

References
1. Chen YS, Menon R, Jayaraman A, Lee K, Huang Y, Dashwood WM, Zhang K, Sun D,

Dashwood RH. Dietary spinach reshapes the gut microbiome in an Apc-mutant genetic

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95

background: mechanistic insights from integrated multi-omics. Gut Microbes, 2021, 13,1972756.
2. Chen, YS, Li J, Neja S, Kapoor S, Tovar Perez JE, Tripathi C, Menon R, Jayaraman A, Lee K,
D a s h w o o d W M , Wa n g S , Z h a n g K , K o b a y a s h i K , R a j e n d r a n P, D a s h w o o d R .
Metabolomics of acute vs. chronic spinach intake in an Apc-mutant genetic background:linoleate and butanoate metabolites targeting HDAC activity and IFN-γ signaling. Cells,

2022, 11, 573.
3. Amos-Landgraf JM, Kwong LN, Kendziorski CM, Reichelderfer M, Torrealba J, Weichert

J, Haag JD, Chen K-S, Waller JL, Gould MN, Dove WF. A target-selected Apc-mutant rat
kindred enhances the modeling of familial human colon cancer. Proc. Natl. Acad. Sci.
U.S.A., 2007, 104, 4036-4041.
4. Liu F, Zuo X, Liu Y, Deguchi Y, Moussalli MJ, Chen W, Yang P, Wei B, Tan L, Lorenzi PL,
Gao S, Jaoude JC, Mehdizadeh A, Valentin LA, Wei D, Shureiqi I. Suppression of
membranous LRP5 recycling, Wnt/β-catenin signaling, and colon carcinogenesis by 15LOX-1 peroxidation of linoleic acid in PI3P. Cell Rep, 2020, 32, 108049.
5. Ulusan AM, Rajendran P, Dashwood WM, Yavuz OF, Kapoor S, Gustafson TA, Savage
MI, Brown PH, Sei S, Mohammed A, Vilar E, Dashwood RH. Optimization of erlotinib
plus sulindac dosing regimens for intestinal cancer prevention in an Apc-mutant modelof familial adenomatous polyposis (FAP). Cancer Prev Res (Phila), 2021, 14, 325-336.
6. Samadder NJ, Foster N, McMurray RP, Burke CA, Stoffel E, Kanth P, Das R, Cruz-Correa
M, Vilar E, Mankaney G, Buttar N, Thirumurthi S, Turgeon DK, Sossenheimer M,
Westover M, Richmond E, Umar A, Della’Zanna G, Rodriguez LM, Szabo E, Zahrieh D,
Limburg PJ. Phase II trial of weekly erlotinib dosing to reduce duodenal polyp burden
associated with familial adenomatous polyposis. Gut, 2023, 72, 256-263.

Roderick H. Dashwood, PhD, FRSB
1983-1986
1986-1990
1990-1998
1998-20132013-now
2023

PhD, University of Portsmouth, UK.
Postdoctoral Associate, Oregon State University,
Corvallis, Oregon, USA.
Faculty member, University of Hawaii, Honolulu,
Hawaii, USA.
Director, Cancer Chemoprotection Program, Linus
Pauling Institute, Corvallis, Oregon, USA.
Director, Center for Epigenetics & Disease Prevention,
Texas A&M HEALTH, Houston, Texas, USA.
University Distinguished Professor.

96

ADDUCTOMIC STRATEGIES FOR EMERGING RISK FACTORSIN LIVER CANCERJohn D. Groopman
Department of Environmental Health and Engineering Johns Hopkins Bloomberg School of
Public Health Sidney Kimmel Comprehensive Cancer Center
Johns Hopkins School of Medicine 615 North Wolfe Street
Baltimore, MD 21205, USA(jgroopm1@jhu.edu)

Liver cancer and its impact on different populations and communities. An epidemiologictransition underpinning etiology of liver cancer is underway impacting concepts of cancer
prevention and control interventions for this nearly always fatal disease. The proportional

contributions of traditional etiological factors in HCC - hepatitis B virus (HBV), hepatitis C

virus (HCV), alcohol, aflatoxin and smoking - are changing with the emergence of metabolic
syndrome, obesity, non- alcoholic fatty liver disease (NAFLD), air pollution, and diabetes.

Overall, liver cancer, about 85% is a hepatocellular carcinoma (HCC) diagnosis, accounts for

up to 8.2% of all reported cancer deaths and is among the top three causes of cancer
mortality worldwide (1).

The global distribution of liver cancer incidence varies enormously and unfortunately theburden of this nearly always fatal disease is frequently much greater in low- and middle

income countries (2). HCC has risen rapidly over the past 20 years in the US and Central
America, and this poses an important public health and clinical problem for its diagnosis

and management (3-6). In our continuing work in Central America where liver cancer is highand HBV and HCV infections are low, aflatoxin continues to be a major risk factor for HCC(7). In the most recent report on Cancer Health Disparities in the US the American

Association for Cancer Research outlined that Hispanics have twice the rate of liver cancer
mortality compared to other ethnic groups (8).

Sex differences in liver cancer incidence have been well documented and worldwide thenumber of cases is almost 600,000 among men and 250,000 among women in the latest datareported in 2020 (1). Sex differences are also evident in experimental animal data for liver
carcinogens, exemplified by aflatoxin, where male rats have been found to have an earlierPrecision Prevention

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onset and higher incidence of cancer compared to female animals (9, 10). Thus, theconsistency of the experimental animal and human data points to the important role thatenvironmental exposures play in sex differences in HCC risk. In many high-risk populationsfor HCC in Asia and Africa there has been a 2 to 8-fold greater prevalence of HCC in men
compared to women (gco.iarc.fr). Remarkably in Central American countries, where livercancer rates are the highest in the Western Hemisphere, the incidence in men and women isalmost equivalent (gco.iarc.fr) (11). These findings compel more detailed investigations todiscern environmental, dietary, and genetic risk factors that contribute to the rise of HCC
and its remarkable impact for women.

An emerging risk for liver cancer risk comes from several epidemiologic studies reportingthat ambient air pollution exposure, particularly particulate matter PM2.5, increases the riskof liver cancer (12-15). A US-based ecological study found positive associations with ambient
PM2.5 levels (adjusted IRR per 10 µg/m3: 1.26, 95% CI 1.08-1.47) (15). Similar results arereported from a prospective cohort study in Taiwan, with increased risk for HCC (adjusted

HR per IQR [0.73 µg/m3]: 1.22, 95% CI 1.02-1.47) (12). As part of the European Study of
Cohorts for Air Pollution Effects (ESCAPE) project, there were suggestive associations for air
pollution exposure and liver cancer risk for NO2 and PM2.5 (13). Finally, the Effects of Low-

Level Air Pollution: A Study in Europe (ELAPSE) project showed increased risks with higher
levels of exposure to NO2 (adjusted HR per 10 μg/m3: 1.17, 95% CI 1.02-1.35), and PM2.5(adjusted HR per 5 μg/m3: 1.12, 95% CI 0.92-1.36) (14). In Central America both indoor andoutdoor air pollution events have been well documented (16, 17). Collectively, these reports
motivate the proposed research utilizing our albumin adductomics technology to assess air
pollution exposures (18, 19).

Our team has developed an albumin adductomics approach employing nanoflow liquid

chromatography-high resolution mass spectrometry and parallel reaction monitoring
capable of simultaneously monitoring dozens of Cys34 and other amino acid adducts, in the

low femtomolar range and using less than 2.5 μL of plasma (18, 19). We characterized themagnitude and impact of ambient outdoor air pollution exposures with three repeatedmeasurements over 84 days in non- smoking women. In concordance with seasonally risingambient concentrations of NO2, SO2, and PM10 measured at stationary monitors, we observed

elevations in concentrations of Cys34 adducts of benzoquinone (p<0.05), benzene diol

epoxide (BDE; p<0.05), crotonaldehyde (p<0.01), and oxidation (p<0.001). Regression

analysis revealed significant elevations in oxidation and BDE adduct concentrations of 300%
to nearly 700% per doubling of ambient airborne pollutant levels (p<0.05). Notably, the ratioof irreversibly oxidized to reduced Cys34 rose more than 3-fold during the 84-day period,
revealing a dramatic perturbation of serum redox balance; potentially serving as a portent of

increased pollution-related mortality risk. These oxidation biomarkers are now validated for98

J. D. Groopman

these proposed investigations.

Aflatoxin B1 remains a major risk factor for liver cancer. Aflatoxin B1 (AFB1 ) is notdirectly carcinogenic but must first be transformed in vivo to its ultimate carcinogenic

derivative. The ultimate carcinogenic metabolite of AFB1, AFB1-exo-8,9-epoxide, is producedin the liver through oxidation by cytochrome P450s (20). These activating enzymes are notrate limiting and there is a linear increase in adduct formation across dietary exposures (21).
The AFB1-exo-8,9-epoxide intercalates with DNA, facilitating electrophilic attack on guanine
residues at the N7 position. While the cytochrome P450s produces both an AFB1 -8,9-exo- and

AFB1 -8,9-endo-epoxide, the exo epoxide is ~103 times as mutagenic as the endo, which exhibits
negligible reactivity towards double-stranded DNA (22). The AFB 1 -exo-8,9-epoxide isultimately responsible for the production of the codon 249 mutation in p53 (10). This
pathway also is critical in the production of a metabolically activated aflatoxin adduct withlysine in albumin and is a biomarker that tracks with DNA damage (23-26).

Many case-control studies have examined AFB1 exposure and HCC, recently summarized(7). Data obtained from cohort studies have the greatest power to determine a true
relationship between an exposure and disease. Using the aflatoxin biomarkers developed in
our laboratory we demonstrated the important role of this AFB1 exposure in the etiology of
HCC. This work, comprising more than 18,000 men in Shanghai, revealed a statisticallysignificant increase in the adjusted relative risk of 3.4 for HCC when AFB1 biomarkers weredetected. For subjects with positive HBsAg detection (a marker of infection with HBV), therelative risk was 7, but for individuals with both urinary aflatoxins and positive HBsAg
status, the relative risk was 59 (27, 28). These results contributed to IARC classifying

aflatoxin as a Group 1 human carcinogen (10). Further, these findings promote thehypothesis that other aflatoxin risk factor interactions could exist that contribute to HCC.

Mechanistic studies have demonstrated that AFB1 mediated DNA damage causes

induction of a guanine to thymine (G→T) transversion mutation in both human samples andexperimental models. This lesion produces a characteristic mutational signature, particularlyin p53 (29,30). Two initial reports found the presence of a G→T transversion hotspot at thethird position of codon 249 (5’- CGG AGG CCC-3’) of the human TP53 gene (31, 32), which

results in the non-synonymous substitution of arginine with serine (R249S) within the p53
protein. These findings have been replicated in many settings of high AFB1 exposure (33, 34)

and data from our team found these same p53 mutations in HCCs from Guatemala and in acollaborative study in Mexico (35, 36).

Aflatoxin-albumin biomarkers document a reduction of liver cancer incidence by morethan 50% in the absence of HBV vaccination. Validated aflatoxin specific biomarkers not
only define exposure but also can define risk and in turn this work has led to the applicationof these biomarkers in preventive intervention trials (37, 38). The aflatoxin-albumin

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biomarkers permitted us to characterize a concordance of a nearly 4000% reduction in
exposure and a 50% reduction in liver cancer incidence over a 30-year time frame in Qidong
China (39). Indeed, the reduction in aflatoxin exposure documented in this work preceded

HBV vaccination in the population. Thus, the discovery of the powerful biologic role ofaflatoxin in liver cancer development sets the stage for multiple public health-based
prevention strategies. Given the data that we have ascertained in Guatemala to date

implementing both primary and secondary (40) strategies in this high aflatoxin exposure,
but low HBV and HCV prevalent region, could mitigate liver cancer.

Cell-free DNA analysis for early detection of liver cancer. Over the past ten years thetranslation of the underlying mechanistic knowledge of cancer has led to an emergence ofliquid biopsy strategies for early detection and clinical care follow up (41). Recently
Velculescu and Foda has pioneered the detection and measurement of genome wide
landscape of cfDNA fragmentation (fragmentomes) and DELFI (DNA evaluation offragments for early interception) (42, 43) was demonstrated to be strongly predictive for livercancer in samples obtained from Europe, the US and Hong Kong (44). This machine learningmodel incorporated multi-feature fragmentome data to evaluate 724 individuals; thesensitivity for detecting liver cancer was 88% in an average risk population at 98% specificity,and 85% among high-risk individuals at 80% specificity (44). An independent study reported
in 2022 in China found similar results using a technically similar fragmentomics strategy
predicting liver cancer risk (45). In this recent study the ultimate model showed a 97%

sensitivity and 96% specificity (45). Collectively the proposed work here provides anopportunity for extension and validation of these biomarkers to a different etiological risk
group for early detection.

In summary, despite the recent successes in the prevention and treatment of HBV andHCV through vaccination and chemotherapy, the incidence of liver cancer continues to rise
across many different populations. To affect future success in liver cancer prevention andcontrol the identification of new underlying and modifiable risk factors are required. Wellannotated epidemiologic studies and concomitant biorepositories will provide thefoundational resource for this research.

References
1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global

Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for
36 Cancers in 185 Countries. CA Cancer J Clin 2021;71:209-49
2. Wild CP, Miller JD, Groopman JD. Mycotoxin Control in Low- and Middle-Income
Countries. 2015

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3. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence
and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int
J Cancer 2015;136:E359-86. doi: 10.1002/ijc.29210. Epub 2014 Oct 9.

4. El-Serag HB, Kanwal F. - Epidemiology of hepatocellular carcinoma in the United States:
where are we? - Hepatology 2014 Nov;60(5):1767-75 doi: 101002/hep27222 Epub 2014
Aug 25 2014:- 1767-75

5. Petrick JL, Kelly SP, Altekruse SF, McGlynn KA, Rosenberg PS. Future of Hepatocellular
Carcinoma Incidence in the United States Forecast Through 2030. J Clin Oncol 2016
6. McGlynn KA, Petrick JL, London WT. Global epidemiology of hepatocellular carcinoma:
an emphasis on demographic and regional variability. Clin Liver Dis 2015;19:223-38. doi:

10.1016/j.cld.2015.01.001. Epub Feb 26.
7. Groopman JD, Smith JW, Rivera-Andrade A, Alvarez CS, Kroker-Lobos MF, Egner PA, etal. Aflatoxin and the Etiology of Liver Cancer and Its Implications for Guatemala. World
Mycotoxin J 2021;14:305-17
8. Cancer Disparities Progress Report. American Association for Cancer Research; 2022.
200p.
9. Wogan GN, Newberne PM. Dose-response characteristics of aflatoxin B1 carcinogenesis
in the rat. Cancer Res 1967;27:2370-6

10. Kensler TW, Roebuck BD, Wogan GN, Groopman JD. Aflatoxin: a 50-year odyssey ofmechanistic and translational toxicology. Toxicol Sci 2011;120 Suppl 1:S28-48
11. Pineros M, Frech S, Frazier L, Laversanne M, Barnoya J, Garrido C, et al. Advancing
Reliable Data for Cancer Control in the Central America Four Region. J Glob Oncol
2018;4:1-11
12. Pan W-C, Wu C-D, Chen M-J, Huang Y-T, Chen C-J, Su H-J, Yang H-I. Fine particle
pollution, alanine transaminase, and liver cancer: A Taiwanese prospective cohort study(REVEAL-HBV). Journal of the National Cancer Institute 2016;108:djv341
13. Pedersen M, Andersen ZJ, Stafoggia M, Weinmayr G, Galassi C, Sørensen M, et al.

Ambient air pollution and primary liver cancer incidence in four European cohorts
within the ESCAPE project. Environmental Research 2017;154:226-33
14. So R, Chen J, Mehta AJ, Liu S, Strak M, Wolf K, et al. Long-term exposure to air pollution
and liver cancer incidence in six European cohorts. Int J Cancer 2021
15. VoPham T, Bertrand KA, Tamimi RM, Laden F, Hart JE. Ambient PM2.5 air pollution
exposure and hepatocellular carcinoma incidence in the United States. Cancer Causes
Control 2018;29:563-72

16. Smith KR, McCracken JP, Weber MW, Hubbard A, Jenny A, Thompson LM, et al. Effect of
reduction in household air pollution on childhood pneumonia in Guatemala (RESPIRE):
a randomised controlled trial. The Lancet 2011;378:1717-26

17. Landrigan PJ, Fuller R, Acosta NJR, Adeyi O, Arnold R, Basu NN, et al. The Lancet

Precision Prevention

101

Commission on pollution and health. Lancet 2018;391:462-512
18. Smith JW, O'Meally RN, Ng DK, Chen JG, Kensler TW, Cole RN, Groopman JD.
Biomonitoring of Ambient Outdoor Air Pollutant Exposure in Humans Using TargetedSerum Albumin Adductomics. Chem Res Toxicol 2021;34:1183-96
19. Smith JW, O'Meally RN, Burke SM, Ng DK, Chen JG, Kensler TW, et al. Global Discovery

and Temporal Changes of Human Albumin Modifications by Pan-Protein Adductomics:
Initial Application to Air Pollution Exposure. J Am Soc Mass Spectrom 2023;34:595-607
20. Kamala A, Kimanya M, Haesaert G, Tiisekwa B, Madege R, Degraeve S, et al. Local post-

harvest practices associated with aflatoxin and fumonisin contamination of maize inthree agro ecological zones of Tanzania. Food additives & contaminants Part A,
Chemistry, analysis, control, exposure & risk assessment 2016;33:551-9

21. Wild CP, Jiang YZ, Allen SJ, Jansen LA, Hall AJ, Montesano R. Aflatoxin-albumin adducts
in human sera from different regions of the world. Carcinogenesis 1990;11:2271-4

22. Smela ME, Currier SS, Bailey EA, Essigmann JM. The chemistry and biology of aflatoxin
B(1): from mutational spectrometry to carcinogenesis. Carcinogenesis 2001;22:535-45
23. Wild CP, Garner RC, Montesano R, Tursi F. Aflatoxin B1 binding to plasma albumin andliver DNA upon chronic administration to rats. Carcinogenesis 1986;7:853-8

24. Wild CP, Hudson GJ, Sabbioni G, Chapot B, Hall AJ, Wogan GN, et al. Dietary intake ofaflatoxins and the level of albumin-bound aflatoxin in peripheral blood in The Gambia,
West Africa. Cancer Epidemiol Biomarkers Prev 1992;1:229-34
25. Groopman JD, Hall AJ, Whittle H, Hudson GJ, Wogan GN, Montesano R, Wild CP.
Molecular dosimetry of aflatoxin-N7-guanine in human urine obtained in The Gambia,
West Africa. Cancer Epidemiol Biomarkers Prev 1992;1:221-7

26. Groopman JD, Egner PA, Schulze KJ, Wu LS, Merrill R, Mehra S, et al. Aflatoxin exposure
during the first 1000 days of life in rural South Asia assessed by aflatoxin B(1)-lysine
albumin biomarkers. Food Chem Toxicol 2014;74:184-9

27. Ross RK, Yuan JM, Yu MC, Wogan GN, Qian GS, Tu JT, et al. Urinary aflatoxin biomarkers
and risk of hepatocellular carcinoma. Lancet 1992;339:943-6

28. Qian GS, Ross RK, Yu MC, Yuan JM, Gao YT, Henderson BE, et al. A follow-up study ofurinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's
Republic of China. Cancer Epidemiol Biomarkers Prev 1994;3:3-10
29. Chawanthayatham S, Valentine CC, 3rd, Fedeles BI, Fox EJ, Loeb LA, Levine SS, et al.
Mutational spectra of aflatoxin B1 in vivo establish biomarkers of exposure for humanhepatocellular carcinoma. Proc Natl Acad Sci U S A 2017;114:E3101-E9

30. Fedeles BI, Essigmann JM. Impact of DNA lesion repair, replication and formation on themutational spectra of environmental carcinogens: Aflatoxin B1 as a case study. DNA
Repair (Amst) 2018;71:12-22

31. Ozturk M. p53 mutation in hepatocellular carcinoma after aflatoxin exposure. Lancet

102

J. D. Groopman

1991;338:1356-9

32. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC. Mutational hotspot in the p53
gene in human hepatocellular carcinomas. Nature 1991;350:427-8

33. Kucab JE, Zou X, Morganella S, Joel M, Nanda AS, Nagy E, et al. A Compendium of
Mutational Signatures of Environmental Agents. Cell 2019;177:821-36 e16
34. Zhang W, He H, Zang M, Wu Q, Zhao H, Lu LL, et al. Genetic Features of Aflatoxinassociated Hepatocellular Carcinomas. Gastroenterology 2017
35. Alvarez CS, Ortiz J, Bendfeldt-Avila G, Xie Y, Wang M, Wu D, et al. Analysis of TP53

aflatoxin signature mutation in hepatocellular carcinomas from Guatemala: A crosssectional study (2016- 2017). Health Sci Rep 2020;3:e155
36. Lino-Silva LS, Lajous M, Brochier M, Santiago-Ruiz L, Melchor-Ruan J, Xie Y, et al.

Aflatoxin levels and prevalence of TP53 aflatoxin-mutations in hepatocellular carcinomas
in Mexico. Salud Publica Mex 2022;64:35-40
37. Wang JS, Shen X, He X, Zhu YR, Zhang BC, Wang JB, et al. Protective alterations in phase
1 and 2 metabolism of aflatoxin B1 by oltipraz in residents of Qidong, People's Republicof China. J Natl Cancer Inst 1999;91:347-54
38. Egner PA, Wang JB, Zhu YR, Zhang BC, Wu Y, Zhang QN, et al. Chlorophyllinintervention reduces aflatoxin-DNA adducts in individuals at high risk for liver cancer.
Proc Natl Acad Sci U S A 2001;98:14601-6
39. Chen JG, Egner PA, Ng D, Jacobson LP, Munoz A, Zhu Y-R, et al. Reduced Aflatoxin
Exposure Presages Decline in Liver Cancer Mortality in an Endemic Region of China.
Cancer Prevention Research 2013;6:1038-45
40. Chemoprevention Working G, Sporn MB. Prevention on Cancer in the Next Millennium:

Report of the Chemoprevention Wokring Group to the American Association for Cancer
Research. Cancer Res 1999;59:4743-58
41. Mattox AK, Bettegowda C, Zhou S, Papadopoulos N, Kinzler KW, Vogelstein B.
Applications of liquid biopsies for cancer. Sci Transl Med 2019;11
42. Mathios D, Johansen JS, Cristiano S, Medina JE, Phallen J, Larsen KR, et al. Detection andcharacterization of lung cancer using cell-free DNA fragmentomes. Nat Commun
2021;12:5060

43. Cristiano S, Leal A, Phallen J, Fiksel J, Adleff V, Bruhm DC, et al. Genome-wide cell-free
DNA fragmentation in patients with cancer. Nature 2019;570:385-9

44. Foda ZH, Annapragada AV, Boyapati K, Bruhm DC, Vulpescu NA, Medina JE, et al.
Detecting liver cancer using cell-free DNA fragmentomes. Cancer Discov 2023;13:616-31

45. Zhang X, Wang Z, Tang W, Wang X, Liu R, Bao H, et al. Ultrasensitive and affordable
assay for early detection of primary liver cancer using plasma cell-free DNA
fragmentomics. Hepatology 2022;76:317-29

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John D. Groopman, PhD
1974-1979
1979-1980
1980-1981
1981-1989
1989-present

Massachusetts Institute of Technology(Toxicology/Biological Engineering)
Postdoctoral fellow, Massachusetts Institute ofTechnology
Staff Fellow, Laboratory of Human Carcinogenesis,
National Cancer InstituteBoston University School of Public Health
Edyth H. Schoenrich Professor of Preventive Medicine,Johns Hopkins School of Public Health andComprehensive Cancer Center

104

INHIBITION OF COLORECTAL CARCINOGENESIS IN
FAMILIAL ADENOMATOUS POLYPOSIS

Michihiro Mutoh
Department of Molecular-Targeting Prevention
Graduate School of Medical Science Kyoto Prefectural University of Medicine
Kawaramachi-Hirikoji, Kamigyou-ku, Kyoto 602-8566, Japan(mimutoh@koto.kpu-m.ac.jp)

To date, many candidate chemopreventive agents against colorectal cancer (CRC) havebeen presented from researchers in the field of basic science and epidemiological science.
Although CRC is the most common cancer in Japan, no preventive drugs are in clinical use

yet. The reason for this can be partly explained by the system of public health insurance forthe whole nation, which does not allow the use of drugs for a presymptomatic status, i.e. thestatus before diagnosis. From the view of scientific research, the reason for this can be partly
explained by the lack of infrastructure to perform cancer prevention trials in Japan.

It was reported, by Dr. Thun in 1991, that aspirin reduced the risk of either colorectal

polyps or cancer . Afterwards, many studies using NSAIDs were performed, whichsuggested their usefulness as cancer chemopreventive agents. Aspirin is an inhibitor of

cyclooxygenase that catalyzes the conversion of arachidonic acid to prostaglandins. In
addition, Aspirin has been reported to possess many other functions, such as inhibition of
NF-kappa B and beta-catenin transcriptional activities.

Now, CRC chemoprevention trials have been performed in Japan. We investigated theeffects of low-dose (100 mg/day), enteric coated aspirin tablets administered for 2 years in a

double-blinded, randomized, placebo-controlled clinical trial for patients with a single/
multiple colorectal adenoma (1). As a result, we obtained the result that low-dose enteric-coated aspirin tablets reduced the recurrence of colorectal tumor development in an Asian
population (after adjustment for potential confounders, we obtained a marginal andsignificant OR of 0.60). Afterwards, a CRC chemoprevention trial using metformin, an anti-

diabetes drug, was performed at other facilities, and showed reduction of the recurrence ofcolorectal tumor development (2).

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105

We established mode of action for the proof of concept (POC) of aspirin that it inhibits

intestinal polyp development, before moving to the human trial. The areas being consideredin this trial were, (i) Anti-inflammation, (ii) Anti-oxidative stress, and (iii) Improvement ofabnormal beta-catenin signaling.

We believe that the use of aspirin as a cancer chemopreventive agent has advantages

because it has a long history of clinical use and its adverse effects are well known. An

overseas clinical trial investigated the effects of aspirin in familial adenomatous polyposis(FAP), using high-dose aspirin (CAPP1). This trial was conducted with a 2 × 2 factorial

design (a type of randomized controlled trial) using aspirin (600 mg/day), resistant starch,o r a re s p e c t i v e p l a c e b o i n y o u n g FA P p a t i e n t s a g e d 1 0 t o 2 1 y e a r s a t l o w r i s k o f
cardiovascular disorder. In this study, resistant starch had no effect on the occurrence ofcolorectal polyps, but aspirin showed a tendency to reduce, though not significantly, thenumber of polyps in the area from the sigmoid colon to the rectum.

We first performed a Phase I study using low-dose enteric-coated aspirin tablets (100 mg/day) for 6 months, and also evaluated the occurrence of new intestinal polyps in Japanesepatients after polyp removal (J-FAPP Study II) (3). In this trial, 17 patients were allocated toeach of the aspirin and placebo groups. The results showed that the aspirin group had ahigher number of participants showing a decreased number of colorectal polyps (primary
endpoint) (risk ratio = 2.33 [95% CI: 0.72-7.55]), which only suggested that aspirin appeared
to be effective.

In the next trial, we performed a chemoprevention trial using low-dose aspirin, a 2 x 2factorial, randomised, double-blind, placebo-controlled, multicenter trial (11 centers) for FAP
in Japan. FAP is characterized by the development of more than 100 polyps in thecolorectum, and thus, is a high-risk status/condition for CRC. FAP and Lynch syndrome are
well-known hereditary colorectal cancers for which causative genes have been identified.

FAP is an autosomal dominant inherited condition, and the APC tumor suppressor gene has

been identified as the causative gene. Hereditary colorectal cancer accounts forapproximately 5% of all diagnosed cases of colorectal cancer.1) FAP accounts for less than 1%of all diagnosed cases of CRC. To avoid development of CRC, FAP patients undergo colonicresection at the age of around 20. From our results, administration of low-dose aspirin for 8
months safely inhibited the growth of colorectal polyps of more than 5.0 mm in FAP (4).

We also confirmed the beneficial effects of intensive downstaging polypectomy (IDP; an

endoscopic method that resects all colorectal polyps more than 5.0 mm) in FAP patients at a
young age (5). For this endoscopic treatment, bipolar snare polypectomy is used in general.
Hot biopsy or argon-dye laser is not used. We removed 100 to 400 polyps during one

polypectomy procedure (in about 90 minutes), and repeated this several times. We recruited

223 patients in this trial. The patients were followed up for 5 years. During the 5 years, the106M. Mutohaverage number of endoscopies was 6.6 times in the non-colectomy group (n=150) and 6.2

times in the colectomy group (n=47). The average number of removals was 524 in the noncolectomy group and 132 in the colectomy group. There was no bleeding requiringtransfusion, and no development of advanced CRC during the 5 years. IDP showed its safety
and effectiveness in preventing colonic carcinogenesis.

I believe that a combination treatment of IDP & aspirin will be a useful method for FAP

patients who decline to undergo surgery due to personal or social reasons, and for reducing
frequent diarrhea that decreases quality of life (QOL) after colectomy. IDP could be used in
combination with colectomy. The problem might be the cost and manpower caused by the

number of endoscopy specialists required. However, aspirin will postpone the interval of
IDP treatment, and may solve these problems. In 2022, IDP was approved in the system of

public health insurance for the whole nation. Use of low-dose aspirin for cancer prevention

is not allowed yet. Use of low-dose aspirin has been approved only as an antithrombotic or
anticoagulant agent. Therefore, we are now planning our final study aimed towards

obtaining National Health Insurance coverage using low-dose aspirin (J-FAPP Study V). In

this multicenter, single-arm interventional clinical study, we will administer low-dose
enteric-coated aspirin (100 mg/day) for 2 years in patients with FAP and evaluate its effects
in reducing the incidence of colorectal polyps of 5.0 mm or greater in diameter.

Our results will propose novel remedies, alternative to colorectal resection, that can beoffered to FAP patients who decline to undergo surgery, and may provide future prospects
for chemoprevention of CRC with respect to the future development of precision medicine.
References
1. Ishikawa H, Mutoh M＊, Suzuki S, Tokudome S, Saida Y, Abe T, Okamura S, Tajika M, Joh
T, Tanaka S, Kudo S, Matsuda T, Iimuro M, Yukawa T, Takayama T, Sato Y, Lee K,
Kitamura S, Mizuno M, Sano Y, Gondo N, Sugimoto K, Kusunoki M, Goto C, Matsuura

N, Sakai T, Wakabayashi K. The preventive effects of low-dose enteric-coated aspirintablets on the development of colorectal tumours in Asian patients: a randomized trial.
Gut 2014, 63, 1755-1759. ＊corresponding author
2. Higurashi T, Hosono K, Takahashi H, Komiya Y, Umezawa S, Sakai E, Uchiyama T,

Taniguchi L, Hata Y, Uchiyama S, Hattori A, Nagase H, Kessoku T, Arimoto J,
Matsuhashi N, Inayama Y, Yamanaka S, Taguri M, Nakajima A. Metformin forchemoprevention of metachronous colorectal adenoma or polyps in post-polypectomy

patients without diabetes: a multicentre double-blind, placebo-controlled, randomised
phase 3 trial. Lancet Oncol 2016, 17, 475-483.
3. Ishikawa H, Wakabayashi K, Suzuki S, Mutoh M, Hirata K, Nakamura T, Takeyama I,

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Kawano A, Gondo N, Abe T, Tokudome S, Goto C, Matsuura N, Sakai T. Preventive
effects of low-dose aspirin on colorectal adenoma growth in patients with familial
adenomatous polyposis: Double-blind, randomized clinical trial. Cancer Med 2013, 2, 50-

56.
4. Ishikawa H, Mutoh M＊, Sato Y, Doyama H, Tajika M, Tanaka S, Horimatsu T, Takeuchi Y,
Kashida H, Tashiro J, Ezoe Y, Nakajima T, Ikematsu, H Hori S, Suzuki S, Otani T,
Takayama T, Ohda Y, Mure K, Wakabayashi K, Sakai T. Chemoprevention with low-dose
aspirin, mesalazine, or both in patients with familial adenomatous polyposis without
previous colectomy (J-FAPP Study IV): a multicentre, double-blind, randomised, two-bytwo factorial design trial. Lancet Gastroenterol Hepatol. 2021, 6, 474-481. ＊corresponding
author

5. Ishikawa H, Yamada M, Sato Y, Tanaka S, Chino A, Tajika M, Doyama H, Takayama T,
Ohda Y, Horimatsu T, Sano Y, Tanakaya K, Ikematsu H, Saida Y, Ishida H, Takeuchi Y,
Kashida H, Kiriyama S, Hori S, Lee K, Tashiro J, Kobayashi N, Nakajima T, Suzuki S,
Mutoh M＊, and J-FAPP Study III Group. Intensive endoscopic resection for downstaging
of polyp burden in patients with familial adenomatous polyposis (J-FAPP Study III): A
multicenter prospective interventional study. Endoscopy 2023, 55, 344-352. ＊corresponding
authorMichihiro Mutoh, MD, PhD
1998-2001
2001-2003
2003-2020
2020-present

Graduated from Yamaguchi University, Graduate Schoolof University of Tsukuba
Visiting fellow, the Laboratory of Cellular
Carcinogenesis and Tumor Promotion, NCI, NIH
Researcher, National Cancer Center
Professor, Department of Molecular-Targeting Prevention
Graduate School of Medical Science, Kyoto Prefectural
University of Medicine

108

MICROBES AND COLON CANCER: WHERE ARE WE ?

Cynthia L. Sears
Division of Infectious Diseases
Departments of Medicine, Oncology & Molecular Microbiology & Immunology
Johns Hopkins University School of Medicine
Bloomberg School of Public Health1550 Orleans Street
CRB2 Building, Suite 1M.05
Baltimore, MD 21231, USA(csears@jhmi.edu)
Disclosures: CLS receives grant support to her institution from Janssen and Bristol Myer

Squibb. CLS receives royalties for authorship and reviews for Up-to-Date. CLS serves as the
Editor-in-Chief of The Journal of Infectious Diseases.

Acknowledgements. CLS thanks all her laboratory members, past and present, for theircontributions to the work described herein. All figures were created with BioRender.com.
Introduction

Colon cancer is a leading cause of cancer morbidity and mortality of women and men

across the globe (Siegel et al., 2023). How the vast microbial world of the colon impacts theinitiation and/or progression of human colon cancer is an evolving field of investigation.
Most of the focus to date—and the focus of this presentation—is on the putative contributionof the trillions of colon bacteria to colon carcinogenesis. From a broad perspective, key

considerations are how single bacteria, presumably through specific virulence factors, or
how bacterial communities, presumably through their interactions, may initiate and/or

augment the progression of colon cancer (Figure 1). Most likely, both specific bacteria andcommunity interactions govern colon carcinogenesis in combination with a broad array ofhost factors (e.g., obesity, smoking, alcohol intake, diabetes mellitus, prior antibiotic exposure

among others), each known to also exert impact on the colon microbiome of individuals(Knippel et al., 2021). Thus, ultimately, integrating considerations of the host’s colon

microbiome into preventive strategies may prove to be a powerful way to improve coloncancer prevention. Herein, a brief presentation of both the power of individual bacterial

Microbiome and Cancer

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species and the colon bacterial community to influence colon carcinogenesis will bepresented.

The Power of Individual Bacterial Species to Impact Colon Carcinogenesis

To date, we estimate that published data on 11 bacterial species suggest their association

with colon cancer (Figure 2)(White & Sears, 2023). Among these species, Bacteroides fragilis

toxin (BFT)-secreting B. fragilis strains, termed enterotoxigenic B. fragilis (ETBF), polyketide
synthase-positive Escherichia coli strains (pks+ E. coli) and Fusobacterium nucleatum and its

subspecies have garnered the most attention. Historically, detection of bloodstream infection
with Streptococcus gallolyticus (formerly S. bovis, type I) was the first bacterium recognized toserve as a clinical signal worrisome for underlying colon cancer. In fact, it remains standard-of-care that detection of S. gallolyticus bloodstream infection requires a follow-up

colonoscopy to rule out the co-occurrence of colon cancer (White & Sears, 2023). Herein, thedata supporting the potential of ETBF and pks+ E. coli to enhance colon carcinogenesis will

be described. The review by White and Sears (2023) provides a recent discussion of thespectrum of bacteria associated with colon carcinogenesis.

Through a series of elegant deductive experimental work conducted by Dr. Lyle Myers ofMontana State University, ETBF was first identified as a cause of diarrhea in husbandry

animals including lambs, calves and piglets among others; work that was subsequently
replicated in lamb and rabbit animal models [reviewed in (Sears et al., 1995)]. Dr. Myers first

identified that the culture supernatants of ETBF contained a heat-labile protein (BFT) thatwas capable of stimulating secretion in the ligated ileal loops of lambs (Sears et al., 1995).
Subsequent work by the Sears laboratory and others identified an in vitro assay for detectionof the BFT that assisted with identification of the B. fragilis gene encoding bft and purificationof biologically active BFT protein [reviewed in (Sears et al., 2014)]. Through these results andothers, BFT was defined as a pre-pro-zinc-dependent metalloprotease protein with, inparticular, the pro-protein domain of BFT being critical to folding and delivery of
biologically-active BFT to the external environment. This work led to the recognition that B.

fragilis were composed of, at least, two molecular subtypes, ETBF and non-toxigenic B.

fragilis (NTBF). Overall, NTBF is considered to contribute to colon health and acts as a
symbiote whereas ETBF is recognized to be associated with inflammatory diarrheal disease,inflammatory bowel disease and human sporadic and hereditary colon cancer (Sears et al.,2014)(Boleij et al., 2015))(Dejea et al., 2018).

Key features of the pathogenesis of ETBF-associated colon tumorigenesis are shown in

Figure 3. This model of the mechanisms by which ETBF, through BFT secretion, promotes

colon tumor formation is based on extensive studies of ETBF pathogenicity using themultiple intestinal neoplasia (Min, Apc+/-) murine model of colon tumorigenesis (White &110

C. L. Sears

Sears, 2023) (Sears et al., 2014) (Chung et al., 2018). While mucosal adherence is undoubtedly
critical to the pathogenicity of ETBF, in-frame deletion of bft is sufficient to eliminate thecolon tumor-promoting activity of ETBF (Chung et al., 2018). Other mechanisms associatedwith ETBF colon tumorigenesis include cleavage of E-cadherin, induction of c-Myc

expression leading to colon epithelial cell proliferation, induction of DNA damage andactivation of Stat3 with a marked increased in IL-17 production by the colon mucosal
immune cells (both Th17 and γδ-T cells) (Housseau et al., 2016). Feedback of IL-17 onto thecolon epithelial cells results in NF-κB activation, chemokine secretion and distal colon

infiltration by pro-tumor myeloid cells (e.g., myeloid-derived suppressor cells, MDSCs), also

contributors to ETBF colon tumorigenesis (Chung et al., 2018). Collectively, in the Min mousecolon, these molecular sequelae rapidly lead to microadenoma formation that evolves tomacroadenoma formation. More recently, the combined presence of colon cancer-associatedoncogenes (Apc, BRAF) in a murine model has been identified to further modify ETBF

pathogenesis (Destefano Shields et al., 2021), suggesting that there is the potential for
dynamic interactions of colon microbial members, the oncogenes of colon cancer and its
immune microenvironment (Dzierozynski et al., 2023). A key gap in knowledge is that thecolon epithelial cell receptor for BFT remains to be identified.

An impressive series of publications have highlighted the potential contributions of pks+

E. coli to colon cancer development (Pleguezuelos-Manzano et al., 2020)(Wilson et al., 2019)(Dziubańska-Kusibab et al., 2020)(Xue et al., 2019)(Arthur et al., 2012.). The critical virulence

determinant of pks+ E. coli is the synthesis of colibactin by the genes of the pks island. The

structural elucidation of colibactin and the recognition of its striking, capacity to cross-link

DNA leading to a specific mutational signature has generated much interest in theepidemiology of pks+ E. coli and their potential to initiate colon cancer. While it ishypothesized that the mutagenic capacity of, at least, some pks+ E. coli contribute to humancolon cancer, longitudinal data defining the association of pks+ E. coli DNA mutagenesis toonset of neoplasia in the human colon are not yet available. However, pks+ E. coli can

asymptomatically colonize children, raising the potential for putative long-term colonization

with pks+ E. coli. This possibility has led to the hypothesis that, combined with host risk

factors associated with human colon cancer, pks+ E. coli may contribute to the increase inearly onset colorectal cancer, defined as a colon or rectal cancer diagnosis prior to age 50,

when preventive colonoscopy is not routinely performed. Another clue to the importance ofboth ETBF and pks+ E. coli to colon cancer pathogenesis are observations in the hereditarycolon cancer syndrome, familial adenomatous polyposis (FAP; autosomal dominant
inheritance of mutation in one APC allele). In individuals with FAP, both ETBF and pks+ E.
coli have been identified as prominent colonizers of mucus-invasive colon biofilms (seebelow) (Dejea et al., 2018).

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The Power of Colon Bacterial Communities to Impact Colon Carcinogenesis

Visually colon mucosal biofilms display dense bacterial invasion of the normally sterile,

Muc-2-dominated inner mucus layer (Figure 1). Specific criteria to identify a mucus-invasive

biofilm are > 20 bacterial cells within 1 µm of the colon epithelial cell surface over a mucosal
surface distance of, at least, 150-200 µm. These parameters define that ~109 or more bacterial

cells are in proximity to the cell-of-origin for colon cancer, the colon epithelial cell. Available

data have identified that biofilms are strongly associated with colon cancer (present on, atleast, 50% of colon cancers both in the USA and Malaysia), are polymicrobial and are procarcinogenic in the Min mouse model (Dejea et al., 2014) (Drewes et al., 2017) (Tomkovich etal., 2019). In contrast, biofilms are found infrequently (~10-15%) in individuals undergoingpreventive colonoscopy (Dejea et al., 2014). Whether individuals displaying mucus-invasive

polymicrobial biofilms are at increased risk for development of colon neoplasia is, at present,
unknown.

Recently, a consortium of bacteria isolated from the mucosa of a human colon cancer

displaying a mucus-invasive, polymicrobial biofilm was studied (Drewes et al., 2022). This
relatively small community of bacteria (N=30 strains) was shown to be pro-carcinogenic inboth germ-free and specific pathogen-free Min mouse models. Additional studies,
unexpectedly, identified that within this bacterial community Clostridioides difficile, throughits secreted toxin B, was the key bacterial driver of the tumorigenesis observed in the Min

murine model. Beyond toxin B secreted by C. difficile, the available data support that colon
epithelial cell and immune mechanisms as well as biologic changes in C. difficile-associatedmurine colon biofilms contribute to the putative oncogenicity of C. difficile. Importantly,

compared to Min mouse colonization with the biofilm isolates in the absence of C. difficile,inclusion of C. difficile in the colonizing consortium leads to biofilms that not only invade theinner mucus layer but invade the colon crypts, enhancing contact with colon stem cells. The

proximity of toxin B-secreting C. difficile to colon stem cells is hypothesized to contribute toits tumorigenic ability in Min mice. Importantly, while C. difficile is the leading cause of

nosocomial infection and a critical organism as defined by the Centers for Disease Control(Antibiotic Resistance Threats in the United States, 2019), its potential to contribute to colon

carcinogenesis represents a new area for further investigation.
Summary and Conclusions

Given the complexity of the colon microbiota, it is not a surprise that multiple individual

bacteria displaying or secreting oncogenic virulence factors and dense communities ofbacteria, such as in colon mucosal biofilms, likely contribute to colon cancer pathogenesis.
Dense bacterial communities are well-known to modify their function through inter-species
signaling (such as by quorum sensing) (Oliveira et al., 2023); by extension, changes in the112

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function of nearby host cells as well as reciprocal interactions have been reported (Liou et al.,2022). While important strides have been made in unraveling the complex factors governing
how the colon microbiota impact colon carcinogenesis, there remain substantive gaps in our
understanding of the epidemiology, virulence, persistence and tumor-inducing potential of

specific bacteria and communities in humans (White & Sears, 2023) (Knippel et al., 2021).
Progress to utilization of colon microbiota biomarkers in the global quest to prevent coloncancer requires that we can discern the host context and bacterial strains or associatedmolecules that pose an oncogenic risk to an individual. These discoveries may offer a
gateway to a new era in colon cancer prevention.

References
1. Antibiotic resistance threats in the United States, 2019. (2019). https://doi.org/10.15620/

cdc:82532
2. Arthur, J. C., Perez-Chanona, E., Mühlbauer, M., et al. (2012) Intestinal Inflammation
Targets Cancer-Inducing Activity of the Microbiota Science, 338, 120-123. http://science.
sciencemag.org/
3. Boleij, A., Hechenbleikner, E. M., Goodwin, A. C., et al. (2015). The Bacteroides fragilis
toxin gene is prevalent in the colon mucosa of colorectal cancer patients. Clinical
Infectious Diseases, 60(2), 208–215. https://doi.org/10.1093/cid/ciu787
4. Chung, L., Thiele Orberg, E., Geis, A. L., et al. (2018). Bacteroides fragilis Toxin Coordinatesa Pro-carcinogenic Inflammatory Cascade via Targeting of Colonic Epithelial Cells. Cell
Host and Microbe, 23(2), 203-214.e5. https://doi.org/10.1016/j.chom.2018.01.007

5. Dejea, C. M., Fathi, P., Craig, J. M., et al (2018). Patients with familial adenomatous
polyposis harbor colonic biofilms containing tumorigenic bacteria. Science, 359(6375),
592–597. https://doi.org/10.1126/science.aah3648
6. Dejea, C. M., Wick, E. C., Hechenbleikner, E. M., et al. (2014). Microbiota organization isa distinct feature of proximal colorectal cancers. Proceedings of the National Academy of
Sciences of the United States of America, 111(51), 18321–18326. https://doi.org/10.1073/

pnas.1406199111
7. Destefano Shields, C. E., White, J. R., Chung, L., et al. (2021). Bacterial-driven

inflammation and mutant braf expression combine to promote murine colon
tumorigenesis that is sensitive to immune checkpoint therapy. Cancer Discovery, 11(7),1792–1807. https://doi.org/10.1158/2159-8290.CD-20-0770
8. Drewes, J. L., Chen, J., Markham, N. O., et al. (2022). Human Colon Cancer–Derived
Clostridioides difficile Strains Drive Colonic Tumorigenesis in Mice. Cancer Discovery, 12(8),1873–1885. https://doi.org/10.1158/2159-8290.CD-21-1273
9. Drewes, J. L., White, J. R., Dejea, et al. (2017). High-resolution bacterial 16S rRNA geneMicrobiome and Cancer

113

profile meta-analysis and biofilm status reveal common colorectal cancer consortia. Npj
Biofilms and Microbiomes, 3(1). https://doi.org/10.1038/s41522-017-0040-3

10. Dzierozynski, L., Queen, J., & Sears, C. L. (2023). Subtle, persistent shaping of the gut
microbiome by host genes: A critical determinant of host biology. In Cell Host and Microbe(Vol. 31, Issue 10, pp. 1569–1573). Cell Press. https://doi.org/10.1016/j.chom.2023.09.007
11. Dziubańska-Kusibab, P. J., Berger, H., Battistini, F., et al. (2020). Colibactin DNA-damage
signature indicates mutational impact in colorectal cancer. Nature Medicine, 26(7), 1063–

1069. https://doi.org/10.1038/s41591-020-0908-2
12. Housseau, F., Wu, S., Wick, E. C., et al. (2016). Redundant Innate and Adaptive Sourcesof IL17 Production Drive Colon Tumorigenesis. Cancer Research, 76(8), 2115–2124.

https://doi.org/10.1158/0008-5472.CAN-15-0749
13. Knippel, R. J., Drewes, J. L., & Sears, C. L. (2021). The cancer microbiome: Recent
highlights and knowledge gaps. Cancer Discovery, 11(10), 2378–2395. https://doi.

org/10.1158/2159-8290.CD-21-0324
14. Liou, M. J., Miller, B. M., Litvak, Y., et al. (2022). Host cells subdivide nutrient niches into
discrete biogeographical microhabitats for gut microbes. Cell Host and Microbe, 30(6), 836847.e6. https://doi.org/10.1016/j.chom.2022.04.012
15. Oliveira, R. A., Cabral, V., Torcato, I., & Xavier, K. B. (2023). Deciphering the quorumsensing lexicon of the gut microbiota. In Cell Host and Microbe (Vol. 31, Issue 4, pp. 500–
512). Cell Press. https://doi.org/10.1016/j.chom.2023.03.015
16. Pleguezuelos-Manzano, C., Puschhof, J., Rosendahl Huber, A., et al. (2020). Mutationalsignature in colorectal cancer caused by genotoxic pks + E. coli. Nature, 580(7802), 269–
273. https://doi.org/10.1038/s41586-020-2080-8
17. Sears, C. L., Geis, A. L., & Housseau, F. (2014). Bacteroides fragilis subverts mucosal

biology: From symbiont to colon carcinogenesis. In Journal of Clinical Investigation (Vol.
124, Issue 10, pp. 4166–4172). American Society for Clinical Investigation. https://doi.
org/10.1172/JCI72334
18. Sears, C. L., Myers, L. L., Lazenby, A., & Van Tassell, R. L. (1995). Enterotoxigenic
Bacteroides fragilis. Clinical Infectious Diseases: An Official Publication of the Infectious
Diseases Society of America, 20 Suppl 2, S142-8. https://doi.org/10.1093/clinids/20.

supplement_2.s142
19. Siegel, R. L., Miller, K. D., Wagle, N. S., & Jemal, A. (2023). Cancer statistics, 2023. CA: A
Cancer Journal for Clinicians, 73(1), 17–48. https://doi.org/10.3322/caac.21763
20. Tomkovich, S., Dejea, C. M., Winglee, K., et al. (2019). Human colon mucosal biofilms
from healthy or colon cancer hosts are carcinogenic. Journal of Clinical Investigation,

129(4), 1699–1712. https://doi.org/10.1172/JCI124196
21. White, M. T., & Sears, C. L. (2023). The microbial landscape of colorectal cancer. In Nature
Reviews Microbiology. Nature Research. https://doi.org/10.1038/s41579-023-00973-4

114

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22. Wilson, M. R., Jiang, Y., Villalta, P. W., et al. (2019). The human gut bacterial genotoxin
colibactin alkylates DNA. Science, 363(6428). https://doi.org/10.1126/science.aar7785
23. Xue, M., Kim, C. S., Healy, A. R., et al. (2019). Structure elucidation of colibactin and its
DNA cross-links. Science, 365(6457). https://doi.org/10.1126/science.aax2685

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C. L. Sears

Cynthia L. Sears, MD
1975
1977
1985
2001
2018
2019
2023

BS, Pennsylvania State University
MD, Thomas Jefferson University
Infectious Diseases Fellowship, University of Virginia
Professor, Johns Hopkins Univ SOM
Bloomberg~Kimmel Professor of Immunotherapy, Johns
Hopkins Univ SOM
President, Infectious Diseases Society of America
Editor-in-Chief, The Journal of Infectious Diseases

117

COMPREHENSIVE GENOMIC PROFILING AND CLONALEVOLUTION OF NEUROENDOCRINE CARCINOMAS OF THE
GASTROINTESTINAL SYSTEM

Shinichi Yachida
Department of Cancer Genome Informatics
Graduate School of Medicine, Osaka University
2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan(syachida@cgi.med.osaka-u.ac.jp)

Neuroendocrine neoplasms (NENs) are rare disorders that can be diagnosed by

exhibiting positive staining for neuroendocrine markers, such as synaptophysin andchromogranin A. NENs arise in a cross-organic manner. The diseases were classified into

well-differentiated NENs (i.e., neuroendocrine tumors [NETs]), and poorly differentiatedNENs (i.e., neuroendocrine carcinoma [NEC]), based on histopathology in the 2019 WHOclassification.

Neuroendocrine carcinoma of the gastrointestinal system (GIS-NEC) is an uncommon yethighly aggressive tumor. Due to the frequent occurrence of distant metastases at diagnosis in

GIS-NEC cases, surgical intervention is recommended in select patients. Consequently, there
needs to be a more comprehensive analysis of genomic and epigenomic data obtained fromfrozen surgical samples. Due to the scarcity of frozen samples, we collected samples fromJapan, the United States, and the Netherlands.The GIS-NEC cases were analyzed using

whole-genome/exome sequencing, RNA-seq, DNA methylation assays, and the assay oftransposase-accessible chromatin sequencing (ATAC) [1].

It is presumed that the majority of GIS-NECs originate from precursor lesions thatusually give rise to non-neuroendocrine carcinomas of the corresponding organ, as
evidenced by the detection of organ-specific genetic events (such as KRAS in pancreaticNECs, APC in colorectal NECs, and ELF3 in ampullary NECs) [2]. Distinct genomic
disparities were observed between pancreatic NECs (Panc-NECs) and non-pancreatic GIS-

NECs (Nonpanc-NECs). The number of structural variations (SV) was significantly morenotable in Nonpanc-NECs. Alterations in TP53 and RB1 were prevalent in GIS-NECs, while
Nonpanc-NECs with intact Rb showed exclusive amplification of either CCNE1 or MYC.

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S. Yachida

Abnormalities in the Notch family genes that originate from non-pancreatic digestiveorgans are observed in NECs. Therefore, the experiments utilized organoids that were
derived from colonic and gastric mucosa. The organoid, which underwent genome editing

in normal colon mucosa to delete TP53 and RB1, exhibits minimal synaptophysin staining.

This suggests that the deletion of TP53 and RB1 alone does not result in NEC. The
administration of DAPT, a Notch inhibitor, leads to a significant increase in synaptophysin

expression. The gastric organoid also yielded a similar observation. There is a notable
upregulation of PTF1A and RBPJL in specific Panc-NECs, suggesting their derivation frompancreatic acinar cells.

In most GIS-NECs, there was an observed overexpression of transcription factors, notably

the SOX2 gene. The unexpected finding in this study was the high expression of SOX2 in

GIS-NECs, which seemed to be regulated by hypermethylation of the promoter region of
SOX2, resulting in a phenomenon known as paradoxical gene activation. A notable positive
correlation was observed between the methylation of SOX2’s promoter region and theexpression of the gene. In order to further investigate this, we utilized ATAC-seq analysis.

This technique helps map open chromatin structures throughout the genome by sequencing.
The ATAC-seq analysis found that NECs with high SOX2 expression have open chromatin,
which helps with gene transcription.

We have analyzed the epigenomic features of GIS-NEC and small cell lung carcinoma(SCLC) by combining motif enrichment analysis from ATAC-seq with enhancer profilingfrom a new CUT&Tag assay for H3K27ac [3]. We have found ELF3 to be a transcription

factor connected to super-enhancers in NEC in cell lines affected by these diseases. By
combining CUT&Tag and knockdown RNA sequencing methods, we unveiled thetranscriptional network governed by ELF3 and identified its distinct gene signatureincluding AURKA, CDC25B, CLDN4, ITGB6, and YWAHB. Furthermore, an assay

investigating loss-of-function demonstrated that the depletion of ELF3 resulted in decreased
cell viability. By analyzing gene expression in clinical samples, we effectively divided GIS-

NEC patients into two separate groups using the ELF3 signature. Our research findings
indicate that pathways promoting tumor growth were activated in the group exhibiting ahigh ELF3 signatur and provide insight into the transcriptional regulation of ELF3 as acancer-causing factor in NEC [4].

A gastric NEC with Merkel cell polyomavirus (MCPyV) appeared to be caused by the

monoclonal integration of MCPyV. The MCPyV large T antigen can directly bind to anddeactivate RB1. A rectal NEC specimen showed intact Rb and p53, with no CCNE1/MYC
amplification. It was determined that the sample tested positive for high-risk humanpapillomavirus (HPV-18). The results indicate that viral infection may be a contributing
factor to Nonpanc-NECs.

Microbiome and Cancer

119

Furthermore, there exists a potential for bidirectional development between NEC and

Non-NEC components. The Rb protein was occasionally absent in both the NEC and NonNEC components. Among these cases, it was observed that four driver mutations were
present in both components, and oncogenic genes, including KRAS (p.G12A), exhibited an

accumulation of driver mutations, specifically in the Non-NEC component. In addition, theevolutional lineage tree of our autopsied case indicated that the Non-NEC component(adenocarcinoma) was linked to a unique WGD (whole-genome duplication) -positive
subclone.

These comprehensive studies of genomic alterations in GIS-NECs uncovered several key

biological processes underlying the genesis of this very lethal form of cancer.
References

1. Yachida S, Totoki Y, Noë M, Nakatani Y, Horie M, Kawasaki K, Nakamura H, SaitoAdachi M, Suzuki M, Takai E, Hama N, Higuchi R, Hirono S, Shiba S, Kato M, Furukawa
E, Arai Y, Rokutan H, Hashimoto T, Mitsunaga S, Kanda M, Tanaka H, Takata S,
Shimomura A, Oshima M, Hackeng WM, Okumura T, Okano K, Yamamoto M, Yamaue
H, Morizane C, Arihiro K, Furukawa T, Sato T, Kiyono T, Brosens LAA, Wood LD,
Hruban RH, Shibata T. Comprehensive genomic profiling of neuroendocrine carcinomasof the gastrointestinal system. Cancer Discov, 2022, 12, 692-711.
2. Yachida S, Wood LD, Suzuki M, Takai E, Totoki Y, Kato M, Luchini C, Arai Y, Nakamura H,
Hama N, Elzawahry A, Hosoda F, Shirota T, Morimoto N, Hori K, Funazaki J, Tanaka H,
Morizane C, Okusaka T, Nara S, Shimada K, Hiraoka N, Taniguchi H, Higuchi R, Oshima
M, Okano K, Hirono S, Mizuma M, Arihiro K, Yamamoto M, Unno M, Yamaue H, Weiss
MJ, Wolfgang CL, Furukawa T, Nakagama H, Vogelstein B, Kiyono T, Hruban RH,
Shibata T. Genomic sequencing identifies ELF3 as a driver of ampullary carcinoma.
Cancer Cell, 2016, 29, 229-240.
3. Horie M, Tanaka H, Suzuki M, Sato Y, Takata S, Takai E, Miyashita N, Saito A, Nakatani Y,
Yachida S. An integrative epigenomic approach identifies ELF3 as an oncogenic regulator
in ASCL1-positive neuroendocrine carcinoma. Cancer Sci, 2023, 114, 2596-2608.
4. Suzuki M, Saito-Adachi M, Arai Y, Fujiwara Y, Takai E, Shibata S, Seki M, Rokutan H,
Maeda D, Horie M, Suzuki Y, Shibata T, Kiyono T, Yachida S. E74-like factor 3 is a key
regulator of epithelial integrity and immune response genes in biliary tract cancer.
Cancer Res, 2021, 81, 489-500.

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S. Yachida

Shinichi Yachida, MD, PhD
1988-1994
1994-1998
2007-20102017-present

MD, Tottori University, Faculty of Medicine
PhD, Kagawa Medical University (Surgery)
Postdoctoral fellow, Department of Pathology, Johns
Hopkins Medical Institutions, Baltimore, USA
Professor, Department of Cancer Genome Informatics,
Graduate School of Medicine, Osaka University

121

THE ROLE OF BRCA1 TUMOR SUPPRESSOR IN
HELICOBACTER PYLORI-MEDIATED GASTRICCARCINOGENESIS

Masanori Hatakeyama
Laboratory of Microbial Carcinogenesis
Institute of Microbial Chemistry, Microbial Chemistry Research Foundation
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan(mhata@m.u-tokyo.ac.jp; mhatakeyama@bikaken.or.jp)
Center for Infection-associated Cancer, Institute of Genetic Medicine, Hokkaido University
Kita 15, Nishi 7, Kita-ku, Sapporo 060-0815, Japan(masahata@igm.hokudai.ac.jp)
Gastric cancer is the fourth most common cancer and the third leading cause of cancer-

related deaths worldwide. Compelling evidence suggests that chronic infection with

Helicobacter pylori, especially with cagA-positive strains, plays a vital role in the developmentof more than 80% of all human gastric cancer cases. Hence, H. pylori is the only known

bacterium causally associated with human cancer. The H. pylori cagA-encoded CagA protein,produced inside the bacterium, is directly injected into the attached gastric epithelial cells
via a bacterial microsyringe known as the type IV secretion system (T4SS) [1]. Once delivered

into gastric epithelial cells, the H. bacterial CagA protein acts as a pro-oncogenic scaffold thatpromiscuously interacts with and functionally perturbs several host proteins, most notably
the prooncogenic phosphatase SHP2 and polarity-regulating kinase PAR1b [2, 3, 4].

Although H. pylori infection is established in early childhood, gastric cancer generally

develops in older adults, indicating that the direct oncogenic action of CagA does not work
effectively at a younger age. Moreover, once established, gastric cancer cannot be cured by H.

pylori eradication, indicating that H. pylori-driven gastric carcinogenesis proceeds through a“hit-and-run” mechanism, in which the maintenance of gastric cancer no longer requires H.
pylori CagA [4].

Importantly, we and others recently found that the delivered CagA induces DNA double-

strand breaks (DSBs) that underlie genomic instability in host gastric epithelial cells [5].
Mechanistically, the CagA-PAR1b interaction inhibits the kinase activity of PAR1b andthereby prevents the phosphorylation of the BRCA1 tumor suppressor, which is required forthe cytoplasmic-to-nuclear translocation of BRCA1 to prevent fork stalling/collapse andsubsequent DSB induction, while promoting error-free homologous recombination (HR)-122

M. Hatakeyama

mediated DSB repair. Hence, CagA-delivered gastric epithelial cells undergo a status termed“BRCAness”, characterized by excessive DSB formation that can only be repaired by error-

prone non-HR mechanisms [5]. As long as the p53 tumor suppressor is functionally active ingastric epithelial cells, however, CagA-induced DSBs elicit premature cell senescence, which

may promote the development of gastritis and ulceration in young adults infected with
cagA-positive H. pylori. Functional inactivation of p53, mainly through aging-associated TP53

mutations, allows for the expansion of cells with excess mutations by CagA-induced

BRCAness, from which cancer precursor cells that no longer require cancer-promoting CagA
activity may arise [5].

Indeed, sustained expression of CagA in gastric epithelial cells lacking TP53 gives rise toa BRCAness-associated genome mutation signature characterized by SBS3 and ID6. The

close connection between CagA and BRCAness was corroborated by a recent large-scale
case-control study showing that the risk of gastric cancer in individuals carrying pathogenic
variants of genes that induce BRCAness (such as BRCA1, BRCA2, PALB2, and ATM) increases

dramatically upon infection with cagA-positive H. pylori [6].
In conclusion, our results indicate the following.

1) The CagA-PAR1b interaction induces BRCAness by inhibiting cytoplasmic-to-nuclear
translocalization of BRCA1.

2) CagA-mediated BRCAness induces genomic instability, which causes mutations.
3) CagA-induced DSBs provoke p53-dependent G1 cell cycle arrest.

4) Loss of functional p53, most probably due to aging-associated TP53 mutation in normal
gastric epithelial cells, cancels G1 arrest and thereby enables the expansion of CagA-

expressing cells with genomic instability, from which CagA-independent cancer precursor
cells that accomplish Hit-and-Run carcinogenesis may arise.

5) The presence of pathogenic BRCA variants dramatically increases the incidence in H.
pylori-associated gastric cancer.

References
1. Murata-Kamiya N, Kikuchi K, Hayashi T, Higashi H, Hatakeyama M. Helicobacter pylorie x p l o i t s h o s t m e m b r a n e p h o s p h a t i d y l s e r i n e f o r d e l i v e r y, l o c a l i z a t i o n , a n dpathophysiological action of the CagA oncoprotein. Cell Host Microbe, 2010;7:399-411.
2. Higashi H, Tsutsumi R, Muto S, Sugiyama T, Azuma T, Asaka M, Hatakeyama M. SHP-2
tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.
Science, 2002; 295:683-686.
3. Saadat I, Higashi H, Obuse C, Umeda M, Murata-Kamiya N, Saito Y, Lu H, Ohnishi N,
Azuma T, Suzuki A, Ohno S, Hatakeyama M. Helicobacter pylori CagA targets PAR1/

Microbiome and Cancer

123

MARK kinase to disrupt epithelial cell polarity. Nature, 2007;447:330-333.
4. Hatakeyama M. Helicobacter pylori CagA and gastric cancer: a paradigm for hit-and-run

carcinogenesis. Cell Host Microbe, 2014;15:306-316.
5. Imai S, Ooki T, Murata-Kamiya N, Komura D, Tahmina K, Wu W, Takahashi-Kanemitsu A,
Knight CK, Kunita A, Suzuki N, Del Valle AA, Tsuboi M, Hata M, Hayakawa Y, Ohnishi
N, Ueda K, Fukayama M, Ushiku T, Ishikawa S, Hatakeyama M. Helicobacter pylori CagA
elicits BRCAness to induce genome instability that may underlie bacterial gastriccarcinogenesis. Cell Host Microbe, 2021;29:941-958.
6. Usui Y, Taniyama Y, Endo M, Koyanagi YN, Kasugai Y, Oze I, Ito H, Imoto I, Tanaka T,
Tajika M, Niwa Y, Iwasaki Y, Aoi T, Hakozaki N, Takata S, Suzuki K, Terao C,
Hatakeyama M, Hirata M, Sugano K, Yoshida T, Kamatani Y, Nakagawa H, Matsuda K,
Murakami Y, Spurdle AB, Matsuo K, Momozawa Y. Helicobacter pylori, homologousrecombination genes, and gastric cancer. N Engl J Med, 2023;388:1181-1190.

Masanori Hatakeyama, MD, PhD
1982-1986
1986-1991
1991-1994
1995-1999
1999-20092009-2022
2022
2022-present
2022-present

Graduated from Graduate School of Medicine, Hokkaido
University (Internal Medicine)Research Associate, Institute for Molecular and Cellular
Biology, Osaka University
Postdoctoral Research Fellow, Whitehead Institute, MIT
Member and Chief, Department of Viral Oncology,
Cancer Institute, Japanese Foundation of Cancer
Research (JFCR)
Professor, Division of Molecular Oncology, Institute for
Genetic Medicine, Hokkaido University
Professor, Division of Microbiology, Graduate School of
Medicine, The University of Tokyo
Professor Emeritus, The University of Tokyo
Head, Laboratory of Microbial Carcinogenesis, Instituteof Microbial Chemistry
Specially Appointed Professor, Institute for Genetic
Medicine, Hokkaido University

124

SOMATIC EVOLUTION, CANCER, AND OUR INEVITABLE
DECLINE WITH AGE – INEXTRICABLY LINKED

James DeGregori
Department of Biochemistry and Molecular Genetics
School of Medicine
University of Colorado Anschutz Medical Campus
Aurora, Colorado, USA(james.degregori@cuanschuz.edu)

Abstract:

Why do we get cancer? Why is cancer highly associated with old age? Of course, aging is

associated with the accumulation of more mutations, and some of these mutations cancontribute to cancer phenotypes. But we now understand that carcinogenesis is much more
complex than originally appreciated. In particular, there are tissue environmental forces thatboth impede and promote cancer evolution. Just as organismal evolution is known to be
driven by environmental changes, cellular (somatic) evolution in our bodies is similarly

driven by changes in tissue environments, whether caused by the normal process of aging,by lifestyle choices or by extrinsic exposures. Environmental change promotes selection for
new phenotypes that are adaptive to the new context. In our tissues, aging or insult-driven

alterations in tissues drives selection for adaptive mutations, and some of these mutations
can confer malignant phenotypes (reviewed in (1)).

We have been using mouse models of cancer initiation, mathematical models of cellular

evolution, and analyses of human tissue samples to better understand the evolutionary

forces that control somatic cell evolution and thus cancer risk. We have shown that aging

and inflammation dependent changes in stem cells and their tissue environments
dramatically dictate whether cancer-causing mutations are advantageous to stem cells in our

tissues, starting the cells down the path to cancer (e.g. (2-4)). Importantly, recent studies frommany labs have shown how as we age our tissues become dominated by clones bearing
mutations in known cancer-associated mutations (5, 6).

Clonal Evolution (1)125

Cigarette smoking and old age are the strongest risk factors for the development of lungcancer. We analyzed mutational profiles from non-malignant biopsies of lung from multiple
different cohorts [PEACE (NCT03004755), TRACERx (7)( NCT01888601) and CT-screened

Colorado cohorts (8)]. For these analyses, we used the DuplexSeq method (9) and a ~50 kb

panel covering the most commonly mutated regions in lung cancers, which allows for thedetection of rare mutations with a sensitivity of less than 1 in 107. Using this method, we
have identified large numbers of mutations in each brushing or biopsy, many of which arecataloged in the Catalogue Of Somatic Mutations In Cancer (COSMIC) (10), and most ofthese are predicted to disrupt protein function. We also observe pervasive positive selection

acting on mutations in many but not all of the cancer-associated genes, with different
patterns of selection in the lungs of ever-smokers and never-smokers. These clonal

expansions not only provide potential initiating clones for lung cancer development, but arealso predicted to perturb tissue function and integrity, as the genes mutated are known key

players in cell specification, proliferation and function. Ongoing studies are using primary
human and mouse lung epithelial cultures and mouse models to study how these mutations
interact with smoking and age to alter associated clonal expansions, tissue integrity, andmalignant progression. Our central hypothesis, to be further explored in the coming years, isthat mutation-driven clonal expansions are promoted by both aging and cigarette smoking,that these clonal expansions can engender lung tissue decline, which in turn further promotes
these clonal expansions. Thus, this feedforward loop can lead to accelerating lungdysfunction with age and exposures such as smoking. We further hypothesize that some (butnot all) of these mutation-driven clonal expansions, together with the perturbations in thetissue microenvironment, can contribute to the evolution of lung cancers. Finally, wehypothesize that interventions that restore a healthier tissue microenvironment can interrupt
this vicious cycle of clonal expansion and tissue decline, thus reducing disease risk.

In all, we propose a model whereby aging and contexts like smoking can promoteselection for cells with adaptive mutations, which can contribute to not only cancer risk but
also tissue aging. Thus, while young tissues impede selection for such adaptive mutations,old age or exposures such as from smoking are associated with a feed-forward loop of

perturbed tissue-mediated selection for mutant clones that then increase tissue aging. Thus,
understanding the forces controlling clonal selection as we age and due to lifetime exposures
could be critical for controlling multiple diseases of old age.
References
1. Marongiu F, DeGregori J. The sculpting of somatic mutational landscapes by

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J. DeGregori

evolutionary forces and their impacts on aging-related disease. Mol Oncol. 2022.
2. Henry CJ, Casas-Selves M, Kim J, Zaberezhnyy V, Aghili L, Daniel AE, et al. Aging-associated inflammation promotes selection for adaptive oncogenic events in B cellprogenitors. The Journal of clinical investigation. 2015;125(12):4666-80.
3. Higa KC, Goodspeed A, Chavez JS, De Dominici M, Danis E, Zaberezhnyy V, et al.

Chronic interleukin-1 exposure triggers selection for Cebpa-knockout multipotent
hematopoietic progenitors. Journal of Experimental Medicine. 2021;218(6).
4. Rozhok A, DeGregori J. A generalized theory of age-dependent carcinogenesis. eLife.

2019;8:e39950.
5. Kakiuchi N, Ogawa S. Clonal expansion in non-cancer tissues. Nature reviews Cancer.
2021.
6. Evans EJ, Jr., DeGregori J. Cells with Cancer-associated Mutations Overtake Our Tissues
as We Age. Aging Cancer. 2021;2(3):82-97.
7. Al Bakir M, Huebner A, Martínez-Ruiz C, Grigoriadis K, Watkins TBK, Pich O, et al. The
evolution of non-small cell lung cancer metastases in TRACERx. Nature.
2023;616(7957):534-42.
8. Assante A, Lkhagvadorj K, Clambey ET, Danhorn T, Merrick DT, Keith RL, et al.
Reduced Progenitor Function and Altered Immune Landscape Contribute to Field
Cancerization of Lung Adenocarcinoma. Am J Respir Crit Care Med. 2023;208(8):903-5.
9. Kennedy SR, Schmitt MW, Fox EJ, Kohrn BF, Salk JJ, Ahn EH, et al. Detecting ultralowfrequency mutations by Duplex Sequencing. Nature protocols. 2014;9(11):2586-606.
10. Forbes SA, Tang G, Bindal N, Bamford S, Dawson E, Cole C, et al. COSMIC (theCatalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations
in human cancer. Nucleic Acids Res. 2010;38(Database issue):D652-7.

James DeGregori, PhD
1987
1993
1993-1997
1997-present

Graduated with B.A. in Microbiology from University ofTexas, Austin, TX
Graduated with PhD in Biology from Massachusetts
Institute of Technology
Postdoctoral Fellow, Duke University
Faculty, University of Colorado School of Medicine, andcurrently Professor and Deputy Director of the
University of Colorado Cancer Center

127

USING ULTRA-SENSITIVE SEQUENCING TO STUDY CLONALEVOLUTION AND CANCER RISK

Rosana Risques
Department of Laboratory Medicine and Pathology, UW Medicine
1959 NE Pacific St, Box 357470, Seattle, WA 98195, USA
Lab: E.506, Office: E.504a(rrisques@uw.edu)

Cancer evolves through mutation, selection, and clonal expansions. However, clonal

expansions driven by cancer mutations are not exclusive of cancer: they also occur inhistologically normal tissue during normal aging1, 2. Little is known about this process in partdue to the difficulty to detect low frequency mutations in histologically normal tissue. Ourgroup pioneered the use of ultra-sensitive duplex sequencing3 to interrogate cancer driver

mutations in non-cancerous human samples. We discovered prevalent TP53 somatic

evolution in individuals with and without cancer4–6 and recently observed that an excess of

TP53 and KRAS clonal expansions in the normal colon may be associated with early-onset
colorectal cancer7. Here, I will present novel data using ultra-sensitive duplex sequencing tocharacterize the landscape of clonal evolution in normal bladder during human lifespan.

We analyzed normal bladder urothelium collected via urothelial brushing at the top of

the bladder (dome) and bottom of the bladder (trigone) from 26 individuals (16 males and 10
females), ages 24 through 86, who underwent autopsy at the University of Washington. We

used ultra-deep duplex sequencing (~6,000x) to perform high sensitivity mutation detectionin the most commonly mutated genes in normal urothelium and bladder cancer (16 genes).
Sequencing reads were processed with in-house pipelines and mutations were characterized

by gene and patient, compared to COSMIC, and analyzed for positive selection andmutational signatures.

Hundreds of mutations were identified in most samples, with some genes more

frequently mutated than others, in close similarity to prior studies. The overall mutation

128

R. Risquesfrequency increased with age, both in the dome and trigone samples, and the mutationallandscape of both sample types was remarkably similar for most patients. The location of

mutations within genes closely resembled the location of mutations reported for bladdercancer in COSMIC, and analyses of selection based on functional impact showed positive
selection for most genes. Mutational signatures revealed a predominance of age, APOBEC,

and smoking related signatures showcasing the contribution of these mutational processes to
bladder clonal evolution.

Our results provide a novel, ultra-high resolution picture of the mutational landscape ofthe normal human bladder which, remarkably, was obtained by ultra-deep sequencing of
single biopsies demonstrating the feasibility of this high-throughput approach for the studyof somatic evolution at large scale. We confirm that clonal evolution is a prevalent process
through human life and highlight the need to understand the promoting factors that enable
the progression of initiated clones into cancer.

References
1. Kennedy SR, Zhang Y, Risques RA. Cancer-Associated Mutations but No Cancer:
Insights into the Early Steps of Carcinogenesis and Implications for Early Cancer
Detection. Trends Cancer 2019;5:531–540.
2. Risques RA, Kennedy SR. Aging and the rise of somatic cancer-associated mutations innormal tissues. PLoS Genet 2018;14:e1007108.
3. Kennedy SR, Schmitt MW, Fox EJ, et al. Detecting ultralow-frequency mutations by

Duplex Sequencing. Nat Protoc 2014;9:2586–2606.
4. Krimmel JD, Schmitt MW, Harrell MI, et al. Ultra-deep sequencing detects ovariancancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous
tissues. Proc Natl Acad Sci U A 2016;113:6005–10.
5. Krimmel-Morrison JD, Ghezelayagh TS, Lian S, et al. Characterization of TP53 mutationsin Pap test DNA of women with and without serous ovarian carcinoma. Gynecol Oncol

2019.
6. Salk JJ, Loubet-Senear K, Maritschnegg E, et al. Ultra-Sensitive TP53 Sequencing forCancer Detection Reveals Progressive Clonal Selection in Normal Tissue over a Centuryof Human Lifespan. Cell Rep 2019;28:132-144 e3.
7. Matas J, Kohrn B, Fredrickson J, et al. Colorectal Cancer Is Associated with the Presenceof Cancer Driver Mutations in Normal Colon. Cancer Res 2022;82:1492–1502.

Clonal Evolution (1)129Rosana Risques, PhD
1992-1996
1996-2001
2001-2003
2003-2007
2007-20132013-20192019-present

BS in Biology. Autonomous University of Barcelona,Spain
PhD in Molecular and Cell Biology. Cancer ResearchInstitute and Autonomous University of Barcelona, Spain
Post-doctoral Associate. Department of Molecular
Biology, Sidney Kimmel Cancer Center, San Diego, CA,USA
Post-doctoral Fellow. Department of Pathology,
University of Washington (UW), Seattle, WA
Acting Instructor/Acting Assistant Professor, Departmentof Pathology, UW, Seattle, WA
Assistant Professor, Department of Pathology, UW,
Seattle, WA
Associate Professor, Department of Laboratory Medicine
and Pathology, UW, Seattle, WA

130

UNDERSTANDING OF GI CANCER INITIATION AND
PROGRESSION USING ORGANOIDS

Toshiro Sato
Department of Integrated Medicine and Biochemistry
Keio University School of Medicine
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan(t.sato@keio.jp)

In homeostatic adult tissues, niche factors play a crucial role in governing the long-term

self-renewal and diverse differentiation potential of tissue stem cells. By recapitulating theseniche factors in an in vitro environment, tissue stem cells have demonstrated the capacity toassemble into stereotypic organoid structures, sustaining long-term self-renewal. Notably, aspectrum of tissue-specific niche factors has been identified by our team and others,
facilitating the successful propagation of organoids derived from various adult tissues.

Human tissue-derived organoids have exhibited the remarkable ability to retain the genetic

and epigenetic alterations inherent in the original tissues. Moreover, these organoids haveshown disease-relevant biological characteristics both in vitro and in vivo.

Our comprehensive phenotypic analysis of patient-derived organoids has unveiled

intricate molecular mechanisms governing genotype-phenotype correlations in humandigestive tissue cancers. In parallel, the insights gleaned from genotype-phenotype
correlations are harnessed in reverse through genome editing technology. The strategic
introduction of genetic mutations into normal organoids has enabled the faithfulrecapitulation of tumor phenotypes. In this symposium, we will present our recent research

showcasing genotype-phenotype associations in patient-derived organoids and engineeredcounterparts.

References
1. Ohta Y, Fujii M, et al., Sato T＊, Cell-matrix interface regulates dormancy in human coloncancer stem cells. Nature. 2022; 608:784-794.

Clonal Evolution (1)131

2. Sugimoto S, Kobayashi E＊, et al., Sato T. ＊ An organoid-based organ-repurposing
approach to treat short bowel syndrome. Nature. 2021;592:99-104.
3. Kawasaki K, et al, Sato T＊. An Organoid Biobank of Neuroendocrine Neoplasms Enables
Genotype-Phenotype Mapping. Cell. 2020; 183: 1420-1435.
4. Nanki K, Fujii M, et al., Sato T ＊. Somatic inflammatory gene mutations in humanulcerative colitis epithelium. Nature. 2020;577:254-259.
5. Nanki K, Toshimitsu K, et al., Sato T＊. Divergent Routes toward Wnt and R-spondin
Niche Independency during Human Gastric Carcinogenesis. Cell. 2018;174:856-869.
6. Shimokawa M, Ohta Y, et al., Sato T＊. Visualization and targeting of LGR5+ humancolon cancer stem cells. Nature 2017;545:187-192.

Toshiro Sato, MD, PhD
1991-1997
1997-1999
1999-2003
2004
2004-2005
2005-2006
2006-2007
2007-20112011-20132013-20182018-2023
2023-Present

MD Keio University School of Medicine
Internship, Keio University HospitalInternal Medicine, Graduate school, School of Medicine,
Keio University
PhD
COE Postdoctoral Researcher, Keio University School of
Medicine (Supervisor: Hideyuki Okano)
Resident, TEPCO Hospital
Postdoctoral Researcher, Stowers Institute, Kansas City
(Supervisor: Linheng Li)
Postdoctoral Researcher, Hubrecht Institute, Utrecht, theNetherlands (Supervisor: Hans Clevers)
Assistant Professor, Department of Gastroenterology,
Keio University School of Medicine
Associate Professor, Department of Gastroenterology,
Keio University School of Medicine
Professor, Department of Organoid Medicine, Keio
University School of Medicine
Professor, Department of Integrated Medicine andBiochemistry, Keio University School of Medicine

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DECODING NORMAL BREAST TISSUE BIOLOGY AND THE
EVOLUTION OF PREMALIGNANT BREAST CANCER

Nicholas E. Navin
Department of Systems Biology
MD Anderson Cancer Center
Houston, Texas 77030, USA(nnavin@mdanderson.org)

To understand the transition from normal breast tissues to premalignant breast cancer, wemust first understand the variation of cell types across women from different backgrounds

and biological variables. To achieve this goal, we have led the Human Breast Cell Atlas(HBCA) that has analyzed normal breast tissues from over 126 women using single celltranscriptomics and spatial genomic approaches (Kumar et al., 2023, Nature). Our single-celltranscriptomics data profiled 714,331 cells and identified 12 major cell types and 58 biologicalcell states. These data revealed abundant pericyte, endothelial and immune cell populations,and highly diverse luminal epithelial cell states. Spatial mapping using four different
technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, aswell as distinct molecular differences between ductal and lobular regions. Collectively, thesedata provide an unprecedented reference of the adult normal breast tissue for studying
mammary biology and diseases such as breast cancer.

Ductal Carcinoma in Situ (DCIS) is the most common form of premalignant breast cancer

and is often detected during annual mammography. DCIS presents a major clinicalchallenge for treatment decisions, since only about 10-30% of women recur with invasive
disease within 10 years. This has led to significant over-treatment in many women with

DCIS that may never recur with invasive breast cancer, who could have avoided the side-

effects of therapy and elected ‘active surveillance’ with no treatment. However currently,
there are no effective biomarkers that can be used to stratify DCIS patients into low-risk andhigh-risk groups. Here, we developed an archival single cell DNA-seq method (arc-well) andapplied it to study FFPE tissues of DCIS samples and matched recurrences that occurredyears to decades later (Wang et al., 2023, Cell).

Clonal Evolution (2)133Analysis of 10 patients with matched DCIS and cancers that recurred 2-16 years later

showed that many primary DCIS had already undergone whole-genome-doubling, clonal

diversification and shared genomic lineages with persistent subclones in the recurrences.

Evolutionary analysis suggests that most DCIS cases underwent an evolutionary bottleneck,revealing chromosome aberrations in the persistent subclones that are associated withrecurrence. In future work, we plan to use scRNA-seq and spatial transcriptomics to also

understand the co-evolution of the TME together with the genetic changes which willundoubtedly lead to great insights into our understanding of breast cancer progression.
References
1. Kumar, T., Nee, K., Wei, R., He, S., Nguyen, Q.H., Bai, S., Blake, K., Pein, M., Gong, Y.,
Sei, E., et al. (2023). A spatially resolved single-cell genomic atlas of the adult humanbreast. Nature. 10.1038/s41586-023-06252-9.
2. Wang, K., Kumar, T., Wang, J., Minussi, D.C., Sei, E., Li, J., Tran, T.M., Thennavan, A., Hu,
M., Casasent, A.K., et al. (2023). Archival single-cell genomics reveals persistentsubclones during DCIS progression. Cell. 10.1016/j.cell.2023.07.024.

Nicholas E. Navin, PhD
2005-20102010-20112011-present
2022-present

Ph.D. at the Cold Spring Harbor Laboratory & Stony
Brook University
Postdoctoral researcher, Cold Spring Harbor Laboratory
Distinguished Professor MD Anderson Cancer Center
Chair, Department of Systems Biology MD Anderson
Cancer Center

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LIFE HISTORIES OF BREAST CANCER AND RELATED CLONES

Seishi Ogawa
Department of Pathology and Tumor Biology
Institute for the Advanced Study of Human Biology (WPI-ASHBi)
Kyoto University
Yoshida-Konoe-Cho, Sakyo-ku, Kyoto 606-8501, Japan(sogawa-tky@umin.ac.jp)

Recent studies have documented frequent evolution of clones carrying common cancer

mutations in apparently normal tissues, which are implicated in cancer development.
However, our knowledge is still missing regarding what additional driver events take placein what order, before one or more of these clones in normal tissues ultimately evolve tocancer. Here, using phylogenetic analyses of multiple microdissected samples from bothcancer and non-cancer lesions, we show unique evolutionary histories of breast cancers
harbouring der(1;16), a common driver alteration found in approximately 20% of breast
cancers. The approximate timing of early evolutionary events was estimated from themutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative
chromosome was acquired from early puberty to late adolescence, followed by the

emergence of a common ancestor by the patient’s early 30s, from which both cancer andnon-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following

years, these clones occupied a large area within the premenopausal breast tissues by the timeof cancer diagnosis. Unexpectedly, evolution of multiple independent cancer founders fromthe non-cancer ancestors was common, contributing to intratumour heterogeneity. Ourfindings provide new insight into the evolution of breast cancer.
References
1. Nishimura T, Kakiuchi N, Yoshida K, Sakurai T, Kataoka TR, Kondoh E, Chigusa Y,
Kawai M, Sawada M, Inoue T, Takeuchi Y, Maeda H, Baba S, Shiozawa Y, Saiki R,
Nakagawa MM, Nannya Y, Ochi Y, Hirano T, Nakagawa T, Inagaki-Kawata Y, Aoki K,

Clonal Evolution (2)135

Hirata M, Nanki K, Matano M, Saito M, Suzuki E, Takada M, Kawashima M, Kawaguchi
K, Chiba K, Shiraishi Y, Takita J, Miyano S, Mandai M, Sato T, Takeuchi K, Haga H, Toi M,

Ogawa S. Evolutionary histories of breast cancer and related clones.
Nature;620(7974):607-614, 2023.
2. K a k i u c h i N , O g a w a S . C l o n a l e x p a n s i o n i n n o n - c a n c e r t i s s u e s . N a t R e v
Cancer;21(4):239-256, 2021.
3. Yokoyama A, Kakiuchi N, Yoshizato T, Nannya Y, Suzuki H, Takeuchi Y, Shiozawa Y,
Sato Y, Aoki K, Kim SK, Fujii Y, Yoshida K, Kataoka K, Nakagawa MM, Inoue Y, Hirano T,
Shiraishi Y, Chiba K, Tanaka H, Sanada M, Nishikawa Y, Amanuma Y, Ohashi S, Aoyama
I, Horimatsu T, Miyamoto S, Tsunoda S, Sakai Y, Narahara M, Brown JB, Sato Y, Sawada
G, Mimori K, Minamiguchi S, Haga H, Seno H, Miyano S, Makishima H, Muto M,
Ogawa S. Age-related remodelling of oesophageal epithelia by mutated cancer drivers.
Nature;565(7739):312-317, 2019.

Seishi Ogawa, MD, PhD
1989-1993
2006-2008
2008-20132013-present

MD, PhD program, University of Tokyo
Associate Professor, The 21st century COE program,
Graduate School of Medicine, University of Tokyo
Associate Professor, Cancer Genomics Project,
University of Tokyo
Professor, Department of Pathology and Tumor Biology,
Kyoto University

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CONCLUDING REMARKS

Hiroyuki Tsuda
Nanotoxicology Project
Nagoya City University Graduate School of Medical Sciences
3-1 Tanabedohri, Mizuho-ku, Nagoya 467-8603, Japan(htsuda@phar.nagoya-cu.ac.jp)

In 2000, Hanahan and Weinberg made a marked impact on the field of cancer researchwith the publication of their seminal paper "The Hallmarks of Cancer". This review

described cancer from the point of view of a complex tissue and enumerated biologicalcapabilities that they argued were shared by virtually all cancers. Douglas Hanahan was

invited to the 35th International Symposium of the Princess Takamatsu Cancer ResearchFund, which was held in 2004, and Robert Weinberg was invited to the 37th International

Symposium of the Princess Takamatsu Cancer Research Fund, which was held in 2006. Since

then, tremendous progress has been made in our understanding of the development ofcancers. This has forged remarkable advances in the early detection and treatment of several

types of cancers. However, while the detection and treatment of early stage cancers is highly

successful and treatment of advanced cancers that have metastasized to other tissues has

improved, the 5-year survival rate of patients with advanced cancers remains poor. Thisemphasizes the continued critical importance of foundations such as the Princess Takamatsu
Cancer Research Fund.

The Keynote Lecture of the 51st International Symposium of the Princess Takamatsu

Cancer Research Fund was given by Curtis C. Harris. This Symposium examined several

aspects of carcinogenesis, ranging from identification of environmental carcinogens to tumor
promotion, DNA mutational signatures, and the multiomics of precision medicine, andincluded the association of the microbiome with different cancers.

It is well known that limiting exposure to environmental and workplace carcinogens is of

the utmost importance in cancer prevention. Two speakers discussed the use of animalmodels to identify human carcinogens. Work with humanized-liver mice found that

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137

acetoaceto-o-toluidide (AAOT) is metabolized to o-toluidine (OTD) by humanized-liver
cells, and since OTD is a known human bladder carcinogen, the presence of OTD in the

bladder of these mice identifies AAOT as a probable human bladder carcinogen. Anotherstudy using intratracheal instillation, a method known as TIPS, found that all tested carbon

nanotubes were carcinogenic to the rat lung. Implementation of regulations for the safe
manufacture and use of these materials could have a tremendous impact on human cancers.

Another topic of this symposium concerned the pathways by which carcinogens induce

cancer. Prostate cancer is the fifth most lethal cancer for males. A talk on prostate cancer
described how many environmental toxins target prostate stem-progenitor cells, and due tothe long-lived nature of these cells, the effects of these toxins increase prostate carcinogenic

potential over extended time periods. Gastric cancer is the fourth leading cause of cancer
mortality. Helicobacter pylori infection is a known risk factor for gastric cancer. Up to 96% of H.
pylori express CagA, which interacts with PAR1b and inhibits the kinase activity of PAR1b.

This prevents phosphorylation and activation of BRCA1, resulting in an increase in DNA
double strand breaks and an increase in mutation levels. p53 protects against CagA induced

stomach cancer. As people age, they have decreased p53, and consequently, young people
infected with H. pylori often develop gastric cancer as they age. Notably, these pathways not

only increase our understanding of carcinogenesis, but can be used clinically. H. pylori
infection also induces inflammation and the methylation level of a single marker gene,miR124a-3, accurately predicts gastric cancer risk. Another pathway that was shown to haveprobable clinical utility was the histone H3.3 specific chaperone HIRA and its target histoneH3.3. This pathway is a promising target to prevent cancer progression.

Another major topic of this symposium was the clinical use of mutational signatures.

Mutational signatures are patterns of mutations characteristic of exposure to different
carcinogens. The Nakahara Memorial Lecture was delivered by Arthur P. Grollman. In thislecture he discussed the mutational signature of aristolochic acid (AA), a powerfulnephrotoxin and human carcinogen. This mutational signature has been detected in several
different cancer types and in healthy tissue, identifying persons exposed to AA in theabsence of tumor development. AA is bioactivated in the liver and active AA metabolites

complex with human serum albumin in the circulatory system. These complexes aretransported to a variety of tissues where they elicit mutational effects. Dr. Grollman thendescribed the development of a test known as UroSEEK which surveys mutations and copy
number changes and is able to detect urothelial cancer cells shed into the urine, allowing
early detection of urothelial cancers.

Technology that identifies patterns of DNA adducts, which can result in contextual

mutational signatures, was also described. Knowledge of DNA adduct and DNA mutational

profiles allows identification of driver adducts and driver mutations associated with

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H. Tsuda

carcinogenesis, and this significantly advances our knowledge of the events that cause
cancer. Advancements in metabolic profiling and technology able to detect all DNA adducts
induced by exposure to various chemicals allows characterization the effect of specificchemicals and their metabolites on DNA and has important implications for genotoxicity

testing, biomonitoring, and improved cancer prevention and treatment. Use of mutationalsignatures and DNA adductomics to improve detection and therapy of cancer in patientswith gastric cancer, liver cancer, and renal cancer was described. The DNA landscape ofdriver events in gastric cancer allows identification of individuals at risk of developing
gastric cancer. This in turn allows extensive endoscopic screening of these patients andapplication of preventive interventions. For liver cancer, the third most lethal cancer,

adductomics was used to identify risk factors. Finally, DNA mutation profiles associatedwith renal cancer indicated the existence of multiple, geographically variable, mutagenic
exposures and promises to expand our knowledge of the causes of renal cancer. In addition,
the use of mutational signatures to uncover an inflammatory pathway driven by IL1B

associated with non-small cell lung cancer in never smokers induced by exposure to air
pollution particulate matter was discussed.

Epidrivers can be considered a type of driver mutation signature. The epigenome consistsof chemical compounds that modify, or mark, the genome and affect its function, forexample, DNA methylation. Genes encoding proteins that directly regulate the epigenome

are known as epigenetic regulator genes (ERGs). Analysis of ERGs in 11,000 tumor andnormal tissue specimens identified a subset of ERGs that were altered at the genetic or RNA
expression level across many malignancies. These epidrivers confer traits associated with thehallmarks of cancer onto cells, indicating that epidrivers can act as drivers of tumorigenesis.

DNA methylation is part of the epigenome, and as noted above, the methylation level of asingle marker gene, miR124a-3, accurately predicts gastric cancer risk. Neuroendocrine
carcinoma of the gastrointestinal system (GIS-NEC) is a rare but highly malignant cancer
with extremely poor patient outcomes. A talk describing comprehensive studies of thealterations in GIS-NECs noted that epigenetic aberrations play a significant role in GIS-

NECs, especially hypermethylation of the promoter region of SOX2 resulting in highexpression of SOX2 in most GIS-NECs.

Driver mutations can occur years, even decades, before events that promote thedevelopment of cancer. Mouse skin tumors were induced in different mouse models and atdifferent times in the life of the mice by exposure to different regimens of mutagens followedby exposure to strong inflammatory processes. Whole genome sequencing of over 100 of thetumors revealed that cells with mutations sufficient to initiate carcinogenesis could remainin the skin for an extended period of the life of the mouse until activated by a promoting
factor. The promoting factors did not necessarily cause mutations. This suggests the probable

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139

existence of unidentified factors that promote human cancers. Another talk discussed the useof ultra-sensitive duplex DNA sequencing to characterize clonal evolution in normal human

bladder urothelium. Clonal evolution is a process that occurs throughout human life, andmutation frequency increased with age. Mutational signatures in normal urothelium often

resembled those found in bladder cancers, highlighting the need to understand promoting
factors and the mechanism by which they enable the progression of initiated clones into

cancer. Another talk discussed age and changes in the tissue environment, and how thesechanges drive selection of cells with adaptive mutations, thus driving clonal evolution as weage. The talk concluded that young tissues and old tissues respond differently to the tissue
environment, and a tissue environment that doesn't promote tumorigenesis in young tissue
may promote tumorigenesis in old tissue. Overall, these talks highlighted the need tounderstand the exact mechanism of tumor promotion.

In agreement with the conclusion that driver mutations can occur many years prior totumor promotion, phylogenetic analysis of microdissected samples from cancer and non-cancer lesions in the breast identified driver mutations that were acquired in early puberty tolate adolescence from which both cancer and non-cancer clones evolved. These clones

occupied a large area within the premenopausal breast tissue by the time of cancer diagnosis.
Evolution of multiple independent cancer founders from non-cancer ancestors was common,

resulting in intratumor heterogeneity, suggesting a role for epigenetic changes and/or the
microenvironment in cancer development. As noted above, the microenvironment is apotential non-mutagenic promoter of cancer. Another potential non-mutagenic cancer

promoter is obesity, which was the topic of another talk. Metabolomic and proteomic studies
have the potential to unravel the obesity-cancer link, enabling informed preventative

strategies and the formulations of effective public health policies, as well as enriching ourunderstanding of the mechanisms of tumor promotion.

It is well known that the gut microbiota impacts colorectal cancer (CRC). One talkdiscussed the relationship between specific bacteria and their communities and CRC. While

challenging, it was proposed that translational research on the manipulation of the
microbiota for the therapeutic benefit of cancer patients is feasible. Another talk discussed

the effect of spinach on the microbiome. In a rat model, spinach increased microbiome
diversity and exhibited significant antitumor activity. It was determined that the antitumor

activity was linked to spinach derived linoleate bioactives and altered butanoate metabolism
arising from increased alpha-diversity of the gut microbiome. Linoleate and butanoate

metabolites inhibited histone deacetylase (HDAC), induced apoptosis, and altered IFNgamma signaling in human and mouse colon cancer cells. In addition, IL-2 secretion wasincreased in T-cell hybridomas co-cultured with the colon cancer cells. Thus, therapeutictargeting of the microbiome is feasible. Another talk discussed the relationship of oral

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H. Tsuda

microbiome dysbiosis with orodigestive cancer, including pancreatic cancer, in humans.
Cancer-associated oral microbiota play a pivotal role in metabolic production of carcinogens.

In addition, the gut microbiome was shown to predict therapy response in non-small celllung cancer patients and melanoma patients. In addition, another talk discussed the utilityof the Collaborative Cross mouse as an optimal model for deciphering gene-environmentmicrobiome interactions in carcinogenesis. This mouse can be used to develop novel

therapies, including microbiome-targeted therapies, for the prevention and treatment ofcancer.

Two talks discussed the use of in vitro testing systems. One talk discussed determining

the mutational signatures of test agents. It was determined that the mutational signatures of

4 different carcinogens were qualitatively similar in human tissue organoids derived fromthe stomach, colon, liver, pancreas, and kidney. The mutational signatures of an additional

16 environmental carcinogens were investigated in human gastric organoids, and 79 agentswere tested using induced human pluripotent stem cells (hIPSCs). The 16 carcinogens

produced 12 different signatures in the organoids and the 79 agents produced 41 different
signatures in hIPSCs, providing insights into processes underlying the causes of humancancer. The other talk discussed the use of patient derived organoids to examine genotypephenotype associations in human digestive tissue cancers. This technique allows
development of therapeutic strategies to combat cancer.

Two talks discussed the use of aspirin for cancer prevention. In young people, but notolder people, aspirin decreases prostaglandin metabolites in the urine, deceasing
inflammation and cancer; aspirin also decreases kynurenate and colorectal cancer; andaspirin also decreases hepatic fat and liver cancer. The other talk described the use of entericcoated aspirin (100 mg/day). In a clinical trial it was found that 100 mg/day enteric-coatedaspirin decreased the recurrence of CRC. In a second clinical trial, aspirin inhibited thegrowth of colorectal polyps of more than 5 mm in diameter in patients with familial
adenomatous polyposis.

One of the final talks of the symposium described the development of a single-cell DNA

sequencing method that could be used with archived formalin-fixed paraffin-embedded
tissue samples from patients with Ductal Carcinoma in Situ (DCIS). Analysis of the genome
profile of 10 patients with DCIS and breast cancers that recurred 2-16 years later enabled theidentification of chromosome aberrations that were associated with recurrence. Since only
about 10-30% of women with DCIS develop invasive breast cancer, this DNA sequencing

method can be used to identify women at high-risk of developing invasive breast cancer andwomen with DCIS that may never develop invasive breast cancer, allowing treatment of thewomen at high-risk and allowing women at low-risk to choose either therapy or active
surveillance with no treatment to avoid the side-effects of therapy.

Concluding Remarks

141

The 51st International Symposium of the Princess Takamatsu Cancer Research Funddemonstrated several advancements in our understanding of the mechanisms ofcarcinogenesis and the detection and treatment of cancer. Advanced, metastatic cancers stillpresents a tremendous challenge, and the 5-year survival rate of patients with advanced

cancers remains poor. We look forward to the 52nd International Symposium of the Princess
Takamatsu Cancer Research Fund and the further advancement of cancer therapies.

I would like to express my sincere gratitude to the organizers of this symposium, Drs.

Allan Balmain, Hitoshi Nakagama, Fuyuki Ishikawa, and Yukari Totsuka. I would also liketo express my sincere gratitude to the secretary general of the symposium Dr. Michihiro

Mutoh. I would also like thank the speakers and all of the participants for the excellent
presentations and fruitful discussions, which greatly contributed to the success of thesymposium. Finally, I would also like to express my deep gratitude to the the Foundation for
its support and the work that was put into this symposium.