Extended Abstracts forthe 48th International Symposium of
the Princess Takamatsu Cancer Research FundCOMPLEXITY IN CANCER-HOST
CROSSTALK
November 7-9, 2017
Tokyo, Japan
Edited by

Hitoshi Nakagama
Michael Karin
Yoshinori Murakami
Mutsuhiro Takekawa
Kohei Miyazono

Princess Takamatsu Cancer Research Fund

CopyrightⒸ2018 by the Princess Takamatsu Cancer Research Fund
No part of this Extended Abstracts may be reproduced, stored in retrieval
systems or transmitted, in any form or by any means, without permission
from the Princess Takamatsu Cancer Research Fund.

Princess Takamatsu Cancer Research Fund

1-14-15-102 Takanawa, Minato-ku, Tokyo 108-0074, Japan
Telephone: +81-3-3441-0111
Facsimile: +81-3-3441-0112
E-mail:info@ptcrf.or.jp
Printed in Japan
ISSN 2186-8603

B

CONTENTS
KEYNOTE LECTURE

Metabolic and Immune Control of Liver Carcinogenesis:
Nash Promoted Generation of Immunosuppressive B Cells that
Antagonize Anti-tumoral CD8+ T Cells are a Critical Promoter ofInflammation-Driven HCC
Michael Karin....................................................................................................................................1

CELLULAR NETWORK OF CANCER TISSUES

Cell Context-dependent Roles of a Cell Adhesion Molecule, CADM1,
in Human Oncogenesis
Yoshinori Murakami ....................................................................................................................8Chemotherapy Induces Enrichment of Immune-evasive Breast Cancer
Cells
Gregg L. Semenza ........................................................................................................................12

NOVEL STRATEGIES TARGETING CANCER NETWORK

Tumor-derived Exosomes Promote Organotropic Metastasis and Systemic
DiseaseDavid Lyden ....................................................................................................................................16iExtracellular Vesicles as a Novel Platform for Cancer Diagnosis andTherapeutics
Takahiro Ochiya ............................................................................................................................21
The Functional Role of Tumor Stroma and Exosomes in Biology andTreatment of Pancreatic Cancer
Raghu Kalluri ..................................................................................................................................24

INFLAMMATION AND CANCER MICROENVIRONMENTMetabolic Crosstalk by Autophagy Adaptors in the Tumor Stroma
Jorge Moscat.....................................................................................................................................29

Comprehensive Phenotypic Characterization of Intestinal Cancer with
Multiple Driver Mutations
Masanobu Oshima ......................................................................................................................32
Cell Plasticity in Colorectal Carcinogenesis
Florian R. Greten...........................................................................................................................37

CANCER-STROMAL INTERACTION

Tumour-stroma Interactions in Breast Cancer Progression
Clare M. Isacke ...............................................................................................................................44
The role of SASP in Tumor Microenvironment of Obesity-associatedLiver Cancer
Naoko Ohtani .................................................................................................................................49ii

Stromal Cells: Essential Regulators of Tumor Progression and Metastasis
and Targets for Therapy
Zena Werb .........................................................................................................................................52

HETEROGENEITY AND PLASTICITY OF CANCER

Development and Maintenance of the Stemness of Colon Cancer
Koji Okamoto ..................................................................................................................................56
Unearthing Mechanisms of Malignant Progression and Resistance ofCancer Stem Cells
Elaine Fuchs .....................................................................................................................................60

Role of Actin Dynamics in Reprogramming and Cell Fate Determinationof Cancer and Microenvironmental Cells
Hideyuki Saya ................................................................................................................................67

NOVEL APPROACHES TO ANALYZE CANCER NETWORK

Role of the Microenvironment in Cancer Invasion and Therapy Response
Erik Sahai ...........................................................................................................................................70
Challenges for Complexity of Cancer by Supercomputer – from Genome
to Network
Satoru Miyano ................................................................................................................................73
Pathology from the Molecular Scale on Up
Garry P. Nolan ................................................................................................................................77
iii

IMMUNE SYSTEMS AND CANCERMICROENVIRONMENTMicrobiome, Metabolism and Mucosal Immunity
Ramnik J. Xavier ...........................................................................................................................79
Immunogenomics in the Cancer Precision Medicine Era; Characterizationof T Cell Repertoires in Tumor Immune Microenvironment by TCR
Analysis
Yusuke Nakamura .......................................................................................................................83
At the Cross-road between the Gut Microbiome and Cancer
Immunosurveillance
Conrad Rauber ...............................................................................................................................87
Breast Tumor Evolution: Heterogeneity and Selection
Kornelia Polyak .............................................................................................................................91

Genetic Screens to Identify Immunotherapy Targets
W. Nicholas Haining ..................................................................................................................94

NAKAHARA MEMORIAL LECTURE

Latency, Immune Evasion and Outbreak of Metastatic Stem Cells
Joan Massagué................................................................................................................................96iv

SIGNALING DIVERSITY IN CANCER

Aberrant Regulation of MAPK Signaling Pathways in Cancer
Mutsuhiro Takekawa ...............................................................................................................106
PI 3-Kinase Links Obesity, Insulin Resistance and Cancer
Lewis C. Cantley .........................................................................................................................111

TGF-β Signaling in Cancer
Kohei Miyazono ..........................................................................................................................114
The Dependence Receptor Paradigm: from an Original Mechanism ofCell Death to Clinical Trial
Patrick Mehlen .............................................................................................................................120
ASK Family Kinases in Stress Signaling and Cancer
Hidenori Ichijo .............................................................................................................................126

CONCLUDING REMARKS

Hitoshi Nakagama ....................................................................................................................130

List of Participants ................................................................................................................................135
List of Previous International Symposium Series .......................................................157v

1

METABOLIC AND IMMUNE CONTROL OF LIVERCARCINOGENESIS: NASH PROMOTED GENERATION OF
IMMUNOSUPPRESSIVE B CELLS THAT ANTAGONIZE ANTITUMORAL CD8+ T CELLS ARE A CRITICAL PROMOTER OF
INFLAMMATION-DRIVEN HCC
Michael Karin

Departments of Pharmacology and Pathology
UC San Diego School of Medicine
9500 Gilman Drive, Leichtag Building MC0723
La Jolla, CA 92093 USA(mkarin@ucsd.edu)
Hepatocellular carcinoma (HCC), the major form of liver cancer, is currently the second

leading cause of cancer deaths worldwide. The most common causes of HCC are hepatitis
virus B and C (HBV, HCV) infections, nonalcoholic steatohepatitis (NASH), and alcoholicsteatohepatitis (ASH). Due to the current obesity epidemic and the advent of HBV vaccinesand effective HCV therapies, NASH will soon become the leading cause of HCC in the US
and Western Europe. NASH is the most severe manifestation of nonalcoholic fatty liverdisease (NAFLD), currently affecting 30-40% of American adults. All of the above

hepatides (HBV and HCV infections, NASH, and ASH) are chronic inflammatory diseasesof the liver, and inflammation driven by the cytokines TNF and IL-6 plays a key role in
HCC pathogenesis [1,2].

To understand how obesity, NAFLD, and NASH increase HCC risk, we initiallycombined exposure to the hepatic carcinogen diethylnitrosamine (DEN) with high-fat diet
(HFD) or genetic obesity and have shown that both dietary and genetic obesity strongly

enhance HCC induction [3]. Subsequently, we found that autocrine IL-6 signaling is critical
for the genesis of HCC progenitor cells (HcPC), which serve as the precursors for HCC4.

Although HcPC exhibit a similar transcriptomic profile to bile-duct-derived bipotential
hepatobiliary progenitors, they actually originate from differentiated zone 3 (pericentral)

hepatocytes [4]. Furthermore, we have shown that both DEN-induced and NASH-derived(see below) HCC are initiated by differentiated zone 3 hepatocytes which express the

enzymes needed for metabolic activation of DEN and de novo lipogenesis (DNL) [5]. In
response to chronic stress and damage, these cells accumulate oncogenic mutations,
undergo de-differentiation, acquire proliferative potential, and give rise to cancer.

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M. Karin

Since HFD-fed or genetically obese mice exhibit so-called bland steatosis but do not

manifest classical signs of NASH and require treatment with a carcinogen (i.e. DEN) to

form HCC, we sought to develop a more accurate model for NASH and its progression to
HCC. Studies of human NASH led to the so-called two-hit and multiple-hit hypotheses,
according to which lipid droplet accumulation in hepatocytes is the first hit, leading toappearance of bland steatosis, whereas mitochondrial dysfunction or endoplasmic

reticulum (ER) stress are likely to constitute secondary hits that lead to liver damage,inflammation, and NASH [6]. We therefore chose to use MUP-uPA transgenic mice, whichdisplay transient liver damage due to ER stress caused by high expression of urokinase

plasminogen activator (uPA) in their hepatocytes [7]. Early liver damage in these mice,
which peaks between 4-6 weeks of age, is transient because liver regeneration results indownregulation of uPA expression. Nonetheless, by placing the mice on HFD for several
months we found that ER stress is re-ignited and that the mice display classical NASH
signs, including ballooning degeneration of hepatocytes, inflammatory and immune

infiltrates, and a “chicken-wire” pattern of peri-cellular fibrosis [8]. NASH development is
totally dependent on ER stress and can be prevented by treating the mice with the chemical

chaperons phenyl butyric acid (PBA) or tauroursodeoxycholic acid (TUDCA) or by
overexpression of the chaperon GRP78/BiP. Importantly, after 8-9 months on HFD, 85-90%of the mice display aggressive, poorly differentiated HCC [8]. Exome sequencing of 16 suchtumors had revealed 50-100 coding region mutations [9], a mutation frequency similar tothat of human HCC [10]. The mutational signature was also nearly identical between themouse and human tumors [9]. Further validating the usefulness of the MUP-uPA model forstudying the pathogenic pathways involved in human HCC was the observation that manyof the mutations were not recurrent, affecting a number of different pathways [9]. By
contrast, exome sequencing of DEN-induced HCC revealed an overwhelming abundanceof a particular Braf mutation, replacing valine with glutamate at position 600 [4]. Curiously,
the same mutation has been identified in 22% of human cholangiocarcinomas but not inhuman HCC [11].

Given the authenticity of the MUP-uPA + HFD model we invested much effort inunderstanding the underlying pathogenic mechanisms. One of the earliest molecular andcellular events to take place in HFD-fed MUP-uPA mice is a large increase in TNF

production, mainly by newly recruited liver macrophages [8]. TNF signaling via the type 1

TNF receptor (TNFR1) was found to be of importance in NASH pathogenesis, operating

through the killing of cholesterol-loaded hepatocytes, which promotes subsequent
compensatory proliferation. In addition, TNFR1 engagement is critical for early tumor

growth acting through IKKβ-driven NF-κB activation [8]. Consistent with these results,
constitutive IKKβ activation in hepatocytes promotes HCC development [12].

Keynote Lecture

3

Another important player in NASH-induced HCC is the Sqstm1 gene, which codes forthe autophagy adaptor protein p62 [13]. Hepatocyte-specific Sqstm1 ablation results in amarked reduction in HCC development in HFD-fed MUP-uPA mice [14]. Curiously, theoncogenic function of p62 is not related to its role as an autophagy adaptor. Instead, it ismediated through the activation of NRF2 [14]. Exactly how NRF2 activation promotes HCC
development is a matter of intense investigation but it should be noted that mutations in

the genes encoding NRF2 and its negative regulator KEAP1 have been detected in nearly
15% of human HCC [10]. However, in HFD-fed MUP-uPA mice, NRF2 activation is non-mutational. Of further note, p62 accumulation is a hallmark of many chronic liver diseases
that increase HCC risk [13].

Most recently we found that the adaptive immune system plays a critical role in thecontrol of NASH to HCC progression. We were first intrigued by the finding that human

NASH and ASH patients exhibit elevated circulating amounts of immunoglobulin A (IgA)[15]. We confirmed that HFD-fed MUP-uPA mice and other mouse models of chronic liver
inflammation and fibrosis also exhibit elevated amounts of circulating IgA and have shown
it to be produced by liver-resident IgA+ plasmablasts and plasma cells, collectively referred

to as IgA + plasmocytes [9]. Importantly, liver IgA + plasmocytes express theimmunosuppressive molecules IL-10 and PD-L1 and are also present in human NASHafflicted liver but are barely detectable in healthy livers [9]. Immunosuppressive

plasmocytes start accumulating in the chronically inflamed and damaged liver long before
visible HCC nodules are detected and their generation depends on TGF-β-driven classswitch recombination, as well as IL-21 and other cytokines. In the livers of HFD-fed MUP-

uPA mice, IL-21 is mainly made by follicular helper cells (Tfh), whose accumulation also
correlates with NASH development [9]. Importantly, previous work in our lab on drugresistant prostate cancer had shown that IgA+ IL-10- and PD-L1-expressing plasmocytes

accumulate in castration-resistant tumors in response to myofibroblast activation and leadto exhaustion of tumor-directed cytotoxic T cells (CTL) [16]. Interestingly, NASH and ASH
are also accompanied by activation of liver resident myofibroblasts, also known as hepatic

stellate cells (HSC). Activated HSC and other myofibroblasts produce large amounts of
CXCL13, an important B-cell chemoattractant [17]. Most importantly, IgA ablation andother genetic manipulations that prevent the development of IgA+ immunosuppressive

plasmocytes severely attenuate NASH to HCC progression in MUP-uPA mice without

affecting NASH development [9]. The decrease in NASH to HCC progression is due toenhanced activation of anti-tumorigenic CD8+ CTL, whose ablation results in dramatic
acceleration (7- to 8-fold reduction in tumor latency) and enhancement of HCC

development [9]. Furthermore, introduction of T cells without B cells into HCC-bearing
Rag1-/- mice that are deficient in both B and T cells results in tumor regression. Conversely,

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M. Karin

CD8+ T-cell depletion from IgA-deficient mice enhances HCC development. Collectively,

these results strongly suggest that chronic liver damage and inflammation also promote

HCC development through indirect effects on the adaptive immune system, in addition totheir effect on the proliferation of early HCC progenitors (Figure 1). IgA +immunosuppressive plasmocytes carry out their tumor-promoting function by inducing
the exhaustion of HCC-directed CTL, which play an important protective role in

Figure 1

The pathogenesis of NASH-driven HCC.
NASH is initiated in response to metabolic alterations that promote the accumulation oflipids in hepatocytes and cause mitochondrial abnormailities that enhance the production ofreactive oxygen species (ROS) that lead to genetic alterations that result in accumulation of
oncogenic mutations as well as hepatocyte death. Release of DAMPs by dying hepatocytes
triggers an inflammatory response that supports the growth of HCC progenitor cells(HcPC), leading to appearance of early malignant lesions. Such lesions are kept in check by
HCC-directed cytotoxic T lymphocytes (CTL). However, the chronic inflammation that
accompanies NASH also results in generation of immunosuppressive plasmocytes thatinterfere with CTL-mediated immunosurveillance, thereby promoting immune escape andtumor growth.

Keynote Lecture

5

suppressing HCC development. Indeed, sequencing of T-cell receptor (TCR) α and β chainsfrom HCC-associated CD8+ T cells indicates that these cells have undergone clonal selectionand expansion, suggesting that the majority of these cells recognize HCC-associatedantigens. Importantly, our results provide the strongest support thus far for the

immunosurveillance hypothesis, which was first proposed more than 50 years ago by

Burnet and Thomas, according to which our immune system is responsible for rejection of

nascent tumors. Our work also indicates that inflammation-driven immunosuppression is a
key tumor-promoting mechanism.

Importantly, by treating HCC-bearing mice with a neutralizing PD-L1-specific antibody

we were able to re-invigorate exhausted HCC-directed CTL and achieve rather impressive

tumor regression. Rinvigoration of HCC-directed CTL was accompanied by a considerable
reduction in the liver content of immunosuppressive IgA+ plasmocytes whose development
depends on their ability to engage PD-1 expressed by Tfh [9]. These results explain why

antibodies that disrupt PD-1:PD-L1 interactions are quite effective in inducing theregression of aggressive stage 4 human HCC [18]. We all hope that the recent clinicalapproval of such drugs will revolutionize the treatment of non-resectable HCC, one of themost lethal cancers that is remarkably resistant to conventional chemotherapy.
References
1. Maeda, S., Kamata, H., Luo, J.L., Leffert, H. & Karin, M. IKKbeta couples hepatocyte
death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121, 977-990 (2005).
2. Naugler, W.E., Sakurai, T., Kim, S., Maeda, S., Kim, K., Elsharkawy, A.M. & Karin, M.
Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 317, 121-124 (2007).
3. Park, E.J., Lee, J.H., Yu, G.Y., He, G., Ali, S.R., Holzer, R.G., Osterreicher, C.H.,
Takahashi, H. & Karin, M. Dietary and genetic obesity promote liver inflammation andtumorigenesis by enhancing IL-6 and TNF expression. Cell 140, 197-208 (2010).
4. He, G., Dhar, D., Nakagawa, H., Font-Burgada, J., Ogata, H., Jiang, Y., Shalapour, S.,
Seki, E., Yost, S.E., Jepsen, K., Frazer, K.A., Harismendy, O., Hatziapostolou, M.,
Iliopoulos, D., Suetsugu, A., Hoffman, R.M., Tateishi, R., Koike, K. & Karin, M.
Identification of liver cancer progenitors whose malignant progression depends on
autocrine IL-6 signaling. Cell 155, 384-396 (2013).
5. Font-Burgada, J., Shalapour, S., Ramaswamy, S., Hsueh, B., Rossell, D., Umemura, A.,
Taniguchi, K., Nakagawa, H., Valasek, M.A., Ye, L., Kopp, J.L., Sander, M., Carter, H.,
Deisseroth, K., Verma, I.M. & Karin, M. Hybrid Periportal Hepatocytes Regenerate the
Injured Liver without Giving Rise to Cancer. Cell 162, 766-779 (2015).

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M. Karin

6. Tilg, H. & Moschen, A.R. Evolution of inflammation in nonalcoholic fatty liver disease: themultiple parallel hits hypothesis. Hepatology 52, 1836-1846 (2010).
7. Sandgren, E.P., Palmiter, R.D., Heckel, J.L., Daugherty, C.C., Brinster, R.L. & Degen, J.L.
Complete Hepatic Regeneration after Somatic Deletion of an Albumin-Plasminogen
Activator Transgene. Cell 66, 245-256 (1991).
8. Nakagawa, H., Umemura, A., Taniguchi, K., Font-Burgada, J., Dhar, D., Ogata, H.,
Zhong, Z., Valasek, M.A., Seki, E., Hidalgo, J., Koike, K., Kaufman, R.J. & Karin, M. ER
stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC
development. Cancer Cell 26, 331-343 (2014).
9. Shalapour, S., Lin, X., Bastian, I., Brain, J., Burt, A.D., Aksenov, A.A., Vrbanac, A.F., Li, W.,
Perkins, A., Matsutani, T., Zhong, Z., Dhar, D., Navas-Molina, J.A., Xu, J., Loomba, R.,
Downes, M., Yu, R., Evans, R.M., Dorrestein, P.C., Knight, R., Benner, C., Anstee, Q.M. &Karin, M. Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
Nature In Press (2017).
10. Schulze, K., Imbeaud, S., Letouze, E., Alexandrov, L.B., Calderaro, J., Rebouissou, S.,
Couchy, G., Meiller, C., Shinde, J., Soysouvanh, F., Calatayud, A.L., Pinyol, R., Pelletier, L.,
Balabaud, C., Laurent, A., Blanc, J.F., Mazzaferro, V., Calvo, F., Villanueva, A., Nault, J.C.,
Bioulac-Sage, P., Stratton, M.R., Llovet, J.M. & Zucman-Rossi, J. Exome sequencing of
hepatocellular carcinomas identifies new mutational signatures and potential therapeutictargets. Nat Genet 47, 505-511 (2015).
11. Tannapfel, A., Sommerer, F., Benicke, M., Katalinic, A., Uhlmann, D., Witzigmann, H.,
Hauss, J. & Wittekind, C. Mutations of the BRAF gene in cholangiocarcinoma but not in
hepatocellular carcinoma. Gut 52, 706-712 (2003).
12. Finkin, S., Yuan, D., Stein, I., Taniguchi, K., Weber, A., Unger, K., Browning, J.L.,
Goossens, N., Nakagawa, S., Gunasekaran, G., Schwartz, M.E., Kobayashi, M., Kumada,H., Berger, M., Pappo, O., Rajewsky, K., Hoshida, Y., Karin, M., Heikenwalder, M., BenNeriah, Y. & Pikarsky, E. Ectopic lymphoid structures function as microniches fortumor progenitor cells in hepatocellular carcinoma. Nat Immunol 16, 1235-1244 (2015).
13. Moscat, J., Karin, M. & Diaz-Meco, M.T. p62 in Cancer: Signaling Adaptor Beyond
Autophagy. Cell 167, 606-609 (2016).
14. Umemura, A., He, F., Taniguchi, K., Nakagawa, H., Yamachika, S., Font-Burgada, J.,
Zhong, Z., Subramaniam, S., Raghunandan, S., Duran, A., Linares, J.F., Reina-Campos, M.,
Umemura, S., Valasek, M.A., Seki, E., Yamaguchi, K., Koike, K., Itoh, Y., Diaz-Meco,
M.T., Moscat, J. & Karin, M. p62, Upregulated during Preneoplasia, Induces
Hepatocellular Carcinogenesis by Maintaining Survival of Stressed HCC-Initiating
Cells. Cancer Cell 29, 935-948 (2016).

Keynote Lecture

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15. McPherson, S., Henderson, E., Burt, A.D., Day, C.P. & Anstee, Q.M. Serum
immunoglobulin levels predict fibrosis in patients with non-alcoholic fatty liver disease. J Hepatol 60, 1055-1062 (2014).
16. Shalapour, S., Font-Burgada, J., Di Caro, G., Zhong, Z., Sanchez-Lopez, E., Dhar, D.,
Willimsky, G., Ammirante, M., Strasner, A., Hansel, D.E., Jamieson, C., Kane, C.J.,
Klatte, T., Birner, P., Kenner, L. & Karin, M. Immunosuppressive plasma cells impede Tcell-dependent immunogenic chemotherapy. Nature 521, 94-98 (2015).
17. Ammirante, M., Shalapour, S., Kang, Y., Jamieson, C.A. & Karin, M. Tissue injury andhypoxia promote malignant progression of prostate cancer by inducing CXCL13
expression in tumor myofibroblasts. Proc Natl Acad Sci U S A 111, 14776-14781 (2014).
18. El-Khoueiry, A.B., Sangro, B., Yau, T., Crocenzi, T.S., Kudo, M., Hsu, C., Kim, T.Y.,
Choo, S.P., Trojan, J., Welling, T.H.R., Meyer, T., Kang, Y.K., Yeo, W., Chopra, A.,
Anderson, J., Dela Cruz, C., Lang, L., Neely, J., Tang, H., Dastani, H.B. & Melero, I.
Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an
open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet(2017).

Michael Karin, PhD

1975-1979
1979-1980
1980-1982
1982-1985
1986-1989

1989-2006
2006-present

PhD, Molecular Biology, Univ. of California,
Los Angeles
Postdoctoral Fellow, Fox Chase Institute for Cancer
Research
Postdoctoral Fellow, Department of Medicine andBiochemistry, Univ. of California, San Francisco
Assistant Professor of Microbiology,
School of Medicine, Univ. of Southern California
Associate Professor, Departments of Medicine andPharmacology, Univ. of California, San Diego (UCSD)
Professor, Department of Pharmacology, UCSD
Distinguished Professor, Department of Pharmacology& Pathology, Moores Cancer Center, UCSD School of
Medicine

8

CELL CONTEXT-DEPENDENT ROLES OF A CELL ADHESION
MOLECULE, CADM1, IN HUMAN ONCOGENESIS

Yoshinori Murakami

Division of Molecular Pathology
The Institute of Medical Science, The University of Tokyo
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan(ymurakam@ims.u-tokyo.ac.jp)

Disruption of cell adhesion is a crucial step in invasion and metastasis of human cancer.

The concept of this symposium is complexity in cancer – host crosstalk, because cancer isessentially a systemic disease. Once cancer cells develop solid tumors, they need tomaintain the neoplastic tissues by cooperating with a variety of stromal components,

including fibroblasts, lymphocytes, neutrophils, macrophages, endothelial cells andextracellular matrices (Figure 1). Here, we focus on the complexity in cancer from theviewpoint of complexity in cell to cell adhesion.
Lymphocytes
Fibroblasts

Neutrophils, Macrophages

Cancer cells

ExtracellularmatrixFigure 1

Blood vessels

Endothelial cells(Modified from Joyce JA and Pollard JW, Nat Rev Cancer, 2009)

Complexity in cancer-host crosstalk from the view point of cell to cell adhesion

Figure 1. Complexity in cancer-host crosstalk from the view point of cell to cell adhesion

Cellular Network of Cancer Tissues

9

A variety of cell adhesion molecules, including cadherins, selectins, integrins, andimmunoglobulin superfamily cell adhesion molecules (IgCAM), is shown to be involved incell to cell interaction in epithelial tissue and solid tumors. An IgCAM, CADM1/Necl-2, isoriginally identified as a tumor suppressor by its suppression activity of tumorigenicity innude mice [1]. CADM1 is involved in the formation and maintenance of an epithelial cellstructure, whereas it is frequently inactivated in various cancers, including non-small celllung cancer (NSCLC), in their advanced stages, suggesting that loss of CADM1 function

promotes cancer invasion and/or metastasis [2]. This is supported by development of moreinvasive lung tumors when Cadm1-/- mice is crossed with mutant K-ras knock-in mice.

Here, we demonstrate that CADM1 inhibits HGF-Met-induced epithelial mesenchymaltransition of MDCK cells by associating with Met on the cell membrane. CADM1 also

suppresses Src-signaling by associating with Csk-binding protein (CBP) in the lipid raft on
the membrane of colon cancer cells. Intervention of growth factor signaling by celladhesion molecules, such as CADM1, on the cell membrane could provide a novel
approach to suppress oncogenic pathways in cancer cells.

In contrast, CADM1 is overexpressed in adult T-cell leukemia (ATL) cells, conferring aninvasive phenotype characteristic to ATL. CADM1 is now established as one of the specificcell surface markers to detect circulating malignant ATL cells in the patients by flow

cytometry [3]. We have demonstrated that CADM1 directly associates with Tiam1 andconstitutively activates Rac signaling in ATL cells, whereas invasive phenotype isdrastically suppressed by introducing dominant-negative Rac mutants into ATL cells.

CADM1 and Tiam1 are co-localized at the leading edge of ATL cells, playing the essential

roles in formation of lamellipodia in ATL cell lines [4]. Moreover, we have found that
CADM1 is overexpressed in small cell lung cancer (SCLC). Immunohistochemistrydemonstrates that 33 of 45 (75%) primary SCLC tumors express CADM1 protein. Western

blotting and RT-PCR analyses have revealed that CADM1 is significantly expressed in 11 of
14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing
attached growth to plastic dishes, suggesting that CADM1 is involved in anchorageindependent growth in SCLC. Interestingly, CADM1 shows enhanced tumorigenicity innude mice when transfected into SCLC cells lacking CADM1. Inversely, suppression of

CADM1 expression by shRNA reduced spheroid-like cell aggregation of SCLC cells thatwere expressing a high amount of CADM1. These findings suggest that CADM1 enhances
the malignant features of SCLC, as is observed in ATL [5].

These findings indicate that CADM1 has dual roles in oncogenesis; a tumor suppressor

in epithelial cancer and an oncoprotein in ATL or SCLC. Our studies demonstrate that oneof the possible molecular mechanisms underlying the distinct roles of CADM1 in epithelial
cancers versus in ATL or SCLC is the distinct downstream cascades triggered from a10

Y. Murakami

membrane protein, CADM1. In NSCLC and most other cancers of epithelial origin,

CADM1 associates with a member of protein 4.1s and a member of MAGuKs and form a
tripartite complex involved in cytoskeleton organization and epithelia-like cell structureand functions as a tumor suppressor [6]. By contrast, in ATL, CADM1 associates with
Tiam1, one of the guanine nucleotide exchange factor (GEF) of Rac protein, which

promotes cell mobility and invasive growth in ATL. Distinct downstream cascade we
found here would provide a clear molecular basis to dual roles of CADM1 in oncogenesis.

In addition to the phenotypic regulation of epithelial cells, it has been reported that

CADM1 is involved in the immunological responses between tumor cells and NK cells, aswell as antigen-presenting cells and CD8+ T cells. Three independent groups havedemonstrated that the CADM1 in tumor cells forms highly speciﬁc heterodimers withanother IgCAM, class I-restricted T-cell-associated molecule (CRTAM), whose expression is

only induced on the membrane of activated NK cells or CD8+ T cells [7]. This heterophilictrans-interaction between CADM1 and CRTAM enhances the cytotoxicity of NK cells andthe secretion of γ-interferon from CD8+ T cells to attack the CADM1-expressing cells. Thus,

it is possible to speculate that CADM1 could act as a tumor antigen, whereas loss of
CADM1 expression might be advantageous for cancer cells to escape the immunologicalsurveillance conducted by the host system.

CADM1 is a unique tumor suppressor that is involved in both adhesion-mediatedepithelial like cell structure and immunological responses in most cancers of epithelial

origin, whereas CADM1 inversely promotes invasive and metastatic growth in somespecific types or context of cells, like ATL or SCLC. CADM1 provides an example toconsider the importance of cancer complexity and cancer-host complexity from the view
point of cell adhesion.

References
1. Kuramochi M et al, TSLC1 is a tumor suppressor gene in human non-small cell lungcancer. Nat Genet, 2001;27:427-430.
2. Sakurai-Yageta M et al, Dynamic regulation of a cell adhesion protein complex including
CADM1 by combinatorial analysis of FRAP with exponential curve-fitting. PLoS ONE,2015;10,:e0116637.
3. Kobayashi et al, CADM1 Expression and Stepwise Downregulation of CD7 are closely
associated with clonal expansion of HTLV-I–infected cells in adult T-cell leukemia/
lymphoma. Clin Cancer Res, 2014;20:2851-2861.
4. Masuda M et al, CADM1 interacts with Tiam1 and promotes invasive phenotype of
HTLV-I transformed cells and ATL cells. J Biol Chem, 2010;285:15511-15522.

Cellular Network of Cancer Tissues

11

5. Kikuchi S et al, Expression of a splicing variant of the CADM1 specific to small cell lungcancer. Cancer Science, 2012;103:1051-1057.
6. Murakami S et al. Intercellular adhesion of CADM1 activates PI3K by forming a complex
with MAGuK-family proteins MPP3 and Dlg. PLoS One, 2014;9:e82894.
7. Takeuchi A et al. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage. J Exp
Med, 2016;213:123-138.

Yoshinori Murakami, MD, PhD

1983
1992
1994

2007-present
2015-present

MD, Faculty of Medicine, The Univ. of Tokyo
PhD, Graduate School of Medicine, The Univ. of Tokyo
Postdoctoral Fellow, Department of Human Genetics,
Univ. of Utah
Section Head, National Cancer Center ResearchInstitute, Japan
Professor, Division of Molecular Pathology,
The Institute of Medical Science, The Univ. of Tokyo
Dean, Institute of Medical Science, The Univ. of Tokyo

12

CHEMOTHERAPY INDUCES ENRICHMENT OFIMMUNE-EVASIVE BREAST CANCER CELLSGregg L. Semenza

Institute for Cell EngineeringJohns Hopkins University School of Medicine
Miller Research Building, Suite 671
733 N. Broadway, Baltimore, MD 21205, USA(gsemenza@jhmi.edu)
Breast cancer is a leading cause of cancer death in women, with an incidence of 250,000

new cases and > 40,000 deaths in the United States annually [1]. Chemotherapy is used inselected early stage and many advanced breast cancers [2]. Many patients initially benefit

from chemotherapy but experience recurrence, metastasis, and ultimately death [3]. This isparticularly true for triple-negative breast cancers, which lack expression of the estrogen
receptor, progesterone receptor, and human epidermal growth factor receptor 2. Breast

cancer stem cells, which are defined by their infinite proliferative potential and tumor

initiating properties, contribute critically to tumor recurrence and metastasis [4, 5]. Breast
cancer stem cells have increased chemoresistance through a variety of mechanisms,
including increased drug inactivation, increased expression of drug transporter proteins,and enhanced DNA repair activity [6]. Chemotherapy may eliminate the bulk of cancer
cells within a tumor, but leave behind breast cancer stem cells that are the source ofrecurrent and metastatic disease. Thus, novel treatment options that target Breast cancer
stem cells are needed.

Hypoxia-inducible factors (HIFs) are associated with resistance to chemotherapy inbreast cancer [7, 8]. HIFs are transcription factors consisting of a highly regulated HIF-1α or
HIF-2α subunit and a constitutively expressed HIF-1β subunit [9]. In order to form asecondary (recurrent or metastatic) tumor, a breast cancer cell must have tumor-initiatingor stem cell-like properties. HIFs play important roles in promoting specification and/or

maintenance of the breast cancer stem cell phenotype in response to hypoxia [10-15].

Chemotherapy also induces enrichment of breast cancer stem cells in a HIF-dependent
manner [8]. Chemotherapy induces HIF-dependent activation of the glutathione

Cellular Network of Cancer Tissues

13

biosynthesis pathway, leading to increased intracellular glutathione levels. Glutathione
inhibits MEK-ERK signaling through copper chelation, leading to nuclear localization of

FoxO3 and transcriptional activation of the NANOG gene, which encodes a pluripotency

factor that specifies the breast cancer stem cells phenotype [16]. Chemotherapy also induces
the HIF-mediated expression of glutathione S-transferase omega 1 (GSTO1) [17].

Knockdown of GSTO1 expression abrogates carboplatin-induced breast cancer stem cellenrichment, decreases tumor initiation and metastatic capacity, and delays tumor

recurrence after chemotherapy. GSTO1 interacts with the ryanodine receptor RYR1 andpromotes calcium release from the endoplasmic reticulum. Increased cytosolic calcium
levels activate PYK2 → SRC → STAT3 signaling, leading to increased expression ofpluripotency factors and enrichment of breast cancer stem cells [17].

In order to form a secondary (recurrent or metastatic) tumor, a breast cancer cell must

evade the innate and adaptive immune systems. CD47 enables cancer cells to evade killingby macrophages [18], whereas CD73 and PDL1 mediate two independent mechanisms ofevasion of cytotoxic T lymphocytes [19, 20]. Expression of CD47, CD73, and PDL1 is
induced by hypoxia in certain cell types [21-25]. Treatment of human or murine triplenegative breast cancer cells with carboplatin, doxorubicin, gemcitabine or paclitaxel

induces the coordinate transcriptional induction of CD47, CD73 and PDL1 mRNA andprotein expression, leading to a marked increase in the percentage of CD47+CD73+PDL1+

breast cancer cells [26]. Genetic or pharmacologic inhibition of HIFs blockedchemotherapy-induced enrichment of CD47+CD73+PDL1+ TNBC cells, which were alsoenriched in the absence of chemotherapy by incubation under hypoxic conditions, leading
to T cell anergy or death and increased dissemination of cancer cells to the lungs ofimmunocompetent mice [26]. Our results delineate a HIF-dependent transcriptional

mechanism contributing to triple-negative breast cancer progression and suggest thatcombining chemotherapy with a HIF inhibitor may improve patient survival by preventing
the counter-therapeutic induction of proteins that mediate immune evasion.
References
1. Siegel RL, Miller KD, Jemal, A. Cancer Statistics, 2016. CA Cancer J Clin. 2016;66:7-30.
2. Anampa J, Makower D, Sparano JA. Progress in adjuvant chemotherapy for breast cancer:
an overview. BMC Med. 2015;13:195.
3. Abdullah LN, Chow EK. Mechanisms of chemoresistance in cancer stem cells. Clin
Transl Med. 2013;2:3.
4. Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S, Bernard L, Viale G,
Pelicci PG, Di Fiore PP. Biological and molecular heterogeneity of breast cancers correlateswith their cancer stem cell content. Cell. 2010;14:62-73.

14

G. L. Semenza

5. Oskarsson T, Batlle E, Massagué J. Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell. 2014;14:306-321.
6. Dean M, Fojo T, Bates S. Tumor stem cells and drug resistance. Nat Rev Cancer.
2005;5:275–284.
7. Flamant L, Notte A, Ninane N, Raes M, Michiels C. Anti-apoptotic role of HIF-1 and AP1 in paclitaxel exposed breast cancer cells under hypoxia. Mol Cancer. 2010;9:191–205.
8. Samanta D, Gilkes DM, Chaturvedi P, Xiang L, Semenza GL. Hypoxia-inducible factors
are required for chemotherapy resistance of breast cancer stem cells. Proc Natl Acad Sci
USA 2014;111:E5429–E5438.
9. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3:721-732.
10. Conley SJ, Gheordunescu E, Kakarala P, Newman B, Korkaya H, Heath AN, ClouthierSG, Wicha MS. Antiangiogenic agents increase breast cancer stem cells via the generationof tumor hypoxia. Proc Natl Acad Sci USA. 2012;109:2784-2789.
11. Xiang L, Gilkes DM, Hu H, Takano N, Luo W, Lu H, Bullen JW, Samanta D, Liang H,
Semenza GL. Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype. Oncotarget. 2014;5:12509-12527.
12. Zhang H, Lu H, Xiang L, Bullen JW, Zhang C, Samanta D, Gilkes DM, He J, Semenza GL.
HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells. Proc Natl Acad Sci USA. 2015;112:E6215-6223.
13. Samanta D, Park Y, Andrabi SA, Shelton LM, Gilkes DM, Semenza GL. PHGDH expression is required for mitochondrial redox homeostasis, breast cancer stem cell maintenance, and lung metastasis. Cancer Res. 2016;76:4430-4442.
14. Zhang C, Samanta D, Lu H, Bullen JW, Zhang H, Chen I, He X, Semenza GL. Hypoxia
induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediatedm6A-demethylation of NANOG mRNA. Proc Natl Acad Sci USA. 2016:113:E2047-2056.
15. Zhang C, Zhi WI, Lu H, Samanta D, Chen I, Gabrielson E, Semenza GL. Hypoxiainducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5mediated modulation of RNA methylation in breast cancer cells. Oncotarget.
2016;7:64527-64542.
16. Lu H, Samanta D, Xiang L, Zhang H, Hu H, Chen I, Bullen JW, Semenza GL.
Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelationthat induces the breast cancer stem cell phenotype. Proc Natl Acad Sci USA.
2015;112:E4600-4609.
17. Lu H, Chen I, Shimoda LA, Park Y, Zhang C, Tran L, Zhang H, Semenza GL.
Chemotherapy-induced Ca2+ release stimulates breast cancer stem cell enrichment.
Cell Rep. 2017;18:1946-1957.
18. Chao MP, Jaiswal S, Weissman-Tsukamoto R, Alizadeh AA, Gentles AJ, Volkmer J,
Weiskopf K, Willingham SB, Raveh J, Park CY, Majeti R, Weissman IL. Calreticulin is thedominant pro-phagocytic signal on multiple human cancers and is counterbalanced by
CD47. Sci Transl Med. 2010;2:63ra94.

Cellular Network of Cancer Tissues

15

19. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev
Cancer. 2012;12:252–264.
20. Hatfield SM, Kjaergaard J, Lukashev D, Schreiber TH, Beilikoff B, Abbott R,
Sethumadhavan S, Philbrook P, Ko K, Cannici, R, Thayer M, Rodig S, Kutok JL, Jackson
EK, Karger B, Podack ER, Ohta A, Sitkovsky MV. Immunological mechanisms of theantitumor effects of supplemental oxygen. Sci Transl Med. 2015;7:277ra30.
21. Zhang H, Lu H, Xiang L, Bullen JW, Zhang C, Samanta D, Gilkes DM, He J, Semenza GL.
HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells. Proc Natl Acad Sci USA. 2015;112:E6215E6223.
22. Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Hansen KR,
Thompson LF, Colgan SP. Ecto-5’-nucleotidase (CD73) regulation by hypoxia-inducible
factor 1 mediates permeability changes in intestinal epithelia. J Clin Invest.
2002;110:993-1002.
23. Hatfield SM, Kjaergaard J, Lukashev D, Belikoff B, Schreiber TH, Sethumadhavan S,
Abbott R, Philbrook P, Thayer M, Shujia D, Rodig S, Kutok JL, Ren J, Ohta A, Podack ER,
Karger B, Jackson EK, Sitkovsky MV. Systemic oxygenation weakens the hypoxia andhypoxia-inducible factor 1α-dependent and extracellular adenosine-mediated tumor
protection. J Mol Med. 2014;92:1283-1292.
24. Barsoum IB, Smallwood CA, Siemens DR, Graham CH. A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells. Cancer Res. 2014;74:665-674.
25. Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, Bronte V, Chouaib S.
PDL1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced
MDSC-mediated T cell activation. J Exp Med. 2014;211:781-790.
26. Samanta D, Park Y, Ni X, Li H, Zahnow CA, Gabrielson E, Pan F, Semenza GL.
Chemotherapy induces enrichment of CD47+/CD73+/PDL1+ immune evasive triple-negative breast cancer cells. Proc Natl Acad Sci USA 2018; doi: 10.1073/pnas.1718197115.

Gregg L. Semenza, MD, PhD

1986-1990
1990-1994
1994-1999

1999-present
2003-present
2008-present
2011-present

Postdoctoral Fellow in Medical Genetics, Johns
Hopkins University School of Medicine (JHUSOM)
Assistant Professor of Pediatrics and Medicine,
JHUSOM
Associate Professor of Pediatrics and Medicine,
JHUSOM
Professor of Pediatrics and Medicine, JHUSOMFounding Director, Vascular Program,
Institute for Cell Engineering, JHUSOM
C. Michael Armstrong Professor of Genetic Medicine,
JHUSOM
American Cancer Society Research Professor

16

TUMOR-DERIVED EXOSOMES PROMOTE ORGANOTROPICMETASTASIS AND SYSTEMIC DISEASE

Ayuko Hoshino, Ayako Hashimoto, Haiying Zhang and David Lyden
Departments of Pediatrics and Cell and Developmental Biology
Children’s Cancer and Blood Foundation Laboratories
Drukier Institute for Children’s Health, Meyer Cancer Center
Belfer Research Building, BB-1228 Box 284, 413 East 69th Street
Weill Cornell Medicine, New York, NY 10021, USA(dcl2001@med.cornell.edu)

Metastasis to distant vital organs such as lung, liver, and brain is the most devastating

feature of cancer progression, responsible for over 90% of cancer-associated deaths. In 1889,

Stephen Paget first proposed that organ distribution of metastases is a non-random event,yet metastatic organotropism remains one of the greatest mysteries in cancer biology. Our
recent studies uncovered that tumor-derived exosomes, alter the microenvironment at
future sites of metastasis to form pre-metastatic niches, creating a favorable “soil” forincoming metastatic “seeds” [1,2]. However, by what mechanism this occurs, and the roleof exosomes in tumor metastasis, remains unknown. To investigate the role of exosomes inorganotropic metastasis, we have used two established organotropic human tumor models:the MDA-MB-231 breast cancer (BC) cell line, and its variants known to metastasize to thelung, brain and bone, respectively, as well as two liver metastatic pancreatic cancer (PC)cell lines, BxPC3 and HPAF-2 [3]. We first analyzed the biodistribution of fluorescently-

labeled exosomes derived from lung metastatic, brain metastatic or bone metastatic MDA-

MB-231 BC variants or PC cell lines, and found that BC exosomes follow the organ-specificdistribution of the cells of origin, while PC exosomes home to the liver (Figure 1a). In eachtarget organ exosomes are taken up by different cell types: fibroblasts/epithelial cells in thelung, Kupffer cells in the liver, and endothelial cells in the brain (Figure 1b). In theorganotropic MDA-MB-231 model, prior education with the lung tropic exosomes

redirected metastasis of the bone tropic cells to the lung, demonstrating the unique capacityof exosomes to determine the site of metastasis. Unbiased proteomic profiling of exosomes
revealed distinctive integrin expression patterns, and analysis of plasma exosomes from BC
and PC patients that later developed site-specific metastasis revealed that specific exosomal

Novel Strategies Targeting Cancer Network

17

integrins could predict metastatic spread (Figure 2). Recently, our group has identified
unique exosome subpopulations and novel nanoparticles (exomeres) which packaged

specific protein and genomic contents (Figure 3) [4]. The biodistribution patterns of theseparticles suggest distinct functional roles in the metastatic process (Figure 4).

a.

b.

Figure 1

Figure 2

Blood sample analysis fromPatients with lung mets or liver metsa. Exosomes from MDA-MB-231 sub-lines exhibit organ tropism
Exosomes injected( MDA-MB-231)
b. Distinct cell types uptake organotropic exosomesĮ6 ȕ4 integrinĮv ȕ5 integrin

Blood sample analysis from Patients with lung mets or liver mets

Figure 2

18

D. Lyden et al.

Identification of different subsets of exosomes by AF4 analysis
Hydrodynamic Radius (Rh) vs. time

P3

Correlation Function (P3)

P5

P4
P2
P1

Input

Figure 3

Exomere (<50nm) Exo-S (60-80nm) Exo-L (90-120nm)

Identification of different subsets of exosomes by AF4 analysis

B16F10 Mock

Exomere

ExoͲS

ExoͲL

Input

Mock

ExomereFigure 3

ExoͲS

ExoͲL

Input

Spleen

Lung

Mock
Mock

Exomere

ExoͲS

ExoͲL

Exomere

ExoͲS

ExoͲL

Input

Input

Liver

Lymph
nodes

Mock Exomere ExoͲS
Mock

Exomere

ExoͲS

ExoͲL

ExoͲL

Input

Input

KidneyBones

Figure 4

OdysseyImaging

Novel Strategies Targeting Cancer Network

19

References
1. Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G,
Hergueta-Redondo M, Williams C, Garcia-Santos G, Ghajar CM, Nitadori-Hoshino A,
Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN,
Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, and Lyden D. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.
Nature Medicine, 2012. 18(6): p. 883-91.
2. Costa-Silva B. Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, Becker A, Hoshino A,
Mark MT, Molina H, Xiang J, Zhang T, Theilen TM, Garcia-Santos G, Williams C,
Ararso Y, Huang Y, Rodrigues G, Shen TL, Labori KJ, Lothe IMB, Kure EH, Hernandez J,
Doussot A, Ebbesen SH, Grandgenett PM, Hollingsworth MA, Jain M, Mallya K, Batra SK,
Jarnagin WR, Schwartz RE, Matei I, Peinado H, Stanger B, Bromberg J, and Lyden D.
Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nature Cell
Biology, 2015. 17(6): p. 816-26.
3. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Mark MT, Molina H,
Kohsaka S, Di Giannatale A, Ceder S, Singh S, Williams C, Soplop N, Uryu K, Pharmer L,
King T, Bojmar L, Davies AE, Ararso Y, Zhang T, Zhang H, Hernandez J, Weiss JM,
Dumont-Cole VD, Kramer K, Wexler LH, Narendran A, Schwartz GK, Healey JH,
Sandstrom P, Labori KJ, Kure EH, Grandgenett PM, Hollingsworth MA, de Sousa M,
Kaur S, Jain M, Mallya K, Batra SK, Jarnagin WR, Brady MS, Fodstad O, Muller V,
Pantel K, Minn AJ, Bissell MJ, Garcia BA, Kang Y, Rajasekhar VK, Ghajar CM, Matei I,
Peinado H, Bromberg J, and Lyden D. Tumour exosome integrins determine organotropic
metastasis. Nature, 2015. 527(7578): p. 329-35.
4. Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, Mark MT, Molina H, Martin AB,
Bojmar L, Fang J, Rampersaud S, Hoshino A, Matei I, Kenific C, Nakajima M, Mutvei AP,
Sansone P, Buehring W, Wang H, Jimenez JP, Cohen-Gould L, Paknejad N, Brendel M,
Manova-Todorova K, Magalhaes A, Ferreira JA, Osorio H, Silva AM, Massay A,
Cubillos-Ruiz JR, Galletti G, Giannakakou P, Cuervo AM, Blenis J, Schwartz R, Brady MS,
Peinado H, Bromberg J, Matsui H, Reis CA, and Lyden D. Identification of distinct
nanoparticles and subsets of extracellular vesicles by asymmetric-flow field-flow fractionation.
Nature Cell Biology, Nature Cell Biology, 2018. 20(3): p. 332-343.

20

D. Lyden et al.

David Lyden, MD, PhD

1981-1986
1985-1989
1989-1992
1992-1997
1998
2002-2004
2004-20132008-20132013-20142013-present

PhD, Univ. of Vermont
MD, Brown Univ.
Pediatric Residency, Duke Univ. Medical Center
Pediatric Hem/Onc Fellow/Chief
Fellow/Research Fellow, MSKCC/Weill-Cornell
Clinical Assistant Pediatrician, MSKCC
Assistant Professor of Pediatrics,
Weill Cornell Medical College
Associate Professor of Cell and Developmental
Biology, Weill Cornell Medical College
Associate Professor of Pediatrics,
Weill Cornell Medical College
Stavros S. Niarchos Associate Professor in Pediatric
Cardiology, Weill Cornell Medical College
Professor of Cell and Developmental Biology,
Weill Cornell Medical College
Stavros S. Niarchos Professor in Pediatric Cardiology
Professor of Pediatrics, Weill Cornell Medical College

21

EXTRACELLULAR VESICLES AS A NOVEL PLATFORM FOR
CANCER DIAGNOSIS AND THERAPEUTICS

Takahiro Ochiya

Division of Molecular and Cellular Medicine
National Cancer Center Research Institute5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan(tochiya@ncc.go.jp)

Research has increased significantly on small vesicles secreted by healthy and diseased

cells. Recent discoveries have revealed their functional and biomarker roles in cancer.

Whether and how this knowledge of extracellular vesicles (EVs), known as exosomes andmicrovesicles, affects translational research and clinical practices have become pertinent
questions.

EVs have been shown to be involved in such cell-cell communication between cancer

cells and the surrounding microenvironment and to be important for the development ofcancer metastasis [1, 2]. In addition, these cancer cell-derived EVs carry pathogenic
components, such as proteins, messenger RNAs (mRNAs), microRNAs (miRNAs), DNAs,
lipids, and transcriptional factors that can mediate paracrine signaling in the tumor

microenvironment [3]. Understanding the metastatic mechanisms through EV transfer may
open up a new avenue for cancer therapeutic strategies [4].

Our current understanding of cancer-EVs functions, cancer-EVs destroy our body’s

natural barriers. One example is EVs-mediated brain metastasis of breast cancer cells. A

key event during brain metastasis is the migration of cancer cells through blood-brain

barrier (BBB). However, the molecular mechanism behind the passage through this naturalbarrier remains unclear. We showed that cancer-derived EVs trigger the breakdown ofblood-brain barrier (BBB) [5]. Importantly, EV-miR-181c promotes the destruction of BBB
through the abnormal localization of actin via the downregulation of its target gene,

PDPK1. PDPK1 degradation by downregulation of phosphorylated cofilin and the resultant

activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate thatsystemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis ofbreast cancer cell lines and are preferentially incorporated into the brain in vivo.

22

T. Ochiya

We also demonstrate that EVs derived from highly metastatic ovarian cancer cells

promote peritoneal dissemination in vivo [6]. The EVs from highly metastatic cells strongly

induce metastatic behaviour in moderately metastatic tumours. Notably, the cancer EVs

efficiently induce apoptotic cell death in human mesothelial cells in vitro and in vivo, thus

resulting in the destruction of the peritoneal mesothelium barrier. Whole transcriptome
analysis shows that MMP1 is significantly elevated in mesothelial cells treated with highly
metastatic cancer EVs and intact MMP1 mRNAs are selectively packaged in the EVs.
Importantly, MMP1 expression in ovarian cancer is tightly correlated with a poor prognosis.

We currently found that prostate cancer-derived EVs are important for bone metastasis

via differentiation and proliferation of osteocrast cells (unpublished). Prostate cancer-derived
EVs play a significant role for bone metastasis by regulating bone remodeling process.

Furthermore, the circulating EVs have also been of interest as a source for liquidbiopsies. EVs in body fluids provide a reliable source of microRNAs and proteins forcancer biomarkers. The tumor specific components in EVs effectively provide various
messages on the physiological and pathological status of cancer patients. Our ExoScreen
technology [7] allows us for developing a novel liquid biopsy for early detection of cancer.

EVs are secreted from cancer cells and delivered to recipient cells, modulating theirphenotype. For example, EVs from cancer cells are delivered to endothelial cells, which

enhances angiogenesis to obtain the oxygen and nutrition required for continued growth ofthe cancer. We propose the following therapeutic applications: inhibition of cancer cell EV
production, disruption of EV uptake by recipient cells, and elimination of circulating cancercell–derived EVs as shown in the Figure 1. These therapeutic strategies will prevent thedelivery of EVs from cancer cells to microenvironmental cells, leading to the developmentof novel antiangiogenic and anticancer drugs. Thus, EVs will provide us the multiple
strategies to fight against cancer, however, it is important to confirm safety issues andovercome technical problems to bring EVs in practical use.

EVs-targeted Cancer Therapy

Figure 1

Therapeutic applications: inhibition of cancer cell EV production, disruption of EV uptake byrecipient cells, and elimination of circulating cancer cell – derived EVs.

Novel Strategies Targeting Cancer Network

23

References
1. Kosaka, N., Iguchi, H., Yoshioka, Y., Takeshita, F., Matsuki, Y., Ochiya, T.: Secretorymechanisms and intercellular transfer of microRNAs in living cells. J. Biol. Chem., 285:
17442-17452, 2010.
2. Ono M, Kosaka N, Tominaga N, Yoshioka Y, Takeshita F, Takahashi RU, Yoshida M,
Tsuda H, Tamura K, Ochiya T. Exosomes from bone marrow mesenchymal stem cells
contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci
Signal, 7:ra63, 2014 (2015 Signaling Breakthrough of the Year)
3. Kawamura Y, Yamamoto Y, Sato TA, Ochiya T. Extracellular vesicles as trans-genomic
agents: Emerging roles in disease and evolution. Cancer Sci. 108: 824-830, 2017
4. Kosaka N, Fujita Y, Yoshioka Y, Ochiya T. Versatile roles of extracellular vesicles incancer. J Clin Invest, 126(4):1163-72., 2016
5. Tominaga N, Kosaka N, Ono M, Katsuda T, Yoshioka Y, Tamura K, Lötvall J,
Nakagama H, Ochiya T. Brain metastatic cancer cells release microRNA-181c-containing
extracellular vesicles capable of destructing blood-brain barrier. Nat Commun,
6:6716,2015
6. Yokoi A, Yoshioka Y, Yamamoto Y, Ishikawa M, Ikeda SI, Kato T, Kiyono T, Takeshita F,
Kajiyama H, Kikkawa F, Ochiya T. Malignant extracellular vesicles carrying MMP1mRNA facilitate peritoneal dissemination in ovarian cancer. Nature Commun, 8, 14470,
2017
7. Yoshioka Y, Kosaka N, Konishi Y, Ohta H, Okamoto H, Sonoda H, Nonaka R,
Yamamoto H, Ishii H, Mori M, Furuta K, Nakajima T, Hayashi H, Sugisaki H,
Higashimoto H, Kato T, Takeshita F, Ochiya T. Ultra-sensitive liquid biopsy of circulating
extracellular vesicles using ExoScreen. Nat Commun, 5:3591, 2014

Takahiro Ochiya, PhD

1988
1988-1992
1991-1992
1992

1992-1998
1998- 20112011-present
2004-present
2008-20122014-present
2014-present
2017-present

PhD, Osaka University
Research Assistant, Osaka University
Post-doctoral Fellow, La Jolla Cancer Research
Foundation (The SF Burnham Institute)
Chief Researcher, Genetics Div., National Cancer
Center Research Institute (NCCRI), Japan
Section Head, Genetics Div., NCCRI
Head, Section for Studies on Metastasis, NCCRI
Chief, Division of Molecular and Cellular Medicine,
NCCRI
Invited Professor, Waseda University
Invited Professor, Tokyo Institute Technology
Invited Professor, Hoshi University
Invited Professor, Showa University
Invited Professor, Tokyo Medical University

24

THE FUNCTIONAL ROLE OF TUMOR STROMA AND
EXOSOMES IN BIOLOGY AND TREATMENT OFPANCREATIC CANCER

Raghu Kalluri

Department of Cancer Biology
The University of Texas MD Anderson Cancer Center
1881 East Road, Unit 1906, Houston, TX 77054, USA(rkalluri@mdanderson.org)

Trillions of exosomes and extracellular vesicles are naturally present in the blood andtissue [1]. Exosomes are nano-sized extracellular vesicles (40-150 nm), likely of endosomal

origin, with a membrane lipid bilayer [2]. Although their precise physiological role remains
largely unknown, is it suggested that exosomes mediate specific cell-cell communication
and activate signaling pathways in cells with which they fuse or interact. Exosomes aredetected in the tumor microenvironment, and emerging evidence suggests they play a rolein facilitating tumorigenesis by regulating angiogenesis, immunity, and metastasis [1, 3].

While exosome production and cargo may vary depending on the cellular source, studies

have identified that exosomes contain proteins, lipids, metabolites, mRNA, non-coding
RNAs (ncRNAs), microRNAs (miRNAs) and genomic DNA [4-7]. Notably, circulating

exosomes could be used as liquid biopsies and non-invasive biomarkers to potentially alert
for early detection and diagnosis of cancer patients [1].

The process of vesicle shedding, by which exosomes are released into the extracellularspace, appears to be amplified in proliferating cancer cells [1]. Tumor-secreted exosomes

have been the focus of recent investigative efforts for their role as “vehicles”, facilitating
local intercellular communication (e.g., autocrine and paracrine) as well as distal (i.e.,

endocrine) signaling, through the transfer of their contents. Unlike liposomes and othersynthetic drug nanoparticle carriers, exosomes contain many transmembrane andmembrane-anchored proteins. These proteins likely enhance endocytosis and/or direct

fusion with the plasma membrane of the recipient cells, thus enhancing potential content
delivery [8-10]. Our studies have focused on harnessing the potential of exosomes astherapeutic vehicles, in particular for the treatment of pancreatic ductal adenocarcinoma

Novel Strategies Targeting Cancer Network

25(PDAC) [11].

Despite current standard of care therapies, the prognosis for patients with pancreatic

ductal adenocarcinoma (PDAC) is dismal [12-15]. The emergence of PDAC is on the riseand patients are in urgent need of effective therapies. Genetic analyses of PDAC show that

mutations in a small GTPase, Kras, are encountered in the majority of patients [16-19] andare key drivers for initiation, progression, and metastasis [20-26]. While it is recognized that
the Ras-mediated signaling pathway is a key mediator of pancreatic cancer progression, a
direct and specific targeting of Ras has been elusive, dubbing Ras as an undruggable target.
The generation of Ras pathway-inhibiting drugs and oncogenic Ras-specific RNAi

molecules (siRNA and shRNA) have encountered targeting challenges and Ras remains an

intractable pharmacological target for therapy and different approaches must be explored[27].

Genetic manipulation of oncogenic Kras in mice determined that dampening oncogenic

Kras inhibits tumor progression despite the presence of other genetic defects [20, 22, 23, 28].
Effective delivery of RNAi molecules to non-liver parenchymal organs, especially thepancreas, remains a challenge. A major hurdle in employing specific RNAi constructs toinhibit Ras is the lack of an optimal delivery system [29, 30]. Using exosomes engineered tocontain siRNA (iExosomes) that target the mutant form of the GTPase Kras found inpancreatic cancer, efficient and specific targeting could be achieved to control cancer
progression in various mouse models of PDAC. The anti-cancer efficacy of iExosomes was
associated with their enhanced retention of in circulation when compared to liposomes.

iExosomes, prepared from fibroblast culture supernatant, were noted to present with the‘don’t eat me’ signal CD47, which enabled their protection from phagocytosis. Further, theenhanced macropinocytic activity of Kras mutant PDAC cells fascilitated the iExosomes

uptake to the pancreas, contributing to the efficacy of iExosomes treatment in suppressing
cancer and increasing the survival of mice with PDAC. These studies are prompting theengineering and use of exosomes in personalized therapy for cancer patients. This lecture

will highlight some of the recent advances in the area of exosomes biology and tumor
microenvironment, and its utility in the diagnosis and treatment of cancer.
References
1. Kalluri R. The biology and function of exosomes in cancer. The Journal of clinical investigation. 2016;126(4):1208-15.
2. Kowal J, Tkach M, Thery C. Biogenesis and secretion of exosomes. Current opinion in cellbiology. 2014;29:116-25.

26

R. Kalluri

3. Kahlert C, Kalluri R. Exosomes in tumor microenvironment influence cancer progression
and metastasis. J Mol Med (Berl). 2013;91(4):431-7.
4. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediatedtransfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between
cells. Nature cell biology. 2007;9(6):654-9.
5. Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, et al.
Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic
phenotype through MET. Nature medicine. 2012;18(6):883-91.
6. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, et al. Exosomes
mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell
migration. Cell. 2012;151(7):1542-56.
7. Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, et al. Identification of
Double Stranded Genomic DNA Spanning all Chromosomes with Mutated KRAS and
p53 DNA in the Serum Exosomes of Patients with Pancreatic Cancer. The Journal of
biological chemistry. 2014.
8. Marcus ME, Leonard JN. FedExosomes: Engineering Therapeutic Biological
Nanoparticles that Truly Deliver. Pharmaceuticals (Basel). 2013;6(5):659-80.
9. van den Boorn JG, Dassler J, Coch C, Schlee M, Hartmann G. Exosomes as nucleic acidnanocarriers. Advanced drug delivery reviews. 2013;65(3):331-5.
10. Johnsen KB, Gudbergsson JM, Skov MN, Pilgaard L, Moos T, Duroux M. A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. Biochimica et biophysica acta. 2014;1846(1):75-87.
11. Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017;546(7659):498-503.
12. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians.
2014;64(1):9-29.
13. Howlader N NA, Krapcho M, Garshell J, Miller D, Altekruse SF, Kosary CL, Yu M,
Ruhl J, Tatalovich Z,Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA. SEER
Cancer Statistics Review, 1975-2011, National Cancer Institute. Bethesda, MD.
http://seercancergov/csr/1975_2011/. 2013.
14. Tuveson DA, Hingorani SR. Ductal pancreatic cancer in humans and mice. Cold Spring
Harbor symposia on quantitative biology. 2005;70:65-72.
15. Hidalgo M, Von Hoff DD. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clinical cancer research : an official journal of the American
Association for Cancer Research. 2012;18(16):4249-56.

Novel Strategies Targeting Cancer Network27

16. Biankin AV, Waddell N, Kassahn KS, Gingras MC, Muthuswamy LB, Johns AL, et al.
Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature.
2012;491(7424):399-405.
17. Hruban RH, van Mansfeld AD, Offerhaus GJ, van Weering DH, Allison DC, Goodman
SN, et al. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of
82 carcinomas using a combination of mutant-enriched polymerase chain reaction
analysis and allele-specific oligonucleotide hybridization. The American journal ofpathology. 1993;143(2):545-54.
18. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell. 1988;53(4):549-54.
19. Chang DK, Grimmond SM, Biankin AV. Pancreatic cancer genomics. Current opinion in
genetics & development. 2014;24:74-81.
20. Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al.
Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose
metabolism. Cell. 2012;149(3):656-70.
21. Hingorani SR, Wang L, Multani AS, Combs C, Deramaudt TB, Hruban RH, et al.
Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely
metastatic pancreatic ductal adenocarcinoma in mice. Cancer cell. 2005;7(5):469-83.
22. Collins MA, Bednar F, Zhang Y, Brisset JC, Galban S, Galban CJ, et al. Oncogenic Kras isrequired for both the initiation and maintenance of pancreatic cancer in mice. The
Journal of clinical investigation. 2012;122(2):639-53.
23. Collins MA, Brisset JC, Zhang Y, Bednar F, Pierre J, Heist KA, et al. Metastatic pancreaticcancer is dependent on oncogenic Kras in mice. PloS one. 2012;7(12):e49707.
24. Eser S, Schnieke A, Schneider G, Saur D. Oncogenic KRAS signalling in pancreatic cancer.
British journal of cancer. 2014;111(5):817-22.
25. Ji B, Tsou L, Wang H, Gaiser S, Chang DZ, Daniluk J, et al. Ras activity levels control thedevelopment of pancreatic diseases. Gastroenterology. 2009;137(3):1072-82, 82 e1-6.
26. Guerra C, Barbacid M. Genetically engineered mouse models of pancreatic adenocarcinoma. Molecular oncology. 2013;7(2):232-47.
27. Gysin S, Salt M, Young A, McCormick F. Therapeutic strategies for targeting ras proteins.
Genes & cancer. 2011;2(3):359-72.
28. Smakman N, Veenendaal LM, van Diest P, Bos R, Offringa R, Borel Rinkes IH, et al.
Dual effect of Kras(D12) knockdown on tumorigenesis: increased immune-mediatedtumor clearance and abrogation of tumor malignancy. Oncogene. 2005;24(56):8338-42.
29. van der Meel R, Fens MH, Vader P, van Solinge WW, Eniola-Adefeso O, Schiffelers RM.
Extracellular vesicles as drug delivery systems: Lessons from the liposome field.
Journal of controlled release : official journal of the Controlled Release Society. 2014.

28

R. Kalluri

30. Gomes-da-Silva LC, Fonseca NA, Moura V, Pedroso de Lima MC, Simoes S, Moreira
JN. Lipid-based nanoparticles for siRNA delivery in cancer therapy: paradigms andchallenges. Accounts of chemical research. 2012;45(7):1163-71.

Raghu Kalluri, MD, PhD

1992

2009
1994-1997
1997-2000
2000-2006
2005-2012
2006-20122012-present

PhD, Univ. of Kansas Medical Center (Biochemistry/
Molecular Biology)
MD, Brown Medical School (Medicine)Research Associate, Department of Medicine,Univ. of Pennsylvania Medical School
Assistant Professor of Medicine, Harvard Medical
School (HMS), Beth Israel Deaconess Medical Center(BIDMC)
Associate Professor of Medicine, HMS, BIDMC
Faculty, Harvard-MIT Division of Health Sciences andTechnology
Faculty, Department of Biological Sciences andMolecular Pharmacology
Chief, Division of Matrix Biology
Professor of Medicine, Harvard Medical School,
Beth Israel Deaconess Medical Center
Professor and Chairman, RE Bob Smith Endowed Chair
in Cancer Biology, Department of Cancer Biology,
The Univ. of Texas MD Anderson Cancer Center
Director, Metastasis Research Center,
The Univ. of Texas MD Anderson Cancer Center

29

METABOLIC CROSSTALK BY AUTOPHAGY ADAPTORSIN THE TUMOR STROMA

Jorge Moscat

Sanford Burnham Prebys Medical Discovery Institute10901 N. Torrey Pines Road, La Jolla, CA 92037, USA(jmoscat@sbpdiscovery.org)

The analysis of the tumor genomic and transcriptomic landscape reveals the extreme

difficulty in many occasions to distinguish between cancer-causing driver mutations frompassenger mutations, which impairs our ability to design effective oncogene-selective

therapies. Importantly, cancer cells create non-oncogenic addictions, irrespective of themutational load of the tumor, which are required for the tumor to survive stressful

conditions. This generates non-oncogenic vulnerabilities that can be identified as a possible
way to target cancer cells in an oncogene agnostic manner. p62 is induced in many types ofcancers, and specifically in prostate cancer and hepatocellular carcinoma (HCC), in which it
has been shown to play a critical role as a non-oncogenic addiction [1]. We have recently

discovered that the accumulation of p62 is necessary and sufficient for HCC induction in

mice, through a mechanism that involves the activation of NRF2 and mTORC1 inhepatocytes, independently of autophagy [2]. We have also shown that p62 is required forprostate cancer through a mechanism that also implicates the ability of p62 to activate

mTORC1 in response to nutrient sensing [3]. Interestingly, although p62 is upregulated in
the liver parenchyma in patients of non-alcoholic steatohepatitis (NASH) and HCC, as wellas in the prostate cancer epithelium, it is dramatically downregulated in the stroma of bothtypes of cancer. This is important because the selective inactivation of stromal p62 creates a
microenvironment that is conducive to tumorigenesis [4,5]. Results will be presented inwhich the detailed mechanisms whereby p62 acts as a tumor suppressor in the stroma will
be discussed. Specifically, we will present the newly identified ability of p62 to regulate

nuclear events in cancer associated fibroblasts, which has important implications in thedesign of novel therapies aimed at reprogramming the tumor stroma. In addition, we have

30

J. Moscatrecently discovered that p62 also impacts tumor metabolism and progression through theregulation of metabolic and signaling cascades in the adipose tissue. These novel findings
are central to our understanding of the metabolism in cancer patients.

Another important aspect of the stroma-epithelium crosstalk is the signaling andmetabolic pathways that confer tumors the ability to withstand situations of nutrient stress.
This is a common stage in many poorly vascularized and aggressive tumors that,surprisingly, correlates with poor patient prognosis. Importantly, it is becoming apparent

that the stromal fibroblasts may exert a critical role in the control of nutrient utilization andmetabolism in the tumor microenvironment. That is, cancer associated fibroblasts (CAFs)
can provide nutrients to support the epithelial cells in the tumor and also modulate

immune responses, which are critical for tumor regression upon immunotherapy.

Interestingly, new results will be discussed that demonstrate that p62 deficiency in stromal
fibroblasts promotes resistance to glutamine deprivation not only of stromal cells but alsoof the tumor epithelium. This is very important because glutamine is the most commonly
depleted amino acid in solid tumors, and serves as the obligate nitrogen donor for

numerous biosynthetic pathways. Our recent studies demonstrate that p62 directly controls

the stability of the transcription factor ATF4. The precise mechanism involves theregulation of its stability through a process that involves the p62-mediatedpolyubiquitination of ATF4. In this way, the loss of p62 in stromal fibroblasts, a common

event in cancer, results in the upregulation of ATF4. This is critical for the survival andproliferation of stromal and tumor epithelial cells, because p62 deficiency in the stroma

activates glucose carbons flux through a pyruvate carboxylase-asparagine synthase cascadethat is central to the generation of asparagine as a source of nitrogen for stroma and tumor
epithelial proliferation. These most recent results, together with our data demonstrating theimportant role of p62 in the regulation of the vitamin D receptor in hepatic stellate cells
demonstrate the existence of a new paradigm whereby p62 directly targets nuclear

transcription factors to control metabolic reprogramming in the microenvironment andrepress tumorigenesis.

References
1. Moscat, J., Karin, M., and Diaz-Meco, M. T. (2016) p62 in Cancer: Signaling Adaptor
Beyond Autophagy. Cell 167, 606-609
2. Umemura, A., He, F., Taniguchi, K., Nakagawa, H., Yamachika, S., Font-Burgada, J.,
Zhong, Z., Subramaniam, S., Raghunandan, S., Duran, A., Linares, J. F., Reina-Campos,
M., Umemura, S., Valasek, M. A., Seki, E., Yamaguchi, K., Koike, K., Itoh, Y., DiazMeco, M. T., Moscat, J., and Karin, M. (2016) p62, Upregulated during Preneoplasia,
Induces Hepatocellular Carcinogenesis by Maintaining Survival of Stressed HCCInitiating Cells. Cancer Cell 29, 935-948

Inflammation and Cancer Microenvironment

31

3. Linares, J. F., Duran, A., Reina-Campos, M., Aza-Blanc, P., Campos, A., Moscat, J., andDiaz-Meco, M. T. (2015) Amino Acid Activation of mTORC1 by a PB1-Domain-Driven
Kinase Complex Cascade. Cell Rep 12, 1339-1352
4. Duran, A., Hernandez, E. D., Reina-Campos, M., Castilla, E. A., Subramaniam, S.,
Raghunandan, S., Roberts, L. R., Kisseleva, T., Karin, M., Diaz-Meco, M. T., and Moscat, J.
(2016) p62/SQSTM1 by Binding to Vitamin D Receptor Inhibits Hepatic Stellate Cell
Activity, Fibrosis, and Liver Cancer. Cancer Cell 30, 595-609
5. Valencia, T., Kim, J. Y., Abu-Baker, S., Moscat-Pardos, J., Ahn, C. S., Reina-Campos, M.,
Duran, A., Castilla, E. A., Metallo, C. M., Diaz-Meco, M. T., and Moscat, J. (2014)
Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis. Cancer Cell 26, 121-135

Jorge Moscat, PhD

2008-20112011-present
2013-present
2016-present

Professor and Chair, Department of Cancer and Cell
Biology, Univ. of Cincinnati College of Medicine
Professor, Cancer Metabolism and Signaling Networks
Program, SBP NCI-Cancer Center, SBMRI
Director, Cancer Metabolism and Signaling Networks
Program, SBP NCI-Cancer Center, SBMRI
Deputy Director, SBP NCI-Cancer Center

32

COMPREHENSIVE PHENOTYPIC CHARACTERIZATION OF
INTESTINAL CANCER WITH MULTIPLE DRIVER MUTATIONS

Masanobu Oshima

Division of Genetics
Cancer Research InstituteKanazawa University
Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan(oshimam@staff.kanazawa-u.ac.jp)
Colorectal cancer (CRC) is a leading cause of cancer-related death in the world. It has

been shown by genome-wide analyses that APC, KRAS, TGFBR2, TP53 are found in themost frequently mutated genes in CRC, which is consistent with a concept of classicalmultistep tumorigenesis. However, it has not yet been fully understood which

combinations of driver mutations are responsible for induction of each process ofmalignant progression. To investigate the in vivo biological mechanism of the respective

driver mutations in combination, we have generated compound mutant mice that carried
driver gene mutations by crossing and comprehensively examined their phenotypes.

Apc simple mutant mice, Apc∆716 mice, developed benign intestinal adenomas caused by

Wnt signaling activation [1]. On the other hand, Apc ∆716 Smad4 -/- and Apc ∆716 Tgfbr2 -/-compound mutant mice developed invasive adenocarcinomas, indicating that suppressionof TGF-β signaling induces submucosal invasion of intestinal tumors [2, 3]. TGF-β signaling
is important for differentiation of intestinal epithelial cells. Notably, organoid culture ofintestinal epithelial cells derived from Tgfbr2-/- mice showed long crypt formation withincreased number of CD44 positive undifferentiated cells, indicating that disruption of
TGF-β signaling induces invasion of tumor glands through suppression of differentiation(Figure 1). Moreover, macrophages infiltrated to microenvironment express MT1-MMP,

Figure 1

Intestinal epithelial cells from X-ray irradiated Tgfbr2-/- mice developed long crypt-likeorganoids in Matrigel with increased number of EdU labeled/CD44 positive cells (right),
while wild-type mouse-derived cells formed typical mini crypt structure (left).

Inflammation and Cancer Microenvironment

33

resulting in activation of MMP2. It is therefore
possible that inflammatory microenvironment
supports invasion of TGF-β -suppressedmucosa by degradation of basement
membrane component (Figure 2).

About 75% of p53 mutations in cancers are

missense type, suggesting a gain-of-function

mechanism of mutant p53 for cancer

development. Importantly, we found thatApc∆716 Trp53R270H compound mice developed

invading adenocarcinomas in the intestine,
which was not found in Apc∆716 Trp53-/- mice [4]. Moreover, mutant p53 expression caused

drastic morphological changes of tumor

Figure 2

Figure 3

Inflammatory microenvironment
activates MMP2 by macrophagederived MT1-MMP, while TGB-βinhibition induces invasivenessby suppression of differentiation.
These process may cooperate toinduce submucosal invasion.

organoids to complex glandular structure(Figure 3), which was associated withacquisition of invasiveness. Consistently, inthe human CRC with p53 mutation at R273,

we found increased irregular branchingcompared with p53 wild-type tumors. We

also found that mutant p53 expression

induces global transcriptome shift, and(A) Mutant p53 R270H induces complex glandular structure of tumor organoids by gain-offunction mechanism. (B) Increased irregular branching in human colon cancer with p53
mutation around codon 273 compared with that in p53 wild-type tumors.

34

M. Oshimaingenuity pathway analysis using expression data indicated that Wnt signaling pathway as
well as inflammatory pathways are significantly activated by mutant p53 R270H expression(Figure 4). These results suggest that mutant p53 induces submucosal invasion of intestinal

tumors through irregular gland formation possibly caused by Wnt acivation, and mutant
p53-induced inflammatory microenvironment generation may also contribute to malignant
phenotypes. However, Tgfbr2 disruption and p53 R270H expression are not sufficient for

more advanced stages of the colon cancer.

To further examine the malignant phenotypes induced by multiple driver mutations,

we crossed mice carrying genetic alterations in Apc (A), Kras (K), Tgfbr2 (T), Fbxw7 (F) and

Trp53 (P) in various combinations [5]. Consistent with the above studies, AT and AP

mutations induced submucosal invasion of intestinal tumors. However, additional Kras

mutation, namely AKT or AKP combination, is required for intravasation and EMT-likemorphology in the primary tumors (Figure 5). These results indicate that activation of Wnt
and Kras pathways together with TGF-β suppression or mutant p53 expression (AKT or

AKP) is a minimum requirement of malignant progression of CRC. Therefore, activatedpathways by these combinations will be an effective preventive strategy for CRC
progression.

Figure 4(A) PCA analysis of expression profiles of intestinal tumor organoids with different p53
status. (B) Differentially expressed genes in mutant p53 tumor organoids. (C) Significantly
activated pathways by mutant p53 expression.

Inflammation and Cancer Microenvironment

Figure 535

Schematic drawing of genotype-phenotype relationship in the mouse primary intestinal
tumors.

References
1. Oshima M, Oshima H, Kitagawa K, Kobayashi M, Itakura C, Taketo M. Loss of Apc
heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carryig
a truncated Apc gene. Proc Natl Acad Sci USA, 1995;92:4482-4486.
2. Kitamura T, Kometani K Hashida H, Matsunaga A, Miyoshi H, Hosogi H, Aoki M,
Oshima M, Hattori M, Takabayashi A, Minato N Taketo MM. SMAD4-deficient intestinal tumors recruit CCR1+ myeloid cells that promote invasion. Nat Genet, 2007;39:467-475.
3. Oshima H, Nakayama M, Han TS, Naoi K, Ju X, Maeda Y, Robine S, Tsuchiya K, Sato T,
Taketo MM, Oshima M. Suppressing TGF-β signaling in regenerating epithelia in an
inflammatory microenvironment is sufficient to cause invasive intestinal cancer. Cancer
Res, 2015;75:754-765.
4. Nakayama M, Sakai E, Echizen K, Yamada Y, Oshima H, Han TS, Ohki R, Fujii F,
Ochiai A, Robine S, Voon DC, Ta naka T, Taketo MM, Oshima M. Intestinal cancer progression by mutant p53 through the acquisition of invasiveness associated with complex
glandular formation. Oncogene, 2017;36:5885-5896.

36

M. Oshima

5. Sakai E, Nakayama M, Oshima H, Kouyama Y, Niida A, Fujii S, Ochiai A, Nakayama KI,
Mimori K, Suzuki Y, Hong CP, Ock CY, Kim SJ, Oshima M. Combined mutatino ofApc, Kras and Tgfbr2 effectively drives metastasis of intestinal cancer. Cancer Res,
2018; in press.

Masanobu Oshima, DVM, PhD

1989-1991
1992-1997
1997-2000
2000-2005

2005-present
2012-2016

Research staff, Chugai Pharmaceutical Co. Ltd
Research staff, Banyu Merck Research Institute,
Banyu Pharmaceutical Co. Ltd
Research Associate (Posdoc), Human Genetics,
Merck Research Laboratories
Associate Professor, Dept. of Pharmacology,
Kyoto Univ. Grad. School of Medicine
Professor, Division of Genetics, Cancer Res. Inst.,
Kanazawa Univ.
Director, Cancer Res Inst, Kanazawa Univ.

37

CELL PLASTICITY IN COLORECTAL CARCINOGENESIS

Florian R. Greten

Institute for Tumor Biology and Experimental Therapy
Georg-Speyer-Haus
Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany(greten@gsh.uni-frankfurt.de)

The tumor microenvironment potentially impacts on different aspects of tumor stem cellbiology. Different subpopulations of tumor stem cells might be able to interconvert in

response to environmental triggers. Microenvironmental signals influence the de-differentiation process of transformed cells into cancer stem cells. Furthermore stromalderived factors can lead to the activation of a transdifferentiation program resulting inepithelial-to-mesenchymal transition (EMT) and metastatic spread. We have recently

developed a new mouse model of invasive colon cancer that closely recapitulates late stage
human CRC [1]. Mice with an IEC-restricted deletion of p53 (Tp53 ∆IEC mice) that are

repetitively challenged with the pro-carcinogen azoxymethane (AOM) develop invasivecancer in the distal colon within three months. In about 30 % of Tp53∆IEC mice lymph nodemetastases can be detected after 5-6 months. Interestingly, p53 controlled invasion does notdepend on well-described downstream functions of p53 such as apoptosis, cell cycle control
or genomic stability but rather on the development of an NF-κB dependent inflammatory
microenvironment supporting the induction of EMT. Loss of IKKβ in IEC significantly

blocks tumor invasion in this model. Interestingly, loss of IKKβ in myeloid cells does notdecrease the incidence of p53-deficient invasive tumors, but instead it reduces the size ofinvasive lesions and decreases the proliferation rate of invading epithelia. This is paralleledby a diminished paracrine activation of STAT3 in invading tumor cells due to lower

expression levels of STAT3 activating cytokines in recruited IKKβ-deficient myeloid cells.
Strikingly, mice with myeloid specific IKKβ deletion are completely protected from lymph

node metastases. The interleukin (IL)-6 family of cytokines is defined by the shared use of
the gp130 receptor β-subunit. Engagement of the gp130 receptor by either IL-6 or IL-11

38

F. R. Greten

induces transient activation of Janus kinases (JAK) and the latent transcription factor
STAT3. To determine the contribution of Stat3 signaling to the development of lymph nodemetastases in AOM-challenged Tp53∆IEC mice, we generated intestinal epithelial cell (IEC)

specific knockout mice of the common IL-6 cytokine family receptor β subunit gp130(gp130 ∆IEC ) and crossed these to Tp53 ∆IEC mice. Expectedly, the loss of gp130 in IEC

substantially decreased the formation of lymph node metastases. However, this was notaccompanied by a loss of Stat3 activation in p53 deficient IEC, suggesting alternative gp130
independent activation of Stat3 in these tumor cells.

Over the past 30-40 years basically every review on the role of reactive oxygen species(ROS) in cancer states that ROS can induce DNA damage and mutations. It had beensuggested that during chronic inflammation increased ROS production may cause

mutations. Myeloid cells are a major source of ROS in acute and chronic inflammation as

well as in tumor induced inflammation. Surprisingly - and despite the firm statement in ahuge number of reviews - nobody has ever shown and formally proven using

a genetic model that myeloid derived ROS indeed can caute to enhanced tumor
progression. TNFuse mutations in neighboring epithelial cells in vivo. Moreover, it has evenbeen speculated that mutagenesis is not a direct consequence of myeloid derived ROS but

rather an effect of ROS production in epithelial cells in response to pro-inflammatory
cytokines such as TNFα. Using relevant genetic models we have now systematicallyaddressed this question.

We used a conditional Gpx4 knockout in myeloid cells which causes spontaneously

increased ROS production in myeloid cells that is comparable to wt cells during an

inflammatory condition (Figure 1). Importantly, loss of Gpx4 does not affect myeloid cell

survival and function allowing us to use these mice in tumor models. We intended to use
these mice as a genetic tool to increase ROS production in myeloid cells rather than todecipher GPx4-dependent functions in macrophages during tumor development. We were

Figure 1

Myeloid specific deletion of Gpx4 (LysM-Cre)Inflammation and Cancer Microenvironment

39

then indeed able to demonstrate that these mice develop invasive cancer in a model of

AOM-initiated intestinal tumorigenesis, which is not detected in wt animals (Figure 2).

Invasive cancer can even be found when Gpx4 is deleted from myeloid cells in establishedtumors using a newly developed tamoxifen-inducible LysM-CreERT2 mouse.

Mechanistically we could show that increased mutational load in IEC is dependent on
myeloid derived H 2 O 2 rather than cytokine mediated effects in IEC (Figure 3, 4).

Nevertheless, we provide evidence that also cytokines contribute to enhanced tumor

progression. TNFα induces a feed-forward loop that culminates in enhanced macrophage

recruitment and therefore increased ROS levels in tumors (Figure 5). Importantly, however,

Figure 2

Loss of Gpx4 in myeloid cells promotes tumor invasion

Figure 3

Increased mutational load in the absence of myeloid Gpx4

40

F. R. Greten

Figure 4

Catalase can block invasion

Figure 5

Loss of Tnfr1 does not affect mutational load

TNFα is not directly involved in DNA damage. We complemented our in vivo data withextensive ex vivo analysis of intestinal organoids and we can impressively demonstrate that
repetitive treatment of wt organoids with H2O2 (but not TNFα) leads to the formation of alarge number of mutations (verified by whole exome sequencing) closely mimicking the invivo situation (Figure 6, 7). Importantly, we could further demonstrate that Gpx4-deleted

mice develop tumors in a model of chronic inflammation whereas wt animals do not in thesame time (Figure 8) and most importantly that Gpx4 ko mice spontaneously develop

tumors at various locations, lung being the most frequently affected organ (Figure 9).

Inflammation and Cancer Microenvironment

Figure 6

Repetitive H2O2 treatment induces mutations in organoids

Figure 7

Loss of Tnfr1 does not prevent mutations upon repetitive H2O2 treatment

Figure 8

Gpx4Dmye mice develop tumors after chronic inflammation without AOM

41

42

F. R. Greten

Figure 9

Spontaneous tumorigenesis in mice with myeloid Gpx4 loss

Figure 10 Summary

Inflammation and Cancer Microenvironment

43

In summary, our study [2] actually proves for the very first time that chronic

inflammation (=increased ROS production in myeloid cells) can initiate cancer and does not
only contribute to tumor promotion and progression by shaping a pro-tumorigenic
cytokine milieu in the tumor microenvironment. Moreover, we provided a detailed

molecular mechanism and could distinguish ROS-dependent events from cytokine

mediated effects: we could rule out a direct role of TNFR-dependent signaling, MyD88dependent signaling as well as RAGE-dependent signaling for epithelial mutagenesis(Figure 10).

References
1. Schwitalla, S., P.K. Ziegler, D. Horst, V. Becker, I. Kerle, Y. Begus-Nahrmann, A. Lechel,
K.L. Rudolph, R. Langer, J. Slotta-Huspenina, F.G. Bader, O. Prazeres da Costa, M.F.
Neurath, A. Meining, T. Kirchner, and F.R. Greten, Loss of p53 in Enterocytes Generates an
Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis ofCarcinogen-Induced Colorectal Tumors. Cancer Cell, 2013. 23(1): p. 93-106.
2. Canli Ö., Nicolas AM., Gupta J., Finkelmeier F., Goncharova O., Pesic M., Neumann T.,
Horst D., Löwer M., Sahin U., Greten FR. Myeloid Cell-Derived Reactive Oxygen Species
Induce Epithelial Mutagenesis. Cancer Cell, 2017, in press

Florian R. Greten, MD

1998
1998-2000
2001-2004
2004-20082010-20132013-present

MD, University Hamburg
Internship and Residency, University Hospital Ulm
Postdoctoral Fellow, University of California,
San Diego
Residency, Klinikum rechts der Isar,
Technical University Munich
Professor of Molecular Gastrointestinal Oncology,
Klinikum rechts der Isar, Technical University Munich
Chair of Tumor Biology, Medical Faculty, Goethe
University Frankfurt and Scientific Director,
Georg-Speyer-Haus, Institute for Tumor Biology andExperimental Therapy, Frankfurt

44

TUMOUR-STROMA INTERACTIONS IN BREAST CANCERPROGRESSION

Clare M. Isacke

Breast Cancer Now Research Centre
The Institute of Cancer Research
237 Fulham Road, London SW3 6JB, UK(clare.isacke@icr.ac.uk)

The interaction of tumour cells with stromal cells and the extracellular matrix in thetumour microenvironment not only promotes tumour progression and metastasis (Figure1), but also influences response to chemotherapy, endocrine therapy and targeted agents).

As a consequence, there is an urgent need to identify strategies to efficiently target theseinteraction pathways for the prevention or suppression of metastatic disease and to

overcome treatment-resistant progression in advanced breast cancer. We use a combinationof in vivo RNAi screening, profiling of stromal cells in the tumour microenvironment, andin vivo, ex vivo and in vitro assays, particularly 3D in vitro co-culture assays, to identify key

determinants promoting breast cancer metastasis.

Figure 1

Cancer-Stromal Interaction

45

As examples of these approaches, we developed an in vivo shRNA screen to identify

novel metastasis suppressor genes. A top hit in this screen was the sialyltransferase

ST6GalNAc2 [1]. Mechanistically, ST6GalNAc2 silencing alters the profile of O-glycans on
the tumour cell surface, facilitating binding of the soluble lectin galectin-3. This thenenhances tumour cell retention and emboli formation at metastatic sites leading toincreased metastatic burden, events that can be completely blocked by galectin-3 inhibition(Figure 2). Critically, elevated ST6GALNAC2 expression significantly correlates with
reduced frequency of metastatic events and improved survival in human patients.

Subsequently, we have adapted this shRNA screening strategy to identify modulators ofchemotherapy response. Chemotherapy remains the mainstay of treatment for advanced

breast cancer; however, resistance is an inevitable event for the majority of patients withmetastatic disease. Moreover, there is little information available to guide stratification of
first line chemotherapy, crucial given the common development of multidrug resistance.

From this approach, we identified JNK signalling as a key modulator of chemotherapy

response. In agreement with the published literature, we demonstrated that JNK inhibition
both promotes tumour cell cytostasis and blocks activation of the proapoptotic protein Bax,

thereby antagonizing chemotherapy-mediated cytotoxicity [2]. To investigate the clinicalrelevance of this dual role of JNK signalling, we developed a proliferation-independent

JNK activity transcriptional signature. Consistent with the dual role of JNK signalling,high-level JNK pathway activation is associated both with poor outcome in untreatedpatients and is predictive of enhanced chemotherapy response. These data highlight thepotential of monitoring JNK activity as early biomarker of response to chemotherapy andemphasizethe2 importance of rational treatment regimes, particularly when combining
Figurecytostatic and chemotherapeutic agents.

!"#$%&'()*+,-+.

!"#$%&'()*&69.

Figure 2$%&/)0123.
,1+,4,5678.

46

C. M. Isacke

Although these studies provide valuable information as to how tumour cells modulate

their response to the microenvironment and to therapeutics, they do directly address a key
feature of breast cancers, namely 'how do the cancer cells drive the recruitment andactivation of stromal cells?' and 'what are the molecular mechanisms driving stromal

heterogeneity in breast cancers'. By profiling tumour cells and fibroblasts directly isolated
from aggressive and non-aggressive mouse mammary tumours, we identified Wnt7a as a

key factor secreted exclusively by aggressive breast tumour cells, which induces cancer-

associated fibroblast (CAF) recruitment and activation [3] (Figure 3). Functionally, thisresults in extracellular matrix remodelling to create a permissive environment for tumour

cell invasion and promotion of distant metastasis. Mechanistically, Wnt7a-mediatedfibroblast activation is not dependent on classical Wnt signalling. Instead, we demonstratethat Wnt7a potentiates TGFb receptor signalling both in 3D in vitro and in vivo models,thus highlighting the interaction between t wo of the key signalling pathways indevelopment and disease. Importantly, in clinical breast cancer cohorts, tumour cell Wnt7a
expression correlates with a desmoplastic, poor-prognosis stroma and poor patient
outcome.

Finally we have used genetically engineered mouse models to address how stromal cell

activation promotes metastatic dissemination. Within the stroma of the primary tumour

stroma, pericytes associated with the blood vessels and myofibroblasts share a number oftraits, including the upregulated expression of the transmembrane receptor endosialin(CD248). By implanting mouse mammary cancer cells into wild-type and endosialin-

deficient mice, in collaboration with Helmut Augustin's laboratory, we revealed thatstromal endosialin does not affect primary tumour growth but strongly promotes

spontaneous metastasis [4]. Mechanistically, endosialin-expressing pericytes in the primary
tumour facilitate distant site metastasis by promoting tumour cell intravasation in a cell

contact-dependent manner,Figure
resultingin elevated numbers of circulating tumour cells
3=15>%2+,-+.
5?@6?78.
aggressive! :(;.
tumour%)0<%061.

=15>%2&69.
5?@6?78.

Figure 3!! :(;.
non-aggressive
tumour%)0<%061.

Cancer-Stromal Interaction

47(Figure 4). Corresponding to these preclinical experiments, in independent cohorts ofprimary human breast cancers, upregulated endosialin expression significantly correlateswith increased metastasis and poorer patient survival.

Our current studies are focused on (a) understanding the different functional roles

played by activated and non-activated cancer-associated fibroblasts, and how thesedifferent fibroblasts interact with both tumour cells and other stromal cell populations, (b)the mechanisms by which a productive microenvironmental niche is initiated at metastatic
sites, and (c) the mechanisms by which dormant breast cancer cells at secondary sites
become reawakened leading to late metastatic relapse.
Figure 4A/7,)B5/2/CD7/88/E.
/1E68,%&,1.D76@65/8.
,157%<%8%061.

Figure 4

References
1. *Murugaesu, N., *Iravani, M., et al., and Isacke, C.M. (2014) An in vivo functional screen
identifies ST6GalNAc2 sialyltransferase as a breast cancer metastasis suppressor Cancer
Discovery 4:204-317
2. Ashenden, M.A., van Weverwijk, A., Murugaesu, N., Fearns, A., Campbell, J., Gao, Q.,
Iravani, M. and Isacke, C.M. (2017) An in vivo functional screen identifies JNK signaling asa modulator of chemotherapeutic response in breast cancer. Mol. Cancer Ther. 16:19671978
3. *Avgustinova, A., *Iravani, M., Robertson, D., Fearns, A., Gao, Q., Klingbeil, P., Hanby, A.,
Speirs, V., Sahai, E., Calvo, F. and Isacke, C. M. (2016) Tumour cell-derived Wnt7a
recruits and activates fibroblasts to promote tumour aggressiveness. Nature
Communications 7:10305
4. *Viski, C., *Konig, K., Kijewska, M., Mogler, C., *Isacke, C.M. and *Augustin, H.G.
(2016) Endosialin-expressing pericytes promote metastatic dissemination. Cancer
Research 76:5313-5325

48

C. M. Isacke

Clare M. Isacke, DPhil

1984-1988
1988-1991
1991-2001

2001-present

Postdoctoral Fellow, The Salk Institute, San Diego
Fellow, Department of Biochemistry,
Imperial College London
Faculty, Department of Biology,
Imperial College London
Professor of Molecular Cell Biology,
The Institute of Cancer Research, London

49

THE ROLE OF SASP IN TUMOR MICROENVIRONMENT OFOBESITY-ASSOCIATED LIVER CANCER

Naoko Ohtani

Department of Pathophysiology
Graduate School of Medicine, Osaka City University
1-4-3 Asahi-machi, Abeno-ku, Osaka, Japan(ohtani.naoko@med.osaka-cu.ac.jp)

Obesity has become a worldwide health problem, and is known to increase a risk ofseveral types of cancers. We previously identified that the enterohepatic circulation of theobesity-induced Gram positive gut microbiota metabolite, deoxycholic acid (DCA) induce

the cellular senescence and senescence-associated secretory phenotype (SASP) of hepatic
stellate cells, a phenotype that senescent cells secrete inflammatory cytokines, chemokines,
proteases, growth factors and so on. Accumulating evidence has indicated that SASP isregulated by a combination of several transcription factors, epigenetic regulators andmetabolic pathways, in response to DNA damage. DCA can create DNA damage by

elevating the levels of reactive oxygen species (ROS), thereby inducing cellular senescenceand the SASP to promote obesity-associated HCC. However, the trigger that initiates the

SASP signals may vary depending on the physiological status, and thus, the precise
mechanisms regulating the expression of SASP factors need to be elucidated in eachbiological and pathophysiological setting.

One of the mechanisms that may trigger the SASP could be Toll-like receptor (TLR)

signaling. Previous in vivo studies have demonstrated that TLR4-mediated inflammatory

signals induced by the Gram-negative bacterial component lipopolysaccharide (LPS),
which is categorized as a pathogen-associated molecular pattern (PAMP) protein from thegut microbiota, are important for promoting liver fibrosis and fibrosis-associated liver
tumorigenesis. However, in our obesity-associated HCC model, Gram-positive bacteria,

but not Gram-negative bacteria, were found to be dramatically increased in obese mice fed

an HFD, consistent with previous reports. Moreover, we did not observe the reduction ofobesity-associated liver tumor formation in TLR4-deficient mice as compared with those in

50

N. Ohtaniwild-type mice when we performed the same protocol of neonatal DMBA treatment with

HFD, suggesting that TLR4-mediated signals are unlikely to be involved in the accelerationof HCC development in our experimental setting.

Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA),a Gram positive gut microbial component, promotes HCC development by enhancing

SASP factor expression of hepatic stellate cells through Toll-like receptor 2 collaboratively
with DCA. LTA also upregulated the expression of cyclooxygenase-2 (COX-2).

Interestingly, COX-2-mediated prostaglandin E 2 (PGE 2 ) production suppresses theantitumor immunity in the tumor microenvironment, thereby contributing to HCC

progression. Moreover, COX-2 overexpression and excess PGE2 production were detected
in HSCs in human HCCs with non-cirrhotic, non-alcoholic steatohepatitis (NASH),
indicating that a similar mechanism could function in humans.

We showed the importance of gut-liver axis in obesity-associated HCC. Gut microbiota-

driven COX-2 pathway produced lipid mediator, PGE2 in the senescent HSCs in the tumor

microenvironment which plays a pivotal role to suppress antitumor immunity, suggestingthat PGE2 and its receptor could be novel therapeutic targets for non-cirrhotic NASHassociated HCC.

References
1. Loo TM, Kamachi F, Watanabe Y, Yoshimoto S, Kanda H, Arai Y, Nakajima-Takagi Y,
Iwama A, Koga T, Sugimoto Y, Ozawa T, Nakamura M, Kumagai M, Watashi K, Taketo
MM , Aoki T, Narumiya S, Oshima M, Arita M, Hara E and Ohtani N. Gut microbiota
promotes obesity-associated liver cancer through PGE2-mediated suppression of antitumor immunity. Cancer Discovery 7, 522-538, 2017 doi: 10.1158/2159-8290.CD-16-0932
2. Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S, Oyadomari S, Iwakura Y, Oshima K,
Morita H, Hattori M, Honda K, Ishikawa Y, Hara E, Ohtani N. Obesity-induced gut
microbial metabolite promotes liver cancer through senescence secretome. Nature 499, 97101 (2013)

Cancer-Stromal Interaction

Naoko Ohtani, MD, PhD

1995-1998
1998-2003
2003-2007
2008-20142014-20172017-present

51

Assistant Professor, Kyoto Prefectural University of
Medicine
Postdoctoral Fellow, University of Manchester,
Paterson Institute for Cancer Research
Lecturer and Associate Professor, Institute for Genome
Research, University of Tokushima
Senior Staff Scientist, Cancer Institute,Japanese Foundation for Cancer Research
Professor, Dept. Applied Biological Science, Faculty of
Science and Technology, Tokyo University of Science
Professor, Department of Pathophysiology,
Graduate School of Medicine, Osaka City University

52

STROMAL CELLS: ESSENTIAL REGULATORS OF TUMORPROGRESSION AND METASTASIS AND TARGETS FOR
THERAPY

Zena Werb

Department of Anatomy andHelen Diller Family Comprehensive Cancer Center
University of California, San Francisco
513 Parnassus Ave., San Francisco CA 94143-0452, USA(zena.werb@ucsf.edu)
Breast cancer is the leading cancer affecting women worldwide and metastatic breast

cancer accounts for over 400,000 deaths per year. Metastatic burden is the main cause of
mortality related to breast cancer. Myeloid cells are critical regulators of breast cancer
tumor growth and metastatic progression and can have either a pro- or anti-tumoral
phenotype [1-3].

Tumor-infiltrating inflammatory cells comprise a major part of the stromal

microenvironment and support cancer progression by multiple mechanisms. High

numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and arecoupled with the angiogenic switch and malignant progression. However, the specific roles
and regulation of heterogeneous tumor myeloid populations are incompletely understood.

CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also
linked to poor outcomes. To characterize myeloid cell function in tumors, we combined

confocal intravital microscopy with depletion of CSF-1R–dependent cells using aneutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-

driven polyoma middle T antigen breast cancer model [2]. The depleted cells sharedmarkers of tumor-associated macrophages and dendritic cells (M-DCs), matching thephenotype of tumor dendritic cells that take up antigens and interact with T cells. We

defined functional subgroups within the M-DC population by imaging endocytic andmatrix metalloproteinase activity. Anti–CSF-1R treatment altered stromal dynamics andimpaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr1+ neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal
effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor

Cancer-Stromal Interaction

53

growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate
in metastatic lungs differed significantly from that in mammary tumors, the reduction inmetastasis may result from the impact on primary tumors. The combination of function

alanalysis by intravital imaging with cellular characterization has refined our understandingof the effects of experimental targeted therapies on the tumor microenvironment.

There is growing evidence indicating that neutrophils have an important role in tumor

development—from the inception of tumor formation and throughout the malignant
progression [3]. Neutrophils both promote and prevent tumor progression. The

contradictory role of neutrophils in tumor development may be explained be the fact thatneutrophils can have both pro- or anti-inflammatory phenotypes. Depending on theirphenotype, neutrophils are either antitumoral (N1) or protumoral (N2). The occurrence of

N1/N2 neutrophils has only been shown in murine tumor models and will have to be

confirmed more importantly, in humans. However, neutrophils with an activatedphenotype, producing pro-inflammatory factors and stimulating T cell proliferation havebeen described in early human lung cancer, while immunosuppressive neutrophils has

been reported in human colorectal cancer. Tumor-associated neutrophils (TANs) with a N1
phenotype have a pro-inflammatory cytokine profile, producing nitrate oxide andhydrogen peroxide (H2O2), and are cytotoxic towards tumor cells. N2 TANs, on the other

hand, are characterized by high levels of arginase 1 (ARG1) and by their ability to inhibit
effector T-cell functions. Our understanding of the polarization of neutrophils and how

neutrophils contribute to tumor development has largely been elucidated fromexperimental mouse models.

Importantly, some breast cancer patients never develop metastatic breast cancer. The

identification of a monocyte, macrophage and/or neutrophil subpopulation that prevents
metastatic breast cancer could provide the backbone for the development of novel
immunotherapeutic target molecules. We have shown that production of atypical T cell-

suppressive neutrophils occurs during early tumor progression, at the onset of malignant

conversion and that these cells preferentially accumulate in peripheral tissues, but not in
the primary tumor, leading to metastasis [3]. Production of these cells results from

activation of a myeloid differentiation program in bone marrow (BM) by a novel
mechanism in which tumor-derived G-CSF directs expansion and differentiation ofhematopoietic stem cells to skew hematopoiesis toward the myeloid lineage. With patientderived xenograft (PDX) models, and large breast cancer tissue microarrays, we haverevealed that tumor-derived cytokines may also inhibit metastatic progression through therecruitment of anti-tumoral activated monocytes.

54

Z. Werb

Fibroblasts also regulate the microenvironment, producing the extracellular matrix aswell as chemokines that recruit myeloid cells [4,5]. Increased collagen expression in tumors

is associated with increased risk of metastasis, and triple negative breast cancer (TNBC) has
the highest propensity to develop distant metastases when there is evidence of central

fibrosis. Transforming growth factor-β (TGF-β) ligands regulated by cancer-associatedfibroblasts (CAFs) promote accumulation of fibrosis and cancer progression. We haveevaluated TNBC tumors with enhanced collagen to determine whether we can reduce

metastasis by targeting the CAFs with Pirfenidone (PFD), an anti-fibrotic agent as well as a

TGF-β antagonist [4]. In patient-derived xenograft models, TNBC tumors exhibited
accumulated collagen and activated TGF-β signaling, and developed lung metastasis. We

derived primary CAFs from 4T1 TNBC homograft tumors, TNBC xenograft tumors andtumor specimens of breast cancer patients. CAFs promoted primary tumor growth withmore fibrosis and TGF-β activation and lung metastasis. We then examined the effects of
PFD in vitro and in vivo. We found that PFD had inhibitory effects on cell viability andcollagen production of CAFs in 2D culture. Furthermore, CAFs enhanced tumor growthand PFD inhibited the tumor growth induced by CAFs by causing apoptosis in the 3D coculture assay of tumor cells and CAFs. In vivo, PFD alone inhibited tumor fibrosis and
TGF-β signaling but did not inhibit tumor growth and lung metastasis. However, PFD

inhibited tumor growth and lung metastasis synergistically in combination withdoxorubicin. Thus, PFD has great potential for a novel clinically applicable TNBC therapy
that targets tumor-stromal interaction.

Thus, the microenvironment contributes critically to drug response via regulation ofvascular permeability and innate immune cell infiltration [6,7]. Clinically applicable

therapy that targets tumor-stromal interaction may have important actions in reducing or
preventing breast cancer progression.

References
1. Hagerland, C. & Z. Werb (2016). Neutrophils: critical components in experimental animalmodels of cancer. Semin. Immunol. 28: 197-204.
2. Lohela, M., A.-J. Casbon, A. Olowa, L. Bonham, D. Branstetter, N. Weng, J. Smith & Z.
Werb (2014). Intravital imaging reveals distinct responses of depleting dynamic tumorassociated macrophage and dendritic cell subpopulations. Proc. Natl. Acad. Sci. U.S.A.
111:E5086-5095.
3. Casbon, A.-J., D. Reynaud, C. Park, E. Khuc, D. D. Gan, K. Schepers, E. Passegué & Z.
Werb (2015). Tumors reprogram early myeloid differentiation in the bone marrow to
generate immunosuppressive neutrophils. Proc. Natl. Acad. Sci. U.S.A. 112:E566-E575.

Cancer-Stromal Interaction

55

4. Takai, K., A. Le, V. M. Weaver & Z. Werb (2016). Targeting the cancer-associatedfibroblasts as a treatment in triple-negative breast cancer. Oncotarget. 7:82889-82901
5. Allaoui, R., C. Bergenfelz, S. Mohlin, C. Hagerling, K. Salari, Z. Werb, R. Anderson, S. P.
Ethier, K. Jirström, S. Påhlman, D. Bexell, B. Tahin, M. E. Johansson, C. Larsson & K.
Leandersson (2016). Cancer-associated fibroblast-secreted CXCL16 attracts monocytes
to promote stroma activation in triple negative breast cancers. Nat. Commun. 7:13050.
6. Nakasone, E., H. A. Askautrud, T. Kees, V. Plaks, A. J. Ewald, M. G. Rasch, Y. X. Tan, J.
Qin, M. Fein, J. Park, P. Sinha, M. J. Bissell, E. Frengen, Z. Werb & M. Egeblad (2012).
Imaging tumor-stroma interactions during chemotherapy reveals microenvironmental
contributions to chemoresistance. Cancer Cell. 21:488-503.
7. Gonzalez, H., I. Robles & Z. Werb (2017). Innate and acquired immune surveillance in the
post-dissemination phase of metastasis. FEBS J. 2017 Nov 13. doi: 10.1111/febs.14325.
[Epub ahead of print]

Zena Werb, PhD

1971-1973
1973-1975
1975-1976
1976-1980

1980-1983
1983-present
1999-present
2016-present

Postdoctoral Fellow, Strangeways Research Laboratory
Research Fellow, Strangeways Research Laboratory
Visiting Assistant Professor, Dartmouth Medical School
Assistant Professor, University of California, San
Francisco (UCSF)
Associate Professor, UCSF
Professor, UCSF
Vice-Chair, Department of Anatomy, UCSF
Associate Director for Basic Science, UCSF Helen
Diller Family Comprehensive Cancer Center

56

DEVELOPMENT AND MAINTENANCE OF THE STEMNESSOF COLON CANCER

Koji Okamoto

Division of Cancer Differentiation
National Cancer Center Research Institute5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan(kojokamo@ncc.go.jp)

Cancer is composed of heterologous cells, and its heterogeneity is largely attributed tothe cellular hierarchy generated by cancer stem cells (CSCs) or related cells, which are
likely to be responsible for the refractory nature of cancer such as chemo-resistance ormetastatic traits. Here we focused on mechanism of generation and maintenance of CSCs.

Lgr5 is one of the most representative markers of stem cells in various normal tissues,

including the intestine. The importance of Lgr5 as a stem cell marker can be extended tocolon tumors. In fact, Lgr5 likely serves as a hallmark of cancer stem cells (CSCs) not onlyin adenoma but also in advanced colon cancer. Therefore, it is likely that Lgr5 stem cells or
related progenitors, in normal tissues, after oncogenic mutations and exposure totumorigenic insults, develop into tumorigenic stem cells, or CSCs.

To obtain a dynamic picture of how CSCs are generated from non-tumor epithelial cells

during colon carcinogenesis, we performed gene expression analyses at the single cell levelin a mouse model of inflammation-induced colon cancer. Apc(Min/+) mice were treatedwith dextran sulfate sodium to generate colon tumors, and the epithelial cells from tumors

and non-tumors were used for multiple-gene qPCR and RNA-seq analyses at the single-celllevel. Statistical analyses of the qPCR data indicated that Lgr5-positive stem cells werestratified into several groups, and comparison of the cells obtained from tumor and nontumor tissues revealed the emergence of a novel Lgr5-positive stem cell group in tumor

epithelia [1].

This tumor-specific group of Lgr5-positive cells showed enhanced tumorigenicity andgene expression profiles that are related to CSCs, including the activation of Wnt signaling.
Functional analyses by CRISPR)/Cas9-mediated gene knockout revealed that, among theHeterogeneity and Plasticity of Cancer

57

Wnt targets that were specifically expressed in the tumor-specific stem cells, the long

isoform of Tcf1 (Tcf1-LF) was required for the proliferation of colon tumor organoids. In
fact, Tcf1-LF was required for a unique Wnt target gene expression profile. Furthermore,

Tcf1-LF expression increased at an early stage of colon carcinogenesis and was associatedwith the nuclear accumulation of β-catenin, underscoring the importance of the inductionof Tcf1-LF expression for the generation of colon cancer stem-like cells (Figure 1) [1].

We also identified several new genes that were specifically expressed in the tumor-

specific stem cells, and their functional characterization is currently underway. To further
investigate the roles of the identified CSC-related factors in full-blown human cancer, we
examined their expression patterns in mouse xenograft models derived from an in vitro

spheroid culture from surgical specimens of colorectal cancer, which was described below.

In addition to mouse models of inflammation-associated colon cancer, we studied

tumor-derived human spheroids to study CSCs. Using the in vitro 3D culture (spheroid
culture) from human colon cancer and serous ovarian cancer, we isolated and expanded

CSCs (Figure 2). Through the transplantation of the in vitro cultivated CSCs into immunocompromized NOG mice, we established in vivo models of colon and ovarian cancer (PDX
models) [2,3]. By applying single-cell gene expression analyses, intra- and interheterogeneity of xenograft tumors were examined.

Colon CSCs

E-catenin+
TCF1

CSC-type Wnt targets

Figure 1

the generation of colon CSCs via the induction of TCF1-LF leads to colon tumorigenesis
Colon tumorigenesis

Figure 1 thegenerationofcolonCSCsviatheinductionofTCF1ͲLFleadstocolontumorigenesis

58

K. Okamoto

CSCs
in vitro cultivation
Surgical specimen

NOG mice

PDX tumors

CSCs
CSCs
CSCs

Spheroid culture

Figure 2

isolation and expansion of colon CSCs as spheroids and establishment of PDX models

Figure 2 isolation and expansion of colon CSCs as spheroids and establishment of PDX models

References
1. Shiokawa D, Sato A, Ohata H, Mutoh M, Sekine S, Kato M, Shibata T, Nakagama H,
Okamoto K. The Induction of Selected Wnt Target Genes by Tcf1 Mediates Generation ofTumorigenic Colon Stem Cells. Cell Reports 19, 981-994. doi: 10.1016/j.celrep.2017.04.
017, 2017
2. Ishiguro T, Sato A, Ohata H, Ikarashi Y, Takahashi R, Ochiya T, Yoshida M, Tsuda H,
Onda T, Kato T, Kasamatsu T, Enomoto T, Tanaka K, Nakagama H, Okamoto K.
Establishment and characterization of an in vitro model of ovarian cancer stem-like cellswith an enhanced proliferative capacity. Cancer Res. 76, 150-160, 2016
3. Ohata H, Ishiguro T, Aihara Y, Sato A, Sakai H, Sekine S, Taniguchi H, Akasu T, Fujita S,
Nakagama H, Okamoto K. Induction of the stem-like cell regulator CD44 by Rho kinase
inhibition contributes to the maintenance of colon cancer-initiating cells. Cancer Res. 72,
5101-5110, 2012

Heterogeneity and Plasticity of Cancer

Koji Okamoto, MD, PhD

1980-1986
1986-1988
1988-1992
1991-1996
1996-2000
2000-20102010-present

Graduated from University of Tokyo,
School of Medicine
Resident, Tokyo University Hospital
Graduated from University of Tokyo,
Graduate School of Medicine
Postdoctoral Fellow, Cold Spring Harbor Laboratory
Postdoctoral Fellow, Columbia University
Section Head, National Cancer Center ResearchInstitute
Chief, National Cancer Center Research Institute

59

60

UNEARTHING MECHANISMS OF MALIGNANT
PROGRESSION AND RESISTANCE OF CANCER STEM CELLSElaine Fuchs

Laboratory of Mammalian Cell Biology and Development
Howard Hughes Medical Institute, The Rockefeller University
1230 York Avenue, Box 300, New York, NY 10065, USA(fuchs@rockefeller.edu)

Adult tissue stem cells have the ability to self-renew long term and differentiate into one

or more tissues. Many stem cells are used sparingly to replenish cells during normal
homeostasis. However, even stem cells that are quiescent must be able to respond quickly
to injury in order to fuel rapid tissue regeneration. How stem cells balance self-renewal anddifferentiation is of fundamental importance to our understanding of normal tissue

maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry
governing this balancing act is at the root of some types of cancers.

The hair follicle is an excellent model system to understand how stem cells remainquiescent during times of minimal wear and tear, how these cells become mobilized during

the cyclical bouts of hair growth and during the re-epithelialization of the epidermis that
occurs in wound-repair, and how the normal process of stem cell activation goes awry incancer. Hair follicles have the remarkable capacity to undergo cycles of growth (anagen),degeneration (catagen) and rest (telogen). In mice, the first several cycles are synchronized,

making hair follicles an ideal system for understanding how stem cells interact withprogeny and heterologous cell types in their niche to transition between quiescence andactive tissue (hair) regeneration.

The quiescent hair follicle stem cells reside in a single layer of the bulge niche, located at

the bottom of the non-cycling portion of the hair follicle. Within the bulge niche is a layer of

BMP-expressing differentiated cells that anchor the hair and transmit inhibitory signals tomaintain stem cell quiescence [1]. The stem cells adhere to a basement membrane flanked

by a dermal sheath. Beneath the bulge is a mesenchymal stimulus called the dermal papilla.

Stem cells at the base of the bulge, closest to this stimulus are the first to be activated at theHeterogeneity and Plasticity of Cancer

61

start of the hair cycle [2]. These cells communicate with the dermal papilla. As Valentina

Greco, a former postdoc in my lab, now at Yale, showed, the ‘primed’ stem cells makeincreasing levels of WNTs; the dermal papilla makes increasing levels of BMP inhibitory

signals, and together these two signals are needed to overcome the inhibitory BMP signalthat otherwise keeps the stem cells in quiescence [3-6].

When the activating threshold is reached, the stem cells at the base of the bulge begin toproliferate and generate short-term progeny called matrix cells. The first group of thesecells produces Sonic Hedgehog (SHH). As shown by Ya-Chieh Hsu, a former postdoc in mylab, now at Harvard, showed, SHH then acts in two ways: first it acts on the dermal papilla,
which elevates BMP inhibitors and pro-activating FGFs; second, it acts on the quiescent

upper bulge stem cells, which self-renew and produce a cellular shaft (the outer root

sheath) which pushes the SHH-DP signaling center downward, resulting in a return of thebulge to quiescence until the next hair cycle [7]. Meanwhile the signaling center fuels theproliferation of the short lived progeny to produce the hair and its channel, the inner root

sheath. After a period of hair growth, the lower two thirds of the hair follicle differentiates
and degenerates, bringing the DP upward to make contact again with the bulge. In thisway, the hair follicle becomes self-propelling to undergo these cyclical bouts of activity.

Over the years, we developed methods to purify the bulge stem cells and their progeny

and we carried out RNA-seq on these populations straight from fluorescence activating cellsorting (FACS). The stem cells expressed high levels of SOX9, LHX2, NFATc1, FOXC1, IDs,

TCF3 and TCF4 transcription factors, which distinguished them from their committed
counterparts and from the epidermal stem cells residing in the basal layer of the epidermis[4,5,8-13]. We used conditional knockout technology to ablate each of these factors in theskin. Our findings showed that some factors, e.g. NFATc1, IDs and FOXC1, acted
downstream from BMP signaling, and promoted stem cell quiescence. We found thatTCF3/4 antagonize WNT-beta catenin to repress HF fate differentiation, while LHX2
suppresses sebaceous gland differentiation and SOX represses epidermal differentiation. In
carrying out a combination of in vivo chromatin immunoprecipitation and high throughput
DNA sequencing (ChIP-seq) with antibodies against not only the transcription factors but
also epigenetic marks including H3K27ac, Med1, H3K27me3, H3K4me1, H3K4me3 andH3K36me2, we identified ~350 HFSC genes which are controlled by large open chromatin
domains, or ‘super-enhancers,’ [10,14-18] a word coined by Rick Young at MIT. Withinthese super-enhancers are small regulatory elements, which we call ‘epicenters,’ that

contain clustered binding sites for the cohort of HFSC transcription factors. In work carriedout by two of my graduate students, Rene Adam and Hanseul Yang, we’ve cloned theseregulatory elements and tested them in mice and found that they act to target reporter gene
expression to the bulge stem cells [18].

62

E. Fuchs

In looking at the endogenous group of HFSC genes regulated by super-enhancers, wefound that they encode the majority of the stemness genes, including the genes regulating
SOX9, TCF3/4 and LHX2 as well as Integrins alpha 6 and beta4, LGR5, BMP6 and thereceptors for BMPs, WNTs and FGFs. There are many other human disease genes that arefound on this short-list comprising ~5% of the total number of genes expressed by HFSCs.

Intriguingly, when HFSCs were cultured, their chromatin landscape changed

dramatically as a new cohort of super-enhancers were gained while others were lost. In
seeking mechanisms, we discovered that many HFSC transcription factor genes weresilenced in vitro while new wound-induced transcription factors appeared. Moreover, the
new super-enhancers were regulated by these wound-induced factors, corresponding to

the new proliferative migratory state of the cultured hair follicle stem cells. Interestingly,

when HFSCs become activated to re-epithelialize the epidermis, they undergo a fate switch
in which they turn off the expression of SOX9, LHX2, NFATc1 and other HFSC

transcription factors and switch on the expression of KLF5, ELK3 and other epidermaltranscription factors. In transit, however, they enter a state where they express certain

features of both HFSCs and epidermal SCs (EpdSCs). We refer to this state as “lineage
infidelity” [19]. In the work of Yejing Ge, a postdoctoral fellow in my laboratory, we showthat lineage infidelity is a transient but essential transitionary state that both EpdSCs and
HFSCs undergo during wound repair. Intriguingly, lineage infidelity is also seen during
malignant progression, and in SCCs, it becomes permanent, a feature which we trace tovery high levels of phosphorylated ETS2.

Published in Cell earlier this year, this work gave us new insights into the sensitivity ofstem cells to their microenvironment and the importance of niche-stem cell interactions in
dictating stem cell behavior. Hanseul Yang along with Daniel Schramek, a former postdoc

in my lab now at the Lunefeld Cancer Center in Toronto, then asked the question of how
the chromatin landscape changes as hair follicle stem cells acquires the mutations that leadto malignant progression. When they carried out ChIP seq analyses on the tumor-initiating(stem) cells of squamous cell carcinoma chromatin, they unearthed new super-enhancers

regulating oncogenes such as Src and Myc. These super-enhancers were regulated by a newcohort of transcription factors whose genes were also regulated by super-enhancers,

thereby generating a feed forward loop. Notably were ETS2 and ELK3, because thesesuper-enhancer regulating transcription factors are known to be phosphorylated andactivated by RAS-MAPK, the pathway upregulated in the SCCs. Indeed, mice engineered
to express a phosphomimic ETS2, the epidermis became hyperproliferative, invasive andgenerated a chromatin landscape that was similar to the SCC state [20]. Again, thesefindings underscored the importance of a now new microenvironment characteristic of the
SCC state. Moreover, in our recent study on lineage infidelity, Yejing cloned and tested

Heterogeneity and Plasticity of Cancer

63

these strongly ETS2 sensitive regulatory elements and identify one that becomes

specifically activated upon malignant transformation [19]. This new driver now provides a
new tool with which to probe deeper into the nature of SCCs in the future.

In addition to exploring oncogenic regulation at the chromatin level, we’ve used in vivo

ribosomal profiling to look into translational controls on the oncogenic state. In work

performed by Ataman Sendoel, a current postdoctoral fellow in my laboratory, we’ve
learned that global protein synthesis is downregulated in EpdSCs when they activate asingle oncogene, in this case, ectopically expressed Sox2 [21]. We were intrigued by our

discovery that a small cohort of mRNAs are still efficiently translated in this condition, andtheir go-terms suggest that they encode cancer promoting proteins. We performed an RNAi
screen to assess the relative importance of various translational initiation factors andribosomal proteins on WT versus oncogenic stem cells. Surprisingly, the canonicalinitiation complex is more important for WT than for oncogenic stem cell growth. The

reason turns out to be that the canonical initiator Eif2α is phosphorylated and repressed in
SOX2-expressing EpdSCs. Under these conditions, the non-canonical EIF2A can compete

for translation. It turns out to be important, as without EIF2A, SCCs fail to be sustained. We
published this work earlier this year in Nature.

Finally, we looked at how the microenvironment changes during malignant

progression. We discovered that wherever blood vessels invade the stroma and approach

the tumor, the tumor-initiating cancer stem cells at the tumor-stroma interface respond to
TGF-beta brought in by the perivasculature, rich in immune cells such as monocytes. The

outcome is striking—these stem cells become slow-cycling and break down the basement

membrane and invade the stroma. By contrast, stem cells more distant from blood vessels
proliferate rapidly, generating the bulk of the tumor [22]. Does this matter? To test thispossibility, we treated mice with cisplatin, the drug of choice for many human squamouscell carcinomas. Most of the tumor cells died, except for those cancer stem cells responding

to TGF-beta. Moreover, these stem cells evaded the chemotherapy and regrew the cancer
when cisplatin was washed out. This was the work of Naoki Oshimori, who has now
moved on to start his own lab at Oregon Health Science Cancer Center [23]. The studies

beautifully underscore the importance of heterogeneity in the tumor microenvironment.

In another recent twist on the impact of the microenvironment, we turned to exploring

the role of stem cells in the memory that skin exhibits when it receives an inflammatory

assault e.g. from a microbial infection, irritant or wound. We learned that epidermal stem

cells, which experience an inflammatory assault and are present long after the pathology
has returned to normal, are able to retain a memory of inflammation. Upon a secondary

assault, e.g. wounding, the epidermis repairs the wound quicker than naïve skin. We showthat this memory is not dependent upon B or T cells or macrophages, but rather is exhibited

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E. Fuchs

at the chromatin level. The EpdSCs retain ~2000 open chromatin domains that act as
inflammation sensors. Many of the genes associated with these domains are activatedquickly upon receiving a secondary assault. This provocative work is that of Shruti Naik, a
postdoctoral fellow in my laboratory, and Samantha Larsen, a graduate student, and was
published in Nature in 2017 [24].

I don’t want to leave you with the notion that genetic variation in tumor progression

doesn’t matter. Clearly, it does, as there are hundreds of mutations in these solid tumors.
To this end, we’ve used our powerful in utero lentiviral technology [25] to carry out insome cases, genome-wide screens for oncogenic drivers [26], tumor suppressors [27] andoncogenic microRNAs involved in SCC progression [28]. Together, these multipronged

approaches should be useful in our ever growing endeavors to understand the underlying
basis for squamous cell carcinomas, which are not only one of the most common but also
one of the most life-threatening cancers world-wide.
References
1. Hsu, Y. C., Pasolli, H. A. & Fuchs, E. Dynamics between stem cells, niche, and progeny inthe hair follicle. Cell. 2011; 144: (92-105).
2. Tumbar, T. et al. Defining the epithelial stem cell niche in skin. Science. 2004; 303: (359-363).
3. Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration.
Cell Stem Cell. 2009; 4: (155-169).
4. Nguyen, H. et al. Tcf3 and Tcf4 are essential for long-term homeostasis of skin epithelia.
Nat Genet. 2009; 41: (1068-1075).
5. Genander, M. et al. BMP signaling and its pSMAD1/5 target genes differentially regulate
hair follicle stem cell lineages. Cell Stem Cell. 2014; 15: (619-633).
6. Lien, W. H. et al. In vivo transcriptional governance of hair follicle stem cells by canonicalWnt regulators. Nat Cell Biol. 2014; 16: (179-190).
7. Hsu, Y. C., Li, L. & Fuchs, E. Transit-amplifying cells orchestrate stem cell activity and tissue regeneration. Cell. 2014; 157: (935-949).
8. Nowak, J. A., Polak, L., Pasolli, H. A. & Fuchs, E. Hair follicle stem cells are specified andfunction in early skin morphogenesis. Cell Stem Cell. 2008; 3: (33-43).
9. Rhee, H., Polak, L. & Fuchs, E. Lhx2 maintains stem cell character in hair follicles.
Science. 2006; 312: (1946-1949).
10. Kadaja, M. et al. SOX9: a stem cell transcriptional regulator of secreted niche signaling factors. Genes Dev. 2014; 28: (328-341).
11. Folgueras, A. R. et al. Architectural niche organization by LHX2 is linked to hair follicle
stem cell function. Cell Stem Cell. 2013; 13: (314-327).

Heterogeneity and Plasticity of Cancer

65

12. Horsley, V., Aliprantis, A. O., Polak, L., Glimcher, L. H. & Fuchs, E. NFATc1 balances quiescence and proliferation of skin stem cells. Cell. 2008; 132: (299-310).
13. Lay, K., Kume, T. & Fuchs, E. FOXC1 maintains the hair follicle stem cell niche andgoverns stem cell quiescence to preserve long-term tissue-regenerating potential. Proc Natl
Acad Sci U S A. 2016; 113: (E1506-1515).
14. Ezhkova, E. et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009; 136: (1122-1135).
15. Ezhkova, E. et al. EZH1 and EZH2 cogovern histone H3K27 trimethylation and areessential for hair follicle homeostasis and wound repair. Genes Dev. 2011; 25: (485-498).
16. Lien, W. H. et al. Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Cell Stem Cell. 2011; 9: (219-232).
17. Keyes, B. E. et al. Nfatc1 orchestrates aging in hair follicle stem cells. Proc Natl Acad Sci U
S A. 2013; 110: (E4950-4959).
18. Adam, R. C. et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity
and lineage choice. Nature. 2015; 521: (366-370).
19. Ge, Y. et al. Stem cell lineage infidelity drives wound repair and cancer. Cell. 2017; 169:
(636-650 ).
20. Yang, H. et al. ETS family transcriptional regulators drive chromatin dynamics andmalignancy in squamous cell carcinomas. Elife. 2015; 4: (e10870).
21. Sendoel, A. et al. Translation from unconventional 5' start sites drives tumour initiation.
Nature. 2017; 541: (494-499).
22. Guasch, G. et al. Loss of TGFbeta signaling destabilizes homeostasis and promotes
squamous cell carcinomas in stratified epithelia. Cancer Cell. 2007; 12: (313-327).
23. Oshimori, N., Oristian, D. & Fuchs, E. TGF-beta promotes heterogeneity and drug
resistance in squamous cell carcinoma. Cell. 2015; 160: (963-976).
24. Naik, S. et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage.
Nature. 2017; 550: (475-480).
25. Beronja, S., Livshits, G., Williams, S. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat Med. 2010; 16:(821-827).
26. Beronja, S. et al. RNAi screens in mice identify physiological regulators of oncogenic
growth. Nature. 2013; 501: (185-190).
27. Schramek, D. et al. Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor ofsquamous cell carcinomas. Science. 2014; 343: (309-313).
28. Ge, Y., Zhang, L., Nikolova, M., Reva, B. & Fuchs, E. Strand-specific in vivo screen of cancer-associated miRNAs unveils a role for miR-21( *) in SCC progression. Nat Cell Biol.
2016; 18: (111-121).

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E. Fuchs

Elaine Fuchs, PhD

1977-1980
1980-1985
1985-1988
1988-1993

1989-2002
1993-present
2002-present

Postdoctoral Fellow, Massachusetts Institute ofTechnology
Assistant Professor, Univ. of Chicago
Associate Professor, Univ. of Chicago
Associate Investigator, Howard Hughes Medical
Institute
Professor, Univ. of Chicago
Investigator, Howard Hughes Medical Institute
Professor, The Rockefeller Univ.

67

ROLE OF ACTIN DYNAMICS IN REPROGRAMMING AND
CELL FATE DETERMINATION OF CANCER AND
MICROENVIRONMENTAL CELLS

Hideyuki Saya

Division of Gene Regulation
Institute for Advanced Medical Research (IAMR)
School of Medicine, Keio University
35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan(hsaya@a5.keio.jp)
Cancer-associated fibroblasts (CAFs) are cellular components in the cancer stroma andconsidered to contribute to progression and survival of cancer cells due to its plasticity.

CAF is characterized as an α-smooth muscle actin (α-SMA) positive cell and speculated tooriginate from not only resident fibroblast but also various cells such as adipocytes,
epithelial cells, endothelial cells, bone marrow derived mesenchymal stem cells andhematopoietic stem cells. Among them, we have special interest in adipocytes because, in a
subset of tumors, growth and metastasis occur as a result of the adipocyte-richmicroenvironments. Cancer cells are known to stimulate lipolysis of adipocytes, leading todelipidation and acquisition of a fibroblast-like phenotype in adipocytes. This transition of
adipocytes to CAFs are accompanied by loss of expression of adipocyte terminal

differentiation markers and are associated with functional changes in the cells, which
support aggressive phenotypes of tumors. However, the molecular mechanism of such
adipocyte-CAF transition remains to be elucidated.

We previously reported that inactivation of MKL1, a monomeric G-actin bindingtranscriptional coactivator, via actin cytoskeleton dynamics drives adipocyte differentiationof multipotent fibroblastic preadipocytes (Nobusue et al., 2014). In addition, we found thatthe expression of ACTA2 which encodes α-smooth muscle actin is induced by MKL1

activation and is required for the maintenance of the fibroblastic phenotype of
preadipocytes. We here investigate the role of MKL1 in the adipocyte-CAF transition. By
using Tet-On system, we induced expression of MKL1 in matured adipocytes and foundthat they lost the expression of adipocyte differentiation markers and became expandable

multipotent fibroblastic progenitor cells both in vitro and in vivo. In addition, withdrawal

68

H. Sayaof doxycycline to suppress the expression of MKL1 did not cause a reversal of the
fibroblastic phenotypes, suggesting that MKL1 is a factor for reprogramming adipocytes

into a fibroblastic progenitor state. We also demonstrated that MKL1 induction reprograms
matured chondrocytes into fibroblastic progenitors.

Given that inactivation of MKL1 potentially suppresses the adipocyte-CAF transition intumor tissue, we investigated the effect of Rho-kinase inhibitors which prevents nuclear

translocation of MKL1 on the tumor formation in mouse osteosarcoma inoculation model(Shimizu et al., 2010). Blocking the adipocyte-CAF transition or promoting adipocyte

differentiation in CAFs may provide a novel therapeutic approach for modulating tumor
microenvironment.

Figure 1

Cancer-associated fibroblasts (CAFs) are converted from various cells. CAFs are plastic
and activated fibroblastic progenitor cells characterized by α-SMA expression. Adipocytes
and the cancer-associated adipocytes (CAAs) are considered to be major sources of CAFs.

Heterogeneity and Plasticity of Cancer

69

References
1. Nobusue H, Onishi N, Shimizu T, Sugihara E, Oki Y, Sumikawa Y, Chiyoda T, Akashi K,
Saya H and Kano K: Regulation of MKL1 via actin cytoskeleton dynamics drives
adipocyte differentiation. Nat Commun 5: 3368, 2014
2. Shimizu T, Ishikawa T, Sugihara E, Kuninaka S, Miyamoto T, Mabuchi Y, Matsuzaki Y,
Tsunoda T, Miya F, Morioka H, Nakayama R, Kobayashi E, Toyama Y, Kawai A,
Ichikawa H, Hasegawa T, Okada S, Ito T, Ikeda Y, Suda T, and Saya H: c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma
accompanied by loss of adipogenesis. Oncogene 29: 5687-5699, 2010

Hideyuki Saya, MD, PhD

1987-1988
1988-1994
1994-2006

2007-present
2015-present

Postdoctoral Fellow, Brain Tumor Research Center,
UCSF
Assistant Professor, Department of Neuro-Oncology
MD Anderson Cancer Center
Professor and Chairman, Department of Tumor
Genetics and Biology, Kumamoto University School of
Medicine
Professor, Institute for Advanced Medical Research
Keio University School of Medicine
Director, Clinical and Translational Research Center
Vice President, Keio University Hospital

70

ROLE OF THE MICROENVIRONMENT IN CANCERINVASION AND THERAPY RESPONSE

Esther Arwert, Eishu Hirata, Anna Labernadie, Takuya Kato, Emma Milford,
Stefanie Derzsi, Cedric Gaggioli, Xavi Trepat and Erik Sahai
Tumour Cell Biology Laboratory
The Francis Crick Institute
1 Midland Road, London NW1 1AT, UK(erik.sahai@crick.ac.uk)

The two most lethal aspects of cancer are its ability to invade into the surrounding tissue

and its resistance to chemo- and radio-therapies. We are interested in understanding how

communication between cancer cells and stromal cells contributes to these phenotypes. In
particular, we will present data on the mechanism of cooperation between cancer cells andstromal fibroblasts (also known as Cancer-Associated Fibroblasts - CAFs). Many studies

have identified roles for soluble factors, such as HGF and SDF-1, and exosomes, in cancer

cell – CAF crosstalk, but we have focused on the role of the extracellular matrix (ECM) anddirect contact in tumour – stroma communication.

Imaging of collectively invading co-cultures of carcinoma cells and patient derived

stromal fibroblasts reveals that the leading cell is always a fibroblast, and that carcinoma

cells move within tracks in the extracellular matrix behind the fibroblast. By using spheroid
and organotypic invasion assays, we show that the generation of these tracks by fibroblasts
is sufficient to enable the collective invasion of the squamous cell carcinoma (SCC) cells andrequires both protease- and force-mediated matrix remodeling [1]. This study emphasizes
the importance of matrix remodeling by stromal fibroblasts for the promotion of local
invasion.

Many tumors show an initial response to targeted therapies before genetic resistanceemerges, however little is known about how tumor cells tolerate therapy before genetic

resistance dominates. Building on our knowledge of how fibroblasts can remodel the ECM
to promote invasion, we show how the ECM can also generate a ‘safe haven’ in whichmelanoma cells can tolerate targeted therapy [2]. The Ras-MAPK pathway is mutated in
the majority of melanoma, with BRAF mutations being most common. Tumors with BRAF

Novel Approaches to Analyze Cancer Network

71

mutations show an initial response to BRAF inhibitors before genetic resistance emerges.
However little is known about the spatio-temporal response to BRAF inhibitors in vivo andhow this relates to the failure of targeted therapy. We have used both intravial ratiometic

FRET and FLIM of an ERK/MAP kinase biosensor to investigate heterogeneity in signaling
in both murine syngeneic and human melanoma models. We have longitudinallymonitored responses to targeted therapy and identified areas that become refractory to

drug action. BRAF mutant melanoma cells can rapidly become tolerant to PLX4720 in areasof high stroma. The rapid kinetics of this process indicate that it is not caused by genetic

events. We demonstrate that PLX4720 has an unexpected effect on the melanomaassociated fibroblasts, leading to enhanced matrix remodeling. The remodeled matrix thenprovides signals that enable melanoma cells to tolerate PLX4720. Blocking these signals

using a FAK inhibitor greatly enhances the efficacy of BRAF inhibition in several preclinical models. We propose that this safe haven enhances the population of cancer cells

from which genetically resistance emerges by supporting a population of cancer cells fromwhich genetically resistance emerges.

We have recently uncovered a novel mechanism of tumor – stroma cross-talk involving

a pathological heterotypic cell-cell contact. Our early imaging studies had revealed a close
physical association between fibroblasts and cancer cells [1]. This was intriguing because innormal skin, epithelial cells and fibroblasts do not contact because they are separated by abasement membrane. However, a signature feature of tissue damage and invasivesquamous cell carcinoma is breakdown of the basement membrane; in this context,

epithelial cells and fibroblasts can contact each other. In collaboration with Xavi Trepat inBarcelona, we now show that CAFs exert a physical force on cancer cells that enables theircollective invasion. Force transmission is mediated by a heterophilic adhesion involving Ncadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesionis mechanically active; when subjected to force it triggers β-catenin recruitment andadhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-

cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell

migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the

CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer

cell/CAF interface and CAF repolarization is afadin dependent. Furthermore, heterotypicjunctions between CAFs and cancer cells are observed in squamous cell carcinoma, but notadjacent normal mucosa. Taken together, these findings show that a mechanically active

heterophilic adhesion between CAFs and cancer cells enables cooperative tumor invasion[3].

We have now extended our analysis of heterotypic cancer cell – CAF junctions toinvestigate downstream changes in gene expression. This has revealed that heterotypic

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E. Sahai et al.

contact triggers dramatic changes in chemokine, cytokine, and other inflammatory
modulators triggering anti-microbial and anti-viral responses. We propose that thisrepresents a tissue level damage sensing mechanism analogous to molecular DAMPs. We
speculate that heterotypic epithelial – fibroblast contact originally evolved to promotewound healing and prophylactically trigger host defense to infection, but that these become
co-opted by cancer enabling invasion and modulating therapy responses.
References

1. Fibroblast-led collective invasion of carcinoma cells with differing roles for
RhoGTPases in leading and following cells. Gaggioli C, Hooper S, et al., Nat Cell Biol 2007
2. Intravital imaging reveals how BRAF inhibition generates drug-tolerant microenvironments with high integrin β1/FAK signaling. Hirata E, Girotti MR, et al., Cancer Cell. 2015
3. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts todrive cancer cell invasion. Labernadie A, Kato T, et al., Nat Cell Biol. 2017

Erik Sahai, PhD

1998
2003
2004

2015-present

PhD in Biochemistry, University College London
Postdoctoral Fellow, Institute of Cancer Research
Postdoctoral Fellow, Albert Einstein College ofMedicine
Established lab at the London Research Institute,
Cancer Research UK
Group Leader, the Francis Crick Institute

73

CHALLENGES FOR COMPLEXITY OF CANCER BY
SUPERCOMPUTER – FROM GENOME TO NETWORK

Satoru Miyano

Human Genome Center
The Institute of Medical Science, The University of Tokyo
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan(miyano@ims.u-tokyo.ac.jp)

The drastic cost down of sequencing has been accelerating the world-wide productionof cancer omics data, e.g. TCGA (The Cancer Genome Atlas), ICGC (International Cancer
Genome Consortium). It is now usual to sequence and analyze several hundreds ofsamples for one study. This situation made us face with cancer big data whose analysis
should require large-scale computational resources (high performance computing, high-speed massive storage, data transfer, etc.) and new big data analysis methodologies. For
example, US NIH “The Big Data to Knowledge Program” (B2K) and NCI “High

Performance Computing Program” have been building the infrastructure and services forbiomedical big data. This is a common public policy in leading countries.

In Japan, we have been using two supercomputer systems for cancer research. One is

the supercomputer SHIROKANE at Human Genome Center of University of Tokyo(http://hgc.jp/english/). It is designed for life science applications, especially, sequence

data analysis. The total computation power at peak is 550TFLOPS generated by two typesof computation nodes (thin node: 5GB memory/core, fat not: 2TB memory/node). The
30PB Lustre File System enables fast data analysis for big data. It processes 4 million

jobs/month. The other is “K computer” at RIKEN Advanced Institute of Computational

Science (http://www.riken.jp/en/research/labs/aics/). Its computing power 10PFLOPS isgenerated by 705,024 CPU cores on 88,128 nodes that are interconnected as a torus fusion
structure (similar to 3D Möbius band). The combined use of these two supercomputerscreates an extraordinarily super computational platform for cancer omics data analysis.For
these supercomputers, we developed and implemented various computational methods forCancer Systems Biology from genome to network.

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S. Miyano

The first is genome data analysis. There are several well-established variant calling

pipelines, e.g. GATK (https://software.broadinstitute.org/gatk/), the EMBL workflow forstructural variation, the DKFZ workflow for SNVs, indels, and copy number(https://github.com/ICGC-TCGA-PanCancer/DEWrapperWorkflow), etc. Independentlyof these pipelines, our open source pipeline Genomon (https://github.com/Genomon-

Project/) has been developed in collaboration with Dr. Seishi Ogawa (currently, Kyoto
University School of Medicine) for blood cancer. It is realized as a suite of bioinformatics
tools for analyzing cancer genome data (WGS, WES, RNA-seq). It enables us to perform

very sensitive and accurate detection of most types of genomic variants (single nucleotide
variants, short indels, mid-size indels and large scale structural variations), andtranscriptomic changes (gene fusions, aberrant splicing patterns). It adopts an efficient job
scheduling framework that realizes easy analysis of several hundreds of genome andtranscriptome sequencing data simultaneously. The first contribution of Genomon was thediscovery of aberrations of splicing machinery in myelodysplasia [1]. Then several tens ofcontributions came out by Genomon pipeline using supercomputers, including clonal

hematopoiesis in aplastic anemia [2]. Detection of ITD (internal tandem duplication) is also
in the scope of Genomon [3]. One of recent advancements of Genomon is the ability ofaccurate detection of mid-size (10bp-1000bp) indels which is considered as a “blind spot” of
Illumina short read sequencing. It played an essential role in the study of adult T cellleukemia/lymphoma (ATL) that was directed by Dr. Seishi Ogawa [4]. 426 ATL patient

samples were analyzed (48 WGS, 81 WES, 57 RNA-seq, 109 methylation analyses, 343

samples for validation) and the study revealed comprehensive understanding how/where
HTLV-1 virus damages human genomes and leads to ATL. K computer helped WGS data
analysis and SHIROKANE did other data analysis. Major driver alterations are found in

TCR/NFκ-B pathway, T cell trafficking and other T cell-related pathways as well as
immunosurveillance. Within them, Genomon found 2,857 structural variations from 48
WGS sequence data and recurrent intragenic deletions causing exon skipping in IKZF2,

CARD11, TP73. Notably, Genomon identified structural variations (SVs) in the 3’UTR

region of PD-L1 for 27% WGS samples that is located at the immune checkpoint. Gene

expressions of the samples with these SVs were found high. Then, 10,210 samples (mostly
RNA-seq data) of 33 tumor types from TCGA were analyzed with Genomon on

SHIROKANE. The data analysis and human investigation revealed SVs in the 3’UTR regionof PD-L1 for 12 tumor types. Through biological validations including genome-editing, aunique genetic mechanism of immune escape caused by SVs commonly disrupting the
3’URT region of PD-L1 was discovered [5].

The second is computational strategy for unraveling gene networks and their diversity

lying over genetic variations, mutations, environments and diseases from gene expression
profiles of cancer cells. We developed methods for exhibiting how gene networks varyNovel Approaches to Analyze Cancer Network

75

from patient to patient according to a modulator, which is any score representing

characteristics of cells, e.g. survival, drug resistance [5,6]. SiGN-L1 (http://sign.hgc.jp/
signl1/) is network estimation software using sparse learning. It uses L1-regularization forsimultaneous parameter estimation and model selection of statistical graphical models suchas graphical Gaussian models and vector autoregressive models. SiGN-L1 also implements

the NetworkProfiler [5] that realizes the analysis of individual differences of gene networks
with respect to the extra individual index called modulator. Network analysis with
NetworkProfiler revealed system changes in epithelial-mesenchymal transition and KLF5

(Krueppel-like factor 5) is identified as a new regulator of EMT (validated by Dr. Takashi
Takahashi, Nagoya University). A further advancement was done by employing adaptive

penalized logistic regression with which drug-sensitivity prediction was drastically
improved [6]. We also developed a microRNA/mRNA gene network analysis with

Bayesian network method that revealed subnetworks with hub genes (miR-30d, miR-195,

TTF-1/NKX2-1, etc.) that switch cancer survival from 126 non-small cell lung cancer
samples. We implemented the method as a software SiGN-BN that is gene network

estimation software using the Bayesian network model and nonparametric regression. It iscapable of estimating regulatory dependencies between genes as gene networks fromexpression data such as individual cell samples, gene knocked-down cell samples, drug-

stimulated time series (time course) samples, and so on. SiGN series are implemented bothon SHIROKANE and K computer. On-going gene network research is also presented,
including a discovery of the first lncRNA, named MYMLR, modulating MYC gene forselective target regulation using K computer (validated by Dr. Takashi Takahashi, Nagoya
University School of Medicine). This study analyzed 7988 gene networks for each

“modulator” non-coding RNA lung cancer samples from 40 studies. The computational

methodology named GIMLET for this study will be published soon [8]. Before K computer,we could not even imagine such large-scale network analysis.
References
1. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, Sato Y, SatoOtsubo A, Kon A, Nagasaki M, Chalkidis G, Suzuki Y, Shiosaka M, Kawahata R,
Yamaguchi T, Otsu M, Obara N, Sakata-Yanagimoto M, Ishiyama K, Mori H, Nolte F,
Hofmann WK, Miyawaki S, Sugano S, Haferlach C, Koeffler HP, Shih LY, Haferlach T,
Chiba S, Nakauchi H, Miyano S, Ogawa S. Frequent pathway mutations of splicing
machinery in myelodysplasia. Nature. 2011; 478(7367):64-69.
2. Yoshizato T, Dumitriu B, Hosokawa K, Makishima H, Yoshida K, Townsley D, SatoOtsubo A, Sato Y, Liu D, Suzuki H, Wu CO, Shiraishi Y, Clemente MJ, Kataoka K,
Shiozawa Y, Okuno Y, Chiba K, Tanaka H, Nagata Y, Katagiri T, Kon A, Sanada M,
Scheinberg P, Miyano S, Maciejewski JP, Nakao S, Young NS, Ogawa S. Somatic mutations and clonal hematopoiesis in aplastic anemia. N Engl J Med. 2015; 373(1):35-47.

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S. Miyano

3. Chiba K, Shiraishi Y, Nagata Y, Yoshida K, Imoto S, Ogawa S, Miyano S. Genomon
ITDetector: a tool for somatic internal tandem duplication detection from cancer
genome sequencing data. Bioinformatics. 2015;31(1):116-118.
4. Kataoka K, Shiraishi Y, Takeda Y, Sakata S, Matsumoto M, Nagano S, Maeda T, Nagata Y,
Kitanaka A, Mizuno S, Tanaka H, Chiba K, Ito S, Watatani Y, Kakiuchi N, Suzuki H,
Yoshizato T, Yoshida K, Sanada M, Itonaga H, Imaizumi Y, Totoki Y, Munakata W,
Nakamura H, Hama N, Shide K, Kubuki Y, Hidaka T, Kameda T, Masuda K, Minato N,
Kashiwase K, Izutsu K, Takaori-Kondo A, Miyazaki Y, Takahashi S, Shibata T,
Kawamoto H, Akatsuka Y, Shimoda K, Takeuchi K, Seya T, Miyano S, Ogawa S.
Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers. Nature.
2016; 534(7607):402-406.
5. Shimamura T, Imoto S, Shimada Y, Hosono Y, Niida A, Nagasaki M, Yamaguchi R,
Takahashi T, Miyano S. A novel network profiling analysis reveals system changes inepithelial-mesenchymal transition. PLoS One. 2011; 6(6): e20804.
6. Park H, Shiraishi Y, Imoto S, Miyano S. A novel adaptive penalized logistic regression for
uncovering biomarker associated with anti-cancer drug sensitivity. IEEE/ACM Trans
Comput Biol Bioinform. 2017; 14(4):771-782.
7. Arima C, Kajino T, Tamada Y, Imoto S, Shimada Y, Nakatochi M, Suzuki M, Isomura H,
Yatabe Y, Yamaguchi T, Yanagisawa K, Miyano S, Takahashi T. Lung adenocarcinoma
subtypes definable by lung development-related miRNA expression profiles in association with clinicopathologic features. Carcinogenesis. 2014; 35(10): 2224-2231.
8. Shimamura T, Matsui Y, Kajino T, Ito S, Takahashi T and Miyano S. GIMLET:
Identifying biological modulators in context-specific gene regulation using local energy
statistics. In press.

Satoru Miyano, PhD

1979-1981
1981-1985
1985-1987
1987-1993
1993-1996

1996-present
2014-present

Assistant Professor, Research Institute of Fundamental
Information Science, Kyushu University
Assistant Professor, Department of Mathematics,
Kyushu University
Postdoctoral Fellow, Alexander von Humboldt
Foundation, FB Informatik, Universität-GH Paderborn,
W. Germany
Associate Professor, Research Institute of Fundamental
Information Science, Kyushu University
Professor, Research Institute of Fundamental
Information Science, Kyushu University
Professor, Laboratory of DNA Information Science,
Human Genome Center, The Institute of Medical
Science, The University of Tokyo
Director, Human Genome Center, The Institute ofMedical Science, The University of Tokyo

77

PATHOLOGY FROM THE MOLECULAR SCALE ON UP

Garry P. Nolan

Department of Microbiology & Immunology
Baxter Laboratory for Stem Cell Biology
Stanford University School of Medicine, Stanford, CA 94305, USA(gnolan@stanford.edu)

High parameter single cell analysis has driven deep understanding of immune

processes. Using a next-generation single-cell “mass cytometry” platform we quantify
surface and cytokine or drug responsive indices of kinase target with 45 or more parameter

analysis (e.g. 45 antibodies, viability, nucleic acid content, and relative cell size). Similarly,
we have developed two advanced technologies that enable deep phenotyping of solid
tissue in both fresh frozen and FFPE formats (50 – 100 markers).

We have recently

extended this parameterization to mRNA with the capability to measure down to 5
molecules per cell in combination with any other set of previously created markers.

I will present evidence of deep internal order in immune functionality demonstratingthat differentiation and immune activities have evolved with a definable “shape”. This

shape is altered during immune surveillance and “imprinted” during, and after, pathogen

attack, traumatic injury, or auto-immune disease. Hierarchies of functionally defined trans-

cellular modules are observed that can be used for mechanistic and clinical insights. I will
focus upon pathogen attack, traumatic surgical intervention in human, and auto-immune
processes for the presentation.

78

G. P. Nolan

Garry P. Nolan, PhD

1983
1989
1990
1993

1993-1999
1995-1999
1999-20092009-present
2011-present

BS, Cornell University, Biology/Genetics
PhD, Stanford University, Genetics
Postdoctoral Fellowship, Stanford University, Genetics
Postdoctoral Fellowship, MIT, David Baltimore
Laboratory, Biochemistry
Assistant Professor, Department of Molecular
Pharmacology, Stanford University School of Medicine
Assistant Professor, Joint Appointment, Department ofMicrobiology and Immunology, Stanford University
School of Medicine
Associate Professor (Tenure), Department of Molecular
Pharmacology, Stanford University School of Medicine
Professor (Tenure), Department of Microbiology &Immunology, Stanford University School of Medicine
Rachford and Carlota A. Harris Professor, Departmentof Microbiology and Immunology, Stanford University
School of Medicine

79

MICROBIOME, METABOLISM AND MUCOSAL IMMUNITY

Ramnik J. Xavier

Harvard University and Broad Institute(xavier@molbio.mgh.harvard.edu)

Complex and dynamic interactions exist between the gut microbiome, metabolism, andmucosal immunity to maintain intestinal health. Sufficient perturbations to theseinteractions contribute to inflammatory and autoimmune diseases. Environmental

conditions and diets, for instance, that accompany western lifestyles profoundly impact thecomposition and function of the gut microbiome, which may lead to breaks in immune

tolerance and a “broken circuit” of inflammation in genetically susceptible individuals. The
consequences of changes in the gut microbiome are dictated by the mechanisms that sense

and respond to dietary and microbial signals. We are utilizing several novel approaches to
examine interactions between host and microbes with the goal of understanding themechanisms by which microbes and their metabolites contribute to mucosal homeostasis

and intestinal immunity. Here, we focus on examples of how chemical, human genetic,
multi’omic, and computational approaches have contributed to this goal.

We and others characterized short-chain fatty acid (SCFA) sensing and signaling in theintestine [1-2]. Produced by microbial metabolism of dietary fiber, SCFAs are sensed by the
G-protein coupled receptor GPR43 and signal to regulate immune responses and protect

against inflammation. Butyrate, but not other SCFAs, was shown to inhibit intestinal stem
cell proliferation and inflammation. Butyrate is further metabolized by host cells, raising

the possibility that a butyrate byproduct was the signaling molecule. Using stem cellproliferation and cytokine production as function assays, we developed a chemical

screening approach to test the activity of structurally similar molecules [3]. Our results

suggested that butyrate, and not predicted byproducts of butyrate metabolism, is directly
involved in the mechanism inhibiting stem cell proliferation and inflammation.

80

R. J. Xavier

Employing a longitudinal study of human cohorts, we recently tested the hygiene

hypothesis, which posits that changes in early microbial exposure lead to altered immune
maturation and increased incidence of autoimmune diseases [4]. The incidences of type 1

diabetes and allergy are much higher in Finland and Estonia than in neighboring Russia.

Taking advantage of this “living laboratory”, the DIABIMMUNE project collected clinicalmetadata, serum, and monthly stool samples from birth to age 3 of 1,000 children born inthese three countries. While the children in the cohort were genetically similar, they were
exposed to vastly different living environments. We analyzed microbiome samples to

identify early-life environmental triggers of autoimmune and allergic diseases. Our study

found that in the first year of life, Bacteroidetes were more common in microbiomes of

Finnish and Estonian children, while Actinobacteria were highly abundant in those of
Russian children. We determined that the largest functional differences between

microbiomes of Finnish and Russian children were in lipopolysaccharide (LPS)

biosynthesis pathways. We further demonstrated that E. coli was the major contributor tolipid A biosynthesis in all three countries. In Finland and Estonia, Bacteroidetes were also
large contributors. Next, we compared the immunogenicity of LPS from these species,

finding that LPS from Bacteroidetes is less immunostimulatory compared to LPS from E.

coli. Structural changes in LPS were shown to influence immunogenicity, suggesting thatthe more potent E. coli LPS contributes to immune education and decreased risk of
autoimmune disease in Russian children.

Because IBD is influenced by the composition of and interactions between microbes, theintestinal epithelium, and the immune system, we use multi’omics approaches to integrate

multiple factors that contribute to disease [5]. We are part of the group leading the second
phase of the Human Microbiome Project (HMP2), an IBD data resource that collects avariety of host and microbial multi’omics data on stool, biopsy, and serum samples from

IBD and non-IBD control subjects. By comparing metagenomes and metatranscriptomes,
we showed that on average, the functional potential of a microbial species is proportional

to its functional activity (that is, DNA levels generally correlate to RNA levels) and that
more abundant microbes show a higher DNA to RNA correlation. For example, P. merdae

was the species with the highest correlation and was detected across Crohn’s disease (CD),ulcerative colitis (UC), and non-IBD samples. This indicated that relative abundance of P.

merdae coincided with increased transcriptional contributions from this species. D. invisus,

an oral bacterium with similar relative abundance as P. merdae, showed the lowest DNA to
RNA correlation, suggesting D. invisus is not actively transcribing in the gastrointestinal

tract. HMP2 data also allows us to contrast metabolic function potential and activity. We

identified specialized pathways that are contributed by specific microbes in the contexts of
IBD and non-IBD. F. prausnitzii almost entirely contributed to the super-pathway ofhexuronide and hexuronate degradation across samples. Importantly, metatranscriptomics

Immune Systems and Cancer Microenvironment

81

allows for the identification of disease-specific changes in microbial transcriptomes. For

instance, the B. vulgatus L-lysine biosynthesis VI pathway was more abundant on thetranscriptional level in IBD samples than non-IBD samples. Our data highlight theimportance of analyzing both the abundances and activities of gut microbes throughmetatranscriptomics. Similar analyses of metabolomics and metagenomics reveal microbial

metabolites associated with disease. We identified classes of microbial metabolites that are
differentially abundant in IBD and correlated this with species relative abundances.

Finally, we use computational approaches to predict microbial function in maintaining

intestinal homeostasis. For example, we hypothesized that identifying gut microbes

genetically capable of utilizing mucins for energy might provide insight into whichmicrobes are critical to maintain intestinal homeostasis [6]. Computational analyses

identified mucin-utilization genes in metagenomics samples from HMP subjects. These

species were shown to robustly utilize mucin as an energy source in functional assays. We
demonstrated that one of these species, Peptostreptococcus russellii, protects against DSS-

induced colitis in vivo. Several Peptostreptococcus species contain a gene cluster that allows
the microbes to metabolize tryptophan into indoleacrylic acid (IA). We showed that IA

promotes intestinal epithelial barrier function and anti-inflammatory responses. A

diminished ability of gut microbes to produce IA was found in stool samples from IBD
subjects.

We are using these novel approaches to ultimately address challenges in IBD treatment.

Currently, anti-integrin therapies produce suboptimal patient responses. In a prospective
study with CD and UC patients receiving anti-integrin therapies, we associated specificmicrobes and their functions with clinical remission [7]. Seven bacteria, including R.

inulinivorans and a Burkholderiales species, and clinical factors such as smoking status werepredictive of outcome. This study demonstrated that microbiome changes occurring earlyin disease course predict response to treatment.

References
1. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS,
Mackay F, Artis D, Xavier RJ, Teixeira MM, Mackay CR. Regulation of inflammatory
responses by gut microbiota and chemoattractant receptor GPR43. Nature.
2009;461(7268):1282-6. PMID: 19865172.
2. Kaiko GE, Ryu SH, Koues OI, Collins PL, Solnica-Krezel L, Pearce EJ, Pearce EL, Oltz EM,
Stappenbeck TS. The Colonic Crypt Protects Stem Cells from Microbiota-Derived
Metabolites. Cell. 2016;167(4):1137. PMID: 27814510.
3. Verma MS, Fink, MJ, Salmon GL, Fornelos N, Ohara TE, Ryu SS, Vlamakis H, Xavier RJ,
Stappenbeck TS, Whitesides GM. A common mechanism links activities of butyrate in thecolon. ChemRxiv. 2017. https://doi.org/10.26434/chemrxiv.5414065.v1

82

R. J. Xavier

4. Vatanen T, Kostic AD, d’Hennezel E, Siljander H, Franzosa E, Yassour M, Kolde R,
Vlamakis H, Arthur TD, Hämäläinen AM, Peet A, Tillmann V, Uibo R, Mokurov S,
Dorshakova N, Ilonen J, Virtanen SM, Szabo SJ, Porter JA, Lähdesmäki H, Huttenhower
C, Gevers D, Cullen TW, Knip M, DIABIMMUNE Study Group, Xavier RJ. Variation in
Microbiome LPS Immunogenicity Contributes to Autoimmunity in Humans. Cell.
2016;165(4):842-53. PMID: 27133167.
5. Schirmer M, Franzosa EA, Lloyd-Price J, McIver LJ, Schwager R, Poon TW,
Ananthakrishnan AN, Andrews E, Barron G, Lake K, Prasad M, Sauk J, Stevens B,
Wilson RG, Braun J, Denson LA, Kugathasan S, McGovern DPB, Vlamakis H, Xavier RJ,
Huttenhower C. Dynamics of metatranscription in the inflammatory bowel disease gut
microbiome. Nat Microbiol. 2018. doi: 10.1038/s41564-017-0089-z. PMID: 29311644.
6. Wlodarska M, Luo C, Kolde R, d'Hennezel E, Annand JW, Heim CE, Krastel P, Schmitt
EK, Omar AS, Creasey EA, Garner AL, Mohammadi S, O'Connell DJ, Abubucker S,
Arthur TD, Franzosa EA, Huttenhower C, Murphy LO, Haiser HJ, Vlamakis H, Porter
JA, Xavier RJ. Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species
Suppresses Inflammation. Cell Host Microbe. 2017;22(1):25-37.e6. PMID: 28704649.
7. Ananthakrishnan AN, Luo C, Yajnik V, Khalili H, Garber JJ, Stevens BW, Cleland T,
Xavier RJ. Gut Microbiome Function Predicts Response to Anti-integrin Biologic
Therapy in Inflammatory Bowel Diseases. Cell Host Microbe. 2017;21(5):603-610.e3.
PMID: 28494241.

Ramnik J. Xavier, MD

2008-present
2010-present
2010-present
2010-present
2014-present
2014-present

Director, Center for the Study of Inflammatory Bowel
DiseasePhysician, Massachusetts General Hospital
Chief, Gastrointestinal Unit, Massachusetts General
Hospital
Kurt Isselbacher Chair in Medicine, Harvard Medical
SchoolInstitute Member, Broad Institute of Harvard & MIT
Co-Director, Center for Microbiome Informatics andTherapeutics, MIT83

IMMUNOGENOMICS IN THE CANCER PRECISION
MEDICINE ERA; CHARACTERIZATION OF T CELLREPERTOIRES IN TUMOR IMMUNE MICROENVIRONMENTBY TCR ANALYSIS
Yusuke Nakamura

Section of Hematology/Oncology
Department of Medicine and Surgery
University of Chicago
th900E 57 Street, KCBD 6130, Chicago, IL 60637, USA(ynakamura@bsd.uchicago.edu)
The recent development of high-throughput DNA sequencing technologies has driven

the rapid progress in genomics research and enabled us to address a multitude of biologicaland pathological conditions, such as cancer, autoimmune diseases, and drug-induced
toxicities. In addition, the development of cancer immunotherapies, particularly drugs
modulating the immune checkpoint molecules, clearly demonstrated the importance of

host immune cells in the fight against cancer. However, the molecular mechanisms bywhich these new therapies kill tumor cells still remain unclear. Hence, we have been
proposing the significance of the new filed “ immunopharmacogenomiccs” tocharacterizing the systemic and tumor immune environment including T cell and B cell
repertoires by next-generation sequencers. This field has been enormously helpful for
better understanding of the changes/alterations of our immune responses during thecourse of various disease conditions and treatments. I here report the deep sequencing of Tcell receptors that enabled us to capture the immune microenvironment in tumors.

We have analyzed T cell changes in melanoma tumors before and after anti-PD-1

antibody treatment and found that tumors in responders exhibited a substantial increase ofCD8, GZMA and perforin 1 (PRF1) expression levels as well as oligoclonal expansion oftumor-infiltrating T lymphocytes (TILs) in the tumor tissues of the responders. In one

patient who showed myocarditis, myositis, and myasthenic crisis after one-shot of anti-PD-

1 antibody treatment, we identified infiltration of clonally expanded T cell populations inthe skeletal muscle, implying the very strong T cell immune response against muscular
cells.

84

Y. Nakamura

Secondly we analyzed tumor microenvironment in 22 kidney cancer tissues before andat 3 months after cryoablation. We found that proportions of abundant TCRB clonotypes(defined as clonotypes with ≥ 1% frequency among total TCRB reads) were significantly
increased in the post-cryoablation tissue samples than those of pre-cryoablation tumor

samples. Expression analysis of immune-related genes showed significantly elevatedtranscriptional levels of CD8, CD4, Granzyme A (GZMA), and CD11c along with highCD8/FOXP3 ratio in the post-cryoablation tissue samples, suggesting that cryoablation

could induce strong immune reactions in tumors with oligoclonal expansion of anti-tumor
T cells, which probably circulate systemically.

Furthermore, we collected surgically-resected tumor tissues from five breast cancer andsix mesothelioma patients, and characterized 3 different portions of individual tumors
through somatic mutation analysis by whole exome sequencing, T cell receptor beta (TCRB)
repertoire analysis of tumor-infiltrating lymphocytes (TILs), and found that tumors
revealed significant correlation between the number of predicted neoantigens and thediversity of TILs (P = 0.0009), suggesting that certain TILs might recognize the cancer-

specific antigens including neoantigens derived from non-synonymous somatic mutationsof cancer cells.

In addition, to investigate the similarity/difference in TILs in original tumor tissues andthose of in vitro expanded populations in squamous cell carcinoma of head and neck
(SCCHN), we performed whole exome analysis, expression profile analysis of immune-

related genes, and T cell receptor (TCR) analysis for TILs in original tumors as well as invitro expended TILs in surgically-resected HPV-negative fresh tumors with SCCHN. Threeof eight SCCHN tumors we examined were found to have an unusually high number of
non-synonymous somatic mutations (900-4,000 mutations) probably due to mutations in

mismatch repair genes, MSH2 or MSH4, or a DNA polymerase gene, POLE. Highly
frequent TCR clonotypes in expended CD8+ TILs from these three tumors revealed highsimilarity to those in original tumors. However, for remaining tumors with the lower
mutational load, T cell clonotypes between TILs in original tumor tissues and those

expanded in vitro were almost entirely different. Our findings might provide clinicallyuseful information for identification of tumor-antigen-specific T cell clones.

This kind of immunogenomics analysis is extremely important to uncover the changes

in immune microenvironment during the cancer treatment and may improve the clinicaloutcome of immunotherapy including development of TCR-engineered T cell therapy.

Immune Systems and Cancer Microenvironment

85

References
1. Choudhury NJ, Kiyotani K, Yap KL, Campanile A, Antic T, Yew PT, Steinberg G, Park JH,
Nakamura Y, O’Donnell PH: Low T cell receptor diversity, high somatic mutation burden,
and high neoantigen load as predictors of clinical outcome in muscle-invasive bladdercancer. European Urology Focus, doi:10.1016/j.euf.2015.09.007, 2015
2. Kimura T, Fukushima S, Miyashita A, Aoi J, Jinnin M, Kosaka T, Matsukawa M, Inoue H,
Kiyotani K, Park JH, Nakamura Y, Ihn H: Myasthenic crisis and polymyositis induced by
one dose of nivolumab. Cancer Science, 107:1055–1058, 2016
3. Inoue H, Park JH, Kiyotani K, Zewde M, Miyashita A, Masatoshi J, Kiniwa Y, Okuyama
R, Tanaka R, Fujisawa Y, Kato H, Morita A, Asai J, Katoh N, Yokota K, Akiyama M, Ihn
H, Fukushima S, Nakamura Y: Intratumoral expression levels of PD-L1, GZMA, and
HLA-A associated with oligoclonal T cell expansion predict response to nivolumab inmetastatic melanoma. OncoImmunology, doi.org/10.1080/2162402X.2016.1204507,
2016
4. Kato T, Iwasaki T, Uemura M, Nagahara A, Higashihara H, Osuga K, Ikeda Y, Kiyotani K,
Park JH, Nonomura N, Nakamura Y: Characterization of the cryoablation-induced
immune response in kidney cancer. OncoImmunology, doi.org/10.1080/2162402X.
2017.1326441, 2017
5. Kiyotani K, Park JH, Inoue H, Husain A, Olugbile S, Zewde M, Nakamura Y,
Vigneswaran WT: Integrated analysis of somatic mutations and immune microenvironment in malignant pleural mesothelioma. Oncoimmunology, doi.org/10.1080/
2162402X.2016.1278330, 2017
6. Ikeda Y, Kiyotani K, Yew PY, Sato S, Imai Y, Yamaguchi R, Miyano S, Fujiwara,
Hasegawa K, Nakamura Y: Clinical significance of T cell clonality and expression levels of
immune-related genes in endometrial cancer. Oncology Reports, DOI:
10.3892/or.2017.5536, 2017
7. Saloura V, Fatima A, Zewde M, Kiyotani K, Brisson R, Park JH, Ikeda Y, Vougiouklakis T,
Seiwert T, Cipriani N, Lingen M, Vokes E, Nakamura Y: Characterization of the T-cell
receptor repertoire in patients with locoregionally advanced squamous cell carcinoma of
the head and neck. Clinical Cancer Research, DOI: 10.1158/1078-0432.CCR-17-0103,
2017

86

Y. Nakamura

Yusuke Nakamura, MD, PhD

1977-1984
1984-1989
1987-1989
1989-1994
1995-20122005-20112011

2012-present

Resident, Second Department of Surgery,
Osaka University School of Medicine
Research Associate, HHMI, University of Utah
Research Assistant Professor, Department of Human
Genetics, University of Utah
Laboratory Head, Biochemistry Department,
Cancer Institute, Tokyo
Director, Human Genome Center, Institute of Medical
Science, The University of Tokyo
Director, RIKEN Center for Genomic Medicine
Special Advisor to the Japanese Cabinet and Secretary
General, Office of Medical Innovation,
Cabinet Secretariat, Government of Japan
Professor, Department of Medicine and Surgery,
The University of Chicago

87

AT THE CROSS-ROAD BETWEEN THE GUT MICROBIOME
AND CANCER IMMUNOSURVEILLANCE

Rauber C1,2,3, Routy B1,2,3, Le Chatelier E4, Derosa L1,2,3, Duong CPM1,2,5,
Tidjani Alou M1,2,3, Daillère R1,2,3, Fluckiger A1,2Roberti MP1,2,5,Fidelle M1,3,5, Flament C1,2,5, Poirier-Colame V1,2,5, Opolon P6, Klein C7,
Iribarren K8,9,10,11,12, Mondragón L8,9,10,11,12, Jacquelot N1,2,3, Qu B1,2,3,Ferrere G1,2,3, Clémenson C1,13, Mezquita L1,14, Masip JR1,14, Naltet C15,
Brosseau S15, Kaderbhai C16, Richard C16, Rizvi H17, Levenez F4,Galleron N4, Quinquis B4, Pons N4, Ryffel B18, Minard-Colin V1,19,
Gonin P1,20, Soria JC1,14, Deutsch E1,13, Loriot Y1,3,14, Ghiringhelli F16,
Zalcman G15, Goldwasser F9,21,22, Escudier B1,14,23, Hellmann MD24,25,
Eggermont A1,2,14, Raoult D26, Albiges L1,3,14, Kroemer G27,9,10,11,12,28,29, Zitvogel L30,2,3,5.
Gustave Roussy Cancer Campus (GRCC), Villejuif, France.
Institut National de la Santé et de la Recherche Medicale (INSERM) U1015 andEquipe Labellisée-Ligue Nationale contre le Cancer, Villejuif, France.
3
Univ. Paris-Sud, Université Paris-Saclay, Gustave Roussy, Villejuif, France.
4
MGP MetaGénoPolis, INRA, Université Paris-Saclay, Jouy-en-Josas, France.
5
Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 1428, Villejuif, France.
6
Gustave Roussy, Laboratoire de Pathologie Expérimentale, 94800 Villejuif, France.
7
Centre de Recherche des Cordeliers, INSERM, Université Paris Descartes,
Sorbonne Paris Cité, UMRS 1138, Université Pierre et Marie Curie Université Paris 06,
Sorbonne Universités, Paris, France.
8
Metabolomics and Cell Biology Platforms, GRCC, Villejuif, France.
9
Paris Descartes University, Sorbonne Paris Cité, Paris, France.
10
Equipe 11 Labellisée-Ligue Nationale contre le Cancer,
Centre de Recherche des Cordeliers, Paris, France.
11
INSERM U1138, Paris, France.
12
Université Pierre et Marie Curie, Paris, France.
13
Department of Radiation Oncology, INSERM U1030, and Molecular Radiotherapy,
Gustave Roussy, Université Paris-Saclay, F-94805 Villejuif, France.
1

2

88

C. Rauber et.al.

Department of Medical Oncology, Gustave Roussy, Villejuif, France.
15
Thoracic Oncology Department-CIC1425/CLIP2 Paris-Nord,
Hospital Bichat-Claude Bernard, AP-HP, Université Paris-Diderot, Paris, France.
16
Department of medical oncology, Center GF Leclerc, Dijon, France.
17
Druckenmiller Center for Lung Cancer Research,
Memorial Sloan Kettering Cancer Center, New York, NY, USA.
18
Molecular lmmunology and Embryology, UMR 7355, CNRS,
University of Orleans, Orléans, France.
19
Department of Pediatric Oncology, GRCC, Villejuif, France.
20
Preclinical Research Platform, GRCC, Villejuif, France.
21
Department of Medical Oncology, Cochin Hospital,
Assistance Publique-Hôpitaux de Paris, Paris, France.
14

22

Immunomodulatory Therapies Multidisciplinary Study Group (CERTIM), Paris, France.
23
INSERM U981, GRCC, Villejuif, France.
24
Department of Medicine, Thoracic Oncology Service,
Memorial Sloan Kettering Cancer Center, New York, NY, USA.
25
Department of Medicine, Weill Cornell Medical College, New York, NY, USA.
26
URMITE, Aix Marseille Université, UM63, CNRS 7278, IRD 198, INSERM 1095,
IHU-Méditerranée Infection, 13005 Marseille, France.
27
Metabolomics and Cell Biology Platforms, GRCC, Villejuif, France.
laurence.zitvogel@gustaveroussy.fr kroemer@orange.fr.
28
Pôle de Biologie, Hôpital Européen Georges Pompidou,
Assistance Publique-Hôpitaux de Paris, Paris, France.
29
Department of Women's and Children's Health, Karolinska University Hospital,
17176 Stockholm, Sweden.
30
Gustave Roussy Cancer Campus (GRCC), Villejuif, France.
laurence.zitvogel@gustaveroussy.fr kroemer@orange.fr.

The tumor microenvironment is influenced by anticancer therapies, and even more soby those affecting the gut homeostasis. We reported that a deviated repertoire of theintestinal microbiome called « dysbiosis », caused by broad spectrum antibiotics,

compromised the efficacy of cyclophosphamide (CTX), an immunomodulatory alkylating
agent exerting cytotoxic effects against cancer [1].

Cancer immunotherapy is successful against different hematologic and solid tumor

entities.

Administration of immune checkpoint inhibitors (ICI) unleash T lymphocyte-mediatedimmune responses by suppressing the interaction of T cell inhibitory receptors with theircognate ligands on tumor or stromal cells. The most widely used ICI are monoclonal

Immune Systems and Cancer Microenvironment

89

antibodies (mAb) targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1.
PD-1 blockade is highly efficacious against advanced melanoma, non-small-cell-lungcancer (NSCLC), head neck cancer and renal cell carcinoma (RCC).

Recent work in mice has highlighted the key role of the gut microbiota in mediatingtumor responses to chemotherapeutic agents and immunotherapies targeting PD-L1 or
cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) [2].

Therefore, we explored the possibility that dysbiosis in the context of cancer patients

could influence primary resistance to PD-1 blockade in tumor bearers.

Antibiotics altered the gut microbiome composition, severely compromising survival ofpatients diagnosed with stage IV lung and kidney cancer patients in second line therapy[3]. Metagenomic analyses of fecal samples performed by shot gun DNA sequencing ofpatients stools at diagnosis predicted best clinical response and progression free survival at
3 months to anti-PD-1 mAb in lung cancer patients and best outcome in both cohorts [3].

The commensal bacterium that was most significantly enriched at diagnosis in patients
predisposed to respond was Akkermansia muciniphila, a mucine consuming metagenomic

species associated with lean healthy volunteers and epithelial homeostasis. The fecal
presence of A. muciniphila was detectable in 69% (11/16), versus 34% (15/44) of patients
showing a partial response or primary progression respectively (p=0,007).

Accordingly, preclinical models of dysbiosis induced by broad spectrum ATB in tumor

bearing mice or enforced gavage with non-responding patients’ stools in germ-free miceled to the loss of animal responsiveness to anti-PD1 Ab therapy while administration of

responders feces induced anticancer efficacy of the mAb. Similarly, compensating dysbiotic

feces with A. muciniphila restored the capacity of recipient mice to respond to PD1
blockade [3]. Our data suggest that central memory CD4+ T-cells expressing CCR9 andCXCR3 accumulate 48h after PD-1 blockade in mesenteric lymph nodes and later on intumor beds, accompanied by a loss of Treg infiltration. These antituùor effects of PD-1

blockade in eubiotic animals or in dysbiotic recipients compensated by A. muciniphila
were dependent on IL-12p70, a Th1 cytokine [3].

Taken together our data suggest the importance of a healthy gut in the therapeutic

efficacy of various anticancer therapies including immune checkpoint blockers,

cyclophosphamide or platinum compounds. Restoration of a healthy gut architecture relies
on microbial homeostasis. Modulation of the gut microbiome composition in cancer

patients holds great promise in resetting the cancer-immune set point and circumventing

primary resistance in advanced malignancies. We show compelling preclinical and clinicalevidence to propose fecal microbial transplantation or the use of live biotherapeutics forprospective clinical investigations in oncology [4].

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C. Rauber et.al.

References
1. Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D, Enot DP, Pfirschke C,
Engblom C, Pittet MJ, Schlitzer A, Ginhoux F, Apetoh L, Chachaty E, Woerther P-L,
Eberl G, Bérard M, Ecobichon C, Clermont D, Bizet C, Gaboriau-Routhiau V, CerfBensussan N, Opolon P, Yessaad N, Vivier E, Ryffel B, Elson CO, Doré J, Kroemer G,
Lepage P, Boneca IG, Ghiringhelli F, Zitvogel L. The intestinal microbiota modulates
the anticancer immune effects of cyclophosphamide. Science. 2013 Nov
22;342(6161):971–6.
2. Vétizou M, Pitt JM, Daillère R, Lepage P, Waldschmitt N, Flament C, Rusakiewicz S,
Routy B, Roberti MP, Duong CPM, Poirier-Colame V, Roux A, Becharef S, Formenti S,
Golden E, Cording S, Eberl G, Schlitzer A, Ginhoux F, Mani S, Yamazaki T, Jacquelot N,
Enot DP, Bérard M, Nigou J, Opolon P, Eggermont A, Woerther P-L, Chachaty E,
Chaput N, Robert C, Mateus C, Kroemer G, Raoult D, Boneca IG, Carbonnel F,
Chamaillard M, Zitvogel L. Anticancer immunotherapy by CTLA-4 blockade relies on thegut microbiota. Science. 2015 Nov 27;350(6264):1079–84.
3. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A,
Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V,
Opolon P, Klein C, Iribarren K, Mondragón L, Jacquelot N, Qu B, Ferrere G, Clémenson C,
Mezquita L, Masip JR, Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F,
Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria J-C, Deutsch E,
Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD,
Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L. Gut microbiome influences
efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2017 Nov 2;
4. Zitvogel L, et al Nat Rev Microbiol 2017

Conrad Rauber, MD

2006-20132009-201020102012-20132013– 20162017-present

School of medicine at University of Heidelberg (Human
Medicine)
Clinical Exchange Istanbul/Turkey (Erasmus program)
University of Istanbul, Cerrahpasa Tip Fakültesi
Doctoral Thesis “A Novel TLR7/8 Agonist as Adjuvansin Radiotherapy of Gastrointestinal Tumors”
Clinical Cooperation Unit Radiotherapy DKFZ (Prof.
Huber) and Visceral Surgery (Prof. Büchler)Internship (PJ): Surgery, Internal Medicine,
Neurology in Heidelberg and Straßbourg/France
Research Fellow in Biliary Disease (Prof. Gotthardt)
Biliary Disease in Liver Transplantation
Immunology of Biliary Diseases
PostDoc in Immunotherapy at INSERM U1015(Prof. Zitvogel)
Immunotherapy of Cancer

91

BREAST TUMOR EVOLUTION:HETEROGENEITY AND SELECTION

Kornelia Polyak

Department of Medical Oncology
Dana-Farber Cancer Institute
Harvard Medical School
450 Brookline Ave., Boston, MA 02215, USA(kornelia_polyak@dfci.harvard.edu)
The immune system and the tumor microenvironment play key roles in shaping tumor

evolution. They can promote the selection for subpopulations of cancer cells and eliminate
less fit clones. Immunotherapy is one of the most promising cancer therapies and almost
the only one that can effectively cure even distant metastatic disease in a relatively non-toxic manner. However, its success has been limited to a subset of patients. In breast cancer,
only ~20% of patients with triple negative breast cancer (TNBC) benefit from immune

checkpoint inhibitor (e.g., anti-PD-L1) therapy potentially due to heterogeneity among

tumors for mechanisms by which they escape immune surveillance. Escape from immune

surveillance is a prerequisite for invasive and subsequently metastatic progression. Thus,
we hypothesized that the in situ to invasive carcinoma transition is a critical step for

immune escape in breast cancer and understanding mechanisms of this immune escapewould facilitate the design of more effective immunotherapies for both early and advanced

stage disease. In DCIS (ductal carcinoma in situ) cancer cells are physically separated fromthe stroma by the basement membrane and the myoepithelial cell layer that form the milkducts and intra-epithelial leukocytes are rarely detected [1]. In contrast, in invasive andmetastatic tumors cancer cells and leukocytes are intermingled, thus, only cancer cells thatcan evade the immune system and survive in this environment will play a role in diseaseprogression hence shaping subsequent tumor evolution.

To begin dissecting mechanisms of immune escape during early stages of breast tumor

progression, we have analyzed the composition of leukocytes in normal breast tissues,

DCIS, and invasive ductal breast carcinomas (IDC) of all major breast tumor subtypes (e.g.,luminal, HER2+, and TNBC) by polychromatic FACS. We found significant differences in

multiple cell types including decreased CD8+ to CD4+ T cell ratio and increased neutrophils

92

K. Polyak

in tumors, and higher fraction of macrophages in TNBC [2]. We have also analyzed thegene expression profiles of T cells from each of these tissue types and identified
progression stage-specific differences. We detected an overall decrease in the frequencies ofactivated granzyme B positive (GZMB+) CD8+ T cells and an increase in suppressor Tregs astumors progress. Multicolor immunofluorescence of DCIS and IDC, including matched

DCIS and locally recurrent IDC cases in the same patient, confirmed a decline in activated

GZMB+CD8+ T cells with invasive progression. We also found that TCR clonotype diversity

was higher in DCIS than in IDCs. Interestingly, we detected a few relatively frequent TCR
clones that were shared among different DCIS patients, one of which was predicted torecognize a protein from the Epstein-Bar virus.

In order to investigate potential mechanisms underlying the more immune suppressive

environment in IDC, we analyzed the expression and copy number of immune checkpoint

genes and proteins by immunofluorescence and FISH, respectively. We found highepithelial expression of PD-L1 in triple negative IDC with amplification of CD274(encoding PD-L1) observed in 3/10 cases, whereas tumor epithelial cells in DCIS had low
expression of PD-L1 and no amplification of CD274. These results suggest a selection forbreast cancer cells with CD274 copy number gain during invasive progression. To identify

other genomic regions encoding for immune regulatory proteins that may also show

differential amplification between DCIS and IDC, we analyzed the copy number of 17q12
chemokine cluster located in close proximity of ERBB2 (encoding HER2). By analyzing

HER2+ tumors in the TCGA, we found that co-amplification of the 17q12 chemokine cluster(CC) with ERBB2 was enriched in HER2+ER+ luminal tumors, whereas there was either noFigure 1

Hypothetical model of immune escape during in situ to invasive carcinoma transition.
In DCIS with intact myoepithelial cell layer and basement membrane occasional invasive cells
are eliminated by the immune cells keeping tumors in the DCIS stage. In microinvasive
lesions there may be an equilibrium between cancer cells and anti-tumor immune
response. Tumor cells invading the surrounding tissue have successfully escaped withimmune cells either by upregulating immune suppressive mechanisms or loosing neoantigens that trigger a response.

Immune Systems and Cancer Microenvironment

93

gain but even a loss of the CC cluster region in HER2+ER- breast tumors. Interestingly, we

found higher expression of T cell activation and exhaustion-related genes in tumors thatlack CC gain. Moreover, when assessing a cohort of HER2+ samples by multicolor FISH and
immunofluorescence, we found an inverse correlation between CC amplification andfrequency of GZMB+CD8+ T cells.

Overall our results show co-evolution of cancer cells and the immune

microenvironment during tumor progression. Our results demonstrate that analyzing

immune-related changes during DCIS to IDC transition will improve our understanding of
immune editing in breast cancer, which will aid the design of more effective
immunotherapies and selection of patients who are more likely to respond to treatment.
References
1. Allinen, M., R. Beroukhim, L. Cai, C. Brennan, J. Lahti-Domenici, H. Huang, D. Porter, M.
Hu, L. Chin, A. Richardson, S. Schnitt, W.R. Sellers, and K. Polyak, Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell, 2004. 6: 17-32.
2. Gil Del Alcazar, C.R., S.J. Huh, M.B. Ekram, A. Trinh, L.L. Liu, F. Beca, X. Zi, M. Kwak, H.
Bergholtz, Y. Su, L. Ding, H.G. Russnes, A.L. Richardson, K. Babski, E. Min Hui Kim, C.
McDonnell, J. Wagner, R. Rowberry, G.J. Freeman, D. Dillon, T. Sorlie, L.M. Coussens, J.E.
Garber, R. Fan, K. Bobolis, D.C. Allred, J. Jeong, S.Y. Park, F. Michor, and K. Polyak,
Immune Escape in Breast Cancer During In Situ to Invasive Carcinoma Transition.
Cancer Discov, 2017.

Kornelia Polyak, MD, PhD

1995-1998
1998-2005
2006-20112010-present
2011-present
2013-present

Postdoctoral Fellow, Johns Hopkins Oncology Center
Assistant Professor of Medicine, Dana-Farber Cancer
Institute, Harvard Medical School
Associate Professor of Medicine, Dana-Farber Cancer
Institute, Harvard Medical School
Principle Faculty, Harvard Stem Cell Institute
Professor of Medicine, Dana-Farber Cancer Institute,
Harvard Medical School
Associate Member, Broad Institute

94

GENETIC SCREENS TO IDENTIFY IMMUNOTHERAPYTARGETS

W. Nicholas Haining

Departments of Pediatric Oncology &Cancer Immunology and Virology
Dana-Farber Cancer Institute450 Brookline Ave, Boston, MA 02215, USA(nicholas_haining@dfci.harvard.edu)
Despite the dramatic clinical success of cancer immunotherapy with PD-1 checkpoint

blockade, most patients don’t experience sustained clinical benefit, suggesting that

additional therapeutic strategies are needed. To discover new immunotherapy targets, wedeveloped a pooled, loss-of-function in vivo genetic screening approach using

CRISPR/Cas9 genome editing in mouse transplantable tumors treated with vaccination
and PD-1 checkpoint blockade. We tested 2,400 genes expressed by melanoma cells for

those that synergize with or cause resistance to checkpoint blockade, and recovered the
known immune evasion molecules, PD-L1 and CD47. Loss of function of multiple genes

required to sense interferon-γ caused resistance to immunotherapy. Deletion of Ptpn2, apleotropic protein tyrosine phosphatase, improved response to immunotherapy. Cellular,
biochemical, transcriptional and genetic epistasis experiments demonstrated that loss offunction of Ptpn2 sensitizes tumors to immunotherapy by enhancing interferon-γ-mediatedeffects on antigen-presentation and growth suppression. Thus, strategies that increaseinterferon-γ sensing by tumor cells could improve the efficacy of immunotherapy. Moregenerally, in vivo genetic screens in tumor models can identify new immunotherapy
targets, model resistance mechanisms, and accelerate the rational selection of combination
immunotherapy.

Immune Systems and Cancer Microenvironment

W. Nicholas Haining, BM, BCh

1989
1992
1997-1999
1999-present
2011-present
2016-present

95

BA, Physicologic Sciences, University of Oxford
BM, BCh, University of Oxford
Post-doctoral Fellow, MIT
Physician-scientist, Dana-Farber Cancer Institute
Associate Member, Broad Institute of Harvard and MIT
Associate Professor of Pediatrics, Harvard Medical
School96

LATENCY, IMMUNE EVASION ANDOUTBREAK OF METASTATIC STEM CELLS

Joan Massagué

Sloan Kettering Institute
Memorial Sloan Kettering Cancer CenterBox 116, 1275 York Avenue, New York, NY 10065, USA(j-massague@ski.mskcc.org)

Metastasis frequently occurs long after an apparently successful treatment of a primarytumor. Cancer patients with no clinical evidence of active disease may relapse with distantmetastasis months, years or even decades later. Metastasis grows from disseminated cancer
cells (DTCs) that remain latent until unknown conditions allow their outgrowth [1]. It isthought that tumors release large numbers of cancer cells into the circulation since earlystages of tumor growth, long before diagnosis and treatment. Although a majority of thedispersed cells are eliminated in the circulation or soon after infiltrating distant tissues, aminority may survive as latent seeds in target tissues. People who are clinically considered
free of disease after treatment of a primary tumor may still carry thousands of DTCs in thebone marrow and other organs. DTCs survive predominantly in a growth-arrested statethat is resistant to cytotoxic and targeted anti-tumor therapies that are effective against

proliferating cancer cells. The presence of these pathogenic cells does not represent an
existence of metastasis. However, DTCs constitute latent metastasis, which is a major
concern in the clinic. A better understanding of the biology of latent metastasis should
yield therapeutic strategies for its elimination and the prevention of cancer relapse.

We approached the problem metastasis through the analysis of mechanisms that

Approaching Metastasis through Mechanistic Biology

regulate cell behavior and are altered in cancer. Our attention has been focused for many
years on delineating the TGF-β pathway and the basis for its contextual action in normal
and cancer cells [2]. TGF-β signaling regulates differentiation in embryonic stem cells,

lineage identity and cell cycle progression in progenitor cells, death in pre-malignant cells,
and metastasis in carcinoma cells. Previously we showed that the TGF-β receptors activate

Nakahara Memorial Lecture

97

SMAD transcription factors that directly control lineage identity genes [3]. We recently

defined a regulatory network involving the Wnt pathway under the control of p53 tumor

suppressor family, and the TGF-β/Nodal pathway [4]. This network drives mesendoderm
differentiation of mouse and human embryonic stem cells. Wnt3 and its receptor Fzd1 arecritical p53 family target genes in this context. WNT3-activated TCF3 and nodal-activatedSmad2/3 depend on each other for co-occupancy of target enhancers in mesendoderm

differentiation genes. The p53 family coordinates essential WNT-TCF and Nodal-SMAD
inputs for mesendoderm differentiation in the early embryo [4].

The role of TGF-β as a regulator of lineage identity and differentiation is also manifest in

adult stem and progenitor cells. In adult pancreatic progenitors TGF-β-activated SMAD

signaling enforces epithelial identity through induction of SOX4 expression and regulates
the cell cycle by inducing CDK inhibitors [5] (Figure 2). However, in mutant KRAS premalignant cells, TGF-β triggers an identity crisis by simultaneously inducing SOX4 andSnail, a master effector of the epithelial-to-mesenchymal transition (EMT). This

contradictory combination triggers apoptosis and acts as a tumor suppressor mechanism[5] (Figure 2). Tumor progression requires the elimination of the tumor suppressive effects
of TGF-β, which pancreatic cells can achieve by mutational inactivation of SMAD4 or TGFβ receptors, or alternatively, by downstream alterations in TGF-β/SMAD signaling thatmonths/decades㻾㼤㻾㼤
A. Seeding

Figure 1

B. Latency

C. Outbreak

Metastatic colonization steps.
Massagué,Fig.1
In our working model, cancer cells suffer extensive clearance after extravasation.
MetSCs cansurvive by entering quiescence (G0) or initiate outgrowth by spreading along capillaries.
However, growth is inhibited by NK cells and by other defenses. Emergence of immune suppressive clones with organ-specific colonization traits eventually triggers aggressive outbreak.
Targeted therapy leaves resistant clones that resort to metastasis re-initiation mechanisms.
Our Aims target key mediators of lung adenoCa metastatic colonization.

98

J. Massagué

remove the tumor suppressive effects. Carcinoma cells can then repurpose the TGF-β-

SMAD pathway for metastatic dissemination, latency, or outbreak (Figure 2). For example,
TGF-β-SMAD signaling stimulates breast cancer cells to express angiopoietin-like 4 forextravasation in lung [6] and interleukin 11 and osteopontin for osteoclast activation inbone [7]. Tumor-derived TGF-β can also act on the stroma to impart immune suppression

or to induce the expression of tenascin C and other extracellular matrix proteins that build
a metastatic niche [8]. These insights shed light on the role of TGF-β as a key mediator inphysiology and disease, and as a potential target for therapy.

Although metastatic progression through the latent stage is a major concern in the clinic,

Latent Metastasis

little is known about the identity of latent DTCs and the mechanisms that allow them toremain quiescent, evade immunity, retain tumor-initiating capacity, and evolve into
aggressive metastasis [1]. The lack of suitable experimental models has inhibited progressin this area. We recently developed mouse models of latent metastasis using cancer cells

from early-stage lung and breast tumors [9]. These models are allowing us to definemechanisms that suppress the outgrowth, support the long-term survival, and enable theeventual outbreak of latent metastatic cells.

Latency-competent lung and breast cancer cells have stem cell-like properties. We referto these cells as metastatic stem cells (MetSCs) [10] (Figure 1). SOX2 and SOX9 lineage㻱㼜㼕㼠㼔㼑㼘㼕㼍㼘㻌㼜㼞㼛㼓㼑㼚㼕㼠㼛㼞㻱㼜㼕㼠㼔㼑㼘㼕㼍㼘㻌㼜㼞㼛㼓㼑㼚㼕㼠㼛㼞㻼㼞㼑㻙㼙㼍㼘㼕㼓㼚㼍㼚㼠

TGF-E

TGF-E

㻯㼍㼞㼏㼕㼚㼛㼙㼍
TGF-E

T,NK

escapeKRAS*CDKN SOX4SNAIL SOX4㼀㻺㻯㻸㼕㼚㼑㼍㼓㼑㻌㼕㼐㼑㼚㼠㼕㼠㼥㻯㼥㼠㼛㼟㼠㼍㼟㼕㼟

Figure 2㻱㻹㼀㻌㼍㼚㼐㻌㼕㼐㼑㼚㼠㼕㼠㼥㻌㼏㼞㼕㼟㼕㼟㻌㻭㼜㼛㼜㼠㼛㼟㼕㼟

㻯㻭㻲

TGF-E

T,NK
䈄㻌㻿㼚㼍㼕㼘㻌㻔㻱㻹㼀㻕䈄㻌㻭㻺㻳㻼㼀㻸㻠䈄㻌㻵㻸㻙㻝㻝㻘㻌㻻㻼㻺㻘㻌

The TGF-β – SMAD pathway in tumor suppression and metastasis.
A basis for the dual role of TGF-β in certain types of cancer was recently elucidated. In
pancreatic epithelial progenitors the TGF-β–SMAD signaling pathway regulates lineage
identity (e.g. though induction of SOX4 epithelial identity transcription factor) and growth
homeostasis (through induction of CDK inhibitors). However, TGF-β is a tumor suppressor
Massagué,Fig.2
for pre-malignant pancreatic progenitors that harbor the obligate oncogene
KRAS. KRAS
primes these cells for a strong induction of the EMT master transcriptional regulator
SNAIL. An EMT occurring side-by-side SOX4 expression in these cells creates an identity crisis of simultaneous pro-mesenchymal and pro-epithelial signals, leading to apoptosis.
Oncogenic progression requires the inactivation of this tumor suppressive response by loss of
SMAD4 or by downstream loss of apoptotic responses. Once free from TGF-β tumor suppressive responses, carcinoma cells can use the altered TGF-β responsive to their benefit during different stages of metastatic progression.

Nakahara Memorial Lecture

99

determining transcription factors impart a progenitor state to MetSCs. These cells reside inperivascular niches and can self-impose a slow-cycling state by avoiding WNT growth

stimuli and responding to TGF-β in this context [10] (Figure 3). This state includes thedownregulation of including MICA and ULBP family members, membrane proteins thatserve as ligands for the receptor NKG2D on NK cells and cytotoxic T cells. Through theseand other mechanisms, quiescent metastatic cells cam evade NK cell-mediated clearance.

Latent MetSCs frequently enter the cell cycle, but are cleared by natural killer (NK) cells

unless they gain additional immune evasive capacities. We propose that metastatic latency
is a highly dynamic stage, in which frequent attempts by disseminated MetSCs to reinitiate

outgrowth are thwarted by immune surveillance. Our working hypothesis is that the latent
metastatic state does not necessarily involve a long-term permanence of MetSCs inreplicative quiescence, but rather involves MetSCs that frequently enter the proliferative
state but fail to form a growing colony because NK cells detect and eliminate the incipient
growth. Proliferation entails the exposure of MetSCs to recognition and elimination byimmune cells, whereas cancer cells that re-enter quiescence are spared (Figure 3). As a
A. Seeding
2

1

B. Latency
3

Figure 3

Metastatic seeding and latency.
(A) NK cells and astrocytes clear a majority of infiltrated cancer cells. MetSCs survive byentering immune evasive G0 and, in the brain, by producing serpin inhibitors of plasminogenactivator. Inset: MetSCs spread on capillaries after extravasation (1), and enter G0Massagué,Fig.3
andround up near capillaries (2), or spread and proliferate along the capillary surface (3);
L1CAM mediates spreading and is required for outgrowth. (B) In this model, MetSCs thatenter G0 constitute a highly dynamic latent state, with ongoing cycles of growth re-initiation
and NK-mediated clearance. Entry into G0 leads to downregulation of ULBPs, ligands for NK
and CTL receptors. This cycling would allow selection of organ-specific colonization traits.

100

J. Massagué

corollary, antibody-mediated depletion of functional NK cells in mice immediately allows
the accumulation of over metastasis, providing us with a functional assay for the study ofregulators of metastatic outbreak. In sum, latency is not created by MetSCs that remainquiescent for long periods but rather by a equilibrium between transiently proliferative
disseminated MetSCs and immune effectors that cull MetSCs that reinitiate proliferation.

Metastatic outbreaks and formation of aggressive metastatic colonies have other

Organ-specific Metastatic Colonization

requirements besides the evasion of immune surveillance. A requirement that may be

relevant for the initiation of metastatic colonization in different organ sites is the spreadingof MetSCs on the surface of capillaries. MetSCs that remain in contact with capillaries after
extravasation are observed to spread on the abluminal surface of the vessels as a condition
for proliferation [11]. The localization of MetSCs in the perivascular niche ensures theiraccess to oxygen, nutrients and paracrine factors. However, spreading on capillaries

licences these cells for the initiation of growth in the rich microenvironment (Figure 4).
Spreading is mediated by the L1 cell adhesion molecule (L1CAM, also known as CD171).

L1CAM-mediated spreading on capillaries provides cancer cells with stimuli that are
critical for outgrowth immediately upon infiltration of target organs as well as after exitingfrom a period of quiescence under conditions of latency. These results identify an

obligatory step in the initiation of metastatic colonization, and provide an explanation for
the widespread association of L1CAM with metastatic relapse in the clinic.
C

FN1-5

Figure 4

Exon 27

1.0

High

0.8

Low

0.6

p<0.0001

T3G

PGK

Control shL1CAM

0.4
0.2
0.0 HR:1.2(1.1-1.3)
0 2 4 6 8 10
Years

6x106

Ig 1-6Overall Survival

Exon 2

L1CAM expression

3x107 Venus

B
Neuronal Non-neuronal
L1CAM
L1CAM

dsRed

A

L1CAM and vascular cooption in metastasis.
(A) Schematic representation of neuronal and non-neuronal L1CAM isoforms. Red ovals represent exons 2 and 27, which are spliced out from the non-neuronal isoforms isoform. (B)Overall survival plot for patients with high and low L1CAM expression bifurcated atmedian L1CAM expression level. High L1CAM expression is associated with poor patient
survival. (C) L1CAM is necessary for metastatic outgrowth post extravasation. L1CAM
knockdown using a doxycycline-inducible construct (top) 9 days after intracardiac injection of lung adenoCa cells reduces metastatic outgrowth (bottom).
shL1CAM + dox

Massagué,Fig.4

Nakahara Memorial Lecture

101

Overt metastasis in different organ involves different mediators of organ-specificcolonization. Many such mediators have been identified in recent years that are expressedby the cancer cells or the affected host stroma [1]. For example, osteolytic bone metastasisby breast cancer cells depends on osteoclast activating factors, several of which are induced
by stromal TGF-β [7]. It also depends on SRC amplification of CXCR4 signaling to activate
the PI3K pathway form cancer cell survival [12,13]. In contrast, colonization of the lungs isenhanced by the expression of monocyte-binding receptors [14] and extracellular matrix
proteins that support stem cell survival [8].

We recently focused on metastasis to the central nervous system. Brain metastases occur

in 20-40% of advanced stage cancers and represent the most prevalent intracranial

malignancy in adults. Current clinical management of brain metastases provides limited
disease control and most patients succumb to tumor progression within one year of
diagnosis. Recent work has elucidated cellular and molecular interactions responsible forbrain metastasis. Circulating cancer cells infiltrate the brain parenchyma by expressing
specific mediators of cell passage across the blood-brain barrier (BBB) [15]. The vast
majority of cancer cells that infiltrate the brain perish, rejected by the most abundant cell

type in the brain, the astrocyte. The astrocyte network, coupled together via gap junctions,serves a protective role in the CNS. In brain metastasis, reactive astrocytes generate theprotease plasmin and cytotoxic cytokines. Brain metastatic cells counter this defense with
serpin inhibitors of plasminogen activator [11] (Figure 3).

Astrocyte-cancer cell interactions are not uniformly antagonistic. Brain metastases

contain abundant reactive astrocytes, and astrocytes can exert a beneficial effect on cancer

cell co-cultures. We recently identified a role of astrocytes in promoting brain metastasis[16]. Breast and lung cancer cells express protocadherin 7 (PCDH7) to favor the assembly of
carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with theastrocyte gap-junctional network, brain metastatic cancer cells employ these channels totransfer the second messenger cGAMP to astrocytes for activation of STING, an innate

immune response pathway to cytosolic double-stranded DNA (dsDNA) (Figure 5A).
STING activation stimulates the production of inflammatory cytokines IFNα and TNF in

the astrocytes. As paracrine signals, these factors activate the STAT1 and NF-κ B pathways

in brain metastatic cells, which in this context support tumor growth and chemoresistance.
We tested the orally bioavailable modulators of gap junctions meclofenamate andtonabersat as inhibitors of this paracrine loop, providing proof-of-principle for theapplicability of this therapeutic strategy to treat established brain metastasis [16]. We areconducting clinical trials of meclofenamate in patients with relapsing brain metastasis ofbreast and lung cancers.

102

J. MassaguéA.

Figure 5

B.

Metastatic colonization of the CNS.
(A) Brain metastatic cells coopt astrocytes via Cx3/PCDH7 gap junctions and cGAMPtransfer. cGAMP triggers production of inflammatory cytokines in astrocytes that support
BrM growth and chemoresistance. (B) Leptomeningeal metastatic cells that reach the CSF
through the choroid plexus (CP) and produce complement C3, which inhibits the blood-CP
barrier for entry of plasma growth factors (e.g. amphiregulin, AR).
Massagué,Fig.5

The leptomeninges surround the brain and spinal cord and contain the cerebrospinal

fluid (CSF). Cancer spread into the CSF compartment, called leptomeningeal metastasis,presents a formidable clinical challenge. Metastases to this fluid-filled space disseminate

rapidly over the entirety of the central nervous system, growing on and invading into thebrain, spinal cord, cranial and spinal nerves, resulting in rapid neurologic disability anddeath. The majority of leptomeningeal metastases arise from primary breast and lungcancers. Approximately 5-10% of patients with solid tumors harbor leptomeningeal
metastasis, and this number is expected to rise. The molecular basis of this morbid,

increasingly prevalent complication of cancer remains unknown. To address this problem,

we selected lung and breast cancer cell lines for the ability to infiltrate and grow in the CSF[17]. Complement component 3 (C3) was upregulated in four leptomeningeal metastatic

models and proved necessary for cancer growth within the leptomeningeal space. In

human disease, cancer cells within the CSF produced C3 in correlation with clinical course.
C3 expression in primary tumors was predictive of leptomeningeal relapse.
Mechanistically, we found that cancer cell-derived C3 activates the C3a receptor in thechoroid plexus epithelium to disrupt the blood-CSF barrier (Figure 5B). This effect allows
plasma components including amphiregulin and other mitogens to enter the CSF andpromote cancer cell growth. Pharmacologic interference with C3 signaling proved

therapeutically beneficial to suppress leptomeningeal metastasis in these preclinical models[17].

Nakahara Memorial Lecture103

By exploiting mouse models of metastasis and patient-derived samples in combination

Perspectives

with a mechanistic dissection of cancer-relevant cell regulation pathways like the TGF-β

pathway and others, we were able to identify genes and mechanisms whose expression

mediates metastasis in mouse models and associates with organ-specific relapse in patients.
Some of these pro-metastatic pathways are mutationally altered in cancer, whereas others

are altered at the epigenetic level [18]. This work additionally illuminated the closemechanistic links between drug resistance and metastasis [19,20]. The ability to biologicallydissect and molecularly deconstruct the basic determinants of metastasis is allowing thedesign and clinical testing of innovative therapeutic strategies for the prevention andtreatment of metastasis.

References
1. Massagué J, Obenauf A Metastatic colonization by circulating tumor cells. Nature
2016;529:298-306.
2. Massagué J. TGFβ signaling in context. Nature Rev Mol Cell Biol. 2012;18:5521-5525.

3. Shi Y and Massagué J. Mechanisms of TGFβ signaling from cell membrane to the nucleus.
Cell. 2003;113:685-700.
4. Wang Q, Zou Y, Nowotschin S, Kim SY, Soh CL, Su J, Zhang C, Shu W, Xi Q, Huangfu D,
Hadjantonakis AK, Massagué J. The p53 family coordinates Wnt and nodal inputs inmesendoderm differentiation of embryonic stem cells. Cell Stem Cell. 2017;20:70-86.
5. David CJ, Huang YH, Chen M, Su J, Bardeesy N, Iacobuzio-Donahue C, Massagué J.
TGF-β tumor suppression through a lethal EMT. Cell. 2016;164:1015-1030.
6. Padua D, Zhang XHF, Wang Q, Nadal C, Gerald WL, Gomis R, Massagué J. TGFß
primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell.
2008;133:66-77.
7. Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen S, Cordón-Cardó C, Guise TA,
Massagué J. A multigenic program mediating breast cancer metastasis to bone. Cancer
Cell. 2003;3:537-549.
8. Oskarsson T, Acharyya S, Zhang XHF, Tavazoie S, Morris PG, Downey R, ManovaTodorova K, Brogi E, Massagué J. Breast cancer cells produce tenascin-C as a metastatic
niche component to colonize the lungs. Nature Med. 2011;17:867-874.
9. Malladi S, Macalinao D, Jin X, He L, de Stanchina E, Massagué J. Metastatic latency andimmune evasion through autocrine inhibition of WNT. Cell. 2016;165:45-60.
10. Oskarsson T, Batlle E, Massagué J. Metastatic stem cells. Cell Stem Cell. 2014;14:306-321.

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11. Valiente M, Obenauf AC, Jin X, Chen Q, Zhang XHF, Lee DJ, Chaft JE, Kris MG, Huse JT,
Brogi E, Massagué J. Serpins promote cancer cell survival and vascular cooption inbrain metastasis. Cell. 2014;156:1002-1016.
12. Zhang XHF, Wang Q, Gerald WL, Hudis CA, Norton L, Smid M, Foekens JA, Massagué J.
Latent bone metastasis in breast cancer tied to Src-dependent survival signals. Cancer
Cell. 2009;16:67-78.
13. Zhang XHF, Jin X, Malladi S, Kim JY, Wen YH, Brogi E, Smid M, Foekens J, Massagué J.
Selection of bone metastasis seeds by mesenchymal signals in the primary tumor stroma.
Cell 2013;154:1060-1073.
14. Chen Q, Zhang XHF, Massagué J. Macrophage binding to receptor VCAM-1 transmits survival signals in metastatic breast cancer cells that invade the lungs. Cancer Cell.
2011;20:538-549.
15. Bos PD, Zhang XHF, Nadal C, Shu W, Gomis RR, Nguyen DX, Minn AJ, Van de Vijver M,
Gerald WL, Foekens JA, Massagué J. Genes that mediate breast cancer metastasis to thebrain. Nature. 2009;459:1005-1009.
16. Chen Q, Boire A, Jin X, Valiente M, Er EE, Lopez-Soto A, Pawta R, Shah H, Xu K, Cross JR,
Massagué J. Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMPtransfer. Nature. 2016;533:493–498.
17. Boire A, Zou Y, Shieh J, Macalinao D, Pentsova E, Massagué J. Complement component 3
adapts the cerebrospinal fluid for leptomeningeal metastasis. Cell. 2017 168, 1101-1113(2017)
18. Vanharanta S, Shu W, Brenet F, Hakimi AA, Heguy A, Viale A, Reuter VE, Hsieh JJD,
Scandura JM, Massagué J. Epigenetic expansion of VHL-HIF signal output drives
multi-organ metastasis in renal cancer. Nature Med. 2013;19:50-56.
19. Acharyya S, Oskarsson T, Vanharanta S, Kim J, Morris PG, Manova-Todorova K,
Leversha M, Hogg N, Norton L, Brogi E, Massagué J. A CXCL1 paracrine network
links cancer chemoresistance and metastasis. Cell. 2012;150:165-178.
20. Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, Kong X, Bosenberg MC,
Wiesner T, Rosen N, Lo RS, Massagué J. Therapy induced tumour secretomes promoteresistance and tumour progression. Nature. 2015;520:368-372.

Nakahara Memorial Lecture

Joan Massagué, PhD

1975
1978

1979-1982
1982-1985
1985-1989

1989-present
1989-2003
1990-20132003-20132006-present
2014-present

105

BS, Pharmacy, Univ. of Barcelona
PhD, Biochemistry, Univ. of Barcelona
Mentor: Joan J. Guinovart
Postdoc Fellow, Brown Univ.
Mentor: Michael P. Czech
Assistant Professor of Biochemistry, Univ. ofMassachusetts Medical School
Associate Professor of Biochemistry, Univ. ofMassachusetts Medical School
Professor, Weill-Cornell Graduate School of Medical
Sciences
Alfred P. Sloan Chair, Memorial Sloan Kettering
Cancer Center, (MSKCC)
Chair, Cell Biology Program, MSKCC
Investigator, Howard Hughes Medical Institute
Chair, Cancer Biology and Genetics Program, MSKCC
Professor, Gerstner Sloan Kettering Graduate School of
Biomedical Sciences
Director, Sloan Kettering Institute, MSKCC
Provost, Gerstner Sloan Kettering Graduate
School of Biomedical Sciences

106

ABERRANT REGULATION OF MAPK SIGNALINGPATHWAYS IN CANCER

Mutsuhiro Takekawa

Division of Cell Signaling and Molecular Medicine
Institute of Medical Science
The University of Tokyo
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan(takekawa@ims.u-tokyo.ac.jp)
Mitogen-activated protein kinase (MAPK) cascades are the major signaling systems that

dictate cell fate decisions such as proliferation, survival, and apoptosis. There are at least
three subfamilies of MAPKs, named ERK, p38, and JNK in human cells (Figure 1). Whilethe classical ERK MAPK is preferentially activated in response to mitogenic stimuli andplays a central role in cell growth and survival, two relatively newly identified types of

Figure 1

Human MAP kinase signaling cascades

Signaling Diversity in Cancer

107

MAPKs, p38 and JNK, are more potently activated by a variety of environmental stresses(e.g., UV- and γ−irradiation, DNA-damaging reagents, oxidative stress, osmotic stress, heatshock and etc.) and are thus collectively called stress-activated protein kinase (SAPK).

Besides cellular stresses, the SAPK pathways are also activated by cytokines such as IL-1,
TNFα, and TGF-β. Perturbation of these critical cellular signaling systems is involved in avariety of life-threatening diseases, including cancer. Therefore, these signaling systems areof clinical importance.

The ERK cascade is a key transducer of mitogenic signals, and is frequently hyper-

activated by various oncogenes in human cancer [1]. This pathway not only upregulates
growth-related genes, but also down-regulates several anti-proliferative and tumor

suppressive genes (e.g., E-cadherin) in various cellular processes including the epithelial-to-mesenchymal transition (EMT). EMT is a cellular trans-differentiation program whereby
epithelial cells lose their epithelial characteristics and acquire a migratory, mesenchymal

phenotype. This phenomenon is involved in a wide range of biological process, including
embryonic development, tissue repair, and tissue fibrosis. Furthermore, inappropriate

reactivation of the EMT program in malignant epithelial cells is considered to be a majormechanism for the induction of tumor invasion and metastasis. Therefore, comprehensive
understanding of the molecular basis of EMT is crucial for the development of novel
therapeutic interventions for cancer. Although TGF-β signaling is a prominent mediator of

EMT, accumulating evidence has revealed that various other signaling pathways also playa role in this process. In particular, it has been reported that hyper-activation of the ERK
pathway is sufficient to induce EMT in many, if not all, types of cells. Besides ERK
signaling, C-terminal binding protein (CtBP), which is a core component of thetranscriptional co-repressor complex that contains histone modifying enzymes (e.g., histone
deacetylases and methyltransferases), is also involved in epigenetic gene silencing of E-

cadherin during EMT. However, the functional relationship, if any, between ERK signaling
and CtBP remains obscure.

By developing a novel technique to isolate ERK substrate proteins, we identified apreviously uncharacterized protein of unknown function, designated MAPK-regulated
corepressor interacting protein 1 (MCRIP1), as a novel and specific substrate of ERK.

MCRIP1 is conserved in all vertebrates from fish to human, and is expressed in all tissues

examined. Interestingly, we found that MCRIP1 bridged ERK signaling and CtBP-mediatedgene silencing [2]. CtBP is recruited to promoter elements of target genes by interactingwith the DNA-binding transcriptional repressor ZEB1. We found that MCRIP1 bound to
CtBP, thereby competitively inhibiting CtBP-ZEB1 interaction. When phosphorylated by

ERK, MCRIP1 dissociates from CtBP, allowing CtBP to interact with ZEB1. In this manner,

the CtBP co-repressor complex is recruited to, and silences the E-cadherin promoter by

108

M. Takekawa

inducing histone modifications. Our findings demonstrate MCRIP1-mediated functional

interaction between ERK signaling and CtBP during EMT, and delineate a molecularmechanism as to how oncogenic ERK signaling induces epigenetic silencing of tumor
suppressor genes such as E-cadherin (Figure 2).

The p38 and JNK MAPKs play pivotal roles in cellular stress responses such as

apoptosis [3]. Cell fate decisions that are influenced by the p38 and JNK pathways might beimportant for minimizing the chance of tumorigenesis. There is increasing evidence thatthese pathways are involved in tumor suppression, and are indeed aberrantly regulated incancer. In this talk, we also report an unexplored role of the p38 and JNK pathways in theregulation of centrosome number [4].

Centrosomes, which consist of a pair of centrioles surrounded by an amorphous

pericentriolar material, serve as the microtubule-organizing centers that are essential forthe formation of mitotic spindles in animal cells. In order for cells to undergo normal

bipolar cell division, the single interphase centrosome must duplicate precisely once percell cycle. The strict control of centrosome numbers is indispensable for accurate

chromosome segregation at cell division and for maintenance of the stability of genomes.

The presence of more than two centrosomes (i.e., centrosome amplification) results in theformation of multipolar mitotic spindles and consequentially in chromosome segregation
errors. Centrosome amplification significantly increases the frequency of lagging
chromosomes during anaphase, thereby promoting chromosome missegregation. Since

Figure 2

A novel ERK substrate, MCRIP1, plays an important role in epigenetic gene silencing of Ecadherin during EMT by regulating CtBPSignaling Diversity in Cancer

109

chromosome missegregation results in both numerical and structural abnormalities ofchromosomes, ablation of the numeral integrity of centrosomes induces chromosomal
instability, and thus is considered to be a major cause of cancer development andprogression.

We have recently demonstrated a direct functional link between the SAPK pathways

and PLK4. We found that, upon stress stimuli, stress-responsive MAPKKKs such as MTK1

and TAK1 directly phosphorylated and activated polo-like kinase 4 (PLK4), a master
regulator of centrosome duplication. Stress-induced, MAPKKK-mediated, PLK4 activation
provides survival signaling and promoted centrosome duplication. At the same time,

however, the p38 and JNK SAPKs and the tumor suppressor p53 protein, which are also

activated by various stress stimuli, cooperated to counteract PLK4 activity, thereby
preventing centrosome amplification. Importantly, we demonstrated that simultaneous

inactivation of SAPKs and p53 in cells exposed to stress allowed unchecked activation of
PLK4, leading to centrosome overduplication and chromosomal instability, both of which
are hallmarks of cancer cells. This co-operation between SAPKs and p53 explains why both

p53 and the MKK4 MAPKK (a SAPK activator) are frequently mutated simultaneously inhuman cancer cells, in which centrosome number is often increased after stress (Figure 3).

Figure 3

Combined inactivation of p53 and p38/ JNK induces centrosome over-duplication

110

M. Takekawa

Mutational inactivation of the MKK4 MAPKK has been shown to drive carcinogenesis(known as “driver” mutations) and is indeed frequently observed in a variety of humancancers. However, the mechanism by which MKK4 prevents carcinogenesis remains to beelucidated. Our results provide the first evidence that centrosome integrity is
synergistically regulated by the two important tumor suppressors, MKK4 and p53, andreveal a molecular mechanism that underlies centrosome amplification in cancer cells.
Based on these findings, we proposed that MKK4 is a novel type of tumor suppressor
whose function is manifested particularly when p53 is also inactivated.
References
1. Kubota Y, O’ Grady P, Saito H and Takekawa M: Oncogenic Ras abrogates MEK
SUMOylation that suppresses the ERK pathway and cell transformation. Nat Cell Biol
2011;13: 282-291.
2. Ichikawa K, Kubota Y, Nakamura T, Weng JS, Tomida T, Saito H and Takekawa M.
MCRIP1, an ERK substrate, mediates ERK-induced gene silencing during epithelialmesenchymal transition by regulating the co-repressor CtBP. Mol Cell 2015;58: 35-46.
3. Arimoto-Matsuzaki K, Saito H and Takekawa M: TIA1 oxidation inhibits stress granuleassembly and sensitizes cells to stress-induced apoptosis. Nat Commun 2016; 7: 10252
doi:10.1038/ncomms10252.
4. Nakamura T, Saito H and Takekawa M: SAPK pathways and p53 cooperatively regulate
PLK4 activity and centrosome integrity under stress. Nat Commun 2013; 4: 1775 doi:10.
1038/ncomms2752.

Mutsuhiro Takekawa, MD, PhD

1994-1998
1998-2000
2000-2003
2003-20092009-20122012-present

Postdoctoral Fellow, Div. of Cancer Immunology and
AIDS Research, Dana-Farber Cancer Institute,
Harvard Medical School
Assistant Professor, First Dept. of Internal Medicine,
Sapporo Medical University School of Medicine
Assistant Professor, Div. of Molecular Cell Signaling,
Institute of Medical Science, The University of Tokyo
Associate Professor, Div. of Molecular Cell Signaling,
Institute of Medical Science, The University of Tokyo
Professor, Department of Cell Signaling and Molecular
Medicine, Research Institute of Environmental
Medicine, Nagoya University
Professor, Division of Cell Signaling and Molecular
Medicine, Institute of Medical Science,
The University of Tokyo

111

PI 3-KINASE LINKS OBESITY,
INSULIN RESISTANCE AND CANCER

Lewis C. Cantley

Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine
413 East 69th St., 13th Floor, Box 50, New York, NY 10021, USA(lcantley@med.cornell.edu)

Retrospective studies have shown that cancers in certain tissues occur at higher rates inindividuals who are obese and/or insulin resistant. Many possibilities for explaining thiscorrelation have been proposed. Of particular interest, some of the cancers that correlatewith obesity and insulin resistance occur in tissues that express the insulin receptor at

relatively high levels, including the liver, pancreas, breast and endometrium. Thisobservation raises the possibility that the ambient high levels of serum insulin that arecharacteristic of patients with insulin resistance might be contributing to the growth oftumors in these organs. Consistent with this idea, agents that lower serum insulin,

including the anti-diabetic drug metformin have been shown to reduce cancer incidents ordeaths in patients with insulin resistance/type 2 diabetes [1-4]. Phosphoinositide 3-Kinase(PI3K) is activated by insulin and other growth factors to mediate cell growth [5]. The PI3K

enzyme encoded by the PIK3CA gene is one of the most frequently mutated oncogenes inhuman cancer. This same enzyme mediates insulin responses in liver, muscle, fat and othertissues. The most common mutations in this gene enhance the ability of PI3K to bind to theinsulin receptor substrates, IRS1 and IRS2 and thereby enhance the ability of PI3K to be

activated by insulin and IGF1. This observation raises the possibility that elevated levels of

serum insulin could enhance the growth of tumors that express the insulin receptor,

especially when the tumor expresses a mutant form of PIK3CA. Consistent with this idea,
retrospective studies have shown that cancers that correlate with obesity and insulin

resistance (conditions where serum insulin levels are high), such as endometrial, breast andcolorectal cancers, frequently have activating mutations in PIK3CA. These observations
suggest a model in which tumors with mutations in PIK3CA are highly sensitized to112

L. C. Cantley

insulin-dependent growth and that the elevated serum insulin levels in individuals withinsulin resistance in liver, muscle and fat promotes anabolic metabolism in the tumor [6].

This condition would allow the tumor to take up glucose more readily than muscle or fat ininsulin resistant individuals.

We have generated mouse models to interrogate the role of PI3Ks in metabolic control

and in the generation of cancers. These studies indicate that inhibitors of PI3Ks could be
effective in treating cancers in specific mutational backgrounds. More than 20 PI3K

inhibitors that target the gene product of PIK3CA have entered clinical trials for treating

solid cancers. Although these inhibitors look promising and two have been approved, avariety of toxicities have created challenges [7]. Some of the toxicities, such as
hyperglycemia, are on target since inhibition of the PIK3CA gene product is expected tocause insulin resistance. Importantly, the acute insulin resistance that occurs in response to
PI3K inhibitors not only raises serum glucose levels, but also induces insulin release fromthe pancreas, which raises insulin levels. As discussed above, the increased serum insulin
has the potential to activate PI3K in the tumor, and it is possible that very high insulinlevels could reactivate PI3K in the tumor despite the presence of a PI3K inhibitor. We are
exploring therapeutic interventions that might prevent this rebound of insulin-dependent
PI3K reactivation in tumors.

References
1. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin andreduced risk of cancer in diabetic patients. BMJ. 2005 Jun 4;330(7503):1304-5. Epub 2005
Apr 22. PMID: 15849206
2. Muti P, Berrino F, Krogh V, Villarini A, Barba M, Strano S, Blandino G.Metformin, dietand breast cancer: an avenue for chemoprevention. Cell Cycle. 2009 Aug 15;8(16):2661.
Epub 2009 Aug 8. PMID: 19571669
3. Libby G, Donnelly LA, Donnan PT, Alessi DR, Morris AD, Evans JM. New usersof metformin are at low risk of incident cancer: a cohort study among people with type 2
diabetes. Diabetes Care. 2009 Sep;32(9):1620-5. doi: 10.2337/dc08-2175. Epub 2009 Jun 29.
PMID: 19564453
4. Cao X, Wen ZS, Wang XD, Li Y, Liu KY, Wang X. The Clinical Effect of Metformin on theSurvival of Lung CancerPatients with Diabetes: A Comprehensive Systematic Review andMeta-analysis of Retrospective Studies. J Cancer. 2017 Aug 2;8(13):2532-2541. doi:
10.7150/jca.19750. eCollection 2017. PMID: 28900491
5. Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT. The PI3K
Pathway in Human Disease. Cell. 2017 Aug 10;170(4):605-635. doi: 10.1016/j.cell.
2017.07.029. Review. PMID: 28802037

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6. Hopkins BD, Goncalves MD, Cantley LC. Obesity and Cancer Mechanisms: Cancer
Metabolism. J Clin Oncol. 2016 Dec 10;34(35):4277-4283. Epub 2016 Nov 7. Review.
PMID: 27903152
7. Mayer IA, Abramson VG, Isakoff SJ, Forero A, Balko JM, Kuba MG, Sanders ME, Yap JT,
Van den Abbeele AD, Li Y, Cantley LC, Winer E, Arteaga CL. Stand up to cancer phase
Ib study of pan-phosphoinositide-3-kinase inhibitor buparlisib with letrozole in estrogen
receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast
cancer. J Clin Oncol. 2014 Apr 20;32(12):1202-9. doi: 10.1200/JCO.2013.54.0518. Epub
2014 Mar 24. PMID: 24663045

Lewis C. Cantley, PhD

1978-1981
1981-1985
1985-1992
1992-2003
1992-2012

2003-20122007-20122012-present
2012-present

Assistant Professor, Dept. of Biochemistry andMolecular Biology, Harvard Univ.
Associate Professor, Dept. of Biochemistry andMolecular Biology, Harvard Univ.
Professor of Physiology, Tufts University School of
Medicine
Professor of Cell Biology, Harvard Medical School
Chief, Division of Signal Transduction,
Beth Israel Deaconess Medical Center
Professor of Systems Biology, Harvard Medical School
Director, Beth Israel Deaconess Cancer Center
Professor of Medicine, Weill Cornell Medical College
Director, Sandra and Edward Meyer Cancer Center at
Weill Cornell Medical College/ Ronald P. Stanton
Clinical Cancer Program at NewYork-Presbyterian

114

TGF-β SIGNALING IN CANCER

Kohei Miyazono

Department of Molecular Pathology
Graduate School of Medicine, The University of Tokyo
Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan(miyazono@m.u-tokyo.ac.jp)

Transforming growth factor-β (TGF-β) elicits both tumor promoting and suppressivefunctions during progression of cancer. We here demonstrate our recent findings on TGF-βsignaling in progression of cancer, focusing on the induction of epithelial-mesenchymaltransition (EMT) by TGF-β. TGF-β induces EMT through activation of Smad as well as nonSmad signaling pathways. Inhibition of TGF-β signaling by inhibitory Smad, Smad7, or

small-molecular-weight TGF-β receptor inhibitors results in suppression of cancer

metastasis in mouse models [1]. Multiple transcription factors are involved in TGF-β-induced EMT. In addition, cross-talk with other signaling pathways plays important rolesin the induction of EMT. Mesenchymal-epithelial transition (MET) is the inverse process of
EMT; TGF-β-mediated EMT is a crucial event in the invasion of cancer, while MET occurs
when cancer cells form metastatic foci at distant organs.

Snail, a key regulator of EMT, is induced by TGF-β in cancer cells. We showed that

Cooperation of Ras and TGF-β Signaling in the Progression of Cancer

induction of Snail by TGF-β was dependent on functional cooperation with active Ras

signals, and knockdown of Ras strongly attenuated the Snail induction by TGF-β inpancreatic cancer Panc-1 cells [2]. Overexpression of active Ras into HeLa cells resulted instrong induction of Snail expression by TGF-β, while most other direct targets of TGF-β,

including SMAD7 and SERPINE1, were not increased by Ras signaling. These observations

indicate that Ras and TGF-β signaling cooperate in the induction of Snail and progressionof the EMT process.

We then performed global mapping of the accessible chromatin regions using

formaldehyde-assisted isolation of regulatory element (FAIRE)-sequencing [3]. To study

Signaling Diversity in Cancer

115

the functional cooperation between TGF-β and Ras, we used the mouse mammary

epithelial cell line EpH4 and its Ras-transformed derivative, EpRas. TGF-β and Ras

modulated chromatin accessibility either cooperatively or independently; chromatin
accessibility to the epithelial marker gene regions, including Cdh1 (encoding E-cadherin)

and Esrp2, was reduced upon TGF-β and Ras stimulation, while that to some mesenchymal

marker gene regions, including Cdh2 (encoding N-cadherin) and Mmp13 (encoding matrix
metalloproteinase 13), was increased (Figure 1). We have also found that Etv4 and Etv5,

ETS family oncogenic transcription factors, bound to more than one-third of the accessible
chromatin regions in EpRas cells upon TGF-β treatment. Expression of Mmp13 and cell

Figure 1

invasiveness induced by TGF-β appeared to be regulated by Etv4 and Etv5. Thus,
transcriptional regulation during EMT induced by Ras and TGF-β involves alterations ofaccessible chromatin regions, which is, in part, regulated by Etv4 and Etv5.
TGF-E

Ras
EpH4 cells

Epithelial
Mesenchymal

Figure 1

EpRas cells

EpRas cells+ TGF-E

Cdh1/E-cadherin
Esrp2
Mmp13
Cdh2/N-cadherin

Regulation of EMT-related gene expression and alteration of chromatin accessibility by Ras
and TGF-β. Chromatins are closed by Ras and TGF-β at the Cdh1/E-cadherin and Esrp2
gene loci. In contrast, Cdh2/N-cadherin and Mmp13 gene loci become accessible upon Ras
and/or TGF-β stimulation. Modified from Arase et al. [3]Thyroid transcription factor-1 (TTF-1/Nkx2-1) is a tissue-specific transcription factor in

Roles of TTF-1/Nkx2-1 in Progression of Lung Adenocarcinomalung epithelial cells, and expressed in lung cancer. TTF-1 is used as a diagnostic marker oflung cancer; TTF-1-positive lung cancer patients show better prognosis than TTF-1-negative
patients. We have found that TTF-1 inhibited the TGF-β-induced EMT, induced theexpression of E-cadherin, and restored epithelial phenotype in lung adenocarcinoma cells.
This effect of TTF-1 was accompanied by down-regulation of TGF-β target genes, including

SNAI1 and SNAI2 (encoding Snail and Slug, respectively) [4]. Knockdown of TTF-1
expression enhanced TGF-β-mediated EMT. Thus, TTF-1 exhibits a tumor-suppressive

effect through inhibition of response to TGF-β. In addition, we have shown that TTF-1
repressed the production of TGF-β2 in A549 cells, while TGF-β in turn decreased theexpression of TTF-1, suggesting that modulation of TTF-1 expression is a potentiallyinteresting therapeutic strategy for treatment of lung adenocarcinoma (Figure 2A).

116

K. Miyazono

Figure 2
(A)

TTF-1

TGF-E
E

MET

Cancer cells

InvasionE-cadherinĻ

EMT

Snail ĹSlug Ĺ
TGF-E
E2 Ĺ
(B)
Smad4

TTF-1

Co-activator

Smad3

Smad3
P P

SNAI1, etc

TTF-1

LMO3, etc

TGF-E
E

Figure 2

TGF-β signaling and TTF-1 in the induction of EMT and MET.
(A) TGF-β signaling induces EMT, whereas TTF-1 induces MET in lung adenocarcinoma
cells. (B) Smad4-independent functions of Smad3 regulated by TTF-1. The Smad3/Smad4
complex interacts with other transcription factors and transcriptional co-activators (or corepressors), and regulates transcription of certain target genes, including SNAI1. In thepresence of TTF-1, Smad3 interacts with TTF-1 and other transcription factors, and regulates
certain other genes, including LMO3 [5].
Transcription factors

We have further investigated the mechanism through which TTF-1 inhibits thefunctions of TGF-β. TTF-1 disrupted the nuclear Smad3/Smad4 complex without affecting
the nuclear localization of phospho-Smad3. Genome-wide analyses by ChIP-seq revealed

that TTF-1 co-localized with Smad3 on the chromatin and modulated the binding patternsof Smad3 throughout the genome. Intriguingly, Smad3 bound to the chromatin with TTF-1,
but not with Smad4, at some Smad3-binding regions, which could be observed

independently of TGF-β stimulation (Figure 2B). Thus, TTF-1 competes with Smad4 forinteraction with Smad3, and the Smad3/TTF-1 complex regulates the transcription ofcertain genes independently of Smad4. These findings propose a new model of Smadsignaling with TTF-1 [5].

Our ChIP-seq analyses in lung adenocarcinoma cells using next generation sequencers

led to identification of new target genes of TGF-β and TTF-1, which appeared to be
involved in cancer cell metabolism and cell growth and survival through cross-talk withother signaling pathways. RNA-binding motif protein 47 (RBM47) is a target of TGF-β inSignaling Diversity in Cancer

117

cells that have undergone EMT. Expression of RBM47 was inhibited by TGF-β in lungcancer cell lines. RBM47 suppressed the expression of cell metabolism-related genes, which
are the direct targets of Nrf2, suggesting tumor-suppressive roles for RBM47 throughsuppression of the Nrf2 activity [6].

Another novel target of TGF-β and TTF-1, identified by the ChIP-seq analyses in lungadenocarcinoma cells, was tuftelin 1 (TUFT1), which turned out to be a key regulator of themTOR complex 1 (mTORC1) signaling pathway [7]. Expression of TUFT1 was induced by
TGF-β stimulation, and TUFT1 induced cell proliferation and motility, leading to tumor
growth and cancer metastasis in mouse models in vivo. Silencing of TUFT1 caused

dispersion of the lysosomes in the cytoplasm, and inhibition of mTORC1 signaling. TUFT1
promoted perinuclear lysosomal localization by interaction with RABGAP1, a GAP for

certain Rab GTPases, suggesting that TUFT1 may also be a potential therapeutic target forlung and some other carcinomas.

We have recently reported a new imaging technology, termed CUBIC-based cancer

Whole-body Profiling of Cancer Metastasis by CUBIC-based Cancer Analysis(CUBIC-Cancer) analysis, which allowed transparantization of animal tissues and wholebody/organ imaging of cancer (collaboration with Prof. Hiroki R. Ueda). By CUBIC-Cancer
analyses, spatiotemporal quantification of metastatic cancer progression at single-cellresolution is possible. Although importance of the EMT in the invasion and intravasation ofcancer cells has been demonstrated, involvement of the EMT in the extravasation of cancer
cells has not been clearly determined. Immunodeficient mice were intravenously injectedwith A549 lung adenocarcinoma cells pre-treated with or without TGF-β. CUBIC-Cancer

analyses clearly showed that colonization of the metastatic cells gradually progressed over

14 days after elimination of the cells at the early time points. Statistical analysis revealed
that TGF-β stimulation significantly increased the number of metastatic foci from 1 to 14

days, demonstrating that EMT promotes not only intravasation but also cell survival andextravasation of cells at metastatic sites (Figure 3) [8].

I would like to thank Prof. Hiroki R. Ueda, the University of Tokyo, for collaboration in

Acknowledgments

CUBIC-Cancer analyses. I also thank all my colleagues at the Department of MolecularPathology, Graduate School of Medicine, the University of Tokyo, for collaboration. This

work was supported by KAKENHI Grants-in-Aid for Scientific Research (S) (15H05774)
and the Yasuda Medical Foundation.

Figure 3
118

K. Miyazono

epithelial cells

mesenchymal
cells

intravasation

invasion

TGF-E
E

ᾐ₶

TGF-E
E

extravasationcolonization

Figure 3

metastatic tumor

EMT in cancer progression. TGF-β signaling induces invasion and intravasation. In addition,
CUBIC-Cancer analyses suggested that TGF-β signaling supports the cell survival andextravasation [8]. E-cadherin expression can be observed in metastatic cells, suggestingthat MET occurs at the metastatic sites.

References
1. Azuma H, Ehata S, Miyazaki H, Watabe T, Maruyama O, Imamura T, Sakamoto T,
Kiyama S, Kiyama Y, Ubai T, Inamoto T, Takahara S, Itoh Y, Otsuki Y, Katsuoka Y,
Miyazono K, Horie S. Effect of Smad7 expression on metastasis of mouse mammary
carcinoma JygMC(A) cells. J Natl Cancer Inst. 2005;97:1734-1746.
2. Horiguchi K, Shirakihara T, Nakano A, Imamura T, Miyazono K, Saitoh M. Role of Ras
signaling in the induction of Snail by transforming growth factor-β. J Biol Chem.
2009;284:245-253.
3. Arase M, Tamura Y, Kawasaki N, Isogaya K, Nakaki R, Mizutani A, Tsutsumi S,
Aburatani H, Miyazono K, Koinuma D. Dynamics of chromatin accessibility during
TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells. Sci Rep.
2017;7:1166.
4. Saito RA, Watabe T, Horiguchi K, Kohyama T, Saitoh M, Nagase T, Miyazono K.
Thyroid transcription factor-1 inhibits transforming growth factor-β-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells. Cancer Res. 2009;69:27832791.

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5. Isogaya K, Koinuma D, Tsutsumi S, Saito RA, Miyazawa K, Aburatani H, Miyazono K. A
Smad3 and TTF-1/NKX2-1 complex regulates Smad4-independent gene expression.
Cell Res. 2014;24:994-1008.
6. Sakurai T, Isogaya K, Sakai S, Morikawa M, Morishita Y, Ehata S, Miyazono K,
Koinuma D. RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor
growth in lung adenocarcinoma. Oncogene. 2016;35:5000-5009.
7. Kawasaki N, Isogaya K, Dan S, Yamori T, Takano H, Yao R, Morishita Y, Taguchi L,
Morikawa M, Heldin CH, Noda T, Ehata S, Miyazono K, Koinuma D. TUFT1 interacts
with RABGAP1 and regulates mTORC1 signaling. Cell Discov. 2018;4:1.
8. Kubota SI, Takahashi K, Nishida J, Morishita Y, Ehata S, Tainaka K, Miyazono K, Ueda
HR. Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep.
2017;20:236-250.

Kohei Miyazono, MD, PhD

1988-1990
1990-1995
1995-2000

2000-present

Assistant of Professor, Third Department of Internal
Medicine, The Univ. of Tokyo
Assistant Member, Ludwig Institute for Cancer
Research, Uppsala, Sweden
Member and Chief, Department of Biochemistry,
The Cancer Institute, Japanese Foundation for Cancer
Research
Professor, Department of Molecular Pathology,
Graduate School of Medicine, The Univ. of Tokyo

120

THE DEPENDENCE RECEPTOR PARADIGM:FROM AN ORIGINAL MECHANISM OF CELL DEATH TO
CLINICAL TRIAL

Patrick Mehlen

Léon Bérard Comprehensive Cancer Center
28 Rue Laennec, 69008, Lyon, France(patrick.mehlen@lyon.unicancer.fr)

A few years ago, an original concept of cell biology was proposed: whereas the classic

dogma postulates that transmembrane receptors are inactive unless bound by their specificligand, they proposed that some receptors may be active not only in the presence of theirligand, but also in their absence. In this latter case, the signaling downstream of theseunbound receptors leads to apoptosis. These receptors were consequently named“dependence receptors”, as their cell expression renders the cell’s survival dependent on
the presence in the cell environment of its respective ligand [1]. To date, we have identified
or have been involved in the identification of most known dependence receptors.

Beyond the basic interest of studying a receptor capable of transducing two

antagonizing signals –i.e., one "positive" in the presence of ligand leading to celldifferentiation, proliferation or migration and one "negative" in the absence of ligand

leading to cell suicide-, we have proposed that this dual function could lead these receptors
to have key roles both during embryonic development and in the regulation oftumorigenesis.

In the context of their involvement in embryonic development, we hypothesized thatthe pro-apoptotic activity of these dependence receptors is crucial for the development of
the nervous system as a mechanism to “authorize” neuronal guidance, migration or

localization in settings of ligand presence. Along this line, we found that the Sonic

Hedgehog (Shh) receptor Patched is a dependence receptor and its ability to induce

apoptosis in the absence of Shh is crucial for the adequate development of the neural tube
[2,3]. We also showed that the netrin-1 receptors DCC and UNC5H regulate death/survivalof specific neurons during the development of the nervous system [4,5]. This implication isSignaling Diversity in Cancer

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not limited to the developing nervous system as we showed the importance of theapoptosis induced by UNC5H in the formation of blood vessels - angiogenesis- [6,7].

In the context of cancer, we have formulated the hypothesis that these receptors aretumor suppressors that would limit tumor progression by inducing apoptosis of tumor
cells outside of settings of ligand accessibility/availability [8,9]. We are particularlyinterested in the receptors that bind netrin-1 –i.e., DCC and UNC5H-. We showed that both
DCC and UNC5H are dependence receptors in cancer cells: whereas in the presence of theirligand netrin-1, they transduce classic “positive” signals, in the absence of netrin-1, they
actively trigger apoptosis [1,7,10-14]. Interestingly, DCC and UNC5H are considered astumor suppressors because their expression is lost in many cancers [8,15,16], suggestingthat the presence of these receptors is a constraint for tumor progression. This was actuallyformally proven by showing that in mice the invalidation of UNC5H3, the overexpressionof netrin-1 in the digestive tract or the specific inactivation of the pro-apoptotic activity of
DCC resulted, in agreement with the dependence receptors paradigm, in a similar
tumorigenesis [17-19]. Thus, aggressive cancers that develop are cancers for which thedependence receptor pathways are blocked through mechanisms including the genetic lossof these receptors (Figure 1).

However, a loss of dependence receptors is not always the selective advantage used bytumor cells to escape this survival dependence on the presence of the ligand. Indeed, we
showed that in many cancers such as metastatic breast cancer, lung cancer, neuroblastoma,

lymphoma or melanoma [20-24] tumor cells acquire the preferred autocrine expression of
netrin-1. This selective advantage for the tumor is much more appealing in terms ofputative therapeutic strategy. Indeed, the titration of the ligand by a molecule thatinterferes with the interaction between a dependence receptor and its ligand should lead to

Figure 1

The dependence receptor paradigm and the associated therapeutic strategy.

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P. Mehlen

tumor cell death (Figure 1). Along this line, we showed that titration of netrin-1 by a drugcandidates allows tumor cell death in vitro and triggers regression of tumors andmetastases in mice [20-24]. Of interest, this gain of ligand is probably not limited to netrin-1

but may possibly be extended to the other ligands of other dependence receptors. Indeed,

we recently showed that an autocrine expression of NT-3, a ligand of the TrkC dependence
receptor is detected in various cancer types and can be targeted to trigger tumor growthand metastasis inhibition [25]. A similar observation was made for the pairSema3E/PlexinD1 where Sema3E, the ligand of the dependence receptor PlexinD1 is up-

regulated in breast cancer [26]. Of interest, this gain of ligand is not only promoting
survival of the cancer cells but also in some case of the endothelial cells as recently

demonstrated with Jagged 1 being up-regulated in lung cancer as a mechanism to block the
pro-death activity of Notch3 behaving as a dependence receptor in this settings [27]. We

thus have been trying to develop drugs based on the interference on the interaction
between dependence receptors and their ligands as anti-cancer strategies. A humanizedanti-netrin-1 antibody has been preclinically developed [28] and the first human trial(Phase I) using an agent interfering between netrin-1 and its receptors has started in 2017 in

all solid tumors (https://clinicaltrials.gov/ct2/show/NCT02977195). Thus, from a basic
cell biology concept, we hope to be able to provide evidence that targeting
ligand/dependence receptor interaction is beneficial to treat cancer.
References
1. Mehlen P., Rabizadeh S., Snipas S.J., Assa-Munt N., Salvesen G.S., and D.E Bredesen(1998). The DCC gene product induces apoptosis by a mechanism requiring receptor
proteolysis. Nature 395: 801-804.
2. Thibert C., Teillet M.A., Lapointe F., Mazelin L., LeDouarin, N., and P. Mehlen (2003).
Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog. Science
301:843-6.
3. Mille F., Thibert C., Fombonne J., Rama N., Guix C., Hayashi H., Corset V., Reed JC, and
P. Mehlen (2009). The Patched dependence receptor triggers apoptosis through a
DRAL-caspase-9 complex. Nat Cell Biology 11, 739-46.
4. Furne C., Rama N., Corset V., Chédotal A. and P. Mehlen (2008). Netrin-1 is a survival factor during commissural neuron navigation. Proc. Natl. Acad. Sci. USA 105, 1446514470.
5. Tang X., Jang S.W., Okada M., Chan C.B., Feng Y., Luo S.W., Hong Y., Liu Y., Rama N.,
Xiong W.C., Mehlen P. and K. Ye (2008). Netrin-1 Mediates Neuronal Survival ThroughPIKE-L Interaction With the Dependence Receptor UNC5B. Nature Cell Biology 10,698-706.

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6. Castets M., Coissieux M.M., Delloye-Bourgeois C., Bernard L., Delcros J.G, Bernet A.,
Laudet V. and P. Mehlen (2009). Inhibition of endothelial cell apoptosis by netrin-1 during
angiogenesis. Dev. Cell 16, 614-620.
7. Guenebeaud C., Goldschneider D., Castets M., Guix C, Chazot G., Delloye-Bourgeois
C., Eisenberg-Lerner A., Shohat G., Zhang M., Laudet V., Kimchi A., Bernet A. and P.
Mehlen (2010). The dependence receptor UNC5H2 triggers apoptosis via PP2A-mediateddephosphorylation of DAP kinase. Mol. Cell 40:863-76.
8. Mehlen P., Puisieux A (2006). Metastasis: a question of life or death. Nature Rev.
Cancer. 6, 449-458.
9. Mehlen P., Delloye-Bourgeois C., and A. Chedotal (2011). Novel roles for Slits andnetrins: axon guidance cues as anti-cancer targets? Nature Rev. Cancer. 11:188-97.
10. Corset V., Tuyen Nguyen-Ba-Charvet K., Forcet C., Moyse E., Chédotal A. and P.
Mehlen (2000). Netrin-1 induced growth cone attraction and cAMP production is mediated via interaction with the Adenosine A2b receptor. Nature 407, 747-450.
11. Forcet C., Stein E., Pays L., Corset V., Llambi F., Tessier-Lavigne M., and P. Mehlen(2002). DCC-dependent MAPK activation is required for netrin-1 mediated axon outgrowth. Nature 417, 443-448.
12. Llambi F., Causeret F., Bloch-Gallego E., and P. Mehlen (2001). Netrin-1 acts as a survival
factor via its receptors UNC5H and DCC. EMBO J. 20, 2715-2722.
13. Llambi F., Calheiros F., Gozuacik D., Guix C., Pays L., Del Rio G., Kimchi A. and P.
Mehlen. (2005). The dependence receptor UNC5H2 mediates apoptosis through DAP
kinase. EMBO J., 24, 1192-1201.
14. Dominici C., Moreno-Bravo JA., Puiggros SR., Rappeneau Q., Rama N., Vieugue P.,
Bernet A., Mehlen P., Chédotal A. (2017). Floor-plate-derived netrin-1 is dispensable forcommissural axon guidance. Nature. 545(7654):350-354. doi: 10.1038.
15. Gibert B and P. Mehlen (2015). Dependence receptors and cancer: addiction to trophic ligands. Cancer Research. 75(24):5171-5. doi: 10.1158/0008-5472.CAN-14-3652.
16. Thiébault K., Mazelin, L., Pays L., Joly., M.O., Scoazec J.Y., Saurin J.C., Romeo G., and P.
Mehlen (2003). The netrin-1 receptors UNC5H are putative tumor suppressors controllingcell death commitment. Proc. Natl. Acad. Sci. USA 7, 4173-4178.
17. Mazelin L., Bernet A., Bidaud-Bonod C., Pays L., Arnaud S., Gespach C., Bredesen D.E.,
Scoazec J.Y., and P. Mehlen. (2004). Netrin-1 controls colorectal tumorigenesis by regulating apoptosis. Nature 431, 80-84.
18. Bernet A., Mazelin L., Coissieux MM., Ackerman SA, Scoazec J.Y and P. Mehlen. (2007).
Inactivation of the netrin-1 receptor UNC5C is associated with tumor progression incolorectal malignancies. Gastroenterology 133, 1840-1848.

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P. Mehlen

19. Castets M., Broutier L., Molin Y., Brevet M., Chazot G., Gadot N., Paquet A., Mazelin L.,
Jarrosson-Wuilleme L., Scoazec J.-Y., Bernet A. and P. Mehlen (2011). DCC constrains
tumor progression via its dependence receptor activity. Nature. 482(7386):534-7.
20. Fitamant J., Guenebeaud C., Coissieux M.M, Guix C., Treilleux I., Scoazec J.Y., Bachelot T.,
Bernet A., and P. Mehlen (2008). Netrin-1 expression confers a selective advantage fortumor cell survival in metastatic breast cancer. Proc. Natl. Acad. Sci. USA 105, 4850-5.
21. Delloye C., Brambilla E., Coissieux M.M., Guenebeaud C, Pedeux R., Firlej V., Cabon F.,
Brambilla C., Mehlen P. and A. Bernet (2008). Interference with Netrin-1 and Tumor
Cell Death in Non–Small Cell Lung Cancer. J. Natl. Cancer Inst. 101,237-247.
22. Delloye-Bourgeois C., Fitamant J., Paradisi A., Cappellen D., Douc-Rasy S., RaquinMA., Stupack D, Nakagawara A., Combaret V., Puisieux A., Couanet-Valteau D.,
Bénard J., Bernet A. and P. Mehlen (2009b). Netrin-1 acts as a survival factor for aggressive neuroblastoma. J. Exp. Medicine 206, 833-847.
23. Broutier L., Creveaux M., Vial J., Tortereau A., Chazot G., MacCarron M., Léon S,
Pangault C., Gadot N., Colombe A., Boulland ML., Blachier J., Marie J., TraverseGlehen A., Donzé O., Chassagne-Clément C., Salles G., Tarte K., P. Mehlen* and M.
Castets* (2015). Targeting netrin-1/DCC interaction in Diffuse Large-B and Mantle Cell
Lymphoma. EMBO Mol Med. 8(2):96-104. doi: 10.15252.
24. Boussouar A., Tortereau A., Manceau A., Gadot N., Vial J., Neves N., Larue L., LeboeufC., Janin A., and Patrick Mehlen. The pair netrin-1/DCC is causally implicated inmelanoma progression. Submitted
25. Bouzas-Rodriguez J., Ruben Cabrera J., Delloye-Bourgeois C., Ichim G., Delcros J.G.,
Raquin M.A., Rousseau R., Combaret V., Bénard J., Tauszig-Delamasure, S., and P.
Mehlen (2010). NT-3 production promotes neuroblastoma cell survival by inhibiting
TrkC-induced apoptosis. J. Clin Investigation, 120(3):850-8.
26. Luchino J, Hocine M, Amoureux MC, Gibert B, Bernet A, Royet A, Treilleux I, Lécine P,
Borg JP, Mehlen P, Chauvet S, Mann F. (2013). Semaphorin 3E Suppresses Tumor Cell
Death Triggered by the Plexin D1 Dependence Receptor in Metastatic Breast Cancers.
Cancer Cell. 24(5):673-85.
27. Lin S., Negulescu A., Bulusu S., Gibert B., Delcros JG., Ducarouge B., Rama N., Gadot N.,
Treilleux I., Saintigny P., Meurette O., Mehlen P. (2017). Non-canonical NOTCH3 signalling limits tumour angiogenesis. Nat Commun. 8:16074. doi: 10.1038/ncomms16074.
28. Grandin M., Meier M., Delcros JG., Nikodemus D., Reuten R., Patel TR, Goldschneider D.,
Orriss G, Okun N., Boussouar A., Abes R, Dean Y., Neves D., Bernet A., Depil S.,
Schneiders F., Poole K., Dante R., Koch M, P. Mehlen* and J. Stetefeld* (2015).
Structural decoding of the Netrin-1-UNC5 interaction and its therapeutical implications incancers. Cancer Cell. 29(2):173-85. doi: 10.1016.

Signaling Diversity in Cancer

Patrick Mehlen, PhD

1992-1995
1997-1998
1998-2004
2004-2006
2007-20112009-20142007-present
2011-present
2011-present
2011-present
2013-present
2014-present
2017-present

125

PhD, Thesis, University Claude Bernard
Sabbatical within the Burnham Institute for Medical
Research, program on "Aging and Cell Death" - Pr. D.
E. BredesenGroup leader of the "Apoptosis and Differentiation"
Laboratory - CNRS UMR5534, CGMC
Research Director of the unit "Apoptosis, Cancer andDevelopment" - CNRS FRE2870, CGMC
Director of the laboratory "Apoptosis, Cancer andDevelopment" - CNRS UMR5238, Centre Léon Bérard
Co-Director of the Institute for Clinical Science,Centre Léon Bérard
Adjunct Professor at the Buck Institute for Age
Research
Head of the Tumor Escape Department,
Research Cancer Center of Lyon
Deputy Director of The Research Cancer Center of
Lyon
Director of the Laboratory of Excellence DEVweCAN
Director of Research “Classe Exceptionnelle”, CNRS
Director of the Translational Research and Innovation atCentre Léon Bérard
Director of the Institut Rabelais for Interdisciplinary
Research on Cancer

126

ASK FAMILY KINASES IN STRESS SIGNALING AND
CANCER

Hidenori Ichijo

Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1 Bunkyo-ku, Tokyo 113-0033, Japan(ichijo@mol.f.u-tokyo.ac.jp)

Stress-responsive signaling cascades function as a rheostat for appropriate cellularresponses; therefore, their dysfunction culminates in tumor initiation and progression [1].
The most important stress-responsive signaling cascades are mitogen-activated protein
kinase (MAPK) pathways, composed of extracellular signal-regulated kinase (ERK), p38

MAPK, and c-Jun N-terminal kinase (JNK). Apoptosis signal-regulating kinase 1 (ASK1) is
a Ser/Thr kinase that is activated in response to various stressors, such as cytokines andoxidative stress and is responsible for activation of p38 and JNK [2]. Although ASK1

reportedly exerts both tumor-promoting and tumor-suppressive effects on tumor
progression [3], it remains unclear whether ASK1 modulates tumor metastasis and/or

serves as an upstream regulator of p38 and JNK. In our presentation, we reported drastic
attenuation of lung tumor metastasis in ASK1 knockout mice [4]. Intriguingly, tumor

metastasis was also attenuated in wild-type mice that received bone marrow from ASK1

knockout mice but not myeloblast-specific (i.e., granulocytes, monocytes, andmacrophages) ASK1 knockout mice. We, therefore, focused on platelets among the non-myeloblastic bone marrow-derived cell types. Importantly, platelet-specific ASK1 knockout

mice exhibited attenuated tumor metastasis, as well as unstable hemostasis and impaired
thrombosis, indicating the involvement of platelet-intrinsic ASK1. Additional analysis of invitro platelet aggregation suggested that ASK1 regulates adenosine diphosphate (ADP)signaling in platelets. ADP signaling is mediated by two types of G protein-coupled

receptors (GPCRs), P2Y 1 and P2Y 1 2 , in platelets [5]. We newly identified thephosphorylation site (Thr345 in mice) of P2Y12 as a target of ASK1-JNK/p38 axis. This site
was found to be located at the C-terminal putative postsynaptic density 95/discs

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large/zonula occludens-1 (PDZ)-binding motif and is required for sustained Akt activation.
Hence, the results in our study [4] offer insight into the positive regulation of ADP
signaling through P2Y12 phosphorylation and ASK1-mediated MAPK signaling in platelets.

Further study will be needed to investigate the precise molecular mechanism by which

the newly identified P2Y12 phosphorylation site affects P2Y12 function, as well as otherdownstream pathways, apart from Akt (e.g., inhibition of adenylate cyclase). In general,
GPCR phosphorylation by GPCR kinases (GRKs) leads to desensitization, arrestin
recruitment, and internalization followed by either receptor recycling or degradation [6].

P2Y12 was shown to be phosphorylated by GRK2 and GRK6, resulting in desensitization;

however, the phosphorylation sites targeted by these kinases have not been identified [5].
Thus, GRKs may also phosphorylate Thr345 (in mice) or other sites of P2Y12. It would beinteresting to examine the relationship between ASK1-JNK/p38 axis and GRKs in theregulation of P2Y12 phosphorylation and function. Moreover, it remains unknown whether
P2Y12 Thr345-phosphorylation is relevant to arrestin recruitment, receptor internalization,

and/or resensitization, similar to the bleeding-patient mutation (P341A) at thecorresponding PDZ motif of human P2Y12 [5].

Of note, ASK1 knockout mice that received bone marrow from wild-type mice also

showed attenuated tumor metastasis, suggesting that ASK1 may regulate tumor metastasis

in X-ray-resistant recipient cells. We reported in this symposium that ASK1 in endothelial
cells (ECs), which are one of the X-ray-resistant cells, also facilitates tumor lung metastasis.
Hence ASK1 may mediate adhesion of tumor cells to ECs followed by tumor cell128

H. Ichijotransmigration and/or EC death induced by direct interaction with tumor cells.

Furthermore, we also observed promising results suggesting that metastasis-suppressive
immunity is enhanced in ASK1 knockout mice compared with wild-type mice. The

responsible cell types and the precise regulatory mechanism of tumor metastasis therein
are now under investigation.

In sum, our findings reveal a previously unidentified function of MAPK pathway in

GPCR fine-tuning in platelets [4]. We hope this knowledge will greatly contribute to thedevelopment of novel molecular mechanism-based strategies against tumor metastasis andthrombosis.

References
1. Chaffer CL, Weinberg RA. A Perspective on Cancer Cell Metastasis. Science 2011;
331:1559-64.
2. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K,
Miyazono K, Gotoh Y. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 1997; 275:90-4.
3. Ryuno H, Naguro I, Kamiyama M. ASK family and cancer. Adv. Biol. Regul. 2017, inpress.
4. Kamiyama M, Shirai T, Tamura S, Suzuki-Inoue K, Ehata S, Takahashi K, Miyazono K,
Hayakawa Y, Sato T, Takeda K, Naguro I, Ichijo H. ASK1 facilitates tumor metastasis
through phosphorylation of an ADP receptor P2Y12 in platelets. Cell Death Differ. 2017, inpress.
5. Cunningham MR, Aungraheeta R, Mundell SJ. Pathophysiological consequences ofreceptor mistraffic: Tales from the platelet P2Y12 receptor. Mol. Cell Endocrinol. 2017;
5:74-81.
6. Ritter SL, Hall RA. Fine-tuning of GPCR activity by receptor-interacting proteins. Nat.
Rev. Mol. Cell Biol. 2009; 10:819-30.

Signaling Diversity in Cancer

Hidenori Ichijo, DDS, PhD

1990-1993
1992-1995
1995-1997
1998-2003

2002-present

129

Postdoctoral Fellow, The Ludwig Institute for Cancer
Research, Biomedical Center, Uppsala, Sweden
Research assistant, Department of Oral Pathology,
Tokyo Medical and Dental University (TMDU)
Associate, Department of Biochemistry,
The Cancer Institute, Tokyo
Professor, Laboratory of Cell Signaling,
Graduate School, TMDU
Professor, Laboratory of Cell Signaling,
Graduate School of Pharmaceutical Sciences,
The University of Tokyo130

CONCLUDING REMARKS

Hitoshi Nakagama

National Cancer Center Research Institute5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan(hnakagam@ncc.go.jp)

The 48th International Symposium of the Princess Takamatsu Cancer Research Fund has

provided cancer researchers here with a great opportunity to discuss and decipher the

biological features of cancer from the aspect of cancer-host crosstalk and its complexity innature.

Cancer arises by having genetic and/or epigenetic alterations caused by exposure tovarious environmental insults of either endogenous or exogenous origins. As aconsequence of the evolutional process, cancer cells further acquire an invasive andmetastatic nature after the stepwise accumulation of multiple genetic and epigenetic

alterations. Heterogeneity (complexity) and plasticity are other characteristic features ofcancer cells, and acquisition of these phenotypic characters in cancer cells is considered tobe achieved, in part, by complex interactions between cancer cells and other cellularsurrounding components, including fibroblasts, endothelial cells of blood and lymphatic

vessels, immune cells and macrophages. Direct or indirect interaction with metabolites ofintestinal microbiota could also be involved in initiation and promotion of cancer cells.
Drastic therapeutic outcomes of immune checkpoint inhibitors, for example, have proved
the substantial contribution of a variety of immune cells for development of human cancers
in an in vivo setting. These facts surpass our previous comprehension regarding thebiological nature of cancer, and nowadays it is conceivable that complex cellularinteractions within cancer tissues in vivo are indispensable for survival and progression ofcancer cells.

Cancer is now defined as a systemic disease that occurs and develops under complex

cellular networks in vivo, and understanding these complex cellular networks from various

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131

aspects by means of comprehensive and integrative approaches would enable us todecipher the complex nature of cancer. In light of the importance of various types of cancer
networks, we created a program for the 48th International Symposium of the Princess

Takamatsu Cancer Research Fund under the title of “Complexity in Cancer-host Crosstalk”.

Throughout the symposium, a considerable number of important subjects on cancer-hostcrosstalk were presented, and fruitful and constructive discussions were exchanged on thisoccasion. Some of the representative issues that have been extensively discussed during the
symposium can be summarized as follows.

Heterogeneity in cancer cells is widely known to be evolutionally achieved through the

1. Heterogeneity in cancer cells and cancer stem cells.

process of tumorigenesis, and pathogenesis of heterogeneity was also revealed to be acomplex and diverse process. Single cell transcriptome analysis demonstrated the presenceof heterogeneity in colon cancer stem cells as well. Cell-cell interaction in tumors wasshown to play a pivotal role in stomach carcinogenesis using genetically-engineered
compound animal models in mice. Diverse biological functions of HIF-1 and metabolic

reprogramming by autophagy adaptor p62 was also presented. TGF-β-induced quiescentstem cells at the perivascular region were reported to be present in squamous cell

carcinoma (SCC), and these heterologous cells could be responsible for acquisition ofresistance to chemotherapy.

Functional crosstalk among various intracellular signals could also confer cells with

2. Signal diversity in cancer cells.

differential responses to extra-cellular stimuli. ASK1/ASK2 signals regulate tumorigenesis
via apoptosis and innate immunity. High insulin levels could reactivate PI3K in tumors
despite the presence of a PI3K inhibitor. Therefore, a ketogenic diet along with a PI3K

inhibitor could be more potent in suppressing tumor growth. TGF-β and TTF-1 reciprocallycooperate in epithelial mesenchymal transition (EMT), and TTF-1 disrupts the
Smad3/Smad4 complex. Dependence receptors (DRs) are regulators of tumor growth andmetastasis, and Netrin-1, a DR ligand, was shown to be up-regulated in cancer-associatedfibroblast (CAF).

Interaction between epithelial tumor cells and mesenchymal stroma cells is considered

3. Stromal – epithelial tumor cell interactions

to play key roles in tumor development, as described earlier. Activation of hepatic stellate

cells in the liver plays a key role in development of non-alcoholic steatohepatitis (NASH).

Intriguingly, inactivation (senescence) of hepatic stellate cells was also reported to be

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H. Nakagama

involved in a subset of liver carcinogenesis related to fatty liver. Crucial roles of CADM1 intumor epithelial – stromal cell communications, tumor-stromal interaction by MLC
phosphorylation and metabolic reprogramming by p62 downregulation in cancer

development were also reported in this symposium. In addition, MKL1, which modulates
actin dynamics, was demonstrated to be involved in adipocyte-CAF transition. Presence ofheterogeneity in CAFs, which was revealed by single cell transcriptome analysis, was also
beautifully demonstrated. More detailed mechanistic analyses are warranted in the future

to dissect molecular mechanisms involved in epithelial tumor cell-mesenchymal fibroblast
interaction.

As evidently proved by beneficial clinical outcomes of immunocheckpoint inhibitors,

4. Immune microenvironment:such as anti-PD-1 and anti-PDL-1 antibodies, the immune microenvironment plays a keyrole in regulating tumor cell survival. Many novel mechanisms involved in the
immunologic microenvironment of cancers were presented during the symposium. IgA+

plasmocytes that express PDL-1 and IL-10, NK activation by CADM1, chemotherapyinduced immune evasion through HIF-1, and immune-modulation by CAFs were alsopresented. Metastatic stem cells (MetSCs) were reported to stay in a slow-cycling state andthereby evade immune surveillance.

Furthermore, immune-pharmacogenomic approaches to characterize T-/B-cell

repertoires in tumor tissues, comprehensive genetic screens for identification of
immunotherapy targets, dynamic changes in neo-antigen/TCR profiles between ductal

carcinoma in situ (DCIS) and invasive ductal breast carcinoma (IDC), and switching to amore suppressive immune environment in IDC were demonstrated in an attractive andenthusiastic manner. As for other cellular components related to immune reaction of cancer
cells, T-cell suppressive neutrophils, activation of a myeloid differentiation in bonemarrows, G-CSF-directed expansion and differentiation of myeloid lineage hematopoietic
stem cells (HSCs), and dynamic changes in leukocyte and macrophage populations werealso presented in this symposium. Future explosive expansion of this field of cancer
research is further warranted to dissect the complexity of tumor cell-immune cell networks
and to provide us with novel therapeutic modalities.

Metabolites from microbiota could work as novel factors to modulate or to intermediate

5. Gut microbiota and their metabolites

epithelial cell transformation in several organs, such as the gastrointestinal tract organs andthe liver as partly detailed in the previous section. Moreover, understanding host-microbe
chemical interaction, a crossroads between gut microbiome and cancer immune

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133

surveillance, dysbiosis, T-cell response against gut microbiome could be responsible foracquisition of novel characteristic phenotypes in cancer cells, such as resistance to anti-PD1/PDL-1 antibody treatment.

Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition

6. EMT-MET transition, invasion, metastasis and metastatic stem cells(MET) are essential phenotypic changes in cancer cells for their invasion and metastasis.

Transcriptional regulation of MMPs and L1CAM by HIF-1 and also the MAPK pathway

were reported to be involved in this process. Modulation of metastasis and cell plasticity by
gp130-dependent- or –independent activation of Stat3 and biological roles of exosomes invarious aspects of metastatic events (namely; dormancy, pre-metastatic niche formation,

extravasation, and dissemination) were presented. Mouse models of latent metastasis bymetastatic stem cells (MetSCs) was quite a novel and exciting finding. ASK1 in platelets
and vascular endothelial cells were demonstrated to coordinately promote lung metastasis.

Since cancer-host crosstalk was revealed to be highly complex, novel analytical

7. Novel approaches to clarification of cancer network

methodologies need to be invented. A computational approach to dissect complex

networks, molecular imaging using a next-generation single-cell mass cytometry with highresolution, and an in vivo RNAi screening have been introduced. In vivo imaging of

p21/p16 and MetSCs, and also a new in vivo imaging technology with transparentization

seem to be two of the most promising and attractive technologies for resolution of cancerhost crosstalk in vivo. Further technical innovation in this field is eagerly awaited.

Throughout the symposium, we were extremely delighted and stimulated to have been

exposed to many more innovative researches, approaches and technologies to decipher the“Complexity in Cancer-host Crosstalk”, which is one the most complex natures of cancer invivo. Comprehensive understanding of the complex and heterogeneous nature of cancer
pathogenesis could lead us to conquering this enemy and future implementation of

personalized precision medicine, especially in cancer detection at early stages, effective
treatment and prevention of metastasis.

Lastly, on behalf of the organizers, I would like to express my sincere thanks to the

Princess Takamatsu Cancer Research Fund for hosting this invaluable occasion, and also

thank our distinguished speakers and guests for bringing us great success and new ideas.

Finally, I as a chairman of the organizing committee would like to see all the speakers andparticipants again in the not-too-distant future to discuss and tackle these difficult subjects.

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H. Nakagama

Hitoshi Nakagama, MD, DMSc

1982
1988
1991
1995

1997
2007
2011
2016-present

University of Tokyo, Faculty of Medicine, MD
Research Associate, University of Tokyo, Faculty of
Medicine
Postdoctoral Fellow, Center for Cancer Research,
Massachusetts Institute of Technology, USA
Section Head, Carcinogenesis Division, National
Cancer Center Research Institute (NCCRI)
Chief, Biochemistry Division, NCCRI
Deputy Director, NCCRI
Director, NCCRI
President, National Cancer Center