bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the β-Catenin/T-Cell–Specific Transcription Factor/Lymphoid Enhancer Factor-1 Pathway by Plasminogen Activators in ECV304 Carcinoma Cells
Authors: Maupas-Schwalm Françoise , Robinet Catherine , Augé Nathalie , Thiers Jean-Claude , Garcia Virginie , Cambus Jean-Pierre , Salvayre Robert , Nègre-Salvayre Anne
Published: June 2022

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. β-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non–serine-threonin phosphorylated, nonubiquitinated) form of β-catenin in ECV304 carcinoma cells. tPA-dependent β-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of β-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and β-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. β-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3β through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of β-catenin/T-cell–specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. β-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the β-catenin pathway was triggered by amino-terminal fragment, the NH2-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the β-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.
Alendronate Inhibits Intraperitoneal Dissemination in In vivo Ovarian Cancer Model
Authors: Hashimoto Kae , Morishige Ken-ichirou , Sawada Kenjiro , Tahara Masahiro , Kawagishi Rikako , Ikebuchi Yoshihide , Sakata Masahiro , Tasaka Keiichi , Murata Yuji
Published: June 2022

Ovarian cancer is characterized by diffuse peritoneal carcinomatosis and often by large volumes of ascites. We previously reported that alendronate, a nitrogen-containing bisphosphonate, inhibited ovarian cancer cell migration by attenuating the activation of Rho through inhibiting the mevalonate pathway. However, questions remain about the ability of alendronate to inhibit the invasiveness of cancer cells to the adherent tissues and the growth of disseminated ovarian cancer in vivo. We established an in vivo ovarian cancer model with i.p. carcinomatosis in athymic immunodeficient mice. In the prevention model, in which alendronate administration started from the day after tumor inoculation, alendronate prevented the stromal invasion, reduced the tumor burden, and inhibited ascites accumulation. Histologic observation revealed that alendronate treatment decreased the stromal invasion of the i.p. tumor while inhibiting the metalloproteinase-2 activity in ascites. This antitumor effect might result from the inhibition of cancer cell migration and proteolytic activity. In the treatment model, in which alendronate was given from 10 days after tumor inoculation when macroscopic tumors are already implanted in the peritoneum, the antitumor effect was weaker but still significant. Furthermore, alendronate administration decreased the serum CA-125 levels of mice bearing disseminated ovarian cancer compared with those of nontreated mice. The potent effects of alendronate in reducing stromal invasion, tumor burden, and ascites suggest that it will be of value in regimens for treatment of women with ovarian cancer.
Effects of Hypoxia on Radiation-Responsive Stress-Activated Protein Kinase, p53, and Caspase 3 Signals in TK6 Human Lymphoblastoid Cells
Authors: Samuni Ayelet M. , Kasid Usha , Chuang Eric Y. , Suy Simeng , DeGraff William , Krishna Murali C. , Russo Angelo , Mitchell James B.
Published: June 2022

Despite significant evidence of a role of hypoxia in cellular resistance to ionizing radiation–induced toxicity, the underlying molecular mechanisms remain unclear. This study focused on the influence of hypoxia on radiation-induced signals in TK6 human lymphoblastoid cells. Hypoxic (<10 ppm oxygen) and aerobic cells were exposed to equilethal doses of ionizing radiation, radiation dose ratio, 3:1 (hypoxia:air). Hypoxia alone or radiation treatment under aerobic or hypoxic conditions led to increased levels of phospho-p44/42 mitogen-activated protein kinase. Levels of phospho-p38 mitogen-activated protein kinase did not change as a result of either hypoxia or irradiation. Hypoxia alone had no effect on expression of phospho-stress-activated protein kinase (SAPK), wild-type p53, or cleaved caspase 3. Irradiation under aerobic conditions resulted in an increase in the phospho-SAPK signal, whereas hypoxia suppressed the irradiation-induced increase in the level of phospho-SAPK. Both hypoxic and aerobic cells showed increases in p53 levels in response to radiation. Hypoxia blocked radiation-induced cleavage of caspase 3 and poly-ADP-ribose polymerase. Irradiation of aerobic and hypoxic TK6 cells using 6 and 18 Gy, respectively, resulted in a similar and significant increase in fraction of apoptotic cells within 24 hours postirradiation. In contrast, basal levels of apoptosis were observed at 24 hours postirradiation in aerobic and hypoxic NH32 cells, a p53 null derivative of TK6 cells. These results suggest that radiation-induced apoptosis under hypoxia occurs independent of phospho-SAPK and caspase 3, and the p53 response is an obligatory apoptotic signal in TK6 cells.
Retinoic Acid Inhibits the Proliferative Response Induced by CD40 Activation and Interleukin-4 in Mantle Cell Lymphoma
Authors: Guidoboni Massimo , Zancai Paola , Cariati Roberta , Rizzo Silvana , Dal Col Jessica , Pavan Alessandro , Gloghini Annunziata , Spina Michele , Cuneo Antonio , Pomponi Fabrizio , Bononi Antonio , Doglioni Claudio , Maestro Roberta , Carbone Antonino , Boiocchi Mauro , Dolcetti Riccardo
Published: June 2022

Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here, we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [3H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G0-G1 together with a marked up-regulation of p27Kip1 by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21Cip1 inhibitor was also up-regulated by RA in Granta 519 cells, whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27Kip1 was bound to cyclin D1/cyclin-dependent kinase 4 complexes, probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor α cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably, RA isomers, and particularly 9-cis-RA, also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting.
Inhibition of Glycolysis in Cancer Cells: A Novel Strategy to Overcome Drug Resistance Associated with Mitochondrial Respiratory Defect and Hypoxia
Authors: Xu Rui-hua , Pelicano Helene , Zhou Yan , Carew Jennifer S. , Feng Li , Bhalla Kapil N. , Keating Michael J. , Huang Peng
Published: June 2022

Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser112, relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently induced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications.
Interactions between Prostaglandin E2, Liver Receptor Homologue-1, and Aromatase in Breast Cancer
Authors: Zhou Jiong , Suzuki Takashi , Kovacic Agnes , Saito Ryoko , Miki Yasuhiro , Ishida Takanori , Moriya Takuya , Simpson Evan R. , Sasano Hironobu , Clyne Colin D.
Published: June 2022

Local synthesis of estrogens within breast adipose tissue by cytochrome P450 aromatase contributes to the growth of postmenopausal breast cancers. One of the major stimulators of aromatase expression in breast is prostaglandin E2 (PGE2) derived from tumorous epithelium and/or infiltrating macrophages. Recently, the orphan nuclear receptor, liver receptor homologue-1 (LRH-1), has also been shown to regulate aromatase expression in breast adipose tissue. We therefore examined the expression of, and correlations between, aromatase and LRH-1 mRNA in a panel of breast carcinoma tissues and adjacent adipose tissue. LRH-1 mRNA expression was low in normal breast tissue but markedly elevated in both breast carcinoma tissue and adipose tissue surrounding the tumor invasion (thereby paralleling aromatase expression). Laser capture microdissection localized the site of LRH-1 expression to tumor epithelial cells but not to intratumoral stromal cells. A strong correlation between LRH-1 and aromatase mRNA levels was observed in tumor-containing adipose tissue but not in tumor tissue. Ectopic expression of LRH-1 in primary human adipose stromal cells strongly activated endogenous aromatase mRNA expression and enzyme activity. Finally, treatment of adipose stromal cells with PGE2 induced expression of both LRH-1 and aromatase. We suggest that PGE2 derived from breast tumor tissue may increase aromatase expression in the surrounding adipose stroma in part by inducing LRH-1 in these cells. The roles of LRH-1 in breast cancer proliferation merit further study.
Genomic Amplification of Orthodenticle Homologue 2 in Medulloblastomas
Authors: Boon Kathy , Eberhart Charles G. , Riggins Gregory J.
Published: June 2022

To better understand the genetic basis of medulloblastoma development, we sought genomic amplifications and deletions in these tumors using digital karyotyping in combination with expression analysis. Five medulloblastoma genomes were karyotyped by sequencing an average of 195,745 genomic DNA tags for each analysis. Tags were tallied at unique positions and mapped to the human genome to determine DNA copy numbers in high resolution along each chromosome. Genomic alterations normally associated with medulloblastomas, including MYC amplification and isochromosome 17q, were easily detected. Surprisingly, analysis of only five genomes revealed novel amplicons on chromosome 14q, one of which contained the orthodenticle homologue 2 (OTX2) homeobox gene. DNA copy number analysis showed that OTX2 had undergone genomic amplification in 2 of 11 medulloblastoma cell lines and 8 of 42 primary tumors. The three genes and a predicted open reading frame flanking OTX2 in the 14q amplicon were not amplified in at least one of the other nine amplicons, implicating OTX2 as the gene target conferring a selective advantage. The degree of OTX2 amplification ranged from 8 copies to over 50 copies of the gene. OTX2 transcript was highly and specifically expressed in medulloblastoma or developing cells. Serial analysis of gene expression of 240 different human tumors or normal tissues revealed that 96% of all 783 OTX2 transcripts sequenced were in medulloblastomas or embryonic stem cells. OTX2 functions to specify the fate of neuroectoderm in various regions of the developing brain. This developmental role is consistent with the evidence suggesting that OTX2 is a medulloblastoma oncogene.
p300 Modulates Nuclear Morphology in Prostate Cancer
Authors: Debes Jose D. , Sebo Thomas J. , Heemers Hannelore V. , Kipp Benjamin R. , Haugen De Anna L. , Lohse Christine M. , Tindall Donald J.
Published: June 2022

Alterations in nuclear structure distinguish cancer cells from noncancer cells. These nuclear alterations can be translated into quantifiable features by digital image analysis in a process known as quantitative nuclear morphometry. Recently, quantitative nuclear morphometry has been shown to predict metastasis and biochemical recurrence of prostate cancer. However, little is known about the cellular mechanisms underlying these nuclear morphometric changes. Alterations of nuclear matrix proteins are frequently involved in changes of nuclear structure. A number of co-activators interact with these nuclear structure–related proteins, suggesting that they might be involved in quantitative nuclear morphometry changes. We have shown previously that the transcriptional co-activator p300 is involved in prostate cancer progression. However, the ability of a transcriptional regulator like p300 to modulate nuclear morphology has not been described previously. In the present study, we show that p300 expression in prostate cancer biopsy tissue from 95 patients correlates with quantifiable nuclear alterations. Moreover, we show that transfection of p300 into prostate cancer cells in culture induces quantifiable nuclear alterations, such as diameter, perimeter, and absorbance among others, as assessed by digital image analysis. These alterations correlate individually with aggressive features in prostate cancer, such as expression of the proliferation marker Ki-67 and extraprostatic extension of the tumor. Finally, we found that transfection of p300 into prostate cancer cells specifically increases mRNA and protein levels of nuclear matrix peptides lamins A and C, suggesting that these proteins mediate the p300-induced effects. These findings reveal a new insight into the transcriptional and structural regulation of prostate cancer.
Carboxyl-Terminal Repressor Domain of MBP-1 is Sufficient for Regression of Prostate Tumor Growth in Nude Mice
Authors: Ghosh Asish K. , Steele Robert , Ray Ratna B.
Published: June 2022

Prostate cancer is the most frequently diagnosed cancer in men and the second leading cause of male cancer death in the United States. Early detection and improved procedures for surgical intervention and radiation therapy have reduced the fatalities; however, there is no effective cure for men with advanced disease and additional therapy is urgently needed. We have previously shown that MBP-1 acts as a general transcriptional repressor and exerts an antiproliferative effect on several human cancer cells. MBP-1 possesses two repressor domains, located at the amino and carboxyl termini. In this study, we have examined the potential of the repressor domains of MBP-1 as a gene therapeutic candidate in regression of prostate tumor growth. Our results suggested that replication-deficient adenovirus-mediated delivery of amino-terminal (MBP-AR) or carboxyl-terminal (MBP-CR) repressor domain of MBP-1 exerted an antiproliferative effect, like the full-length MBP-1, and induced caspase-independent apoptosis in prostate cancer cells. Next, we investigated the therapeutic effectiveness of MBP-1 repressor domain on prostate tumors. When tested in human tumor xenografts in nude mice, MBP-CR suppressed prostate tumor growth more effectively than full-length MBP-1, whereas MBP-AR delayed prostate tumor growth. Together, these results suggested that MBP-CR expression has an antiproliferative effect in human prostate cancer cells, being more effective than the full-length MBP-1 in preventing tumor growth.
Genetic Linkage of Prostate Cancer Risk to the Chromosome 3 Region Bearing FHIT
Authors: Larson Garry P. , Ding Yan , Cheng Li S-C. , Lundberg Cathryn , Gagalang Virgil , Rivas Guillermo , Geller Louis , Weitzel Jeffrey , MacDonald Deborah , Archambeau John , Slater Jerry , Neuberg Donna , Daly Mary B. , Angel Irene , Benson Al B. , Smith Kimberly , Kirkwood John M. , O'Dwyer Peter J. , Raskay Barbara , Sutphen Rebecca , Drew Rosalind , Stewart James A. , Werndli Jae , Johnson David , Ruckdeschel John C. , Elston Robert C. , Krontiris Theodore G.
Published: June 2022

We conducted linkage analysis of 80 candidate genes in 201 brother pairs affected with prostatic adenocarcinoma. Markers representing two adjacent candidate genes on chromosome 3p, CDC25A and FHIT, showed suggestive evidence for linkage with single-point identity-by-descent allele-sharing statistics. Fine-structure multipoint linkage analysis yielded a maximum LOD score of 3.17 (P = 0.00007) at D3S1234 within FHIT intron 5. For a subgroup of 38 families in which three or more affected brothers were reported, the LOD score was 3.83 (P = 0.00001). Further analysis reported herein suggested a recessive mode of inheritance. Association testing of 16 single nucleotide polymorphisms (SNP) spanning a 381-kb interval surrounding D3S1234 in 202 cases of European descent with 143 matched, unrelated controls revealed significant evidence for association between case status and the A allele of single nucleotide polymorphism rs760317, located within intron 5 of FHIT (Pearson's χ2 = 8.54, df = 1, P = 0.0035). Our results strongly suggest involvement of germline variations of FHIT in prostate cancer risk.
CDKN2A Common Variants and Their Association with Melanoma Risk: A Population-Based Study
Authors: Dębniak Tadeusz , Scott Rodney J. , Huzarski Tomasz , Byrski Tomasz , Rozmiarek Andrzej , Dębniak Bogusław , Załuga Elżbieta , Maleszka Romuald , Kładny Józef , Górski Bohdan , Cybulski Cezary , Gronwald Jacek , Kurzawski Grzegorz , Lubinski Jan
Published: June 2022

The population frequencies of the CDKN2A variants remain undetermined. In Poland there are three common variants of CDKN2A: an alanine to threonine substitution (A148T), Nt500c>g and Nt540c>t, which have been detected in other populations. To establish if they are associated with an increased malignant melanoma (MM) risk we did an association study based on genotyping 471 patients with MM and 1,210 random control subjects from the same Polish population. We found a significantly increased frequency of the A148T variant among patients with MM (7.0%) in comparison with the general population (2.9%). The incidence of the A148T variant remained greater in both unselected and familial melanoma subgroups. A statistically significant positive association was seen for unselected MM (odds ratio, 2.529; P = 0.0003), especially in patients diagnosed under 50 years of age (odds ratio, 3.4; P = 0.0002). The A148T carrier population (heterozygous G/A alleles) was more likely to have a relative with malignancy compared with the noncarrier population (57% versus 36%, respectively; P = 0.03). Further examination of the CDKN2A promoter sequence done in 20 melanoma patients with the A148T change (heterozygous G/A alleles) and 20 patients with MM without this alteration identified it was in linkage disequilibrium with a polymorphism in the promoter region at position P-493. We found no statistically significant overrepresentation of the Nt500c>g and the Nt540c>t polymorphisms in the Polish melanoma population. In conclusion, the A148T variant of the CDKN2A gene seems to be associated with an increased risk of development of MM. Additional studies are required to confirm whether this particular change is associated with increased risk of other nonmelanoma malignancies.
Tissue Inhibitor of Metalloproteinase-1 Protects Human Breast Epithelial Cells from Extrinsic Cell Death: A Potential Oncogenic Activity of Tissue Inhibitor of Metalloproteinase-1
Authors: Liu Xu-Wen , Taube Marcus E. , Jung Ki-Kyung , Dong Zhong , Lee Yong J. , Roshy Stefanie , Sloane Bonnie F. , Fridman Rafael , Kim Hyeong-Reh Choi
Published: June 2022

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases and some members of a disintegrin and metalloproteinase domain (ADAM) family. In addition, recent studies unveiled novel functions of TIMPs in the regulation of apoptosis. TIMP-1 inhibits intrinsic apoptosis by inducing TIMP-1 specific cell survival pathways involving focal adhesion kinase (FAK). TIMP-3, however, was shown to enhance extrinsic cell death by inhibiting the shedding of the cell surface death receptors mediated by tumor necrosis factor-α converting enzymes (TACE/ADAM-17). Here, we examined whether TIMP-1, an inhibitor of some of the ADAM family members, enhances the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)–induced extrinsic apoptotic pathway. Surprisingly, we found that TIMP-1 effectively protects human breast epithelial cells from TRAIL-induced apoptosis, demonstrating opposite roles of TIMP-1 and TIMP-3 for the regulation of extrinsic apoptosis. TIMP-1 inhibition of TRAIL-induced apoptosis does not depend on its ability to inhibit matrix metalloproteinases or ADAM activities and is unrelated to its ability to stabilize active or decoy death receptors. Importantly, inhibition of PI 3-kinase signaling by wortmannin and down-regulation of FAK expression using siRNA significantly diminish TIMP-1 protection of human breast epithelial cells against TRAIL-induced extrinsic apoptosis. In addition, the in vitro three-dimensional culture studies showed that TIMP-1 inhibits lumen formation and apoptosis during morphogenesis of MCF10A acini. Taken together, these studies suggest that TIMP-1 may exert oncogenic activity in breast cancer through inhibition of both intrinsic and extrinsic apoptosis involving the FAK survival signal transduction pathway.
Spontaneous Feline Mammary Carcinoma Is a Model of HER2 Overexpressing Poor Prognosis Human Breast Cancer
Authors: De Maria Raffaella , Olivero Martina , Iussich Selina , Nakaichi Munekazu , Murata Tomoaki , Biolatti Bartolomeo , Di Renzo Maria Flavia
Published: June 2022

Companion animal spontaneous tumors are suitable models for human cancer, primarily because both animal population and the tumors are genetically heterogeneous. Feline mammary carcinoma (FMC) is a highly aggressive, mainly hormone receptor–negative cancer, which has been proposed as a model for poor prognosis human breast cancer. We have identified and studied the feline orthologue of the HER2 gene, which is both an important prognostic marker and therapeutic target in human cancer. Feline HER2 (f-HER2) gene kinase domain is 92% similar to the human HER2 kinase. F-HER2–specific mRNA was found 3- to 18-fold increased in 3 of 3 FMC cell lines, in 1 of 4 mammary adenomas and 6 of 11 FMC samples using quantitative reverse transcription-PCR. Western blot showed that an anti-human HER2 antibody recognized a protein comigrating with the human p185HER2 in FMC cell lines. The same antibodies strongly stained 13 of 36 FMC archival samples. These data show that feline HER2 overexpression qualifies FMC as homologous to the subset of HER2 overexpressing, poor prognosis human breast carcinomas and as a suitable model to test innovative approaches to therapy of aggressive tumors.
Novel Roles of Unphosphorylated STAT3 in Oncogenesis and Transcriptional Regulation
Authors: Yang Jinbo , Chatterjee-Kishore Moitreyee , Staugaitis Susan M. , Nguyen Hannah , Schlessinger Karni , Levy David E. , Stark George R.
Published: June 2022

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated on tyrosine residue 705 in response to growth factors or cytokines to form activated homodimers that drive gene expression. Because the stat3 promoter has a binding site for STAT3 dimers, the amount of STAT3 protein increases when STAT3 is activated (e.g., in response to interleukin 6). Unphosphorylated STAT1 is known to drive the expression of certain genes. To explore the possibility of a similar role for the induced expression of unphosphorylated STAT3, we overexpressed either Y705F STAT3, which can not be phosphorylated on residue 705, or wild-type STAT3 in normal human mammary epithelial cells or STAT3-null mouse cells. The levels of many mRNAs were affected strongly by high levels of either form of STAT3. Some genes whose expression was increased by overexpressed STAT3, but not by activated STAT3 dimers, encode well-known oncoproteins (e.g., MRAS and MET). In many tumors, STAT3 is activated constitutively, and thus the unphosphorylated form is likely to be expressed highly, driving oncogene expression by a novel mechanism. In addition, expression of the stat3 gene is increased strongly in response to interleukin 6, and the high levels of unphosphorylated STAT3 that result drive a substantial late phase of gene expression in response to this cytokine. Thus, unphosphorylated STAT3, which activates gene expression by a novel mechanism distinct from that used by STAT3 dimers, is very likely to be an important transcription factor both in cancer and in responses to cytokines.
Silencing of CXCR4 Blocks Breast Cancer Metastasis
Authors: Liang Zhongxing , Yoon Younghyoun , Votaw John , Goodman Mark M. , Williams Larry , Shim Hyunsuk
Published: June 2022

RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.
Ras Inhibition in Glioblastoma Down-regulates Hypoxia-Inducible Factor-1α, Causing Glycolysis Shutdown and Cell Death
Authors: Blum Roy , Jacob-Hirsch Jasmine , Amariglio Ninette , Rechavi Gideon , Kloog Yoel
Published: June 2022

Active Ras and phosphatidylinositol-3-kinase–dependent pathways contribute to the malignant phenotype of glioblastoma multiformes (GBM). Here we show that the Ras inhibitor trans-farnesylthiosalicylic acid (FTS) exhibits profound antioncogenic effects in U87 GBM cells. FTS inhibited active Ras and attenuated Ras signaling to extracellular signal-regulated kinase, phosphatidylinositol-3-kinase, and Akt. Concomitantly, hypoxia-inducible factor 1α (HIF-1α) disappeared, expression of key glycolysis pathway enzymes and of other HIF-1α–regulated genes (including vascular endothelial growth factor and the Glut-1 glucose transporter) was down-regulated, and glycolysis was halted. This led to a dramatic reduction in ATP, resulting in a severe energy crisis. In addition, the expression of E2F-regulated genes was down-regulated in the FTS-treated cells. Consequently, U87 cell growth was arrested and the cells died. These results show that FTS is a potent down-regulator of HIF-1α and might therefore block invasiveness, survival, and angiogenesis in GBM.
The Kinetics and (Dys)kinetics of Cancer Chronotherapy
Authors: Field Jeffrey M. , Sehgal Amita
Published: June 2022

Circadian rhythms are the daily cycles that time almost all aspects of physiology, but treatments of the clock or by the clock are rarely tested in the clinic. We develop a framework for identifying interventions that may benefit from administration at the appropriate time of day (chronotherapy). Typically, pharmacokinetics is an important consideration for chronotherapy, with short half-life drugs deemed optimal for such treatments. However, recent data suggest long-lived antibodies can show time-of-day specific effects. Examples include both tumor-targeted antibodies as well as immunotherapies with antibodies that activate T cells. Clues to the immunotherapy mechanism come from animal vaccination studies, which demonstrate circadian responses of T cells to a single dose that leads to long-lasting T-cell activation. Conversely, some studies have challenged the efficacy of chronotherapy, underscoring the need to rigorously investigate its application for each drug and tumor type.
Abstract 6293: Genomic characteristics of nivolumab/ipilimumab with or without CBM-588 supplementation in patients with metastatic renal cell carcinoma
Authors: Castro Daniela V. , Dizman Nazli , Zengin Zeynep B. , Malhotra Jasnoor , Meza Luis A. , Muddasani Ramya , Govindarajan Ameish , Chawla Neal S. , Chehrazi-Raffle Alex , Hsu JoAnn , Bergerot Paulo G. , Bergerot Cristiane D. , Dorff Tanya B. , Lyou Yung , Pal Sumanta K.
Published: June 2022

Background: In a previous randomized phase I trial, addition of CBM-588 to the nivolumab/ipilimumab (N/I) regimen showed improved objective response rate, clinical benefit rate, and progression free survival compared to N/I alone in patients (pts) with metastatic renal cell carcinoma (mRCC; Meza et al., ASCO 2021). Furthermore, genomic alterations such as PBRM1 have been associated with clinical benefit to anti-PD-1 monotherapy in patients with mRCC (Miao et al., Science 2018). The primary aim of this study was to investigate tumor genomic characteristics according to treatment arms.

Methods: We retrospectively identified pts with mRCC who received N/I alone or with CBM-588 supplementation along with whole exome and transcriptome sequencing (Ashion Analytics). Responses were measured according to RECIST v1.1. A two-tailed Fischer’s exact test was performed to compare genomic characteristics across arms.

Results: In this study, 29 mRCC pts were randomized to receive N/I +/- CBM-588 and 21 (72%) pts (71% in N/I with CBM-588 arm and 29% N/I arm) had available genomic data. Within this cohort, the median age was 66.8 (range 46-90) and 71% of pts were male. Eleven (52.4%) pts had clear-cell histology and 10 (47.6%) pts had sarcomatoid features; 15 pts received N/I with CBM-588 and 6 pts received N/I alone. The most commonly mutated genes in the overall cohort were VHL (61.9%), PBRM1 (42.9%), and SETD2 (33.3%). Alterations in VHL, PBRM1, and SETD2 were seen in 66.7% vs. 73.3% (p=0.115), 50.0% vs. 40.0% (p=0.523) and 33.3% vs. 33.3% (p=0.686), in N/I vs. N/I with CBM-588 arm, respectively.

Conclusions: There was no significant difference observed in clinically relevant genomic features across study arms. The clinical benefit from CBM-588 appears to be independent of tumor genomic characteristics. More extensive investigations are needed to characterize the determinants of benefit from CBM-588 supplementation.

Citation Format: Daniela V. Castro, Nazli Dizman, Zeynep B. Zengin, Jasnoor Malhotra, Luis A. Meza, Ramya Muddasani, Ameish Govindarajan, Neal S. Chawla, Alex Chehrazi-Raffle, JoAnn Hsu, Paulo G. Bergerot, Cristiane D. Bergerot, Tanya B. Dorff, Yung Lyou, Sumanta K. Pal. Genomic characteristics of nivolumab/ipilimumab with or without CBM-588 supplementation in patients with metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6293.
Abstract ND14: Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers
Authors: Hentemann Murphy
Published: June 2022

Pharmacological profile and anti-tumor properties of FHD-286, a novel BAF inhibitor for the treatment of transcription factor-driven cancersMartin Hentemann, Rishi G. Vaswani, Lan Xu, Richard C. Centore, Luis M. M. Soares, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Sarah Reilly, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Liyue Huang, Jessica Piel, Johannes Voigt, David S. Millan, Ho Man Chan, Ryan G. Kruger, Carl P. Decicco, Samuel Agresta, Steven F. BellonFoghorn Therapeutics, 500 Technology Square, Suite 700, Cambridge, MA 02139The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a compound that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that this SMARCA4/SMARCA2 inhibitor acts through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. FHD-286 is orally bioavailable and demonstrates robust efficacy in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. In preclinical efficacy and toxicology studies, this first in class molecule demonstrates an acceptable therapeutic index which should allow for flexibility in clinical designs. FHD-286 is currently in two Phase I trials for both relapsed refractory AML and MDS, and metastatic uveal melanoma.

Citation Format: Murphy Hentemann. Pharmacological profile and anti-tumor properties of FHD-286: A novel BAF inhibitor for the treatment of transcription factor-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND14.
Abstract PL01-03: From population to personalized prevention: Lessons from Barrett's esophagus
Authors: Fitzgerald Rebecca
Published: June 2022

Cancer medicine is undergoing a radical shift away from a reactive approach in patients presenting with symptomatic disease towards a more proactive approach aimed at early diagnosis, including at the pre-cancerous stage. Oesophageal adenocarcinoma develops gradually from the pre-malignant condition Barrett’s oesophagus which presents an opportunity to radically improve outcomes from this highly aggressive cancer. Work to unravel the: clinical-demographic and inherited risk factors; the cell of origin of this disease; and the molecular landscape of Barrett’s samples over time and space, in patients with different outcomes, is shedding light on the disease pathogenesis. Our improved understanding of the disease can be used to develop novel approaches to risk-based population screening and personalised prevention. One emerging paradigm is a triage system that comprises a risk assessment, followed by targeted screening using novel non-endoscopic detection devices coupled with molecular biomarkers to identify individuals at high risk of invasive cancer needing monitoring or preventative treatment. In patients with a Barrett’s lesion undergoing monitoring, molecular strategies can further distinguish between those with indolent disease versus those likely to progress. As we redesign clinical pathways for this disease we must be sure to maximise patient and clinician acceptability, affordability and ease of access to a given technology while balancing the risks for over-diagnosis and over-treatment.

Citation Format: Rebecca Fitzgerald. From population to personalized prevention: Lessons from Barrett's esophagus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr PL01-03.
Abstract SY05-02: Applications of base editor technology in small molecule: Target validation
Authors: Griffith Audrey , Sangree Annabel , Roy Priyanka , Hanna Ruth , Doench John
Published: June 2022

CRISPR screens that focus on the gene as the unit of information - whether using knockout, interference, or activation - have reinvigorated the field of functional genomics. The recent development of Base Editor technology allows nucleotide-level manipulation, enabling fine-scale investigation of endogenous loci. The low upfront costs of generating a pooled base editing library, coupled with the small scale and relative ease of executing such screens, makes the financial barrier to entry for such experiments low while also not requiring deep experience with screening.

One application of this technology is screens that aim to identify resistance mutations in putative targets of small molecules, which provides excellent evidence that the phenotype is caused by on-target activity of the small molecule. This has heretofore typically been an extraordinarily laborious and time-consuming experimental path, by either slowly cultivating spontaneous drug resistant mutants or by generating and screening large scale ORF-variant pools. Base editor screens are well-suited to vastly accelerate this process.

Using both A>G and C>T base editors, coupled with variants of the SpCas9 enzyme, we have conducted drug resistance screens with small molecules that target the anti-apoptotic proteins MCL1, BCL2L1, and BCL2, as well as the DNA repair regulator PARP1. These screens identified resistance residues that were also observed in clinical samples, illustrating the utility of this approach. Additionally, base editor screens are excellent tools for studying genetic variants, as we will demonstrate with screens identifying loss-of-function mutations in BRCA1, BRCA2, and TP53. We anticipate that the tools and methodologies described here will facilitate the investigation of impactful variants at a finer and deeper resolution for any locus of interest.

Citation Format: Audrey Griffith, Annabel Sangree, Priyanka Roy, Ruth Hanna, John Doench. Applications of base editor technology in small molecule: Target validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY05-02.
Abstract SY18-02: Beyond tumor antigen immunogenicity: Determinants of protective immunity induced by therapeutic cancer vaccines
Authors: Delamarre Lelia
Published: June 2022

Cancer-specific mutations can generate neoantigens that are recognized as foreign by the immune system and elicit potent T cell responses against cancer. The identification of neoantigens as drivers of effective anti-tumor immunity has promoted the development of neoantigen-based therapeutic strategies, including vaccines and cell-based therapies. One challenge for these approaches is to accurately predict neoantigens. Most mutations are unique to each patient and only a fraction are immunogenic. The generation of large-scale mass spectrometry-based immunopeptidomics training data for the development of deep learning models has resulted in significantly improved computational methods for prediction of neoantigen presentation on HLA. In addition, on-going efforts to characterize antigen determinants involved in TCR recognition may also contribute to further improving immunogenicity prediction. However, induction of neoantigen-specific T cells may not be sufficient for efficacy. We used the MC38 cancer model to characterize the factors associated with effective neoantigen-specific T cell responses after vaccination. We designed two RNA-LPX vaccines (Deca-1 and Deca-2), each encoding 10 MC38 neoantigens. The two vaccines elicited T cell responses with comparable magnitude and functionality. Prophylactic vaccination with either vaccine prevented establishment of MC38 tumors, confirming that vaccine-induced T cells can recognize and kill tumor cells. Surprisingly, while vaccination with Deca-2 was protective in therapeutic settings, vaccination with Deca-1 was unable to reverse growth of established tumors. Neoantigen-specific CD8 T cell migration to the tumor was comparable between the two vaccines, but the phenotypes of the T cells were significantly different. Gene expression analysis revealed specific activation states of T cells that differed according to their antigenic specificity and clonal expansion, suggesting intrinsic differences across different T cell specificities. Remarkably, T cells elicited by Deca-1 exhibited a gene signature of reduced functionality at the tumor site and also in the spleen, the primary site of vaccine activity. Our study has important implications for the development of neoantigen therapies, as it suggests that the properties of the neoantigen/TCR pairs influence the phenotype of vaccine-induced T cells at the tumor site and therapeutic outcome. Furthermore, our results indicate that the tumor may influence the phenotype of vaccine-induced T cells outside the tumor microenvironment. Additional experiments are on-going to further define the exact nature of these determinants.

Citation Format: Lelia Delamarre. Beyond tumor antigen immunogenicity: Determinants of protective immunity induced by therapeutic cancer vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY18-02.
Abstract SY19-01: Investigating cancer determinants of tumor immune composition
Authors: Brown Brian
Published: June 2022

The cellular composition, spatial architecture, and tissue localization of a tumor have a major impact on a cancer’s progression and response to therapy. These factors are particularly relevant to tumor immunity as immune cell composition and localization within the tumor microenvironment (TME) is one of the major determinants of response to immunotherapy and patient outcome. Spatially resolved single cell genome sequencing has revealed that many genetically distinct sub-clones exist in proximity in tumors and can have different TME compositions, but how specific genes influence the TME, or how neighboring clones influence one another is not well-defined. Indeed, while some genes involved in orchestrating the TME have been identified, the potential roles of many genes in influencing the architecture and immune composition of different tumors are not established. To enable scale up studies to identify determinants of TME biology and tumor immunity, we established a first-of-its-kind in vivo spatial functional genomics platform called Perturb-map. We applied Perturb-map to knockout (KO) dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each KO influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. In contrast to in vitro CRISPR screens, we found KO of positive regulators of IFNγ signaling led to a major reduction of tumor burden, with varying impact from different pathway components. KO of Socs1, a negative regulator of IFNγ signaling, provided a growth advantage though was accompanied by increased T cell infiltration and notably, Socs1 KO tumors were more responsive to PDL1 blockade. Perturb-map also found that KO of the TGFβ Receptor 2 (Tgfbr2) resulted in a growth advantage, but unlike Socs1 KO, T cells were excluded from the lesions and the TME converted to a fibro-mucinous state. Spatial transcriptomics revealed TGFβ signaling was upregulated in Tgfbr2 KO tumors. Further analysis indicated this signal was from TGFβ-mediated activation of fibroblasts in tumors where cancer cells lost Tgfbr2, suggesting the mechanism for TME remodeling and T cell exclusion in Tgfbr2 KO tumors. These studies establish Perturb-map for functional genomics within tissue at single cell-resolution with spatial architecture preserved and provide insight into how TGFβ responsiveness of cancer cells can affect the TME (Dhainaut, Rose et al. BioRxiv 2021, doi.org/10.1101/2021.07.13.451021).

Citation Format: Brian Brown. Investigating cancer determinants of tumor immune composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY19-01.
Abstract SY41-02: Epigenetic influence of our (fatty) diet on metastatic-initiating cells: Role of intra-tumor Schwann cells
Authors: Aznar-Benitah Salvador
Published: June 2022

We have recently identified the cells uniquely responsible for the formation of metastasis in different types of human tumors. These cells have intriguing characteristics: i) they form primary lesions as efficiently as their tumor-initiating counterparts, but are exclusive in their ability to generate metastases; ii) they express the fatty acid translocase CD36, and are characterized by a unique lipid metabolic signature; iii) they are exquisitely sensitive to the levels of fat in circulation, and consequently, they relate the predisposition of metastasis directly to the content of dietary fat; iv) they are highly sensitive to CD36 inhibition, which almost completely abolishes their metastatic potential in preclinical models. In my talk, I will present our latest data on the molecular characterization of metastatic-initiating cells. I will place special emphasis showing how specific dietary lipids exert a striking long-term epigenetic effect over metastatic-initiating cells related to activation of intra-tumor Schwann cells, putting forward a novel concept of ¨metastatic epigenetic memory¨ elicited by our diet.

Citation Format: Salvador Aznar-Benitah. Epigenetic influence of our (fatty) diet on metastatic-initiating cells: Role of intra-tumor Schwann cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY41-02.
Rise of the Clones: Myelodysplastic Syndrome to Secondary Acute Myeloid Leukemia
Authors: Romine Kyle A. , van Galen Peter
Published: June 2022

Myelodysplastic syndrome (MDS) describes a family of blood disorders driven by the clonal expansion of mutated blood cells that can evolve into secondary acute myeloid leukemia (sAML). Two new studies use single-cell and deep sequencing to elucidate the progression of MDS to AML, revealing discrete clonal architectures and the driving role of signaling mutations.

See related article by Guess et al., p. 316 (8) .

See related article by Menssen et al., p. 330 (9) .