bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2427: Identification of transcription factors activated in glioblastoma multiforme
Authors: Wu Zexun , Schreiner Shannon , Farnham Peggy J. , Rhie Suhn K.
Published: July 2021

Introduction Glioblastoma multiforme (GBM) is the most common intrinsic brain tumor in adults and is universally fatal. Our strategy to study the molecular mechanisms leading to GBM is to investigate the role of transcription factors (TFs) that are differentially expressed between normal and GBM. Previous studies have shown that ZFX is a key TF that shows increased expression in GBM. However, the role of ZFX, and the identity of TFs that may work with ZFX, in driving GBM carcinogenesis is not yet understood.

Materials and methods: ChIP-seq was performed to determine ZFX binding sites in the U87 GBM cancer cell line. RNA-seq datasets generated from GBM tissues by The Cancer Genome Atlas (TCGA) were analyzed to identify TFs showing increased expression in GBM. Publicly available H3K27ac chromatin immunoprecipitation (ChIP-seq) and Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) datasets generated from GBM tissues were analyzed to identify active enhancers and TFs that bind to GBM enhancers.

Results: We found that a majority of ZFX binding sites in GBM are located at promoters, with ~1000 binding sites being at non-promoter regions. An overlap analysis of ZFX binding sites in multiple cancer cell types showed that a high percentage of ZFX-binding sites at promoter regions are shared across cell types whereas hundreds of GBM-specific binding sites are located at non-promoter regions. Using TCGA GBM RNA-seq datasets, we next identified differentially expressed TFs. For example, we found that MTY1L and NEUROD2, which have been reported to be tumor suppressors, are downregulated while HOX family TFs are upregulated in GBM tissues, as compared to normal tissues. We also identified thousands of enhancers specifically active in GBM, as compared to normal cells, using H3K27ac ChIP-seq and ATAC-seq datasets. TF motif analysis of the identified enhancers revealed that motifs of Basic Leucine Zipper Domain (bZIP), CAAT box-binding transcription factor (CTF), homeobox (HOX), signal transducer and activator of transcription (STAT), and helix-turn-helix (HTH) family were enriched.

Conclusion: By integrating transcriptomic and epigenomic datasets, we identified and characterized TFs activated in GBM. We are currently investigating how ZFX cooperates with the TFs we identified as bound to the GBM-specific enhancers. These studies will provide insight into the molecular mechanisms underlying GBM.

Citation Format: Zexun Wu, Shannon Schreiner, Peggy J. Farnham, Suhn K. Rhie. Identification of transcription factors activated in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2427.
Abstract 2428: NR4A1 ligands inhibits PSPC1 regulated EMT and stemness markers in breast, lung and liver cancer cells
Authors: Mohankumar Kumaravel , Shrestha Rupesh , Safe Stephen
Published: July 2021

The orphan nuclear receptor 4A1 (Nur77, TR3, NR4A1) is highly expressed in solid tumors and is a negative prognostic factor for survival of breast, lung, and liver cancer patients (1) and ongoing studies in our laboratory show that NR4A1 is pro-oncogenic in multiple tumor types. We have also identified a series of bis-indole derived compounds including 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl) methane (DIM-C-pPhOH; CDIM8) that act as NR4A1 antagonists that inhibit pro-oncogenic NR4A1-regulated genes and pathways in multiple cancer cell lines (2). Mechanistic studies show that many of the NR4A1-regulated genes involve interactions of NR4A1 with gene promoter DNA or with Sp-bound to GC-rich sequences and similar mechanisms have previously been observed for steroid hormone and other nuclear receptors. Our studies have identified paraspeckle component 1 (PSPC1) as an NR4A1 regulated gene in breast, lung and liver cancer cells and PSPC1 gene expression and protein levels are suppressed after treatment with C-DIM/NR4A1 antagonists. PSPC1 promotes tumorigenesis, epithelial-to-mesenchymal transition (EMT), stemness and metastasis and is a master activator of pro-metastatic switches and a potential target for anti-metastatic drugs. Knockdown of PSPC1 downregulates EMT and stemness markers in breast, lung and liver cancer cells. The novel buttressed analog of CDIM8 (DIM-C-pPhOH-3-Br-5-OCF3) at a concentration of 10 uM downregulates PSPC1 expression and its downstream targets (Snail, Slug, Nanog, OCT4, SOX2, c-myc). Knockdown of NR4A1 or SP1 inhibits PSPC1 expression in MDA-MB-231, H1299 and SNU449 cell lines respectively. These results identify PSPC1 as a novel gene that regulates EMT and invasion in these cancer cells and NR4A1 is an upstream regulator of PSPC1 gene expression. Thus, NR4A1 ligands act as an antimetastatic mimic by inhibiting PSPC1 and the mechanism of PSPC1 regulation involves ligand induced interactions of NR4A1/SP1 with a GC-rich region of the PSPC1 gene promoter.

References:1.Safe S, Jin UH, Hedrick E, Reeder A, Lee SO.

Minireview: Role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol. 2014;28(2):157-72.

2.Mohankumar K, Li X, Sridharan S, Karki K, Safe S. Nuclear receptor 4A1 (NR4A1) antagonists induce ROS-dependent inhibition of mTOR signaling in endometrial cancer. Gynecol Oncol. 2019; 154(1):218-227.

Citation Format: Kumaravel Mohankumar, Rupesh Shrestha, Stephen Safe. NR4A1 ligands inhibits PSPC1 regulated EMT and stemness markers in breast, lung and liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2428.
Abstract 2430: HOXC6 promotes tumorigenesis of right-sided colon cancer
Authors: li Xin , Xia Jinhong , Ji Dengbo , Gu Jin
Published: July 2021

Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, with high heterogeneity. The significant difference in prognosis and therapeutic response of left and right colon cancer is a hot spot in the field of colorectal cancer (CRC) in recent years. The prognosis and drug response of right-sided colon cancer were significantly worse than that of the left-sided colon cancer. Clinical trials have proved that MSI-H colon cancer is mainly seen in the right-sided colon cancer, and the prognosis is better than that of MSS. Because MSS and MSI-H patients have different molecular characteristics and prognosis, we searched for key genes of RCC and LCC in MSS and MSI-H groups respectively. The difference between the prognosis and the curative effect of the left and right-sided colon cancer is essentially the difference between the molecular pathology and the immune environment. Therefore, the aim of this study is to explore the molecular mechanism related to the prognosis of left and right-sided colon cancer in order to provide a molecular target for accurate treatment of left and right-sided colon cancer. First of all, bioinformatics methods were used to screen the right and left-sided CRC differentially expressed genes in MSS and MSI-H. The expression levels of these key genes in clinical samples and cell lines were detected by RT-qPCR and IHC, and their correlation with survival prognosis was analyzed. Homeobox C6(HOXC6)were selected. First, HOXC6 stable overexpressed and knockout colorectal cancer cell lines were established and its biological function was preliminarily studied, including transwell, wound-healing assay and CCK-8 tests. Finally, the ChIP-seq data were used to analyze the carcinogenic mechanism of HOXC6. Bioinformatics methods were used to screen 30 differentially expressed genes in MSS right and left CRC, of which HOXC6 in the right colon cancer was significantly higher than that in the left-sided colon cancer, which was positively correlated with TNM staging and lymph node metastasis. Kaplan-Meier survival analysis showed that the expression of HOXC6 was negatively correlated with the total survival of CRC patients (p=0.002). RT-qPCR and IHC showed that the expression of HOXC6 in the right-sided colon cancer was higher than that in the left, P<0.05. Functional experiments showed that Hoxc6 promoted the growth, invasion and migration. ChIP-seq data and real-time PCR results showed that SPOCD1 and S100A1 were the target genes of HOXC6.

Citation Format: Xin li, Jinhong Xia, Dengbo Ji, Jin Gu. HOXC6 promotes tumorigenesis of right-sided colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2430.
Abstract 2435: Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression
Authors: Zhang Keqiang , Sun Ting , Li Wendong , Pangeni Rajendra , Raz Dan
Published: July 2021

Ubiquitin-specific peptidase 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in various human cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in a number of human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown. We set out to characterize and identify potential transcription factors that regulate USP22 expression in human non-small cell lung cancers (NSCLC). A luciferase reporter driven by the promoter region from -5085 to +55 of USP22 gene was constructed to screen a customized siRNA library targeting about 100 selected transcription factors that are expected to bind to the USP22 promoter. Luciferase reporter assays have revealed that knockdown of SP1 and activating transcription factor 3 (ATF3) promote USP22 expression, while knockdown of transcription factor 2α, 2β (TFAP2α and TFAP2β), cMyc, and PPAR α suppress USP22 expression. The specific binding of TFAP2α and TFAP2β to the USP22 promoter was further validated by binding site-directed mutagenesis and chromosome immunoprecipitation (Chip) assays. Furthermore, overexpression of TFAP2α and TFAP2β significantly up-regulate USP22 expression and downstream molecules of USP22, concurrently enhance the proliferation, migration and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. In addition, TFAP2 was demonstrated to be associated with a poor prognosis of NSCLC patients. Moreover, TFAP2 protein expression correlated with USP22 protein expression in human NSCLC tissues. Taken together, the findings of this study indicate TFAP2 is an important transcription factor driving USP22 gene expression, and it promotes the progression of NSCLC partially through enhancing USP22 expression.

Citation Format: Keqiang Zhang, Ting Sun, Wendong Li, Rajendra Pangeni, Dan Raz. Transcription Factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2435.
Abstract 2437: Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods
Authors: Buzy Armelle , Paul Pascal , Hamon Stephanie , Venier Olivier , Guillemot Jean-Claude , Mikol Vincent , Moll Jurgen , Debussche Laurent , Valtingojer Iris
Published: July 2021

The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors

Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.
Abstract 2438: Genetic activation of JUN fuels the core regulatory circuitry of de-differentiated liposarcoma
Authors: Chen Ye , Dakle Pushkar , Tan Tuan Zea , Lin Ruby Yu-Tong , Puhaindran Mark E. , Gery Sigal , Xu Liang , Koeffler Phillip H.
Published: July 2021

Background: As a high-grade neoplasm of connective tissues, de-differentiated liposarcoma (DDLPS) is featured by genomic amplification of MDM2, CDK4 and JUN. Super-enhancer-driven master transcription factors (RUNX1, FOSL2 and MYC) establish a core feed-forward transcriptional circuit underpinning the DDLPS malignancy. However, an important question remains unanswered: how do genomic insults interact with epigenetic dysregulation in this disease?

Methods: We performed ChIP-seq analysis to map loading of transcription factors and histone marks on chromatin in DDLPS cells. Functional assays following genetic manipulation of target genes were conducted to explore their disease relevance.

Results: Genomic activation of JUN represents a promising prognostic marker for DDLPS. Meanwhile, JUN locus engaged excessive H3K27ac signals in both DDLPS tissues and cells, while a markedly lower level of H3K27ac deposition was observed in normal adipose tissues, adipocytes and MSCs. JUN formed a complex with FOSL2 and other co-factors in active chromatin, especially super-enhancers. Silencing of either JUN or its co-factors disrupted the core regulatory circuitry, which was accompanied by strong deleterious impacts on both cell viability and tumorigenicity. As BET bromodomain proteins dictate transcriptionally active chromatin, targeted degradation of BET proteins by PROTAC compounds exerted potent inhibitory effects on both JUN cistrome and DDLPS development.

Conclusion: Our study reveals that amplification and overexpression of JUN contribute to DDLPS development. DDLPS-associated genomic abnormalities collaborate with BET proteins to fuel disease-driving core regulatory circuitry.

Citation Format: Ye Chen, Pushkar Dakle, Tuan Zea Tan, Ruby Yu-Tong Lin, Mark E. Puhaindran, Sigal Gery, Liang Xu, Phillip H. Koeffler. Genetic activation of JUN fuels the core regulatory circuitry of de-differentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2438.
Abstract 2447: TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes
Authors: Kirschnerova Viktoria , Vaishampayan Prajakta , Khawam Maria , Curiel-Lewandrowski Clara , Wondrak Georg T. , Dickinson Sally E.
Published: July 2021

Toll-like receptor 4 (TLR4) is gaining attention for its role in several cancer-associated signaling pathways, including the regulation of inflammation, DNA repair and epithelial-mesenchymal transition (EMT). There is evidence that TLR4 contributes to ultraviolet (UV) radiation-induced stress signaling in skin cells, including keratinocytes. Blockade of TLR4 activity using the pharmacological antagonist resatorvid (TAK-242) inhibits not only acute UV-induced signaling in vitro and in vivo, but blocks UV-induced skin carcinogenesis in mouse models. In order to genetically explore the role of TLR4 in keratinocyte responses to UV-induced stress, we recently utilized CRISPR/Cas9 techniques to delete TLR4 from the genome of human HaCaT keratinocyte cells in culture. First, using differential gene expression analysis (employing the EMTRT2 Profiler PCR array technology) it was observed that TLR4 KO status is associated with pronounced downregulation of EMT-related gene expression (MMP9, VIM, FN1, SNAI1, TWIST1) confirmed by independent RT-qPCR analysis. TLR4 was also a determinant of UV-induced inflammatory enzyme expression including PTGS2. Phenotypically, TLR4 KO cells displayed an attenuation of Matrigel transwell migration. TLR4 KO cells demonstrated increased sensitivity to UV-induced apoptosis as detected by flow cytometry of annexin V/propidium iodide stained cells, and by immunodetection of increased PARP cleavage. Moreover, NanoString nCounter expression analysis of murine skin chronically exposed to tumorigenic UV indicated that topical resatorvid treatment was associated with MMP9 downregulation. Future studies will employ this novel TLR4 KO keratinocyte cell line as a mechanistic tool to dissect the role of TLR4 in inflammatory signaling and EMT response in the context of skin photodamage and carcinogenesis. Supported by NIH grants ES029579, R03CA230830, P01CA229112.

Citation Format: Viktoria Kirschnerova, Prajakta Vaishampayan, Maria Khawam, Clara Curiel-Lewandrowski, Georg T. Wondrak, Sally E. Dickinson. TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2447.
Abstract 2459: Altered bioenergetics profile in prostate cancer cells treated with andrographolide
Authors: Sosa Jesus , Vázquez María Sánchez , Ayala-Peña Sylvette , Ramos Carlos Torres , Ferrer Magaly Martínez
Published: July 2021

Prostate cancer (PCa) is the second leading cause of cancer deaths in the United States alone. An emerging area of cancer therapeutics is the combination of the standard-of-care treatment with natural products. Andrographolide, a labdane diterpenoid and the main bioactive component of the medicinal plant Andrographis paniculata, has shown a wide range of therapeutic potential, including cancer. Previous studies from our laboratory have demonstrated that Andrographolide induces a DNA damage response, including ATM gene upregulation, involving cell growth suppression, cell cycle arrest, and apoptosis in PCa in vitro. The ATM gene is an important regulator of p53 and is consider a master switch for the p53-family member p63. The p63 is a natural marker of the basal lamina in the normal prostate, while its expression is lost in 99% of prostatic adenocarcinomas. Like p53, the p63 protein is implicated in tumor suppression and inhibition of metastasis, and also plays a role in energy metabolism. In this study, we aim to (1) determine the role of Andrographolide on the expression of p63, and (2) determine the bioenergetics profile of PCa cells. Androgen-dependent (22Rv1) and androgen-independent (PC3) PCa cell lines were treated with Andrographolide (25µM) for 24 h and 48 h. The gene expression of TP63 was assessed by real-time quantitative PCR (RT-qPCR). At the protein level, the expression of p63 was evaluated by immunofluorescence. The bioenergetics profile was performed using the Agilent Seahorse XF Real-Time ATP Rate Assay to determine the ATP production from glycolysis and mitochondria in living PC3 and 22Rv1 PCa cells. RT-PCR analysis showed a significant upregulation of the TP63 gene with a fold-change of 2.2 (p< 0.05) and immunofluorescence analysis showed an increase in expression of p63 protein with a fold-change of 1.8 (p< 0.05) in 22Rv1 cells treated with Andrographolide. The bioenergetics profile of PC3 cells, 48 h after Andrographolide treatment, revealed a shift in the cytoplasmic to mitochondrial ATP production rate from 54.4% to 72.6% and from 45.6% to 27.4%, respectively. Similar results were obtained in 22Rv1 cells. These results confirmed that Andrographolide induces the expression of the tumor protein p63 in androgen-dependent 22Rv1 PCa cells and alters the bioenergetics profile of both PC3 and 22Rv1 PCa cells. Our findings suggest a novel mechanism in which Andrographolide promotes the expression of p63 as in normal cells, and transforms the bioenergetics by increasing glycolytic-ATP.

Citation Format: Jesus Sosa, María Sánchez Vázquez, Sylvette Ayala-Peña, Carlos Torres Ramos, Magaly Martínez Ferrer. Altered bioenergetics profile in prostate cancer cells treated with andrographolide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2459.
Abstract 2461: SIRT1 regulates ovarian cancer metastasis via altering the exosome release and glycogen deposition
Authors: Roy Sraddhya , Das Ananya , Vernekar Manisha , Das Subhadip , Chatterjee Nabanita
Published: July 2021

Ovarian cancer is the lethal most gynaecological malignancy which exhibits high metastatic rate in peritoneal cavity, especially leading to metastasis including omentum. According to the histopathological segregation of ovarian cancer, epithelial ovarian cancer (EOC) is considered as the most metastatic in both pre- or post-menopausal state, thus several changes in hormonal level such as estrogen, progesterone etc., increase the risk of developing ovarian cancer in women. Obesity or fat deposition are observed to be the after effects of hormonal transition mainly due the depletion of estrogen level. In this context, stored glycogen or deposited fat in the cancer cells has recently drawn the research concern. Few studies suggested that hypoxic environment (HIF-1) could regulate the accumulation of glycogen in cancer cells leading the metastatic micro-environment. The level of HIF-1 could coordinate with the SIRT1 status in tumor dormancy via regulating oxidative stress and metabolic alterations that confers the ovarian cancer stem cell like properties. Interestingly, SIRT1 also controls the secretion of pro-tumorigenic exosomes in cancer invasion. Exosomes, small bioactive extracellular vesicles, can migrate expediently through body barriers and promotes the homing of migrating cancer cells. We observed that the overexpressed SIRT1 in EOC patients has increased concentration of secreted exosomes as compared to heathy individuals. Simultaneously, we have shown how SIRT1 level in ovarian cancer cells co-relate with exosome secretion in regulating glycogen deposition of post-menopausal females. Furthermore, we also found that post-menopausal EOC patients have shown an increased expression of lipids and glycogen in the cancer cells. Therefore, we can conclude that ovarian cancer cell derived exosomes might facilitate fat deposition via regulating their oncogenic cargoes to metastasize ovarian cancer.

Citation Format: Sraddhya Roy, Ananya Das, Manisha Vernekar, Subhadip Das, Nabanita Chatterjee. SIRT1 regulates ovarian cancer metastasis via altering the exosome release and glycogen deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2461.
Abstract 247: Identification of an ovarian cancer selective splice variant of mesothelin utilizing the Kiromic proprietary search engine CancerDiff
Authors: Piccotti Lucia , Mirandola Leonardo , Chiriva-Internati Maurizio
Published: July 2021

The advancement of cures for cancer needs the development of novel, more efficacious, and more specific immunotherapeutic approaches through the discovery of novel target candidates displaying differential expression between healthy and malignant tissues. CancerDiff is a proprietary software module for the identification of potential new immunotherapeutic cancer targets that originate from differentially expressed, alternatively spliced transcripts. When utilized to analyze Ovarian Cancer (OV) datasets, CancerDiff identified a selectively upregulated mesothelin (MSLN) splice variant translated into a protein isoform (IsoMSLN) bearing a distinct unique peptide absent in the canonical protein sequence. To validate this prediction and to confirm the upregulation of IsoMSLN in OV, datasets from publicly available proteomic repositories were searched for its unique signature peptide. In agreement with CancerDiff prediction, IsoMSLN peptide was detected in 71% of OV samples and 61% of adjacent normal tissues. Molecular modeling tools predicted this peptide to be part of the extracellular portion of the protein in an antibody accessible region. These results indicate IsoMSLN unique peptide as a suitable target for immunotherapy for OV cancer.

Citation Format: Lucia Piccotti, Leonardo Mirandola, Maurizio Chiriva-Internati. Identification of an ovarian cancer selective splice variant of mesothelin utilizing the Kiromic proprietary search engine CancerDiff [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 247.
Abstract 2476: The oncogenic role of p63 in altered-FGFR3 bladder tumors
Authors: Zambrano Macarena , Moreno-Vega Aura I. , Dufour Florent , Sciacca Marianela , Langle Yanina V. , Radvanyi François , Eiján Ana M. , Bernard-Pierrot Isabelle , Lodillinsky Catalina
Published: July 2021

Recently, a new classification of muscle-invasive bladder cancer (MIBCs) has been proposed (Kamoun, 2020). There are 6 classes of MIBC: the over-expression or activating mutations in Fibroblast Growth Factor Receptor 3 (FGFR3) are associated with luminal subtypes. As typically expected, basal tumors exhibit increased TP63 activity, but interestingly, this is also observed in luminal-papillary (LumP) tumors. LumP are enriched in both, FGFR3 mutations and p63 activation. The regulatory gene pathways and p63 functional role in these tumors remains poorly characterized. Therefore, this is our main objective, together with the study of the link with FGFR3 mutations. We hypothesize that p63 drives a protumoral program in bladder cancer (BC) commanded by the regulation of altered FGFR3. We used human BC cell lines bearing FGFR3 mutations with inducible silencing for p63 in order to identify the functional role of p63 in this context. Loss of p63 led to a decreased 2D and 3D growth (p<0.001). Gelatin degradation and migration ability were significantly lower in p63 depleted cells compared to CRL (p<0,01). In vivo assays showed that tumor volume was significantly lower in p63 depleted tumors compared to CRL (p<0.01), accompanied by a reduction of proliferating cell nuclear antigen (PCNA) expression, indicating that p63 is an essential protein through which the cell mediates its proliferative and migratory mechanism. In order to study the link between both, FGFR3 alterations and p63 activity, we used FGFR3 Knocked down cells and pharmacological inhibitors. FGFR3 inhibition decreased p63 expression associated with a reduction in TP63 activity. It reduced proliferation and cell migration in similar level to those observed by directly silencing p63 in FGFR3 altered cells. Conversely, FGFR3 wt cells were not affected by the inhibitor. Overall, these results show that FGFR3 inhibition blunted the carcinogenic program carried out by p63, suggesting there is a regulation of FGFR3 over p63. Supporting the regulation of p63 by FGFR3, analysis of mRNA levels in human bladder tumors showed that p63 expression was significantly higher in FGFR3 mutated tumors in two different sets of samples. In summary, we experimentally demonstrated the regulation of FGFR3 over the protumoral transcription factor p63 that induces proliferation, migration and tumor growth in bladder cancer.

Citation Format: Macarena Zambrano, Aura I. Moreno-Vega, Florent Dufour, Marianela Sciacca, Yanina V. Langle, François Radvanyi, Ana M. Eiján, Isabelle Bernard-Pierrot, Catalina Lodillinsky. The oncogenic role of p63 in altered-FGFR3 bladder tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2476.
Abstract 2483: Expression of microRNA and mRNA in U87 glioblastoma cells transfected with PTEN
Authors: Stefanik David , Goldblatt Sidney , Jordan Rick
Published: July 2021

Background: U87 glioblastoma cells (ATCC HTB-14) were transfected with a plasmid containing the wild type PTEN gene. Untransfected U87 cells and U87 cells transfected with a plasmid containing only a fluorescent marker served as controls.

Methods: Cells were assessed for neurosphere formation and growth in soft agar. Genes were assessed for expression of microRNA and messenger RNA by the NanoString nCounter platform.

Results: The PTEN transfectants exhibited reduced neurosphere formation and diminished growth in soft agar. The PTEN transfectants exhibited a 10-fold or greater change in 101 of 799 microRNA species assessed compared to untransfected cells. The change in microRNA expression was strongly influenced by growth in hypoxic conditions. Expression of mRNA was altered in 260 of 760 genes assessed with 8% of the genes showing a 10-fold or greater difference in expression level. A detailed summary of the NanoString results is provided.

Conclusions: (1) The PTEN transfectants demonstrate growth characteristics in vitro suggestive of decreased tumorigenicity. (2) The expression level of multiple genes is affected in the PTEN transfectants. (3) Hypoxic conditions bring out the greatest change in the expression level of microRNA in the PTEN transfected cells.

Citation Format: David Stefanik, Sidney Goldblatt, Rick Jordan. Expression of microRNA and mRNA in U87 glioblastoma cells transfected with PTEN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2483.
Abstract 2488: Elucidating the role of G0S2 in breast cancer
Authors: Corbet Andrea K. , Bikorimana Emmanuel , Singh Ratnakar , Fazal Zeeshan , Boyd Raya I. , Freemantle Sarah , Spinella Michael J.
Published: July 2021

Breast cancer has the second-highest cancer mortality rate for women in the United States. There are effective therapies available and a growing list of biomarkers for prognosis. However, new breast cancer targets are critically needed for patients refractory to available therapies. The present study evaluates the role of gap 0 switch 2 (G0S2), in breast cancer. Using online databases, we observed G0S2 expression is downregulated in patients with estrogen receptor-positive (ER+) breast cancer and patients with high G0S2 expression have a lower rate of relapse after treatment. Previously, our lab showed G0S2 overexpression in ER+ breast cancer cell lines resulted in decreased proliferation. Further knockout of G0S2 in murine embryonic fibroblast resulted in higher oncogenic transformation and resistance to mTOR inhibitors. These results suggest that G0S2 has a tumor suppressive function in ER+ breast cancer. To test whether G0S2 has a role in ER-negative breast cancer global knockout of G0S2 was engineered in the ER-negative MMTV-PyMT transgenic mouse model. Slower tumor progression and decreased lung metastasis was observed in G0S2-null transgenic females compared to wild-type transgenic females. These data suggest that the role of G0S2 in breast cancer in dependent on ER-status, where G0S2 functions as a tumor suppressor in ER-positive breast cancer and a tumor promoter in ER-negative breast cancer. Molecular and genetic studies manipulating G0S2 and ER in various breast cancer contexts are ongoing and will help elucidate that precise role of G0S2 in breast cancer and provide clarity on the potential of G0S2 as a novel prognostic marker and therapeutic target.

Citation Format: Andrea K. Corbet, Emmanuel Bikorimana, Ratnakar Singh, Zeeshan Fazal, Raya I. Boyd, Sarah Freemantle, Michael J. Spinella. Elucidating the role of G0S2 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2488.
Abstract 2496: CD82 regulation of c-Met in metastatic prostate cells
Authors: Sridhar Suganthi , Brandes Emily , Aller Patrick , Barrie Eddie
Published: July 2021

Metastatic prostate cancer is the second leading cause of cancer deaths among men in the U.S. Metastasis involves regulators, which involve complex cell signaling processes and our current knowledge on how prostate cancer metastasis develops is still under investigation. If these regulators can be identified and the mechanisms fully elucidated, they can be potential targets for oncologic treatment. CD82 (KAI1), a tetraspanin first identified as a metastasis tumor suppressor in prostate cells is one such regulator involved in the metastatic process. Currently, CD82 downregulation is not only observed in prostate but also in a variety of many invasive epithelial tumors which include tumors of the gastric, colon, cervix, breast, skin, bladder, lung, pancreas, liver, and thyroid. CD82 has been well documented as an inhibitor of cell motility and invasion in cancer cells, with varied inhibitory mechanisms. By introducing CD82 in metastatic cell lines (PC3) that do not express CD82, we have shown CD82 to regulate c-Met, a growth factor receptor, a protooncogene that is over expressed and or over activated in prostate tumors. Overactivation of c-Met promotes cell proliferation, migration and invasion. However, it is still not well understood how exactly CD82 regulates c-Met. Using metastatic prostate cell lines (PC3) and comparing them with PC3 clones generated to express CD82, we are using immunofluorescence, immunoprecipitation, flow cytometry, and western blot techniques to analyze the exact mechanism by which CD82 regulates c-Met. Preliminary data indicate CD82 does not colocalize with CD82, nor does it downregulate c-Met expression on the cell surface. However, CD82 expression seems to cause varied distribution of c-Met in the cell and is currently being investigated. Preliminary indications are that CD151, another tetraspanin may be involved in this regulation. CD151 is a known tumor promoter and has been shown to associate with c-Met. Currently, our lab is exploring the redistribution pattern of c-Met upon CD82 expression as a regulatory mechanism, in addition to exploring the CD82 and CD151 expression dynamics as part of or as alternate mechanism to c-Met regulation. The results observed will provide insights into the role CD82 plays in c-Met regulation as well as it may overall play in tumor progression and metastasis in prostate cancer. Notably, the results may also shed more light on CD82 regulation in other tumors where CD82 downregulation is observed.

Citation Format: Suganthi Sridhar, Emily Brandes, Patrick Aller, Eddie Barrie. CD82 regulation of c-Met in metastatic prostate cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2496.
Abstract 251: Data distribution for easy pancancer analysis
Authors: Lindgren Caleb M. , Minor Chelsie , Olsen Lindsey K. , Henderson Brittany , Investigators CPTAC , Payne Samuel H.
Published: July 2021

A growing number of datasets characterize tumors with quantitative proteomic and genomic data. These emerging datasets are typically specific to a single cancer type. However, analysis of proteogenomic patterns across multiple cancer types allows us to identify common mechanisms in the function of cancer, as well as mechanisms that are unique to specific cancer types. Here we present how a Python data API from the NCI's CPTAC program bridges the gap between different cancer datasets to facilitate pancancer analysis. While we have previously presented this API as a general model for data dissemination, we have yet to display the specific pancancer applications of the tool. Several key aspects of its design optimize its use from a pancancer point of view, including parsing data to a single consistent format for all datasets, and automatically managed data updates for each dataset. Our analyses demonstrate the efficacy of our data model for generalizing an analysis pipeline, applying it to multiple cancer types, and comparing the results. This enables unique discoveries that characterize the overall proteogenomic landscape of cancer.

Citation Format: Caleb M. Lindgren, Chelsie Minor, Lindsey K. Olsen, Brittany Henderson, CPTAC Investigators, Samuel H. Payne. Data distribution for easy pancancer analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 251.
Abstract 2558: Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer
Authors: Wilson Mike R. , Reske Jake J. , Chandler Ronald L.
Published: July 2021

Up to 20% of all cancer deaths in women can be attributed to excess body weight. Endometrial cancer was the first cancer identified with an increased risk among obese populations, and incidence of endometrial cancer is rising steadily due to the obesity epidemic. Obesity is associated with over 60% of endometrial cancer occurring in the United States, but like many cancers, the causes of endometrial cancer are multi-faceted. Increased estrogen abundance in obese women is considered the primary driver, but obesity-linked complications, including hyperinsulinemia, are also known to contribute to endometrial cancer. However, obesity alone is not sufficient for tumorigenesis. Genetic mutations driving endometrial cancer are well characterized, but these mutations alone are not sufficient for EC pathogenesis as they exist in uteri without cancer. In normal cells, the PI3K pathway maintains metabolic homeostasis, but in cancer the PI3K pathway is activated to promote cell survival. There is an urgent need to understand the mechanisms by which obesity and genetic mutation interact to promote EC. Metabolic signaling influences protein production and folding. The unfolded protein response pathway acts to maintain protein quality control, and is activated upon endoplasmic reticulum stress. During endoplasmic reticulum stress, if proteostasis cannot be achieved, the unfolded protein response is activated, resulting in cell death. Obesity results in systemic endoplasmic reticulum stress, with overabundant estrogen and hyperinsulinemia likely as contributing factors. In our preliminary studies, we have identified a role for PI3K pathway mutations in regulating endoplasmic reticulum stress in the endometrial epithelium. By antagonizing endoplasmic reticulum stress, PI3K pathway mutations may promote survival of endometrial epithelium through resistance to unfolded protein response-mediated cell death mechanisms. Through understanding endoplasmic reticulum stress resistance in the endometrial epithelium, we hope to develop preventative interventions for obese women at risk for endometrial cancer.

Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2558.
Abstract 2569: miR520a inhibits Fzd9 in bronchial epithelial cells and NSCLC
Authors: Smith Alex J. , Do Paulina , Sompel Kayla M. , Elango Alamelu
Published: July 2021

Non-small cell lung cancer (NSCLC) continues to be the most diagnosed cancer type worldwide and has the highest mortality rate. Treatment is highly researched, but 5 year survival remains around 20%. Chemoprevention could combat the development of lung cancer in individuals who are at high risk, such as former cigarette smokers. Iloprost is a prostacyclin analogue demonstrated to reduce the presence of endobronchial dysplasia in former smokers. Increased Frizzled-9 (Fzd9) expression is induced by Iloprost, resulting in activation of peroxisome proliferator activated receptor γ (PPARγ) and inhibition of transformed growth. The goal of this study is to elucidate the relationship of cigarette smoke, NSCLC, and miRNA regulation of Fzd9 expression. We hypothesized that miRNA regulate Fzd9 expression when NSCLC cells are exposed to both cigarette smoke condensate (CSC) and Iloprost. miR520a5p was predicted in silico to bind Fzd9 and we validated binding with a miR520a5p mimic and Fzd9 3'UTR luciferase in an immortalized human bronchial epithelial cell line (HBEC) and NSCLC cell lines. A549 and H322 NSCLC cells and HBEC were exposed to cigarette smoke condensate (CSC) and Iloprost. CSC increased miR520a expression, while the opposite was observed in Iloprost treated cells. We also measured Fzd9 3'UTR activity and demonstrated that CSC decreases both Fzd9 3'UTR and PPRE activity, while Iloprost does the opposite. We investigated the effect of miR520a5p on targets downstream of Fzd9, including PPARγ response element (PPRE), a direct measure of PPARγ activity, and epithelial to mesenchymal transition (EMT) gene expression, including Cox2, E-cadherin, and CRB3. We found that when Fzd9 is inhibited by miR520a5p, PPRE activity and epithelial gene expression are inhibited, while mesenchymal gene expression is stimulated. Rescue experiments combining CSC with a miR520a5p inhibitor or iloprost with a miR520a5p mimic supported a role for miR-520a-5p in inhibition of Fzd9 in a CSC carcinogenic pathway in lung epithelial cells that is reversed with iloprost treatment. Overall, these findings suggest a mechanism for miRNA regulation of Fzd9 in lung epithelial cells exposed to cigarette smoke and a potential chemoprevention target.

Citation Format: Alex J. Smith, Paulina Do, Kayla M. Sompel, Alamelu Elango. miR520a inhibits Fzd9 in bronchial epithelial cells and NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2569.
Abstract 2605: A natural history study of men with high-risk genetics for prostate cancer (PCa) using multiparametric MRI (mpMRI)
Authors: Karzai Fatima , Couvillon Anna , McKinney Yolanda , Lee-Wisdom Katherine , Choyke Peter L. , Giri Veda N. , Morgan Todd M. , Cheng Heather H. , Merino Maria J. , Pinto Peter A. , Turkbey Baris , Dahut William L.
Published: July 2021

Introduction: The understanding of molecular genetics in PCa provides insight into disease progression and treatment. Less is known about PCa development in men with known high-risk germline pathogenic/likely pathogenic variants in PCa related genes, particularly DNA damage repair (DDR). mpMRI can localize and detect PCa lesions in this population. We are conducting a multicenter natural history study of male participants (prts) with documented germline variant(s) in BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, EPCAM, HOXB13, ATM, NBN, TP53, CHEK2, PALB2, RAD51D, BRIP1, or FANCA who do not have a PCa diagnosis using mpMRI (NCT03805919).

Methods: Up to 500 men, 30-75 years old (y/o) will undergo mpMRI evaluation at baseline and every 2 years (yrs). Prts are followed at 12-mo intervals to determine PSA, PCa diagnosis, and/or disease/survival status until death. Indication for prostate biopsy includes PSA >2.0 for 30 - 49 y/o; >2.5 ng/mL for 50 - 75 y/o and/or PIRADS 3+ MRI lesion or clinical discretion. Tissue obtained will undergo full transcriptome analysis.

Results: 100 prts have enrolled. Median age is 47 y/o. Median on-study PSA is 0.87 (0.16-10.05 ng/mL). Median PSA density is 0.029. Gene mutations are BRCA2 (40%), BRCA1 (33%), MSH2 (9%), and CHEK2 (4%). Mutations in ATM, HOXB13, MLH1, MSH6, PMS2, and EPCAM are ≤3%. No significant AEs have been observed. Of 96 prts, 25 (26%), had indication for biopsy [PIRADS 4 lesion: 16 (64%), PIRADS 3 lesion: 4 (16%), elevated PSA: 6 (24%), or clinical indication 1: (4%)]. 22 prostate biopsies were performed and 6 prts were diagnosed with PCa. One prt was upstaged on radical prostatectomy (RP).

Conclusions: We found mpMRI-based PCa screening in men with high-risk gene mutations, especially DDR genes, is feasible and can be used to identify clinically significant PCa and monitor for disease progression. Future guidelines should consider age, mutation specificity and mpMRI. Accrual and correlative studies (biomarkers, PBMCs) are on-going.

AgeMutationPSA (ng/mL)Biopsy Gleason ScoreTreatment65BRCA22.253+3RP52BRCA10.693+3Active Surveillance (AS)62CHEK22.493+3AS59PMS21.033+3AS59CHEK22.243+4Radiation55BRCA21.683+3RP: 4+4=8

Citation Format: Fatima Karzai, Anna Couvillon, Yolanda McKinney, Katherine Lee-Wisdom, Peter L. Choyke, Veda N. Giri, Todd M. Morgan, Heather H. Cheng, Maria J. Merino, Peter A. Pinto, Baris Turkbey, William L. Dahut. A natural history study of men with high-risk genetics for prostate cancer (PCa) using multiparametric MRI (mpMRI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2605.