High-grade Serous Ovarian Carcinoma Cell Lines Research Articles

Abstract 2418: Copy number variation status of DNA in extracellular vesicles as novel biomarkers of high-grade serous ovarian carcinoma

Abstract Objective: Ovarian cancer is a gynecologic malignancy with a dismal prognosis due to the lack of accurate methods for early diagnosis methods and effective therapies. High-grade serous ovarian carcinoma (HGSOC) is the most common subtype and copy number variations (CNVs) are the dominant genomic events in HGSOC. Extracellular vesicles (EVs) circulate in body fluids and carry pathological genomic information positively. In this study, we evaluated the significance of EV-DNA as novel biomarkers for HGSOC patients. Methods: A total of 124 samples from HGSOC patients and cell lines were analyzed. EVs were isolated using the ultracentrifuge method. Comprehensive CNV statuses were analyzed by whole genome sequencing (WGS) and SNP array. In reference to the Ovarian Cancer Moon Shot database, 30 dominant CNVs in HGSOC were selected as dominant targets for analysis by droplet digital PCR (ddPCR). For predicting the response to PARP inhibitors, HGSOC patients who were treated with olaparib were collected, and DNA was extracted from FFPE tissue at the time of surgery. Results: NTA assays and cryo-electron microscopic analyses showed that the EVs were successfully isolated from all samples. Both WGS and SNP arrays detected correlating CNVs in cell-DNA and EV-DNA for cell lines, and in tissue-DNA and EV-DNA in ascites for patients. The CNV status was also accurately measured by ddPCR. Copy numbers of RAD51, BRCA1, AKT2, CCNE1, and MSH6 were significantly higher in malignant tissue than in benign tissue (p<0.0001, p<0.0001, p=0.0012, p=0.002. p=0.0328, respectively), and were detectable in EV-DNA from ascites. Additionally, the amount of DNA in malignant ascites was significantly higher than in benign ascites (p=0.016), and increased DNA in ascites itself suggested the presence of malignancy. Furthermore, EV-DNA more accurately reflected the CNV status of tumor DNA than cell-free DNA in ascites. Regarding the prediction of PARP inhibitor response, the CNV status in EV-DNA from patients who were treated with olaparib, a PARP inhibitor, demonstrated a marked difference between responders and non-responders. The equation calculated based on the combination of six genes (RB1, GABRA6, CTNNB1, MYC, RAD51, BARD1) could predict the response to olaparib (AUC: 1.0). The selected genes were validated in vitro using EV-DNA, and responders could be identified using the prediction equation calculated from CNV status of the selected genes in EV-DNA from ascites. Conclusion: CNV status of EV-DNA was correctly evaluated and can serve as novel diagnostic and prognostic biomarkers for HGSOC. Citation Format: Ryosuke Uekusa, Akira Yokoi, Kosuke Yoshida, Juntaro Matsuazki, Yusuke Yamamoto, Hiroaki Kajiyama. Copy number variation status of DNA in extracellular vesicles as novel biomarkers of high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2418.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Abstract 3058: Colocalization of cancer-associated biomarkers on single extracellular vesicles for early-cancer detection

Abstract Introduction: Extracellular vesicles (EVs) are abundant in biological fluids and increasingly recognized for their diagnostic and therapeutic potential. Unlike circulating tumor DNA which scales in abundance with tumor size and is a product of tumor cell death; tumor EVs are secreted continuously, are remarkably stable, and used by cancer cells as messengers to benefit tumor growth and metastasis, suggesting they may be a better indicator of early-stage cancer. Tumor-derived EVs reflect their cell of origin, making them an ideal analyte for liquid biopsy diagnostic assays. We describe a platform technology, Mercy Halo™, which interrogates millions of individual EVs within a plasma or serum sample to capture and detect cancer cell derived EVs. Using panels of antibodies targeting up to three cancer-associated biomarkers simultaneously, we demonstrate Mercy Halo enables sensitive and specific detection of early-stage high-grade serous ovarian carcinoma (HGSOC) with excellent reproducibility in a proof-of-concept (PoC) study. Methods: Following purification of a sample by size-exclusion chromatography, EVs were captured using antibody-functionalized beads and interrogated using proximity ligation qPCR. EVs were characterized and biomarker expression confirmed by traditional immunoassay and Mercy Halo. The colocalization of specific biomarkers on single EVs was measured using a panel of antibodies against biomarkers upregulated in HGSOC and human cancer cell lines. Optimized antibody panels were then used in a feasibility study using EVs purified from plasma samples from early- and late-stage HGSOC, benign ovarian masses, and healthy controls. Results: Mercy Halo detects colocalized biomarkers specifically and sensitively on the surface of tumor derived EVs. Surface biomarkers expressed by cancer cells were detected in our assay and signal strength correlated with protein expression, demonstrating that EVs can be used as a surrogate to indicate the presence or absence of cancer. We demonstrate that biomarkers present on cancer cells are colocalized on EVs derived from a tumor and are readily detectable using the Mercy Halo platform. A positive signal was dependent on the presence of all biomarkers on the same EV. Importantly, there was significant improvement in both the specificity and sensitivity of the assay as the number of biomarker antibodies used to interrogate the EVs increases. Finally, in a small clinical cohort PoC study, we demonstrate improved detection of early stage HGSOC relative to CA125 ELISA. Conclusions: Tumor-derived EVs are a robust analyte for liquid-biopsy assays which can readily be detected using our platform technology. This PoC study suggests Mercy Halo can discriminate all stages of HGSOC from benign and healthy samples in human plasma with high sensitivity and specificity and is being investigated for extensibility in other cancers and diseases. Citation Format: Daniel P. Salem, Laura T. Bortolin, Anthony D. Couvillon, Dan Gusenleitner, Jonian Grosha, Sanchari Banerjee, Kelly M. Biette, Ibukunoluwapo O. Zabroski, Christopher R. Sedlak, Delaney M. Byrne, Peter A. Duff, Bilal F. Hamzeh, MacKenzie S. King, Lauren T. Cuoco, Emily S. Winn-Deen, Eric K. Huang, Joseph C. Sedlak. Colocalization of cancer-associated biomarkers on single extracellular vesicles for early-cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3058.

Loss of CBX2 causes genomic instability and Wnt activation in high grade serous ovarian carcinoma cells.

High grade serous ovarian carcinoma (HGSOC) is lethal with insidious onset, rapid progression, poor prognosis, and limited treatment options. Polycomb repressor complexes (PRC) 1 and 2 are intimately involved in progression of many types of cancer including HGSOC. Unlike the consistent constitution of PRC2, PRC1 consists of diverse components whose clinical significance in HGSOC are not entirely clear. Here, prognosis-associated PRC1 components were identified through data-mining. CBX2 promoted proliferation and reduced apoptosis of HGSOC cell lines OVCAR4, OVCAR3, and CAOV3. Complete loss of CBX2 by CRISPR-cas9 editing (CBX2KO ) destabilized genome stability with increased spontaneous chromosomal breaks and tendency to polyploidy accompanied by disrupted cell cycle especially stalled G2/M transition and caused severe cell death. Wnt/β-catenin/LEF1/TCF7L1 was activated in surviving OVCAR4-CBX2KO clones to bypass the crisis caused by loss of CBX2. The relieve of TCF7L1 core-promoter region occupied by CBX2 might be one of the possible explanations to TCF7L1 increase in OVCAR4-CBX2KO clones. Subcutaneous tumor model further validated that depletion of CBX2 repressed HGSOC cell line derived tumor growth. High immunohistochemistry score of CBX2 in primary ovarian cancer tissue associated with advanced clinical stage (p = 0.033), poor overall survival (HR = 3.056, 95% CI: 1.024-9.123), and progression free survival (HR = 4.455, 95% CI: 1.513-13.118) in HGSOC. Overall, our results suggested that CBX2 was a promising prognostic factor and therapeutic target in HGSOC.

Upregulation of the Long Noncoding RNA CASC10 Promotes Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Despite initial responses to first-line treatment with platinum and taxane-based combination chemotherapy, most high-grade serous ovarian carcinoma (HGSOC) patients will relapse and eventually develop a cisplatin-resistant fatal disease. Due to the lethality of this disease, there is an urgent need to develop improved targeted therapies against HGSOC. Herein, we identified CASC10, a long noncoding RNA upregulated in cisplatin-resistant ovarian cancer cells and ovarian cancer patients. We performed RNA sequencing (RNA-seq) in total RNA isolated from the HGSOC cell lines OVCAR3 and OV-90 and their cisplatin-resistant counterparts. Thousands of RNA transcripts were differentially abundant in cisplatin-sensitive vs. cisplatin-resistant HGSOC cells. Further data filtering unveiled CASC10 as one of the top RNA transcripts significantly increased in cisplatin-resistant compared with cisplatin-sensitive cells. Thus, we focused our studies on CASC10, a gene not previously studied in ovarian cancer. SiRNA-mediated CASC10 knockdown significantly reduced cell proliferation and invasion; and sensitized cells to cisplatin treatment. SiRNA-mediated CASC10 knockdown also induced apoptosis, cell cycle arrest, and altered the expression of several CASC10 downstream effectors. Multiple injections of liposomal CASC10-siRNA reduced tumor growth and metastasis in an ovarian cancer mouse model. Our results demonstrated that CASC10 levels mediate the susceptibility of HGSOC cells to cisplatin treatment. Thus, combining siRNA-mediated CASC10 knockdown with cisplatin may represent a plausible therapeutic strategy against HGSOC.

Functional interaction between endothelin-1 and ZEB1/YAP signaling regulates cellular plasticity and metastasis in high-grade serous ovarian cancer

BackgroundEpithelial-to-mesenchymal transition (EMT) encompasses a highly dynamic and complex key process which leads to metastatic progression. In high-grade serous ovarian carcinoma (HG-SOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling promotes EMT driving tumor progression. However, the complex nature of intertwined regulatory circuits activated by ET-1 to trigger the metastatic process is not fully elucidated.MethodsThe capacity of ET-1 pathway to guide a critical transcriptional network that is instrumental for metastatic growth was identified in patient-derived HG-SOC cells and cell lines through immunoblotting, q-RT-PCR, co-immunoprecipitation, in situ proximity ligation, luciferase reporter, chromatin immunoprecipitation assays and publicly available databases. Functional assays in HG-SOC cells and HG-SOC xenografts served to test the inhibitory effects of ET-1 receptors (ET-1R) antagonist in vitro and in vivo.ResultsWe demonstrated that ET-1/ETAR axis promoted the direct physical ZEB1/YAP interaction by inducing their nuclear accumulation in HG-SOC cells. Moreover, ET-1 directed their engagement in a functional transcriptional complex with the potent oncogenic AP-1 factor JUN. This led to the aberrant activation of common target genes, including EDN1 (ET-1) gene, thereby creating a feed-forward loop that sustained a persistent ET-1/ZEB1 signaling activity. Notably, ET-1-induced Integrin-linked kinase (ILK) signaling mediated the activation of YAP/ZEB1 circuit driving cellular plasticity, invasion and EMT. Of therapeutic interest, treatment of HG-SOC cells with the FDA approved ET-1R antagonist macitentan, targeting YAP and ZEB1-driven signaling, suppressed metastasis in vivo in mice. High gene expression of ETAR/ILK/YAP/AP-1/ZEB1 was a strong predictor of poor clinical outcome in serous ovarian cancer patients, indicating the translational relevance of this signature expression.ConclusionsThis study provides novel mechanistic insights of the ET-1R-driven mediators that support the ability of HG-SOC to acquire metastatic traits which include the cooperation of YAP and ZEB1 regulatory circuit paving the way for innovative treatment of metastatic ovarian cancer.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

Simple SummaryHigh-grade serous ovarian carcinoma (HGSOC) has poor prognosis for patients due to its high rate of recurrence and acquired resistance to therapy. MEK1/2-ERK1/2 signaling pathway that controls cell proliferation and survival is active in the majority of HGSOC cases, but its functional impact is unclear. We suggest that inhibition of MEK1/2 with specific inhibitor trametinib may exert anti-cancer effects upon HGSOC cells. Here we demonstrate that trametinib treatment of HGSOC cells indeed prominently inhibits cell proliferation and tumor growth, and that cisplatin-resistant cells displaying high MEK1/2 activity are particularly sensitive to trametinib. However, we also discovered that trametinib treatment of HGSOC cells has no cytotoxic effects and promotes cancer stem-like characteristics. We therefore suggest to use MEK1/2 inhibitors with other treatment strategies targeting cancer stem-like cells, like aldehyde dehydrogenase 1 inhibition that might show together strong synergy.High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.

Candidate Markers of Olaparib Response from Genomic Data Analyses of Human Cancer Cell Lines.

Simple SummaryOlaparib is an oral medication typically used to treat certain advanced ovarian and breast cancers with mutations in BRCA1 or BRCA2 genes. Mutations in these genes can increase the risk of developing breast, ovarian, and other types of cancer. Olaparib is the first clinically approved drug that specifically targets a vulnerability of cancers with these mutations. Genetic alterations in cancer tumors can affect response to treatment in cancer patients. Cancer models such as cell lines, which are cancer cells derived from patients and have been grown in the laboratory over time, can be used to identify these alterations which may contribute to sensitivity or resistance to treatment. We analyzed data from two independent groups of cancer cell lines and identified alterations in additional genes (PUM3, EEF1A1 and ELP4) that potentially increase sensitivity to olaparib. Further experimental and clinical investigations are required to validate our findings.The benefit of PARP inhibitor olaparib in relapsed and advanced high-grade serous ovarian carcinoma (HGSOC) is well established especially in BRCA1/2 mutation carriers. Identification of additional biomarkers can help expand the population of patients most likely to benefit from olaparib treatment. To identify candidate markers of olaparib response we analyzed genomic and in vitro olaparib response data from two independent groups of cancer cell lines. Using pan-cancer cell lines (n = 896) from the Genomics of Drug Sensitivity in Cancer database, we applied linear regression methods to identify statistically significant gene predictors of olaparib response based on mRNA expression. We then analyzed whole exome sequencing and mRNA gene expression data from our collection of 18 HGSOC cell lines previously classified as sensitive, intermediate, or resistant based on in vitro olaparib response for mutations, copy number variation and differential expression of candidate olaparib response genes. We identify genes previously associated with olaparib response (SLFN11, ABCB1), and discover novel candidate olaparib sensitivity genes with known functions including interaction with PARP1 (PUM3, EEF1A1) and involvement in homologous recombination DNA repair (ELP4). Further investigations at experimental and clinical levels are required to validate novel candidates, and ultimately determine their efficacy as potential biomarkers of olaparib sensitivity.

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian cancer

BackgroundIntrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC.MethodsHerein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC.ResultsWe demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo.ConclusionsOur findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.

Ovarian cancer cells direct monocyte differentiation through a non-canonical pathway

BackgroundAlternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC). Increased levels of AAMs are correlated with poor HGSOC survival rates, and AAMs increase the attachment and spread of HGSOC cells in vitro. However, the mechanism by which monocytes in the HGSOC tumor microenvironment are differentiated and polarized to AAMs remains unknown.MethodsUsing an in vitro co-culture device, we cultured naïve, primary human monocytes with a panel of five HGSOC cell lines over the course of 7 days. An empirical Bayesian statistical method, EBSeq, was used to couple RNA-seq with observed monocyte-derived cell phenotype to explore which HGSOC-derived soluble factors supported differentiation to CD68+ macrophages and subsequent polarization towards CD163+ AAMs. Pathways of interest were interrogated using small molecule inhibitors, neutralizing antibodies, and CRISPR knockout cell lines.ResultsHGSOC cell lines displayed a wide range of abilities to generate AAMs from naïve monocytes. Much of this variation appeared to result from differential ability to generate CD68+ macrophages, as most CD68+ cells were also CD163+. Differences in tumor cell potential to generate macrophages was not due to a MCSF-dependent mechanism, nor variance in established pro-AAM factors. TGFα was implicated as a potential signaling molecule produced by tumor cells that could induce macrophage differentiation, which was validated using a CRISPR knockout of TGFA in the OVCAR5 cell line.ConclusionsHGSOC production of TGFα drives monocytes to differentiate into macrophages, representing a central arm of the mechanism by which AAMs are generated in the tumor microenvironment.

Abstract B27: Cellular retinoic acid binding protein 2 (CRABP2) is a novel biomarker and potential therapeutic target for high-grade serous ovarian carcinomas

Abstract Ovarian cancer ranks fifth in cancer deaths among women in the United States, accounting for more deaths than any other gynecologic cancer. There is an unmet medical need for biomarkers that can detect high-grade serous ovarian carcinoma (HGSOC) at an early stage. Our laboratory evaluated the biomarker potential of cellular retinoic acid binding protein 2 (CRABP2). Using Western blots (WB) and immunohistochemistry (IHC), we show that CRABP2 is expressed and secreted by HGSOC cells and tissues and is absent in normal fallopian tube (FT) epithelium. CRABP2 expression was associated with poor overall survival in patients with HGSOC. Proteomic analyses quantified specific secretion of CRABP2 in conditioned media from HGSOC cell lines, primary ascites-derived tumor cells, and serum from HGSOC patients. Expression of CRABP2 was positively associated with CRABP2 copy number amplifications by mining The Cancer Genome Atlas database. In addition, DNA methylation studies identified a putative enhancer downstream of CRABP2 that is hyper-methylated in fallopian tube epithelia (FTE) tissue and hypomethylated in HGSOC cells and tissues that expressed CRABP2. Inhibition of DNA methylation with DNA methytransferase (DNMT) inhibitors resulted in robust expression of CRABP2 protein in FT cell lines. Finally, a finding supported by CRABP2 knockdown experiments showed that CRABP2 loss triggers cell death in HGSOC cell lines. In conclusion, CRABP2 may serve as a novel biomarker for HGSOC with clinical potential as a therapeutic target. Citation Format: Yi Feng, Michael Gillette, Eric Kuhn, David Klinkebiel, Marilyn A. Mitchell, Kai Doberstein, Daniele Chaves-Moreira, Sho Sato, Haineng Xu, Brett Bomwell, Michelle S. Hirsch, Carolina Reyes, Adam R. Karpf, Michael J. Birrer, Steven J. Skates, Steven A. Carr, Ronny Drapkin. Cellular retinoic acid binding protein 2 (CRABP2) is a novel biomarker and potential therapeutic target for high-grade serous ovarian carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B27.

Abstract A45: Quinacrine induces necrosis of ovarian high-grade serous carcinoma cells by breaking the mitochondria ROS balance

Abstract Ovarian high-grade serous ovarian carcinoma (HGSC) was found to be initiated and promoted by repeated exposure of fimbrial epithelium to ovulatory ROS with resultant intolerable stress of oxidative damages and DNA repair load. Consequential cell apoptosis is overcome on the one hand by quenching excessive ROS by hemoglobin contributed by retrograde menstruation and on the other by upregulation of antioxidant machinery. A dangerous balance is achieved between the oxidative stress and antioxidation and between DNA damage stress and repair. Breaking of this balance on the stress side such as by DNA chelating agent such as cisplatin or inhibitors targeting on DNA repair enzyme such as PARP leads to therapeutic cell killing. In this report, we show ROS enhancing drug such as quinacrine effectively kill HGSC cells and its tubal precursor cells with p53 and Rb defect. Quinacrine is a traditional antimalarial drug with wide antitumor effect in either a single agent or combined with chemotherapy drugs. We show here that quinacrine has a cytotoxic effect in two HGSC cell lines (KURAMOCHI, OVSAHO) and in an HPV E6/E7-immortalized human fimbrial epithelial cells, which are readily fully transformed by ovulatory follicular fluid. Cytotoxicity was observed dose-dependently at concentrations ranging from 2.5μM to 25μM. Flow cytometry analysis indicated a cell necrosis (PI +, annexin V-) 48hr post-treatment. We further found a significant induction of the mitochondrial ROS upon quinacrine treatment, and ROS inhibitor N-acetyl-l-cysteine considerably reversed this cytotoxic effect. The study proves the concept of breaking the balance of mitochondria-ROS is a feasible strategy for the development of a new drug for ovarian HGSC. Note: This abstract was not presented at the conference. Citation Format: Ching-I Huang, Tang-Yuan Chu, Hsuan-Shun Huang. Quinacrine induces necrosis of ovarian high-grade serous carcinoma cells by breaking the mitochondria ROS balance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A45.

Abstract A77: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma

Abstract The purpose of this study is to examine the contribution of epigenetics to PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC). To identify epigenetic mechanisms of PARPi resistance in HGSOC we utilized unbiased exploratory techniques, including mass spectrometry profiling of histone modifications and RNA-sequencing. Our data show that, compared to sensitive cells and tumors, PARPi-resistant HGSOC cell lines and in vivo patient-derived ascites display a global increase of histone H3 lysine 9 dimethylation (H3K9me2) accompanied by overexpression of histone methyltransferases EHMT1 and EHMT2. EHMT1/2 catalyze H3K9me2 and have canonical roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and have previously been implicated in erlotinib resistance in non-small cell lung cancer. The role of EHMT1/2 in PARPi response has not been evaluated in HGSOC. Using immunohistochemistry, we stained a tissue microarray of over 100 primary ovarian tumors for H3K9me2. High H3K9me2 correlates with significantly worse overall survival. We then used genetic and pharmacologic methods to disrupt EHMT1/2 and examined PARPi sensitivity, apoptosis, DNA repair, and cell cycle. EHMT1/2 disruption reduced H3K9me2 and sensitizes HGSOC cells to PARPi. EHMT1/2 disruption does not increase PARPi-induced apoptosis, suggesting a cytostatic effect, rather than cytotoxic. Functional assays of DNA repair show that disruption of EHMT1/2 ablates both homologous recombination and nonhomologous end joining. Consistently, immunofluorescent staining of phosphorylated histone gamma-H2AX shows increases in DNA damage following EHMT1/2 inhibition. Propidium iodide staining and flow cytometry analysis of cell cycle shows PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of Cyclin A, Cyclin B1, and M-phase inducer CDC25C. We also observed reduced phosphorylation of CDC25C and mitotic markers MPM2 and histone H3 serine 28. These data suggest EHMT1/2 increases DNA damage repair and regulates cell cycle progression, both of which contribute to PARPi resistance. In summary, this study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi and suggests a potential mechanism through DNA damage and cell cycle dysregulation. Identification of factors promoting PARPi resistance, such as EHMT1/2 and H3K9me2, will facilitate development of novel prognostic and therapeutic strategies to successfully treat resistant HGSOC. Citation Format: Zachary L. Watson, Tomomi M. Yamamoto, Connor J. Hughes, Lindsay J. Wheeler, Miriam D. Post, Kian Behbakht, Benjamin G. Bitler. Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A77.

Long Non-Coding RNA HAND2-AS1 Acts as a Tumor Suppressor in High-Grade Serous Ovarian Carcinoma.

Long non-coding RNAs (lncRNAs) are increasingly being identified as crucial regulators in pathologies like cancer. High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer (OC), one of the most lethal gynecological malignancies. LncRNAs, especially in cancers such as HGSC, could play a valuable role in diagnosis and even therapy. From RNA-sequencing analysis performed between an OC cell line, SKOV3, and a Fallopian Tube (FT) cell line, FT194, an important long non-coding RNA, HAND2 Anti sense RNA 1 (HAND2-AS1), was observed to be significantly downregulated in OCs when compared to FT. Its downregulation in HGSC was validated in different datasets and in a panel of HGSC cell lines. Furthermore, this study shows that the downregulation of HAND2-AS1 is caused by promoter hypermethylation in HGSC and behaves as a tumor suppressor in HGSC cell lines. Since therapeutic relevance is of key importance in HGSC research, for the first time, HAND2-AS1 upregulation was demonstrated to be one of the mechanisms through which HDAC inhibitor Panobinostat could be used in a strategy to increase HGSC cells’ sensitivity to chemotherapeutic agents currently used in clinical trials. To unravel the mechanism by which HAND2-AS1 exerts its role, an in silico mRNA network was constructed using mRNAs whose expressions were positively and negatively correlated with this lncRNA in HGSC. Finally, a putative ceRNA network with possible miRNA targets of HAND2-AS1 and their mRNA targets was constructed, and the enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified.

GATA3 as a master regulator for interactions of tumor-associated macrophages with high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic cancer. Emerging evidence suggests that tumor-associated macrophages (TAMs) play an immunosuppressive role in the tumor microenvironment and promote tumor growth, angiogenesis, and metastasis in ovarian cancer. Therefore, targeting TAMs in patients with ovarian cancer is an appealing strategy; however, all trials to date have failed. To improve the efficacy of this approach, we sought to elucidate the underlying mechanisms of the role of TAMs in ovarian cancer. We found that the developmental transcription factor GATA3 was highly expressed in HGSOC cell lines but not in the fallopian tube, which is the main origin of HGSOC. GATA3 expression was associated with poor prognosis in HGSOC patients (P < .05) and was found to promote proliferation and migration in HGSOC cell lines. GATA3 was released abundantly from TAM cells via exosomes and contributed to tumor growth in the tumor microenvironment. Moreover, GATA3 acted as a regulator for macrophage polarization and interactions between TAMs and HGSOC to support proliferation, motility, and cisplatin chemoresistance in mutant TP53 HGSOC cell lines. Furthermore, GATA3 played a critical role in the interactions between TAMs and mutant TP53 HGSOC to promote angiogenesis and epithelial-mesenchymal transition with epigenetic regulation. Targeting GATA3 using GATA3siRNA in TAMs impeded GATA3-driven proliferation, migration, cisplatin chemoresistance, and angiogenesis in mutant TP53 HGSOC cell lines. Our findings indicate that GATA3 plays a novel role in immunoediting of HGSOC and demonstrate that GATA3 may serve as a prognostic marker for HGSOC and a promising target in the treatment of HGSOC.

Abstract A057: The MEK1/2 pathway as a therapeutic target in high-grade serous ovarian carcinoma

Abstract Rationale: High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to high rates of recurrence and acquired chemoresistance. Recurrent tumors are suggested to originate from cancer stem-like cells (CSCs) which have been shown to have high MEK1/2 activity signaling pathway to survive and proliferate. Present work investigates the potential of selective MEK1/2 inhibition as a CSC-targeting HGSOC therapy approach. Methods: MEK1/2 pathway activity was evaluated in clinical HGSOC tissue samples and ovarian cancer cell lines using tissue microarray-based immunohistochemistry, immunoblotting and RT-qPCR. OVCAR8 and PEO4 HGSOC cell lines were used to assess the effect of MEK1/2 inhibition on cell viability, proliferation rate and stem-like characteristics in vitro. Mouse xenografts were used to investigate MEK1/2 inhibition effect on tumor growth in vivo. Drug washout experimental model was used to study the lasting effects of MEK1/2 inhibition therapy. Results: MEK1/2 signaling is active in majority of HGSOC tissue samples and cell lines and is further stimulated by cisplatin treatment. MEK1/2 inhibitor trametinib drastically inhibits activity of MEK1/2 downstream signaling, causes prominent cell cycle arrest in G1/0-phase in vitro and reduces tumor growth rate in vivo, but does not induce cell death. Cells treated with trametinib display high CD133+ fraction and increased expression of stemness-associated genes. Transient trametinib treatment causes long-term increase in high ALDH1 activity cells that possess capability of surviving and growing in non-adherent conditions. Conclusions: MEK1/2 inhibition in HGSOC cells efficiently inhibits cell proliferation and tumor growth and therefore may be promising approach to suppress ovarian cancer cell growth. As MEK1/2 inhibition promotes stem-like properties, including ALDH activity, it should be used in combination with CSC-targeting drugs, especially ALDH1 inhibitors. Citation Format: Mikhail Chesnokov, Abdelrahman Yousif, ilana Chefetz. The MEK1/2 pathway as a therapeutic target in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A057. doi:10.1158/1535-7163.TARG-19-A057

Activation of Wnt signaling promotes olaparib resistant ovarian cancer.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease-free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib-sensitive vs -resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi-resistant cells. Forced activation of canonical Wnt signaling in several PARPi-sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to promote downregulation of β-catenin. In both an HGSOC cell line and a patient-derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.

PBK, targeted by EVI1, promotes metastasis and confers cisplatin resistance through inducing autophagy in high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma (HGSOC) is the most lethal type of gynecologic malignancy. Chemoresistance is the main reason for the poor prognosis of HGSOC. PDZ-binding kinase (PBK) promotes the malignant progression of various carcinomas. However, the roles and clinical significance of PBK in HGSOC remain unclear. Here, we reported that PBK was overexpressed in HGSOC tissues and cell lines. High PBK expression was associated with a poor prognosis, metastasis, and cisplatin resistance of HGSOC. Overexpression of PBK promoted autophagy and enhanced cisplatin resistance via the ERK/mTOR signaling pathway. Further study showed that inhibition of autophagy by chloroquine or bafilomycin A1 reversed PBK-induced cisplatin resistance. Overexpression of PBK decreased ovarian cancer responsiveness to cisplatin treatment through inducing autophagy in vivo. We also demonstrated that the PBK inhibitor OTS514 augmented the growth inhibition effect of cisplatin in vitro and in vivo. Moreover, ecotropic viral integration site-1 (EVI1) could regulate PBK expression through directly targeting the PBK promoter region. In conclusion, high PBK expression was correlated with a poor prognosis, metastasis, and cisplatin resistance through promoting autophagy in HGSOC. PBK might be a promising target for the early diagnosis and individual treatment of ovarian cancer.

CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer

High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. We hypothesize that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, we observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.