Serous Ovarian Cancer Cell Research Articles

In vivo selection of highly metastatic human ovarian cancer sublines reveals role for AMIGO2 in intra-peritoneal metastatic regulation.

The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres.

ABCA1 is associated with the development of acquired chemotherapy resistance and predicts poor ovarian cancer outcome.

Aim: This study investigated the ATP binding cassette (ABC) transporter (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) expression in high grade serous ovarian cancer (HGSOC) tissues, cell lines and primary cells to determine their potential relationship with acquired chemotherapy resistance and patient outcome.Methods: ABC transporter mRNA and protein expression (ABCA1, ABCB1, ABCB3, ABCC2 and ABCG2) was assessed in publicly available datasets and in a tissue microarray (TMA) cohort of HGSOC at diagnosis, respectively. ABC transporter mRNA expression was also assessed in chemosensitive ovarian cancer cell lines (OVCAR-5 and CaOV3) versus matching cell lines with acquired carboplatin resistance and in primary HGSOC cells from patients with chemosensitive disease at diagnosis (n = 10) as well as patients with acquired chemotherapy resistance at relapse (n = 6). The effects of the ABCA1 inhibitor apabetalone in carboplatin-sensitive and -resistant cell lines were also investigated.Results: High ABCA1 mRNA and protein expression was found to be significantly associated with poor patient outcome. ABCA1 mRNA and protein levels were significantly increased in ovarian cancer cell lines (OVCAR-5 CBPR and CaOV3 CBPR) with acquired carboplatin resistance. ABCA1 mRNA was significantly increased in primary HGSOC cells obtained from patients with acquired chemotherapy resistance. Apabetalone treatment reduced ABCA1 protein expression and increased the sensitivity of both parental and carboplatin-resistant ovarian cancer cells to carboplatin.Conclusion: These results suggest that inhibiting ABCA1 transporter may be useful in overcoming acquired chemotherapy resistance and improving outcome for patients with HGSOC.

PAR2 induces ovarian cancer cell motility by merging three signalling pathways to transactivate EGFR.

Specific cellular functions mediated by GPCRs are often associated with signalling through a particular G protein or β-arrestin. Here, we examine signalling through a GPCR, protease-activated receptor 2 (PAR2), in a high-grade serous ovarian cancer cell line (OV90). Human ovarian cancer tissues (n = 1,200) and nine human ovarian cancer cell lines were assessed for PAR2 expression. PAR2 signalling mechanisms leading to cell migration and invasion were dissected using cellular assays, western blots, CRISPR-Cas9 gene knockouts, pharmacological inhibitors of PAR2 and downstream signalling proteins in OV90 cancer cells. PAR2 was significantly overexpressed in clinical ovarian cancer tissues and in OV90 ovarian cancer cells. PAR2 agonists, an endogenous protease (trypsin) and a synthetic peptide (2f-LIGRL-NH2 ), induced migration and invasion of OV90 ovarian cancer cells through activating a combination of Gαq/11 , Gα12/13 and β-arrestin1/2, but not Gαs or Gαi . This novel cooperative rather than parallel signalling resulted in downstream serial activation of Src kinases, then transactivation of epidermal growth factor receptor (EGFR), followed by downstream MEK-ERK1/2-FOS/MYC/STAT3-COX2 signalling. Either a PAR2 antagonist (I-191), CRISPR-Cas9 gene knockouts (PAR2 or Gα proteins or β-arrestin1/2), or inhibitors of each downstream protein attenuated human ovarian cancer cell motility. This study highlights a novel shared signalling cascade, requiring each of Gαq/11 , Gα12/13 and β-arrestin1/2 for PAR2-induced ovarian cancer cell migration and invasion. This mechanism controlling a cellular function is unusual in not being linked to a specific individual G protein or β-arrestin-mediated signalling pathway.

Reduced Gonadotrophin Receptor Expression Is Associated with a More Aggressive Ovarian Cancer Phenotype.

Follicle-stimulating hormone (FSH) and luteinising hormone (LH) play important roles in regulating cell growth and proliferation in the ovary. However, few studies have explored the expression of FSH and LH receptors (FSHR and LHCGR) in ovarian cancer, and their functional roles in cancer progression remain inconclusive. This study investigated the potential impact of both mRNA (FSHR, LHCGR) and protein (FSHR, LHCGR) expression on ovarian cancer progression using publicly available online databases, qRT-PCR (high grade serous ovarian cancers, HGSOC, n = 29 and benign ovarian tumors, n = 17) and immunohistochemistry (HGSOC, n = 144). In addition, we investigated the effect of FSHR and LHCGR siRNA knockdown on the pro-metastatic behavior of serous ovarian cancer cells in vitro. High FSHR or high LHCGR expression in patients with all subtypes of high-grade ovarian cancer was significantly associated with longer progression-free survival (PFS) and overall survival (OS). High FSHR protein expression was associated with increased PFS (p = 0.050) and OS (p = 0.025). HGSOC patients with both high FSHR and high LHCGR protein levels had the best survival outcome, whilst both low FSHR and low LHCGR expression was associated with poorest survival (p = 0.019). Knockdown of FSHR significantly increased the invasion of serous ovarian cancer cells (OVCAR3 and COV362) in vitro. LHCGR knockdown also promoted invasion of COV362 cells. This study highlights that lower FSHR and LHCGR expression is associated with a more aggressive epithelial ovarian cancer phenotype and promotes pro-metastatic behaviour.

HDAC6 Degradation Inhibits the Growth of High-Grade Serous Ovarian Cancer Cells.

Simple SummaryThe objective of this study was, firstly, to investigate the relationship between Histone deacetylase 6 (HDAC6) expression and survival in patients with ovarian cancer and, secondly, to test the effects of histone deacetylase 6 (HDAC6) inhibition on ovarian cancer cells in vitro. A meta-analysis of the correlation between HDAC6 gene expression and survival was performed on 3573 ovarian tumors from 19 datasets showed that high HDAC6 gene expression was associated with a decreased risk of death. Knockdown of HDAC6 gene expression with small interfering RNA (siRNA) and protein expression with a HDAC6 targeting protein degrader decreased ovarian cell proliferation, migration, and viability. Conversely, the selective inhibition of HDAC6 catalytic activity did not produce a robust inhibition of HDAC6 protein function. In summary, we demonstrated, for the first time, that HDAC6 over-expression in ovarian cancers is a favorable prognostic marker. We provide evidence to suggest that inhibition of HDAC6 catalytic activity has limited efficacy as a monotherapy in ovarian cancers.Histone deacetylase 6 (HDAC6) is a unique histone deacetylating enzyme that resides in the cell cytoplasm and is linked to the modulation of several key cancer related responses, including cell proliferation and migration. The promising anti-cancer response of the first-generation HDAC6 catalytic inhibitors continues to be assessed in clinical trials, although its role in high grade serous ovarian cancer is unclear. This study investigated HDAC6 tumor expression by immunohistochemistry in high-grade serous ovarian cancer (HGSOC) tissue samples and a meta-analysis of HDAC6 gene expression in ovarian cancer from publicly available data. The pharmacological activity of HDAC6 inhibition was assessed in a patient-derived model of HGSOC. HDAC6 was found to be highly expressed in HGSOC tissue samples and in the patient-derived HGSOC cell lines where higher HDAC6 protein and gene expression was associated with a decreased risk of death (hazard ratio (HR) 0.38, (95% confidence interval (CI), 0.16–0.88; p = 0.02); HR = 0.88 (95% CI, 0.78–0.99; p = 0.04)). Similarly, the multivariate analysis of HDAC6 protein expression, adjusting for stage, grade, and cytoreduction/cytoreductive surgery was associated with a decreased risk of death (HR = 0.19 (95% CI, 0.06–0.55); p = 0.002). Knock-down of HDAC6 gene expression with siRNA and protein expression with a HDAC6 targeting protein degrader decreased HGSOC cell proliferation, migration, and viability. Conversely, the selective inhibition of HDAC6 with the catalytic domain inhibitor, Ricolinostat (ACY-1215), inhibited HDAC6 deacetylation of α-tubulin, resulting in a sustained accumulation of acetylated α-tubulin up to 24 h in HGSOC cells, did not produce a robust inhibition of HDAC6 protein function. Inhibition of HGSOC cell proliferation by ACY-1215 was only achieved with significantly higher and non-selective doses of ACY-1215. In summary, we demonstrated, for the first time, that HDAC6 over-expression in HGSOC and all ovarian cancers is a favorable prognostic marker. We provide evidence to suggest that inhibition of HDAC6 catalytic activity with first generation HDAC6 inhibitors has limited efficacy as a monotherapy in HGSOC.

The Treatment of Clear Cell Ovarian Cancer with the Poly(ADP- Ribose) Polymerase (PARP1) Inhibitors (AG14361,Veliparib,Olaparib) as Chemosensitizers.

Introduction: Most of those who get ovarian cancer will die from this cancer. Of the major types of ovarian cancer clear cell carcinoma is the most aggressive and chemoresistant type of epithelial ovarian cancer. Here the sensitivity of clear cell ovarian carcinoma to poly adenosine diphosphate [ADP-ribose] polymerase (PARP) inhibitors is tested. Methodology: Ovarian cancer cell lines were treated with the PARP inhibitors AG14361, Veliparib, or Olaparib alone or in combination with cisplatin, carboplatinum, doxorubicin, 5-fluorouracil (5-FU), gemcitabine and paclitaxel for 72 hours. The IC50 concentrations were calculated. Each experiment was replicated 10 times. Results: As single agents the PARP inhibition of ovarian cancer among serous, endometroid and clear cell ovarian cancer cell lines was similar. Clear cell ovarian cancer seemed particularly susceptible to chemo-sensitization by PARP inhibitors with paclitaxel, 5-FU, carboplatin, doxorubicin and/or cisplatin. Antagonism was seen with gemcitabine. Conclusion: PARP inhibitors are exceptional chemosensitizers of clear cell ovarian cancer to treatment with most standard chemotherapy agents.

Single-cell analysis of copy-number alterations in serous ovarian cancer reveals substantial heterogeneity in both low- and high-grade tumors

ABSTRACT Unusually high aneuploidy is a hallmark of epithelial serous ovarian cancer (SOC). Previous analyses have focused on aneuploidy on average across all tumor cells. With the expansion of single-cell sequencing technologies, however, an analysis of copy number heterogeneity cell-to-cell is now technically feasible. Here, we describe an analysis of single-cell RNA sequencing (scRNA-seq) data to infer arm-level aneuploidy in individual serous ovarian cancer cells. By first clustering high-quality sequenced epithelial versus non-epithelial cells, high-confidence tumor cell populations were identified. InferCNV was used to predict segmented copy-number alterations (CNAs), which were then used to determine arm-level aneuploidy at the single-cell level. Control comparisons of normal cells to normal cells showed zero arm-level aneuploidy, whereas a median of four aneuploid events were detectable in cancer cells. A heterogeneity analysis of high-grade tumor cells compared to low-grade tumor cells showed similar levels of cell-to-cell variation between cancer grades. Metastatic tumors potentially showed selection pressure with reduced cell-to-cell variation compared to cells from primary tumors. Minor cell populations with CNAs similar to metastatic cells were identified within the matched primary tumors. Taken together, these results provide a minimum estimate for single-cell aneuploidy in serous ovarian cancer and demonstrate the utility of single-cell sequencing for CNA analysis.

Simvastatin is a potential candidate drug in ovarian clear cell carcinomas.

Ovarian clear cell carcinomas (OCCC) constitute a rare subtype of epithelial ovarian cancer, lacking efficient treatment options. Based on previous studies, we assessed the anti-proliferative effect of simvastatin, a Rho GTPase interfering drug, in three OCCC cell lines: JHOC-5, OVMANA and TOV-21G, and one high-grade serous ovarian cancer (HGSOC) cell line, Caov3. We used the Rho GTPase interfering drug CID-1067700 as a control. All OCCC cell lines were more sensitive to single-agent simvastatin than the HGSOC cells, while all cell lines were less sensitive to CID-1067700 than to simvastatin. Combinations of carboplatin and simvastatin were generally antagonistic. Most treatments inhibited migration, while only simvastatin and CID-1067700 also disrupted actin organization in the OCCC cell lines. All treatments induced a G1 arrest in JHOC-5 and TOV-21G cells. Treatments with simvastatin consistently reduced c-Myc protein expression in all OCCC cell lines and displayed evidence of causing both caspase-mediated apoptotic cell death and autophagic response in a cell line dependent manner. Differences between cell lines in response to the treatments were observed and such differences, including e. g. prior treatment, should be investigated further. Conclusively, simvastatin efficiently controlled OCCC proliferation and migration, thus showing potential as a candidate drug for the treatment of OCCC.

Functional targeting of the TGF-βR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-β-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-β-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-β-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8–10 carbons. Functional inhibition of the TGF-β receptor (TGF-βR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-βR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-βR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-βR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-βR1 in order to antagonize TGF-β-mediated signaling that is required for ovarian cancer cell invasion and metastasis.

ATM inhibition synergizes with fenofibrate in high grade serous ovarian cancer cells

While therapies targeting deficiencies in the homologous recombination (HR) pathway are emerging as the standard treatment for high grade serous ovarian cancer (HGSOC) patients, this strategy is limited to the ~50% of patients with a deficiency in this pathway. Therefore, patients with HR-proficient tumors are likely to be resistant to these therapies and require alternative strategies. We found that the HR gene Ataxia Telangiectasia Mutated (ATM) is wildtype and its activity is upregulated in HGSOC compared to normal fallopian tube tissue. Interestingly, multiple pathways related to metabolism are inversely correlated with ATM expression in HGSOC specimens, suggesting that combining ATM inhibition with metabolic drugs would be effective. Analysis of FDA-approved drugs from the Dependency Map demonstrated that ATM-low cells are more sensitive to fenofibrate, a PPARα agonist that affects multiple cellular metabolic pathways. Consistently, PPARα signaling is associated with ATM expression. We validated that combined inhibition of ATM and treatment with fenofibrate is synergistic in multiple HGSOC cell lines by inducing senescence. Together, our results suggest that metabolic changes induced by ATM inhibitors are a potential target for the treatment of HGSOC.

Abstract 5682: Synergism of PARP inhibitor and MET inhibitor in multiple cancer types with intrinsic and acquired PARP inhibitor resistances

Abstract Leveraging compromised DNA damage repair (DDR) pathways commonly found in tumor cells, a classic strategy in cancer therapy is inducing excessive DNA damage to cause cancer cell death. Small molecule poly(ADP-ribose) polymerase (PARP) inhibitors (PARP-is) have been approved for clinical use in treating breast cancer and ovarian cancer patients bearing DDR-deficient tumors with mutations in breast cancer susceptibility proteins (BRCAm). However, accumulating evidences show that both intrinsic and acquired resistances to PARP-is exist in clinic and pre-clinical animal models. Therefore, we developed panels of cells with acquired PARP-is resistance from PARP-is-sensitive triple negative breast cancer (TNBC) and estrogen receptor positive breast cancer cell lines, and used these cells to screen for common traits that can be targeted with feasible therapeutic agent combinations to overcome PARP-is resistance. Since TNBC lacks of effective targeted therapy so far, we focused on using the panel of PARP-is-resistant TNBC cells in this study. Among the molecular mechanisms known contribute to PARP-is resistance, oncogenic kinase activations, including several hyper-activated receptor tyrosine kinases (RTKs), are involved in enhancing DNA damage repair and decreasing affinity of PARP-is to PARP1. In this study, we systematically screened for activated RTKs in the PARP-is-resistant cells we developed by antibody arrays. We then identified that activations of MET, Axl and EphA2 were common traits in TNBC cells with acquired PARP-is resistance, but not in estrogen receptor positive cells. Among the three RTKs, MET has more small molecules inhibitors that can target it, and thus, we made it a priority in investigating synergism between MET inhibitor and PARP inhibitor in multiple cancer types including TNBC with intrinsic and acquired PARP-is-resistance, high-grade serous ovarian cancer (HGSOC) and liver cancer cells. Here, we demonstrated that combinations of PARP-is and MET inhibitors (MET-is) possess moderate to strong synergism in the different cancer types we studied. As we previously reported that MET translocates into cell nucleus and phosphorylates PARP1 at tyrosine (Y) 907 residue in TNBC with intrinsic PARP-is resistance, we found that this MET-mediated PARP1-Y907 phosphorylation also exist in and can serve as marker to indicate PARP-is resistance among the HGSOC, liver cancer cells and the TNBC cells with acquired PARP-is resistance. We further found that MET phosphorylation is high (immunohistological staining H-score greater than 200) in breast cancer (23 out of 31, 70%) and ovarian cancer (8 out of 23, 35%) patient-derived xenograft mouse model tissue microarrays, suggesting that the combination of MET-is and PARP-is is likely to benefit a huge population of cancer patient in multiple cancer types. Citation Format: Mei-Kuang Chen, Weiya Xia, Qiongzhu Dong, Yi Du, Hung-Ling Wang, Coya Tapia, Yongkun Wei, Ye Han, Yu-Yi Chu, Clinton Yam, Yuan Gao, Yu-Han Wang, Funda Meric-Bernstam, Jinsong Liu, Shao-Chun Wang, Dihua Yu, Mien-Chie Hung. Synergism of PARP inhibitor and MET inhibitor in multiple cancer types with intrinsic and acquired PARP inhibitor resistances [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5682.

Abstract 4144: A tissue-microfluidic derived aptamer displays prognostic and therapeutic characteristics in serous ovarian carcinoma through heat shock protein 70(HSP70)

Abstract Background: Previously, we have identified aptamers through ovarian cancer (OC) tissue microfluidic platform which show high specificity to ovarian cancer tissue and cells. However, the clinical diagnostic and therapeutic roles of these aptamers are still largely unknown. Materials and Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to find binding candidates of aptamer, Tx-01, from serous type ovarian cancer cells. Immunohistochemistry (IHC), clinical survival analysis, western blotting and confocal microscopic images were performed to detect the expression of Tx-01 and HSP70/ Notch1 intracellular domain (NICD) in OC tissues. The functions of Tx-01 were studied in vitro and in vivo experiments. Inhibitors treatment, nuclear protein extraction and western blotting were carried out to demonstrate the possible mechanisms of Tx-01 function. Results: High expression Tx-01 in OC tissues was correlated to poor patients' survival in serous ovarian carcinoma. In vitro experiments showed that Tx-01 has a feature of recognizing membrane-bound HSP70. In the meantime, Tx-01 inhibit HSP70 signal activation via interrupting intracellular HSP70/NICD interaction to reduce cell migration and invasion. Furthermore, Tx-01 inhibited the tumor growth in the mice experiment. Conclusion: Our data showed that Tx-01 acted as a prognostic predictor in serous OC through recognizing the membrane-bound HSP70. Also, Tx-01 reduced the tumor cell migration, invasion through blocking HSP70 signal activation. Thus, our work demonstrated that Tx-01 might be a prognostic biomarker and potential inhibitor in serous OC therapy. Citation Format: Chang-Ni Lin, Yu-Ling Liang, Pei-Ying Wu, Gwo-Bin Vincent Lee, Keng-Fu Hsu. A tissue-microfluidic derived aptamer displays prognostic and therapeutic characteristics in serous ovarian carcinoma through heat shock protein 70(HSP70) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4144.

Abstract 2565: NANOG sustains ovarian cancer cell survival and disrupts ER stress signaling through BiP/PERK under glutamine depletion

Abstract Metabolic reprogramming is a common hallmark of multiple cancers which functions to meet their high proliferation needs. High demand for the amino acid glutamine is frequently observed in many cancers. Catalyzed by glutaminase, glutamine is readily converted to glutamate which then further fuels the tricarboxylic acid cycle. In ovarian cancer, depletion of glutamine significantly inhibited cell growth suggesting that targeting glutamine metabolism might be an effective therapeutic strategy for ovarian cancer. However, the molecular mechanisms involved were not completely understood. In this study, we aim to identify molecular targets and the signaling involved in ovarian cancer cells under glutamine depletion. We observed that glutamine depletion stimulated the expression of the stem cell transcriptional factor NANOG in the serous ovarian cancer cell line OVCAR3. Stable knockdown of NANOG in OVCAR3 (shNANOG-OVCAR3) enhanced the level of reactive oxygen species under glutamine depletion. It was also found that under glutamine depletion, growth rate was not altered in shNANOG-OVCAR3 when compared to control shCtrl-OVCAR3 cells. However, shNANOG-OVCAR3 were more sensitive to glutamine depletion compared to shCtrl-OVCAR3 suggesting that NANOG is in part required to sustain growth of ovarian cancer cells under glutamine depletion. To further decipher the signaling involved in NANOG under glutamine depletion, expressions of markers of ER stress were detected by western blot. We found that shNANOG-OVCAR3 cells expressed lower levels of BiP, a master regulator of ER stress. Under glutamine depletion, knockdown of NANOG further enhanced the expression of phosphorylated PERK but did not alter the levels of cleaved ATF6 or phosphorylated IRE-1a, suggesting NANOG regulates ER stress pathway through BiP/PERK signaling in glutamine-depleted environment. Immunohistochemical studies were performed to determine the expressions of NANOG and BiP in our ovarian cancer patient cohort. NANOG expression was positively correlated with the expression of nuclear BiP. Total BiP expression was significantly higher in cancerous tissue than in benign and borderline ovarian tumor tissue. Altogether, these findings suggested a hitherto unreported role of the stem cell transcription factor NANOG in regulating ER stress through BiP/PERK signaling under glutamine depleted environment. Citation Format: Astrid Man-Yee Suen, Ivy Tsz-Lo Wong, Cindy Yin-Si Tse, Ka-Kui Chan, Oscar Gee-Wan Wong, Annie Nga-Yin Cheung. NANOG sustains ovarian cancer cell survival and disrupts ER stress signaling through BiP/PERK under glutamine depletion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2565.

Abstract 4320: Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival

Abstract 75% of women suffering from ovarian cancer are diagnosed at late stage of the disease often associated with cancer cell infiltration into the peritoneal cavity. Standard therapy is tumor resection and subsequent platin-based chemotherapy. Relapse is frequently observed caused by persisting cancer cells. Persister cells comprise a therapy-tolerant subpopulation of cancer cells and repeated therapies presumably select for increased tolerance. The present project aims to find molecular markers for the identification and targeted eradication of persister ovarian cancer cells. Persister cells of the cisplatin-sensitive high-grade serous ovarian cancer cell line, OVCAR-3, were selected after cisplatin incubation with clinically relevant doses. Individual clones were isolated from control or cisplatin challenged OVCAR-3 cells. A subpopulation of the resulting clonal persister cells showed a distinct morphological phenotype characterized by increased migration and high viability in short- and long-term survival after cisplatin treatment. A significant upregulation (p<0.001) of the endoplasmic reticulum (ER) stress marker, ATF3, was identified by RNA sequencing and this ER stress phenotype is maintained for more than 20 passages. Additionally, the ovarian cancer OVCAR-8 luciferase reporter cell line was transduced with the “Watermelon” (WM) library [1]. The WM library is a complex barcode library that enables simultaneous tracing of cell lineage and the cellular transcriptional and proliferative states. To study cancer relapse in a CDX model, WM-labelled OVCAR-8-Luc cells were injected into mice and pulse-treated with carboplatin. Single cell RNA sequencing of 900 cells isolated from solid metastases, ascites and treatment-naïve mice revealed an increase in multiple ER stress markers including ATF3, ATF4, JUN and XBP1 in the most abundant solid relapse lineages. Furthermore, analysis of clinical data from GEO, EGA and TCGA showed that low ATF3 expression is associated with increased 5-year-survival of patients with high-grade serous ovarian cancer (Logrank p=6.3e-6). We found that increased ER stress correlates with survival of cancer persister cells and incidence of metastases. This phenomenon was observed in cell lines, a CDX model and clinical data from human ovarian cancer samples. We propose ER stress response, exemplified by ATF3, as a molecular marker to identify and therapeutic targeting of ovarian cancer persister cells. [1] Metabolic switching underlies the ability of cancer persister cells to cycle under drug treatment, Oren Y et al., unpublished Citation Format: Kathrin Böpple, Meng Dong, Markus Kleih, Andrea Gaißler, Yaara Oren, Whitney S. Henry, Moshe Oren, Yael Aylon, Bernd Winkler, Aviv Regev, Robert A. Weinberg, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Ovarian cancer persister cells are characterized by enhanced ER stress gene expression correlating with poor survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4320.

Abstract 2840: Development of a model of dihomo-gamma-linolenic acid interference with platelet promotion of ovarian cancer

Abstract Background: Cancer is the second leading cause of deaths worldwide after cardiovascular diseases. A commonality between cancer and cardiovascular diseases is disregulation of platelets. Platelets and cancer are known to promote each other in a feedforward mechanism that is poorly understood. A long chain polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA) was shown to reduce cardiovascular events in a platelet dependent manner while the exact mechanism of DGLA-mediated cancer cells death is yet to be explored. Objectives: The objective of our study was to develop an experimental model to study interventions that could disrupt the cancer/platelets loop. Methods: The effects of a range of DGLA concentrations on platelet aggregation and growth of high grade serous ovarian cancer cell lines (OVCAR3, MesOV, OVSAHO), normal human fallopian tube secretory epithelial cells (hFTSECs) primary culture and an immortalized hFTSEC cultures were determined with an MTT assay. Cocultures were used to evaluate effects of platelets on ovarian cancer cell line spheroid formation and size and effects of ovarian cancer cells and hFTSECs on platelet aggregation. Results: The minimal cytotoxic DGLA concentrations for cancer and normal epithelial cells were above physiologically-achievable concentrations, and the half maximal inhibitory concentrations (IC50's) were similar across epithelial cell types with the IC50's for hFTSEC primary culture (261 µM) and immortalized culture (206 µM) falling within the range of cancer cell IC50's (153-275 µM). DGLA inhibited platelet aggregation at concentrations ≥250 µM. Platelets increased cancer spheroid sizes in a concentration-dependent manner and cancer cells, but not hFTSECs, induced platelet aggregation in cocultures. Conclusions: Our model incorporates both platelet effects on cancer cells and cancer cell effects on platelets. Physiologically-achievable DGLA concentrations that affect platelet aggregation without affecting epithelial cells were identified. Minimal doses of DGLA needed to cause cytotoxicity against cancer and normal cells are above levels that are physiologically achievable. Future studies will utilize DGLA doses that can interfere with platelet aggregation without directly causing cytotoxicity to cancer or healthy cells. This experimental model will allow study of DGLA's potential for interfering with platelet promotion of ovarian cancer in the absence of direct effects of DGLA on cancer cells. Funded by NCI R01 CA196200 Citation Format: Zitha Redempta Isingizwe, Doris M. Benbrook. Development of a model of dihomo-gamma-linolenic acid interference with platelet promotion of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2840.

Abstract A23: Verteporfin as a new treatment paradigm for platinum-resistant ovarian cancer cells

Abstract Ovarian cancer is the most lethal of the gynecologic malignancies and has poor survival rates due to a combination of diagnosis at advanced stages and disease recurrence due to platinum chemotherapy resistance. It is of utmost importance that novel chemotherapeutic agents are identified to increase or restore chemosensitization to platinum-based chemotherapy. Verteporfin (VP, VisudyneTM) is a benzoporphyrin derivative used for treating adult macular degeneration. Verteporfin has recently been identified as an inhibitor of YAP/TEAD interaction. Yes-associated protein (YAP), the main downstream target of the HIPPO pathway, plays an important role in cell proliferation and apoptosis. We investigated the efficacy of VP alone or in combination with cisplatin (CDDP) or carboplatin (CP) or paclitaxel (Taxol) on two serous ovarian cancer cell lines (OV90, COV504). The effect of VP, CDDP, CP or Taxol was studied either alone or in combination after 72 hours of treatment. Cell viability-based cytotoxicity measurements indicate dose-dependent increase in cytotoxicity for all the drugs. IC50 values of the drugs were calculated based on dose-effect curves using Compusyn software following the Chou-Talalay method. For OV90 cells, the calculated IC50 values were 29.33μM for VP, 2.17μM for cisplatin, 96.21μM for carboplatin, and 89.05μM for paclitaxel. For COV504 cells, the calculated IC50 values were 8.37μM for VP, 1.79μM for cisplatin, 117.23μM for carboplatin, and 7.16μM for paclitaxel. Based on the IC50 values of single agents, we performed cell proliferation assay using VP and CDDP/CP/Taxol in combination. We observed synergistic effects of VP with CDDP/CP/Taxol based on combination index plots. Based on the dose-reduction index (DRI) values of the drugs in combination treatments, we performed cytotoxicity and invasion assays. The combination treatments of these drugs at DRI values induced cytotoxicity and inhibited invasion of cancer cells. To check the chemosensitization of VP in cancer cells, we used cisplatin-resistant ovarian cancer cells, A2780Cis. Similar to ovarian cancer cells, VP effectively inhibited proliferation of A2780Cis cells. Based on these results, we hypothesize that VP is inducing chemosensitivity in platinum resistant ovarian cancer cells and increasing the efficacy of either platin drugs or Taxol. We suggest that our preclinical data in combination with previously available human safety data support the study of VP in combination with platinum in a clinical phase 2 trial of serous ovarian cancer patients. Note: This abstract was not presented at the conference. Citation Format: Radhika P. Gogoi, Venkata Ramesh Dasari. Verteporfin as a new treatment paradigm for platinum-resistant ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A23.

Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.

In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response.

Abstract B68: Characterization of microtentacle phenotype and function in ovarian carcinomas

Abstract Ovarian carcinomas are categorized into five major histotypes, each characterized by distinct differences in grade at diagnosis, presentation with metastatic disease, and clinical responses to treatments. Microtentacles (McTNs), microtubule-based extensions of the plasma membrane, were initially described on cells of nongynecologic primaries with high metastatic potential but have not been extensively explored in ovarian carcinomas. In this study, we investigated whether ovarian cancer cells exhibit McTNs as well as the effect of microtubule-targeted drugs on ovarian cancer McTNs. We analyzed 3 immortalized ovarian surface epithelium cell lines (IOSE), 8 clear-cell (OCCC), 3 low-grade serous (LGS), and 6 high-grade serous (HGS) ovarian cancer cells for McTN phenotype, length, and number, using a novel tethering platform in which cells are suspended but stationary, allowing for analysis via confocal microscopy. Additionally, selected cell lines were also treated with microtubule-targeting agents, including paclitaxel, ixabepilone, vinblastine, and colchicine and the effects on McTN dynamics and functions were analyzed. Tubulin subtype expression and post-translational modifications (PTM) were characterized by Western blot. Unpaired t-tests were used to describe differences between McTN length and number. We observed 4 patterns of McTN expression: absent (A), symmetric-short (SS), symmetric-long (SL), and tufted (T). In some cases, multiple morphologies were observed within the same cell line. LGS and OCCC expressed fewer McTNs per cell than HSC. McTNs were shorter among OCCC compared to HGS; whereas LGS expressed a range of McTN lengths similar to those observed in HGS. We also observed differences in the expression of the PTM between the different ovarian cancer subtypes, including alterations in alpha tubulin-stabilizing modifications such as glu-tubulin and acetylated tubulin, as well as proteins involved in actin cortex stability. Increased acetylated tubulin was associated with increased McTN number and length in HGS cells. Microtubule-destabilizing drugs such as colchicine and vinblastine led to a decrease in McTN formation. Ovarian cancer metastasis typically occurs through shedding of the main tumor into the peritoneal space, and the spread of disease is the leading cause of mortality. Studying McTN function and response to chemotherapeutics allows us to improve our understanding of ovarian cancer metastasis and the effect of microtubule-targeting compounds on the spread of ovarian cancer. Citation Format: Cong (Ava) Fan, Sulan Wu, Jocelyn Reader, Eleanor Claire-Higgins Ory, Cornell Lee, McMillan Ching, Trevor Mathias, Julia Ju, Rachel Lee, Michele Vitolo, Stuart Martin, Christopher Jewell, Amy Fulton, Gautam Rao, Mark Carey, Dana M. Roque. Characterization of microtentacle phenotype and function in 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 B68.

Abstract B63: Lysophosphatidic acid as a mediator of ovarian cancer cell stemness

Abstract Epithelial ovarian cancer (OC) is the deadliest gynecologic malignancy. Recurrence of advanced, multidrug-resistant metastatic disease after initially good response to standard lines of chemotherapy occurs in the majority of OC patients. Chemoresistant, recurrent OC is essentially fatal. Disease relapse is due to survival and expansion of a small pool of chemorefractory ovarian cancer stem cells (OCSCs). OC evolution to a platinum-resistant state is associated with aberrant epigenetic modifications, including altered DNA methylation and histone modifications. In addition, OC cells residing in the peritoneal cavity are exposed to a variety of external cues arising from the ascitic microenvironment, including the bioactive molecule lysophosphatidic acid (LPA), which potentially contribute to cell stemness and chemoresistance. LPA in the intraperitoneal fluid of OC patients has been strongly implicated in ovarian carcinogenesis and metastasis through a variety of mechanisms. However, regulation of OC cells by LPA is incompletely understood, and the role of LPA in OCSC acquisition/chemoresistance remains unclear. In the current study, we tested the hypothesis that LPA is a mediator of OCSC promotion and survival. Expression of aldehyde dehydrogenase (ALDH), an accepted functional marker of cancer stem cells, was assessed in a panel of high-grade serous ovarian cancer (HGSOC) cell lines (OVSAHO, OVCAR3, OVCAR5, PEO1, and Kuramochi) using an Aldefluor assay. Treatment of HGSOC cells with increasing (0-80uM, 72h) doses of LPA enriched the ALDH+ population in a cell line-specific dose-dependent manner. In PEO1 and Kuramochi cells, intermediate LPA doses (1-10uM) resulted in a 1.5-2.5-fold increase (P<0.05) of ALDH+ cells (from ∼4% to ∼8% in PEO1 and from ∼4% up to ∼10% in Kuramochi, respectively). However, 80uM LPA treatment was necessary to increase (P<0.05) the percentage ALDH+ cells in OVCAR3 (from ∼17% to 34%) and OVSAHO cells (from ∼10% up to 29%). In accordance with the results of the flow cytometry analysis, cancer stem cell properties after LPA treatment (0-80uM, every 72hr, up to 21 days) were enhanced (P<0.05), based on clonogenic assay. In particular, moderate (10-20uM) LPA treatment increased (P<0.05) cell clonogenic survival of PEO1 and Kuramochi, while a greater (80uM) LPA dose was needed for OVCAR3 and OVSAHO. Furthermore, LPA (0-80uM, 14 days) augmented spheroid formation ability in PEO1 cells (3-4-fold increase; P<0.05). Global transcriptomic and epigenomic assessment of altered genes/pathways induced by LPA are currently being examined to assess stemness-promoting pathways and epigenetic changes, which we believe will identify new biologic insights and potential therapeutic targets to eradicate OCSC in patients. Citation Format: Yuliya Klymenko, Kenneth P. Nephew. Lysophosphatidic acid as a mediator of ovarian cancer cell stemness [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 B63.

Abstract A31: The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors

Abstract Background: Poly (ADP-ribose) polymerase (PARP) inhibitors have transformed treatment of ovarian cancer in the past decade. Most of the currently active PARP inhibitors have similar ability to inhibit PARP’s enzymatic activity. However, it has been previously reported that velipairb, rucaparib, olaparib, niraparib, and talazoparib vary in their PARP trapping potency levels. Veliparib was also characterized to be mainly a catalytic inhibitor while the other four drugs were determined to be both PARP trappers and catalytic inhibitors, with talazoparib being the most potent PARP trapper. Responses in many patients with HGSC tumors are limited, particularly those women without known mutations in BRCA1 or BRCA2. Recently, PARP inhibitors have been reported to influence innate immune responses. For these reasons, there are ongoing clinical trials testing the safety and efficacy of PARP inhibitors in combination with various immune checkpoint inhibitors. The objective of this study is to compare the effect of the five most clinically active PARP inhibitors on type 1 interferon regulated genes and the PD-L1 gene. Methods: Two high-grade serous ovarian cancer cell lines were used in this study: CAOV3 (TP53 mutant) and OVCAR3 (TP53 mutant, CCNE1-amplified, and EMSY-amplified). The cell lines were treated for 48 hours with the PARP inhibitors listed above as well as a vehicle control (DMSO). Gene expression was measured using TaqMan assays. CXCL10 (IP-10) protein expression levels were determined via an ELISA assay. Summary of the Data: Talazoparib upregulated CXCL10 gene expression in both OVCAR3 and CAOV3 cell lines. These results were translated to the protein level, as we observed an increase in IP-10 protein expression levels after talazoparib treatment in the OVCAR3 cell line. IP-10 protein levels were also significantly increased when treated with the second most potent PARP inhibitor, niraparib. A statistically significant effect was not observed after treatment with the three other PARPis. Futhermore, CD274 (PD-L1) gene expression levels were significantly increased after talazoparib and not veliparib treatment in the CAOV3 cell line. Conclusions: Our data suggest that there are distinct effects exhibited among PARP inhibitors on innate immune pathways. The two most potent PARP inhibitors, niraparib and talazoparib, had the greatest effect on these pathways, specifically in BRCA wild-type HGSCs, compared to the three other drugs. These finding support the idea that we need to define the best PARP inhibitor to use in future clinical trials when combining this group of anticancer agents with immune checkpoint inhibitors. Citation Format: Monica Wielgos-Bonvallet, Danhui Weng, Petar Jelinic, Douglas A. Levine. The two most potent PARP inhibitors increase expression levels of innate immune response genes in BRCA wild-type HGSC tumors [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 A31.