OVCAR-3 Cells Research Articles

Abstract 5341: Data mining of PubChem bioassay data reveals OXPHOS inhibitors with anti-cancer activity

Abstract High Grade Serous Ovarian Cancer (HGSOC) is a fatal disease that is typically detected at an advanced stage when there are limited modalities for effective treatment. Treatment efficacy against cancer is dependent on tumor metabolism and its adaptation for most therapies. Warburg effect is an important adaptation found in tumors where cells primarily catabolize glucose even in presence of sufficient oxygen and nutrients. Nevertheless, recent evidence suggests cells in tumor microenvironments remain dependent on mitochondrial ATP production via oxidative phosphorylation (OXPHOS). OXPHOS also represents a major metabolic pathway in cancer stem cells and tumor cells of relapse patients. Molecular signatures of glycolysis and OXPHOS are both detected in tumors. This points to the OXPHOS pathway as a potential therapeutic target in cancer. Here we apply a data mining approach to identify OXPHOS inhibitors to evaluate as candidates for therapies against HGSOC. High-Throughput Screening (HTS) on mechanistic targets like OXPHOS are cost prohibitive for academic laboratories. As an alternative, we deployed a data mining pipeline to find active chemical classes among 8415 available OXPHOS-related bioassays in the PubChem data repository. Active compound clusters frequently contain α,β-unsaturated carbonyl groups, which are also observed in both endogenous quinone electron carriers and atovaquone–a potent inhibitor of OXPHOS. Out of 312041 unique compounds tested in the assays, we selected 6 representative molecules from each of the 6 largest active chemical clusters to test for OXPHOS inhibition, ROS production, and reduced cancer cell proliferation. We assayed cell viability (MTT) to check for proliferation of human (OVCAR3, OVCAR5, OVISE) and mouse (ID8) ovarian cancer cells against 6 compounds, Oryzalin, Allylyestrenol, Esbiothrin, Lacidipine, Coumatetralyl, and CID929419 (MLS000713029), 72 h post treatment. ID8 and OVCAR5 significantly decreased cell proliferation after Lacidipine and Oryzalin treatment. Significant increase in intracellular ROS, a frequent consequence of OXPHOS inhibition, was observed in all cell lines after Lacidipine treatment. Elevated ROS was also observed in ID8 and OVISE cells after Esbiothrin treatment. To confirm OXPHOS inhibition as the mechanism of action, we performed Mito Stress Test on the Seahorse Analyzer and measured Oxygen Consumption Rate. Treatment with Oryzalin, Allylyestrenol, Esbiothrin, and Lacidipine significantly decreased basal respiration rate and ATP production in ID8 and OVCAR3 cells. This suggests that 4 of the 6 compound classes prioritized by the data mining pipeline inhibits OXPHOS in ovarian cancer cells. Thus, our pipeline provides an efficient alternative to HTS for identifying OXPHOS inhibitors. Next, we plan to test the in-vivo efficacy of these drugs against ovarian tumors in a mouse model and investigate their molecular mechanisms. Citation Format: Sejal T. Sharma, Liping Feng, Nicha Boonpattrawong, Lisa M. Barroilhet, Manish Patankar, Spencer S. Ericksen. Data mining of PubChem bioassay data reveals OXPHOS inhibitors with anti-cancer activity. [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 5341.

Abstract 4576: 3D in vitro models uncover malignant cell intrinsic and extrinsic mechanisms of CAR-T cell resistance in high grade serous ovarian cancer

Abstract Chimeric antigen receptor (CAR)-T cell therapies have promising outcomes in hematologic malignancies but limited activity in solid tumors. We used 3-dimentional (3D) in vitro human cell models of increasing complexity to investigate resistance to CAR-T cell therapy in high grade serous ovarian cancer (HGSOC), a disease with poor response to immunotherapy. We identified mucin-1 (MUC1) and TnMUC1 as target antigens in HGSOC omental metastasis and the HGSOC cell lines, OvCAR3 and G164. We then generated CAR-T cells against these targets and tested them in spheroids, collagen gels and microfluidic models. In spheroids, OvCAR3 cells were killed by CAR-T cells whereas, G164 cells were resistant to CAR-T cell cytotoxicity. RNA sequencing suggested that the impaired apoptotic signaling in G164 cells caused malignant cell-intrinsic resistance to CAR-T cell cytotoxicity. Treating G164 spheroids with birinapant, an antagonist of cellular inhibitor of apoptosis protein, induced CAR-T cell cytotoxicity. Moreover, when resistant cells were co-cultured as spheroids with primary omental fibroblasts from ovarian cancer patients, CAR-T cells were activated and cytotoxic. G164 spheroids cultured in fibroblast-conditioned medium also induced CAR-T cell cytotoxicity due to C-C motif chemokine ligand 2 (CCL2) produced by fibroblasts. CCL2 activated CCR2/4+ CAR-T cells to induce cytotoxicity in an antigen-dependent manner. We then investigated CAR-T cell migration and cytotoxicity in malignant cell/fibroblast co-culture collagen gels. CAR-T cell migrated into OvCAR3 gels and were cytotoxic against the malignant cells. However, a dense extracellular matrix (ECM) produced primarily by fibroblasts prevented CAR-T cell migration and cytotoxicity in G164 gels. Treating G164 gels with transforming growth factor beta (TGFβ) receptor inhibitor SB431542 reduced ECM content and altered ECM structure, thus stimulating CAR-T cell penetration and restoring cytotoxicity against G164 cells. We also developed an ovarian cancer-on-a-chip model which involved culturing cell-seeded collagen gels in vascularized tri-channel microfluidic devices. CAR-T cells successfully penetrated and killed malignant cells in co-culture collagen gels when delivered through vascularized microfluidic devices. To our knowledge, this is the first report to use microfluidic tumor models to successfully deliver CAR-T cells. Using these different human 3D in vitro models, we uncovered malignant cell-intrinsic factors and novel mechanisms involving fibroblasts which may influence CAR-T cell activity. Complex human cell models may accelerate preclinical research into CAR-T cell therapies in solid tumors. Citation Format: Joash Dominic Joy, Beatrice Malacrida, Florian Laforêts, Panoraia Kotantaki, Eleni Maniati, Sarah Hopkins, Ianire Garrobo-Calleja, Julien Gautrot, Frances Balkwill. 3D in vitro models uncover malignant cell intrinsic and extrinsic mechanisms of CAR-T cell resistance in high grade serous ovarian cancer. [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 4576.

Abstract 5804: Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells

Abstract Ovarian cancer (OC) is one of the deadliest gynecologic cancers in US. Platinum (Pt)-based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to incurable recurrence. We have previously shown that Pt-containing drugs, e.g., cisplatin, enrich for OCSCs and Pt-induced alterations in DNA methylation enhanced OCSC malignant properties. CSCs have been shown to be dependent on methionine (Met) in many cancers, and in the current study, we investigated the role of Met and the interplay between methylation and Met metabolism in OCSC enrichment. In high grade serous ovarian cancer cell lines (OVCAR5, OVCAR3), cisplatin treatment (IC50, 16hr) increased (p<0.01) the percentage of cells that were positive for aldehyde dehydrogenase (ALDH+ cells), a known OCSCs marker. Cisplatin treatment also increased (p<0.05) the whole cell level of 5-methylcytosine, indicating Pt-induced methylation changes. In addition, Met metabolism was altered by cisplatin, demonstrated by decreased (p<0.01) level of Met derivative, S-adenosyl-methionine (SAM), and increased (p<0.01) expression of Met utilization enzyme, methionine adenosyltransferase 2A (MAT2A). To investigate a possible role of Met metabolism in Pt-induced enrichment of OCSCs, OVCAR5 cells were cultured in complete or Met-depleted (Met-) media, treated with cisplatin or vehicle and examined for CSC properties, including ALDH+ cells and the ability to form spheroids under low-attachment conditions. Met depletion blocked (p<0.01) both the Pt-induced increase of ALDH+ cells and spheroid formation. Both Met- only and Met- in combination with cisplatin activated p38, determined by increased phosphorylated p38, which may have responded to Met depletion and promoted cell cycle arrest. In addition, MAT2A expression was higher (p<0.01) in ALDH+ compared to ALDH- OC cells. MAT2A inhibitor FIDAS-5 combined with cisplatin blocked the Pt-induced increase in ALDH+ cells (p<0.01), demonstrating a requirement for Met utilization in Pt-induced OCSC enrichment. As MAT2A is a therapeutic target in cancer, MAT2A inhibition could represent a therapeutic approach for inhibiting CSC in ovarian cancer. We are further investigating the mechanism of MAT2A-mediated inhibition on Pt-induced OCSCs enrichment by examining the OC methylome and cell cycle arrest. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methyl donor synthesis inhibits platinum-induced enrichment of ovarian cancer stem cells. [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 5804.

Abstract 870: Glutamine metabolism drives some mechanisms of PARP inhibitor persistence in ovarian cancer

Abstract Ovarian cancer is the most lethal gynecological cancer with a five-year survival rate of 49.7%. Almost 1 in 6 patients dies within the first three months of diagnosis. Targeted therapies such as poly (ADP-ribose) polymerase (PARP) inhibitors have gained popularity due to their reduced toxicities. However, only 41% of patients respond to PARP inhibitors and most patients develop resistance. Here, we explored mechanisms of PARP inhibitor resistance in ovarian cancer at single-cell resolution to characterize potentially transient transcriptomic phenotypes associated with resistance. Parental (sensitive) and PARP inhibitor-persistent populations from two ovarian cancer cell lines (OVCAR4 and OVCA3) were sequenced by single-cell RNA sequencing (10X Genomics). Count matrices were log-normalized and scaled to remove variation due to read counts. Dimension reduction was performed using principal component analysis (PCA); cells were clustered phenotypically with graph-based clustering (Louvian algorithm) and visualized with UMAP projections. Downstream analysis includes characterization of the cell cycle phase, calculation of single-cell metabolic activity scores across 37 KEGG metabolic pathways, and pseudotime evolutionary trajectory analysis was performed to study the transition from the sensitive to the persistent phenotype. Across sensitive populations, most cells were in G1-phase; however, after developing persistence to PARP inhibitors, the OVCAR4 cells arrested at G2M phase, while the OVCA3 cells continued to arrest in the G1 phase. Our metabolic single cell RNA seq analysis pipelines revealed that OVCA3 sensitive population expressed high glycolysis metabolic scores compared to their persistent population, while the OVCAR4 persistent population showed a shift toward oxidative phosphorylation compared to the sensitive population implying the heterogenous metabolic adaptation across cancer cells. Persistent cells have high glutamine metabolism scores, suggesting that this pathway is upregulated in these populations compared to the parental ancestors. These findings were experimentally validated in cell lines treated with glutamine deprived media treated with different concentrations of PARP inhibitor. We used Palantir to characterize the evolutionary trajectories of the cells based on predictions of cell fate probabilities. OVCAR4 cells were predicted to develop into two distinct transcriptomic phenotypes, which could be related with sensitivity or tolerance to PARP inhibitors. However, OVCA3 cell did not have distinct, clear differentiation trajectories suggesting that transcriptomic information alone is not sufficient to explain the development of resistance in this cell line. These approaches further our understanding PARP inhibitor resistance in ovarian cancer, which is needed to improve current treatment strategies. Citation Format: Adriana Del Pino Herrera, Meghan Ferrall-Fairbanks, Mehdi Damaghi, Joon-Hyun Song, Snigdha Kanadibhotla. Glutamine metabolism drives some mechanisms of PARP inhibitor persistence in ovarian cancer [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 870.

Abstract 5020: IRAK1 is a critical mediator of hyaluronic acid induced stemness

Abstract Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due in part to late-stage diagnoses. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease, characterized by resistance to standard chemotherapy. There is convincing evidence that cancer stem cells (CSCs) have implications in resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing and identified TLR-ILR1 (TIR) pathways as highly activated in cisplatin resistant OvCa and stem-cell populations. Subsequently, using a tumor microarray, we observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling, is upregulated in OvCa patient tissues compared to normal. This expression further correlated with younger diagnosis age and shorter overall survival, suggesting a role in OvCa tumorigenesis. Knockdown of IRAK1 by specific shRNA in OvCa cells, significantly impaired CSC enriched spheroid growth and orthotopic tumor growth in mouse models replicating advanced peritoneal OvCa. RNA sequencing of IRAK1 knocked down cells identified downregulation of CSC related genes including STAT3, MYC, NOTCH1, and NOTCH3. In addition, there was a reduction in the cisplatin efflux transporter ABCC1 and stemness marker gene CD44. Advanced OvCa is often associated (~90% in stage III and IV) with the development of malignant ascites. We demonstrate for the first time that significant amounts of low molecular weight hyaluronic acid fragments (LMW HA) are present in ascites. LMW HA induced IRAK1 dependent activation of non-canonical signaling through PKC-β leading to induction of STAT3 phosphorylation and MYC. This was further accompanied by increased spheroid formation, demonstrating a critical role for IRAK1 in HA-induced stemness. We have further identified a selective IRAK1 inhibitor, TCS2210 (1,2-Dihydro-N-hydroxy-2-oxo-3-(3-phenylpropyl)-6-quinoxalinecarboxamide). TCS2210 abrogated LMW HA induced activation of IRAK1 and expression of stemness genes MYC, NOTCH1, and NOTCH3. Lastly, we found that TCS2210 effectively suppresses OvCa cell growth in vitro and in vivo, synergizing with standard-of-care cisplatin. Our data suggests a role of IRAK1 in OvCa, by enhancing cancer cell growth and stemness. It further suggests that IRAK1 is a putative target for advanced OvCa. Citation Format: David Standing, Prasad Dandawate, Jaimie Johnson, Sumedha Gunewardena, Michele T. Pritchard, Harsh Pathak, Dineo Khabele, Katherine Roby, Andrew Godwin, Roy Jensen, Scott Weir, Shrikant Anant. IRAK1 is a critical mediator of hyaluronic acid induced stemness. [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 5020.

Abstract 2739: Genetic modification of mesothelial cells with full length membrane-bound TRAIL for ovarian cancer treatment

Abstract Mesothelial cells lining the peritoneum play an important role in epithelial ovarian cancer (EOC) locoregional spreading by promoting cancer cell proliferation, adhesion, and invasion into the peritoneal wall and organs. Peritoneal dissemination represents a critical first step in EOC progression, and we posit that genetic modification of mesothelial cells with full length membrane-bound TRAIL is a practical approach to intervention and will provide local control of EOC progression. Here, we stably overexpressed full length tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with a lentiviral vector in the immortalized mesothelial cell line, MeT5A. Overexpression of TRAIL on the cell surface of MeT5A was confirmed by flow cytometry, and no obvious changes in proliferation or morphology were observed when MeT5A/TRAIL cells were grown in culture. In co-culture experiments, however, MeT5A/TRAIL cells induced apoptosis of surrounding cancer cells and continued proliferating until the culture surface was confluent with mesothelial cells alone. Interestingly, MeT5A/TRAIL not only induced cell death of TRAIL-sensitive cancer cells, such as OVCAR3 and H460, but it also induced significant apoptosis of cancer cells previously deemed resistant to soluble TRAIL, such as A2780, and OVCAR8. This finding suggests that full length TRAIL (amino acid 1-281) expressed on the cell surface is capable of activating death receptor-mediated apoptotic pathways that are not reactive to the soluble form of TRAIL (amino acid 114-281), which was previously developed as a cancer therapeutic and thoroughly evaluated in animal models and clinical trials. When co-cultured with MeT5A/TRAIL, primary ovarian cancer cells from patients also underwent apoptosis as detected by flow cytometry and the upregulation of cleaved caspase-3, caspase-8, caspase-9, PARP, and Bid on Western blots. Next, we assessed the anticancer potential of mesothelial cells expressing membrane-bound TRAIL in a NSG xenograft mouse model of ovarian cancer. Intraperitoneal inoculation of MeT5A/TRAIL cells significantly reduced overall cancer burden in mice where OVCAR3, A2780, and OVCAR8 cancer cells were pre-established. Intraperitoneal gene delivery of TRAIL to resident peritoneal mesothelial cells utilizing a conditionally replicative adenovirus vector also prolonged survival of NSG mice bearing A2780 xenografts. Our data suggest that genetic modification of mesothelial cells with full length membrane-bound TRAIL is feasible and has therapeutic potential for disrupting EOC progression. Citation Format: Er Yue, Tonya Walser, Tamara Mirzapoiazova, Ravi Salgia, Edward W. Wang. Genetic modification of mesothelial cells with full length membrane-bound TRAIL for ovarian cancer treatment [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 2739.

Abstract 4971: Identification of statins as a potential treatment for ovarian cancer

Abstract Late-stage diagnoses make ovarian cancer extremely hard to treat. This leads to ovarian cancer accounting for 5% of female cancer deaths and 2.5% of female cancer incidence. Since there are few options for early detection of ovarian cancer, targeted therapies could lead to better outcomes. One target that has been implicated in ovarian cancer, is the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). STAT3 function is associated with tumor growth, metastasis, drug resistance, angiogenesis, and resistance to apoptosis. These many STAT3 controlled, cancer-associated cellular functions, make it an ideal and versatile target for cancer therapy. Using the Broad institute’s online C.L.U.E. tool and a STAT3 gene signature, we identified statin drugs as potential inhibitors of STAT3 activity. To assess the ability of statins to inhibit STAT3, we treated ovarian cancer spheroids with statins and analyzed STAT3 activity. We have found that statins do not induce a change in the phosphorylation of STAT3 on either tyrosine 705 or serine 727 residues, the two major sites of phosphorylation on STAT3. However, there are other poorly understood post-translational modifications of STAT3, that have previously shown effects on STAT3 function, that may be involved. Importantly, we have observed that statin treatment reduces the viability of Ovcar8 and Heya8 ovarian cancer cell spheroids. We also found that statin treatment induced apoptosis of ovarian spheroids by measuring Annexin V staining. A major problem for ovarian cancer patients is metastasis. Our preliminary evidence suggests that statins reduce the ability of ovarian cancer spheroids to clear a mesothelial monolayer of cells, an early step in ovarian cancer metastasis. There are several membrane-associated signaling proteins, including RAF and RAS, that affect STAT3 activity. Both Ovcar8 and Heya8 spheroids showed a decrease in cell viability with statin treatment, which is partially rescued by geranylgeranyl pyrophosphate (GGPP), a downstream metabolite in the mevalonate pathway. The partial recovery by GGPP indicates a mechanism involving the prenylation of membrane associated proteins. This data shows statins may provide effective treatments for ovarian cancer and defining the mechanisms behind the effects of statins may lead to better therapies and outcomes for ovarian cancer patients Citation Format: Brendan M. Reilly, Sarah J. Miseirvitch, Sarah R. Walker. Identification of statins as a potential treatment for ovarian cancer . [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 4971.

Abstract 1823: Overcoming siglec-9-mediated immunosuppression by using glycomimetics that block the binding of this receptor with its natural ligands

Abstract MUC16, a 3-5 million Da mucin overexpressed by ovarian cancer cells binds to Siglec-9, an inhibitory receptor on immune cells. Upon binding to Siglec-9, MUC16 triggers inhibitory signaling in the immune cells that allows ovarian cancer cells to evade cytolytic immune responses. Ovarian and other tumors express a plethora of cell surface as well as secreted glycoproteins with glycans that are terminated with the Siglec-9 ligand, α-2,3-linked sialic acid. Therefore, targeting the sialic acid-Siglec-9 axis has been of major interest to augment antitumor immunity. Here, we demonstrate that synthetic glycomimetic Siglec-9 ligands have the potential to enhance the immune recognition of ovarian cancer cells. We first demonstrate, using siglec-9 chimeras, that the glycomimetics developed by our group inhibit the binding of Siglec-9 to its natural ligands. Our data showed that 50-fold excess of the glycomimetics over Siglec-9 chimera prevented the Siglec-9 chimera from binding to the OVCAR-3 cell line (P.value, 0.039). Additionally, we demonstrate that glycomimetics (#153 and 157) developed by our group, enhance the recognition of ovarian cancer cell targets by human NK cells. In healthy donor 1, CD107a expression was increased (21, 17, 19 %) when 0.01, 0.025, 0.05 mM concentration of glycomimetic 153 was used, respectively. A statistically significant increase (5%) in CD107a expression was observed when the NK cells were treated with 0.05 mM concentration of glycomimetic 157. At higher concentrations of glycomimetic 153 and 157, inhibition of CD107a was repeatedly observed. These experiments, therefore, suggest that there is likely a certain concentration range of the glycomimetic that can be used to overcome NK cell suppression occurring via Siglec-9. To our knowledge, these data are among the first to demonstrate that specially designed glycomimetics can be used to overcome the suppression of NK cells against ovarian cancer cells. Future studies will employ murine and humanized models to demonstrate the therapeutic potential of the Siglec-9-targeting glycopeptides in enhancing immune responses against epithelial ovarian cancer. Citation Format: Lojain I. AlJohani, Manish Patankar, Weiping Tang, Christopher Stevens, Peng Teng. Overcoming siglec-9-mediated immunosuppression by using glycomimetics that block the binding of this receptor with its natural ligands [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 1823.

Abstract 5797: NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer

Abstract Background: DNA methylation causes the silencing of tumor suppressor and differentiation-associated genes being linked to chemoresistance and stemness. Previous studies demonstrated that hypomethylating agents (HMA) re-sensitize ovarian cancer (OC) cells to chemotherapy, however, translation to the clinic has been slow. NTX 301 (PinotBio) is a novel, highly potent, and orally bioavailable HMA, in early clinical development. Methods: We assessed the anti-tumor effects of NTX301 in OC models and studied the underlying molecular mechanisms by using cell proliferation, stemness, and ferroptosis assays, RNA sequencing, and validation. Results: OC cells (SKOV3, IC50=5.089nM; OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p<0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p<0.05). Treatment with low dose NTX301 (100nM) induced significant transcriptomic reprogramming with 15,000 differentially expressed genes (DEGs) compared to DMSO (p<0.05). Among the NTX301 down-regulated DEGs, Gene Ontology (GO) enrichment analysis identified pathways related to the regulation of alcohol, cholesterol, and fatty acid biosynthetic process and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM) reduced the ALDH(+) cell population and expression of stemness-associated transcription factors (Oct4 and Sox2). Additionally, stearoyl-Coenzyme A desaturase 1 (SCD), a key enzyme that regulates unsaturated fatty acid homeostasis in the lipogenesis process was found among the top DEGs downregulated in response to NTX3 (fold change=9.36, FDR<0.05). As blockade of SCD had been shown to induce ferroptosis, oxidized lipids levels were measured by C11-BODIPY staining. NTX301 treatment increased the levels of oxidized lipids compared to DMSO and this was blunted by deferoxamine, indicating that NTX301 promoted cell death via ferroptosis. NTX301-induced ferroptosis was successfully rescued by the addition of oleic acid (200μM) into the culture medium, supporting that it was mediated through SCD depletion. In vivo, monotherapy with NTX301 (0.5mg/kg), significantly inhibited subcutaneous OC xenograft tumor growth (p<0.05). Decreased SCD levels and increased oxidized lipids were recorded in NTX301-treated xenografts. Conclusions: NTX301 is a potent HMA active in OC models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death. Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei, Ellie Siu, Sayedabdulrazzaq A. Alhunayan. NTX301, a novel DNA hypomethylating agent, targets lipid metabolism in ovarian cancer [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 5797.

Abstract 2668: Long non-cording RNA TUG1 promotes cisplatin resistance in ovarian cancer via DNA polymerase eta

Abstract Ovarian cancer (OVCA) is the most malignant gynecologic cancer. Despite the use of surgery and platinum-based chemotherapy, the 5-year survival rate is approximately 30% at the time of diagnosis. Cisplatin inhibits DNA synthesis by cross-linking DNA and then induces cell apoptosis. However, cisplatin resistance often causes treatment failure and tumor relapse in OVCA patients. To overcome the cisplatin resistance in OVCAs, we studied the mechanism of chemoresistance by a long non-coding RNA (lncRNA), TUG1 (taurine upregulated gene 1), which is associated with carcinogenesis and drug sensitivity in OVCAs. The expression level of TUG1 in cisplatin-resistant OVCA clinical samples is significantly higher than that of cisplatin-sensitive samples by public database. A depletion of TUG1 increased the sensitivity of OVCA cells to cisplatin together with upregulation of a set of miRNAs in vitro. We examined the targets of miRNAs and found that TUG1 depletion downregulates DNA polymerase eta (Polη) by upregulating the miRNAs. Polη is a major translesion DNA synthesis (TLS) DNA polymerase, which bypasses DNA lesions produced by DNA damaging agents and helps DNA replication. The expression level of Polη showed a positive correlation with that of TUG1 in OVCA clinical samples. Overexpression of Polη recovered cisplatin resistance in TUG1-depleted cells. Finally, cisplatin treatment combined with TUG1 depletion effectively inhibited tumor growth in xenograft mouse models, indicating the efficacy of TUG1 depletion in vivo. Our data provide a strong rationale for targeting TUG1 as a novel therapeutic approach to overcome cisplatin resistance in OVCA. Citation Format: Ryosuke Sonobe. Long non-cording RNA TUG1 promotes cisplatin resistance in ovarian cancer via DNA polymerase eta [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 2668.

OTX1 silencing suppresses ovarian cancer progression through inhibiting the JAK/STAT signaling

BackgroundThe aim of our study was to investigate the roles and the underlying mechanisms of orthodenticle homolog 1 (OTX1) in ovarian cancer. MethodsOTX1 expression was obtained from TCGA database. OTX1 expression in ovarian cancer cells was detected using qRT-PCR and western blot assay. The cell viability and proliferation was detected by CCK-8 and EdU assays. Cell invasion and migration were detected by transwell assay. Flow cytometry was utilized to determine cell apoptosis and cycle. In addition, western blot assay was used to detect the expression of cell cycle related protein (Cyclin D1 and p21), epithelial-mesenchymal transition (EMT) related protein (E-cadherin, N-cadherin, Vimentin, and Snail), apoptosis related protein (Bcl-2, Bax, and cleaved caspase-3), and JAK/STAT pathway related protein (p-JAK2, JAK2, STAT3, and p-STAT3). ResultsOTX1 was highly expressed in ovarian cancer tissues and cells. OTX1 silencing blocked the cell cycle and repressed cell viability, proliferation, invasion, and migration, while OTX1 silencing facilitated the apoptosis of OVCAR3 and Caov3 cells. OTX1 silencing increased the protein levels of p21, E-cadherin, Bax, and cleaved caspase-3, while the protein levels of Cyclin D1, Bcl-2, N-cadherin, Vimentin, and Snail were decreased by OTX1 silencing. Furthermore, OTX1 silencing suppressed the protein levels of p-JAK2/JAK2 and p-STAT3/STAT3 in OVCAR3 and Caov3 cells. Moreover, overexpression of OTX1 promoted cell proliferation and invasion and inhibited apoptosis in Caov3 cells, but AG490 (an inhibitor of JAK/STAT pathway) reversed the influences on cell biological behavior induced by overexpression of OTX1. ConclusionsOTX1 silencing repressed ovarian cancer cell proliferation, invasion, and migration and induced cell apoptosis, which might be involved in JAK/STAT signaling pathway. OTX1 may be considered as a novel therapeutic target for ovarian cancer.

Next Generation of Ovarian Cancer Detection Using Aptamers

Ovarian cancer is among the seven most common types of cancer in women, being the most fatal gynecological tumor, due to the difficulty of detection in early stages. Aptamers are important tools to improve tumor diagnosis through the recognition of specific molecules produced by tumors. Here, aptamers and their potential targets in ovarian cancer cells were analyzed by in silico approaches. Specific aptamers were selected by the Cell-SELEX method using Caov-3 and OvCar-3 cells. The five most frequent aptamers obtained from the last round of selection were computationally modeled. The potential targets for those aptamers in cells were proposed by analyzing proteomic data available for the Caov-3 and OvCar-3 cell lines. Overexpressed proteins for each cell were characterized as to their three-dimensional model, cell location, and electrostatic potential. As a result, four specific aptamers for ovarian tumors were selected: AptaC2, AptaC4, AptaO1, and AptaO2. Potential targets were identified for each aptamer through Molecular Docking, and the best complexes were AptaC2-FXYD3, AptaC4-ALPP, AptaO1-TSPAN15, and AptaO2-TSPAN15. In addition, AptaC2 and AptaO1 could detect different stages and subtypes of ovarian cancer tissue samples. The application of this technology makes it possible to propose new molecular biomarkers for the differential diagnosis of epithelial ovarian cancer.

Cancer-associated mesothelial cell-derived ANGPTL4 and STC1 promote the early steps of ovarian cancer metastasis.

Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin-mediated ablation in Msln-Cre mice significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietin-like 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell-induced monocyte migration, endothelial cell vessel formation, and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell-induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with Abs reduced the ex vivo colonization of 3 different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8p53-/-Brca2-/- cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis and that the crosstalk between mesothelial cells and the tumor microenvironment promotes OvCa metastasis through the secretion of ANGPTL4.

Heracleum persicum Desf. ex Fisch., C.A.Mey. & Avé-Lall. fruit essential oil: content, antimicrobial activity and cytotoxicity against ovarian cancer cell line

BackgroundOne of the most important aromatic species of the Apiaceae family is Heracleum persicum Desf. ex Fisch., C.A.Mey. & Avé-Lall, which used as a spice and condiment in food. It is widely used in traditional Iranian medicine due to its anti-toxic properties. The aim of this study was to evaluate the essential oil of this plant in terms of content and ingredients, cytotoxicity and antimicrobial activity.MethodsThe fruit of H. persicum was collected in June 2019 from Maragheh region of Kashan, Iran. The essential oil was extracted by water distillation using Clevenger for 3 h. Identification analysis of H. persicum fruit essential oil (HPFEO) components was performed using gas chromatography-mass spectrometry (GC–MS). Evaluation of the effect of the HPFEO on the growth and proliferation of ovarian cancer cell line (OVCAR-3) was performed using MTT colorimetric method. Its antimicrobial activity was evaluated by agar diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal/fungal lethality concentration (MBC/MFC).ResultsThe results of analysis of the HPFEO by GC–MS showed that there were 35 compounds with 99.54% relative content. Hexyl butyrate (35.24%), octyl 2-methylbutyrate (11.65%), octyl isobutyrate (9.23%), and octyl acetate (8.42%) were the predominant compounds. Examination of cell survival showed that the viability of cells decreased depending on the concentration of the HPFEO in 24 h and had value of IC50 ~ 12.08 μg/ml against OVCAR-3 cell line. The strongest inhibitory activity of the HPFEO was against Gram-negative bacteria Pseudomonas aeruginosa and Salmonella paratyphi-A serotype (MIC < 62.50 μg/mL). Also, the strong inhibitory and lethal activity of this essential oil against Candida albicans (MIC and MBC 250 μg/mL was one degree weaker compared to nystatin (MIC = 125 μg/mL).ConclusionsThus, HPFEO, in addition to its traditional use, may have a strong and potential potential for the production of anti-proliferative and antimicrobial drugs.

An environmentally safe approach for the facile synthesis of anti-mutagenic fluorescent quantum dots: Property investigation and the development of novel antimicrobial applications

Quantum dots as nanocrystals of semiconductor materials are synthesized with different methods. The green synthesis of fluorescent quantum dots using natural stabilizing agents has attracted much attention. This research aims to study the ability of Teucrium polium L. extract to green synthesize fluorescent silver quantum dots (AgQDs). The green synthesis of AgQDs was proved using UV–Visible spectrophotometer, Fourier Transform Infrared spectroscopy (FTIR), X-Ray Diffraction (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDX), Photoluminescence (PL) analysis, and Dynamic Light Scattering (DLS).According to the findings, the shape of fluorescent AgQDs was spherical, and the maximum abundance of particle size distribution was between 3 and 5 nm.Thereupon, antimicrobial, anti-mutagenicity, anticancer, and antioxidant activities were evaluated to distinguish the biological features of AgQDs. Gram-positive bacteria (S. epidermidis, MIC: 31.25 μg/ml) and fungi (C. albicans, MIC: 31.25 μg/ml) were the most susceptible to AgQDs. Moreover, the AgQDs solution had higher antimicrobial activity than AgNO3. The Ames test demonstrated that AgQDs have considerable anti-mutagenic activities (% mutagenicity inhibition greater than 40) and are not mutagenic. The anticancer activity of AgQDs was distinguished using MTT and brine shrimp lethality (BSL) assays. The analysis indicated that AgQDs had a strong potential for cytotoxicity (BSL: LC50 = 2.4 μg/ml, MTT: IC50 = 0.8 μg/ml). Further, AgQDs were used for bioimaging human ovarian OVCAR3 cells, and their biocompatibility was remarkable. In the DPPH assay, AgQDs displayed high antioxidant activity (89.9% inhibition). Using AgQDs to coat materials to develop antibacterial agents revealed significant activity against C. albicans and S. epidermidis strains. Our results indicate that T. polium is a promising source for synthesizing AgQDs. Due to the potent bioactivities of AgQDs, they can be used in medicine and industry.

VEGFR3 suppression through miR-1236 inhibits proliferation and induces apoptosis in ovarian cancer via ERK1/2 and AKT signaling pathways.

Vascular endothelial growth factor receptor 3 (VEGFR3) is expressed in cancer cell lines and exerts a critical role in cancer progression. However, the signaling pathways of VEGFR3 in ovarian cancer cell proliferation remain unclear. This study aimed to demonstrate the signaling pathways of VEGFR3 through the upregulated expression of miR-1236 in ovarian cancer cells. We found that the messenger RNA and protein of VEGFR3 were expressed in the ovarian cancer cell lines, but downregulated after microRNA-1236 (miR-1236) transfection. The inhibition of VEGFR3, using miR-1236, significantly reduced cell proliferation, clonogenic survival, migration, and invasion ability in SKOV3 and OVCAR3 cells (p < 0.01). The flow cytometry results indicated that the rate of apoptotic cells in SKOV3 (38.65%) and OVCAR3 (41.95%) cells increased following VEGFR3 inhibition. Moreover, VEGFR3 stimulation (using a specific ligand, VEGF-CS) significantly increased extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation (p < 0.01), whereas VEGFR3 suppression reduced p-ERK1/2 (67.94% in SKOV3 and 93.52% in OVCAR3) and p-AKT (59.56% in SKOV3 and 78.73% in OVCAR3) compared to the VEGF-CS treated group. This finding demonstrated that miR-1236 may act as an endogenous regulator of ERK1/2 and AKT signaling by blocking the upstream regulator of VEGFR3. Overall, we demonstrated the important role of the miR-1236/VEGFR3 axis in ovarian cancer cell proliferation by regulating the ERK1/2 and AKT signaling that might be an effective strategy against ovarian cancer.

Cytotoxicity Evaluation of Unmodified Paddlewheel Dirhodium(II,II)-Acetate/-Formamidinate Complexes and Their Axially Modified Low-Valent Metallodendrimers.

Two diphenyl formamidine ligands, four dirhodium(II,II) complexes, and three axially modified low-valent dirhodium(II,II) metallodendrimers were synthesized and evaluated as anticancer agents against the A2780, A2780cis, and OVCAR-3 human ovarian cancer cell lines. The dirhodium(II,II) complexes show moderate cytotoxic activity in the tested tumor cell lines, with acetate and methyl-substituted formamidinate compounds displaying increased cytotoxicity that is relative to cisplatin in the A2780cis cisplatin resistant cell line. Additionally, methyl- and fluoro-substituted formamidinate complexes showed comparable and increased cytotoxic activity in the OVCAR-3 cell line when compared to cisplatin. The low-valent metallodendrimers show some activity, but a general decrease in cytotoxicity was observed when compared to the precursor complexes in all but one case, which is where the more active acetate-derived metallodendrimer showed a lower IC50 value in the OVCAR-3 cell line in comparison with the dirhodium(II,II) tetraacetate.

Evodiamine inhibits malignant progression of ovarian cancer cells by regulating lncRNA-NEAT1/miR-152-3p/CDK19 axis.

Evodiamine (EVO) has been demonstrated to promote apoptosis of ovarian cancer cells, and upregulate miR-152-3p level in colorectal cancer. Here, we explore part of the network mechanism of EVO and miR-152-3p in ovarian cancer. The bioinformatics website, dual luciferase reporter assay, and quantitative real-time polymerase chain reaction were applied to analyze the network among EVO, lncRNA, miR-152-3p, and mRNA. The effect and mechanism of EVO on ovarian cancer cells were determined using cell counting kit-8, flow cytometry, TUNEL, Western blot, and rescue experiments. As a result, EVO dose-dependently attenuated cell viability, induced G2/M phase arrest and apoptosis, promoted miR-152-3p level (4.5- or 2-fold changes), and inhibited expressions of NEAT1 (0.225- or 0.367-fold changes), CDK8 (0.625- or 0.571-fold changes), and CDK19 (0.25- or 0.147-fold changes) in OVCAR-3 and SKOV-3 cells. In addition, EVO decreased Bcl-2 expression, but increased the expressions of Bax and c-caspase-3. NEAT1 targeted miR-152-3p which bound to CDK19. The impacts of EVO on cell viability, cycle, apoptosis, and apoptosis-related proteins were partially reversed by miR-152-3p inhibitor, NEAT1 overexpression, or CDK19 overexpression. Furthermore, miR-152-3p mimic offset the effects of NEAT1 or CDK19 overexpression. The role of NEAT1 overexpression in the biological phenotype of ovarian cancer cells was counteracted by shCDK19. In conclusion, EVO attenuates ovarian cancer cell progression via the NEAT1-miR-152-3p-CDK19 axis.

Evaluation of Biological Activities of Twenty Flavones and In Silico Docking Study

This work aimed to evaluate the biological activities of 20 flavones (M1 to M20) and discuss their structure-activity relationships. In vitro assays were established to assess their numerous biological activities (anti-α-amylase, anti-acetylcholinesterase, anti-xanthine oxidase, anti-superoxide dismutase, and anticancer cell lines (HCT-116, MCF7, OVCAR-3, IGROV-1, and SKOV-3 cells lines)). An in silico docking study was also established in order to find the relationship between the chemical structure and the biological activities. In vitro tests revealed that M5 and M13 were the most active in terms of anti-α-amylase activity (IC50 = 1.2 and 1.4 µM, respectively). M17 was an inhibitor of xanthine oxidase (XOD) and performed better than the reference (allopurinol), at IC50 = 0.9 µM. M7 presented interesting anti-inflammatory (IC50 = 38.5 µM), anti-supriode dismutase (anti-SOD) (IC50 = 31.5 µM), and anti-acetylcholinesterase (IC50 = 10.2 µM) activities. Those abilities were in concordance with its high scavenging activity in antioxidant ABTS and DPPH assays, at IC50 = 6.3 and 5.2 µM, respectively. Selectivity was detected regarding cytotoxic activity for those flavones. M1 (IC50 = 35.9 µM) was a specific inhibitor to the MCF7 cancer cell lines. M3 (IC50 = 44.7 µM) and M15 (IC50 = 45.6 µM) were particularly potent for the OVCAR-3 cell line. M14 (IC50 = 4.6 µM) contributed more clearly to inhibiting the colon cancer cell line (HCT116). M7 (IC50 = 15.6 µM) was especially active against the ovarian SKOV human cancer cell line. The results of the biological activities were supported by means of in silico molecular docking calculations. This investigation analyzed the contribution of the structure-activity of natural flavones in terms of their biological properties, which is important for their future application against diseases.

Overexpression of CLDN16 in ovarian cancer is modulated by PI3K and PKC pathways

Epithelial ovarian cancer (EOC) is the gynecological malignant tumor of poorest prognosis and higher mortality rate. Chemotherapy is the base of high-grade serous ovarian cancer (HGSOC) treatment; however, it favors the emergence of chemoresistance and metastasis. Thus, there is an urge to search for new therapeutic targets, such as proteins related to cellular proliferation and invasion. Herein, we investigated the expression profile of claudin-16 (CLDN16 protein and CLDN16 transcript) and its possible functions in EOC. In silico analysis of CLDN16 expression profile was performed using data extracted from GENT2 and GEPIA2 platforms. A retrospective study was carried out with 55 patients to evaluate the expression of CLDN16. The samples were evaluated by immunohistochemistry, immunofluorescence, qRT-PCR, molecular docking, sequencing, and immunoblotting assays. Statistical analyzes were performed using Kaplan-Meier curves, one-way ANOVA, Turkey posttest. Data were analyzed using GraphPad Prism 8.0. In silico experiments showed that CLDN16 is overexpressed in EOC. 80.0% of all EOC types overexpressed CLDN16, of which in 87% of the cases the protein is restricted to cellular cytoplasm. CLDN16 expression was not related to tumor stage, tumor cells differentiation status, tumor responsiveness to cisplatin, or patients' survival rate. When compared to data obtained from in silico analysis regarding EOC stage and degree of differentiation, differences were found in the former but not in the later, neither in survival curves. CLDN16 expression in HGSOC OVCAR-3 cells increased by 1.95-fold (p < 0.001), 2.32-fold (p < 0.001), and 6.57-fold (p < 0.001) via PKC, PI3K, and estrogen pathways, respectively. Altogether, our results suggest that despite the low number of samples included in our in vitro studies, adding to the expression profile findings, we provided a comprehensive study of CLDN16 expression in EOC. Therefore, we hypothesize that CLDN16 is a potential target in the diagnosis and treatment of the disease.