Serous Ovarian Cancer Cell Research Articles

The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

Protein kinase C iota promotes glycolysis via PI3K/AKT/mTOR signalling in high grade serous ovarian cancer.

Epithelial ovarian cancer, especially high grade serous ovarian cancer (HGSOC) is by far, the most lethal gynecological malignancy with poor prognosis and high relapse rate. Despite of availability of several therapeutic interventions including poly-ADP ribose polymerase (PARP) inhibitors, HGSOC remains unmanageable and identification of early detection biomarkers and therapeutic targets for this lethal malady is highly warranted. Aberrant expression of protein kinase C iota (PKCί) is implicated in many cellular and physiological functions involved in tumorigenesis including cell proliferation and cell cycle deregulation. Two high grade serous ovarian cancer cells SKOV3 and COV362 were employed in this study. PKCί was genetically knocked down or pharmacologically inhibited and several functional and biochemical assays were performed. We report that PKCί is overexpressed in HGSOC cells and patient tissue samples with a significant prognostic value. Pharmacological inhibition of PKCί by Na-aurothiomalate or its shRNA-mediated genetic knockdown suppressed HGSOC cell proliferation, EMT and induced apoptosis. Moreover, PKCί positively regulated GLUT1 and several other glycolytic genes including HK1, HK2, PGK1, ENO1 and LDHA to promote elevated glucose uptake and glycolysis in HGSOC cells. Mechanistically, PKCί drove glycolysis via PI3K/AKT/mTOR signalling. Na-aurothiomalate and highly selective, dual PI3K/mTOR inhibitor dactolisib could serve as novel anti-glycolytic drugs in HGSOC. Taken together, our results indicate PKCί/PI3K/AKT/mTOR signalling cascade could be a novel therapeutic target in a lethal pathology like HGSOC.

Aulosirazole Stimulates FOXO3a Nuclear Translocation to Regulate Apoptosis and Cell-Cycle Progression in High-Grade Serous Ovarian Cancer (HGSOC) Cells.

Ovarian cancer, the fifth leading cause of cancer-related mortality in women, is the most lethal gynecological malignancy globally. Within various ovarian cancer subtypes, high-grade serous ovarian cancer is the most prevalent and there is frequent emergence of chemoresistance. Aulosirazole, an isothiazolonaphthoquinone alkaloid, isolated from the cyanobacterium Nostoc sp. UIC 10771, demonstrated cytotoxic activity against OVCAR3 cells (IC50 = 301 ± 80 nM). Using immunocytochemistry, OVCAR3 cells treated with aulosirazole demonstrated increased concentrations of phosphorylated protein kinase B and phosphorylated c-Jun N-terminal kinase with subsequent accumulation of forkhead box O3a (FOXO3a) in the nucleus. The combination of aulosirazole with protein kinase B inhibitors resulted in the most nuclear accumulation of FOXO3a aulosirazole-induced apoptosis based on cleavage of poly(ADP-ribose) polymerase, annexin V staining, and induction of caspase 3/7 activity in OVCAR3, OVCAR5, and OVCAR8. The expression of downstream targets of FOXO3a, including B-cell lymphoma 2 (BCL2) and p53-upregulator modulator of apoptosis, increased following aulosirazole treatment. Aulosirazole upregulated the FOXO3a target, cyclin-dependent kinase inhibitor 1, and increased cell-cycle arrest in the G0/G1 phase. The downregulation of FOXO3a by short hairpin RNA (shRNA) reduced the cytotoxicity after aulosirazole treatment by 3-fold IC50 (949 ± 16 nM) and eliminated its ability to regulate downstream targets of FOXO3a. These findings underscore FOXO3a as a critical mediator of aulosirazole-induced cytotoxicity. Additionally, aulosirazole was able to decrease migration and invasion while increasing cell death in 3D tumor spheroids. However, in vivo OVCAR8 tumor burden was not reduced by aulosirazole using an intraperitoneal tumor model. Given the mechanism of action of aulosirazole, this class of alkaloids represents promising lead compounds to develop treatments against FOXO3a-downregulated cancers. SIGNIFICANCE STATEMENT: Aulosirazole, an isothiazolonaphthoquinone alkaloid, exhibits potent cytotoxic effects against high-grade serous ovarian cancer by promoting forkhead box O3a (FOXO3a) nuclear accumulation and modulating downstream targets. These findings highlight the potential of aulosirazole as a promising therapeutic intervention for cancers characterized by FOXO3a downregulation.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of the Long Noncoding RNA HOTAIR and Lysine Methyltransferase EZH2.

Persistence of cancer stem cells (CSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse in ovarian cancer, the fifth leading cause of cancer-related death among US women. HOXC transcript antisense RNA (HOTAIR) is a long noncoding RNA (lncRNA) overexpressed in high-grade serous ovarian cancer and linked to chemoresistance. However, HOTAIR impacts chromatin dynamics in ovarian CSCs and how this oncogenic lncRNA contributes to drug resistant disease are incompletely understood. Here we generated HOTAIR knock-out (KO) high-grade serous ovarian cancer cell lines using paired CRISPR guide RNA design to investigate the function of HOTAIR. We show loss of HOTAIR function re-sensitized ovarian cancer cells to platinum treatment and decreased the population of ovarian CSCs. Furthermore, HOTAIR KO inhibited development of stemness-related phenotypes, including spheroid formation ability, as well as expression of key stemness-associated genes ALDH1A1, NOTCH3, SOX9, and PROM1. HOTAIR KO altered both the cellular transcriptome and chromatin accessibility landscape of multiple oncogenic-associated genes and pathways, including the NF-kB pathway. HOTAIR functions as an oncogene by recruiting enhancer of zeste 2 (EZH2) to catalyze H3K27 tri-methylation to suppress downstream tumor suppressor genes, and it was of interest to inhibit both HOTAIR and EZH2. In vivo, combining a HOTAIR inhibitor with an EZH2 inhibitor and platinum chemotherapy decreased tumor formation and increased survival. These results suggest a key role for HOTAIR in ovarian CSCs and malignant potential. Targeting HOTAIR in combination with epigenetic therapies may represents therapeutic strategy to ameliorate ovarian cancer progression and resistance to platinum-based chemotherapy.

Fallopian Tube-Derived High-Grade Serous Cancers Influence Ovarian Production of Norepinephrine and Generate Specific Metabolomic Signatures.

High-grade serous ovarian cancer is the most common and lethal gynecologic malignancy, which is often attributed to the lack of available screenings, allowing the disease to progress unnoticed until it is diagnosed at more aggressive stages. As such, identifying signals in the tumor microenvironment involved in the primary metastasis of tumorigenic fallopian tube epithelial (FTE) cells to the ovary could provide new avenues for prevention, diagnostics, or therapeutic intervention. Since our previous work identified that the interaction of tumorigenic FTE and the ovary causes the release of norepinephrine (NE) from the ovary, we intended to determine the effects of ovarian NE on signaling and invasion of tumorigenic FTE models and high-grade serous ovarian cancer cell lines. We demonstrate that NE does not universally enhance migration, invasion, or adhesion by using multiple cell types but does alter specific oncogenic protein expression in certain models. In vivo, we found that blocking NE signaling via slow-release propranolol pellets significantly increased survival time in mice injected intraperitoneally with murine FTE cells engineered to stably express shRNA for PTEN and an activated KRAS expression construct. Finally, we identified that the metabolome released from the ovary is variable depending upon which cell type it is cocultured with, suggesting that distinct driver mutations in fallopian tube epithelial tumor models and early lesions can alter specific metabolomes within the surrounding ovarian microenvironment. These metabolomes provide the next frontier for evaluating local signals of the tumor microenvironment that facilitate ovarian spread of FTE lesions.

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate, demonstrates in vitro and in vivo antitumor activity against primary and metastatic ovarian tumors overexpressing HER2.

High-grade serous ovarian cancer (HGSOC) and ovarian clear cell carcinoma (CC), are biologically aggressive tumors endowed with the ability to rapidly metastasize to the abdominal cavity and distant organs. About 10% of HGSOC and 30% of CC demonstrate HER2 IHC 3 + receptor over-expression. We evaluated the efficacy of trastuzumab deruxtecan (T-DXd; DS-8201a), a novel HER2-targeting antibody-drug conjugate (ADC) to an ADC isotype control (CTL ADC) against multiple HGSOC and CC tumor models. Eleven ovarian cancer cell lines including a matched primary and metastatic cell line established from the same patient, were evaluated for HER2 expression by immunohistochemistry and flow cytometry, and gene amplification by fluorescence in situ hybridization assays. In vitro experiments demonstrated T-DXd to be significantly more effective against HER2 3 + HGSOC and CC cell lines when compared to CTL ADC (p < 0.0001). T-DXd induced efficient bystander killing of HER2 non-expressing tumor cells when admixed with HER2 3 + cells. In vivo activity of T-DXd was studied in HER2 IHC 3 + HGSOC and CC mouse xenograft models. We found T-DXd to be significantly more effective than CTL ADC against HER2 3 + HGSOC (KR(CH)31) and CC (OVA10) xenografts with a significant difference in tumor growth starting at day 8 (p = 0.0003 for KR(CH)31, p < 0.0001 for OVA10). T-DXd also conferred a survival advantage in both xenograft models. T-DXd may represent an effective ADC against primary and metastatic HER2-overexpressing HGSOC and CC.

The G-Protein-Coupled Estrogen Receptor Selective Agonist G-1 Attenuates Cell Viability and Migration in High-Grade Serous Ovarian Cancer Cell Lines.

The G-protein-coupled estrogen receptor (GPER; G-protein-coupled estrogen receptor 30, also known as GPR30) is a novel estrogen receptor and has emerged as a promising target for ovarian cancer. GPER, a seven-transmembrane receptor, suppresses cellular viability and migration in studied ovarian cancer cells. However, its impact on the fallopian tube, which is the potential origin of high-grade serous (HGSC) ovarian cancer, has not been addressed. This study was conducted to evaluate the relationship of GPER, ovarian cancer subtypes, i.e., high-grade serous cell lines (OV90 and OVCAR420), as well as the cell type that is the potential origin of HGSC ovarian cancer (i.e., the fallopian tube cell line FT190). The selective ligand assessed here is the agonist G-1, which was utilized in an in vitro study to characterize its effects on cellular viability and migration. As a result, this study has addressed the effect of a specific GPER agonist on cell viability, providing a better understanding of the effects of this compound on our diverse group of studied cell lines. Strikingly, attenuated cell proliferation and migration behaviors were observed in the presence of G-1. Thus, our in vitro study reveals the impact of the origin of HGSC ovarian cancers and highlights the GPER agonist G-1 as a potential therapy for ovarian cancer.

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

AMIGO2 is involved in the spread of peritoneal metastasis in serous ovarian cancer via promoting adhesion to the peritoneal mesothelial cells.

Amphoterin-induced gene and open reading frame 2 (AMIGO2) have been reported to be related to the prognosis of colorectal, gastric, and cervical cancer. However, their association with ovarian cancer remains unclear. This study aimed to elucidate the role of AMIGO2 in ovarian cancer. AMIGO2 expression was evaluated using immunohistochemistry in patients with ovarian serous carcinoma. We validated in vitro studies using four serous ovarian cancer cell lines and in vivo studies using a murine model. The AMIGO2-high group had significantly shorter progression-free survival (PFS) than the AMIGO2-low group. The predictive index of the AMIGO2-high group was considerably higher than that of the AMIGO2-low group. The rate of complete cytoreductive surgery was lower in the AMIGO2-high group than in the AMIGO2-low group. Moreover, in vitro studies revealed that four serous ovarian cancer cell lines exhibited AMIGO2 expression and adhesion to mesothelial cells. Adhesion to mesothelial cells was attenuated by AMIGO2 knockdown in SKOV3 and SHIN3 cells. Furthermore, AMIGO2 downregulation in SKOV3 cells significantly suppressed peritoneal dissemination in the murine model. These results suggest that high AMIGO2 expression in serous ovarian carcinoma cells contributes to a poor prognosis by promoting peritoneal metastasis through enhanced cell adhesion to mesothelial cells.

NB compounds are potent and efficacious FOXM1 inhibitors in high-grade serous ovarian cancer cells

BackgroundGenetic studies implicate the oncogenic transcription factor Forkhead Box M1 (FOXM1) as a potential therapeutic target in high-grade serous ovarian cancer (HGSOC). We evaluated the activity of different FOXM1 inhibitors in HGSOC cell models.ResultsWe treated HGSOC and fallopian tube epithelial (FTE) cells with a panel of previously reported FOXM1 inhibitors. Based on drug potency, efficacy, and selectivity, determined through cell viability assays, we focused on two compounds, NB-73 and NB-115 (NB compounds), for further investigation. NB compounds potently and selectively inhibited FOXM1 with lesser effects on other FOX family members. NB compounds decreased FOXM1 expression via targeting the FOXM1 protein by promoting its proteasome-mediated degradation, and effectively suppressed FOXM1 gene targets at both the protein and mRNA level. At the cellular level, NB compounds promoted apoptotic cell death. Importantly, while inhibition of apoptosis using a pan-caspase inhibitor rescued HGSOC cells from NB compound-induced cell death, it did not rescue FOXM1 protein degradation, supporting that FOXM1 protein loss from NB compound treatment is specific and not a general consequence of cytotoxicity. Drug washout studies indicated that FOXM1 reduction was retained for at least 72 h post-treatment, suggesting that NB compounds exhibit long-lasting effects in HGSOC cells. NB compounds effectively suppressed both two-dimensional and three-dimensional HGSOC cell colony formation at sub-micromolar concentrations. Finally, NB compounds exhibited synergistic activity with carboplatin in HGSOC cells.ConclusionsNB compounds are potent, selective, and efficacious inhibitors of FOXM1 in HGSOC cells and are worthy of further investigation as HGSOC therapeutics.

Identification of MAD2L1 and BUB1B as Potential Biomarkers Associated with Progression and Prognosis of Ovarian Cancer.

Ovarian cancer develops insidiously and is frequently diagnosed at advanced stages. Screening for ovarian cancer is an effective strategy for reducing mortality. This study aimed to investigate the molecular mechanisms underlying the development of ovarian cancer and identify novel tumor biomarkers for the diagnosis and prognosis of ovarian cancer. Three databases containing gene expression profiles specific to serous ovarian cancer (GSE18520, GSE12470, and GSE26712) were acquired. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were analyzed for the differentially expressed gene (DEGs). The protein-protein interaction (PPI) network was constructed using the STRING database. The pivotal genes in the PPI network were screened using the Cytoscape software. Survival curve analysis was performed using a Kaplan-Meier Plotter. The cancer genome atlas and Gene Expression Omnibus databases were used to find the relationship between Hub gene and serous ovarian cancer. PCR and immunohistochemistry were used to detect the expression of Hub gene in serous ovarian cancer tissues and cells. Downstream pathways of the candidate tumor marker genes were predicted using Gene Set Enrichment Analysis. In this study, 252 DEGs were screened for pathway enrichment. 20 Hub genes were identified. Survival analysis suggested that Aurka, Bub1b, Cenpf, Cks1b, Kif20a, Mad2l1, Racgap1, and Ube2c were associated with the survival of patients with serous ovarian cancer. MAD2L1 and BUB1B levels were significantly different in serous ovarian cancer at different stages. Finally, Mad2l1 was found to play a role in the cell cycle, oocyte meiosis, and ubiquitin-mediated proteolysis. Meanwhile, Bub1b may play a role in the cell cycle, ubiquitin-mediated proteolysis, and spliceosome processes. Mad2l1 and Bub1b could be used as markers to predict ovarian carcinogenesis and prognosis, providing candidate targets for the diagnosis and treatment of serous ovarian cancer.

Deletion of PTP4A3 phosphatase in high grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.

The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and ERK. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3; to assess their tumorigenicity; to examine their cellular phenotype; and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/Cas9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by Western blotting in the clonal cell lines compared to the sham transfected wildtype population. The wildtype and polyclonal knockout cell lines shared similar monolayer growth rates, while the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal PTP4A3 knockout cell lines also had substantially lower in vitro colony formation efficiencies compared to the wildtype cells and were less tumorigenic in vivo The clonal knockout cells were markedly less responsive to IL-6-stimulated migration in a scratch wound assay compared to the wildtype cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the PTP4A3 deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. Significance Statement Clones of high grade serous ovarian cancer cells were isolated in which the oncogenic phosphatase PTP4A3 was deleted using CRISPR/Cas9 methodologies. The PTP4A3 null cells exhibited loss of in vitro proliferation, colony formation, and migration, and reduced in vivo tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed PTP4A3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity and cell signaling.

Abstract 952: Ex vivo drug testing identifies a potentially effective drug in 94% of cases for 403 patients in 42 tumor types

Abstract Objective: To determine whether patient derived tumor cells (PDTC) could be derived from a variety of solid tumors and used to select effective therapies, we developed a CLIA certified assay, the PARIS® test, to quantify drug sensitivities using PDTCs. Methods: Oncologists referred patients for the PARIS® test and patients had the option to consent to SEngine Precision Medicine’s IRB protocol. Samples were obtained from multiple cancer centers across the US and abroad. Patients were at all stages in their clinical journey ranging from treatment naïve to late stage, recurrent/refractory disease settings. Fresh specimens obtained from surgical resections, core biopsies, or body fluids arrived at our CLIA laboratory within 48 hrs from collection. Tumor origin and driver mutations were confirmed by IHC or DNA sequence in 88% of the cases. Results: PDTCs derived from 403 patients diagnosed with 42 different solid tumor types, including both common and rare tumors, were subjected to the PARIS® assay. An average of 67 oncology drugs (ranging from 6-307), including chemotherapies and targeted agents, were tested for each patient sample. A report ranking drug sensitivities was provided to the oncologist with an average turnaround time of less than 3 weeks. Drug sensitivity was classified as exceptional/good, moderate/low and no responses. Remarkably, 94.2% of the PDTCs exhibited selective exceptional/good responses to at least one, and typically several drugs, despite the majority of PDTCs being derived from patients with drug resistant, metastatic disease. The drug sensitivity profiles showed highly individualized responses as well as tumor type specificity. While known biomarkers/drug sensitivity associations were confirmed, many samples exhibited sensitivity to targeted agents in the absence of known biomarkers. For example, HER2+ breast cancers demonstrated sensitivity to HER2/EGFR inhibitor and a subset of luminal B and triple negative breast cancers demonstrated HER2/EGFRi responses without underlying genetic alterations. Functional testing was also useful in the rare tumor types. For example, low grade serous ovarian cancer (LGSOC) cells, differentiated from high grade by their response to specific receptor tyrosine kinase inhibitors and estrogen antagonists, but a poor response to standard of care, as is observed in the clinic. Three patients with LGSOC that were treated with the PARIS test top scoring drugs had a positive clinical response and cases from other tumor types will be presented. Conclusions: Functional testing of PDTCs from both common and rare tumor types is feasible, even for those that have failed standard of care. We provide an unprecedented view of patient-specific, actionable vulnerabilities to current oncology drugs. Personalization of cancer care will require improved access to off-label drugs. Citation Format: Rachele Rosati, Alex C. Rajevski, Payel Chatterjee, Lauren R. Appleyard, Ana Cristina Cordero Schmidt, Shalini Pereira, Robert L. Diaz, Brady Bernard, Brady Bernard, Carla Grandori, Christopher J. Kemp. Ex vivo drug testing identifies a potentially effective drug in 94% of cases for 403 patients in 42 tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 952.

Abstract 626: EGFL6 induced HER2-HER3 signaling controls accurate centrosome deposition of activated ERK and mitotic spindle formation in ovarian cancer cells

Abstract Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer (HGSOC) cells and ovarian tumor vasculature. EGFL6 acts on tumor cells to drive tumor cell proliferation and migration. To better understand EGFL6 signaling and develop therapeutic strategies to target EGFL6 signaling in HGSOC, we evaluated phosphorylation of putative EGFL6 receptors and their downstream signaling. We demonstrated that EGFL6 induced phosphorylation and activation of ERBB/HER family of receptors; EGFL6 treatment of ovarian cancer cells led to rapid and transient phosphorylation of EGFR while inducing prolonged activation of both HER2 and HER3, with subsequent phosphorylation of ERK. Interestingly, we found that in EGFL6 stimulated ovarian cancer cells undergoing mitosis, pERK localized to the centrosome and the contractile ring. EGFL6 neutralizing antibodies reduce ERK phosphorylation and resulted in pERK being aberrantly localized. This resulted in altered mitotic spindle alignment and an increased number of cells undergoing mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro. Consistent with these findings, dual anti-EGFL6 and neratinib therapy significantly restricted tumor growth in vivo, resulting in increased tumor cell death and a reduction in angiogenesis. Combined our data identify an unexplored role for pERK at the centrosome and suggest that dual targeting of the EGFL6/HER signaling axis maybe an effective therapeutic strategy in ovarian cancer. Citation Format: Shoumei Bai, Navneet Gupta, Qi Jiang, Ronald J. Buckanovich. EGFL6 induced HER2-HER3 signaling controls accurate centrosome deposition of activated ERK and mitotic spindle formation in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 626.

Abstract 6320: CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant ovarian cancer

Abstract Ovarian cancer is the second leading cause of cancer-related mortality among gynaecological cancers in the Western world. CAR-T cell therapy is an adoptive immunotherapy used to treat some blood cancers. This study has investigated whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a suitable target for CAR-T cell therapy for ovarian cancer. We assessed the cytotoxic effects of LGR5-targeting CAR-T cells on a range of ovarian cancer cell lines and primary serous ovarian cancer cells derived from patient ascites (at diagnosis and following relapse with chemotherapy resistant disease) in monolayer culture, 3D spheroid assays and patient-derived explant assays. OVCAR3 and a chemoresistant patient-derived xenograft (PDX) models have been used to assess whether LGR5-targeting CAR-T cells can inhibit growth and metastasis in vivo. We found that LGR5 is highly expressed in high-grade serous ovarian carcinoma (HGSOC) cancer cell lines and LGR5 expression was increased in chemotherapy resistant primary ovarian cancer cells. We demonstrated that LGR5-targeting CAR-T cells were cytotoxic and significantly inhibited the survival of ovarian cancer cell lines and chemotherapy-resistant primary ovarian cancer cells with high LGR5 expression levels in both monolayer culture and 3D spheroids compared to un-transduced CD3+ T cells. In patient-derived explant assays, we observed increased apoptosis in HGSOC tissues expressing high LGR5 following treatment with LGR5-targeting CAR-T cells. In vivo, LGR5-targeting CAR-T cell treatment decreased the tumour burden in mice with OVCAR3 xenografts and a chemotherapy-resistant PDX model with high LGR5 expression, compared to CD3+ T cells. In summary, LGR5-targeting CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer. Citation Format: Wanqi (Jady) Wang, Veronika Bandara, Noor A. Lokman, Zoe K K. Price, Tannith M. Noye, Silvana Napoli, Batjargal Gundsambuu, Martin K. Oehler, Simon C. Barry, Carmela Ricciardelli. CAR-T cells targeting LGR5: An effective treatment for chemotherapy resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6320.

Abstract 1556: AMIGO2 is associated with the peritoneal dissemination in serous ovarian cancer by enhancing its adhesion to peritoneal mesothelial cells

Abstract Amphoterin-induced gene and open reading frame 2 (AMIGO2) has been associated with the prognosis of colorectal, gastric, and cervical cancer. However, its relationship with ovarian cancer remains unclear. This study aimed to clarify the role of AMIGO2 in ovarian cancer. The research involved evaluating AMIGO2 expression in patients with ovarian serous carcinoma using immunohistochemistry. The AMIGO2-high group exhibited significantly shorter progression-free survival (PFS) compared to the AMIGO2-low group. The predictive index of the AMIGO2-high group was considerably higher than that of the AMIGO2-low group. The rate of complete cytoreductive surgery was lower in the AMIGO2-high group than in the AMIGO2-low group. Furthermore, in vitro studies showed that four serous ovarian cancer cell lines expressed AMIGO2 expression and adhesion to mesothelial cells. Adhesion to mesothelial cells was attenuated by AMIGO2 knockdown in SKOV3 cells. Moreover, in a murine model, AMIGO2 downregulation in SKOV3 cells significantly suppressed peritoneal dissemination. These findings suggest that high AMIGO2 expression in serous ovarian carcinoma cells contributes to a poor prognosis by promoting peritoneal metastasis through enhanced cell adhesion to mesothelial cells. Citation Format: Yuki Iida, Shinya Sato, Koji Yamamoto, Kohei Hikino, Masayo Okawa, Masayo Hosokawa, Mayumi Sawada, Hiroaki Komatsu, Akiko Kudoh, Hee Kyung Seong, Runa Izutsu, Mitsuhiko Osaki, Futoshi Okada, Fuminori Taniguchi. AMIGO2 is associated with the peritoneal dissemination in serous ovarian cancer by enhancing its adhesion to peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1556.

Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 3 Expression and Its Correlation with Prognosis and Growth of Serous Ovarian Cancer: Correlation of DYRK3 with Ovarian Cancer Survival.

Epithelial ovarian cancer, primarily serous ovarian cancer (SOC), stands as a predominant cause of cancer-related mortality among women globally, emphasizing the urgent need for comprehensive research into its molecular underpinnings. Within this context, the dual-specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3) has emerged as a potential key player with implications for prognosis and tumor progression. This study conducted a meticulous retrospective analysis of 254 SOC cases from our medical center to unravel the prognostic significance of DYRK3. Survival analyses underscored DYRK3 as an independent adverse prognostic factor in SOC, with a hazard ratio of 2.60 (95% CI 1.67-4.07, P < 0.001). Experimental investigations involved DYRK3 knockdown in serous ovarian cancer cell lines (CAOV3 and OVCAR-3) through a shRNA strategy, revealing substantial decreases in cell growth and invasion capabilities. Bioinformatics analyses further hinted at DYRK3's involvement in modulating the tumor immune microenvironment. In vivo experiments with DYRK3-knockdown cell lines validated these findings, demonstrating a notable restriction in the growth of ovarian cancer xenografts. Our findings collectively illuminate DYRK3 as a pivotal tumor-promoting oncogene in SOC. Beyond its adverse prognostic implications, DYRK3 knockdown exhibited promising therapeutic potential by impeding cancer progression and potentially influencing the tumor immune microenvironment. This study establishes a compelling foundation for further research into DYRK3's intricate role and therapeutic potential in ovarian cancer treatment. As we unravel the complexities surrounding DYRK3, our work not only contributes to the understanding of SOC pathogenesis but also unveils new prospects for targeted therapeutic interventions, holding promise for improved outcomes in ovarian cancer management.

Plasma membrane SK2 channel activity regulates migration and chemosensitivity of high-grade serous ovarian cancer cells.

No data are currently available on the functional role of small conductance Ca2+-activated K+ channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non-cell-permeant SK2 inhibitor Lei-Dab7, we identified functional SK2 channels at the plasma membrane, regulating store-operated Ca2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei-Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei-Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro-migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression-free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.

Abstract A110: Targeting methionine metabolism inhibits ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of gynecologic cancer death. Platinum (Pt)- based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to disease recurrence. In the current study, we demonstrated that platinum drugs can enrich for OCSCs. In high grade serous ovarian cancer (HGSOC) cell lines, cisplatin treatment (IC50 for 16hrs) increased the percentage of cells with high aldehyde dehydrogenase (ALDH+) activity, a known OCSCs marker, and altered methionine (Met) metabolism. Extracellular Met uptake is converted to S-adenosyl-methionine (SAM) by methionine adenosyltransferase 2A (MAT2A), the rate-limiting enzyme of Met utilization. Short-term cisplatin treatment decreased SAM level and increased expression of MAT2A. It has been reported that CSCs have a high-flux metabolic state and are dependent on Met metabolism. We observed higher MAT2A expression in ALDH+ cells compared to the whole cell population, and Met depletion blocked the cisplatin-induced increase in %ALDH+ cells and spheroid formation ability. Furthermore, both MAT2A siRNA knockdown and the MAT2A inhibitor FIDAS-5 prevented cisplatin-induced increase in %ALDH+ cells, suggesting that blocking Met metabolism phenotypically inhibited the platinum enrichment of OCSCs. MAT2A inhibition also downregulated Cyclin E and arrested HGSOC cells in G1/S phase, indicating that a lower level of Met utilization re-established G1/S checkpoint in p53-mutant OC. As SAM is the universal methyl-group donor, it was of interest to examine whether the observed cisplatin-induced alterations in SAM level resulted in changes in DNA methylation. Methylation specific-PCR analysis demonstrated that siMAT2A or FIDAS-5 treatment of HGSOC cells inhibited cisplatin-induced hypermethylation of CpG islands of BRCA1 and ALDH1L1, a folate metabolic enzyme of which promoter hypermethylation is associated to tumor progression. Collectively, these results demonstrated a crucial role of Met metabolism in cisplatin-induced OCSCs enrichment. MAT2A has been recognized as a therapeutic target for the treatment of solid tumors and Met inhibition by targeting MAT2A could represent a therapeutic approach for inhibiting CSC in ovarian cancer. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methionine metabolism inhibits ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A110.

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response

Abstract B012: Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response