Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. Implications: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.

Background: Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown.Results: To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR.Conclusions: This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.

Gynecologic malignancies, including ovarian cancer, endometrial cancer, and cervical cancer, affect hundreds of thousands of women worldwide every year. Wnt signaling, specifically Wnt/β-catenin signaling, has been found to play an essential role in many oncogenic processes in gynecologic malignancies, including tumorigenesis, metastasis, recurrence, and chemotherapy resistance. As such, the Wnt/β-catenin signaling pathway has the potential to be a target for effective treatment, improving patient outcomes. In this review, we discuss the evidence supporting the importance of the Wnt signaling pathways in the development, progression, and treatment of gynecologic malignancies.

High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. Implications: This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

Identifying novel, durable treatments for high-grade serous ovarian cancer (HGSOC) is paramount to extend both progression-free survival (PFS) and overall survival (OS) in patients afflicted with this disease. Dual-specificity phosphatase 1 (DUSP1) was identified as one of seven genes that may significantly affect prognosis in patients with HGSOC; however, the role of DUSP inhibition (DUSPi) in the treatment of HGSOC remains largely unknown. In this study, we show that DUSP1 is highly expressed in HGSOC and confers worse PFS and OS. Further, we corroborate data that show DUSP1 expression is directly associated with therapy resistance. Using a tissue microarray of 137 different serous ovarian carcinomas, we demonstrate the high expression of DUSP1 in primary and recurrent serous ovarian cancer. In both acquired and de novo therapy HGSOC-resistant models, DUSPi both inhibited cellular proliferation and promoted cell death. RPPA analysis of HGSOC cells revealed DUSPi led to the differential regulation of several pathways, including AMPK and mTORC. Further, in a patient-derived xenograft HGSOC model, DUSPi significantly inhibited tumor progression.