Ovarian cancer (OVCA) is the most lethal gynecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as targeted therapies and immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long term therapeutic success. Therefore, there is an urgent need to understand the cellular and molecular mechanisms of immune dysregulation in chemoresistant ovarian cancer in order to harness the host’s immune system to improve cancer survival. Early diagnosis and residual disease are key determinants of favorable survival in OVCA; however, CA125 which is the conventional marker is not reliable and has modest diagnostic accuracy. There is therefore an urgent need to discover reliable biomarkers to optimize individualized treatment and diagnostic recommendations. Plasma gelsolin (pGSN; an actin binding protein) is the secreted isoform of the gelsolin (GSN) gene implicated in inflammatory disorders, colon cancer and prostate cancer. Increased expression of total GSN is associated with poor survival of patients with gynecological cancers. As to whether this is due to pGSN is yet to be investigated. Increased expression of pGSN is significantly associated with the down-regulation of immune cell markers; however, the exact mechanism has not been explored. If and how pGSN is involved in the cellular and molecular mechanisms of OVCA remains to be determined. In our current research, we have demonstrated that pGSN is involved in the regulation of immune cells, early diagnosis, tumor recurrence and chemoresistance in OVCA, using standard in vitro techniques and human clinical samples (North America, Asia and public datasets). We have shown that pGSN is highly expressed and secreted in chemoresistant OVCA cells than their chemosensitive counterparts. pGSN, secreted and transported via exosomes, upregulated HIF1α–mediated pGSN expression in chemoresistant OVCA cells in an autocrine manner as well as conferred cisplatin resistance in otherwise chemosensitive OVCA cells. pGSN also induced the OVCA expression of the antioxidant and tumor growth promoter, glutathione (GSH), by activating Nuclear factor erythroid 2-related factor 2 (NRF2), a response that attenuated cisplatin (CDDP)-induced apoptosis. In human tumor tissues, increased pGSN mRNA and protein expressions were significantly associated with advanced tumor stage, suboptimal residual disease, tumor recurrence, chemoresistance and poor survival regardless of patients’ ethnic background and histologic subtypes. Increased Infiltration of CD8+ T cells was significantly associated with favorable patient survival; however, increased pGSN hindered the survival impact of these infiltrated CD8+ T cells. Further investigation revealed that pGSN induced CD8+ T cell death via caspase-3 activation, an action that resulted in decreased IFNγ levels. Increased epithelial pGSN expression was significantly associated with reduced survival benefits of infiltrated M1 macrophages, through caspase-3-dependent apoptosis as well as reduced production of TNFα and iNOS. The clinical application of circulatory pGSN as a biomarker for early detection and patients’ survival was investigated. Pre-operative circulating pGSN presented as a favorable and independent biomarker for early disease detection and residual disease prediction compared with CA125. The test accuracy of pGSN was significantly enhanced when combined with CA125 in multianalyte index assay. The findings suggest that pGSN is a potential target for chemoresistant OVCA and presents as a diagnostic marker for early stage disease and surgical outcomes, interventions that could maximize the therapeutic success of immunotherapies.

Ovarian cancer (OVCA) is the most lethal gynecologic cancer. Chemoresistance remains a major hurdle to successful therapy and patient survival. The secreted isoform of the actin-associated protein, gelsolin (plasma gelsolin; pGSN), is highly expressed in chemoresistant than chemosensitive OVCA cells, although the mechanism underlying the differential expression is not known. Also, its overexpression significantly correlates with shortened survival of OVCA patients. DNA methylation plays a key role in the regulation of genes expression and contributing to cancer development and chemoresistance with the help of DNA methyltransferases (DNMTs) or Ten eleven translocation (TETs) enzymes. TET1 is the most studied isoform of TETs family and primarily responsible for 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) oxidation to initiate demethylation and increase in the expression of methylated genes. Whether pGSN expression in OVCA cells is regulated by DNA methylation and TET1 regulates the differential pGSN expression between chemosensitive and resistant OVCA cells is not known. In this study, we hypothesized pGSN overexpression in chemoresistant OVCA cells is due to the hypomethylation at its promoter region by TET1. Our objective was to investigate whether DNA methylation and specifically TET1 plays a role in the regulation of differential pGSN expression and chemosensitivity in OVCA. Chemosensitive and resistant OVCA cell lines of different histological subtypes were used in this study to measure pGSN and TET1 mRNA abundance and protein contents by qPCR and Western blotting respectively. Cisplatin-induced chemoresponsiveness was morphologically assessed by Hoechst staining (apoptosis). Infinium HumanMethylation450 BeadChip assay was used for global methylation analysis of twelve (12) different OVCA cells and to investigate the role of DNA methylation specifically in pGSN regulation and pGSN-induced chemoresistance. DNMTs and TETs were pharmacologically inhibited in sensitive and resistant OVCA cell using specific inhibitors. Gain-and-loss-of-function assays were carried to identify the relationship between TET1 and pGSN in OVCA chemoresponsiveness. Differential protein and mRNA expressions of pGSN and TET1 were observed between sensitive and resistant OVCA cells and cisplatin reduced their expression in sensitive but not in resistant cells. Global methylation analysis revealed hypomethylation in resistant cells compared to sensitive cells. Pharmacological inhibition of DNMTs increased pGSN protein levels in sensitive OVCA cells and decreases their responsiveness to cisplatin, however we did not observe any difference in methylation level at pGSN promoter region. TETs inhibition resulted in hypermethylation at multiple CpG sites and decreased pGSN protein level in resistant OVCA cells which was also associated with enhanced response to cisplatin, findings that suggested the methylation role of TETs in the regulation of pGSN expression in OVCA cells. Further, we found that TET1 is inversely related to pGSN and positively related to chemoresponsiveness of OVCA cells. This project does not only broaden our knowledge about the mechanistic insights into the epigenetic regulation of pGSN in OVCA chemoresistance, but it also reveals a new potential target to re-sensitize chemotherapy resistant OVCA cells. This may provide a future strategy to improve overall OVCA patient survival.