Tumor-Derived Exosomes Mediate Platinum Resistance in Ovarian Cancer Through Regulation of EMT

Abstract

Nano-sized vesicles, termed exosomes, have been implicated in the transfer of oncogenic proteins and genetic material from one cell to another. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we have utilized the platinum-sensitive A2780 cell line and independent platinum-resistant derivatives, e.g., CP70 and C30, as well as a non-related platinum-resistant cell line, OVCAR10. We find A2780 cells treated with exosomes derived from highly resistant cells demonstrate up to a ~2-fold increase (p<0.05) in resistance to carboplatin as compared to treatment with isolated autologous exosomes. Importantly, this exosome-associated phenotype is stable and associated with increased epithelial to mesenchymal transition (EMT) characteristics. In addition, we identified previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4), only in cells (OVCAR10, C30, and CP70) that demonstrated robust acquired resistance after platinum therapy. Cells displaying mutations in SMAD4 exhibited significant changes in EMT-related markers following treatment with carboplatin. Interestingly, exosomes derived from A2780 cells engineered to exogenously express specific SMAD4 mutations resulted in ~1.7-fold (p<0.05) increase in resistance as compared to exosomes isolated from exogenous wildtype SMAD4 (SMAD4WT) expressing A2780 cells, suggesting these mutations are contributing to the development of a resistant phenotype. Additionally, cells expressing mutations in SMAD4 exhibit a loss of phosphorylation of SMAD2 but retain activated SMAD3, which is important for EMT. Importantly, inhibition of SMAD3 via the small molecule inhibitor, SIS3, reversed the EMT phenotype and acted synergistically with carboplatin to enhance cell death. Lastly, we identified a clinically relevant inhibitor of SMAD3, Eribulin Mesylate (Halaven®), which also acted synergistically with carboplatin in vitro. Altogether, our findings provide the first evidence that ovarian tumor cells use exosomes as a vehicle to achieve tumor cell-cell crosstalk and this exchange advantageously impacts the recipient cells response to platinum. We continue to describe a novel mechanism of action by which an EMT phenotype is perpetuated via exchange of tumor-derived exosomes, ultimately leading to the development of a subpopulation of chemotherapy refractory cells. Importantly, we present a novel therapeutic strategy that targets resistant cells and ‘tricks’ them into responding once again to the most effective therapeutics to date, cisplatin and carboplatin.