Abstract B75: Increased basal autophagy decreases sensitivity to cisplatin in chemoresistant ovarian cancer.
Authors: Silvers Thomas R. , Dottino Peter R. , Martignetti John A. , Evans Brad R. 
Published: May 2016

Ovarian cancer (OvCa) is the 5th leading cause of cancer-related mortalities among women in the US. The majority of patients diagnosed with OvCa are initially responsive to first-line chemotherapy; however, 75% will suffer a recurrence characterized by progressive drug resistance. Overcoming chemoresistance is a major obstacle in treating patients with OvCa. Autophagy is a conserved biological process involved in multiple hematologic and solid malignancies, including OvCa, and is a potential target for overcoming chemoresistance. Autophagy has a context-dependent role in cancer, where, initially, it offers a protective role by preventing the initiation of tumorigenesis and, subsequently, is used by the tumor for survival. A number of chemotherapeutics, including cisplatin, are potent inducers of autophagy; taken together, these indicate a significant role for autophagy in cancer. The role of autophagy in OvCa is additionally complex, with increased levels of major autophagic components associated with contradictory outcomes: increased levels of Beclin-1 and LC3-II correlate with low grade and increased survival, yet increased levels of p62 in the cytoplasm correlate with advanced stage and decreased survival. Further, inhibition of autophagy by pharmacologic treatment or knockdown of key autophagic components induces OvCa cell death and re-sensitizes CP-resistant cells to cisplatin. These results indicate that autophagy has a nonobvious role in chemoresistance, which may be distinguished by increased basal autophagy and/or a more acute autophagic response. In the present study, we investigated whether chemoresistant OvCa cells are more susceptible to autophagy modulation relative to sensitive cells, and if this increased sensitivity was due to higher levels of basal autophagy occurring in resistant cells or due to a more robust autophagic response.

First, to evaluate the effect of autophagy on chemosensitivity, MTTs were performed with cisplatin and in combination with autophagy activation or inhibition in two pairs of isogenic cisplatin (CP)-resistant OvCa cell lines: A2780 and CP70; OVCAR-3 and CP_OVCAR-3, an isogenic line developed using intermittent exposure to cisplatin [IC20] over 180 days. Inhibition of autophagy enhanced CP sensitivity in both pairs of cell lines, though resistant cells were more sensitive to autophagic inhibition with a greater increase in cisplatin sensitivity. Conversely, autophagy induction decreased sensitivity to cisplatin in the sensitive lines though not in the resistant lines. Beclin-1 and p62 protein were increased in resistant lines relative to the parental lines. Next, we measured autophagic flux to compare levels of basal autophagy. Increased LC3 turnover and p62 degradation in resistant lines indicated increased basal autophagic flux in resistant lines. Autophagy induction was measured temporally to determine the maximal turnover rate and earliest time of response. Resistant lines had no difference in interval between treatment and start of autophagy after treatment, but did reach a greater level of autophagy flux compared to sensitive lines. These results indicate there is an increase in basal autophagy rather than an increase in response in resistant cells relative to sensitive cells, which in turn contributes to a greater level of autophagy achieved by resistant cells. Finally, we investigated the role of autophagy inhibition in OvCa patient-derived cell lines from our biobank of 400+ cell lines that span CP sensitivities. There was a synergistic cytotoxic response with co-treatment of cisplatin and autophagy inhibition, and, as with the isogenic models of CP-resistance, patient-derived lines that were less sensitive to cisplatin were more susceptible to autophagy inhibition. We are currently examining the basal levels of autophagy in this cohort. These results indicate that inhibition of autophagy is a promising strategy to overcome chemoresistance in OvCa patients.

Citation Format: Thomas R. Silvers, Peter R. Dottino, John A. Martignetti, Brad R. Evans. Increased basal autophagy decreases sensitivity to cisplatin in chemoresistant ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B75.