Abstract 4418: Autophagy induction contributes to cisplatin resistance in ovarian cancer and is independent of AKT pathway activity
Authors: Kipps Emma L. , Walton Mike I. , Banerji Udai , Kaye Stan B. , Garrett Michelle D. 
Published: August 2015

Abstract
Advanced ovarian cancer eventually becomes resistant to platinum based chemotherapy. We studied mechanisms of platinum resistance using two paired ovarian cancer cell lines: A2780; A2780Cis and PE01; PE04 (relative resistance 5.2 & 3.6 respectively by 96h SRB assay). While cisplatin dependent DNA damage and repair govern initial disease sensitivity, adaptation to stress by upregulation of pro-survival mechanisms, such as autophagy, may underpin resistance. Analysis of A2780Cis and PE04 platinum resistant cells by western blot showed a significant increase in expression of autophagy proteins LC3BII and Beclin (P&lt;0.05, n = 4). Cisplatin treatment caused a PepA/E64D dependent increase in LC3BII expression in A2780 and A2780Cis cell lines demonstrating an increase in autophagic flux. Furthermore the use of two autophagy inhibitors, 3-MA and hydroxychloroquine, enhanced the cytotoxicity of cisplatin when used in combination (p&lt;0.05 & p&lt;0.01 respectively, n = 3, SRB). These data demonstrate autophagy is upregulated by cisplatin as a pro-survival mechanism in two ovarian cancer cell lines and platinum sensitivity can be partially restored through the use of autophagy inhibitors. AKT has been implicated in platinum resistance and upregulation of AKT has been reported to negatively regulate autophagy. We therefore compared the status of the AKT signalling pathway in the A2780 and A2780Cis cells. Phosphorylation of S473 AKT and its direct substrates, GSK-3β and FOX01, were significantly increased in platinum resistant A2780Cis cells (p&lt;0.05, p&lt;0.05 & p&lt;0.01, respectively, n≤3). This is consistent with upregulation of the AKT pathway and increased survival through inhibition of FOX01 dependent apoptosis. Despite this, the phosphorylation status of AKT substrates PRAS40 and TSC2 (that regulate mTOR) were unchanged in A2780Cis cells suggesting no apparent AKT-driven upregulation of mTOR activity. Moreover, expression of the mTOR substrates S6RP, p70S6K and 4E-BP1 were decreased in A2780Cis cells when compared to the parental A2780 cells (p&lt;0.001, p&lt;0.05 & p&lt;0.001, respectively, n≤3). In addition, ULK1, a substrate of mTOR, which negatively regulates autophagy when phosphorylated on S757, was hypophosphorylated at S757 in A2780Cis cells (p&lt;0.05, n = 3) consistent with downregulation of mTOR activity and consequent upregulation of autophagy function. In conclusion we present a novel relationship between autophagy and AKT and show that differential activation of the AKT substrates which control mTOR, has resulted in the upregulation of autophagy as a pro-survival mechanism, despite the active AKT status. This likely provides cells with an increased ability to cope with cisplatin mediated cellular damage. Our study reveals a novel insight into the regulation of autophagy and provides a rationale for the use of autophagy inhibitors in the treatment of platinum resistant ovarian cancer.
Citation Format: Emma L. Kipps, Mike I. Walton, Udai Banerji, Stan B. Kaye, Michelle D. Garrett. Autophagy induction contributes to cisplatin resistance in ovarian cancer and is independent of AKT pathway activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4418. doi:10.1158/1538-7445.AM2015-4418