Abstract 2334: Examining the contribution of autophagy to TRAIL resistance in an <i>in vitro</i> model of circulating breast cancer cells
Authors: Julianne D. Twomey, Baolin Zhang
Journal: Cancer Research (2017)

Abstract Circulating tumor cells (CTCs) are cancer cells which dissociate from the primary tumor and circulate within the peripheral blood, initiating metastasis at a distant location. CTCs present a “non-invasive, liquid biopsy”, making them attractive targets for the development of cancer biomarkers and therapies. In order to form metastases, these cells must undergo a phenotypic shift acquiring the ability to survive anoikis within the blood stream and evade immune surveillance involving death events mediated by death receptors (DRs) expressed on the surface of cancer cells. Under physiological conditions, these DRs can be activated by circulating cytokines such as TNF, Fas ligand, and TNF-related apoptosis inducing ligand (TRAIL), triggering cell death. In vitro studies have demonstrated that environmental stress caused by non-adherent suspension culture conditions, similar to circulation, will increase autophagy in cancer cell lines aiding anoikis survival. Our lab has previously identified a link between increased autophagic rates and decreased DR surface expression. Based on this we were interested in determining whether increased autophagy due to non-adherent conditions caused breast cancer cell lines to decrease death receptor expression, evading immune surveillance and resisting death-ligand induced apoptosis. To this end, breast cancer cell (BCC) lines were cultured in monolayer or a non-adherent suspension condition for 7 days. Following 7 days of culture, death receptor expression and autophagy initiation were measured using immunoblot and flow cytometry microscopy. BCCs were treated with TRAIL and analyzed over 24 hours for apoptosis and caspase activation. We found that the BCC lines decreased total and surface expression of DR5 and total expression of TNFR1 over the 7 days of suspension condition. The reduced expression of DR5 in suspension cultured BCCs resulted in a delayed response to TRAIL mediated apoptosis, with delayed caspase activation and PARP cleavage compared to monolayer cultured parental cells. Under suspension condition, autophagy was initiated by increased LC3-II/ LC3-I turnover and p62 degradation. This initiation of autophagy is potentially due to increased expression of the mitochondrial related proteins BNIP3 and BNIP3L, which have been implicated in anoikis survival. This work warrants additional studies investigating the therapeutic potential of targeting CTCs within the blood to limit metastatic spread and disease progression in breast cancer. Our findings suggest the potential to sensitize CTCs to anoikis and circulating death ligands through autophagy inhibition. Citation Format: Julianne D. Twomey, Baolin Zhang. Examining the contribution of autophagy to TRAIL resistance in an in vitro model of circulating breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2017-2334