Abstract 4110: Measurement of autophagy in sensitive versus resistant breast cancer cells

Abstract

Abstract In 2011, there will be almost 192,000 newly diagnosed cases of invasive breast cancer in the U.S. with estrogen receptor positive ≤ positive (ER+) tumors. For these patients, endocrine therapy, administered as an antiestrogen, e.g., Tamoxifen (TAM) or Faslodex (Fulvestrant; ICI 182,780) or an aromatase inhibitor (AI), is the least toxic and most effective means to manage their hormone-dependent breast cancer. Tamoxifen (TAM) produces a 26% proportional reduction in mortality, however, advanced ER+ breast cancer largely remains an incurable disease and resistance to endocrine therapy remains a significant clinical problem. We and others have shown that antiestrogen resistant breast cancer cells have higher level of basal autophagy, a process that allows the cells to survive stress by digesting its own organelles. However, the precise role of this sustained autophagy in antiestrogen resistance is unclear and the measurement of autophagy flux, the dynamic measurement of passage of substrates through the autophagic pathway, is essential to determine levels of autophagy in a cell. In this study, we investigate the rate of autophagosome formation and degradation in antiestrogen sensitive versus antiestrogen resistant breast cancer cells. Autophagosome formation was measured using autophagosome specific fluorescent dye incorporation (Cyto-IDTM). Progression of autophagy was indicated by the degradation SQSTM1/p62 and NBR1 in cell lysates as measured by colorimentric enzyme immunoassays (ELISAs). The ELISAs were validated to provide biologically relevant sensitivity, dilutional linearity, reproducibility and recovery in native samples. Cells further progressing into apoptosis and cell death were identified by fluorescence using a combination of yellow emitting dye labeled Annexin V and a red emitting viability dye (GFP-Certified™Apoptosis/Necrosis detection kit). We hypothesize that combination of sustained autophagosome formation and inhibition of degradation are crucial in promoting pro-survival autophagy in antiestrogen resistant breast cancer cells. We show that while autophagosome formation and concurrent degradation of SQSTM1/p62 and NBR1 leads to cell death in sensitive cells, resistant cells have higher levels of autophagosomes, lower levels of degradation of SQSTM1/p62 and NBR1 and decreased cell death. Our data will highlight the importance of determining autophagy flux in pro-death versus pro-survival autophagy in drug resistance in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4110. doi:1538-7445.AM2012-4110