Abstract 4845: TONDU, a transcription factor that regulates prosurvival autophagy and the development of antiestrogen resistance in breast cancer

Abstract

In recent studies, we have demonstrated that autophagy is essential for the survival of estrogen receptor positive (ER+) breast cancer cells during anti-estrogen therapy and facilitates the development of antiestrogen resistance. For these studies, we subjected ER+ MCF-7 cells to a step-wise drug selection protocol, with 4 hydroxytamoxifen (4-OHT) as the selecting drug and established an antiestrogen resistant subline, designated TR5 (4-OHT resistant to 5μM). TR5 cells, unlike the parent MCF-7 cells, do not die in response to 4-OHT treatment and tolerate high levels of antiestrogen-induced autophagosome formation. Microarray analysis of 4-OHT-treated TR5 cells shows an approximate 5-10 -fold increase in TONDU (TDU) mRNA compared to that of the parent MCF-7 cells. TDU, also known as vestigial like gene-1, was initially identified as a transcription factor required for wing development in Drosophilia, but specific roles for TDU in mammalian cells have not been identified. Based on the upregulation of TDU in antiestrogen-resistant TR5 cells, we hypothesized that TDU played a role in pro-survival autophagy. To test this hypothesis, we analyzed the levels of TDU in cultured MCF-7 and TR5 cells under conditions that induce autophagy and further determined how modulation of TDU levels (TDU cDNA overexpression and TDU attenuation by RNAi targeting) affected autophagy and death. 4-OHT and rapamycin, an mTOR inhibitor that induces macroautophagy, increased TDU expression within 24 hours of treatment and increased TDU expression persisted for 96 hours. Further, quantitative Real-Time (RT) PCR analysis of cells overexpressing TDU revealed that TDU upregulated the transcription of autophagy genes (i.e. upregulation of LC3, Atg5 and Atg6). Importantly, the TDU-mediated increased expression of autophagy genes was blocked by TDU RNAi. Consistent with this apparent TDU-induced autophagy, TDU overexpression significantly increased autophagic proteolysis (long-lived protein turnover). In addition, down regulation of TDU by RNAi induced cell death in E2- and 4-OHT-treated MCF-7 and TR5 cells, with increased mitochondrial membrane permeability, cleavage of PARP, and cleavage of lamin-A as read-outs of cell death. To our knowledge, these studies uniquely identify TDU as a transcription factor involved in the regulation of autophagy in ER+ breast cancer cells and as a potential molecular target to block pro-survival autophagy and enhance the cytotoxicity of antiestrogen therapy. Our current studies are aimed at determining whether TDU expression levels can serve as a prognostic indicator of breast cancer response to hormonal therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4845.