Introduction In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1. Methods IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors. Results IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER + breast cancer cells. Conclusion his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER + breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.

In recent studies, we and others showed that autophagy is one of the prosurvival mechanisms during anti-estrogen therapy that plays a significant role in the development of resistance. In this study, we sought to determine if autophagy plays a role in the protective effects of insulin like growth factor 1 (IGF-1)/MEK1 signaling on breast cancer cells treated with hormonal therapy. In initial studies, we established that IGF1 protects estrogen receptor-positive (ER+) breast cancer cells from cell death induced by the anti-estrogen 4-hydroxytamoxifen (4-OHT) and the anti-progestin mifepristone (MIF) and that this protection was mediated by MEK1 signaling. To determine if the mechanism of MEK1-mediated protection involved modulation of autophagy, ER+ breast cancer cells alone or transfected with MEK1-GFP or MEK1 dominant negative (MEK1-DN) expression vectors were treated with 4-OHT and/or MIF, in the presence or absence of IGF1. Effects on autophagy were monitored using immunoblotting with antibodies to key autophagy proteins, electron microscopy, GFP-LC3 reporter-based expression analysis, quantitative PCR, and autophagic proteolysis (turnover of long-lived proteins). Apoptosis was monitored with the JC-1 assay (mitochondrial membrane permeability determination), trypan blue exclusion, cytokeratin 18 cleavage assay, and determination of the levels of cleaved PARP and lamin A. The results showed that IGF1 significantly increased the levels of several autophagy genes, including Atg8, Atg6, and Atg5. However, even with the increased levels of these autophagy proteins, IGF-1 was shown to clearly suppress hormonally-induced long-lived protein turnover or macroautophagy. Thus, the levels of key autophagy proteins were increased, while the catabolic function of the mature autolysosomes was reduced. MEK1 overexpression also significantly protected breast cancer cells from death, while MEK1DN expression significantly enhanced 4-OHT and/or MIF induced apoptotic cell death. Our interpretation of the concomitant attenuation of macroautophagy and cell death by IGF1/MEK signaling is not that autophagy is a prerequisite to cell death, but that autophagic catabolism is a readout of the significant stress experienced by MEK1 inhibition in breast cancer cells. Based on our results, we are now addressing three key questions: (1) will blockade of autophagy enhance the cytotoxicity of MEK1 inhibitors; (2) is the elevated expression of autophagy genes in IGF-1-treated breast cancer cells mediated by MEK1 signaling; and (3) is mitophagy enhanced in breast cancer cells overexpressing MEK1. This knowledge will be used to support the proposed use of MEK1 blockade to enhance the cytotoxic action of hormonal therapy and to circumvent the occurrence of anti-estrogen resistance of breast cancer, particularly in patients with high circulating levels of IGF-1. 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 4846.

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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.

Paclitaxel functions by preventing microtubule degradation, leading to mitotic arrest and apoptotic death. Of particular interest, paclitaxel-induced death in breast cancer cells is dependent, in part, on the levels of BimEL, a pro-apoptotic member of the Bcl-2 family of proteins. In addition, our recent studies demonstrated that BimEL is required for 4-hydroxytamoxifen-induced apoptosis of estrogen receptor positive (ER+) MCF-7 breast cancer cells [Breast Cancer Res. 2012 Mar 19;14(2):R52]. In contrast, we demonstrated low-level BimEL expression in ER+ T47D breast cancer cells that do not undergo antiestrogen-induced apoptosis. Thus, low-level BimEL expression in ER+ breast cancer may predict a poor apoptotic threshold which ultimately would facilitate the development of acqured resistance to paclitaxel, as well as antiestrogen therapy. Based on the ability of HDAC inhibitors to increase the transcription of pro-apoptotic genes, we hypothesized that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would increase BimEL expression in T47D breast cancer cells and induce a robust apoptotic response to paclitaxel chemotherapy and antiestrogen treatment. In this study, we now demonstrate that SAHA does significantly up-regulate BimEL expression in T47D cells, as well as in MCF-7 cells. Concomitant with BimEL upregulation, SAHA sensitizes T47D and MCF-7 cells to paclitaxel-induced apoptosis. Similarly, SAHA sensitizes T-47D cells to antiestrogen-induced apoptosis, while augmenting the level of antiestrogen-induced apoptosis in MCF-7 cells. These studies indicate that the pro-apoptotic protein BimEL is required for SAHA-induced sensitization of breast cancer cells to paclitaxel and/or antiestrogen-induced apoptosis. Currently, siRNA studies are being conducted to determine if BimEL is a key death effector in response to SAHA treatment and if the increased death from SAHA and paclitaxel or SAHA and antiestrogens is synergistic or additive. Our results provide strong support for the use of HDAC inhibitors when designing novel combination therapies to reduce the emergence of acquired resistance in breast cancer cells undergoing chemo- or antihormonal therapy. Acknowledgement: this work was supported by teh MCG foundation and NIHRO1 CA121438 to P.V.S. Citation Format: Aric Berning, Alexander Eason, Nathan Gilley, Suchreet Takhar, Sally ElShafey, Muthusamy Thangaraju, Patricia V. Schoenlein. HDAC inhibition induces Bim expression and apoptosis in breast cancer cells undergoing paclitaxel or antiestrogen treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2013-1725