Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis Anatasha C. Crawford, Rebecca B. Riggins, Ayesha N. Shajahan, Alan Zwart, Robert Clarke* Lombardi Comprehensive Cancer Center and Department of Oncology, School of Medicine, Georgetown University, Washington, District of Columbia, United States of America Abstract BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W6BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis. Citation: Crawford AC, Riggins RB, Shajahan AN, Zwart A, Clarke R (2010) Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis. PLoS ONE 5(1): e8604. doi:10.1371/journal.pone.0008604 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received November 11, 2009; Accepted December 2, 2009; Published January 6, 2010 Copyright: ß 2010 Crawford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by awards from the Department of Defense Breast Cancer Research Program (BC050389) to Dr. Anatasha Crawford, and from the U.S. Department of Health and Human Services (R01-CA131465) and Department of Defense (BC073977) to Dr. R. Clarke. Flow Cytometry, Microscopy and Imaging, and Tissue Culture Shared Resources are supported by P30-CA-51008-16. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]. Introduction membrane depolarization and caspase activation [6]. BCL-W acts by binding to pro-apoptotic BCL2 family members and Approximately 70% of all newly diagnosed breast cancers preventing mitochondria-mediated apoptosis [7]. Overexpression express estrogen receptor-alpha (ER) [1], many of which are of BCL-W can prevent cell death [6] but its role(s) in affecting sensitive to antiestrogens. The steroidal antiestrogen ICI 182,780 breast cancer cell fate decisions or antiestrogen responsiveness is (ICI; Faslodex, Fulvestrant) is a selective ER downregulator unknown. BCL2 also blocks the induction of apoptosis by (SERD) that acts as an ER antagonist and enhances ubiquitin- inhibiting the activation of pro-apoptotic family members such mediated ER degradation. ICI is an effective second-line as BAX and preventing mitochondrial membrane depolarization treatment for TAM resistant, ER-positive (ER+) tumors, and is [8,9]. Overexpression of BCL2 is a potential mediator of resistance as effective as some aromatase inhibitors [2,3]. One limitation of to several chemotherapeutic drugs [10]. antiestrogen therapy is the prevalence of de novo and acquired BCL2 family members also play essential roles in autophagy resistance in breast cancer. Acquired antiestrogen resistance (macroautophagy), a process characterized by the presence of occurs when a tumor has an initially beneficial response to autophagosomes that engulf damaged organelles for subsequent antiestrogen treatment but the remaining tumor cells stop lysosomal degradation. Several anti-apoptotic BCL2 family mem- responding [4,5]. We report the roles of BCL2L2 (BCL-W), bers inhibit the activity of BECN1 [11], a key regulator of BCL2, and Beclin-1 (BECN1) in affecting responsiveness to ICI- autophagy [12] that binds to PIK3C3 to facilitate autophagosome resistance, and describe how anti-apoptotic BCL2 family members production [13]. However, the precise relationships between are involved in determining breast cancer cell fate. apoptosis and autophagy are unclear. Apoptosis or autophagy can BCL2 family proteins are essential regulators of apoptosis. each lead to cell death, but in some cellular contexts autophagy is a BCL2 and BCL-W are both antiapoptotic members of this family. pro-survival process, for example, in the face of nutrient deprivation BCL-W maintains cell viability by preventing mitochondrial [11]. While autophagy can contribute to TAM resistance in some PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8604 BCL2, BCL-W, and AE Resistance breast cancer cells [14–16], its role in response to other and BCL2 improves ICI sensitivity in antiestrogen-sensitive cells antiestrogens is unknown. In ER+ MCF-7 breast cancer cells by increasing apoptosis. Therefore, BCL-W, BCL2, and BECN1 treated with camptothecin, autophagy prolongs survival and delays integrate central functions in determining ICI responsiveness likely apoptosis [17]. In marked contrast, autophagy promotes apoptosis by regulating functional autophagy to dictate the balance between in MCF-7 cells treated with the cytotoxic diterpenoid oridonin, apoptotic and necrotic cell death. where an inhibition of autophagy increases cell survival [18]. We determined whether BCL-W and BCL2 regulate ICI Results response in human breast cancer cells, and whether any effects involve changes in apoptosis and/or BECN1-associated autoph- We measured endogenous BCL-W and BCL2 expression in agy. We used three estrogen-independent cell lines: MCF-7/ control and ICI treated resistant and sensitive cells. BCL2 expression LCC1 (ICI sensitive) [19], and LY2 and MCF-7/LCC9 cells that was significantly higher in ethanol control and ICI treated (resistant) are crossresistant to TAM and ICI [20,21]. We show that co- MCF-7/LCC9 cells when compared to (sensitive) MCF-7/LCC1 inhibition of BCL-W and BCL2 restores sensitivity to the growth- cells (Figure 1A; ANOVA p = 0.002). BCL-W expression was lower inhibitory effects of ICI in both MCF-7/LCC9 and LY2 cells. In in MCF-7/LCC1 cells after 24 hr of ICI treatment and increased in re-sensitized cells, ICI treatment increases the levels of autophagy both sensitive and resistant cells after 72 hr of ICI treatment. and necrosis but has no effect on apoptosis. Inhibition of However, the levels in resistant cells remained higher than in autophagy by 3-methyladenine (3MA) or BECN1 shRNA under sensitive cells (Figure 1B; ANOVA p = 0.004;). these conditions reduces necrosis and increases apoptosis. Thus, To determine if BCL2 transcription is regulated in antiestrogen- restoration of ICI sensitivity with BCL-W+BCL2 inhibition resistant cells, we measured basal BCL2 promoter activity using a appears to occur through increasing an autophagy-associated BCL2-luciferase promoter-reporter assay. Basal BCL2 promoter necrotic cell death. Finally, we show that co-inhibiting BCL-W activity was increased 14-fold in MCF-7/LCC9 cells (Figure S1; Figure 1. Increased expression of BCL-W and BCL2 in MCF-7/LCC9 cells. Whole cell lysates were subjected to Western blot analysis with a specific BCL2 or BCL-W antibody. (A) Bars represent the mean6SE of the relative BCL2:actin ratio (normalized to control cells) for three independent experiments. Inset, a representative blot.(B) Bars represent the mean6SE of the relative BCL-W:actin ratio (normalized to control cells) for three independent experiments. Inset, a representative blot. doi:10.1371/journal.pone.0008604.g001 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8604 BCL2, BCL-W, and AE Resistance p,0.003) when compared to MCF-7/LCC1 cells, suggesting that Total cell number was significantly decreased after treatment with the transcriptional regulation of basal BCL2 expression is altered both concentrations of ICI and/or YC137 in MCF-7/LCC1 cells in MCF-7/LCC9 cells. (ANOVA p,0.001; Fig. 2A). RI = 2.56 (20 nmol/L ICI)