Abstract B50: MEK inhibitors mount a two-pronged attack to kill estrogen receptor positive (ER+) breast cancer cells undergoing hormonal therapy: Attenuated autophagy and induction of apoptosis
Authors: Takhar S. , Manning M. , Eason A. , Dix M. , Periyasamy-Thandavan S. , Padi R. , Bieberich E. , Hill W. , Browning D. , Ganapathy V. , Thangaraju M. , Schoenlein P. V. 
Published: May 2015

In a recent study, we identified the dephosphorylated form of BimEL as a key death effector of antiestrogen treatment of ER+ breast cancer cells and further showed that MEK1/MAPK1/2 blockade was required to produce high levels of dephosphorylated BimEL, particularly under conditions of insulin like growth factor 1 (IGF1) stimulation (Periyasamy-Thandavan et al., Breast Cancer Res. 14, 2012). Studies by others have identified MEK1/MAPK1/2 activation as essential to autophagy, a catabolic process induced by multiple stresses including ROS, ceramide accumulation, and nutrient deprivation. Autophagy induction results in autophagosome formation, trafficking of damaged proteins and mitochondria to the autophagosomes, and ultimately fusion with the lysosomes resulting in autolysosome formation. The autolysosome and its contents are degraded by the hydrolytic enzymes of the lysosome. Of particular interest to antiestrogen treatment of breast cancer, we and others have shown that pro-survival autophagy facilitates the emergence of antiestrogen resistant breast cancer cells. Thus, we are keenly interested in how MEK1/MAPK1/2 signaling affects pro-survival autophagy and if MEK blockade would be an effective approach toward blocking pro-survival autophagy in ER+ breast cancer cells undergoing hormonal treatment. In this study, we hypothesized that the requirement of MEK1/MAPK1/2 for pro-survival autophagy is due, in part, to its role in blocking the intracellular accumulation of dephosphorylated BimEL. To test this hypothesis, we modulated the expression of dephosphorylated BimEL with either a BimEL cDNA expression vector, siRNA targeting of BimEL, or MEK1 blockade with the small molecule inhibitor U0126 and determined the levels of the autophagic flux in ER+ breast cancer cells undergoing antiestrogen treatment. The determination of autophagic flux was made by comparing the levels of two proteins involved in autophagy -the LC3 /Atg8 and p62 (SQSTM1) proteins- in cell populations undergoing the different treatments in the presence or absence of chloroquine (CQ). The lipidated form of LC3, designated LC3II, is typically increased in cells undergoing autophagy, facilitates the formation of the mature autophagosomal membranes, and is subsequently degraded in the autolysosome. The p62 protein is required for the delivery of ubiquitinated protein complexes to the autophagosome and is degraded along with the ubiquitinated complex of proteins. CQ is a lysosomotrophic agent routinely used in autophagic flux assays because it blocks the turnover of autolysosomes with accumulation of LC3 II and p62, allowing the total levels of LC3II and p62 to be ascertained under all treatment conditions. These studies showed that siRNA targeting of BimEL increased basal and tamoxifen-induced autophagy in ER+ MCF-7 breast cancer cells. In contrast, the overexpression of dephosphorylated BimEL led to an increase in LC3 II and p62 levels due to a significant attenuation of autophagic flux (approximately 50%) in antiestrogen-treated cell populations. Current studies are focused on the mechanism of BimEL-mediated blockade of pro-survival autophagy, with the long term goal of optimizing this “downstream effector” function of MEK1/MAPK1/2 blockade in ER+ breast cancer cells for improved therapeutic outcome.

Citation Format: S. Takhar, M. Manning, A. Eason, M. Dix, S. Periyasamy-Thandavan, R. Padi, E. Bieberich, W. Hill, D. Browning, V. Ganapathy, M. Thangaraju, P. V. Schoenlein. MEK inhibitors mount a two-pronged attack to kill estrogen receptor positive (ER+) breast cancer cells undergoing hormonal therapy: Attenuated autophagy and induction of apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B50. doi: 10.1158/1557-3125.RASONC14-B50