Abstract 1046: BAD is a multifunctional protein in breast cancer cells

Abstract

Abstract BAD is a multifunctional protein in breast cancer cells R. Fernando1, M. Cekanova2, J. Woraratphoka2, M. Sukhthankar2, J. Bahn2, S. Dharmawardhana3, N. Siriwardana2, N. Moustaid-Moussa2, A. Strom4, S. Baek2, Q. Wong5, M. Zou5, R. Donnell2, J. Wimalasena2 1NCI, Bethesda, MD; 2University of Tennessee, Knoxville, TN; 3University of Puerto Rico Medical Sciences, San Juan, PR; 4University of Houston, Houston, TX; 5University of Oklahoma Medical Center, Oklahoma City, OK. BAD is a typical BH3 protein and, like many other BCL-2 family proteins, regulates apoptosis. However, several recent studies suggest that BAD has non-apoptotic roles. Previously, we had shown that BAD inhibits the MCF7 cell cycle via abrogation of cyclin D1 synthesis (JBC 282, 28864, 2007). Using tetracycline-regulated stable BAD overexpression in MCF7 cells, we now report that BAD regulates a wide variety of functions: since BAD binds to c-Jun (JBC, 2007), we investigated the ability of BAD to regulate the AP1 reporter activity. BAD inhibited MEK and activated RAS (V12, S35) regulated AP1 activity, but not the increase due to AKT. BAD overexpression blocked ERK activation by MEK1, but not by active AKT. Wildtype BAD inhibited estrogen- or serum-induced activation of c-Jun, JNK, and ERK, but not p38 MAPK. However, the S112/S136 double mutant BAD had no inhibitory effects, suggesting a requirement for phosphorylation. We then explored if BAD can regulate signaling pathways and found that it can inhibit beta catenin mRNA and protein expression. Several signaling proteins, including total AKT, active GSK 3 beta, and apoptosis-related soluble FasL, TNF alpha, TRAIL, EGFR, and RBM5, which may regulate FasL were significantly (p<0.01) regulated by BAD. BAD decreased invasion of MCF7 cells through Matrigel (p<0.05) and decreased cellular VEGF (p50% (p<0.01). Surprisingly, BAD decreased protein expression of PPAR gamma, active AMPK, LKB1, and active acetylCoA carboxylase (all at p<0.01). We analyzed the expression of several markers in breast cancer specimens: phospho (p)-BAD, BAD and cyclin D1 using tissue microarrays containing normal and neoplastic tissue. In addition, we checked the expression levels of ERK-1, phospho (p)-ERK1, AKT, and phospho-AKT in surgical pathology blocks with normal and neoplastic breast epithelial cells. As expected, cytoplasmic p-BAD expression was significantly and positively correlated with nuclear BAD, nuclear p-BAD, as well as nuclear cyclin D1. Our data showed that cytoplasmic cyclin D1 was positively correlated with cytoplasmic ERK-1, and cytoplasmic cyclin D1 was negatively correlated with nuclear AKT expression. Nuclear BAD and AKT were negatively correlated with nuclear cyclin D1 and nuclear ER-beta. Cytoplasmic expression of p-ERK1 was negatively correlated with nuclear AKT and positively correlated with nuclear cyclin D1, and nuclear ER-beta. Taken together, these results suggest that BAD may be a multifunctional protein. 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 1046.