Abstract A136: Proteome analysis reveals candidate genes regulated by BLID, a novel drug-inducible apoptotic target, in breast cancer cells.

Abstract

Abstract BLID, BH-3 Like motif containing Inducer of cell Death, is a strong prognostic factor in invasive breast cancer (1). Frequent lack of BLID in breast cancer has been associated with triple-negative breast cancer (TNBC), African American ethnicity and younger women. Significant correlations exist between BLID negative breast cancer and declines in overall survival, local relapse-free survival and distant metastasis-free survival. In dose response and time course studies, BLID expression was found to be induced by chemotherapeutic drugs including doxorubicin, 5-fluorouracil and cisplatin in MCF-7 breast cancer cells. In addition, exogenous expression of BLID cDNA nanocomplex led to significant increase in chemosensitivity in SKBr3 (ER-ve/PR-ve) and MDA-MB231 (TNBC) breast cancer cell lines. Here we have investigated the effects of BLID knockdown on protein expression profiles in representative hormone responsive (MCF-7) and TNBC breast cancer cells (MDA-MB231) with a goal to identify a core translational component signifying BLID activity in breast cancer cells. BLID shRNA lentiviral particles were generated using pLKO.1-TRC containing BLID shRNA clone 47 according to the supplier's manual (Sigma-Aldrich). Breast cancer cells were stably transfected with BLID shRNA or scrambled shRNA. The pooled stable clones were tested for BLID expression by western blot analysis. Significant knockdown of BLID protein was achieved in breast cancer cells transfected with BLID shRNA (% inhibition of BLID expression vs. scrambled shRNA: MCF-7, 69%; MDA-MB231, 63%). The BLID proteome analysis was performed using antibody microarrays as we reported earlier (2). We have observed distinct and overlapping BLID-centric protein profiles in MCF-7 and MDA-MB231 cells. Among the top-ranked genes, BLID knockdown correlated with increased expression of PI3-kinase catalytic subunit α and DEK oncogene in MCF-7 cells, and increased expression of myeloid cell leukemia sequence 1 (MCL-1) in MDA-MB231 cells. Remarkably, BLID depletion correlated with decreased expression of tumor protein p73 and cAMP-dependent protein kinase (PKA Type Iα) in both cell lines. Thus, we have identified a working platform of genes signifying BLID expression in breast cancer cells. Validation of the top-ranked genes in various models of breast cancer is ongoing and data will be presented. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A136. Citation Format: Sivaramakrishna Yadavalli, Ofer Eidelman, Meera Srivastava, Usha N. Kasid. Proteome analysis reveals candidate genes regulated by BLID, a novel drug-inducible apoptotic target, in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A136.