Abstract P4-06-03: TMEPAI a Novel Prognostic Marker for TGF-β Dependency of Metastatic Breast Cancers: Converts Tumor Suppressive TGF-β into a Tumor Promoter

Abstract

Abstract Background: The dependency of many aggressive and treatment resistant breast cancers on transforming growth factor-beta (TGF-β) for their growth and invasiveness is paradoxical because TGF-β is a classical tumor suppressor that inhibits the growth of normal and early neoplastic breast epithelium. Therefore, we addressed the question how does TGF-β signaling transform from its expected role as a tumor suppressor in early cancer to that of a tumor promoter that facilitates growth and invasion in the late stages? Materials and methods: TMEPAI RNA was quantitated by for qPCR with SYBR green in an Applied Biosystems 7500 Real Time PCR System. pLKO. 1 based lentiviral vector was used to knockdown TMEPAI. Immunoblotting, invasion assays and tumor xenografts were performed using standard methods. Results: TMEPAI expression is observed in ER/PR-negative and HER2-negative breast tumors compared to their matched normal/benign tissues. TMEPAI protein was found to be highly expressed in aggressive and invasive breast cancer cells compared to non-invasive ER-positive and HER2-negative breast cancer cells. Importantly, TGF-β induced late growth spurt in MDA-MB-231 cells and inhibited cell growth in normal mammary epithelial cells (HME). While TGF-β stimulated TMEPAI RNA by ∼40 fold and protein by ∼8-10 fold in MDA-MB-231 cells, it had minimal effect on HME. Both basal expression and TGF-β induction of TMEPAI were completely abolished by SB431542, a pharmacological inhibitor of TGF-β signaling as well as by overexpression of Smad7 or dominant negative TGF-β RI (DN Alk-5). We confirmed TMEPAI as a direct target gene of TGF-β signaling when breast cancer cells MDA-MB-468 and MCF-7 that do not express TMEPAI and are defective in either Smad4 or TGF-β receptor I respectively, by exogenously expressing Smad4 in MDA-MB-468 and Alk-5 in MCF-7 to restore TMEPAI induction by TGF-β. Notably, knockdown of TMEPAI has profound corrective effects on cancer cell growth in vitro and in vivo. These important biological effects were accompanied by increase of cellular levels of the tumor suppressor PTEN and p27kip1, decreased activation of the PI3K signaling mediator Akt, and diminished expression of the oncogenic proteins Hif-1a, and VEGF. Discussion: Our results indicate that increase of TMEPAI protein — further exacerbated by TGF-β — may play a crucial role in TGF-β mediated tumor promotion. We believe that increased expression of TMEPAI by genomic amplification and/or epigenetic events contributes to the cancer phenotype and oncogenic progression through regulation of critical proteins involved in growth, epithelial mesenchymal transition (EMT), and angiogenesis, thereby increasing the aggressiveness of breast carcinomas. Thus, increase of TMEPAI and its induction by TGF-β to pathological levels may be the key lynchpin that explains the conversion of tumor suppressive TGF-β into a tumor promoter and TGF-β dependent cancer cell aggressiveness. Moreover, the data suggest that TMEPAI might serve as a novel prognostic marker for aggressiveness, invasiveness and TGF-β dependency of metastatic breast cancers. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-03.