P2-11-04: TMEPAI Is a Feedback Regulator of TGF-b Signaling during Breast Cancer Progression.

Background: Despite lack of hormone receptors (ER/PR) and HER2, many triple negative breast cancers (TNBC) depend on transforming growth factor beta (TGF-β) signaling activity for late growth and metastasis. Thus TGF-β signaling is a major potential target to treat TNBC patients. However, inhibition of TGF-β signaling pathway carries the risk of disturbing the tumor suppressive activity of TGF-β in normal tissues and early cancers. Hence, present study was undertaken with a goal to identify suitable markers which will enable to develop drugs that retard only the “oncogenic activity” of TGF-β while preserving its growth suppressive activities. Materials and Methods: All cell lines were cultured according to the recommended standard procedures. Lentiviral mediated expression vector was used to stably knockdown endogenous TMEPAI expression. DNA transfections and luciferase assays were performed according to vendor instructions. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting and immunohistochemical analysis were performed using standard methods. Results: Previously we have shown that Transmembrane prostate androgen-induced protein (TMEPAI), a TGF-β inducible gene has the potential to convert tumor suppressive TGF-β into a tumor promoter. In the present study, we show that Smad binding elements driven luciferase reporter activity, a measure of TGF-β signaling, was dramatically increased in TMEPAI knockdown cells. While wild type cancer cells showed transient raise in phosphorylation of Smad2 and Smad3 with TGF-β treatment, TMEPAI knockdown resulted in increased and sustained levels of phosphorylated Smad2 and Smad3. In normal mammary epithelial cells, continuous presence of TGF-β blocked their growth. In contrast, breast cancer cells showed a biphasic growth response (moderate inhibition up to 72h followed by strong growth stimulation) to TGF-β, while TMEPAI-deficiency blocked this response to TGF-β. Furthermore, exogenous expression of TMEPAI in normal mammary epithelial cells and TMEPAI-knockdown cells resulted in reduced TGF-β-dependent Smad activity. Additionally, we found that TMEPAI subverts tumor suppressive TGF-β dependent Smad signaling into tumor promotive non-Smad signaling through stimulation of stress activated protein kinases. Inhibition of stress kinases also reduced the growth of cancer cells similar to TMEPAI deficiency. To evaluate the translational importance of TMEPAI as predictive marker, immunohistochemical analysis on human breast cancer specimens revealed the gain of TMEPAI expressions in aggressive human breast tumor samples but not in normal human breast tissue specimens. Conclusions: Our results show that TMEPAI, which subverts tumor suppressive Smad signaling, may serve as a novel prognostic and predictive marker for aggressive and TGF-β dependent of metastatic breast cancers. These studies will further provide new clues to develop an effective and decisive anti-TGF-β therapy against aggressive breast cancers without disturbing the growth suppression by TGF-β in normal tissues and early tumors. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-04.