Abstract P1-01-22: TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer

Background: In the absence of mutations and/or deletions in the TGF-β signaling molecules, how triple negative breast cancer (TNBC) cells become resistant to growth inhibitory signaling of TGF-β was addressed by screening of several TNBC cell lines for Smad proteins involved in TGF-β signaling. Unexpectedly, altered Smad2 and Smad3 protein levels were detected in breast cancer cells relative to normal mammary epithelial cells, suggesting a novel mechanism to escape from TGF-β mediated growth inhibition. In order to reflect the true functional status of the endogenous molecules, Smad2 or Smad3 were individually knocked down and tested its effect on pro-oncogenic behavior of TGF-β. Materials and Methods: All cell lines were cultured according to the recommended standard procedures. TMEPAI knockdown was achieved by using lentiviral vectors and knockdown of Smad2 and Smad3 was achieved by using retroviral vectors. DNA transfections and luciferase assays were performed according to vendor instructions. Cell proliferation was measured by quantitation of total cellular DNA. Immunoblotting, invasion and immunohistochemical assays were performed using standard methods. Bioinformatics: Triple negative breast cancer patient dataset (GSE58812) was used to compare Smad2, Smad3, Smad4 and TMEPAI/PMEPA1 expressions correlated with overall survival using the PROGgene tool by using median gene expression value as a dividing point. Results: While Smad2 deficiency has no effect on breast cancer cell behavior, Smad3 deficiency reduced growth and invasion capacity of breast cancer cells. Interestingly, Smad3 deficiency was associated with reduced gene expressions of transmembrane prostate androgen induced (TMEPAI or PMEPA1) gene and EMT inducing transcription factors and increased expression of cell cycle inhibitors. In contrast, Smad2 deficiency had opposite effect on these regulators. Importantly, the decreased growth and invasion and their associated gene expressions in Smad3 knockdown cells were largely reversed by overexpression of TMEPAI. Meta-analysis of Jezequel dataset of triple negative breast cancer patients suggested higher TMEPAI/PMEPA1 and lower Smad2 expressions are significantly associated with decreased survival. Our results support the idea that development of several triple negative breast cancers may involve expansion of cell populations with altered Smad2 and Smad3 levels resulting in Smad3 dependent expression of TMEPAI, which provides a competitive advantage for cancer cells to grow and metastasize in presence of TGF-β. Moreover, following meta-analysis of published microarray datasets in lung and gastric cancers also revealed that increased TMEPAI and Smad3 expression and decreased Smad2 expressions were significantly associated with poorer patient prognosis in non-small cell lung adenocarcinomas and gastric cancers suggesting universality of this phenomenon. Conclusion: We identified TGF-β-Smad3-TMEPAI signaling axis as a driver of tumor progression in triple negative breast cancer. Our results suggest that novel therapeutics targeting TMEPAI will selectively inhibit oncogenic activity of TGF-β and promote its tumor suppressive activity in treating TNBC. Citation Format: Saikumar P, Singha PK, Pandeswara S, Manjeri VA. TGFβ-Smad3-TMEPAI axis drives the tumor progression of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-22.