Abstract P2-06-08: Interplay of Smad2 and Smad3 during TGF-β induced TMEPAI/ PMEPA1 mediated triple negative breast cancer cell growth

Background: Despite the known effects of TGF-β mediated canonical and non-canonical Smad signaling in cancer cell growth and metastasis, the role played by individual R-Smads in mediating TGF-β dependent late growth and metastasis remained enigmatic. Previously we have reported that transmembrane prostate androgen induced (TMEPAI/PMEPA1), a TGF-β target gene has unique role in subverting TGF-β mediated growth suppression into growth promotion of triple negative breast cancer (TNBC) cells. But the relationship between Smads and TGF-β mediated TMEPAI induction and TNBC proliferation is not known. Therefore, we undertook the present study to know the differential role played by Smads in TGF-β mediated induction of TMEPAI and TNBC growth. Materials and Methods: Cells were cultured as recommended by ATCC. Knockdown of TMEPAI and Smad2, Smad3 proteins was achieved by lentiviral or retroviral shRNA vectors. Transfections, luciferase assays, RT-qPCR and immunoblotting were performed according to standard methods. Cell proliferation was measured by quantitation of total cellular DNA. Results: Although no significant differences were found in mRNA levels of Smad2 and Smad3 in normal human mammary epithelial cells (HMEC) and different TNBC cell lines, at the protein level aggressive TNBC cells expressed more Smad3 protein than Smad2 compared to normal cells. TMEPAI knockdown did not modify this profile in TNBC cells. However, HMEC that expressed more Smad2 protein than Smad3, produced little TMEPAI and growth arrested in response to TGF-β. In contrast, MDA-MB-231 (231) cells, which contained more Smad3 over Smad2 produced high levels of TMEPAI and grew robustly in response to TGF-β. To delineate the role of individual R-Smads in TGF-β mediated growth regulation and TMEPAI expression, Smad2 or Smad3 were selectively knocked down using shRNAs. Knockdown of either Smad2 or Smad3 rescued HMEC from TGF-β mediated growth arrest, suggesting that Smad signaling is growth suppressive in HMEC. In contrast, selective Smad2 or Smad3 knockdown had distinctive effects on 231 cell proliferation and TMEPAI expression. Interestingly Smad3 knockdown, which showed diminished TMEPAI expression in 231 cells, greatly inhibited their growth both in the absence or presence of TGF-β. In contrast, Smad2 knockdown in 231 cells caused augmented TMEPAI expression in response to TGF-β and increased cell proliferation at the rates similar to control cells either in the absence or presence of TGF-β. Notably, individual R-Smad deficiency caused a compensatory increase in the complementary R-Smad and its associated signaling in 231 cells. Therefore, the effect of Smad3 shRNA on TNBC proliferation was similar to that of TMEPAI shRNA reported by us earlier. Like TMEPAI knockdown, Smad3 knockdown also elevated PTEN protein levels with reduced Akt phosphorylation that reduced growth both in the absence or presence of TGF-β. Conclusion: Our results suggest that growth of TNBC in the presence of TGF-β is unique to cancer cells and is pathological in a TGF-β-Smad3-TMEPAI axis dependent manner. Smad3 plays an important role in growth promoting TGF-β dependent non-canonical signaling not only with respect to the induction of TMEPAI but also by decreasing the PTEN. Citation Format: Singha PK, Pandeswara S, Venkatachalam MA, Saikumar P. Interplay of Smad2 and Smad3 during TGF-β induced TMEPAI/ PMEPA1 mediated triple negative breast cancer cell growth. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-08.