Abstract 505: Role of TMEPAI-PTEN-PI3K/Akt axis in TGF-β mediated growth and metastasis of breast cancer cells

The tumor suppressive transforming growth factor-β (TGF-β) plays a key role in breast cancer by paradoxically promoting tumor growth and metastasis. The molecular basis for this dual function of TGF-β is poorly understood. Earlier, we have reported that transmembrane prostate androgen induced (TMEPAI), a direct target of TGF-β signaling plays an important role in converting tumor suppressor TGF-β to a tumor promoter in breast cancer progression. We have also shown that TMEPAI knockdown had elevated PTEN levels. In the present study we show that TMEPAI stimulates TGF-β mediated non-canonical PI3K/Akt signaling by decreasing PTEN. Increased PTEN protein in TMEPAI knockdown (TMKD) cells is dramatically decreased, when mouse TMEPAI was expressed. The half-life of PTEN was drastically decreased from ∼ 178 h to ∼14h probably due to increased proteasomal degradation as MG132, a broad spectrum proteasome inhibitor, prevented PTEN loss. Even though there is direct interaction between TMEPAI and PTEN, but both proteins coprecipitated with NEDD4, a WW-domain containing HECT E3 ligase. TGF-β treatment caused increased association between TMEPAI and NEDD4, while PTEN disappeared from the complex. Through mutagenesis, we showed that PY motifs of TMEPAI are essential for NEDD4 binding and PTEN degradation. Reduced PTEN in TMEPAI expressing cells caused increased Akt activation through phosphorylation (pAkt) and Snail induction, a transcription factor that reduces cell-cell contacts, causing growth and epithelial-to-mesenchymal transition (EMT). Addition of LY294002, a PI3K inhibitor or knockdown of TMEPAI blocked pAkt formation and snail induction in TEMPAI expressing breast cancer cells. When dominant negative mutants of PTEN (lipid and protein phosphatase inactive [C124S], and lipid phosphatase alone inactive [G129E] mutants) were expressed in knockdown cells harboring TMEPAI shRNA, they reversed growth retardation, pAkt and Snail protein levels and cell morphology in TMEPAI knockdown cells, suggesting that TMEPAI mediated reduction in PTEN is responsible for growth and metastasis of breast cancer. Moreover, TMEPAI knockdown inhibited tumor lung metastases in vivo. Finally, immunohistochemistry of human breast cancer tissues revealed an inverse correlation between TMEPAI and PTEN proteins. While normal/ benign human breast tissues showed high PTEN and low or no staining of TMEPAI, about 69% of triple negative breast cancer tissues showed low PTEN and high TMEPAI staining. Hence our results suggest that low PTEN in TMEPAI expressing breast cancer cells may be responsible for TGF-β mediated proliferation and metastasis through pAkt and Snail upregulation. Citation Format: Prajjal K. Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. Role of TMEPAI-PTEN-PI3K/Akt axis in TGF-β mediated growth and metastasis of breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 505. doi:10.1158/1538-7445.AM2015-505