Differential regulation of mTORc1 and mTORc2 by expression of BTG2/TIS21/PC3 inhibits breast cancer cell growth and malignancy

Abstract

12-O-tetradecanoyl phorbol-13-acetate (TPA)-inducible sequences 21 (TIS21)-an ortholog of B-cell translocation gene 2 (BTG2) in humans and pheochromocytoma cell-3 (PC3) in rats- is an antiproliferative and early growth response gene and belongs to a family of six proteins which includes BTG1-4 and Tob1, Tob2. All these proteins contain APRO domain at the N-terminus with highly conserved sequences termed Box A and Box B. However the C-terminus is less conserved except for the presence of PAM2 motif (Poly-A binding protein Motif 2) in Tob 1 and 2. BTG2 is induced following DNA damage in a p53-dependent and -independent manner by protein kinase C-δ. BTG2 induces cell cycle arrest by inhibiting cyclin D1 and E, as well as CDK4, depending on pRB activity, and by interacting with cyclin B1/CDK1. BTG2 is constitutively expressed in all human tissues but lost during the early stage of carcinogenesis and loss of BTG2 expression is associated with the size and grade of ER-positive breast cancers along with breast tumor progression. We previously reported that BTG2 inhibits invadopodia formation by downregulating reactive oxygen species (ROS) through targeting of the mDia genes and regulated AKT phosphorylation at S473 . We therefore tried to explore the mechanism of AKT activation by the BTG2 gene and its outcome in breast carcinogenesis. It has been reported that AKT1 inhibits cancer progression, but maintains cancer cell growth; however, here, we proposed novel mechanisms of AKT1 for inhibiting both cancer progression and cancer growth following expression of the tumor suppressor, BTG2, in triple negative breast cancer cells via differential regulation of mTORc1 and mTORc2. Expression of BTG2 inhibited the mTOR-Raptor interaction, and increased mTOR binding to Rictor. BTG2 selectively induced phosphorylation of AKT1, not AKT2, by mTORc2 and reduced expression of PHLPP2, an AKT1-specific phosphatase. BTG2-induced pAKT1S473 degraded NFAT1, which regulates transcription of cytokines, chemokine receptors and possibly PHLPP2, resulting in modification of the tumor microenvironment. In human breast cancer tissues, expression of NFAT1 and PHLPP2 was significantly higher compared to the low expression of BTG2. In tsc-null and p53-null mouse embryonic fibroblasts (MEFs), BTG2 was substituted for the tsc gene, -a negative regulator of mTORc1 activation- , and was also evident in -null mice. Constitutive expression of BTG2 in the basal cells of normal mammary ducts was maintained in ductal-carcinoma in situ (DCIS) of human breast cancer but absent in infiltrating breast cancer, as examined by immunohistochemistry. Indeed, the relative risk of lymph node invasion in BTG2+ breast cancer patients was approximately 1/10th that of BTG2- patients, and overall survival of ER-LN+ breast cancer patients was significantly higher among BTG2 high expressers than low expressers. Indeed, BTG2 expression significantly inhibited the in vitro colony forming ability of triple negative breast cancer cells. We present here the signaling pathways that regulate cancer growth and progression by expression of BTG2, which inhibits cancer growth via the BTG2-tsc1/2-mTORc1-p70S6K axis and downregulates cancer progression via the BTG2-mTORc2-AKT1-NFAT1-PHLPP2 axis. Therefore, we suggest the BTG2 gene is a promising candidate to combat intractable breast cancer.