Insulin-like growth factor-I inhibits transforming growth factor-b (TGF-b) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3-kinase (PI3K)/Aktdependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF-b responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (Akt WT and Akt Myr ) suppress TGF-b-induced phospho-activation of S3 but not Smad2 (S2), whereas kinasedead Akt1 (Akt1 K179M ) or dominant-negative PI3K enhances TGF-b-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-b type I receptor (ALK5), we demonstrate that mammalian target of Rap (mTOR) mediates Akt1 suppression of phospho-activation of S3. These and further data on Akt1-S3 binding do not support a recently proposed model that Akt blocks S3 activation through physical interaction and sequestration of S3 from TGF-b receptors. We propose a novel model whereby Akt suppresses activation of S3 in an Akt kinase-dependent manner through mTOR, a likely route for loss of tumor suppression by TGF-b in cancers.

Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-b (TGF-b). We show that TGF-b rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/ E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-b response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-b alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-b/Rb/survivin axis with a putative role in the functional switch of TGF-b from tumor suppressor to tumor promoter.

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named ARA55, androgen receptor-associated protein 55) can bind to the TGF-β signaling regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-β responses. We now show that Hic-5 can also control TGF-β responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-β signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-β signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-β-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated shRNA silencing of endogenous Hic-5 reduced TGF-β responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell type-specific activator of TGF-β signaling through its ability to physically interact with and neutralize Smad7.

The androgen receptor cross-talks with transforming growth factor-B (TGF-B) through mechanisms that remain poorly understood. Here we provide strong evidence that 5A-dihydrotestosterone (DHT) intercepts the ability of prostate epithelial cells to undergo TGF-B-induced apoptosis, and present a new model for this androgenic effect. We report that DHT decreases the level of TGF-B receptor II (TBRII) through a transcriptional mechanism, leading to suppression of the ability of TGF-B to down-regulate expression of Bcl-xL and cyclin Ds, activate caspase-3, and induce apoptosis. Promoter analysis, DNA pulldown, and electrophoretic mobility shift assays support that transcriptional down-regulation of TBRII by DHT occurs through Sp1/Sp3 response elements, with the binding of Sp1 to the TBRII promoter being suppressed by DHT, largely driven by loss of Sp1 protein and/or activity. These results provide fresh insight on the mechanism of growth control by androgens and the progression of prostate cancer to androgen independence.

Androgens suppress TGF-β responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5α-dihydrotestosterone (DHT) suppresses the ability of TGF-β to inhibit proliferation and induce apoptosis of prostatic epithelial cells and provided evidence that such suppression was fueled by transcriptional down-regulation of TGF-β receptor II (ΤβRII). We now show that androgen receptor (AR) activated by DHT suppresses the TGF-β-induced phosphorylation of Sma- and Mad-related protein (Smad)3 in LNCaP cells overexpressing TβRII under the control of a cytomegalovirus promoter, which is not regulated by DHT, suggesting that transcriptional repression of TβRII alone does not fully account for the impact of DHT on TGF-β responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. We provide evidence that DHT down-regulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-β-induced apoptosis in NRP-154+AR, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-β-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-β through transcriptional suppression of Smad3, thereby providing new mechanistic insight into the development of castration-resistant prostate cancer.