Kyung Song

Novel roles of Akt and mTOR in suppressing TGF‐β/ALK5‐mediated Smad3 activation

Insulin‐like growth factor‐I inhibits transforming growth factor‐β (TGF‐β) signaling by blocking activation of Smad3 (S3), via a phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent pathway. Here we provide the first report that the kinase activity of Akt is necessary for its ability to suppress many TGF‐β responses, including S3 activation and induction of apoptosis. Wild‐type and myristoylated Akts (AktWT and AktMyr) suppress TGF‐β‐induced phospho‐activation of S3 but not Smad2 (S2), whereas kinase‐dead Akt1 (Akt1K179M) or dominant‐negative PI3K enhances TGF‐β‐induced phospho‐activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12‐resistant and constitutively active TGF‐β 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‐β 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‐β in cancers.

Insulin-like Growth Factor-I Inhibits Transcriptional Responses of Transforming Growth Factor-β by Phosphatidylinositol 3-Kinase/Akt-dependent Suppression of the Activation of Smad3 but Not Smad2

Insulin-like growth factor-I (IGF-I) and transforming growth factor-β (TGF-β) have been shown to be oncogenic and tumor suppressive, respectively, on prostate epithelial cells. Here we show that IGF-I inhibits the ability of TGF-β to regulate expression of several genes in the non-tumorigenic rat prostatic epithelial line, NRP-152. In these cells, IGF-I also inhibits TGF-β-induced transcriptional responses, as shown by several promoter reporter constructs, suggesting that IGF-I intercepts an early step in TGF-β signaling. We show that IGF-I does not down-regulate TGF-β receptor levels, as determined by both receptor cross-linking and Western blot analyses. However, Western blot analysis reveals that IGF-I selectively inhibits the TGF-β-triggered activation Smad3 but not Smad2, while not altering expression of total Smads 2, 3, or 4. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY29004 reverses the ability of IGF-I to inhibit TGF-β-induced transcriptional responses and the activation of Smad3, suggesting that the suppression of TGF-β signaling by IGF-I is mediated through activation of PI3K. Moreover, we show that enforced expression of dominant-negative PI3K (DN-p85α) or phosphatidylinositol 3-phosphate-phosphatase, PTEN, also reverse the suppressive effect of IGF-I on TGF-β-induced 3TP-luciferase reporter activity, whereas constitutively active PI3K (p110αCAAX) completely blocks TGF-β-induced 3TP-luciferase reporter activity. Further transfection experiments including expression of constitutively active and dominant-negative Akt and rapamycin treatment suggest that suppression of TGF-β signaling/Smad3 activation by IGF-I occurs downstream of Akt and through mammalian target of rapamycin activation. In summary, our data suggest that IGF-I inhibits TGF-β transcriptional responses through selective suppression of Smad3 activation via a PI3K/Akt-dependent pathway.

Protein kinase C-mediated down-regulation of cyclin D1 involves activation of the translational repressor 4E-BP1 via a phosphoinositide 3-kinase/Akt-independent, protein phosphatase 2A-dependent mechanism in intestinal epithelial cells.

We reported previously that protein kinase Cα (PKCα), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCα-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr45 and Ser64 on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCα-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an ∼2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H2O2 and ceramide, two naturally occurring PKCα agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCα-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCα-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.

Rb/E2F4 and Smad2/3 link survivin to TGF-β-induced apoptosis and tumor progression

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-β (TGF-β). We show that TGF-β 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-β 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-β alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-β/Rb/survivin axis with a putative role in the functional switch of TGF-β from tumor suppressor to tumor promoter.

Smad7 is inactivated through a direct physical interaction with the LIM protein Hic-5/ARA55

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-β (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 small-hairpin RNA 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.

DHT Selectively Reverses Smad3-Mediated/TGF-β-Induced Responses through Transcriptional Down-Regulation of Smad3 in Prostate Epithelial Cells

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.

Androgenic Control of Transforming Growth Factor-{beta} Signaling in Prostate Epithelial Cells through Transcriptional Suppression of Transforming Growth Factor-{beta} Receptor II

The androgen receptor cross-talks with transforming growth factor-beta (TGF-beta) through mechanisms that remain poorly understood. Here we provide strong evidence that 5alpha-dihydrotestosterone (DHT) intercepts the ability of prostate epithelial cells to undergo TGF-beta-induced apoptosis, and present a new model for this androgenic effect. We report that DHT decreases the level of TGF-beta receptor II (TbetaRII) through a transcriptional mechanism, leading to suppression of the ability of TGF-beta 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 TbetaRII by DHT occurs through Sp1/Sp3 response elements, with the binding of Sp1 to the TbetaRII 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. [Cancer Res 2008;68(19):8173-82].

Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization.

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

Novel Permissive Role of Epidermal Growth Factor in Transforming Growth Factor β (TGF-β) Signaling and Growth Suppression MEDIATION BY STABILIZATION OF TGF-β RECEPTOR TYPE II

Transforming growth factor β (TGF-β) signals through TGF-β receptor serine/threonine kinases (TβRI and TβRII) and Smads, regulating cell growth and apoptosis. Although loss of TGF-β receptor levels is strongly selected for during the progression of most cancers, tumor cells frequently escape from complete loss of TGF-β receptors through unknown mechanisms. Here, we provide the first evidence that epidermal growth factor (EGF) signaling, which is generally enhanced in cancer, is permissive for regulation of gene expression and growth suppression by TGF-β in LNCaP prostate adenocarcinoma cells. Our results support that these permissive effects occur through enhanced stability of TβRII mRNA and reversal of TGF-β-mediated TβRII mRNA loss. Changes in stability of TβRII mRNA occur soon after EGF or TGF-β1 addition (optimal within 3 h) and are independent of de novo protein synthesis or transcription. Remarkably, such loss of TβRII by TGF-β can be mediated by a kinase-dead TβRII (K277R), as well as by other forms of this receptor harboring mutations at prominent autophosphorylation sites. Moreover, Smad3 small interfering RNA, which blocks TGF-β-induced AP-1 promoter activity, does not block changes in the expression of TβRII by EGF or TGF-β. We have also shown that changes in TβRII levels by EGF are EGF receptor-kinase-dependent and are controlled by signals downstream of MEK1/2. Our findings provide invaluable insights on the role of the EGF receptor-kinase in enhancing TGF-β responses during prostate carcinogenesis.

Insulin-Like Growth Factor I Suppresses Bone Morphogenetic Protein Signaling in Prostate Cancer Cells by Activating mTOR Signaling

Insulin-like growth factor (IGF) I and bone morphogenetic proteins (BMP) are critical regulators of prostate tumor cell growth. In this report, we offer evidence that a critical support of IGF-I in prostate cancer is mediated by its ability to suppress BMP4-induced apoptosis and Smad-mediated gene expression. Suppression of BMP4 signaling by IGF-I was reversed by chemical inhibitors of phosphoinositide 3-kinase (PI3K), Akt, or mTOR; by enforced expression of wild-type PTEN or dominant-negative PI3K; or by small hairpin RNA-mediated silencing of mTORC1/2 subunits Raptor or Rictor. Similarly, IGF-I suppressed BMP4-induced transcription of the Id1, Id2, and Id3 genes that are crucially involved in prostate tumor progression through PI3K-dependent and mTORC1/2-dependent mechanisms. Immunohistochemical analysis of non-malignant and malignant prostate tissues offered in vivo support for our model that IGF-I-mediated activation of mTOR suppresses phosphorylation of the BMP-activated Smad transcription factors. Our results offer the first evidence that IGF-I signaling through mTORC1/2 is a key homeostatic regulator of BMP4 function in prostate epithelial cells, acting at two levels to repress both the proapoptotic and pro-oncogenic signals of BMP-activated Smads. We suggest that deregulation of this homeostatic control may be pivotal to the development and progression of prostate cancer, providing important implications and new potential targets for the therapeutic intervention of this malignancy.