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2.
Cancer Res ; 70(22): 9106-17, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-21062988

ABSTRACT

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.


Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Profiling , Humans , Inhibitor of Differentiation Protein 1/genetics , Insulin-Like Growth Factor I/pharmacology , Male , Oligonucleotide Array Sequence Analysis , Phosphorylation/drug effects , Prostate/cytology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology , Smad Proteins/genetics , Smad Proteins/metabolism , TOR Serine-Threonine Kinases/genetics , Transcriptional Activation/drug effects , Transforming Growth Factor beta/pharmacology
3.
Mol Endocrinol ; 24(10): 2019-29, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20739403

ABSTRACT

Androgens suppress TGF-ß responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5α-dihydrotestosterone (DHT) suppresses the ability of TGF-ß to inhibit proliferation and induce apoptosis of prostatic epithelial cells and provided evidence that such suppression was fueled by transcriptional down-regulation of TGF-ß receptor II (ΤßRII). We now show that androgen receptor (AR) activated by DHT suppresses the TGF-ß-induced phosphorylation of Sma- and Mad-related protein (Smad)3 in LNCaP cells overexpressing TßRII under the control of a cytomegalovirus promoter, which is not regulated by DHT, suggesting that transcriptional repression of TßRII alone does not fully account for the impact of DHT on TGF-ß 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-ß-induced apoptosis in NRP-154+AR, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-ß-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-ß through transcriptional suppression of Smad3, thereby providing new mechanistic insight into the development of castration-resistant prostate cancer.


Subject(s)
Dihydrotestosterone/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/physiology , Gene Expression Regulation/drug effects , Prostate/cytology , Smad3 Protein/genetics , Transforming Growth Factor beta/pharmacology , Animals , Cell Line , Epithelial Cells/cytology , Humans , Male , Rats , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Smad3 Protein/metabolism , Transcription, Genetic
4.
Cancer Res ; 68(19): 8173-82, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18829577

ABSTRACT

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].


Subject(s)
Androgens/pharmacology , Epithelial Cells/drug effects , Prostate/drug effects , Protein Serine-Threonine Kinases/genetics , Receptors, Transforming Growth Factor beta/genetics , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Apoptosis/drug effects , Caspase 3/metabolism , Cells, Cultured , Cyclin D , Cyclins/genetics , Cyclins/metabolism , Cytoprotection/drug effects , Dihydrotestosterone/pharmacology , Down-Regulation/drug effects , Epithelial Cells/metabolism , Humans , Male , Plasminogen Activator Inhibitor 1/genetics , Plasminogen Activator Inhibitor 1/metabolism , Prostate/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/genetics , Transcription, Genetic/drug effects , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta/physiology , bcl-X Protein/genetics , bcl-X Protein/metabolism
5.
J Biol Chem ; 281(12): 7765-74, 2006 Mar 24.
Article in English | MEDLINE | ID: mdl-16428382

ABSTRACT

Transforming growth factor beta (TGF-beta) signals through TGF-beta receptor serine/threonine kinases (TbetaRI and TbetaRII) and Smads, regulating cell growth and apoptosis. Although loss of TGF-beta receptor levels is strongly selected for during the progression of most cancers, tumor cells frequently escape from complete loss of TGF-beta 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-beta in LNCaP prostate adenocarcinoma cells. Our results support that these permissive effects occur through enhanced stability of TbetaRII mRNA and reversal of TGF-beta-mediated TbetaRII mRNA loss. Changes in stability of TbetaRII mRNA occur soon after EGF or TGF-beta1 addition (optimal within 3 h) and are independent of de novo protein synthesis or transcription. Remarkably, such loss of TbetaRII by TGF-beta can be mediated by a kinase-dead TbetaRII (K277R), as well as by other forms of this receptor harboring mutations at prominent autophosphorylation sites. Moreover, Smad3 small interfering RNA, which blocks TGF-beta-induced AP-1 promoter activity, does not block changes in the expression of TbetaRII by EGF or TGF-beta. We have also shown that changes in TbetaRII 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-beta responses during prostate carcinogenesis.


Subject(s)
Epidermal Growth Factor/metabolism , Gene Expression Regulation, Neoplastic , Transforming Growth Factor beta/metabolism , Adenoviridae/genetics , Adenoviridae/metabolism , Blotting, Northern , Blotting, Western , Cell Line, Tumor , Culture Media, Serum-Free/metabolism , Down-Regulation , Gene Transfer Techniques , Humans , Luciferases/metabolism , MAP Kinase Kinase 1/metabolism , Male , Mutation , Phosphorylation , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad Proteins/metabolism , Time Factors , Transcriptional Activation , Transfection , Transforming Growth Factor beta1
6.
EMBO J ; 25(1): 58-69, 2006 Jan 11.
Article in English | MEDLINE | ID: mdl-16362038

ABSTRACT

Insulin-like growth factor-I inhibits transforming growth factor-beta (TGF-beta) 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-beta responses, including S3 activation and induction of apoptosis. Wild-type and myristoylated Akts (Akt(WT) and Akt(Myr)) suppress TGF-beta-induced phospho-activation of S3 but not Smad2 (S2), whereas kinase-dead Akt1 (Akt1K179M) or dominant-negative PI3K enhances TGF-beta-induced phospho-activation of both S2 and S3. Using siRNA, rapamycin (Rap), and adenoviral expression for FKBP12-resistant and constitutively active TGF-beta 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-beta 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-beta in cancers.


Subject(s)
Activin Receptors, Type I/antagonists & inhibitors , Protein Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Smad3 Protein/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Animals , Apoptosis , Cells, Cultured , Humans , Mutation , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Rats , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Sirolimus/pharmacology , Smad2 Protein/metabolism , TOR Serine-Threonine Kinases , Tacrolimus Binding Protein 1A/metabolism , Transforming Growth Factor beta/metabolism
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