RESUMO
Androgens play a central role in prostate development and prostate cancer proliferation. Induction of telomerase is an early event in prostate carcinogenesis and is considered as a marker for both primary tumors and metastases. Interestingly, several reports suggest that telomerase activity is regulated by androgens in vivo. Here, we show that the wild-type (WT) human androgen receptor (AR) inhibits the expression of the human telomerase reverse transcriptase (hTERT) and telomerase activity via inhibition of hTERT promoter activity in the presence of androgen receptor agonists. However, pure androgen antagonists failed to repress hTERT transcription. The androgen-mediated repression of hTERT is abrogated in a human prostate cancer cell line exhibiting hormone-dependent growth, which expresses a mutant AR (T877A) frequently occurring in prostate cancer. We reveal that this single amino acid exchange is sufficient for the lack of transrepression. Interestingly, chromatin immunoprecipitation data suggest that, in contrast to the WT AR, the mutant AR is recruited less efficiently to the hTERT promoter in vivo, indicating that loss of transrepression results from reduced chromatin recruitment. Thus, our findings suggest that the WT AR inhibits expression of hTERT, which is indicative of a protective mechanism, whereas the T877A mutation of AR not only broadens the ligand spectrum of the receptor but abrogates this inhibitory mechanism in prostate cancer cells. This novel role of AR mutations in prostate cancer development suggests the benefit to a search for new AR antagonists that inhibit transactivation but allow transrepression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Telomerase/genética , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Subunidades Proteicas/genética , Telomerase/metabolismo , Ativação TranscricionalRESUMO
Recently, using a proteomic approach we have identified the corepressor Alien as a novel interacting factor of the cell cycle regulator E2F1. Unclear was whether this interaction influences cell proliferation and endogenous E2F1 target gene expression. Here, we show by chromatin immunoprecipitation (ChIP) that Alien is recruited in vivo to the E2F binding sites present in the E2F1 gene promoter, inhibits the transactivation of E2F1 and represses endogenous E2F1 gene expression. Interestingly, using synchronized cells to assess the expression of Alien profile during cell cycle the levels of endogenous Alien are increased during G1, G1/S and G2 phase. Furthermore, stable transfection of Alien leads to reduction of cell proliferation. Thus, the data suggest that Alien acts as a corepressor for E2F1 and is involved in cell cycle regulation.
Assuntos
Proliferação de Células , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/fisiologia , Expressão Gênica , Proteínas Repressoras/fisiologia , Sítios de Ligação , Complexo do Signalossomo COP9 , Ciclo Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Fator de Transcrição E2F1/genética , Fase G1/fisiologia , Fase G2/fisiologia , Células HeLa , Humanos , Masculino , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Ligação Proteica , Fase S/fisiologia , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
Prostate cancer cell growth is initially androgen dependent. Androgen antagonists are used in prostate cancer therapy to inactivate the transcriptional activity of the human androgen receptor (hAR) and to inhibit the proliferation of prostate cancer. Here, we have characterized Alien with characteristics of a corepressor as a novel interacting factor for the antagonist bound hAR. Alien is recruited to hAR in the presence of the AR antagonist cyproterone acetate (CPA). The interaction of Alien with hAR is verified in vivo and in vitro by a modified mammalian two-hybrid system, coimmunoprecipitation, chromatin immunoprecipitation, and in vitro binding assays. In contrast to other nuclear receptors, Alien binds to the amino-terminus of hAR with the receptor SUMOylation (small ubiquitin modifier) sites being involved. Furthermore, cellular localization of Alien is changed towards a predominant nuclear localization upon treatment of prostate cancer cells with CPA. Notably, stable expression of Alien in LNCaP cells inhibits both endogenous prostate-specific antigen expression and proliferation of these cells in the presence of CPA but not in the presence of an AR agonist. These findings underline the importance of corepressors for inhibition of prostate cancer cell growth by androgen antagonists.
Assuntos
Acetato de Ciproterona/farmacologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/fisiologia , Antagonistas de Androgênios/farmacologia , Androgênios/fisiologia , Divisão Celular/efeitos dos fármacos , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Neoplasias da Próstata/prevenção & controle , RNA Mensageiro/genética , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The Sin3 proteins are evolutionarily conserved co-repressors (CoR) that function as mediators of gene repression for a variety of transcriptional silencers. The paired amphipathic helices of Sin3A were identified and studied as protein-protein interacting domains. Previously we have shown the interaction of Sin3A with the CoR Alien in vivo and in vitro. Here, we show that Alien and Sin3A reside together in vivo with the vitamin D3 receptor on the human 24-hydroxylase (CYP24) promoter containing vitamin D3 response elements by chromatin immunoprecipitation. We delineated and characterized the interaction domains of Sin3A with Alien. Interestingly, the highly conserved region (HCR) of Sin3A, which has not yet been functionally characterized, interacts with Alien. The HCR encompasses only 134 amino acids, shares more than 80% identity with Sin3B and binds to the N-terminus of Alien, which harbours a transferable silencing function. Functionally, co-expression of Sin3A enhances Alien-mediated gene repression and overexpression of the HCR alone leads to the inhibition of Alien-mediated repression and to the induction of the endogenous CYP24 promoter. Our results therefore indicate a novel functional role of the Sin3 HCR and give novel insights into Alien-mediated gene repression.
Assuntos
Sequência Conservada , Inativação Gênica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Complexo do Signalossomo COP9 , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Elementos de Resposta/genética , Complexo Correpressor Histona Desacetilase e Sin3 , Esteroide Hidroxilases/genética , Vitamina D3 24-HidroxilaseRESUMO
The repression of the androgen receptor (AR) activity is a major objective to inhibit prostate cancer growth. One underlying mechanism for efficient hormone therapy is based on corepressors that inactivate the AR. In line with this, castration-resistant prostate cancer is associated with malfunction or reduced corepressor action. To overcome this, the overexpression of endogenous corepressors, however, affects many other transcription factors. Therefore, an AR-specific corepressor could be of advantage. Using a yeast peptide aptamer two-hybrid screen with the full-length human AR, we identified a short amino acid-stretch that binds specifically to the human AR in yeast and in mammalian cells and not to the closely related progesterone or glucocorticoid receptors. Furthermore, fused to a silencing domain, this aptamer-based corepressor (AB-CoR) exhibits corepressor activity by inhibiting both the AR-mediated transactivation and expression of the AR target gene PSA. Furthermore, stable expression of the AB-CoR inhibits growth of human LNCaP prostate cancer cells. Moreover, we generated a cell-permeable AB-CoR by fusing a protein transduction domain to establish a vector-free transport system. Treatment of LNCaP cells with the bacterially expressed and affinity-purified cell-permeable AB-CoR peptide resulted in a significant inhibition of both AR-mediated transactivation and prostate cancer cell proliferation. Thus, generation of a novel AR-specific aptamer-based corepressor may present a vector-free inhibition of AR-dependent prostate cancer growth as a novel approach.