RESUMO
Androgen receptor (AR) activation is critical for prostate cancer (PCa) development and progression, including castration resistance. The nuclear export signal of AR (NESAR) has an important role in AR intracellular trafficking and proteasome-dependent degradation. Here, we identified the RNA helicase DHX15 as a novel AR co-activator using a yeast mutagenesis screen and revealed that DHX15 regulates AR activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity. DHX15 and Siah2 form a complex with AR, through NESAR. DHX15 stabilized Siah2 and enhanced its E3 ubiquitin-ligase activity, resulting in AR activation. Importantly, DHX15 was upregulated in PCa specimens and its expression was correlated with Gleason scores and prostate-specific antigen recurrence. Furthermore, DHX15 immunostaining correlated with Siah2. Finally, DHX15 knockdown inhibited the growth of C4-2 prostate tumor xenografts in mice. Collectively, our data argue that DHX15 enhances AR transcriptional activity and contributes to PCa progression through Siah2.
Assuntos
Recidiva Local de Neoplasia/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , RNA Helicases/metabolismo , Receptores Androgênicos/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos SCID , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/patologia , Sinais de Exportação Nuclear/genética , Proteínas Nucleares/metabolismo , Próstata/patologia , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , RNA Helicases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor signaling in prostate cancers. ELL-associated factor 2 (EAF2) is an androgen-regulated tumor suppressor and its intracellular localization can be modulated by ultraviolet light, suggesting a potential role for EAF2 in androgen regulation of DNA repair in prostate cancer cells. Here we show that knockdown of EAF2 or its homolog EAF1 sensitized prostate cancer cells to DNA damage and the sensitization did not require p53. EAF2 knockout mouse prostate was also sensitized to γ-irradiation. Furthermore, EAF2 knockdown blocked androgen repression of LNCaP or C4-2 cells from doxorubicin induction of γH2ax, a DNA damage marker. In human prostate cancer specimens, EAF2 expression was inversely correlated with the level of γH2ax. Further analysis showed that EAF2 and EAF1 are required for the recruitment and retention of Ku70/Ku80 to DNA damage sites and play a functional role in nonhomologous end-joining DNA repair. These findings provide evidence for EAF2 as a key factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.
Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Androgênios/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Autoantígeno Ku/genética , Masculino , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismoRESUMO
Multiple genetic alterations are associated with prostate carcinogenesis. Tumor-suppressor genes phosphatase and tensin homolog deleted on chromosome 10 (Pten) and androgen upregulated gene 19 (U19), which encodes ELL-associated factor 2 (EAF2), are frequently inactivated or downregulated in advanced prostate cancers. Previous studies showed that EAF2 knockout caused tumors in multiple organs and prostatic intraepithelial neoplasia (PIN) in mice. However, EAF2-knockout mice did not develop prostate cancer even at 2 years of age. To further define the roles of EAF2 in prostate carcinogenesis, we crossed the Pten+/- and EAF2+/- mice in the C57/BL6 background to generate EAF2-/-Pten+/-, Pten+/-, EAF2-/- and wild-type mice. The prostates from virgin male mice with the above four genotypes were analyzed at 7 weeks, 19 weeks and 12 months of age. Concomitant loss of EAF2 function and inactivation of one Pten allele induced spontaneous prostate cancer in 33% of the mice. Prostatic tissues from intact EAF2-/- Pten+/- mice exhibited higher levels of phospho-Akt, -p44/42 and microvessel density. Moreover, phospho-Akt remained high after castration. Consistently, there was a synergistic increase in prostate epithelial proliferation in both intact and castrated EAF2-/-Pten+/- mice. Using laser-capture microdissection coupled with real-time reverse transcription-PCR, we confirmed that co-downregulation of EAF2 and Pten occurred in >50% clinical prostate cancer specimens with Gleason scores of 8-9 (n=11), which is associated with poor prognosis. The above findings together demonstrated synergistic functional interactions and clinical relevance of concurrent EAF2 and Pten downregulation in prostate carcinogenesis.
Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Transativadores/genética , Alelos , Animais , Carcinogênese/patologia , Modelos Animais de Doenças , Regulação para Baixo , Deleção de Genes , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microvasos/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Gradação de Tumores , Próstata/irrigação sanguínea , Próstata/enzimologia , Próstata/patologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Inactivation of U19/EAF2 has been shown previously to lead to tumorigenesis in multiple organs; however, the mechanism of U19/EAF2 tumor suppression remains unclear. In this paper, we report that the expression of an anti-angiogenic protein, thrombospondin-1 (TSP-1) is down-regulated in the prostate and liver of U19/EAF2 knockout mouse. The U19/EAF2 knockout liver displayed increased CD31-positive blood vessels, suggesting that the TSP-1 down-regulation can contribute to increased angiogenesis. TSP-1 is reported to be a p53-target gene and p53 is a known binding partner of ELL, which binds to U19/EAF2. Here, we show that U19/EAF2 can co-localize and co-immunoprecipitate with p53 in transfected cells. In a TSP-1 promoter-driven luciferase reporter assay, p53 transfection suppressed the TSP-1 promoter activity and U19/EAF2 co-transfection blocked the p53 suppression of TSP-1 promoter. However, U19/EAF2 transfection alone had little or no effect on the TSP-1 promoter. The above observations together suggest that U19/EAF2 regulates the expression of TSP-1 via blocking p53 repression of the TSP-1 promoter.
