Transgelin Functions As A Suppressor Via Inhibition Of ARA54-enhanced Androgen Receptor Transactivation And Prostate Cancer Cell Growth

Introduction and objectiveProstate cancer is the second leading cause of malignancy in aging males. Androgen and its receptor play pivotal roles in the carcinogenesis and progression of prostate cancer. Surgical or chemical castration in combination with antiandrogens, known as complete androgen blockade, has been widely used in the treatment of this disease. However, androgen ablation therapy eventually fails, and prostate cancer progresses to a hormone refractory state in spite of consistent androgen receptor (AR) expression in the majority of patients in this androgen independent stage. Dissecting the precise molecular mechanism of how androgen receptor signaling is regulated and how it contributes to the prostate cancer progression may, therefore, greatly help in battling this disease.The androgen receptor requires coregulators for its optimal function. Several androgen receptor coregulators have been identified and their potential pathophysiological roles in the prostate cancer progression have been studied. ARA54 is an important coregulator of androgen receptor. It enhances androgen receptor function in a ligand-dependent manner and co-expression of ARA54 with other androgen receptor coactivators like SRC-1 or ARA70 additively enhances androgen receptor function.The detailed mechanisms of the androgen receptor transcriptional machinery still remain to be further elucidated. It is unknown whether these androgen receptor coregulators need interacting proteins to modulate their coregulator activity. The objective of this study is to explore the effect of putative interacting protein(s) of coactivator ARA54 on the transcativation of androgen receptor and the growth of prostate cancer cell lines.Materials and methodsA fusion protein containing GAL4 DNA-binding domain and full length of ARA54 was used as bait to screen 1.26×10~6 transformants of human prostate cDNA library. The full-length cDNA of transgelin was generated by PCR using the marathon-ready cDNA library as template. Co-immunoprecipitation was further applied to confirm its interaction with ARA54. Transient transfection and reporter assay were used to study the effect of transgelin on the ARA54-enhanced androgen receptor signal pathway. The molecular mechanism of its suppressive effect on androgen receptor transactivation was further explored by immunofluorescent staining and mammalian two-hybrid assay. The stable transfection of transgelin resulted in the suppression of LNCaP cell growth and its prostate-specific antigen expression. Finally, the expression of transgelin in the normal and malignant prostate tissue was assayed using immunohistochemistry and tissue microarray.ResultsHere we describe transgelin as the first ARA54-associated negative modulator for androgen receptor. Transgelin suppressed ARA54-enhanced androgen receptor function in ARA54-positive but not in ARA54-negative cells. Transgelin suppressed androgen receptor transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of androgen receptor and ARA54. Stable transfection of transgelin in LNCaP cells suppressed androgen receptor-mediated cell growth and prostate-specific antigen expression, while this suppressive effect was abolished by the addition of ARA54-siRNA. Results from tissue surveysshowing decreased expression of transgelin in prostate cancer specimens further strengthened the suppressor role of transgelin.Conclusions1. Transgelin is an ARA54-associated secondary modulator for androgen receptor.2. Transgelin suppressed androgen receptor transactivation via interruption of ARA54 homodimerization and AR-ARA54 heterodimerization, resulting in the cytoplasmic retention of androgen receptor and ARA54.3. Transgelin suppressed androgen receptor transactivation and prostate cancer cell growth.4. The expression level of transgelin was significantly lower in the prostate cancer tissue than the normal prostate tissue.5. The expression level of ARA54 was significantly higher in the prostate cancer tissue than the normal prostate tissue.6. Androgen receptor coregulators, like ARA54, may have dual in vivo roles to both function as a coactivator directly and as a mediator for its interacting protein's influence on the androgen receptor function indirectly.