Abstract
Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.
Highlights
Prostate cancer remains the second leading cause of cancer death among American men due to the progression after androgen deprivation therapy of initially hormone dependent prostate cancer
Immunoblotting confirmed that protein levels of truncated androgen receptor (AR) withY267F and Y363F mutations were similar to that of wild type truncated AR (Fig 1C), all three truncated AR proteins were expressed at lower levels than full length AR
The potential functional significance of the N-terminal phosphorylation sites Tyr267 and Tyr-363 in ligand-dependent and—independent activation of AR was investigated by characterizing the effect of tyrosine to phenylalanine substitution mutants in the context of full length and truncated AR
Summary
Prostate cancer remains the second leading cause of cancer death among American men due to the progression after androgen deprivation therapy of initially hormone dependent prostate cancer. Current evidence indicates that reactivation of androgen receptor (AR) in tumor cells may play a critical role in the development of castration resistant prostate cancer (CRPC) [1]. Multiple mechanisms of AR activation in CRPC tumor cells have been characterized. These include increased expression of AR mRNA, AR gene amplification, and point mutations in AR [2,3,4,5]. Modest overexpression of AR in prostate cancer cells was sufficient to promote the castrate resistant growth of xenograft tumors [2]. Intratumoral de novo biosynthesis of androgen in CRPC tumor cells may provide for ligand-dependent activation of AR [3,7,8]. Emergence of constitutively active AR variants may mediate growth and progression of prostate cancer in the castrate host and may confer resistance to new potent antiandrogens [13,14]
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