Ack1: A Critical Regulator of Hormone-Refractory Prostate Cancer

描述(申请人提供)：雄激素受体(AR)在前列腺癌的进展中起关键作用。我们最近已经证明，AcK1(也称为TNK2)调节AR Tyr267-反式激活区域内的磷酸化。虽然多个酪氨酸激酶的AR转录激活是AR激活的一种替代方式，但AcK1介导的AR Tyr267-磷酸化在AR募集到雄激素反应增强子(ARE)和雄激素非依赖性AR反应基因转录中的确切作用尚不完全清楚。在这项拨款提案中，我们证明了激活的AcK1(pTyr284-AcK1)的表达随着前列腺癌的进展而上调，并且这种激活与前列腺癌患者的生存呈负相关。由于AcK1通过其Tyr267的磷酸化来调节雄激素非依赖性AR的活性，我们产生了特异性识别pTyr267-AR的抗体。PTyr267-AR的表达和转录激活均不受抗雄激素药物如比卡鲁胺/卡索地塞米松和氟他胺的影响。然而，AcK1的小分子抑制剂4-氨基-5，6-联芳基-呋喃并[2，3-d]嘧啶(YL3-026)不仅能抑制AcK1的激活，而且能抑制pTyr267-AR的磷酸化，与PSA、NKX3.1和TMPRS2启动子结合，并抑制AR转录活性，表现为PSA基因表达显著下降。我们的证据表明，在前列腺癌患者中靶向AcK1激酶可能是一种非常有效的治疗策略。在这项提案中，我们将确定AR Tyr267-磷酸化如何调节雄激素非依赖性生长。进一步，我们将检测AcK1抑制剂YL3-026抑制雄激素非依赖性转录和异种移植瘤形成的能力。此外，我们将评估AcK1抑制剂YL3-026在ProB-AcK1转基因小鼠中抑制AR Tyr267磷酸化和前列腺上皮内瘤(PINs)形成的能力。 公共卫生相关性：荷尔蒙耐药前列腺癌是美国男性癌症死亡的重要原因。在这里，我们证明，随着前列腺癌进展到雄激素非依赖性，激活的AcK1表达上调，并且这种激活与前列腺癌患者(n=267例)的生存呈负相关。使用针对Tyr267-磷酸化AR(pTyr267-AR)的特异性抗体，我们已经证明pTyr267-AR的转录激活不受抗雄激素如比卡鲁胺/肌松和氟他胺的影响。为了准确了解AcK1在无雄激素条件下AR转录激活中的作用，我们合成了4-氨基-5，6-联芳基-呋喃并[2，3-d]嘧啶(简称YL3-026)。YL3-026不仅能抑制AcK1的激活，而且能抑制pTyr267-AR的磷酸化及其与PSA、NKX3.1和TMPRS2启动子的结合，并抑制AR的转录活性，表现为PSA基因表达的显著降低。在这项提案中，我们将识别一组受pTyr267-AR调控的基因。进一步，我们将评估YL3-026对转基因小鼠前列腺上皮内瘤形成的影响。总体而言，这项建议将使我们能够检查针对前列腺癌患者的AcK1激酶是否是一种合适的治疗策略。

DESCRIPTION (provided by applicant): Androgen receptor (AR) plays a critical role in progression of prostate cancer. We have recently demonstrated that Ack1 (also known as TNK2) regulates AR Tyr267-phosphorylation within the transactivation domain. While AR transcriptional activation by multiple tyrosine kinases is emerging as an alternate mode of AR activation, the precise role of Ack1 mediated AR Tyr267- phosphorylation in AR recruitment to the androgen responsive enhancers (ARE) and androgen-independent AR-responsive gene transcription is not fully understood. In this grant proposal we demonstrate that activated Ack1(pTyr284-Ack1) expression is upregulated as prostate cancer progresses and this activation is inversely correlated with the survival of prostate cancer patients. Since Ack1 regulates androgen-independent AR activity by its phosphorylation at Tyr267, we generated antibodies that specifically recognize pTyr267-AR. Neither pTyr267-AR expression nor its transcriptional activation was affected by anti-androgens e.g. bicalutamide/casodex and flutamide. However, a small molecule inhibitor of Ack1, 4-Amino-5,6- biaryl-furo[2,3-d]pyrimidine (YL3-026) not only inhibited Ack1 activation, but was able to suppress pTyr267-AR phosphorylation, binding to PSA, NKX3.1 and TMPRS2 promoters and inhibit AR transcription activity as seen by significant decrease in PSA gene expression. Our evidence indicates that targeting Ack1 kinase in prostate cancer patients could therefore be highly effective therapeutic strategy. In this proposal we will determine how AR Tyr267-phosphorylation regulates Androgen-Independent growth. Further, we will examine the ability of Ack1 inhibitor, YL3-026, to suppress androgen-independent transcription and xenograft tumor formation. Moreover, we will assess the ability of Ack1 inhibitor YL3-026 to suppress AR Tyr267-phosphorylation and prostatic intraepithelial neoplasia (PINs) formation in Prob-Ack1 transgenic mice. PUBLIC HEALTH RELEVANCE: A Hormone refractory prostate cancer is a significant cause of cancer death among American men. Here we demonstrate that that activated Ack1 expression is upregulated as prostate cancer progresses to androgen independence and this activation is inversely correlated with the survival of prostate cancer patients (n=267 patients). Using specific antibodies against Tyr267-phosphorylated AR (pTyr267-AR), we have shown that transcriptional activation of pTyr267-AR was unaffected by anti-androgens e.g. bicalutamide/casodex and flutamide. To precisely understand role of Ack1 in AR transcriptional activation in absence of androgens, we synthesized 4-Amino-5,6-biaryl-furo[2,3- d]pyrimidine (termed here as YL3-026). YL3-026 not only inhibited Ack1 activation, but was able to suppress pTyr267-AR phosphorylation, its binding to PSA, NKX3.1 and TMPRS2 promoters and inhibit AR transcription activity as seen by significant decrease in PSA gene expression. In this proposal we will identify a set of genes that are regulated by pTyr267-AR. Further, we shall assess effect of YL3-026 on formation of prostatic intraepithelial neoplasia in transgenic mice. Overall, this proposal would allow us to examine whether targeting Ack1 kinase in prostate cancer patients is an appropriate therapeutic strategy.