Ack1: A Critical Regulator of Hormone-Refractory Prostate Cancer

描述（由申请人提供）：雄激素受体（AR）在前列腺癌进展中起关键作用。我们最近已经证明，Ack 1（也称为TNK 2）调节反式激活结构域内的AR Tyr 267-磷酸化。虽然多种酪氨酸激酶的AR转录激活正在成为AR激活的替代模式，但Ack 1介导的AR Tyr 267-磷酸化在AR募集至雄激素应答增强子（ARE）和雄激素非依赖性AR应答基因转录中的确切作用尚未完全了解。在这项资助提案中，我们证明了激活的Ack 1（pTyr 284-Ack 1）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率呈负相关。由于Ack 1通过其在Tyr 267的磷酸化调节雄激素非依赖性AR活性，我们产生了特异性识别pTyr 267-AR的抗体。pTyr 267-AR表达及其转录激活均不受抗雄激素药物（如比卡鲁胺/casodex和氟替卡松）的影响。然而，Ack 1，4-氨基-5，6-联芳基-呋喃并[2，3-d]嘧啶（YL 3 -026）的小分子抑制剂不仅抑制Ack 1活化，而且能够抑制pTyr 267-AR磷酸化，与PSA、NKX3.1和TMPRS 2启动子结合，并抑制AR转录活性，如PSA基因表达显著降低所示。我们的证据表明，靶向前列腺癌患者的Ack 1激酶可能是非常有效的治疗策略。在这个提议中，我们将确定AR Tyr 267-磷酸化如何调节雄激素非依赖性生长。此外，我们将检查Ack 1抑制剂YL 3 -026抑制雄激素非依赖性转录和异种移植肿瘤形成的能力。此外，我们将评估Ack 1抑制剂YL 3 -026抑制Prob-Ack 1转基因小鼠中AR Tyr 267磷酸化和前列腺上皮内瘤形成（PIN）的能力。 公共卫生相关性：激素难治性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，激活的Ack 1表达上调前列腺癌进展到雄激素非依赖性，这种激活与前列腺癌患者的生存率呈负相关（n=267例）。使用针对Tyr 267-磷酸化AR（pTyr 267-AR）的特异性抗体，我们已经证明pTyr 267-AR的转录激活不受抗雄激素（例如比卡鲁胺/casodex和氟替卡松）的影响。为了精确地理解Ack 1在雄激素不存在下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（在此称为YL 3 -026）。YL 3 -026不仅抑制Ack 1活化，而且能够抑制pTyr 267-AR磷酸化，其与PSA、NKX3.1和TMPRS 2启动子的结合，并抑制AR转录活性，如PSA基因表达的显著降低所示。在这个建议中，我们将确定一组基因的调控pTyr 267-AR。此外，我们将评估YL 3 -026对转基因小鼠中前列腺上皮内瘤形成的影响。总的来说，这项建议将使我们能够检查靶向前列腺癌患者的Ack 1激酶是否是一种适当的治疗策略。

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.