Loss of Filamin A Nuclear Localization in Prostate Cancer Progression

描述(由申请人提供)： 项目摘要/摘要前列腺癌被列为大约16%的老年男性VHA使用者的主要诊断，这对这些人的发病率和死亡率有很大影响。大多数患有转移性前列腺癌的VA患者，与美国大多数非VA患者一样，接受激素治疗，这种治疗最初是有效的，但经常失败，表明发生了去势抵抗前列腺癌(CRPC)。尽管许多临床试验测试了多种药物，但激素治疗失败的患者的治疗选择有限。因此，我们的实验室采取了创新的整体策略，即确定可以防止前列腺癌进展到去势抵抗的治疗靶点，而不是在前列腺癌已经发展之后试图治愈它。在之前的资助期间进行的研究，导致了14篇相关主题的出版物，有助于理解细胞生存调节因子Akt在前列腺癌发生和发展中的重要性。虽然一些Akt抑制剂已经商业化开发，但在人类患者身上使用时，由于诱导包括非肿瘤前列腺区在内的正常组织中的细胞凋亡，其有效性受到了损害。因此，该项目的一个目标是确定Akt的天然拮抗剂，这些拮抗剂可以选择性地抵消肿瘤中Akt磷酸化增加的影响，而不影响其他细胞中Akt激活的基础水平。我们最近发表的两篇论文表明，丝氨酸A(Flna)可能就是这样的一种蛋白质。体外研究表明，Flna定位于细胞核可导致前列腺癌细胞的去势敏感生长，这种生长是通过拮抗Akt磷酸化及其下游效应来实现的。Flna的核定位受其磷酸化的调节，而磷酸化又受PKA活性的调节。FLNA的核定位效应可能是由其与雄激素受体(AR)的相互作用引起的，AR是一种核类固醇受体，介导雄激素对细胞机制的影响。因此，在目前的应用中，我们将研究核FLNA在调节去势敏感细胞行为中所起的作用。我们假设Flna抵消了Akt磷酸化及其下游效应器的影响，其定位反过来又受其磷酸化的调节。以下目标将检验这一假设。具体目的1：通过阻止Akt对AR活性和细胞存活的影响，验证Fla16-24核定位通过抑制AKT对AR活性和细胞存活的影响而使前列腺癌细胞对雄激素戒断敏感的假设。我们将确定FlnA16-24是否通过拮抗Akt依赖的信号通路和阻止AR/TIF2相互作用来诱导CRPC细胞对雄激素阻断的敏感性。我们还将研究Flna 16-24的核定位是否通过抑制NF-kB的激活和刺激AR依赖的p21Cip1转录来诱导细胞凋亡。具体目的2：验证假设，在去势抵抗的前列腺癌中，Flna的磷酸化是阻止Flna核定位的关键因素。我们将研究在表达功能性AR的前列腺癌细胞系中，FLNA的磷酸化是否阻止了FLNA的核定位，并导致了去势抵抗。我们还将确定抑制Flna磷酸化是否能阻止复发前列腺癌小鼠模型的去势抵抗的发展。具体目的3：验证假设，在未接受根治性前列腺癌切除术的VA转移性前列腺癌患者中，FLNA磷酸化增加和核定位减少预测治疗失败的时间。我们已经确定了北加州退伍军人管理局系统内表现为转移性前列腺癌的患者。我们将在这些患者的前列腺活检组织中检测细丝素A、Flna磷酸化、AR水平和Akt磷酸化的定位，并确定他们是否对雄激素停药治疗有反应。

DESCRIPTION (provided by applicant): Project Summary/Abstract Prostate cancer is listed as a primary diagnosis for approximately 16% of older male VHA users, and contributes significantly to morbidity and mortality rates in these individuals. The majority of VA patients with metastatic prostate cancer, similar to most non-VA patients in the United States, receives hormonal treatment which is initially effective, but frequently fail indicative of the development of castration resistant prostate cancer (CRPC). The treatment options for patients who fail hormonal therapy are limited, despite numerous clinical trials testing multiple drugs. Hence, our laboratory has undertaken the innovative overall strategy of identifying therapeutic targets that can prevent prostate cancer progression to castration resistance, rather than trying to cure it after it has already developed. Studies performed during the previous funding period, which resulted in 14 publications in related topics, contributed to understanding the importance of the cell survival regulator Akt in prostate cancer development and progression. While a number of Akt inhibitors have been developed commercially, their utility has been compromised by high levels of toxicity when used in human patients, caused by the induction of apoptosis in normal tissue including non-tumor prostate regions. As a result, a goal of the project was to identify natural antagonists of Akt that selectively counteract the effects of the increase in Akt phosphorylation in the tumor without affecting basal levels of Akt activation in other cells. Two of our recent publications demonstrate that Filamin A (FlnA) may be one such protein. In vitro studies showed that FlnA localization to the nucleus resulted in castration sensitive growth of prostate cancer cells which was mediated by antagonism of Akt phosphorylation and its downstream effectors. FlnA nuclear localization is regulated by its phosphorylation, which in turn is regulated by PKA activity. The effects of FlnA nuclear localization are likely caused by its interaction with the androgen receptor (AR), a nuclear steroid receptor mediating the effects of the androgens on cell mechanisms. Therefore, in the current application, we will investigate the role nuclear FlnA plays in regulating castration sensitive cell behavior. We hypothesize that FlnA counters the effects of Akt phosphorylation and its downstream effectors, and that its localization is in turn regulated by its phosphorylation. The following aims will test this hypothesis. Specific Aim 1: To test the hypothesis that FlnA 16-24 nuclear localization sensitizes prostate cancer cells to androgen withdrawal by preventing the effects of Akt on AR activity and cell survival. We will determine whether FlnA16-24 induces sensitivity to androgen blockade in CRPC cells by antagonizing an Akt- dependent signaling pathway and preventing AR/TIF2 interactions. We will also investigate whether nuclear localization of FlnA 16-24 induces apoptosis by repressing NF-kB activation and stimulating AR-dependent p21Cip1 transcription. Specific Aim 2: To test the hypothesis that FlnA phosphorylation is a key factor responsible for preventing FlnA nuclear localization in castrate resistant prostate cancer. We will examine whether FlnA phosphorylation prevents FlnA nuclear localization and causes castration resistance in prostate cancer cell lines expressing a functional AR. We will also determine whether inhibition of FlnA phosphorylation prevent the development of castration resistance in a mouse model of recurrent prostate cancer. Specific Aim 3: To test the hypothesis that increased FlnA phosphorylation and decreased FlnA nuclear localization predict time to treatment failure in VA patients with metastatic prostate cancer on androgen withdrawal therapy who did not undergo radical prostatectomy. We have identified patients within the VA Northern California System who presented with metastatic prostate cancer. We will determine in biopsies of the prostates of these patients the localization of Filamin A, FlnA phosphorylation, AR levels and Akt phosphorylation and determine whether they respond to androgen withdrawal therapy.