Loss of Filamin A Nuclear Localization in Prostate Cancer Progression

描述（由申请人提供）： 前列腺癌被列为大约16%的老年男性VHA使用者的主要诊断，并显著增加这些个体的发病率和死亡率。大多数患有转移性前列腺癌的VA患者，与美国的大多数非VA患者相似，接受激素治疗，最初有效，但经常失败，表明发生去势抵抗性前列腺癌（CRPC）。激素治疗失败的患者的治疗选择是有限的，尽管有许多临床试验测试多种药物。因此，我们的实验室已经采取了创新的整体策略，即确定可以防止前列腺癌进展为去势抵抗的治疗靶点，而不是在它已经发展之后试图治愈它。在上一个资助期进行的研究，导致14篇相关主题的出版物，有助于理解细胞存活调节剂Akt在前列腺癌发展和进展中的重要性。虽然已经商业开发了许多Akt抑制剂，但是当用于人类患者时，它们的效用已经被高水平的毒性所损害，所述高水平的毒性是由在正常组织（包括非肿瘤前列腺区域）中诱导细胞凋亡引起的。因此，该项目的目标是鉴定Akt的天然拮抗剂，其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活化的基础水平。我们最近的两个出版物表明，细丝蛋白A（FlnA）可能是这样一种蛋白质。体外研究表明，FlnA定位于细胞核导致前列腺癌细胞的去势敏感性生长，这是通过拮抗Akt磷酸化及其下游效应物介导的。FlnA核定位受其磷酸化调节，磷酸化又受PKA活性调节。FlnA核定位的作用可能是由其与雄激素受体（AR）的相互作用引起的，雄激素受体是一种核类固醇受体，介导雄激素对细胞机制的作用。因此，在本申请中，我们将研究核FlnA在调节去势敏感细胞行为中的作用。我们假设FlnA对抗Akt磷酸化及其下游效应物的影响，并且其定位反过来受其磷酸化调节。以下目标将检验这一假设。具体目标1：检验FlnA 16-24核定位通过阻止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FlnA 16 -24是否通过拮抗Akt依赖性信号传导途径和阻止AR/TIF 2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-κ B活化和刺激AR依赖性p21 Cip 1转录诱导细胞凋亡。具体目标二：验证FlnA磷酸化是阻止去势抵抗性前列腺癌中FlnA核定位的关键因素的假设。我们将研究是否FlnA磷酸化阻止FlnA核定位，并导致去势抵抗前列腺癌细胞系表达功能性AR。我们还将确定是否抑制FlnA磷酸化防止去势抵抗的发展，在复发性前列腺癌的小鼠模型。具体目标3：检验FlnA磷酸化增加和FlnA核定位减少预测未接受根治性前列腺切除术的转移性前列腺癌VA患者雄激素戒断治疗的至治疗失败时间的假设。我们已经确定了VA北方加州系统内的转移性前列腺癌患者。我们将在这些患者的前列腺活检中确定细丝蛋白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戒断治疗有反应。 公共卫生相关性： 前列腺癌影响大约16%的65岁或以上的男性VA患者，其中许多人发展为转移性前列腺癌并接受激素治疗。这种治疗最初是有效的，但最终失败;因此，本项目的总体目标是确定治疗的目标，以防止治疗失败。在上一个资助期内，我们在14篇发表的论文中表明，Akt蛋白促进了对激素治疗的失败反应，而另一种蛋白质细丝蛋白A如果存在于细胞核中，可以对抗这种作用。因此，在目前的应用中，我们将确定FIlamin A如何发挥这种作用，以及现有的药物是否可以用于维持FIlamin A在细胞核中的作用。此外，我们将确定细丝蛋白A，Akt和其他蛋白质在患有转移性前列腺癌的VA患者的前列腺中的表达，这些患者没有手术切除前列腺，是否可以帮助预测患者是否会对激素治疗产生反应，从而通过防止不必要的治疗来节省VA的时间，金钱和精力。

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. PUBLIC HEALTH RELEVANCE: Project Narrative Prostate cancer affects approximately 16% of male VA patients age 65 or older, and many of them develop metastatic prostate cancer and are placed on hormonal therapy. This treatment is initially effective but ultimately fails; hence the overall goal of this project is to identify targets of therapy to prevent treatment failure. During the last funding period, we showed in 14 published papers that the protein Akt promotes failure to respond to hormonal therapy, and that another protein, Filamin A, can counter this effect if it is present in the nucleus. Therefore, in the current application, we will determine how FIlamin A has that effect and whether existing drugs can be used to maintain FIlamin A in the nucleus. Further, we will determine whether the expression of Filamin A, Akt and other proteins in the prostate of VA patients with metastatic prostate cancer, who did not have surgery to remove the prostate, can help predict whether the patients will or will not respond to hormonal therapy, thereby saving the VA time, money and effort by preventing needless therapy.