Loss of Filamin A Nuclear Localization in Prostate Cancer Progression

 描述(由申请人提供)： 这一新的应用解决了支架蛋白Filamin A(FLNA)在前列腺癌(CAP)进展中的作用，并测试了一种假说，即在CAP的管腔上皮细胞中FLNA核定位的丢失意味着肿瘤对用于治疗去势抵抗前列腺癌(CRPC)的抗雄激素比卡鲁胺、醋酸阿比特龙或苯扎鲁胺具有耐药性。这一假说基于初步的和已发表的数据，该数据表明，在核FLNA存在的情况下，帽细胞和动物模型对抗雄激素产生反应，而在没有核FLNA的情况下，它们不起作用。根据已有的初步数据，我们认为，在核FLNA存在的情况下，一些AR转录靶点，如TMPRSS2、VEGFA、GDF15、IL32和Jun(在RNA-Seq分析中被鉴定为FLNA调节的AR转录靶点)，仅在雄激素刺激下选择性地被转录。由于这些基因调节细胞的存活，这将导致雄激素成瘾，从而使细胞变得容易被抗雄激素诱导凋亡。 在目标1中，我们将确定受FLNA核定位影响的AR靶点。由于其中许多也是转录因子Sp1的靶标，我们假设FLNA通过将AR和选定的转录因子(如Sp1)结合在一起来调节AR靶标的选择性。此外，我们假设FLNA通过与AR铰链区结合从而控制AR的结合伙伴来调节这种选择性和整体转录活性。因此，核FLNA可以通过阻止与鼓励配体非依赖转录活动的辅助激活物的结合来调节雄激素敏感性。这些研究的意义在于，他们将确定在CRPC细胞中，AR是否失去了其靶向特异性，而Flna定位于细胞核能够恢复这种特异性。 在目标2中，我们将调查为什么在一些CRPC肿瘤中FLNA从上皮细胞中丢失，而在其他肿瘤中则不是。Flna的核定位涉及Calpains对该分子的切割，其中一个切割产物移位到细胞核；然而，S2152位的Flna的磷酸化阻止了这些蛋白酶的切割，从而阻止了核定位。因此，我们将确定Flna在细胞核中的表达或缺失，是由于Calain活性和Flna磷酸化之间失去平衡所致。这一假说将通过将患者来源的异种移植瘤系原位移植到免疫受损的NOD-SCID IL-2Rγ缺失(Nsg)小鼠的前列腺中进一步验证，并将确定对这些药物的反应是否与细胞核中flna的表达相关。 最后，在目标3中，我们将确定是否可以使用Flna调节的AR转录活性的靶标来确定患有CRPC的人类患者是否对阿比特龙或苯扎鲁胺有反应。这是必要的，因为CRPC患者通常没有血液以外的组织可供分析，因此，我们将确定这些标记物是否存在于血清或血浆中，以及这些标记物是否与FLNA核定位相关。50名患有CRPC的VA患者的血液将用于这些研究，这些患者将接受比卡鲁胺、阿比特龙+泼尼松或苯扎鲁胺的标准治疗。血液中选定的标志物水平将与这些患者对药物的反应相关。拟议研究的意义在于，它们将为退伍军人管理局医生提供一种工具，以确定患者是否会对比卡鲁胺、阿比特龙或苯扎鲁胺的护理使用标准做出反应。此外，拟议的研究将确定FLNA核定位对AR转录活性的作用，并赋予共同调节因子一个新的角色。此外，拟议的全基因组分析研究将使识别新的AR转录靶点成为可能。

 DESCRIPTION (provided by applicant): This renewal application addresses the role of the scaffolding protein Filamin A (FlnA) in the progression of prostate cancer (CaP), and tests the hypothesis that loss of FlnA nuclear localization in the luminal epithelial cells of CaP signifies resistance of the tumor to anti-androgens bicalutamide, abiraterone acetate or enzalutamide, that are used to treat patients with castration resistant prostate cancer (CRPC). This hypothesis is based on preliminary and published data demonstrating that in the presence of nuclear FlnA, CaP cells and animal models respond to anti-androgens whereas in the absence of nuclear FlnA, they do not respond. Based on available preliminary data, we propose that in the presence of nuclear FlnA, some AR transcriptional targets, such as TMPRSS2, VEGFA, GDF15, IL32, and JUN (which were identified in a RNA-Seq analysis as FlnA-regulated AR transcriptional targets), are selectively targeted for transcription only upon androgen stimulation. Since these genes regulate cell survival, this will cause androgen-addiction, whereby the cells become susceptible to apoptosis induction by anti-androgens. In Aim 1, we will identify targets of AR affected by FlnA nuclear localization. Since many of these are also targets of the transcriptional factor Sp1, we hypothesize that FlnA regulates the selectivity of AR targets by bringing AR together with selected transcriptional factors such as Sp1. Further, we hypothesize that FlnA regulates this selectivity and overall transcriptional activity by binding to the AR hinge region and thereby controlling the binding partners of AR. Therefore, nuclear FlnA can regulate androgen sensitivity by preventing binding to co-activators that encourage ligand-independent transcriptional activity. The significance of these studies is that they will determine whether in CRPC cells, AR loses its target specificity and that FlnA localization to the nucleus is able to restore that specificity. In Aim 2, we will investigate why FlnA is lost from the epithelia in some CRPC tumors and not in others. FlnA nuclear localization involves the cleavage of this molecule by calpains whereby one of the cleaved products translocates to the nucleus; however, phosphorylation of FlnA at S2152 impedes cleavage by these proteases, thereby preventing nuclear localization. Therefore, we will determine whether expression of FlnA in the nucleus or the lack thereof, results from a loss of equilibrium between calpain activity and FlnA phosphorylation. The hypothesis will be further tested using patient derived xenograft (PDX) tumor lines orthotopically implanted in the prostates of immunocompromised NOD-SCID IL-2Rγ-null (NSG) mouse, and will determine whether response to these drugs correlate with the expression of FlnA in the nuclei. Finally, in Aim 3, we will determine whether targets of FlnA-regulated AR transcriptional activity may be used to determine whether human patients with CRPC will respond to abiraterone or enzalutamide. This will be necessary because no tissue other than blood is usually available from CRPC patients for analysis, therefore, we will determine whether such markers exist in the serum or plasma, and whether these markers correlate with FlnA nuclear localization. Blood from 50 VA patients with CRPC, who will be treated with bicalutamide, abiraterone acetate+prednisone or with enzalutamide, as standard-of-care, will be used for these studies. The level of selected markers in the blood will be correlated with the response of these patients to the drugs. The significance of the proposed studies is that they will provide VA physicians with a tool to determine whether a patient will respond to the standard of care use of bicalutamide, abiraterone or enzalutamide. In addition, the proposed studies will determine the role of FlnA nuclear localization on AR transcriptional activity and confer a novel role for a co-regulator. Further, the genome wide analyses studies proposed will enable the identification of novel AR transcriptional targets.