Loss of Filamin A Nuclear Localization in Prostate Cancer Progression

 描述（由申请人提供）： 本更新申请阐述了支架蛋白细丝蛋白A（FlnA）在前列腺癌（CaP）进展中的作用，并检验了CaP腔上皮细胞中FlnA核定位缺失表示肿瘤对抗雄激素比卡鲁胺、醋酸阿比特龙或恩杂鲁胺的抗性的假设，这些药物用于治疗去势抵抗性前列腺癌（CRPC）患者。 该假设基于初步和公开的数据，这些数据表明，在存在核FlnA的情况下，CaP细胞和动物模型对抗雄激素有反应，而在不存在核FlnA的情况下，它们没有反应。基于现有的初步数据，我们提出，在核FlnA的存在下，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），仅在雄激素刺激时被选择性地靶向转录。由于这些基因调节细胞存活，这将导致雄激素成瘾，由此细胞变得容易受到抗雄激素诱导的凋亡。 在目标1中，我们将鉴定受FlnA核定位影响的AR靶点。由于其中许多也是转录因子Sp1的靶点，我们假设FlnA通过将AR与选定的转录因子如Sp1结合在一起来调节AR靶点的选择性。此外，我们假设FlnA通过与AR铰链区结合从而控制AR的结合伴侣来调节这种选择性和总体转录活性。因此，核FlnA可以通过阻止与促进配体非依赖性转录活性的共激活因子结合来调节雄激素敏感性。这些研究的意义在于，它们将确定在CRPC细胞中，AR是否失去其靶向特异性，以及FlnA定位于细胞核是否能够恢复该特异性。 在目标2中，我们将研究为什么FlnA在一些CRPC肿瘤中从上皮细胞中丢失，而在其他肿瘤中没有。 FlnA核定位涉及钙蛋白酶对该分子的切割，由此切割产物之一易位至细胞核;然而，FlnA在S2152处的磷酸化阻碍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乏是否是由于钙蛋白酶活性和FlnA磷酸化之间平衡的丧失。将使用原位植入免疫受损NOD-SCID IL-2R γ-null（NSG）小鼠前列腺中的患者来源的异种移植物（PDX）肿瘤系进一步测试该假设，并将确定对这些药物的应答是否与细胞核中FlnA的表达相关。 最后，在目标3中，我们将确定FlnA调节的AR转录活性的靶点是否可用于确定CRPC人类患者是否对阿比特龙或恩杂鲁胺有反应。这将是必要的，因为除了血液，通常没有其他组织可从CRPC患者进行分析，因此，我们将确定这些标志物是否存在于血清或血浆中，以及这些标志物是否与FlnA核定位相关。这些研究将使用50例接受比卡鲁胺、醋酸阿比特龙+泼尼松或Enzalutamide标准治疗的VA CRPC患者的血液。血液中选定标志物的水平将与这些患者对药物的反应相关。拟定研究的意义在于，它们将为VA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Enzalutamide的标准治疗产生应答。此外，拟议的研究将确定FlnA核定位对AR转录活性的作用，并赋予一个新的作用的辅助调节。此外，提出的全基因组分析研究将能够鉴定新的AR转录靶点。

 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.