Regulation of androgen receptor in prostate cancer cells in an aged mouse model

描述（由申请人提供）：前列腺癌是美国最常见的非皮肤恶性肿瘤，是美国男性癌症相关死亡的第二大原因，并且与任何人类恶性肿瘤的年龄关系最密切。晚期前列腺癌最常见的治疗方法之一是通过化学或物理去势剥夺雄激素。不幸的是，大多数患者复发并继续经历使人衰弱的骨痛、泌尿系统症状和性功能障碍。在这个阶段，癌症被称为雄激素非依赖性或去势抵抗（CRPC）;然而，已经表明，这个阶段的癌症仍然依赖于雄激素受体（AR）信号传导。与这种增强的AR转录活性有关的蛋白质之一是Vav 3，一种Rho GT鸟嘌呤核苷酸交换因子。在雄激素非依赖性人前列腺癌和年龄相关性前列腺癌的Nkx3.1;Pten突变小鼠模型中，Vav 3在体外和体内均上调。Vav 3在生理和亚纳摩尔雄激素浓度下均增强AR活性，与CRPC中证实的前列腺微环境水平相似。因此，Vav 3水平升高似乎在雄激素非依赖性癌症中持续的AR信号传导中是关键的。在雄激素的存在下，Vav 3增强AR转录活性需要Vav 3普列克底物蛋白同源（PH）结构域，但不需要鸟嘌呤核苷酸交换因子（GEF）活性。此外，初步数据表明，PH结构域中的突变导致Vav 3被排除在细胞核之外，并且不能募集到AR靶基因中的雄激素应答元件。另一方面，组成型活性的Vav 3已显示通过GEF活性和Rho GTdR Rac 1增强配体非依赖性AR活化。通过利用功能分离的Vav 3突变体的方法，可以确定AR的配体非依赖性Vav 3 GEF活性和配体依赖性Vav 3共活化的贡献。在这项研究中，各种Vav 3突变体将稳定转染到Vav 3缺陷型前列腺癌细胞系LNCaP和他们的基因表达谱将被检查，以确定通过Vav 3增强AR转录活性的分子途径。然后将Vav 3功能分离突变体原位注射到免疫受损小鼠的前列腺中，以更好地重建前列腺微环境。将评价这些细胞的体外和体内侵袭和细胞生长的差异，以检查不同的AR激活途径在年龄相关的前列腺肿瘤形成和转移中的作用。此外，Nkx3.1; Pten突变小鼠是衰老依赖性前列腺癌的忠实模型，将在与可用的Vav 3缺失小鼠交配后进行检查，以确定Vav 3信号传导在该模型中的贡献。将鉴定的在各种形式的Vav 3稳定转染的前列腺癌细胞中上调或下调的哨兵基因表达途径与Nkx3.1;Pten和Nkx;Pten; Vav 3敲除小鼠模型中所得的表达模式进行比较。此外，通过将通过遗传分析鉴定的哨兵基因簇与突变小鼠中年龄依赖性肿瘤的预后结果进行比较，可以鉴定新的Vav 3信号传导途径，从而发现新的诊断标志物。 公共卫生相关性：前列腺癌是美国最常见的非皮肤恶性肿瘤，是美国男性癌症相关死亡的第二大原因，并且是与任何人类恶性肿瘤年龄关系最密切的疾病之一。雄激素受体（AR）及其共激活蛋白在增加前列腺癌的恶性程度、转移潜力和使人衰弱的身体症状中起关键作用。这项研究试图描绘AR激活蛋白（Vav 3）增加前列腺癌恶性程度的能力背后的途径，最终目的是为前列腺癌药物和前列腺癌诊断标志物提供新的靶点。

DESCRIPTION (provided by applicant): Prostate cancer is the most common non-dermatological malignancy in the United States, the second leading cause of cancer-related death in American males, and has one of the strongest relationships with age of any human malignancy. One of the most common therapies for advanced prostate cancer is androgen deprivation via chemical or physical castration. Unfortunately, most patients relapse and continue to experience debilitating bone pain, urinary symptoms, and sexual dysfunction. At this stage, the cancer is termed androgen-independent or castration resistant (CRPC); however, it has been shown that this stage of cancer remains dependent on androgen receptor (AR) signaling. One of the proteins implicated in this enhanced AR transcriptional activity is Vav3, a Rho GTPase guanine nucleotide exchange factor. Vav 3 is up-regulated both in vitro and in vivo in androgen-independent human prostate cancer and in the Nkx3.1;Pten mutant mouse model of age-related prostate cancer. Vav3 enhances AR activity at both physiological as well as at subnanomolar androgen concentrations, similar to prostatic microenvironment levels demonstrated in CRPC. Thus, elevated levels of Vav3 seem to be pivotal in the continued AR signaling in androgen-independent cancers. In the presence of androgen, Vav3 potentiation of AR transcriptional activity requires the Vav3 pleckstrin homology (PH) domain, but not guanine nucleotide exchange factor (GEF) activity. Furthermore, preliminary data suggests that a mutation in the PH domain causes Vav3 to be excluded from nuclei and incapable of recruitment to the androgen responsive element in an AR target gene. On the other hand, constitutively active Vav3 has been shown to enhance ligand-independent AR activation via GEF activity and the Rho GTPase Rac1. Through an approach utilizing separation-of-function Vav3 mutants, contributions of ligand-independent Vav3 GEF activity and ligand-dependent Vav3 coactivation of AR can be determined. In this study, various Vav3 mutants will be stably transfected into the Vav3 deficient prostate cancer cell line LNCaP and their gene expression profiles will be examined in order to identify the molecular pathways through which Vav3 enhances AR transcriptional activity. The Vav3 separation-of-function mutants will then be orthotopically injected into the prostates of immunocompromised mice in order to better recreate the prostatic microenvironment. These cells will be evaluated for differences of in vitro and in vivo invasion and cell growth in order to examine the role of distinct AR activation pathways in age-related prostate tumor formation and metastasis. In addition, Nkx3.1;Pten mutant mice, which are faithful models of aging-dependent prostate cancer, will be examined following mating with available Vav3 null mice to define the contribution of Vav3 signaling in this model. Identified sentinel gene expression pathways that are up or down-regulated in various forms of Vav3 stably transfected prostate cancer cells will be compared to the resulting expression patterns in both the Nkx3.1;Pten and Nkx;Pten;Vav3 knock out mouse models. Furthermore, by comparing the sentinel gene clusters identified via genetic profiling to the prognostic outcomes of the age-dependent tumors in the mutant mice, novel Vav3 signaling pathways may be identified and thus novel diagnostic markers uncovered. PUBLIC HEALTH RELEVANCE: Prostate cancer is the most common non-dermatological malignancy in the United States, the second leading cause of cancer-related death in American males, and has one of the strongest relationships with age of any human malignancy. Androgen receptor (AR) and its co-activating proteins play a key role in increasing prostate cancer's malignancy, metastatic potential and debilitating physical symptoms. This research endeavors to delineate the pathways behind the ability of an AR activating protein (Vav3) to increase the malignancy of prostate cancer with the eventual aim of providing a new target for a prostate cancer drug and a prostate cancer diagnostic marker.