Regulation of androgen receptor activity in prostate cancer cells in an aged mous

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，也是与年龄关系最密切的人类恶性肿瘤之一。晚期前列腺癌最常见的治疗方法之一是通过化学或物理阉割来剥夺雄激素。不幸的是，大多数患者复发并继续经历衰弱的骨痛、泌尿系统症状和性功能障碍。在这个阶段，癌症被称为雄激素不依赖型或去势抵抗型（CRPC）；然而，研究表明，这一阶段的癌症仍然依赖于雄激素受体（AR）信号。这种增强AR转录活性的蛋白之一是Vav3，一种Rho GTPase鸟嘌呤核苷酸交换因子。在体外和体内，在雄激素不依赖型人前列腺癌和Nkx3.1中，vav3均上调；Pten突变小鼠衰老相关性前列腺癌模型。Vav3在生理和亚纳摩尔雄激素浓度下增强AR活性，与CRPC中显示的前列腺微环境水平相似。因此，Vav3水平的升高似乎在雄激素非依赖性癌症中持续的AR信号传导中起着关键作用。在雄激素存在的情况下，Vav3增强AR转录活性需要Vav3 pleckstrin同源结构域（PH），而不是鸟嘌呤核苷酸交换因子（GEF）活性。此外，初步数据表明，PH结构域的突变导致Vav3被排除在细胞核之外，无法招募到AR靶基因中的雄激素反应元件。另一方面，组成活性Vav3已被证明通过GEF活性和Rho GTPase Rac1增强配体非依赖性AR激活。通过利用功能分离Vav3突变体的方法，可以确定不依赖于配体的Vav3 GEF活性和依赖于配体的Vav3 AR共激活的贡献。本研究将各种Vav3突变体稳定转染到Vav3缺陷前列腺癌细胞系LNCaP中，并检测其基因表达谱，以确定Vav3增强AR转录活性的分子途径。然后将Vav3功能分离突变体原位注射到免疫功能低下小鼠的前列腺中，以便更好地重建前列腺微环境。为了研究不同的AR激活途径在年龄相关性前列腺肿瘤形成和转移中的作用，我们将评估这些细胞在体外和体内的侵袭和细胞生长的差异。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Vav3缺失小鼠交配后进行检查，以确定Vav3信号在该模型中的贡献。在Vav3稳定转染的各种形式的前列腺癌细胞中，已确定的前哨基因表达途径上调或下调，将与Nkx3.1的结果表达模式进行比较；Pten和Nkx；Vav3敲除小鼠模型。此外，通过将基因谱鉴定的前哨基因簇与突变小鼠年龄依赖性肿瘤的预后结果进行比较，可能会发现新的Vav3信号通路，从而发现新的诊断标志物。

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.