Hormone-regulated Prostate Cancer Metastasis

描述(申请人提供)：一种新的观点支持类固醇激素，就像许多肽生长因子一样，可能同时具有促分裂和促运动的特性。这一观点得到了最近发现的支持，即蜕皮激素调节果蝇胚胎边界细胞的入侵。这些结果表明，类固醇激素可以刺激侵袭行为，而不是对细胞增殖的任何可察觉的影响。肿瘤细胞的运动性是局部疾病向转移发展的关键决定因素。最近发现一种新的p21激活蛋白6(PAK6)，它是一种雄激素受体(AR)相互作用蛋白。PAK6的表达仅限于激素敏感的组织，包括前列腺、睾丸和胎盘。初步研究表明，PAK6与AR之间的相互作用是雄激素依赖性事件。最重要的是，我们最近的研究结果表明，pak6的表达升高与前列腺癌的转移进展有关。有趣的是，在正常和恶性上皮细胞中，PAK6的亚细胞定位似乎是不同的；而在正常上皮细胞中，PAK6定位于细胞膜，而在癌细胞中观察到强烈的胞浆染色。虽然PAK6似乎不是调节AR转录活性的辅助因子，但我们实验室的结果表明，它在调节激素依赖型前列腺癌细胞的运动和生存方面发挥了作用。PAK6结构活性突变体的异位表达促进了人前列腺癌LNCaP细胞的细胞运动和不依赖锚定的软琼脂细胞生长。相反，PAK6显性负性激酶死亡突变体的表达抑制了细胞的运动和软琼脂集落的生长。最有趣的是，这些结果进一步表明，PAK6和AR之间的相互作用至少在一定程度上导致了前列腺癌细胞中PAK6的激活，以响应雄激素的刺激。我们的模型预测，雄激素受体和PAK6之间的相互作用可能在控制激素依赖型前列腺癌转移中发挥关键作用。假设：我们的工作假设是雄激素依赖的AR介导的PAK6激活促进了前列腺癌激素调节的转移表型。本研究的目的是明确PAK6在雄激素受体介导的前列腺癌进展中的生理作用。具体目的和研究设计：目的1：确定AR-PAK6相互作用在调节雄激素刺激的PAK6激活中的结构-功能需求。这一目的是为了在分子水平上表征AR和PAK6之间的相互作用。这一目标的重点是确定PAK6是如何通过与AR相互作用来响应雄激素刺激而被激活的。两个分子的相互作用区域将被绘制出来，并将确定对相互作用起作用的关键残基。将使用传统的分子生物学技术、哺乳动物双杂交分析和激酶分析。目的2：鉴定和鉴定PAK6下游促进细胞运动和侵袭的分子靶点。将使用串联亲和标记PAK6和串联质谱学的蛋白质组学方法来识别PAK6下游的候选靶标。目的：利用裸鼠移植瘤模型，研究PAK6激活对LNCaP细胞成瘤和转移的影响。相关性：PAK6的激活是雄激素刺激的AR介导的事件，这表明它是干预激素调节的前列腺癌转移的潜在靶点。充分剖析信号通路上的分子相互作用将促进我们开发控制雄激素调节的前列腺癌转移的新方法的长期目标。 公共卫生相关性：尽管临床证据表明雄激素促进前列腺癌细胞的转移进展，但其潜在机制仍不清楚。目前的建议旨在研究一种新的信号分子PAK6，它在控制雄激素刺激的细胞运动中起主导作用。PAK6的激活被确认为雄激素刺激的AR介导的事件，这表明它是干预激素调节的前列腺癌转移的潜在靶点。充分剖析这一信号通路上的分子相互作用将有助于理解雄激素信号与前列腺癌转移进展之间的基础生物学。

DESCRIPTION (provided by applicant): An emerging view supports that steroid hormones, like many peptide growth factors, may possess both mitogenic and motogenic properties. This notion is backed by the recent finding that ecdysone regulates the embryonic border cell invasion in Drosophila. These results demonstrate that steroid hormones can stimulate invasive behavior independent of any discernable effects on cell proliferation. Tumor cell motility is a critical determinant in the progression of localized disease to metastasis. A novel p21-activated kinase 6 (PAK6) was recently identified to be an androgen receptor (AR) interacting protein. The expression of PAK6 is restricted to the hormone-sensitive tissues including prostate, testis and placenta. Initial characterization indicates that the interaction between PAK6 and AR is an androgen-dependent event. Most importantly, our recent results demonstrate an elevated expression of Pak6 associated with prostate cancer metastatic progression. Intriguingly, subcellular localization of PAK6 appears to be heterogeneous between normal and malignant epithelial; while PAK6 localized to the cell membrane in normal epithelia, intense cytoplasmic staining was observed in carcinoma cells. Although PAK6 does not appear to function as a cofactor in modulating AR transcriptional activity, results from our lab indicate its role in regulating hormone-dependent prostate cancer cell motility and survival. Ectopic expression of a PAK6 constitutive active mutant promotes cell motility and anchorage-independent soft agar cell growth of human prostate cancer LNCap cells. Conversely, expression of a PAK6 dominant negative kinase dead mutant inhibits cell motility and soft agar colony growth. Most intriguingly, these results further indicate that the interaction between PAK6 and AR is responsible, at least in part, for the activation of PAK6 in response to androgen stimulation in prostate cancer cells. Our model predicts that interaction between androgen receptor and PAK6 may play a pivotal role in controlling hormone-dependent prostate cancer metastasis. Hypothesis: Our working hypothesis is that androgen-dependent AR-mediated PAK6 activation promotes hormone-regulated metastatic phenotypes in prostate cancer. The goal of the current proposal is to define the physiological role of PAK6 in androgen receptor mediated prostate cancer progression. Specific Aims and Study Design: Aim 1: To determine the structural-functional requirement of the AR-PAK6 interaction in regulating androgen-stimulated PAK6 activation. This aim is designed to characterize the interaction between AR and PAK6 at the molecular level. The focus of this aim is to determine how PAK6 is activated by interacting with AR in response to androgen stimulation. The interacting domains of the two molecules will be mapped and critical residues contributing to the interaction will be identified. Conventional molecular biological techniques, mammalian two-hybrid assays and kinase assays will be employed. Aim 2: to identify and characterize molecular targets downstream of PAK6 pertinent to promote cell motility and invasion. A proteomic approach employing tandem affinity tagged PAK6 and tandem mass spectrometry will be used to identify PAK6 downstream candidate targets. Aim 3: To characterize the effect of PAK6 activation on LNCap cell tumorigenicity and metastasis using a nude mouse xenograft model. Relevance: The identification of PAK6 activation as an androgen-stimulated AR- mediated event suggests a potential target for intervention of hormone regulated prostate cancer metastasis. Fully dissecting the molecular interactions along the signal pathway will foster our long-term goal of development of new modality to control androgen-regulated prostate cancer metastasis. PUBLIC HEALTH RELEVANCE: Despite clinical evidence indicating that androgen promotes the metastatic progression of prostate cancer cells, the underlying mechanism remains unclear. The current proposal aims at studying a novel signal molecule PAK6 that functions as a dominant factor in controlling androgen-stimulated cell motility. The identification of PAK6 activation as an androgen-stimulated AR-mediated event suggests a potential target for intervention of hormone-regulated prostate cancer metastasis. Fully dissecting the molecular interactions along this signal pathway will fulfill the programmatic interest of understanding the basic biology between androgenic signals and prostate cancer metastatic progression.