Understanding the RhoGDI2 metastasis suppressor gene

描述(申请人提供)：在所有患者中，有一半的局部晚期膀胱癌发展为致命的转移性疾病，通常是肺部转移。使用临床和实验室方法，我们确定了一种新的分子途径，调节转移性肺定植(微转移到临床疾病的生长)，其成分在人类疾病中发生改变。该通路涉及RhoGDI2(ARHGDIB，Ly-GDI)、c-Src、rac1、ET-1和versican。对不同肺转移能力的谱系相关的人膀胱癌细胞系进行微阵列分析，结合对不同阶段的人膀胱癌的分析，确定RhoGDI2是转移定植的候选抑制因子。我们随后发现，RhoGDI2蛋白表达降低是膀胱癌患者转移发展的独立预测因子。我们还发现，c-Src部分通过磷酸化RhoGDI2来调节这一途径，这增加了它作为肺定植抑制因子的效力。与这些数据一致的是，c-Src的表达随着膀胱癌的分期而降低，并且这种降低与RhoGDI2的表达降低似乎是互斥的。RhoGDI2和其他RhoGDi一样，结合和抑制Rho家族的GTP酶。对这些蛋白质的研究得出了令人惊讶的结果，即RhoGDI2抑制转移与rac1的激活有关。在同时进行的工作中，为了寻找这一途径的效应者，基因阵列研究引导我们确定内皮素-1和万西肯是由RhoGDI2下调和在高阶段人类膀胱癌中上调的mRNAs。这两种蛋白都是已知的巨噬细胞趋化剂，其中一种蛋白的耗尽减少了巨噬细胞向转移性膀胱癌细胞的迁移。RhoGDI2在膀胱癌细胞中的重新表达也与小鼠肺转移的巨噬细胞浸润减少有关。由于肿瘤相关巨噬细胞(TAMs)可促进多种类型肿瘤的转移定植，内皮素-1和维西康对巨噬细胞的募集可能介导了RhoGDI2降低的细胞的转移生长增强。因此，我们的指导假设是，活性的RhoGDI2通过下调内皮素-1和versican的表达来抑制转移定植，从而减少肿瘤相关巨噬细胞向转移部位的募集。为了验证这一假说，为晚期膀胱癌开发新的预后工具，并为患者确定新的治疗靶点，提出了具体的目标。目的1：评价RhoGDI2作为肿瘤转移的调节和预测因子的作用。目的2：阐明RhoGDI2激活rac1抑制转移的机制；目的3：评价RhoGDI2抑制转移的效应分子。这些目标的完成将有助于临床有用的知识，并为抑制膀胱癌转移的新途径提供机制上的见解。这样做还将为转移抑制蛋白如何通过影响天然免疫发挥作用提供一个新的范例，并促进旨在为膀胱癌患者开发合理治疗方法的翻译方法。 公共卫生相关性：膀胱癌每年导致1.3万美国人死亡，每个患者从诊断到死亡的成本约为15万美元，是所有癌症中最大的，但不幸的是，针对这种疾病的研究资源极少。对于这些患者中的大多数，死亡原因可归因于转移性扩散，通常是肺转移。该项目的目标是了解人类膀胱癌肺转移的潜在机制，并利用这一知识预测和治疗患者的这种致命疾病。

DESCRIPTION (provided by applicant): Locally advanced bladder cancer develops into lethal metastatic disease, commonly to the lung, in half of all patients. Using both clinical and laboratory methodologies, we identified a new molecular pathway that regulates metastatic lung colonization (growth of micrometastases into clinical disease), components of which are altered in human disease. This pathway involves RhoGDI2 (ARHGDIB, Ly-GDI), c-Src, Rac1, endothelin-1 and versican. Microarray profiling of lineage related human bladder cancer cell lines of differing lung metastatic ability coupled with analysis of human bladder cancers at different stages identified RhoGDI2 as a candidate inhibitor of metastatic colonization. We subsequently found decreased protein expression of RhoGDI2 is an independent predictor of metastasis development in patients with bladder cancer. We also found that c-Src regulates this pathway in part by phosphorylating RhoGDI2, which increases its potency as a suppressor of lung colonization. Consistent with these data, c-Src expression decreases as a function of bladder tumor stage, and this decrease appears mutually exclusive with reduced RhoGDI2 expression. RhoGDI2, like other RhoGDIs, binds and inhibits Rho family GTPases. Investigation of these proteins yielded the surprising result that inhibition of metastases by RhoGDI2 correlated with activation of Rac1. In concurrent work, aimed at finding effectors of this pathway, gene array studies led us to identify endothelin-1 and versican as mRNAs downregulated by RhoGDI2 and upregulated in high stage human bladder cancer. Both are known macrophage chemoattractants and depletion of either protein reduced macrophage migration toward metastatic bladder cancer cells. RhoGDI2 re-expression in bladder cancer cells was also associated with decreased macrophage infiltration of lung metastases in mice. Since tumor associated macrophages (TAMs) have been found to promote metastatic colonization in many types of cancer, recruitment of macrophages by endothelin-1 and versican may mediate enhancement of metastatic growth in cells with decreased RhoGDI2. Hence, our Guiding Hypothesis is that active RhoGDI2 inhibits metastatic colonization by downregulating endothelin-1 and versican expression, which reduces tumor associated macrophage recruitment to the metastatic site. Specific Aims are proposed to test this hypothesis, develop novel prognostic tools for advanced bladder cancer and identify new targets for therapy in patients. Aim 1: Evaluate the role of RhoGDI2 as a regulator and predictor of metastasis. Aim 2: Elucidate the mechanism by which RhoGDI2 activates Rac1 to inhibit metastasis; Aim 3: Evaluate effectors of metastasis suppression by RhoGDI2. Completion of these aims will contribute clinically useful knowledge as well as providing mechanistic insights into a novel pathway that suppresses bladder cancer metastasis. Doing so will also provide a new paradigm of how metastasis suppressor proteins can function by affecting innate immunity and facilitate translational approaches aimed at development of rational therapies for patients with bladder cancer. PUBLIC HEALTH RELEVANCE: Bladder cancer kills 13,000 Americans each year and is associated with a cost per patient from diagnosis to death of ~$150K, the greatest of any cancer but, unfortunately, disproportionately few research resources are targeted to this disease. For most of these patients, the cause of death is attributable to metastatic spread, commonly to the lungs. The goal of this project is to understand the mechanisms that underlie lung metastasis in human bladder cancer and use this knowledge to predict and treat this lethal condition in patients.