Understanding the RhoGDI2 metastasis suppressor gene

描述（由申请人提供）：局部晚期膀胱癌发展为致死性转移性疾病，通常转移至肺部，占所有患者的一半。使用临床和实验室方法，我们确定了一个新的分子途径，调节转移性肺定植（微转移生长为临床疾病），其中的组成部分在人类疾病中发生改变。该通路涉及RhoGDI 2（ARHGDIB，Ly-GDI）、c-Src、Rac 1、内皮素-1和多功能蛋白聚糖。不同肺转移能力的谱系相关人膀胱癌细胞系的微阵列分析结合不同阶段人膀胱癌的分析鉴定RhoGDI 2为转移性定殖的候选抑制剂。我们随后发现RhoGDI 2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RhoGDI 2来调节该途径，这增加了其作为肺定植抑制剂的效力。与这些数据一致，c-Src表达作为膀胱肿瘤分期的函数而降低，并且这种降低似乎与RhoGDI 2表达降低相互排斥。RhoGDI 2与其他RhoGDIs一样，结合并抑制Rho家族GTP酶。对这些蛋白质的研究产生了令人惊讶的结果，即RhoGDI 2对转移的抑制与Rac 1的激活相关。在同时进行的工作中，旨在寻找该途径的效应子，基因阵列研究使我们鉴定出内皮素-1和versican作为RhoGDI 2下调的mRNA，并在高阶段人膀胱癌中上调。两者都是已知的巨噬细胞化学引诱物，并且任一蛋白质的消耗减少了巨噬细胞向转移性膀胱癌细胞的迁移。RhoGDI 2在膀胱癌细胞中的再表达也与小鼠肺转移的巨噬细胞浸润减少相关。由于已经发现肿瘤相关巨噬细胞（TAM）促进许多类型癌症中的转移性定植，因此内皮素-1和versican对巨噬细胞的募集可能介导RhoGDI 2降低的细胞中转移性生长的增强。因此，我们的指导假设是活性RhoGDI 2通过下调内皮素-1和versican表达抑制转移性定植，这减少了肿瘤相关巨噬细胞向转移部位的募集。具体目标是测试这一假设，开发新的晚期膀胱癌的预后工具，并确定新的治疗靶点。目的1：评估RhoGDI 2作为转移的调节剂和预测剂的作用。目的2：阐明RhoGDI 2激活Rac 1抑制肿瘤转移的机制;目的3：评价RhoGDI 2抑制肿瘤转移的效应因子。这些目标的完成将有助于临床有用的知识，以及提供一个新的途径，抑制膀胱癌转移的机制的见解。这样做还将提供一个新的范例，转移抑制蛋白如何通过影响先天免疫发挥作用，并促进旨在为膀胱癌患者开发合理疗法的翻译方法。

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.