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命的转移性疾病，通常转移到肺部，在所有患者中有一半。使用临床和实验室方法，我们确定了一种调节转移性肺定植（微转移到临床疾病的生长）的新分子途径，其成分在人类疾病中发生改变。该途径涉及RhoGDI2 (ARHGDIB, Ly-GDI), c-Src, Rac1，内皮素-1和versican。对不同肺转移能力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，发现RhoGDI2是转移定落的候选抑制剂。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，RhoGDI2磷酸化RhoGDI2增加了其作为肺定植抑制因子的效力。与这些数据一致，c-Src表达随着膀胱肿瘤分期的变化而降低，并且这种降低与RhoGDI2表达的降低是相互排斥的。RhoGDI2与其他rhogdi一样，结合并抑制Rho家族gtpase。对这些蛋白的研究得出了令人惊讶的结果，RhoGDI2对转移的抑制与Rac1的激活相关。同时，为了寻找这一通路的效应物，我们通过基因阵列研究确定了内皮素-1和versican是在高阶段人类膀胱癌中被RhoGDI2下调和上调的mrna。这两种蛋白都是已知的巨噬细胞趋化剂，其中一种蛋白的缺失会减少巨噬细胞向转移性膀胱癌细胞的迁移。RhoGDI2在膀胱癌细胞中的再表达也与小鼠肺转移灶巨噬细胞浸润减少有关。由于肿瘤相关巨噬细胞（tumor associated macrophages, tam）已被发现在许多类型的癌症中促进转移定植，内皮素-1和versican募集巨噬细胞可能介导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RhoGDI2通过下调内皮素-1和versican的表达来抑制转移性定植，从而减少肿瘤相关巨噬细胞向转移部位的募集。我们提出了具体的目标来验证这一假设，为晚期膀胱癌患者开发新的预后工具，并确定新的治疗靶点。目的1：评估RhoGDI2作为转移调节因子和预测因子的作用。目的2：阐明RhoGDI2激活Rac1抑制肿瘤转移的机制；目的3：评价RhoGDI2抑制肿瘤转移的效应。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见解。这样做也将为转移抑制蛋白如何通过影响先天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转化方法。

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. 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.