RAl GTPases and partners in human bladder cancer metastasis

膀胱癌每年导致13，000名美国人死亡，但很少有研究项目针对这种疾病。这些死亡大多数是由于转移性扩散，通常是肺部。更全面地了解膀胱癌生长和扩散的分子机制可能会提供新的治疗机会。RalA和RalB是两种同源的GTP酶，在人膀胱癌移植瘤的生长和肺转移中起重要作用。此外，这些旁系同源物通过Aurora-A和PKC调节的位点处的磷酸化激活，Aurora-A和PKC是与人膀胱癌生长和进展相关的两种激酶。为了确定Ral下游的信号传导途径，进行Ral依赖性基因表达的全基因组分析，并揭示RalA和RalB调节CD 24的表达，CD 24是一种GPI连接的糖蛋白和膀胱癌中转移发展的生物标志物。此外，该数据鉴定了锌指转录因子RREB 1作为CD 24表达的推定调节剂，并且RalBP 1（两种Ral旁系同源物的效应物）作为RREB 1活性的调节剂。基于这些数据，我们提出了指导假设，即RalA和RalB调节由RalBP 1、RREB 1和CD 24组成的新的下游信号传导途径，通过该途径，它们有助于膀胱癌生长和肺转移，此外，RalA和RalB活化的生物标志物如磷酸化或转录特征是患者膀胱癌转移发展的预后。三个特定目的检验该假设：在目的1中，将使用磷酸失活突变体确定RalA和RalB磷酸化在膀胱癌生长、迁移和肺转移中的作用。将在来自患有局部晚期膀胱癌的患者的组织中评价Ral副磷酸化与转移发展的预后相关性。目的2将确定RalBP 1如何调节RREB 1活性以及RREB 1如何反过来调节CD 24表达。由于Ral依赖性基因表达的全基因组分析导致了CD 24的发现，我们将使用来自Aim 1的组织和先进的计算工具来产生Ral表达的特异性特征。将评价这些特征预测患者转移性疾病发展的能力。在目标3中，我们评估了CD 24对膀胱癌发生和进展的需求。将使用CD 24敲除小鼠研究CD 24在化学诱导的膀胱癌发生和/或随后的所得肿瘤的侵袭和转移中的必要性。一项单独的研究将测试抗CD 24抗体是否可以抑制已建立的膀胱癌肺转移的生长。建立Ral磷酸化与膀胱癌肺转移的生物学相关性以及RalBP 1、RREB 1和CD 24之间的调节联系为治疗提供了机会。与该机制工作相关的转化研究提供了患者转移发展的生物标志物预测因子，而临床前研究为针对已确定的转移性疾病的新型疗法的近期开发提供了基础。膀胱癌每年导致13，000名美国人死亡，但很少有研究项目针对这种疾病。对于这些患者中的大多数，死亡原因可归因于转移性扩散，通常转移至肺部。该项目的目标是了解人类膀胱癌肺转移的机制，并利用这些知识来预测和治疗患者的这种情况。

DESCRIPTION (provided by applicant): Bladder cancer kills 13,000 Americans each year, but very few research projects target this disease. Most of these deaths are due to metastatic spread, commonly to the lungs. A fuller understanding of the molecular mechanisms driving growth and dissemination of bladder cancer is likely to provide new therapeutic opportunities. RalA and RalB, two homologous GTPases play an important role in growth and lung metastasis in xenograft models of human bladder cancer. Furthermore, these paralogs are activated by phosphorylation at sites regulated by Aurora-A and PKC, two kinases associated with human bladder cancer growth and progression. To define signaling pathways downstream of Ral, genome-wide analysis of Ral dependent gene expression was carried out and revealed that RalA and RalB regulate the expression of CD24, a GPI-linked glycoprotein and biomarker of metastasis development in bladder cancer. Moreover, this data identified the zinc finger transcription factor RREB1 as a putative regulator of CD24 expression and RalBP1, an effector of both Ral paralogs, as a regulator of RREB1 activity. Based on this data, we formulate the Guiding Hypothesis that RalA and RalB regulate a new downstream signaling pathway consisting of RalBP1, RREB1 and CD24 via which they contribute to bladder cancer growth and lung metastasis and furthermore, that biomarkers of RalA and RalB activation such as phosphorylation or transcriptional signatures are prognostic for development of bladder cancer metastasis in patients. Three Specific Aims test this hypothesis: In Aim 1, the role of RalA and RalB phosphorylation in bladder cancer growth, migration and lung metastasis will be determined using phospho inactive mutants. The prognostic relevance of Ral paralog phosphorylation to metastasis development will be evaluated in tissues from patients with locally advanced bladder cancer. Aim 2 will determine how RalBP1 regulates RREB1 activity and how RREB1 in turn, regulates CD24 expression. Since genome-wide profiling of Ral dependent gene expression led to the discovery of CD24, we will use tissues from Aim 1 and advanced computational tools to generate paralog specific signatures of Ral expression. The ability of these signatures to predict the development of metastatic disease in patients will be evaluated. In Aim 3 we evaluate the requirement of CD24 for bladder carcinogenesis and progression. A CD24 knockout mouse will be used to study the necessity of CD24 in chemically induced bladder carcinogenesis and/or subsequent invasion and metastasis of resultant tumors. A separate study will test whether anti-CD24 antibodies can inhibit growth of established bladder cancer lung metastases. Establishing the biological relevance of Ral phosphorylation to bladder cancer lung metastasis and the regulatory connections between RalBP1, RREB1 and CD24 provide opportunities for therapy. Translational studies associated with this mechanistic work provide biomarker predictors for metastasis development in patients while a preclinical study provides a foundation for the near term development of novel therapies directed at established metastatic disease. PUBLIC HEALTH RELEVANCE: Bladder cancer kills 13,000 Americans each year, but few research projects 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 condition in patients.