RAl GTPases and partners in human bladder cancer metastasis

描述（由申请人提供）：膀胱癌每年导致13000名美国人死亡，但很少有研究项目针对这种疾病。这些死亡大多是由于转移性扩散，通常是肺部。对驱动膀胱癌生长和传播的分子机制的更全面的了解可能会提供新的治疗机会。RalA和RalB两种同源gtpase在人膀胱癌异种移植模型的生长和肺转移中起重要作用。此外，这些类似物是由Aurora-A和PKC调控位点的磷酸化激活的，这两种激酶与人类膀胱癌的生长和进展有关。为了确定Ral下游的信号通路，我们对Ral依赖基因的表达进行了全基因组分析，发现RalA和RalB调节CD24的表达，CD24是一种gpi相关的糖蛋白和膀胱癌转移发展的生物标志物。此外，这些数据表明锌指转录因子RREB1可能是CD24表达的调节剂，而RalBP1是RREB1活性的调节剂，RalBP1是两种RREB1的效应因子。基于这些数据，我们提出了RalA和RalB调节由RalBP1、RREB1和CD24组成的新的下游信号通路，参与膀胱癌生长和肺转移的指导假设，并且RalA和RalB激活的生物标志物如磷酸化或转录特征是膀胱癌患者转移发展的预后。三个特定目标验证了这一假设：在Aim 1中，RalA和RalB磷酸化在膀胱癌生长、迁移和肺转移中的作用将通过磷酸化失活突变体来确定。将在局部晚期膀胱癌患者的组织中评估Ral平行磷酸化与转移发展的预后相关性。Aim 2将确定RalBP1如何调节RREB1活性，以及RREB1如何反过来调节CD24的表达。由于Ral依赖基因表达的全基因组分析导致了CD24的发现，我们将使用来自Aim 1的组织和先进的计算工具来生成Ral表达的平行特异性签名。这些特征预测患者转移性疾病发展的能力将被评估。在目的3中，我们评估了膀胱癌发生和发展对CD24的要求。CD24敲除小鼠将用于研究CD24在化学诱导膀胱癌发生和/或随后的肿瘤侵袭和转移中的必要性。一项单独的研究将测试抗cd24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RalBP1、RREB1和CD24之间的调节联系为治疗提供了机会。与此机制工作相关的转化研究为患者转移发展提供了生物标志物预测指标，而临床前研究为近期开发针对已确定转移性疾病的新疗法提供了基础。公共卫生相关性：膀胱癌每年导致13000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，通常是肺部。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.