Function of the Ras Related Ral Protein

描述(由申请人提供)：这项建议的总体目标是利用我们对ral信号级联的理解来揭示它如何在肿瘤细胞和邻近的间质成纤维细胞中促进癌症。Rala和RalB是Ras超家族的成员，它们被一组不同的鸟嘌呤核苷酸交换因子激活，如RalGDS，以响应各种细胞外信号。一旦被激活，Ral蛋白就会影响一组独特的下游信号分子，这些分子调控多种细胞过程，包括囊泡运输、细胞凋亡、细胞迁移和细胞增殖。RAL信号级联的一个关键特性是它受到RAS蛋白的刺激。在许多研究中，包括那些涉及RalGDS基因敲除小鼠的研究中，Ral信号级联支持RAS诱导的致癌转化，但涉及的机制尚不清楚。因此，人们对揭示RAR信号级联如何导致癌症产生了浓厚的兴趣。为了了解Ral GTP酶在皮肤鳞状细胞癌中的作用，激活的RAS通常是一个重要的组成部分，我们使用了一个人类皮肤的生物工程组织模型，它允许我们操纵上皮和间质间隔中的Ral信号级联。我们发现Rala在RAS介导的鳞状细胞癌中起着细胞类型依赖的作用。在上皮的角质形成细胞中，Rala抑制而不是支持肿瘤的发生，因为抑制Rala的表达至少部分是通过促进细胞侵袭性，通过其效应蛋白Exo84和降低E-钙粘附素的稳定性来促进肿瘤的进展。此外，在这个模型系统中，肿瘤的进展与Rala水平的下调有关，并且需要下调。RalB基因下调通过促进角质形成细胞的增殖来补充Rala抑制的作用。具体目标1将阐明Rala和RalB如何发挥这些令人惊讶的肿瘤抑制活性，并揭示肿瘤进展如何下调细胞中的Rala水平以允许肿瘤进展。在真皮成纤维细胞中，Rala具有相反的功能。它支持肿瘤的发生，因为这些细胞中的Rala基因敲除会阻止邻近上皮细胞的侵袭特性。具体目标2将揭示这一惊人现象背后的机制，并检验这一假设，即RalGDS基因敲除小鼠的肿瘤耐药表型至少有一部分是由于真皮成纤维细胞中该蛋白的丢失造成的。最后，我们将测试令人兴奋的可能性，即在遗传稳定的成纤维细胞中，Rala信号级联的组件代表着新的药物靶点，不仅可以阻止皮肤鳞癌的形成，还可以阻止乳腺癌的形成。 公共卫生相关性：有大量证据表明，组织微环境对癌症进展有重要影响。因此，这种现象背后的机制，以及如何利用它们来开发新的策略来阻止肿瘤的发展，正在积极地被探索。在这项建议中，我们通过使用皮肤鳞状细胞癌的生物工程化人类组织模型以及这种疾病的动物模型来研究al GTP酶在角质形成细胞和邻近间质成纤维细胞中的作用来解决这些问题。预计通过靶向上皮细胞和/或间质成纤维细胞中的Ral GTPase信号级联来阻止肿瘤发展的新策略将从本提案中描述的实验结果中出现。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to exploit our understanding of the Ral signaling cascade to reveal how it contributes to cancer in both tumor cells and adjacent stromal fibroblasts. RalA and RalB are members of the Ras superfamily that become activated by a distinct set of guanine nucleotide exchange factors, like RalGDS, in response to a variety of extracellular signals. Once activated, Ral proteins influence a unique set of downstream signaling molecules that regulate multiple cellular processes including vesicle trafficking, apoptosis, cell migration and cell proliferation. A key property of the Ral signaling cascade is that it is stimulated by Ras proteins. In many studies, including those involving RalGDS knockout mice, the Ral signaling cascade supports Ras-induced oncogenic transformation, however the mechanisms involved are poorly understood. Thus, there is intense interest in revealing how the Ral signaling cascade contributes to cancer. In order to understand how Ral GTPases function in squamous carcinoma of the skin, where activated Ras is often an important component, we used a bioengineered tissue model of human skin that allows us to manipulate the Ral signaling cascade in both epithelial and stromal compartments. We found that RalA plays a cell-type dependent role in Ras-mediated squamous cell carcinoma. In keratinocytes of the epithelium, RalA inhibits, rather than supports tumorigenesis, since suppression of RalA expression enhances tumor progression at least in part by promoting cell invasiveness, through its effector protein the exocyst subunit Exo84 and decreased E-cadherin stability. Moreover, tumor progression in this model system is associated with, and requires, down-regulation of RalA levels. RalB knock-down complements the effects of RalA inhibition by enhancing keratinocyte proliferation. Specific Aim 1 will elucidate how RalA and RalB play these surprising tumor-suppressing activities, and reveal how tumor progression down-regulates RalA levels in cells to allow tumor progression. In fibroblasts of the dermis, RalA has the opposite function. It supports tumorigenesis, since RalA knock-down in these cells blocks the invasive properties of adjacent epithelial cells. Specific Aim 2 will reveal the mechanism behind this striking phenomenon and test the hypothesis that at least part of the tumor- resistant phenotype of RalGDS knockout mice is due to the loss of this protein in dermal fibroblasts. Finally, we will test the exciting possibility that the components of a RalA signaling cascade in genetically stable fibroblasts represent new drug targets to block the formation of not only skin squamous carcinoma but also breast adenocarcinoma. PUBLIC HEALTH RELEVANCE: There is abundant evidence that the tissue microenvironment has a major effect on carcinoma progression. As such, the mechanisms underlying this phenomenon, and how to exploit them to develop new strategies to block tumor progression are aggressively being pursued. In this proposal, we address these issues by studying the role of Ral GTPases in both keratinocytes and adjacent stromal fibroblasts using a bioengineered human tissue model of skin squamous cell carcinoma, as well as an animal model of this disease. It is anticipated that new strategies to block tumor development by targeting the Ral GTPase signaling cascade in epithelial cells and/or stromal fibroblasts will emerge from the result of experiments described in this proposal.