Regulation of tumor promotion by RasGRP1

描述（由申请人提供）：小鼠皮肤癌模型用于研究肿瘤发生的多阶段性质，识别新的致癌基因和肿瘤抑制基因，以及与癌症相关的信号通路。在经典的两阶段癌变模型中，Ras被致癌物诱导的突变激活，随后用佛波酯治疗诱导肿瘤促进。然而，导致由Ras启动的肿瘤细胞形成的机制-以及磷脂酯信号通路的参与-仍然知之甚少。通过小鼠皮肤模型，我们发现RasGRP1是一种新的皮肤癌变调节因子。RasGRP1连接磷酯和Ras，因为它既是高亲和的磷酯受体又是Ras激活剂。我们在转基因小鼠实验中发现，在两期癌变模型中，RasGRP1通过野生型Ras的生化激活参与肿瘤的发生和发展。此外，表皮过表达RasGRP1的转基因小鼠容易自发发生皮肤肿瘤。综上所述，我们推测RasGRP1通过介导Ras的生化激活并协同致癌Ras参与肿瘤形成。在本应用中，我们拟确定Ras和RasGRP1在皮肤癌变过程中的合作机制，无论是在Ras致癌突变缺失还是存在的情况下。此外，我们计划将我们在小鼠模型中的观察扩展到使用人体皮肤的3-D器官型培养的人体模型。具体目的是：(1)确定在Ras致癌基因突变不存在的情况下，RasGRP1诱导的肿瘤发生机制：根据我们近期对RasGRP1转基因小鼠创伤诱导肿瘤的研究结果，我们将重点研究细胞因子和EGFR配体在皮肤中激活RasGRP1-Ras轴的潜在作用；(2)探讨RasGRP1与致癌Ras在皮肤肿瘤进展中的协同作用：我们的假设是，RasGRP1激活野生型Ras亚型，特别是N-Ras亚型，并与致癌Ras在肿瘤进展中协同作用；(3)阐明RasGRP1在人角质形成细胞稳态和转化中的作用：我们将利用人体外模型验证和了解RasGRP1在癌变中的功能。这些研究的意义在于，确定RasGRP1作为Ras在癌变过程中调控的相关靶点，可用于开发化学预防和/或癌症治疗的新干预措施。此外，从该提案中得出的发现也有助于推进我们对Ras信号通路的理解，而不仅仅是皮肤癌，并且可能应用于RasGRP1也表达的其他系统。

DESCRIPTION (provided by applicant): The mouse model of skin carcinogenesis has served to investigate the multistage nature of tumorigenesis and to identify novel oncogenes and tumor suppressor genes, as well as signaling pathways relevant in cancer. In the classic two-stage carcinogenesis model, Ras is activated by mutations induced by a carcinogen, and subsequent treatment with phorbol esters induces tumor promotion. However, the mechanisms that lead to tumor formation of cells initiated by Ras -and the participation of phorbol ester signaling pathways- are still poorly understood. Using the mouse skin model, we have identified RasGRP1 as a novel regulator of skin carcinogenesis. RasGRP1 links phorbol esters and Ras, since it is both, a high affinity phorbol ester receptor and a Ras activator. Using genetically modified mice we have shown that RasGRP1 participates in tumor initiation and progression in the two-stage carcinogenesis model through the biochemical activation of wild type Ras. Additionally, transgenic mice overexpressing RasGRP1 in the epidermis are prone to develop skin tumors spontaneously. Taken together, we hypothesize that RasGRP1 participates in carcinogenesis by mediating biochemical Ras activation and cooperating with oncogenic Ras in tumor formation. In this application, we propose to identify the mechanisms of cooperation between Ras and RasGRP1 during skin carcinogenesis, both in the absence and presence of Ras oncogenic mutations. In addition, we plan to extend our observation in mouse models to a human model using the 3-D organotypic culture of human skin. The specific aims are: (1) Determine the mechanism of tumorigenesis induced by RasGRP1 in the absence of Ras oncogenic mutations: following findings from our recent studies on wounding-induced tumors in the RasGRP1 transgenic mice, we will focus on the potential role of cytokines and EGFR ligands in activation of the RasGRP1-Ras axis in the skin; (2) Investigate the cooperation between RasGRP1 and oncogenic Ras in tumor progression in the skin: our hypothesis is that RasGRP1 activates wild type Ras isoforms, particularly N-Ras, and cooperates with oncogenic Ras in tumor progression; and (3) Elucidate the role of RasGRP1 in human keratinocyte homeostasis and transformation: we will use human in vitro models to validate and understand the functions of RasGRP1 in carcinogenesis. The significance of the studies is that the identification of RasGRP1 as a relevant target in Ras modulation in carcinogenesis can be used to develop new interventions for chemoprevention and/or cancer therapy. Moreover, the findings derived from this proposal should also contribute to advance our understanding of Ras signaling pathways beyond skin carcinogenesis, and may have applications to other systems where RasGRP1 is also expressed.