Regulation of tumor promotion by RasGRP1

描述(由申请人提供)：皮肤癌的小鼠模型已用于研究肿瘤发生的多阶段性质，并识别新的癌基因和肿瘤抑制基因，以及与癌症相关的信号通路。在经典的两阶段致癌模型中，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，RAS启动细胞肿瘤形成的机制以及佛波酯信号通路的参与仍然知之甚少。利用小鼠皮肤模型，我们已经确定RASGRP1是一种新的皮肤癌发生调节因子。RASGRP1连接佛波酯和RAS，因为它既是一种高亲和力的佛波酯受体，又是一种RAS激活剂。利用转基因小鼠，我们已经证明RASGRP1通过野生型RAS的生化激活参与了两阶段致癌模型中肿瘤的发生和发展。此外，在表皮中过度表达RASGRP1的转基因小鼠容易自发地发生皮肤肿瘤。综上所述，我们假设RASGRP1通过介导生化RAS激活和与致癌RAS在肿瘤形成中的协同作用而参与肿瘤的发生。在这项应用中，我们建议确定RAS和RASGRP1在皮肤癌发生过程中的合作机制，无论是在RAS癌基因突变的缺失还是存在。此外，我们计划将我们在小鼠模型上的观察扩展到使用人体皮肤的3D器官型培养的人类模型。其具体目的是：(1)确定RASGRP1在无Ras癌基因突变的情况下诱导肿瘤发生的机制：根据我们最近对RASGRP1转基因小鼠创伤诱发肿瘤的研究结果，我们将重点关注细胞因子和EGFR配体在激活皮肤RASGRP1-RAS轴中的潜在作用；(2)探讨RASGRP1和致癌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，并在肿瘤进展中与癌基因RAS协同作用；(3)阐明RASGRP1在人角质形成细胞动态平衡和转化中的作用：我们将利用人类体外模型来验证和了解RASGRP1在致癌中的作用。这些研究的意义在于，RASGRP1作为RAS调控肿瘤发生的相关靶点，可用于开发新的化学预防和/或癌症治疗的干预措施。此外，从这一建议中得出的发现也应该有助于促进我们对RAS信号通路的理解，而不仅仅是皮肤癌的发生，并可能应用于其他表达RASGRP1的系统。

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.