Molecular Control of EGF Receptor Down-Regulation

描述（申请人提供）：EGF受体是ErbB受体酪氨酸激酶家族的一员，通过响应特定配体产生细胞增殖、存活、分化和迁移信号，在上皮组织的发育和维持中发挥重要的生理作用。ErbB受体的激活与人类癌症的病因学有关，包括肿瘤的发生和发展。ErbB受体也介导化疗和放疗的细胞保护，并代表肿瘤放射和化疗感知的靶点。因此，阐明控制ErbB受体信号效力的分子机制是癌症生物学的主要目标。配体诱导的下调，代表了溶酶体降解和细胞表面再循环之间的平衡，构成了ErbB受体信号效力的主要决定因素。我们和其他人已经确定，通过促进受体泛素化，cbl家族泛素连接酶蛋白在EGFR下调中起关键作用。后者被内体ESCRT蛋白复合物识别为受体溶酶体分选的信号。Cbl及其密切相关的功能减少的家族成员Cbl-b生理运作的精确内噬步骤和机制仍有待确定。Cbl蛋白对EGFR生理和致癌作用的负调控的生物学后果仍有待发现。该提案将利用独特的Cbl/Cbl-b缺失的小鼠MEF和乳腺上皮细胞以及Cbl/Cbl-b敲低的人乳腺上皮细胞来解决Cbl蛋白是否选择性地控制EGFR的溶酶体分选，或者它们是否也是初始内化所必需的；Cbl/Cbl-b缺陷细胞的重构将为Cbl蛋白对EGFR的负调控提供结构基础。通过蛋白质组学和基因表达分析，我们将研究Cbl蛋白是否对某些信号模块提供全局或选择性调控，并将这些调控与EGFR信号的生物学输出联系起来。最后，我们将在小鼠中使用乳腺靶向Cbl/Cbl-b敲除，以解决在乳腺生理性发育和EGFR介导的乳腺癌发生过程中，Cbl蛋白调节EGFR的功能后果。从这些研究中获得的见解将进一步加深我们对受体酪氨酸激酶下调的理解，并可能确定新的靶点，以合理设计抗癌药物，并通过对抗受体酪氨酸激酶介导的细胞保护来增强现有的化疗和放疗癌细胞杀伤。

DESCRIPTION (provided by applicant): EGF receptor, a member of the ErbB family of receptor tyrosine kinases, plays essential physiological roles in development and mainatenance of epithelial tissues by genrating cell proliferation, survival, differentiation and migration signals in response to specific ligands. Activation of ErbB receptors is linked to etiology of human cancer, including tumor initiation and progression. ErbB receptors also mediate cytoprotection from chemotherapy and radiation, and represent targets for radio- and chemo-sentization of tumors. Elucidating molecular mechanisms that control signaling potency of ErbB receptos is therefore a major goal in cancer biology. Ligand-induced downregulation, representing a balance between lysosomal degradation and recycling to cell sureface, constitutes a major determinant of signaling potency of ErbB receptors. We and others have established that Cbl-family ubiquitin ligase proteins play a crucial role in EGFR down-regulation by faciliatating receptor ubiquitination. The latter is recognized by endosomal ESCRT protein complexes as a signal for lysosomal sorting of the receptor. The precise endocytic step(s) and mechanisms by which Cbl and its closely related, functionally reducndant, family member Cbl-b physiologically operate remain to be established. The biological consequence of negative regulation by Cbl proteins on physiological and oncogenic roles of EGFR also remain to be uncovered. This proposal will utilize unique Cbl/Cbl-b null murine MEF and mammary epithelial cells as wel as Cbl/Cbl-b knockdown human mammary epithelial cells to address whether Cbl proteins selectively control lysosomal sorting of EGFR or if they are also required for initial internalization; reconstitiution of Cbl/Cbl-b deficient cells will provide structural basis for negative regulation of EGFR by Cbl proteins. By using proteomic and gene expression analyses, we will investigate if Cbl proteins provide global or selective regulation of certain signaling modules and link these to biological outputs of EGFR signaling. Finally, we will use mammary gland targeted Cbl/Cbl-b knockout in mice to address the functional consequences of EGFR regulation by Cbl proteins during physiological mammary galnd development and in EGFR-mediated mammary oncogenesis. Insights gained from these studies should further our understanding of receptor tyrosine kinase down-regulation, and may identify newer targets to rationally design anti-cancer agents and to potentiate existing chemotherapeutic and radiation-based cancer cell killing, by countering receptor tyrosine kinase-mediated cytoprotection.