Role of G1-Cdks in immortalization and oncogenesis

描述(申请人提供)：大量人类癌症表现出基因突变，异常激活G1控制的激酶，细胞周期蛋白D相关的CDK4和CDK6，提示这些蛋白在致癌转化中起关键作用。摆脱衰老是转化所必需的。由RAS激活或体外持续增殖引起的衰老伴随着p16INK4a和ARF的表达增加。P16INK4a抑制CDK4和CDK6，从而增强Rb的生长抑制作用。ARF稳定P53，导致另一种CDK抑制剂p21Cip1/Waf1的诱导。这些与衰老相关的通路对肿瘤抑制至关重要。CDK4缺失的小鼠胚胎成纤维细胞(MEF)正常增殖，但在Ras激活并抑制P53或INK4a/ARF中断后，对致癌转化具有抵抗力。缺乏ARF-P53功能的衰老是缺乏转化潜力的原因。此外，CDK4基因缺失的小鼠对致癌物或辐射诱导的肿瘤形成的敏感性降低。因此，通过在通常使细胞永生化的条件下诱导衰老，CDK4的破坏可能有效地使细胞对转化不敏感。其长期目标是为肿瘤转化的治疗干预奠定基础。这项建议将评估一种假说，即细胞需要细胞周期蛋白D/CDK4和细胞周期蛋白D/CDK6的异常激活，以克服衰老依赖的肿瘤抑制机制，导致永生化和肿瘤形成。其具体目标是：(1)通过研究CDK4缺失的MEF中的衰老/永生化相关蛋白，确定CDK4干扰如何导致MEF在ARF-p53途径抑制下衰老；(2)通过检测带有乳腺特异性CDK4中断的MMTV-RAS转基因小鼠，确定CDK4是否对于RAS诱导的小鼠乳腺上皮癌变是必需的；(3)通过检测CDK6缺失的MEF和小鼠，确定CDK4和CDK6在永生化、转化和肿瘤发生中如何相互作用。这些研究有望阐明细胞周期蛋白D依赖的激酶在肿瘤发生过程中的重要作用。这个项目采取了创新的方法，应该引导我们更好地理解CDK4和CDK6与衰老依赖的肿瘤抑制途径的致癌相互作用，使用独特的小鼠和细胞模型。

DESCRIPTION (provided by applicant): A large number of human cancers display genetic alterations that aberrantly activate the G1-control kinases, cyclin D-associated Cdk4 and Cdk6, suggesting critical roles for these proteins in oncogenic transformation. Escape from senescence is requisite for transformation. Senescence induced by Ras activation or continuous in vitro proliferation is accompanied by increased expression of p16ink4a and ARF. p16ink4a inhibits Cdk4 and Cdk6, leading to enhance growth-inhibitory action of Rb. ARF stabilizes p53, resulting in induction of another Cdk inhibitor, p21Cip1/Waf1. These senescence-associated pathways are critical for tumor suppression. Cdk4-null mouse embryonic fibroblasts (MEF) proliferate normally, but are resistant to oncogenic transformation upon Ras activation with p53 inhibition or Ink4a/ARF disruption. Senescence without ARF-p53 function accounts for this lack of transformation potential. Furthermore, Cdk4-null mice display reduced susceptibility to carcinogen- or radiation-induced tumorigenesis. Thus, Cdk4 disruption may effectively render cells insensitive to transformation, by inducing senescence under conditions that normally immortalize cells. The long-term goal is to establish the basis for therapeutic intervention of oncogenic transformation. This proposal will evaluate the hypothesis that aberrant activation of cyclin D/Cdk4 and cyclin D/Cdk6 is required for a cell to overcome the senescence-dependent tumor suppressive mechanism, leading to immortalization and tumorigenesis. The specific aims are: (1) Determine how Cdk4 disruption leads MEF to senescence under inhibition of the ARF-p53 pathway, by investigating senescence/immortalization-associated proteins in Cdk4-null MEF; (2) Determine whether Cdk4 is required for Ras-induced carcinogenesis of the mammary epithelium in mice, by examining MMTV-Ras transgenic mice with mammary-specific Cdk4 disruption; (3) Determine how Cdk4 and Cdk6 interact in immortalization, transformation and oncogenesis, by examining Cdk6-null MEF and mice. These studies are expected to clarify essential roles of the cyclin D-dependent kinases during oncogenesis. This program takes innovative approaches that should lead us to better understanding of the oncogenic interplay of Cdk4 and Cdk6 with the senescence-dependent tumor suppressive pathways, using unique mouse and cell models.