Inhibition of colon tumor progression by Ink4a/Arf

描述(申请人提供)：结肠癌是人类第二常见的致命性恶性肿瘤，也是研究肿瘤进展的原型。近年来，血管密度已成为实体瘤发展的一个限制因素，并在结肠肿瘤的发现、复发和治疗中具有重要的临床意义。Ink4a/Arf基因座编码p16INK4a(P16)和p19Arf(Arf；可选阅读框架)。这两种蛋白都是肿瘤抑制因子，也是体外细胞周期停滞和衰老的有力中介。在结肠腺瘤和结肠癌中，Ink4a/Arf基因座经常因甲基化而沉默。这一观察表明，该基因座可能抑制结肠肿瘤的发生，但缺乏直接证据。我们研究了该基因缺失对多发性肠道肿瘤(Min)小鼠的影响。我们发现，在缺乏p16和aft的情况下，Min结肠肿瘤表现出加速的肿瘤进展。这些肿瘤更大，显示出癌症的组织学特征，最突出的是，更多的血管。后一特征伴随着血管内皮生长因子(VEGF)含量的增加。我们建议在这里剖析表型的分子基础。在目标1和目标2中，我们将使用选择性缺乏这两种蛋白的小鼠，确定p16和Arf对小鼠结肠癌进展的单独影响。我们将分析p16和Arf在Min结肠肿瘤中的表达，并评估p16或Arf启动子的甲基化和沉默是否与肿瘤进展相关。我们将评估p16和Arf对肿瘤细胞周期停滞和衰老的影响。在目标3中，我们将探讨血管表型对肿瘤进展的意义。我们将在Ink4a/Arf缺失和野生型背景下，在肠道上皮中靶向缺失血管内皮生长因子基因的Min小鼠，并研究其对结肠癌进展的影响。在目标4中，我们将检测体内和体外对Min结肠肿瘤细胞中血管内皮生长因子表达的调节，并将检测针对血管内皮生长因子的抗体是否破坏肿瘤细胞招募血管内皮细胞的能力。综上所述，这些研究将确定结肠癌进展和血管的关键决定因素，提高我们对p16和Arf在肿瘤抑制的生理环境中的功能的理解，并解决血管增强对结肠癌进展的意义。

DESCRIPTION (provided by applicant): Colon carcinoma is the second most common fatal human malignancy and has served as a prototype for studies of tumor progression. Recently, vascularity has emerged as a limiting factor in solid tumor development and a clinically important feature in the detection, recurrence, and therapy of colon tumors. The Ink4a/Arf locus encodes p16Ink4a (p16) and p19Arf (Arf; alternative reading frame). Both proteins are tumor suppressors and potent mediators of cell cycle arrest and senescence in vitro. The Ink4a/Arf locus is frequently silenced by methylation in colon adenoma and carcinoma. This observation suggests that the locus may suppress colon tumorigenesis, but direct evidence has been lacking. We examined the effect of a deletion of the locus in mice with multiple intestinal neoplasia (Min). We have found that Min colon tumors show accelerated tumor progression in the absence of p16 and Aft. These tumors are larger, show histologic features of carcinoma, and, most prominently, are more vascular. The latter feature is accompanied by increased vascular endothelial growth factor (VEGF) content. We propose here to dissect the molecular basis of the phenotype. In Aims 1 and 2, respectively, we will determine the individual impacts of p16 and Arf on Min colon tumor progression, using mice selectively deficient in each protein. We will analyze expression of p16 and Arf in Min colon tumors and assess whether methylation and silencing of the p16 or Arf promoters correlates with features of tumor progression. We will assess the impacts of p16 and Arf on tumor cell cycle arrest and senescence. In Aim 3 we will explore the significance of the vascular phenotype for tumor progression. We will generate Min mice with targeted deletion of the VEGF gene in the intestinal epithelium, in Ink4a/Arf-null and -wild type backgrounds, and examine the consequences for colon tumor progression. In Aim 4, we will examine the regulation of VEGF expression in Min colon tumor cells in vivo and in vitro and will test whether antibodies directed against VEGF disrupt the ability of tumor cells to recruit vascular endothelial cells. In summary, these studies will define key determinants of colon tumor progression and vascularity, improve our understanding of p16 and Arf function in physiologic settings of tumor suppression, and address the significance of the enhanced vascularity for colon tumor progression.