Inhibition of colon tumor progression by Ink4a/Arf

描述（由申请人提供）：结肠癌是第二常见的致命性人类恶性肿瘤，并已作为肿瘤进展研究的原型。最近，血管已成为实体瘤发展的限制因素，也是结肠肿瘤检测、复发和治疗的重要临床特征。Ink 4a/Arf基因座编码p16 Ink 4a（p16）和p19 Arf（Arf;交替阅读框架）。这两种蛋白质都是肿瘤抑制因子，也是体外细胞周期停滞和衰老的有效介质。Ink 4a/Arf基因座在结肠腺瘤和结肠癌中常因甲基化而沉默。这一观察结果表明，该位点可能抑制结肠肿瘤发生，但缺乏直接证据。我们研究了在多发性肠肿瘤（Min）小鼠中基因座缺失的影响。我们发现Min结肠肿瘤在p16和Aft缺失的情况下显示加速的肿瘤进展。这些肿瘤较大，显示癌的组织学特征，最显著的是血管较多。后一特征伴随着血管内皮生长因子（VEGF）含量的增加。我们建议在这里解剖的表型的分子基础。在目标1和2中，我们将分别使用每种蛋白质选择性缺陷的小鼠来确定p16和Arf对Min结肠肿瘤进展的个体影响。我们将分析Min结肠肿瘤中p16和Arf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的特征相关。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Ink 4a/Arf缺失和野生型背景下产生肠上皮中VEGF基因靶向缺失的Min小鼠，并检查结肠肿瘤进展的后果。在目的4中，我们将检查体内和体外Min结肠肿瘤细胞中VEGF表达的调节，并将测试针对VEGF的抗体是否破坏肿瘤细胞募集血管内皮细胞的能力。总之，这些研究将确定结肠肿瘤进展和血管分布的关键决定因素，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，并解决结肠肿瘤进展中血管分布增强的意义。

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.