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人类恶性肿瘤，并已作为肿瘤进展研究的原型。近年来，血管性已成为实体瘤发展的一个限制因素，也是结肠肿瘤检测、复发和治疗的一个重要临床特征。Ink4a/Arf基因座编码p16Ink4a （p16）和p19Arf （Arf；备选阅读框）。这两种蛋白都是肿瘤抑制因子和体外细胞周期阻滞和衰老的有效介质。在结肠腺瘤和癌中，Ink4a/Arf位点经常被甲基化沉默。这一观察结果表明，该位点可能抑制结肠肿瘤的发生，但缺乏直接证据。我们研究了该基因座缺失对多发性肠肿瘤小鼠的影响（Min）。我们发现，在缺乏p16和Aft的情况下，Min结肠肿瘤的肿瘤进展加快。这些肿瘤更大，表现出癌的组织学特征，最显著的是血管性更强。后一种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Aims 1和Aims 2中，我们将分别使用选择性缺乏p16和Arf的小鼠，确定p16和Arf对Min结肠肿瘤进展的个体影响。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的特征相关。我们将评估p16和Arf对肿瘤细胞周期阻滞和衰老的影响。在目的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-null和-野生型背景下，在肠上皮中产生靶向缺失VEGF基因的Min小鼠，并研究其对结肠肿瘤进展的影响。在Aim 4中，我们将在体内和体外研究Min结肠肿瘤细胞中VEGF表达的调控，并测试针对VEGF的抗体是否会破坏肿瘤细胞招募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性的关键决定因素，提高我们对p16和Arf在肿瘤抑制生理环境中的功能的理解，并解决血管性增强对结肠肿瘤进展的意义。

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.