TGFBR1 Signaling in Colorectal Cancer

描述(由申请人提供)：我们为TGFBR1的靶向零突变产生了一个新的杂合子小鼠模型。当与携带APC肿瘤抑制基因突变的小鼠杂交时，这些小鼠患上的肠道肿瘤是野生型小鼠的两倍。具有人类结肠癌特征的浸润性腺癌仅在ApcMin/+；TGFBR1+/-小鼠中发现，而在ApcMin/+中不存在；TGFBR1+/+小鼠和肿瘤在TGFBR1基因座上没有杂合性丢失。转化生长因子介导的生长抑制、Smad2和Smad3的磷酸化以及总的转化生长因子？在单倍体不足的胚胎成纤维细胞中，信号轻微减少。在ApcMin/+；TGFBR1+/-小鼠的结肠上皮腺中观察到Smad2和Smad3的磷酸化水平降低。Smad信号的减少与肠粘膜和肠道肿瘤隐窝中细胞增殖的增加有关。因此，结构性降低的TGFBR1介导的信号是结直肠癌发生的一个有效的修饰物。为了确定这些发现在人类中的相关性，我们分析了生殖系外周血中TGFBR1的表达。我们发现242例结直肠癌患者中有29例(12.0%)，而195例对照组中只有3例(1.5%)有TGFBR1(TGFBR1酶)的种系特异性表达。这些结果表明，TGFR1ASE是结直肠癌最常见的遗传原因之一，它使癌症风险增加约870%。携带野生型TGFBR1的人淋巴样细胞和携带TGFBR1酶的人淋巴样细胞的Smad2和Smad3在转化生长因子-β介导的磷酸化水平上的差异与Tgbr1+/+和TGFBR1+/-小鼠胚胎成纤维细胞之间的差异相似。本申请概述的研究目的是通过执行三个特定目标来揭示TGFBR1单倍体缺陷在结直肠癌中的分子机制：第一：确定TGFBR1+/+和TGFBR1+/-小鼠与结肠癌的表型：将在三个不同的背景(129Sv/J、C57BL/6和FVB/N)下评估TGFBR1+/+和TGFBR1+/-小鼠的表型。使用偶氮甲烷方案并将转基因菌株与Apc1638N/+和KRASV12G；Apc1638N/+小鼠杂交，将诱导结肠肿瘤。第二，为了确定TGFBR1单倍体缺陷在肿瘤干细胞和肠道干细胞发育中的作用以及间质和淋巴上皮细胞TGFBR1信号在肿瘤形成中的作用：我们将检测TGFBR1+/+和TGFBR1+/-肿瘤在裸鼠体内的生长情况，以评估TGFBR1单倍体缺陷对肿瘤干细胞发育的贡献。利用Lgr5小鼠模型，我们还将评估TGFBR1单倍体不足是否促进肠道干细胞的发育。TGFBR1单倍体不足在间质和淋巴上皮细胞中的作用将被评估，因为它与结肠癌的发展有关。第三：评估TGFBR1单倍体缺失的致癌特性：使用来自三个不同遗传背景(129Sv/J、C57BL/6和FVB/N)的MEF，我们将确定TGFBR1单倍体缺失促进肿瘤发生的程度。

DESCRIPTION (provided by applicant): We generated a novel model of mice heterozygous for a targeted null mutation of Tgfbr1. When crossed with mice carrying a mutation in the Apc tumor suppressor gene, these mice develop twice as many intestinal tumors as wild-type littermates. Invasive adenocarcinoma with features of human colon cancer is only identified among ApcMin/+; Tgfbr1+/- mice, not among ApcMin/+; Tgfbr1+/+ mice and tumors do not exhibit loss of heterozygosity at the Tgfbr1 locus. TGF-?-mediated growth inhibition, phosphorylation of Smad2 and Smad3, and overall TGF-? signaling are mildly decreased in haploinsufficient embryonic fibroblasts. Decreased Smad2 and Smad3 phosphorylation is observed in the colonic epithelium crypts of ApcMin/+; Tgfbr1+/- mice. Decreased Smad signaling is associated with increased cell proliferation in the crypts of the intestinal mucosa and intestinal tumors. Thus, constitutively reduced Tgfbr1-mediated signaling is a potent modifier of colorectal cancer development. To determine the relevance of these findings in humans, we analyzed germline peripheral blood for TGFBR1 expression. We found that 29 of 242 (12.0%) patients with colorectal cancer but only three of 195 (1.5%) controls had evidence of germline allele-specific expression of TGFBR1 (TGFBR1 ASE). These results indicate that TGFR1 ASE is one of the most commonly inherited cause of colorectal cancer, which increases cancer risk by approximately 870%. Differences in TGF-?-mediated phosphorylation of SMAD2 and SMAD3 between human lymphoid cells from individuals with wild-type TGFBR1 and individuals with TGFBR1 ASE were comparable to those found between Tgbr1+/+ and Tgfbr1+/- mouse embryonic fibroblasts. It is the purpose of the studies outlined in this application to unravel the molecular mechanisms of Tgfbr1 haploinsufficiency in colorectal cancer through execution of three Specific Aims: First: To characterize the phenotype of Tgfbr1+/+ and Tgfbr1+/- mice with respect to colon cancer: The phenotype of Tgfbr1+/+ and Tgfbr1+/- mice will be evaluated in three different backgrounds (129Sv/J, C57BL/6 and FVB/N). Colon tumors will be induced using the azoxymethane protocol and by crossing the transgenic strains with Apc1638N/+ and KRASV12G; Apc1638N/+ mice. Second: To determine the role of Tgfbr1 haploinsufficiency in development of cancer stem cells and intestinal stem cells as well as the contribution of stromal and lymphoepithelial Tgfbr1 signaling to tumor formation: We will determine the growth of Tgfbr1+/+ and Tgfbr1+/- tumors in nude mice to evaluate the contribution of Tgfbr1 haploinsufficiency on tumor stem cell development. Using the Lgr5 mouse model, we will also assess whether Tgfbr1 haploinsufficiency enhances the development of intestinal stem cells. The role of Tgfbr1 haploinsufficiency within stromal and lymphoepithelial cells will be evaluated as it relates to colon cancer development. Third: To assess Tgfbr1 haploinsufficiency oncogenic properties: Using MEFs from three distinct genetic backgrounds (129Sv/J, C57BL/6 and FVB/N) we will determine the extent to which Tgfbr1 haploinsufficiency enhances oncogenesis.