TGFBR1 Signaling in Colorectal Cancer

描述（由申请人提供）：我们为Tgfbr1的靶向零突变生成了一种新的小鼠杂合模型。当与携带Apc肿瘤抑制基因突变的小鼠杂交时，这些小鼠的肠道肿瘤数量是野生型小鼠的两倍。具有人类结肠癌特征的侵袭性腺癌仅在ApcMin/+中被发现；Tgfbr1+/-小鼠，不在ApcMin/+小鼠中；Tgfbr1+/+小鼠和肿瘤在Tgfbr1位点未表现出杂合性缺失。TGF - ?-介导的生长抑制，Smad2和Smad3的磷酸化，以及TGF-？在单倍体不足的胚胎成纤维细胞中，信号传导轻度减少。在ApcMin/+的结肠上皮隐窝中观察到Smad2和Smad3磷酸化降低；Tgfbr1 + / -老鼠。Smad信号的减少与肠粘膜隐窝和肠肿瘤细胞增殖增加有关。因此，组成性减少的tgfbr1介导的信号是结直肠癌发展的有效调节剂。为了确定这些发现在人类中的相关性，我们分析了种系外周血中TGFBR1的表达。我们发现242例结直肠癌患者中有29例（12.0%）存在TGFBR1 （TGFBR1 ASE）的种系等位基因特异性表达，而195例对照中只有3例（1.5%）存在TGFBR1 ASE。这些结果表明，TGFR1 ASE是结直肠癌最常见的遗传原因之一，可使患癌风险增加约870%。TGF-？来自野生型TGFBR1和TGFBR1 ASE个体的人淋巴样细胞之间SMAD2和SMAD3介导的磷酸化与TGFBR1 +/+和TGFBR1 +/-小鼠胚胎成纤维细胞之间的磷酸化相似。本申请中概述的研究目的是通过三个特定目的来揭示Tgfbr1单倍不足在结直肠癌中的分子机制：第一：表征Tgfbr1+/+和Tgfbr1+/-小鼠与结肠癌的表型：Tgfbr1+/+和Tgfbr1+/-小鼠的表型将在三种不同的背景（129Sv/J， C57BL/6和FVB/N）中进行评估。通过偶氮甲烷方案和将转基因菌株与Apc1638N/+和KRASV12G杂交诱导结肠肿瘤；Apc1638N / +老鼠。二是确定Tgfbr1单倍不全在癌症干细胞和肠道干细胞发育中的作用，以及基质和淋巴上皮Tgfbr1信号通路对肿瘤形成的贡献：我们将在裸鼠中测定Tgfbr1+/+和Tgfbr1+/-肿瘤的生长情况，以评估Tgfbr1单倍不全对肿瘤干细胞发育的贡献。通过Lgr5小鼠模型，我们还将评估Tgfbr1单倍不足是否会促进肠道干细胞的发育。Tgfbr1单倍不全在基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单倍性不足的致癌特性：使用来自三种不同遗传背景（129Sv/J、C57BL/6和FVB/N）的mef，我们将确定Tgfbr1单倍性不足在多大程度上促进了肿瘤的发生。

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. PUBLIC HEALTH RELEVANCE: We have shown that decreased expression of the type I TGF-? receptor gene is emerging as the most common cause of colorectal cancer reported to date. The primary goal of this project is to understand the mechanisms linking decreased gene expression and colorectal cancer development. Experiments will be conducted with mice that only express one copy of the gene, which will be bred together with mice that spontaneously develop colon cancer.