TGFBR1 Signaling in Colorectal Cancer

描述（由申请人提供）：我们生成了一种新的Tgfbr 1靶向无效突变杂合小鼠模型。当与携带Apc肿瘤抑制基因突变的小鼠杂交时，这些小鼠产生的肠道肿瘤是野生型同窝小鼠的两倍。具有人结肠癌特征的浸润性腺癌仅在ApcMin/+; Tgfbr 1 +/-小鼠中发现，而在ApcMin/+; Tgfbr 1 +/+小鼠中未发现，并且肿瘤未表现出Tgfbr 1基因座的杂合性缺失。转化生长因子？介导的生长抑制，Smad 2和Smad 3的磷酸化，和整体TGF-？信号转导在单倍不足的胚胎成纤维细胞中轻度降低。在ApcMin/+; Tgfbr 1 +/-小鼠的结肠上皮隐窝中观察到Smad 2和Smad 3磷酸化降低。Smad信号的减少与肠粘膜和肠肿瘤的隐窝中细胞增殖的增加相关。因此，组成性减少Tgfbr 1介导的信号传导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，我们分析了生殖系外周血的TGFBR 1表达。我们发现，242例结直肠癌患者中有29例（12.0%）有TGFBR 1生殖系等位基因特异性表达（TGFBR 1 ASE）的证据，而195例对照中只有3例（1.5%）有TGFBR 1生殖系等位基因特异性表达的证据。这些结果表明，TGFR 1 ASE是结直肠癌最常见的遗传原因之一，可使癌症风险增加约870%。TGF-？-的差异来自具有野生型TGFBR 1的个体和具有TGFBR 1 ASE的个体的人淋巴细胞之间的SMAD 2和SMAD 3介导的磷酸化与在Tgbr 1 +/+和Tgfbr 1 +/-小鼠胚胎成纤维细胞之间发现的那些相当。本申请中概述的研究的目的是通过执行三个具体目的来阐明结肠直肠癌中Tgfbr 1单倍不足的分子机制：第一：表征Tgfbr 1 +/+和Tgfbr 1 +/-小鼠关于结肠癌的表型：将在三种不同背景（129 Sv/J、C57 BL/6和FVB/N）下评价Tgfbr 1 +/+和Tgfbr 1 +/-小鼠的表型。将使用氧化偶氮甲烷方案并通过将转基因品系与Apc 1638 N/+和KRASV 12 G; Apc 1638 N/+小鼠杂交来诱导结肠肿瘤。第二个：为了确定Tgfbr 1单倍不足在癌症干细胞和肠干细胞发育中的作用以及间质和淋巴上皮Tgfbr 1信号传导对肿瘤形成的贡献：我们将确定裸鼠中Tgfbr 1 +/+和Tgfbr 1 +/-肿瘤的生长，以评估Tgfbr 1单倍不足对肿瘤干细胞发育的贡献。使用Lgr 5小鼠模型，我们还将评估Tgfbr 1单倍不足是否会增强肠道干细胞的发育。将评估Tgfbr 1单倍不足在间质细胞和淋巴上皮细胞中的作用，因为它与结肠癌的发展有关。第三：为了评估Tgfbr 1单倍不足的致癌特性：使用来自三种不同遗传背景（129 Sv/J，C57 BL/6和FVB/N）的MEFs，我们将确定Tgfbr 1单倍不足增强肿瘤发生的程度。

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.