Epigenetics and Infection-induced EMT of Colonic Crypts - Target for Chemoprevention

描述（由申请人提供）：上皮细胞向间充质细胞的反分化，一个被称为上皮-间充质转化（EMT）的过程发生在整个胚胎发育过程中，并在上皮组织损伤和癌症进展过程中重演。这种可塑性是由表观遗传调控的潜在转变所实现的。然而，我们对这些途径如何协同抑制上皮表型并诱导产生具有干细胞特性的细胞的间充质程序以及在遗传易感个体中获得EMT表型是否可能是由肠道病原体触发的早期事件的理解知之甚少。啮齿柠檬酸杆菌（Citrobacter rodentium， CR）引起感染性结肠炎，与肠致病性（EPEC）和肠出血性（EHEC）大肠杆菌在促进附着和消隐（a /E）病变形成方面具有显著的功能相似性，是组织靶向和感染的主要机制。我们之前已经证明，Wnt/b-catenin、Notch和NF-kB通路之间的功能性交叉对话调节CR感染时的隐窝增生和/或肿瘤发生，而炎症和/或结肠炎则由MEK/ERK和NF-kB通路调节。我们还发现干细胞标记物Dclk1和Lgr5在隐窝增生的进展期（6-12天）和退行期（20-34天）的差异表达。CR感染的隐窝上皮细胞和腺瘤呈波形蛋白、纤维连接蛋白和Dclk1阳性，但e -钙粘蛋白呈阴性，因此表现出类似emt的现象。这些事件似乎受到表观遗传调控，因为分离的隐窝表现出EZH2 （Zeste同源物-2的增强子）水平升高，通过组蛋白甲基化和去乙酰化转录抑制e -钙粘附蛋白和WIF1 （Wnt抑制因子1）的表达。在感染cr的ApcMin/+小鼠的腺瘤中，EZH2和b-catenin水平升高，同时伴有WIF1表达的降低，这与BLT1-/-ApcMin/+小鼠（结肠癌自发性模型）、AOM/DSS模型和人腺癌中记录的变化相似。最后，口服三丁酸甘油酯（TBT）阻断EZH2，上调WIF1，抑制隐窝增生。基于这些发现，我们假设CR感染诱导的表观遗传信号通过EZH2在遗传易感小鼠的干细胞和/或祖细胞中促进结肠隐窝的EMT， TBT/丁酸盐通过上调WIF1来减轻表观遗传相关的EMT和/或转移过程。我们提出三个具体目标来检验这一假设。在Aim 1中，我们将研究干细胞内EZH2/WIF1轴对细菌感染的反应是否先于或伴随EMT、局部侵袭或转移。Aim 2将尝试建立结肠对CR感染的表观遗传和EMT变化的特异性，Aim 3将检查TBT是否阻断干细胞驱动和表观遗传调节的EMT和/或肿瘤扩散过程。新发现的TBT作为EZH2抑制剂的作用有望最终为开发TBT作为一种消除癌症启动细胞的疗法铺平道路，从而帮助防止肿瘤复发或远处转移。

DESCRIPTION (provided by applicant): The trans-differentiation of epithelial cells into mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT) occurs throughout embryonic development and is recapitulated during epithelial tissue injury and in carcinoma progression. This plasticity is enabled by underlying shifts in epigenetic regulation. Little is known however, regarding our understanding of how these pathways coordinately suppress the epithelial phenotype and induce a mesenchymal program that generates cells with properties of stem cells and whether the acquisition of EMT phenotype in genetically predisposed individuals could be an early event triggered by enteric pathogens. Citrobacter rodentium (CR) causes infectious colitis and shares a remarkable functional similarity to enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli in promoting attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. We have shown previously that functional cross-talks between Wnt/b-catenin, Notch and NF-kB pathways, regulate crypt hyperplasia and/or tumorigenesis in response to CR infection while inflammation and/or colitis is regulated by the MEK/ERK and NF-kB pathways. We have also discovered differential expression of stem cell markers Dclk1 and Lgr5 during progression (days 6-12) and regression (days 20-34) phases of crypt hyperplasia. CR- infected crypt epithelial cells and adenomas stain positive for vimentin, fibronectin and Dclk1 but negative for E-cadherin thereby exhibiting an EMT-like phenomenon. These events seem to be epigenetically regulated as isolated crypts exhibit elevated levels of EZH2 (Enhancer of Zeste Homolog-2) that transcriptionally represses E-cadherin and WIF1 (Wnt Inhibitory Factor 1) expression via histone methylation and deacetylation. Elevated levels of EZH2 and b-catenin with concomitant decrease in WIF1 expression in the adenomas of CR-infected ApcMin/+ mice also parallel changes recorded in BLT1-/-ApcMin/+ mice, a spontaneous model of colon cancer, in AOM/DSS model and in human adenocarcinomas. Finally, orally administered Tributyrin (TBT) blocks EZH2, upregulates WIF1 and inhibits crypt hyperplasia. Based on these findings, we hypothesize that CR infection- induced epigenetic signaling via EZH2 in stem and/or progenitor cells of genetically susceptible mice will promote EMT of colonic crypts and that TBT/butyrate will mitigate epigenetically-linked EMT and/or metastatic process by upregulating WIF1. We propose three specific aims to test this hypothesis. In Aim 1, we will investigate whether alteration in EZH2/WIF1 axis within the stem cells in response to bacterial infection precedes or accompanies EMT, local invasion or metastasis. Aim 2 will attempt to establish specificity of the epigenetic and EMT changes in the colon in response to CR infection and, Aim 3 will examine if TBT blocks stem cell-driven and epigenetically regulated process of EMT and/or tumor spread. The new found role for TBT as an inhibitor of EZH2 is expected to eventually pave the way for developing TBT as a therapy to eliminate cancer-initiating cells thereby helping to prevent tumor recurrence or distant metastasis.