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

描述（由申请人提供）：上皮细胞转分化为间充质细胞，这一过程称为上皮-间充质转化（EMT），发生在整个胚胎发育过程中，并在上皮组织损伤和癌进展过程中重演。这种可塑性是由表观遗传调控的潜在变化所实现的。然而，关于我们对这些途径如何协调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的理解，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，知之甚少。啮齿类柠檬酸杆菌（CR）引起感染性结肠炎，与肠致病性大肠杆菌（EPEC）和肠出血性大肠杆菌（EHEC）具有显著的功能相似性。大肠杆菌作为组织靶向和感染的主要机制，在促进附着和消退（A/E）损伤形成中起重要作用。我们先前已经表明，Wnt/β-连环蛋白、Notch和NF-kB通路之间的功能性交叉对话调节了对CR感染的应答中的隐窝增生和/或肿瘤发生，而炎症和/或结肠炎由MEK/ERK和NF-kB通路调节。我们还发现了干细胞标志物Dclk 1和Lgr 5在隐窝增生的进展（第6-12天）和消退（第20-34天）阶段的差异表达。CR感染的隐窝上皮细胞和腺瘤对波形蛋白、纤连蛋白和Dclk 1染色呈阳性，但对E-钙粘蛋白染色呈阴性，从而表现出EMT样现象。这些事件似乎是表观遗传学调节的，因为分离的隐窝表现出升高水平的EZH 2（Zeste同源物增强子-2），其通过组蛋白甲基化和脱乙酰化转录抑制E-钙粘蛋白和WIF 1（Wnt抑制因子1）表达。在CR感染的ApcMin/+小鼠的腺瘤中EZH 2和b-连环蛋白水平升高以及伴随的WIF 1表达降低也与BLT 1-/-ApcMin/+小鼠（结肠癌的自发模型）、AOM/DSS模型和人腺癌中记录的变化平行。最后，口服三丁酸甘油酯（TBT）阻断EZH 2，上调WIF 1并抑制隐窝增生。基于这些发现，我们假设在遗传易感小鼠的干细胞和/或祖细胞中通过EZH 2的CR感染诱导的表观遗传信号传导将促进结肠隐窝的EMT，并且TBT/丁酸盐将通过上调WIFl来减轻表观遗传相关的EMT和/或转移过程。我们提出了三个具体目标来检验这一假设。在目的1中，我们将研究干细胞内EZH 2/WIF 1轴对细菌感染的反应是否先于或伴随EMT、局部侵袭或转移。目标2将试图建立特异性的表观遗传和EMT的变化在结肠中响应CR感染，目标3将检查TBT是否阻断干细胞驱动和表观遗传调节的EMT和/或肿瘤扩散的过程。TBT作为EZH 2抑制剂的新发现的作用预计最终将为开发TBT作为消除癌症起始细胞的疗法铺平道路，从而有助于预防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.