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

描述(申请人提供)：上皮细胞向间充质细胞的转分化，这一过程被称为上皮-间充质转化(EMT)，发生在整个胚胎发育过程中，在上皮组织损伤和癌症进展过程中反复出现。这种可塑性是由表观遗传调控的潜在变化实现的。然而，关于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质程序，以及遗传易感个体获得EMT表型是否可能是肠道病原体触发的早期事件，我们知之甚少。轮状柠檬酸杆菌(CR)可引起感染性结肠炎，在促进附着和消除(A/E)病变形成方面与肠源性(EPEC)和肠出血性(EHEC)大肠杆菌有显著的相似之处，是组织靶向和感染的主要机制。我们以前已经证明，Wnt/b-catenin、Notch和NF-kB通路之间的功能交叉对话，调节CR感染后隐窝的增殖和/或肿瘤的发生，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)和退化期(20-34天)中的表达存在差异。感染CR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，E-钙粘蛋白阴性，表现为EMT样现象。这些事件似乎是表观遗传调节的，因为分离的隐窝表现出高水平的EZH2(增强型Zust Homolog-2)，通过组蛋白甲基化和去乙酰化在转录上抑制E-钙粘素和WIF1(Wnt抑制因子1)的表达。在CR感染的ApcMin/+小鼠的腺瘤中，EZH2和b-catenin水平的升高伴随着WIF1表达的降低，在BLT1-/-ApcMin/+小鼠(一种自发的结肠癌模型)、AOM/DSS模型和人类腺癌中也记录了类似的变化。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感染通过EZH2在遗传易感小鼠的干细胞和/或祖细胞中诱导表观遗传信号将促进结肠隐窝的EMT，TBT/丁酸盐将通过上调WIF1来缓解表观遗传连锁的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。在目标1中，我们将研究干细胞内EZH2/WIF1轴的变化是否发生在EMT、局部侵袭或转移之前或伴随着细菌感染。Aim 2将尝试确定CR感染后结肠中表观遗传学和EMT变化的特异性，Aim 3将研究TBT是否阻止干细胞驱动和表观遗传学调节的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作为一种治疗方法铺平道路，从而消除致癌细胞，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.