Role of the microbiota in DNA methylation and CRC development

结肠微生物组与结直肠癌(CRC)的发病机制有关，但确切的机制 在这些观察的基础上，人们仍然没有完全理解。我们观察到了惊人的联想 结直肠癌中特定宿主微生物和DNA甲基化异常之间的关系。例如，梭杆菌属 在受CpG岛甲基化表型(CIMP)影响的癌症中大量富含。初步数据 基于测序和定量聚合酶链式反应的验证也显示了与疾病有关的细菌的丰富 在人或小鼠模型中(E.colisp.，Klebsiella sp.等)。高水平的这些致病细菌是 与CIMP结肠癌的复发有关。这一出人意料的联系在结肠微生物区系和 表观遗传控制也见于对生殖细胞结肠粘膜DNA甲基化全基因组的分析。 自由(GF)小鼠与常规小鼠进行比较，在常规小鼠中，我们发现细菌的重新引入导致了 正常未甲基化的CpG岛的高甲基化(主要异常见于CIMP病例)。因此， 我们的初步数据支持一种新的假设，即胃肠道微生物区系通过 诱导或调节异常的DNA甲基化和表观遗传控制。从机制上讲，我们建议 多个平行机制可能有助于这种联系，包括与DNA损伤相关的招募 沉默复合体，以及细菌分泌代谢物和/或毒素扩散的代谢障碍 并直接影响DNA甲基化(例如，2-羟基戊二酸[2-HG]，它抑制 Tet DNA去甲基酶)或间接(例如丁酸，已知通过 组蛋白脱乙酰酶的抑制)。为了检验这些假设，我们提出了三个具体目标：(1)定义 CIMP肿瘤的微生物群。我们将使用16S RNA基因组测序技术 广泛的肿瘤(癌症、先兆和邻近正常)同时具有CIMP基因突变的特征 和基因表达。(2)CIMP相关细菌对小鼠肿瘤发生及DNA甲基化的影响 老鼠。Gf Il10-/-；Apcmin/小鼠将与候选细菌(例如：E.Coli、F.nuatum、K.肺炎)一起定植 以及正常组和肿瘤组的肿瘤发生率、严重程度、生存期以及DNA甲基化和基因表达 将对组织进行评估。我们还将测试针对DNA甲基化的药物是否在预防中有效 与细菌相关的肿瘤发生。(3)研究细菌影响DNA甲基化的代谢产物 配置文件。我们将在CIMP相关细菌的培养和CIMP的裂解物上使用代谢组学 和CIMP-癌症，以确定可能调节DNA甲基化的代谢物。这些(如2HG、丁酸盐) 将被测试对DNA甲基化(在细胞培养和GF小鼠中)和肿瘤形成(在小鼠中)的影响。这个 拟议的研究测试了微生物组相关肿瘤发生的新机制，并具有重要的 对发现、预防和治疗癌症的影响。

The colonic microbiome has been implicated in colorectal cancer (CRC) pathogenesis but the exact mechanisms underlying these observations remain incompletely understood. We have observed striking associations between specific host microbes and aberrant DNA methylation in CRC. For example, Fusobacterium species are substantially enriched in cancers affected by the CpG Island Methylator Phenotype (CIMP). Preliminary data based on sequencing and qPCR validation also show enrichment of bacteria that have been linked to disease in humans or mouse models (E.coli sp., Klebsiella sp. etc.). High levels of these pathogenic bacteria are associated with recurrences in CIMP+ colon cancers. This unexpected link between colonic microbiota and epigenetic control was also seen in an analysis of DNA methylation genome wide in the colonic mucosa of germ free (GF) mice compared to conventionalized mice, where we found that reintroduction of bacteria led to hypermethylation of normally unmethylated CpG island sites (the main anomaly seen in CIMP+ cases). Thus, our preliminary data support a new hypothesis, that the GI microbiota affects colonic neoplasia through inducing or modulating aberrant DNA methylation and epigenetic control. Mechanistically, we propose that multiple parallel mechanisms may be contributing to this link including DNA damage associated recruitment of silencing complexes, and metabolic disturbances whereby bacteria secrete metabolites and/or toxins that diffuse into colonic epithelial cells and affect DNA methylation directly (e.g. 2-hydroxyglutarate [2-HG], which inhibits the TET DNA demethylase enzymes) or indirectly (e.g. butyrate, which is known to modulate epigenetics through inhibition of histone deacetylases). To test these hypotheses, we propose three specific aims: (1) Define the microbiome across the spectrum of CIMP+ tumors. We will use 16S RNA genomic sequencing in an extensive tumor (cancer, precursors and adjacent normal) set simultaneously characterized for CIMP, mutations and gene expression. (2) Impact of CIMP+ associated bacteria on tumorigenesis and DNA methylation in mice. GF Il10-/-;Apcmin/+ mice will be colonized with bacterial candidates (e.g. E.coli, F.nucleatum, K.pneumonia) and tumor incidence, severity, survival as well as DNA methylation and gene expression in normal and tumor tissues will be evaluated. We will also test whether drugs targeting DNA methylation are effective in prevention of bacteria-associated tumorigenesis. (3) Study metabolites by which bacteria influence DNA methylation profiles. We will use metabolomics on cultures of bacteria associated with CIMP and on lysates from CIMP+ and CIMP- cancers to identify metabolites that potentially modulate DNA methylation. These (e.g. 2HG, butyrate) will be tested for effects on DNA methylation (in cell culture and in GF mice) and tumorigenesis (in mice). The proposed research tests a new mechanism for microbiome-associated tumorigenesis and has important implications for detection, prevention and treatment of CRCs.