Role of the microbiota in DNA methylation and CRC development

结肠微生物群与结直肠癌 (CRC) 发病机制有关，但确切机制尚不清楚 这些观察结果的背后仍然不完全清楚。我们观察到了惊人的关联 CRC 中特定宿主微生物与异常 DNA 甲基化之间的关系。例如，梭杆菌属 在受 CpG 岛甲基化表型 (CIMP) 影响的癌症中显着富集。初步数据 基于测序和 qPCR 验证还显示与疾病相关的细菌富集 在人类或小鼠模型中（大肠杆菌、克雷伯氏菌等）。这些致病菌的含量很高 与 CIMP 结肠癌的复发有关。结肠微生物群与肠道菌群之间的这种意想不到的联系 在对细菌结肠粘膜全基因组 DNA 甲基化的分析中也发现了表观遗传控制。 与传统小鼠相比，自由（GF）小鼠，我们发现重新引入细菌会导致 正常未甲基化的 CpG 岛位点过度甲基化（CIMP 病例中出现的主要异常）。因此， 我们的初步数据支持一个新的假设，即胃肠道微生物群通过以下方式影响结肠肿瘤： 诱导或调节异常 DNA 甲基化和表观遗传控制。从机制上来说，我们建议 多种平行机制可能促成了这种联系，包括与 DNA 损伤相关的招募 沉默复合物和代谢紊乱，细菌分泌代谢物和/或毒素扩散 进入结肠上皮细胞并直接影响 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（例如丁酸盐，已知它可以通过调节表观遗传学） 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 肿瘤范围内的微生物组。我们将使用 16S RNA 基因组测序 广泛的肿瘤（癌症、前体癌和邻近正常肿瘤）同时具有 CIMP、突变特征 和基因表达。 (2) CIMP相关菌对肿瘤发生和DNA甲基化的影响 老鼠。 GF Il10-/-;Apcmin/ 小鼠将被候选细菌定植（例如大肠杆菌、具核梭菌、肺炎克雷伯氏菌） 正常和肿瘤中肿瘤的发生率、严重程度、存活率以及 DNA 甲基化和基因表达 将评估组织。我们还将测试针对DNA甲基化的药物是否能有效预防 细菌相关的肿瘤发生。 (3) 研究细菌影响DNA甲基化的代谢物 配置文件。我们将对与 CIMP 相关的细菌培养物和 CIMP 裂解物使用代谢组学 和 CIMP-癌症，以识别可能调节 DNA 甲基化的代谢物。这些（例如 2HG、丁酸盐） 将测试对 DNA 甲基化（在细胞培养物和 GF 小鼠中）和肿瘤发生（在小鼠中）的影响。的 拟议的研究测试了微生物组相关肿瘤发生的新机制，具有重要意义 对 CRC 的检测、预防和治疗的影响。

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.