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 这些观察结果的根本原因仍然不完全清楚。我们观察到惊人的关联 特定宿主微生物与结直肠癌中异常DNA甲基化之间的关系。例如，梭杆菌属物种 在受CpG岛甲基化表型（CIMP）影响的癌症中大量富集。初步数据 基于测序和qPCR验证的结果也显示， 在人或小鼠模型中（E.coli sp.，克雷伯氏菌属等）。高水平的这些致病细菌是 与CIMP+结肠癌复发相关。结肠微生物群和 表观遗传控制也见于细菌结肠粘膜DNA甲基化全基因组分析中， 自由（GF）小鼠与常规小鼠相比，我们发现重新引入细菌导致 正常未甲基化的CpG岛位点的高甲基化（在CIMP+病例中观察到的主要异常）。因此，在本发明中， 我们的初步数据支持了一个新的假设，即胃肠道微生物群通过以下途径影响结肠肿瘤 诱导或调节异常DNA甲基化和表观遗传控制。从机制上讲，我们建议， 多个平行机制可能有助于这种联系，包括DNA损伤相关的招募， 沉默复合物和代谢紊乱，由此细菌分泌代谢物和/或毒素， 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]，其抑制 泰特DNA脱甲基酶）或间接地（例如丁酸，已知其通过 组蛋白脱乙酰酶的抑制）。为了验证这些假设，我们提出了三个具体目标：（1）定义 CIMP+肿瘤谱中的微生物组。我们将使用16 S RNA基因组测序， 广泛的肿瘤（癌症，前体和相邻正常）集同时表征为CIMP突变 和基因表达。(2)CIMP+相关细菌对肿瘤发生和DNA甲基化的影响 小鼠GF II 10-/-;Apcmin/+小鼠将用候选细菌（例如大肠杆菌、具核梭菌、肺炎克雷伯菌）定殖 与肿瘤的发病率、严重程度、生存率以及正常和肿瘤组织中DNA甲基化和基因表达的关系 将对组织进行评价。我们还将测试靶向DNA甲基化的药物是否能有效预防 与细菌相关的肿瘤(3)研究细菌影响DNA甲基化的代谢物 数据区.我们将在与CIMP相关的细菌培养物和CIMP+的裂解物上使用代谢组学 和CIMP-癌症，以鉴定可能调节DNA甲基化的代谢物。这些（例如2 HG、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， 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.