Epigenetic mechanisms of carcinogenesis by Parvimonas micra, an oral cavity commensal turned colon cancer pathogen

项目摘要摘要 结直肠癌是世界范围内最常见的恶性肿瘤之一，具有很高的死亡率。在……里面 尽管我们对结直肠癌的遗传学和免疫学的了解取得了进展，但它在很大程度上仍然对 心理治疗。口腔微生物对大肠的定植在健康人中很常见。其中许多 共生体对结直肠癌患者有病理生理影响。然而，他们的作用机制尚不清楚。 我们最近的研究确定微小副单胞菌是结直肠癌患者粪便和结肠中最丰富的口腔细菌 相对于健康个体的粘膜。大肠癌粪便中的P.Micra和其他口腔共生菌的网络 患者排除保护性共生关系。结肠中一组主要基因DNA甲基化的变化 患者的粘膜和血液可预测结直肠癌的风险。将结直肠癌粪便移植给经治疗的无菌小鼠 AOM导致宿主的DNA甲基化和异常隐窝病灶的形成 通过从健康个体转移微生物区系来观察。我们提供了初步数据，P.Micra可以 人结肠肿瘤细胞在低氧条件下共培养时直接甲基化。在此基础上 在这些发现中，我们假设以副单胞菌为例的口腔共生改变了DNA甲基化 宿主DNA以适应肿瘤和促进结直肠癌。我们将在两个具体目标中解决这一问题。1.我们将测试 假设在自发性结直肠癌小鼠模型中，P.Micra改变DNA甲基化和表达 影响结直肠癌生长和肿瘤相关免疫的宿主基因。易患自发性结直肠癌的小鼠 无论有没有P.Micra或嗜酸乳杆菌，都可以得到健康的人体微生物群作为比较。减少的代表性 亚硫酸氢盐测序(RRBS)、ATACseq、RNAseq、免疫分析和组织病理学将如何确定 DNA甲基化的变化影响：(1)结直肠癌的生长和侵袭，(2)肿瘤相关 炎症和免疫反应；(3)肿瘤黏膜和粪便微生物群落组成。2.我们 将检验P.Micra和细菌群落网络调节肿瘤生长和免疫的假设 改变宿主细胞DNA甲基化对结直肠癌的反应。为了测试这一点，我们将(1)识别粪便和 结直肠癌患者的组织贴壁细菌，并与患者结肠和结肠组织的DNA高甲基化有关 跨不同CMS亚类的血液，(2)确定肿瘤DNA甲基化变化与血液的关系 对于突变负荷和免疫反应，(3)区分DNA甲基化的致病和保护模式 在结肠上皮类器官中，是由于暴露于P.Micra而不是嗜酸乳杆菌。

Project Summary Abstract Colorectal cancer (CRC) is among the most common malignancy worldwide and has a high mortality rate. In spite of advances in our understanding of the genetics and immunology of CRC, it remains largely resistant to therapy. Colonization of the large intestine by oral microbes is common among healthy individuals. Many of these commensals have pathophysiological effects in CRC patients. However, their mechanism of action is unclear. Our recent study identified Parvimonas micra as the most enriched oral bacteria in CRC patient stool and colon mucosa relative to healthy individuals. Networks of P. micra and other oral commensal in the stool of CRC patients excluded protective commensals. Changes in DNA methylation of a set of cardinal genes in the colon mucosa and blood of the patients predicted CRC risk. Transfer of CRC stool to germ free mice that were treated with AOM resulted in DNA methylation of the host and formation of aberrant crypt foci, over and above that observed with transfer of microbiota from healthy individuals. We provided preliminary data that P. micra can directly methylate human colon tumor cells when co-cultured together under hypoxic conditions. On the basis of these findings we hypothesize that oral commensals exemplified by Parvimonas alter DNA methylation of host DNA to adapt to tumors and promote CRC. We will address this in two Specific Aims. 1. We will test the hypothesis that in mouse models of spontaneous CRC, P. micra alters DNA methylation and expression of host genes that affect CRC tumor growth and tumor associate immunity. Mice prone to spontaneous CRC will get healthy human microbiota with or without P. micra, or L. acidophilus for comparison. Reduced representation bisulfite sequencing (RRBS), ATACseq, RNAseq, immune assays, and histopathology will determine how changes in DNA methylation impacts, (1) the growth and invasion of CRC tumors, (2) tumor associated inflammation and immune response, (3) microbial community composition of the tumor mucosa and stool. 2. We will test the hypothesis that P. micra and bacterial community networks regulate tumor growth and immune response in CRC by altering DNA methylation of host cells. To test this we will, (1) Identify clusters of fecal and tissue-adherent bacteria in CRC patients and relate these to the DNA hypermethylation of patient colon and blood across different CMS subclasses, (2) determine how altered DNA methylation of tumor and blood relate to mutation load and immune response, (3) distinguish pathogenic versus protective patterns of DNA methylation in colon epithelial organoids, that result from exposure to P. micra, versus L. acidophilus.