Folic Acid Supplementation and Colitis-associated Colon Carcinogenesis

项目总结/摘要 溃疡性结肠炎是一种炎症性肠病， 结肠直肠癌虽然在治疗这种疾病方面已经取得了进展， 对这一高危人群制定癌症预防战略的关注较少。 溃疡性结肠炎患者经常出现叶酸缺乏症，需要补充叶酸（FA）， 叶酸合成形式。FA对结直肠癌风险的影响尚不清楚，最近的建议 FA补充剂可能有助于预防结肠炎相关的结肠直肠癌，这一点备受关注。 该小组的初步研究提供了第一个证据，即补充FA（8 mg/kg饮食）会导致 AOM/DSS诱导的结肠炎小鼠中结直肠肿瘤形成的剂量依赖性增加。结果 相关的RNASeq和体外分析表明，FA通过激活ERK促进肿瘤发生， 在p53功能失调的结肠上皮细胞中诱导NF-κB信号传导， 结肠炎相关癌症的发展。拟议研究的假设是， 在具有功能障碍/突变型p53的细胞中促进UC相关的癌症，但在具有野生型p53的细胞中不促进UC相关的癌症， ERK和NF-κB B介导的炎症通路。FA诱导肿瘤形成的机制将 在Aim 1中使用具有不同p53的CRISPR工程化的同基因人RKO结肠癌细胞进行研究 状态（p53+/+、p53-/-和p53+/Mut）。FA和功能失调的p53对ERK、NF-κB信号通路的激活， 以及细胞周期进程。将在目标2中验证体外结果，其中 补充的体内分析将检查FA和突变型p53对DSS诱导的细胞凋亡的联合作用。 p53+/+和p53+/515 A（突变）小鼠中结肠炎相关肿瘤发生和相关生物标志物。此外，本发明还提供了一种方法， 将检查在诱导结肠炎之前给予的高剂量FA的影响。肿瘤发生率及 多重性以及肠道炎症程度将作为这些研究的主要终点。基于 关于肠道微生物群对结肠炎的重要作用及其重新合成叶酸的能力， FA给药对粪便和粘附细菌多样性和相对丰度的影响 目的3中将检查患有DSS诱导的结肠炎的p53+/515 A小鼠的肠。小鼠将接受以下处理： 5-氨基水杨酸（5-阿萨），在FA期间溃疡性结肠炎的常用维持治疗 暴露以概括诊断为结肠炎后患者的临床治疗。的组成 根据16 S rRNA和宏基因组测序数据确定的粪便和结肠粘附微生物组， 与结肠炎症、屏障功能和肿瘤发生率/多样性相关。结果预计 提供关于FA补充对结肠炎相关肿瘤发生的影响的重要见解， 告知溃疡性结肠炎患者使用FA补充剂的第一个指南。

Project Summary/Abstract Patients with ulcerative colitis, a form of inflammatory bowel disease, face an increased risk of developing colorectal cancer. Although advances have been made in the therapeutic management of this disease, much less attention has been given to the development of cancer preventive strategies for this high-risk population. Ulcerative colitis patients often develop folate deficiencies that require supplementation with folic acid (FA), a synthetic form of folate. The effect of FA on risk for colorectal cancer remains unclear and the recent suggestion that FA supplements may be useful in preventing colitis-associated colorectal cancer is of great concern. Preliminary studies from this group provide the first evidence that FA supplementation (8 mg/kg diet) causes a dose-dependent increase in the formation of colorectal tumors in mice with AOM/DSS-induced colitis. Results from associated RNASeq and in vitro analyses suggest that FA promotes tumorigenesis by activating ERK and inducing NF-κB signaling in colonic epithelial cells with dysfunctional p53, the gatekeeping event in the development of colitis-associated cancers. The hypothesis of the proposed studies is that FA supplementation promotes UC-associated cancer in cells with dysfunctional/mutant p53, but not in cells with wild type p53, via an inflammatory pathway mediated by ERK and NF-κB. The mechanism by which FA induces tumor formation will be investigated in Aim 1 using CRISPR engineered isogenic human RKO colon carcinoma cells with varying p53 status (p53+/+, p53-/- and p53+/Mut). The impact of FA and dysfunctional p53 on activation of ERK, NF-κB signaling, as well as cell cycle progression will be evaluated. In vitro findings will be validated in Aim 2, where complimentary in vivo analyses will examine the combined effect of FA and mutant p53 on DSS-induced colitis-associated tumorigenesis and associated biomarkers in p53+/+ and p53+/515A (mutant) mice. In addition, the impact of high dose FA given prior to the induction of colitis will be examined. Tumor incidence and multiplicity, as well as degree of intestinal inflammation, will serve as primary endpoints of these studies. Based on the important contribution of intestinal microbiota to colitis and their ability to synthesize folate de novo, the impact of FA administration on the diversity and relative abundance of fecal and adherent bacteria within the intestine of p53+/515A mice with DSS-induced colitis will be examined in Aim 3. Mice will be treated with 5-aminosalicylic acid (5-ASA), a common maintenance therapy for ulcerative colitis, for the duration of FA exposure to recapitulate the clinical therapy of a patient following a diagnosis of colitis. The composition of the fecal and colon-adherent microbiomes, determined from 16S rRNA and metagenomic sequencing data, will be correlated with colonic inflammation, barrier function and tumor incidence/multiplicity. The results are expected to provide significant insight into the impact of FA supplementation on colitis-associated tumorigenesis and inform the first guidelines for the use of FA supplements by patients with ulcerative colitis.