Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

Abstract

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10−/− mice. Germ-free AOM-treated Il10−/− mice showed normal colon histology and were devoid of tumors. Il10−/−; Myd88−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.

Mu Opioid Signaling Protects Against Acute Murine Intestinal Injury in a Manner Involving Stat3 Signaling

“…Mice were euthanized at days 8 and 12. Colonoscopies were performed as described previously, 24 on days 5, 8, and 12.…”

“…2,25,26 Immunohistochemistry (IHC) staining for pStat3(Y705) (Cell Signaling Technology Inc., Beverly, MA) was performed according to the manufacturer's specifications at a 1:50 dilution as previously described. 24 Ki-67 staining was performed as previously described. 24 All sections were counterstained with hematoxylin.…”

“…24 Ki-67 staining was performed as previously described. 24 All sections were counterstained with hematoxylin. Two different sections (per animal), approximately 200 m apart, were assessed for staining, and representative images are shown.…”

“…RNA isolation from colonic tissues and CMT-93 cells and subsequent mRNA analysis were performed as previously described, 24 using an ABI Prism HT7700. Specificity and linearity of amplification for each primer set were determined by melting curve analysis and calculation of the slope from serial diluted samples.…”

“…Values were normalized to the internal control glyceraldehyde-3-phosphate dehydrogenase. Primers were Tnf (5=-ATGAGCACAGAAAGCATGATC-3= and 5=-TACAGGCTTGTCACTCGAATT-3=), 24 Il6 (5=-CGGAGGC-TTAATTACACATGTT-3= and 5=-CTGGCTTTGTCTTTCTT-GTTATC-3=), 29 Il1b (5=-GCCCATCCTCTGTGACTCAT-3= and 5=-AGGCCACAGGTATTTTGTCG-3=), GAPDH (5=-GGTGAAGGTCGGAGTCAACGGA-3= and 5=-GAGGGA-TCTCGCTCCTGGAAGA-3=), 29 and Cox2 (Ptgs2) (5=-TG- AGTACCGCAAACGCTTCTC-3= and 5=-TGCAGCCATTT-CCTTCTCTCCT-3=). 30 Cyclin D (Ccnd1), Ifng, Myc, and Reg3b are QuantiTect primers (Qiagen, Valencia, CA).…”