Epigenetic repression of DNA mismatch repair by inflammation and hypoxia in inflammatory bowel disease-associated colorectal cancer

Abstract

Sporadic human mismatch repair (MMR)-deficient colorectal cancers account for approximately 12.5% of all cases of colorectal cancer. MMR-deficient colorectal cancers are classically characterized by right-sided location, multifocality, mucinous histology, and lymphocytic infiltration. However, tumors in germ-line MMR-deficient mouse models lack these histopathologic features. Mice lacking the heterotrimeric G protein alpha subunit Gialpha2 develop chronic colitis and multifocal, right-sided cancers with mucinous histopathology, similar to human MMR-deficient colorectal cancer. Young Gialpha2-/- colonic epithelium has normal MMR expression but selectively loses MLH1 and consequently PMS2 expression following inflammation. Gialpha2-/- cancers have microsatellite instability. Mlh1 is epigenetically silenced not by promoter hypermethylation but by decreased histone acetylation. Chronically inflamed Gialpha2-/- colonic mucosa contains patchy hypoxia, with increased crypt expression of the hypoxia markers DEC-1 and BNIP3. Chromatin immunoprecipitation identified increased binding of the transcriptional repressor DEC-1 to the proximal Mlh1 promoter in hypoxic YAMC cells and colitic Gialpha2-/- crypts. Treating Gialpha2-/- mice with the histone deacetylase inhibitor suberoylanilide hydroxamic acid significantly decreased colitis activity and rescued MLH1 expression in crypt epithelial cells, which was associated with increased acetyl histone H3 levels and decreased DEC-1 binding at the proximal Mlh1 promoter, consistent with a histone deacetylase-dependent mechanism. These data link chronic hypoxic inflammation, epigenetic MMR protein down-regulation, development of MMR-deficient colorectal cancer, and the firstmouse model of somatically acquired MMR-deficient colorectal cancer.

Figure 1. A.) Acquired suppression of MLH1 and PMS in Giα2-/- crypts

Purified 8 week old (top) and older than 20 week (bottom) WT and Giα2-/- crypt epithelial cells were immunoblotted for MLH1, PMS2, and MSH2. B.) qPCR for MLH1 and PMS2 in young and old crypts (n=3 each) reveals a significant downregulation of MLH1 in older Giα2-/- crypts. Results shown are representative of three separate experiments. **, p<0.01.

Figure 2. Inflamed Giα2-/- crypts are hypoxic

(A), YAMC cells grown in 1% O2 (right) upregulate the hypoxia markers DEC-1 and BNIP3, which are also upregulated in purified crypt lysates from colitic Giα2-/- mice (left). The data shown are representative of three separate YAMC and crypt isolation experiments. (B), Frozen sections from EF5-treated WT and Giα2-/- mice (age 20-27 weeks) were stained with Cy3-conjugated anti EF5. WT colon (A) had minimal EF5 staining, while colitic Giα2-/- mucosa (B) was profoundly hypoxic in surface epithelium and in some deeper crypt cells. EF5 staining of Giα2-/- cancers (C.) was patchy, and involved primarily malignant epithelial cells and the adjacent stroma. As a negative control, pre-adsorbed anti EF-5 (D.) revealed no staining.

Figure 3. Hypoxia modulates histone H3 acetylation, and DEC-1 and HDAC1 binding to the proximal MLH1 promoter

Hypoxia induces histone H3-deacetylation at the MLH1 promoter in a HDAC-dependent manner (A), enhances DEC-1 and HDAC1 binding to the proximal MLH1 promoter in YAMC cells (B) In colitic Giα2-/- crypts, DEC-1 binding to the MLH1 promoter is also enhanced.

Figure 4. HDACi treatment improves colitis and rescues MLH1 expression by decreasing DEC-1 occupancy at the proximal MLH1 promoter

(A), Age matched 33-36 week old Giα2-/- mice were treated with vehicle or 50 mg/kg SAHA (n=4 each) for 8 days, then assessed for disease severity using a histologic activity scoring index. *, p < 0.05; data are representative of three separate experiments. Purified crypt lysates were evaluated for MLH1 protein (B) and mRNA (C) by western blotting and qPCR, respectively. (D), ChIP reveals an increase in acetyl H3 levels and decreased DEC-1 binding in SAHA-treated crypts.