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. 2017 Jul 1;77(13):3467-3478.
doi: 10.1158/0008-5472.CAN-17-0056. Epub 2017 May 18.

Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis

Ashley R Maiuri  1 , Michael Peng  1 , Ram Podicheti, Shruthi Sriramkumar  1 , Caitlin M Kamplain  1 , Douglas B Rusch, Christina E DeStefano Shields  2 , Cynthia L Sears  2   3   4 , Heather M O'Hagan  5   6
Affiliations

Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis

Ashley R Maiuri et al. Cancer Res. .

Erratum in

Abstract

Aberrant silencing of genes by DNA methylation contributes to cancer, yet how this process is initiated remains unclear. Using a murine model of inflammation-induced tumorigenesis, we tested the hypothesis that inflammation promotes recruitment of epigenetic proteins to chromatin, initiating methylation and gene silencing in tumors. Compared with normal epithelium and noninflammation-induced tumors, inflammation-induced tumors gained DNA methylation at CpG islands, some of which are associated with putative tumor suppressor genes. Hypermethylated genes exhibited enrichment of repressive chromatin marks and reduced expression prior to tumorigenesis, at a time point coinciding with peak levels of inflammation-associated DNA damage. Loss of MutS homolog 2 (MSH2), a mismatch repair (MMR) protein, abrogated early inflammation-induced epigenetic alterations and DNA hypermethylation alterations observed in inflammation-induced tumors. These results indicate that early epigenetic alterations initiated by inflammation and MMR proteins lead to gene silencing during tumorigenesis, revealing a novel mechanism of epigenetic alterations in inflammation-driven cancer. Understanding such mechanisms will inform development of pharmacotherapies to reduce carcinogenesis. Cancer Res; 77(13); 3467-78. ©2017 AACR.

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Conflict of interest statement

COI statement: The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Inflammation-induced tumors have unique DNA hypermethylation alterations
A) Unsupervised hierarchical clustering of MBD-seq z-scores of the 239 regions with DNA hypermethylation in one of the tumor groups relative to mock epithelium (rows). Each column corresponds to the indicated epithelium or tumor sample. The color of each cell reflects the degree of methylation. All tissue samples were collected 8 weeks after inoculation (N=3 mock and ETBF epithelium and mock tumors; N=5 ETBF tumors). B) Tukey box plots of z-scores of regions with gains or losses of methylation in ETBF or mock tumors relative to mock epithelium. C) MBD-seq data at representative regions for indicated epithelium and tumors. In vitro methylated DNA (IVD) serves as a positive control. D) Pyrosequencing of bisulfite treated DNA from indicated tissue in promoters of candidate genes. Mean +/− SEM. N≥6. *p<0.05. E) qMSP of samples as in D. *p<0.05 compared to mock epithelium. #p<0.05 compared to ETBF epithelium. F) Gene expression by qPCR of candidate genes relative to mock epithelium. Mean +/− SEM. N≥6. *, # as in E. See also Figure S1.
Figure 2
Figure 2. Genes that undergo methylation in tumors have reduced expression at the time point of highest oxidative damage
A) 8-oxoG dot blot of DNA extracted from distal colon epithelium from mock or ETBF-inoculated mice, N=3. Bar graph of densitometry determined using ImageJ for dots. Mean +/− SEM. B) 8-oxoG staining by flow cytometry of Epcam-positive cells from mice treated as in A. C) Expression of candidate genes by qPCR in distal colon epithelium from mice at the indicated days post-mock or ETBF. Mean +/− SEM. N=5. *p<0.05. D) EZH2, IgG and E) H3K27me3 enrichment by ChIP relative to input at promoters of indicated genes in distal colon epithelium from mice 2 days post-mock or ETBF. Mean +/− SEM. N=3. *p<0.05. See also Figure S2.
Figure 3
Figure 3. Acute inflammation-induced epigenetic changes are dependent on MSH2
A) IgG or Ezh2 co-IP from distal colon epithelial cells 2 days post-mock (M) or ETBF (E) inoculation. B) Anti-MSH2 IHC of the distal colon of WT or Msh2l/lVC mice 2 days post-mock or ETBF. Images are representative of staining from 3 mice per group. C) Proteins that are tightly bound to chromatin (tight chromatin) from epithelium as in A. D) Expression of candidate genes by qPCR in distal colon epithelial cells from mice at 2 days post-mock or ETBF. Mean +/− SEM. N=5–7. *p<0.05. E) Anti-γ-H2AX IHC of the same tissue sections as in B with quantification of γ-H2AX foci per crypt. N≥45 crypts. Mean+/−SEM. *p<0.05 by Tukey’s multiple comparison test. See also Figure S3.
Figure 4
Figure 4. Inflammation-induced tumorigenesis is increased in the distal colon of mice with altered Msh2 expression
A) PCA analysis of 16S microbiome sequencing of DNA from stool samples from WT/Min or Msh2l/lVC/Min mice 8 weeks post mock or ETBF. B) Heatmap representing the unsupervised hierarchical clustering of 65 OTUs found to be differentially abundant by one pair-wise comparison (rows) in individual stool samples (columns) from indicated mice treated as in A. C) ETBF abundance in stool relative to total bacterial DNA by qPCR. Symbols represent data from individual mice. Horizontal line is mean +/− SEM. N≥13. *p<0.05. D) Tukey box plots of tumor counts by cm in WT/Min or Msh2l/lVC/Min mice 8 weeks after mock or ETBF. N≥8. *p<0.05. E) Whole cell protein lysate from distal colon epithelium or tumors from mice of the indicated genotypes 8 weeks post-mock or ETBF inoculation, respectively, were blotted for the indicated proteins. See also Figure S4.
Figure 5
Figure 5. Msh2 deficiency abrogates inflammation-induced epigenetic changes in tumors
A) Unsupervised hierarchical clustering of MBD-seq z-scores of regions with increased DMRs in one of the tumor groups relative to mock epithelium (rows). Each column corresponds to the indicated epithelium or tumor sample. The color of each cell reflects the degree of methylation. B) Numbers of DMRs for the indicated tumor group relative to the corresponding mock epithelium that overlap between comparisons. Green and red numbers are hypomethylated and hypermethylated regions, respectively. C) MBD-seq data at representative regions for indicated epithelium and tumors 8 weeks post-inoculation. D) Expression of candidate genes by qRTPCR relative to WT/Min ETBF tumors. Mean +/− SEM. N=5. *p<0.05. E) Total 5-mC content of DNA. Mean +/− SEM. N=3 mock, N=6 tumor. *p<0.05. See also Figure S5.
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