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Epigenetic Repression of DNA Mismatch Repair by Inflammation and Hypoxia in Inflammatory Bowel Disease–Associated Colorectal Cancer

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Epigenetic Repression of DNA Mismatch Repair by Inflammation and Hypoxia in Inflammatory Bowel Disease–Associated Colorectal Cancer Published OnlineFirst July 28, 2009; DOI: 10.1158/0008-5472.CAN-09-1285 Cell, Tumor, and Stem Cell Biology Epigenetic Repression of DNA Mismatch Repair by Inflammation and Hypoxia in Inflammatory Bowel Disease–Associated Colorectal Cancer Robert A. Edwards,1 Mavee Witherspoon,2 Kehui Wang,1 Kambiz Afrasiabi,2 Trang Pham,2 Lutz Birnbaumer,3 and Steven M. Lipkin2 Departments of 1Pathology and 2Medicine, University of California-Irvine, Irvine, California; and 3Laboratory of Neurobiology/Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina Abstract increased likelihood of right-sided colon cancer location, non- Sporadic human mismatch repair (MMR)–deficient colorectal polyposis pathology, mucinous histology, and presence of a cancers account for f12.5% of all cases of colorectal cancer. Crohn’s-like lymphocytic infiltrate in tumors (2, 3). MMR-deficient colorectal cancers are classically characterized MMR-deficient colorectal cancers arise through three distinct by right-sided location, multifocality, mucinous histology, and genetic mechanisms. First, monoallelic germ-line MMR mutation lymphocytic infiltration. However, tumors in germ-line MMR- and somatic loss of the second MMR allele causes hereditary deficient mouse models lack these histopathologic features. nonpolyposis colon cancer, also known as Lynch syndrome. Lynch Mice lackingthe heterotrimeric G protein A subunit GiA2 syndrome carriers body tissues are MMR-proficient because of the develop chronic colitis and multifocal, right-sided cancers other allele. As predicted by the Knudson hypothesis, the second with mucinous histopathology, similar to human MMR- MMR gene allele is somatically lost in some cells, which then À À deficient colorectal cancer. YoungGiA 2 / colonic epithelium become MMR-deficient and transform into colorectal cancer and has normal MMR expression but selectively loses MLH1 and other cancers. The second mechanism is biallelic germ-line consequently PMS2 expression followinginflammation. constitutive MMR gene mutation, where all body tissues are À À GiA2 / cancers have microsatellite instability. Mlh1 is MMR-deficient. These patients have a different clinical phenotype, epigenetically silenced not by promoter hypermethylation with very early-onset hematologic, brain, and small intestinal but by decreased histone acetylation. Chronically inflamed cancers (4–6). Although colorectal cancers can develop, penetrance À À GiA2 / colonic mucosa contains patchy hypoxia, with is low and arise more commonly in the left colon (4, 5). increased crypt expression of the hypoxia markers DEC-1 The third genetic mechanism, de novo sporadic MMR-deficient f and BNIP3. Chromatin immunoprecipitation identified in- colorectal cancers, accounts for 12.5% of colorectal cancer (6). Both creased bindingof the transcriptional repressor DEC-1 to the MMR alleles are somatically mutated or epigenetically inactivated in proximal Mlh1 promoter in hypoxic YAMC cells and colitic some cells, which transform into cancer. Mlh1 promoter hyper- À À À À GiA2 / crypts. TreatingGi A2 / mice with the histone methylation is the most common cause of sporadic MMR-deficient deacetylase inhibitor suberoylanilide hydroxamic acid signif- colorectal cancer (7–9). In vitro studies have shown that epigenetic icantly decreased colitis activity and rescued MLH1 expression Mlh1 inactivation can also occur via hypoxia-induced transcriptional in crypt epithelial cells, which was associated with increased repression of the proximal Mlh1 promoter by histone deacetylases acetyl histone H3 levels and decreased DEC-1 bindingat the (HDAC; ref. 9). However, whether this mechanism is relevant to proximal Mlh1 promoter, consistent with a histone deacety- in vivo colorectal cancer tumorigenesis is unknown. lase–dependent mechanism. These data link chronic hypoxic Multiple biallelic MMR germ-line knockout mouse models have inflammation, epigenetic MMR protein down-regulation, been created (Supplementary Table S1). These mutations primarily development of MMR-deficient colorectal cancer, and the cause lymphomas and small intestine adenomas (10–16) Only first mouse model of somatically acquired MMR-deficient rarely do these models develop colorectal cancer, and mucinous, colorectal cancer. [Cancer Res 2009;69(16):6423–9] nonpolyposis, or proximal colon colorectal cancers essentially never arise. These models are therefore more similar to human biallelic germ-line constitutive MMR deficiency than somatically Introduction acquired MMR deficiency syndromes. Gia2 is a heterotrimeric G protein a subunit, the deletion of DNA mismatch repair (MMR)–deficient colorectal cancers have which in mice leads to spontaneous colitis and nonpolyposis, distinct features that distinguish them from other colorectal cancer right-sided, multifocal colorectal cancers with mucinous histology types (1). These include increased frameshift mutation rates and a Crohn’s-like inflammatory infiltrate (17–19). The similarities [commonly called microsatellite instability (MSI)], multifocality, À À between Gia2 / mouse colorectal cancer and somatically acquired human MMR-deficient colorectal cancer (Supplementary Note: Supplementary data for this article are available at Cancer Research Online Table S1) led us to investigate whether acquired MMR silencing (http://cancerres.aacrjournals.org/). might contribute as a mechanism of colorectal cancer tumorigen- Requests for reprints: Robert A. Edwards, Department of Pathology, University of a À/À California-Irvine, D449 Med Sci I, Irvine, CA 92697-4800. Phone: 949-824-8576; Fax: 949- esis in Gi 2 mice. We found that MLH1 and PMS2 protein levels 824-2160; E-mail: [email protected] and Steven M. Lipkin, Department of Genetic in crypts are initially normal but are suppressed following the onset Medicine, Weill Cornell Medical College, Room 718 HSS, New York, NY 10021. of hypoxic colitis. Hypoxia induces colonic expression of the bHLH Phone: 212-774-7160; E-mail: [email protected]. I2009 American Association for Cancer Research. transcriptional repressor DEC-1 and enhances its binding to the doi:10.1158/0008-5472.CAN-09-1285 proximal Mlh1 promoter, thereby repressing MLH1 expression and www.aacrjournals.org 6423 Cancer Res 2009; 69: (16). August 15, 2009 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst July 28, 2009; DOI: 10.1158/0008-5472.CAN-09-1285 Cancer Research À À leading to MSI. Treatment of Gia2 / mice with HDAC inhibitor forward 5¶-GTTTTACTCCATTCGGAAGCAGT-3¶, murine MLH1 reverse (HDACi) decreases colitis and relieves epigenetic repression of 5¶-TGTGAGCGGAAGGCTTTATAGAT-3¶,murinePMS2forward ¶ ¶ ¶ MLH1 expression. In summary, we describe the first mouse model 5 -ACCTCACGCAGGTTGACTT-3 , murine PMS2 reverse 5 -AGTCGAGTCC- ¶ ¶ of somatically acquired MMR deficiency and show that hypoxia- CAACGCTTG -3 , murine BNIP3 forward 5 -CGAAGCGCACAGCTACTCTC- 3¶, murine BNIP3 reverse 5¶-GCCTTCCAATGTAGATCCCCAAG-3¶, murine induced Mlh1 silencing may be another important epigenetic DEC-1 forward 5¶-TACAAGCTGGTGATTTGTCGG-3¶, and murine DEC-1 mechanism contributing to the development of MMR-deficient reverse 5¶-CTGGGAAGATTTCAGGTCCCG-3¶. Normalization was done colorectal cancer. using murine RPA2 (forward 5¶-ATACCCAGACAACAGAGGG-3¶ and reverse 5¶-GCCAGTCCGCTCAATCACC-3¶). In vitro hypoxia treatment. Subconfluent cultures of YAMC cells Materials and Methods (a kind gift of Dr. Robert Whitehead) were grown in RPMI with 5% fetal À À Mice. Wild-type (WT) and Gia2 / mice on a cross-bred 129Sv/C57BL6 bovine serum, 100 ng/AL IFN-g, and 1Â insulin/transferrin/selenium and j background were genotyped as described previously (19). All experiments incubated in a Biospherix hypoxia chamber at 37 Cin1%O2 for 24 h and were approved by the Institutional Animal Care and Use Committee at total RNA was analyzed by quantitative real-time PCR. À À University of California-Irvine. Suberoylanilide hydroxamic acid treatment of GiA2 / mice. À À In vivo assessment of apoptosis induction. Age-matched WT and Groups of 32- to 36-week-old Gia2 / mice (n = 4) were treated with À À Gia2 / mice were injected i.p. with 20 mg/kg 1,2-dimethylhydrazine for daily gavage feedings with a 5 mg/mL solution of suberoylanilide 24 h. Sections (5 Am) of fixed control and 1,2-dimethylhydrazine–treated hydroxamic acid (SAHA; Cayman Chemical) solubilized in PBS containing colons were stained for apoptotic cells using an Apoptag staining kit a 5 molar excess of h-hydroxypropyl cyclodextrin (Sigma) for 7 days (final (Chemicon). The data are expressed as the mean F SE of the normalized dose 50 mg/kg). Control animals received only cyclodextrin solution. Colons ratio of terminal deoxynucleotidyl transferase–mediated dUTP nick end were harvested and crypts isolated as described above and analyzed for labeling–positive/propidium iodide–positive cells. MLH1 expression by Western blot analysis. Colonic crypt isolation, SDS-PAGE, and Western blotting. Rinsed Chromatin immunoprecipitation. Cells and crypts were cross-linked small intestine and colon segments were reverted on a Labconco vibratory in 1% formaldehyde for 10 min and then lysed in SDS lysis buffer and stirrer and shaken in 30 mmol/L EDTA in HBSS. Crypts liberated between sonicated to a fragment length of 300 to 1,000 bp. The sonicate was 20 and 40 min (>98% pure by cytospin analysis) were used for Western precleared and the supernatant was incubated overnight at 4jC
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