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Upregulation of DNA Methyltransferase–Mediated Gene Silencing, Anchorage-Independent Growth, and Migration of Colon Cancer Cells by Interleukin-6

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Upregulation of DNA Methyltransferase–Mediated Gene Silencing, Anchorage-Independent Growth, and Migration of Colon Cancer Cells by Interleukin-6 Published OnlineFirst March 30, 2010; DOI: 10.1158/1541-7786.MCR-09-0496 Molecular Angiogenesis, Metastasis, and the Cellular Microenvironment Cancer Research Upregulation of DNA Methyltransferase–Mediated Gene Silencing, Anchorage-Independent Growth, and Migration of Colon Cancer Cells by Interleukin-6 Eilis Foran1, Megan M. Garrity-Park3, Coralie Mureau1, John Newell2, Thomas C. Smyrk3, Paul J. Limburg3, and Laurence J. Egan1,2 Abstract Inflammatory bowel disease is characterized by chronic inflammation which predisposes to colorectal cancer. The mechanisms by which inflammation promotes tumorigenesis are not fully known. We aimed to investi- gate the links between colonic inflammation and tumorigenesis via epigenetic gene silencing. Colon cancer specimens were assessed for the expression of DNA methyltransferase-1 (DNMT-1) using immunohistochem- istry. Colorectal carcinoma cell lines were assessed for DNMT1 expression, methylcytosine content, promoter methylation, gene expression, and tumorigenesis in response to interleukin (IL)-6. DNMT1 was expressed at higher levels in both the peritumoral stroma and tumor in inflammatory bowel disease–associated cancers compared with sporadic colon cancers. IL-6 treatment of colon cancer cells resulted in an increase in DNMT1 expression, independent of de novo gene expression. IL-6 increased the methylation of promoter regions of genes associated with tumor suppression, adhesion, and apoptosis resistance. Expression of a subset of these genes was downregulated by IL-6, an effect that was prevented by preincubation with 5-azadeoxycytidine, a DNMT1 inhibitor. Anchorage-independent growth and migration of colon cancer cells was also increased by IL-6 in a 5-azadeoxycytidine–sensitive manner. Our results indicate that DNMT-mediated gene silencing may play a role in inflammation-associated colon tumorigenesis. Mol Cancer Res; 8(4); 471–81. ©2010 AACR. Introduction CD4+ T-lymphocytes at sites of inflammation is mediated in part by interleukin (IL)-6 (4). IL-6 is a pleiotropic cy- Chronic inflammation of gastrointestinal organs elevates tokine secreted by many cell types which exerts proinflam- the risk of carcinomas developing in the affected organ. matory effects in IBD (5-7). High concentrations of IL-6 The relationship between inflammation and cancer has are associated with disease severity, and increased serum been extensively studied in patients with inflammatory IL-6 levels during remission are predictive of disease re- bowel disease (IBD) who have a 2- to 3-fold greater life- lapse (6). Treatment with anti–IL-6 receptor antibody time risk of developing colorectal cancer (CRC) compared ameliorated disease in the CD45RBhigh/SCID adoptive with the general population (1). Duration and extent of transfer model of colitis (8), and the results of a small clin- disease, as well as severity of colonic inflammation, are risk ical trial in Crohn's disease suggested possible therapeutic factors for colon carcinogenesis in patients with IBD (2). efficacy (9). Other studies have recorded high IL-6 serum Specific preventive or therapeutic strategies for colitis- levels in patients with certain cancers (breast, lung, and associated cancer have not yet been developed because of lymphoma; refs. 10-12), and have correlated elevated se- the absence of a clear understanding of the molecular patho- rum IL-6 levels with poor prognosis in colorectal cancers genesis of this disease complication. (10). These findings and the results from experiments in Ulcerative colitis and Crohn's disease are characterized an animal model of colitis-associated cancer point to the by a cytokine-driven mixed inflammatory infiltrate in the possible involvement of IL-6 in the pathogenesis of inflam- intestinal mucosa (3). The accumulation of pathogenic mation-associated cancer (11-13). The mechanisms by which IL-6 might promote car- cinogenesis are not well understood. One study reported Authors' Affiliations: 1Department of Pharmacology and Therapeutics, that overexpression of IL-6 in cholangiocarcinoma cells National University of Ireland and 2Health Research Board Clinical Research Facility, Galway, Ireland; and 3Division of Gastroenterology increased the expression of DNA methyltransferase-1 and Hepatology, Mayo Clinic, Rochester, Minnesota (DNMT1; ref. 14). This suggests that IL-6, acting in an Corresponding Author: Laurence J. Egan, University Hospital Galway, autocrine manner, might interact with the CpG island Newcastle Road, Galway, Ireland. Phone: 353-9149-5355; Fax: 353- methylator mechanism of carcinogenesis. The hyper- 9149-5572. E-mail: [email protected] methylation of CpG islands, and concomitant loss of gene doi: 10.1158/1541-7786.MCR-09-0496 expression, is the best characterized epigenetic change to ©2010 American Association for Cancer Research. occur in tumors and is catalyzed by DNMT1, DNMT3a, www.aacrjournals.org 471 Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst March 30, 2010; DOI: 10.1158/1541-7786.MCR-09-0496 Foran et al. and DNMT3b (15, 16). Classically, DNMT1 is consid- were resolved on 6% SDS-PAGE gels and transferred ered a maintenance methyltransferase whereas DNMT3b to polyvinylidene difluoride membranes. Membranes may be more important for de novo CpG methylation, al- were incubated overnight at 4°C in the appropriate pri- though this distinction is not absolute (17, 18). DNMT1 mary antibody. Membranes were washed and incubated overexpression has been observed in cancers (19), but with goat anti-rabbit horseradish peroxidase or goat an- whether cytokines expressed during chronic inflammation ti-mouse horseradish peroxidase secondary antibodies have any role in regulating its expression is unclear. (Pierce). A G:BOX chemi-system (Syngene) was used to In this study, we investigated whether IL-6 might link capture luminescent signals. colonic inflammation with carcinogenesis through the reg- ulation of epigenetic tumor suppressor gene silencing. Our Real-time PCR results suggest that IL-6, a component of the inflammatory RNA was extracted from cells using kits (Qiagen) and tumor microenvironment, might promote colonic tumori- cDNA was prepared using Superscript II (Invitrogen). genesis through DNMT1-mediated tumor suppressor gene Primer sequences were designed using the Primer 3 algo- silencing. rithm (available on request). Real-time PCR was done us- ing Power SYBR Green master mix on a 7500 Real-time Materials and Methods PCR system (Applied Biosystems). GAPDH was used as an internal control, and relative expression levels were cal- Patient Selection culated using the ΔCT method (20). The Institutional Review Board of the Mayo Clinic ap- proved this research protocol. IBD-CRC cases were select- Promoter Analysis ed from the Mayo Clinic diagnostic index (1976-2006). Luciferase assays were done on P1, P2, P3, and P4 of the Sporadic-CRC controls were identified through the Mayo DNMT1 promoter. D1P1 contained 1,270 bp of the pro- pathology index. Data with regard to age, gender, and du- moter sequence preceding exon 1 of DNMT1. D1P1x was ration of IBD were abstracted from the records. constructed by deleting the 5′ 786 bp of D1P1. D1P2F and D1P2R consisted of the 1,656 bp fragment encom- Reagents and Media passing promoters P2 to P4 cloned into pT81luc in for- McCoy's 5A, DMEM, RPMI 1640, and fetal bovine se- ward (D1P2F) or reverse (D1P2R) orientation. These rum (FBS) were from Life Technologies. IL-6, LY294002, constructs were gifts from Prof. Dr. Wolfgang Schulz triciribine, SP600125, and curcubitacin were from Calbio- (21). For DNMT1 promoter experiments, cells were cotrans- chem. Antibodies to DNMT1 were from Santa Cruz Bio- fected with Renilla and either DNMT1-P1, DNMT1-P1x, technology,Inc.AntibodiestoAKT,STAT3,andERK DNMT1-P2-P4-F, or DNMT1-P2-P4-R in a 96-well plate were from Cell Signaling. Anti-Lamin B1 was from Ab- at a 1:10 ratio. IL-6 was added 24 h posttransfection, and cam. Unless otherwise stated, reagents were obtained from cells were incubated for a further 24 h. Luciferase assays Sigma (Sigma Aldrich). were done using the Dual-glo luciferase assay (Promega). Immunohistochemistry Individual 5-μm sections were processed following stan- DNMT1-V5 Tet-off System dard protocols using DNMT1 antibody (AB19905, dilu- pTRE-tight/DNMT1-V5 was constructed as follows: tion 1:300; Abcam) and CD68 (dilution 1:100; DAKO). the stop codon in pORF9-hDNMT1 (Invivogen) was re- Slides were scored and determined by two pathologists moved by site-directed mutagenesis, and hDNMT1 was (T. Smyrk and M. Garrity-Park) using an ordinal 0-3 scale excised, purified, and subcloned into pcDNA3.1-V5 (Invi- for staining intensity, which was multiplied by the percent- trogen). DNMT1-V5 was excised and subcloned into the age of positive cells to give an overall abundance score pTRE-tight vector (Clontech). The construct was electro- which ranges from 0 to 300. porated into HeLa Tet-off cells. Twenty four hours later, doxycycline (2 μg/mL) was added to the medium in the Cell Lines presence or absence of IL-6 (100 ng/mL). In certain experi- μ HT29, HCT15, and HCT116 cell lines were used ments, cells were pretreated with 5 mol/L of triciribine, μ μ (American Type Culture Collection). HeLa Tet-off cells 50 mol/L of SP600125, 1 mol/L of curcubitacin, or ve- were from Clontech. HCT116 cells were cultured in hicle control (DMSO) for 30 min. Cells were then treated μ McCoy's 5A medium with 10% FBS. HT29 cells were with 2 g/mL of doxycycline, 100 ng/mL of IL-6, or both cultured in DMEM with 10% FBS. HCT15 cells were together. cultured in RPMI 1640 with 10% FBS. HeLa Tet-Off cells were maintained in DMEM with 10% FBS and 5-Methylcytosine Assay 100 μg/mL of G418. Cells were treated with 5 aza-CdR or IL-6 for 72 h, and genomic DNA was obtained using a kit (Qiagen). One mi- Western Blotting crogram of DNA was digested using McrBC (New Eng- Nuclear proteins were extracted using the NE-PER nu- land Biolabs) for 2 h. DNA was resolved on a DNA clear extraction kit (Pierce).
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