Cellular Oncology 28 (2006) 259–272 259 IOS Press ADAMTS1, CRABP1,andNR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis Guro E. Lind a, Kristine Kleivi b,∗, Gunn I. Meling c, Manuel R. Teixeira d, Espen Thiis-Evensen e, Torleiv O. Rognum f and Ragnhild A. Lothe a,g,∗∗ a Department of Cancer Prevention, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway b Department of Genetics, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway c Surgical Department, Faculty Division Akershus University Hospital, University of Oslo, Oslo, Norway d Department of Genetics, Portuguese Oncology Institute, Porto, Portugal e Medical Department, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway f Institute of Forensic Medicine, Institute for Cancer Research, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway g Department of Molecular Biosciences, University of Oslo, Oslo, Norway Abstract. Background: Gene silencing through CpG island hypermethylation is a major mechanism in cancer development. In the present study, we aimed to identify and validate novel target genes inactivated through promoter hypermethylation in colorectal tumor development. Methods: With the use of microarrays, the gene expression profiles of colon cancer cell lines before and after treatment with the demethylating agent 5-aza-2 -deoxycytidine were identified and compared. The expression of the responding genes was compared with microarray expression data of primary colorectal carcinomas. Four of these down- regulated genes were subjected to methylation-specific PCR, bisulphite sequencing, and quantitative gene expression analysis using tumors (n = 198), normal tissues (n = 44), and cell lines (n = 30). Results: Twenty-one genes with a CpG island in their promoter responded to treatment in cell lines, and were simultaneously down-regulated in primary colorectal carcinomas. Among 20 colon cancer cell lines, hypermethylation was subsequently identified for three of four analyzed genes, ADAMTS1 (85%), CRABP1 (90%), and NR3C1 (35%). For the latter two genes, hypermethylation was significantly associated with absence or reduced gene expression. The methylation status of ADAMTS1, CRABP1,andNR3C1 was further investigated in 116 colorectal carcinomas and adenomas. Twenty-three of 63 (37%), 7/60 (12%), and 2/63 (3%) adenomas, as well as 37/52 (71%), 25/51 (49%), and 13/51 (25%) carcinomas were hypermethylated for the respective genes. These genes were unmethylated in tumors (n = 82) from three other organs, prostate, testis, and kidney. Finally, analysis of normal colorectal mucosa demonstrated that the observed promoter hypermethylation was cancer-specific. Conclusion: By using a refined microarray screening approach we present three genes with cancer-specific hypermethylation in colorectal tumors, ADAMTS1, CRABP1, and NR3C1. Keywords: ADAMTS1, adenomas, bisulphite sequencing, colorectal carcinomas, CRABP1, gene expression, methylation, microarrays, NR3C1, 5-aza-2 deoxycytidine 1. Introduction *Current address: Medical Biotechnology, VTT Technical Re- search Centre of Finland, Turku, Finland. Impaired epigenetic regulation is as common as ** Corresponding author: Ragnhild A. Lothe, Department of gene mutations in human cancer [22]. These mech- Cancer Prevention, Institute for Cancer Research, Rikshospitalet- Radiumhospitalet Medical Centre, Montebello, 0310 Oslo, Nor- anisms lead to quantitative and qualitative gene ex- way. Tel.: +47 22934415; Fax: +47 22934440; E-mail: Ragnhild. pression changes causing a selective growth advantage, [email protected]. which may result in cancerous transformation. Aber- 1570-5870/06/$17.00 2006 – IOS Press and the authors. All rights reserved 260 G.E. Lind et al. / ADAMTS1, CRABP1, and NR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis rantly hypermethylated CpG islands in the gene pro- were cultured in DMEF-12 medium (GIBCO, Invit- moter associated with transcriptional inactivation are rogen Carlsbad, CA) with 10% fetal bovine serum among the most frequent epigenetic changes in cancer. (GIBCO), 2 mM L-glutamine (GIBCO), 100 U/ml In the large bowel, the adenoma-carcinoma se- penicillin G, and 100 µg/ml streptomycin (GIBCO) quence is paralleled by several genetic and epigenetic and harvested before confluency. Two MSI cell lines changes. One of these is hypermethylation of MLH1, (HCT15, SW48) and two MSS cell lines (HT29, SW480) were cultured in parallel with and without which leads to inactivation of the DNA mismatch re- pair system and subsequent microsatellite instability 10 µM 5-aza-2 -deoxycytidine (Sigma-Aldrich Com- (MSI) [20]. Approximately 15% of all sporadic col- pany Ltd., Dorset, UK) in the medium for 72 hours. DNA was extracted from the cell lines and their 5- orectal carcinomas display MSI seen as insertions and deletions of short nucleotide repeats in their near- aza-2 -deoxycytidine treated counterparts by a stan- dard phenol-chloroform procedure, and total RNA was diploid genome [1]. The microsatellite stable (MSS) isolated using Trizol (Invitrogen, Carlsbad, CA). The tumors have a functional mismatch repair system but RNA quality was measured by a 2100 bioanalyzer (Ag- usually display chromosomal aberrations, including ilent Technologies, Palo Alto, CA) and the RNA con- several common regions of loss or gain [42]. Sub- centration was determined using ND-1000 Nanodrop groups of both types of colorectal carcinomas reveal (NanoDrop Technologies, Wilmington, DE). aberrant methylation of tumor suppressor genes asso- DNA from 53 colorectal carcinomas (25 MSS and ciated with lack of expression, although the methyla- 28 MSI) from 52 patients, including one patient with tion frequencies are generally higher in the MSI tumor hereditary non-polyposis colorectal cancer (HNPCC), group [29]. 63 adenomas (61 MSS and 2 MSI) from 52 patients, Furthermore, Toyota and co-workers suggested the normal mucosa samples from 22 colorectal cancer pa- CpG island methylator phenotype (CIMP) as the third tients (taken from distant sites from the primary car- pathway of colorectal tumorigenesis [56]. In 1999, cinoma), and another 22 normal colorectal mucosa they identified two epigenetically distinct tumor groups, samples from cancer-free individuals was subjected to one in which methylation was extremely rare (CIMP methylation analysis. The carcinomas (mean age 68 negative) and a second, in which the tumors were years) and the first group of normal mucosa samples methylated at multiple loci (CIMP positive). This ini- (mean age 64 years) were taken from colorectal cancer tial concept has been supported by some reports [45, patients admitted between 1987–1989 to one of seven 47,59,60], but not by others [3,26,61]. hospitals located in the south-east region of Norway Aberrant DNA methylation changes have also been [34]. For nine of these 22 normal mucosa samples the reported to occur early in the development of colorectal corresponding primary tumor was also included in the tumors [26,44] and are therefore promising as early di- present study. The adenomas were obtained from vol- agnostic markers of existing disease. The potential re- unteers (mean age 67 years) participating in a polyp versibility of DNA methylation patterns by epigenetic screening program [55]. The normal mucosa samples therapy further suggests that these are viable targets for from cancer-free individuals were obtained from de- treatment of cancer. ceased persons (mean age 54 years, including 8 in- dividuals 60 years). The majority of the total set In the present study we have used a genome-wide of normal samples (27/44) consisted of mucosa only, approach to identify novel epigenetically regulated tar- whereas the remaining samples were taken from the get genes in colorectal tumorigenesis, followed by bowel wall. The research bio-bank is registered at the technical, biological, and clinical validation analyses. Norwegian Institute of Public Health according to the Norwegian biobank legislation and the project was ap- proved for filing and processing personal health data 2. Material and methods by the national Data Inspectorate. Ten cell lines from various tissues were also in- 2.1. Cell cultures and tissue samples cluded in the present study (prostate, n = 3; testicu- lar germ cell tumor, n = 3; and ovary, n = 4). Addi- Twenty colon cancer cell lines were included in the tionally, clinical series of prostate (n = 20) and kid- present study, nine MSI: Co115, HCT15, HCT116, ney (n = 20) carcinomas from the University Hospi- LoVo, LS174T, RKO, SW48, TC7, and TC71, and tal of Porto, and a series of testicular germ cell tumors 11 MSS: ALA, Colo320, EB, FRI, HT29, IS1, IS2, (n = 42) from patients admitted to the Norwegian Ra- IS3, LS1034, SW480, and V9P [23]. All cell lines dium Hospital, Oslo, were screened for methylation. G.E. Lind et al. / ADAMTS1, CRABP1, and NR3C1 identified as epigenetically deregulated genes in colorectal tumorigenesis 261 2.2. Gene expression microarrays 2.3. Bisulphite treatment and methylation-specific PCR Eighteen cell lines and four 5-aza-2-deoxycytidine Bisulphite treatment of DNA leads to sequence vari- treated counterparts were analyzed by cDNA microar- ations as unmethylated but not methylated cytosines rays. Labeled cDNA was synthesized from 30 µgto- are converted to uracil [8]. DNA from cell lines and tal RNA in an oligo dT-primed polymerization with TM colorectal carcinomas was bisulphite treated as previ-