Kim A.D. Wouters

N-Myc Downstream-Regulated Gene 4 (NDRG4): A Candidate Tumor Suppressor Gene and Potential Biomarker for Colorectal Cancer

BACKGROUND Identification of hypermethylated tumor suppressor genes in body fluids is an appealing strategy for the noninvasive detection of colorectal cancer. Here we examined the role of N-Myc downstream-regulated gene 4 (NDRG4) as a novel tumor suppressor and biomarker in colorectal cancer. METHODS NDRG4 promoter methylation was analyzed in human colorectal cancer cell lines, colorectal tissue, and noncancerous colon mucosa by using methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. NDRG4 mRNA and protein expression were studied using real-time-PCR and immunohistochemistry, respectively. Tumor suppressor functions of NDRG4 were examined by colony formation, cell proliferation, and migration and invasion assays in colorectal cancer cell lines that were stably transfected with an NDRG4 expression construct. Quantitative methylation-specific PCR was used to examine the utility of NDRG4 promoter methylation as a biomarker in fecal DNA from 75 colorectal cancer patients and 75 control subjects. All P values are two-sided. RESULTS The prevalence of NDRG4 promoter methylation in two independent series of colorectal cancers was 86% (71/83) and 70% (128/184) compared with 4% (2/48) in noncancerous colon mucosa (P < .001). NDRG4 mRNA and protein expression were decreased in colorectal cancer tissue compared with noncancerous colon mucosa. NDRG4 overexpression in colorectal cancer cell lines suppressed colony formation (P = .014), cell proliferation (P < .001), and invasion (P < .001). NDRG4 promoter methylation analysis in fecal DNA from a training set of colorectal cancer patients and control subjects yielded a sensitivity of 61% (95% confidence interval [CI] = 43% to 79%) and a specificity of 93% (95% CI = 90% to 97%). An independent test set of colorectal cancer patients and control subjects yielded a sensitivity of 53% (95% CI = 39% to 67%) and a specificity of 100% (95% CI = 86% to 100%). CONCLUSIONS NDRG4 is a candidate tumor suppressor gene in colorectal cancer whose expression is frequently inactivated by promoter methylation. NDRG4 promoter methylation is a potential biomarker for the noninvasive detection of colorectal cancer in stool samples.

GATA4 and GATA5 are Potential Tumor Suppressors and Biomarkers in Colorectal Cancer

Purpose: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. Experimental Design: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. Results:GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74–95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37–65%) and specificity of 93% (95% CI, 84-100%) in the validation set. Conclusions: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

Early life exposure to famine and colorectal cancer risk: A role for epigenetic mechanisms

Background Exposure to energy restriction during childhood and adolescence is associated with a lower risk of developing colorectal cancer (CRC). Epigenetic dysregulation during this critical period of growth and development may be a mechanism to explain such observations. Within the Netherlands Cohort Study on diet and cancer, we investigated the association between early life energy restriction and risk of subsequent CRC characterized by the (promoter) CpG island methylation phenotype (CIMP). Methodology/Principal Findings Information on diet and risk factors was collected by baseline questionnaire (n = 120,856). Three indicators of exposure were assessed: place of residence during the Hunger Winter (1944–45) and World War II years (1940–44), and fathers employment status during the Economic Depression (1932–40). Methylation specific PCR (MSP) on DNA from paraffin embedded tumor tissue was performed to determine CIMP status according to the Weisenberger markers. After 7.3 years of follow-up, 603 cases and 4631 sub-cohort members were available for analysis. Cox regression was used to calculate hazard ratios (HR) and 95% confidence intervals for CIMP+ (27.7%) and CIMP- (72.3%) tumors according to the three time periods of energy restriction, adjusted for age and gender. Individuals exposed to severe famine during the Hunger Winter had a decreased risk of developing a tumor characterized by CIMP compared to those not exposed (HR 0.65, 95%CI: 0.45–0.92). Further categorizing individuals by an index of ‘0–1’ ‘2–3’ or ‘4–7’ genes methylated in the promoter region suggested that exposure to the Hunger Winter was associated with the degree of promoter hypermethylation (‘0–1 genes methylated’ HR = 1.01, 95%CI:0.74–1.37; ‘2–3 genes methylated’ HR = 0.83, 95% CI:0.61–1.15; ‘4–7 genes methylated’ HR = 0.72, 95% CI:0.49–1.04). No associations were observed with respect to the Economic Depression and WWII years. Conclusions This is the first study indicating that exposure to a severe, transient environmental condition during adolescence and young adulthood may result in persistent epigenetic changes that later influence CRC development.

Genetic Variants of Methyl Metabolizing Enzymes and Epigenetic Regulators: Associations with Promoter CpG Island Hypermethylation in Colorectal Cancer

Aberrant DNA methylation affects carcinogenesis of colorectal cancer. Folate metabolizing enzymes may influence the bioavailability of methyl groups, whereas DNA and histone methyltransferases are involved in epigenetic regulation of gene expression. We studied associations of genetic variants of folate metabolizing enzymes (MTHFR, MTR, and MTRR), DNA methyltransferase DNMT3b, and histone methyltransferases (EHMT1, EHMT2, and PRDM2), with colorectal cancers, with or without the CpG island methylator phenotype (CIMP), MLH1 hypermethylation, or microsatellite instability. Incidence rate ratios were calculated in case-cohort analyses, with common homozygotes as reference, among 659 cases and 1,736 subcohort members of the Netherlands Cohort Study on diet and cancer (n = 120,852). Men with the MTHFR 677TT genotype were at decreased colorectal cancer risk (incidence rate ratio, 0.49; P = 0.01), but the T allele was associated with increased risk in women (incidence rate ratio, 1.39; P = 0.02). The MTR 2756GG genotype was associated with increased colorectal cancer risk (incidence rate ratio, 1.58; P = 0.04), and inverse associations were observed among women carrying DNMT3b C→T (rs406193; incidence rate ratio, 0.72; P = 0.04) or EHMT2 G→A (rs535586; incidence rate ratio, 0.76; P = 0.05) polymorphisms. Although significantly correlated (P < 0.001), only 41.5% and 33.3% of CIMP tumors harbored MLH1 hypermethylation or microsatellite instability, respectively. We observed inverse associations between MTR A2756G and CIMP among men (incidence rate ratio, 0.58; P = 0.04), and between MTRR A66G and MLH1 hypermethylation among women (incidence rate ratio, 0.55; P = 0.02). In conclusion, MTHFR, MTR, DNMT3b, and EHMT2 polymorphisms are associated with colorectal cancer, and rare variants of MTR and MTRR may reduce promoter hypermethylation. The incomplete overlap between CIMP, MLH1 hypermethylation, and microsatellite instability indicates that these related “methylation phenotypes” may not be similar and should be investigated separately. (Cancer Epidemiol Biomarkers Prev 2009;18(11):3086–96)

A let-7 microRNA SNP in the KRAS 3'UTR is prognostic in early-stage colorectal cancer

Purpose: Colorectal cancer (CRC) is a common cause of death worldwide. Tumor-node-metastasis-system stage is currently used to guide therapy decisions but lacks precision. Prognostic biomarkers are needed to refine stratification of patients for chemotherapy but validated biomarkers are not yet available. Recently, a SNP in a lethal-7 (let-7) miRNA complementary site (LCS6) in the KRAS 3′untranslated region was suggested to affect survival in metastatic CRC. Effects in early-stage CRC are however unknown. We studied KRAS-LCS6 genotype, hypothesizing that it might identify early-stage cases with a poor prognosis, and could potentially be used in therapy decision-making. Experimental Design: We studied 409 early stage, 182 stage III, and 69 stage IV cases, and 1,886 subcohort members from the Netherlands Cohort Study. KRAS-LCS6 genotype was assessed with TaqMan PCR. Kaplan–Meier analyses or Cox regression were used to assess associations between genotype and CRC risk or cause-specific survival. Results: Early-stage cases with the KRAS-LCS6 variant had a lower CRC risk (incidence-rate ratio 0.68; 95% CI: 0.49–0.94) and a better survival (log-rank P = 0.038; HR 0.46; 95% CI: 0.18–1.14). In patients with KRAS-mutated CRC carrying the KRAS-LCS6 variant, the better outcome was enhanced as no patients died of CRC (log-rank P = 0.017). In advanced patients, no clear association between genotype and CRC risk or survival was observed. Conclusions: Our results indicate that early-stage CRC cases with the KRAS-LCS6 variant have a better outcome. In advanced disease, the better outcome no longer exists. For early-stage patients, KRAS-LCS6 genotype combined with KRAS mutations merits validation as a prognostic biomarker and consideration in therapy decision-making. Clin Cancer Res; 17(24); 7723–31. ©2011 AACR.

Genetic and epigenetic alterations in the von hippel-lindau gene: the influence on renal cancer prognosis.

Background: Inactivation of the von Hippel-Lindau (VHL) gene is considered as an early event in renal cancer tumorigenesis. The prognostic relevance of these changes, however, is not clear and previous results are contradictory. We have evaluated the influence of (epi)genetic alterations in VHL on cause-specific survival in clear-cell renal cell cancer (ccRCC) in a large, population-based group of cases. Methods: One hundred and eighty-five cases of ccRCC, identified in the Netherlands Cohort Study on diet and cancer diagnosed in the period 1986 to 1997, were included in the analyses. Mortality information until December 2005, including causes of death, were obtained for all cases through linkage with the Central Bureau of Statistics. VHL mutations were determined with PCR single-strand conformational polymorphism and direct sequencing. VHL methylation was determined with methylation-specific PCR. Kaplan-Meier analyses and Cox proportional hazards models were used to assess associations between VHL alterations and cause-specific mortality. Results: Median follow-up in our population was 6 years. The frequency of loss of function mutations and methylation, separately or combined, did not differ statistically significant between different cancer stages or between tumors with different sizes. We observed no influence of loss of function mutations or methylation of the VHL gene on cause-specific mortality (hazard ratio, 1.08; 95% confidence interval, 0.69-1.68, P = 0.735) as compared with patients with a wild-type or silent mutation in VHL. Discussion: Our results indicate that (epi)genetic alterations in the VHL gene do not have prognostic value in ccRCC.

MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events

BACKGROUND To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

An Inactivating Mutation in HDAC2 Leads to Dysregulation of Apoptosis Mediated by APAF1

BACKGROUND & AIMS Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing tumor suppressor genes. We examined mutation of HDAC2 and examined consequences of HDAC2 loss. METHODS Colon cancer cell lines and primary cancers were examined for mutations in HDAC2 by direct sequencing and capillary electrophoresis. Promoter methylation was determined using methylation-specific polymerase chain reaction in primary tumors. Sensitivity to HDAC inhibitors and resistance in vitro used colon cancer cell lines. RESULTS HDAC2 mutations in the poly(A) tract in exon 1 result in a frameshift and premature stop codon. These were found in microsatellite instability (MSI) cell lines and 43% of MSI colon cancers, but only 7% of microsatellite stable cancers. Mutation was associated with complete or regional tumor specific loss of HDAC2 protein. Inactivation of HDAC2 was not associated with large-scale changes in promoter region methylation, but rather is a consequence of epigenetic MLH1 inactivation leading to MSI. HDAC2 mutation results in apoptotic resistance to HDAC inhibitors (trichostatin A or suberoylanilide hydroxamic acid), despite induction of global histone acetylation. Differential induction of apoptosis by HDAC inhibitors is mediated by the proapoptotic gene APAF1, as shown by specific APAF1 induction only in cell lines with functional HDAC2, HDAC2 protein localization to the APAF1 promoter by chromatin immunoprecipitation, siRNA knockdown of HDAC2 leading to up-regulation of APAF1, and stable knockdown of APAF1 reducing apoptotic response to HDAC inhibitors. CONCLUSIONS Frequent HDAC2 mutations are found in MSI tumors and HDAC2 plays a major role in mediating apoptotic response to HDAC inhibitors through direct regulation of APAF1.

Early onset MSI-H colon cancer with MLH1 promoter methylation, is there a genetic predisposition?

BackgroundTo investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer.MethodsWe studied a set of 46 MSI-H colon tumors cases with MLH1 promoter methylation which was enriched for patients with an age of onset below 50 years (n = 13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: BRAF, KRAS, GADD45A and the MLH1 -93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for GADD45A mutations and germline MLH1 methylation. SNP array analysis was performed on a subset of tumors.ResultsWe identified two cases (33 and 60 years) with MLH1 germline promoter methylation. BRAF mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to BRAF mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. GADD45A sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity.ConclusionSomatic or germline GADD45A mutations did not explain sporadic MSI-H colon cancer. Although germline MLH1 methylation was found in two individuals, locus-specific somatic MLH1 hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of BRAF.

Promoter CpG island methylation of RET predicts poor prognosis in stage II colorectal cancer patients

Improved prognostic stratification of patients with TNM stage II colorectal cancer (CRC) is desired, since 20–30% of high‐risk stage II patients may die within five years of diagnosis. This study was conducted to investigate REarranged during Transfection (RET) gene promoter CpG island methylation as a possible prognostic marker for TNM stage II CRC patients.