Veerle Melotte

The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications

The N‐myc downstream regulated gene (NDRG) family of proteins consists of 4 members, NDRG1–4, which are well conserved through evolution. The first member to be discovered and responsible for the family name was NDRG1, because its expression is repressed by the proto‐oncogenes MYCN and MYC. All family members are characterized by an α/β hydrolase‐fold motif; however, the precise molecular and cellular function of these family members has not been fully elucidated. Although the exact function of NDRG family members has not been clearly elucidated, emerging evidence suggests that mutations in these genes are associated with diverse neurological and electrophysiological syndromes. In addition, aberrant expression as well as tumor suppressor and oncogenic functions affecting key hallmarks of carcinogenesis such as cell proliferation, differentiation, migration, invasion, and stress response have been reported for several of the NDRG proteins. In this review, we summarize the current literature on the NDRG family members concerning their structure, origin, and tissue distribution. In addition, we review the current knowledge regarding the regulation and signaling of the NDRG family members in development and normal physiology. Finally, their role in disease and potential clinical applications (their role as detection or prognostic markers) are discussed.—Melotte, V., Qu, X., Ongenaert, M., van Criekinge, W., de BruÏne, A. P., Baldwin, H. S., van Engeland, M. The N‐myc downstream regulated gene (NDRG) family: diverse functions, multiple applications. FASEB J. 24,4153–4166 (2010). www.fasebj.org

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.

DNA methylation of phosphatase and actin regulator 3 detects colorectal cancer in stool and complements FIT

Using a bioinformatics-based strategy, we set out to identify hypermethylated genes that could serve as biomarkers for early detection of colorectal cancer (CRC) in stool. In addition, the complementary value to a Fecal Immunochemical Test (FIT) was evaluated. Candidate genes were selected by applying cluster alignment and computational analysis of promoter regions to microarray-expression data of colorectal adenomas and carcinomas. DNA methylation was measured by quantitative methylation-specific PCR on 34 normal colon mucosa, 71 advanced adenoma, and 64 CRC tissues. The performance as biomarker was tested in whole stool samples from in total 193 subjects, including 19 with advanced adenoma and 66 with CRC. For a large proportion of these series, methylation data for GATA4 and OSMR were available for comparison. The complementary value to FIT was measured in stool subsamples from 92 subjects including 44 with advanced adenoma or CRC. Phosphatase and Actin Regulator 3 (PHACTR3) was identified as a novel hypermethylated gene showing more than 70-fold increased DNA methylation levels in advanced neoplasia compared with normal colon mucosa. In a stool training set, PHACTR3 methylation showed a sensitivity of 55% (95% CI: 33–75) for CRC and a specificity of 95% (95% CI: 87–98). In a stool validation set, sensitivity reached 66% (95% CI: 50–79) for CRC and 32% (95% CI: 14–57) for advanced adenomas at a specificity of 100% (95% CI: 86–100). Adding PHACTR3 methylation to FIT increased sensitivity for CRC up to 15%. PHACTR3 is a new hypermethylated gene in CRC with a good performance in stool DNA testing and has complementary value to FIT. Cancer Prev Res; 5(3); 464–72. ©2011 AACR.

The emerging role of GATA transcription factors in development and disease.

The GATA family of transcription factors consists of six proteins (GATA1-6) which are involved in a variety of physiological and pathological processes. GATA1/2/3 are required for differentiation of mesoderm and ectoderm-derived tissues, including the haematopoietic and central nervous system. GATA4/5/6 are implicated in development and differentiation of endoderm- and mesoderm-derived tissues such as induction of differentiation of embryonic stem cells, cardiovascular embryogenesis and guidance of epithelial cell differentiation in the adult.

Promoter CpG island methylation markers in colorectal cancer: the road ahead.

Despite increasing knowledge on the biology, detection and treatment of colorectal cancer (CRC), the disease is still a major health problem. Hypermethylation of promoter regions of genes has been studied extensively as a contributor in CRC carcinogenesis. In addition, it is the topic of many studies focusing on biomarkers for the early detection, prediction of prognosis and treatment outcome. Methylation markers may be preferred over current screening and test methods as they are stable and easy to detect. However, almost no methylation marker is currently being used in clinical practice, often due to a lack of sensitivity, specificity, or validation of the results. This review summarizes the current knowledge of hypermethylation biomarkers for CRC detection, progression and treatment outcome.

Epigenetics in radiotherapy: where are we heading?

Radiotherapy is an important component of anti-cancer treatment. However, not all cancer patients respond to radiotherapy, and with current knowledge clinicians are unable to predict which patients are at high risk of recurrence after radiotherapy. There is therefore an urgent need for biomarkers to guide clinical decision-making. Although the importance of epigenetic alterations is widely accepted, their application as biomarkers in radiotherapy has not been studied extensively. In addition, it has been suggested that radiotherapy itself introduces epigenetic alterations. As epigenetic alterations can potentially be reversed by drug treatment, they are interesting candidate targets for anticancer therapy or radiotherapy sensitizers. The application of demethylating drugs or histone deacetylase inhibitors to sensitize patients for radiotherapy has been studied in vitro, in vivo as well as in clinical trials with promising results. This review describes the current knowledge on epigenetics in radiotherapy.

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.

Emerging evidence for CHFR as a cancer biomarker: from tumor biology to precision medicine

Novel insights in the biology of cancer have switched the paradigm of a “one-size-fits-all” cancer treatment to an individualized biology-driven treatment approach. In recent years, a diversity of biomarkers and targeted therapies has been discovered. Although these examples accentuate the promise of personalized cancer treatment, for most cancers and cancer subgroups no biomarkers and effective targeted therapy are available. The great majority of patients still receive unselected standard therapies with no use of their individual molecular characteristics. Better knowledge about the underlying tumor biology will lead the way toward personalized cancer treatment. In this review, we summarize the evidence for a promising cancer biomarker: checkpoint with forkhead and ring finger domains (CHFR). CHFR is a mitotic checkpoint and tumor suppressor gene, which is inactivated in a diverse group of solid malignancies, mostly by promoter CpG island methylation. CHFR inactivation has shown to be an indicator of poor prognosis and sensitivity to taxane-based chemotherapy. Here we summarize the current knowledge of altered CHFR expression in cancer, the impact on tumor biology and implications for personalized cancer treatment.

CHFR Promoter Methylation Indicates Poor Prognosis in Stage II Microsatellite Stable Colorectal Cancer

Purpose: Data on the prognostic significance of promoter CpG island methylation in colorectal cancer (CRC) are conflicting, possibly due to associations between methylation and other factors affecting survival such as genetic alterations and use of adjuvant therapy. Here, we examine the prognostic impact of promoter methylation in patients with CRC treated with surgery alone in the context of microsatellite instability (MSI), BRAF and KRAS mutations. Experimental Methods: One hundred and seventy-three CRCs were analyzed for promoter methylation of 19 tumor suppressor and DNA repair genes, the CpG island methylator phenotype (CIMP), MSI, the exon 15 V600E BRAF mutation and KRAS codon 12 and 13 mutations. Results: Unsupervised hierarchical clustering based on methylation status of 19 genes revealed three subgroups: cluster 1 [CL1, 57% (98/173) of CRCs], cluster 2 [CL2, 25% (43/173) of CRCs], and cluster 3 [CL3, 18% (32/173) of CRCs]. CL3 had the highest methylation index (0.25, 0.49, and 0.69, respectively, P = <0.01) and was strongly associated with CIMP (P < 0.01). Subgroup analysis for tumor stage, MSI, and BRAF status showed no statistically significant differences in survival between CL1, CL2, and CL3 nor between CIMP and non-CIMP CRCs. Analyzing genes separately revealed that CHFR promoter methylation was associated with a poor prognosis in stage II, microsatellite stability (MSS), BRAF wild-type (WT) CRCs: multivariate Cox proportional HR = 3.89 [95% confidence interval (CI), 1.58–9.60, P < 0.01; n = 66] and HR = 2.11 (95% CI, 0.95–4.69, P = 0.068, n = 136) in a second independent population-based study. Conclusions: CHFR promoter CpG island methylation, which is associated with MSI, also occurs frequently in MSS CRCs and is a promising prognostic marker in stage II, MSS, BRAF WT CRCs. Clin Cancer Res; 20(12); 3261–71. ©2014 AACR.