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Promoter methylation correlates with reduced NDRG2expression in advanced colon tumour
© Piepoli et al; licensee BioMed Central Ltd. 2009
Received: 20 June 2008
Accepted: 03 March 2009
Published: 03 March 2009
Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current study to validate our previous microarray data and to identify novel hypermethylated gene promoters.
The validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour tissues from 30 patients with CRC.
Data from our previous genome search have received confirmation in the new set of 8 patients with CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated.
The findings highlight the usefulness of combining gene expression patterns and epigenetic data to identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being associated with a more advanced stage.
Colorectal cancer (CRC) is the third most common cancer in men and women, accounting for 11% of all cancer-related deaths. The majority of cases are diagnosed in advanced stages when a curative treatment is less likely to occur and chemotherapy is the only option . The identification of the molecular, genetic, and epigenetic changes underlying the adenoma-carcinoma sequence  leading to CRC has been the focus of many researches . It is now widely accepted that sporadic CRC frequently arises from preneoplastic lesions through the activation of proto-oncogenes, such as K-ras, and the inactivation of tumor suppressor genes (TSG), such as APC, p53, DCC, and the mismatch repair genes [4, 5]. Apart from mutations, gene expression may also be modified by altering of DNA methylation . Two general phenomena have until now been observed. The first one is global DNA hypomethylation with decreased 5-methylcytosine content which results in both enhanced expression of proto-oncogenes  and genomic instability . The second event is represented by local DNA hypermethylation of CpG islands, short sequences rich in CpG dinucleotides in the 5'untranscribed region (5'-UTR). This event occurs in approximately half of all human genes [9, 10] silences specific TSG, and accelerates cancer formation [11, 12]. Treatment with DNA demethylating drugs, such as 5-aza-2'-deoxycytidine (5-Aza-CdR or Decitabine), was shown to reverse the hypermethylation and restore expression of TSG . Therefore, cancer-specific promoter methylation may by itself serve as a valuable clue to uncover novel TSG.
In the present study, we aimed to uncover novel targets of promoter methylation in CRC, by combining gene expression profile data, already highlighted by our group , with results of demethylating assay and in silico screening for CpG islands.
Clinical data of colorectal cancer (CRC) patients
n = 30 (%)
Mean (± SD)
59 ± 14
Mean age <50
41 ± 7
Mean age >50
66 ± 9
RNA extraction from fresh frozen tissue
About 150–200 mg fresh frozen tissues were used to isolate total RNA by phenol extraction (TRIzol Reagent, Invitrogen Corporation, Carlsbad, CA, USA) which was subsequently purified by column chromatography (RNeasy Mini Kit, Qiagen, Valencia, CA, USA). RNA integrity was monitored using MOPS gel electrophoresis.
Cell culture, 5-Aza-CdR Treatments
The HCT116, CaCo2 and SW480 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA), and maintained in DMEM medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2 atmosphere at 37°C. For demethylation studies, cells were seeded at a density of 1 × 106 cells per 100-mm dish, and incubated for 24 hrs in a growth media. Subsequently, 5-Aza-CdR (Merck Chemicals Ltd., Nottingham, UK) was added to the incubation mixture following two different protocols. In the acute treatment 1 μM 5-Aza-CdR was added to incubation mixture for 24 hrs; afterwards, the medium was changed once daily for 3 consecutive days; DNA and RNA content were checked at 2nd, 4th and 6th days [15, 16]. In the chronic treatment, 2 μM 5-Aza-CdR was added for 24 hrs at day 1st, 3rd and 5th ; at each experimental day, the cells were placed in fresh medium and harvested at day 6th to isolate DNA and RNA.
Primer Sequences and Conditions for qRT-PCR, Bisulfite sequencing and MSP Analysis
Primer Forward (5'-->3')
Primer Reverse (5'-->3')
Quantitative Real Time PCR (qPCR) assay
Bisulfite Sequencing Analysis (BSA)
Methylation-Specific PCR (MSP) assay ?
In SilicoSearch and Bisulfite Sequencing Analysis (BSA)
The presence of CpG islands, overlapping the 5'-UTR, was examined by means of the MethPrimer http://www.urogene.org/methprimer/, according to CpG islands definition.
Bisulfite modification of DNA from colon cancer cell lines, peripheral blood and frozen tissues of patients was assayed, as reported by Herman et al. ; normal lymphocytes (NL) and in vitro methylated DNA (IVD) were used as negative and positive controls, respectively. In the assay, 1 μg of DNA was denaturated by treatment with NaOH at 37°C for 10 min, followed by incubation with hydroquinone and sodium bisulfite at 50°C for 16–17 h in the dark. After treatment, DNA was purified using DNA cleanup kit (Promega Corporation, Madison, WI), incubated with NaOH at 37°C for 15 min, precipitated with ammonium acetate and 100% ethanol, washed with 70% ethanol and, finally, re-suspended in 25 μl of distilled water. DNA methylation patterns in the CpG islands were determined by BSA using the primers listed in Table 2. The PCR conditions were 3 min at 94°C, 30 cycles of 94°C for 30 sec, specific annealing temperature for 30 sec, and 72°C for 60 sec. The sequence of the PCR products was analysed by using Sequencing Analysis 3.4.1 (Applied Biosystems, Applera, Foster City, CA, USA).
Qualitative analysis of CpG islands in the promoter region of the NDRG2, p16, APC, and MLH1 genes in 30 patients of CRC, in cell lines, in NL and IVD was carried out by MSP assay . The primers for unmethylated and methylated DNA are listed in Table 2. For the NDRG2 promoter region we used two different primers (NDRG2_UnM/NDRG2_M and NDRG2_UnM2/NDRG2_M2) to cover the same region sequenced by the bisulfite assays. PCR reaction was carried out in a 25 μl mixture containing 0,2 mM each dNTP, 1.5 mM MgCl2, primers (10 μM each), bisulfite-modified DNA (50 ng), and 0.75 U of Amplitaq Taq Gold polymerase (Applied Biosystems, Applera, Foster City, CA) for 35 cycles (95°C for 12 min, 94°C for 1 min, TA for 1 min, then 72°C for 1 min, followed by a final extension at 72° for 5 min) and analysed on a 3% agarose gel stained with ethidium bromide. All reactions were run in duplicate to ensure consistent and reproducible results.
We carried out three separate statistical analyses. In the initial analysis, qPCR data were used to validate our previous array results . Calculations were made using the Comparative CT method [19, 20]. We used three genes, that is hEEF1A1, hGAPDH and hHRPT1, to normalize input cDNA for each sample, with cDNA of normal tissue used as calibrator. The chi-square method with one degree of freedom (χ2 1), calculated by BMDP Statistical Software (BMDP Statistical Software, Cork Technology Park, Model Farm Road, Cork, Ireland) , was used to asses statistical significance of expression difference for each gene of the 8 paired samples. The second analysis concerned the variation of gene expression before and after 5'-Aza-CdR treatment by means of the T test, calculated by BMDP Statistical Software. Expression level of the post-treatment specimen compared to the pre-treatment specimen was calculated as a log-transformed ratio. A gene was classified as up-regulated following the 5-Aza-CdR treatment when relative mRNA expression was greater or equivalent to 1.65-fold in at least one treatment condition in one cell line. Genes with no change or very low expression levels in post treatment specimens were no further considered in the analysis.
The final part of our analysis evaluated the BSA data. The median number of full CpG islands, present in normal and tumour tissues, was calculated and compared in tumour (T) matched normal (N) tissue of the same patients. The χ2 1 method was used to asses significant difference; a number of CpG islands higher than 5 was taken as statistically significant.
qPCR validation of deregulated genes
List of genes selected from microarrays analysis comparing normal mucosa matched tumour colon tissue. The different expression in tumoural tissue was showed.
Insulin Receptor Signaling
Serum glucocorticoid regulated kinase (SGK)
chloride channel, calcium activated, family member 4
solute carrier family 26, member 3 (SLC26A3)
sodium channel, nonvoltage-gated 1, beta (S. Liddle)
Transport (ATP binding)
ATP-binding cassette, sub-family A (ABC1), member 8
Receptor activity (mineralcorticoid)
nuclear receptor subfamily 3, group C, member 2
Cell cycle (proliferation)
MAX-interacting protein 1 (MXI1)
Cell cycle (differentiation)
N-myc downstream-regulated gene 2
hydroxyprostaglandin dehydrogenase 15-(NAD)
Prostaglandin and leukotriene Metabolism
peroxidase, acidic calcium-independent phospholipase A2
serumglucocorticoid regulated kinase (SGK)
forkhead box M1 (FOXM1)
GTF2I repeat domain-containing 1 (GTF2IRD1)
Sex determining region Y-box 9
heat shock 105 kD (HSP105B)
Transport solute carrier
solute carrier family 12
Cell cycle (proliferation)
restricted expressed proliferation associated protein
Cell cycle (progression)
ubiquitin carrier protein E2-C (UBCH10)
Cell cycle (proliferation)
CSE1 chromosome segregation 1-like (yeast)
nebulette protein (NEBL, actin-binding Z-disc protein)
Replication and repair
replication factor C (activator 1) 3 (38 kD)
qPCR assay was applied to analyse mRNA expression of the 24 selected genes, as well as of three control housekeeping genes (GAPDH, EEF1A1 and HRPT1) in the tumour and normal tissues taken from new cohort of 8 patients with CRC.
Gene expression before and after 5-Aza-CdR in colon cell lines
To investigate the role of methylated CpG islands in the modulation of gene expression, HCT-116, CaCo2 and SW480 human colon cancer cell lines were cultured with different doses of 5-Aza-CdR to induce a demethylation event, and gene expression levels were measured by means of qPCR. Two 5-Aza-CdR challenge regimens were used to obtain expression data under different cellular conditions: the acute treatment focused on moderate DNA demethylation to minimize cell viability, and the chronic treatment to maximize DNA demethylation (see Additional file 1).
In silicosearch verification and Bisulfite Sequencing Analysis (BSA)
The methylation status of promoter regions of these putative tumour-suppressor genes was assessed by BSA in untreated cell lines, in tumour matched to normal tissues of one patient, in vitro methylated DNA (IVD) and in normal lymphocytes (NL). PRDX6 showed dense methylation only in IVD; NDRG2 showed a significant methylation in cell lines and in tumour tissue compared to normal tissue, suggesting a potential epigenetic regulation of the gene (Fig. 3B and Additional file 3). Full methylation in all 16 CpG sites of the NDRG2 gene was found in HCT116 and CaCo2 cell lines, and partial methylation at the 3th CpG site in the SW480 cell line (Fig. 3B).
Quantitation of NDRG2 methylation in paired tumour and normal tissue samples of CRC patients
Methylation-specific PCR (MSP) assay in colon cancer cell lines and primary CRC samples
Comparison of the clinicopathological features of 30 CRC patients according to the presence of NDRG2 methylation
Number of CRC
with NDRG2 methylation (%)
Number of CRC
without NDRG2 methylation (%)
MSI status 1
Promoter gene methylation rates in tumour and normal tissue from patients with colorectal cancer (CRC) sorted by tumour location
Proximal colon CRC (n = 9)
Distal colon CRC (n = 21)
When relating the NDRG2 methylation status to clinical pathologic features, no association with age, gender, tumour site, and MSI status was observed. Conversely, a significant correlation was found between the NDRG2 methylation and the AJCC stage of the cancer (Z test, p < 0,05) (Table 4).
Carcinogenesis is a complex event characterized by the progressive development of genetic and epigenetic aberrations which ultimately result in loss of physiological control of cell growth and differentiation. The two most important epigenetic mechanism are represented by the DNA methylation, the conversion of cytosine into methyl-cytosine catalyzed by the DNA methyltransferase And histone modifications . Changes in the DNA methylation pattern may occur everywhere in the DNA molecule. Global DNA hypomethylation generally occurs in centromeric repeats and repetitive sequences and contributes to carcinogenesis by causing chromosomal instability, reactivation of transposable elements, and loss of imprinting . Hypermethylation is especially frequent in CpG islands, i.e. short DNA sequences rich in CpG dinucleotides, mostly located in the 5'-untranslated region (5'-UTR) of genes . When CpG islands are heavily methylated, transcriptional gene silencing generally occurs. Although the fine mechanisms of regulation of the "epigenetic" machinery are still poorly understood, the DNA methylation may switch on or off several genes and, in particular, those regulating important biological phenomena, such as cell growth and differentiation . In normal cells, this epigenetic mechanism is involved in several physiological events, such as the inactivation of X chromosome in female cells, silencing either paternal or maternal alleles of "imprinted" genes, and transcriptional blocking of exogenous integrated genes potentially dangerous for the cell life. However, aberrant DNA methylation is also relatively common in cancer cells and is likely to play an important role in cancer initiation and progression .
Since the pioneer studies of Baylin et al. , it has been widely recognized that cancer cells are characterised by two opposite events: a global hypomethylation which results in either up-regulation of proto-oncogenes and induction of genomic instability, favouring both uncontrolled cell growth  and mutations, and CpG islands hypermethylation of other genes, the so-called tumour-suppressor-genes (TSG), which contributes to loss of the negative control of the cell cycle . Searching for up-regulated oncogenes and down-regulated TSG is important in basic science, especially when an epigenetic mechanism (hypomethylation or hypermethylation) is suspected. In fact, oncogenes and TSG not only may elucidate the highly complex molecular derangement in cancer cells, but also may be used as potential targets for new therapeutic approaches. DNA methylation is a reversible phenomenon which can be modulated by specific agents. An example is represented by demethylating drugs which can globally reduce the DNA methylation level of TSG promoters, restoring their normal activity. Interestingly, some in vitro experiments have shown that cancer cell lines reverted to normal phenotype after treatment with demethylating agent.
The current study was carried out with a three-step design. First, we specifically looked at up- and down-regulated genes not yet firmly associated with colon carcinogenesis, and selected 24 genes for validation with qPCR. A straight correlation between results obtained from qPCR and those from DNA microarray was found, implying that DNA microarray technology is a reliable tool to search for new genes significantly deregulated in cancer . Second, we selected 10 of 21 genes (ABCA8, AQP8, HPGD, PRDX6, SLC26A3, STX12, NDRG2, MXI1, SGK2, and SCNNB1) as possible targets of epigenetic modifications in colon cancer, and after treatment with a demethylating agent, seven of them showed a significant increase of mRNA expression (AQP8, HPGD, PRDX6, MXI1, SCNNB1, SGK2 and NDRG2). From an in silico screening, only 2 genes (PRDX6 and NDRG2) were considered as possible candidates for the presence of CpG islands in their 5'-UTR. For the excluded genes, additional mechanisms of transcriptional regulation were hypothesized to be responsible for their differential expression. Third, to evaluate the methylation status of PRDX6 an NDRG2 genes in normal and cancer tissues, as well as in colon cancer cell lines, bisulphite sequencing analysis was used. In the PDRX6 gene the methylation status was not different from that observed in normal tissue. In the NDRG2 gene a significant methylation status either in colon cancer cell lines and in tumour tissue compared to normal tissue was observed. The underexpression of the PRDX6 protein responsible for the red-ox regulation of the cell, was found to be correlated with loss of function of NKX3.1 gene, known as TSG .
Using these approaches, the NDRG2 gene was selected for further analysis because: (i) it was suppressed in all colon cancer cell lines, (ii) its expression may be up-regulated in all cell lines by 5Aza-CdR treatments, and (iii) it is involved in important biological process such as cell growth , differentiation  and apoptosis . The NDRG2 gene is a new member of the N-myc downstream-regulated gene (NDRG) family, that is located on chromosome 14q11.2 and encodes for a 41 kDa protein. It has been proposed that the NDRG2 gene is a candidate TSG, and its expression is low or undetectable in several primary tumour and tumour cell lines [30, 33, 34]. Liu et al.  revealed that the down-regulation reported in cancer be driven by promoter methylation, mutation, and genomic deletion of the NDRG2 gene. Recently, it has been shown that expression of the NDRG2 protein is modulated by the insulin-stimulated Akt-dependent phosphorylation . Several studies have suggested that the NDRG2 mRNA is down-regulated or undetectable in a number of human primary cancers, such as squamous cell carcinoma, pancreatic cancer , glioblastoma , and cancer cell-lines. Recently, Zhang et al.  have demonstrated that c-Myc represses NDRG2 gene expression via Miz-1-dependent interaction with NDRG2 core promoter region, and this inverse regulatory relationship induces cell differentiation and proliferation.
The MSP assay was used to check for NDRG2 methylation status in 30 primary colon tumour tissues compared to normal colonic mucosal samples. After sorting colon cancer patients by age, gender, tumour site, and MSI status, no statistically significant association was observed between these features and the NDRG2 methylation. Nevertheless, there was a trend towards NDRG2 methylation status with an advanced tumour stage of the CRC samples, with significant value detected in patients with AJCC stage IV (p < 0.05). These results are in agreement with those reported in other cancer types [34, 39] where NDRG2 expression is reduced in high-grade compared to low-grade tumours. In particular, Lorentzen et al.  suggested that in CRC samples the down-regulation of NDRG2 expression occurs during the progression from adenoma to carcinoma.
In conclusion, we showed that NDRG2 expression is frequently suppressed in colon cancer cell lines in conjunction with aberrant DNA methylation, and that the loss of expression of this gene could be related to advanced colon tumour stage.
We thank Dr. Pierluigi Di Sebastiano for colon cancer sample collection; Dr. Mirco Fanelli for critical reading of the manuscript. This work was supported by "Ministry of Italian Health" grants RC0502GA07 and RC0604GA52, through Research Unit of Gastroenterology, "Casa Sollievo della Sofferenza" IRCCS, San Giovanni Rotondo (FG), Italy.
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