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The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth

Abstract

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2′-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer in both men and women and accounts for 10% of all new cancer cases and cancer deaths (Jemal et al., 2005). At diagnosis, 19% of CRC cases are metastatic, and the overall 5-year survival rate for patients with metastatic CRC is less than 10% (Jemal et al., 2005). Finding potential diagnostic and therapeutic target molecular markers for CRC would thus be invaluable to physicians and patients for early detection and treatment of CRC.

Epigenetic alterations have been widely recognized in the past decade to play an important role in the development of cancer in addition to genetic events. Hypermethylation of gene promoter and corresponding loss of gene expression has been recognized as one of the hallmarks of cancer (Herman and Baylin, 2003). In addition, genes that display cancer-specific methylation may serve as biomarkers for early detection, diagnosis and prognosis of cancer (Belinsky et al., 1998; Palmisano et al., 2000; Sidransky, 2002; Mandelker et al., 2005).

The N-methyl-D-aspartate receptors (NMDARs) are heteromeric ligand-gated ion channels that interact with multiple intracellular proteins through different subunits. Among the essential NMDARs (NMDAR2A, NMDAR2B, NMDAR1), NMDAR2A has the greatest structural and functional similarity with NMDAR2B (Schito et al., 1997). The NMDAR2A coding sequence shows 78% homology to human NMDAR2B but no significant homology with the human NMDAR1 subunit which is essential for the function of NMDARs (Schito et al., 1997). NMDAR expression is present in the brain and in peripheral tissues including the adrenal medulla, pancreatic islet cells (Inagaki et al., 1995), lung (Said et al., 1996) and male lower urogenital tract (Gonzalez-Cadavid et al., 2000). Gene expression profiling of human primary glioblastoma multiforme showed that NMDARs were downregulated in tumors compared to normal brain tissue samples (Markert et al., 2001).

We previously identified genes harboring cancer-specific methylation in esophageal squamous cell carcinoma (ESCC) by pharmacological unmasking and subsequent microarray analysis (Yamashita et al., 2002). One of those genes identified was NMDAR2B; it was methylated in primary human ESCC tissues and exhibited tumor-suppressive activity in ESCC cell lines (Kim et al., 2006). In this study, we examine the NMDAR2A promoter in both ESCC and CRC cell lines as well as in primary human tumor tissues, corresponding normal adjacent mucosa, and nonmalignant normal tissues. NMDAR2A was methylated in both types of cancer cell lines, but unlike NMDAR2B, the methylation frequency of NMDAR2A was very low in human primary ESCC (2/20, 10%). Interestingly, NMDAR2A was more frequently and more specifically methylated in primary human CRC tissues, and forced expression markedly suppressed growth in cell lines.

Results

Promoter methylation often results in silencing of the gene product. Thus, before investigating the methylation status of the NMDARs (2A, 2B and 1) promoters, we examined the expression of the NMDARs in ESCC and CRC cell lines by performing reverse transcription (RT)–PCR. We previously reported that all 12 ESCC cell lines examined exhibited silencing of NMDAR2B mRNA expression (Kim et al., 2006). NMDAR1-1a expression was also silenced in 9 of 12 ESCC cell lines. Silenced or downregulated transcription of the genes in ESCC cell lines was reactivated by treatment of the demethylating agent, 5-Aza-dC (Figure 1a). In contrast, NMDAR2B and NMDAR1-1a were ubiquitously expressed in CRC cells lines tested (Figure 1b). Interestingly, however, the basal expression of NMDAR2A was absent or weak in most ESCC cell lines, and undetectable in all CRC cell lines tested. TE5 was the only cell line that showed a high level of NMDAR2A expression at baseline. Silenced NMDAR2A was also robustly reactivated by 5-Aza-dC in both types of cell lines. In seven primary esophageal tissue cDNAs, NMDAR2A and NMDAR1-1a were expressed in normal corresponding mucosa. Unlike NMDAR2B, downregulation of NMDAR2A and NMDAR1-1a in ESCC tissues was barely detectable (Figure 1c). In contrast, NMDAR2A was the only gene whose expression was downregulated in colon cancer tissue cDNA (Figure 1d). By immunohistochemical staining of a colon cancer tissue microarray with normal colon tissue controls, we found that the three major NMDARs were expressed in all nonmalignant normal tissues and adjacent normal colon mucosa (Figure 2 and Table 1). In colon adenocarcinoma, NMDAR1-1a expression was very strong in 10/10 cases, and NMDAR2B expression was also observed (mild or moderate positivity in 8/10 cases). Interestingly, however, NMDAR2A was barely detectable in almost all primary cancers (weak positivity only in 3 of 10 cases).

Figure 1
figure1

N-methyl-D-aspartate receptors (NMDAR) expression in ESCC and CRC cell lines. (a) The expression of NMDAR2A was lost or decreased in all 12 ESCC cell lines except TE5 (left). Downregulated transcription of these genes was reactivated by treatment of the demethylating agent, 5 μM 5-Aza-dC (right) (m, no treatment; α, 5-Aza-dC treatment; L, 1 kb plus DNA ladder). Arrows indicate splice variants of NMDAR1-1a that were determined by sequencing of RT–PCR products after gel extraction. β-actin was used as a loading control. PCR products were loaded on 10% acrylamide gel. Gels were stained with ethidium bromide and visualized under UV light. (b) The expression of NMDAR in all seven CRC cell lines. HCT116, HCT116−/− (p53-null), RKO, RKO-E6 (functional p53-deficient). PCR products were loaded on 4% agarose gel. (c) Expression of NMDAR2A and NMDAR1-1a in seven primary esophageal tissues (N, normal corresponding mucosa; T, tumor; numbers, primary tissue cDNA samples from cases. PCR products were loaded on 1.2% agarose gel). (d) RT–PCR was performed to test for NMDAR expression in a cDNA panel of human adult normal placenta (NP), normal colon and cancer tissues.

Figure 2
figure2

Immunohistochemical staining of NMDAR in colon cancer tissue microarray with normal colon tissue controls. (A) NMDAR2A expression in nonmalignant normal tissues (a), colon adenocarcinoma with tumor grade I (b), II (c) and III (d). (B) NMDAR1-1a (a, b) and NMDAR2B (c, d) expression in nonmalignant normal tissues (a, c) and colon adenocarcinoma with tumor grade II (b, d). NMDAR2A was barely detected in the tissues from colon cancer patients.

Table 1 Immunohistochemical analysis of NMDA receptors in colon cancer tissue microarray with normal tissue controls

To examine whether gene expression correlates with methylation of the NMDAR promoters, we performed bisulfite sequencing. Representative sequencing results of NMDAR2A are shown in Supplementary Figure 2. ECSS cell lines (11/12) and all 7 colon cell lines tested were methylated in the NMDAR2A promoter region (Figures 3a and b). All 29 CpGs examined were completely methylated in the cell lines. Promoter hypermethylation of the NMDAR2A was absent only in TE5, the cell line that expressed NMDAR2A at baseline, indicating that promoter methylation of NMDAR2A was correlated well with gene expression. However, NMDAR1 was found to be methylated in all cell lines of both types, indicating that NMDAR1-1a expression was not dependent on the specific pattern in cell lines. Both F1 and F3 regions of NMDAR2B were methylated in all ESCC cell lines tested (Kim et al., 2007). Interestingly, the F1 region of NMDAR2B was methylated in all CRC cell lines tested, whereas methylation of the F3 region was not detected by COBRA (Kim et al., 2007), suggesting that NMDAR2B expression in CRC cell lines might be through promoter activity of the F3 region that remained unmethylated in the cell lines. Transcriptional activity of the F3 region was confirmed by a promoter assay with F3 promoter–reporter constructs (Kim et al., 2007).

Figure 3
figure3

N-methyl-D-aspartate receptors (NMDAR) promoter methylation in cell lines and primary tissues determined by sequencing and COBRA. (a, b) The NMDAR2A promoter was methylated in all esophageal squamous cell carcinoma (ESCC) and colorectal carcinoma (CRC) cell lines except TE5. NMDAR1 and the F1 region of NMDAR2B were found to be methylated in all cell lines tested (black, the presence of methylation; white, the absence of methylation). (c) Methylation frequency of NMDAR2A was low in primary ESCC. (d) NMDAR2A was more frequently methylated in primary colon cancer tissues, whereas NMDAR2B methylation was barely detected in CRC tissues. NMDAR1 was methylated in both types of cancer and also methylated with high frequency in corresponding normal esophageal and colon mucosa (numbers, primary tissue samples from patients).

To investigate promoter methylation of the NMDARs, we first searched CpG islands in each gene promoter within a 5 kb upstream region and 2 kb downstream region of the transcription start site by Methprimer software (Supplementary Figure 1A). The details of each promoter are described in Supplementary Materials. To analyse the NMDAR methylation status in primary tumors, we examined 20 cases of ESCC and corresponding normal-appearing tissues, and 10 cases of randomly selected colon cancer and matched normal adjacent mucosa tissues by bisulfite sequencing. Previously, we reported that NMDAR2B was methylated in primary ESCC with high frequency (19/20) (Kim et al., 2006). However, unlike NMDAR2B, methylation of the NMDAR2A promoter in primary ESCC tissues was detected at a very low frequency (2 of 20 ESCC, 10%) (Figure 3c), but was more frequently methylated in primary colon cancer tissues (7 of 10 tumors, 70%) (Figure 3d). In the normal esophagus tissues, most cases (3–11, 14–20) did not harbor any CpG methylation in the promoter, but some cases (12, 13) harbored both methylated and unmethylated alleles together in less than five CpG sites. Cases 1 and 2 harbored three methylated CpGs of a total of 29 CGs. Thus, they were all considered ,methylation negative,. In normal colon tissues, only case 99 harbored methylation in more than 10 CpG sites, whereas the other samples harbored no CpG methylation in the promoter.

In contrast, NMDAR2B promoter methylation was very rare in 10 pairs of CRC (1/10, 10%) and matched normal tissues (1/10, 10%). No methylation of NMDAR2A was found in all 20 matched normal-appearing esophageal mucosa, and methylation of the gene was detected only in 1 of 10 matched normal colon tissue. NMDAR1 was methylated (more than 5 CpGs) in all 20 ESCC and 5 colon cancer tissues tested, and also methylated with high frequency in corresponding normal esophageal mucosa (16/20) and colon mucosa (10/10). These data suggest that NMDAR2A is more frequently methylated in primary CRC than ESCC. Therefore, we decided to focus on NMDAR2A methylation and its function in CRC. Sequencing results were confirmed by combined bisulfite restriction analysis (COBRA) after gel extraction of the NMDAR2A PCR product of bisulfite-treated DNA in randomly selected samples from both types of tissues and cell lines as described in Supplementary Figure 3.

To quantify NMDAR2A promoter methylation, we performed TaqMan methylation-specific PCR (TaqMan MSP) in 11 normal colon epithelial tissues from patients without cancer, 100 pairs of normal colon mucosa and primary CRC as well as 4 colon cancer cell lines that included samples previously analysed by bisulfite sequencing and COBRA. In 100 pairs of colon samples, methylation values (TaqMeth V) in tumor ranged from 0 to 292.77 (median value 1.22), and in normal colon from 0 to 28.24 (median value 0.01) (Table 2). The overall TaqMeth V levels detected in primary CRC (36.55±58.72, mean±s.d., n=100) were also significantly higher than that in corresponding normal tissues (0.94±4.39, mean±s.d., n=100) (P<0.001, t-test) (Figure 4a).

Table 2 Sensitivity and specificity of NMDAR2 A promoter methylation at different cutoff values
Figure 4
figure4

Quantitative Taqman MSP of the N-methyl-D-aspartate receptor (NMDAR)-2A promoter. (a) The overall TaqMeth V detected in primary CRC were significantly higher than that in corresponding normal tissues (P<0.001, t-test). (b) ROC curve analysis of TaqMeth V of NMDAR2A. The area under ROC (AUROC, 0.907) conveys the accuracy in distinguishing matched normal colon from CRC in terms of its sensitivity and specificity (P<0.001) (solid line, NMDAR2A; dash line, no discrimination). (c) Scatter plot of NMDAR2A promoter methylation. CRC primary tumors (82/100) harbor values above the optimal cutoff value, 0.18. Only 1 of 11 normal colon epithelial tissues and 15 of 100 corresponding normal colon tissues were above the cutoff. CRC cell lines tested were HCT116, DLD1, SW480 and RKO cells. Arrow (*) indicates the cutoff value of 0.18. Samples with a ratio equal to zero could not be plotted correctly on a log scale, so presented here as 0.001.

We investigated cutoff lines for TaqMeth V, and cutoff values from 0.1 to 10 were all statistically significant by χ2 analysis (normal vs tumor and methylation vs cases without methylation (Table 1). Methylation of NMDAR2A showed highly discriminative receiver-operating characteristic (ROC) curve profile, clearly distinguishing CRC from corresponding normal mucosa (P<0.001) (Figure 4b). The optimal cutoff value (0.18) was calculated from the ROC curve in order to maximize sensitivity and specificity. At this cutoff, the specificity was 85% (85/100) and sensitivity was 82% (82/100) (P<0.001) (Table 1). Only 1 of 11 normal colon epithelial components and 15 of 100 of paired normal colon mucosa displayed a TaqMeth V over the cutoff (Figure 4c). A high level of NMDAR2A promoter methylation was also found in all CRC cell lines tested (4/4, 100%), consistent with the bisulfite-sequencing results. In addition to a simple frequency, the comparison of methylation level of normal and tumor tissues from the same individual patients revealed that the majority of the tumor tissues harbored much higher values than matched normal colon mucosa (Supplementary Figure 4A). Taken together, NMDAR2A was frequently methylated in primary CRC tissues, but at minimal levels in corresponding normal tissue.

Since NMDAR2A expression was silenced in CRC cell lines and its promoter was specifically methylated in primary colon cancer tissues, we tested its growth suppressive activity. We transfected the plasmids carrying NMDAR2A (NMDAR2A-pcDNA1) and NMDAR1-1a (NMDAR1-1a-pRc/CMV) into HCT116 in which basal expression of NMDAR2A was barely detectable (Figure 1b), and then treated cells with NMDAR agonists, L-glutamate/L-glycine (200/50 μM). We performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay first to compare the growth in cells with or without expression of NMDARs. Cell growth decreased about 70% of control in cells co-overexpressing NMDAR2A and NMDAR1-1a (Figure 5a, P<0.05, t-test). NMDAR2A or NMDAR1-1a alone also decreased cell growth, but the inhibition was less than 35%. To determine if HCT116 cells experiencing NMDAR-induced death were undergoing apoptosis, the TUNEL assay was performed. As shown in Figure 5b, NMDAR-transfected HCT116 cells were found to display the early stage of apoptosis. We then performed a colony focus assay in the presence of G418 and NMDAR agonists after transfection of the two NMDAR in HCT116 cells. There were multiple colonies in the control cells transfected with empty vector only (194.33±20.82 colonies), whereas colony numbers in the cells with forced expression of the NMDAR was markedly decreased (4.33±2.52 colonies) (P<0.001, t-test) (Figure 5c). Similar effects were observed in RKO and DLD-1 after co-transfection of NMDAR2A and NMDAR1-1a (Supplementary Figure 5).

Figure 5
figure5

Reexpression of NMDAR2A inhibits cell growth and induces apoptosis. (a) MTT assay was performed in HCT116 cell lines transfected with plasmid carrying the NMDAR2A and/or NMDAR1-1a genes, or empty vector alone (Control). NMDAR agonists, L-glutamate/L-glycine (200 μM per 50 μM) were added to cells after transfection. Percent cell growth compared to mock plasmid-transfected cells (100%) (NR, NMDAR). (b) NMDAR-transfected HCT116 cells undergo apoptosis as determined by the TUNEL assay (upper panel). All nuclei were stained with DAPI (lower panel) (× 100). (c) Colony focus assays were performed after co-transfection of NMDAR2A and NMDAR1-1a in HCT116 cells. Cells were incubated in the presence of G418 (1 mg ml−1) and NMDAR agonists for 2 weeks and stained with 0.4% crystal violet solution (MeOH/Acetic acid, 3:1). After air-drying, colonies were photographed under the microscope (upper right), and counted (left). Values are expressed as a mean±s.d. and derived from experiments done in triplicate. The t-test was performed for statistical analysis. To confirm the expression of NMDAR, cells were harvested 48 h after transient transfection and RT–PCR was performed (lower right panel). (d) Calcein-AM assay was performed in TE5 cells after transient transfection of siRNAs targeting NMDAR2A (right). Experiments were done in triplicate, and values indicate means±s.d. *P<0.05. NMDAR2A gene knockdown was examined by RT–PCR (left). β-actin expression is shown as a loading control. (c) Control siRNA; 1, siRNA-NMDAR2A-1; 2, siRNA-NMDAR2A-2; 3, siRNA-NMDAR2A-3; 4, siRNA-NMDAR2A-4.

Next, we transfected transiently four individual siRNAs targeting NMDAR2A or a nontargeting control siRNA into TE5 cells, and performed the calcein-AM assay for the cell growth assay. We observed a significant increase of cell growth in TE5 cells transfected with each siRNA after 4 days of incubation (Figure 5d, left). Gene knockdown was confirmed by RT–PCR in each transfected cells (Figure 5d, right). These results show that loss of NMDAR2A expression could increase cell growth.

Discussion

We previously reported cancer-specific methylation of the NMDAR2B promoter in human primary ESCC (Kim et al., 2006). In this study, we extended our studies by examining whether the three major NMDARs (NMDAR2A, NMDAR2B, and NMDAR1) also exhibited gene silencing and promoter methylation in human CRC. We found that among the three NMDARs, only NMDAR2A expression was not observed in most cases of colon cancer. Although the NMDAR2A promoter, like NMDAR2B, was completely methylated in the majority of the ESCC cell lines (Kim et al., 2006), only a very small number of primary ESCC tissues harbored NMDAR2A promoter methylation. This result is another example of the discrepancy between gene methylation status observed in cell lines and primary tumors. Despite the observation that NMDAR2A might play only a minor role in esophagus squamous cell tumorigenesis compared to NMDAR2B, we found that NMDAR2A was specifically methylated in CRC. NMDAR2B methylation was not detected in CRC and matched normal tissues by bisulfite sequencing, suggesting a tissue-specific methylation pattern for these receptors. In addition, we showed a tight correlation between promoter methylation and silencing of gene expression in ESCC and CRC cell lines, implying that promoter methylation of NMDAR2A is one of the mechanisms of gene silencing in CRC. mRNA expression of all the three NMDARs was detected in normal esophageal as well as colon tissue cDNA, and protein expression was also observed in normal colon tissue sections. Interestingly, in contrast to NMDAR2B that exhibited decreased expression in primary ECSS tissues (Kim et al., 2006), little difference between normal and tumor esophageal tissues was found for NMDAR2A. Overexpression of NMDAR1 has been detected in patients with oral squamous cell carcinoma (Choi et al., 2004). In our results, higher expression of NMDAR1 expression in tumor than in normal tissues was also detected in four of seven cases of ESCC patients by RT–PCR. However, overexpression of NMDAR1 in colon cancer was not observed.

NMDAR2A methylation was quantified by a sensitive and quantitative assay (Sidransky, 2002). Specificity increased to over 90% when we used 1 as the cutoff since only 7% of cases harbored a methylation level over 1 in corresponding normal tissues. The optimal cutoff value of 0.18 was calculated by ROC analysis to minimize the background methylation in matched normal tissues. Since matched normal tissues often harbor rare tumor clones not really visible by standard tissue sectioning and morphology, we tested normal colon tissues from patients without cancer. Only 1 of 11 cases harbored a methylation level over 0.18, which may be due to a small population of inflammatory cells and/or stromal cells in normal colon epithelial components. The methylation level in all 10 remaining normal tissues was undetectable.

The high level and frequency of NMDAR2A methylation in primary CRC has important clinical implications. Cancer-specific methylation serves as an important biomarker for the early detection of cancer. Such markers are often needed to supplement the cytopathological assessment of tissues. For example, methylation of genes that encode p16, DAPK and MGMT have proven useful in the detection of lung and head and neck cancer (Sanchez-Cespedes et al., 2000; Weaver et al., 2006). Similarly, methylation levels of glutathione-S-transferase placental enzyme 1 were found to be elevated in prostate cancer cells compared to normal tissues (Jeronimo et al., 2001). In colon cancers, there have been several hypermethylated genes reported including APC, CDKN2A, MGMT, MLH1, DAPK and TIMP-3 (Esteller et al., 2001; Fang et al., 2004; Ebert et al., 2005). Most of these genes are involved in cell proliferation and apoptosis, and some of them are defined as tumor suppressors. The high specificity and sensitivity of NMDAR2A methylation in this study supports additional development of NMDAR2A as a biomarker for CRC.

Ion channels are fundamental components of living cells and their role in the cell proliferation and development of cancer has been extensively studied (Kunzelmann, 2005). In addition, abnormal expression of certain ion channels has been reported in some cancers. For example, the human eag-related gene (HERG) channel is constitutively activated in human acute myeloid leukemias, and HERG-mediated K+ current stimulates cell proliferation of normal and leukemic hemopoietic progenitors (Pillozzi et al., 2002). In contrast, expression of Ca2+-activated chloride channels (hCLCA2) in normal mammary epithelium is consistently lost in human breast cancer and in all tumorigenic breast cancer cell lines, and reexpression of hCLCA2 in human breast cancer cells abrogates invasiveness and tumorigenicity in nude mice (Gruber and Pauli, 1999).

Interestingly, it was reported that moderate activity of NMDAR is implied in anti-apoptotic signaling (Ikonomidou et al., 1999), and NMDAR antagonist MK-801 inhibits Erk-1/2 pathway and decreases cell growth of lung adenocarcinoma cells (Stepulak et al., 2005), suggesting that NMDAR increases cell survival. However, it is more possible that the antiproliferative activity of MK-801 in the study is independent of the action as a general inhibitor of NMDA receptors. The concentrations of MK-801 applied for decrease of the cell growth was much higher (200–400 μM) than those for general inhibition of NMDA receptors (10–100 μM) (Rootwelt et al., 1998). They also mentioned in discussion that 10 μM of MK-801 did not influence proliferation or levels of phosphorylated extracellular regulated kinase and CAMP responsive element binding protein when given in the absence of the growth factors. In addition, it was reported that 10 μM MK-801 decreased NMDAR2B-induced cell death in ESCC cell lines (Kim et al., 2006), and the cell death induced by introduction of NMDAR in HEK293 and CHO cells was blocked by 100 μM MK-801 treatment (Anegawa et al., 2000). These results support that NMDAR increases cell death, which can be blocked by NMDAR inhibition.

The silencing of NMDAR2A in CRC is most likely to harbor some selection advantage of the tumor cells since forced expression of NMDAR2A markedly inhibited cell growth and colony formation. However, it is not clear how methylation-induced silencing of NMDAR2A plays a role in key aspects of cancer cell growth. A clue could be driven from previous reports about APC–PSD-95–NMDAR complex and/or hDlg–APC–β-catenin complex; the major postsynaptic density protein, PSD-95 specifically binds to the C terminus of the NMDAR2A and 2B subunits (Bacon et al., 1995) as well as to the colorectal tumor-suppressor protein APC (Yanai et al., 2000). Moreover, the human homologue of the Drosophila disks large tumor-suppressor protein, hDlg, interacts with APC–β-catenin complex (Azim et al 1995; Matsumine et al., 1996), and PSD-95 interacts with the hDlg-associated protein DAP-1, suggesting a link between hDlg and the NMDAR–PSD-95–APC complex (Haraguchi et al., 2000). Interestingly, PSD-95 is expressed in normal human epithelial cells (Ludford-Menting et al., 2002) and rat colon (Saur et al., 2002). hDlg is downregulated during colon cancer development, and loss of the protein is associated with lack of epithelial cell polarity, one of the hallmarks of malignant carcinomas, and disorganized tissue architecture (Gardiol et al., 2006). Although PSD-95 expression and the existence of APC–PSD-95–NMDAR complex in human colon have not been reported yet, all these results suggest that the NMDAR2A, PSD-95, hDlg, APC and/or β-catenin may be involved in maintaining normal function of colon. Thus, the promoter methylation-induced silencing of the NMDAR2A might play a role in loss of function of the TSG complex by disturbing its binding to PSD-95, which might contribute CRC progression. Future work will focus on the investigation of specific mechanisms in NMDAR2A function in human cancers.

In summary, NMDAR2A was commonly hypermethylated and downregulated in primary human CRC. Full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A possesses tumor-suppressive activity.

Materials and methods

5-Aza-dC/TSA treatment and RT–PCR

Cell treatment, RNA extraction and cDNA sythesis were performed as described (Kim et al., 2006). For amplification of NMDARs, and β-actin touchdown PCR or standard PCR was performed as reported previously (Kim et al., 2006). The primer sequences for NMDARs are shown in Supplementary Table 1 or reported previously (Kim et al., 2006). PCR products were gel extracted and sequenced to verify true expression of the genes.

Immunohistochemistry

Tissue microarrays with sections (5 μm) of colon cancer tissues, adjacent tissues 1.5 cm away from tumor and nonmalignant normal colon tissues were deparaffinized and incubated with anti-NMDAR1 rabbit polyclonal Ab (1:50 dilution, AB1516), anti-NMDAR2A (1:50 dilution, MAB5216) and anti-NMDAR2B (1:50 dilution, MAB5220) mouse monoclonal antibodies at 4 °C overnight. Antibodies were purchased from Chemicon (Temecula, CA, USA). Secondary antibody was used at a dilution of 1:500 and incubated for 1 h. After washing the slides in phosphate-buffered saline we stained them with freshly prepared diaminobenzidin solution. We exposed control slides to secondary antibody alone, and they did not show any nonspecific staining. Sections were counterstained in Harry’s hematoxyline. Matched isotopic controls were used in each case.

TaqMan MSP

For quantitative methylation analysis, all protocols for TaqMan MSP were performed as reported (Kim et al., 2006), and all reactions were performed in duplicate. The methylation ratio was defined as the quantity of fluorescence intensity derived from the NMDAR2A promoter amplification divided by fluorescence intensity from β-actin amplification, and multiplied by 100 (TaqMan methylation value: TaqMeth V).

Statistical analysis

We used the methylation levels (TaqMeth V) for NMDAR2A to construct ROC curves for the detection of CRC. Using this approach, the AUROC (area under ROC) identified optimal sensitivity and specificity levels (that is, cutoffs) at which to distinguish normal from malignant CRC tissues, and corresponding TaqMeth V threshold was calculated. The cutoff values determined from ROC curve was applied to determine the frequency of NMDAR2A methylation. Samples with TaqMeth V, 0.18 or higher were designated as methylated, and samples containing less than TaqMeth V of 0.18 were designated as unmethylated. Statistical analyses were conducted using STATA version 9 (STATA Inc., College Station, TX, USA).

Transfection

The rat NMDAR2A expression plasmid (NMDAR2A-pcDNA1) was kindly provided by Dr John J Woodward (Medical University of South Carolina) and the rat NMDAR1-1a expression plasmid (NMDAR1-1a-pRc/CMV) was kindly provided by Dr David Lynch (University of Pennsylvania). Cells were transfected using Fugene-6 (Roche, Basel, Switzerland) in OPTI-MEM (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions.

TUNEL assay

Detection of free 3′-OH was performed with the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI, USA) according to the manufacturer's protocol. Fluorescence was detected with an inverted fluorescence microscope (Nikon TE200, HG-100 W mercury lamp). These experiments were done in duplicate and repeated twice.

Colony focus assay

Colony focus assays were performed using transfected cells in the presence of G418 (1 mg ml−1 for HCT116 and 125 μg ml−1 for KYSE140) and 50 μM L-glycine per 200 μM L-glutamate for 2 weeks.

Knockdown of NMDAR2A and calcein-AM assay

Four individual siRNAs targeting NMDAR2A genes and nontargeting control siRNA were purchased from Dharmacon (Chicago, IL, USA). On-Target plus set of four duplex (0508) was relabeled as siRNA–NMDAR2A-1−4, respectively. A total of 50 nM of each siRNA was transiently transfected to TE5 using LipofectamineRNAiMax transfection reagent (Invitrogen) in OPTI-MEM. After 24 h of transfection, cells were added with growth medium, incubated and calcein-AM assay (Invitrogen) was performed at indicated time points.

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Acknowledgements

Under a licensing agreement between OncoMethylome Sciences, SA and the Johns Hopkins University, Dr Sidransky is entitled to a share of royalty received by the University on sales of products described in this article. Dr Sidransky owns OncoMethylome Sciences, SA stock, which is subject to certain restrictions under University policy. Dr Sidransky is a paid consultant to OncoMethylome Sciences, SA and is a paid member of the company's Scientific Advisory Board. The term of this arrangement is being managed by the Johns Hopkins University in accordance with its conflict of interest policies.

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Correspondence to D Sidransky.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Kim, M., Chang, X., Nagpal, J. et al. The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth. Oncogene 27, 2045–2054 (2008). https://doi.org/10.1038/sj.onc.1210842

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Keywords

  • NMDAR2A
  • promoter hypermethylation
  • colon cancer
  • 5-aza-2′-deoxycytidine
  • tumor suppressor
  • biomarker

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