Putative tumour-suppressor gene DAB2is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma

NPC cell lines (C666-1, HK1 and HONE1), xenografts (X2117, X666, C15, C17, X1915) were maintained as described previously [20]. An SV40 large T oncogene immortalized normal nasopharyngeal epithelial cell line NP69 was also included in this study [21]. The NPC cell line C666-1 and all the NPC xenografts harbour EBV, whereas HK1, HONE1 and NP69 cells are EBV-negative. Forty-six formalin-fixed paraffin-embedded primary NPC biopsy samples were retrieved from tissue bank of the Department of Anatomical and Cellular Pathology at Prince of Wales Hospital, Hong Kong. All specimens were taken before treatment and were histologically evaluated to be EBV-positive undifferentiated carcinomas as demonstrated by EBER in situ hybridization. The male to female ratio of the patients was 3.1:1. The age of the patients ranged from 16 to 84 years with the median age of 48. Based on UICC staging classification, 5 patients had Stage I disease (11%), 14 patients had Stage II (30%), 10 patients had Stage III (22%), 17 patients had Stage IV disease (37%). The median follow-up time was 57.8 months (range 13.8-95.5 months). The study protocol was approved by the Joint CUHK-NTE Clinical Research Ethics Committee, Hong Kong.

NPC cell line C666-1 was treated with 1, 5, 10, 15 μM of 5-Aza-2'-deoxycytidine (Sigma, St. Louis, MO) for 3 days. Culture medium with fresh 5-aza was replenished every 24 hours. For TSA treatment, cells were incubated with 50, 100 and 200 ng/ml TSA (Sigma) for 24 hours. Synergistic effect of 5-aza and TSA was tested by treating cells with 5 μM of 5-aza for 3 days followed by 100 ng/ml of TSA for 24 hours.

Immunohistochemistry was performed on the Ventana Nex ES automated stainer (Ventana Corporation, Tucson, AZ) using anti-DAB2 antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA). The cytoplasmic expression of DAB2 was assessed by assigning a proportion score and an intensity score. The proportion score was according to proportion of tumour cells with positive cytoplasmic staining (0, none; 1, < = 10%; 2, 10 to < = 25%; 3, > 25 to 50%; 4, > 50%). The intensity score was assigned for the average intensity of positive tumour cells (0, none; 1, weak; 2, intermediate; 3, strong). The cytoplasmic score of DAB2 was the product of proportion and intensity scores, ranging from 0 to 12. The cytoplasmic expression was categorized into low (score 0 to 3), intermediate (score 4-6), and high (score 7-12). The scoring was independently assessed by two pathologists (K.F.T. and A.W.C.)

Bisulfite sequencing and MSP has been described previously [22, 23]. A total of 47 CpG sites spanning approximately 800-bp on the 5'CpG island of DAB2 gene were analyzed by bisulfite sequencing (Additional file 1 Figure S1). This region covered the critical transcriptional regulatory domains sufficient for DAB2 expression in epithelial cells [24, 25]. The PCR Primers are listed in Table 1.

Full length DAB2 was amplified from TrueORF cDNA clone (Origene, Rockville, MD) and inserted into pcDNA3.1(+) (Invitrogen) to produce DAB2 expression vector. pcDNA3.1(+)/DAB2 were transfected into C666-1 cells using FuGENE HD (Roche, Mannheim, Germany). Restoration of DAB2 expression was confirmed by western blot analysis using DAB2 antibody (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Cells transfected with pcDNA3.1(+) or pcDNA3.1(+)/DAB2 were subjected to flow cytometric analysis as described previously [22].

Cell proliferation of DAB2 transfected C666-1 cells was assessed using CellTiter 96^® Non-Radioactive Cell Proliferation Assay (MTT) (Promega, Madison, WI) according to the manufacturer's instructions. For monolayer colony formation assay, C666-1 cells were culture overnight in 60 mm plates and transfected with pcDNA3.1(+)/DAB2 or empty vector. Forty-eight hours later the transfectants were re-plated in triplicate and cultured for 21 days in selection medium containing G418 (500 μg/ml). Surviving colonies were stained with Gentian violet after methanol fixation and counted. The experiments were repeated in triplicate.

Gene expression analysis of C666-1 transiently transfected with pcDNA3.1(+)/DAB2 for 24 hours as compared with cells transfected with empty vectors as controls were subjected to microarray analysis using 4 × 44 K Whole Human Genome Oligo Microarray system (Agilent Technologies, Santa Clara, CA). The experiment was done in duplicate. Microarray slides were scanned using Agilent G2505C microarray scanner (Agilent). The images were quantified with Feature Extraction Software (Agilent Technologies). The raw data were quantile normalized by robust multiarray average (RMA) algorithm and analyzed in Partek Genomics Suite 6.4 (Partek, St. Charles, MO). Differential gene expression was evaluated using one-way ANOVA. A fold-change cut-off of 1.5 and p < 0.01 was set to identify differentially expressed genes. The Ingenuity Pathways Analysis (IPA, Ingenuity^® Systems, http://www.ingenuity.com) was utilized to identify network of interacting genes and the biological functions that were most significant to the data set. Fisher's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone. A p-value of less than 0.01 was considered significant.

QRT-PCR was performed using premixed primers and probes from TaqMan^® Gene Expression Assays (Appliedbiosystems, Foster city, CA) and run on ABI 7500 real time fast PCR system (Appliedbiosystems). All reactions were done in triplicate. The relative expression level was normalized with beta-actin and calculated using the 2^(-Delta Delta Ct) method [26].

MAP kinase pathways were studied by western blotting according to standard procedures as described previously [22, 23]. Anti-ERK1/2, anti-phospho-ERK1/2 and anti-c-Fos antibodies came from Cell Signalling Technology, Inc. (Beverly, MA). Anti-GAPDH (Calbiochem, La Jolla, CA) was used as a loading control.

Mann-Whitney U test was used to compare the data and p value < 0.05 was considered as statistically significant. Statistical analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).

We used QRT-PCR assay to assess the DAB2 mRNA expression level in NPC cell lines, xenografts and immortalized normal nasopharyngeal epithelial cell line. As shown in Figure 1, DAB2 expression was undetectable in NPC cell line C666-1 and all 5 NPC xenografts (X2117, X666, C15, C17 and X1915). In NPC cell lines HK1 and HONE1, the DAB2 expression levels were significantly reduced comparing with the expression level of immortalized normal nasopharyngeal epithelial cell line.

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Immunohistochemistry was performed in 46 NPC biopsy samples to determine the DAB2 protein expression in primary NPC. We first optimized the staining condition using human ovarian tissue. Intense DAB2 immunoreactivity was observed in the ovarian surface epithelial cells (Figure 2A). In normal nasopharyngeal epithelium, intermediate to high level of cytoplasmic stain was observed as demonstrated in Figure 2B. In NPC biopsy samples, thirty-three cases (72%, 33 out of 46) were negative for DAB2 stain. Focal immunoreactivity was observed in the remaining 13 NPC biopsies and all were scored as low level expression (score <4). Examples of positive and negative DAB2 immunoreactivity in NPC biopsies were shown in Figurea 2C and 2D, respectively. The dendritic cells distributed in the stroma showed strong immunoreactivity for DAB2. It has been reported that macrophage and fibroblast-like cells in the stroma expressed DAB2 [12, 27]. Therefore, they served as internal positive controls for the staining as shown in Figure 2D. DAB2 immunoreactivity was not associated with age, gender, stage of disease, disease specific survival or overall survival of the patients.

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Sheng et al. characterized human DAB2 gene and identified a CpG island (39 CpG sites) spanning 420-bp sequence 5' of exon 1. They found that this 420-bp putative promoter contains the transcription start site and is sufficient for active transcription in epithelial cells [24]. Since the expression of DAB2 was down-regulated in NPC, we examined whether the gene was silenced by promoter hypermethylation. Bisulfite sequencing was performed to determine the methylation pattern of the 5'CpG island of DAB2 in NPC cell lines and xenografts. Two regions were amplified for analysis: region 1 with 24 CpG sites and region 2 with 23 CpG sites (Figure 3A and Additional file 1 Figure S1). As shown in Figure 3B, dense methylation in both region 1 and region 2 was observed in NPC cell line C666-1, NPC xenografts X2117 and X666, in which no DAB2 mRNA expression was detected. In NPC cell lines HK1 and HONE1, and immortalized normal nasopharyngeal epithelial cell line NP69, where the DAB2 expression was retained, the promoter region was unmethylated. These results indicated that aberrant methylation contributed to the loss of DAB2 expression in NPC cell lines and xenografts.

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We further analyzed the DAB2 promoter methylation status in primary NPC tissues by MSP. Aberrant methylation on the promoter region of DAB2 was detected in 65.2% (30/46) of NPC primary tumour biopsy samples. Representative results were shown in Figure 3C. Our data suggested that aberrant promoter methylation is a common event that contributes to the down regulation of DAB2 expression in NPC. However, no correlation could be demonstrated between DAB2 hypermethylation and clinicopathologic features including age, gender, stage of disease, disease specific survival or overall survival of the patients.

We treated NPC cell line C666-1, which shown complete loss of DAB2 expression, with demethylation agent 5-aza-2'-deoxycytidine. DAB2 mRNA expression was restored in a dose dependent manner as demonstrated by quantitative real time PCR (Figure 4A). Using bisulfite sequencing, partial demethylation of DAB2 promoter was detected in C666-1 cells after 5-aza treatment (Figure 4B). The promoter region we analyzed included the major GATA6 cis elements that are critical for the transcriptional regulatory of DAB2 gene [25]. The finding further confirmed that DAB2 down regulation was mediated by promoter methylation. Induction of DAB2 transcription by histone deacetylase inhibitor has been reported previously [19]. In C666-1 cells, histone deacetylases inhibitor (HDACi) trichostatin A (TSA) could also restore the DAB2 mRNA expression (Figure 4A). However, we didn't observe a synergistic induction of DAB2 transcript after combined treatment with 5-aza and TSA in comparison to TSA treatment alone (Figure 4A).

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To evaluate the potential tumour suppressor effect of DAB2 in NPC, we transfected full length DAB2 cDNA in pcDNA3.1(+) expression vector into C666-1 cells. The expression of transfected cDNA was confirmed by western blot analysis. The endogenous DAB2 expression was completely loss in wild type C666-1 cells. Re-expression of DAB2 could be detected at 12 hours after transfection and remained at high level after 72 hours (Figure 5A). MTT assay was done to assess the proliferation rate of the transfectants (Figure 5B). DAB2 suppressed the proliferation of C666-1 cells in comparison to vector controls and mock transfection controls (transfection reagent only). We further evaluated the growth inhibitory effect of DAB2 in NPC cells using anchorage-dependent colony formation assay. C666-1 cells transfected with pcDNA3.1(+)/DAB2 or empty vector were selected with G418 for 21 days. Exogenous DAB2 resulted in 35% reduction in colony formation compared to vector-transfected control (Figure 5C). Since a growth inhibitory effect was observed in DAB2 transfected NPC cells, we analyzed the transfectants for cell cycle parameters using flow cytometry. Twenty-four hours after transfected with pcDNA3.1(+)/DAB2, empty vector, or mock transfection, the C666-1 cells were harvested and analyzed. Under our experimental condition, DAB2 did not cause significant change in cell cycle parameters, although a slightly reduction (3.9%) of S phase cells was observed in DAB2 expressing cells (Figure 5D).

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DAB2 was identified as a mitogen responsive phosphoprotein and its growth suppression function is thought to be secondary to the inhibition of MAP kinase pathway [28]. To explore the mechanism for tumour suppression by DAB2 in NPC, we studied the effect of DAB2 on serum-induced MAP kinase signalling in NPC cells C666-1. We transfected C666-1 cells with empty vector or DAB2 expression vector. The cells were allowed to recover for 24 hours, followed by starvation in serum-free medium for another 18 hours, and then stimulated with 20% serum in culture medium. The cells were harvested at various time points and assayed for MAP kinase pathway by western blotting. C666-1 cells exhibited no basal MAP kinase activity after starvation (Figure 6). Activation of MAP kinase, as demonstrated by phospho-ERK1/2 immunoblot, was observed at 5 minutes after serum stimulation and attenuated after 30 minutes. MAP kinase activation was not affected by DAB2 expression. The p-ERK1/2 activities were unchanged upon serum stimulation in transient DAB2-transfected cells compared with vector-transfected cells. C-Fos was induced at 30 minutes after serum stimulation and reached the maximal level at 60 minutes. In DAB2 expressing C666-1 cells, a much weaker c-Fos induction was observed compared with vector-transfected control cells. C-Fos is an immediate early gene that is required for malignant tumour growth. DAB2 might exert the tumour suppression function by inhibiting c-Fos expression in NPC.

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To search for potential genes and pathways regulated by DAB2 in NPC, we compared the expression profile of C666-1 cells overexpressing DAB2 to vector control-transfected cells. The expression of DAB2 protein after transfection was confirmed by western blotting. The expression profile was performed using Agilent 4 × 44K whole human genome oligonucleotide (60 mer) array. Differential gene expression was evaluated using one-way ANOVA. Within the ANOVA model, linear contrasts were used to compare the transiently transfected cells carrying DAB2 to the vector control. To adjust for multiple testing errors, a step-up Benjamini false discovery rates (FDR) adjustment was used. FDR was set to be less than 20% to produce a list of significantly differentially expressed genes for each contrast described above.

A total of 628 differentially expressed genes were identified at expression cutoff of 1.5 fold and p = 0.01. Top 50 significantly up-regulated and down-regulated genes were listed in Additional file 2 Table S1. Some of the genes were confirmed by real time QRT-PCR (Additional file 2 Table S2). Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. Two hundred and nine of these genes, called focus genes, were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of these focus genes were then algorithmically generated based on their connectivity. A total of 32 partially overlapped networks were identified. Of which 9 networks involved more than 1 focus gene. Top functions of these genes were related to cancer, cell death, endocrine systems disorders, cellular and organismal development, cell-to-cell signalling and interaction, grow and proliferation, cell cycle and cell morphology. Additional file 1 Figure S2 shows the most significant three gene networks. The first Network, which was built from 22 genes and received the highest IPA score, was centred on MAPK, RAS, AKT and IGF1 and associated with cancer, cell death and endocrine system disorders. The second network, incorporating 22 genes, was centred on NFκB and AP1, and associated with endocrine system disorders, metabolic diseases. The third network built on 23 genes was mostly centred around ERK and associated with cardiovascular, cellular and organismal development.

The use of IPA identified the mitotic roles of Polo-like kinase, Wnt/beta-cantenin signalling and cell cycle regulation by BTG family proteins as the most significant canonical pathways altered upon DAB2 transfection (Table 2, Additional file 1 Figure S3). All three canonical pathways were down-regulated in DAB2-expressing C666-1 cells.