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Mucins are high molecular weight glycoproteins that have oligosaccharides attached through O-glycosidic linkages to serine or threonine residues of the core protein. Many human mucin core proteins (MUC1-MUC9, MUC11-13 and MUC15-20) have been identified, (Lapensee et al, 1997; Yonezawa and Sato, 1997; Gum et al, 2002; Pallesen et al, 2002; Yin et al, 2002) including MUC4, a large transmembrane mucin with a very long glycosylated extracellular domain that is expressed in various normal tissues (Audie et al, 1993, 1995; Buisine et al, 1999; Gipson et al, 1999). MUC4 is also often overexpressed in epithelial cancers and our immunohistochemical studies have shown that aberrant expression of MUC4 is associated with invasive proliferation of tumours and a poor outcome for patients (Shibahara et al, 2004; Saitou et al, 2005; Tamada et al, 2006; Tsutsumida et al, 2007).

MUC4 also serves as a novel intramembrane ligand for the receptor tyrosine kinase ErbB2, a transmembrane glycoprotein with a tyrosine kinase domain that is encoded by the c-ErbB-2 proto-oncogene and is highly homologous with the epidermal growth factor receptor (Yamamoto et al, 1986). Furthermore, MUC4 plays an important role in cell proliferation and differentiation of epithelial cells by inducing specific phosphorylation of ErbB2 and enhancing the expression of p27 (Jepson et al, 2002), a cyclin-dependent kinase inhibitor that regulates the G1 and S phases of the cell cycle (Polyak et al, 1994).

We recently described the epigenetic regulation of the MUC1 gene (Yamada et al, 2008). Similarly to MUC1, MUC4 is a factor associated with poor prognosis in several carcinomas (Shibahara et al, 2004; Saitou et al, 2005; Tamada et al, 2006; Tsutsumida et al, 2007), and thus we hypothesised that MUC4 is also regulated by an epigenetic mechanism. To investigate the possible epigenetic regulation of MUC4 gene expression, we mapped the DNA methylation status of the MUC4 promoter region using 10 cancer cell lines derived from carcinomas of four different organs (breast, lung, pancreas and colon). Methylation of cytosine in genomic DNA plays an important role in gene regulation, and especially in gene silencing (Bird, 1992), and generally the promoter region of a transcribed gene is hypomethylated (Wolffe et al, 1999; Stirzaker et al, 2004).

To examine the methylation profiles of 92 CpG sites in the MUC4 promoter in the cancer cell lines, we performed a MassARRAY methylation analysis (Figure 3A). Samples were analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), which permits high-throughput identification of methylation sites and semiquantitative measurement at single or multiple CpG sites. The raw spectrum data for CpG sites 108–112 in NCI-H292 and A427 lung cancer cells are shown in Figure 3B. In the vicinity of the transcriptional start site, MUC4-negative cells or cells with low expression of MUC4 showed high methylation (MCF-7, T-47D, MDA-MB-453, A427, PANC1 and Caco2) compared with MUC4-positive cell lines (NCI-H292, BxPC-3 and HPAFII). In contrast, the MUC4 promoter CpG sites were mostly hypomethylated in LS174T cells, which showed low expression of MUC4. In the 10 cancer cell lines, methylation of CpG sites 108–112 was inversely correlated with MUC4 gene expression, whereas methylation of sites 1–107 was almost unrelated to gene expression. These results indicate that the CpG methylation status near to the MUC4 transcriptional start site may play an important role in gene expression of MUC4.

Figure 3
figure 3

Summary of the CpG methylation status of the MUC4 gene promoter region in 10 cancer cell lines. (A) Quantitative methylation analysis of CpG sites located in the MUC4 promoter using a MassARRAY compact system. Different colours display relative methylation changes in 10% increments (green=0%, red=100% methylated). CpG sites that correlated well with MUC4 expression are boxed in red. (B) Raw spectrum data for CpG sites 108–112 in lung cancer cell lines. Non-methylated and methylated CpG sites generate status-specific mass signals. Methylation status (%) is calculated as (intensity of methylated CpG (red dotted line)/(intensity of methylated CpG (red dotted line)+intensity of unmethylated CpG (green dotted line))). † Indicates methylation in one of the two CpG sites (eg, 109 or 110). ‡ Indicates methylation of both CpG sites (eg, 109 and 110). Asterisks indicate MUC4-positive cell lines.

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Methylation-specific PCR analysis of MUC4 promoter methylation in 10 cancer cell lines

Methylation-specific PCR primers were designed to target CpG sites in the 5′ flanking region of the MUC4 promoter (Table 1) based on the results of MassARRAY analysis. The five CpG sites (Nos. 108–112) included in the MSP primer showed 4–39% methylation in MUC4-positive cells and 63–100% methylation in all MUC4-negative/low cells, except for LS174T cells. The LS174T cells showed 5–19% methylation at the five sites. To confirm the reliability of the MSP primer, MSP analysis was performed on the 10 cell lines (Figure 4). An unmethylated band (lanes indicated by U in Figure 4) was obtained in MUC4-positive NCI-H292 and BxPC-3 cells, whereas a methylation band (lanes indicated by M in Figure 4) was observed in MUC4-negative/low MCF-7, MDA-MB-453, A427 and PANC1 cells. The MSP data were almost consistent with the results of MassARRAY analysis of the methylation status of the cell lines. Therefore, our results suggest that the 5′ flanking region of the promoter, and especially CpG sites 108–112, may play an important role in methylation-related gene silencing of MUC4.

Figure 4
figure 4

Methylation-specific PCR (MSP) analysis of the MUC4 gene promoter region in 10 cancer cell lines. The PCR products labelled as ‘M’ (methylated) were generated by methylation-specific primers and those labelled as ‘U’ (unmethylated) were generated by primers specific for unmethylated DNA. The unmethylated allele was detected in MUC4-positive NCI-H292 and BxPC-3 cells, whereas the methylated allele was detected in MUC4-negative/low MCF-7, MDA-MB-453, A427 and PANC1 cells. Asterisks indicate MUC4-positive cell lines.

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Discussion

We have previously examined the expression profiles of MUC4 in primary tumours of patients with intrahepatic cholangiocarcinoma-mass forming type (n=27; Shibahara et al, 2004), pancreatic adenocarcinoma (n=135) (Shibahara et al, 2004; Saitou et al, 2005; Tamada et al, 2006; Tsutsumida et al, 2007), extrahepatic bile duct carcinoma (n=70; Tamada et al, 2006) and small-sized lung adenocarcinoma (less than 3 cm; n=185; Tsutsumida et al, 2007) and compared MUC4 expression with the survival of the patients. In the four series, survival in MUC4-positive or high-expression cases was significantly worse than that for MUC4-negative or low-expression cases, and multivariate analyses showed that MUC4 expression in the carcinomas was a risk factor for a poor prognosis (Shibahara et al, 2004; Saitou et al, 2005; Tamada et al, 2006; Tsutsumida et al, 2007).

The regulatory mechanism of the MUC4 gene is unclear. Initially, to examine the possible epigenetic regulation of MUC4 expression, we treated MUC4-negative or low-expression cancer cell lines with 5-AzadC and/or TSA (Figure 1B). In four of seven cells (T-47D, PANC1, LS174T and Caco2), treatment with 5-AzadC/TSA significantly restored the MUC4 mRNA expression, compared to the treatment with 5-AzadC or TSA alone. In MCF-7 cells, however, the restoration level of MUC4 mRNA decreased in 5-AzadC/TSA treatments rather than in treatment with 5-AzadC or TSA alone. Yet, it is unclear whether 5-AzadC and TSA caused an antagonistic effect in MCF-7 cells. We showed that treatment with TSA did not restore the MUC4 mRNA in MDA-MB-453 and A427 cells. Kondo et al (2003) showed that 5-AzadC or a combination of 5-AzadC and TSA, but not TSA alone, reactivates tumour suppressor gene expression at silenced loci (eg, p16). Our results in MDA-MB-453 and A427 cells are in good agreement with their observation. Although there were differences in the restoration level of MUC4 mRNA among treated cell lines, our overall results suggested the possibility that MUC4 expression is regulated by epigenetic mechanisms.

Next, to rule out the contribution of SNPs in control of MUC4 expression, the sequences of the MUC4 promoter were verified in all the cells used in the study using primers based on the published sequence (Perrais et al, 2001). In Figure 2, the sequence of part of the promoter (−833 to −647) could not be determined by single-strand sequencing, and we were also unable to obtain reliable results for one promoter region (−1822 to −973) using the MassARRAY compact system. Moreover, we found that bases at some positions differed among the 10 cell lines in a 3.6-kb MUC4 promoter region. However, we found no correlation between these sequence differences and MUC4 expression, and therefore we examined the DNA methylation status of the MUC4 promoter in 10 cancer cell lines.

In this study, we used MassARRAY analysis to examine the methylation status of 92 CpG sites in the promoter of the MUC4 gene. Methylation at CpG sites 108–112 (−170 to −102) was associated with the expression of MUC4 in all cell lines except for LS174T. MUC4-negative cell lines were hypermethylated at these five CpG sites, whereas MUC4-positive cell lines showed hypomethylation at the same sites. We recently reported that MUC1 expression in pancreatic, breast and colon cancer cells is regulated by DNA methylation and histone H3-K9 modification in the 5′ flanking region, also using a MassARRAY compact system. Methylation of CpG near the transcriptional start site (−100 to 100) was inversely correlated with MUC1 gene expression (Yamada et al, 2008). Similarly, MUC4 expression was also regulated by methylation of CpG sites near the transcriptional start site (−170 to −102).

On the other hand, the methylation status of upstream regions clearly differed between MUC1 and MUC4. In MUC1, most CpG sites except for the regulatory region were unmethylated (Yamada et al, 2008); in contrast, in MUC4, almost all CpG sites of the upstream region (until CpG site 100) were methylated (Figure 3A). The reason for the different global methylation levels of these similar transmembrane mucins requires further investigation.

We were unable to show a relationship between DNA methylation status and expression of the MUC4 gene in the LS174T colon cancer cell line, similarly to our results for MUC1 (Yamada et al, 2008). However, expression of MUC4 in Caco2 cells, which are derived from cancer cells of the same organ, was related to the DNA methylation status. This indicates that the regulation of expression by DNA methylation is not organ-specific, but varies in individual cell lines.

Recently, Vincent et al (2008) reported that MUC4 expression is regulated epigenetically through DNA methylation and histone modification in pancreatic and gastric epithelial cancer cell lines, and that CpG sites 110–114 (−121 to −81) were associated with expression of MUC4 in these cells (Vincent et al, 2008). However, in our study, CpG 113 (−93) and 114 (−81) were hypomethylated in all cell lines and unrelated to the expression of MUC4 gene, whereas sites 108 and 109 were associated with MUC4 gene expression. These differences might be due to different methods of detection, and it also possible that these CpG sites (108, 109, 113 and 114) may be sensitive and unstable. This issue may be resolved by the use of different analytical methods in future studies.

MUC4 is a factor associated with a poor prognosis in various carcinomas and particularly in intractable carcinomas, such as pancreatobiliary and lung carcinomas (Saitou et al, 2005; Tamada et al, 2006; Tsutsumida et al, 2007). This study shows that MUC4 expression is regulated by DNA methylation in the promoter region, and we have also shown that MUC1, another prognostic indicator, is regulated by a similar epigenetic mechanism (Yamada et al, 2008). These results indicate that MassARRAY is a powerful tool for detection of methylation of CpG sites, and screening of the DNA methylation status of MUC1 and MUC4 genes by MassARRAY analysis of discharged fluids, such as pancreatic juice, bile or sputum is likely to be of value for early detection of pancreatobiliary and lung carcinomas.