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. Author manuscript; available in PMC: 2012 Mar 21.
Published in final edited form as: Int J Oncol. 2011 Jan 21;38(4):1129–1135. doi: 10.3892/ijo.2011.918

Promoter methylation of heat shock protein B2 in human esophageal squamous cell carcinoma

Xiaofei Chang 1, Keishi Yamashita 2, David Sidransky 1, Myoung Sook Kim 1
PMCID: PMC3309605  NIHMSID: NIHMS358941  PMID: 21258768

Abstract

Hypermethylation of gene promoters and the corresponding loss of gene expression are recognized as a hallmark of human cancer, and DNA methylation has emerged as a promising biomarker for the detection of human esophageal squamous cell carcinoma (ESCC). To identify novel genes methylated in ESCC, we screened 35 candidate genes identified from an oligonucleotide microarray. Among them, the heat shock protein B2 (HSPB2) was methylated in 95.7% (67/70) of primary ESCCs, whereas no methylation was found in normal esophageal tissues from ESCC patients (0%, 0/20). RT-PCR analysis revealed that HSPB2 expression was silenced or weakly expressed in most ESCC cell lines, and re-activated by the demethylating agent 5-aza-2'-deoxycytidine. These results indicate that promoter methylation of HSPB2 is one of the causal factors for loss or down-regulation of HSPB2 expression. mRNA expression of HSPB2 in ESCC tissues was significantly down-regulated compared to normal tissues. Our data suggest that promoter methylation of HSPB2 deserves further attention as a novel molecular biomarker in human ESCC.

Keywords: heat shock protein B2, methylation, esophageal squamous cell carcinoma

Introduction

Esophageal cancer is one of the common malignancies with an increasing incidence and a high mortality worldwide (1,2). Most patients with ESCC are diagnosed at an advanced stage, and metastasis to the regional lymph nodes occurs frequently (3). As a consequence, the prognosis of ESCC patients is usually very poor. Finding molecular markers for ESCC early detection is thus one of the most promising research avenues that might improve the survival of patients with ESCC.

Heat shock protein B2 (HSPB2) is a member of the small heat shock protein family, expressed in heart and skeletal muscles (4). It is also known as heat shock 27-kDa protein 2 (HSP27), located at chromosome 11q22-q23 which is a frequent target for deletion during the development of many solid tumor types (5). HSPB2 is present in the cytosol as granules in association with the outer membrane components of the mitochondria, and thus plays a protective role for cells from deleterious stresses such as oxidative stress, heat shock, radiation and anti-cancer drugs (4–9). It is co-expressed with β-catenin in the cellular compartment of tumor and cell lines, and also interacts with β-catenin. The biological activity of HSPB2 is regulated by phosphorylation of its serines by mitogen-activated protein kinases associated protein kinases (MAPKAPKs) which are activated by phosphorylation of p38 mitogen-activated protein (MAP) kinase (10,11). HSP27 regulates Akt activation (12) and p53 transcriptional activity in doxorubicin-treated fibroblasts and cardiac cells (13). Interestingly, HSP27-overexpression in NIH3T3 cells retards cell migration and invasion, which is correlated with reduced expression of matrix metalloproteinase-2 and Stat3 phophorylation (14).

Although it has been reported that small heat shock proteins including HSPB2 participates in regulation of apoptosis and are involved in tumorigenesis (6,13,15,16), the expression of HSPB2 is frequently reduced in patients with ESCC, which is associated significantly with poor prognosis and postoperative patient survival (17,18). Expression of HSPB2 correlates negatively with lymph node metastases (18) and also correlates positively with lymphocyte infiltration (18) whereas inversely with depth of invasion (17). These findings suggest that HSPB2 is a significant prognostic factor for ESCC.

Hypermethylation of gene promoters and the corresponding loss of gene expression are now recognized as a hallmark of human cancer (19). We have intensively investigated methylation of gene promoters in primary tumors and in body fluids for the development of biomarkers in cancer (20–23). Recently, a combination of several methylation markers was reported to be significantly correlated with patient prognosis in esophageal adenocarcinoma (24). We also reported that PGP9.5 and NMDAR2B were independent prognostic markers for ESCC (25–27). In this study, we examined the methylation status of the HSPB2 gene promoter in human ESCC, and report for the first time that HSPB2 is frequently methylated in primary ESCC but not in normal esophageal tissues. A specific decrease of HSPB2 transcript in ESCC cell lines and tissue was also observed.

Materials and methods

Cell lines and tissues

ESCC cell lines were obtained from the Cell Response Center for Biomedical Research Institute, Department of Aging and Cancer, Tohoku University (TE series) and kindly provided by Dr Y. Shimada in the Department of Surgery, Kyoto University (KYSE series). Cells were grown in RPMI-1640 supplemented with 10% fetal bovine serum. Twenty pairs of ESCC and normal esophagus tissues (patient no. 1–20) were obtained from the Gastroenterology Division, Department of Medicine, University of Maryland. Fifty cases of primary ESCC with clinical features, and cDNAs prepared from six ESCC and one normal esophageal tissue were obtained from patients who underwent surgery at the Medical Institute of Bioregulation Hospital, Kyushu University and the Saitama Cancer Center (26,27). Five normal esophageal epithelial tissues (NN) were obtained from patients without cancer at the Department of Pathology, The Johns Hopkins University.

Bisulfite-sequencing

Bisulfite-modified genomic DNA was amplified by PCR as described (26). Primers were designed in the promoter proximal to transcription start site (TSS), and primer sequences are shown in Table I.

Table I.

PCR primers for bisulfite-sequencing.

Forward Reverse
NM_001541 HSPB2
  Region 1 GAAGAGGAGAAAATGAGGTGTGGAA AAATAAAAACCCTAAATAAAAACAA
  Region 2 AGGGTTTTTATTTTTAGTTTTTTTT AATAATATCTATACCATACCTTCTCC
NM_001666 ARHGAP4 TTAAAACTCTCACACCTACTC CCGTTGCGGTTAGTATTTTG
NM_153613 LOC254531 CTCAACTCCCAAACCCACTC TTTTGGCGTTTTTGTTTTTT
NM_003224 ARFRP1 CTCTTCTATCTAACCAAAAC TTGTTGGGGCGAAAAGGGGG
NM_003224 KCNMA1 CGAATTACCACCTAATAAA GGGGGGAGGGGGATGGAGTG
NM_000517 HBA2 TACAAACTAACTTTCTCCCT GTTAGGGTTTATGTTTGGGG
NM_003004 SECTM1 CACGAACTCTCCTAAAAAAATACTCC AGAGGGAGGGGTTAGGGAGGA
NM_007075 WDRX1 AAACTTAATCTTAACCCAACCCTCC GAGTTTAGGTATTGAATGGTATTTA
NM_007011 ABHD2 TAACCCTCCTCCCCTTTCCC TCGTTTGTTTATTGGGTAGTTTAGT
NM_006159 NELL2 CCATCAACTTAACACCTTAAATTTA GTAAGGAGGAGGGAGGGGT
NM_003080 SMPD2 CCCACCACCTTCACCTACAC TAAAGTTGAGGAAAGGTTTAGT
NM_007074 COROA1 TTCGTACCTACCAAAAAAAAACTAA GATGTTTTTGTTAAGATAATGAATG
NM_006454 MXD4 DTR TCTACATCTATCAACATTCT TTTTAGGTTTTTAAATTGATATATA
NM_001945 (HBEGF) ATCAATAACCCTACCCACAATCAAA TCGGGAATAAGGTTTTAGGAGG
NM_003380 VIM CCATACCCAATCCCAAACCC AGAGTGGTAGAGGATTGGATTT
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The criteria to determine methylation in cell lines and tissues

Bisulfite-sequencing was based on nucleotide sequences in electropherograms, and the criteria to determine methylation positivity was reported previously (28). When methylated CpG was found in >30% of total CpGs in an amplified PCR product, it was considered as methylation-positive.

5-aza-dC and TSA treatment and RT-PCR

Treatment with 5-aza-2'-deocycytidine (5-aza-dC) and/or trichostatin A (TSA), and RNA extraction and cDNA synthesis were performed as described (26). HSPB2 primers were 5'-ATGTCGGGCCG CTCAGTGCC-3' (forward) and 5'-GTCACCTCGTCTGGG GTAAA-3' (reverse). GAPDH primers were 5'-CAACTAC ATGGTTTACATGTTC-3' (forward) and 5'-GCCAGTGGA CTCCACGAC-3' (reverse). PCR products were gel-extracted and sequenced to verify true expression of the gene.

Quantitative RT-PCR

cDNAs from an ESCC patient and from a patient without cancer (NN) were purchased from BioChain Institute, Inc. (Hayward, CA). A pair of normal (PN) and tumor cDNA (PT) was obtained from patients who underwent surgery at the Medical Institute of Bioregulation Hospital, Kyushu University. Real-time RT-PCR was performed as described previously (28).

Results

After ruling out presumed background genes we prioritized candidate genes according to the microarray data described previously (26). We randomly selected 35 genes and further diminished the number of candidate genes to 19 genes by excluding known oncogenes, tumor antigens, genes with high expression in tumor, genes with no CpG islands near the transcription start site (TSS), and genes whose methylation status was already known (Fig. 1). We examined promoter methylation status of these 19 genes by bisulfite-sequencing in three ESCC cell lines (KYSE30, KYSE410, KYSE520) and four tumor tissue samples (PT). To rule out genes methylated in normal tissues, we also examined these genes in 4 normal adjacent esophageal mucosa (PN) samples collected from ESCC patients. As a result, we found that heat shock protein B2 (HSPB2), Rho GTPase activating protein 4 (ARHGAP4), α2 hemoglobin (HBA2), a hypothetical protein (LOC254531), potassium large conductance calcium-activated channel (KCNMA1) and ADP-ribosylation factor related protein 1 (ARFRP1) were methylated in ESCC cell lines and tissues, but not in 4 normal esophageal tissues (Table II). Epsin (HPN) and type 1-troponin I (TNNI1) were found to be methylated in normal esophageal mucosa and thus were excluded. The other eleven genes were not methylated in either ESCC cell lines or primary tissues.

Figure 1.

Figure 1

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Flowchart for selection of candidate genes in ESCC by pharmacologic unmasking and microarray analysis. We identified candidate-methylated genes which were epigenetically silenced in ESCC cell lines using pharmacologic reversal of methylation followed by microarray analysis.

Table II.

Methylation profiles of genes in ESCC cel lines and tissues.

Cell lines PT PN
KYSE30 KYSE410 KYSE520 T5 T6 T7 T8 N2 N4 N5 N6
HSPB2 M M M M M M M U U U U
LOC254531 M M M M M U U U U
ARFRP1 M M M M M M U U
KCNMA1 M M M M M U U
ARHGAP4 M M M M M M M U U U U
HBA2 M M M M U U M U U U U
HPN M M M M M M M
TNNI1 M M M M M M M
DFKZP564008232 U U U U U U U U U U
SECTM1 U U U U U U U U U U
WDRX1 U U U U U U U U U U
ABHD2 U U U U U U U U U U
NELL2 U U U U U U U U U U U
SMPD2 U U U U U U U U U U U
COROA1 U U U U U U U U
MXD4 U U U U U U U U U U U
DTR U U U U U U U U U
VIM U U U U U U U U U U U
IGFBP7 U U U U U U U
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M, methylated; U, unmethylated. PN, normal esophageal mucosa collected from ESCC patients; PT, ESCC tissues. Note, HSPB2 methylaton was examined in the region 1 of the promoter.

Next, we examined the methylation status of the above 6 candidate genes in 20 ESCC tumor tissue samples (PT). ARFRP1 (35%, 7/20), KCNMA1 (45%, 9/20) and HBA2 (40%, 8/20) were methylated with relatively low frequency, whereas HSPB2 (90%, 18/20), LOC254531 (95%, 19/20), and ARHGAP4 (85%, 17/20) were methylated in ESCC with high frequency (Fig. 2A, upper). In addition, HSPB2, LOC254531 and ARHGAP4 were methylated in <20% of the 20 normal matched esophageal tissues, indicating that the methylation was cancer-specific (Fig. 2A, lower). Among the three genes, HSPB2 was selected for further analysis due to the absence of methylation in all 20 matched normal appearing esophageal tissues (PN) (0%). Representative sequencing results of HSPB2 are shown in Fig. 2B. It is noteworthy that the HSPB2 promoter region we examined (region 1) contained no CpG island. We have investigated the region that contained one CpG island (region 2) and found a higher methylation frequency in the normal appearing esophageal tissues (Fig. 2C and D). These data suggested normal and malignant esophageal tissues were distinguished more significantly by the methylation of HSPB2 in region 1 than in region 2.

Figure 2.

Figure 2

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Methylation status of HSPB2 in ESCC and matched normal esophageal tissues. (A) Methylation status of six candidate genes selected in the pilot study were examined in 20 pairs of ESCC (PT) and matched normal esophageal tissues (PN) by bisulfite-sequencing (upper). Bisulfite-sequencing results in region 1 of the HSPB2 promoter. Grey square with M, methylation; white square with U, absence of methylation. (B) Representative sequencing results of HSPB2. Arrowhead, all guanines present after sequencing that are complementary to methyl cytosines on the opposite DNA strand. (C) CpG methylation of the HSPB2 promoter was examined in two regions (1 and 2). The CG sequences of region 1 are underlined and numbered, and the PCR-amplified region is indicated as F1 (forward) and R1 (reverse) primers. (D) Bisulfite-sequencing results in region 2. Grey square with M, methylation; white square with U, absence of methylation.

We numbered the ten CpGs in region 1 of the HSPB2 promoter, and the methylation status of each individual CpG was mapped. Five normal esophageal tissues from patients without cancer (Eso NN) were also included for comparison. Our data showed that none of the CpGs were methylated in Eso NN (Fig. 3A). CpGs in positions 1–7 were found to be commonly methylated in 12 ESCC cell lines and 18 ESCC tissues (PT), (Fig. 3B and C). In matched normal esophageal tissues (PN), only one methylated CpG was found (position 5 or 6) in 7 out of 20 cases. We further examined HSPB2 promoter methylation in 50 ESCC, and found a methylation frequency of 98% (49/50). Taken together, the overall HSPB2 methylation was observed in a total of 67 out of 70 ESCC cases (95.7%, P=0.02).

Figure 3.

Figure 3

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Individual CpG methylation map of the HSPB2 promoter. Individual CpG site methylation was mapped in five normal esophageal tissues from patients without cancer (Eso NN) (A), 12 ESCC cell lines (B), and 20 pairs of ESCC (PT) and matched normal esophageal tissues (PN) (C). Black circle, methylayed CpG; white circle, unmethylated CpG.

To examine the transcriptional level of HSPB2, RT-PCR was performed using primers specific for HSPB2. Weak expression of HSPB2 was observed in the TE series, and no detectable level of HSPB2 was observed in most of the KYSE series (Fig. 4A). Unexpectedly, HSPB2 was expressed in KYSE70 and KYSE520, two cell lines which harbored HSPB2 promoter methylation. However, the mRNA level of HSPB2 was reactivated by treatment with 5-aza-dC, and/or trichostatin A (TSA) in all ESCC cell lines (Fig. 4B, and data not shown), except for the KYSE70 cells where reactivation was minimal. These results indicate that the transcriptional level of HSPB2 is regulated, although not in all cases, by promoter methylation. HSPB2 expression was also examined in cDNA prepared from one normal esophagus (N. Eso) and six ESCC tissues (patients 1–6) by RT-PCR (Fig. 4A, right lanes). HSPB2 expression was observed only in normal esophageal cDNA. In addition, real-time RT-PCR analysis performed in a pair of normal and tumor cDNA prepared from an ESCC patient showed that HSPB2 expression in PN was twice higher than in PT (Fig. 4C, P<0.05). Moreover, the level of HSPB2 in normal cDNA prepared from a patient without cancer was twenty times higher than in tumor cDNA from an ESCC patient (Fig. 4D, P<0.05). These results suggest that HSPB2 expression is markedly down-regulated in ESCC.

Figure 4.

Figure 4

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Promoter hypermethylation downregulates expression of HSPB2 in ESCC cell lines. (A) RT-PCR analysis for HSPB2 expression in cell lines and tissues. GAPDH was used as a loading control. L, 1 kb Plus DNA ladder. (B) Reactivation of HSPB2 by 5 µM 5-Aza-dC and/or TSA treatment. m, mock treatment; a, 5-Aza-dC treatment; a+T, 5-Aza-dC + TSA treatment. (C) HSPB2 expression in a pair of PN and PT cDNA prepared from an ESCC patient by real-time PCR. (D) HSPB2 expression in patients with ESCC (T) or without cancer (NN) by real-time PCR. Relative expression (fold) was calculated by comparing the ratios of mRNA expression of HSPB2 to an internal control gene, GAPDH. Experiments were done in duplicate, and values indicate the means ± SD. *P<0.05 (t-test).

Discussion

Promoter methylation is a promising type of molecular marker in human cancer (29). In previous studies, we have established a set of strategies to screen new potential methylation markers in different types of tumors (26,28,30). We undertook pharmacological unmasking of ESCC cell lines with 5-aza-dC and TSA followed by microarray analysis to comprehensively identify epigenetically inactivated genes in ESCC (26). Among the large number of genes identified by the microarray analysis, we selected in this study candidate genes of which expression was initially absent and then reactivated by treatment with 5-aza-dC. We evaluated the methylation status of the promoter regions in 19 genes by bisulfite-sequencing, and discovered that three genes (HSPB2, LOC254531 and ARHGAP4) harbored methylation in >80% of ESCC tissues.

Based on the differential methylation status of these three genes in human ESCC and normal tissue, we selected HSPB2 to further evaluate its potential as a biomarker in ESCC. The high methylation frequency in ESCC (95.7%, 67/70) strongly supports that HSPB2 is a cancer-specific methylation marker in human ESCC. To the best of our knowledge, this is the first report on HSPB2 promoter methylation in human cancers. Although the region we examined contains no CpG island, discrimination of ESCC from matched normal esophageal tissues was significant for methylation in this region.

HSPB2 belongs to a sub-family of small heat shock proteins that display an enhanced expression in response to various stresses (e.g., heat shock) (4). HSPB2 with other members of HSP family (e.g., Hsp90, Hsp70) has been implicated in tumorigenesis, which may be related to their ability to protect cells against stress-activated programmed cell death (31). The enhanced expression of HSPB2 has been associated with poor prognosis in several tumor types (32–39), however, it also showed a positive correlation with favorable prognosis in many types of cancers (17,18,40–45). These differences seen in the value of HSPB2 as a prognostic factor are thought to be at least partly due to the inherent differences in other biological and molecular features of each cancer type.

Silencing of small heat shock proteins (HSP) has been reported previously; expression of αB-crystallin (CRYAB), a low-molecular-weight HSP, was down-regulated in highly malignant anaplastic thyroid carcinomas (ATC) (46) and ESCC. In addition, αB-crystallin and Hsp25, the mouse homolog of human HSP27, were not expressed in the P388 murine leukemic cell line (47), indicating a defect or a block in expression of these genes. Interestingly, promoter methylation of the αB-crystallin gene occurred in ATC (46) and ESCC cell lines (48), but it was not clear whether DNA-methylation silenced the expression of Hsp25 and αB-crystallin genes in P388 cells since no reactivation was observed in mRNA levels after 5-azacytidine treatment (47).

In our results, expression of the HSPB2 gene was clearly repressed at the mRNA level in most ESCC cell lines tested, but reactivated after 5-aza-dC and/or TSA treatment, indicating regulation of gene expression by promoter methylation of HSPB2 in ESCC cell lines. Real-time RT-PCR analysis revealed that the mRNA level of HSPB2 was >20 times higher in normal esophageal tissue than in ESCC. In addition, previous studies reported that protein expression of HSPB2 was frequently reduced in patients with ESCC (17,18). Taken together, these results suggest that promoter methylation may play an important role in down-regulation of HSPB2 expression in ESCC.

In conclusion, we have found that HSPB2 was methylated in human ESCC in a cancer-specific manner. HSPB2 promoter methylation thus deserves further attention as a potential diagnostic DNA marker in human ESCC. In addition, the biological and molecular consequences of HSPB2 down-regulation in ESCC carcinogenesis remain to be elucidated.

Acknowledgements

This study was supported by National Cancer Institute (U01-CA84986), Oncomethylome Sciences, and the Flight Attendant Medical Research Institute (Myoung S. Kim).

Footnotes

Conflict of interest: Under a licensing agreement between Oncomethylome Sciences and the Johns Hopkins University, D. Sidransky is entitled to a share of royalties received by the University upon sales of any products described in this article. D. Sidransky owns Oncomethylome Sciences SA stock, which is subject to certain restrictions under University policy. D. Sidransky is a paid consultant to Oncomethylome Sciences SA, and is a paid member of the company's Scientific Advisory Board. The Johns Hopkins University in accordance with its conflict of interest policies is managing the terms of this agreement.

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