Background
Aberrant DNA methylation has been recognized as one of the most common molecular alterations in human neoplasia. Hypermethylation of gene-promoter regions is being revealed as one of the most frequent event that causes loss of gene function. DNA methylation usually occurs at a cytosine associated with CpG sites [
1]. DNA (cytosine-5)-methyltransferase (DNA-MTase) catalyzes this reaction by adding a methyl group from S-adenosyl-L-methionine to the fifth carbon position of the cytosine [
1]. Methylation of CpG sites in the promoter region of the genes is known to transcriptionally repress these genes [
2]. CpG sites of a large number of genes that are unmethylated in normal tissue are methylated in human cancers, such as breast, ovarian, colon, and prostate cancers [
3,
4]. Methylation at the promoter region of specific genes depends on tumor type. For example, the mismatch repair gene hMLH1 is silenced by hypermethylation more frequently in colorectal, endometrial, and gastric tumors; while the BRCA1 is methylated in breast and ovarian tumors [
5‐
8]. Recent studies have suggested that CpG methylation of certain genes may be associated with HER2 receptor overexpression and/or hormone status in breast cancer [
8,
9]. It is unclear as to which breast cancer specific genes are transcriptionally silenced and if their silencing is associated with failure in treatment and decrease in disease-free survival (DFS).
CASP8 is an important initiator of apoptosis [
10]. Absent or downregulation of CASP8 could cause resistance to apoptosis and is correlated with unfavorable disease outcome, such as in childhood medulloblastoma and neuroblastoma [
11,
12]. The absence or downregulation of CASP8 may be due to epigenetic changes. Studies have also indicated that methylation and demethylation of maspin promoter may regulate maspin gene expression and that reduced maspin expression is associated with cancer progression [
13].
In the current study we used methylation specific PCR (MSP), and bisulfite sequencing to determine the methylation status of these two genes. We examined the mechanisms associated with transcriptional silencing of CASP8 and maspin by promoter methylation using real-time PCR and by restoring the methylated genes back to their unmethylated status using the demethylating agent, 5-aza-2'-deoxycytidine; TSA (Trichostatin A), inhibitor of histone deacetylase; and chemotherapeutic agent 5-Fu (5-Fluorouracil).
Methods
Cells and culture
The breast cancer cells with varying tissue subtypes selected for our methylation studies were: MCF-7 (ER positive and HER2/neu negative); MDA-MB231 (ER negative and HER2/neu negative); SKBR3 (ER negative and HER2/neu positive); HCC1937 (ER negative, HER2/neu negative and BRCA1 mutated); non-tumorigenic breast epithelial cells (MCF12A), and non-tumorigenic breast fibroblast cells (MCF10). These cell lines were purchased from American Type Culture Collection (Rockville, MD), and unless otherwise stated, the cells were grown and maintained in DMEM/F12 (Fisher Scientific, CA) containing 10% FCS (Invetrogen), 2 mM glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin (Fisher Scientific, CA).
5-aza-2'-deoxycytidine (5-aza-dc) and Trichostatin A (TSA) treatment
The cells were growing in culture medium until 80% confluence; 5 μM 5-aza-dc was added and incubated for 3 to 6 days. The medium containing 5-aza-dc was refreshed every 2 days. For combination treatment cells were first treated with 0.3 μM TSA in combination with 5 μM 5-aza-dc for 2 days and then TSA was removed from culture medium. Treatment with 5 μM 5-aza-dc was continued for 1 more day.
Bisulfite modification and Methylation Specific PCR (MSP)
Bisulfite conversion of genomic DNA was carried out using Zymo EZ DNA Methylation-Gold™ kit (D5005, Zymo Research Corp, Orange, CA) according to the manufacture's instructions. This process converts unmethylated cytosine residues to uracil, while methylated cytosine residues remain unchanged. Bisulfite modified DNA was used as a template, and then MSP was performed to determine the methylation status of CASP8 and maspin. The primer sequences for MSP are as follows:
(a) CASP8, 5'-TGTTGTTTGGGTAACGTATCGA-3' (methylated forward),
5'-CCCTACTTAACTTAACCCTACTCGAC-3' (methylated reverse), and
5'-TTGTTGTTTGGGTAATGTATTGA-3' (unmethylated forward),
5'-CAACCCTACTTAACTTAACCCTACTCA-3' (unmethylated reverse);
(b) maspin, 5'-ATTTTATCGAATATTTTATTTTTCGG-3' (methylated forward),
5'-TAACTCACCTAAACAACACCGCC-3' (methylated reverse), and
5'-TTTTATTTTATTGAATATTTTATTTTTTGG-3' (unmethylated forward),
5'-TAACTCACCTAAACAACACCACC-3' (unmethylated reverse).
The primers for MSP were designed using MethPrimer [
14] according to the sequences provided by Panomics (Fremont, CA). The cover regions were shown in Additional file
1, Figure S1. PCR conditions were as follows: an initial denaturation at 95°C for 5 min was followed by 40 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 7 min.
Bisulfite sequencing for CASP8 promoter
Bisulfite modified DNA (described above) was amplified by two primer sets. The primer sequences were as follows: P1-CASP8 (nt -744 to nt -466), 5'-GGTGGTGGGTGTTTGTAGTTTTAGT-3' (forward) and 5'-CCATCCTTAACCATATTCTCCAATTTA-3' (reverse); P2-CASP8 (nt -486 to nt +51), 5'-TAGATTTTTTGTAAGAAAGAATGGTAT-3' (forward) and 5'-ACAAAAAAACAAAATCTAATCTCC-3' (reverse). PCR conditions were as follows: an initial denaturation at 95°C for 5 min was followed by 40 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 7 min. The PCR products were purified with QIAquick PCR purification kit (#28104, QIAGEN), and then sent to Retrogen Inc (San Diego, CA) for sequencing. The primers for bisulfite sequencing were designed using MethPrimer [
14]. The sequence was used to design the bisulfate sequence primers were as same as the sequence used to design primers for MSP. The specific cover regions for each primer site have been demonstrated in Additional file
1.
Quantitative real-time PCR (Q-PCR)
Total RNA from cultured cells was isolated by using RNeasy micro kit (#74004, QIAGEN) according to the manufacture's instructions, and cDNA was synthesized by reverse transcription (RT) with ThermoScript™ RT-PCR system (Invitrogen) according to the manufacture's instructions. Q-PCR analysis was performed with iCycle iQ real-time PCR detection system (Bio-Rad Lab, Hercules, CA) using SYBR Green Master Mix (#204143, QIAGEN). The primers used were as follows: (a) CASP8, 5'-CCAGAGACTCCAGGAAAAGAGA-3' (forward) and 5'-GATAGAGCATGACCCTGTAGGC-3' (reverse). (b) maspin, 5'-AGATGGCCACTTTGAGAACATT-3' (forward) and 5'-GGTTTGGTGTCTGTCTTGTTGA-3' (reverse). (c) housekeeping control gene, 18 s, 5'-GATCCATTGGAGGGCAAGTC-3' (forward) and 5'-TCCCAAGATCCAACTACGAG-3' (reverse). Reactions were characterized during cycling when amplification of the PCR product was first detected (C
t
). The target message and the housekeeping gene, 18 s, in breast cancer and non-tumorigenic cell lines, were quantified by measuring C
t
. The relative level of target messages in cells was normalized on the basis of its 18 s content by taking the difference of threshold cycles between target gene and 18 s. Using non-tumorigenic breast cancer cells or untreated breast cancer cells as reference, the level of CASP8 and maspin in each sample was normalized with the corresponding reference by taking the difference between threshold cycles. Final results were expressed as N-fold difference in target gene expression relative to the reference.
Immunobloting and Immunofluorescence (IF)
For immunoblot analysis cells were lysed in 1 × lysis buffer (20 mM Tris pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM β-glycerolphosphate; 1 mM Na3VO4; 1 μg/ml leupeptin; 0.1 mM PMSF) and protein concentration was measured using BCA dye (Pierce). Total protein (50 μg) from cell lysates was resolved on SDS-PAGE followed by transfer to nitrocellulose membrane. The membranes were incubated with antibodies specific against Dnmt1 (IMG-261A, IMGENEX), Dnmt2 (IMG-281, IMGENEX), Dnmt3a (IMG-268A, IMGENEX), Dnmt3b (IMG-184A, IMGENEX), and caspase-8 (#9746, Cell Signaling) according to manufacturer's instruction. Detection of antigen-bound antibody was performed with the enhance chemiluminescence reagent. For immunofluorescence analysis cells (2 × 104/each) were mounted by cytospin on polylysine coated glass slides and fixed with 4% paraformaldehyde for 15 min followed by 100% ice-cold acetone for 10 min at 4°C. To detect caspase-8 protein expression immunofluorescence analysis was performed with cleaved caspase-8 antibody (#9496, Cell Signaling) followed by incubation with anti-goat IgG-FITC (Santa Cruz) for 30 min in dark and mounted with DAPI mounting medium (Vector Labs). The cells with positive staining were counted in five different areas and adjusted with total number of cells. To analyze histone H3 methylation and acetylation status in MCF-7 cells double immunofluorescence analysis was performed with antibodies against Di-Methyl Histone H3 (Lys27) (#9755, Cell Signaling) or Acetyl Histone H3 (Lys9) (#9671, Cell Signaling) followed by β-actin antibody (A1975, Sigma-Aldrich). FITC-conjugated secondary antibodies were used to label Di-Methyl Histone H3 (Lys27) and Acetyl Histone H3 (Lys9). The β-actin was labeled with Tex-red-conjugated secondary antibody. After mounting, the cells were viewed under a fluorescence microscope.
CHIP assay
Cells were fixed with formaldehyde (1% final concentration) to cross-link protein to DNA at room temperature for 10 min after treated with or without 5-aza-dc (5 μM, 3 days), and then incubated with glycine (0.125 M final concentration) for 5 min to stop the cross-linking. Soluble chromatin was prepared as described previously [
15]. The chromatin was then diluted 1:10 with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl and a protease inhibitor cocktail set (CalBiochem)), and subjected to immunoprecipitation with anti-dimethylated histone H3 lysine 9 (H3K9me2) antibody (#9753, Cell signaling). Pre-immune serum (Santa Cruz) was also used as a control. The chromatin and antibodies were incubated overnight at 4°C. Chromatin/antibody complexes were recovered by adding 30 μl of protein A/G Plus-agarose beads (Santa Cruz) and incubating at 4°C for 2 h. The beads were sequentially washed for 10 min each in 1 ml of low salt, high salt and LiCl immune complex wash buffer. Immunocomplexes were eluted off the beads by incubation with 300 μl of 1% SDS and 50 mM NaHCO
3. The eluent was incubated at 65°C for 5 h or overnight to reverse the formaldehyde-induced protein-DNA cross-links. The DNA was extracted with phenol and chloroform. Extracted DNA was resuspended in 100 μl of TE buffer and real time PCR was performed by using Bio-Rad thermal cycler with CASP8 primers: forward 5'GGT GCC TGT AGT CCC AGC TAC TC3' and reverse 5'CCT AGA CCC TCC CCT GTT CTG TC3'. For input DNA, the chromatin preparation without incubated with antibodies was subjected to real time PCR.
Discussion
Hypermethylation of the promoter regions of various genes has been recognized as one of the most frequent mechanisms causing loss of gene function. The association between aberrant DNA methylation and carcinogenesis has been demonstrated in several studies [
20‐
26]. However, the association between epigenetic changes with cancer etiology needs to be elucidated.
Several cancer-related genes have been reported to be silenced by aberrant methylation in breast cancer, such as 14-3-3 s, E-cadherin and tissue inhibitor of metalloproteinase 3 (TIMP3) genes. Treatment with 5-aza-dc activated the expression of 14-3-3 s [
27] and E-cadherin genes [
28] in breast carcinoma cells and of TIMP3 in different tumor cell lines [
29,
30].
Hypermethylation of CASP8 has been showed as a frequent feature of relapsed glioblastoma compared with the corresponding primary tumors [
31]. The authors suggested that epigenetic deregulation of the mitochondria-independent apoptosis is a relevant characteristic in recurrent glioblastoma. The development of targeted therapies restoring functional extrinsic apoptosis, as recently shown
in vivo with the synergistic combination of the DNA demethylating agent decitabine and TRAIL [
32], may provide a useful tool to overcome the resistance of glioblastoma to contemporary treatment modalities. Methylation of CASP8 gene has also been reported in some childhood tumors and in neuroendocrine lung tumors [
33]. CASP8 is an important initiator of apoptosis [
10]. Structurally, the promoter region of CASP8 has binding sites for p53, nuclear factor-κB (NF-κB), AP-1, SP-1, IRF-1, and Ets-like transcription factors [
34]. Therefore, CASP8 functions both as a pivotal molecule for death-receptor-induced apoptosis and as a selective signal transducer, such as for NF-κB activation [
35]. Absent or downregulation of CASP8 could cause resistance to apoptosis and is correlated with unfavorable disease outcome, such as in childhood medulloblastoma and neuroblastoma [
11,
12]. Others have also demonstrated that absence or downregulation of CASP8 may be due to epigenetic changes, such as hypermethylation, or mutations [
36,
37].
In current study we have investigated the promoter methylation of CASP8 and maspin in relation to their expression levels as well as the involvement of histone methyltransferases and histone H3K9me2. Using MSP and bisulfite sequence analysis, we have established the relationship between aberrant cytosine methylation and downregulated or loss of CASP8 in breast cancer cells. We confirmed that CpG sites methylation in the promoter region of CASP8 is the mechanistic basis for transcriptional downregulation or silencing of CASP8 in breast cancer cells. The methylation status of CASP8 can be completely or partially reversed by treatment with 5-aza-dc in MB231, SKBR3, and BT474, but not in MCF-7 breast cancer cells. The cells that had fewer methylated CpG sites, such as MB231 and SKBR3 were totally demethylated by 5-aza-dc. This change in demethylation resulted in a significant increase in CASP8 mRNA and protein expression. In contract SKBR3 cells, most CpG sites of CASP8 were methylated in MCF-7 cells. Results from MSP showed that methylation was partially reversed by 5-aza-dc in MCF-7 cells and the mRNA and protein level of CASP8 had no significant increase. We also examined the effect of histone acetylation of CASP8 by treating MCF-7 cells with Trichostatin A (TSA). The TSA treatment alone did not change the methylation status and mRNA expression of CASP8 in MCF-7 cells (data not shown). However, TSA in combination with 5-aza-dc was able to partially demethylate CASP8 promoter and increased CASP8 mRNA and protein expression in MCF-7 cells. The data suggested the involvement of histone acetylation in the regulation of CASP8 gene expression in MCF-7 cells. CHIP analysis (Figure
4B) showed that 5-aza-dc treatment inhibits H3K9me2 occupancy on CAPS8 promoter in SKBR3 and MDA-MB231, but not in MCF-7 cells. These results suggest that CASP8 gene transcription is restored in theses SKBR3 and MB231, but not in MCF-7 cells.
Several studies have demonstrated a link between methylation and histone acetylation in which a family of methyl-CpG-binding proteins is involved [
38]. When these proteins bind to a methylated promoter, they recruit HDAC, and the interaction of these two epigenetic events inhibits gene expression by interfering with the function of transcription factors and the compaction of the chromatin structure [
39‐
41]. Inhibitors of these epigenetic changes can lead to the reactivation of genes that suppress tumorigenesis. In accord with this hypothesis is the report on the synergistic interaction of 5-aza-dc and the HDAC inhibitor, trichostatin A (TSA), in the reactivation of tumor suppressor genes [
42]. This same drug combination was also reported to induce a synergistic reactivation of the estrogen receptor-α in breast carcinoma cells [
43].
A recent study in prostate cancer cells indicated that CASP8 plays a pathway specific role in inhibiting androgen receptor signaling [
44]. This evidence suggests that CASP8 may have the role beyond its role as a cell death protease and may play a role in hormonal receptor cell signaling.
The anticancer drug, fluorouracil (5-Fu), has been commonly used in clinical practice for first line treatment of breast cancer for decades. Up to now, except for interferon-γ and azacytidine, the cytotoxic drugs, 5-Fu and methotrexate, have been shown to upregulate CASP8 and induce cell apoptosis in neuroblastoma, medulloblastoma, Ewing sarcoma, glioblastoma, leukemia, and breast cancer cells [
18,
19]. The mechanism by which 5-Fu regulates CASP8 protein is more likely to involve p53 [
19]. However, studies from Zoli, et al. [
18] suggest that 5-Fu in combination with doxorubicin and paclitaxel regulates CASP8 and induces cell apoptosis by a caspase-dependent mechanism independent of hormonal, p53, bcl-2 or bax status in breast cancer cells [
18]. These observations made us to suspect that 5-Fu induced cell apoptosis may involve a demethylation process. We then tested the expression CASP8 mRNA, as well as its methylation status, in MCF-7, MB231 and SKBR3 cells treated with 5-Fu for 3 days. Compared to untreated cells, the cells treated with 5-Fu showed a significant increase mRNA expression of CASP8 followed by demethylation of its promoter region.
Taken together, our results confirm that expression of CASP8 in breast cancer is epigenetically controlled and the modification may vary in different types of breast cancer cells.
Another gene promoter methylation has been studied was maspin in current study. Maspin belongs to the serpin family [
45]. It acts as a tumor suppressor, increases cell adhesion, induces apoptosis, and inhibits tumor growth and metastasis [
46‐
48]. Maspin is also involved in angiogenesis and mammary gland development [
49]. The expression of maspin is epigenetically controlled by methylation and/or histone acetylation. Studies have also indicated that methylation and demethylation of maspin promoter may regulate maspin gene expression and that reduced maspin expression is associated with cancer progression [
13]. In our study, we have confirmed that the expression of maspin in breast cancer cells is epigenetically controlled by methylation of the CpG sites. The demethylation agent, 5-aza-dc, treatment reversed maspin promoter methylation and increased maspin gene expression in MCF-7, MB231, and SKBR3 cells. An early study from Domann FE et al. [
50] also reported that normal human mammary epithelial cells expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter. In contrast, most breast cancer cell lines had no detectable maspin expression and those maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. In this study we have also examined maspin and CASP8 mRNA levels in 30 breast cancer tissues and 10 non-breast cancer tissues. The mRNA levels of CASP8 and maspin were lower in breast cancer tissue than non-breast cancer tissue (data not shown). The decreased mRNA expressing levels of maspin and CASP8 in patients were associated with positive lymph node status, late stage disease, and HER2 overexpression (3+ and/or FISH positive). The DFS was significant decreased in patients with low CASP8 or maspin expression (data not shown). Inactivation of apoptotic pathways is often critical for the pathogenesis of tumor cells and for their resistance to chemotherapeutic drug treatment and/or irradiation [
51‐
53]. It needs to be determined if the loss of CASP8 and/or maspin expression can be a prognostic marker for Breast Cancer and if it is associated with amplification of MYCN (v-myc myelocytomatosis viral related oncogene), as frequently observed in neuroblastomas.
Limitations of this study are that the most investigation was used cell lines only. To better understand the clinical relevance of CASP8 and/or maspin promoter methylation in breast tumors and if the decreased mRNA expression of CASP8 and/or maspin were correlated to the aberrant pattern of cytosine methylation of the gene promoter further studies with large sample size should be conducted.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YW, MA and JVV were responsible for data collection, analysis, manuscript preparation, and editing. HPK and DS critical review and were involved in study design. All authors read and approved the final manuscript.