DNA methylation of claudin-6 promotes breast cancer cell migration and invasion by recruiting MeCP2 and deacetylating H3Ac and H4Ac
- Yafang Liu†1, 2,
- Xiangshu Jin†1,
- Yanru Li1,
- Yang Ruan1,
- Yan Lu1,
- Minlan Yang1,
- Dongjing Lin1,
- Peiye Song1,
- Yantong Guo1,
- Shuai Zhao1,
- Bing Dong1,
- Yinping Xie1,
- Qihua Dang1 and
- Chengshi Quan1Email author
© The Author(s). 2016
Received: 17 May 2016
Accepted: 13 July 2016
Published: 26 July 2016
Claudin-6 (CLDN6), a member of claudin transmembrane protein family, has recently been reported to be undetectable or at low levels in human breast cancer cell lines and tissues and plays a role in suppression of migration and invasion in breast cancer cells. In addition, it is reported that CLDN6 expression is regulated by DNA methylation in various human cancers and cell lines. However, it is unclear how DNA methylation regulates CLDN6 expression. Here we show the mechanism by which DNA methylation regulates CLDN6 expression in human breast cancer cell line MCF-7.
RT-PCR, Western blot and immunofluorescent staining were utilized to investigate CLDN6 expression in breast cancer tissues and MCF-7 cells. Methylation-Specific PCR (MSP) was applied to determine DNA methylation status in CLDN6 gene promoter region. Wound-healing assay and invasion assay were utilized to test mobility of MCF-7 cells treated with 5-aza-dC (DNA methyltransferase inhibitor). MeCP2 binding, H3Ac and H4Ac in CLDN6 promoter region were analyzed by ChIP assay. Nuclease accessibility assay was performed for analysis of the chromatin conformation of CLDN6 gene. To study the role of CLDN6 in malignant progression, we used RNAi to knockdown CLDN6 expression in MCF-7 cells treated with 5-aza-dC, and examined the mobility of MCF-7 cells by wound-healing assay and invasion assay.
5-aza-dC and TSA (histone deacetylase inhibitor) application induced CLDN6 expression in MCF-7 cells respectively and synergistically. 5-aza-dC treatment induced CLDN6 demethylation, inhibited MeCP2 binding to CLDN6 promoter and increased H3Ac and H4Ac in the promoter. In addition, TSA increased H4Ac, not H3Ac in the promoter. The chromatin structure of CLDN6 gene became looser than the control group after treating with 5-aza-dC in MCF-7 cells. 5-aza-dC up-regulated CLDN6 expression and suppressed migration and invasion in MCF-7 cells, whereas CLDN6 silence restored tumor malignance in MCF-7 cells.
DNA methylation down-regulates CLDN6 expression through MeCP2 binding to the CLDN6 promoter, deacetylating H3 and H4, and altering chromatin structure, consequently promoting migratory and invasive phenotype in MCF-7 cells.
Claudins are structurally and functionally crucial components of tight junctions, which play important roles in regulating paracellular permeability and maintaining cell polarity in epithelial and endothelial cell sheets. Abnormal breast epithelial cell polarity causes epithelial-mesenchymal transition (EMT), and promotes tumor metastasis and invasion [1, 2]. The CLDNs family consists of 27 members, which are expressed in a tissue- and cell-specific manner [3, 4]. Loss of CLDNs expression has also been reported in several malignancies, e.g. loss of claudin 1 expression in breast cancer and in head and neck cancer.
In the present study, the expression of CLDN6 was undetectable or at low levels in human and rat mammary cancer cell lines and tissues . Besides, tissues derived from breast carcinoma with lymph node metastasis were also accompanied with decreasing expression of CLDN6.
It is reported that the CLDN6 over-expression could inhibit migratory and invasive abilities in MCF-7 cells with stable CLDN6 expression . Similarly, CLDN6 expression may suppress breast tumorigenesis . These results revealed that CLDN6 over-expression might contribute to suppression of breast cancer progression. Nevertheless, the mechanism for aberrant CLDNs expression has not been explored yet. Silence of tumor suppressor gene may relate to the epigenetic events. Cancer epigenetic events such as aberrant DNA methylation and histone modification lead to inactivation of specific tumor-suppressor genes [8, 9]. DNA methylation is an important epigenetic modification that affects chromatin structure and gene expression . Tumor suppressor gene silence is involved in carcinogenesis, drug resistance and recurrence [11–13]. High levels of methylation were associated with disease recurrence. Particularly, MLH1, ATM and FHIT gene promoters are found to be significantly hypermethylated in recurring adenomas . DNA methylation leads to transcriptional repression either by methyl groups which interfere with protein binding to its cognate DNA sequence or by recruiting proteins that specifically recognize methylated CpGs (mCpGs) , the prototype of which is methyl-CpG-binding protein 2 (MeCP2). In the enzymatic mode, MeCP2 specifically targets mCpGs and determines repressive heterochromatin by interacting with co-repressor mSin3A, which is a part of a large complex containing histone deacetylases (HDACs) [16, 17]. MeCP2 can also recruit histone methyltransferase that methylates local H3 lysine9 for strengthening the repressive state .
In our study we firstly examined CLDN6 expression and methylation status at CLDN6 gene CpG sites in breast cancer tissues and MCF-7 cells to study relationship between CLDN6 expression and DNA methylation. To explore the molecular mechanism of DNA methylation of CLDN6, we investigated the relevance among CLDN6, MeCP2, H3Ac and H4Ac in MCF-7 cells. Our investigation showed that CLDN6 expression was associated with DNA methylation in breast cancer tissues and cells. Moreover, DNA methylation of CLDN6 promoter down-regulated its expression through binding with MeCP2, deacetylating H3 and H4, and altering chromatin structure, ultimately resulted in malignant phenotype in MCF-7 cells. In addition, we demonstrated that DNA methyltransferase inhibitor 5-aza-dC and histone deacetylase inhibitor TSA treatment induced CLDN6 expression respectively and synergistically, and suppressed migration and invasion in MCF-7 cells. However, knockdown CLDN6 expression with 5-aza-dC treatment would enhance the invasive and migratory abilities.
Patients’ samples and cell culture
Paraffin-embedded tissues from 30 specimens of breast cancer including 19 specimens with lymph node metastasis, 11 specimens without lymph node metastasis and 10 specimens of breast pericarcinomatous tissues were collected from patients at the Third Hospital of Jilin University with approval by the institutional review board (IRB). None of the patients received neo-adjuvant therapy. The patients’ medical records were reviewed to obtain their age, tumor status and clinical stage. All the cancer cases were classified and graded according to the International Union Against Cancer (UICC) staging system for breast cancer.
Human breast epithelial cell line HBL-100, human breast adenocarcinoma cell line MCF-7 and ovarian cancer cell line COC1 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, USA) supplemented with 10 % fetal bovine serum (FBS) (BD, Tokyo, Japan) at 37 °C in a humidified 5 % CO2 atmosphere.
Reverse transcription—PCR (RT- PCR)
Total RNA was isolated using TRIzol reagent (Invitrogen, USA). The concentration of RNA was measured by the absorbance at 260 nm. 0.5 μg total RNA was reverse transcribed using M-MLV reverse transcriptase (TaKaRa, Japan) and random primer (TaKaRa, Japan) at 42 °C for 60 min and 1 μl of the final cDNA reaction was used for subsequent PCR reaction using Taq DNA polymerase (TaKaRa, Japan). Amplification conditions were as follows: PCR reactions were denatured at 94 °C for 2 min, followed by 28 cycles of 94 °C for 30s, 50–60 °C for 30s, and 72 °C for 1 min. This was followed by a final extension of 10 min at 72 °C. β-actin and GAPDH were used as endogenous control.
Primers for PCR assay
Sequence (5’ → 3’)
Reverse transcription PCR primers
Methylation-Specific PCR primers
Nuclease Accessibility primers
Total protein was extracted using 400 μl protein extraction fluid (HuaTe Sheng, China) with 40 μl (10 mM) phenylmethanesulfonyl fluoride (PMSF) according to the supplier’s instructions. The concentration of the total protein was determined using BCA Protein Assay Kit (Pierce Chemical Co, USA). 60 μg of denatured total protein of each sample per lane were resolved by SDS-PAGE (15 % acrylamide), and transferred to a nitrocellulose membrane (Millipore, USA) using a Semi-DRY Transfer cell (Bio-Rad Laboratories Inc, Hercules, CA, USA). The membrane was blocked with 5 % defatted milk (in 25 mM Tris, pH 8.0, 125 mM NaCl, 0.1 % Tween 20) for 1 h at room temperature then incubated with 1:1000 diluted anti-CLDN6 (Santa Cruz Biotechnologies) antibody at 4 °C overnight. After washing three times with PBS, the membrane was incubated with a horseradish peroxidase conjugated anti-goat IgG (1:1000, Santa Cruz Biotechnologies) secondary antibody at room temperature for 1 h. After washing, the immunoreactive bands were visualized using an ECL western blot system (GE, USA) and exposed to X-ray film (Estman Kodak). Anti-β-actin (Santa Cruz Biotechnologies) antibody was used as the endogenous control.
Methylation-specific PCR analysis
Genomic DNA was extracted from MCF-7 cells using Wizard Genomic DNA Purification Kit (Promega), and 1 μg DNA was denatured by 0.3 M NaOH for 10 min at 37 °C. All samples were incubated at 50 °C for 16 h after adding hydroquinone (Sigma, USA) and sodium bisulfate (Sigma, USA). Methylation-specific PCR (MSP) was performed to analyze the methylation status at CpG-rich area of CLDN6 promoter. Primers specific for unmethylated DNA and methylated DNA were summarized in Table 1. PCR was carried out for 30 cycles at 95 °C for 30s, 58 °C for 30s, followed by a final extension at 72 °C for 10 min. PCR products were electrophoresed on 1.5 % agarose gel stained with ethidium bromide.
5-Aza-2’-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) Treatment
MCF-7 cells were treated with various concentrations (0, 2.5, 5, 10, 15, and 20 μM) of DNA methyltransferase inhibitor, 5-aza-dC (Sigma, USA) for 24 h or 48 h, and various concentrations (0, 0.075, 0.125, 0.25, 0.5, and 1 μM) of histone deacetylase inhibitor, TSA (Sigma, USA) for 48 h respectively, and treated with 20 μM of 5-aza-dC with 0.125 μM TSA for 48 h.
MCF-7 cells were seeded onto small coverslips and treated with 17-β-estradiol at 0.005 μM for 24 h. After washing three times with PBS, the cells were fixed with 4 % paraformaldehyde for 30 min at room temperature. The cells were incubated with 0.3 % H2O2 and 30 μl goat serum 30 min respectively. The cells were incubated with 1:500 diluted anti-CLDN6 antibodies (Santa Cruz Biotechnologies) at 4 °C overnight and 1:50 diluted Rhodamine (red) -conjugated anti-goat IgG was used as a secondary antibody. The cells were photographed by confocal microscope (OLYMPUS, Japan).
Cancer cells were cultured in monolayer at 90 % confluence on gridded plastic dishes, and then wounded by scratching them with a 200 μl pipette tip, and then washed 3 times. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, USA) supplemented with fetal bovine serum free (BD, Tokyo, Japan) at 37 °C in a humidified 5 % CO2 atmosphere. The wounds were photographed at 0 and 24 h.
The invasive potential of cancer cells was determined using Boyden chamber. MCF-7 cells were first cultured in DMEM with 10 % fetal bovine serum to 90 % confluence, and then washed 2 times with PBS. Then the cells were cultured in FBS free medium for 24 h at 37 °C in a humidified 5 % CO2 atmosphere. The medium containing chemotactic factors from the cell culture was collected. Matrigel was added to the upper boyden chamber of 24-well and incubated at 37 °C for 30 min. Then it was followed by adding medium with chemotactic factor to 24-well, putting MCF-7 cells suspension onto the matrigel and incubating at 37 °C in a humidified 5 % CO2 atmosphere 24 h. In the end, the invasive cells were counted.
The chromatin immunoprecipitation was performed using the EZ Magna ChIP G chromatin immunoprecipitation kit (Millipore). Briefly, MCF-7 cells were treated with 20 μM 5-aza-dC. The protein-DNA complexes of cells were cross-linked using formaldehyde at room temperature for 10 min. The genomic DNA of lysed cells was sheared to 200–1000 bp by sonication. The final lysate was incubated with MeCP2-, and MBD2-specific antibodies and precipitated with protein G magnetic beads. After washing three times, DNA-protein complexes were reversely cross-linked, and genomic DNA was extracted with Wizard Genomic DNA Purification Kit (Promega, USA), and eluted in 50 μl of TE buffer. 0.5 μg DNA was used for PCR as described above. The PCR primers were summarized in Table 1. The products of PCR amplification were separated on 2.0 % agarose gel and visualized using ethidium bromide and UV light. The results were analyzed as described above.
To quantify amplified signals from the CLDN6 5’ flanking region and promoter in MCF-7 cells, quantitative PCR was undertaken by using the iCycleriQ real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and an iQ SYBR green master mix (Bio-Rad, Hercules, CA, USA). The PCR cycles were 95 °C for 2 min and then 40 cycles of 95 °C for 15 s, 60 °C for 30s. The primers were designed to amplify 100 bp per primer pair and summarized in Table 1. The specificity of amplification for each pair was confirmed by separation of amplified fragments by agarose electrophoresis and by performing melting curve analysis. PCR assays were run in triplicate.
Nuclease accessibility assay
The nuclei of MCF-7 cells were prepared as described . Nuclei (100 μg DNA) were treated with 500 units of nuclease S7 (TaKaRa, Japan) for 10 min at 37 °C to produce mononucleosomal fragments. The mixture was treated with proteinase K, and DNA was purified using phenolchloroform. The digested product was electrophoresed on 1.2 % agarose gel stained with ethidium bromide. To analyze the extent to which different parts of the CLDN6 promoter region were digested by S7 nuclease, we used real-time PCR as described above. PCR assays were run in triplicate and the relative abundance of CLDN6 promoter regions was estimated from the threshold amplification cycle number, Ct, using software supplied with iCycler IQ. The differences between Ct values obtained from MCF-7 cells treated with 20 μM 5-aza-dC and control cells were plotted against corresponding primer pairs that amplified small, overlapping sequences within the 5’flanking region of the CLDN6 gene (primer sequences used in this assay are available upon request). The primers were summarized in Table 1.
Short hairpin RNA (shRNA) transfection
Four shRNAs were designed, based on the claudin-6 mRNA sequence NM_021195 and were constructed into the vector pGCsilencer™U6/Neo/GFP (GeneChem, Shanghai, China). MCF-7 cells were transfected with shRNA-CLDN6 using SuperFect Transfection Reagent (QIAGEN, USA). A negative control cell line was generated by transfecting cells with the vector constructed by targeting a sequence that did not yield any appreciable knockdown of the protein production. shRNA sequences were as follows: i) caGTCAAGCTATGGAACTAATTTCAAGAGAATTAGTTCCATCATAGCTTGACtg; ii) aaCTGAGCCAAGGTGTTGACTTTCAAGAGAAGTCAACACCTTGGCTCAGtt; iii) gcCAGATGCAGTGCAAGGTGTTTCAAGAGAACACCTTGCAACTGCATCTGgc and iv) caGTGCAAGGTGTACGACTCATTCAAGAGATGAGTCGTACACCTTGCACtg. shRNA-mediated suppression of CLDN6 expression was confirmed using RT-PCR and Western blot as described above.
Counts and rates were calculated for categorical variables. The Chi-square test was used to compare categorical variables among different groups. All statistical analyses were performed using the SPSS software package (version 13.0.0). P values less than 0.05 were considered statistically significant.
CLDN6 expression is down-regulated in breast cancer tissues and MCF-7 cells, correlating with DNA methylation
Clinical pathological characteristic of breast cancer patients associating with CLDN6 expression
Breast cancer tissues
7 (23.33 %)
23 (76.67 %)
10 (100 %)
0 (0 %)
P < 0.05
2 (22.22 %)
7 (77.78 %)
3 (21.43 %)
11 (78.57 %)
2 (28.57 %)
5 (71.43 %)
P > 0.05
2 (15.38 %)
11 (84.62 %)
4 (33.33 %)
8 (66.67 %)
1 (20 %)
4 (80 %)
P > 0.05
4 (28.57 %)
10 (71.43 %)
3 (18.75 %)
13 (81.25 %)
P > 0.05
2 (10.53 %)
17 (89.47 %)
5 (45.45 %)
6 (54.55 %)
P < 0.05
The comparison of mRNA and protein level of CLDN6 expression among breast cancer MCF-7 cells, HBL-100 cells (human normal breast cells) and COC1cells (human cervical cancer cells as tissue-specific control) showed that CLDN6 expression was decreased in MCF-7 as compared with HBL-100 and COC1, indicating that reduction of CLDN6 expression had tissue- and cell-specificity, which was consistent with clinical assays (Fig. 1b).
DNA methylation of CLDN6 in breast pericarcinomatous tissues and cancer tissues
Type of tissues
DNA methylation associating with CLDN6 expression in breast pericarcinomatous tissues and cancer tissues
P < 0.05
5-aza-dC application induces CLDN6 expression and inhibits migratory and invasive abilities in MCF-7 cells
TSA application induces CLDN6 expression in MCF-7 cells
Methylated DNA recruits MeCP2, and deacetylates H3Ac and H4Ac at CLDN6 promoter
CLDN6 promoter is more accessible after micrococcal nuclease digestion in MCF-7 cells with 5-aza-dC treatment
DNA demethylation restores CLDN6 expression, inhibiting invasion and migration in MCF-7 cells
All together, we demonstrated that DNA methylation down-regulated CLDN6 expression via recruiting MeCP2 and deacetylating H3Ac and H4Ac, indicating loss of CLDN6 expression could further destroy integrity of cells by losing cell polarity and cell adhesion, and promote malignant progression. It is interesting that DNA methyltransferase inhibitor 5-aza-dC and histone deacetylase inhibitor TSA could induce CLDN6 re-expression partially and 5-aza-dC could inhibit invasion and migration of MCF-7 cells. Overall, these data suggest that CLDN6 plays a critical role in suppressing malignant progression in MCF-7 cells.
In our previous work, we have identified CLDN6 as a potential mammary cancer suppressor gene, which may contribute to mammary cancer resistant phenotype observed in human and rat mammary cancer cell lines [5, 6]. However, its regulation mechanisms are elusive. Therefore, investigation of the mechanism of regulating CLDN6 expression may help in understanding of breast tumorigenesis and provide new opportunities for detection and therapy of this disease. Promoter hypermethylation, with MeCPs binding and histone deacetylation, has been identified as a major epigenetic event associated with the loss of tumor suppressor gene expression during cancer progression [23–25]. DNA methylation-dependent repression of transcription can be mediated by several mechanisms. At first, DNA methylation at binding sites of transcription factors can repress the transcription factor. A methylated target sequence can interfere transcription factors such as Sp1/Sp3, E2F and CREB to bind to their binding sites and to prevent transcriptional activation [17, 26]. A more elaborate mechanism is that methyl-CpG binding proteins such as MeCP2 and MBD2, a member of the MeCP1 complex , are recruited to the methyl-CpG sequence and then these methyl-CpG binding proteins are associated with various chromatin modifiers to establish a repressive chromatin environment [18, 27–29].
In this report, we examined CLDN6 expression in HBL-100, MCF-7 and COC1 cells by RT-PCR and western blot assay (Fig. 1b) and the methylation status at the CLDN6 gene CpG sites and found that MCF-7 cells did not express CLDN6 with methylation at the CLDN6 CpG sites. HBL-100 and COC1 cells with CLDN6 expression exhibited unmethylation at CLDN6 CpG sites. To determine the contribution of DNA methylation and histone modification in CLDN6 expression, we treated MCF-7 cells with 5-aza-dC and TSA. The CLDN6 expression was up-regulated by treating with 5-aza-dC and TSA respectively and synergistically, indicating that DNA methylation and histone deacetylation play an essential role in CLDN6 silence.
Our data indicate that the methylation of CpG dinucleotides in the CLDN6 promoter may not directly interfere with the binding ability of transcription factor, but methyl-CpG binding protein may interfere with it. However, the role of methyl-CpG-binding proteins in the decrease of transcription factor binding in the methylated CLDN6 promoter is not clear. Nevertheless, our ChIP assay suggested that MeCP2 did interact with CLDN6 proximal promoter in MCF-7 cells in vitro, but not in 5-aza-dC treatment group. These results demonstrated that methylated CLDN6 promoter interfered transcription of CLDN6, which was likely intermediated by MeCP2 protein.
Methyl-CpG binding proteins (MBPs) bind to regions of methylated DNA and form complexes with HDACs, then resulting in histone deacetylation downstream of the methylated CpG sequence and transcriptional silencing of that region of the genome . Similarly, proteins that either bind modified histones such as heterochromatin protein 1 (HP1) or directly modify histones such as lysine specific demethylase 1 (LSD1) and protein arginine methyltransferase 5 (PRMT5), can recruit DNA methyltransferases to induce DNA methylation in the silenced gene [31–33]. Furthermore, our data showed TSA could up-regulate CLDN6 expression, indicating that not only DNA methylation, but also histone deacetylation play an essential role in CLDN6 silencing in MCF-7 cells. We determined H3Ac and H4Ac in CLDN6 promoter in MCF-7 cells with 20 μM 5-aza-dC and 125nM TSA treatment for 48 h respectively and simultaneously. H4Ac in CLDN6 promoter was induced by using 5-aza-dC and TSA respectively and simultaneously, but H3Ac in CLDN6 was induced only with 5-aza-dC treatment, suggesting that deacetylation of H3Ac might depend on DNA methylation to repress CLDN6 expression, but H4Ac might not rely on DNA methylation, and might independently regulate CLDN6 expression. Histone modification may repress transcriptional complexes to combine with gene promoter by changing the chromatin structure in nucleosomes and in turn making the complexes inaccessible to the transcriptional machinery. Our nucleosomal mapping experiment indicated that indeed the DNA methylation of CLDN6 promoter in MCF-7 cells existed in a more organized nucleosomal form that is more resistant to endonuclease digestion. Collectively, our results indicated that 5-aza-dC and TSA treatment in MCF-7 cells could increase the expression of CLDN6, and suggested that the deacetylase inhibitor TSA may partly relax demethylated chromatin and activate transcription.
Additional studies are needed to determine the exact molecular mechanism of DNA methylation, and whether there is a link between DNA methylation and histone deacetylation. Our study showed MeCP2 was a key protein that bound to methylated DNA regions and recruited HDACs, and formed complexes that leaded to histone deacetylation and silenced the transcriptional activity of that region of the genome [17, 28, 27]. Our ChIP assay experiments indicated that MeCP2 did interact with the CLDN6 promoter in MCF-7 cells in vitro, we presumed that methylated DNA might recruit MeCP2, which can recruit HDACs to repress CLDN6 expression. It is possible to have other potential recruited complexes for methylation.
CLDN6 expression is related to DNA methylation in breast cancer tissues and MCF-7 cells. DNA methylation of CLDN6 gene down-regulates its expression by recruiting MeCP2, deacetylating H3 and H4, and altering chromatin structure. Down-regulation of CLDN6 expression could facilitate migratory and invasive phenotype in breast cancer cells.
CLDN6, Claudin-6; TJ, Tight junction; 5-aza-dC, 5-Aza-2’-deoxycytidine; TSA, Trichostatin A; MSP, Methylation-Specific PCR; EMT, Epithelial-mesenchymal transition; MeCP2, methyl-CpG-binding protein 2; JAM, Junctional adhesion molecules; mCpGs, methylated CpGs; HDAC, histone deacetylase; H3Ac, Histone 3 acetylation; H4Ac, Histone 4 acetylation; MBP, Methyl-CpG binding protein; HP1, Heterochromatin protein 1; LSD1, Lysine specific demethylase 1; PRMT5, Protein arginine methyltransferase 5.
This study was supported by grant from the Key laboratory of Pathobiology, Ministry of Education School of Basic Medical Sciences, Jilin University, Changchun, People’s Republic of China.
YL and XJ were involved in the study conception and design. YL, YR and YL were participated in data acquisition, analysis and interpretation. DL, PS, YG, SZ, BD, YX and QD performed Immunohistochemistry, PCR and Western blot. YL and XJ wrote the manuscript. All authors have read and approved the manuscript for publication.
The authors declare that they have no competing interests.
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