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Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells
Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 91–97
Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells Ming Zhang a , Qing Wang a , Wei Yuan a , Shuang Yang a , Xu Wang a , Ji-Dong Yan a , Jun Du a , Jian Yin b , Song-Yuan Gao b , Bao-Cun Sun b , Tian-Hui Zhu a,∗ a
Laboratory of Molecular Medicines, Medical College, Nankai University, Tianjin 300071, China b Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China Received 9 October 2006; accepted 25 January 2007
Abstract Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER+ ) breast cancer cell line MCF-7, but not in ER negative (ER− ) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5 flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER− cell line MDA-MB-231 was relatively lower than that in ER+ MCF-7 and T47D cell lines. After the treatment with 5-aza-2 -deoxycytidine (5-aza-dC, especially in the concentration of 10 M), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer. © 2007 Published by Elsevier Ltd. Keywords: Epigenetic regulation; 5-aza-2 -Deoxycytidine (5-aza-dC); Methylation-sensitive enzyme restriction PCR (MSRE-PCR); Bisulfite sequencing (BSG); Methylation-specific PCR (MSP); Estrogen receptor (ER)
1. Introduction Bone morphogenetic protein-6 (BMP-6), which belongs to the transforming growth factor- (TGF-) superfamily, is a multifunctional molecule that regulates bone growth and organ development. In addition to BMP-6’s pivotal role in endochondral bone formation, more and more researches have shown that the expression of BMP-6 is closely correlated with tumor differentiation and metastasis. Recent researches found that BMP-6 is overexpressed in both breast cancer cells and tumor samples from breast carcinoma patients ∗
Corresponding author. Fax: +86 22 23505501. E-mail address: [email protected] (T.-H. Zhu).
0960-0760/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.jsbmb.2007.01.002
[1,2]. BMP-6 overexpression has been detected in advancedstage prostate cancer with skeletal metastasis by in situ hybridization (ISH) and immunohistochemistry [3], suggesting a possible association of BMP-6 expression with skeletal metastasis. Our recent studies demonstrated that BMP-6 can be activated dose-dependently by estrogen in human estrogen receptor positive (ER+ ) breast cancer cell line MCF-7 through estrogen interacting with a 1/2 ERE element and 3 Sp1 sites on BMP-6 promoter [4]. Considering previous observations that patients with ER+ breast tumors are more likely to develop skeletal metastasis, we hypothesized that BMP-6 plays an important role in the process of estrogen-induced breast cancer skeleton metastasis [5,6].
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On the other hand, alterations of DNA methylation status have been observed in many types of tumors including breast [7], prostate [8], lung [9], and pancreatic adenocarcinoma [10]. Genes in abnormal methylation status, activated by demethylation or silenced by hypermethylation, are important for development [11], progression [12], and metastasis of tumors [13]. Resent research found that BMP6 is up-regulated in advanced prostate cancers with metastasis through demethylation of the CpG loci in the promoter region [14], suggesting that epigenetic regulation mechanism plays an important role in activating the BMP-6 expression during cancer progression and giving cancer cells a changeable trait. In this study, we report that BMP-6 promoter methylation status is closely correlated with ER status in breast cancer. Through computational analysis by MethPrimer (http://www.urogene.org/methprimer/index1.html), CpG islands in human BMP-6 gene promoter region have been identified. In the present study, we first examined the relationship between the methylation status of the BMP-6 gene promoter and its steady-state expression in three breast cancer cell lines MDA-MB-231 (ER− ), MCF-7 and T47D (ER+ ) by real-time PCR and 5-aza-2 -deoxycytidine (5-azadC) treatment studies. Using methylation-sensitive enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), we further analyzed the different BMP-6 methylation status in these three breast cancer cell lines. BMP-6 promoter methyaltion status were also detected in 33 breast tumor specimens (17 ER positive; 16 ER negative) through methylation-specific PCR (MSP). This study contributes to the understanding of the role that BMP-6 plays in breast carcinogenesis and skeleton metastasis.
2. Materials and methods 2.1. Tumor samples Fresh breast tissues were obtained from the Tianjin Medical University Cancer Institute and Hospital. The patients (17 ER positive and 16 ER negative) had a mean age of 52.8 ± 12.1 years and were recruited in the same department. 2.2. Cell culture and treatment with 5-aza-2 -deoxycytidine Human breast cancer cell lines MCF-7, MDA-MB-231, T47D and normal human epithelial cell line 293T were maintained in RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone, Logan, Utah, USA), and 100 U/ml Penicillin and Streptomycin (growth medium) at 37 ◦ C in a humidified atmosphere with 5% CO2 . The breast cancer cell lines MCF-7, MDA-MB-231, T47D were seeded at 105 cells in six-well plates, allowed to attach for 24 h and treated with 0, 1, 10 or 30 M of the demethylating agent, 5-aza-2 -deoxycytidine (5-aza-dC,
Sigma Chemical Co., St. Louis, MO) in serum-containing medium for 5 days. Medium and 5-aza-dC were changed daily. 2.3. RNA isolation and real-time PCR Total RNA, extracted from three breast cancer cell lines and 293T cell line, was isolated using TRIzol Reagent (Life Technologies Inc., Grand Island, NY, USA) according to the manufacturer’s protocol. After reverse transcription reaction (20 l) using 2 g of total RNA, real-time PCR was carried out in a 25 l final volume by an ABI PRISM 7000 (ABI, USA) sequence detection system according to the manufacturer’s protocol. The reaction mixture contained 1 × SYBR Green I, 0.5 pmol/L of each primer, 2.5 mM MgCl2 , and 0.5 l cDNA from (20 l of) reverse transcription reaction. The conditions of real-time PCR were as follows: 94 ◦ C 4 min followed by 40 cycles at 94 ◦ C for 30 s, 68 ◦ C for 1.5 min. There is no non-specific amplification determined by dissolve curve. There are four repeat tubes per cDNA specimen, three cDNA specimens independently for each data point. The real-time PCR results were reported as the fold of relative light units for breast cancer cell lines (MCF-7, MDA-MB-231 and T47D) compared with those for 293T after normalization to GAPDH expression. Error bars represent the standard errors for three independent experiments, with each data point done in quadruple. The primers used for real-time PCR are as follows: • BMP-6: ◦ Upstream primer: 5 -CAACAGAGTCGTAATCGCTCTACC-3 (+1323 to +1346). ◦ Downstream primer: 5 -TTAGTGGCATCCACAAGCTCT-3 (+1701 to +1721) PCR product is 308bp. • GAPDH: ◦ Upstream primer: 5 -ACCACAGTCCATGCCATCAC3 (+526 to +545). ◦ Downstream primer: 5 -TCCACCACCCTGTTGCTGTA-3 (+958 to +977) PCR product is 451bp. 2.4. Expression plasmid construction, transient transfection study and luciferase assay A human 1.2 kb (−1119/+37) BMP-6 promoter-luciferase plasmid (Promega, Madison, WI, USA) was constructed as previously described [4]. The plasmid construct was transfected into the three human breast cancer cell lines by LipofectAMINE reagents (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Twenty four hours after transfection, luciferase activity was detected using a Kit from Promega Corp. (Madison, WI, USA) with a luminometer. The efficiency of transfection was monitored by cotransfection with phRL-null vector (0.5 g/well). The luciferase results were reported as relative light units of firefly luciferase activity normalized with respect to the Renilla luciferase activity. Error bars represent the standard errors for
M. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 91–97
three independent experiments, with each data point done in triplicate. 2.5. Genomic DNA extraction and methylation-sensitive enzyme restriction PCR (MSRE-PCR) Genomic DNA was purified from the three breast cancer cell lines and tumor samples according to the method mentioned by Melnikov et al. [15]. Digestions were performed with methylation sensitive restriction enzyme HpaII or methylation insensitive restriction enzyme MspI (recognition site CC|GG; TaKaRa, Dalian, China). Typically, 1 g of genomic DNA were digested with 40 U of the enzyme at 37 ◦ C for 72 h under a layer of mineral oil; the final volume of the reaction was 50 l. Control samples were treated in the same way without the enzyme. After incubation, digested samples were purified using phenol and chlorformisoamyl alcohol and eluted in 50 l of ddH2 O. Control samples were ethanol precipitated and dissolved in 50 l of ddH2 O. Genomic fragments containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. ExTaq (TaKaRa, Dalian, China) was used 0.2 U per reaction. The conditions of MSRE-PCR were as follows: 94 ◦ C 3 min followed by 25 cycles at 94 ◦ C for 30 s, 57 ◦ C for 30 s, 72 ◦ C for 30 s. Primers used in this study are as follows: • Upstream primer: 5 -AGAGAAAGCTTCAGATCGGG-3 (−959 to −940). • Downstream primer: 5 -CGGCGCCGGAATTCGGTC-3 (−628 to −611) PCR product is 348 bp.
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• Upstream primer: 5 -CGTTTTTATTGGTTGTTAGTTTGTC-3 (−762 to −737). • Downstream primer: 5 -CAACGTAAACTTTCTACTACGCG-3 (−685 to −663) PCR product is 99 bp. The sequences of methylation-sensitive primers were: • Upstream primer: 5 -TGTTTTTATTGGTTGTTAGTTTGTTG-3 (−762 to −737). • Downstream primer: 5 -CCAACATAAACTTTCTACTACACACC-3 (−687 to −662) PCR product is 100 bp. The PCR was performed under the following conditions: 94 ◦ C 5 min, followed by 30 cycles at 94 ◦ C for 30 s, 66 ◦ C for 30 s, 72 ◦ C for 30 s. To ensure completion of bisulfite chemical modification, universal methylated DNA and genomic DNA from normal placenta tissue were include in each sodium bisulfite treatment experiment. For each run of MSP test samples, they served as methylation-positive and unmethylation-positive controls. Genomic DNA extracted from normal placenta tissue untreated with bisulfite modification was used as reaction negative control. Bisulfite sequencing was carried out using methylation unbiased primers on sodium bisulfite modified DNA, the primer sequences were as follows: • Upstream primer: 5 -GGGTTGGAGTGAGGTATTTGAG3 (−712 to −691). • Downstream primer: 5 -TATCTCTCATAATCATCC-3 (+18 to +35) PCR product is 747 bp. The PCR was performed under the following conditions: 94 ◦ C 5 min, followed by 30 cycles at 94 ◦ C for 30 s, 60 ◦ C for 30 s, 72 ◦ C for 1 min. 2.7. Statistical analysis
2.6. Sodium bisulfite treatment, methylation-specific PCR and sequencing The bisulfite reaction was carried out according to the procedure of Frommer M et al. [16]. The 1 g of DNA in a volume of 50 l of Tris-EDTA (TE) was denatured by NaOH (final concentration, 0.2 M) for 10 min at 37 ◦ C. Freshly prepared 30 l of 10 mM hydroquinine and 520 l of 3 M sodium bisulfite at pH 5 were added to the samples. Each sample was incubated under mineral oil at 50 ◦ C for 16 h. Modified DNA was purified using the Wizard DNA purification resin according to the manufacturer’s recommended protocol (Promega, Madison, WI, USA) and eluted into 50 l of ddH2 O. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 min at room temperature and then by ethanol precipitation. DNA was resuspended in 20 l of ddH2 O and used immediately or stored at −20 ◦ C. Sodium bisulfite-treated DNA was subjected to PCR using the following primer: BMP-6 region between −762 and −662 bp upstream of the translation initiation site of the exon 1 was amplified. The sequences of primers used to amplify and detect unmethylated BMP-6 promoter were:
Statistical significance was determined by ANOVA and Scheffe’s test, and the levels of probability are noted. Results are expressed as means ± standard error (S.E.) for at least three separate experiments.
3. Results 3.1. Alternated gene expression of BMP-6 in breast cancer cells Real-time PCR was used for detecting the BMP-6 expression on mRNA level in three breast cancer cell lines: MCF-7 and T47D which are both estrogen receptor positive (ER+ ) cell lines; MDA-MB-231 which is an estrogen receptor negative (ER− ) cell line. As shown in Fig. 1(A), BMP-6 mRNA expression level was relatively higher both in MCF-7 and T47D (about 2-fold) compared with the level of BMP-6 in normal human epithelial cell line 293T; while in MDA-MB231, the BMP-6 expression level was about 50% lower than that in 293T cell line.
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Fig. 1. The BMP-6 mRNA expression detection in the breast cancer cell lines: MDA-MB-231, MCF-7 and T47D, after the 5-aza-dC treatment. (A) Real-time analysis of BMP-6 mRNA expression in three breast cancer cell lines, using normal epithelial cell lines 293T as control, house-keeping gene GAPDH was used to normalize the BMP-6 level. (B) Comparison of BMP-6 promoter activity in three cell lines. The 1.2 kb BMP-6 promoter-luciferase plasmid was transient transfected, luciferase activities were detected 24 h later. The results are shown as relative light units of firefly luciferase activity normalized with respect to the Renilla luciferase activity. (C) Five days after the 5-aza-dC treatment with different concentrations (0, 1, 10 and 30 M), BMP-6 mRNA expression levels were detected by real-time PCR, using the house-keeping gene GAPDH to normalize the BMP-6 level. The demethylation experiment was repeated on three independent occasions and the measurement of the BMP-6 expression level was quadrupled. The means and standard errors were indicated by the bars and errors bars. Significant induction by 5-aza-dC compared with 0 M group in each type of cell line (* P < 0.05; ** P < 0.01).
The BMP-6 promoter (−1119/+37, 1.2 kb) activities in three cell lines were detected through luciferase assay. The BMP-6 promoter activity in MDA-MB-231 cell line was relatively higher than that in the other two breast cancer cell lines MCF-7 and T47D (Fig. 1(B)). The discrepancy between the expression level of BMP-6 mRNA and BMP-6 promoter activity in MDA-MB-231 suggested that epigenetic mechanism plays a role in regulating the BMP-6 expression. Treatment by the demethylating drug 5-aza-dC on the cell lines was successfully performed. As shown in Fig. 1(C), using quantitative real-time PCR, the mRNA expression level of BMP-6 was found to increase after the 5-aza-dC treatment in ER− cell line MDA-MB-231. MDA-MB-231 was found to have the highest elevated expression of BMP-6 after treatment by 5-aza-dC at the optional concentration of 10 M (about 3.9-fold of the 0 M group) (Fig. 1(C)). There was no significant change in BMP-6 mRNA expression after the treatment with 5-aza-dC in ER+ cell line MCF-7 and T47D, suggesting a demethylation status of BMP-6 promoter in these two cell lines. 3.2. MSRE-PCR and bisulfite sequencing (BSG) analysis demonstrate the BMP-6 promoter methylation status in breast cancer cells Methylation-sensitive enzyme restriction PCR (MSREPCR) was used to investigate the methylation status of BMP-6 promoter in three breast cancer cell lines. Genomic fragments
(−959/−611) containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. As shown in Fig. 2, in MDA-MB-231, successful amplification was detected in the genomic DNA digested by the methyaltion-sensitive enzyme HpaII, not in that digested by the methylation-insensitive enzyme MspI. This result implied the presence of the methylated CpGs in these methylation-sensitive enzyme restriction sites in MDAMB-231. On the contrary, BMP-6 promoter methylation was not detected in MCF-7 and T47D breast cancer cell lines, since no amplification was detected in the DNA digested by HpaII in these two cell lines.
Fig. 2. MSRE-PCR detecting the methylation status of BMP-6 gene promoter in three breast cancer cell lines MDA-MB-231, MCF-7 and T47D. Genomic DNA were digested with methylation-sensitive enzyme Hpa II (lanes 2, 5, 8) or methylation-insensitive enzyme Msp I (lanes 3, 6, 9). Genomic fragments (−959/−611) containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. Genomic DNA extracted from the three cell lines (undigested) were used as PCR control (lanes 1, 4, 7); Genomic DNA digested by Msp I were used as digestion control to make sure the digestion reaction was thorough (lanes 3, 6, 9).
M. Zhang et al. / Journal of Steroid Biochemistry & Molecular Biology 105 (2007) 91–97
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and T18–T33 are ER negative), analyzed the methylation status of BMP-6 promoter by methylation-specific PCR. As shown in Fig. 4, MSP results revealed that 16 of 16 ER negative cases (100%) of the breast cancer DNAs were positive for methylation, while ER positive cases had a significantly lower frequency of BMP-6 methylation (3/17, 18%), indicating the BMP-6 promoter methylation status is correlated with ER status in breast cancer.
4. Discussion
Fig. 3. Sequence analysis of genomic DNA extracted from the breast cancer cell lines after sodium bisulfite conversion. CpG dinucleotides are in boxes. In MDA-MB-231 cells, four CpG loci are all methylated; on the contrary, in MCF-7 and T47D cells, four CpG loci remain in demethylated status.
To further comfirm different methylation patterns of these three breast cancer cell lines, bisulfite treatment and sequencing (BSG) were performed. Bisulfite treatment displayed positive 5 -methylcytosines as cytosines, while unmethylated cytosines appeared as thymines in the final sequence. As shown in Fig. 3, BSG revealed methylation of the promoter region of BMP-6 gene in ER− cell line MDA-MB-231, while BMP-6 promoter region remained demethylated status in the other two ER+ breast cancer cell lines MCF-7 and T47D, indicating different BMP-6 promoter methylation staus are correlated with ER status in breast cancer cell lines. 3.3. BMP-6 promoter methylation status detection in breast tumor specimens To determine whether there is a relationship between BMP-6 promoter methylation and ER status in breast cancer, we collected 33 tumor specimens (T1–T17 are ER positive
In the present study, we first analyzed the regulatory mechanism of BMP-6 gene expression in breast cancer cell lines. Significantly higher levels of BMP-6 mRNA were observed in ER+ breast cancer cell lines MCF-7 and T47D when compared with that in ER− cell line MDA-MB-231. Treatment with 5-aza-dC reversed the methylation status, which was associated with increased expression of BMP6 mRNA in MDA-MB-231, indicating that the methylation status of BMP-6 gene promoter was an important epigenetic mechanism regulating the level of its steady-state mRNA expression. However, this DNA methyltransferase inhibitor failed to restore BMP-6 gene expression in MCF-7 and T47D. Through MSRE-PCR and BSG, we further investigated the BMP-6 methylation status of its 5 -flanking region in three human breast cancer cell lines. Hypermethylation of BMP-6 was detected in the ER− breast cancer cell line MDA-MB-231, while BMP-6 promoter remained demethylation status in the ER+ breast cancer cell lines MCF-7 and T47D. Together with the MSP results of the tumor specimens, our research indicates that different BMP-6 promoter methylation status are associated with ER status in breast cancer. Our previous research demonstrated that BMP-6 can be activated dose-dependently by estrogen in ER+ breast cancer cell line MCF-7; while in ER− cell line MDA-MB-231, estrogen failed to regulate the expression of BMP-6, even when the ER␣ was transfected (data not shown) [4]. We presume that due to the different methylation status of BMP-6 promoter, the responses of BMP-6 promoter in ER+ /ER− breast cancer cells to estrogen stimulation are different. The hypermethylation status of BMP-6 promoter in MDA-MB-231 blocks the interaction of estrogen receptor with the 1/2 ERE element and Sp1 sites on the BMP-6 promoter, while because of the demethylation status of BMP-6 promoter in ER+ cell lines, BMP-6 can be activated dose-dependently by estrogen. DNA methylation is an important regulatory mechanism during the development and metastasis of breast cancer. Hypermethylation silences tumor suppressor genes such as Runx3 [17], p16INK4A [18], and TIMP3 [19], and results in uncontrolled growth; whereas hypomethylation leads to activation of genes required for metastasis such as uPA [20], heparanase [21] and S1004A [22]. Different expression levels of BMP-6 due to demethylation status in its promoter region in ER+ breast cancer and hypermethylation status in ER− breast cancers indicate a potential role BMP-6 plays
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Fig. 4. Methylation-specific PCR analysis for BMP-6 methylation in 33 breast tumor specimens. Universal methylated DNA and genomic DNA from normal placenta tissue treated with sodium bisulfite served as positive controls for methylated (M) and unmethylated (U) genes, respectively; genomic DNA from normal placenta tissue untreated with sodium bisulfite served as negative controls. Figure is a composite of several gels and shows data for 19 ER positive cases (T1–T17) and 16 ER negative cases (T18–T33).
in breast cancer development and metastasis. The estrogen receptor (ER) is present in nearly two-third of breast tumors, and estrogen plays an important role in the cell proliferation and invasiveness in breast cancer. Although ER− tumors exhibit a more aggressive metastatic phenotype, ER+ breast tumor cells spread easily to bone. Recent research revealed that the presence of high estrogen receptor concentration in breast cancer tissue might induce a deleterious effect on bone mass particularly in pre-menopausal women [23]. Although the molecular mechanism remains unclear, ER functions as a versatile transcription factor to either activate or repress specific gene expression. Fotovati A et al. found that 17estradiol induces down-regulation of differentiation-related and metastasis suppressor gene Cap43/NDRG1/Drg-1 in human breast cancer cells [24]. Besides, estradiol metabolite 4-hydroxyestradiol activates the MMP-2 and -9, and this activation is considered to be a crucial step in metastases development [25]. BMPs constitute a subfamily of the TGF- superfamily that induce bone formation in vivo. Among them, BMP-6 expression is associated with maturation of chondrocytes, and BMP-6 also is expressed in various cancer cells including breast, prostate, esophagus and osteosarcoma. Dai et al. found that BMP-6 contributes to the osteoblastic activity of prostate cancer in vivo, indicating that BMP-6 plays an important role in promoting the ability of tumor cells to invade the bone microenvironment [26]. The new findings of different methylation status of BMP-6 in ER− and ER+ breast cancer cell lines and tumor specimens in this experiment provide evidence to the potential role of BMP-6 in tumor metastasis. Breast cancer skeleton metastasis is a quite complicated process. Considering the fact that ER+ breast cancer spread more
easily to bone and BMP-6 in ER+ breast cancer cells can be activated by estrogen through its demethylated promoter, we speculate that the overexpressed BMP-6 which is activated by estrogen, may regulate specific target genes through Smad signaling pathway, to stimulate osteoclastogenesis and bone resorption. Furthermore, we plan to collect and analyze tumor samples of breast cancer with bone metastasis to confirm our findings.
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Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells Ming Zhang a , Qing Wang a , Wei Yuan a , Shuang Yang a , Xu Wang a , Ji-Dong Yan a , Jun Du a , Jian Yin b , Song-Yuan Gao b , Bao-Cun Sun b , Tian-Hui Zhu a,∗ a
Laboratory of Molecular Medicines, Medical College, Nankai University, Tianjin 300071, China b Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China Received 9 October 2006; accepted 25 January 2007
Abstract Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER+ ) breast cancer cell line MCF-7, but not in ER negative (ER− ) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5 flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER− cell line MDA-MB-231 was relatively lower than that in ER+ MCF-7 and T47D cell lines. After the treatment with 5-aza-2 -deoxycytidine (5-aza-dC, especially in the concentration of 10 M), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer. © 2007 Published by Elsevier Ltd. Keywords: Epigenetic regulation; 5-aza-2 -Deoxycytidine (5-aza-dC); Methylation-sensitive enzyme restriction PCR (MSRE-PCR); Bisulfite sequencing (BSG); Methylation-specific PCR (MSP); Estrogen receptor (ER)
1. Introduction Bone morphogenetic protein-6 (BMP-6), which belongs to the transforming growth factor- (TGF-) superfamily, is a multifunctional molecule that regulates bone growth and organ development. In addition to BMP-6’s pivotal role in endochondral bone formation, more and more researches have shown that the expression of BMP-6 is closely correlated with tumor differentiation and metastasis. Recent researches found that BMP-6 is overexpressed in both breast cancer cells and tumor samples from breast carcinoma patients ∗
Corresponding author. Fax: +86 22 23505501. E-mail address: [email protected] (T.-H. Zhu).
0960-0760/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.jsbmb.2007.01.002
[1,2]. BMP-6 overexpression has been detected in advancedstage prostate cancer with skeletal metastasis by in situ hybridization (ISH) and immunohistochemistry [3], suggesting a possible association of BMP-6 expression with skeletal metastasis. Our recent studies demonstrated that BMP-6 can be activated dose-dependently by estrogen in human estrogen receptor positive (ER+ ) breast cancer cell line MCF-7 through estrogen interacting with a 1/2 ERE element and 3 Sp1 sites on BMP-6 promoter [4]. Considering previous observations that patients with ER+ breast tumors are more likely to develop skeletal metastasis, we hypothesized that BMP-6 plays an important role in the process of estrogen-induced breast cancer skeleton metastasis [5,6].
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On the other hand, alterations of DNA methylation status have been observed in many types of tumors including breast [7], prostate [8], lung [9], and pancreatic adenocarcinoma [10]. Genes in abnormal methylation status, activated by demethylation or silenced by hypermethylation, are important for development [11], progression [12], and metastasis of tumors [13]. Resent research found that BMP6 is up-regulated in advanced prostate cancers with metastasis through demethylation of the CpG loci in the promoter region [14], suggesting that epigenetic regulation mechanism plays an important role in activating the BMP-6 expression during cancer progression and giving cancer cells a changeable trait. In this study, we report that BMP-6 promoter methylation status is closely correlated with ER status in breast cancer. Through computational analysis by MethPrimer (http://www.urogene.org/methprimer/index1.html), CpG islands in human BMP-6 gene promoter region have been identified. In the present study, we first examined the relationship between the methylation status of the BMP-6 gene promoter and its steady-state expression in three breast cancer cell lines MDA-MB-231 (ER− ), MCF-7 and T47D (ER+ ) by real-time PCR and 5-aza-2 -deoxycytidine (5-azadC) treatment studies. Using methylation-sensitive enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), we further analyzed the different BMP-6 methylation status in these three breast cancer cell lines. BMP-6 promoter methyaltion status were also detected in 33 breast tumor specimens (17 ER positive; 16 ER negative) through methylation-specific PCR (MSP). This study contributes to the understanding of the role that BMP-6 plays in breast carcinogenesis and skeleton metastasis.
2. Materials and methods 2.1. Tumor samples Fresh breast tissues were obtained from the Tianjin Medical University Cancer Institute and Hospital. The patients (17 ER positive and 16 ER negative) had a mean age of 52.8 ± 12.1 years and were recruited in the same department. 2.2. Cell culture and treatment with 5-aza-2 -deoxycytidine Human breast cancer cell lines MCF-7, MDA-MB-231, T47D and normal human epithelial cell line 293T were maintained in RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone, Logan, Utah, USA), and 100 U/ml Penicillin and Streptomycin (growth medium) at 37 ◦ C in a humidified atmosphere with 5% CO2 . The breast cancer cell lines MCF-7, MDA-MB-231, T47D were seeded at 105 cells in six-well plates, allowed to attach for 24 h and treated with 0, 1, 10 or 30 M of the demethylating agent, 5-aza-2 -deoxycytidine (5-aza-dC,
Sigma Chemical Co., St. Louis, MO) in serum-containing medium for 5 days. Medium and 5-aza-dC were changed daily. 2.3. RNA isolation and real-time PCR Total RNA, extracted from three breast cancer cell lines and 293T cell line, was isolated using TRIzol Reagent (Life Technologies Inc., Grand Island, NY, USA) according to the manufacturer’s protocol. After reverse transcription reaction (20 l) using 2 g of total RNA, real-time PCR was carried out in a 25 l final volume by an ABI PRISM 7000 (ABI, USA) sequence detection system according to the manufacturer’s protocol. The reaction mixture contained 1 × SYBR Green I, 0.5 pmol/L of each primer, 2.5 mM MgCl2 , and 0.5 l cDNA from (20 l of) reverse transcription reaction. The conditions of real-time PCR were as follows: 94 ◦ C 4 min followed by 40 cycles at 94 ◦ C for 30 s, 68 ◦ C for 1.5 min. There is no non-specific amplification determined by dissolve curve. There are four repeat tubes per cDNA specimen, three cDNA specimens independently for each data point. The real-time PCR results were reported as the fold of relative light units for breast cancer cell lines (MCF-7, MDA-MB-231 and T47D) compared with those for 293T after normalization to GAPDH expression. Error bars represent the standard errors for three independent experiments, with each data point done in quadruple. The primers used for real-time PCR are as follows: • BMP-6: ◦ Upstream primer: 5 -CAACAGAGTCGTAATCGCTCTACC-3 (+1323 to +1346). ◦ Downstream primer: 5 -TTAGTGGCATCCACAAGCTCT-3 (+1701 to +1721) PCR product is 308bp. • GAPDH: ◦ Upstream primer: 5 -ACCACAGTCCATGCCATCAC3 (+526 to +545). ◦ Downstream primer: 5 -TCCACCACCCTGTTGCTGTA-3 (+958 to +977) PCR product is 451bp. 2.4. Expression plasmid construction, transient transfection study and luciferase assay A human 1.2 kb (−1119/+37) BMP-6 promoter-luciferase plasmid (Promega, Madison, WI, USA) was constructed as previously described [4]. The plasmid construct was transfected into the three human breast cancer cell lines by LipofectAMINE reagents (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Twenty four hours after transfection, luciferase activity was detected using a Kit from Promega Corp. (Madison, WI, USA) with a luminometer. The efficiency of transfection was monitored by cotransfection with phRL-null vector (0.5 g/well). The luciferase results were reported as relative light units of firefly luciferase activity normalized with respect to the Renilla luciferase activity. Error bars represent the standard errors for
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three independent experiments, with each data point done in triplicate. 2.5. Genomic DNA extraction and methylation-sensitive enzyme restriction PCR (MSRE-PCR) Genomic DNA was purified from the three breast cancer cell lines and tumor samples according to the method mentioned by Melnikov et al. [15]. Digestions were performed with methylation sensitive restriction enzyme HpaII or methylation insensitive restriction enzyme MspI (recognition site CC|GG; TaKaRa, Dalian, China). Typically, 1 g of genomic DNA were digested with 40 U of the enzyme at 37 ◦ C for 72 h under a layer of mineral oil; the final volume of the reaction was 50 l. Control samples were treated in the same way without the enzyme. After incubation, digested samples were purified using phenol and chlorformisoamyl alcohol and eluted in 50 l of ddH2 O. Control samples were ethanol precipitated and dissolved in 50 l of ddH2 O. Genomic fragments containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. ExTaq (TaKaRa, Dalian, China) was used 0.2 U per reaction. The conditions of MSRE-PCR were as follows: 94 ◦ C 3 min followed by 25 cycles at 94 ◦ C for 30 s, 57 ◦ C for 30 s, 72 ◦ C for 30 s. Primers used in this study are as follows: • Upstream primer: 5 -AGAGAAAGCTTCAGATCGGG-3 (−959 to −940). • Downstream primer: 5 -CGGCGCCGGAATTCGGTC-3 (−628 to −611) PCR product is 348 bp.
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• Upstream primer: 5 -CGTTTTTATTGGTTGTTAGTTTGTC-3 (−762 to −737). • Downstream primer: 5 -CAACGTAAACTTTCTACTACGCG-3 (−685 to −663) PCR product is 99 bp. The sequences of methylation-sensitive primers were: • Upstream primer: 5 -TGTTTTTATTGGTTGTTAGTTTGTTG-3 (−762 to −737). • Downstream primer: 5 -CCAACATAAACTTTCTACTACACACC-3 (−687 to −662) PCR product is 100 bp. The PCR was performed under the following conditions: 94 ◦ C 5 min, followed by 30 cycles at 94 ◦ C for 30 s, 66 ◦ C for 30 s, 72 ◦ C for 30 s. To ensure completion of bisulfite chemical modification, universal methylated DNA and genomic DNA from normal placenta tissue were include in each sodium bisulfite treatment experiment. For each run of MSP test samples, they served as methylation-positive and unmethylation-positive controls. Genomic DNA extracted from normal placenta tissue untreated with bisulfite modification was used as reaction negative control. Bisulfite sequencing was carried out using methylation unbiased primers on sodium bisulfite modified DNA, the primer sequences were as follows: • Upstream primer: 5 -GGGTTGGAGTGAGGTATTTGAG3 (−712 to −691). • Downstream primer: 5 -TATCTCTCATAATCATCC-3 (+18 to +35) PCR product is 747 bp. The PCR was performed under the following conditions: 94 ◦ C 5 min, followed by 30 cycles at 94 ◦ C for 30 s, 60 ◦ C for 30 s, 72 ◦ C for 1 min. 2.7. Statistical analysis
2.6. Sodium bisulfite treatment, methylation-specific PCR and sequencing The bisulfite reaction was carried out according to the procedure of Frommer M et al. [16]. The 1 g of DNA in a volume of 50 l of Tris-EDTA (TE) was denatured by NaOH (final concentration, 0.2 M) for 10 min at 37 ◦ C. Freshly prepared 30 l of 10 mM hydroquinine and 520 l of 3 M sodium bisulfite at pH 5 were added to the samples. Each sample was incubated under mineral oil at 50 ◦ C for 16 h. Modified DNA was purified using the Wizard DNA purification resin according to the manufacturer’s recommended protocol (Promega, Madison, WI, USA) and eluted into 50 l of ddH2 O. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 min at room temperature and then by ethanol precipitation. DNA was resuspended in 20 l of ddH2 O and used immediately or stored at −20 ◦ C. Sodium bisulfite-treated DNA was subjected to PCR using the following primer: BMP-6 region between −762 and −662 bp upstream of the translation initiation site of the exon 1 was amplified. The sequences of primers used to amplify and detect unmethylated BMP-6 promoter were:
Statistical significance was determined by ANOVA and Scheffe’s test, and the levels of probability are noted. Results are expressed as means ± standard error (S.E.) for at least three separate experiments.
3. Results 3.1. Alternated gene expression of BMP-6 in breast cancer cells Real-time PCR was used for detecting the BMP-6 expression on mRNA level in three breast cancer cell lines: MCF-7 and T47D which are both estrogen receptor positive (ER+ ) cell lines; MDA-MB-231 which is an estrogen receptor negative (ER− ) cell line. As shown in Fig. 1(A), BMP-6 mRNA expression level was relatively higher both in MCF-7 and T47D (about 2-fold) compared with the level of BMP-6 in normal human epithelial cell line 293T; while in MDA-MB231, the BMP-6 expression level was about 50% lower than that in 293T cell line.
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Fig. 1. The BMP-6 mRNA expression detection in the breast cancer cell lines: MDA-MB-231, MCF-7 and T47D, after the 5-aza-dC treatment. (A) Real-time analysis of BMP-6 mRNA expression in three breast cancer cell lines, using normal epithelial cell lines 293T as control, house-keeping gene GAPDH was used to normalize the BMP-6 level. (B) Comparison of BMP-6 promoter activity in three cell lines. The 1.2 kb BMP-6 promoter-luciferase plasmid was transient transfected, luciferase activities were detected 24 h later. The results are shown as relative light units of firefly luciferase activity normalized with respect to the Renilla luciferase activity. (C) Five days after the 5-aza-dC treatment with different concentrations (0, 1, 10 and 30 M), BMP-6 mRNA expression levels were detected by real-time PCR, using the house-keeping gene GAPDH to normalize the BMP-6 level. The demethylation experiment was repeated on three independent occasions and the measurement of the BMP-6 expression level was quadrupled. The means and standard errors were indicated by the bars and errors bars. Significant induction by 5-aza-dC compared with 0 M group in each type of cell line (* P < 0.05; ** P < 0.01).
The BMP-6 promoter (−1119/+37, 1.2 kb) activities in three cell lines were detected through luciferase assay. The BMP-6 promoter activity in MDA-MB-231 cell line was relatively higher than that in the other two breast cancer cell lines MCF-7 and T47D (Fig. 1(B)). The discrepancy between the expression level of BMP-6 mRNA and BMP-6 promoter activity in MDA-MB-231 suggested that epigenetic mechanism plays a role in regulating the BMP-6 expression. Treatment by the demethylating drug 5-aza-dC on the cell lines was successfully performed. As shown in Fig. 1(C), using quantitative real-time PCR, the mRNA expression level of BMP-6 was found to increase after the 5-aza-dC treatment in ER− cell line MDA-MB-231. MDA-MB-231 was found to have the highest elevated expression of BMP-6 after treatment by 5-aza-dC at the optional concentration of 10 M (about 3.9-fold of the 0 M group) (Fig. 1(C)). There was no significant change in BMP-6 mRNA expression after the treatment with 5-aza-dC in ER+ cell line MCF-7 and T47D, suggesting a demethylation status of BMP-6 promoter in these two cell lines. 3.2. MSRE-PCR and bisulfite sequencing (BSG) analysis demonstrate the BMP-6 promoter methylation status in breast cancer cells Methylation-sensitive enzyme restriction PCR (MSREPCR) was used to investigate the methylation status of BMP-6 promoter in three breast cancer cell lines. Genomic fragments
(−959/−611) containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. As shown in Fig. 2, in MDA-MB-231, successful amplification was detected in the genomic DNA digested by the methyaltion-sensitive enzyme HpaII, not in that digested by the methylation-insensitive enzyme MspI. This result implied the presence of the methylated CpGs in these methylation-sensitive enzyme restriction sites in MDAMB-231. On the contrary, BMP-6 promoter methylation was not detected in MCF-7 and T47D breast cancer cell lines, since no amplification was detected in the DNA digested by HpaII in these two cell lines.
Fig. 2. MSRE-PCR detecting the methylation status of BMP-6 gene promoter in three breast cancer cell lines MDA-MB-231, MCF-7 and T47D. Genomic DNA were digested with methylation-sensitive enzyme Hpa II (lanes 2, 5, 8) or methylation-insensitive enzyme Msp I (lanes 3, 6, 9). Genomic fragments (−959/−611) containing three HpaII/MspI recognition sites and located within corresponding CpG islands were selected for amplification. Genomic DNA extracted from the three cell lines (undigested) were used as PCR control (lanes 1, 4, 7); Genomic DNA digested by Msp I were used as digestion control to make sure the digestion reaction was thorough (lanes 3, 6, 9).
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and T18–T33 are ER negative), analyzed the methylation status of BMP-6 promoter by methylation-specific PCR. As shown in Fig. 4, MSP results revealed that 16 of 16 ER negative cases (100%) of the breast cancer DNAs were positive for methylation, while ER positive cases had a significantly lower frequency of BMP-6 methylation (3/17, 18%), indicating the BMP-6 promoter methylation status is correlated with ER status in breast cancer.
4. Discussion
Fig. 3. Sequence analysis of genomic DNA extracted from the breast cancer cell lines after sodium bisulfite conversion. CpG dinucleotides are in boxes. In MDA-MB-231 cells, four CpG loci are all methylated; on the contrary, in MCF-7 and T47D cells, four CpG loci remain in demethylated status.
To further comfirm different methylation patterns of these three breast cancer cell lines, bisulfite treatment and sequencing (BSG) were performed. Bisulfite treatment displayed positive 5 -methylcytosines as cytosines, while unmethylated cytosines appeared as thymines in the final sequence. As shown in Fig. 3, BSG revealed methylation of the promoter region of BMP-6 gene in ER− cell line MDA-MB-231, while BMP-6 promoter region remained demethylated status in the other two ER+ breast cancer cell lines MCF-7 and T47D, indicating different BMP-6 promoter methylation staus are correlated with ER status in breast cancer cell lines. 3.3. BMP-6 promoter methylation status detection in breast tumor specimens To determine whether there is a relationship between BMP-6 promoter methylation and ER status in breast cancer, we collected 33 tumor specimens (T1–T17 are ER positive
In the present study, we first analyzed the regulatory mechanism of BMP-6 gene expression in breast cancer cell lines. Significantly higher levels of BMP-6 mRNA were observed in ER+ breast cancer cell lines MCF-7 and T47D when compared with that in ER− cell line MDA-MB-231. Treatment with 5-aza-dC reversed the methylation status, which was associated with increased expression of BMP6 mRNA in MDA-MB-231, indicating that the methylation status of BMP-6 gene promoter was an important epigenetic mechanism regulating the level of its steady-state mRNA expression. However, this DNA methyltransferase inhibitor failed to restore BMP-6 gene expression in MCF-7 and T47D. Through MSRE-PCR and BSG, we further investigated the BMP-6 methylation status of its 5 -flanking region in three human breast cancer cell lines. Hypermethylation of BMP-6 was detected in the ER− breast cancer cell line MDA-MB-231, while BMP-6 promoter remained demethylation status in the ER+ breast cancer cell lines MCF-7 and T47D. Together with the MSP results of the tumor specimens, our research indicates that different BMP-6 promoter methylation status are associated with ER status in breast cancer. Our previous research demonstrated that BMP-6 can be activated dose-dependently by estrogen in ER+ breast cancer cell line MCF-7; while in ER− cell line MDA-MB-231, estrogen failed to regulate the expression of BMP-6, even when the ER␣ was transfected (data not shown) [4]. We presume that due to the different methylation status of BMP-6 promoter, the responses of BMP-6 promoter in ER+ /ER− breast cancer cells to estrogen stimulation are different. The hypermethylation status of BMP-6 promoter in MDA-MB-231 blocks the interaction of estrogen receptor with the 1/2 ERE element and Sp1 sites on the BMP-6 promoter, while because of the demethylation status of BMP-6 promoter in ER+ cell lines, BMP-6 can be activated dose-dependently by estrogen. DNA methylation is an important regulatory mechanism during the development and metastasis of breast cancer. Hypermethylation silences tumor suppressor genes such as Runx3 [17], p16INK4A [18], and TIMP3 [19], and results in uncontrolled growth; whereas hypomethylation leads to activation of genes required for metastasis such as uPA [20], heparanase [21] and S1004A [22]. Different expression levels of BMP-6 due to demethylation status in its promoter region in ER+ breast cancer and hypermethylation status in ER− breast cancers indicate a potential role BMP-6 plays
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Fig. 4. Methylation-specific PCR analysis for BMP-6 methylation in 33 breast tumor specimens. Universal methylated DNA and genomic DNA from normal placenta tissue treated with sodium bisulfite served as positive controls for methylated (M) and unmethylated (U) genes, respectively; genomic DNA from normal placenta tissue untreated with sodium bisulfite served as negative controls. Figure is a composite of several gels and shows data for 19 ER positive cases (T1–T17) and 16 ER negative cases (T18–T33).
in breast cancer development and metastasis. The estrogen receptor (ER) is present in nearly two-third of breast tumors, and estrogen plays an important role in the cell proliferation and invasiveness in breast cancer. Although ER− tumors exhibit a more aggressive metastatic phenotype, ER+ breast tumor cells spread easily to bone. Recent research revealed that the presence of high estrogen receptor concentration in breast cancer tissue might induce a deleterious effect on bone mass particularly in pre-menopausal women [23]. Although the molecular mechanism remains unclear, ER functions as a versatile transcription factor to either activate or repress specific gene expression. Fotovati A et al. found that 17estradiol induces down-regulation of differentiation-related and metastasis suppressor gene Cap43/NDRG1/Drg-1 in human breast cancer cells [24]. Besides, estradiol metabolite 4-hydroxyestradiol activates the MMP-2 and -9, and this activation is considered to be a crucial step in metastases development [25]. BMPs constitute a subfamily of the TGF- superfamily that induce bone formation in vivo. Among them, BMP-6 expression is associated with maturation of chondrocytes, and BMP-6 also is expressed in various cancer cells including breast, prostate, esophagus and osteosarcoma. Dai et al. found that BMP-6 contributes to the osteoblastic activity of prostate cancer in vivo, indicating that BMP-6 plays an important role in promoting the ability of tumor cells to invade the bone microenvironment [26]. The new findings of different methylation status of BMP-6 in ER− and ER+ breast cancer cell lines and tumor specimens in this experiment provide evidence to the potential role of BMP-6 in tumor metastasis. Breast cancer skeleton metastasis is a quite complicated process. Considering the fact that ER+ breast cancer spread more
easily to bone and BMP-6 in ER+ breast cancer cells can be activated by estrogen through its demethylated promoter, we speculate that the overexpressed BMP-6 which is activated by estrogen, may regulate specific target genes through Smad signaling pathway, to stimulate osteoclastogenesis and bone resorption. Furthermore, we plan to collect and analyze tumor samples of breast cancer with bone metastasis to confirm our findings.
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