- Research article
- Open Access
- Open Peer Review
BMP-6 promotes E-cadherin expression through repressing δEF1 in breast cancer cells
© Yang et al; licensee BioMed Central Ltd. 2007
- Received: 13 April 2007
- Accepted: 13 November 2007
- Published: 13 November 2007
Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. Recent studies indicate that BMP-6 is closely related to tumor differentiation and metastasis.
Quantitative RT-PCR was used to determine the expression of BMP-6, E-cadherin, and δEF1 at the mRNA level in MCF-7 and MDA-MB-231 breast cancer cells, as well as in 16 breast cancer specimens. Immunoblot analysis was used to measure the expression of δEF1 at the protein level in δEF1-overexpressing and δEF1-interfered MDA-MB-231 cells. Luciferase assay was used to determine the rhBMP-6 or δEF1 driven transcriptional activity of the E-cadherin promoter in MDA-MB-231 cells. Quantitative CHIP assay was used to detect the direct association of δEF1 with the E-cadherin proximal promoter in MDA-MB-231 cells.
MCF-7 breast cancer cells, an ER+ cell line that expressed high levels of BMP-6 and E-cadherin exhibited very low levels of δEF1 transcript. In contrast, MDA-MB-231 cells, an ER- cell line had significantly reduced BMP-6 and E-cadherin mRNA levels, suggesting an inverse correlation between BMP-6/E-cadherin and δEF1. To determine if the same relationship exists in human tumors, we examined tissue samples of breast cancer from human subjects. In 16 breast cancer specimens, the inverse correlation between BMP-6/E-cadherin and δEF1 was observed in both ER+ cases (4 of 8 cases) and ER- cases (7 of 8 cases). Further, we found that BMP-6 inhibited δEF1 transcription, resulting in an up-regulation of E-cadherin mRNA expression. This is consistent with our analysis of the E-cadherin promoter demonstrating that BMP-6 was a potent transcriptional activator. Interestingly, ectopic expression of δEF1 was able to block BMP-6-induced transactivation of E-cadherin, whereas RNA interference-mediated down-regulation of endogenous δEF1 in breast cancer cells abolished E-cadherin transactivation by BMP-6. In addition to down-regulating the expression of δEF1, BMP-6 also physically dislodged δEF1 from E-cadherin promoter to allow the activation of E-cadherin transcription.
We conclude that repression of δEF1 plays a key role in mediating BMP-6-induced transcriptional activation of E-cadherin in breast cancer cells. Consistent with the fact that higher level of δEF1 expression is associated with more invasive phenotype of breast cancer cells, our collective data suggests that δEF1 is likely the switch through which BMP-6 restores E-cadherin-mediated cell-to-cell adhesion and prevents breast cancer metastasis.
- Breast Cancer
- Breast Cancer Cell
- Breast Cancer Specimen
- Clinical Tumor Specimen
- Quantitative Chip Assay
Breast cancer is the most common neoplasm in women. The unique histological features of breast cancer are prominent proliferation of epithelial cells and the formation of ectopic mesenchymal tissue, including cartilage and bone, especially in complex adenomas and benign mixed tumors [1, 2]. The association between loss or down-regulation of E-cadherin, an epithelial cell-cell adhesion protein, and progression of breast cancer has been extensively documented [3, 4]. Tumor cells acquire invasive properties when E-cadherin-mediated adhesion is inhibited [5, 6]. In line with these findings, ectopic expression of E-cadherin in a transgenic mouse model prevented tumor cell invasion and metastasis . Several epigenetic mechanisms are implicated in E-cadherin loss during breast cancer, including hypermethylation of the E-cadherin promoter region at CpG islands  and transrepression by specific transcriptional factors. Several zinc finger transcription factors, such as Twist [9, 10], Snail1 [11–14], Snail2 [15, 16], SIP1 , and E12/E47 , have been found to bind to the E-box elements in the proximal E-cadherin promoter and repress its transcription. Moreover, a few factors, including ErbB2 , TGF-β , and estrogen , were reported to regulate E-cadherin expression.
Bone morphogenetic protein-6 (BMP-6), a member of TGF-β superfamily, has been characterized as a multifunctional molecule with a distinct ability to induce ectopic cartilage and bone formation [1, 21]. In vitro, BMP-6 inhibits cell division, promotes cell differentiation, induces ectopic bone formation, and regulates epithelial-mesenchymal interaction [21–24]. Furthermore, a number of recent studies have shown that BMP-6 expression is associated with progression of tumorigenesis. BMP-6 is detected in several human neoplastic epithelial cells including breast, prostate, salivary, rectal, and thyroid carcinomas, and is speculated to be closely associated with tumor metastasis [21, 25–29].
δEF1, a member of the zinc finger-homeodomain family of transcription factors [30, 31], was originally identified as a binding protein of the lens-specific δ1-crystalline enhancer in chicken . Studies revealed that δEF1 is a widely expressed transcriptional repressor, working through its zinc finger clusters binding to consensus E-box-like sequences, 5'-CA(G/C)(G/C)TG-3' [33–35]. Several recently reported properties mark δEF1 as a potential regulatory factor in various cellular processes during tumor progression. In lung and breast tumor cells, δEF1 has been implicated in epithelial to mesenchymal transition (EMT) [36–38], a process associated with tumor metastasis . Moreover, δEF1 can itself be regulated by hormones in target tissues [40–42]. In one example, δEF1 is up-regulated by estrogen in the chick oviduct and is responsible for estrogen-mediated regulation of the ovalbumin gene [40, 41, 43]. In human T47D breast carcinoma cells, δEF1 is up-regulated by progesterone via both isoforms of the progesterone receptor (PR) .
In this communication, we report that BMP-6 up-regulates the expression of E-cadherin at the mRNA level in breast cancer cells, determined by quantitative RT-PCR and luciferase assay. We demonstrate that this effect is a direct result of BMP-6-indcued down-regulation of δEF1. The inverse relationship between BMP-6/E-cadherin and δEF1 were validated in 16 breast cancer specimens using quantitative RT-PCR. This finding contributes significantly to our understanding of the potential role of BMP-6 and δEF1 in breast tumorigenesis and metastasis.
Fresh breast cancer tissues of invasive ductal carcinoma (stage II) were obtained from the Tissue Banking Facility Jointly Supported by TMUCIH (Tianjin Medical University Cancer Institute and Hospital) & NFCR (National Foundation for Cancer Research). The pathological stage and nodal status were obtained from the primary pathology reports. The patients (8 ER positive and 8 ER negative) had a mean age of 52.8 ± 12.1 years and were recruited in the same department. This study was approved by the institutional ethics committee.
MCF-7 and MDA-MB-231 cells were maintained in DMEM-high glucose medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% FBS (Hyclone, Logan, Utah, USA), penicillin, and streptomycin according to the recommendations of the American Type Culture Collection (ATCC). MDA-MB-231 cells were plated at a density of 2 × 104 cells/well in 24-well plates and 8 × 104 in 6-well plates for use in quantitative RT-PCR and luciferase assays, respectively. The cells were cultured in the presence or absence of 200 ng/ml rhBMP-6 (R&D Systems, Minneapolis, MN, USA) in DMEM supplemented with 5% FBS.
cDNA fragment encoding the full-length δEF1 sequence was prepared by PCR using the forward primer, 5'-CGCGGATCCAGGATCATGGCGGATGGC-3', and reverse primer, 5'-GAACCTCGAGCGAGCTTCATTTGTCTTCTCTTC-3'. The PCR products were digested with BamHI/XhoI, and cloned into pcDNA6B (Life Technologies, Grand Island, NY, USA). The E-cadherin promoter sequence (-308/+21) was obtained by PCR from human blood genomic DNA and cloned into the pGL4.10 vector (Promega, Madison, WI, USA) as described by Comijn et al. . Mutagenesis of the δEF1 sites in the human E-cadherin promoter was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with the forward primer E2 box 1: 5'-gctgtggccggCAGATGaaccctcag-3' and reverse primer E2 box 1: 5'-ctgagggttCATCT Gccggccacagc-3'; forward primer E2 box 3: 5'-gctccgggctCATCTGgctgcag c-3' and reverse primer E2 box 3: 5'-gctgcagcCAGATGagccccggagc-3', as described by Comijn et al. .
RNA Extraction and Quantitative RT-PCR
Total RNA was extracted from MCF-7 and MDA-MB-231 cells treated with or without 200 ng/ml rhBMP-6 for various times or from 16 breast cancer specimens using the TRIzol Reagent (Life Technologies, Grand Island, NY, USA). Total RNA (0.5 μg) from each sample was used for first strand cDNA synthesis (M-MLV Reverse Transcriptase, Promega, Madison, WI, USA). Specific products of human δEF1, human E-cadherin, and human BMP-6 were amplified by quantitative PCR using the following primers: δEF1, 5'-GGCCCCAGGTGTAAGCGC-3' (forward), and 5'-CAGGCCCCAGGATTTCTTG C-3' (reverse); E-cadherin, 5'-TGCTGCAGGTCTCCTCTTGG-3' (forward), and 5'-AGT CCCAGGCGTAGACCAAG-3' (reverse); BMP-6, 5'-CAACAGAGTCGTAATCA-3' (forward), and 5'-TTAGTGGCATCCACAAGCTCT-3' (reverse). GAPDH was used as an internal control. Verification of the expression levels of genes was performed by quantitative RT-PCR using EvaGreen (Botium, Hayward, CA, USA). The expression level was expressed as the threshold cycle (CT) values of the target and reference gene-GAPDH, which is constitutively expressed and not regulated by treatment with rhBMP-6. Comparison and calculation of CT values was used to determine the relative mRNA expression expressed as the fold change of target genes relative to the reference gene.
The following antibodies (Abs) were used: mouse monoclonal Ab against c-Myc (sc-40, Santa Cruz Biotechnology, CA, USA); goat polyclonal Ab against the N-terminal epitope of δEF1 (ZEB-E20, Santa Cruz Biotechnology, CA, USA); a mouse monoclonal Ab against E-cadherin (610181, BD Transduction Laboratories, KY, USA); and a mouse monoclonal Ab against FLAG-M2 (F-3165, Sigma, MO, USA).
Western Immunoblot Analysis
MDA-MB-231 cells were solubilized in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton ×-100, 0.5% sodium deoxycholate) supplemented with aprotinin (10 μg/ml), leupeptin (10 μg/ml), and PMSF (1 mM). The lysates were cleared by centrifugation and the protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). Western blotting was performed using standard techniques and immunoreactive bands were detected by chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ, USA).
MDA-MB-231 cells were co-transfected with wild-type or mutant human E-cadherin promoter constructs and different amounts of δEF1 expression plasmids (1.5, 3.0, and 6.0 μg/well) in 6-well plates using Lipofectamine 2000 (Invitrogen, Austin, TX, USA). Cells were treated with rhBMP-6 (200 ng/ml) for 24 h after transfection. Lysates were prepared 24 h after treatment. The luciferase activity was then measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer's instructions. Luciferase activity was normalized using the Renilla luciferase activity.
Preparation of small interfering RNAs and Transfection
The target sequence of the siRNA is 5'-TGATCAGCCTCAATCTGCA-3' for human δEF1 as previously reported . The sense and antisense oligonucleotides with the internal loop were synthesized (TaKaRa, Shiga, Japan). These were annealed and ligated into the BamHI and HindIII sites of pSilencer 4.1-CMVneo (Ambion, Carlsbad, CA, USA) to construct the δEF1-specific siRNA expression plasmid according to the manufacturer's instructions. pSilencer 4.1-CMVneo expressing a scrambled siRNA (Ambion, Carlsbad, CA, USA) was used as a control. Transient transfection with siRNAs was performed with Lipofectamine 2000 (Invitrogen, Austin, TX, USA) in MDA-MB-231 cells. G418-resistent clones were isolated over a period of 3–4 weeks. Down-regulation of δEF1 was confirmed by western immunoblot analysis.
Quantitative CHIP Assays
MDA-MB-231 cells were grown for 24 h up to 80% confluence in the presence or absence of 200 ng/ml rhBMP-6. Cells were cross-linked with 1% formaldehyde and processed using the Chromatin Immunoprecipitation (ChIP) Assay Kit (Updates, Lake Placid, NY, USA). The following antibodies (10 μg) were used: anti-ZEB, a polyclonal antibody raised against N-terminal epitopes of δEF1, or unrelated anti-FLAG control antibody (F-3165, Sigma, MO, USA) with 100 μg of chromatin per CHIP. Purified immunoprecipitated DNA was used for quantitative PCR reactions. The primers used for this analysis were as follows: 5'-AGGCTAGAGGGTCACCGCGTC-3' (forward), and 5'-GCTTTGCAGTTCCGACGCCAC-3' (reverse). Copy numbers for the DNA fragments (-175 to +21) of E-cadherin promoter in each anti-ZEB sample with or without BMP-6 induction were determined and compared to copy numbers of the DNA fragment without IP (input DNA). Anti-FLAG antibody was used as a control for IP reactions. The percentage of the input was then calculated. The final value was the percentage input obtained with specific antibody minus the percentage input obtained with anti-FLAG control antibody. The dissociation curve was determined for each quantitative PCR to ensure that a single band was produced. Each data point represents three independent samples.
Expression levels of BMP-6 and E-cadherin are inversely related to that of δEF1 in breast cancer cell lines and in clinical breast cancer specimens
BMP-6 down-regulated δEF1 and concurrently promoted E-cadherin transcription in breast cancer cells
To verify these findings, western blot was performed to determine BMP-6-modulated expression of δEF1 and E-cadheirn at the protein level. This time, MDA-MB-231 cells were cultured in the presence or absence of 200 ng/ml rhBMP-6 and total protein lysates were collected at 24 and 48 h following treatment. Western blot analysis revealed that BMP-6 treatment for 48 h significantly reduced the level of δEF1 protein (Figure 3c) with the expression of E-cadherin being up-regulated concurrently (Figure 3d).
BMP-6-induced transcription of endogenous E-cadherin was suppressed by δEF1 in MDA-MB-231 cells
BMP-6 attenuates the association of endogenous δEF1 with E-cadherin promoter
The progressive and metastatic nature of breast cancer has been well recognized, yet the mechanisms through which breast cancer cells acquire their invasive properties have not been clearly elucidated. Several studies have reported that breast cancer produces a variety of growth factors that can play a role in the progression and metastasis of breast cancer. In the current study, we have uncovered that one of these factors, BMP-6, contributes to the regulation of E-cadherin-mediated epithelial-mesenchymal transition of breast cancer in vitro. In addition, we have shown that the stimulatory effect of BMP-6 on E-cadherin transcription occurs through reducing the expression and activity of δEF1, which we have found to be a strong transcriptional repressor of the E-cadherin gene. Importantly, the reverse relationship between BMP-6/E-cadherin and δEF1 expressions in cancer cell lines has been verified in clinical tumor specimens. Our results provide the first evidence, at the cellular level, to support the hypothesis that breast cancers may progress and metastasize through the regulation of E-cadherin expression by BMP-6 and δEF1.
Expression of BMPs has been reported to increase with breast cancer progression. Several studies on the overexpression of BMPs, such as BMP-2, BMP-4, and BMP-7 in mammary tumor cells [45–47] are suggestive of a role of BMPs in breast cancer development. Clement et al. were the first to report that BMP-6 is detectable, not only in breast cancer cell lines, such as MCF-7, SK-BR-3, MDA-MB-453, BT-20, and ZR-75-1, but also in most tumor specimens, using RT-PCR and immunohistochemistry . Importantly, BMP-6 expression was significantly increased and was most intense in the vicinity of chondroid matrix of complex adenomas and mixed benign tumors of canine mammary glands [1, 2]. In agreement with these reports, we present here our finding that significantly higher levels of BMP-6 were observed in breast cancer cell lines and clinical tumor specimens, using quantitative RT-PCR. Moreover, BMP-6 expression is higher in the ER+ breast cancer cell line, MCF-7, compared to the ER- breast cancer cell line, MDA-MB-231. Although BMP-6 expression levels varied widely among tumor specimens, it was relatively higher in ER+ cases than in ER- cases. This coincides with our previous finding that BMP-6 promoter methylation status is correlated with ER status in breast cancer. In that study, we observed significantly lower levels of BMP-6 mRNA in ER- breast cancer cells compared with ER+ breast cancer cells, an effect attributed to hypermethylation status in the ER- breast cancer cells . In addition to breast cancer, BMP-6 has been found in a variety of other cancer cell types, including prostate, kidney, esophagus, and osteosarcoma [25, 49–51], suggesting its association with the progression of tumorigenesis.
E-cadherin is ubiquitously expressed by epithelial cells, from which most cancers are derived. E-cadherin-mediated cell-cell adhesion prevents cells in a primary tumor from breaking away and invading near or distant sites. It has been well documented that loss of E-cadherin in mammary epithelial cells can promote breast cancer progression and metastasis . Recently, several factors, including ErbB2 , TGF-β , and estrogen , were reported to regulate E-cadherin expression through different mechanisms. In this study, we have determined that BMP-6 is a novel stimulus of E-cadherin expression in breast cancer cells, providing evidence for a potential role of BMP-6 in tumor progression and metastasis. In addition, our results indicate that BMP-6-induced expression of E-cadherin is correlated with ER status. Higher levels of BMP-6 and E-cadherin transcripts were observed in ER+ breast cancer cells, while lower amounts were detected in ER- cells. These observations are in line with a previous finding that loss of E-cadherin expression was associated with the lack of ER expression and a more aggressive phenotype of breast cancer with poor clinical prognosis . Thus, our observations lead us to propose that BMP-6 may cooperate with other factors, such as estrogen, to participate in attenuating breast caner progression.
Several epigenetic mechanisms have been implicated in E-cadherin regulation in breast cancer , among these, a few zinc-finger transcription factors are known to bind to E-box elements of the E-cadherin promoter and repress transcription, including Snail1 [11–14], Snail2 [15, 16], and SIP1 . δEF1, a close homolog of SIP1 , was previously shown to directly bind to E-box elements of the E-cadherin promoter and inhibit the expression of endogenous E-cadherin mRNA and protein in mammary epithelial cells . These previous observations are consistent with our results demonstrating that overexpression of δEF1 is sufficient to block transactivation of E-cadherin in MDA-MB-231 cells. In line with our findings, a recent paper has also supported a role of δEF1 in E-cadherin repression in lung cancer cells . In spite of these findings, the upstream factors responsible for regulating the δEF1/E-cadherin loop in breast cancer have not yet been identified. Our current work has provided two novel findings in this context, (1) δEF1 can repress BMP-6-mediated up-regulation of E-cadherin and (2) the integrity of a single δEF1-binding element in the proximal E-cadherin promoter is sufficient for the repression effect of δEF1, providing evidence that BMP-6 may affect E-cadherin-mediated progression and metastasis through the regulation of specific genes, such as δEF1, during breast tumorigenesis. Furthermore, the fact that artificial removal of δEF1 by RNAi enhances gene expression of E-cadherin supports the important role of δEF1 to be down-regulated by BMP-6 in the native state in order for BMP-6 to induce E-cadherin expression. RNAi does it by mediating mRNA degradation, whereas BMP-6 does it by (1) inhibiting the expression of δEF1, and (2) by dislodging δEF1 that has already bound to the E-cadherin gene. Our overall data demonstrates that δEF1 is a native repressor of E-cadherin gene. Factors that remove/decrease δEF1 level would subsequently increase the expression of E-cadherin.
Previous research from our group indicated that δEF1 represses BMP-2-induced differentiation of C2C12 myoblasts into the osteoblast lineage, an effect that is not mediated via the canonical BMP/Smad signaling pathway, but instead by differential regulation of the AP-1 pathway . In the study reported here, we found that overexpression of R-Smads (Smad 1 and 5) failed to augment BMP-6-induced transactivation of E-cadherin, which is mediated by suppression of δEF1 (data not shown). However, further studies will be required for a better understanding of the signal transduction mechanisms regulated by BMP-6 and δEF1 in breast cancer cells.
Breast cancer metastasis is a complicated process, during which the important functions of estrogen and the estrogen receptor have been consistently recognized [56, 57]. In this study, we observed that, in breast cancer cell lines and clinical tumor specimens, δEF1 was expressed at a higher level in ER- cells compared to ER+ cells, indicating that δEF1 might contribute to the malignant conversion of breast cancer cells towards invasive and metastatic phenotypes. On the other hand, the contribution of BMP-6 to tumor metastasis has been recently studied in prostate cancer, suggesting that BMP-6 plays an important role in regulating tumor cell invasion . We recently showed that BMP-6 in ER+ breast cancer cells could be activated by estrogen through promoter demethylation [29, 48]. Considering our finding that BMP-6-induced E-cadherin transactivation occurs indirectly, through the reduction of δEF1 expression, we speculate that overexpression of BMP-6 by breast cancer cells may represent a novel mechanism that regulates specific target genes such as E-cadherin and δEF1, in modulating metastasis and invasion of breast cancer.
Our data suggest that the stimulatory effect of BMP-6 on E-cadherin transcription in breast cancer cells occurs indirectly, through the reduced expression and activity of δEF1. Repressors of BMP-6 and E-cadherin, such as δEF1, may regulate breast tumor progression and metastasis at different stages, including the initial de-differentiation of primary tumor cells and the maintenance of migratory and/or undifferentiated phenotypes. Further studies should be focused on better understanding of the signal transduction mechanisms and the relationship between BMP-6 and δEF1 in breast cancer formation, progression, and metastasis.
This work is supported by a grant from the National Nature Science Foundation of China to S. Yang (No. 30700471). We thank Dr. D. Ji at Global Bioscience, Procter and Gamble Company, USA, for his critical reading and comments on the manuscript We thank Dr. X. Zheng at Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Center, Canada, for his critical reading of the manuscript.
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