Abstract
BackgroundBone 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.MethodsQuantitative 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.ResultsMCF-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.ConclusionWe 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.
Highlights
Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes
Our preliminary studies indicated that expressions of BMP-6 and δEF1 were inversely related in MCF-7 and MDA-MB-231 cells
FExigpurersesi1on of BMP-6, E-cadherin, and δEF1 in MCF-7 and MDA-MB-231 breast cancer cells is inversely correlated Expression of BMP-6, E-cadherin, and δEF1 in MCF-7 and MDA-MB-231 breast cancer cells is inversely correlated. (a) Transcript levels of BMP-6, E-cadherin, and δEF1 in MCF-7 and MDA-MB-231 breast cancer cells were detected by RT-PCR analysis
Summary
Bone morphogenetic protein-6 (BMP-6) is critically involved in many developmental processes. 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 [7]. A few factors, including ErbB2 [18], TGF-β [19], and estrogen [20], were reported to regulate E-cadherin expression
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