Quantitative RT-PCR was used to determine the expression of
BMP-6,
E-cadherin, and deltaEF1 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 deltaEF1 at the
protein level in deltaEF1-overexpressing and deltaEF1-interfered MDA-MB-231 cells.
Luciferase assay was used to determine the rhBMP-6 or deltaEF1 driven transcriptional activity of the
E-cadherin promoter in MDA-MB-231 cells. Quantitative CHIP assay was used to detect the direct association of deltaEF1 with the
E-cadherin proximal promoter in MDA-MB-231 cells.
RESULTS: MCF-7
breast cancer cells, an ER+ cell line that expressed high levels of
BMP-6 and
E-cadherin exhibited very low levels of deltaEF1 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 deltaEF1. 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 deltaEF1 was observed in both ER+ cases (4 of 8 cases) and ER- cases (7 of 8 cases). Further, we found that
BMP-6 inhibited deltaEF1 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 deltaEF1 was able to block BMP-6-induced transactivation of
E-cadherin, whereas RNA interference-mediated down-regulation of endogenous deltaEF1 in
breast cancer cells abolished
E-cadherin transactivation by
BMP-6. In addition to down-regulating the expression of deltaEF1,
BMP-6 also physically dislodged deltaEF1 from
E-cadherin promoter to allow the activation of
E-cadherin transcription.
CONCLUSION: We conclude that repression of deltaEF1 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 deltaEF1 expression is associated with more invasive phenotype of
breast cancer cells, our collective data suggests that deltaEF1 is likely the switch through which
BMP-6 restores
E-cadherin-mediated cell-to-cell adhesion and prevents
breast cancer metastasis.