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Background: PTHrP, a mediator of humoral hypercalcemia of malignancy, is considered as a potential activator to induce breast cancer cells metastasizing to bone. However, recent clinical evidences and basal research results prove that PTHrP expression in primary tumors indicates good prognosis. BMP-6, as a member of TGF-β superfamily, is closely correlated with tumor differentiation and skeletal metastasis. Purpose: These experiments were designed to investigate the molecular mechanism of PTHrP regulating BMP-6 in breast cancer cells. Methods and results: Through detecting mRNA expression levels of PTHrP and BMP-6 in 35 breast cancer specimens, the two genes' expression were proved to be negatively correlated. Moreover, PTHrP (1-40), instead of PTHrP (107-139), inhibited BMP-6 mRNA expression in MCF-7 cells, indicating that PTHrP exerts its effect on BMP-6 through membranous PTHrP receptor. Inhibitors against signaling pathways downstream of PTHrP were utilized. H89, the PKA pathway inhibitor, eliminated the inhibitory effect of PTHrP on BMP-6. In addition, silencing of BMP-6 strengthened the antimitogenic effect of PTHrP. Conclusions: These results suggest that PTHrP acts as the upstream molecule of BMP-6, and exerts antimitogenic effect via reducing BMP-6 mRNA expression through PKA signaling pathway in breast cancer cells. [ABSTRACT FROM AUTHOR]

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 10μM), 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. [Copyright &y& Elsevier]