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Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

Adrenocortical tumors (ACT) are rare and heterogeneous, but their pathogenesis is unclear. The oncoprotein parathyroid hormone-related protein (PTHrP), found in many common tumors, can regulate their growth in an autocrine/paracrine fashion through the PTH-R1 receptor. Little is known about the role of PTHrP in ACT. We monitored the synthesis of PTHrP and PTH-R1 in a series of 25 ACT: 12 adrenocortical carcinomas (ACC) and 13 adrenocortical adenomas (ACA), and investigated the effects of PTHrP(1-34) on H295R cells derived from an ACC. PTH-R1 mRNA and proteins were detected by real-time PCR and Western blotting in all the ACT samples and in H295R cells. Their concentrations did not differ significantly from one ACT to another. PTHrP mRNA was assayed by quantitative real-time PCR. It was detected in 90% of ACC, and in 10% of ACA. There was a positive correlation with the prognostic factors, McFarlane stage and Weiss score. Tissue-specific PTHrP protein processing was shown by Western blotting. Immunohistochemical staining revealed numerous, dense foci of PTHrP-containing cells in ACC, but few positive cells in ACA or normal tissue. PTHrP stimulated the growth of H295R cells, whereas a specific anti-PTHrP antibody and a PTHrP-R1 antagonist both enhanced their apoptosis. PTHrP activated both adenylate cyclase/protein kinase A and the intracellular calcium/protein kinase C pathways via PTHrP-R1. The active synthesis of PTHrP is linked to poor prognosis in ACC, in which it may act as an autocrine/paracrine factor in tumor growth and malignancy.

Parathyroid hormone-related protein (PTHrP) is produced by many tumors including breast cancer. We have reported that Ets1 factor activates P3 PTHrP promoter in our model of tumorigenic breast cancer cell and not in pre- or non-tumorigenic cell lines, thus contributing to an increased PTHrP production. In this study, gel retardation assays revealed that Etsl and its promoter binding site (EBS) specifically formed complexes whose abundance correlates with Ets1 levels in the three cell lines. Coexpression of Etsl and CBP induced a synergistic activation of the P3 promoter only in the tumorigenic cell line. This synergism required the integrity of the EBS and was abrogated by E1A. All breast cancer cell lines showed high basal concentrations of phosphorylated CREB. Moreover a CRE-like sequence was also required for Ets1/CBP synergy and, finally, CREB expression was found to enhance the PTHrP P3 promoter activity. Thus a multipartite complex of transcription factors and coactivators seems to regulate PTHrP transcription and contribute to the alterations that promote tumorigenic behavior in breast epithelial cells.