We evaluated the capacity of estradiol (E2) to regulate PTHrP production, cell growth, tumor growth, and metastasis to the skeleton in breast cancer. In estrogen receptor (ER)-negative human breast cancer cells, MDA-MB-231, and cells transfected with full-length cDNA encoding ER (S-30), E2 caused a marked decrease in cell growth and PTHrP production, effects that were abrogated by anti-E2 tamoxifen. E2 also inhibited PTHrP promoter activity in S-30 cells. For in vivo studies, MDA-MB-231 and S-30 cells were inoculated into the mammary fat pad of female BALB/c nu.nu mice. Animals receiving S-30 cells developed tumors of significantly smaller volume compared with MDA-MB-231 tumor-bearing animals. This change in tumor volume was reversed when S-30 cells were inoculated into ovariectomized (OVX) hosts. Inoculation of MDA-MB-231 cells into the left ventricle resulted in the development of lesions in femora and tibia as determined by x-ray analysis. In contrast, these lesions were significantly smaller in volume and number in animals inoculated with S-30, and this lower incidence was reversed in OVX animals. Bone histological analysis showed that the tumor volume to tissue volume ratio was comparable with that seen by x-ray. Immunohistochemical analysis showed that PTHrP production was inhibited in S-30 group and restored to levels comparable to that seen in MDA-MB-231 tumor-bearing animals when S-30 cells were inoculated in OVX animals. Collectively these studies show that E2 production is inversely correlated with PTHrP production and that the growth-promoting effect of PTHrP has a direct impact on tumor growth at both nonskeletal and skeletal sites.

PTHrP is the major pathogenetic factor for hypercalcemia in several malignancies including prostate cancer. In the current study, we have assessed the ability of androgens to regulate PTHrP production in androgen-insensitive human prostate cancer cells PC-3 and cells transfected with androgen receptor (PC-3T). Androgen responsiveness caused a marked decrease in PC-3T cell growth, and treatment of these cells with dihydrotestosterone led to inhibition of PTHrP production. These inhibitory effects were readily reversed by androgen receptor antagonist flutamide. To determine the effect of androgens on tumor growth and PTHrP production in vivo, PC-3 and PC-3T cells were injected into the right flank of male BALB/c nu/nu mice. Animals inoculated with PC-3 and PC-3T cells developed palpable tumors at wk 2 and 4, respectively. Inoculation of PC-3T cells into castrated animals resulted in rapid tumor growth in PC-3T tumors, effects that were reversed in PC-3T tumors grown in castrated hosts. Using PTHrP promoter luciferase reporter, a 30% decrease in luciferase activity was seen following treatment with dihydrotestosterone. These results indicate that PC-3 cell growth correlates inversely with androgen sensitivity and directly with PTHrP production in vitro and in vivo, androgens can regulate PTHrP production, and the androgen effect on PTHrP is mediated at least in part by transcriptional regulation via the androgen receptor.