Hedgehog Signaling Regulates PTHrP Expression in Osteolytic Breast Tumor Metastases:
The skeleton is the most common site of metastasis in patients with advanced breast cancer. Once established in the skeleton, breast tumors have catastrophic effects on the skeleton inducing a vicious cycle in which tumor produced factors stimulate bone resorption resulting in the release of growth factors from the bone which further stimulate tumor growth and bone destruction. One such factor is parathyroid hormone-related peptide (PTHrP) secreted by breast tumors that have metastasized to bone leads to tumor induced osteolysis and bone destruction. It has been demonstrated previously that inhibition of PTHrP transcription by small molecules reduces bone lesions and tumor burden. It is therefore clearly important to understand the molecular mechanisms responsible for PTHrP expression by breast cancer cells. However, the mechanisms responsible for increased PTHrP expression by breast cancer cells remains largely unknown. We have found that the Gli family of transcriptional regulators are responsible for regulating PTHrP transcription in breast cancer. Specifically we have found that Gli2 mRNA expression in various different cancer cell lines. We found that Gli2 was expressed in osteolytic cell lines that express PTHrP (MDA-MB-231, RWGT2, and PC-3), but not in non-osteolytic cell lines which do not secrete PTHrP (MCF-7, ZR-75, and T47D). When breast cancer cell lines were transiently transfected with a PTHrP promoter luciferase constructs and expression vectors for the Gli family, we found that Gli2 specifically increased PTHrP promoter activity, while Gli3 and Gli1 did not significantly affect basal promoter activity. Using a proteasomally processed, repressor form of Gli3 or a dominant negative Gli2 construct (Gli2-EnR) suppressed the Gli2 stimulated-PTHrP transcription in human MDA-MB-231 breast cancer cells. Furthermore, when MDA-MB-231 human breast cancer cells stably overexpressing Gli2 were inoculated into the left cardiac ventricle of athymic nude mice, enhanced osteolysis as determined by lesion area and trabecular bone volume was observed when compared with mice injected with the empty vector control. Taken together, these data indicate that the Gli2 regulates PTHrP expression and subsequent tumor-induced osteolysis.