Background: PTHrP is produced by various osteotropic malignancies and promotes progression of bone metastasis. PTHrP(12-48) is a unique, proteolytically-derived fragment of full length PTHrP and is the first PTHrP fragment, identified in the serum of breast cancer patients. Serum PTHrP(12-48) levels in breast cancer patients, correlate with a high sensitivity and specificity with the presence of bone metastasis. The purpose of this study was to define the role of PTHrP(12-48) in the metastatic breast tumor microenvironment. PTHrP(12-48) activity at the cellular level, was examined by PTHrP(12-48) agonist/antagonist activity at the PTH1 receptor (PTH1R), examination of subcellular localization and the effects of PTHrP(12-48) on cell proliferation, survival and osteoclast differentiation.
Methods: To test the agonist/antagonist activity of PTHrP(12-48), c-AMP and IP3 assays were performed in cells expressing the PTH1R, in the presence of potent PTH1R agonists (PTH 1-34) and antagonists (PTH7-34). Immunolocalization of PTHrP(12-48) was examined by immunohistochemistry (IHC) using a specific anti-PTHrP(12-48) antibody and by immunofluorescence in PTH1R expressing human cells and human peripheral blood mononuclear cell (PBMC)-derived osteoclasts, which lack PTH1R. Cellular proliferation was measured in PTH1R expressing and PTH1R lacking cells. In addition, the effect of PTHrP(12-48) on human osteoclast precursor survival and osteoclast differentiation was assessed in receptor activator of nuclear factor kappa beta ligand (RANKL) treated PBMC cultures.
Results: The data demonstrate that PTHrP(12-48) is not an agonist or an antagonist of the PTH1R receptor. IHC demonstrated positive immunostaining in primary breast tumors and bone metastases, whereas normal breast tissue was negative. Immunofluorescence successfully identified the cytoplasmic localization of PTHrP(12-48), 90 minutes after addition of ligand in PTH1R expressing cells. In contrast, no cytoplasmic staining was observed in PTH1R lacking cells. In addition PTHrP(12-48) significantly inhibited serum-induced proliferation of PTH1R expressing cells, but not PTH1R lacking cells. Interestingly, although human osteoclast precursors lack PTH1R expression, PTHrP(12-48) treatment strongly inhibited the survival and proliferation of PBMCs as well as their differentiation into osteoclasts.
Conclusions: PTHrP(12-48) is a circulating bone metastasis biomarker that is expressed by primary breast tumors as well as bone metastasis. PTHrP(12-48) does not interact productively with the PTH1R in terms of second messenger activation. However, treatment of cells with PTHrP(12-48) results in cellular uptake by PTH1R expressing cells, with a resultant inhibition of serum-induced cell proliferation and osteoclastogenesis.
Citation Format: Varinder Kaur, Archana Kamalakar, Nisreen Akel, Kim Leitzel, Allan Lipton, Larry J. Suva. PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2015-1041