Abstract 2386: MMP processing of bone metastatic prostate cancer-derived PTHrP yields novel osteogenic peptides
Authors: Frieling Jeremy Steven , Shay Gemma , Lynch Conor C. 
Published: August 2015

Abstract
Introduction: Bone metastatic prostate cancer lesions are incurable and contain areas of extensive osteoblast (osteogenic) and osteoclast (osteolytic) activity. Understanding how prostate cancer cells elicit these bone effects can identify new therapeutic targets.
Rationale/Hypothesis: Parathyroid hormone-related peptide (PTHrP) stimulates osteoblasts to promote osteoclast mediated bone resorption. Mature PTHrP consists of 36 amino-acids and is susceptible to proteolytic processing. For the first time, we demonstrate that matrix metalloproteinases (MMP-2, -3, -7, and 9), can process PTHrP1-36 into distinct peptides (PTHrP 1-17, PTHrP 18-26, and PTHrP 27-36). We posit that MMP generated PTHrP peptides possess distinct, biological properties and focused our efforts on osteoblasts and osteoclasts based on the well documented effects of tumor derived PTHrP 1-36 on these cell types.
Methods: Osteoblast proliferation was determined by trypan blue and MTT assay. Boyden chamber assays were used to study PTHrP peptide effects on mesenchymal stem cell (MSC) and osteoblast (MC3T3) migration. Morphological changes (10nM peptides for 1 hour) were determined by confocal imaging of actin (phalloidin-488). ERK activity was analyzed by Western blot while cyclic AMP and calcium flux assays were used to examine PTHrP signaling. In vitro osteogenic assays were carried out for 16-days in the presence of PTHrP peptides (10nM) followed by alizarin red staining/quantification. For in vivo calvarial assays, 2μg of PTHrP peptide was injected over the calvaria every 6 hours for 72 hours. Calvariae were subsequently isolated and processed for histological analysis.
Results: MMP generated peptides did not impact osteoblast proliferation. However, migration assays demonstrated that PTHrP 1-17 and PTHrP 1-36 significantly enhanced MSC and MC3T3 migration compared to control (p&lt;0.05) and had profound effects on morphology whereas PTHrP 18-26 and PTHrP 27-36 had no effect on these processes. Focusing further on PTHrP 1-17, we observed rapid ERK phosphorylation and calcium flux within 10 minutes of peptide treatment (10nM), indicating potent biological activity of PTHrP 1-17. PTHrP 1-17 and PTHrP 1-36 also significantly stimulated osteoblast differentiation compared to control (1.6 and 2-fold respectively, p&lt;0.05), but had little or no effect on direct osteoclast formation or activity in vitro. However, in vivo we observed that PTHrP 1-36 induced a robust osteolytic response, an effect that was not observed with PTHrP 1-17.
Conclusion: Our data demonstrate that MMPs can generate distinct PTHrP peptides, of which PTHrP 1-17 significantly stimulates osteoblast migration and differentiation. However, unlike PTHrP 1-36, PTHrP 1-17 does not impact osteoclast activity suggesting a novel role for MMPs in regulating the osteolytic-osteogenic responses commonly observed in bone metastatic prostate cancer.
Citation Format: Jeremy Steven Frieling, Gemma Shay, Conor C. Lynch. MMP processing of bone metastatic prostate cancer-derived PTHrP yields novel osteogenic peptides. [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 2386. doi:10.1158/1538-7445.AM2015-2386