Abstract A54: PTHR1 signaling regulates the invasion and differentiation stage of osteosarcoma
Authors: Ho Patricia WM , Russell Megan , Goradia Ankita , Chalk Alistair , Slavin John , Dickins Ross , Martin T. John , Walkley Carl 
Published: May 2015

Osteosarcoma (OS) is the most common tumor of bone. Parathyroid hormone (PTH) is a major endocrine regulator of skeletal development and homeostasis. We have sought to understand the contribution of PTH and parathyroid hormone related protein (PTHrP) in the regulation of osteosarcoma behaviour. In the present study we have used shRNA to knock-down the PTH receptor (PTHR1) to understand the role of PTH/PTHrP signaling in OS.

Methods: shRNA to PTHR1 were designed and screened for activity using cyclic AMP (cAMP) radioimmunoassay following stimulation with PTH(1-34) (1). The most effective shRNA was designated PTHR1.358. OS cell lines derived from Osx-Cre p53fl/flpRbfl/fl mice (2) were stably infected with PTHR1.358 or Ren.1309 (negative control) and tested for gene expression changes, cellular invasion and cell cycle parameters. Cells were tagged with luciferase and grafted subcutaneously to determine the in vivo effects of PTHR1 signaling.

Results: We have made use of an engineered murine model in which p53 and Rb have been deleted in an osteoblast-specific manner using Osx-Cre. Tumors in the mouse model share the cardinal features of human OS including histology, metastatic behavior and gene expression profiles. PTHR1 and PTHrP are expressed in the primary and metastatic lesions, and OS cell cultures derived from the tumors show cAMP induction in response to PTH or PTHrP treatment. Expression analysis confirmed PTHR1 expression in human OS. PTHR1 knock-down did not affect viability, but impaired the invasive capacity of the cells and altered gene expression, notably a reduction in RANKL RNA, an increase in OPG, and increased mRNA for Phex and matrix gla protein (MGP), with the latter effects likely related to the increased mineralization in knockdown cells. Strikingly, PTHR1.358 expressing cells showed a dramatically reduced proliferation in vivo which did not appear due to profound death of the cells but rather, as revealed by microCT, the acquisition of a more differentiated phenotype in the tumors. The Pthr1.358 tumors were smaller and more highly mineralized. PTH treatment did not influence tumor growth in vivo, nor did treatment with neutralizing antibody against PTHrP (3), the likely in vivo ligand for PTHR1 in OS cells. These results together establish clearly the importance of signaling through the PTHR1 in maintaining OS proliferation, but indicate that extracellular PTHrP is not significantly contributing to this effect, making it likely that an intracrine action of PTHrP contributes to this aspect of OS biology.

Conclusions: Collectively these studies demonstrate that PTHR1 signaling plays a cell autonomous role in OS, acting in an intracrine manner. Inhibition of PTHR1 signaling reduced in vitro cell invasion but most notably profoundly reduces OS proliferation in vivo and drives the acquisition of a mineralized phenotype. Therapeutic inhibition of PTHR1 may be useful at impairing OS proliferation and/or increasing differentiation.

1. R. A. Dickins et al., Nat Genet 37, 1289 (Nov, 2005).

2. C. R. Walkley et al., Genes Dev 22, 1662 (Jun 15, 2008).

3. E. Onuma et al., Anticancer Res 24, 2665 (Sep-Oct, 2004).

Citation Format: Patricia WM Ho, Megan Russell, Ankita Goradia, Alistair Chalk, John Slavin, Ross Dickins, T. John Martin, Carl Walkley. PTHR1 signaling regulates the invasion and differentiation stage of osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A54.