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Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo

Oncogene volume 34, pages 2922–2933 (2015)Cite this article

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Abstract

Osteosarcoma (OS) is the most common cancer of bone. Parathyroid hormone (PTH) regulates calcium homeostasis and bone development, while the paracrine/autocrine PTH-related protein (PTHrP) has central roles in endochondral bone formation and bone remodeling. Using a murine OS model, we found that OS cells express PTHrP and the common PTH/PTHrP receptor (PTHR1). To investigate the role of PTHR1 signaling in OS cell behavior, we used shRNA to reduce PTHR1 expression. This only mildly inhibited proliferation in vitro, but markedly reduced invasion through collagen and reduced expression of RANK ligand (RANKL). Administration of PTH(1–34) did not stimulate OS proliferation in vivo but, strikingly, PTHR1 knockdown resulted in a profound growth inhibition and increased differentiation/mineralization of the tumors. Treatment with neutralizing antibody to PTHrP did not recapitulate the knockdown of PTHR1. Consistent with this lack of activity, PTHrP was predominantly intracellular in OS cells. Knockdown of PTHR1 resulted in increased expression of late osteoblast differentiation genes and upregulation of Wnt antagonists. RANKL production was reduced in knockdown tumors, providing for reduced homotypic signaling through the receptor, RANK. Loss of PTHR1 resulted in the coordinated loss of gene signatures associated with the polycomb repressive complex 2 (PRC2). Using Ezh2 inhibitors, we demonstrate that the increased expression of osteoblast maturation markers is in part mediated by the loss of PRC2 activity. Collectively these results demonstrate that PTHR1 signaling is important in maintaining OS proliferation and undifferentiated state. This is in part mediated by intracellular PTHrP and through regulation of the OS epigenome.

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Acknowledgements

We thank the SVH BioResources Centre; P Kocovski and S Taylor for technical assistance, L Purton and J Heierhorst for comments and discussion. This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC; to CRW and CRW/TJM); Cancer Council of Victoria (to CRW and EKB); NHMRC Career Development Award (CRW); Cure Cancer Australia Foundation Fellowship (EKB); in part by the Victorian State Government Operational Infrastructure Support Program (to St. Vincent’s Institute). CRW is the Philip Desbrow Senior Research Fellow of the Leukaemia Foundation. The authors thank Chugai Pharmaceutical Company for providing the α-PTHrP neutralizing antibody; The Structural Genomics Consortium for generously providing GSK343 and UNC1999.

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  1. T J Martin and C R Walkley: These authors contributed equally to this work.

Authors and Affiliations

  1. St. Vincent’s Institute of Medical Research and Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Fitzroy, Victoria, Australia

    P W M Ho, A Goradia, M R Russell, A M Chalk, E K Baker, M Walia, B Crimeen-Irwin, T J Martin & C R Walkley

  2. School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia

    K M Milley & J A Danks

  3. Department of Pathology, St Vincent's Hospital, Fitzroy, Victoria, Australia

    J L Slavin

  4. Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    R A Dickins

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  1. P W M Ho
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Ho, P., Goradia, A., Russell, M. et al. Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo. Oncogene 34, 2922–2933 (2015). https://doi.org/10.1038/onc.2014.217

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