title: Knockdown of PTHR1 in osteosarcoma cells decreases invasion and growth and increases tumor differentiation in vivo.
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.
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: P W M
authors.initials: PW
authors.lastname: Ho
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Goradia
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: M R
authors.initials: MR
authors.lastname: Russell
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: A M
authors.initials: AM
authors.lastname: Chalk
authors.affiliation: School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
authors.forename: K M
authors.initials: KM
authors.lastname: Milley
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: E K
authors.initials: EK
authors.lastname: Baker
authors.affiliation: School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
authors.forename: J A
authors.initials: JA
authors.lastname: Danks
authors.affiliation: Department of Pathology, St Vincent's Hospital, Fitzroy, Victoria, Australia.
authors.forename: J L
authors.initials: JL
authors.lastname: Slavin
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Walia
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: B
authors.initials: B
authors.lastname: Crimeen-Irwin
authors.affiliation: Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
authors.forename: R A
authors.initials: RA
authors.lastname: Dickins
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: T J
authors.initials: TJ
authors.lastname: Martin
authors.affiliation: St. Vincent's Institute of Medical Research and Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.
authors.forename: C R
authors.initials: CR
authors.lastname: Walkley
mesh_terms: D000818:Animals; D001859:Bone Neoplasms / Q000235:genetics* / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D012516:Osteosarcoma / Q000235:genetics* / Q000473:pathology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D014407:Tumor Cells, Cultured
chemical_list: C473449:PTH1R protein, human; D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1