Poster Presentation 25th Lorne Cancer Conference 2013

Hedgehog Signaling Maintains a Primitive Osteoblast Phenotype in Osteosarcoma (#130)

Jason Cain 1 , Samantha Jayasekara 1 , Andrew McCaw 1 , Luciano Martelotto 1 , James Deane 2 , Maya Kansara 3 , William Matsui 4 , David Ashley 1 5 , David Thomas 3 , Neil Watkins 1
  1. MIMR, Monash University, Clayton, Vic, Australia
  2. The Ritchie Centre, MIMR, Monash University, Clayton, Vic, Australia
  3. Peter MacCallum Cancer Centre, East Melbourne, Vic, Australia
  4. The Sidney Kimmel Comprehensive Cancer Centre, Johns Hopkins, Baltimore, MD, USA
  5. Deakin University / Barwon Health, Geelong, Vic, Australia
Hedgehog (Hh) signaling regulates patterning, cell-fate and self-renewal in development. Hh proteins signal via Smoothened (Smo), a G-Protein Coupled Receptor whose activity is dependent on translocation to the primary cilium, a single immotile tubulin-based structure present on most mammalian cells. Activation of Smo results in the stabilization of GLI activator transcription factors, inducing Hh pathway gene expression. During development, Hh signaling is required for the specification of osteoblast precursor cells, but is down regulated during terminal differentiation. We hypothesized that deregulation of the Hh pathway perturbs normal osteoblast differentiation, maintains cells in an undifferentiated state leading to tumorigenesis. We show that during development, constitutive activation of Smo in the osteoblast cell lineage induces a dramatic failure of terminal osteoblast differentiation. Furthermore, we demonstrated marked upregulation of Hh ligand expression, and Smo localization to the primary cilia, is a common feature of human osteosarcoma in vivo and in vitro. Inhibition of Hh signaling in human osteosarcoma cell lines using siRNA targeted against SMO, GLI2 and the ciliary kinesin KIF3A lead to a marked reduction in cell growth. Inhibition of SMO activity with the small molecule antagonist LDE225 (Novartis) blocked growth, self-renewal capacity, and induced a gene expression profile consistent with terminal osteoblast differentiation in vitro. Remarkably, this effect was only seen in osteosarcoma cell lines in which primary cilia could be identified by confocal microscopy. In mouse xenograft models, continuous treatment with LDE225 induced sustained cytostatic tumour response. These data suggest that aberrant activation of Hh signaling disrupts normal osteoblast development, and that sustained Hh signaling driven by HH ligand overexpression is a key feature of osteosarcoma. We propose targeting Hh signaling may be of therapeutic benefit in osteosarcoma, and that identification of primary cilia in these tumors could serve as a potential biomarker of Hh pathway dependence.