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.