The Hedgehog (Hh) signalling pathway acts as a key mediator of many fundamental processes, such as cell fate specification, proliferation and patterning, as well as tissue morphogenesis and homeostasis. Misregulation of Hh signalling has been described in many malignancies, including breast and ovarian cancer, melanoma and medulloblastoma. Pathway activation is initiated by binding of one of the three secreted and lipid-modified ligands to Patched (Ptch1), a 12-pass transmembrane-spanning receptor (Fig. 1). In the absence of a ligand, Ptch constitutively represses the activity of Smoothened (Smo), a 7-pass transmembrane-spanning protein with homology to G-protein-coupled receptors. Following Hh ligand binding to Ptch, the repression of Smo is released, and the expression and/or post-translational processing of the three Gli zinc-finger transcription factors is modulated. Gli1 acts as a transcriptional activator and Gli3 as a repressor, whereas Gli2 can either activate or repress gene expression. The balance between the activating and repressive forms of the Glis results in the expression of the incompletely described target genes, including Ptch1 and Gli1.
The screening for small molecules inhibitors of Hh signalling will provide valuable tools for dissecting the biochemistry and biology of the pathway, and will be the basis for the development of novel drug discovery programs aimed at cancer.
The C3H/10T1/2 differentiation assay was used as a model for Hh signalling. In the C3H/10T1/2 cells, activation of Hh signalling leads to osteogenic differentiation which can be quantified by the measurement of the osteogenic marker alkaline phosphatase. In the assay, the Hh pathway is induced in the cells using the small molecule purmorphamine. After 5 days, the level of alkaline phosphatase is measured using a luminogenic substrate (CDP-Star, Roche). Cell viability is measured in parallel. Hit criteria were at least a 50% reduction in the Hh signal and a minimum of 80% cell viability. Dose response analysis was carried out for selected hit compounds.
Fig. 1: Principle of the Hedgehog osteogenesis assay.