The hedgehog pathway on malignant mesothelioma

[Truncated] Malignant mesothelioma is an invasive, locally aggressive tumor, predominantly of the pleura and peritoneum, which is associated with asbestos exposure. Its incidence is increasing worldwide and it has an extremely poor prognosis, due to resistance to conventional treatment modalities, with a median survival of less than one year after diagnosis. Clearly, novel therapeutic strategies are required to improve survival of mesothelioma patients. Mounting evidence supports the aberrant hyperactivation of the Hedgehog (Hh) signaling pathway as crucial to the pathogenesis of certain cancers, including mesothelioma. The Hh pathway is critical for embryonic development and adult homeostasis but hyperactivation of the pathway, through mutations in the Hh pathway genes or overexpression of ligand or receptors, have been shown to drive tumorigenesis.

This thesis examines the general hypothesis that targeting the Hh pathway can be a therapeutic strategy against mesothelioma. More specifically, this thesis aims to:
1. Identify mutations in Hh pathway genes in mesothelioma
2. Functionally characterize the mutations identified through in silico and in vitro analysis
3. Determine the preclinical efficacy of Hh pathway inhibitors, in particular the Gli inhibitor GANT61, on mesothelioma cells in vitro
4. Characterize the biological effects of GANT61 using the mesothelioma cell line LO68.

Real-time PCR analysis of Hh pathway genes PTCH1, GLI1 and GLI2 were performed on seven human mesothelioma cell lines. Exon sequencing of 13 Hh pathway genes was also performed in cell lines and human mesothelioma tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in mesothelioma cells, indicative of active Hh signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 mesothelioma cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function, however, no functional effect
of this mutation on Gli activity was demonstrated. Deletion of exons of the PTCH1 gene and a 3- bp insertion (69_70insCTG) in SMO was also identified in JU77 cells and predicted to alter protein function. Although Hh pathway mutations are relatively rare in mesothelioma, these data suggest a possible role for a dysfunctional Hh pathway in the pathogenesis of a subgroup of mesothelioma patients and help rationalize the exploration of Hh pathway inhibitors for mesothelioma therapy.