Molecular complexities of patched signaling in cancer development

Sammanfattning: Basal Cell Cancer (BCC) is the most common cancer in the Western world. Although BCCs hardly ever metastasize, invasive growth may cause considerable local tissue destruction. BCCs mainly occur as sporadic tumors but can also be found in a hereditary form in the Nevoid Basal Cell Carcinoma Syndrome (NBCCS). The gene underlying the NBCCS is Patched1 (PTCH1), a tumor suppressor gene that is mutated or deleted in these patients. This thesis is focusing in deciphering complexities that are associated with signal tranduction in BCCs and was initiated by the cloning of a second receptor of the Hedgehog (HH) ligand, Patched2 (PTCH2). In similarity to PTCH1, PTCH2 was found to be up-regulated in BCCs, however in contrast to PTCH1 no mutations have been detected in this gene in BCCs. Thus PTCH2 overexpression cannot compensate for a defective PTCH1 allele implying that either the inherent properties of these proteins or the timing and cellular specificity of expression are distinct. An interesting observation during the analysis of PTCH2 expression was that in tissues that included skin and testis a number of PTCH2 splice variants have been detected. Some of these variant mRNA forms appeared to be of potential functional significance as the open reading frame was conserved. Consequently efforts were focused into the analysis of the functional properties of the proteins encoded by the PTCH2 splice variants as well as their relation to PTCH1. Although cellular localization assays or the capacity to internalize Sonic Hedgehog (SHH) did not reveal major differences, the splice variants had distinct properties in influencing signaling as revealed by co-transfection of a reporter gene. In fact the reporter gene used was PTCH2 providing unambiguous evidence that it represents a direct target of HH signaling. These transfection assays established that PTCH1 is a stronger inhibitor of SHH than the PTCH2 splice variants and inclusion of both the internal exon domains and the alternative 3 end is necessary for increased PTCH2 inhibition. Using a Ptch1-/- fibroblast cell line it was also shown that the PTCH2 splice variant with the strongest inhibitory activity is also most effective into reconstituting Desert Hedgehog (DHH) signaling, albeit to a lower extent than that of PTCH1. PTCH1 gene expression is characterized by the presence of three distinct first exons. Each of these is individually spliced with exon two resulting in mRNAs with unique 5 ends. Interestingly in BCCs, overexpression of only one of the three PTCH1 mRNAs is observed implying that signaling dysregulation does not affect PTCH1 expression at large but solely the production of a specific mRNA. By the use of transfection assays it was shown that the PTCH1 protein encoded by the up-regulated mRNA is most effective as an inhibitor of signaling initiated by the PTCH1 interacting protein, Smoothened (SMO). An unexpected finding in the analysis of HH signaling was the observation that gene activation by the transcription factor GLI1 is apparently down-regulated by PTCH1. Although activation by GLI1 is known to be inhibited by the signaling component SU(FU) and the critical amino acids for this interaction have been mapped in GLI1, these are not involved in the observed PTCH1 mediated down-regulation. Deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180 to 786 and 1058 to 1210 are of highest significance in inhibiting GLI1 gene activation.