Analysis of Sonic Hedgehog signalling pathway gene expression in Basal Cell Carcinoma and in GLII induced systems

Abstract

Embryonic development is regulated by a number of signalling pathways, Which are critical for normal growth. Many of these genes then continue to play an important role in the regulation of cell growth and differentiation in adult. One such pathway is the sonic hedgehog (SHH) pathway; SHH protein is secreted which binds to its receptor patched (PTCH), leading to the activation and repression of target genes via zinc-finger GLI family transcription factors. Deregulation of this pathway, leads to a number of human birth defects and diseases such as Basal Cell Carcinoma (BCC) of the skin. In transgenic mouse model systems activation of GLI1 by SHH-signalling is a key step in initiating BCC formation. However, there is limited understanding of the molecular mechanisms involved in response to hedgehog signalling and GLI activity in human BCC formation and how this pathway interacts with other pathways. The aim of this thesis was to establish in vitro and in vivo model systems to investigate the molecular events leading to BCC formation. I have shown that Gl.Il , Gi12, Gi13, PTCH, SMO and KlF4 were induced and a-TUB was repressed in BCC relative to normal skin. Using an in vitro model I further showed that Gl.ll , Gi12, Gi13, PTCH, SMO and a-TUB were induced and KlF4 was repressed in GLII expressing keratinocytes. Collaborative work with Dr Fritz Aberger's laboratory in Salzburg showed that Gl.Il and FOXEI are direct targets of GLI2 and I showed that Gl.I? and FOXEI were expressed in the interfollicular epidermis and the outer root sheath of hair follicles in normal skin as well as in BCC tumour islands suggesting a possible link between hair follicle and BCe. I further showed that epidermal growth factor (EGF) signalling reduces transcription activity of GLI1 by shuttling GLII out of nucleus and altering the expression of PTCH, SMO, Gil2 and Gl.B SHH genes. In addition, I demonstrated that whilst EGF induced Vimentin and Snail2 expression and GLII repressed their expression suggesting that GLI is able to counter epithelial-mesenchymal transition associated with EGF and this may in part explain why Bee very rarely metastasise. Furthermore, GLI1 appears to upregulate stem cell like signature and EGF downregulates this signature. Finally, we were able to generate a KRTI4- Floxed-GFP-Gill transgenic mouse but were unable to activate the target gene (KRTI4-GFP-GiIl). In conclusion, I have identified possible targets of GLI activity and shown interactions between EGF signalling and GLI that will help us to understand the potential molecular actions of SHH signalling with the goal of developing better therapeutic strategies.