Characterisation of a novel in vitro model of Basal cell carcinoma (BCC) through stable PTCH1 suppression in immortalised human keratinocytes
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Basal cell carcinoma (BCC) of the skin is predominantly associated with mutational inactivation of the PTCH1 tumour suppressor gene resulting in constitutive activation of the Hedgehog (HH) developmental pathway. Tumour formation is linked to induction of the GLI (GLI1 and GLI2) transcription factors via a pathway that is thought to require the transmembrane protein SMOOTHENED (SMO) and accordingly, SMO is attracting much interest as a drug target in cancer therapy. However, although there has been a high degree of success in treating some BCCs with anti-SMO compounds, many tumours are only partially or unresponsive which indicates that SMO-independent mechanisms may contribute to tumour formation and/or viability. To further understand how loss of (or reduced) PTCH1 function contributes to BCC, RNAi (retroviral shRNA) was employed to suppress PTCH1 in NEB1 and N/Tert immortalised human keratinocyte cells. Compared to control (shCON) cells, PTCH1 knockdown (shPTCH1) cells displayed more compact colony formation as well as increased GLI1 (but not GLI2) expression however, whereas the increase of GLI1 was suppressed upon transfection with SMO siRNA in shPTCH1 cells, it was insensitive to the presence of the SMO antagonists Cyclopamine-KAAD and SANT1 in shCON cells. The reason for this is unclear but SMO levels were increased and more nuclear in shPTCH1 cells indicating that SMO may have nuclear signalling capability that is unresponsive to certain SMO antagonists. Indeed, cDNA microarray profiling revealed that 80% of the genes that were differentially expressed in NEB1-shPTCH1 cells (>2-fold, p<0.01) remained differentially expressed in the presence of Cyclopamine-KAAD; this includes the chemokines CXCL10 and CXCL11 which were recently shown to be over-expressed in BCC thus helping validate the efficacy of NEB1-shPTCH1 cells as an in vitro tumour model. In addition, functional gene grouping has predicted novel biological processes downstream of PTCH1 that may be important in BCC biology including NF-κB signalling. In summary, the data presented in this thesis suggests that the mechanism(s) through which the loss of PTCH1 leads to BCC formation may be more complex than has been inferred from previous studies.
AuthorsRahman, Muhammad Mahmudur
- Theses