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Title: Identification of novel molecular targets for cutaneous squamous cell carcinoma
Author: Robinson, Kim
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2014
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Cutaneous Squamous Cell Carcinoma (cSCC) is the most common human tumour with malignant potential which is responsible for over 1 in 4 skin cancer deaths in the UK. High risk groups exist such as, immunosuppressed patients and those with the genetic condition recessive dystrophic epidermolysis bullosa whom are burdened with increased cSCC incidence, metastasis and mortality. Current clinically available molecular targets for cSCC are limited. Moreover, the key molecular events in cSCC remain poorly defined, presumably including both loss/gain of DNA or through gene silencing by DNA modifications. Understanding the molecular events which lead to cSCC is paramount for developing successful, novel molecular targets for therapeutic purpose. Preceding this thesis, our group used two independent methods to identify potential molecular targets: microarray technology (Watt 2011) and Sanger sequencing (Wang 2011), of which some of the targets identified from these studies, formulate the basis of this thesis. Using microarray technology, a comparison between gene expression profiles of cultured cSCC and normal keratinocytes, identified a gene signature specific for cSCC. From this signature, five genes were identified as being over-expressed in cSCC compared to normal and whose gene expression was important for cell survival. Subsequently, three of these genes were further explored within this thesis: germ cell associated 2 (GSG2), bradykinin receptor B1 (BDKRB1) and protease serine 21 (PRSS21). To begin, genes were confirmed to be over-expressed at both mRNA and protein level. Following this conformation, several endpoints were monitored using standard assays of cell viability, proliferation and apoptosis, in cSCC keratinocytes using gene specific siRNA. Using this validation method, PRSS21 showed the most promise as a molecular target. Depletion of PRSS21 resulted in decreased cell viability through an increase in cytotoxicity and apoptosis. Therefore PRSS21 was further validated as a molecular target through stable overexpression in an immortalised normal human cell line with no endogenous PRSS21 expression. Subsequently, using 3D organotypic cultures, it was shown that the expression of PRSS21 clearly transformed these cells into an invasive phenotype, compared to the control. Finally, PRSS21 was shown to interact with the tumour suppressor maspin, potentially negatively regulating maspin dependant apoptosis. This data strongly suggests that both PRSS21 and maspin are potential targets in cSCC. The second method of identifying molecular targets was direct sequencing of tumour DNA, which identified loss of function notch mutations in ~75% of cSCC. It has been postulated that loss of notch function contributes to cSCC through a reduction in differentiation. Using three cSCC cell lines of known notch status (wild type/loss of function/notch null), cells were grown in organotypic culture, and the expression of markers associated with differentiation studied. With the aid of a gamma secretase inhibitor to inhibit notch signalling the contribution of notch to the expression of differentiation markers was studied in cSCC cells compared to normal human keratinocytes (NHK). There was a significant reduction in differentiation within the cSCC cell lines compared to NHK, regardless of notch status, and differentiation could be further reduced by the presence of GSI in the cSCC with wild type/loss of function notch expression. Furthermore, overexpression of a gamma secretase regulatable intracellular notch1 (ICN1) construct, showed an induction of involucrin in these cells, compared to cells overexpressing an empty vector control, confirming that notch1 contributes to differentiation. Overall this work highlights the importance of validating targets before embarking on expensive, time consuming experiments. In doing so, it reveals two potential molecular targets which could be important for the progression of cSCC, PRSS21 and maspin. Additionally it confirms the potential mechanism of notch loss of function in suppressing differentiation, suggesting that its re-activation would be is a valid approach to cSCC therapy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available