AbstractEpidermolysis Bullosa encompasses a group of inherited heterogeneous diseases involving trauma induced blistering of the skin. Recessive Dystrophic Epidermolysis Bullosa (RDEB) is one of the most debilitating variants of the disease and patients are predisposed to developing aggressive cutaneous Squamous Cell Carcinoma (SCC). Unlike SCC in the general population, the primary cause of RDEB associated SCC is not UV-radiation. SCC in RDEB patients has poor prognosis due to a high frequency of recurrence and metastasis. 70% of all severe generalized RDEB patients die from SCC by the age of 45, compared to only 1.25% of all patients with UV-induced SCC in the general population (Fine et al. 2008), making SCC the leading cause of death in these RDEB patients. The aim of this investigation was to identify therapeutic targets for RDEB associated SCC. Global gene expression studies identified 36 candidate genes which were differentially regulated in RDEB SCC (n=4) compared with non-RDEB SCC (n=5) primary keratinocyte cultures. The validation of these genes by qRT-PCR in replicate cultures of RDEB SCC (n=3), non-RDEB SCC (n=3) keratinocytes and normal human keratinocytes as a control, deduced 5 genes to be significantly differentially regulated. Of particular interest, is the over-expression of SLCO1B3 by 6.25 fold in RDEB SCC keratinocytes (p = 0.035). SLCO1B3 encodes the organic anion transporter OATP1B3, which is normally exclusively expressed on the basolateral membrane of hepatocytes. qRT-PCR revealed the mRNA expression level of SLCO1B3 is reduced in RDEB SCC keratinocyte cultures when COL7A1, the causal gene mutated in RDEB, is re-expressed, suggesting that COL7A1 which encodes the Type VII Collagen protein and is secreted into the extracellular matrix, may suppress the transcription of SLCO1B3. Immunofluorescent staining of RDEB SCC keratinocytes and tissue identified OATP1B3 expression, whilst qRT-PCR using mRNA isolated from freshly frozen skin and SCC tissue samples from both RDEB and non-RDEB individuals identified increased SLCO1B3 mRNA expression in RDEB SCC in vivo. Over expression of SLCO1B3 and increased activity of OATP1B3 is associated with breast, colon and pancreatic cancer and is a known transporter of chemotherapeutic agents, such as Methotrexate and Paclitaxel. These observations have led to speculation that, as a transporter over expressed in cancer and capable of introducing drugs into cells, OATP1B3 represents a potential therapeutic target. Preliminary results from a cell viability assay suggest that exposing RDEB SCC cells to Microcystin-LR specifically reduces cell viability in a SLCO1B3 dependent manner. This supports the conclusion that SLCO1B3 represents a viable RDEB SCC specific therapeutic target and provides a pathway which can be exploited to deliver anti-cancer drugs directly to tumour cells whilst reducing the systemic toxicity of these agents.
|Date of Award||2011|
|Supervisor||Andrew South (Supervisor) & Irene Leigh (Supervisor)|
Identification of OATP1B3 as a potential therapeutic target in Recessive Dystrophic Epidermolysis Bullosa Associated Squamous Cell Carcinoma
Cole, C. L. (Author). 2011
Student thesis: Doctoral Thesis › Doctor of Philosophy