Chaudhry, S.I.; (2010) Analysis of squamous cell carcinoma invasion identifies a role for TRAF6. Doctoral thesis , UCL (University College London).

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Abstract

The principal objectives of the work in this thesis were: (a) to investigate cancer cell invasion in squamous cell carcinoma (SCC) by examining the role of ubiquitin-linked pathways in the regulation of the actin cytoskeleton and (b) to establish a human tissue bank of SCCHN specimens matched to adjacent non-cancerous tissue. An siRNA screen was performed with the premise that genes that regulate the actin cytoskeleton would be involved in the invasion of cancer cells. 289 E3 ubiquitin ligases and 137 de-ubiquitinating enzymes (DUBs) were screened yielding 3 gene hits (RKHD2, TRAF6 and ZA20d1) capable of regulating both the F-actin cytoskeleton of SCC cells as well as their collective invasion in a three-dimensional organotypic invasion system. A secondary morphological and functional screen of 20 E3 ligases and 9 DUBs in carcinoma-associated fibroblasts demonstrated that 5 genes (MKRN2, RKHD2, TRAF6, ZA20d1 and USP6) influenced the ability of stromal fibroblasts to promote SCC invasion. Following transcriptional analysis using qPCR, the E3 ligase TRAF6 and its complementary DUB ZA20d1 emerged as candidate genes for detailed study. TRAF6 was shown to influence NF-ĸB signalling in SCC cells by modulating the levels of IĸBα. Given the importance of NF-ĸB activation in the pathogenesis of SCC, I investigated its significance in our organotypic system. Using TNFα, IL-1α and several IKK inhibitors, I demonstrated that SCC invasion was dose-responsive and NF-ĸB dependent. Furthermore, infliximab (a monoclonal antibody to TNFα in clinical use) inhibited carcinoma invasion at a therapeutic dose. Ethical and R&D approval for the establishment of a human tissue bank was obtained and SCCHN tumour samples with patient matched control tissue are now being banked. Analysis of these specimens and commercial tissue microarrays indicates that TRAF6 is overexpressed in a subset of SCC.

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