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Title: The WWOX gene : investigation of its function and its role in ovarian tumourigenesis
Author: Gourley, Charlie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Two approaches were taken to the investigation of the role of WWOX in carcinogenesis. Firstly, the HCT 116 colorectal and PEO1 and A2780 ovarian cancer cell lines were transfected with sense and antisense constructs to the WWOX open reading frame and the level of expression of WWOX in the resultant transfectants was quantified using real-time PCR. No in vivo or in vitro phenotypic change was detected in the HCT116 antisense clones. In all PEO1 clones transfected with the WWOX gene there was complete abolition of tumourigenicity in nude mice. The PEO1 parent line is homozygously deleted for the WWOX gene, expresses no full length transcript and produces tumours in nude mice. The abolition of tumourigenicity in the WWOX-transfected PEO1 clones suggested that a functional WWOX pathway was present in the PEO1 cell line system so an in vitro phenotype for the gene was sought. No difference in growth (either in plastic or soft agar), clonogenicity (in presence or absence of cytotoxic drugs), cell aggregation or cell invasion was found. However, the WWOX transfectants displayed decreased cell migration towards fibronectin and decreased attachment to Matrigel compared to controls. In parallel, the expression of the mRNA isoforms of the WWOX gene were quantified using real-time PCR in 71 human ovarian tumours, 13 normal ovaries and in a panel of 37 human cancer cell lines. Full length WWOX expression was significantly lower in the tumours than in the normal ovaries. Two tumours expressed no full-length WWOX mRNA. The WWOX D6-8 mRNA isoforms was expressed at low levels, was significantly associated with high grade and advanced stage ovarian cancer, but was also identified in non-malignant ovarian tissue. Patients whose tumours expressed abundant full-length WWOX had significantly worse survival if they expressed WWOX D6-8. Analysis of the cell lines identified two prostate cancer lines and one further ovarian cancer line with no or extremely low WWOX expression. It also identified cell lines with robust expression of both full length WWOX and one of the alternative transcripts. These latter cell line would be suitable for RNA interference-based knockout of the various isoforms to investigate whether the alternate transcripts act in a dominant negative fashion. In addition, a real-time PCR-based study of HCT 116 colorectal cancer cells exposed to hyaluronidase or cytotoxic agents suggested that induction of WWOX mRNA occurred via both p53-dependent and p53 independent pathways. Therefore, a cell line system with a functional WWOX pathway was identified and the use of clones expressing quantified levels of replaced WWOX mRNA have allowed the identification of an in vitro phenotype for the WWOX gene for the first time in ovarian cancer. Replacement of WWOX decreases tumour cell migration and attachment to extra-cellular matrix components. Hence loss of WWOX in the parent tumour may have increased its ability to migrate and attach to the extra-cellular matrix. The mechanisms by which this may promote ovarian tumourigenesis are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available