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Title: Characterisation of Wnt signalling pathway in rhabdomyosarcoma
Author: Annavarapu, Srinivas Rao
ISNI:       0000 0004 6495 8218
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Introduction: Rhabdomyosarcoma (RMS) remains one of the most challenging tumours in paediatric oncology, accounting for around 5% of all malignant paediatric tumours. Of the two major subtypes of RMS, embryonal and alveolar, the latter portends a poorer clinical outcome. Canonical Wnt signalling pathway is an important evolutionarily conserved signalling pathway that is required for muscle development and embryonal somite patterning. β-Catenin is a potent nuclear transcriptional activator and is the central effector of the canonical Wnt signalling pathway. Interestingly, constitutional activation of Wnt signalling also promotes tissue invasion and metastasis in various tumours. Aims: This study aims to characterise the canonical Wnt/β-catenin signalling in paediatric RMS to assess its functional relevance to the tumourigenesis of RMS and to investigate if modulation of this pathway could provide a therapeutic target for RMS. Results: When we evaluated the immunohistochemical expression of β-catenin in the paraffin-embedded tissues derived from 44 RMS patients, we found positive expression in 26 cases. There was positive expression of β-catenin in the cytoplasm or the cell membrane of alveolar (9/14) and embryonal RMS (15/30); nuclear staining was only seen in two embryonal RMS cases. Next, we assessed β-catenin expression by immunoblot analysis in four RMS cell lines - RD and RD18 (embryonal); Rh4 and Rh30 (alveolar). We were able to demonstrate expression of major canonical Wnt proteins in all cell lines that included: β-catenin, glycogen synthase kinase-3β, disheveled, axin-1, naked and LRP-6. To assess the functional significance of these proteins, we incubated the RMS cell lines with human recombinant Wnt3a to stimulate the Wnt signalling pathway. Thereafter, by using cellular fractionation and immunofluorescence experiments, we demonstrated change in the phosphorylation status of β-catenin, stabilisation of its active form and its nuclear translocation. By employing a TOP/FOP flash reporter gene assay, we showed a T-cell factor/lymphoid-enhancing factor (TCF/LEF)-mediated transactivation. In addition, we found a significant decrease in the proliferation rate of the alveolar RMS cells after Wnt3a stimulation. This decrease in proliferation rate was thought to be due to the concomitant activation of myodifferentiation as seen by the immunoblot expression of myogenin, MyoD1 and myf5. Our data indicates that the major regulatory proteins of the canonical Wnt/β-catenin signalling are expressed in RMS and that functional activation of this pathway, at least in a subset of RMS, may represent a novel therapeutic target.
Supervisor: Helliwell, Tim R. Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral