The role of FGFR3 mutation in tumour initiation, progression and invasion of urothelial cell carcinoma in mice

Foth, Mona (2014) The role of FGFR3 mutation in tumour initiation, progression and invasion of urothelial cell carcinoma in mice. PhD thesis, University of Glasgow.

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Abstract

Bladder cancer is the 5th most common and the 9th most lethal cancer in the UK. Based on histopathological and genomic analysis, a model of two independent pathogenesis pathways has been suggested, resulting in either non-invasive superficial or invasive urothelial tumours with potential to metastasise. Prominently, the fibroblast growth factor receptor 3 (FGFR3) is found mutated in up to 84% of non-invasive superficial tumours. Alterations in FGFR3 such as mutation or wild type receptor overexpression are also found in 54% of muscle-invasive tumours. FGFR3 is a tyrosine kinase receptor for fibroblast growth factors (FGFs), which stimulates both the RAS/MAPK and the PI3K/AKT pathways and regulates a range of cellular processes such as cell growth and division during development. In this study we examined the role of FGFR3 in bladder cancer by using mice as a model organism. Firstly, we addressed whether combination of Fgfr3 and Pten mutation, UroIICre Fgfr3+/K644E Ptenflox/flox, is able to drive non-invasive superficial bladder cancer. We observed that the thickness of the double mutant urothelium was significantly increased compared to singly mutated Fgfr3 or Pten, UroIICre Fgfr3+/K644E and UroIICre Ptenflox/flox. Moreover, several cellular abnormalities were detected that were accompanied by differential expression of layer-specific markers, which strongly suggested that they were caused cooperatively by Fgfr3 mutation and Pten deletion. The results supported the hypothesis that FGFR3 activation can play a causative role in urothelial pathogenesis of non-invasive superficial bladder cancer together with upregulated PI3K-AKT signalling. Secondly, we aimed to identify mutations that cooperate with Fgfr3 and with other common bladder cancer mutations such as Pten and Ras, in promoting urothelial tumourigenesis by Sleeping Beauty (SB) insertional mutagenesis in mice. The SB system may constitute an inefficient tool in the bladder to induce urothelial tumourigenesis, since it failed to produce bladder tumours in Fgfr3 as well as in Hras mutant mice. In mice with Pten deletion, one tumour was generated and general hypertrophy with cellular abnormalities was observed in all samples. No direct association between Fgfr3 and Pten mutations was found; however, SB mutagenesis supported that Fgfr3 and Pten cooperation may merge at the signalling downstream. Thirdly, we examined the role of the most common mutation in FGFR3, S249C, in the urothelium and in tumour progression and invasion by subjecting Fgfr3 mutant mice to a bladder-specific carcinogen, N-butyl-N-(hydroxybutyl)-nitrosamine (OH-BBN). We showed that FGFR3 S249C mutation by itself does not lead to urothelial abnormalities. However, in OH-BBN-induced tumours the presence of S249C increased the number of animals that formed bladder tumours by 4.4-fold. Our results present for the first time an effect of FGFR3 S249C mutation in invasive bladder cancer. Lastly, we sought to establish methods to generate and assess invasive bladder tumours using in vivo and in vitro techniques. First we examined the effectiveness of a Cre-expressing adenovirus (AdenoCre) to generate mouse models of bladder cancer with different combinations of genetic mutations. p53 deletion or mutation together with Pten loss led to formation of aggressive bladder tumours; however the origin of these tumours was likely to be the bladder muscle. Hras activation in combination with Pten deletion did not produce tumours or any cellular abnormalities by 8 months. AdenoCre-mediated tumour induction was successful in the presence of β-catenin and Hras mutation. However, an issue of AdenoCre transduction was the frequent observation of tumours in various other tissues such as the pelvic soft tissue, liver, pancreas and lung. Using an optimised AdenoCre procedure, the technique would allow lineage tracing of cancer stem cells in a developing bladder tumour and potentially during metastatic spread. Secondly, we tested imaging techniques in the living animals and validated ultrasound as a functional method to detect bladder wall thickening, as well as to monitor tumour growth in vivo. Thirdly, with the aim to assess cell transformation, migration and response to drug treatment, we tested essential ex vivo techniques and assays such as 3D sphere culture, organotypic slice culture as well as a Collagen-I invasion assay. The 3D tumour sphere culture was successful with murine Wnt-activated tumours as well as with invasive human cell lines. The organotypic slice culture was assessed as a system to test the effect of therapeutic drugs on the tumour cells; however, an issue of tissue disintegration has yet to be overcome. The Collagen-I assay successfully recapitulated invasion of a human bladder cancer cell line; however, the system needs to be adapted to murine bladder tumours. Taken together, this study presents for the first time evidence that support the functional role of FGFR3 signalling in the early stages of non-invasive urothelial carcinoma as well as in tumour progression of established neoplasms in mice. Given the wide availability of inhibitors specific to FGF signalling, our FGFR3 mouse models in conjunction with optimised ex vivo assays and imaging systems may open the avenue for FGFR3-targeted translation in urothelial disease.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Urothelial cell carcinoma, transgenic mouse model, fibroblast growth factor, bladder cancer, Sleeping Beauty mutagenesis, AdenoCre, organotypic cell culture
Subjects: Q Science > Q Science (General)
R Medicine > R Medicine (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences > Beatson Institute of Cancer Research
Funder's Name: UNSPECIFIED
Supervisor's Name: Iwata, Dr. Tomoko and Sansom, Prof. Owen
Date of Award: 2014
Depositing User: Ms Mona Foth
Unique ID: glathesis:2014-5642
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 06 Nov 2014 10:37
Last Modified: 06 Nov 2014 10:38
URI: http://theses.gla.ac.uk/id/eprint/5642

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