The role of Wnt signalling in urothelial cell carcinoma.
PhD thesis, University of Glasgow.
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Urothelial cell carcinoma (UCC) of the bladder is a common malignancy worldwide, causing considerable morbidity and mortality. It is unique among epithelial carcinomas in respect of the fact that it has divergent pathways of tumourigenesis. Low-grade papillary tumours which frequently recur, but seldom convert to muscle invasive tumours harbour mutations that activate the MAPK pathways, as a consequence of oncogenic mutations in FGFR3 or HRAS. However, in contrast, the high-grade muscle invasive tumours that readily metastasise have been shown to have defects in the p53 and retinoblastoma (RB) protein pathways
Transgenic mice have allowed us to analyse the molecular basis of initiation, invasion and progression of many human cancers. These mouse models increase our understanding of the disease process as well as providing targets for developing novel therapeutic approaches. In UCC there has been a paucity of models that readily mimic the human disease.
Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. Recent studies have demonstrated the importance of this pathway in UCC progression, thus I endeavoured to test its importance both as a “driver” mutation, as well as a “progressor” mutation in more established UCC mutations. Thus I targeted expression of an activated form of β-catenin to the urothelium of transgenic mice using Cre-Lox technology. Expression of this activated form of β-catenin led to the formation of localised hyperproliferative lesions by 3 months, which did not progress to malignancy. Furthermore expression in UroIICRE+ β-cateninexon3/+ mice showed marked upregulation of the PTEN tumour suppressor protein that appears to be a direct consequence of activating Wnt signalling in the bladder. I therefore combined PTEN deficiency with β-catenin activation, which resulted in rapid formation of papillary UCC by 6 months. These tumours had increased pAKT signalling and were dependent on mTOR. Importantly in human UCC, there was a significant correlation between high levels of β-catenin and pAKT (and low levels of PTEN) (p<0.01, n=80). Taken together, these data suggest that deregulated Wnt signalling plays a role in driving UCC, and human UCC that have high levels of Wnt and PI3 kinase signalling may be responsive to mTOR inhibition.
I next expressed oncogenic K-Ras or H-Ras in the urothelium alone, and in urothelial cells expressing an activated β-catenin. Although Ras activation was not sufficient to drive tumourigenesis, Ras activation combined with β-catenin activation in UroIICRE+ β-cateninexon3/exon3 K-RasG12D/+ and UroIICRE+ β-cateninexon3/exon3 H-RasQ61L mice rapidly developed UCC. These tumours had upregulation of pERK1/2 with minimal levels of pAKT. Importantly in human UCC, there was a significant correlation between high levels of β-catenin and pERK1/2 (p<0.01, n=80). This data further supports the role of deregulated Wnt signalling and its co-operation with Ras in bladder carcinogenesis.
I observed upregulated p21 expression in our UroIICRE+ β-cateninexon3/+urothelial lesions and thus postulated that p21 may be acting as a block to tumourigenesis in the lesion. It was highly relevant to observe tumour formation in the double mutant UroIICRE+ β-cateninexon3/exon3 p21-/- mice.
FGFR3 gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 mutations as a “driver” of UCC, expression of mutated Fgfr3 was targeted to the murine urothelium using UroIICRE+ promoter. These FGFR3 mutations (K644E and K644M) had no effect on bladder homeostasis or tumourigenesis up to 18 months of ages. Even when these mutations were combined with β-Catenin or Ras activating mutations, no urothelial dysplasia/hyperplasia or UCC was observed. This suggests that other alterations are required that can cooperate with FGFR3 activation to cause UCC. Interestingly, however, due to sporadic ectopic Cre recombinase expression in the lung and skin of these mice, FGFR3 mutation caused skin papilloma and promoted lung tumourigenesis in cooperation with K-Ras and β-Catenin activation, respectively. This confirmed that the mutant Fgfr3 was functional and that FGFR3 cooperates with other genetic events involving Ras and Wnt pathways to promote tumourigenesis in a context dependent manner and support the hypothesis that activation of FGFR signaling contributes to human cancer.
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