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The von Hippel-Lindau (VHL) tumour suppressor gene is central to the development of sporadic conventional clear cell renal cell carcinoma (ccRCC). The role VHL plays as part of a ubiquitin ligase targeting HIF-a for proteasomal degradation underpins many changes seen in ccRCC but the importance of other VHL functions and the clinical significance of specific genetic/epigenetic changes are not clear. Genetic and epigenetic analysis of VHL gene in 86 tumours from patients with ccRCC was carried out, adding to the 96 tumours already analysed in an ongoing study within the group. Overall, loss of heterozygosity (LOH) was found in 89.2%, mutation in 74.6% and methylation in 30.9%. Evidence of biallelic inactivation (LOH and mutation or methylation alone) was "found in 84.9% whilst no involvement of VHL was found in only 4% of samples, consistent with VHL involvement in the majority of conventional ccRCCs. Associations between mutation and gender (p=0.0189), LOH and grade (p=0.0097) and methylation and grade (p=0.0159) were found with a possible association between methylation and gender (p=0.0835). There was a suggestion of LOH and mutation correlating with a better overall survival compared to patients with no VHL involvement and a similar relationship was seen with methylation; however sample numbers were small in the no VHL group and neither reached statistical significance.

The aim was to measure the expression levels of hypoxia inducible factor 1-α (HJF-lα), glucose transporter one (GLUT-1), and vascular endothelial growth factor (VEGF) isoforms 165 and 189 mRNA in patients with renal cell carcinoma (RCC). Patients with RCC underwent radical nephrectomy at Derriford Hospital, Plymouth. Tumour as well as matched normal tissue from the kidney was harvested, snap frozen and stored in liquid nitrogen, and used to quantitate VEGF 165, VEGF 189, GLUT-1 and HJF-1α mRNA expression using ribonuclease protection assays, and quantified using a Phosphor-imager system. The VEGF 165 isoform was increased in the tumour tissue in comparison with the adjacent normal tissue (3.05 vs. 1.56 P=0.00002) as was the VEGF 189 isoform (2.41 vs. 1.43 P=0.0002). Forty four patients were analysed for the expression of HIF-lα and GLUT-1 with statically significant differences seen between the tumour tissues with respect to the normal tissue for both HIF-lα (1.34 vs. 1.10 P=0.01) and GLUT-1 (1.99 vs. 1.63 P=0.003). Hypoxia inducible factor I (HIF-1) is a key regulator of genes involved in the hypoxic response. HIF consists of alpha and beta subunits, with the alpha subunit being degraded under normoxic conditions and stabilised under hypoxia. Polymorph isms in exon 12 of the HIF gene have been recently been identified consisting of nucleotide changes (C+ 1772T and G+ 1790A) resulting in an amino acid substitution from Proline 582 to Serine, and Alanine 588 to Threonine respectively. These polymorphisms are found within the oxygen-dependent degradation domain (ODD) of the HIF-lα protein when transcribed which is important in the oxygen regulation of the protein via hydroxylation of the proline residue at position 564 (P564) by HIF-α- prolylhydroxylase (HIF-PH). The regulation of HIF-1α by this method is a novel way of regulating the levels of the protein, and polymorphisms in the ODD of HIF-lα may affect the ability of VHL to direct the alpha subunit for destruction. We have genotyped 146 patients and 288 controls for the G+ 1790A, and 160 patients and 162 controls for the C+ 1772T polymorphisms respectively. We found an increase in both the GA and CC (P<0.00001 and P= 0.00002) genotypes in our patients with renal cell carcinoma, and a decrease in GG and CT (P<0.00001 and P=0.00002) genotypes respectively. Haplotype analysis revealed there to be an increase in the T-A and C-A haplotypes (P=0.00008, and P=0.02) and a decrease in the T-G haplotype P=0.01. No statistical difference was found for the other haplotypes. We have shown that these HIF-lα polymorphisms are present in RCC with increased frequency and may play an important role in the disease process, leading to increased angiogenesis in the tumour. Vascular endothelial growth factor (VEGF) is a highly specific mitogen that is able to stimulate the proliferation of endothelial cells. There have been a number of findings linking the expression of VEGF with RCC, with it also being used to assess the prognosis of the disease. Polymorphisms in the VEGF gene have been recently identified. A possible link between promoter polymorphisms and expression of mRNA isoforms has been found in a variety of cytokines. Certain polymorphisms in renal cell carcinoma patients can lead to an up regulation of the expression of the mRNA, and may be a factor in the highly vascularized nature of the tumours studied. The aim was to investigate the frequency of polymorphisms within the VEGF gene (C-2578A) in 173 patients with RCC and 142 normal controls. No differences were seen between the patients and control populations, and the polymorphism did not correlate with Robson stage, Fuhrman grade or age and gender. Although a trend was seen between the C- 2578A polymorphism and expression of VEGF mRNA species in RCC patients.

Hypoxia, a state of low oxygen, is a feature of most solid tumours as a consequence of poor tumour vascularisation. The mechanisms, which allow cancer cells to survive and continue to grow in hypoxia, are coordinated by the transcription factor HIF. The tumour suppressor gene van Hippel-Lindau (vHL) that targets HIF for degradation is mutated in the vast majority of renal cell carcinomas (RCCs), highlighting the importance of hypoxia to tumour biology. There is, therefore, an important need to understand the adaptive changes mediated by hypoxia and to target this clinically. One class of genes regulated by HIF are microRNAs (miRNAs). MiRNAs are short, single stranded RNA that primarily inhibit protein expression from target m RNA. The first aim of this project was to identify novel hypoxia regulated miRNAs in bladder cancer and assess their functional significance. It was found that a number of miRNAs were induced in hypoxic conditions. The hypoxic induction of miR-210 was conserved in all cell lines tested. MiR-145 was found to be highly induced by hypoxia in RT4, a cell line derived from a low- grade, non-muscle invasive tumour. We showed that miR-145 was a novel, HIF target gene with two hypoxia response elements identified within the promoter. Functionally we found that miR-145 induces apoptosis in RT4 cells. MiR-100 was downregulated in hypoxia, but this downregulation did not involve HIF. Regulation of miR-100 was of interest, as it is known to target FGFR3, a gene commonly overexpressed or mutated in bladder cancer. Concomitant with a decrease in miR-100, both the mRNA and protein level of FGFR3 were found to increase in hypoxia in RT4 and RT112 cells. Increased FGFR3 expression in hypoxia was involved in sustaining activation of the downstream signaling targets phospho-PKB and phospho-ERK. In addition, we demonstrate a role for FGFR3 in regulating both 2D and 3D growth and of miR-100 in regulating 3D growth of RT4 cells. We also showed that miR-100 decreased the protein levels of mammalian target of rapamycin (mTOR). However, transfection of miR-l00 into RT4 cells did not affect the sensitivity of this cell line to rapamycin. The genetic and biochemical changes that occur in (hypoxic) tumours may alter their responsiveness to chemotherapeutic agents such as rapamycin. The second aim of this project was to investigate the responsiveness of RCCs to clinically approved chemotherapeutic agents, with the goal of correlating any differences in response to alterations in expression of specific genes. Although hypoxia regulated miR-100 did not affect sensitivity to rapamycin, we extended these studies and investigated the role of vHL status on response of renal cancer cell lines to sorafenib, sunitinib, rapamycin and metformin. We found that the presence of vHL increased resistance to rapamycin. Sensitivity to these drugs was also tested in 10 primary cell lines. There was varying sensitivity to these drugs across the cell lines representing the heterogeneity of renal cancer. We analysed the expression of a number of genes in the m TOR and hypoxic pathways in these tumours, we found the expression of a known hypoxic gene REDDl correlated with sensitivity to rapamycin. REDDl expression levels were also higher in tumour tissue when compared to normal renal parenchymal tissue and was associated with other prognostic markers such as CA9, miR-210 and vascular invasion suggesting a role as a diagnostic or prognostic marker and in patient selection for treatment with rapamycin.

The type 1 insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that mediates diverse cellular functions including growth, differentiation, migration and apoptosis protection. IGF-1R signalling has been implicated in tumorigenesis in a variety of cancers, and IGF-1R inhibitory drugs are currently undergoing clinical evaluation. Previous work in our laboratory has shown IGF-1R over-expression in urological cancers at both the mRNA and protein level, thus making it a potential therapeutic target. The first aim of this project was to develop a protocol for IGF-1R immunohistochemistry, investigate the expression and cellular distribution of the IGF-1R receptor in clear cell renal cell carcinomas (ccRCC), and assess correlation with clinical parameters. In tissue microarray analysis, IGF-1R was detected in ~90% of 195 ccRCCs, with signal in the plasma membrane, cytoplasm and also in the nucleus. The presence of nuclear IGF-1R in up to 50% of ccRCCs and its association with adverse prognosis was a novel finding, and suggests that nuclear IGF-1R may influence ccRCC biology. Further investigations will clarify its role in the nucleus and its potential as a prognostic biomarker. The second aim was to investigate effects of IGF-1R inhibition on radiosensitivity and DNA repair, following previous work in our laboratory showing that IGF-1R depletion enhances chemo- and radio-sensitivity, delays double strand break (DSB) resolution, and may play a role in the homologous recombination (HR) pathway of DNA DSB repair. However, the repair defect seen in these early experiments was larger than could be entirely explained by a defect in HR. The current project used a small molecule IGF-1R tyrosine kinase inhibitor AZ12253801 (AstraZeneca), which blocked IGF-1 induced IGF-1R activation and inhibited cell survival. AZ12253801 enhanced the radiosensitivity of prostate cancer cells, which appeared to be independent of effects of IGF-1R inhibition on cell cycle distribution and apoptosis induction. IGF-1R inhibition delayed the resolution of γH2AX foci, supporting a potential role for the IGF-1R in DSB repair. This delay in focus resolution was apparent at early time-points (less than 4 hr), and was epistatic with DNA dependent protein kinase (DNAPK) inhibition in prostate cancer cells and DNAPK deficiency in glioblastoma cells. These results suggest a role for the IGF-1R in the non-homologous end-joining (NHEJ) pathway of DNA DSB repair. A cell-based reporter assay in HEK-293 cells confirmed that IGF-1R inhibition suppressed DSB repair by NHEJ, helping to explain the radiosensitization demonstrated upon IGF-1R inhibition. There was lack of support for a transcriptional effect, with no significant change observed in gene expression on microarray analysis. Although the mechanism of this effect remains unclear, the observed inhibition of NHEJ has implications for the use of IGF-1R inhibitors in combination with DNA damaging agents in cancer treatment.