A STUDY OF THE MOLECULAR GENETICS OF RENAL CELL CARCINOMA IN MAN

Abstract

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.