VHL GENE ALTERATIONS PROMOTE CANCER PROGRESSION IN EARLY-STAGE CLEAR CELL RCC THROUGH NUCLEAR LOCALIZZATION OF HYPOXIA-INDUCIBLE FACTOR-1ALFA

INTRODUCTION AND OBJECTIVE: Von Hipple-Lindau gene VHL (VHL) inactìvation represents the most frequent abnormality in clear cell RCC. VHL encodes two different proteins: VHL30 and VHL19 arising as a result of an alternative translation initiation. Hypoxia-inducible factor-1 (HIF-1) is a heterodìmer composed of a alfa and beta subunit. HIF-1beta is constitutively expressed, while HIF-1alfa expression is regulated by 02 level. In normal 02 conditions VHL binds after hydroxylation in ODD (oxygen-dependent degradation domain) and it allows HIF-1alfa proteasomal degradation. In this domain a SNP at codon 582 is located. In hypoxia, VHL/HIF-1 alfa interaction is abolished and HIF-1alfa activates target genes in the nucleus. The study analiyzes the VHL and codon 582 of HIF1alfa gene (HIF-1alfa) status in clear cell RCC, correlating genetic alterations, protein expression and subcellular localization with prognosis. METHODS: Tissues microarray from 136 intracapsular clear cell RCC cases were constructed and two anti-pVHL Ab (Ig32 and Ig33) and a polyclonal anti-HIF-1alfa Ab were used for IHC analysis. Mean follow up was 117 months. It was performed mutational analysis for the whole VHL and for HIF-1alfa SNP (C1772T), promoter methylation of VHL and LOH (3p25). The results were correlated with survival (TSS). RESULTS: VHL gene alteration was found in 57% of cases. VHL mutations, methylation and LOH were detected in 51%, 11% and 17% of cases. Patients with biallelic VHL alteration had a shorter TSS (p=0.01). We found a statistical association between biallelic VHL alteration and no pVHL expression (p=0.004) and nuclear localization of HIF-1alfa (1 (p=0,04). The SNP was associated only with a cytoplasmic localization of HIF-1alfa. (p=0.007). pVHL negativity and HIF-1alfa nuclear positivity were correlated with a short TSS. CONCLUSIONS: VHL and HIF-1alfa alterations influence their protein expression and localization. Biallelic VHL gene alterations with loss of pVHL expression are associated to nudear localization of HIF-1alfa and poor prognosis in early-stage clear cell RCC. Alteration of pVHL/HIF-1alfa pathway is an early event in clear cell RCC carcinogenesis and it is involved in cancer progression through nuclear localizzation of HIF-1alfa.