Loss of expression of TIMP3 in clear cell renal cell carcinoma.

In clear cell renal cell carcinoma (CCRCC), vascular endothelial growth factor (VEGF) represents the central positive mediator of tumour angiogenesis while VEGF receptor (VEGFR) is the primary target of anti-angiogenic therapies. TIMP3 is a physiological VEGFR-2 antagonist and thus could be considered as an anti-angiogenic factor. We therefore determined the status of this physiological inhibitor in CCRCC.Archival tumour from 105 patients was studied. TIMP3 expression was analysed using immuno-histochemistry and real-time RT-PCR. Results were correlated with clinicopathological variables. To analyse the mechanisms of gene silencing involved, we performed Multiplex Ligation-dependent Probe Amplification (MLPA) and methylation-specific MLPA (MS-MLPA). At last, we evaluated the main upstream pathway described implicating TGFbetaRII, which induces TIMP3 expression.A down-expression of TIMP3, determined by immunohistochemistry, affected 100/105 renal cancers (95.2%). TIMP3 mRNA levels were significantly lower in high-grade tumours. Loss of heterozygosity of the TIMP3 gene was observed in 8 tumours (7.6%) and the 5'CpG island of the TIMP3 promoter was found to be methylated in 25 tumours (23.8%). A down-expression of TGFbetaRII was found in 85/105 CCRCCs (80.9%). A significant correlation was found between TIMP3 expression and TGFbetaRII expression.This is the first demonstration that the loss of TIMP3 expression is observed in almost all CCRCCs. This loss of expression is a common molecular event in CCRCC. It may be an important initiation step for tumour development in a complex process implicating loss of heterozygosity on chromosome 22q, promoter hyper-methylation and inactivation of the TGFbetaRII pathway.