Amplification of 6p22 occurs in about 10-20% of bladder cancers and is associated with enhanced tumour cell proliferation. Candidate target genes for the 6p22 amplicon include E2F3 and the adjacent gene NM_017774. To clarify which gene is representing the main target, we compared the prevalence of the amplification and the functional role of both genes. Amplification of E2F3 and NM_017774 was analysed by fluorescence in situ hybridization on a bladder cancer tissue microarray composed of 2317 cancer samples. Both genes showed amplification in 104 of 893 (11.6%) interpretable tumours and were exclusively found co-amplified. Additional gene expression analysis by real-time polymerase chain reaction in 12 tumour-derived cell lines revealed that amplification of 6p22 was always associated with co-overexpression of E2F3 and NM_017774. Furthermore, RNA interference was used to study the influence of reduced gene expression on cell growth. In tumour cells with and without the 6p22 amplicon, knockdown of E2F3 always lead to unequivocal reduction of proliferation, whereas knockdown of NM_017774 was only capable to slow down cell proliferation in non-amplified cells. Our findings point out that E2F3 but not NM_017774 is driving enhanced proliferation of 6p22 amplified tumour cells. We conclude that E2F3 must be responsible for the growth advantage of 6p22 amplified bladder cancer cells.
