E2F3 is responsible for frequent amplification of 6p22.3 in human bladder cancer

The 6p22 is generally regarded as one of the most important amplification sites in urinary
bladder cancer. Investigations, encouraged by these findings, subsequently lead to the
delineation of the amplicon. During this process the genomic region was narrowed down to
1.7 Mb at 6p22.3, including presumably 13 different genes. Some of these genes were
withdrawn from additional investigations due to low-level or absent expression in 6p22.3-
amplified bladder cancers. Two genes, however, showed unquestionable correlation
between high-level amplification and subsequent overexpression. But the relevant target
gene that drives the amplification remained unidentified, yet.
This work was ultimately aimed in a comprehensive comparison of the two remaining
candidate oncogenes. The major findings were:
- By performing FISH on a large bladder cancer TMA we show that NM_017774 is
amplified in 11.6% of 893 tested human bladder cancer samples. Thus, the gene
reaches an amplification level that is comparable to E2F3.
- Following case-by-case re-evaluation of a large-section FISH analysis, exhibiting
104 6p22.3-amplified bladder cancers, demonstrates that both genes are 100% coamplified.
- Furthermore, we show that both candidate oncogenes are always co-overexpressed
in 6p22.3-amplified bladder cancer cell lines, presumably as a consequence of the
amplification.
- Experimentally decreased expression levels of NM_017774 and/or E2F3 similarly
lead to strongly inhibited cell proliferation (observed in normal bladder cancer cells
CRL-7930; without 6p22.3-amplification).
- This finding suggests that NM_017774 -the gene of hitherto unknown function –must
be functionally connected to the cell cycle regulatory machinery.
- Besides, decreased E2F3-expression results in proliferation-reduction, and thus
confirms the previously predicted essential role of this transcriptionfactor in cell
cycle progression.
- Finally, functional analysis performed in the 6p22.3-amplified HTB-5 cell line,
demonstrate that E2F3 –but not NM_017774 -captures a limiting role for enhanced
cellular proliferation in 6p22.3-amplified bladder cancer cells.
- Hence, our results suggest that NM_017774 is only accidentally co-amplified
because of its spatial neighbourhood to E2F3 (like other genes in the area), but does
not have a functional role in 6p22.3-amplification, whatsoever.
Conclusively, the findings of this study consistently document that amplification of 6p22.3
leads to upregulated mRNA expression, and increased protein production of the
transcriptionfactor E2F3. While also other genes localized in the amplified region may be coamplified
and co-overexpressed as a by-product of the amplification, E2F3 represents the
main target gene and is therefore responsible for the frequent amplification of 6p22.3 in
urinary bladder cancer.