Abstract
BackgroundAmplifications at 6p22.3 are prevalent in advanced stage bladder cancer (TCC). Previous studies have identified SOX4, CDKAL, and E2F3 as targets of this amplification and therefore potential oncogenes, but the more telomeric DEK gene too has been reported as overexpressed and amplified. We have therefore investigated whether the intermediate region harboring the oncogene candidate ID4 is also part of the amplicon.ResultsExpression of E2F3, DEK, and ID4 was investigated by real-time RT-PCR in 28 TCC compared to 6 normal bladder tissues and in 15 TCC cell lines compared to cultured normal urothelial cells. Expression of E2F3 as well as DEK increased on average in tumor vs. normal tissues (3-fold and 2.5-fold, resp.), but only the increase for E2F3 was statistically significant (p = 0.039). ID4 overexpression was observed in selected specimens. Each of the three genes was overexpressed in several cell lines, up to 150-fold (ID4), 30-fold (E2F3), and 9-fold (DEK), but these increases were not correlated to each other. Instead, moderate (DEK) to excellent (ID4) correlations were observed with copy number increases of microsatellites near each gene. Microsatellite copy number increases were highly heterogeneous across the investigated several Mb region revealing at least three subregions of amplification.ConclusionExtending previous reports, our data indicate that the 6p22.3 amplicon in TCC is highly heterogeneous and targets several genes in a variable fashion. Among these, expression of E2F3 and DEK appear to be generally increased in TCC, with additional increases caused by amplifications. In contrast, over-expression of ID4, which is normally predominantly expressed in testes and brain, appears to depend more strictly on gene amplification. Accordingly, the effect of amplifications at 6p22.3 in bladder cancer is expected to be non-uniform, thereby contributing to the highly variable biological and clinical behavior of advanced stage tumors. ID4 is a potential oncogene in a small subset of bladder cancers.
Highlights
Amplifications at 6p22.3 are prevalent in advanced stage bladder cancer (TCC)
Expression of 6p22 genes in bladder cancer cell lines First, expression of ID4 mRNA in comparison to E2F3 and DEK mRNAs was investigated by real-time PCR in 16 TCC cell lines (Figure 2)
The divergence is strikingly illustrated by the cell lines HT1376 and 5637, which presented increased expression levels for all three genes, but with either ID4 or E2F3 displaying pronounced increases (Figure 2)
Summary
Tissues Cancerous and normal bladder tissues were used from a previous study [39]. Normal tissues were identified by gross morphology, with microscopic verification in case of extended tumors. RNA from testicular normal and cancer tissues used as a control for ID4 expression was prepared in the course of a former study [40]. For several 6p22 genes subject to amplification, it is plausible to assume that their overexpression may confer a more aggressive phenotype to bladder cancer cells. SOX factors determine cell fate and cell differentiation [33], so SOX4 overexpression might lead to further dedifferentiation. Specific functional studies on the role of these proteins in urothelial cells are lacking.
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