Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) has been associated with tumour suppression in various cancers. However, its putative role in bladder cancer is completely unknown. Therefore, we initiated a study analysing ITIH5 expression as well as its prognostic and functional impact on human urothelial cancers (UCs). Expression analysis showed a clear down-regulation of ITIH5 mRNA in 61% (n = 45) of UCs, especially in muscle-invasive tumours (P < 0.001). ITIH5 loss in UCs was further evident on protein level (65.5%, n = 55) as detected by immunohistochemistry. DNA methylation analysis demonstrated tumour-specific ITIH5 promoter methylation in 50% of papillary none-invasive pTa (n = 30) and 68% of invasive (n = 28) UCs. Aberrant ITIH5 promoter methylation in bladder tumours was tightly linked (P < 0.001) with loss of ITIH5 mRNA expression, which was furthermore functionally confirmed by demethylation analysis in cell lines. Pyrosequencing analysis revealed that ITIH5 promoter hypermethylation was closely associated with progressive bladder cancers. Subsequently, a large cohort (n = 120) of clinically challenging pT1 high-grade UC was analysed for ITIH5 expression. Of clinical significance, we found an association between loss of ITIH5 expression and unfavourable prognosis of UC patients without distant metastasis at first diagnosis (recurrence-free survival; hazard ratio: 4.35, P = 0.048). Functionally, ITIH5 re-expression in human RT112 bladder cancer cells led to both suppression of cell migration and inhibition of colony spreading. Hence, we provide evidence that down-regulation of ITIH5 by aberrant DNA hypermethylation may provoke invasive phenotypes in human bladder cancer. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high-grade UC patients.

No abstract

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 µm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.

FGFR amplifications are rare events in bladder cancer, with FGFR3 amplification being the most prevalent (3.4% of cases). Concomitant FGFR3 mutations and protein overexpression indicate that FGFR3-mediated signalling in these tumours would probably be highly active. This patient subgroup may be particularly suited to FGFR-targeted pharmacotherapy.