Introduction Intermediate filaments (IF), principally keratins (K), are key components of epithelial cytoskeleton. K8, 18 and 19 are expressed in intestinal epithelial cells and play a role in cell-death signalling pathways, in particular apoptosis mediated by tumour necrosis factor-α. Reduced K8 and K20 expression is linked to epithelial-to-mesenchymal transition indicative of increased tumour aggressiveness. We investigated the change in levels of insoluble IF proteins in well-characterised groups of patients at differing risk of UC-associated cancer.
Methods Rectal biopsies were obtained from patients with inactive UC with: (1) Long-standing (20–40 years) pancolitis (LSPC) (n=10); (2) Recent onset (<5 years) UC (ROUC) (n=8); (3) UC with primary sclerosing cholangitis (PSC) (n=7); (4) pancolitis with dysplasia (n=4) and 10 controls, with additional biopsies from dysplastic/neoplastic lesions and snap frozen. An iTRAQ (isobaric tagging for relative and absolute quantification)-compatible extraction and solubilisation protocol for IF proteins was developed. Labelled peptides from pooled patients were analysed by SCX-LC-MS/MS (strong cation exchange-reverse phase HPLC tandem mass spectrometry) and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction.
Results Tandem mass spectrometry (MS/MS) identified 52 proteins; 32 (61.5%) were matched by two or more peptides. Abstract PMO-249 figure 1A shows the log fold change in IF levels compared to control, with significant increase in levels of K8, K19 and vimentin in those with LSPC, but marked reduction in IF levels in areas of dysplasia (DT) and rectal mucosa distant from this (DR). Marked increase in levels of keratins was noted in patients with LSPC compared to those with ROUC (Abstract PMO-249 figure 1B), suggesting an effect of disease duration on IF levels.
Conclusion This is the first study using a quantitative proteomic approach with an iTRAQ based proteomic workflow to analyse changes in IF levels in patients with UC with differing colon cancer risk. LSPC is associated with enhanced mucosal levels of keratins, spectrin and xin, which is reduced in dysplasia and in distant rectal mucosa of those with dysplasia—suggesting a field change. These changes need further characterisation including of post-translational modifications, which may help better understanding of the pathogenesis of colitis associated cancer.
Competing interests None declared.