Introduction Gastro-oesophageal reflux disease is the main risk factor for Barrett’s oesophagus (BE), the precursor lesion to oesophageal adenocarcinoma. In BE, GORD leads to chronic inflammation and to NF-κB pathway activation.
SIRT2 is a histone deacetylase involved in deacetylation of key players in the cell, including p65, one subunit of the NF-κB transcription complex. SIRT2 is part of our previously published gene signatures in which loss of SIRT2 confers a poor prognosis and over-expression a good prognosis in keeping with its known role as a tumour suppressor.
We hypothesised that exerts its protective effect through recruitment of inflammatory cells to the tumour site via the NF-κB pathway The aim of this study was to assess the inflammatory infiltrate in positive tumoursto assess the relationship betweenthe NF-κB pathway.
Methods 76 surgical resection specimen of oesophageal adenocarcinoma were immunostained for SIRT2. An in-depth analysis of the nature of inflammatory cells localised to high SIRT2 areas was done in 5 cases using immune cell markers (CD3, CD4, CD8, CD20, CD56 and CD68). NF- κB and SIRT2 luciferase reporter assays were used with SIRT2 overexpression and TNFα stimulation to study the interplay between the NF- κB pathway and SIRT2. A panel of SIRT2 promoter mutants with mutations of one or two or both NF-κB putative binding sites, identified through an in silico analysis, were also used.
Results 32% of the cases were strongly positive for SIRT2 (+3 and +2 on a scale from 0 to +3 where 0 is negative). A higher number of inflammatory cells were identified in SIRT2-positive cases compared to SIRT2 negative cases. In particular, SIRT2 positive cases showed strong staining for CD68 indicating an enrichment in the number of macrophages. SIRT2 overexpression significantly down-regulated NF-κB activity (p = 0.0011). Immunoblotting suggests that this downregulation is probably conferred by the deacetylation of Lysine 310 at the p65 subunit of NF- κB. Luciferase assays with the full-length SIRT2-promoter reporter revealed that the SIRT2 promoter was induced by TNFα stimulation (activates NF- κB pathway). This stimulation resulted in decreased luciferase activity when the NF- κB binding sites mutants were used, suggesting a direct action of NF- κB on SIRT2.
Conclusion In oesophageal adenocarcinoma, SIRT2 expression is linked with an increased inflammatory infiltrate, especially macrophages. Luciferase reporter assays suggest that SIRT2 and NF-κB regulate each other. Taken together, downregulation of NF- κB by SIRT2 could be an explanation for the protective effect of SIRT2 overexpression in oesophageal adenocarcinoma. Further work is required to confirm these findings.
Disclosure of Interest None Declared