Introduction Increased numbers of adherent, invasive E.coli (AIEC) have been reported within intestinal epithelium of patients with Crohn’s disease (CD) and colorectal cancer (CRC).1 Genotoxicity and angiogenic activity of AIEC have been described by our group and others.2–4 We hypothesised that a key contribution of cancer-promoting activity of AIEC may also be through their ability to activate Wnt/b-catenin signalling, and reported that Wnt target-genes were up-regulated in colonocytes at mRNA and protein level, including cyclooxygenase-2 (COX-2).5 Here, we further investigated the ability of AIEC to activate Wnt transcription and nuclear translocation of b-catenin. We sought also to confirm our findings in vivo using an AIEC mono-association mouse model.
Methods Activation of Wnt transcription activity in response to E.coli isolates1 (MOI: 10; for 4 h) was assessed using a TCF/LEF HeLa cell luciferase reporter assay. Infected cells were also pre-treated with and without COX inhibitors. Nuclear translocation of b-catenin was assessed by immunofluorescence in CRC cell-lines SW480 and DLD1. Following 6 week mono-association of Il10 -/-129 SvEv mice with CRC AIEC isolate HM44, intestinal tissue was fixed, Cox-2 and β-catenin expression assessed by immunohistochemistry and compared to germ-free controls.
Results Mucosa-associated E.coli isolated from CRC (HM44, HM358, HM545), Crohn’s disease (HM95, HM605), and ulcerative colitis (HM250, HM374) patients significantly increased Wnt TCF/LEF signalling in HeLa cells, ranging from 1.56±0.11 to 2.60±0.06 fold above uninfected controls (1.0±0.03); all p < 0.001, Kruskal-Wallis. Infection of SW480 and DLD1 showed significant increases in b-catenin nuclear translocation as per prostaglandin E2 treatment (1–10 μM). These responses were blocked using COX inhibitors (diclofenac>indomethecin>aspirin; 1–100 μM). Increased intestinal Cox-2 expression and Wnt signalling was observed in vivo in Il10 -/- mice infected with E. coli HM44 (n = 15) compared to germ-free mice (n = 5), with Cox-2 elevated 2.04±0.10 fold, and nuclear localisation of β-catenin elevated 1.98±0.13 fold; both p < 0.001; Mann-Whitney U.
Conclusion IBD and CRC mucosa-associated E.coli activate intestinal Wnt-signalling in vitro and in vivo. The specific bacterial factors triggering early cancer-promoting signals such as elevated COX-2 and Wnt pathway activation are currently being investigated using a validated CRC E.coli fosmid-library screening approach,3 with 12 confirmed positive clones currently undergoing sequence analysis.
References 1 Martin HM, et al. Gastroenterology 2004;127:80–93
2 Arthur JC, et al. Science 2012;338:120–23
3 Prorok-Hamon M, et al. Gut 2014;63:761–70
4 Buc E, et al. PLoS One 2013;8(2):e56964
5 Meehan B, et al. Gut 2015;64(Supp 1):A362–A363 (abstract)
Disclosure of Interest B. Meehan: None Declared, B. Campbell Conflict with: Has received honoraria from Amgen, Falk and Enterome, J. Rhodes Conflict with: Is/has been a member of advisory boards for Atlantic, Procter & Gamble and Falk, has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter & Gamble and Schering Plough.