OC-044 Mucosa-Associated E.coli Isolates from Inflammatory Bowel Disease and Colorectal Cancer Patients Activate Wnt/Beta-Catenin Signalling in vitro and in vivo
Authors: B Meehan, BJ Campbell, JM Rhodes
Journal: Gut (2016)

<h3>Introduction</h3> Increased numbers of adherent, invasive <i>E.coli</i> (AIEC) have been reported within intestinal epithelium of patients with Crohn’s disease (CD) and colorectal cancer (CRC).<sup>1</sup> Genotoxicity and angiogenic activity of AIEC have been described by our group and others.<sup>2–4</sup> 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).<sup>5</sup> 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. <h3>Methods</h3> Activation of Wnt transcription activity in response to <i>E.coli</i> isolates<sup>1</sup> (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 <i>Il10</i><sup>-/-</sup>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. <h3>Results</h3> Mucosa-associated <i>E.coli</i> 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 &lt; 0.001, Kruskal-Wallis. Infection of SW480 and DLD1 showed significant increases in <i>b</i>-catenin nuclear translocation as per prostaglandin E2 treatment (1–10 μM). These responses were blocked using COX inhibitors (diclofenac&gt;indomethecin&gt;aspirin; 1–100 μM). Increased intestinal Cox-2 expression and Wnt signalling was observed <i>in vivo</i> in <i>Il10</i><sup>-/-</sup> mice infected with <i>E. coli</i> 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 &lt; 0.001; Mann-Whitney U. <h3>Conclusion</h3> 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,<sup>3</sup> with 12 confirmed positive clones currently undergoing sequence analysis. <h3>References</h3> 1 Martin HM, <i>et al</i>. <i>Gastroenterology</i> 2004;<b>127</b>:80–93 2 Arthur JC, <i>et al</i>. <i>Science</i> 2012;<b>338</b>:120–23 3 Prorok-Hamon M, <i>et al</i>. <i>Gut </i>2014;<b>63</b>:761–70 4 Buc E, <i>et al</i>. <i>PLoS One</i> 2013;<b>8</b>(2):e56964 5 Meehan B, <i>et al</i>. <i>Gut</i> 2015;<b>64</b>(Supp 1):A362–A363 (abstract) <h3>Disclosure of Interest</h3> 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 &amp; Gamble and Falk, has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter &amp; Gamble and Schering Plough.