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PWE-347 Adherent, invasive mucosa-associated e. coli isolates from colorectal cancer patients activate WNT signalling
  1. B Meehan,
  2. B Campbell,
  3. J Rhodes
  1. Gastroenterology, Institute of Translational Medicine, Liverpool, UK

Abstract

Introduction Increased numbers of adherent, invasive E.coli (AIEC) have been reported on and within intestinal epithelial cells of patients with colorectal cancer (CRC).1Cancer-inducing activity of AIEC have recently been described by our group and others, including potential for genotoxicity and promotion of angiogenesis.2,3,4We hypothesise that CRC-associated AIEC may also trigger key cancer signalling pathways that support development and progression of early carcinogenesis, including (Wingless) Wnt/b-catenin cell signalling pathway.5

Method Two human CRC cell-lines SW-480 and DLD1 were infected with CRC mucosa-associated AIEC isolates HM44 and HM3581for 4h at multiplicity of infection of 10. RNA was isolated, and synthesised cDNA subjected to RT2PCR Wnt Target Array (84 Wnt-relevant genes). Gene expression changes were confirmed by Roche Lightcycler qPCR and protein expression by immunoblot (0.5h to 4h infections).

Results In response to 4h infection with CRC-associated AIEC isolates HM44 and HM358, 11 Wnt target-genes were significantly up-regulated and 7 significantly down-regulated, many regulating cell cycle, cell proliferation, migration and angiogenesis. Key genes up-regulated in SW480 cells included cyclooxygenase-2 (PTGS-2/COX2), increased 8.1 ± 0.9 and 9.2 ± 1.0 fold (both P = 0.0002), Fos-related antigen 1 (FOSL1) increased 6.1 ± 0.6 and 7.9 ± 1.9 fold (P < 0.01), and vascular endothelial growth factor-A (VEGFA) upregulated 4.6 ± 0.3 and 4.2 ± 0.2 fold respectively (P < 0.0001). Down-regulated genes (>2 fold change) included a known CRC tumour suppressor, Wnt-1 inducible signalling pathway 2 (WISP2); P < 0.05. Similar gene changes were seen using DLD-1 cells, and confirmed by qPCR. AIEC also significantly increased cellular protein levels; e.g. COX2, up 5.7 ± 0.7 and 2.8 ± 0.04 fold respectively (both P < 0.01; N=3, n = 3). In addition, b-catenin increases were 2–3 fold at 2h and 4h post-infection (P < 0.05).

Conclusion Early indications suggest a key contribution of cancer-promoting activity of CRC-mucosa-associated E.coli events may be through ability to activate the Wnt signalling pathway. Confirmation of the importance of these findings is being investigated in vivousing a mono-association mouse model,2along with a CRC E.coli fosmid-library screening approach3to identify specific bacterial factors triggering these early cancer-promoting signals.

Disclosure of interest B. Meehan: None Declared, B. Campbell Conflict with: Received honoraria from Amgen and Falk., J. Rhodes Conflict with: Is/has been a member of advisory boards for Atlantic, Procter and Gamble and Falk, has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble and Schering Plough.

References

  1. Martin, 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, et al. PLoS One 2013;8(2):e56964

  5. Schneikert J, Behrens J. Gut2007;56:417–25

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