ADTU-07 5-aminosalicylic acid targets colorectal cancer stemness in vitro. (8th June 2018)

• Record Type: Journal Article
• Authors: Dixon, Steven; Mortensson, Eleanor; Creed, Tom; Williams, Ann C
• Is Part Of: Gut. Volume 67(2018)Supplement 1
• Page Start: A185
• Page End: A186
• Publication Date: 2018-06-08
• Subjects: Gastroenterology -- Periodicals; 616.33
• DOI: 10.1136/gutjnl-2018-BSGAbstracts.369
• Languages: English
• ISSNs: 0017-5749

Abstract

Abstract : Introduction: The mechanism by which 5-aminosalicylic acid (5-ASA) reduces colorectal cancer risk in ulcerative colitis is not fully understood and appears to involve multiple targets. Although previously reported to target wnt signalling, the functional consequence of inhibition is not fully understood. Using a 3-dimensional (3D) organoid model we report that 5-ASA can prevent the formation of adenoma and carcinoma derived spheroids and propose that it does so through targeting the cancer stem cells. Methods: Human colorectal cancer (CRC) and adenoma (CA) cell lines were seeded into Matrigel (Corning, USA), treated with 5-ASA (Sigma Life Sciences, UK) in culture medium and imaged at multiple timepoints. Z-stacks through the Matrigel hemispheres were analysed using an in-house MatLab (Mathworks, USA) programme to estimate organoid area. In parallel cells were also grown in T25 flasks and treated with 5-ASA and expression of β-catenin targets assessed by western blotting. Wnt/β-catenin activity was assayed using a TOPflash TCF-luciferase construct in 24-well plates. Results: All cell lines tested formed organoids in Matrigel. 5-ASA slowed the growth of CRC and CA cell organoids in all lines tested at physiologically relevant concentrations (5 and 10 mm) when treatment was initiated 7 days after seeding into Matrigel. Importantly, treatment with 5-ASA within 24 hours of seeding at low doses (2 mM) prevented any organoid growth (figure 1). In these cultures, Wnt/β-catenin signalling, active in the intestinal crypt stem compartment and constitutively active in the majority of CRC, was reduced in CRC cells by 24% (10 mM) – 38% (20 mM) and CA cells by 22% (10 mM) – 44% (20 mM) by treatment with 5-ASA. Furthermore expression of the colonic stem cell marker leucine-rich G-coupled receptor-5 (LGR5) and Wnt-targets c-Myc, Axin-2 and dephosphorylated beta-catenin was suppressed in both CRC and CA cell lines by treatment with 5-ASA. Conclusions: Here we demonstrate that 5-ASA impairs growth of CRC and CA in a 3D model. That 5-ASA arrests the growth of organoid if treated at the time of seeding suggests that 5-ASA may be affecting cell stemness. This is likely to be due to – at least in-part – reduced Wnt/β-catenin activity. Further work is being undertaken to elucidate the mechanism by which of 5-ASA could target cancer stemness to inform on potential clinical trials of 5-ASA as a prophylactic agent for patients at high risk of CRC.