Search in the Abstract Database

Abstracts Search 2019

OP31 TP53 mutation in human colonic organoids acquires resistance to in vitro long-term inflammation

K. Tsuchiya*1, S. Watanabe1, T. Shirasaki1, R. Nishimura1, N. Katsukura1, S. Hibiya1, R. Okamoto1, T. Nakamura1, M. Watanabe1

1Tokyo Medical and Dental Universityl and Dental Universityl and, Tokyo, Japan

Background

In colitis-associated cancer (CAC), tumour protein p53 (TP53) mutation often occurs in the early phase of colon carcinogenesis known as dysplasia–carcinoma sequence. Although there are some reports about the relation between TP53 mutation and colon carcinogenesis in mice model, the function of TP53 mutation on colonic epithelial cells in the patients with inflammatory bowel disease (IBD) has remained unknown. We therefore aimed to assess the influence of TP53 mutation by using a CRISPR Cas9 system on human colon epithelial organoids under long-term inflammation model which we originally generated.

Methods

TP53 was mutated by using CRISPR Cas9 system (LentiCRISPR v2®) in human colonic epithelial organoids derived from normal mucosa. Written informed consent was obtained and this study was approved by the Ethics Committee of Tokyo Medical and Dental University. The guide RNA was designed to bind exon 10 of TP53 according to previous report (Matano et al. Nat Med. 2015). The long-term inflammation model was established by culturing organoids with inflammatory factors (TNF-α, Flagellin, and IL-1β) for 60 weeks. Inflammatory response in the organoids was assessed by gene expression of inflammatory-related genes and the level of reactive oxygen species (ROS). Phenotypes of each organoids were assessed by MTS Assay, sphere formation assay for cell proliferation and stemness, respectively.

Results

We successfully established TP53 mutation in three different human colon epithelial organoids. Mutant TP53 was strongly expressed in nuclei as often shown in dysplastic lesion of IBD, whereas wild type (WT)–TP53 was not expressed in naive organoids. We assessed the effect of mutant TP53 with or without inflammatory stimulation for 60 weeks. Long-term inflammation impaired cell proliferation and sphere formation of the organoids with WT-TP53. Mutant TP53, however, enhanced cell growth and stemness with increased gene expression of c-myc and Lgr5 compared with WT–TP53 under the inflammatory situation; nevertheless, inflammatory response in the organoids with mutant TP53 was equal to that in the organoids with WT-TP53.

Conclusion

We, for the first time, showed TP53 mutation maintains cell proliferation and stemness of human colonic organoids even under in vitro long-term inflammation. Mutant TP53 acquired resistance to cell damage by chronic inflammation, suggesting that these results might mimic cell phenotype at the early step of colitis associated carcinogenesis.