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Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

ABSTRACT: OBJECTIVE:Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. DESIGN:We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. RESULTS:Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. CONCLUSION:We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

SUBMITTER: Lannagan TRM

PROVIDER: S-EPMC6192855 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

Lannagan Tamsin R M TRM Lee Young K YK Wang Tongtong T Roper Jatin J Bettington Mark L ML Fennell Lochlan L Vrbanac Laura L Jonavicius Lisa L Somashekar Roshini R Gieniec Krystyna K Yang Miao M Ng Jia Q JQ Suzuki Nobumi N Ichinose Mari M Wright Josephine A JA Kobayashi Hiroki H Putoczki Tracey L TL Hayakawa Yoku Y Leedham Simon J SJ Abud Helen E HE Yilmaz Ömer H ÖH Marker Julie J Klebe Sonja S Wirapati Pratyaksha P Mukherjee Siddhartha S Tejpar Sabine S Leggett Barbara A BA Whitehall Vicki L J VLJ Worthley Daniel L DL Woods Susan L SL

Gut 20180417 4

<h4>Objective</h4>Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, <i>BRAF</i>, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most importa ...[more]

PMID: 29666172

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Project description:BackgroundIndividuals with serrated polyps (SP) are at higher risk for synchronous colorectal advanced neoplasms (AN) and cancers. However, it remains unclear whether there is a unique involvement of the serrated pathway and/or the classical adenoma-carcinoma sequence in this setting.MethodsColorectal ANs, which include tubular adenomas ? 10 mm, adenomas with villous histology, high-grade intraepithelial neoplasms, and cancers, were collected retrospectively. The groups included ANs with (AN+SP) or without (AN-only) coexisting SPs. Clinicopathological findings were compared between groups. BRAF and KRAS mutations in ANs and SPs, and methylation levels at long interspersed element-1 (LINE-1) in adjacent mucosa were determined by pyrosequencing.ResultsSeventy-five ANs from 40 patients in the AN+SP group, and 179 ANs from 119 patients in the AN-only group were analyzed. There were no significant differences in clinicopathological findings between the two groups, except that intraepithelial neoplasia in the AN+SP group was more likely to be located in the right colon (P=0.018). BRAF mutations were significantly more frequent in the AN+SP group (P=0.003), while KRAS mutations showed no significant differences between groups (P=0.142). The majority of high-grade intraepithelial neoplasms in both groups showed a contiguous component of conventional adenoma. Individuals with large and right-sided SPs had significantly more conventional adenomas compared to those without such SPs (P=0.027 and P=0.031, respectively). Adjacent mucosa from individuals with multiple and large SPs showed significantly lower methylation levels at LINE-1 compared to individuals without such associated SPs (P=0.049 and P=0.015, respectively).ConclusionOur data suggest that both the adenoma-carcinoma sequence and the serrated pathway are operational in individuals with coexisting ANs and SPs. The reduced methylation levels at LINE-1 in the background mucosa suggest the possibility of an underlying 'field defect'.

Dataset Information

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

Publications

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

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