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

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.

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia.

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.

Supplementation with Brazil nuts and green tea extract regulates targeted biomarkers related to colorectal cancer risk in humans.

Se and green tea have been shown in epidemiological, observational and preclinical studies to be inversely related to the risk of developing colorectal cancer (CRC). However, there are limited studies to evaluate their regulatory effects on genes/proteins that relate to CRC oncogenesis in human subjects, such as selenoproteins, WNT signalling pathway, inflammation and methylation. This study examined the effects of supplementation of Se using Brazil nuts and green tea extract (GTE) capsules, alone and in combination, on targeted biomarkers. In total, thirty-two volunteers (>50 years of age) with plasma Se≤1·36 µmol/l were randomised to one of three treatment groups: nine to Se (approximately 48 µg/d) as six Brazil nuts, eleven to four GTE capsules (800 mg (-)-epigallocatechin-3-gallate) and twelve to a combination of Brazil nuts and GTE. Blood and rectal biopsies were obtained before and after each intervention. Plasma Se levels, rectal selenoprotein P (SePP) and ß-catenin mRNA increased significantly in subjects consuming Brazil nuts alone or in combination, whereas rectal DNA methyltransferase (DNMT1) and NF-κB mRNA were reduced significantly in subjects consuming GTE alone or in combination. None of the interventions significantly affected rectal acetylated histone H3 or Ki-67 expression at the protein level or plasma C-reactive protein. Effects of the combination of Brazil nuts and GTE did not differ from what would be expected from either agent alone. In conclusion, supplementation of Brazil nuts and/or GTE regulates targeted biomarkers related to CRC oncogenesis, specifically genes associated with selenoproteins (SePP), WNT signalling (ß-catenin), inflammation (NF-κB) and methylation (DNMT1). Their combination does not appear to provide additional effects compared with either agent alone.

Manipulation of the gut microbiota using resistant starch is associated with protection against colitis-associated colorectal cancer in rats.

This study evaluated whether dietary resistant starch (RS) and green tea extract (GTE), which have anti-inflammatory and anticancer properties, protect against colitis-associated colorectal cancer (CAC) using a rat model, also investigated potential mechanisms of action of these agents including their effects on the gut microbiota. Rats were fed a control diet or diets containing 10% RS, 0.5% GTE or a combination of the two (RS + GTE). CAC was initiated with 2 weekly azoxymethane (AOM) injections (10mg/kg) followed by 2% dextran sodium sulphate in drinking water for 7 days after 2 weeks on diets. Rats were killed 20 weeks after the first AOM. Colon tissues and tumours were examined for histopathology by H&E, gene/protein expression by PCR and immunohistochemistry and digesta for analyses of fermentation products and microbiota populations. RS and RS + GTE (but not GTE) diets significantly (P< 0.05) decreased tumour multiplicity and adenocarcinoma formation, relative to the control diet. Effects of RS + GTE were not different from RS alone. RS diet caused significant shifts in microbial composition/diversity, with increases in Parabacteroides, Barnesiella, Ruminococcus, Marvinbryantia and Bifidobacterium as primary contributors to the shift. RS-containing diets increased short chain fatty acids (SCFA) and expression of the SCFA receptor GPR43 mRNA, and reduced inflammation (COX-2, NF-kB, TNF-α and IL-1ß mRNA) and cell proliferation P< 0.05. GTE had no effect. This is the first study that demonstrates chemopreventive effects of RS (but not GTE) in a rodent CAC model, suggesting RS might have benefit to patients with ulcerative colitis who are at an increased risk of developing CRC.