BACKGROUND:Colorectal cancer (CRC) progression from adenoma to adenocarcinoma is associated with global reduction in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). DNA hypomethylation continues upon liver metastasis. Here we examine 5hmC changes upon progression to liver metastasis. RESULTS:5hmC is increased in metastatic liver tissue relative to the primary colon tumour and expression of TET2 and TET3 is negatively correlated with risk for metastasis in patients with CRC. Genes associated with increased 5-hydroxymethylcytosine show KEGG enrichment for adherens junctions, cytoskeleton and cell migration around a core cadherin (CDH2) network. Overall, the 5-hydroxymethylcyosine profile in the liver metastasis is similar to normal colon appearing to recover at many loci where it was originally present in normal colon and then spreading to adjacent sites. The underlying sequences at the recover and spread regions are enriched for SALL4, ZNF770, ZNF121 and PAX5 transcription factor binding sites. Finally, we show in a zebrafish migration assay using SW480 CRISPR-engineered TET knockout and rescue cells that reduced TET expression leads to a reduced migration frequency. CONCLUSIONS:Together these results suggest a biphasic trajectory for 5-hydroxymethyation dynamics that has bearing on potential therapeutic interventions aimed at manipulating 5-hydroxymethylcytosine levels.

Supplementary Figure 2 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figure 1 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figure 8 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Methods, Figure Legends 1-8 from Dvl2 Promotes Intestinal Length and Neoplasia in the <i>Apc</i>^<i>Min</i> Mouse Model for Colorectal Cancer

Supplementary Methods, Figure Legends 1-8 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

<p>PDF file - 4151K, Fig S1 Molecular targets of beta-catenin inhibitors. Fig S2 Efficacy and toxicity of beta-catenin inhibitors. Fig S3 Effects of TNKSi on Wnt signaling components. Fig S4 TNKSi-induced Axin degradasomes. Fig S5 TNKSi treatment of COLO320 cells. Fig S6 Prolonged TNKSi treatment and beta-catenin stability. Fig S7 TNKSi of chronically Wnt-stimulated HeLa cells. Fig S8 TNKSi responses of Wnt-stimulated cells. Fig S9 CA treatment of mice. Fig S10 Comparison of LEF1 antibodies.</p>

Supplementary Figure 7 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figure 2 from Dvl2 Promotes Intestinal Length and Neoplasia in the <i>Apc</i>^<i>Min</i> Mouse Model for Colorectal Cancer

Supplementary Figures 3-6 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figure 1 from Dvl2 Promotes Intestinal Length and Neoplasia in the <i>Apc</i>^<i>Min</i> Mouse Model for Colorectal Cancer

Supplementary Figure 8 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Supplementary Figures 3-6 from Dvl2 Promotes Intestinal Length and Neoplasia in the ApcMin Mouse Model for Colorectal Cancer

Bcl9 and Pygo are Wnt enhanceosome components that effect β-catenin-dependent transcription. Whether they mediate β-catenin-dependent neoplasia is unclear. Here we assess their roles in intestinal tumourigenesis initiated by Apc loss-of-function ( Apc Min ), or by Apc 1322T encoding a partially-functional Apc truncation commonly found in colorectal carcinomas. Intestinal deletion of Bcl9 extends disease-free survival in both models, and essentially cures Apc 1322T mice of their neoplasia. Loss-of-Bcl9 synergises with loss-of-Pygo to shift gene expression within Apc -mutant adenomas from stem cell-like to differentiation along Notch-regulated secretory lineages. Bcl9 loss also promotes tumour retention in Apc Min mice, apparently via relocating nuclear β-catenin to the cell surface, but this undesirable effect is not seen in Apc 1322T mice whose Apc truncation retains partial function in regulating β-catenin. Our results demonstrate a key role of the Wnt enhanceosome in β-catenin-dependent intestinal tumourigenesis and reveal the potential of BCL9 as a therapeutic target during early stages of colorectal cancer.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.

Colorectal cancer (CRC) shows variable underlying molecular changes with two major mechanisms of genetic instability: chromosomal instability and microsatellite instability. This review aims to delineate the different pathways of colorectal carcinogenesis and provide an overview of the most recent advances in molecular pathological classification systems for colorectal cancer. Two molecular pathological classification systems for CRC have recently been proposed. Integrated molecular analysis by The Cancer Genome Atlas project is based on a wide-ranging genomic and transcriptomic characterisation study of CRC using array-based and sequencing technologies. This approach classified CRC into two major groups consistent with previous classification systems: (1) ∼16 % hypermutated cancers with either microsatellite instability (MSI) due to defective mismatch repair (∼13 %) or ultramutated cancers with DNA polymerase epsilon proofreading mutations (∼3 %); and (2) ∼84 % non-hypermutated, microsatellite stable (MSS) cancers with a high frequency of DNA somatic copy number alterations, which showed common mutations in APC, TP53, KRAS, SMAD4, and PIK3CA. The recent Consensus Molecular Subtypes (CMS) Consortium analysing CRC expression profiling data from multiple studies described four CMS groups: almost all hypermutated MSI cancers fell into the first category CMS1 (MSI-immune, 14 %) with the remaining MSS cancers subcategorised into three groups of CMS2 (canonical, 37 %), CMS3 (metabolic, 13 %) and CMS4 (mesenchymal, 23 %), with a residual unclassified group (mixed features, 13 %). Although further research is required to validate these two systems, they may be useful for clinical trial designs and future post-surgical adjuvant treatment decisions, particularly for tumours with aggressive features or predicted responsiveness to immune checkpoint blockade.

Colorectal cancer screening using conventional colonoscopy lacks molecular information and can miss dysplastic lesions. We tested here the ability of fluorescently labelled lectins to distinguish dysplasia from normal tissue when sprayed on to the luminal surface epithelium of freshly resected colon tissue from the Apc(min) mouse and when applied to fixed human colorectal tissue sections. Wheat germ agglutinin (WGA) showed significantly decreased binding to adenomas in the mouse tissue and in sections of human colon from 47 patients. Changes in WGA binding to the human surface epithelium allowed regions containing normal epithelium (NE) or hyperplastic polyps (HP) to be distinguished from regions containing low-grade dysplasia (LGD), high-grade dysplasia (HGD) or carcinoma (C), with 81% sensitivity, 87% specificity and 93% positive predictive value (PPV). Helix pomatia agglutinin (HGA) distinguished epithelial regions containing NE from regions containing HP, LGD, HGD or C, with 89% sensitivity, 87% specificity and 97% PPV. The decreased binding of WGA and HPA to the luminal surface epithelium in human dysplasia suggests that these lectins may enable more sensitive detection of disease in the clinic using fluorescence colonoscopy.

Background The discovery of cytosine hydroxymethylation (5hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behaviour in colon cancer. 5hmC is globally reduced in proliferating cells such as colon tumours and the gut crypt progenitors, from which tumours can arise. Results Here, we show that colorectal tumours and cancer cells express Ten-Eleven-Translocation ( TET ) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. Conclusions Together our results indicate that promoters that acquire 5hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5hmC in tumours. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation.

Abstract Objective Screening for colorectal cancer (CRC) is currently performed using bright-field colonoscopy, which has low specificity and sensitivity for the detection of early dysplasia in the colon. Fluorescently labeled lectins have been applied topically for detecting glycosylation changes in early dysplastic lesions in the esophagus, providing a route for improved endoscopic detection using fluorescence endoscopy [1]. We have identified here fluorescently labeled lectins that can detect changes in glycosylated biomarkers on the surface epithelium of early dysplasia in CRC. These probes may enable fluorescence-guided resection of dysplastic lesions at colonoscopy and subsequent automated assessment of colon histopathology. Study Design Ninety-nine colon lesions in 48 patients (34 males, 14 females; mean patient age 68.8 ± 8.3 yr, range 53-95 yr) were identified during routine colonoscopy at Addenbrooke's Hospital, Cambridge, UK. Lesions suspected of being dysplastic or neoplastic, were detected using magnification colonoscopy or surgery, and removal performed at biopsy (11.7%) and/or polypectomy (62.1%), or partial colon resection (11.7%). Colon histological sections were stained with fluorescently labeled lectins, and the lectin binding to surface epithelium, which would be observable in colonoscopy, was compared with conventional histopathology for the presence of disease and disease stage. Results Normal colonic epithelium (NE) occupied 40.1% of the area of all sections, hyperplastic polyps (HP) occupied 10.2%, low- (LGD) dysplasia 25.6%, high-grade (HGD) dysplasia 13.9%, and adenocarcinoma (C) 10.2%, as assessed by conventional histopathology based on H&E staining. Lesions were located in the ascending (10.4%), transverse (15.6%), descending (14.3%), sigmoid (42.9%) colon and rectum (7.8%), with the mean lesion size being 16.0 ± 14.2 mm (range 2-60 mm). The lectin wheat germ agglutinin (WGA), when fluorescently-labeled, was capable of distinguishing epithelial regions containing NE from regions containing LGD, HGD and cancer, with >81% sensitivity, >79% specificity and >93% positive predictive value. Conclusions Automated analysis of fluorescently-labeled WGA binding to the surface epithelium of excised colon sections may be used for improving the assessment of early dysplasia in CRC. The same fluorescent lectin may also be useful as a topical imaging agent for improving detection of early dysplasia using fluorescence colonoscopy, increasing the early diagnosis of CRC and improving patient survival. References 1. Bird-Lieberman, E.L., Neves, A.A. et al. (2012). Nat Med. 18(2): p. 315-21. Citation Format: ANDRE NEVES, JOE KUO, Ashraf Ibrahim, KEVIN BRINDLE. molecular imaging of early colorectal dysplasia using fluorescently-labeled lectins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-122. doi:10.1158/1538-7445.AM2015-LB-122

Abstract Hyperactive β-catenin drives colorectal cancer, yet inhibiting its activity remains a formidable challenge. Interest is mounting in tankyrase inhibitors (TNKSi), which destabilize β-catenin through stabilizing Axin. Here, we confirm that TNKSi inhibit Wnt-induced transcription, similarly to carnosate, which reduces the transcriptional activity of β-catenin by blocking its binding to BCL9, and attenuates intestinal tumors in ApcMin mice. By contrast, β-catenin's activity is unresponsive to TNKSi in colorectal cancer cells and in cells after prolonged Wnt stimulation. This TNKSi insensitivity is conferred by β-catenin's association with LEF1 and BCL9-2/B9L, which accumulate during Wnt stimulation, thereby providing a feed-forward loop that converts transient into chronic β-catenin signaling. This limits the therapeutic value of TNKSi in colorectal carcinomas, most of which express high LEF1 levels. Our study provides proof-of-concept that the successful inhibition of oncogenic β-catenin in colorectal cancer requires the targeting of its interaction with LEF1 and/or BCL9/B9L, as exemplified by carnosate. Cancer Res; 74(5); 1495–505. ©2014 AACR.