Catherine E. Bond

Bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer development

Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well‐described functions in bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down‐regulated as assessed by real‐time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hypermethylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aberrant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01), the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001). Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re‐introduction of BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tumor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early and frequent event in colorectal tumors progressing via the serrated and traditional pathways.

Oncogenic PIK3CA mutations in colorectal cancers and polyps

Oncogenic PIK3CA mutations contribute to colorectal tumorigenesis by activating AKT signaling to decrease apoptosis and increase tumor invasion. A synergistic association of PIK3CA mutation with KRAS mutation has been suggested to increase AKT signaling and resistance to antiepidermal growth factor receptor inhibitor therapy for advanced colorectal cancer, although studies have been conflicting. We sought to clarify this by examining PIK3CA mutation frequency in relation to other key molecular features of defined pathways of tumorigenesis. PIK3CA mutation was assessed by high resolution melt analysis in 829 colorectal cancer samples and 426 colorectal polyps. Mutations were independently correlated with clinicopathological features including patient age, sex and tumor location as well as molecular features including microsatellite instability, KRAS and BRAF mutation, MGMT methylation and the CpG Island Methylator Phenotype (CIMP). Mutation of the helical (Exon 9) and catalytic (Exon 20) domain mutation hotspots were also examined independently. Overall, PIK3CA mutation was positively correlated with KRAS mutation (p < 0.001), MGMT methylation (p = 0.007) and CIMP (p < 0.001). Novel, exon‐specific associations linked Exon 9 mutations to a subgroup of cancers characterized by KRAS mutation, MGMT methylation and CIMP‐Low, whilst Exon 20 mutations were more closely linked to features of serrated pathway tumors including BRAF mutation, microsatellite instability and CIMP‐High or Low. PIK3CA mutations were uncommonly, but exclusively, seen in tubulovillous adenomas (4/124, 3.2%) and 1/4 (25.0%) tubulovillous adenomas with a focus of cancer. These data provide insight into the molecular events driving traditional versus serrated pathway tumorigenesis.

Chromosomal Instability in BRAF Mutant, Microsatellite Stable Colorectal Cancers

The BRAF oncogene is mutated in 15% of sporadic colorectal cancers. Approximately half of these BRAF mutant cancers demonstrate frequent frameshift mutations termed microsatellite instability (MSI), but are diploid and chromosomally stable. BRAF wild type cancers are typically microsatellite stable (MSS) and instead acquire chromosomal instability (CIN). In these cancers, CIN is associated with a poor outcome. BRAF mutant cancers that are MSS, typically present at an advanced stage and have a particularly poor prognosis. We have previously demonstrated clinical and molecular similarities between MSS cancers with or without a BRAF mutation, and therefore hypothesised that CIN may also be frequent in BRAF mutant/MSS cancers. BRAF mutant/MSS (n = 60), and BRAF wild type/MSS CRCs (n = 90) were investigated for CIN using loss of heterozygosity analysis over twelve loci encompassing chromosomal regions 5q, 8p, 17p and 18q. CIN was frequent in BRAF mutant/MSS cancers (41/57, 72%), which was comparable to the rate found in BRAF wild type/MSS cancers (74/90, 82%). The greatest loss in BRAF mutant/MSS cancers occurred at 8p (26/44, 59%), and the least at 5q (19/49, 39%). CIN in BRAF mutant/MSS cancers correlated with advanced stage (AJCC III/IV: 15/17, 88%; p = 0.02); showed high rates of co-occurrence with the CpG Island Methylator Phenotype (17/23, 74%); and CIN at 18q and 8p associated with worse survival (p = 0.02, p<0.05). This study demonstrates that CIN commonly occurs in advanced BRAF mutant/MSS colorectal cancers where it may contribute to poorer survival, and further highlights molecular similarities occurring between these and BRAF wild type cancers.

p53 mutation is common in microsatellite stable, BRAF mutant colorectal cancers

The majority of “serrated pathway” colorectal cancers have mutation of the BRAF oncogene and display the CpG island methylator phenotype (CIMP). Half these cancers have microsatellite instability (MSI) and an excellent prognosis. In the absence of MSI (microsatellite stable, MSS), BRAF mutation has been associated with a particularly poor prognosis. “Traditional pathway” cancers are BRAF wild type. Mutation of p53 is common and this correlates with advanced stage. We therefore hypothesized that p53 mutation would be common in MSS/BRAF mutant colorectal cancer. One thousand and eighty‐one colorectal cancers were screened for BRAF mutation to identify two BRAF mutant study groups (MSI: n = 77; MSS: n = 69) and a BRAF wild type control group (n = 101). These were screened for p53 mutation by high resolution melt analysis and classified for CIMP and MGMT methylation by quantitative methylation specific PCR. Molecular data were compared to patient age, gender, tumor location and stage. p53 was mutated significantly more frequently in MSS/BRAF mutant (28/69, 40.6%) compared to MSI/BRAF mutant cancers (13/77, 16.9%), but this mutation rate did not differ from MSS/BRAF wild type cancers (47/101, 46.5%)(p < 0.0001). CIMP was less common in MSS/BRAF mutant (26/47, 55.3%) compared to MSI/BRAF mutant cancers (41/54, 75.9%), but was more common than in MSS/BRAF wild type cancers (3/85, 3.5%) (p < 0.0001). MSS/BRAF mutant cancers were more commonly proximal (38/54, 70.3%), but were similar to MSS/BRAF wild type cancers in terms of patient age, gender distribution and stage at presentation. MSS/BRAF mutant cancers share molecular and clinical features of both the serrated and traditional pathways of colorectal tumorigenesis.

RNF43 and ZNRF3 are commonly altered in serrated pathway colorectal tumorigenesis

Serrated pathway colorectal cancers (CRCs) are characterised by a BRAF mutation and half display microsatellite instability (MSI). The Wnt pathway is commonly upregulated in conventional CRC through APC mutation. By contrast, serrated cancers do not mutate APC. We investigated mutation of the ubiquitin ligases RNF43 and ZNRF3 as alternate mechanism of altering the Wnt signal in serrated colorectal neoplasia. RNF43 was mutated in 47/54(87%) BRAF mutant/MSI and 8/33(24%) BRAF mutant/microsatellite stable cancers compared to only 3/79(4%) BRAF wildtype cancers (p<0.0001). ZNRF3 was mutated in 16/54(30%) BRAF mutant/MSI and 5/33(15%) BRAF mutant/microsatellite stable compared to 0/27 BRAF wild type cancers (p=0.004). An RNF43 frameshift mutation (X659fs) occurred in 80% BRAF mutant/MSI cancers. This high rate was verified in a second series of 25/35(71%) BRAF mutant/MSI cancers. RNF43 and ZNRF3 had lower transcript expression in BRAF mutant compared to BRAF wildtype cancers and less cytoplasmic protein expression in BRAF mutant/MSI compared to other subtypes. Treatment with a porcupine inhibitor reduced RNF43/ZNRF3 mutant colony growth by 50% and synergised with a MEK inhibitor to dramatically reduce growth. This study suggests inactivation of RNF43 and ZNRF3 is important in serrated tumorigenesis and has identified a potential therapeutic strategy for this cancer subtype.

Microsatellite Stable Colorectal Cancers Stratified by the BRAF V600E Mutation Show Distinct Patterns of Chromosomal Instability

The BRAF (V600E) mutation in colorectal cancers that are microsatellite stable (MSS) confers a poor patient prognosis, whereas BRAF mutant microsatellite-unstable (MSI) colorectal cancers have an excellent prognosis. BRAF wild type cancers are typically MSS and display chromosomal instability (CIN). CIN has not been extensively studied on a genome-wide basis in relation to BRAF mutational status in colorectal cancer. BRAF mutant/MSS (BRAFmut/MSS) cancers (n = 33) and BRAF mutant/MSI (BRAFmut/MSI) cancers (n = 30) were compared for presence of copy number aberrations (CNAs) indicative of CIN, with BRAF wild type/MSS (BRAFwt/MSS) cancers (n = 18) using Illumina CytoSNP-12 arrays. BRAFmut/MSS and BRAFwt/MSS cancers showed comparable numbers of CNAs/cancer at 32.8 and 29.8 respectively. However, there were differences in patterns of CNA length between MSS cohorts, with BRAFmut/MSS cancers having significantly greater proportions of focal CNAs compared to BRAFwt/MSS cancers (p<0.0001); whereas whole chromosomal arm CNAs were more common in BRAFwt/MSS cancers (p<0.0001). This related to a reduced average CNA length in BRAFmut/MSS compared to BRAFwt/MSS cancers (20.7 Mb vs 33.4 Mb;p<0.0001); and a smaller average percent of CIN affected genomes in BRAFmut/MSS compared to BRAFwt/MSS cancers (23.9% vs 34.9% respectively). BRAFmut/MSI cancers were confirmed to have low CNA rates (5.4/cancer) and minimal CIN-affected genomes (average of 4.5%) compared to MSS cohorts (p<0.0001). BRAFmut/MSS cancers had more frequent deletion CNAs compared to BRAFwt/MSS cancers on 6p and 17q at loci not typically correlated with colorectal cancer, and greater amplification CNAs on 8q and 18q compared to BRAFwt/MSS cancers. These results indicate that comparable rates of CIN occur between MSS subgroups, however significant differences in their patterns of instability exist, with BRAFmut/MSS cancers showing a ‘focal pattern’ and BRAFwt/MSS cancers having a ‘whole arm pattern’ of CIN. This and the genomic loci more frequently affected in BRAFmut/MSS cancers provides further evidence of the biological distinctions of this important cancer subgroup.

Methylation and expression of the tumour suppressor, PRDM5, in colorectal cancer and polyp subgroups

BackgroundPRDM5 is an epigenetic regulator that has been recognized as an important tumour suppressor gene. Silencing of PRDM5 by promoter hypermethylation has been demonstrated in several cancer types and PRDM5 loss results in upregulation of the Wnt pathway and increased cellular proliferation. PRDM5 has not been extensively investigated in specific subtypes of colorectal cancers. We hypothesized it would be more commonly methylated and inactivated in serrated pathway colorectal cancers that are hallmarked by a BRAF V600E mutation and a methylator phenotype, compared to traditional pathway cancers that are BRAF wild type.MethodsCancer (214 BRAF mutant, 122 BRAF wild type) and polyp (59 serrated polyps, 40 conventional adenomas) cohorts were analysed for PRDM5 promoter methylation using MethyLight technology. PRDM5 protein expression was assessed by immunohistochemistry in cancers and polyps. Mutation of PRDM5 was analysed using cBioPortal’s publicly available database.ResultsBRAF mutant cancers had significantly more frequent PRDM5 promoter methylation than BRAF wild type cancers (77/214,36% vs 4/122,3%; p<0.0001). Serrated type polyps had a lower methylation rate than cancers but were more commonly methylated than conventional adenomas (6/59,10% vs 0/40,0%). PRDM5 methylation was associated with advanced stages of presentation (p<0.05) and the methylator phenotype (p=0.03). PRDM5 protein expression was substantially down-regulated in both BRAF mutant and wild type cancer cohorts (92/97,95% and 39/44,89%). The polyp subgroups showed less silencing than the cancers, but similar rates were found between the serrated and conventional polyp cohorts (29/59, 49%; 23/40, 58% respectively). Of 295 colorectal cancers, PRDM5 was mutated in only 6 (2%) cancers which were all BRAF wild type.ConclusionsSerrated pathway colorectal cancers demonstrated early and progressive PRDM5 methylation with advancing disease. Interestingly, PRDM5 protein expression was substantially reduced in all polyp types and more so in cancers which also indicates early and increasing PRDM5 down-regulation with disease progression. Methylation may be contributing to gene silencing in a proportion of BRAF mutant cancers, but the large extent of absent protein expression indicates other mechanisms are also responsible for this. These data suggest that PRDM5 is a relevant tumour suppressor gene that is frequently targeted in colorectal tumourigenesis.

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.

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

ABSTRACT 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.

Copy number profiles of paired primary and metastatic colorectal cancers

Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases (P = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples (P = 0.003). Amplification of the clinically actionable genes ERBB2, FGFR1, PIK3CA or CDK8 was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences.