Abstract
Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF p.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF p.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF p.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF p.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF p.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF p.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF p.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.
Highlights
Colorectal cancer (CRC) is a frequently occurring malignancy in the Western world with a 5% life-time risk [1] and a high worldwide annual incidence (n = 1,200,000) and mortality (n = 608,000) [2]
For single gene analyses of DNA methylation profiling and validation, 19 paired tumor-normal samples were selected on presence in the ascending colon, including 8 BRAFp.V600E (6 of which were MSI-H and 2 MSS) and 11 BRAF wild types (9 of which were MSS and 2 MSI-H)
Additional tumor characteristics, including histology, mismatch repair status, CpG island methylator phenotype (CIMP), KRAS and p53 mutation status and MLH1 methylation status are shown in Table S2 and were described previously [18]
Summary
CRC is caused by inactivating mutations in tumor suppressor genes and/or activating mutations in proto-oncogenes Such mutated oncogenes include KRAS and BRAF that are mutually exclusive in colon tumors [3] and are both part of the Mitogen Activated Protein Kinase (MAPK) pathway. In this biological route, signals traffic from growth factor receptors present on the cell membrane towards the nucleus and cause cell proliferation [4]. The BRAF gene encodes for the serine/threonine-protein kinase BRaf and the most commonly found BRAF mutation in CRC is present in exon 15 (c.1799TA) [5] This mutation leads to substitution of valine by the negatively charged glutamic acid at position 600 (BRAFp.V600E) and increased protein kinase activity [6]. MSI-H is caused by deficient mismatch repair as a result of MLH1 mutation or promoter methylation [15]
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