| Popis: |
Approximately 10-20% of human colorectal cancers harbour an activating BRAFV600E mutation, which acts as a founder mutation for an alternative, serrated pathway of colorectal carcinogenesis. Conversely, BRAFV600E mutations are detectable in hyperplastic colonic polyps: lesions traditionally considered not to harbour malignant potential. Furthermore, both in vitro and in vivo, activated oncogenic BRAFV600E induces a stable proliferation arrest: oncogene induced senescence, which is a fundamental intrinsic tumour-suppressor mechanism. Thus, for neoplastic transformation to occur, it is clear that additional genetic and epigenetic events are required. BRAFV600E-mutant colorectal cancers are frequently associated with a CpG island methylator phenotype (CIMP), which is proposed to promote neoplastic transformation by bypass of intrinsic tumour-suppressor mechanisms, such as silencing of CDKN2A/INK4A. Consistent with this, neoplastic transformation in the serrated pathway is characterised by the progressive development of a CIMP phenotype. An emerging body of evidence supports a model in which the BRAFV600E mutation directly induces CIMP through the de novo methyltransferase, DNMT3B. Separately, elevated DNMT3B expression has previously been linked to the development of CIMP in both murine and human colorectal cancer. The published data however do not universally support this model, and significant questions over its validity remain. In the present work, the relationships between BRAFV600E mutation, DNMT3B expression and the CpG island methylator phenotype were examined by multiple approaches. A panel of DNMT3B antibodies were first characterised and validated. Significantly, the antibody previously used to link DNMT3B and CIMP in human colon cancer was demonstrated not to react with human DNMT3B. The ability of BRAFV600E to induce CIMP was next tested by whole genome bisulfite sequencing in a primary cell culture model. Surprisingly, activated BRAFV600E repressed expression of DNMT3B and failed to induce a CIMP phenotype. Consistent with this, human colorectal cancer cell lines expressing activated BRAFV600E typically expressed a low level of DNMT3B, and inactivation of DNMT3B in a BRAFV600E-mutant, CIMP-positive cell line did not reverse gene silencing characteristic of CIMP. An in vitro model system was next designed to test functional interactions between BRAFV600E and DNMT3B. Ectopic expression of DNMT3B antagonised BRAFV600E-induced proliferation arrest: a hallmark of senescence. Moreover, ectopic DNMT3B expression was demonstrated to accelerate BrafV600E-induced intestinal carcinogenesis in a mouse model, and conversely, Dnmt3b knockout impaired BrafV600E-induced murine intestinal carcinogenesis. Analysis of human colorectal cancer TCGA data was next undertaken, and confirmed that expression of DNMT3B is frequently elevated in human colorectal cancer and that this is often linked to amplification of the DNMT3B gene. However, more detailed analysis of human TCGA data revealed that BRAFV600E mutation is neither necessary nor sufficient to induce CIMP, and that both BRAFV600E mutations and CIMP are both linked to low expression of DNMT3B. Thus, while both BRAFV600E and DNMT3B both harbour oncogenic potential, they do not appear to cooperate to induce CIMP, and do not appear to cooperate frequently in human colorectal cancer by any mechanism. |
