^V600E Braf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16 ^INK4a

Abstract

The majority of human colorectal cancers (CRCs) are initiated by mutations arising in the adenomatous polyposis coli (APC) tumour suppressor gene. However, a new class of non-APC mutated CRCs has been defined that have a serrated histopathology and carry the V600EBRAF oncogene. Here we have investigated the pathogenesis of serrated CRCs by expressing V600EBraf in the proliferative cells of the mouse gastrointestinal tract. We show that the oncogene drives an initial burst of Mek-dependent proliferation, leading to the formation of hyperplastic crypts. This is associated with β-catenin nuclear localization by a mechanism involving Mapk/Erk kinase (Mek)-dependent, Akt-independent phosphorylation of Gsk3β. However, hyperplastic crypts remain dormant for prolonged periods due to the induction of crypt senescence accompanied by upregulation of senescence-associated β-galactosidase and p16Ink4a. We show that tumour progression is associated with down-regulation of p16Ink4a through enhanced CpG methylation of exon 1 and knockout of Cdkn2a confirms this gene is a barrier to tumour progression. Our studies identify V600EBRAF as an early genetic driver mutation in serrated CRCs and indicate that, unlike APC-mutated cancers, this subtype arises by the bypassing of a V600EBraf driven oncogene-induced senescence programme.

Up and downregulation of p16Ink4a expression in BRAF-mutated polyps/adenomas indicates a senescence barrier in the serrated route to colon cancer

“…This was overcome by methylation of the CDKN2A promoter leading to the development of invasive carcinomas. 28 The mouse model confirms our findings in human serrated polyps.…”

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

“…25,26 We found that induction of BRAF V600K induced significant DNA damage in the intestine, as demonstrated by immunofluorescence analysis of phosphorylated histone γH2A. However, we did not find increased apoptosis in BRAF V600K -induced intestinal tissues, as assessed by cleaved caspase 3 IHC (Supplementary Figure 8).…”

“…This is in contrast to previous mouse models of oncogenic activation of BRAF, which used knock-in alleles. 18,26 As a consequence, BRAF V600K induction in our mouse model results in immediate and strong activation the MAPK cascade. Consequently, MAPK target genes in transgenic BRAF V600K mice were induced to levels characteristic of more advanced stages of tumor progression obtained in BRAF V637E knock-in mice.…”

p16Ink4a overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors

“…However, recent evidence suggests that not all kinds of cells have the ability to enter in senescence when faced with the same stimuli. In fact, recent studies have shown that expression of oncogenic K-RAS in mice induces senescence in colonic cells, while small bowel cells become only hyperplasic (Bennecke et al, 2010;Carragher et al, 2010). These results suggest that something more than oncogenic stimuli is needed to induce senescence, and this secondary event seems to be tissue/cellular background specific.…”

“…In the colon, two different patterns of p16 Ink4a overexpression have been observed. The first pattern is related to senescence in serrated adenomas, which have malignant transformation associated with p16 Ink4a downregulation (Carragher et al, 2010). The second pattern is characterized by a very low p16 Ink4a immunostaining in normal mucosa, with a progressively higher expression in aberrant crypt foci, non-serrated adenomas, primary carcinomas and metastatic tumors (Dai et al, 2000;Zhao et al, 2006).…”