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

Abstract

Colon cancer cells frequently carry mutations that activate the β-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/β-catenin signals encourage ISC identity, we asked whether β-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3β. Similarly, transgenic expression of stabilized β-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF V637E knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/β-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/β-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells

“…With this caveat acknowledged, inhibition of MEK prior to Wnt inhibition blocked proliferation and differentiation of ISCs into TA cells and impaired intestinal homeostasis shortly after C59 administration. This model is consistent with studies showing that increased MAPK signaling in the intestine due to BRAFV600 mutation promotes loss of ISCs and increases differentiation (26,27).…”

RNF43 germline and somatic mutation in serrated neoplasia pathway and its association with BRAF mutation

“…Thus, apart from a predisposition for MSS CRC, whether germline RNF43 mutation carriers have a predisposition for MSI CRC awaits further confirmation. Oncogenic mutation may confer specific stresses that invite particular collaborating mutations, as shown by a previous study whereby induction of BRAF mutation in mouse intestines led to stem cell depletion that could be rescued by Wnt activation, providing a biological explanation for the coordinated alterations in both pathways 35. The preferential co-occurrence of RNF43, instead of APC or β-catenin, with BRAF mutation may be a consequence of complex interactions between environmental mutation load, underlying epigenetic/genetic instability driving susceptibility of specific gene loci, as well as other yet unknown factors.…”

Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer

“…Mutant BRAF activation triggers cellular senescence in some models (Carragher et al, 2010), and intestinal stem cell differentiation in others (Riemer et al, 2015). A third model only observes p21 expression at later stages of tumor progression (Rad et al, 2013).…”

“…While intestinal stem cells are not the only cell capable of serving as the cell of origin (Visvader, 2011), they are by far the most efficient, requiring only a single activating mutation of the WNT/β-catenin pathway - a frequent event associated with the progression of the majority of CRCs, such as APC mutations in adenocarcinomas (Clevers and Nusse, 2012; Sekine et al, 2016). Paradoxically, despite a high prevalence among colorectal tumors, ectopic expression of an oncogenic BRAF mutant transgene promotes either senescence or differentiation of intestinal stem cells (Carragher et al, 2010; Riemer et al, 2015). Thus, while oncogenic activation of BRAF can eventually lead to tumor formation in mouse models, these tumors are slow to arise (Rad et al, 2013; Sakamoto et al, 2017).…”