Summary Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers, however the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors which reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25(OH)2D3 activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, Atp2b1) are also induced by 1,25(OH)2D3 in the distal intestine of KO/TG mice. In addition, Slc30a10, a manganese efflux transporter, was one of the genes most induced by 1,25(OH)2D3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA-seq analysis of human enteroids indicated that the effects of 1,25(OH)2D3 observed in mice are conserved in humans. Using Slc30a10 -/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action.

Wnt signaling has an essential role in embryonic development as well as stem/progenitor cell renewal, and its aberrant activation is implicated in many diseases, including several cancers. ␤-Catenin is a critical component of Wnt-mediated transcriptional activation. Here we show that ARF6 activation during canonical Wnt signaling promotes the intracellular accumulation of ␤-catenin via a mechanism that involves the endocytosis of growth factor receptors and robust activation of extracellular signalregulated kinase (ERK). ERK promotes casein kinase 2-mediated phosphorylation of ␣-catenin, leading to destabilization of the adherens junctions and a subsequent increase in cytoplasmic pools of active ␤-catenin and E-cadherin. ERK also phosphorylates LRP6 to amplify the Wnt transduction pathway. The aforementioned Wnt-ERK signaling pathway initiates lumen filling of epithelial cysts by promoting cell proliferation in three-dimensional cell cultures. This study elucidates a mechanism responsible for the switch in ␤-catenin functions in cell adhesion at the adherens junctions and Wnt-induced nuclear signaling.

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While Wnt signaling is clearly important for the intestinal epithelial homeostasis, the relevance of various sources of Wnt ligands themselves remains incompletely understood. Wnt blockage in distinct stromal cell types suggested obligatory functions of several stromal cell sources and yielded different observations. The physiological contribution of epithelial Wnt to tissue homeostasis remains unclear. We show here that blocking epithelial Wnts affected colonic Reg4+ epithelial cell differentiation, and impaired colonic epithelial regeneration after injury. Single cell RNA analysis of intestinal stroma showed that the majority of Wnt-producing cells were contained in transgelin (Tagln+) and smooth muscle actin alpha 2 (Acta2+) expressing populations. We genetically attenuated Wnt production from these stromal cells using Tagln-Cre and Acta2-CreER drivers, and found that Wnt blockage from either epithelium or Tagln+ and Acta2+ stromal cells impaired colonic epithelial healing after chemical-induced injury. Aggregated Wnt blockage from both epithelium and Tagln+ or Acta2+ stromal cells drastically diminished epithelial repair, increasing morbidity and mortality. These results from two uncharacterized stromal populations suggested that colonic recovery from colitis-like injury depends on multiple Wnt-producing sources.