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BRAF V600E confers poor prognosis and is associated with a distinct subtype of colorectal cancer (CRC). Little is known, however, about the genetic events driving the initiation and progression of BRAF V600E mutant CRCs. Recent genetic analyses of CRCs indicate that BRAF V600E often coexists with alterations in the WNT- and p53 pathways, but their cooperation remains ill-defined. Therefore, we systematically compared small and large intestinal organoids from mice harboring conditional Braf floxV600E , Trp53 LSL-R172H , and/or Apc flox/flox alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAF V600E expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAF V600E alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAF V600E induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAF V600E -driven CRC. Co-expression of p53 R172H delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53 R172H cooperates with BRAF V600E to modulate the abundance of transcripts linked to carcinogenesis, in particular within colonic organoids. Remarkably, WNT-pathway activation by Apc deletion fully protects organoids against BRAF V600E -induced disintegration and confers growth/niche factor independence. Still, Apc-deficient BRAF V600E -mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53 R172H confers partial resistance against this clinically relevant compound. In summary, our systematic comparison of the response of small and large intestinal organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. In addition, our work on BRAF-, p53-, and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-Raf AVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.