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BRAFV600E 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 BRAFV600E mutant CRCs. Recent genetic analyses of CRCs indicate that BRAFV600E 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 BraffloxV600E, Trp53LSL-R172H, and/or Apcflox/flox alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAFV600E expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAFV600E alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAFV600E induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAFV600E-driven CRC. Co-expression of p53R172H delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53R172H cooperates with BRAFV600E 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 BRAFV600E-induced disintegration and confers growth/niche factor independence. Still, Apc-deficient BRAFV600E-mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53R172H 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.

The emerging field of synthetic biology is a novel biological discipline at the interface between traditional biology, chemistry, and engineering sciences. Synthetic biology aims at the rational design of complex synthetic biological devices and systems with desired properties by combining compatible, modular biological parts in a systematic manner. While the first engineered systems were mainly proof-of-principle studies to demonstrate the power of the modular engineering approach of synthetic biology, subsequent systems focus on applications in the health, environmental, and energy sectors. This review describes recent approaches for biomedical applications that were developed along the synthetic biology design hierarchy, at the level of individual parts, of devices, and of complex multicellular systems. It describes how synthetic biological parts can be used for the synthesis of drug-delivery tools, how synthetic biological devices can facilitate the discovery of novel drugs, and how multicellular synthetic ecosystems can give insight into population dynamics of parasites and hosts. These examples demonstrate how this new discipline could contribute to novel solutions in the biopharmaceutical industry.

The Wnt signaling pathway has been associated with many essential cell processes. This study aims to examine the effects of Wnt signaling on proliferation of cultured HEK293T cells. Cells were incubated with Wnt3a, and the activation of the Wnt pathway was followed by analysis of the level of the β-catenin protein and of the expression levels of the target genes MYC and CCND1. The level of β-catenin protein increased up to fourfold. While the mRNA levels of c-Myc and cyclin D1 increased slightly, the protein levels increased up to a factor of 1.5. Remarkably, MTT and BrdU assays showed different results when measuring the proliferation rate of Wnt3a stimulated HEK293T cells. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the applied Wnt3a concentration. Oppositely, this correlation could not be shown in the MTT assays. The MTT results, which are based on the mitochondrial activity, were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting. Taken together, our study shows that Wnt3a activates proliferation of HEK293 cells. These effects can be detected by measuring DNA synthesis rather than by measuring changes of mitochondrial activity.