Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRAS G12V in mouse intestinal organoids, while transgenic BRAF V600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with β-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

BackgroundThe neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.Methodology/Principal FindingsMice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.Conclusion/SignificanceSynthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.

1Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer and are 2 thought to constitutively activate the terminal mitogen-activated protein kinase, ERK. Using mass 3 cytometry, we found graded phosphorylation of ERK anti-correlated with cell differentiation in patient-4 derived colorectal cancer organoids, independent of KRAS mutational status. Reporter, single cell 5 transcriptome and mass cytometry analyses showed that transgenic KRAS G12V activated ERK in a cell 6 type-specific pattern in mouse intestinal organoids. In contrast, transgenic BRAF V600E triggered high ERK 7activity and downstream gene expression in all intestinal cell types, followed by epithelial 8 disorganisation. Quantitative network modelling from perturbation data revealed that activation of 9 ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We 10 identified dual-specificity phosphatases as candidate modulators of ERK activity between intestinal cell 11 types. Furthermore, we found that oncogenic KRAS, together with β-Catenin, favoured expansion of 12 crypt cells with high ERK activity. Our experiments highlight key differences between ERK activity 13 elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected heterogeneity in a 14 signalling pathway with fundamental relevance for cancer therapy. 15