Activation of the epidermal growth factor (EGF) receptor (EGFR) at the cell surface initiates signaling through the RAS-RAF-MAPK/ERK1/2 pathway and receptor endocytosis. Whether this signaling continues from endosomes remains unclear, because RAS is predominantly located on the plasma membrane, and the localization of endogenous RAF kinases, downstream effectors of RAS, is not defined. To examine RAF localization, we labeled endogenous RAF1 with mVenus using gene editing. From 10 to 15% of RAF1-mVenus (<2000 molecules/cell), which was initially entirely cytosolic, transiently translocated to the plasma membrane after EGF stimulation. Following an early burst of translocation, the membrane-associated RAF1-mVenus was undetectable by microscopy or subcellular fractionation, and this pool was estimated to be <200 molecules per cell. In contrast, persistent EGF-dependent translocation of RAF1-mVenus to the plasma membrane was driven by the RAF inhibitor sorafenib, which increases the affinity of Ras-GTP:RAF1 interactions. RAF1-mVenus was not found in EGFR-containing endosomes under any conditions. Computational modeling of RAF1 dynamics revealed that RAF1 membrane abundance is controlled most prominently by association and dissociation rates from RAS-GTP and by RAS-GTP concentration. The model further suggested that the relatively protracted activation of the RAF-MEK1/2-ERK1/2 module, in comparison with RAF1 membrane localization, may involve multiple rounds of cytosolic RAF1 rebinding to active RAS at the membrane.

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Here, we show that hypoxia drives especially long-lasting epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) primarily through a positive-feedback histone methylation-MAPK signaling axis. We find that neoplastic PDAC cells preferentially undergo EMT in hypoxic tumor regions in multiple model systems and that hypoxia drives a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, can last for weeks. We further demonstrate that hypoxia reduces histone demethylase KDM2A activity, suppresses PP2 family phosphatase expression, and activates MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors play only a supporting role. This mechanism can be antagonized in vivo by combinations of MAPK inhibitors that may be effective in multi-drug therapies designed to target EMT.

Introduction Pharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing. Methods We modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing. Results When simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1–2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue. Conclusions These findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds.

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