The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis1-3. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation4-10. To date, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighboring untransfected cells, implicating a noncell autonomous mechanism. We identify the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG), as a transcriptional target of YAP, whose induction contributes to YAPmediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1/2 is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signaling components support the link between these two highly conserved signaling pathways. Thus, YAP-dependent secretion of AREG implicates activation of EGFR signaling as an important non-cell autonomous effector of the Hippo pathway, with implications for the regulation of both physiological and malignant cell proliferation.Normal cells require mitogenic growth signals to proliferate, whereas, tumor cells often generate their own proliferative signals through the secretion of growth factors or the activation of growth factor receptors 11 . We have shown that YAP transduced MCF10A cells proliferate in 3D acinar cultures in the absence of EGF 7 . MCF10A are immortalized, non transformed human mammary epithelial cells, which exhibit dependence on growth factors for proliferation and survival 12 , raising the possibility that YAP itself induces secretion of required growth factors or cytokines in these cells. To test this hypothesis, we performed mixing experiments with cells transduced with either GFP-labeled YAP or Red-Cherry tagged vector. MCF10A cells expressing GFP-YAP, but not Cherry-vector, formed acini in 3D cultures in the absence exogenous EGF. Remarkably, vector transduced cells did produce acini when co-cultured in a 1:1 ratio with YAP expressing cells (Fig. 1a) To identify mediators of this apparent YAP-induced non-cell-autonomous effect, we made use of a constitutively active YAP mutant, YAP-S127A. Mutation in the serine residue targeted for phosphorylation by the negative regulatory kinases LATS1/2 prevents cytoplasmic sequestration of YAP by 14-3-3 proteins 5 , 10 , thus resulting in its exclusively nuclear localization (Fig. S1a). Retroviral vectors containing wild type YAP or YAP-S127A induced similar levels of protein expression (Fig. 1b). Ectopic expression of YAP-S127A induced a strong EMT and cell migration phenotype ( Fig. S1b and S1c),...

Background-Intravenous (IV) amiodarone has proven efficacy in adults. However, its use in children is based on limited retrospective data. Methods and Results-A double-blind, randomized, multicenter, dose-response study of the safety and efficacy of IV amiodarone was conducted in 61 children (30 days to 14.9 years; median, 1.6 years). Children with incessant tachyarrhythmias (supraventricular arrhythmias [nϭ26], junctional ectopic tachycardia [JET, nϭ31], or ventricular arrhythmias [nϭ4]) were randomized to 1 of 3 dosing regimens (low, medium, or high: load plus 47-hour maintenance) with up to 5 open-label rescue doses. The primary efficacy end point was time to success. Of 229 patients screened, 61 were enrolled during 13 months by 27 of 48 centers in 7 countries. Median time to success was significantly related to dose (28.2, 2.6, and 2.1 hours for the low-, medium-, and high-dose groups, respectively; Pϭ0.028). There was no significant association with dose for any arrhythmia subgroup, including JET, but the subgroups were too small for an accurate assessment. Adverse events (AEs) were common (87%), leading to withdrawal of 10 patients. There were 5 deaths in the 30-day follow-up period (2 possibly related to the study drug). Dose-related AEs included hypotension (36%), vomiting (20%), bradycardia (20%), atrioventricular block (15%) and nausea (10%). Conclusions-In children, the overall efficacy of IV amiodarone, as measured by time to success, was dose related but not significantly for any arrhythmia subgroup. AEs were common and appeared to be dose related. Although efficacious for critically ill patients, the dose-related risks of IV amiodarone should be taken into account when treating children with incessant arrhythmias. Prospective, placebo-controlled trials would be helpful in assessing antiarrhythmic drug efficacy in children, because their results may differ from retrospective series and adult studies.

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