YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway

Abstract

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),...

Role of Src Family Kinases in Regulation of Intestinal Epithelial Homeostasis

“…7D). The expression of the YAP target gene for amphiregulin (Areg) (41,42) was also significantly increased in crypts of the mutant mice (Fig. 7E).…”

The Hippo pathway regulates stem cell proliferation, self-renewal, and differentiation

“…Furthermore, Yki-Sd was shown to transcriptionally induce dMyc, a potent promoter of ribosome biogenesis and cell growth (Neto-Silva et al, 2010;Ziosi et al, 2010), which may mediate the cell competition phenomenon observed in tissues with imbalance of Hippo pathway activity. In addition, Yki induces E2F1 (Goulev et al, 2008), which may be involved in cell-autonomous regulation of cell proliferation; the EGFR ligands Vein, Keren, and Spitz (Zhang et al, 2009b;Ren et al, 2010); the Jak/Stat pathway ligands Unpaired1/2/3 (Upd1/2/3) (Karpowicz et al, 2010;Ren et al, 2010;Shaw et al, 2010;Staley and Irvine, 2010), which could mediate non-cell-autonomous function of the Hippo pathway; and the Hippo pathway genes Ex, Kibra, Crb, and Fj (Cho et al, 2006;Hamaratoglu et al, 2006;Genevet et al, 2009;Genevet et al, 2010), which may constitute a signal feedback loop.…”

“…CTGF alone, however, does not account for all YAP phenotypes. YAP and TAZ also induce the expression of AREG (Zhang et al, 2009b) and FGF1 (Hao et al, 2008), which may also mediate non-cell-autonomous function of the Hippo pathway. However, the mechanisms underlying the induction of these genes, including the responsible transcription factors, are mostly unclear.…”

When pathways collide: collaboration and connivance among signalling proteins in development

“…However, this has changed, as a recent study identified the epidermal growth factor receptor (EGFR) ligand Amphiregulin as a transcriptional target of the Hippo pathway in mammals 83 , and showed that activation of YAP leads to proliferation of neighboring cells in an EGRF dependent manner. This study also demonstrated that Yorkie shows genetic interactions with the EGFR in flies, suggesting that alterations in Hippo pathway activity will have nonautonomous effects in both normal development and in cancers.…”