Nailing Zhang

Elucidation of a universal size-control mechanism in Drosophila and mammals.

Coordination of cell proliferation and cell death is essential to attain proper organ size during development and for maintaining tissue homeostasis throughout postnatal life. In Drosophila, these two processes are orchestrated by the Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yorkie (Yki). Here we demonstrate that a single phosphorylation site in Yki mediates the growth-suppressive output of the Hippo pathway. Hippo-mediated phosphorylation inactivates Yki by excluding it from the nucleus, whereas loss of Hippo signaling leads to nuclear accumulation and therefore increased Yki activity. We further delineate a mammalian Hippo signaling pathway that culminates in the phosphorylation of YAP, the mammalian homolog of Yki. Using a conditional YAP transgenic mouse model, we demonstrate that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results uncover a universal size-control mechanism in metazoan.

The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals.

The conserved Hippo signaling pathway regulates organ size in Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using conditional knockout mice, we demonstrate that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotypes in multiple tissues were largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Our studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2.

The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program

Although a developmental role for Hippo signaling in organ size control is well appreciated, how this pathway functions in tissue regeneration is largely unknown. Here we address this issue using a dextran sodium sulfate (DSS)-induced colonic regeneration model. We find that regenerating crypts express elevated Yes-associated protein (YAP) levels. Inactivation of YAP causes no obvious intestinal defects under normal homeostasis, but severely impairs DSS-induced intestinal regeneration. Conversely, hyperactivation of YAP results in widespread early-onset polyp formation following DSS treatment. Thus, the YAP oncoprotein must be exquisitely controlled in tissue regeneration to allow compensatory proliferation and prevent the intrinsic oncogenic potential of a tissue regeneration program.

Spatial Organization of Hippo Signaling at the Plasma Membrane Mediated by the Tumor Suppressor Merlin/NF2

Although Merlin/NF2 was discovered two decades ago as a tumor suppressor underlying Neurofibromatosis type II, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo/Mst. In contrast to the commonly depicted linear model of Merlin functioning through Hpo/Mst, here we show that in both Drosophila and mammals, Merlin promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo/Mst. Instead, Merlin directly binds and recruits the effector kinase Wts/Lats to the plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation by the Hpo-Sav kinase complex. We further show that disruption of the actin cytoskeleton promotes Merlin-Wts interactions, which implicates Merlin in actin-mediated regulation of Hippo signaling. Our findings elucidate an important molecular function of Merlin and highlight the plasma membrane as a critical subcellular compartment for Hippo signal transduction.

A temporal requirement for Hippo signaling in mammary gland differentiation, growth, and tumorigenesis

Despite recent progress, the physiological role of Hippo signaling in mammary gland development and tumorigenesis remains poorly understood. Here we show that the Hippo pathway is functionally dispensable in virgin mammary glands but specifically required during pregnancy. In contrast to many other tissues, hyperactivation of YAP in mammary epithelia does not induce hyperplasia but leads to defects in terminal differentiation. Interestingly, loss of YAP causes no obvious defects in virgin mammary glands but potently suppresses oncogene-induced mammary tumors. The selective requirement for YAP in oncogenic growth highlights the potential of YAP inhibitors as molecular targeted therapies against breast cancers.

Yes-Associated Protein Regulates the Hepatic Response After Bile Duct Ligation

Human chronic cholestatic liver diseases are characterized by cholangiocyte proliferation, hepatocyte injury, and fibrosis. Yes‐associated protein (YAP), the effector of the Hippo tumor‐suppressor pathway, has been shown to play a critical role in promoting cholangiocyte and hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. Therefore, the aim of this study was to examine whether YAP participates in the regenerative response after cholestatic injury. First, we examined human liver tissue from patients with chronic cholestasis. We found more‐active nuclear YAP in the bile ductular reactions of primary sclerosing cholangitis and primary biliary cirrhosis patient liver samples. Next, we used the murine bile duct ligation (BDL) model to induce cholestatic liver injury. We found significant changes in YAP activity after BDL in wild‐type mice. The function of YAP in the hepatic response after BDL was further evaluated with liver‐specific Yap conditional deletion in mice. Ablating Yap in the mouse liver not only compromised bile duct proliferation, but also enhanced hepatocyte necrosis and suppressed hepatocyte proliferation after BDL. Furthermore, primary hepatocytes and cholangiocytes isolated from Yap‐deficient livers showed reduced proliferation in response to epidermal growth factor in vitro. Finally, we demonstrated that YAP likely mediates its biological effects through the modulation of Survivin expression. Conclusion: Our data suggest that YAP promotes cholangiocyte and hepatocyte proliferation and prevents parenchymal damage after cholestatic injury in mice and thus may mediate the response to cholestasis‐induced human liver disease. (HEPATOLOGY 2012;56:1097–1107)

Homeostatic control of Hippo signaling activity revealed by an endogenous activating mutation in YAP

The Hippo signaling pathway converges on YAP to regulate growth, differentiation, and regeneration. Previous studies with overexpressed proteins have shown that YAP is phosphorylated by its upstream kinase, Lats1/2, on multiple sites, including an evolutionarily conserved 14-3-3-binding site whose phosphorylation is believed to inhibit YAP by excluding it from the nucleus. Indeed, nuclear localization of YAP or decreased YAP phosphorylation at this site (S168 in Drosophila, S127 in humans, and S112 in mice) is widely used in current literature as a surrogate of YAP activation even though the physiological importance of this phosphorylation event in regulating endogenous YAP activity has not been defined. Here we address this question by introducing a Yap(S112A) knock-in mutation in the endogenous Yap locus. The Yap(S112A) mice are surprisingly normal despite nuclear localization of the mutant YAP protein in vivo and profound defects in cytoplasmic translocation in vitro. Interestingly, the mutant Yap(S112A) mice show a compensatory decrease in YAP protein levels due to increased phosphorylation at a mammalian-specific phosphodegron site on YAP. These findings reveal a robust homeostatic mechanism that maintains physiological levels of YAP activity and caution against the assumptive use of YAP localization alone as a surrogate of YAP activity.

Yes-Associated Protein impacts adherens junction assembly through regulating actin cytoskeleton organization

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

YAP Is Essential for Treg-Mediated Suppression of Antitumor Immunity

Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their function. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg function are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling, which amplifies TGFβ/SMAD activation in Tregs. YAP deficiency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifier of a Treg-reinforcing pathway with significant potential as an anticancer immunotherapeutic target.Significance: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fighting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026-43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.