Physiological and pathological roles of the Hippo‐YAP/TAZ signaling pathway in liver formation, homeostasis, and tumorigenesis

Abstract The liver plays central homeostatic roles in metabolism and detoxification, and has a remarkable capacity to fully recover from injuries caused by the various insults to which it is constantly exposed. To fulfill these functions, the liver must maintain a specific size and so must regulate its cell numbers. It must also remove senescent, transformed, and/or injured cells that impair liver function and can lead to diseases such as cirrhosis and liver cancer. Despite their importance, however, the mechanisms governing liver size control and homeostasis have resisted delineation. The discovery of the Hippo intracellular signaling pathway and its downstream effectors, the transcriptional coactivators Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), has provided partial elucidation of these mechanisms. The Hippo‐YAP/TAZ pathway is considered to be a cell’s sensor of its immediate microenvironment and the cells that surround it, in that this pathway responds to changes in elements such as the ECM, cell–cell tension, and cell adhesion. Once triggered, Hippo signaling negatively regulates the binding of YAP/TAZ to transcription factors such as TEAD and Smad, controlling their ability to drive gene expression needed for cellular responses such as proliferation, survival, and stemness. Numerous KO mouse strains lacking YAP/TAZ, as well as transgenic mice showing YAP/TAZ hyperactivation, have been generated, and the effects of these mutations on liver development, size, regeneration, homeostasis, and tumorigenesis have been reported. In this review, I summarize the components and regulation of Hippo‐YAP/TAZ signaling, and discuss this pathway in the context of liver physiology and pathology.


| INTRODUC TI ON
During mammalian embryogenesis, the liver develops from the foregut derived from the endoderm and functions as a site of hematopoiesis ( Figure 1). 1,2 In the adult, this hematopoietic function is lost and the liver instead plays a central role in metabolism that involves the synthesis, storage, and redistribution of nutrients. The liver is also a major detoxifying organ, removing waste and xenobiotics through metabolic conversion and biliary excretion. Undesirable substances in the gastrointestinal tract enter the liver by way of the portal vein and diffuse through its structure through small blood vessels known as hepatic sinusoids. These sinusoids wind among the several different cell types composing the liver mass, including hepatocytes, which metabolize and detoxify substances, liver sinusoidal endothelial cells, which form the walls of sinusoids and cover the hepatocytes, and Kupffer cells, which are sinusoid-resident macrophages.
For the liver to properly function and maintain homeostasis, it must achieve and preserve a specific size. To this end, the liver must regulate its cell numbers while removing senescent, transformed, and/or damaged cells that can impair function and lead to liver diseases such as cirrhosis and cancer. The liver has a striking capacity to recover from injuries caused by various insults, such as surgical resection, viral infection, metabolic disorders, and chemical or toxic stresses. Interestingly, the processes underlying liver recovery differ depending on the type of injury. In the case of a partial hepatectomy in which 70% of the liver tissue is surgically removed, the remaining 30% returns the liver to near-original size through cell hypertrophy and cell proliferation. 3,4 However, the precise mechanisms that the liver uses to control its size and maintain everyday homeostasis have been difficult to resolve.
The discovery of the Hippo-YAP/TAZ pathway has provided clues that could solve some of the above mysteries. Genetic studies using KO and transgenic mice have revealed that the Hippo-YAP/ TAZ pathway is a key regulator of organ size, regeneration, and homeostasis in many tissues, and that perturbations in the Hippo-YAP/ TAZ pathway can lead to the development of various cancers. Many excellent review articles have been written about these strains and their phenotypes. [5][6][7][8][9][10][11] So as not to repeat these outstanding efforts, I focus in this review on the roles of the Hippo-YAP/TAZ pathway in mammalian liver physiology and pathology, specifically with respect to this organ's development, regeneration, mechanisms of size control and cell competition, and tumorigenesis.
The major effectors downstream of the Hippo core are the transcriptional coactivators YAP and its paralog TAZ. Activation of the Hippo core components results in the phosphorylation of conserved serine residues in YAP/TAZ. Phosphorylated YAP/TAZ proteins either undergo proteasomic degradation in the cytoplasm, or are retained in the cytoplasm by binding to the phosphoserine/phosphothreoninebinding protein 14-3-3. Thus, Hippo activation negatively regulates YAP/TAZ activity. Conversely, a lack of triggering of Hippo signaling, or inactivation of a pathway element, allows unphosphorylated YAP/TAZ to translocate into the nucleus. These coactivators then bind to various TFs, including TEAD1/2/3/4, Smad1/2/3, p73, KLF5, F I G U R E 1 Liver development, regeneration, and homeostasis. In the mammalian embryo, the endoderm gives rise to the foregut, which in turn gives rise to the fetal liver. In the adult, the liver plays a central role in metabolism and detoxification. The normal adult liver can successfully regenerate even after 70% of its mass is removed by partial hepatectomy. When there is a failure to regulate liver cell number or remove senescent, transformed, and/or damaged cells (gray arrows), abnormalities like hepatomegaly (increased liver size) can impair function and lead to liver diseases, including cirrhosis and cancer include Ctgf, Cyr 61, and Birc5, also known as Survivin. [12][13][14][15] The expression of these TEAD target genes is balanced by VGLL4, which competes with YAP/TAZ for binding to the TEADs and represses TEAD target gene expression. 16,17

| RegulationofHippo-YAP/TAZsignaling
The Hippo-YAP/TAZ pathway is a sensor of the mechanical properties of the extracellular environment surrounding a cell and a F I G U R E 2 Hippo signaling and the regulation of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)-dependent gene expression. (A) Once triggered, the Hippo pathway initiates the activation of the mammalian STE20-like protein kinases (MST1/2; Hpo in Drosophila) supported by their adaptor protein Salvador homolog-1 (Sav1). MST1/2 phosphorylate the large tumor suppressor homolog kinases (LATS1/2) kinases to activate them, supported by their adaptor proteins Mob1a/b. Activated LATS1/2 phosphorylate YAP/TAZ, negatively regulating their translocation from the cytoplasm to the nucleus by either inducing binding to 14-3-3 protein or promoting degradation. In the absence of Hippo signaling, unphosphorylated YAP/TAZ bind to many transcription factors (TFs) and coactivate them to launch transcription of their target genes. In the liver, the main targets of YAP/TAZ binding are the transcriptional enhanced associate domain (TEAD) TFs. Transcription cofactor vestigial-like protein 4 (VGLL4) competes with YAP/TAZ for binding to TEADs and represses TEAD target gene expression. (B) The indicated mechanical cues regulate the Hippo-YAP/TAZ pathway by activating, or inactivating, Rho GTPases. These Rho GTPases promote F-actin formation, which blocks LATS1/2 activity. Rho GTPase-independent inhibition of LATS1/2 activity also exists. In the absence of LATS1/2-mediated phosphorylation (and sometimes independently of LATS1/2), YAP/TAZ are free to enter the nucleus and coactivate transcription by TEADs, inducing the indicated diverse cellular responses. EMT, epithelial-mesenchymal transition; GPCR, G protein-coupled receptor tension, cell stretching, altered cell shape, and cell-cell contact, initiate a chain of events that leads to activation or inactivation of Hippo signaling and thus inactivation or activation of YAP/TAZ, followed by specific effects on cell behavior ( Figure 2B). In particular, cell stresses trigger the activation of intracellular Rho GTPases, which may also be stimulated (or inhibited) following the binding of soluble extracellular factors, such as lysophosphatidic acid and sphingosine-1 phosphate, to surface G protein-coupled receptors. 18,19 Once activated, these Rho GTPases induce the remodeling of F-actin within the cell, which in turn can control the coactivation function of YAP/ TAZ in both LATS1/2-dependent and -independent ways. 20,21 When F-actin inactivates LATS1/2, YAP/TAZ avoid phosphorylation, translocate to the nucleus, and enable TEAD-mediated target gene expression. The functions of these genes then allow the cell to take action to alleviate the stress. For example, consider "cell contact inhibition", which is a well-known phenomenon in which cells in monolayer culture stop proliferating when they reach confluence.
In response to cell-cell contact, the angiomotin complex at the tight junction directly binds to YAP/TAZ. Angiomotin then stimulates LATS-dependent phosphorylation of YAP/TAZ that inhibits their activity. 22 The cells can no longer transcribe the genes driving proliferation and culture overgrowth is prevented. Conversely, when Hippo signaling is inactivated by loss of function of a kinase or adaptor protein, YAP/TAZ activity in the nucleus is uncontrolled, cell proliferation is rampant, and tumorigenesis can initiate. Thus, although the Hippo-YAP/TAZ pathway has properties shared by many other existing signaling pathways, it also displays unique regulatory mechanisms.

| FUN C TI ON SOFHIPP O -YAP/ TA Z S I G NALINGINTHELIVER
Tissues and organs undergo stress that can lead to damaged, senescent, and/or transformed cells requiring elimination. The loss of these cells must then be compensated for by cell proliferation, which maintains the size and functionality of the tissues and organs.
The liver functions normally over a long period of time despite being exposed to more stress than most tissues, suggesting the existence of a variety of mechanisms that maintain liver homeostasis starting from birth. Some of these mechanisms involve Hippo signaling and YAP/TAZ regulation. 23

| Earlyembryogenesisandliverdevelopment
Various animal-based methods of studying the Hippo-YAP/TAZ pathway have been reported (

| Liversizecontrolandregeneration
The In

| Livercancerformationandsuppression
There is now much evidence that dysregulation of the Hippo-YAP pathway promotes liver cancer formation. The overexpression of YAP in mouse liver caused by any one of numerous defects in the Hippo pathway induced not only hepatomegaly but also eventually liver cancer. 15,29,32-36 Liver-specific Mob1a/1b double-deficient mice also developed liver cancer, which could be suppressed by inactivation of the YAP gene in these animals. 29 In general, the liver pheno-

| Cellcompetition
"Cell competition" is a type of cell-cell interaction that was originally discovered in the imaginal wing disc of Drosophila melanogaster. 40 During cell competition, a cell compares its fitness to that of its neighboring cells. Cells that are less fit than their neighbors are "losers" and are eliminated by either apoptosis or apical extrusion; cells that are more fit are the "winners" and survive. For example, within the Drosophila wing disc, cells that were heterozygous for the Minute gene, which encodes a ribosomal protein, underwent apoptosis as losers when they were confronted with WT cells ( Figure 3A). 41,42 Activation of the Src oncogene also turns Drosophila cells into losers, In other words, YAP acts as a stress sensor that induces elimination of injured hepatocytes to maintain tissue and liver homeostasis.

| THER APEUTI CPER S PEC TIVE
The studies described above raise the tempting possibility of ma-  57 Although this latter approach might seem to offer proof-of-concept that differentiation The normal mouse liver maintains its size and functionality through various mechanisms that control cell proliferation and remove senescent, damaged or abnormal cells. For example, if a hepatocyte sustains a change that causes it to constitutively express YAP (red cells), the cell proliferates. "Size control" mechanisms then act to remove the altered cells and restore normal liver size. If these mechanisms fail, the altered cells proliferate uncontrollably and liver cancer may develop. Similarly, if traumatic liver injury damages a hepatocyte, YAP can become activated and "quality control" mechanisms are triggered to return the liver to normalcy. The injured cell may be eliminated by apical extrusion or induction of apoptosis, followed by Kupffer cell-mediated engulfment

| CON CLUS ION
The Hippo-YAP/TAZ pathway is a very attractive target of exploration from the point of view of liver physiology and pathology.
Tellingly, Hippo pathway mutations are extremely rare in human liver cancers. 58 This observation suggests that nongenetic factors, such as the status of elements in the surrounding microenvironment (e.g., stiff ECM), are vital for the activation of YAP/TAZ linked to cancer initiation. Multifaceted investigation of these issues is sure to yield much helpful information on Hippo-YAP/TAZ signaling in liver biology.

CO N FLI C T O F I NTE R E S T
The author declares no conflicts of interest.