The Hippo Pathway: Biology and Pathophysiology.

The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size control, and regeneration. The core of the Hippo pathway in mammals consists of a kinase cascade, MST1/2 and LATS1/2, as well as downstream effectors, transcriptional coactivators YAP and TAZ. These core components of the Hippo pathway control transcriptional programs involved in cell proliferation, survival, mobility, stemness, and differentiation. The Hippo pathway is tightly regulated by both intrinsic and extrinsic signals, such as mechanical force, cell-cell contact, polarity, energy status, stress, and many diffusible hormonal factors, the majority of which act through G protein-coupled receptors. Here, we review the current understanding of molecular mechanisms by which signals regulate the Hippo pathway with an emphasis on mechanotransduction and the effects of this pathway on basic biology and human diseases.

The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity.

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer.

Two decades of studies in multiple model organisms have established the Hippo pathway as a key regulator of organ size and tissue homeostasis. By inhibiting YAP and TAZ transcription co-activators, the Hippo pathway regulates cell proliferation, apoptosis, and stemness in response to a wide range of extracellular and intracellular signals, including cell-cell contact, cell polarity, mechanical cues, ligands of G-protein-coupled receptors, and cellular energy status. Dysregulation of the Hippo pathway exerts a significant impact on cancer development. Further investigation of the functions and regulatory mechanisms of this pathway will help uncovering the mystery of organ size control and identify new targets for cancer treatment.

Alternative Wnt Signaling Activates YAP/TAZ.

The transcriptional co-activators YAP and TAZ are key regulators of organ size and tissue homeostasis, and their dysregulation contributes to human cancer. Here, we discover YAP/TAZ as bona fide downstream effectors of the alternative Wnt signaling pathway. Wnt5a/b and Wnt3a induce YAP/TAZ activation independent of canonical Wnt/ß-catenin signaling. Mechanistically, we delineate the "alternative Wnt-YAP/TAZ signaling axis" that consists of Wnt-FZD/ROR-Gα12/13-Rho GTPases-Lats1/2 to promote YAP/TAZ activation and TEAD-mediated transcription. YAP/TAZ mediate the biological functions of alternative Wnt signaling, including gene expression, osteogenic differentiation, cell migration, and antagonism of Wnt/ß-catenin signaling. Together, our work establishes YAP/TAZ as critical mediators of alternative Wnt signaling.

Acetyl-CoA, protein acetylation, and liver cancer.

Using multi-omics approaches, Park et al. show that reduced cellular acetyl-CoA and protein hypoacetylation promote liver cancer growth and dedifferentiation.

Hippo pathway key to ploidy checkpoint.

Tetraploid cells generated by abnormal cell division are often arrested during the cell cycle or cleared by apoptosis. Evasion of these defense mechanisms leads to genomic instability and tumorigenesis. In this issue, Ganem et al. report that extra centrosome-induced activation of the Hippo pathway kinase LATS2 is a key mechanism of tetraploidy-induced cell-cycle arrest.

Amino Acids License Kinase mTORC1 Activity and Treg Cell Function via Small G Proteins Rag and Rheb.

Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.

Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.

Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.

Critical roles of phosphoinositides and NF2 in Hippo pathway regulation.

The Hippo pathway is a master regulator of tissue homeostasis and organ size. NF2 is a well-established tumor suppressor, and loss of NF2 severely compromises Hippo pathway activity. However, the precise mechanism of how NF2 mediates upstream signals to regulate the Hippo pathway is not clear. Here we report that, in mammalian cells, NF2's lipid-binding ability is critical for its function in activating the Hippo pathway in response to osmotic stress. Mechanistically, osmotic stress induces PI(4,5)P2 plasma membrane enrichment by activating the PIP5K family, allowing for NF2 plasma membrane recruitment and subsequent downstream Hippo pathway activation. An NF2 mutant deficient in lipid binding is unable to activate the Hippo pathway in response to osmotic stress, as measured by LATS and YAP phosphorylation. Our findings identify the PIP5K family as novel regulators upstream of Hippo signaling, and uncover the importance of phosphoinositide dynamics, specifically PI(4,5)P2, in Hippo pathway regulation.

Induction of AP-1 by YAP/TAZ contributes to cell proliferation and organ growth.

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control cell growth and organ size, of which dysregulation yields to tumorigenesis or hypertrophy. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or cell specification. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote FOS transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of FOS to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. FOS induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as Lats1/2-deficient cancer cells as well as Gαq/11 mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. Our findings reveal a feed-forward interplay between immediate early transcription of AP-1 and Hippo pathway function.

Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

OTUB2 Promotes Cancer Metastasis via Hippo-Independent Activation of YAP and TAZ.

The transcriptional regulators YAP and TAZ play important roles in development, physiology, and tumorigenesis and are negatively controlled by the Hippo pathway. It is yet unknown why the YAP/ TAZ proteins are frequently activated in human malignancies in which the Hippo pathway is still active. Here, by a gain-of-function cancer metastasis screen, we discovered OTUB2 as a cancer stemness and metastasis-promoting factor that deubiquitinates and activates YAP/TAZ. We found OTUB2 to be poly-SUMOylated on lysine 233, and this SUMOylation enables it to bind YAP/TAZ. We also identified a yet-unknown SUMO-interacting motif (SIM) in YAP and TAZ required for their association with SUMOylated OTUB2. Importantly, EGF and oncogenic KRAS induce OTUB2 poly-SUMOylation and thereby activate YAP/TAZ. Our results establish OTUB2 as an essential modulator of YAP/TAZ and also reveal a novel mechanism via which YAP/TAZ activity is induced by oncogenic KRAS.

Amino acid signaling in TOR activation.

The target of rapamycin (TOR) is a central cell growth regulator conserved from yeast to mammals. Uncontrolled TOR activation is commonly observed in human cancers. TOR forms two distinct structural and functional complexes, TORC1 and TORC2. TORC1 promotes cell growth and cell size by stimulating protein synthesis. A wide range of signals, including nutrients, energy levels, and growth factors, are known to control TORC1 activity. Among them, amino acids (AA) not only potently activate TORC1 but are also required for TORC1 activation by other stimuli, such as growth factors. The mechanisms of growth factors and cellular energy status in activating TORC1 have been well elucidated, whereas the molecular basis of AA signaling is just emerging. Recent advances in the role of AA signaling on TORC1 activation have revealed key components, including the Rag GTPases, protein kinases, nutrient transporters, and the intracellular trafficking machinery, in relaying AA signals to TORC1 activation.

The multifaceted role of autophagy in cancer.

Autophagy is a cellular degradative pathway that plays diverse roles in maintaining cellular homeostasis. Cellular stress caused by starvation, organelle damage, or proteotoxic aggregates can increase autophagy, which uses the degradative capacity of lysosomal enzymes to mitigate intracellular stresses. Early studies have shown a role for autophagy in the suppression of tumorigenesis. However, work in genetically engineered mouse models and in vitro cell studies have now shown that autophagy can be either cancer-promoting or inhibiting. Here, we summarize the effects of autophagy on cancer initiation, progression, immune infiltration, and metabolism. We also discuss the efforts to pharmacologically target autophagy in the clinic and highlight future areas for exploration.

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

The ribosome and TORC2: collaborators for cell growth.

The target of rapamycin complex 2 (TORC2) is a key regulator of cell growth. Zinzalla et al. (2011) now provide evidence that TORC2 is activated by direct association with the ribosome, which may ensure that TORC2 activity is calibrated to match the cell's intrinsic growth capacity.

Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated Lipid-Protein Interaction.

The Hippo pathway plays a crucial role in organ size control and tumor suppression, but its precise regulation is not fully understood. In this study, we discovered that phosphatidic acid (PA)-related lipid signaling is a key regulator of the Hippo pathway. Supplementing PA in various Hippo-activating conditions activates YAP. This PA-related lipid signaling is involved in Rho-mediated YAP activation. Mechanistically, PA directly interacts with Hippo components LATS and NF2 to disrupt LATS-MOB1 complex formation and NF2-mediated LATS membrane translocation and activation, respectively. Inhibition of phospholipase D (PLD)-dependent PA production suppresses YAP oncogenic activities. PLD1 is highly expressed in breast cancer and positively correlates with YAP activation, suggesting their pathological relevance in breast cancer development. Taken together, our study not only reveals a role of PLD-PA lipid signaling in regulating the Hippo pathway but also indicates that the PLD-PA-YAP axis is a potential therapeutic target for cancer treatment.

Polycystic kidney disease: a Hippo connection.

Mutations in PKD1 and PKD2 are the leading cause of autosomal dominant polycystic kidney disease (ADPKD). In this issue of Genes & Development, a report by Cai and colleagues (pp. 781-793) reveals new insight into the molecular basis by which PKD1 deficiency leads to cystic kidney pathogenesis. By using extensive mouse genetic analyses coupled with in vitro cystic assays, the investigators delineate a RhoA-YAP-c-Myc signaling axis as a key downstream from PKD1 deficiency in ADPKD pathogenesis. Their findings provide evidence that the Hippo pathway could be a potential target for treating ADPKD.

Hippo Signaling in Embryogenesis and Development.

Hippo pathway components are structurally and functionally conserved and are notable for their role in controlling organ size. More diverse functions of the Hippo pathway have been recognized, including development, tissue homeostasis, wound healing and regeneration, immunity, and tumorigenesis. During embryogenesis, different signaling pathways are repeatedly and cooperatively activated, leading to differential gene expression in specific developmental contexts. In this article, we present an overview on the regulation and function of the Hippo pathway in mammalian early development. We introduce the Hippo pathway components and major upstream signals that act through this pathway to influence embryogenesis. We also discuss the roles of Hippo pathway in tissue specification and organ development during organogenesis.