Organ Size Control Research Articles

The Hippo/YAP1 pathway interacts with FGFR1 signaling to maintain stemness in lung cancer

The Hippo pathway plays a critical role in organ size control, tissue homeostasis and tumor genesis through its key transcription regulator Yes-associated protein1 (YAP1), but the mechanism underlying its role in lung cancer is unclear. We hypothesized that YAP1 influences FGFR1 signaling to maintain cancer stem-like cell (CSC) properties in FGFR1-amplified lung cancer. In support of this, our data confirms that expression levels of YAP1 are positively associated with those of FGFR1 in clinical lung carcinoma samples as measured by real-time PCR, western blot, and immunohistochemistry (IHC) staining. Mechanistically, YAP1 up-regulates FGFR1 expression at the level of promoter through the TEAD binding site while bFGF/FGFR1 induces YAP1 expression via large tumor suppressors 1(LATS1). In addition, the absence of YAP1 abolishes self-renewal ability in lung cancer. Furthermore, an orthotropic mouse model highlights the function of YAP1 in the initiation and metastasis of lung cancer. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and FGFR1 expression in lung cancer. Thus, we conclude that YAP1 is a potential therapeutic target for lung cancer. Combined targeting of YAP1 and FGFR1 may provide benefits to patients with FGFR1-amplified lung cancer.

STERILE APETALA modulates the stability of a repressor protein complex to control organ size in Arabidopsis thaliana.

Organ size control is of particular importance for developmental biology and agriculture, but the mechanisms underlying organ size regulation remain elusive in plants. Meristemoids, which possess stem cell-like properties, have been recognized to play important roles in leaf growth. We have recently reported that the Arabidopsis F-box protein STERILE APETALA (SAP)/SUPPRESSOR OF DA1 (SOD3) promotes meristemoid proliferation and regulates organ size by influencing the stability of the transcriptional regulators PEAPODs (PPDs). Here we demonstrate that KIX8 and KIX9, which function as adaptors for the corepressor TOPLESS and PPD, are novel substrates of SAP. SAP interacts with KIX8/9 and modulates their protein stability. Further results show that SAP acts in a common pathway with KIX8/9 and PPD to control organ growth by regulating meristemoid cell proliferation. Thus, these findings reveal a molecular mechanism by which SAP targets the KIX-PPD repressor complex for degradation to regulate meristemoid cell proliferation and organ size.

A Local Auxin Gradient Regulates Root Cap Self-Renewal and Size Homeostasis

Organ size homeostasis, compensatory growth to replace lost tissue, requires constant measurement of size and adjustment of growth rates. Morphogen gradients control organ and tissue size by regulating stem cell activity, cell differentiation and removal in animals. In plants, control of organ size is of specific importance in root caps to protect the growing root tip from mechanical damage. New root cap tissue (columella cells) is formed by the columella stem cells, whose activity is regulated through the quiescent center (qc), non-dividing niche-like cells. Daughter cells in contact with the qc retain the potency to divide, while derivatives oriented towards the mature cap undergo differentiation into columella cells. The outermost columella layers are sequentially separated from the root body involving remodeling of cell walls. Factors regulating the balance between cell division, elongation and separation to keep root cap size constant are currently unknown. Here, we report that stem cell proliferation induced cell separation at the periphery of the root cap resulting in organ size homeostasis. An auxin response gradient with a maximum in the niche cells and a minimum in the detaching layer was established prior to the onset of cell separation. In agreement with a mathematical model, organ size was positively regulated by the amount of auxin released from the source. Auxin transporters localized non-polarly to plasma membranes of the inner cap isolating separating layers from the auxin source. Together, these results are in support of an auxin gradient measuring and regulating organ size.

Lats1/2 inactivation reveals Hippo function in alveolar type I cell differentiation during lung transition to air breathing.

Lung growth to its optimal size at birth is driven by reiterative airway branching followed by differentiation and expansion of alveolar cell types. How this elaborate growth is coordinated with the constraint of the chest is poorly understood. Here, we investigate the role of Hippo signaling, a cardinal pathway in organ size control, in mouse lung development. Unexpectedly, we found that epithelial loss of the Hippo kinase genes Lats1 and Lats2 (Lats1/2) leads to a striking reduction of lung size owing to an early arrest of branching morphogenesis. This growth defect is accompanied by abnormalities in epithelial cell polarity, cell division plane and extracellular matrix deposition, as well as precocious and increased expression of markers for type 1 alveolar epithelial cells (AEC1s), an indicator of terminal differentiation. Increased AEC1s were also observed in transgenic mice with overexpression of a constitutive nuclear form of downstream transcriptional effector YAP. Conversely, loss of Yap and Taz led to decreased AEC1s, demonstrating that the canonical Hippo signaling pathway is both sufficient and necessary to drive AEC1 fate. These findings together reveal unique roles of Hippo-LATS-YAP signaling in the developing mouse lung.

Hippo pathway coactivators Yap and Taz are required to coordinate mammalian liver regeneration

The mammalian liver has a remarkable capacity for repair following injury. Removal of up to two-third of liver mass results in a series of events that include extracellular matrix remodeling, coordinated hepatic cell cycle re-entry, restoration of liver mass and tissue remodeling to return the damaged liver to its normal state. Although there has been considerable advancement of our knowledge concerning the regenerative capacity of the mammalian liver, many outstanding questions remaining, such as: how does the regenerating liver stop proliferating when appropriate mass is restored and how do these mechanisms relate to normal regulation of organ size during development? Hippo pathway has been proposed to be central in mediating both events: organ size control during development and following regeneration. In this report, we examined the role of Yap and Taz, key components of the Hippo pathway in liver organ size regulation, both in the context of development and homeostasis. Our studies reveal that contrary to the current paradigms that Yap/Taz are not required for developmental regulation of liver size but are required for proper liver regeneration. In livers depleted of Yap and Taz, liver mass is elevated in neonates and adults. However, Yap/Taz-depleted livers exhibit profound defects in liver regeneration, including an inability to restore liver mass and to properly coordinate cell cycle entry. Taken together, our results highlight requirements for the Hippo pathway during liver regeneration and indicate that there are additional pathways that cooperate with Hippo signaling to control liver size during development and in the adult.

Deubiquitylase USP9X suppresses tumorigenesis by stabilizing large tumor suppressor kinase 2 (LATS2) in the Hippo pathway

The Hippo pathway plays important roles in controlling organ size and in suppressing tumorigenesis through large tumor suppressor kinase 1/2 (LATS1/2)-mediated phosphorylation of YAP/TAZ transcription co-activators. The kinase activity of LATS1/2 is regulated by phosphorylation in response to extracellular signals. Moreover, LATS2 protein levels are repressed by the ubiquitin-proteasome system in conditions such as hypoxia. However, the mechanism that removes the ubiquitin modification from LATS2 and thereby stabilizes the protein is not well understood. Here, using tandem affinity purification (TAP), we found that anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex, and USP9X, a deubiquitylase, specifically interact with LATS2. We also found that although APC1 co-localizes with LATS2 to intracellular vesicle structures, it does not regulate LATS2 protein levels and activity. In contrast, USP9X ablation drastically diminished LATS2 protein levels. We further demonstrated that USP9X deubiquitinates LATS2 and thus prevents LATS2 degradation by the proteasome. Furthermore, in pancreatic cancer cells, USP9X loss activated YAP and enhanced the oncogenic potential of the cells. In addition, the tumorigenesis induced by the USP9X ablation depended not only on LATS2 repression, but also on YAP/TAZ activity. We conclude that USP9X is a deubiquitylase of the Hippo pathway kinase LATS2 and that the Hippo pathway functions as a downstream signaling cascade that mediates USP9X's tumor-suppressive activity.

Hippo Signaling in the Immune System

Hippo signaling has a pivotal role in organ size control, tissue regeneration, and cancer. Recent studies have demonstrated critical functions of Hippo signaling in cancer immunity, innate immune responses against pathogens, and autoimmune diseases, refreshing our understanding of the implications of this pathway in the context of disease and therapy design.

PaCYP78A9, a Cytochrome P450, Regulates Fruit Size in Sweet Cherry (Prunus avium L.).

Sweet cherry (Prunus avium L.) is an important fruit crop in which fruit size is strongly associated with commercial value; few genes associated with fruit size have, however, been identified in sweet cherry. Members of the CYP78A subfamily, a group of important cytochrome P450s, have been found to be involved in controlling seed size and development in Arabidopsis thaliana, rice, soybean, and tomato. However, the influence of CYP78A members in controlling organ size and the underlying molecular mechanisms in sweet cherry and other fruit trees remains unclear. Here, we characterized a P. avium CYP78A gene PaCYP78A9 that is thought to be involved in the regulation of fruit size and organ development using overexpression and silencing approaches. PaCYP78A9 was significantly expressed in the flowers and fruit of sweet cherry. RNAi silencing of PaCYP78A9 produced small cherry fruits and PaCYP78A9 was found to affect fruit size by mediating mesocarp cell proliferation and expansion during fruit growth and development. Overexpression of PaCYP78A9 in Arabidopsis resulted in increased silique and seed size and PaCYP78A9 was found to be highly expressed in the inflorescences and siliques of transgenic plants. Genes related to cell cycling and proliferation were downregulated in fruit from sweet cherry TRV::PaCYP78A9-silencing lines, suggesting that PaCYP78A9 is likely to be an important upstream regulator of cell cycle processes. Together, our findings indicate that PaCYP78A9 plays an essential role in the regulation of cherry fruit size and provide insights into the molecular basis of the mechanisms regulating traits such as fruit size in P. avium.

Characterization of mice carrying a conditional TEAD1 allele.

The Hippo- yes-associated protein (YAP) pathway is essential for controlling organ size and tumorigenesis. Previous studies have demonstrated that the primary outcome of YAP signaling in the nucleus is achieved by interaction with the transcription factor TEA domain transcription factor (TEAD1). The YAP/TEAD1 complex binds to DNA element and regulates the expression of genes involved in cell growth. However, constitutive knockout of TEAD1 leads to early embryonic lethality in mice. Thus, generation of a floxed TEAD1 mouse becomes crucial for further understanding mid- to late-gestation and post-natal role of TEAD1. Herein, we created and characterized a mouse model that allows for conditional disruption of TEAD1. Embryonic fibroblasts derived from the floxed TEAD1 mice enabled the Cre-mediated deletion of TEAD1 in vitro using virally delivered Cre recombinase. Furthermore, crossing the floxed TEAD1 mouse with a ubiquitously expressing Cre mouse resulted in efficient ablation of the floxed allele in vivo, and the animals recapitulated early embryonic lethality defects. In conclusion, our data demonstrate an important role of TEAD1 in early development in mice, and the floxed TEAD1 mouse model will be a valuable genetic tool to determine the temporal and tissue-specific functions of TEAD1.

Homeostatic Control of Hpo/MST Kinase Activity through Autophosphorylation-Dependent Recruitment of the STRIPAK PP2A Phosphatase Complex

The Hippo pathway controls organ size and tissue homeostasis through a kinase cascade leading from the Ste20-like kinase Hpo (MST1/2 in mammals) to the transcriptional coactivator Yki (YAP/TAZ in mammals). Whereas previous studies have uncovered positive and negative regulators of Hpo/MST, how they are integrated to maintain signaling homeostasis remains poorly understood. Here, we identify a self-restricting mechanism whereby autophosphorylation of an unstructured linker in Hpo/MST creates docking sites for the STRIPAK PP2A phosphatase complex to inactivate Hpo/MST. Mutation of the phospho-dependent docking sites in Hpo/MST or deletion of Slmap, the STRIPAK subunit recognizing these docking sites, results in constitutive activation of Hpo/MST in both Drosophila and mammalian cells. In contrast, autophosphorylation of the Hpo/MST linker at distinct sites is known to recruit Mats/MOB1 to facilitate Hippo signaling. Thus, multisite autophosphorylation of Hpo/MST linker provides an evolutionarily conserved built-in molecular platform to maintain signaling homeostasis by coupling antagonistic signaling activities.

Organ size control via hydraulically gated oscillations.

Hollow vesicular tissues of various sizes and shapes arise in biological organs such as ears, guts, hearts, brains and even entire organisms. Regulating their size and shape is crucial for their function. Although chemical signaling has been thought to play a role in the regulation of cellular processes that feed into larger scales, it is increasingly recognized that mechanical forces are involved in the modulation of size and shape at larger length scales. Motivated by a variety of examples of tissue cyst formation and size control that show simultaneous growth and size oscillations, we create a minimal theoretical framework for the growth and dynamics of a soft, fluid-permeable, spherical shell. We show that these shells can relieve internal pressure by bursting intermittently, shrinking and re-growing, providing a simple mechanism by which hydraulically gated oscillations can regulate size. To test our theory, we develop an in vitro experimental set-up to monitor the growth and oscillations of a hollow tissue spheroid growing freely or when confined. A simple generalization of our theory to account for irreversible deformations allows us to explain the time scales and the amplitudes of oscillations in terms of the geometry and mechanical properties of the tissue shells. Taken together, our theory and experimental observations show how soft hydraulics can regulate the size of growing tissue shells.

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis.

Force Triggers YAP Nuclear Entry by Regulating Transport across Nuclear Pores

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation.

The MADS-Box Gene SlMBP21 Regulates Sepal Size Mediated by Ethylene and Auxin in Tomato.

Normal organ size is achieved by successful co-ordination of cell proliferation and cell expansion, which are modulated by multiple factors such as ethylene and auxin. In our work, SlMBP21-RNAi (RNA interference) tomato exhibited longer sepals and improved fruit set. Histological analysis indicated that longer sepals were attributed to cell expansion. To explore how SlMBP21 regulates sepal size, we compared the transcriptomes of sepals between SlMBP21-RNAi and the wild type by RNA sequencing and found that the differentially expressed genes were dominantly related to cell expansion, ethylene and auxin, and photosynthesis. Down-regulation of SlMBP21 affected ethylene production and the free IAA and IAA-Val intensity in sepals. Hormone treatment further indicated that SlMBP21 was involved in the ethylene and auxin pathways. As reported, ethylene and auxin were important factors for cell expansion. Hence, SlMBP21 negatively regulated cell expansion to control sepal size, and ethylene and auxin may mediate this process. Additionally, the contents of Chl and the activity of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key photosynthetic enzyme, were both increased in SlMBP21-RNAi sepals, which indicated that photosynthesis might be enhanced in transgenic longer sepals. Therefore, the longer sepal, with better protection and enhanced photosynthesis, may contribute to improve fruit set. Altogether, these results suggested that SlMBP21 was a novel factor involved in organ size control. Moreover, our study provided potential application value for improving fruit set.

Research advances in the role of the Hippo signaling pathway in pathogenesis of liver cancer

The Hippo signaling pathway consists of four core components in mammals, i.e., Mst1/2, WW45, Mob1, and LATS1/2, which can inhibit the transcriptional coactivator YAP from entering the nucleus, maintain the balance between cell proliferation and apoptosis, control organ size, and maintain homeostasis. If the core components of the Hippo signaling pathway are inactivated due to gene mutation or epigenetic alterations, YAP is overexpressed and activated in the nucleus, which then induces excessive cell proliferation and inhibits cell apoptosis. It has been confirmed that this process is closely associated with the formation of various tumors including liver cancer. Research on the Hippo signaling pathway may provide new directions for exploring the pathogenesis of liver tumor and developing effective therapies.

LncRNA wires up Hippo and Hedgehog signaling to reprogramme glucose metabolism.

The Hippo pathway plays essential roles in organ size control and cancer prevention via restricting its downstream effector, Yes-associated protein (YAP). Previous studies have revealed an oncogenic function of YAP in reprogramming glucose metabolism, while the underlying mechanism remains to be fully clarified. Accumulating evidence suggests long noncoding RNAs (lncRNAs) as attractive therapeutic targets, given their roles in modulating various cancer-related signaling pathways. In this study, we report that lncRNA breast cancer anti-estrogen resistance 4 (BCAR4) is required for YAP-dependent glycolysis. Mechanistically, YAP promotes the expression of BCAR4, which subsequently coordinates the Hedgehog signaling to enhance the transcription of glycolysis activators HK2 and PFKFB3. Therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 attenuated YAP-dependent glycolysis and tumor growth. The expression levels of BCAR4 and YAP are positively correlated in tissue samples from breast cancer patients, where high expression of both BCAR4 and YAP is associated with poor patient survival outcome. Taken together, our study not only reveals the mechanism by which YAP reprograms glucose metabolism, but also highlights the therapeutic potential of targeting YAP-BCAR4-glycolysis axis for breast cancer treatment.

Palmitic acid dysregulates the Hippo–YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS–STING–IRF3 signaling mechanism

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

Cloning and expression analysis of BmYki gene in silkworm, Bombyx mori.

The transcriptional coactivator Yorkie(Yki), is a critical downstream effector of the Hippo(Hpo) signaling pathway that controls organ size through the regulation of cell proliferation and apoptosis. During the past ten years the biological function of Yki has been studied extensively in Drosophila and a few other insects, however, little is known about it in the silkworm, Bombyx mori, a major research model of lepidopteran insect. Here, we describe the isolation, characterization and expression of the B. mori Yki ortholog, BmYki. The coding sequence of the BmYki was 1314 bp in length, encoding a protein of 437 amino acids containing two conserved WW domains. BmYki transcripts were ubiquitous but not abundant in all detected tissues and developmental stages. Comparatively, it was expressed at pretty high level in silk glands and at the stage of fifth-instar day-3 larvae. Overexpression of BmYki in cultured B. mori embryonic cells significantly promoted transcription of genes associated with cell proliferation and apoptosis, indicating that BmYki functions in the regulation of organ growth-related biological processes. Interestingly, transcription of silk protein-coding genes and transcription factors regulating the synthesis of silk proteins was downregulated remarkably, suggesting that BmYki was involved in the regulation of silk protein synthesis. This study provides new insights into the role of BmYki in Hpo pathway regulation in silkworm.

Regulation of Hippo pathway transcription factor TEAD by p38 MAPK-induced cytoplasmic translocation.

The Hippo pathway controls organ size and tissue homeostasis, with deregulation leading to cancer. The core Hippo components in mammals are composed of the upstream serine/threonine kinases Mst1/2, MAPK4Ks and Lats1/2. Inactivation of these upstream kinases leads to dephosphorylation, stabilization, nuclear translocation and thus activation of the major functional transducers of the Hippo pathway, YAP and its paralogue TAZ. YAP/TAZ are transcription co-activators that regulate gene expression primarily through interaction with the TEA domain DNA-binding family of transcription factors (TEAD). The current paradigm for regulation of this pathway centres on phosphorylation-dependent nucleocytoplasmic shuttling of YAP/TAZ through a complex network of upstream components. However, unlike other transcription factors, such as SMAD, NF-κB, NFAT and STAT, the regulation of TEAD nucleocytoplasmic shuttling has been largely overlooked. In the present study, we show that environmental stress promotes TEAD cytoplasmic translocation via p38 MAPK in a Hippo-independent manner. Importantly, stress-induced TEAD inhibition predominates YAP-activating signals and selectively suppresses YAP-driven cancer cell growth. Our data reveal a mechanism governing TEAD nucleocytoplasmic shuttling and show that TEAD localization is a critical determinant of Hippo signalling output.

The roles and mechanisms of the Hippo/YAP signaling pathway in the nervous system.

The Hippo signaling pathway, consisting of a highly conserved kinase cascade and downstream transcription co-activators YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif), plays a key role in tissue homeostasis and organ size control by regulating the proliferation, differentiation and apoptosis of cells. During normal development, the precise control of neural cell numbers and spatial distributions of these neural cells is important for brain development. Recent studies have shown that the Hippo/YAP signaling pathway is actively involved in the self-renewal of neural stem cells, proliferation of neural progenitor cells, differentiation and activation of glial cells, and myelination of glial cells as well as in the development of neurological diseases. Due to its prominent role in the nervous system, it is necessary to further study on this pathway. In this review, we summarize the recent studies and focus on the roles and mechanisms of the Hippo/YAP signaling pathway in the nervous system, and provide insights for neural development and neural injury diseases.