Hippo signalling pathway mediates oncogenic properties of NAB2::STAT6 in solitary fibrous tumour

The BCL-6 proto-oncogene encodes a transcriptional repressor protein. Among normal tissues, BCL-6 expression is confined to germinal center B-cells and a subpopulation of T-helper cells. Little is known about BCL-6...

Growth Regulation: A Beginning for the Hippo Pathway

Upregulated nicotinic ACh receptor signaling contributes to intestinal stem cell function through activation of Hippo and Notch signaling pathways

Integration of intercellular signaling through the Hippo pathway

Solitary fibrous tumours (SFTs) are known to overexpress insulin-like growth factor 2 (IGF-2). The down-stream oncogenic pathways of IGF-2, however, are not clear. Here we report uniform activation of the insulin receptor (IR) pathway in SFTs, which are mesenchymal tumours frequently associated with hypoglycaemia. Whereas the IR and its downstream signalling pathways were constitutively activated in SFTs, insulin-like growth factor 1 receptor (IGF-1R) was not expressed in these tumours. We also find that SFT cells secrete IGF-2 and proliferate in serum-free medium, consistent with an IGF-2/IR autocrine loop. The aetiological relevance of IGF-2 is supported by expression of IR-A, the IR isoform with high affinity for IGF-2, in all SFTs. Our studies suggest that IR activation plays an oncogenic role in SFTs.

Hippo signaling pathways are evolutionarily conserved signal transduction mechanisms mainly involved in organ size control, tissue regeneration, and tumor suppression. However, in mammals, the primary role of Hippo signaling seems to be regulation of immunity. As such, humans with null mutations in STK4 (mammalian homologue of Drosophila Hippo; also known as MST1) suffer from recurrent infections and autoimmune symptoms. Although dysregulated T cell homeostasis and functions have been identified in MST1-deficient human patients and mouse models, detailed cellular and molecular bases of the immune dysfunction remain to be elucidated. Although the canonical Hippo signaling pathway involves transcriptional co-activator Yes-associated protein (YAP) or transcriptional coactivator with PDZ motif (TAZ), the major Hippo downstream signaling pathways in T cells are YAP/TAZ-independent and they widely differ between T cell subsets. Here we will review Hippo signaling mechanisms in T cell immunity and describe their implications for immune defects found in MST1-deficient patients and animals. Further, we propose that mutual inhibition of Mst and Akt kinases and their opposing roles on the stability and function of forkhead box O and β-catenin may explain various immune defects discovered in mutant mice lacking Hippo signaling components. Understanding these diverse Hippo signaling pathways and their interplay with other evolutionarily-conserved signaling components in T cells may uncover molecular targets relevant to vaccination, autoimmune diseases, and cancer immunotherapies.

The Hippo pathway is recognized as an important regulator of tissue growth and cell fate [1-3]. Originally indentified in Drosophila, the Hippo pathway, also known as the Salvador–Warts–Hippo pathway, contains core kinases cascade, Hippo and Warts(Wts) coupled by the scaffold protein Salvador (Sav), as well as Mats. The activation of the Hippo pathway kinases results in phosphorylation and inactivation of the downstream transcriptional co-activator Yorkie which binds to the sequence-specific DNA-binding protein Scalloped and enhances the expression of proliferative and pro-survival genes. In general, the primary function of the Hippo signaling pathway is to inhibit the activation of Yorkie, inasmuch as deletion of Yki reverses the overgrowth phenotypes resulted from loss of Hippo, Warts, Salvador or Mats. Components of the Hippo pathway are highly conserved throughout evolution. The counterparts for the Hippo pathway in Drosophila can all be found in mammals, although they are more diverse and complex [4]. The Hippo orthologs Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 to regulate the Warts orthologs Lats1/Lats2. Activated Lats kinases phosphorylate the transcriptional regulators TAZ/YAP (Yorkie orthologs) which promotes 14-3-3 binding to YAP, causing YAP nuclear exit, hereby inhibiting its function. In recent years, increasing numbers of mammalian studies have expanded the large proteins network of the Hippo signaling pathway that controls tissues growth during development and regeneration, as well as in pathological states such as cancer [5]. The upstream regulator of the Hippo pathway and the downstream of Mst1/Mst2 have been diversified considerably in mammals compared with the Drosophila Hippo pathway. Multiple cellular stresses can trigger an adaptive response by activating the Hippo signaling pathway, which may, in turn, maintain the cellular homeostasis. The Hippo/Warts/Mats/Yorkie pathway predicated in Drosophila is not universal in all mammalian tissues in which their regulation and function are different in selected cell types. For examples, Mst1/2 negatively regulates YAP1 in mammalian liver, however, Mst1/2 is not required for YAP1 phosporylation and nuclear exclusion resulted from the cell-cell contact in mouse embryonic fibroblasts(MEFs); Mst1/2 is dispensable for Lats1/2 signaling in MEFs, but not in HeLa cells [6]. Independent of YAP, Mst1 negatively regulates naive T cell proliferation upon the T cell receptor stimulation, as well as regulates peripheral naive T cell trafficking and thymus egress [7]. Furthermore, patients with Mst1 deficiency are reported to have a primary immunodeficiency phenotype [8,9]. During the tissues regeneration and tumorigenesis, the Hippo signaling pathway has been shown to cross talk with other signaling players such as Notch and Wnt [10]. Thus, not just for the organ size control, the Hippo pathway receives inputs from multiple extracellular or intracellular signals and interacts with other essential signaling pathways to play critical roles in many aspects for cell fate decisions. In this issue of the Cell & Bioscience, we have provided some updates on the regulations beyond the canonical Hippo signaling, and their implications in pathological states. Qin et al. will review the recent updates of the roles of Mst1/2 on the cellular redox state regulation, the effects of Mst1/2 deficiency on the development process and tumorigenesis in multiple organs, and their involvement in the immune regulation. The review by Hergovich will summarize the current understanding of mammalian Lats1/2 kinases together with their closest relatives, the NDR1/2 kinases. He will focus on discussion about the regulation of the LATS/NDR family of kinases and their currently known substrates, as well as the biological roles of LATS/NDR kinases. Guo and Zhao follows with a discussion of the function of YAP and TAZ as effectors of cell responses to several extracellular signals including mechanical stress, GPCR signaling, and the Wnt signaling pathway, emphasizing that YAP and TAZ might have different role with cell-type specificity in the promotion of specific cancers. Collectively, these reviews have provided additional information to address the complexity of the hippo signaling pathway in response to physiological signals for regulating cellular and tissues homeostasis.

The Hippo signaling pathway is critical for carcinogenesis. However, the roles of the Hippo signaling pathway in the tumor immune microenvironment have been rarely investigated. This study systematically analyzed the relationship between the Hippo signaling pathway and immune cell infiltration across 32 cancer types. Both bioinformatics analyses and biological experiments revealed that the downstream effector of Hippo signaling YAP1 might inhibit CD8+ T cell infiltration by upregulating the expression of the transcription factor CREB1 in uterine corpus endometrial carcinoma. In addition, esophageal carcinoma (ESCA) patients were classified into three subtypes based on the Hippo-immune gene panel. The subtypes of ESCA had distinct characteristics in immune cell infiltration, immune pathways, and prognosis. Thus, this study also reveals a new classification of the immune subtypes with prognostic characteristics in ESCA.

YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment.

Context.—Immunohistochemical staining for β-catenin may be used as an indicator of the integrity of the Wnt signaling and β-catenin degradation pathways. Among mesenchymal tumors, aberrant nuclear localization of β-catenin is seen in desmoid-type fibromatoses but has not been described for solitary fibrous tumors that may mimic the former lesions, especially in small biopsy samples.Objective.—To study the immunohistochemical expression of β-catenin in solitary fibrous tumors.Design.—We performed immunohistochemical staining for β-catenin in 12 solitary fibrous tumors, one of which showed histologic features of malignancy.Results.—All the tumors showed strong and diffuse reactivity for β-catenin. Four tumors (33%) showed nuclear staining for β-catenin, whereas the remaining tumors showed either a membranous or mixed membranous and cytoplasmic pattern of staining. The only histologically malignant tumor of the group showed a mixed membranous and cytoplasmic pattern of staining for β-catenin.Conclusions.—Immunohistochemical staining for β-catenin in solitary fibrous tumors does not show a consistent pattern, which may be due to differences in tumorigenesis. Larger studies with clinical follow-up are required for estimating the impact of the variable staining pattern on clinical behavior of these tumors.

Yap1 Acts Downstream of α-Catenin to Control Epidermal Proliferation

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.

That the Hippo signaling controls organ size was first recognized almosta decadeago.Hipposignalinghasbeendemonstrated asa master regulator in diverse developmental processes. Its malfunction induces mis-regulated organ growth and tumorigenesis. Hippo signaling research has attracted increasing attention worldwide leading to a rapidly evolving of the emerging field. The ChineseHippoConsortiumhasbeenset upbyagroupofleading scientist from top institutes and universities in China. The consortium not only provides a platform for Chinese researchers who are interested in studying Hippo signaling to share resources and communicate ideas but also promotes collaboration. We invited eight leading Chinese researchers who are Chinese Hippo Consortium members to contribute to this special issue by highlighting topics that they think are the burgeoning interests in Hippo signaling. We are happy to present you eight review articles that illustrate some key trends in the area of Hippo signaling. The articles included cover the regulation and the function of the Hippo pathway in regulating organsize, cancer progression, and tissue regeneration as well as its roles in mammalianadaptive immunity. Although the subjects covered in these reviews are not comprehensive, we have attempted to cover the up-to-date key findings of this field. In their review, Yuan and colleagues discuss how Hippo signaling responds to cellular stress and hence maintain homeostasis, while Lei and colleagues focus on the bridge from the extracellular signalsto the Hippo pathway. The Hippo pathway controls diverse developmental processes through the regulation of its downstream effector Yorkie/ YAP. As reviewed by Zhao and colleagues, YAP is tightly regulated by various mechanisms in organ size control, regeneration and tumorigenesis, while it is not yet completely clear how YAP is coordinately regulated by physical signals, matrix stiffness and chemical signals. To help further understand the function of proteins, structural information may help. Zhou and colleagues provide a structural dissection of Hippo signaling to discuss the regulatory mechanisms underlying Hippo signaling derived from structural aspect. Much work is currently focusing on the function of Hippo signalingin stem cellbiologyandtumorigenesis: understanding howthe correct signaling levels are maintained to allow sufficient developmental growth and adult homeostasis, while preventing disease and tumor formation. Yin and Zhang discuss the roles of Hippo signaling in its roles in regulating stem cells in epithelial tissues and its potential implications in related cancers. Zhou and colleagues give more specific details regarding Hippo signaling in liver growth and liver cancers, while Shi and Chen describe its roles in mammary gland development and breast cancer. Though the initial work in Drosophila focused on the roles of Hipposignaling in developmentalsizecontrol, multiple non-canonical Hippo signaling pathways have turned out to implicate in the regulation of various other biological processes. In their review, Tao and colleagues discuss the roles of non-canonical Hippo signaling in lymphocyte developmentandfunctions. Researchreviewedin thisspecial issue indicates that Hippo signaling is an important regulator of various diseases and cancers. Targeting this signaling pathway may hopefully pave the way to new therapeutic interventions. To conclude, this issue coversthe major topics in the field of Hippo signaling and provides up-to-date information in this field. We hope you will enjoy and benefit from the topics that we cover in this special

Acquiring a selective growth advantage by breaking the proliferation barrier established by gatekeeper genes is a centrally important event in tumor formation. Removal of the mammalian Hippo kinase Mst1 and Mst2 in hepatocytes leads to rapid hepatocellular carcinoma (HCC) formation, indicating that the Hippo signaling pathway is a critical gatekeeper that restrains abnormal growth in hepatocytes. By rigorous genetic approaches, we identified an interacting network of the Hippo, Wnt/β-catenin and Notch signaling pathways that control organ size and HCC development. We found that in hepatocytes, the loss of Mst1/2 leads to the activation of Notch signaling, which forms a positive feedback loop with Yap/Taz (transcription factors controlled by Mst1/2). This positive feedback loop results in severe liver enlargement and rapid HCC formation. Blocking the Yap/Taz-Notch positive feedback loop by Notch inhibition in vivo significantly reduced the Yap/Taz activities, hepatocyte proliferation and tumor formation. Furthermore, we uncovered a surprising inhibitory role of Wnt/β-catenin signaling to Yap/Taz activities, which are important in tumor initiation. Genetic removal of β-catenin in the liver of the Mst1/2 mutants significantly accelerates tumoriogenesis. Therefore, Wnt/β-catenin signaling, known for its oncogenic property, exerts an unexpected function in restricting Yap/Taz and Notch activities in HCC initiation. The molecular interplay between the three signaling pathways identified in our study provides new insights in developing novel therapeutic strategies to treat liver tumors.

Malignant mesotheliomas (MMs) are tumors originating from the mesothelial lining of internal organs. Hippo pathway disruptions are common in MMs; up to 50% of cases exhibit NF2 deficiency and other pathway genes such as LATS2 are also mutated (1). This suggests hyperactivity of YAP and TAZ as a likely oncogenic driver in MMs and a potential axis to exploit therapeutically (2). Before committing to such an approach, a dependence of MMs on YAP/TAZ for survival needs to be tested. We investigated this using different MM cell line models cultured in vitro. The impact of Hippo pathway mutations on YAP and TAZ activity in mesothelial cells was assessed using an NF2 mutant isogenic system in an immortalized mesothelial cell line (Met5A.) YAP activation levels were probed through immunoblotting for YAP S127 phosphorylation, immunofluorescence, and RNA sequencing (RNAseq). YAP and TAZ were inhibited through RNAi-mediated depletion or treatment with simvastatin or verteporfin in a panel of 7 MM cell lines, including 3 with mutations in known Hippo pathway genes and 4 lacking such mutations, and cell viability was measured using an Alamar Blue assay. We found that Hippo pathway-mediated phosphorylation of YAP at S127, and YAP cytoplasmic abundance, was reduced in NF2 mutant Met5A cells compared to isogenic nonmutant cells. Consistent with this, RNAseq analysis showed elevated YAP activity in NF2 mutant Met5A cells. However, RNAi-mediated depletion of YAP and TAZ in a panel of mesothelioma cell lines did not reveal a clear correlation between dependence on YAP/TAZ for survival and aberrant Hippo signaling. There was also a comparable level of sensitivity of NF2 wild-type and NF2 mutant Met5A cells to YAP/TAZ knockdown. Additionally, treatment with verteporfin and simvastatin did not show increased toxicity in cell lines with Hippo pathway mutations relative to nonmutant cells. Our findings confirm that Hippo pathway mutations drive YAP/TAZ hyperactivity in mesothelial cells. However, they show that pathway mutation status does not always correlate with a dependence on YAP and TAZ for mesothelioma cell survival, at least when cultured in vitro. Clearly, a more in-depth analysis of the Hippo pathway in MMs is required to determine the potential of this pathway as a therapeutic target for this disease.