Advancements of anticancer agents by targeting the Hippo signalling pathway: biological activity, selectivity, docking analysis, and structure-activity relationship.

The Hippo signalling pathway is prominent and governs cell proliferation and stem cell activity, acting as a growth regulator and tumour suppressor. Defects in Hippo signalling and hyperactivation of its downstream effector's Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play roles in cancer development, implying that pharmacological inhibition of YAP and TAZ activity could be an effective cancer treatment strategy. Conversely, YAP and TAZ can also have beneficial effects in promoting tissue repair and regeneration following damage, therefore their activation may be therapeutically effective in certain instances. Recently, a complex network of intracellular and extracellular signalling mechanisms that affect YAP and TAZ activity has been uncovered. The YAP/TAZ-TEAD interaction leads to tumour development and the protein structure of YAP/TAZ-TEAD includes three interfaces and one hydrophobic pocket. There are clinical and preclinical trial drugs available to inhibit the hippo signalling pathway, but these drugs have moderate to severe side effects, so researchers are in search of novel, potent, and selective hippo signalling pathway inhibitors. In this review, we have discussed the hippo pathway in detail, including its structure, activation, and role in cancer. We have also provided the various inhibitors under clinical and preclinical trials, and advancement of small molecules their detailed docking analysis, structure-activity relationship, and biological activity. We anticipate that the current study will be a helpful resource for researchers.

Astragalus polysaccharide ameliorates bone marrow haematopoietic failure in mice with aplastic anaemia via the Hippo/TERT signalling pathway.

Objective: To study the effects of astragalus polysaccharide (APS) on aplastic anaemia (AA) in mice through modulation of the upstream and downstream effectors of the Hippo pathway. Further, we investigated the underlying mechanisms of APS in the treatment of AA to provide possible therapeutic strategies and drugs for clinical use. Methods: BALB/c mice were subjected to 4 Gy X-ray irradiation and subsequently injected with lymphocytes from DBA/2 donor mice by using the tail vein to establish an AA mouse model. Next BALB/c mice were randomly divided into five groups: control (untreated), AA (model), and three APS remedy groups - LA (low-dose APS, 200 mg/kg), MA (medium-dose APS, 400 mg/kg), and HA (high-dose APS, 800 mg/kg). The mice were sacrificed after 14 d of continuous gavage with different doses of APS or saline, and the bone marrow, spleen and liver were obtained. The haematopoietic condition, apoptosis levels, and the expression of Large Tumour Suppressor 1/2 (LATS 1/2), transcriptional co-activator of activation (TCA) Yes-associated protein (YesAP), transcriptional enhancer of adherence domain (TEAD) protein, and Telomerase Reverse Transcriptase (TERT), were analysed. Results: Compared with control mice, mice in the AA group exhibited a reduction in peripheral blood cell counts, a significant decrease in the haematopoietic area, increased adipocyte infiltration, and overall haematopoietic failure, mirroring the clinical presentation of AA; After 14 days of treatment with different doses of APS, the APS-treated group, in comparison to the untreated AA group, showed an increase in peripheral blood cell counts (P < 0.05), an expansionof the haematopoietic area in the bone marrow, restoration of haematopoietic function (P < 0.05), and a marked reduction in adipocyte infiltration within the bone marrow(P < 0.05). Cell infiltration into the bone marrow was also reduced. Further experiments revealed that the expression levels of TERT, LATS 1/2, YAP, and TEAD were elevated in the treated mice (P < 0.05). Conclusion: X-ray irradiation combined with T-lymphocyte infusion successfully established an AA mouse model, and APS administration modulated the expression of TERT and the Hippo pathway effectors LATS 1/2, YAP, and TEAD, which are implicated in the pathogenesis of AA. This modulation resulted in the restoration of bone marrow haematopoietic function in AA mice, suggesting APS as a promising therapeutic agent for the treatment of AA.

Immunohistochemical evaluation of yes-associated protein molecule in the odontogenic epithelium of different histopathological variants of ameloblastoma and unicystic ameloblastoma.

Background: Ameloblastoma is one of the major odontogenic neoplasms with an invasive and recurrence potential. Its tumourigenesis and proliferative capacity can be attributed to the activation or inactivation of certain molecular signalling pathways. Hippo signalling pathway is known to regulate diverse physiological processes related to mitosis and organ growth and is an emerging tumour suppressor pathway, the dysfunction of which is implicated in various diseases including cancers. Yes-associated protein1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the downstream effectors in the Hippo cascade, which on nuclear activation leads to cellular proliferation in various tumours. Aim: The current study was undertaken to evaluate the expression of YAP in various histopathological variants of ameloblastoma and unicystic ameloblastoma. Materials and Methods: Fifty formalin-fixed paraffin-embedded tissue samples of histopathologically diagnosed cases of ameloblastoma, and 10 histopathologically diagnosed cases of unicystic ameloblastoma were obtained from the departmental archives to evaluate the immunohistochemical expression of YAP both manually and by software analysis. Results: More than 90% of cases of conventional ameloblastoma and unicystic ameloblastoma elicited positive expression of YAP. No statistical difference was found among different histopathological variants of conventional ameloblastoma. Significant difference between the means of all four quantitative score groups was observed. Conclusion: In view of the modulating effect of YAP in tumourigenesis and its higher expression in ameloblastoma, further exploration of this molecule appears to be a promising area of research.

The m6A methyltransferase METTL3 drives thyroid cancer progression and lymph node metastasis by targeting LINC00894.

BACKGROUND: Long noncoding RNAs (lncRNAs) are significant contributors to various human malignancies. The aberrant expression of lncRNA LINC00894 has been reported in various human malignancies. We aimed to illustrate the role of LINC00894 and its underlying mechanism in the development of papillary thyroid carcinoma (PTC). METHODS: We performed bioinformatics analysis of differentially expressed RNAs from TCGA and GEO datasets and selected the target lncRNA LINC00894. SRAMP analysis revealed abundant M6A modification sites in LINC00894. Further analysis of StarBase, GEPIA, and TCGA datasets was performed to identify the related differentially expressed genes METTL3. Colony formation and CCK-8 assays confirmed the relationship between LINC00894, METTL3, and the proliferative capacity of PTC cells. The analysis of AnnoLnc2, Starbase datasets, and meRIP-PCR and qRT‒PCR experiments confirmed the influence of METTL3-mediated m6A modification on LINC00894. The study employed KEGG enrichment analysis as well as Western blotting to investigate the impact of LINC00894 on the expression of proteins related to the Hippo signalling pathway. RESULTS: LINC00894 downregulation was detected in PTC tissues and cells and was even further downregulated in PTC with lymphatic metastasis. LINC00894 inhibits the lymphangiogenesis of vascular endothelial cells and the proliferation of cancer cells. METTL3 enhances PTC progression by upregulating LINC00894 by enhancing LINC00894 mRNA stability through the m6A-YTHDC2-dependent pathway. LINC00894 may inhibit PTC malignant phenotypes through the Hippo signalling pathway. CONCLUSION: The METTL3-YTHDC2 axis stabilizes LINC00894 mRNA in an m6A-dependent manner and subsequently inhibits tumour malignancy through the Hippo signalling pathway.

Genetic and Pharmacological YAP Activation Induces Proliferation and Improves Survival in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocyte loss following myocardial infarction cannot be addressed with current clinical therapies. Cell therapy with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a potential approach to replace cardiomyocyte loss. However, engraftment rates in pre-clinical studies have been low, highlighting a need to refine current iPSC-CM technology. In this study, we demonstrated that inducing Yes-associated protein (YAP) by genetic and pharmacological approaches resulted in increased iPSC-CM proliferation and reduced apoptosis in response to oxidative stress. Interestingly, iPSC-CM maturation was differently affected by each strategy, with genetic activation of YAP resulting in a more immature cardiomyocyte-like phenotype not witnessed upon pharmacological YAP activation. Overall, we conclude that YAP activation in iPSC-CMs enhances cell survival and proliferative capacity. Therefore, strategies targeting YAP, or its upstream regulator the Hippo signalling pathway, could potentially be used to improve the efficacy of iPSC-CM technology for use as a future regenerative therapy in myocardial infarction.

miR-3133 inhibits gastrointestinal cancer progression through activation of Hippo and p53 signalling pathways via multi-targets.

BACKGROUND: Malignant cell growth and chemoresistance, the main obstacles in treating gastrointestinal cancer (GIC), rely on the Hippo and p53 signalling pathways. However, the upstream regulatory mechanisms of these pathways remain complex and poorly understood. METHODS: Immunohistochemistry (IHC), western blot and RT-qPCR were used to analyse the expression of RNF146, miR-3133 and key components of Hippo and p53 pathway. CCK-8, colony formation, drug sensitivity assays and murine xenograft models were used to investigate the effect of RNF146 and miR-3133 in GIC. Further exploration of the upstream regulatory mechanism was performed using bioinformatics analysis, dual-luciferase reporter gene, immunoprecipitation assays and bisulfite sequencing PCR (BSP). RESULTS: Clinical samples, in vitro and in vivo experiments demonstrated that RNF146 exerts oncogenic effects in GIC by regulating the Hippo pathway. Bioinformatics analysis identified a novel miRNA, miR-3133, as an upstream regulatory factor of RNF146. fluorescence in situ hybridization and RT-qPCR assays revealed that miR-3133 was less expressed in gastrointestinal tumour tissues and was associated with adverse pathological features. Functional assays and animal models showed that miR-3133 promoted the proliferation and chemotherapy sensitivity of GIC cells. miR-3133 affected YAP1 protein expression by targeting RNF146, AGK and CUL4A, thus activating the Hippo pathway. miR-3133 inhibited p53 protein degradation and extended p53's half-life by targeting USP15, SPIN1. BSP experiments confirmed that miR-3133 promoter methylation is an important reason for its low expression. CONCLUSION: miR-3133 inhibits GIC progression by activating the Hippo and p53 signalling pathways via multi-targets, including RNF146, thereby providing prognostic factors and valuable potential therapeutic targets for GIC.

Lineage segregation in human pre-implantation embryos is specified by YAP1 and TEAD1.

STUDY QUESTION: Which processes and transcription factors specify the first and second lineage segregation events during human preimplantation development? SUMMARY ANSWER: Differentiation into trophectoderm (TE) cells can be initiated independently of polarity; moreover, TEAD1 and YAP1 co-localize in (precursor) TE and primitive endoderm (PrE) cells, suggesting a role in both the first and the second lineage segregation events. WHAT IS KNOWN ALREADY: We know that polarity, YAP1/GATA3 signalling and phospholipase C signalling play a key role in TE initiation in compacted human embryos, however, little is known about the TEAD family of transcription factors that become activated by YAP1 and, especially, whether they play a role during epiblast (EPI) and PrE formation. In mouse embryos, polarized outer cells show nuclear TEAD4/YAP1 activity that upregulates Cdx2 and Gata3 expression while inner cells exclude YAP1 which upregulates Sox2 expression. The second lineage segregation event in mouse embryos is orchestrated by FGF4/FGFR2 signalling which could not be confirmed in human embryos; TEAD1/YAP1 signalling also plays a role during the establishment of mouse EPI cells. STUDY DESIGN, SIZE, DURATION: Based on morphology, we set up a development timeline of 188 human preimplantation embryos between Day 4 and 6 post-fertilization (dpf). The compaction process was divided into three subgroups: embryos at the start (C0), during (C1), and at the end (C2) of, compaction. Inner cells were identified as cells that were entirely separated from the perivitelline space and enclosed by cellular contacts on all sides. The blastulation process was divided into six subgroups, starting with early blastocysts with sickle-cell shaped outer cells (B0) and further on, blastocysts with a cavity (B1). Full blastocysts (B2) showed a visible ICM and outer cells referred to as TE. Further expanded blastocysts (B3) had accumulated fluid and started to expand due to TE cell proliferation and zona pellucida (ZP) thinning. The blastocysts then significantly expanded further (B4) and started to hatch out of the ZP (B5) until they were fully hatched (B6). PARTICIPANTS/MATERIALS, SETTING, METHODS: After informed consent and the expiration of the 5-year cryopreservation duration, 188 vitrified high quality eight-cell stage human embryos (3 dpf) were warmed and cultured until the required stages were reached. We also cultured 14 embryos that were created for research until the four- and eight-cell stage. The embryos were scored according to their developmental stage (C0-B6) displaying morphological key differences, rather than defining them according to their chronological age. They were fixed and immunostained for different combinations of cytoskeleton (F-actin), polarization (p-ERM), TE (GATA3), EPI (NANOG), PrE (GATA4 and SOX17), and members of the Hippo signalling pathway (YAP1, TEAD1 and TEAD4). We choose these markers based on previous observations in mouse embryos and single cell RNA-sequencing data of human embryos. After confocal imaging (LSM800, Zeiss), we analysed cell numbers within each lineage, different co-localization patterns and nuclear enrichment. MAIN RESULTS AND THE ROLE OF CHANCE: We found that in human preimplantation embryos compaction is a heterogeneous process that takes place between the eight-cell to the 16-cell stages. Inner and outer cells are established at the end of the compaction process (C2) when the embryos contain up to six inner cells. Full apical p-ERM polarity is present in all outer cells of compacted C2 embryos. Co-localization of p-ERM and F-actin increases steadily from 42.2% to 100% of the outer cells, between C2 and B1 stages, while p-ERM polarizes before F-actin (P < 0.00001). Next, we sought to determine which factors specify the first lineage segregation event. We found that 19.5% of the nuclei stain positive for YAP1 at the start of compaction (C0) which increases to 56.1% during compaction (C1). At the C2 stage, 84.6% of polarized outer cells display high levels of nuclear YAP1 while it is absent in 75% of non-polarized inner cells. In general, throughout the B0-B3 blastocyst stages, polarized outer/TE cells are mainly positive for YAP1 and non-polarized inner/ICM cells are negative for YAP1. From the C1 stage onwards, before polarity is established, the TE marker GATA3 is detectable in YAP1 positive cells (11.6%), indicating that differentiation into TE cells can be initiated independently of polarity. Co-localization of YAP1 and GATA3 increases steadily in outer/TE cells (21.8% in C2 up to 97.3% in B3). Transcription factor TEAD4 is ubiquitously present throughout preimplantation development from the compacted stage onwards (C2-B6). TEAD1 displays a distinct pattern that coincides with YAP1/GATA3 co-localization in the outer cells. Most outer/TE cells throughout the B0-B3 blastocyst stages are positive for TEAD1 and YAP1. However, TEAD1 proteins are also detected in most nuclei of the inner/ICM cells of the blastocysts from cavitation onwards, but at visibly lower levels as compared to that in TE cells. In the ICM of B3 blastocysts, we found one main population of cells with NANOG+/SOX17-/GATA4- nuclei (89.1%), but exceptionally we found NANOG+/SOX17+/GATA4+ cells (0.8%). In seven out of nine B3 blastocysts, nuclear NANOG was found in all the ICM cells, supporting the previously reported hypothesis that PrE cells arise from EPI cells. Finally, to determine which factors specify the second lineage segregation event, we co-stained for TEAD1, YAP1, and GATA4. We identified two main ICM cell populations in B4-6 blastocysts: the EPI (negative for the three markers, 46.5%) and the PrE (positive for the three markers, 28.1%) cells. We conclude that TEAD1 and YAP1 co-localise in (precursor) TE and PrE cells, indicating that TEAD1/YAP1 signalling plays a role in the first and the second lineage segregation events. LIMITATIONS, REASONS FOR CAUTION: In this descriptive study, we did not perform functional studies to investigate the role of TEAD1/YAP1 signalling during the first and second lineage segregation events. WIDER IMPLICATIONS OF THE FINDINGS: Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the way for further functional studies. Understanding the gene regulatory networks and signalling pathways involved in early embryogenesis could ultimately provide insights into why embryonic development is sometimes impaired and facilitate the establishment of guidelines for good practice in the IVF lab. STUDY FUNDING/COMPETING INTERESTS: This work was financially supported by Wetenschappelijk Fonds Willy Gepts (WFWG) of the University Hospital UZ Brussel (WFWG142) and the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO, G034514N). M.R. is doctoral fellow at the FWO. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

A conserved role of the Hippo signalling pathway in initiation of the first lineage specification event across mammals.

Our understanding of the molecular events driving cell specification in early mammalian development relies mainly on mouse studies, and it remains unclear whether these mechanisms are conserved across mammals, including humans. We have shown that the establishment of cell polarity via aPKC is a conserved event in the initiation of the trophectoderm (TE) placental programme in mouse, cow and human embryos. However, the mechanisms transducing cell polarity into cell fate in cow and human embryos are unknown. Here, we have examined the evolutionary conservation of Hippo signalling, which is thought to function downstream of aPKC activity, in four different mammalian species: mouse, rat, cow and human. In all four species, inhibition of the Hippo pathway by targeting LATS kinases is sufficient to drive ectopic TE initiation and downregulation of SOX2. However, the timing and localisation of molecular markers differ across species, with rat embryos more closely recapitulating human and cow developmental dynamics, compared with the mouse. Our comparative embryology approach uncovered intriguing differences as well as similarities in a fundamental developmental process among mammals, reinforcing the importance of cross-species investigations.

YAP/TAZ axis was involved in the effects of metformin on breast cancer.

Breast cancer is malignant tumours in women. A large amount of data analysis shows that Metformin has been shown to play a significance role in reducing the risk of breast cancer, but the mechanism remains unclear. The hippo signalling pathway can be involved in the formation, metastasis and recurrence of breast cancer. When YAP/TAZ is activated, cells can overcome contact inhibition and enter a state of uncontrolled proliferation. Therefore, YAP/TAZ is considered a potential therapeutic target for breast cancer. Eighty breast cancer patients, forty cases of triple-negative and forty cases of HER-2+, were included in this study. In vitro and in vivo experiments were used to confirm the YAP/TAZ axis was involved in the effects of metformin on breast cancer. EMT plays an important role in breast cancer, including chemoresistance and tumour metastasis. Our results confirmed that YAP could modulate the activity of EMT, which in turn altered tumour resistance. Therefore, MET can inhibit EMT by reducing the expression of YAP, and finally achieve the therapeutic effect of breast cancer. Our findings support metformin as a novel YAP inhibitor and potentially as a novel breast cancer drug.

Cytoskeletal protein SPTA1 mediating the decrease in erectile function induced by high-fat diet via Hippo signaling pathway.

BACKGROUND: The mechanism of high-fat diet (HFD)-induced decrease in erectile function has not been elucidated, and in previous studies, spectrin alpha, erythrocytic 1 (SPTA1) is a cytoskeletal protein that regulates cellular function, which belongs to a family of proteins that can affect cell and tissue growth and development by regulating YAP, an effector on the Hippo signaling pathway, but its particular role has not been elucidated. OBJECTIVE: To explore the role of SPTA1 in the abnormality of erectile function induced by HFD. METHODS: We analyzed the penile tissues of mice on normal diet and HFD by transcriptomics and screened for differentially expressed genes, further identified closely related target genes in rat penile tissues, and verified target gene expression in in vitro construction of high-glucose (HG)-treated corpus cavernosum endothelial cells (CCECs) and corpus cavernosum smooth muscle cells (CCSMCs) models. The distribution of target genes in various cell populations in penile tissues was retrieved by single-cell sequencing Male Health Atlas database. Moreover, interfering with target genes was further applied to explore the mechanisms involved in erectile function decline. RESULTS: Transcriptomic analysis screened out down-regulated differential gene SPTA1; Western blot and immunohistochemistry results showed that SPTA1 expression significantly decreased in the penile tissues of Sprague-Dawley (SD) rats in the HFD group. Immunofluorescence staining showed a positive expression of CD31 and VWF in CCECs and a positive expression of α-SMA in CCSMCs. The expression level of SPTA1 protein significantly decreased in the HG group of CCECs and CCSMCs. The expression of SPTA1 mRNA significantly decreased in CCSMCs while significantly increased in CCECs. SPTA1 may have various expression patterns and biological functions in different cell populations. Real-time quantitative PCR results showed that the siSPTA1 transfected in CCSMCs had a significant interference effect compared with the control siNC. Transfection of siSPTA1 into CCSMCs resulted in the significant down-regulation of mRNA and protein expression of eNOS, and significant up-regulation of YAP, Caspase-1, GSDMD, GSDMD-N IL-18, and IL-1ß protein expression levels. The expression level of CCSMCs contractile-type protein α-SMA was significantly down-regulated. CONCLUSIONS: The down-regulation of SPTA1 in SD rats fed with HFD may induce cell pyroptosis and lead to the decrease of erectile function by activating the Hippo pathway; these findings may provide new therapeutic targets for improving erectile function.

Advances in prognostic and therapeutic targets for hepatocellular carcinoma and intrahepatic cholangiocarcinoma: The hippo signaling pathway.

Primary liver cancer is the sixth most frequently diagnosed cancer worldwide and the third leading cause of cancer-related death. The majority of the primary liver cancer cases are hepatocellular carcinoma and intrahepatic cholangiocarcinoma. Worldwide, there is an increasing incidence of primary liver cancer cases due to multiple risk factors ranging from parasites and viruses to metabolic diseases and lifestyles. Often, patients are diagnosed at advanced stages, depriving them of surgical curability benefits. Moreover, the efficacy of the available chemotherapeutics is limited in advanced stages. Furthermore, tumor metastases and recurrence make primary liver cancer management exceptionally challenging. Thus, exploring the molecular mechanisms for the development and progression of primary liver cancer is critical in improving diagnostic, treatment, prognostication, and surveillance modalities. These mechanisms facilitate the discovery of specific targets that are critical for novel and more efficient treatments. Consequently, the Hippo signaling pathway executing a pivotal role in organogenesis, hemostasis, and regeneration of tissues, regulates liver cells proliferation, and apoptosis. Cell polarity or adhesion molecules and cellular metabolic status are some of the biological activators of the pathway. Thus, understanding the mechanisms exhibited by the Hippo pathway is critical to the development of novel targeted therapies. This study reviews the advances in identifying therapeutic targets and prognostic markers of the Hippo pathway for primary liver cancer in the past six years.

Empagliflozin Reduces the Progression of Hepatic Fibrosis in a Mouse Model and Inhibits the Activation of Hepatic Stellate Cells via the Hippo Signalling Pathway.

Hepatic fibrosis is the excessive production and deposition of the extracellular matrix, resulting in the activation of the fibrogenic phenotype of hepatic stellate cells (HSCs). The Hippo/Yes-associated protein (YAP) signalling pathway is a highly conserved kinase cascade that is critical in regulating cell proliferation, differentiation, and survival, and controls stellate cell activation. Empagliflozin, a sodium-glucose cotransporter type-2 inhibitor, is an antidiabetic drug that may prevent fibrotic progression by reducing hepatic steatosis and inflammation. However, little is known about its mechanism of action in liver fibrosis. In this study, we used male C57 BL/6 J mice fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) as a model for hepatic fibrosis. For 5 weeks, the mice received either a vehicle or empagliflozin based on their assigned group. Empagliflozin attenuated CDAHFD-induced liver fibrosis. Thereafter, we identified the Hippo pathway, along with its effector, YAP, as a key pathway in the mouse liver. Hippo signalling is inactivated in the fibrotic liver, but empagliflozin treatment activated Hippo signalling and decreased YAP activity. In addition, empagliflozin downregulated the expression of pro-fibrogenic genes and activated Hippo signalling in HSCs. We identified a mechanism by which empagliflozin ameliorates liver fibrosis.

A novel EHD1/CD44/Hippo/SP1 positive feedback loop potentiates stemness and metastasis in lung adenocarcinoma.

BACKGROUND: There is growing evidence that endocytosis plays a pivotal role in cancer metastasis. In this study, we first identified endocytic and metastasis-associated genes (EMGs) and then investigated the biological functions and mechanisms of EMGs. METHODS: Cancer stem cells (CSCs)-like characteristics were evaluated by tumour limiting dilution assays, three-dimensional (3D) spheroid cancer models. Microarray analysis was used to identify the pathways significantly regulated by mammalian Eps15 homology domain protein 1 (EHD1) knockdown. Mass spectrometry (MS) was performed to identify EHD1-interacting proteins. The function of EHD1 as a regulator of cluster of differentiation 44 (CD44) endocytic recycling and lysosomal degradation was determined by CD44 biotinylation and recycling assays. RESULTS: EHD1 was identified as a significant EMG. Knockdown of EHD1 suppressed CSCs-like characteristics, epithelial-mesenchymal transition (EMT), migration and invasion of lung adenocarcinoma (LUAD) cells by increasing Hippo kinase cascade activation. Conversely, EHD1 overexpression inhibited the Hippo pathway to promote cancer stemness and metastasis. Notably, utilising MS analysis, the CD44 protein was identified as a potential binding partner of EHD1. Furthermore, EHD1 enhanced CD44 recycling and stability. Indeed, silencing of CD44 or disruption of the EHD1/CD44 interaction enhanced Hippo pathway activity and reduced CSCs-like traits, EMT and metastasis. Interestingly, specificity protein 1 (SP1), a known downstream target gene of the Hippo-TEA-domain family members 1 (TEAD1) pathway, was found to directly bind to the EHD1 promoter region and induce its expression. Among clinical specimens, the EHD1 expression level in LUAD tissues of metastatic patients was higher than that of non-metastatic patients. CONCLUSIONS: Our findings emphasise that EHD1 might be a potent anti-metastatic target and present a novel regulatory mechanism by which the EHD1/CD44/Hippo/SP1 positive feedback circuit plays pivotal roles in coupling modules of CSCs-like properties and EMT in LUAD. Targeting this loop may serve as a remedy for patients with advanced metastatic LUAD.

Emerging roles for the YAP/TAZ transcriptional regulators in brain tumour pathology and targeting options.

The transcriptional co-activators Yes-associated protein 1/transcriptional co-activator with PDZ-binding motif (YAP/TAZ) have emerged as significant regulators of a wide variety of cellular and organ functions with impact in early embryonic development, especially during the expansion of the neural progenitor cell pool. YAP/TAZ signalling regulates organ size development, tissue homeostasis, wound healing and angiogenesis by participating in a complex network of various pathways. However, recent evidence suggests an association of these physiologic regulatory effects of YAP/TAZ with pro-oncogenic activities. Herein, we discuss the physiological functions of YAP/TAZ as well as the extensive network of signalling pathways that control their expression and activity, leading to brain tumour development and progression. Furthermore, we describe current targeting approaches and drug options including direct YAP/TAZ and YAP-TEA domain transcription factor (TEAD) interaction inhibitors, G-protein coupled receptors (GPCR) signalling modulators and kinase inhibitors, which may be used to successfully attack YAP/TAZ-dependent tumours.

LATS1 K751 acetylation blocks activation of Hippo signalling and switches LATS1 from a tumor suppressor to an oncoprotein.

Large tumor suppressor 1 (LATS1) is the key kinase controlling activation of Hippo signalling pathway. Post-translational modifications of LATS1 modulate its kinase activity. However, detailed mechanism underlying LATS1 stability and activation remains elusive. Here we report that LATS1 is acetylated by acetyltransferase CBP at K751 and is deacetylated by deacetylases SIRT3 and SIRT4. Acetylation at K751 stabilized LATS1 by decreasing LATS1 ubiquitination and inhibited LATS1 activation by reducing its phosphorylation. Mechanistically, LATS1 acetylation resulted in inhibition of YAP phosphorylation and degradation, leading to increased YAP nucleus translocation and promoted target gene expression. Functionally, LATS1-K751Q, the acetylation mimic mutant potentiated lung cancer cell migration, invasion and tumor growth, whereas LATS1-K751R, the acetylation deficient mutant inhibited these functions. Taken together, we demonstrated a previously unidentified post-translational modification of LATS1 that converts LATS1 from a tumor suppressor to a tumor promoter by suppression of Hippo signalling through acetylation of LATS1.

The proliferation role of LH on porcine primordial germ cell-like cells (pPGCLCs) through ceRNA network construction.

BACKGROUND: The transdifferentiation of skin-derived stem cells (SDSCs) into primordial germ cell-like cells (PGCLCs) is one of the major breakthroughs in the field of stem cells research in recent years. This technology provides a new theoretical basis for the treatment of human infertility. However, the transdifferentiation efficiency of SDSCs to PGCLCs is very low, and scientists are still exploring ways to improve this efficiency or promote the proliferation of PGCLCs. This study aims to investigate the molecular mechanism of luteinising hormone (LH) to enhance porcine PGCLCs (pPGCLCs) proliferation. RESULTS: In this study, we dissected the proliferation regulatory network of pPGCLCs by whole transcriptome sequencing, and the results showed that the pituitary-secreted reproductive hormone LH significantly promoted the proliferation of pPGCLCs. We combined whole transcriptome sequencing and related validation experiments to explore the mechanism of LH on the proliferation of pPGCLCs, and found that LH could affect the expression of Hippo signalling pathway-related mRNAs, miRNAs and lncRNAs in pPGCLCs. CONCLUSIONS: For the first time, we found that LH promotes pPGCLCs proliferation through the competing endogenous RNA (ceRNA) regulatory networks and Hippo signalling pathway. This finding may help to elucidate the molecular mechanism by which LH promotes pPGCLCs proliferation.

Discovery of N-aryl sulphonamide-quinazoline derivatives as anti-gastric cancer agents in vitro and in vivo via activating the Hippo signalling pathway.

Hippo signalling pathway plays a crucial role in tumorigenesis and cancer progression. In this work, we identified an N-aryl sulphonamide-quinazoline derivative, compound 9i as an anti-gastric cancer agent, which exhibited potent antiproliferative ability with IC50 values of 0.36 µM (MGC-803 cells), 0.70 µM (HCT-116 cells), 1.04 µM (PC-3 cells), and 0.81 µM (MCF-7 cells), respectively and inhibited YAP activity by the activation of p-LATS. Compound 9i was effective in suppressing MGC-803 xenograft tumour growth in nude mice without obvious toxicity and significantly down-regulated the expression of YAP in vivo. Compound 9i arrested cells in the G2/M phase, induced intrinsic apoptosis, and inhibited cell colony formation in MGC-803 and SGC-7901 cells. Therefore, compound 9i is to be reported as an anti-gastric cancer agent via activating the Hippo signalling pathway and might help foster a new strategy for the cancer treatment by activating the Hippo signalling pathway regulatory function to inhibit the activity of YAP.

Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice.

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

Genetic variants in Hippo signalling pathway-related genes affect the risk of colorectal cancer.

The Hippo signalling pathway plays a crucial role in carcinogenesis. Therefore, we hypothesized that genetic variants in genes related to this pathway are associated with the colorectal cancer risk. A case-control study including 1150 patients and 1342 controls was performed to assess the association of genetic variants of genes involved in the Hippo signalling pathway with the risk of colorectal cancer. The results were corrected for multiple comparisons using the false discovery rate (FDR). We used a regression model to determine the effects of single-nucleotide polymorphisms (SNPs) on the survival of patients with colorectal cancer in The Cancer Genome Atlas (TCGA) datasets. An expression quantitative trait loci (eQTL) analysis was performed using TCGA datasets and the Genotype-Tissue Expression (GTEx) project. Gene Expression Omnibus (GEO) datasets were used to provide additional data on the expression of genes in colorectal cancer. The SCRIB rs13251492 G allele was associated with a significantly decreased risk of colorectal cancer (odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.70-0.89, P = 7.76 × 10-5, P(FDR) = 6.98 × 10-4). Patients with the rs13251492 AG/GG allele experienced a longer recurrence-free survival (RFS) time (hazard ratio (HR) = 0.64, 95% CI = 0.42-0.99, P = 0.049) than patients with the rs13251492 A allele. The eQTL analysis revealed a significant association between rs13251492 and the expression of the SCRIB mRNA in colorectal tumors. Dual-luciferase reporter assays in DLD-1 and HCT116 cells revealed a lower enhancer activity of the rs13251492 G allele than the A allele. In addition, the SCRIB mRNA was expressed at markedly higher levels in colorectal cancer tissues than in normal tissues. Therefore, we identified the SCRIB rs13251492 variant as a novel colorectal cancer susceptibility locus and provided evidence of its functional relevance.

The Hippo in the room: Targeting the Hippo signalling pathway for osteosarcoma therapies.

Osteosarcoma (OS) is a primary malignant bone tumour which usually occurs in children and adolescents. OS is primarily a result of chromosomal aberrations, a combination of acquired genetic changes and, hereditary, resulting in the dysregulation of cellular functions. The Hippo signalling pathway regulates cell and tissue growth by modulating cell proliferation, differentiation, and migration in developing organs. Mammalian STE20-like 1/2 (MST1/2) protein kinases are activated by neurofibromatosis type 2, Ras association domain family member 2, kidney and brain protein, or other factors. Interactions between MST1/2 and salvador family WW domain-containing protein 1 activate large tumour suppressor kinase 1/2 proteins, which in turn phosphorylate the downstream Yes-associated protein 1/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). Moreover, dysregulation of this pathway can lead to aberrant cell growth, resulting in tumorigenesis. Interestingly, small molecules targeting the Hippo signalling pathways, through affecting YAP/TAZ cellular localisation and their interaction with members of the TEA/ATTS domain family of transcriptional enhancers are being developed and hold promise for the treatment of OS. This review discusses the existing knowledge about the involvement of the Hippo signalling cascade in OS and highlights several small molecule inhibitors as potential novel therapeutics.