Large Tumor Suppressor Research Articles

Hippo pathway in cancer cells induces NCAM1+αSMA+ fibroblasts to modulate tumor microenvironment

Cancer cells adeptly manipulate the tumor microenvironment (TME) to evade host antitumor immunity. However, the role of cancer cell-intrinsic signaling in shaping the immunosuppressive TME remains unclear. Here, we found that the Hippo pathway in cancer cells orchestrates the TME by influencing the composition of cancer-associated fibroblasts (CAFs). In a 4T1 mouse breast cancer model, Hippo pathway kinases, large tumor suppressor 1 and 2 (LATS1/2), promoted the formation of neural cell adhesion molecule 1 (NCAM1)+alpha-smooth muscle actin (αSMA)+ CAFs expressing the transforming growth factor-β, which is associated with T cell inactivation and dysfunction. Depletion of LATS1/2 in cancer cells resulted in a less immunosuppressive TME, indicated by the reduced proportions of NCAM1+αSMA+ CAFs and dysfunctional T cells. Notably, similar Hippo pathway-induced NCAM1+αSMA+ CAFs were observed in human breast cancer, highlighting the potential of TME-manipulating strategies to reduce immunosuppression in cancer immunotherapy.

7290 Hippo Signaling Regulates Zona Glomerulosa Cell Fate In The Adrenal Cortex Of Mice

Abstract Disclosure: N. Abou Nader: None. N. Jakuc: None. M. Meinsohn: None. L. Charrier: None. M. Paquet: None. G. St-Jean: None. J. Brind’Amour: None. D. Boerboom: None. J. Mao: None. D. Pepin: None. G. Zamberlam: None. D.T. Breault: None. A. Boyer: None. The Hippo signaling is a major signaling pathway that regulates processes such as cell proliferation and differentiation during both embryogenesis and postnatal homeostatic maintenance in several tissues. Our recent findings, using the Nr5a1-cre model, have highlighted the essential role of Lats1 and Lats2 (large tumor suppressor homolog kinases 1/2), the main kinases of the Hippo signaling cascade, in maintaining proper differentiation of mouse developing adrenocortical cells by suppressing their differentiation into myofibroblast-like cells. However, since the abnormal transdifferentiation of Lats1/2-negative adrenocortical cells occurs before the establishment of zonation when using the Nr5a1-cre model, the role of Hippo signaling in zona glomerulosa (zG) cells could not be assessed with this model. To overcome this limitation, we inactivated Lats1/2 in aldosterone-producing zG cells using the zG cell-specific AS-cre strain (Lats1flox/flox; Lats2flox/flox; AScre/+). We report here that specific loss of Lats1/2 in steroidogenic zG cells results in the accumulation of an important extracellular matrix (ECM) in the zG an outer zona fasciculata (zF) by 4 weeks of age; a phenotype that does not seem to progress with aging. In addition, tracing experiments demonstrate that the inactivation of Lats1/2 leads to the ectopic transdifferentiation of the zG cells into the ECM-producing cells rather than into zF cells. We then performed RNAseq experiments on whole adrenals which further suggested that the ECM producing cells derived from zG cells share characteristics with chondroblast/osteoblast mesenchymal progenitors among other cell lineages and, that the ectopic transdifferentiation of zG cells triggers a significant inflammation/immune response in the adrenal cortex. Our study also demonstrates that the increased activity of the Hippo pathway effectors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) is responsible for the ectopic transdifferentiation of the zG cells as their concomitant inactivation with Lats1/2 (Yapflox/flox; Tazflox/flox; Lats1flox/flox; Lats2flox/flox; AScre/+) rescues the observed phenotype. Finally, we also demonstrate that the expression of a constitutively active form of YAP (YAP5SA) in zG cells (Rosa26YAP5SA/+; AScre/+) does not alter their identity as severely as the loss of Lats1/2 but leads to adrenal hyperplasia. Together, these novel findings underscore the crucial role of Hippo signaling in maintaining the identity of zG cells in the adrenal cortex. Presentation: 6/2/2024

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

ObjectiveTo 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. MethodsBALB/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. ResultsCompared 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). ConclusionX-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.

KLF4 activates LATS2 to promote cisplatin sensitivity in ovarian cancer through DNA damage.

We aimed to investigate the role of large tumor suppressor kinase 2 (LATS2) in cisplatin (DDP) sensitivity in ovarian cancer. Bioinformatic analysis explored LATS2 expression, pathways, and regulators. Quantitative reverse transcription-PCR measured LATS2 and KLF4 mRNA levels. Dual-luciferase and chromatin immunoprecipitation assays confirmed their binding relationship. Cell viability, half maximal inhibitory concentration (IC50) values, cell cycle, and DNA damage were assessed using CCK-8, flow cytometry, and comet assays. Western blot analyzed protein expression. The effect of LATS2 on the sensitivity of ovarian cancer to DDP was verified in vivo. LATS2 and KLF4 were downregulated in ovarian cancer, with LATS2 enriched in cell cycle, DNA replication, and mismatch repair pathways. KLF4, an upstream regulator of LATS2, bound to its promoter. Overexpressing LATS2 increased G1-phase cells, reduced cell viability and IC50 values, and induced DNA damage. Silencing KLF4 alone showed the opposite effect on LATS2 overexpression. Knocking out LATS2 reversed the effects of KLF4 overexpression on cell viability, cell cycle, IC50 values, and DNA damage in ovarian cancer cells. Inhibiting LATS2 inactivated the Hippo-YAP signaling pathway. In vivo experiments showed that overexpression of LATS2 enhanced the sensitivity of ovarian cancer to DDP. KLF4 activates LATS2 via DNA damage to enhance DDP sensitivity in ovarian cancer, providing a potential target for improving treatment outcomes.

Upregulation of Angiomotin-Like 2 Ameliorates Experimental Pulmonary Arterial Hypertension by Inactivating YAP1 Signaling.

Angiomotin-like 2 (AMOTL2) is related to numerous physiological and pathological conditions by affecting signal transduction. However, whether AMOTL2 is linked to pulmonary arterial hypertension (PAH) has not been addressed. This work aimed to investigate the potential role of AMOTL2 in PAH. A decrease in AMOTL2 abundance was observed in the lungs of PAH rats. The upregulation of AMOTL2 significantly decreased right ventricle systolic pressure and right ventricular hypertrophy in PAH rats. Overexpression of AMOTL2 also led to a noteworthy decrease in vascular wall thickness, pulmonary artery area, and collagen deposition in rats with PAH. AMOTL2 was downregulated in hypoxia-stimulated pulmonary arterial smooth muscle cells (PASMCs). Moreover, AMOTL2 overexpression impeded hypoxia-evoked proliferation, migration, and phenotypic transformation in rat PASMCs. Mechanistic investigation revealed that Yes-associated protein 1 (YAP1) activation in PAH rats or hypoxia-stimulated PASMCs was markedly inhibited by AMOTL2 overexpression, which was associated with increased large tumor suppressor 1/2 phosphorylation. The inhibition of large tumor suppressor 1/2 reversed the AMOTL2-mediated inactivation of YAP1. Restoring the activity of YAP1 reversed the inhibitory effect of AMOTL2 on hypoxia-evoked proliferation, migration, and phenotypic transformation of PASMCs. Collectively, these results suggest that AMOTL2 can ameliorate PAH in a rat model by interfering with pulmonary arterial remodeling via the inactivation of YAP1 signaling. Our work indicates that AMOTL2 may be a candidate target for novel drug development for the treatment of PAH.

ITCH inhibits alkaliptosis in human pancreatic cancer cells through YAP1-dependent SLC16A1 activation

Alkaliptosis is a type of pH-dependent cell death and plays an emerging role in tumor suppression. However, the key modulation mechanism of alkaliptosis remains largely unknown. In particular, the nucleus, as the centre of genetic and metabolic regulation, is crucial for the regulation of cellular life. It is not known whether nuclear proteins are involved in the regulation of alkaliptosis. Here, we isolated nuclear proteins to perform a proteomics that identified itchy E3 ubiquitin protein ligase (ITCH) as a natural inhibitor of alkaliptosis in human pancreatic ductal adenocarcinoma (PDAC) cells. The downregulation of ITCH protein is associated with the induction of alkaliptosis in three human PDAC cell lines (SW1990, MiaPaCa2, and PANC1). Functionally, increasing ITCH expression reduces JTC801-induced growth inhibition and cell death. In contrast, knocking down ITCH using specific shRNA increases JTC801-induced cell growth inhibition in the short or long term, resulting in increased cell death. Mechanistically, JTC801-induced ITCH inhibition blocks large tumor suppressor kinase 1 (LATS1) ubiquitination, which in turn suppresses Yes1 associated transcriptional regulator (YAP1)-dependent the transcriptional activation of solute carrier family 16 member 1 (SLC16A1), a proton-linked monocarboxylate transporter that inhibits JTC801-induced alkaliptosis. Additionally, decreased expression of ITCH is associated with longer survival times in patients with PDAC. Collectively, our results establish an ITCH-dependent pathway that regulates alkaliptotic sensitivity in PDAC cells and deepen the understanding of alkaliptosis in targeted therapy.

Targeting YAP Activity and Glutamine Metabolism Cooperatively Suppresses Tumor Progression by Preventing Extracellular Matrix Accumulation.

Cancer cells use multiple mechanisms to evade the effects of glutamine metabolism inhibitors. The pathways that govern responses to alterations in glutamine availability within the tumor may represent therapeutic targets for combinatorial strategies with these inhibitors. Here, we showed that targeting glutamine utilization stimulated Yes-associated protein (YAP) signaling in cancer cells by reducing cyclic adenosine monophosphate/protein kinase A (PKA)-dependent phosphorylation of large tumor suppressor (LATS). Elevated YAP activation induced extracellular matrix (ECM) deposition by increasing the secretion of connective tissue growth factor that promoted the production of fibronectin and collagen by surrounding fibroblasts. Consequently, inhibiting YAP synergized with inhibition of glutamine utilization to effectively suppress tumor growth in vivo, along with a concurrent decrease in ECM deposition. Blocking ECM remodeling also augmented the tumor suppressive effects of the glutamine utilization inhibitor. Collectively, these data reveal mechanisms by which targeting glutamine utilization increases ECM accumulation and identify potential strategies to reduce ECM levels and increase the efficacy of glutamine metabolism inhibitors. Significance: Blocking glutamine utilization activates YAP to promote ECM deposition by fibroblasts, highlighting the potential of YAP inhibitors and antifibrotic strategies as promising approaches for effective combination metabolic therapies in cancer.

Endothelial LATS2 is a suppressor of bone marrow fibrosis

Myelofibrosis and osteosclerosis are fibrotic diseases disrupting bone marrow function that occur in various leukemias but also in response to non-malignant alterations in hematopoietic cells. Here we show that endothelial cell–specific inactivation of the Lats2 gene, encoding Hippo kinase large tumor suppressor kinase 2, or overexpression of the downstream effector YAP1 induce myofibroblast formation and lead to extensive fibrosis and osteosclerosis, which impair bone marrow function and cause extramedullary hematopoiesis in the spleen. Mechanistically, loss of LATS2 induces endothelial-to-mesenchymal transition, resulting in increased expression of extracellular matrix and secreted signaling molecules. Changes in endothelial cells involve increased expression of serum response factor target genes, and, strikingly, major aspects of the LATS2 mutant phenotype are rescued by inactivation of the Srf gene. These findings identify the endothelium as a driver of bone marrow fibrosis, which improves understanding of myelofibrotic and osteosclerotic diseases, for which drug therapies are currently lacking.

LATS1 inhibitor and zinc supplement synergistically ameliorates contrast-induced acute kidney injury: Induction of Metallothionein-1 and suppression of tubular ferroptosis

Contrast-induced acute kidney injury (CI-AKI) is a prevalent cause of renal dysfunction among hospitalized patients, yet the precise pathogenesis and effective therapeutic strategies remain elusive. In this study, we investigated the role of tubular ferroptosis in both experimental CI-AKI models and in primary tubular epithelial cells (PTECs) treated with ioversol. Using whole exome sequencing, we identified metallothioneins (MTs) as being among the most significantly downregulated genes following ioversol exposure. Our findings reveal that overexpression of Mt1 mitigates, whereas suppression of Mt-1 exacerbates, ioversol-induced tubular ferroptosis. Interestingly, the level of MTF1 (metal regulatory transcription factor 1), a principal regulator of Mt1, was found to increase in response to ioversol treatment. We further elucidated that ioversol activates LATS1 (Large tumor suppressor homolog 1), a kinase that promotes the phosphorylation and nuclear translocation of MTF1, thereby inhibiting its transcriptional activity for Mt1. Both genetic and pharmacological inhibition of LATS1 reversed the ioversol-induced suppression of Mt-1. From a therapeutic perspective, the LATS1 inhibitor TDI-011536, in combination with zinc acetate, was administered to a rodent model of CI-AKI. Our data indicate that this combination synergistically upregulates Mt1 expression and provides protection against contrast media-induced tubular ferroptosis. In summary, our study demonstrates that the reduction of Mt-1 contributes to tubular ferroptosis associated with CI-AKI. We show that contrast media activate LATS1, which in turn suppresses the transcriptional activity of MTF1 for Mt1. Herein, the combination of zinc acetate and a LATS1 inhibitor emerges as a potential therapeutic approach for the treatment of CI-AKI.

A Food Odorant, α-Ionone, Inhibits Skin Cancer Tumorigenesis by Activation of OR10A6.

This study aims to investigate the anticancer properties of α-ionone in squamous cell carcinoma (SCC). The expression of OR10A6 together with olfactory receptor signaling components is demonstrated in A431 human SCC cells via RT-PCR and qRT-PCR analysis. OR10A6 activation in A431 cells using the ligand α-ionone inhibits proliferation and migration but induces apoptosis which is confirmed by proliferation assay, colony formation, and western blotting. The mechanism involves the core proteins of the Hippo pathway, where the phosphorylation of large tumor suppressor kinase (LATS), yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) is confirmed by western blotting. However, the anticancer effects of α-ionone are abrogated in A431 cells with OR10A6 gene knockdown. In A431 xenograft mouse model, the injection of α-ionone suppresses tumor growth, induces apoptosis, and increases phosphorylation of the LATS-YAP-TAZ signaling axis in the Hippo pathway. None of these effects are observed in xenografted tumors with OR10A6 gene knockdown. These findings collectively demonstrate that activation of ectopic OR OR10A6 by α-ionone in SCC cells stimulates the Hippo pathway and suppresses tumorigenesis both in vitro and in vivo, suggesting a novel therapeutic candidate for the treatment of SCC.

Hippo pathway inactivation through subcellular localization of NF2/merlin in outer cells of mouse embryos.

The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.

Integrin-Linked Kinase in the Development of Gastric Tumors Induced by Helicobacter pylori: Regulation and Prevention Potential.

Integrin-linked kinase (ILK) is crucial in solid tumors by regulating the Hippo-Yes-associated protein 1 (YAP) pathway. This study aimed to uncover how Helicobacter pylori influences ILK levels and its role in regulating YAP during H. pylori-induced gastric cancer. GES-1 cells with stable Ilk knockdown and overexpression and a mouse carcinogenesis model for H. pylori infection were constructed. And ILK, the phosphorylated mammalian STE20-like protein kinase 1 (MST1), large tumor suppressor 1 (LATS1; S909, T1079), and YAP (S109, S127) were detected in cells, and mice by western blotting, as well as fluorescence intensity of YAP were assayed by immunofluorescence. YAP downstream genes Igfbp4 and Ctgf, the pathological changes and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1beta (IL-1β), and nitric oxide (NO) levels in mice gastric tissues were detected by real-time PCR, H&E, and ELISA assays. In this study, stable Ilk knockdown cells exhibited significantly higher phosphorylated levels of MST1, LATS1, and YAP, as well as increased YAP in the nuclei of GES-1 cells. Conversely, cells with Ilk overexpression showed opposite results. H. pylori infection led to decreased ILK levels in gastric epithelial cells but increased ILK levels in gastric cancer cell lines (MGC803, SGC7901) and gastric cancer tissues in mice. Treatment with the ILK inhibitor OST-T315 elevated the phosphorylated MST, LATS1, and YAP levels, and inhibited the mRNA levels of Igfbp4 and Ctgf at 44, 48 week-aged mice. OST-T315 also reduced the release of TNF-α, IL-6, IL-1β, and NO, as well as the progression of gastric cancer caused by H. pylori and N-Nitroso-N-methylurea (NMU) treatment. Upon initiation of gastric tumorigenesis signals, H. pylori increases ILK levels and suppresses Hippo signaling, thereby promoting YAP activation and gastric cancer progression. ILK can serve as a potential prevention target to impede H. pylori-induced gastric cancer.

Tudor-SN promotes cardiomyocyte proliferation and neonatal heart regeneration through regulating the phosphorylation of YAP

BackgroundThe neonatal mammalian heart exhibits considerable regenerative potential following injury through cardiomyocyte proliferation, whereas mature cardiomyocytes withdraw from the cell cycle and lose regenerative capacities. Therefore, investigating the mechanisms underlying neonatal cardiomyocyte proliferation and regeneration is crucial for unlocking the regenerative potential of adult mammalian heart to repair damage and restore contractile function following myocardial injury.MethodsThe Tudor staphylococcal nuclease (Tudor-SN) transgenic (TG) or cardiomyocyte-specific knockout mice (Myh6-Tudor-SN −/−) were generated to investigate the role of Tudor-SN in cardiomyocyte proliferation and heart regeneration following apical resection (AR) surgery. Primary cardiomyocytes isolated from neonatal mice were used to assess the influence of Tudor-SN on cardiomyocyte proliferation in vitro. Affinity purification and mass spectrometry were employed to elucidate the underlying mechanism. H9c2 cells and mouse myocardia with either overexpression or knockout of Tudor-SN were utilized to assess its impact on the phosphorylation of Yes-associated protein (YAP), both in vitro and in vivo.ResultsWe previously identified Tudor-SN as a cell cycle regulator that is highly expressed in neonatal mice myocardia but downregulated in adults. Our present study demonstrates that sustained expression of Tudor-SN promotes and prolongs the proliferation of neonatal cardiomyocytes, improves cardiac function, and enhances the ability to repair the left ventricular apex resection in neonatal mice. Consistently, cardiomyocyte-specific knockout of Tudor-SN impairs cardiac function and retards recovery after injury. Tudor-SN associates with YAP, which plays important roles in heart development and regeneration, inhibiting phosphorylation at Ser 127 and Ser 397 residues by preventing the association between Large Tumor Suppressor 1 (LATS1) and YAP, correspondingly maintaining stability and promoting nuclear translocation of YAP to enhance the proliferation-related genes transcription.ConclusionTudor-SN regulates the phosphorylation of YAP, consequently enhancing and prolonging neonatal cardiomyocyte proliferation under physiological conditions and promoting neonatal heart regeneration after injury.Graphical

EPEN-21. EXPLORATION OF TUMOR SUPPRESSOR LATS1 LOSS IN POSTERIOR FOSSA GROUP A EPENDYMOMA WITH LOSS OF CHROMOSOME 6Q

Abstract INTRODUCTION Chromosome 6q loss (6q-) has recently been identified as an ultra-high-risk factor in posterior fossa group A ependymoma (PFA). This study seeks to pinpoint the biological mechanism underlying the aggressive biology of PFA 6q-. We hypothesize that loss of chromosome 6q gene large tumor suppressor kinase 1 (LATS1) in PFA 6q- results in increased Hippo pathway activity that drives malignant tumor progression. LATS1 is a key negative regulator of the Hippo pathway which may represent a novel therapeutic vulnerability in 6q- PFA. METHODS We are testing this hypothesis using genomic and transcriptomic analysis of PFA patient samples, and by gene knock-in and pharmaceutical interventions of LATS1 and Hippo pathway components in PFA 6q- models. RESULTS Tumor suppressor LATS1 shows reduced expression in PFA 6q- patient samples. Although complete loss of the entire 6q arm is common in PFA 6q-, partial loss of chromosome 6q is also observed, and in all cases this partial loss encompasses the LATS1 locus on chromosome 6q. We used lentiviral transduction to knock-in LATS1 in PFA 6q- cell lines to determine the effect on tumor growth. This demonstrated loss of viability that was specific for 6q- PFA cell lines versus 6q WT control cell lines. DISCUSSION Data point to LATS1 loss being a key driver of the aggressive phenotype of PFA6q-, implicating the Hippo pathway in PFA 6q- and potential for novel therapeutic strategies.

Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage.

Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

Zinc finger protein 367 exerts a cancer-promoting role in small cell lung cancer by influencing the CIT/LATS2/YAP signaling cascade

A remarkable cancer-related role of zinc finger protein 367 (ZNF367) has been demonstrated in multiple malignancies. However, whether ZNF367 has a role in small-cell lung cancer (SCLC) remains unexplored. The purpose of this work was to explore the potential role and mechanism of ZNF367 in SCLC. In silico analysis using the Gene Expression Omnibus (GEO) dataset revealed high levels of the ZNF367 transcript in SCLC. Examination of clinical tissues confirmed the significant abundance of ZNF367 in SCLC tissues compared with adjacent non-malignant tissues. The genetic depletion of ZNF367 in SCLC cells led to remarkable alterations in cell proliferation, the cell cycle, colony formation and chemosensitivity. Mechanistically, ZNF367 was shown to regulate the activation of yes-associated protein (YAP) associated with the up-regulation of phosphorylated large tumour suppressor kinase 2 (LATS2). Further investigation revealed that ZNF367 affected the LATS2-YAP cascade by regulating the expression of citron kinase (CIT). Re-expression of constitutively active YAP diminished the tumour-inhibiting function of ZNF367 depletion. Xenograft experiments confirmed the tumour-inhibiting effect of ZNF367 depletion in vivo. In summary, our results demonstrate that the inhibition of ZNF367 displays anticancer effects in SCLC by inhibiting YAP activation, suggesting it as a potential druggable oncogenic target.

The hippo-YAP1/HIF-1α pathway mediates arsenic-induced renal fibrosis

Epidemiological evidence reveals that arsenic increases the risk of chronic kidney disease (CKD) in humans, but its mechanism of action has so far been unclear. Fibrosis is the manifestation of end-stage renal disease. Hypoxia is recognized as a vital event accompanying the progression of renal fibrosis. KM mice were exposed to 0, 20, 40, and 80 mg/L NaAsO2 for 12 weeks. HK-2 cells were treated with 1 μM NaAsO2 for 4 weeks. The results showed that arsenic increased the expression of hypoxia-inducible factor 1α (HIF-1α) (P < 0.05), which is involved in inorganic arsenic-induced renal fibrosis. The Hippo signaling pathway is the upstream signal of HIF-1α and the kinase cascade of Large tumor suppressor kinase 1 (LATS1) and Yes-associated protein 1 (YAP1) is the heart of the Hippo pathway. Our results showed that protein expressions of LATS1 and phosphorylated YAP1 were decreased, and dephosphorylated YAP1 expression increased in arsenic-treated mouse kidneys and human HK-2 cells (P < 0.05). Our research manifested that arsenic treatment suppressed the Hippo signaling and induced high expression of YAP1 into the nucleus. We also found that YAP1 was involved in arsenic-induced renal fibrosis by forming a complex with HIF-1α and maintaining HIF-1α stability. Our findings indicate that YAP1 is a potential target for molecular-based therapy for arsenic-mediated renal fibrosis.

Hippo-YAP/TAZ-ROS signaling axis regulates metaflammation induced by SelenoM deficiency in high-fat diet-derived obesity

IntroductionMetabolic inflammation (metaflammation) in obesity is primarily initiated by proinflammatory macrophage infiltration into adipose tissue. SelenoM contributes to the modulation of antioxidative stress and inflammation in multiple pathological processes; however, its roles in metaflammation and the proinflammatory macrophage (M1)-like state in adipose tissue have not been determined. ObjectivesWe hypothesize that SelenoM could effectively regulate metaflammation via the Hippo-YAP/TAZ-ROS signaling axis in obesity derived from a high-fat diet. MethodsMorphological changes in adipose tissue were examined by hematoxylin-eosin (H&E) staining and fluorescence microscopy. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were used to evaluate the impact of SelenoM deficiency on blood glucose levels. RNA-Seq analysis, LC-MS analysis, Mass spectrometry analysis and western blotting were performed to detect the levels of genes and proteins related to glycolipid metabolism in adipose tissue. ResultsHerein, we evaluated the inflammatory features and metabolic microenvironment of mice with SelenoM-deficient adipose tissues by multi-omics analyses. The deletion of SelenoM resulted in glycolipid metabolic disturbances and insulin resistance, thereby accelerating weight gain, adiposity, and hyperglycemia. Mice lacking SelenoM in white adipocytes developed severe adipocyte hypertrophy via impaired lipolysis. SelenoM deficiency aggravated the generation of ROS by reducing equivalents (NADPH and glutathione) in adipocytes, thereby promoting inflammatory cytokine production and the M1-proinflammatory reaction, which was related to a change in nuclear factor kappa-B (NF-κB) levels in macrophages. Mechanistically, SelenoM deficiency promoted metaflammation via Hippo-YAP/TAZ-ROS-mediated transcriptional regulation by targeting large tumor suppressor 2 (LATS2). Moreover, supplementation with N-acetyl cysteine (NAC) to reduce excessive oxidative stress partially rescued adipocyte inflammatory responses and macrophage M1 activation. ConclusionOur data indicate that SelenoM ameliorates metaflammation mainly via the Hippo-YAP/TAZ-ROS signaling axis in obesity. The identification of SelenoM as a key regulator of metaflammation presents opportunities for the development of novel therapeutic interventions targeting adipose tissue dysfunction in obesity.

Research progress on Hippo signaling pathway effector molecules in rheumatic immune system diseases.

The core components of the Hippo signaling pathway encompass upstream regulatory molecules, core kinase cascade complexes, and downstream transcriptional regulation complexes. This pathway modulates cellular behaviors by influencing the effector molecules of its core components and plays a pivotal role in immune regulation. Effector molecules,such as Yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), transcriptional enhanced associate domain transcriptional factor (TEAD), monopolar spindle-one binder (MOB1), large tumor suppressor (LATS), can stimulate fibroblast-like synovial cell migration and invasion in rheumatoid arthritis, regulate osteoarthritis disease progression, promote pathological new bone formation in ankylosing spondylitis, sustain submandibular gland development while delaying Sjogren's syndrome progression, mediate alpha-smooth muscle actin in systemic sclerosis, and refine the regulation of target genes associated with pulmonary fibrosis. This article provides an overview of the regulatory mechanisms involving Hippo signaling pathway-related effector molecules in the pathogenesis and progression of rheumatic immune system diseases, to serve as a reference for exploring novel therapeutic targets of rheumatic immune system diseases.

Exploring the effects of Hippo signaling pathway on rumen epithelial proliferation

BackgroundThe current understanding to the mechanism of rumen development is limited. We hypothesized that the Hippo signaling pathway controlled the proliferation of rumen epithelium (RE) during postnatal development. In the present study, we firstly tested the changes of the Hippo signaling pathway in the RE during an early growing period from d5 to d25, and then we expanded the time range to the whole preweaning period (d10-38) and one week post weaning (d45). An in vitro experiment was also carried out to verify the function of Hippo signaling pathway during RE cell proliferation.ResultsIn the RE of lambs from d5 to d25, the expression of baculoviral IAP repeat containing (BIRC3/5) was increased, while the expressions of large tumor suppressor kinase 2 (LATS2), TEA domain transcription factor 3 (TEAD3), axin 1 (AXIN1), and MYC proto-oncogene (MYC) were decreased with rumen growth. From d10 to d38, the RE expressions of BIRC3/5 were increased, while the expressions of LATS2 and MYC were decreased, which were similar with the changes in RE from d5 to d25. From d38 to d45, different changes were observed, with the expressions of LATS1/2, MOB kinase activator 1B (MOB1B), and TEAD1 increased, while the expressions of MST1 and BIRC5 decreased. Correlation analysis showed that during the preweaning period, the RE expressions of BIRC3/5 were positively correlated with rumen development variables, while LAST2 was negatively correlated with rumen development variables. The in vitro experiment validated the changes of LATS2 and BIRC3/5 in the proliferating RE cells, which supported their roles in RE proliferation during preweaning period.ConclusionsOur results suggest that the LATS2-YAP1-BIRC3/5 axis participates in the RE cell proliferation and promotes rumen growth during the preweaning period.