YAP Activity Research Articles

Epstein-Barr virus infection upregulates extracellular OLFM4 to activate YAP signaling during gastric cancer progression

Extracellular vesicles (EVs) are known to mediate cell communications and shape tumor microenvironment. Compared to the well-studied small EVs, the function of large microvesicles (MVs) during tumorigenesis is poorly understood. Here we show the proteome of MVs in Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC), and identify olfactomedin 4 (OLFM4) is induced by EBV infection and secreted via MVs to promote tumor progression through Hippo signaling. Specifically, OLFM4 is a target gene of the cGAS-STING pathway, and EBV infection activates cGAS-STING pathway and increases OLFM4 expression. Moreover, MV-carried OLFM4 binds with the extracellular cadherin domain of FAT1, thereby impairing its intracellular interaction with MST1 and leading to YAP activation in recipient cells. Together, our study not only reveals a regulatory mechanism though which viral infection is coupled via MVs with intercellular control of the Hippo signaling, but also highlights the OLFM4-Hippo axis as a therapeutic target for EBV-associated cancers.

P4HA2 knockdown prevents the progression of intracranial aneurysm by inducing prolyl hydroxylation of YAP1.

Prolyl 4-hydroxylase subunit alpha 2 (P4HA2), a key enzyme modulating the post-transcription of proteins, was reported to be a causative gene in IA. Nevertheless, the exact function and mechanism of P4HA2 in the formation and rupture of IA is elusive. The current study first explored the expression of P4HA2 and its association with the clinicopathological demonstrations in patients with IA. In addition, an in vitro model of IA was established using H2O2 to stimulate vascular smooth muscle cells (VSMCs). The behaviors of treated VSMCs were evaluated using CCK-8, Wound healing, and Transwell assays. The expression of genes was detected by RT-qPCR and Western blot. Interaction between genes was confirmed using Luciferase Reporter assay and Co-immunoprecipitation (Co-IP) assay. Our results revealed that P4HA2 expression was upregulated in IA, especially ruptured IA; high P4HA2 expression correlates with unfavorable clinicopathological parameters. Through the in vitro experiments, it was discovered that P4HA2 knockdown rescued VSMCs from H2O2-induced viability impairment, enhancement in migration and apoptosis, switch from contractile phenotype, and augmentation of oxidative stress and inflammation. Mechanistically, P4HA2 was found to trigger the prolyl hydroxylation of YAP1 to negatively regulate the transcriptional activity of YAP1 in H2O2-challenged VSMCs. The effect of P4HA2 on H2O2-challenged VSMCs could be annulled by the mutation of YAP1 hydroxylation sites. In summary, P4HA2 served as a contributing factor during IA progression through its suppression on YAP1 activity by prolyl hydroxylation.

Whole-exome sequencing reveals genomic landscape of intrahepatic cholangiocarcinoma and identifies SAV1 as a potential driver.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy after hepatocellular carcinoma, with poor prognosis and limited treatment options. The genomic features of ICC in Chinese patients remain largely unknown. In this study, we perform deep whole-exome sequencing of 204 Chinese primary ICCs and characterize genomic alterations and clonal evolution, and reveal their associations with patient outcomes. We identify six mutational signatures, including Signatures A and F, which are highly similar to previously described signatures linked to aristolochic acid and aflatoxin exposures, respectively. We also identify 13 significantly mutated genes in the ICC samples, including SAV1. We find that SAV1 was mutated in 2.9% (20/672) of 672 ICC samples. SAV1 mutation is associated with lower SAV1 protein levels, higher rates of tumor recurrence, and shorter overall patient survival. Biofunctional investigations reveal a tumor-suppressor role of SAV1: its inactivation suppresses Hippo signaling, leading to YAP activation, thereby promoting tumor growth and metastasis. Collectively, our results delineate the genomic landscape of Chinese ICCs and identify SAV1 as a potential driver of ICC.

Molecular Mechanism: Inhibition of Fusarium oxysporum T-2 Toxin Synthesis by Surfactin in Dried Fish: Induction of Yap1 Nucleation by ROS Accumulation.

(1) T-2 toxin synthesized by Fusarium oxysporum (F. oxysporum) can cause deterioration of dried fish and endanger human health. (2) The molecular mechanism by which antibacterial lipopeptides surfactin inhibited F. oxysporum growth and toxin production was elucidated by investigating the intracellular ROS production pathway and the subcellular distribution and transcriptional activity of the transcription factor Yap1 and its regulation of Tri5 gene in F. oxysporum. (3) Surfactin caused hyphal damage and nucleic acid and protein leakage; thus, the growth of F. oxysporum was disrupted. Surfactin's excessive accumulation of intracellular ROS triggered the translocation of transcription factor Yap1 into the nucleus, resulting in toxin cluster gene Tri5 expression inhibition, thereby blocking T-2 toxin synthesis. (4) This is a novel mechanism by which surfactin inhibits the growth and T-2 toxin synthesis of F. oxysporum from multiple aspects, including cell structural integrity and the ROS-Yap1 signaling pathway. (5) This study provides a theoretical basis for the application of surfactin in the antifungal control of aquatic dry products.

Abstract 4134975: ROS-Traf6-Yap Promotes the Development of Cardiac Hypertrophy

Introduction: Heart failure (HF) is caused by structural and functional abnormalities of the heart and is associated with high morbidity and mortality. Pathological myocardial hypertrophy accounts for a significant portion heart failure. The Yap protein was found to be responsive to mechanotransduction signals and can limit organ size by inducing cell cycle arrest during development. In the heart, pathological activation of Yap has been shown to drive cardiac hypertrophy, but regenerative activation of Yap can promote myocardial cell-cycle re-entry. However, how Yap is pathologically activated in the beating cardiomyocyte remains elusive. Hypothesis: ROS-Traf6 promotes the development of cardiac hypertrophy by activating Yap in a non-canonical fashion. Methods: Traf6 protein levels in heart failure patients biopsies as well as in various murine failing hearts were examined by immunofluorescence. Molecular interrogation of ROS-Traf6-Yap signaling axis was performed in primary neonatal rat cardiomyocytes (NRCM) and human induced pluripotent stem cell derived cardiomyocytes. Yap protein stability and activation by Traf6 was examined by immunoprecipitation, CHX induction, and point mutation. Myocardial specific Traf6 knockout and Yap mutant animals were used to evaluate Traf6-Yap signaling in vivo . Results: We found that Traf6 expression was significantly elevated in multiple heart failure models. Using the transverse aortic constriction (TAC)-induced cardiac hypertrophy model, pressure overload increased ROS levels which activated Traf6. Traf6 overexpression induced myocardial hypertrophy while knockdown blocked myocardial hypertrophy in Ang II treated NRCMs. Mechanistically, Traf6 binds to and ubiquitinates Yap-K234 – this promotes Yap protein stability and nuclear translocation evidenced by increase in phosphorylated Yap. Myocardial Traf6 deficiency prevents TAC-induced cardiac hypertrophy. Moreover, overexpression of Yap-K237 mutant did not restore TAC-induced cardiac hypertrophy in myocardial Yap deficient mice. Conclusions: Our results demonstrate that non-canonical Yap activation is responsible for pressure overload induced cardiac hypertrophy. Targeting of the newly identified ROS-Traf6-Yap signaling axis may provide a new approach to prevent pathogenic cardiac hypertrophy.

CSIG-08. AGGRESSIVE HIGH-GRADE NF2 MUTANT MENINGIOMAS DOWNREGULATE ONCOGENIC YAP SIGNALING VIA THE UPREGULATION OF VGLL4 AND FAT3/4

Abstract Meningiomas are the most common primary brain tumors in adults. Although generally benign, a subset is of higher grade, shows aggressive growth behavior and recurs even after multiple surgeries. Around half of all meningiomas harbor inactivating mutations in NF2. While benign low-grade NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. We and others have previously shown that NF2 inactivation leads to YAP1 activation and that YAP1 acts as the pivotal oncogenic driver in benign NF2 mutant meningiomas. Using bulk and single-cell RNA-Seq data from a large cohort of human meningiomas, we show that aggressive NF2 mutant meningiomas harbor decreased levels YAP1 activity compared to their benign counterparts. Furthermore, decreased expression levels of YAP target genes are significantly associated with an increased risk of recurrence. We then identify the increased expression of the YAP1 competitor VGLL4 as well as the YAP1 upstream regulators FAT3/4 as a potential mechanism for the downregulation of YAP activity in aggressive NF2 mutant meningiomas. High expression of these genes is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight on the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 in NF2 mutant meningiomas.

De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells.

Regeneration after tissue injury is often associated with cell fate plasticity, which restores damaged or lost cells. Here, we examined the de-differentiation of corneal epithelial cells (CECs) into functional limbal epithelial stem cells (LESCs) after the ablation of innate stem cells. The regeneration of LESCs after the ablation of innate LESCs was identified by a set of markers: ApoE+/Cx43low/CK12-. CK14-CreERT2 or Slc1a3-CreERT mice were crossed with reporter mice to trace the fate of CECs. YAP-TEAD inhibitor verteporfin (VTP) and LATS inhibitor TRULI were used to examine the role of Hippo/YAP pathway in the de-differentiation of CECs. LESCs-ablation cornea showed to be functionally normal, including the maintenance of corneal transparency, prevention of conjunctivalization, and wound healing rate equivalent to that of normal cornea. ApoE+/Cx43low/CK12- LESCs regenerated at the limbus at 6 days after the ablation of innate stem cells, and maintained for at least 6 months. Corneal epithelial lineage tracing showed that CECs migrated back to the limbus after the ablation of innate stem cells, and de-differentiated into active and quiescent LESCs (aLESCs and qLESCs), which participated in corneal epithelial homeostasis and wound healing, respectively, like their innate counterparts. However, when the limbus niche was destroyed by NaOH (1 M, 5 seconds), CECs that occupied the limbus could not de-differentiate into ApoE+/Cx43low/CK12- LESCs and cornea developed into conjunctivalization. In addition, the protein level and activity of YAP increased at the early stage (1-2 days) after the ablation of limbal epithelium, and decreased when the de-differentiation occurred. The YAP-TEAD inhibitor VTP promoted the de-differentiation, whereas LATS inhibitor TRULI inhibited the de-differentiation of CECs. However, the persistent activation of YAP prevented the de-differentiation of CECs after an additional NaOH burn to the limbal stroma, and VTP could not rescue the capacity of CECs to de-differentiate into LESCs. These results reveal the de-differentiation of CECs into functional LESCs after the ablation of innate stem cells, and suggest potential role of Hippo/YAP pathway in the de-differentiation of CECs in vivo.

Transient proliferation by reversible YAP and mitogen-control of the cyclin D1/p27 ratio.

Hippo-YAP signaling orchestrates epithelial tissue repair and is therefore an attractive target in regenerative medicine. Yet it is unresolved how YAP integrates with mitogen signaling and contact inhibition to control the underlying transient proliferative response. Here we show that reduced contact inhibition, increased mitogen signaling, and YAP-TEAD activation converge on increasing the nuclear cyclin D1/p27 protein ratio during G1 phase, towards a threshold ratio that dictates whether individual cells enter or exit the cell cycle. YAP increases this ratio indirectly, in concert with mitogen signaling, by increasing EGFR and other receptors that signal primarily through ERK. After a delay, contact inhibition suppresses YAP activity which gradually downregulates mitogen signaling and the cyclin D1/p27 ratio. Increasing YAP activity by ablating the suppressor Merlin/NF2 reveals a balancing mechanism in which YAP suppression and contact inhibition of proliferation can be recovered but only at higher local cell density. Thus, critical for tissue repair, robust proliferation responses result from the YAP-induced and receptor-mediated prolonged increase in the cyclin D1/p27 ratio, which is only reversed by delayed suppression of receptor signaling after contact inhibition of YAP.

YAP signaling orchestrates the endothelin-1-guided invadopodia formation in high-grade serous ovarian cancer.

The high-grade serous ovarian cancer (HG-SOC) is a notoriously challenging disease, characterized by a rapid peritoneal dissemination. HG-SOC cells leverage actin-rich membrane protrusions, known as invadopodia, to degrade the surrounding extracellular matrix (ECM) and invade, initiating the metastatic cascade. In HG-SOC, the endothelin-1 (ET-1)/endothelin A receptor (ETAR)-driven signaling coordinates invadopodia activity, however how this axis integrates pro-oncogenic signaling routes, as YAP-driven one, impacting on the invadopodia-mediated ECM degradation and metastatic progression, deserves a deeper investigation. Herein, we observed that downstream of the ET-1/ET-1R axis, the RhoC and Rac1 GTPases, acting as signaling intermediaries, promote the de-phosphorylation and nuclear accumulation of YAP. Conversely, the treatment with the dual ETA/ETB receptor antagonist, macitentan, inhibits the ET-1-driven YAP activity. Similarly, RhoC silencing, or cell transfection with a dominant inactive form of Rac1, restore the YAP phosphorylated and inhibited state. Mechanistically, the ET-1R/YAP signal alliance coordinates invadopodia maturation into ECM-degrading structures, indicating how such ET-1R-guided protein network represents a route able to enhance the HG-SOC invasive potential. At functional level, we found that the interconnection between the ET-1R/RhoC and YAP signals is required for MMP-2 and MMP-9 proteolytic functions, cell invasion, and cytoskeleton architecture changes, supporting the HG-SOC metastatic strength. In HG-SOC patient-derived xenografts (PDX) macitentan, turning-off the invadopodia regulators RhoC/YAP, halt the metastatic colonization. ET-1R targeting, hindering the YAP activity, weakens the invadopodia machinery, embodying a promising therapeutic avenue to prevent peritoneal dissemination in HG-SOC.

YAP enhances mitochondrial OXPHOS in tumor-infiltrating Treg through upregulating Lars2 on stiff matrix

BackgroundTumor-infiltrating regulatory T cells (TI-Tregs) are well-adapted to thrive in the challenging tumor microenvironment (TME) by undergoing metabolic reprogramming, notably shifting from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) for energy production. The extracellular matrix is an important component of the TME, contributing to the regulation of both tumor and immune cell metabolism patterns by activating mechanosensors such as YAP. Whether YAP plays a part in regulating TI-Treg mitochondrial function and the underlying mechanisms are yet to be elucidated.MethodsTo gain insights into the effect of matrix stiffness on YAP activation in Tregs, alterations in stiffness were performed bothin vitroandin vivo. YAP conditional knockout mice were used to determine the role of YAP in TI-Tregs. RNA-seq, quantitative PCR, flow cytometry, lentivirus infection and mitochondrial function assay were employed to uncover the mechanism of YAP modulating mitochondrial function in TI-Tregs. A YAP inhibitor and a low leucine diet were applied to tumor-bearing mice to seek the potential antitumor strategy.ResultsIn this study, we found that YAP, as a mechanotransducer, was activated by matrix stiffness in TI-Tregs. A deficiency in YAP significantly hindered the immunosuppressive capability of TI-Tregs by disrupting mitochondrial function. Mechanically, YAP enhanced mitochondrial OXPHOS by upregulating the transcription ofLars2(Leucyl-tRNA synthetase 2, mitochondrial), which was essential for mitochondrial protein translation in TI-Tregs. Since Lars2 relied much on its substrate amino acid, leucine, the combination of a low leucine diet and YAP inhibitor synergistically induced mitochondrial dysfunction in TI-Tregs, ultimately restraining tumor growth.ConclusionsThis finding uncovered a new understanding of how YAP shapes mitochondrial function in TI-Tregs in response to mechanical signals within the TME, making the combined strategy of traditional medicine and diet adjustment a promising approach for tumor therapy.

YAP Alleviates Pulmonary Fibrosis Through Promoting Alveolar Regeneration via Modulating the Stemness of Alveolar Type 2 Cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with no cure except transplantation. Abnormal alveolar epithelial regeneration is a key driver of IPF development. The function of Yes1 Associated Transcriptional Regulator (YAP) in alveolar regeneration and IPF pathogenesis remains elusive. Here, we first revealed the activation of YAP in alveolar epithelium 2 cells (AEC2s) from human IPF lungs and fibrotic mouse lungs. Notably, conditional deletion of YAP in mouse AEC2s exacerbated bleomycin-induced pulmonary fibrosis. Intriguingly, we showed in both conditional knockout mice and alveolar organoids that YAP deficiency impaired AEC2 proliferation and differentiation into alveolar epithelium 1 cells (AEC1s). Mechanistically, YAP regulated expression levels of genes associated with cell cycle progression and AEC1 differentiation. Furthermore, overexpression of YAP in vitro promoted cell proliferation. These results indicate the critical role of YAP in alveolar regeneration and IPF pathogenesis. Our findings provide new insights into the regulation of alveolar regeneration and IPF pathogenesis, paving the road for developing novel treatment strategies.

Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

Advanced pancreatic ductal adenocarcinomas (PDACs) respond poorly to all therapies, including the first-line treatment, chemotherapy, the latest immunotherapies, and KRAS-targeting therapies. Despite an enormous effort to improve therapeutic efficacy in late-stage PDAC patients, effective treatment modalities remain an unmet medical challenge. To change the status quo, we explored the key signaling networks underlying the universally poor response of PDAC to therapy. Here, we report a previously unknown chemo-induced symbiotic signaling circuit that adaptively confers chemoresistance in patients and mice with advanced PDAC. By integrating single-cell transcriptomic data from PDAC mouse models and clinical pathological information from PDAC patients, we identified Yap1 in cancer cells and Cox2 in stromal fibroblasts as two key nodes in this signaling circuit. Co-targeting Yap1 in cancer cells and Cox2 in stroma sensitized PDAC to Gemcitabine treatment and dramatically prolonged survival of mice bearing late-stage PDAC, whereas simultaneously inhibiting Yap1 and Cox2 only in cancer cells was ineffective. Mechanistically, chemotherapy triggers non-canonical Yap1 activation by nemo-like kinase in 14-3-3ζ-overexpressing PDAC cells and increases secretion of CXCL2/5, which bind to CXCR2 on fibroblasts to induce Cox2 and PGE2 expression, which reciprocally facilitate PDAC cell survival. Finally, analyses of PDAC patient data revealed that patients who received Statins, which inhibit Yap1 signaling, and Cox2 inhibitors (including Aspirin) while receiving Gemcitabine displayed markedly prolonged survival compared to others. The robust anti-tumor efficacy of Statins and Aspirin, which co-target the chemo-induced adaptive circuit in the tumor cells and stroma, signifies a unique therapeutic strategy for PDAC.

Cardiomyocyte proliferation is promoted by intracellular Gas6-mediated Yap activity during neonatal heart regeneration

Abstract Purpose The adult mammalian heart has limited regenerative capacity after injury, mostly attributable to postnatal cardiomyocyte (CM) cell cycle arrest. The key to unlocking the regenerative potential of the adult hearts may lie within the developmental transitions occurring during neonatal life. Gas6, a secretory protein, stimulates the proliferation of various type cells through binding to TAM receptor family, such as endothelial progenitor cells, tumor cells, and hair follicle stem-cell. However, no study to date has studied the role of Gas6 during the proliferation of CMs. Herein, our study uncovers that Gas6 is transiently expressed during early postnatal heart and regenerative heart, and functions through intracellular pathway by regulating Yap activity to promote CM proliferation. Methods CM isolation was conducted to assess the change of Gas6 expression in both postnatal development and regenerated stages using the CM-specific Tdtomato mice. Cardiac deletion of Gas6 gene mice was used to evaluate the impaired heart regeneration. CM-specific Gas6 overexpression mice were generated using adeno-associated virus system to evaluate Gas6 pro-proliferate and protect effect after ischemic injury. Secreted signal peptide truncation (ΔSP-Gas6) and predicted functional region truncation (ΔCC-Gas6) were generated to identify the intracellular mechanism of Gas6 via Sav1/p-mst1/p-lats1/ p-yap axis. Results We found that Gas6 expression is enriched in CM4 and the expression profile correlates with the expression of the CM4 marker Acta2, as well as cell-cycle genes Mki67 and Aurora B, in individual CM. Gas6 was highly expressed in the neonatal heart and continued to increase during the neonatal heart regenerative process, but was nearly absent in the adult heart under physiological and ischemia conditions. CM-specific Gas6 deletion resulted in significantly reduced CM proliferation rate and increased CM size in P0 hearts, and congestive heart failure in P28 hearts. Conversely, sustained CM-specific overexpression of Gas6 in postnatal hearts increased CM division, elevated CM numbers, and reduced CM size; and reduced infarct scar area and enhanced cardiac function after ischemia injury. Pre-overexpression of Gas6 in adult CMs protected adult cardiac function against ischemia injury, indicated by the activation of mitosis, angiogenesis and reduced oxidative phosphorylation. In primary neonatal CMs, both Gas6 and ΔSP-Gas6promoted CM proliferation with sarcomere disassembly. Mechanistically, Gas6 interacted with Sav1to inhibit Mst1recruitment and Mst1/Lats1/Yap phosphorylation cascade, therefore, promoted Yap translocation into nucleus. Conclusions We demonstrate that Gas6 is an important intrinsic regulator of neonatal heart regeneration, which acts as a promoter of Yap transcriptional activity to enhance CM proliferation. Supplementing Gas6 in the adult heart represents a potential therapeutic approach for cardiac repair.

A ZO-2 scaffolding mechanism regulates the Hippo signalling pathway.

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density-dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO-2 is required for the contact-mediated inhibition of proliferation. We additionally determined that the well-established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO-2 and that the scaffolding function of ZO-2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO-2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO-2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP.

DLAT promotes triple-negative breast cancer progression via YAP1 activation

ABSTRACT Background Breast cancer (BC) is the most prevalent malignant tumor in women globally. Triple-negative breast cancer (TNBC) represents the most malignant and invasive subtype of BC. New therapeutic targets are urgently needed for TNBC owing to its receptor expression characteristics, which render it insensitive to traditional targeted and endocrine therapies for BC. The role and mechanisms of dihydrolipoamide S-acetyltransferase (DLAT) as a crucial molecule in glycometabolism and cuproptosis-related biological processes in tumors remain to be explored. Methods DLAT expression was investigated using bioinformatics methods and quantitative real-time polymerase chain reaction. Subsequently, the MTT assay, colony formation assay, and migration-invasion assay were performed to validate the effect of DLAT on TNBC cell viability, proliferation, and migration. Cytoplasmic-nuclear separation experiments, western blot analysis, and co-immunoprecipitation assays were performed to elucidate the underlying molecular mechanisms. Results This study revealed a robust correlation between elevated DLAT expression in BC and unfavorable prognosis in patients, with higher expression of DLAT compared to other subtypes in TNBC. Functional cytology experiments indicated that DLAT plays a tumor-promoting role in TNBC. Mechanistic studies showed that DLAT directly interacts with YAP1, leading to the dephosphorylation and activation of YAP1 and its increased nuclear translocation, thereby transcriptionally activating and regulating downstream oncogenes, promoting the malignant phenotype of TNBC. Rescue experiments indicated that DLAT promotes the malignant behavior of TNBC through a YAP1-dependent pathway. Conclusions Our research unveiled the significant involvement of DLAT in TNBC, along with the potential for modulating DLAT/YAP1 activity as a targeted treatment strategy for TNBC.

CRB2 Depletion Induces YAP Signaling and Disrupts Mechanosensing in Podocytes.

Focal Segmental Glomerulosclerosis (FSGS) is a histologic lesion caused by a variety of injurious stimuli that lead to dysfunction/loss of glomerular visceral epithelial cells (i.e. podocytes). Pathogenic mutations in CRB2, encoding the type 1 transmembrane protein Crumb 2 Homolog Protein, have been shown to cause early-onset corticosteroid-resistant nephrotic syndrome (SRNS)/FSGS. Here, we identified a 2-generation East Asian kindred (DUK40595) with biopsy-proven SRNS/FSGS caused by a compound heterozygous mutation in CRB2 comprised of the previously described truncating mutation p.Gly1036_Alafs*43 and a rare 9-bp deletion mutation p.Leu1074_Asp1076del. Because compound heterozygous mutations involving the truncating p.Gly1036_Alafs*43 variant have been associated with reduced CRB2 expression in podocytes and autosomal recessive SRNS/FSGS, we sought to define the pathogenic effects of CRB2 deficiency in podocytes. We show that CRB2 knockdown induces YAP activity and target gene expression in podocytes. It upregulates YAP-mediated mechanosignaling and increases the density of focal adhesion and F-actin. Using Elastic Resonator Interference Stress Microscopy (ERISM), we demonstrate that CRB2 knockdown also enhances podocyte contractility in a substrate stiffness-dependent manner. The knockdown effect decreases with increasing substrate stiffness, indicating impaired mechanosensing in CRB2 knockdown cells at low substrate stiffness. While the mechanical activation of CRB2 knockdown cells is associated with increased YAP activity, the enhanced cell contractility is not significantly reduced by the selective YAP inhibitors K-975 and verteporfin, suggesting that multiple pathways may be involved in mechanosignaling downstream of CRB2. Taken together, these studies provide the first evidence that CRB2 deficiency may impair podocyte mechanotransduction via disruption of YAP signaling in podocytes.

SON-dependent nuclear speckle rejuvenation alleviates proteinopathies.

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as a pivotal membrane-less organelle responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate alleviated retina degeneration and reduced tauopathy by promoting autophagy and ubiquitin-proteasome system in a SON-dependent manner without causing cellular stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

Icariin promotes functional recovery in rats after spinal cord injury by inhibiting YAP and regulating PPM1B ubiquitination to inhibiting the activation of reactive astrocytes.

The limited ability to regenerate axons after spinal cord injury (SCI) is influenced by factors such as astrocyte activation, reactive proliferation, and glial scar formation. The TGF-β/Smad (transforming growth factor-β/mothers against decapentaplegic homolog) pathway, associated with astrocytic scarring, plays a crucial role in recovery post-injury. This study aims to investigate how icariin (ICA) interacts with reactive astrocytes in the treatment of spinal cord injury. A rat SCI model was constructed, and the recovery of motor function was observed after treatment with ICA.HE staining, LFB staining, immunofluorescence staining, and Western blotting were employed to assess ICA's ability to inhibit astrocyte proliferation in rats following spinal cord injury by modulating YAP, as well as to evaluate the reparative effects of ICA on the injured spinal cord tissue. Primary astrocytes were isolated and cultured. Immunoprecipitation-Western Blot (IP-WB) ubiquitination and cytoplasm-nuclear separation were employed to assess PPM1B ubiquitination and nuclear translocation. The CatWalk XT gait analysis, BBB (Basso, Beattie, and Bresnahan) score, electrophysiological measurements, HE staining, and LFB staining collectively demonstrated that ICA promotes motor function and tissue recovery following spinal cord injury in rats. Immunofluorescence staining and Western Blot analyses revealed that ICA inhibits astrocyte proliferation in rats post-spinal cord injury by suppressing YAP activity. Furthermore, the activation of YAP by XMU-MP-1 was shown to compromise the efficacy of ICA in these rats after spinal cord injury. Additional immunofluorescence staining and Western Blot experiments confirmed that ICA inhibits TGFβ1-induced astrocyte activation through the regulation of YAP. The knockdown of PPM1B (protein phosphatase, Mg2+/Mn2+-dependent 1B) in astrocytes was found to inhibit TGFβ signaling. Additionally, YAP was shown to regulate PPM1B ubiquitination and nuclear translocation through immunoprecipitation-Western blot analysis, along with the segregation of cytoplasm and nucleus. Icariin promotes functional recovery in rats after spinal cord injury by inhibiting YAP and regulating PPM1B ubiquitination to inhibiting the activation of reactive astrocytes.

6873 Expression of Constitutively Active YAP in Murine Gonadotrope Cells Leads to Morphological Alterations and Dysfunction of the Anterior Pituitary Gland

Abstract Disclosure: N. Jakuc: None. M. Bérubé: None. E. Corrêa Dos Santos: None. G. St-Jean: None. M. Paquet: None. U. Boehm: None. D.J. Bernard: None. D.T. Boerboom: None. A. Boyer: None. G. Zamberlam: None. The Hippo transcriptional coactivators, YAP and TAZ, are expressed in cells throughout the anterior pituitary gland in mice. Although the potential physiological roles of YAP and TAZ in gonadotrope cells was unknown, we recently reported that conditional deletion of YAP and TAZ in gonadotrope cells results in increased circulating levels of LH (in males and females) and FSH (males only), and female hyperfertility in mice. Here, to further study the effects of Hippo signaling in gonadotropes, we conditionally expressed a constitutively active YAP (5SA) in these cells using the Cre-lox system with one of two Cre-driver strains, steroidogenic factor-1 (SF-1)-Cre or GnRH receptor-IRES-Cre (GRIC). Such crosses generated two distinct mouse models, which are referred to herein as YAP5SA;SF1-Cre and YAP5SA;GRIC, respectively. Expression of total YAP (YAP5SA and endogenous Yap) and the classic YAP-TEAD target genes, Ctgf, Cyr61, and Ankrd1 was significantly increased in pituitaries of 6-week-old mutant males and females in both the YAP5SA;SF1-Cre and YAP5SA;GRIC models. Animals from both models also showed significantly reduced serum LH and FSH levels as well as lower body mass and gonadosomatic indices when compared to respective controls. Six-week-old male and female mutants from both models showed significant morphological alterations in the pituitary. YAP5SA;SF1-Cre mutants exhibited proliferation of poorly differentiated cells, with high cellular and nuclear atypia with several mitoses. Similarly, YAP5SA;GRIC pituitaries were characterized by an increased number of poorly differentiated cells as well as follicle or papillary-like structure formation and increased cell proliferation and necrosis. Together, these preliminary findings strongly suggest pituitary blastoma or choriocarcinoma, and confirmatory experiments are underway. In light of gross morphological changes that appeared to extend beyond gonadotropes, we performed qPCR analysis of other cell lineages in 6-week-old YAP5SA;GRIC mutants and corresponding controls. Gh, Prl, Tshb, and Pomc mRNA levels were decreased in pituitaries of mutant animals. In addition, GH and ACTH protein expression, as assessed by immunohistochemistry, was nearly undetectable in pituitaries in mutant animals, suggesting compromised presence or function of somatotropes and corticotropes. To better understand the onset of this pathology, we performed histopathological analysis of YAP5SA;GRIC mutants in early post-natal life. Although pituitaries from 1-day-old mutant animals seemed normal in comparison to controls, we found signs of cellular necrosis and vacuolar degeneration in 1-week-old mutants. Together, our results indicate that constitutively active YAP in murine gonadotropes leads to significant morphological and functional alterations throughout the pituitary. Presentation: 6/1/2024

PRMT1-mediated arginine methylation promotes YAP activation and hepatocellular carcinoma proliferation.

The activity of Hippo signaling is commonly dysregulated in various human malignancies, including hepatocellular carcinoma (HCC). YAP, the key effector of Hippo pathway, is regulated through several posttranslational modifications. However, the mechanism by which YAP is regulated by arginine methylation remains unknown. In this study, immunoprecipitation and mass spectrometry were used to identify the arginine methylation site of YAP in HCC cells. The transcriptional activity of YAP and TEAD were further characterized by real-time qPCR and immunofluorescence assay, and a subcutaneous and orthotopic tumor mouse model was used to assess the effect of PRMT1-knockdown on HCC tumor growth. The result of mass spectrometry analysis identified that YAP was methylated at arginine 124. Moreover, we found that arginine methyltransferase PRMT1 interacted with YAP to mediate its arginine methylation, thus inhibited YAP phosphorylation and promoted YAP activity in the nucleus. PRMT1 was up-regulated in HCC tissues and positively associated with the expressions of YAP target genes. Silencing PRMT1 in HCC cells inhibited cell proliferation and tumor growth, while PRMT1-overexpression promoted HCC growth through YAP methylation. Our study reveals that PRMT1-mediated arginine methylation at R124 is mutually exclusive with YAP S127 phosphorylation, thereby facilitating YAP activity in the nucleus and promoting tumorigenesis in HCC.