YAP Expression Research Articles

Rotating culture regulates the formation of HepaRG-derived liver organoids via YAP translocation.

Liver organoid serves as an alternative model for liver pathophysiology in carbohydrate or lipid metabolism and xenobiotic metabolism transformation. Biomechanical cues including spaceflight mission can affect liver organoid construction and their related functions, but their underlying mechanisms are not fully understood yet. Here, a rotating cell culture device, namely Rotating Flat Chamber (RFC), was specifically designed for adhering cells or cell aggregated to elucidate the effects of altered gravity vector on HepaRG-derived liver organoids construction. The organoids so formed under RFC presented the fast growth rate and large projection area. Meanwhile, the expressions of two pluripotency markers of SOX9 and CD44 were enhanced. This finding was positively correlated with the increased YAP expression and nuclear translocation as well as the elevated α4β6-integrin expression. Inhibition of YAP expression and nuclear translocation decreased the expression of SOX9 and CD44 under RFC, thereby attenuating the pluripotency of HepaRG-derived liver organoids. In conclusion, we proposed a novel liver organoid construction method using rotating culture, by which the pluripotency of liver organoids so constructed is mediated by α4β6-integrin and YAP translocation. This work furthered the understanding in how the gravity vector orientation affects the construction of liver organoids and the related mechanotransductive pathways.

ER stress-induced YAP upregulation leads to chondrocyte phenotype loss in age-related osteoarthritis

BackgroundOsteoarthritis (OA) is a common degenerative joint disease, leading to pain and restricted mobility. Age-related endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of OA, but the underlying mechanisms remain unclear. This study aims to explore the relationship between age-related ER stress, YAP overexpression, and chondrocyte phenotype loss in the development of OA.MethodsCartilage samples were collected from patients undergoing amputation, and age-related ER stress markers and YAP expression were assessed using immunohistochemical staining and qPCR. Transgenic mice with cartilage-specific YAP overexpression (YAPOE) were created, and Pamrevlumab was administered to evaluate its therapeutic effects.ResultsHigher expression of ER stress markers and YAP were showed in aged tissues compared to younger tissues. YAP overexpression led to decreased levels of cartilage phenotype markers and increased osteogenesis-related proteins. In vivo, YAPOE mice exhibited OA-like cartilage degeneration, which was mitigated by Pamrevlumab treatment.ConclusionAge-related ER stress induces YAP overexpression, contributing to OA pathogenesis. Pamrevlumab effectively prevents this phenotype loss in YAPOE mice, suggesting its potential as a therapeutic agent for OA. These findings provide new insights into the molecular mechanisms of OA and highlight the importance of targeting the ER stress-YAP-CTGF signaling pathway in OA treatment and prevention.

TMIC-67. EMERGING ROLE OF EXTRACELLULAR MATRIX STIFFNESS AS A HALLMARK OF GLIOBLASTOMA: INSIGHT TO MECHANICAL MEMORY FORMATION AND PROLONGED INVASION IN THE TUMOR PERIPHERY

Abstract The extracellular matrix (ECM) plays a critical role in cancer progression, with its biophysical properties influencing cancer behavior. ECM stiffness can induce long-lasting adaptations in cancer cells, promoting sustained invasion through a process known as “mechanical memory” (MM). Glioblastoma (GBM) exhibits spatial heterogeneity in stiffness, with stiffer ECM found at the tumor periphery where invasive cells reside and promote recurrence. We hypothesize that increased stiffness at the tumor periphery primes GBM cells for sustained invasion through MM. METHODS Cell lines were established from image-guided human GBM samples from tumor margin and core (n=10 patients, 20 cell lines). Cells from random locations were conditioned in physiologically relevant extracellular matrices for two weeks. Human organotypic assay (n=3), Migration Assays, Atomic Force Microscopy (AFM), Immunocytochemistry, Western Blot, and RNA-sequencing (n=6) were performed. RESULTS GBM cells at higher stiff surfaces displayed increased migration (p<0.01). Magnetic Resonance Elastography (n=9) confirmed GBM heterogeneity, with softer cores (0.2-0.5kPa) transitioning to stiffer margins (>2kPa). Organotypic scaffolds mimicking the tumor margin promoted enhanced migration of GFP-labeled GBM cells. Margin cells and cells conditioned on stiffer matrices demonstrated higher migratory potential compared to their core and soft-conditioned counterparts (p<0.05). These trends persist over time and across multiple passages. ECM stiffness exposure causes nanomechanical, cytoskeletal, and transcriptional adaptations. AFM shows that cells from the margin (n=6) are softer (p<0.01) and more deformable (p<0.01). RNAseq identified upregulation of MM-specific genes in GBM margin cells. The mechanosensitive transcription factor YAP1 emerged as a key driver of MM. Notably, YAP1 is targetable by the FDA-approved drug Verteporfin. YAP1 expression was elevated in margin and stiff-conditioned cells (p<0.05). Stiff-conditioned cells and margin cells displayed increased sensitivity to Verteporfin treatment (p<0.05), with treatment significantly reducing margin migration compared to the core (p<0.001). CONCLUSION Stiffer GBM margins promote the formation of aggressive phenotypes through MM. Targeting YAP1 with Verteporfin, a readily available drug, presents a promising strategy to halt GBM invasion and improve patient outcomes.

CNSC-44. TARGETING OF THE YAP/TAZ-TEAD AXIS DIMINISHES VIABILITY AND DIFFERENTIATION POTENTIAL IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Pediatric high-grade gliomas (pHGGs) are invasive and fast-growing brain malignancies. They account for up to 40% of all brain tumor-related deaths in childhood. Genetic alterations that characterize these gliomas include H3 onco-histone mutations and genetic alterations in receptor-tyrosine kinases (RTKs), including amplification, mutation, and gene fusions in RTKs. However, direct targeting with tyrosine kinase inhibitors (TKIs) has shown at best partial responses in patients due to the molecular heterogeneity within these tumors and the lack of pharmaceutical penetration into the central nervous system (CNS). Thus, alternative therapeutic approaches to treat pHGGs are urgently needed. The YAP/TAZ transcription factors, which cooperate with TEAD co-factors to promote target gene expression, orchestrate stem-cell fate decisions and proliferation downstream of growth factor signaling pathways, such as RTKs. We observed that YAP expression is upregulated in RTK mutant and H3 mutant pHGGs. Depletion of YAP via RNAi methods decreased proliferation, inhibited pHGG stem cell self-renewal, and induced apoptosis of pHGG cells. Use of Verteporfin (VP), a FDA-approved YAP/TAZ small molecule inhibitor, mimicked the effects seen by YAP genetic depletion. Using an in vitro human cortical organoid-tumor model in which tumor cells are engrafted to human brain cortical organoids, we screened for responses in tumor cells and in non-tumor cells in the neural microenvironment upon VP treatment, using single-cell RNA-sequencing and immunofluorescence to visualize responses. These organoid models offer improved models for modeling how the neural CNS microenvironment shapes responses of tumor cells to YAP/TAZ inhibition and VP treatments. Results from our work showing that YAP/TAZ-TEAD inhibition is a promising therapeutic avenue for management of pHGGs will be presented. Further pre-clinical work will follow to correlate observation and understand the therapeutic efficacy of YAP/TAZ-TEAD inhibition for pHGG treatment.

STEM-07. UNVEILING THE ROLE OF HYPOXIA AND TAM-SECRETED TGF-Β1 IN PROMOTING GLIOMA STEM CELL MAINTENANCE VIA YAP/TAZ ACTIVATION IN GLIOBLASTOMA PERI-NECROTIC NICHE

Abstract Glioblastoma (GBM) is a highly malignant brain tumor with rapid growth and poor outcomes. In the tumor microenvironment (TME), necrosis triggers significant reshaping and rapid progression, marked by tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) accumulation in peri-necrotic zones. Analysis of Ivy-GAP and single-cell RNA-Seq data revealed consistent Hippo pathway activation in the hypoxic peri-necrotic zone, despite canonical stem-cell transcription factors such as SOX2, OLIG2, and FOXM1 not being upregulated. We hypothesize that Hippo transcription activation may promote GSC stemness via direct effects of hypoxia and/or TAM-secreted cytokines. Exposing patient-derived GBM neurospheres to 1% hypoxia increased nuclear YAP1 and TAZ expression, along with CYR61, a Hippo pathway readout, compared to normoxia at 48 hours. Single-cell RNA-Seq identified potential hypoxia-regulated genes upstream of YAP/TAZ, including Zyxin, upregulated in 1% hypoxia-treated cultures, possibly facilitating nuclear YAP/TAZ transport. Culturing GBM cells with primary macrophage-conditioned media activated Hippo transcription, with TGF-β1 identified as a hypoxia-specific driver of malignant cell gene expression with single-cell RNA-Seq analysis using NichenetR. Treating GBM cells with TGF-β1 increased CYR61 expression, signifying Hippo transcription activation. Furthermore, we showed that hypoxia and TGF-β1 synergistically maintained GSCs with extreme limiting dilution assays. In the immunocompetent RCAS/tv-a mouse model, Verteporfin, a YAP/TAZ inhibitor, significantly reduced tumor size and prolonged mouse lifespan. TAZ knockdown similarly extended lifespan and reduced tumor growth, with flow cytometry showing a decrease in CD133/CD44 positive GSC populations in knockdown mice. These findings suggest the conclusion that hypoxia and TAM-secreted TGF-β1 directly stimulate YAP/TAZ activation and potentially promote GSC stemness in the GBM peri-necrotic niche.

Dual effects of DLG5 (disks large homolog 5 gene) modulation on chemotherapy-induced thrombocytopenia and nausea/vomiting via the hippo signalling pathway.

The CAPEOX (combination of oxaliplatin and capecitabine) chemotherapy protocol is widely used for colorectal cancer treatment, but it can lead to chemotherapy-induced adverse effects (CRAEs). To uncover the mechanisms and potential biomarkers for CRAE susceptibility, we performed whole-genome sequencing on normal colorectal tissue (CRT) before adjuvant chemotherapy. This is followed by in vivo and in vitro verifications for selected gene and CRAE pair. Our analysis revealed specific germline mutations linked to Grade 2 (or higher) chemotherapy-induced thrombocytopenia (CIT) and nausea/vomiting (CINV). Notably, both CRAEs were associated with mutations in the DLG5 gene. We found that DLG5 mutations related to CIT were associated with increased gene expression, while those associated with CINV were linked to suppressed gene expression, as indicated by the Genotype-Tissue Expression (GTEX) database. In megakaryocytes, overexpression of human DLG5 suppressed the hippo signalling pathway and induced YAP expression. In zebrafish, overexpression of human DLG5 not only reduced platelet production but also inhibited thrombus formation. Subsequent qPCR analysis revealed that DLG5 overexpression affected genes involved in cytoskeleton formation and alpha-granule formation, which could impact the normal generation of proplatelets. We identified a series of germline mutations associated with susceptibility to CIT and CINV. Of particular interest, we demonstrated that induced and suppressed DLG5 expression is respectively related to CIT and CINV. These findings shed light on the involvement of the hippo signalling pathway and DLG5 in the development of CRAEs, providing valuable insights into potential targets for therapeutic interventions.

YAP regulates HER3 signaling-driven adaptive resistance to RET inhibitors in RET-aberrant cancer.

Rearranged during transfection (RET) aberrations represent a targetable oncogene in several tumor types, with RET inhibitors displaying marked efficacy. However, some patients with RET-aberrant cancer are insensitive to RET tyrosine kinase inhibitors (TKIs). Recently, drug-tolerant mechanisms have attracted attention as targets for initial therapies to overcome drug resistance. The underlying mechanisms of drug-tolerant cell emergence treated with RET-TKIs derived from RET-aberrant cancer cells remain unknown. This study investigated the role of YAP-mediated HER3 signaling in the underlying mechanisms of adaptive resistance to RET-TKIs in RET-aberrant cancer cells. Four RET-aberrant cancer cell lines were used to assess sensitivity to the RET-TKIs selpercatinib and pralsetinib and to elucidate molecular mechanisms underlying adaptive resistance using RNA sequencing, phospho-RTK antibody arrays, chromatin immunoprecipitation assay, and luciferase reporter assays. Clinical specimens from patients with RET-fusion-positive lung cancer were analyzed for pre-treatment YAP expression and correlated with treatment outcomes. In high YAP-expressing RET-aberrant cancer cells, YAP-mediated HER3 signaling activation maintained cell survival and induced the emergence of cells tolerant to the RET-TKIs selpercatinib and pralsetinib. The pan-ErBB inhibitor afatinib and YAP/TEAD inhibitors verteporfin and K-975 sensitized YAP-expressing RET-aberrant cancer cells to the RET-TKIs selpercatinib and pralsetinib. Pre-treatment YAP expression in clinical specimens obtained from patients with RET-fusion-positive lung cancer was associated with poor RET-TKI treatment outcomes. The YAP-HER3 axis is crucial for the survival and adaptive resistance of high YAP-expressing RET-aberrant cancer cells treated with RET-TKIs. Combining YAP/HER3 inhibition with RET-TKIs represents a highly potent strategy for initial treatment.

Activation of PAR1 contributes to ferroptosis of Schwann cells and inhibits regeneration of myelin sheath after sciatic nerve crush injury in rats via Hippo-YAP/ACSL4 pathway.

Peripheral nerve injury (PNI) is characterized by high incidence and sequela rate. Recently, there was increasing evidence that has shown ferroptosis may impede functional recovery. Our objective is to explore the novel mechanism that regulates ferroptosis after PNI. LC-MS/MS proteomics was used to explore the possible differential signals, while PCR array was performed to investigate the differential factors. Besides, we also tried to activate or inhibit the key factors and then observe the level of ferroptosis. Regeneration of myelin sheath was finally examined in vivo via transmission electron microscopy. Proteomics analysis suggested coagulation signal was activated after sciatic nerve crush injury, in which high expression of F2 (encoding thrombin) and F2r (encoding PAR1) were observed. Both thrombin and PAR1-targeted activator TRAP6 can induce ferroptosis in RSC96 cells, which can be rescued by Vorapaxar (PAR1 targeted inhibitor) in vitro. Further PCR array revealed that activation of PAR1 induced ferroptosis in RSC96 cells by increasing expression of YAP and ACSL4. Immunofluorescence of sciatic nerve confirmed that the expression of YAP and ACSL4 were simultaneously reduced after PAR1 inhibition, which may contribute to myelin regeneration after injury in SD rats. Inhibition of PAR1 can relieve ferroptosis after sciatic nerve crush injury in SD rats through Hippo-YAP/ACSL4 pathway, thereby regulating myelin regeneration after injury. In summary, PAR1/Hippo-YAP/ACSL4 pathway may be a promising therapeutic target for promoting functional recovery post-sciatic crush injury.

Inhibiting YAP1 reduced abdominal aortic aneurysm formation by suppressing adventitial fibroblast phenotype transformation and migration.

The adventitial fibroblast (AF) is the most abundant cell in the vascular adventitia, a few studies had confirmed that AF contributed to abdominal aortic aneurysm (AAA) development; YAP1 involved in several vascular diseases by promoting AF transformed to myofibroblast, the role of YAP1 in AAA is not clear yet. This study aims to determine whether YAP1 play a role in AAA process by regulating AF function. We found the expression of YAP1was significantly increased in aneurysm tissues of AAA patients compared to normal adjacent vascular tissues and mainly in adventitia. YAP1 also upregulated in elastase-induced and CaCl2-induced mice AAA model. Suppressed YAP1 function with YAP1 inhibitor-Verteporfin declined AAA incident rate remarkably in mice, and the collagen deposition in the adventitia was alleviated obviously. Afterwards, we studied the effect of YAP1 on the function of AF, Verteporfin was used to block YAP1 invitro, the process of AF transforming to myofibroblast and migration were almost completely eliminated after inhibiting YAP1 expression. This study demonstrated that YAP1 may play a key role in AAA development, inhibiting YAP1 significantly reduced AAA formation through suppressed the process of AF transformed to myofibroblast and migration.

Exploring IRGs as a Biomarker of Pulmonary Hypertension Using Multiple Machine Learning Algorithms.

Pulmonary arterial hypertension (PAH) is a severe disease with poor prognosis and high mortality, lacking simple and sensitive diagnostic biomarkers in clinical practice. This study aims to identify novel diagnostic biomarkers for PAH using genomics research. We conducted a comprehensive analysis of a large transcriptome dataset, including PAH and inflammatory response genes (IRGs), integrated with 113 machine learning models to assess diagnostic potential. We developed a clinical diagnostic model based on hub genes, evaluating their effectiveness through calibration curves, clinical decision curves, and ROC curves. An animal model of PAH was also established to validate hub gene expression patterns. Among the 113 machine learning algorithms, the Lasso + LDA model achieved the highest AUC of 0.741. Differential expression profiles of hub genes CTGF, DDR2, FGFR2, MYH10, and YAP1 were observed between the PAH and normal control groups. A diagnostic model utilizing these hub genes was developed, showing high accuracy with an AUC of 0.87. MYH10 demonstrated the most favorable diagnostic performance with an AUC of 0.8. Animal experiments confirmed the differential expression of CTGF, DDR2, FGFR2, MYH10, and YAP1 between the PAH and control groups (p < 0.05); Conclusions: We successfully established a diagnostic model for PAH using IRGs, demonstrating excellent diagnostic performance. CTGF, DDR2, FGFR2, MYH10, and YAP1 may serve as novel molecular diagnostic markers for PAH.

Characterization of Hippo Signaling Components in the Early Dorsal Pancreatic Bud.

YAP1 expression in the early mouse pancreatic anlagen is low until approximately E11.5, when it becomes localized to cell nuclei in multipotent progenitor cells. At E14.5, we find nuclear YAP1 in ductal cells.YAP1 is not expressed in early and midgestation endocrine cells. By contrast, TAZ is expressed in first transition endocrine cells.Hippo regulators MERLIN and LATS1/2 kinases are robustly expressed in the early pancreatic bud by E10.5. Both MERLIN and LATS1/2 exhibit strong apical localization in epithelial cells at nascent microlumens. Using in vitro models of de novo pancreas lumen formation, we show that YAP1 nuclear localization is high in early phases of lumen formation and gradually decreases as lumens matures.

The tumor immune microenvironment of SCLC is not associated with its molecular subtypes

IntroductionSmall-cell lung carcinoma (SCLC) is a high-grade neuroendocrine carcinoma of poor prognosis. Although immune checkpoint blockers have shown promising results in advanced SCLC, the tumor immune microenvironment (TME) remains poorly understood, with no validated prognostic or predictive biomarkers of efficacy. MethodsThis retrospective study included surgically samples from 48 SCLC patients between 2009 and 2018. We assessed the TME using two quantitative 7-plex immunofluorescence panels focusing on T and B cells, and compared it to NSCLC (N = 10). Molecular subtypes were determined by assessing the expression of ASCL1, NEUROD1 and YAP1 using immunohistochemistry. ResultsImmune-hot SCLC were defined as those exhibiting the highest immune cell and immune-related marker densities. They were associated with longer overall survival, significantly more frequently detected at early stages, and exhibited high PD-L1 expression in immune cells, but were not associated with molecular subtypes. Compared to NSCLC, SCLC had significantly lower densities of CD20 + cells and higher density of PD1 + cells, with no significant differences in CD4 + , CD8 + and plasma cell densities. In univariate analysis, the highest OS was significantly associated with early stage (p < 0.001), low expression of NEUROD1 (p = 0.047), high PD1 + cell density (p < 0.001) and high PD-L1 immune cell expression (p = 0.04). Only stage and PD1 + cell density emerged as independent prognostic markers. ConclusionSCLC TME is highly heterogeneous. Immune-hot tumors were associated with OS but not with molecular classification. PD1 expression and PD-L1 expression by immune cells may thus serve as a prognostic marker.

GL-V9 Promotes Autophagy-Mediated YAP1 Degradation and Activates Mitochondrial Apoptosis in PDAC Cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive forms of pancreatic cancer with a poor prognosis. YAP1 expression is markedly elevated in PDAC, but how it works is not clear. GL-V9, a derivative of the natural compound wogonin, effectively fights a variety of tumors; however, its effect on PDAC has not yet been studied. Methods: TCGA database analysis, Western blots, immunofluorescence, and real-time PCR were used to evaluate GL-V9's effect on YAP1 expression and mRNA levels. Immunofluorescence was used to examine the co-location of YAP1 with LAMP2 and p62. Co-immunoprecipitation was used to assess the binding of YAP1 to ubiquitin, p62, and TEAD1. A PDAC graft tumor model was used to test GL-V9's pharmacological effects. Western blots and immunohistochemistry were used to measure apoptosis- and autophagy-related protein expression. Results: GL-V9 effectively promoted the degradation of YAP1, reduced YAP1 nuclear localization, and induced mitochondrial apoptosis in PDAC cells. YAP1 overexpression led to the upregulation of Bcl-2 and attenuated the caspase cascade induced by GL-V9. Furthermore, we demonstrated that GL-V9 induced autophagosome-lysosome fusion via the AKT/mTOR/TFEB pathway, leading to mitochondrial apoptosis in PDAC cells. In vivo studies also confirmed that GL-V9 exerts anti-tumor effects by suppressing YAP1 expression, while also activating autophagy and inducing mitochondrial apoptosis in BXPC-3-bearing BALB/c nude mice. Conclusions: Our findings underscore the importance of autophagy-mediated YAP1 degradation in PDAC, providing a novel molecular rationale (GL-V9) as a promising treatment for this disease.

HIF-1α induced FGF18 alleviates renal ischemia/reperfusion injury via YAP.

Acute kidney injury (AKI) is a devastating clinical condition characterized by an abrupt loss of renal function. The pathophysiology of AKI involves diverse processes and elements, of which survival and regeneration have been established to be significant hallmarks. And early studies have confirmed the fundamental role of FGFs in the regulation of AKI pathology, although the association between FGF18 and AKI still remains elusive. Our study demonstrates a substantial up-regulation of FGF18 in the renal tubules of mice subjected to ischemia. Notably, targeted overexpression of FGF18 effectively mitigates the impairment of kidney function induced by AKI. Mechanistically, FGF18 facilitates cell proliferation and anti-apoptosis in RTECs by enhancing the expression of YAP and facilitating its translocation to the nucleus. Aside from that, we also discovered that the substantial expression of FGF18 under ischemic conditions is HIF-1α dependent. This study aims to uncover the inherent mechanism behind the beneficial effects of FGF18 in attenuating AKI. By doing so, it aims to offer novel insights into the development of therapeutic strategies for AKI.

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

Corosolic acid inhibits EMT in lung cancer cells by promoting YAP-mediated ferroptosis

BackgroundCorosolic acid (CA), a naturally occurring pentacyclic triterpenoid is renowned for its anticancer attributes. Previous studies have predominantly centered on the anticancer properties of CA in lung cancer, specifically its role in inducing apoptosis, however, investigations regarding its involvement in ferroptosis have been scarce. MethodsThe apoptotic and proliferative effects were evaluated by CCK8 and colony formation assay. Cell death and ROS generation were measured to assess the response of CA to iron death induction. Scratch and invasion assays were performed to verify the effect of CA on the invasive ability of lung cancer cells. Protein and mRNA expression were analyzed using Western blotting and qPCR. The CHX assay was carried out to detect protein half-life. Metabolite levels were measured with appropriate kits. Protein expression was detected through IF and IHC. A xenograft tumor model was established to investigate the inhibitory effect of CA on lung cancer in vivo. ResultsThe current findings revealed that CA exerts its anticancer effect by inducing cell death, accompanied by the accumulation of lipid reactive oxygen species (ROS), hinting at the possible involvement of ferroptosis. Our experimental results further substantiated the significance of ferroptosis in the CA anticancer mechanism, as ferroptosis inhibitors were found to effectively rescue CA-induced cell death. Significantly, we demonstrated for the first time that CA could induce ferroptosis further by suppressing EMT in lung cancer cells. Additionally, CA could regulate GPX4 to induce ferroptosis, interestingly, CA downregulated GSH synthetase by inhibiting YAP rather than GPX4, thereby reducing GSH, inducing ferroptosis, and further suppressing EMT in lung cancer cells.We also discovered that GSS is a crucial downstream target of YAP in regulating GSH. Moreover, a xenograft mouse model indicated that CA could trigger ferroptosis in lung cancer cells by regulating YAP expression and GSH levels. ConclusionCA inhibited lung cancer cell metastasis by inducing ferroptosis. Our data offer the first evidence that CA induces ferroptosis in lung cancer cells by regulating YAP/GSS to modulate GSH, thereby further suppressing EMT. These results imply the potential of CA as an inducer of ferroptosis to inhibit lung cancer metastasis.

New insights into zinc alleviating renal toxicity of arsenic-exposed carp (Cyprinus carpio) through YAP-TFR/ROS signaling pathway

Environmental pollution caused by arsenic or its compounds is called arsenic pollution. Arsenic pollution mainly comes from people mining and smelting arsenic compounds. In addition, arsenic compounds' widespread use and production of arsenic-containing pesticides, arsenic-rich water used to irrigate farms, or high arsenic levels in foods caused by coal burning are all sources of arsenic contamination. Arsenic contamination poses a significant threat to global public health. It is reported that exposure to arsenic can induce severe renal injury. However, the underlying mechanism needs to be clarified. In this study, the arsenic exposure model in vivo and in vitro was used to explore the mechanism of arsenic-induced renal injury, especially the role of ferroptosis and its regulatory mechanism, and then to evaluate its anti-pollution effect by supplementing zinc. The results showed that arsenic significantly induced ferroptosis, characterized by up-regulating the expression of YAP and TFR in kidney and CIK cells and then increasing the levels of Fe2+ and ROS, lipid peroxidation, and iron metabolism. Microscopic observation revealed the shrinkage of mitochondria and the increase in membrane density. In addition, molecular docking and inhibitor experiments further confirmed that arsenic is involved in the process of ferroptosis by activating YAP and TFR. These results clarify the harmful effects of arsenic on carp kidneys and its mechanism and highlight the critical interactions between the YAP-TFR pathway, ROS, and ferroptosis. Importantly, this study found that zinc can reduce ferroptosis caused by the arsenic-activated YAP-TFR pathway by inhibiting YAP activation and lipid peroxidation.

Anoikis-related genes in breast cancer patients: reliable biomarker of prognosis

BackgroundBreast cancer (BC) is the most common cancer in women, and its progression is closely related to the phenomenon of anoikis. Anoikis, the specific programmed death resulting from a lack of contact between cells and the extracellular matrix, has recently been recognized as playing a critical role in tumor initiation, maintenance, and treatment. The ability of cancer cells to resist anoikis leads to cancer progression and metastatic colonization. However, the impact of anoikis on the prognosis of BC patients remains unclear.MethodThis study utilized data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to collect transcriptome and clinical data of BC patients. Anoikis-related genes (ARGs) were classified into subtypes A and B through consensus clustering. Subsequently, survival prognosis analysis, immune cell infiltration analysis, and functional enrichment analysis were performed for both subtypes. Using the Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis, a set of 10 ARGs related to prognosis was identified. Immune cell infiltration and tumor microenvironment analyses were conducted on these 10 ARGs to develop a prognostic model. Furthermore, single-cell data analysis and real-time polymerase chain reaction (RT-PCR) analysis were employed to study the expression of the 10 identified prognostic ARGs in BC cells.ResultsOne hundred thirty-five ARGs were identified as differentially expressed genes in the TCGA and GEO databases, with 42 of them associated with the survival prognosis of BC patients. Analyses involving Principal Component Analysis (PCA), t-Distributed Stochastic Neighbor Embedding (t-SNE), and Uniform Manifold Approximation and Projection (UMAP) revealed distinct expression patterns of ARGs between types A and B. Patients in type A exhibited worse survival prognosis and lower immune cell infiltration compared to type B. Subsequent analyses identified 10 key ARGs (YAP1, PIK3R1, BAK1, PHLDA2, EDA2R, LAMB3, CD24, SLC2A1, CDC25C, and SLC39A6) relevant to BC prognosis. Kaplan–Meier analysis indicated that high-risk patients based on these ARGs had a poorer BC prognosis. Additionally, Cox regression analysis established gender, age, T (tumor), N (nodes), and risk score as predictive factors in a nomogram model for BC. The model demonstrated diagnostic value for BC patients at 1, 3, and 5 years. Decision curve analysis (DCA) verified the risk score as a reliable predictor of BC patient survival rates. Moreover, RT-PCR results confirmed differential expressions of YAP1, PIK3R1, BAK1, PHLDA2, CD24, SLC2A1, and CDC25C in BC cells, with SLC39A6, EDA2R, and LAMB3 showing low expression levels.ConclusionARGs markers can be used as BC biomarkers for risk stratification and survival prediction in BC patients. Besides, ARGs can be used as stratification factors for individualized and precise treatment of BC patients.

Poison Turned Panacea: Arsenic Trioxide Loaded Hydrogel for Inhibiting Scar Formation in Wound Healing.

Without intervention, the natural wound healing process can often result in scarring, which can have detrimental effects on both the physical and mental well-being of patients. Therefore, it is crucial to develop biomaterials that can promote healing without scarring. Regulating the Yes-associated protein-1/PDZ-binding motif (YAP/TAZ) signaling pathway is possible to reduce excessive fibrosis of fibroblasts and proliferation of vascular endothelial cells, ultimately impacting scar formation. Arsenic trioxide (ATO), an ancient drug with medicinal and toxic properties, has shown promise in regulating this pathway. An ATO-loaded hydrogel dressing (ATO@CS/SA) was created to facilitate scarless wound healing, utilizing chitosan (CS) and sodium alginate (SA) to prevent direct contact of ATO with the wound tissue and minimize potential side effects. In vitro studies demonstrated that low concentrations of ATO did not impact cell viability and even promoted proliferation and migration. Co-culturing the hydrogel with fibroblasts and vascular endothelial cells led to decreased expression levels of YAP and TAZ. Animal studies over a 90-day period revealed significant inhibition of scar formation with this system. Histological experiments further confirmed that the decreased expression of YAP and TAZ was responsible for this outcome. In conclusion, when administered at the appropriate dose, ATO can be repurposed from a traditional poison to a therapeutic agent, effectively suppressing excessive cell fibrosis and blood vessel proliferation and offering a novel approach to scar-free treatment.

SOX9 promotes hypoxic pulmonary hypertension through stabilization of DPP4 in pulmonary artery smooth muscle cells

Pulmonary hypertension (PH) is a progressive cardiopulmonary disorder characterized by pulmonary vascular remodeling (PVR), primarily due to the excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). This study aimed to investigate the role and molecular mechanism of SOX9 in hypoxic PH in rats. The findings revealed that SOX9 was upregulated in the pulmonary arteries and PASMCs of hypoxia-exposed rats. SOX9 knockdown inhibited hypoxia-induced proliferation and migration of PASMCs, reduced PVR, and subsequently alleviated hypoxia-induced PH in rats, suggesting that SOX9 plays a critical role in PH. Further investigation demonstrated that SOX9 interacted with DPP4, preventing its ubiquitin degradation in hypoxia-exposed PASMCs. DPP4 knockdown inhibited hypoxia-induced PASMC proliferation and migration, and administration of the DPP4 inhibitor sitagliptin (5 mg/kg) significantly reduced PVR and alleviated hypoxia-induced PH in rats, indicating that SOX9 contributes to PH by stabilizing DPP4. The results also showed that hypoxia induced YAP1 expression and dephosphorylation, leading to YAP1 nuclear localization. YAP1 knockdown promoted the degradation of HIF-1α in hypoxia-exposed PASMCs and inhibited hypoxia-induced proliferation and migration of PASMCs. Additionally, HIF-1α, as a transcription factor, promoted SOX9 expression by binding to the SOX9 promoter in hypoxia-exposed PASMCs. In conclusion, hypoxia promotes the proliferation and migration of PASMCs through the regulation of the YAP1/HIF-1α/SOX9/DPP4 signaling pathway, leading to PH in rats. These findings suggest that SOX9 may serve as a potential prognostic marker and therapeutic target for PH.