Hippo Signaling Research Articles

Pan-Transcriptional Enhanced Associated Domain Palmitoylation Pocket Covalent Inhibitor.

In the Hippo signaling pathway, the palmitoylated transcriptional enhanced associated domain (TEAD) protein interacts with the coactivator Yes-associated protein/PDZ-binding motif, leading to transcriptional upregulation of oncogenes such as Ctgf and Cyr61. Consequently, targeting the palmitoylation sites of TEAD has emerged as a promising strategy for treating TEAD-dependent cancers. Compound 1 was identified using a structure-based drug design approach, leveraging the molecular insights gained from the known TEAD palmitoylation site inhibitor, K-975. Optimization of the initial hit compound resulted in the development of compound 3, a covalent pan-TEAD inhibitor characterized by high potency and oral bioavailability.

Dysregulation of R-loop homeostasis shapes the immunosuppressive microenvironment and induces malignant progression in melanoma.

Dysregulated R-loop homeostasis leads to DNA replication stress and genomic instability, a major driver of cancer. However, the role of R-loops in melanoma development remains unclear. We established an R-loop scoring model based on a single-cell RNA sequencing dataset and evaluated the association between the R-loop score with the melanoma immune microenvironment and treatment response. We explored the role of CENPA-mediated changes in R-loop distribution during melanoma progression by DNA/RNA immunoprecipitation and sequencing and a series of functional experiments. We found that malignant cells with high R-loop scores may be involved in melanoma progression by modulating immune evasion, metabolic reprogramming, and cancer-related pathways. A cell communication analysis revealed that high-score R-loops play an important role in altering cell-cell interactions and limiting the CD8 + cytotoxic T cell response and T cell accumulation. CENPA silencing induced changes in R-loop distribution, upregulated Hippo signaling activity, and inhibited tumor cell proliferation and migration. Moreover, the R-loop score can predict the prognosis and immunotherapy effect of melanoma patients. Our work reveals the potential molecular mechanism by which abnormal R-loops promote melanoma progression, which may help develop anticancer therapies based on R-loops or R-loop regulators.

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2

Identification and validation of circulating microRNAs as biomarkers for heart failure with preserved ejection fraction

Abstract Background Heart failure (HF) is a syndrome due to cardiac insufficiency, manifested by excessive volume overload, increased sympathetic activity, and circulatory redistribution [1]. Heart failure with preserved ejection fraction (HFpEF) accounts for approximately 40%-50% of the total HF events, and its development is often accompanied by various complications, including hypertension, obesity, and metabolic syndrome. Biomarker associations in heart failure with reduced ejection fraction were mostly cardiac stretch based, while many biomarkers of HFpEF are also associated with inflammation [2]. To date, treatments that effectively improve the clinical prognosis of HFpEF are relatively limited, thus HFpEF remains one of the challenges that needs to be further explored. A full understanding of the molecular mechanisms of HFpEF is essential to improve the early diagnosis, treatment and prognosis of these patients. Purpose The study aims to explore key circulating miRNAs and related molecular pathways in HFpEF patients through bioinformatics analysis and machine learning, and to further validate them using a double-hit HFpEF mouse model. Methods We downloaded HF datasets including circulating miRNA expression profiles from the GEO database. LASSO regression, SVM-RFE, Random Forest and XGboost algorithm were conducted to identify key miRNAs for diagnostic model construction. We used R software, GO, KEGG and Cytoscape to better understand the functions of key miRNA targets. Besides, the mRNA-miRNA interaction networks were predicted by starbase. We also constructed a HFpEF mouse model subjected to high fat diet and L-NAME (0.5 g/L) in drinking water to discover the correlation between the screened circulating miRNA and HFpEF. Results After differential expression analysis of the combined HFpEF dataset, we identified 37 differentially circulating miRNAs, of which 15 were down-regulated and 22 were up-regulated. Subsequently, we screened by machine learning and identified 4 key miRNAs that may be involved in the progression of HFpEF, including hsa-miR-645, hsa-miR-296-5p, hsa-miR-199b-5p, and hsa-miR-509-3-5p. We validated these miRNAs based on their expression, and the ROC plots suggest that these miRNAs may have diagnostic capability in HFpEF. The results of GO and KEGG enrichment analyses showed that the target genes of the differentiated miRNAs were mainly enriched in the MAPK signaling pathway, mTOR signaling pathway, protein processing in endoplasmic reticulum, hippo signaling pathway, cell cycle, Foxo signaling pathway, hypertrophic cardiomyopathy, dilated cardiomyopathy, and circadian rhythm. The mRNA-miRNA interaction networks suggested that TMEM135 was regulated by both has-miR-199b-5p and hsa-miR-296-5p. The RT-PCR results also suggested that miR-199b-5p was upregulated in the HFpEF mice. Conclusion We validated 4 circulating miRNAs with strong correlation to HFpEF, which provide a more reliable basis for pre-symptomatic diagnosis.

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.

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.

Stromal softness confines pancreatic cancer growth through lysosomal-cathepsin mediated YAP1 degradation

The progression and malignancy of many tumors are associated with increased tissue stiffness. Conversely, the oncogenically transformed cells can be confined in soft stroma. Yet, the underlying mechanisms by which soft matrix confines tumorigenesis and metastasis remain elusive. Here, we show that pancreatic cancer cells are suppressed in the soft extracellular matrix, which is associated with YAP1 degradation through autophagic-lysosomal pathway rather than Hippo signal mediated proteasome pathway. In the soft stroma, PTEN is upregulated and activated, which consequently promotes lysosomal biogenesis, leading to the activation of cysteine-cathepsins for YAP1 degradation. In vitro, purified cathepsin L can directly digest YAP1 under acidic conditions. Lysosomal stress, either caused by chloroquine or overexpression of cystatin A/B, results in YAP1 accumulation and malignant transformation. Likewise, liver fibrosis induced stiffness can promote malignant potential in mice. Clinical data show that down-regulation of lysosomal biogenesis is associated with pancreatic fibrosis and stiffness, YAP1 accumulation, and poor prognosis in PDAC patients. Together, our findings suggest that soft stroma triggers lysosomal flux-mediated YAP1 degradation and induces cancer cell dormancy.Graphical

Goat Milk Protein-Derived ACE Inhibitory Peptide SLPQ Exerts Hypertension Alleviation Effects Partially by Regulating the Inflammatory Stress of Endothelial Cells

Hypertension has always posed a severe threat to people’s health. Food-derived angiotensin-converting enzyme (ACE)-inhibitory peptides have the potential to both prevent and treat hypertension. In the current investigation, two ACE-inhibitory peptides (SLPQ and PYVRYL) from goat milk were studied for their endothelial effects using EA.hy926 cells. PYVRYL outperformed SLPQ, yet neither impacted cell survival below 200 μg/mL. Investigation of SLPQ’s impact on EA.hy926 cell expression revealed 114 differentially expressed genes, with 65 downregulated and 49 upregulated. The genes were enriched in cytokine interactions, coagulation cascades, Hippo signaling, and ECM–receptor interaction. Decreased c-x-c motif chemokine ligand 2 (CXCL2), integrin subunit beta 2 (ITGB2), and fbj murine osteosarcoma viral oncogene homologue (FOS) expression and increased secreted phosphoprotein 1 (SPP1) expression may protect endothelial cells from inflammation. Our findings suggest that beyond ACE inhibition, SLPQ aids blood pressure control by influencing endothelial function, paving the way for its use as an antihypertensive food ingredient.

Adipose-derived mesenchymal stem cells inhibit hepatic stellate cells activation to alleviate liver fibrosis via Hippo pathway

BackgroundLiver fibrosis is a common pathological process of chronic liver disease, characterized by excessive deposition of extracellular matrix (ECM). Mesenchymal stem cells (MSCs) have been found to have potential therapy effect on liver fibrosis, but the mechanism involved was still unclear. The objective of this study is to investigate the therapeutic efficacy of adipose-derived mesenchymal stem cells (ADMSCs) on the treatment of liver fibrosis, with particular emphasis on elucidating the underlying mechanism of action through which ADMSCs inhibit the activation of hepatic stellate cells (HSCs).MethodsADMSCs were isolated from adipose tissue and injected intravenously into hepatic fibrosis model of rats. The histopathological changes, liver function, collagen deposition, the expression of fibroin and Hippo pathway were evaluated. In vitro, ADMSCs were co-cultured with HSCs activated by transforming growth factor beta 1 (TGF-β1), and the inhibitor of Hippo pathway was used to evaluate the therapeutic mechanism of ADMSCs transplantation.ResultsThe results showed that after the transplantation of ADMSCs, the liver function of rats was improved, the degree of liver fibrosis and collagen deposition were reduced, and the Hippo signaling pathway was activated. In vitro, ADMSCs can effectively inhibit the proliferation and activation of HSCs induced by TGF-β1 treatment. However, the inhibitory effect of ADMSCs was weakened after blocking the Hippo signaling pathway.ConclusionsADMSCs inhibit HSCs activation by regulating YAP/TAZ, thereby promoting functional recovery after liver fibrosis. These findings lay a foundation for further investigation into the precise mechanism by which ADMSCs alleviate liver fibrosis.

Dan'e fukang decoction reduces hemorrhage in a rat model of mifepristone induced incomplete abortion and may correlate with cell adhesion molecule signaling interference

Ethnopharmacological relevanceDan'e fukang decoction(DFD) is a traditional Chinese medicine formula. DFD obtains 10 herbs, including Salvia yunnanensis C.H.Wright (Zidanshen) , Curcuma zedoaria (Christm.) Roscoe (Ezhu), Angelica sinensis (Oliv.) Diels (Danggui), Cyperus rotundus L. (Xiangfuzi), Corydalis yanhusuo (Y.H.Chou & Chun C.Hsu) W.T.Wang ex Z.Y.Su & C.Y.Wu, Bupleurum marginatum Wall. ex DC. (Yanhusuo), Sparganium stoloniferum (Buch.-Ham. ex Graebn.) Buch.-Ham. ex Juz. (Sanleng), Panax notoginseng (Burkill) F.H.Chen (Sanqi), Paeonia lactiflora Pall. (Shaoyao) and Glycyrrhiza uralensis Fisch. (Gancao). DFD is now clinically used for the treatment of menstrual irregularities, dysmenorrhea and menstrual discomfort caused by blood stasis and easing of endometriosis. Based on this, it is reasonable to presume that DFD may be effective in treating incomplete abortion and reducing postpartum bleeding, but no specific studies have been reported so far. Aim of the studyTo investigate the efficacy of Dan'e fukang decoction (DFD) in reducing prolonged vaginal bleeding followed by mifepristone induced incomplete abortion and explore the mechanisms of action of DFD in treating incomplete abortion. MethodsAn incomplete abortion model of rat was established by single intragastrically administered 8.5 mg/kg mifepristone on the 7th day of pregnancy. From the 8th day of pregnancy, the abortive rats were administered solvent, a positive control drug, or different doses of DFD, respectively for seven consecutive days. The efficacy of DFD was assessed by measuring the vaginal bleeding volume of the rats. Four coagulation parameters and platelet counts were measured. Hematoxylin and eosin (HE) staining was performed to evaluate pathological changes in the uterine embryos. Serum levels of progesterone and estrogen were measured using ELISA. Network pharmacology and transcriptomics were used to predict potential targets and pathways for DFD to reduce hemorrhage. The levels of mRNA related to cell adhesion molecules (CAMs) were detected by RT-qPCR. The levels of progesterone and estrogen receptors and the proteins associated with CAMs pathway in uterine tissues were detected by Western Blot. ResultsDFD significantly reduced the volume of vaginal bleeding of the abortive rats and significantly downgraded the pathological scores of uterine embryos. DFD significantly increased serum levels of E2, and had no impact on serum levels of P4 and the protein expression of ER and PR in the uteri of the abortive rats. Pathways in cancer, lipid, focal adhesion and immune-related signaling were predicted to be influenced by DFD via the analysis of network pharmacology. The CAMs signaling was found the most critical pathway regulated by both mifepristone and DFD via RNA-seq assay, followed by axon guidance, basal cell carcinoma, hippo signaling pathway and neuroactive ligand-receptor interaction. Combining the two analytical methods, ICAM-1 was predicted likely the key targeted gene by DFD. Finally, DFD was validated to decrease the protein expression of ICAM-1, ITGB2, ITGB7 and RASSF5 in the uterine tissues, which correlated to suppress the CAMs signaling pathway. ConclusionDFD significantly reduced hemorrhage. DFD significantly increased the serum levels of E2 and inhibited CAMs signaling pathway, which was likely to be involved in the mechanism of action of DFD facilitating residual uterine embryo expulsion in the rat model of incomplete abortion.

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

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

Hippo signaling cooperates with p53 to regulate lung airway mucous cell metaplasia.

Airway mucous cell metaplasia is a significant feature of many chronic airway diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma. However, the mechanisms underlying this process remain poorly understood. Here, we employ in vivo mouse genetic models to demonstrate that Hippo and p53 cooperate to modulate the differentiation of club cells into goblet cells. We reveal that ablation of Mst1 and Mst1 (Mst1/2), the core components of Hippo signaling, significantly reduces mucous metaplasia in the lung airways in a lipopolysaccharide (LPS)-induced lung inflammation murine model while promoting club cell proliferation in a Yap-dependent manner. Additionally, we show that deleting Mst1/2 is sufficient to suppress p53 deficiency-mediated goblet cell metaplasia. Finally, single-cell RNA analysis reveals a downregulation of Yap and p53 signaling in goblet cells in the human airways. These findings underscore the important role of Hippo and p53 signaling in regulating airway mucous metaplasia.

Brain-Region-Specific Differences in Protein Citrullination/Deimination in a Pre-Motor Parkinson's Disease Rat Model.

The detection of early molecular mechanisms and potential biomarkers in Parkinson's disease (PD) remains a challenge. Recent research has pointed to novel roles for post-translational citrullination/deimination caused by peptidylarginine deiminases (PADs), a family of calcium-activated enzymes, in the early stages of the disease. The current study assessed brain-region-specific citrullinated protein targets and their associated protein-protein interaction networks alongside PAD isozymes in the 6-hydroxydopamine (6-OHDA) induced rat model of pre-motor PD. Six brain regions (cortex, hippocampus, striatum, midbrain, cerebellum and olfactory bulb) were compared between controls/shams and the pre-motor PD model. For all brain regions, there was a significant difference in citrullinated protein IDs between the PD model and the controls. Citrullinated protein hits were most abundant in cortex and hippocampus, followed by cerebellum, midbrain, olfactory bulb and striatum. Citrullinome-associated pathway enrichment analysis showed correspondingly considerable differences between the six brain regions; some were overlapping for controls and PD, some were identified for the PD model only, and some were identified in control brains only. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified in PD brains only were associated with neurological, metabolic, immune and hormonal functions and included the following: "Axon guidance"; "Spinocerebellar ataxia"; "Hippo signalling pathway"; "NOD-like receptor signalling pathway"; "Phosphatidylinositol signalling system"; "Rap1 signalling pathway"; "Platelet activation"; "Yersinia infection"; "Fc gamma R-mediated phagocytosis"; "Human cytomegalovirus infection"; "Inositol phosphate metabolism"; "Thyroid hormone signalling pathway"; "Progesterone-mediated oocyte maturation"; "Oocyte meiosis"; and "Choline metabolism in cancer". Some brain-region-specific differences were furthermore observed for the five PAD isozymes (PADs 1, 2, 3, 4 and 6), with most changes in PAD 2, 3 and 4 when comparing control and PD brain regions. Our findings indicate that PAD-mediated protein citrullination plays roles in metabolic, immune, cell signalling and neurodegenerative disease-related pathways across brain regions in early pre-motor stages of PD, highlighting PADs as targets for future therapeutic avenues.

YAP Overcomes Mechanical Barriers to Induce Mitotic Rounding and Adult Cardiomyocyte Division.

Many specialized cells in adult organs acquire a state of cell cycle arrest and quiescence through unknown mechanisms. Our limited understanding of mammalian cell cycle arrest is derived primarily from cell culture models. Adult mammalian cardiomyocytes, a classic example of cell cycle arrested cells, exit the cell cycle postnatally and remain in an arrested state for the life of the organism. Cardiomyocytes can be induced to re-enter the cell cycle by YAP5SA, an active form of the Hippo signaling pathway effector YAP. We performed clonal analyses to determine the cell kinetics of YAP5SA cardiomyocytes. We also performed single-cell RNA sequencing, marker gene analysis, and functional studies to examine how YAP5SA cardiomyocytes progress through the cell cycle. We discovered that YAP5SA-expressing cardiomyocytes divided efficiently, with >20% of YAP5SA cardiomyocyte clones containing ≥2 cardiomyocytes. YAP5SA cardiomyocytes re-entered cell cycle at the G1/S transition and had an S phase lasting ≈48 hours. Sarcomere disassembly is required for cardiomyocyte progression from S to G2 phase and the induction of mitotic rounding. Although oscillatory Cdk expression was induced in YAP5SA cardiomyocytes, these cells inefficiently progressed through G2 phase. This is improved by inhibiting P21 function, implicating checkpoint activity as an additional barrier to YAP5SA-induced cardiomyocyte division. Our data reveal that YAP5SA overcomes the mechanically constrained myocardial microenvironment to induce mitotic rounding with cardiomyocyte division, thus providing new insights into the in vivo mechanisms that maintain cell cycle quiescence in adult mammals.

Global A-to-I RNA editing during myogenic differentiation of goat MuSCs.

RNA editing, especially A-to-I editing sites, is a common RNA modification critical for stem cell differentiation, muscle development, and disease occurrence. Unveiling comprehensive RNA A-to-I editing events associated with myogenesis of the skeletal muscle satellite cells (MuSCs) is essential for extending our knowledge of the mechanism underpinning muscle development. A total of 9,632 RNA editing sites (RESs) were screened in the myoblasts (GM), myocytes (DM1), and myotubes (DM5) samples. Among these sites, 4,559 A-to-I edits were classified and further analyzed. There were 3,266 A-to-I sites in the protein-coding region, out of which 113 missense sites recoded protein. Notably, five A-to-I sites in the 3' UTR of four genes (TRAF6, NALF1, SLC38A1, ENSCHIG00000019092) altered their targeted miRNAs. Furthermore, a total of 370 A-to-I sites with different editing levels were detected, including FBN1, MYH10, GSK3B, CSNK1D, and PRKACB genes. These genes were predominantly enriched in the cytoskeleton in muscle cells, the hippo signaling pathway, and the tight junction. Furthermore, we identified 14 hub genes (TUFM, GSK3B, JAK2, RPSA, YARS1, CDH2, PRKACB, RUNX1, NOTCH2, CDC23, VCP, FBN1, RARS1, MEF2C) that potentially related to muscle development. Additionally, 123 stage-specific A-to-I editing sites were identified, with 43 sites in GM, 25 in DM1, and 55 in DM5 samples. These stage-specific edited genes significantly enriched essential biological pathways, including the cell cycle, oocyte meiosis, motor proteins, and hedgehog signaling pathway. We systematically identified the RNA editing events in proliferating and differentiating goat MuSCs, which was crucial for expanding our understanding of the regulatory mechanisms of muscle development.

Substrate stiffness-dependent activation of Hippo pathway in cancer associated fibroblasts

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex three-dimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer.

The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show can regress established tumors in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.

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

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.