YAP Expression Research Articles

Abstract PR06: Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution

Abstract Human papillomavirus-positive head and neck squamous cell carcinoma (HPV+HNSC) constitutes the most common HPV-driven malignancy in the United States, and yet the molecular and cellular mechanisms underlying carcinogenesis in HPV+HNSC remain poorly understood. The decades long latency period and incomplete penetrance linking oral HPV infection to HPV+HNSC suggest that oncogenic events beyond E6-E7 expression are required for carcinogenesis. YAP is a transcriptional co-activator that coordinates cell stemness, growth, proliferation, and survival. We previously identified alterations resulting in YAP activation as a potential oncogenic event in HNSC. We thus hypothesized YAP activation in the context of E6-E7 expression may represent a key tumor initiating event leading to HPV+HNSC. We developed a genetically engineered murine system to investigate the effects of local induction of YAP activation and E6-E7-expression in Keratin14+ (K14+) oral epithelial progenitor cells (OEPCs). In this system, intralingual administration of tamoxifen and dietary administration of doxycycline enables spatiotemporally controlled expression of E6-E7 and/or YAP. Employing this model, we identify combined E6-E7 and YAP activation (E+Y) as sufficient to induce highly penetrant carcinoma formation as early as 10 days after transgene induction. Immunofluorescent analyses showed E+Y resulted in the ectopic expansion of a mitotically active (Ki67+), stem-like (TP63+, SOX2+) epithelial cell population with invasive potential. Bulk RNAseq of microdissected tongue epithelia showed that E+Y uniquely induced transcriptional programs promoting cell cycle progression, epithelial cell plasticity, and acute inflammatory response, which were not observed upon sole activation of either the E6-E7 or YAP transgene. scRNAseq of transgene-induced tongue epithelial cell suspensions identified 8 distinct clusters, including previously defined physiologic oral epithelial cell states and novel transgene-associated clusters. E+Y resulted in the emergence of a unique epithelial cell cluster with transcriptional characteristics consistent with a cancer stem cell population, with enrichment in YAP, mTORC1, E2F, and MYC-driven programs, and pan-cancer cell states including cell cycle, hypoxia, squamous differentiation, and partial EMT. Implantation of E+Y cells resulted in tumor formation in 100% of NSG mice, indicating that E+Y drives the emergence of a cancer stem cell population. We next defined transcriptionally co-expressed modules driving E+Y tumor initiating capacity. Among TCGA-HNSC subjects, 8 of 12 modules were enriched in tumor compared to matched normal tissue. Interestingly, 3 modules (G1/S, cell migration, and focal adhesion) were associated with overall and disease-free survival. Our findings show YAP activation in the context of E6-E7 expression is sufficient for rapid carcinogenesis. We illuminate cellular processes driving malignant conversion at single cell resolution, and show enrichment of these processes in human HNSC exhibiting poor prognosis. Citation Format: Farhoud Faraji, Sydney I. Ramirez, Lauren M. Clubb, Kuniaki Sato, Paola Y. Anguiano Quiroz, William M. G. Galloway, Thomas S. Hoang, Kate Medetgul-Ernar, Pauline Marangoni, Alfredo A. Molinolo, Joseph A. Califano, Quinton Smith, Ophir D. Klein, Pablo Tamayo, J. Silvio Gutkind. Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR06.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

NRP1 transduces mechanical stress inhibition via LATS1/YAP in hypertrophic scars

Hypertrophic scar (HS) is an abnormal fibrous hyperplasia of the skin caused by excessive tissue repair in response to skin burns and trauma, which restricts physical function and impairs patients’ quality of life. Numerous studies have shown that pressure garment therapy (PGT) is an effective treatment for preventing hypertrophic scars. Herein, we found that mechanical stress stimulates the neuropilin 1 (NRP1) expression through screening GSE165027, GSE137210, and GSE120194 from Gene Expression Omnibus (GEO) database and bioinformatics analysis. We verified this stimulation in the human hypertrophic scar, pressure culture cell model, and rat tail-scar model. Mechanical compression increased LATS1 and pYAP enrichment, thus repressing the expression of YAP. Functionally, the knockdown of NRP1 promoted the expression of LATS1, thus decreasing the expression of YAP and inhibiting endothelial cell proliferation. Furthermore, co-immunoprecipitation analysis confirmed that NRP1 binds to YAP, and mechanical compression disrupted this binding, which resulted in the promotion of YAP relocation to nuclear. In conclusion, our results indicated that NRP1 transduces mechanical force inhibition by inhibiting YAP expression. Mechanical pressure can release YAP bound to NRP1, which explains the phenomenon that mechanical stress increases YAP in the nucleus. Strategies targeting NRP1 may promote compression therapy with optimal and comfortable pressures.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Glucose dysregulation promotes oncogenesis in human bladder cancer by regulating autophagy and YAP1/TAZ expression.

Glucose dysregulation is strongly correlated with cancer development, and cancer is prevalent in patients with Type 2 diabetes (T2D). We aimed to elucidate the mechanism underlying autophagy in response to glucose dysregulation in human bladder cancer (BC). 220 BC patients were included in this retrospective study. The expression of YAP1, TAZ and AMPK, EMT-associated markers, and autophagy marker proteins was analysed by immunohistochemistry, western blotting, and quantitative real-time PCR (qPCR). Further, T24 and UMUC-3 BC cells were cultured in media with different glucose concentrations, and the expression of YAP1, TAZ, AMPK and EMT-associated markers, and autophagy marker proteins was analysed by western blotting and qPCR. Autophagy was observed by immunofluorescence and electron microscopy. BC cell viability was tested using MTT assays. A xenograft nude mouse model of diabetes was used to evaluate tumour growth, metastasis and survival. A poorer pathologic grade and tumour-node-metastasis stage were observed in patients with BC with comorbid T2D than in others with BC. YAP1 and TAZ were upregulated in BC samples from patients with T2D. Mechanistically, high glucose (HG) promoted BC progression both in vitro and in vivo and inhibited autophagy. Specifically, various autophagy marker proteins and AMPK were negatively regulated under HG conditions and correlated with YAP1 and TAZ expression. These results demonstrate that HG inhibits autophagy and promotes cancer development in BC. YAP1/TAZ/AMPK signalling plays a crucial role in regulating glucose dysregulation during autophagy. Targeting these effectors exhibits therapeutic significance and can serve as prognostic markers in BC patients with T2D.

YAP Activation Is Associated with a Worse Prognosis of Poorly Cohesive Gastric Cancer.

Poorly cohesive (PC) gastric cancer (GC) is extremely aggressive in progression, and there is an urgent need to identify the molecular pathways involved. We hypothesized the essential role of the RhoA-YAP axis in these mechanisms. The present observational multicenter retrospective study included 133 patients with PC GC treated at two dedicated European surgical centers between 2004 and 2014. YAP nuclear localization was measured by immunohistochemical (IHC) analysis of tissue biopsies. The complete absence of nuclear reactivity was coded as negative expression; we considered "any positive" as low nuclear expression (>0% but <10% of cells) and high nuclear expression (≥10% of cells). Women represented about half of the present series (52%), and the median age was 64 years (p25-p75 range: 53-75). Neoadjuvant and adjuvant treatments were administered to 10% and 54% of the cases, respectively. Extended systemic lymphadenectomy (D2) was the most common (54%). In nearly all cases, the number of retrieved nodes was ≥15, i.e., adequate for tumor staging (94%). An R0 resection was achieved in 80% of the cases. Most patients were pathological T stage 3 and 4 (pT3/pT4 = 79.0%) and pathological N stage 2, 3a, and 3b (pN2/pN3a/pN3b = 47.0%) at the pathological examination. Twenty patients (15%) presented distant metastases. Five-year overall survival (OS) was significantly higher (p = 0.029) in patients with negative YAP (46%, 95% CI 31.1-60.0%) than in the other patients (27%, 17.5-38.1%). Moreover, when controlling for sex, age, pT, pN, and percentage of signet ring cells in the multivariable analysis, YAP expression was a significant predictor of OS (HR 2.03, 95% CI: 1.18-3.51, p = 0.011). Our results provide new insights into the role of the YAP signaling cascade, as its activation was associated with a worse prognosis in PC GC.

Molecular subtype expression and genomic profiling differ between surgically resected pure and combined small cell lung carcinoma

A new molecular subtype classification method has been proposed for small cell lung carcinoma (SCLC). However, little is known about the differences between the pure (P-SCLC) and combined subtypes (C-SCLC). We aimed to compare the molecular subtype expression and genomic profiling in terms of clinical relevance between the two groups. 154 surgically resected SCLCs were analyzed for protein expression of four subtypes (ASCL1, NEUROD1, POU2F3, and YAP1) and two predictive markers (DLL3 and MYC) by immunohistochemistry (IHC). We also performed whole exome sequencing of 60 samples to examine genomic profiles. A total of 113 patients with P-SCLC and 41 with C-SCLC were included. In P-SCLC and C-SCLC, the expression of these markers was 78.8% and 41.5%, 98.2% and 97.6%, 42.5% and 51.2%, 38.9% and 85.4%, 85.0% and 68.3%, and 24.8% and 34.1%, respectively. ASCL1 and DLL3 were highly expressed in P-SCLC (p=0.000 and p=0.021, respectively), and YAP1 expression was significantly enriched in C-SCLC (p=0.000). NGS results, including 45P-SCLCs and 15C-SCLCs, indicated that EGFR gene mutations were mostly observed in C-SCLCs (p=0.000). C-SCLC showed higher CNA burden and wGII than P-SCLC (p<0.01 and p<0.05); conversely, P-SCLC had higher TMB burden and SDI (p<0.05 and p<0.05). YAP1 expression was associated with poor prognosis in P-SCLC but with favorable prognosis in C-SCLC. P-SCLC and C-SCLC are heterogeneous diseases characterized by different molecular subtype expressions and genomic profiles. Our data provide a basis for adopting histological subtype-based treatments, and further prospective studies are required to confirm our conclusions.

Modeling the Maturation of the Vocal Fold Lamina Propria Using a Bioorthogonally Tunable Hydrogel Platform.

Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) are encapsulated in hyaluronic acid (HA)-based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion-controlled interfacial bioorthogonal crosslinking is carried out on the soft cellular construct using HA modified with a ferocious dienophile, trans-cyclooctene (TCO). Cultures are maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restrictscell spreading. Compared to the nLP controls, sLP7 conditions upregulate the expression of fibrous matrix proteins (Col I, DCN, and FN EDA), classic fibroblastic markers (TNC, FAP, and FSP1), and matrix remodeling enzymes (MMP2, TIMP1, and HAS3). Day 7 stiffening also upregulates the catabolic activities, enhances ECM turnover, and promotes YAP expression. Overall, in situ delayed matrix stiffening promotes a fibroblast transition from MSCs and enhances YAP-regulated mechanosensing.

CS-NO suppresses inhibits glycolysis and gastric cancer progression through regulating YAP/TAZ signaling pathway.

Gastric cancer (GC) is a significant contributor to global mortality and is recognized for its elevated prevalence and fatality rates. Nitric Oxide (NO) plays a role in multiple aspects of cancer metastasis and progression. CS-NO is a polysaccharide-based biomaterial with NO-releasing properties that shows promising therapeutic potential. Nonetheless, the action mechanism of CS-NO in GC is still largely unclear. The present study employed various experimental techniques, including CCK-8 assay, colony formation assay, EdU staining, and transwell assays, to evaluate the proliferation, migration, and invasion of GC cells. Additionally, ELISA was utilized to measure glucose uptake, lactate production, and cellular ATP levels in GC cells. In vivo investigations on nude mice were conducted to validate the in vitro results. The present study aimed to examine the potential anti-tumor properties of CS-NO on GC through in vitro and in vivo investigations, while also exploring the underlying mechanisms involved. Our data suggested that CS-NO might prevent GC cell invasion and migration. Decreased expressions of GLUT1, HK2, and LDHA further demonstrated that CS-NO significantly suppressed aerobic glycolysis in GC cells. The administration of CS-NO resulted in a significant reduction of YAP and TAZ levels in GC cells. Our data further show that CS-NO treatment could inhibit GC cancer growth in mice, consistent with the significant decrease in Ki67, GLUT1 and YAP expression levels. These findings could reveal the good effects of CS-NO therapy on inhibiting GC.

Deep annotation of long noncoding RNAs by assembling RNA-seq and small RNA-seq data

Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators in various biological processes. However, due to their low expression, their systematic characterization is difficult to determine. Here, we performed transcript annotation by a newly developed computational pipeline, termed RNA-seq and small RNA-seq combined strategy (RSCS), in a wide variety of cellular contexts. Thousands of high-confidence potential novel transcripts were identified by the RSCS, and the reliability of the transcriptome was verified by analysis of transcript structure, base composition, and sequence complexity. Evidenced by the length comparison, the frequency of the core promoter and the polyadenylation signal motifs, and the locations of transcription start and end sites, the transcripts appear to be full length. Furthermore, taking advantage of our strategy, we identified a large number of endogenous retrovirus-associated lncRNAs, and a novel endogenous retrovirus-lncRNA that was functionally involved in control of Yap1 expression and essential for early embryogenesis was identified. In summary, the RSCS can generate a more complete and precise transcriptome, and our findings greatly expanded the transcriptome annotation for the mammalian community.

Biallelic ANKS6 null variants cause notable extrarenal phenotypes in a nephronophthisis patient and lead to hepatobiliary abnormalities by YAP1 deficiency.

The ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) gene, encoding an inversin compartment protein of the primary cilium, was recently reported as a pathogenic gene of nephronophthisis (MIM PS256100). Extrarenal manifestations are frequently observed in this disease, however, potential genotype-phenotype correlations and the underlying mechanisms remain poorly understood. Here we described an infant with kidney failure, hepatobiliary abnormalities, and heart disease, in whom whole exome sequencing identified compound heterozygous variants in ANKS6, including a novel nonsense variant p.Trp458* and a recurrent splicing variant c.2394+1G > A. mRNA expression studies showed that the splicing variant caused aberrant mRNA splicing with exon 13 skipping and the biallelic variants were predicted to cause loss of ANKS6 function. We systematically characterized the clinical and genetic spectra of the disease and revealed that biallelic null variants in ANKS6 cause more severe kidney disease and more extrarenal manifestations, thus establishing a clear genotype-phenotype correlation for the disease. Further evaluations showed that ANKS6 deficiency reduced YAP1 expression in the patient's bile duct epithelium and ANKS6 promotes YAP1 transcriptional activity in a dose-dependent manner, indicating that loss of ANKS6 function causes hepatobiliary abnormalities through YAP1 deficiency during biliary morphogenesis and development, which may offer new therapeutic targets.

Down-regulation of long noncoding RNA HULC inhibits the inflammatory response in ankylosing spondylitis by reducing miR-556-5p-mediated YAP1 expression

ObjectiveAnkylosing spondylitis (AS) is a progressive systemic disease characterized by a chronic inflammatory response in the sacroiliac joints and spine. Long noncoding RNAs suggest significant actions in the progression of AS. Therefore, a specific lncRNA, highly upregulated in liver cancer (HULC), was studied here regarding its functions and related mechanisms in AS.MethodsMeasurements of miR-556-5p, HULC, and YAP1 expression were performed on AS cartilage tissues and chondrocytes. The interaction between miR-556-5p and HULC or YAP1 was verified. CCK-8, flow cytometry and enzyme-linked immunosorbent assay were used to evaluate the effects of HULC, miR-556-5p, and YAP1 on the proliferation, apoptosis, and inflammatory response of AS chondrocytes. Furthermore, the action of HULC/miR-556-5p/YAP1 was experimentally observed in AS mice.ResultsHULC and YAP1 levels were augmented, while miR-556-5p levels were suppressed in AS cartilage tissues and chondrocytes. Downregulating HULC or upregulating miR-556-5p stimulated chondrocyte proliferation and inhibited apoptosis and inflammation in AS. miR-556-5p was a downstream factor of HULC, and YAP1 was a potential target of miR-556-5p. The improvement effect of downregulated HULC on AS chondrocytes was saved when YAP1 expression was forced. In addition, silence of HULC improved the pathological injury of spinal cartilage in AS mice by enhancing miR-556-5p-related regulation of YAP1.ConclusionHULC inhibition relieves the inflammatory response in AS by reducing miR-556-5p-mediated YAP1 expression.

ENO1 promotes liver carcinogenesis through YAP1-dependent arachidonic acid metabolism.

Enolase 1 (ENO1) is a glycolytic enzyme that plays essential roles in various pathological activities including cancer development. However, the mechanisms underlying ENO1-contributed tumorigenesis are not well explained. Here, we uncover that ENO1, as an RNA-binding protein, binds to the cytosine-uracil-guanine-rich elements of YAP1 messenger RNA to promote its translation. ENO1 and YAP1 positively regulate alternative arachidonic acid (AA) metabolism by inverse regulation of PLCB1 and HPGD (15-hydroxyprostaglandin dehydrogenase). The YAP1/PLCB1/HPGD axis-mediated activation of AA metabolism and subsequent accumulation of prostaglandin E2 (PGE2) are responsible for ENO1-mediated cancer progression, which can be retarded by aspirin. Finally, aberrant activation of ENO1/YAP1/PLCB1 and decreased HPGD expression in clinical hepatocellular carcinoma samples indicate a potential correlation between ENO1-regulated AA metabolism and cancer development. These findings underline a new function of ENO1 in regulating AA metabolism and tumorigenesis, suggesting a therapeutic potential for aspirin in patients with liver cancer with aberrant expression of ENO1 or YAP1.

Elastic modulus of hydrogel regulates osteogenic differentiation via liquid-liquid phase separation of YAP.

Craniofacial bone defects induced by congenital malformations, trauma, or diseases frequently challenge the orthodontic or restorative treatment. Stem cell-based bone regenerative approaches emerged as a promising method to resolve bone defects. Microenvironment physical cues, such as the matrix elastic modulus or matrix topography, regulate stem cell differentiation via multiple genes. We constructed gelatin methacryloyl (GelMA), a well-known scaffold, to investigate the impact of elastic modulus on osteogenic differentiation in a three-dimensional environment. Confocal microscope was used to observe and assess the condensates fission and fusion. New bone formation was evaluated by micro-computed tomography at 6 weeks in calvarial defect rat. We found that the light curing increased elastic modulus of GelMA, and the pore size of GelMA decreased. The expression of osteogenic markers was inhibited in hBMSCs cultured in the low-elastic-modulus GelMA. In contrast, the expression of YAP, TAZ and TEAD was increased in the hBMSCs in the low-elastic-modulus GelMA. Furthermore, YAP assembled via liquid-liquid phase separation (LLPS) into condensates that were sensitive to 1'6-hexanediol. YAP recruit TAZ and TEAD4, but not RUNX2 into the condensates. In vivo, we also found that hBMSCs in high-elastic-modulus GelMA was more apt to form new bone. This study provides new insight into the mechanism of osteogenic differentiation. Reagents that can regulate the elastic modulus of substrate or LLPS may be applied to promote bone regeneration.

Significance of YAP1–MAML2 rearrangement and GTF2I mutation in the diagnosis and differential diagnosis of metaplastic thymoma

Background Metaplastic thymoma (MT) is a very uncommon thymoma type, with biphasic differentiation as one of its histological characteristics. This histological pattern, however, can also be mistaken for type A thymoma and the A component in type AB thymoma. Methods Postoperative specimens were collected from five MT and four type A thymomas with a retrospective analysis involving immunohistochemistry, fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Results The histological morphology of the MT overlapped with that of the type A thymoma. With immunostains, the former’s spindle cell components expressed vimentin and EMA, but not CD20. In MT, 3/5 cases had the nuclear expression of YAP1. The spindle cell component of the type A thymoma was found to express CD20. In all five cases of MT, FISH detection revealed YAP1–MAML2 fusion, which was not found in any type A thymoma cases. NGS sequencing confirmed YAP1–MAML2 rearrangement in all five cases of MT, and mutations in POLE and HRAS were also found in two cases, respectively. GTF2I c.74146970 T > A mutations were found in all cases of type A thymoma, and HRAS and NRAS mutations were found in two cases, but no YAP1–MAML2 rearrangement was evident. Conclusions For the diagnosis and differential diagnosis of challenging cases, the YAP1–MAML2 rearrangement and GTF2I mutation were both significant molecular events specific to MT and type A thymoma, respectively.

The circadian clock protein Cryptochrome 1 is a direct target and feedback regulator of the Hippo pathway

Circadian clock controls daily behavior and physiology. The activity of various signaling pathways affects clock gene expression. Here, we show that the core circadian clock gene CRY1 is a direct target of the Hippo pathway effector YAP. YAP binds to TEADs and occupies the proximal promoter regions of CRY1, positively regulating its transcription. Interestingly, we further identified that CRY1 acts in a feedback loop to fine-tune Hippo pathway activation by modulating the expression of YAP and MOB1. Indeed, loss of CRY1 results in enhanced YAP activation. Consistently, we found that YAP levels and activity control clock gene expression and oscillation in synchronized cells. Furthermore, in breast cancer cells, CRY1 downregulation causes YAP/TAZ hyperactivation and enhanced DNA damage. Together, our findings provide a direct mechanistic link between the Hippo pathway and the circadian clock, where CRY1 and Hippo components form an orchestrated signaling network that influences cell growth and circadian rhythm.

JMJD3 and SNAI2 synergistically protect against Parkinson's disease by mediating the YAP/HIF1α signaling pathway in a mouse model.

This study aimed to characterize the functional relevance and mechanistic basis of the histone demethylase JMJD3 in preserving dopaminergic neuron survival in PD. Mice with MPTP-induced lesions and MN9D dopaminergic neuronal cell lines exposed to 6-OHDA, respectively, were used to simulate in vivo and in vitro PD-like environments. PD-related genes with differential expressions were identified using RNA sequencing of hippocampal tissues collected from MPTP-lesioned mice. A specific lentiviral shRNA vector was used to investigate the effects of JMJD3 on neuron activities in vitro and PD-like phenotypes in vivo. JMJD3 was found to up-regulate the expression of SNAI2 through the inhibition of H3K27me3 enrichment in the SNAI2 promoter region. As a result, the viability of 6-OHDA-exposed MN9D cells was stimulated, and cell apoptosis was diminished. Knockdown of SNAI2 decreased the expression of YAP and HIF1α while also reducing the viability of 6-OHDA-exposed MN9D cells and increasing cell apoptosis. The in vivo experiments demonstrated that JMJD3 activated the SNAI2/YAP/HIF1α signaling pathway, inhibiting PD-like phenotypes in MPTP-lesioned mice. Thus, the findings provide evidence that JMJD3 inhibits the enrichment of H3K27me3 at the SNAI2 promoter, leading to the upregulation of SNAI2 expression and activation of the YAP/HIF1α signaling pathway, ultimately exerting a protective effect on PD mice. This finding suggests that targeting the JMJD3-SNAI2 pathway could be a promising therapeutic strategy for Parkinson's disease. Further in-depth studies are needed to elucidate the underlying mechanisms and identify potential downstream targets of this pathway.

UVB promotes melanogenesis by regulating METTL3.

Ultraviolet (UV) radiation is the primary exogenous inducer of skin pigmentation, although the mechanism has not been fully elucidated. N6-methyladenosine (m6 A) modification is one of the key epigenetic form of gene regulation that affects multiple biological processes. The aim of this study was to explore the role and underlying mechanisms of m6 A modification in UVB-induced melanogenesis. Low-dose UVB increased global m6 A modification in melanocytes (MCs) and MNT1 melanoma cell line. The GEPIA database predicted that methyltransferase METTL3 is positively correlated with the melanogenic transcription factor MITF in the sun-exposed skin tissues. After METTL3 respectively overexpressed and knocked down in the MNT1, the melanin content and melanogenesis-related genes were significantly upregulated after overexpression of METTL3, especially with UVB irradiation, and downregulated after METTL3 knockdown. METTL3 levels were also higher in melanocytic nevi with high melanin content. METTL3 overexpression and knockdown also altered the protein level of YAP1. SRAMP analysis predicted four high-potential m6 A modification sites on YAP1 mRNA, of which three were confirmed by methylated RNA immunoprecipitation. Inhibition of YAP1 expression can partially reverse melanogenesis induced by overexpression of METTL3. In conclusion, UVB irradiation promotes global m6 A modification in MCs and upregulates METTL3, which increases the expression level of YAP1 through m6 A modification, thereby activating the co-transcription factor TEAD1 and promoting melanogenesis.

Mechanism of Huatan Sanjie Fang in improving goiter in Graves' disease mice based on the Hippo signaling pathway

To explore the mechanism of Huatan Sanjie Fang (HTSJ) in regulating goiter in Graves’ disease (GD) mice by detecting key factors of the hippo signaling pathway. A mouse model of GD was established by injecting Ad-TSHR289 adenovirus into the bilateral quadriceps femoris of female mice. Successful mouse models were then randomly divided into a model group, methimazole (MMI) group, and HTSJ group, and fed with deionized water, MMI (4.5 mg/kg per day), and HTSJ (35.10 g/kg per day), respectively, for 10 weeks. Histopathological changes of the thyroid gland were subsequently observed by hematoxylin-eosin staining. Radioimmunoassay was used to detect serum total thyroxine (T4) and thyrotrophin-receptor antibody (TRAb) levels. The relative expression of mRNA of Mst1, YAP, and TAZ was detected by quantitative real-time polymerase chain reaction, while the protein expression of Mst1, YAP, TAZ, pMst1, and pYAP was detected by western blot. After 10 weeks of drug intervention, goiter and other pathological changes in the HTSJ group significantly improved compared with the model group, and the levels of serum T4 and TRAb significantly decreased (P = .002, P < .001, respectively). Decreased mRNA expression of Mst1, YAP, and TAZ, the key factors of the hippo signaling transduction pathway, was also observed (P = .002, P = .022, P < .001, respectively). In contrast, protein expression of Mst1 (P = .046), pMst1 (P = .026), and pYAP (P = .004) increased, while protein expression of YAP and TAZ decreased (P = .041, P < .001, respectively). HTSJ can effectively improve goiter in GD mice through the hippo signaling pathway.

P-395 Protective effect of metformin on the ovarian reserve of prepubertal mice under cyclophosphamide chemotherapy

Abstract Study question Can metformin reduce the side effects of cyclophosphamide on the ovaries of prepubertal mice treated with cyclophosphamide? Summary answer Yes, metformin, as an inhibitor of mTOR and, by affecting the HIPPO pathway, can exert its protective effect in the chemotherapy mice model. What is known already PI3K/AKT and HIPPO pathways are influential pathways in the process of activation of primordial follicles and proliferation of cell growth, which are affected by various agents and drugs such as cyclophosphamide. One of the side effects of cyclophosphamide is the reduction of the ovarian reserve through abnormal changes in the PI3K/AKT and HIPPO pathways. Metformin is a drug that is used to treat type 2 diabetes and at the same time regulates PI3K/AKT and HIPPO pathways and prohibits the proliferation of cancer cells. Study design, size, duration Controlled experimental study. After metformin and cyclophosphamide dose adjustment, 12 female NMRI 14-day-old mice were randomly divided into four groups: the control group (CONT), metformin group (MET), cyclophosphamide group (CYC) and metformin with cyclophosphamide group (MET-CYC). Participants/materials, setting, methods Mice were treated by intraperitoneal injection of a dose of 150mg/kg of metformin for 11 consecutive days, and/or a dose of 65mg/kg of cyclophosphamide every three days (The third, sixth and ninth days). The mice were sacrificed 24 hours after the last metformin injection or 3 days after the last cyclophosphamide injection. The ovaries were collected for stereological evaluation, and expression of Pten, Mtor, Yap-1, p53, Bcl-2, and Bax genes by qRT-PCR method. Main results and the role of chance The stereological evaluation showed that the ovarian volume and primordial follicle number in the CYC group were decreased compared to the other groups (P &amp;lt; 0.05), and also in the MET-CYC group reduced compared to the CONT and MET groups, but increased significantly compared to the CYC group (P &amp;lt; 0.05). A significantly increased number of growing follicles was observed in the CYC group compared to the other groups (P &amp;lt; 0.05). The molecular analysis showed that the Pten expression in the CYC group was lower than CONT and MET groups (P &amp;lt; 0.05). Mtor expression in the CYC group was significantly increased compared to other groups (P &amp;lt; 0.05). The expression of Yap-1 was significantly decreased in the MET group compared to other groups (P &amp;lt; 0.05), and was lower in the MET-CYC than in the CYC group (P &amp;lt; 0.05). The of P53 expression in the MET-CYC group was higher than CONT and MET groups (P &amp;lt; 0.05), and was increased in the CYC compared to the CONT group (P &amp;lt; 0.05). Bcl-2 expression was higher in CONT and MET groups than in MET-CYC and CYC groups (P &amp;lt; 0.05). The expression of Bax in the CYC group was significantly increased compared to CONT and MET groups (P &amp;lt; 0.05). Limitations, reasons for caution Due to ethical issues in less use of mice, dose of metformin was obtained according to the articles used in adult mice and also by using the human to animal dose conversion formula. Wider implications of the findings It can be concluded that metformin during cyclophosphamide treatment can maintains ovarian volume, reserve, and activity in prepubertal mice by regulating PI3K/AKT and HIPPO pathways. Considering the relatively acceptable cost and long-term safety of metformin, it is a promising factor to be used to preserve fertility during chemotherapy. Trial registration number not applicable