Modeling YAP fusions: a paradigm for investigating rare cancers?

Loss of the NF2 tumor suppressor gene is a common finding in meningiomas, and more recently YAP1 fusions have been found in a subset of pediatric NF2 wild-type meningiomas. In the previous issue of Genes & Development, Szulzewsky and colleagues (pp. 857-870) showed that TEAD-dependent YAP1 activity by either the loss of the NF2 gene or YAP1-MAML2 fusion is an oncogenic process promoting meningioma tumorigenesis. Furthermore, pharmacological inhibition of YAP1-TEAD resulted in antitumor activity in both YAP1 fusion-positive and NF2 mutant meningiomas. Together, these data indicate that disruption of the YAP1-TEAD interaction raises a potential therapeutic option for these tumors that requires future investigation.

Both YAP1-MAML2 and constitutively active YAP1 drive the formation of tumors that resemble NF2 mutant meningiomas in mice.

YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.

Comparison of tumor-associated YAP1 fusions identifies a recurrent set of functions critical for oncogenesis.

YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C'-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1-TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

NEDD8 enhances Hippo signaling by mediating YAP1 neddylation.

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling.

Mechanotransduction through hemidesmosomes during aging and longevity.

Hemidesmosomes are structural protein complexes localized at the interface of tissues with high mechanical demand and shear forces. Beyond tissue anchoring, hemidesmosomes have emerged as force-modulating structures important for translating mechanical cues into biochemical and transcriptional adaptation (i.e. mechanotransduction) across tissues. Here, we discuss the recent insights into the roles of hemidesmosomes in age-related tissue regeneration and aging in C. elegans, mice and humans. We highlight the emerging concept of preserved dynamic mechanoregulation of hemidesmosomes in tissue maintenance and healthy aging.

BCAR4 facilitates trastuzumab resistance and EMT in breast cancer via sponging miR-665 and interacting with YAP1.

Breast cancer antiestrogen resistance 4 (BCAR4) has been suggested that can modulate cell behavior, resulting in tumorigenesis and chemoresistance. However, the underlying mechanisms of BCAR4 in trastuzumab resistance (TR) is still elusive. Here, we explored the function and the underlying mechanism of BCAR4 involving in TR. We found that BCAR4 is significantly upregulated in trastuzumab-resistant BC cells. Knockdown of BCAR4 could sensitize the BC cells to trastuzumab and suppress epithelial-mesenchymal transition (EMT). Mechanically, BCAR4 promotes yes-associated protein 1 (YAP1) expression by competitively sponging miR-665, to activated TGF-ß signaling. Reciprocally, YAP1 could occupy the BCAR4 promoter to enhance its transcription, suggesting that there exists a positive feedback regulation between YAP1 and BCAR4. Targeting the BCAR4/miR-665/YAP1 axis may provide a novel insight of therapeutic approaches for TR in BC.

A novel mechanism of ferroptosis inhibition-enhanced atherosclerotic plaque stability: YAP1 suppresses vascular smooth muscle cell ferroptosis through GLS1.

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe-/-) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. In vitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques.

LRG1 loss effectively restrains glomerular TGF-ß signaling to attenuate diabetic kidney disease.

Transforming growth factor (TGF)-ß signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-ß signaling regulators can substantially influence TGF-ß's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-ß-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-ß-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-ß signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-ß signaling to attenuate DKD.

YAP1-CPNE3 positive feedback pathway promotes gastric cancer cell progression.

Hippo-Yes-associated protein 1 (YAP1) plays an important role in gastric cancer (GC) progression; however, its regulatory network remains unclear. In this study, we identified Copine III (CPNE3) was identified as a novel direct target gene regulated by the YAP1/TEADs transcription factor complex. The downregulation of CPNE3 inhibited proliferation and invasion, and increased the chemosensitivity of GC cells, whereas the overexpression of CPNE3 had the opposite biological effects. Mechanistically, CPNE3 binds to the YAP1 protein in the cytoplasm, inhibiting YAP1 ubiquitination and degradation mediated by the E3 ubiquitination ligase ß-transducin repeat-containing protein (ß-TRCP). Thereby activating the transcription of YAP1 downstream target genes, which creates a positive feedback cycle to facilitate GC progression. Immunohistochemical analysis demonstrated significant upregulation of CPNE3 in GC tissues. Survival and Cox regression analyses indicated that high CPNE3 expression was an independent prognostic marker for GC. This study elucidated the pivotal involvement of an aberrantly activated CPNE3/YAP1 positive feedback loop in the malignant progression of GC, thereby uncovering novel prognostic factors and therapeutic targets in GC.

Tumor-associated macrophage-derived exosomal miR21-5p promotes tumor angiogenesis by regulating YAP1/HIF-1α axis in head and neck squamous cell carcinoma.

Extracellular vesicles (EVs) have recently received increasing attention as essential mediators of communication between tumor cells and their microenvironments. Tumor-associated macrophages (TAMs) play a proangiogenic role in various tumors, especially head and neck squamous cell carcinoma (HNSCC), and angiogenesis is closely related to tumor growth and metastasis. This research focused on exploring the mechanisms by which EVs derived from TAMs modulate tumor angiogenesis in HNSCC. Our results indicated that TAMs infiltration correlated positively with microvascular density in HNSCC. Then we collected and identified EVs from TAMs. In the microfluidic chip, TAMs derived EVs significantly enhanced the angiogenic potential of pHUVECs and successfully induced the formation of perfusable blood vessels. qPCR and immunofluorescence analyses revealed that EVs from TAMs transferred miR-21-5p to endothelial cells (ECs). And targeting miR-21-5p of TAMs could effectively inhibit TAM-EVs induced angiogenesis. Western blot and tube formation assays showed that miR-21-5p from TAM-EVs downregulated LATS1 and VHL levels but upregulated YAP1 and HIF-1α levels, and the inhibitors of YAP1 and HIF-1α could both reduce the miR-21-5p enhanced angiogenesis in HUVECs. The in vivo experiments further proved that miR-21-5p carried by TAM-EVs promoted the process of tumor angiogenesis via YAP1/HIF-1α axis in HNSCC. Conclusively, TAM-derived EVs transferred miR-21-5p to ECs to target the mRNA of LATS1 and VHL, which inhibited YAP1 phosphorylation and subsequently enhanced YAP1-mediated HIF-1α transcription and reduced VHL-mediated HIF-1α ubiquitination, contributing to angiogenesis in HNSCC. These findings present a novel regulatory mechanism of tumor angiogenesis, and miR-21-5p/YAP1/HIF-1α might be a potential therapeutic target for HNSCC.

Untying the Gordian knot of composite hemangioendothelioma: Discovery of novel fusions.

Composite hemangioendothelioma is a rare, locally aggressive, and rarely metastasizing vascular neoplasm which affects both children and adults. Recently, a number of gene fusions including YAP1::MAML2, PTBP1::MAML2, and EPC1::PHC2 have been detected in a small subset of cases with or without neuroendocrine expression. Herein, we present four additional cases with novel in-frame fusions. The cohort comprises two females and two males with a wide age range at diagnosis (24-80 years). Two tumors were deep involving the right brachial plexus and mediastinum, while the remaining were superficial (right plantar foot and abdominal wall). The size ranged from 1.5 to 4.8 cm in greatest dimension. Morphologically, all tumors had an admixture of at least two architectural patterns including retiform hemangioendothelioma, hemangioma, epithelioid hemangioendothelioma, or angiosarcoma. The tumors were positive for endothelial markers CD31 (3/3), ERG (4/4), and D2-40 (1/4, focal), while SMA was expressed in 2/3 highlighting the surrounding pericytes. Synaptophysin showed immunoreactivity in 2/3 cases. One patient had a local recurrence after 40 months, while two patients had no evidence of disease 4 months post-resection. Targeted RNA sequencing detected novel in-frame fusions in each of the cases: HSPG2::FGFR1, YAP1::FOXR1, ACTB::MAML2, and ARID1B::MAML2. The two cases with neuroendocrine expression occurred as superficial lesions and harbored YAP1::FOXR1 and ARID1B::MAML2 fusions. Our study expands on the molecular spectrum of this enigmatic tumor, further enhancing our current understanding of the disease.

USP1/UAF1-Stabilized METTL3 Promotes Reactive Astrogliosis and Improves Functional Recovery after Spinal Cord Injury through m6A Modification of YAP1 mRNA.

RNA N6-methyladenosine (m6A) modification is involved in diverse biological processes. However, its role in spinal cord injury (SCI) is poorly understood. The m6A level increases in injured spinal cord, and METTL3, which is the core subunit of methyltransferase complex, is upregulated in reactive astrocytes and further stabilized by the USP1/UAF1 complex after SCI. The USP1/UAF1 complex specifically binds to and subsequently removes K48-linked ubiquitination of the METTL3 protein to maintain its stability after SCI. Moreover, conditional knockout of astrocytic METTL3 in both sexes of mice significantly suppressed reactive astrogliosis after SCI, thus resulting in widespread infiltration of inflammatory cells, aggravated neuronal loss, hampered axonal regeneration, and impaired functional recovery. Mechanistically, the YAP1 transcript was identified as a potential target of METTL3 in astrocytes. METTL3 could selectively methylate the 3'-UTR region of the YAP1 transcript, which subsequently maintains its stability in an IGF2BP2-dependent manner. In vivo, YAP1 overexpression by adeno-associated virus injection remarkably contributed to reactive astrogliosis and partly reversed the detrimental effects of METTL3 knockout on functional recovery after SCI. Furthermore, we found that the methyltransferase activity of METTL3 plays an essential role in reactive astrogliosis and motor repair, whereas METTL3 mutant without methyltransferase function failed to promote functional recovery after SCI. Our study reveals the previously unreported role of METTL3-mediated m6A modification in SCI and might provide a potential therapy for SCI.SIGNIFICANCE STATEMENT Spinal cord injury is a devastating trauma of the CNS involving motor and sensory impairments. However, epigenetic modification in spinal cord injury is still unclear. Here, we propose an m6A regulation effect of astrocytic METTL3 following spinal cord injury, and we further characterize its underlying mechanism, which might provide promising strategies for spinal cord injury treatment.

circELP2 reverse-splicing biogenesis and function as a pro-fibrogenic factor by targeting mitochondrial quality control pathway.

Idiopathic pulmonary fibrosis (IPF) is considered as a chronic, fibrosing interstitial pneumonia with unknown mechanism. The present work aimed to explore the function, biogenesis and regulatory mechanism of circELP2 in pulmonary fibrosis and evaluate the value of blocking circELP2-medicated signal pathway for IPF treatment. The results showed that heterogeneous nuclear ribonucleoprotein L initiated reverse splicing of circELP2 resulting in the increase of circELP2 generation. The biogenetic circELP2 activated the abnormal proliferation and migration of fibroblast and extracellular matrix deposition to promote pulmonary fibrogenesis. Mechanistic studies demonstrated that cytoplasmic circELP2 sponged miR-630 to increase transcriptional co-activators Yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ). Then, YAP1/TAZ bound to the promoter regions of their target genes, such as mTOR, Raptor and mLST8, which in turn activated or inhibited the genes expression in mitochondrial quality control pathway. Finally, the overexpressed circELP2 and miR-630 mimic were packaged into adenovirus vector for spraying into the mice lung to evaluate therapeutic effect of blocking circELP2-miR-630-YAP1/TAZ-mitochondrial quality control pathway in vivo. In conclusion, blocking circELP2-medicated pathway can alleviate pulmonary fibrosis, and circELP2 may be a potential target to treat lung fibrosis.

Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl.

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

Protein Phosphatase 2A with B' specificity subunits regulates the Hippo-Yorkie signaling axis in the Drosophila eye disc.

Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. We show that Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the B‴ subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.

YAP1 regulates esophageal stem cells' self-renewal and differentiation.

Esophageal epithelium is one of the most proliferative and regenerative epithelia in our body, indicating robust stem cell activity. However, the underlying mechanisms regulating the self-renewal and differentiation of esophageal stem cells need to be more elucidated. Here, we identify the role of YAP1 in esophageal stem cells. YAP1 is differentially expressed in the nuclei of esophageal basal cells. Furthermore, the treatment of verteporfin, a YAP1 inhibitor, interfered with esophageal organoid formation. Consistently, YAP1 deletion decreased esophageal organoid formation and the expression of basal genes while increasing the expression of suprabasal genes. Finally, global transcriptomic analysis revealed that YAP1 inhibition induced a significant enrichment of gene sets related to keratinization and cornification, while depleting gene sets related to DNA repair and chromosome maintenance. Our data uncover a novel regulatory mechanism for esophageal stem cells, which could provide a potential strategy for esophageal regenerative medicine.

Overexpression of transcription factor TBX5 inhibits the activation of YAP1-TEAD1 pathway to promote ferroptosis in lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for more than 80 % of lung cancer (LC) cases, making it the primary cause of cancer-related mortality worldwide. T-box transcription factor 5 (TBX5) is an important regulator of embryonic and organ development and plays a key role in cancer development. Here, our objective was to investigate the involvement of TBX5 in ferroptosis within LC cells and the underlying mechanisms. METHODS: First, TBX5 expression was examined in human LC cells. Next, overexpression of TBX5 and Yes1-associated transcriptional regulator (YAP1) and knockdown of TEA domain 1 (TEAD1) were performed in A549 and NCI-H1703 cells. The proliferation ability of A549 and NCI-H1703 cells, GSH, MDA, ROS, and Fe2+ levels were measured. Co-immunoprecipitation (Co-IP) was performed to verify whether TBX5 protein could bind YAP1. Then TBX5, YAP1, TEAD1, GPX4, p53, FTH1, SLC7A11 and PTGS2 protein levels were assessed. Finally, we verified the effect of TBX5 on ferroptosis in LC cells in vivo. RESULTS: TBX5 expression was down-regulated in LC cells, especially in A549 and NCI-H1703 cells. Overexpression of TBX5 significantly decreased proliferation ability of A549 and NCI-H1703 cells, downregulated GPX4 and GSH levels, and upregulated MDA, ROS, and Fe2+ levels. Co-IP verified that TBX5 protein could bind YAP1. Moreover, oe-YAP1 promoted proliferation ability of A549 and NCI-H1703 cells transfected with Lv-TBX5, upregulated GPX4 and GSH levels and downregulated MDA, ROS, and Fe2+ levels. Additionally, oe-YAP1 promoted FTH1 and SLC7A11 levels and inhibited p53 and PTGS2 levels in A549 and NCI-H1703 cells transfected with Lv-TBX5. However, transfection with si-TEAD1 further reversed these effects. In vivo experiments further validated that TBX5 promoted ferroptosis in LC cells. CONCLUSIONS: TBX5 inhibited the activation of YAP1-TEAD1 pathway to promote ferroptosis in LC cells.

Human trophoblast invasion and migration are mediated by the YAP1-CCN1 pathway: defective signaling in trophoblasts during early-onset severe preeclampsia.

The transcription coactivator YAP1 mediates the major effects of the Hippo signaling pathway. The CCN family is a small group of glycoproteins known to be downstream effectors of YAP1 in diverse tissues. However, whether CCN family members mediate the effects of YAP1 in human trophoblasts is unknown. In this study, placental expression of both YAP1 and CCN1 was found to be impaired in pregnancies complicated by early-onset severe preeclampsia (sPE). CCN1 was expressed not only in cytotrophoblasts, trophoblast columns and mesenchymal cells, similar to active YAP1, but also in syncytiotrophoblasts of normal first-trimester placental villi; moreover, decidual staining of active YAP1 and CCN1 was found in both interstitial and endovascular extravillous trophoblasts. In cultured immortalized human trophoblastic HTR-8/SVneo cells, knockdown of YAP1 decreased CCN1 mRNA and protein expression and led to impaired cell invasion and migration. Also, CCN1 knockdown negatively affected HTR-8/SVneo cell invasion and migration but not viability. YAP1 knockdown was further found to impair HTR-8/SVneo cell viability via G0/G1 cell cycle arrest and apoptosis, while CCN1 knockdown had minimal effect on cell cycle arrest and no effect on apoptosis. Accordingly, treatment with recombinant CCN1 partially reversed the YAP1 knockdown-induced impairment in trophoblast invasion and migration but not in viability. Thus, CCN1 mediates the effects of YAP1 on human trophoblast invasion and migration but not apoptosis, and decreased placental expression of YAP1 and CCN1 in pregnancies complicated by early-onset sPE might contribute to the pathogenesis of this disease.