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The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency., (© 2024. The Author(s).)

Cardiac fibroblasts (CFs) are the primary cells tasked with depositing and remodeling collagen and significantly associated with heart failure (HF). TEAD1 has been shown to be essential for heart development and homeostasis. However, fibroblast endogenous TEAD1 in cardiac remodeling remains incompletely understood. Transcriptomic analyses revealed consistently upregulated cardiac TEAD1 expression in mice 4 weeks after transverse aortic constriction (TAC) and Ang-II infusion. Further investigation revealed that CFs were the primary cell type expressing elevated TEAD1 levels in response to pressure overload. Conditional TEAD1 knockout was achieved by crossing TEAD1-floxed mice with CFs- and myofibroblasts-specific Cre mice. Echocardiographic and histological analyses demonstrated that CFs- and myofibroblasts-specific TEAD1 deficiency and treatment with TEAD1 inhibitor, VT103, ameliorated TAC-induced cardiac remodeling. Mechanistically, RNA-seq and ChIP-seq analysis identified Wnt4 as a novel TEAD1 target. TEAD1 has been shown to promote the fibroblast-to-myofibroblast transition through the Wnt signalling pathway, and genetic Wnt4 knockdown inhibited the pro-transformation phenotype in CFs with TEAD1 overexpression. Furthermore, co-immunoprecipitation combined with mass spectrometry, chromatin immunoprecipitation, and luciferase assays demonstrated interaction between TEAD1 and BET protein BRD4, leading to the binding and activation of the Wnt4 promoter. In conclusion, TEAD1 is an essential regulator of the pro-fibrotic CFs phenotype associated with pathological cardiac remodeling via the BRD4/Wnt4 signalling pathway., (© 2024. The Author(s).)

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

Long non-coding RNA (lncRNA) HOXA cluster antisense RNA 3 (HOXA-AS3) regulates the progression of several types of human malignancy. However, the role and potential mechanism of HOXA-AS3 in osteosarcoma (OS) remain unknown. In this study, upregulation of HOXA-AS3 was observed in OS tissues and cell lines and associated with poor clinical outcomes. Silencing of HOXA-AS3 significantly inhibited the proliferation, migration and invasion of OS cells in vitro and suppressed the tumorigenesis of OS cells in vivo. Furthermore, knockdown of HOXA-AS3 inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) and epithelial-to-mesenchymal transition (EMT) in OS. Further investigation of this mechanism revealed that HOXA-AS3 could directly upregulate the expression of TEAD1 via its competing endogenous RNA (ceRNA) activity on miR-1286. This study clarified the oncogenic roles of the HOXA-AS3/miR-1286/TEAD1 axis in OS progression, suggesting a novel therapeutic target for OS., (© 2023. The Author(s).)

Autonomous circadian clocks exist in nearly every mammalian cell type. These cellular clocks are subjected to a multilayered regulation sensitive to the mechanochemical cell microenvironment. Whereas the biochemical signaling that controls the cellular circadian clock is increasingly well understood, mechanisms underlying regulation by mechanical cues are largely unknown. Here we show that the fibroblast circadian clock is mechanically regulated through YAP/TAZ nuclear levels. We use high-throughput analysis of single-cell circadian rhythms and apply controlled mechanical, biochemical, and genetic perturbations to study the expression of the clock gene Rev-erbα. We observe that Rev-erbα circadian oscillations are disrupted with YAP/TAZ nuclear translocation. By targeted mutations and overexpression of YAP/TAZ, we show that this mechanobiological regulation, which also impacts core components of the clock such as Bmal1 and Cry1, depends on the binding of YAP/TAZ to the transcriptional effector TEAD. This mechanism could explain the impairment of circadian rhythms observed when YAP/TAZ activity is upregulated, as in cancer and aging., (© 2023 Abenza et al.)

Transcription of the human papillomavirus (HPV) oncogenes, E6 and E7 , is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed in vitro DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes E6 and E7 play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis., Competing Interests: The authors declare no conflict of interest.

Muscle development is a complex biological process involving an intricate network of multiple factor interactions. Through the analysis of transcriptome data and molecular biology confirmation, this study aims to reveal the molecular mechanism underlying sheep embryonic skeletal muscle development. The RNA sequencing of embryos was conducted, and microRNA (miRNA)-mediated competitive endogenous RNA (ceRNA) networks were constructed. qRT-PCR, siRNA knockdown, CCK-8 assay, scratch assay, and dual luciferase assay were used to carry out gene function identification. Through the analysis of the ceRNA networks, three miRNAs (miR-493-3p, miR-3959-3p, and miR-410-5p) and three genes ( TEAD1 , ZBTB34, and POGLUT1 ) were identified. The qRT-PCR of the DE-miRNAs and genes in the muscle tissues of sheep showed that the expression levels of the TEAD1 gene and miR-410-5p were correlated with the growth rate. The knockdown of the TEAD1 gene by siRNA could significantly inhibit the proliferation of sheep primary embryonic myoblasts, and the expression levels of SLC1A5, FoxO3, MyoD , and Pax7 were significantly downregulated. The targeting relationship between miR-410-5p and the TEAD1 gene was validated by a dual luciferase assay, and miR-410-5p can significantly downregulate the expression of TEAD1 in sheep primary embryonic myoblasts. We proved the regulatory relationship between miR-410-5p and the TEAD1 gene, which was related to the proliferation of sheep embryonic myoblasts. The results provide a reference and molecular basis for understanding the molecular mechanism of embryonic muscle development.

Transcriptional enhanced associate domain (TEAD) transcription factors play important roles in embryonic stem cell (ESC) renewal and differentiation. Four TEAD transcription factors (Tead1, Tead2, Tead3 and Tead4) and their various splice variants have been discovered in mice, but the expression pattern of them during pluripotency state transition is unclear. Here, we investigated the expression of TEADs and their splice variants in mouse ESCs at different pluripotent/differentiating states and adult mouse tissues. Our results preliminarily revealed the diversity and heterogeneity of TEAD family, which is helpful for understanding their overlapping and distinctive functions. Furthermore, a novel splice variant of Tead1 was identified and named Tead1 isoform 4., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Perineural invasion (PNI) driven by the tumor microenvironment (TME) has emerged as a key pattern of metastasis of prostate cancer (PCa), while its underlying mechanism is still elusive. Here, we identified increased CAFs and YAP1 expression levels in patients with metastatic PCa. In the cultured PCa cell line LNCaP, co-culture with cancer-associated fibroblasts (CAFs) could upregulate YAP1 protein expression. Either ectopic overexpression of YAP1 or co-culture with CAFs could promote the infiltration of LNCaPs towards dorsal root ganglia (DRG). This effect could be blocked using an YAP1 inhibitor. In vivo, overexpression of YAP1 could increase PNI in a mouse model of sciatic nerve tumor invasion. Mechanistically, TEAD1 binds to the NGF promotor and YAP1/TEAD1 activates its transcription and consequently increases NGF secretion. In turn, PCa cells treated with CM from CAFs or stable YAP1 overexpression can stimulate DRG to secrete CCL2. The epithelial-to-mesenchymal transition (EMT) of PCa cells is thus activated via CCL2/CCR2. Overall, our data demonstrate that CAFs can activate YAP1/TEAD1 signaling and increase the secretion of NGF, therefore promoting PCa PNI. In addition, EMT induced by PNI suggests a feedback loop is present between neurons and PCa cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Senescence, a stable state of growth arrest, affects many physiological and pathophysiological processes, especially aging. Previous work has indicated that transcription factors (TFs) play a role in regulating senescence. However, a systematic study of regulatory TFs during replicative senescence (RS) using multi-omics analysis is still lacking. Here, we generated time-resolved RNA-seq, reduced representation bisulfite sequencing (RRBS) and ATAC-seq datasets during RS of mouse skin fibroblasts, which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome. Through integrative analysis and genetic manipulations, we found that transcription factors E2F4, TEAD1 and AP-1 are key regulators of RS. Overexpression of E2f4 improved cellular proliferative capacity, attenuated SA-β-Gal activity and changed RS-associated differentially methylated sites (DMSs). Moreover, knockdown of Tead1 attenuated SA-β-Gal activity and partially altered the RS-associated transcriptome. In addition, knockdown of Atf3, one member of AP-1 superfamily TFs, reduced Cdkn2a (p16) expression in pre-senescent fibroblasts. Taken together, the results of this study identified transcription factors regulating the senescence program through multi-omics analysis, providing potential therapeutic targets for anti-aging., (© 2022. The Author(s).)

Aim: The underlying mechanisms by which circular RNAs (circRNAs) regulate non-small-cell lung cancer (NSCLC) progression remain elusive. This study investigated the role of circRNA circTTBK2 in NSCLC tumorigenesis. Materials & methods: Quantitative reverse transcriptase polymerase chain reaction analysis of circTTBK2 in NSCLC tissues and cell lines was performed. Cell proliferation, migration, invasion and tumorigenesis were confirmed in vitro and in vivo using CCK-8, EdU incorporation, Transwell assays and xenograft technique. The circTTBK2/miR-873-5p/TEAD1/DERL1 axis was verified by RNA immunoprecipitation, chromatin immunoprecipitation and luciferase reporter assays. Results: Overexpressed circTTBK2 in NSCLC tissues indicates poor prognosis of NSCLC patients. circTTBK2 harbors miR-873-5p, and miR-873-5p directly targets TEAD1. TEAD1 transcriptionally activates DERL1. Conclusion: This study revealed a novel machinery of circTTBK2/miR-873-5p/TEAD1/DERL1 for NSCLC tumorigenesis.

Background and Aim: Vasculogenic mimicry (VM) is a unique blood supply pattern in malignant tumors that is closely associated with metastasis and poor prognosis. The Hippo signaling effector TAZ is upregulated in several cancers, promoting cancer proliferation and metastasis. This study aimed to identify the function of TAZ and its regulatory mechanism in promoting VM in gastric cancer (GC)., Methods: The expression of TAZ and TEAD4 and their correlations with overall survival and VM-related markers were analyzed in 228 cases of GC. The regulatory mechanism of TAZ and its interaction with TEAD4 in epithelial-mesenchymal transition (EMT) and VM were investigated in vitro and in vivo., Results: TAZ was highly expressed in GC samples and was associated with shorter patient survival time. TAZ expression was positively correlated with TEAD4 and VM in patients with GC. TAZ enhanced the migration and invasion capacity of GC cells through EMT in vitro and upregulated the expression of VM-associated proteins, including VE-cadherin, MMP2, and MMP9, thus promoting VM formation. Overexpression of TAZ accelerated the growth of subcutaneous xenograft and promoted VM formation in vivo. Co-immunoprecipitation assays showed that TAZ can directly bind to TEAD4, and in vitro experiments showed that this binding mediates the function of TAZ in regulating EMT and VM formation in GC., Conclusions: TAZ promotes GC metastasis and VM by upregulating TEAD4 expression. Our findings expand the role of TAZ in VM and provide new theoretical support for the use of antiangiogenic therapy in the treatment of advanced GC., (© 2022 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Aims: Transcriptional enhanced associate domain (TEAD) transcription factor family, a very important family in the hippo signaling pathway, has been found to play oncogenic functions in the occurrence of various malignant tumors. However, the expression of TEADs in pan-cancer and the important role of TEAD4 in clear cell renal cell carcinoma (ccRCC) have not been analyzed. Herein, we aim to evaluate the expression of TEADs in pan-cancer, and focus on analyzing the role of TEAD4 in the progression of ccRCC., Main Methods: Data from the Cancer Genome Atlas (TCGA) was used to analyze the expression of TEADs in pan-cancer and its clinical correlation. TEAD4 expression in ccRCC tissues, biological functions in vitro and in vivo were analyzed by immunohistochemistry (IHC), western blotting, RNAi and Xenograft assay. Mircode, BioGRID and g: Profiler website were used to build a ceRNA network and downstream pathway prediction., Key Findings: TEAD1, TEAD2, TEAD3 and TEAD4 were highly expressed in 3, 6, 5, and 12 types of cancer tissues, respectively, indicating that TEAD4 is most closely related to tumor progression. Among the cancers with high TEAD4 expression, the expression of TEAD4 has the greatest correlation with the poor prognosis of ccRCC. We also found the malignant phenotypes of ccRCC cells in vitro and vivo have been significantly suppressed by silencing TEAD4., Significance: TEADs, especially TEAD4, were overexpressed in many human tumors. This study is the first to show that TEAD4 acts as an oncogene in ccRCC and may be an important factor in progress of ccRCC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Aims: TEAD4 transcription factor belonging to TEAD-family, is a key downstream element of the Hippo Signalling pathway and is very important for YAPinduced tumor progression. YAP-TEAD interaction is required to promote tumor progression and metastasis in various cancers. This study aims to investigate the role of TEAD4 in CRC progression and to compare the TEAD4 expression with different clinicopathological parameters of the study population. We also aim to explore the expression pattern of miR-4269 and miR-1343-3p and their functional role in TEAD4 mediated CRC progression. Furthermore, we intend to evaluate the prognostic significance of TEAD4, miR-4269, and miR-1343-3p in colorectal carcinoma., Methods: Real-time PCR, Immunohistochemical Staining, and Western Blotting were performed on 71 human CRC tissue specimens and their adjacent normal tissues to evaluate the TEAD4 expression and the results were statistically analyzed against the clinicopathological variables of patient data and also with survival data using STATA software. miRNA expression was analyzed by quantitative real-time PCR., Results: TEAD4 expression levels in tumor specimens were significantly higher than their paired normal specimens. The higher protein expression levels showed a significant association with TNM stage, Duke Stage, tumor grade, invasion depth, node status, necrosis of tumor tissue, lymphovascular and perineural invasion. As per the cox-regression model and classification tree analysis, TNM stage and perineural invasion were important predictors for TEAD4 expression and prognosis of CRC patients. Survival analysis indicated that TEAD4 overexpression was associated with poorer overall and disease-free survival. miR-4269 and miR-1343-3p were downregulated in CRC tumors and showed a negative correlation with TEAD4. The nuclear overexpressed TEAD4 and downregulated miR-4269 and miR-1343-3p evaluated for the first time in CRC, are believed to serve as important prognostic markers in CRC., Conclusion: Expression of TEAD4 was increased in CRC and was negatively regulated by miR-4269 and miR-1343-3p. The overexpression of TEAD4 is linked with poor overall and disease-free survival of CRC patients. These findings support prior observations and thus TEAD4 may be a possible prognostic marker in CRC., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The LIM protein Ajuba has been implicated in the development of human cancers. To date, its expression pattern and biological significance in breast cancers (BC) have not been fully investigated. In the current study, we examined Ajuba protein levels in 93 invasive ductal carcinoma specimens by immunohistochemistry. The Ajuba expression level was elevated in breast cancer tissue compared with normal tissue. Ajuba overexpression is correlated with advanced tumor-node-metastasis (TNM) stage, positive node status, and adverse patient outcomes. The Ajuba protein level was also higher in BC cell lines compared to normal breast epithelial cell line MCF-10A. Ectopically expressed Ajuba in MCF-7 cells stimulated in vitro and in vivo cell growth, invasion, cell cycle progression, and decreased paclitaxel-induced apoptosis. RNA-sequencing (RNA-seq) followed by gene set enrichment analysis (GSEA) analysis showed that Ajuba overexpression regulated the Hippo signaling pathway. Ajuba overexpression also increased glucose uptake and increased expression of TAZ, GLUT3, and Survivin. TAZ knockdown abolished the role of Ajuba on GLUT3 and Survivin induction. The ChIP assay showed that TEAD4, a major TAZ binding transcription factor, could bind to the GLUT3 and Survivin promoter regions. In conclusion, our data demonstrated that elevated Ajuba expression is correlated with poor BC prognosis and regulated malignant behavior through TAZ-GLUT3/Survivin signaling in BC cells., Competing Interests: We declare that we have no conflict of interest., (Copyright © 2022 Xiang Li et al.)

Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling., (© 2022. The Author(s).)

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high incidence and mortality. MiR-597-5p is downregulated in tumor tissues of HCC compared with non-tumor tissues. However, its role in HCC is still unknown. This study aims to assess the function of miR-597-5p in HCC development and investigate the underlying mechanism. To perform gain- and loss-of-function studies, SK-HEP-1 cells and Huh-7 cells were transfected with miR-597-5p mimics and inhibitor, respectively. MiR-597-5p markedly reduced the cell viability and the expression of Ki-67 in HCC cells. MiR-597-5p also repressed the cell cycle progression of HCC cells and the protein levels of cyclin D1 and CDK2. Moreover, miR597-5p inhibited the migration and invasion of HCC cells and decreased MMP2 and MMP9 levels. Transcriptional enhancer associate domain transcription factor 1 (TEAD1) was identified as a target of miR-597-5p by luciferase reporter assay. TEAD1 and its downstream target genes, CTGF and CYR61, were downregulated by miR-597-5p in HCC cells. Furthermore, miR-597-5p was demonstrated to function in HCC progression by targeting TEAD1 via TEAD1 expression gain and loss. Our study demonstrates that miR-597-5p represses the proliferation, migration, and invasion of HCC cells through targeting TEAD1, which provides a therapeutic target for HCC treatment., (© 2021. The Society for In Vitro Biology.)

Circular RNAs (circRNAs) act as important regulators in myocardial infarction (MI). This study aimed to explore the regulatory mechanism of circRNA solute carrier family 8 member A1 antisense RNA 1 (circSLC8A1) in hypoxia-induced myocardial injury.Exosomes were isolated by ultracentrifugation and identified by microscopic observation or protein detection. Protein levels were examined by Western blot. CircSLC8A1, microRNA-214-5p (miR-214-5p), and TEA domain transcription factor 1 (TEAD1) levels were determined via quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Oxidative stress was assessed by reactive oxygen species (ROS) production, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity through the corresponding detection kits. Target analysis was performed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, and pull-down assay.Exosomes released circSLC8A1 from hypoxic cardiomyocytes. Exosomal circSLC8A1 exacerbated hypoxia-induced repression of cell viability but promotion of cell apoptosis, inflammation, and oxidative stress. Knockdown of circSLC8A1 ameliorated hypoxia-mediated cell injury. CircSLC8A1 directly targeted miR-214-5p and miR-214-5p downregulation reverted the effects of si-circSLC8A1 on hypoxia-treated cardiomyocytes. TEAD1 was a target of miR-214-5p and circSLC8A1 upregulated TEAD1 level via targeting miR-214-5p. In addition, miR-214-5p inhibited hypoxia-caused cell injury by downregulating the expression of TEAD1.These results suggested that circSLC8A1 aggravated cell damages in hypoxia-treated cardiomyocytes by the regulation of TEAD1 via sponging miR-214-5p.

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs., Competing Interests: Declaration of interests G.R.C. is a founder and stockholder in Foghorn Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Xp11 translocation renal cell carcinoma (tRCC) characterized by the rearrangement of the TFE3 is recently identified as a unique subtype of RCC that urgently requires effective prevention and treatment strategies. Therefore, determining suitable therapeutic targets and fully understanding the biological significance of tRCC is essential. The importance of autophagy is increasingly acknowledged because it shows carcinogenic activity or suppressor effect. Autophagy is a physiological cellular process critical to maintaining cell homeostasis, which is involved in the lysosomal degradation of cytoplasmic organelles and macromolecules via the lysosomal pathway, suggesting that targeting autophagy is a potential therapeutic approach for cancer therapies. However, the underlying mechanism of autophagy in tRCC is still ambiguous. In this review, we summarize the autophagy-related signaling pathways associated with tRCC. Moreover, we examine the roles of autophagy and the immune response in tumorigenesis and investigate how these factors interact to facilitate or prevent tumorigenesis. Besides, we review the findings regarding the treatment of tRCC via induction or inhibition of autophagy. Hopefully, this study will shed some light on the functions and implications of autophagy and emphasize its role as a potential molecular target for therapeutic intervention in tRCC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: This study was carried out to explore the potential involvement of miR-125a-5p in the oncogenic effects of EphA2, TAZ, and TEAD2 and the activity of the Hippo signaling pathway in gastric cancer progression., Methods: In vitro transfection of miR-125a-5p mimics or inhibitors, qRT-PCR, colony formation assays, and cell invasion assays were used to assess the effect of miR-125a-5p on the growth and invasion in gastric cancer (GC). Male nude mice bearing tumors derived from human GC cells were used for evaluating the effects of miR-125a-5p on tumor growth. Luciferase reporter assay, immunofluorescence, immunohistochemistry, qRT-PCR, and immunoblotting were performed to explore the role of miR-125a-5p in the epithelial-mesenchymal transition (EMT) and association among miR-125a-5p, EphA2, TAZ, and TEAD2 in GC cells., Results: MiR-125a-5p enhanced GC cell viability and invasion in vitro, whereas inhibition of miR-125a-5p using a specific inhibitor and antagomir suppressed cancer cell invasion and tumor growth. Moreover, inhibition of miR-125a-5p reversed EMT in vitro. miR-125a-5p upregulated the expression of EphA2, TAZ, and TEAD2, promoted TAZ nuclear translocation, and induced changes in the activity of the Hippo pathway by enhancing the expression of TAZ target genes. Finally, miR-125a-5p was overexpressed in late-stage GCs, and positive correlations were observed with its targets EphA2, TAZ, and TEAD2., Conclusion: miR-125a-5p can promote GC growth and invasion by upregulating the expression of EphA2, TAZ, and TEAD2., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Lung adenocarcinoma (LUAD) is a deadly disease with a hallmark of aberrant metabolism. TEA domain 4 (TEAD4) is involved in the progression of several forms of cancer including LUAD. However, the role of TEAD4 in LUAD glucose metabolism is rarely reported as well as its potential mechanisms. Pyruvate kinase isozymes M2 (PKM2), the key regulatory enzymes in glycolysis, was predicted to be a target for TEAD4 by bioinformatics analysis. Thus, we aimed to explore whether TEAD4/PKM2 axis was related to LUAD glucose metabolism and malignant phenotype. The expression level of TEAD4 and PKM2 was measured by quantitative real-time PCR and Western blot. Luciferase reporter assay were employed to verify the effect of TEAD4 on PKM2 promoter as well as TEAD4/PKM2 axis on reporter activity of hypoxia inducible factor-1α (HIF-1α). Glycolysis was investigated according to glucose consumption, lactate production and the extracellular acidification rate. The present study indicated that TEAD4 and PKM2 were upregulated in LUAD and closely related to prognosis. Mechanistic investigations identified that TEAD4 played a key role as a transcription factor and promoted PKM2 transcription and expression, which further altered the reporter activity of HIF-1α and upregulated HIF-1α-targeted glycolytic genes glucose transporter-1 and hexokinase II. Functional assays revealed that TEAD4 and PKM2 affected glycolytic and 2-DG blocked the positive function of TEAD4 and PKM2 on glycolytic. Besides, TEAD4/PKM2 axis affects LUAD cell viability, apoptosis, migration, and invasion. Together, these data provided evidence that both TEAD4 and PKM2 were poor prognosticator. Targeting TEAD4/PKM2 axis might be an effective therapeutic strategy for LUAD., (© 2021 International Federation for Cell Biology.)

Gastric cancer (GC) is one of the most common types of malignancy worldwide and is accompanied by both high mortality and morbidity rates. Homeobox B13 (HOXB13) has been reported to act as a tumor suppressor gene in multiple types of human cancer. The present study aimed to investigate the effects and potential underlying molecular mechanisms of HOXB13 in the progression of GC. The expression of HOXB13 in GC cells was first examined using the Cancer Cell Line Encyclopedia database and subsequently validated in a number of GC cell lines. Following HOXB13 overexpression (Ov‑HOXB13), HGC‑27 cell proliferation was evaluated by colony formation and Cell Counting Kit‑8 assays. Wound healing and Matrigel assays were used to determine the migratory and invasive abilities, respectively. Additionally, cell apoptosis was assessed using TUNEL staining, and the expression of apoptosis‑related proteins was detected by western blot analysis. Subsequently, TEA domain transcription factor 4 (TEAD4) was overexpressed to evaluate the effects on HGC‑27 cell proliferation, migration, invasion and apoptosis following co‑transfection with Ov‑HOXB13. The potential binding sites of HOXB13 on the vestigial‑like family member 4 (VGLL4) promoter were verified using chromatin immunoprecipitation and dual luciferase reporter assays. Moreover, the expression levels of proteins involved in the Hippo signaling pathway were analyzed using western blotting. The results revealed that the expression of HOXB13 was notably lower in GC cells compared with normal gastric cells. The overexpression of HOXB13 significantly inhibited the proliferation, migration and invasion, but promoted the apoptosis of HGC‑27 cells. Moreover, Ov‑HOXB13 downregulated TEAD4 expression. Notably, Ov‑TEAD4 transfection partially reversed the effects of Ov‑HOXB13 on the cellular behaviors of HGC‑27 cells. HOXB13 was also confirmed to bind with the VGLL4 promoter. The knockdown of VGLL4 restored the inhibitory effects of Ov‑HOXB13 on the expression levels of VGLL4 and Hippo pathway signaling proteins. In conclusion, the findings of the present study suggested that Ov‑HOXB13 may suppress the proliferation, migration and invasion, and promote the apoptosis of GC cells through the transcriptional activation of VGLL4 to inhibit the involvement of TEAD4 in the Hippo signaling pathway. These results may provide novel and potent targets for the treatment of GC.

The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA., (© 2021 The Protein Society.)

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAP on or YAP off cancer classes that express or silence YAP, respectively. YAP off solid cancers are neural/neuroendocrine and frequently RB1 -/- , such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAP off or YAP on cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications., Competing Interests: Declaration of interests B.H.L. reports honoraria and non-financial support from AstraZeneca, and has received research funding from Pfizer and AstraZeneca not related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

UDP-glucose pyrophosphorylase 2 (UGP2), the enzyme that synthesizes uridine diphosphate (UDP)-glucose, rests at the convergence of multiple metabolic pathways, however, the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an important role for UGP2 in the maintenance of pancreatic ductal adenocarcinoma (PDAC) growth in both in vitro and in vivo tumor models. We found that transcription of UGP2 is directly regulated by the Yes-associated protein 1 (YAP)-TEA domain transcription factor (TEAD) complex, identifying UGP2 as a bona fide YAP target gene. Loss of UGP2 leads to decreased intracellular glycogen levels and defects in N-glycosylation targets that are important for the survival of PDACs, including the epidermal growth factor receptor (EGFR). These critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer insights into therapeutic options for otherwise intractable PDACs., Competing Interests: Competing interest statement: A.L.W. has received research funding from Oncogenuity. M.S. has received research funds from Puma Biotechnology, AstraZeneca, Daiichi-Sankyo, Immunomedics, Targimmune, and Menarini Ricerche. He is on the scientific advisory board of Menarini Ricerche and the Bioscience Institute, a cofounder of medendi.org, and an employee and stockholder of AstraZeneca. F.M. is a consultant for Daiichi-Sankyo, Pfizer, Amgen, BridgeBio, Olema, OPNA-IO, PMV, Quanta, and Remedy Plan and has received research funding from Daiichi-Sankyo., (Copyright © 2021 the Author(s). Published by PNAS.)

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The Hippo signaling effector, TEAD1 plays an essential role in cardiovascular development. However, a role for TEAD1 in postmitotic cardiomyocytes (CMs) remains incompletely understood. Herein we reported that TEAD1 is required for postmitotic CM survival. We found that adult mice with ubiquitous or CM-specific loss of Tead1 present with a rapid lethality due to an acute-onset dilated cardiomyopathy. Surprisingly, deletion of Tead1 activated the necroptotic pathway and induced massive cardiomyocyte necroptosis, but not apoptosis. In contrast to apoptosis, necroptosis is a pro-inflammatory form of cell death and consistent with this, dramatically higher levels of markers of activated macrophages and pro-inflammatory cytokines were observed in the hearts of Tead1 knockout mice. Blocking necroptosis by administration of necrostatin-1 rescued Tead1 deletion-induced heart failure. Mechanistically, genome-wide transcriptome and ChIP-seq analysis revealed that in adult hearts, Tead1 directly activates a large set of nuclear DNA-encoded mitochondrial genes required for assembly of the electron transfer complex and the production of ATP. Loss of Tead1 expression in adult CMs increased mitochondrial reactive oxygen species, disrupted the structure of mitochondria, reduced complex I-IV driven oxygen consumption and ATP levels, resulting in the activation of necroptosis. This study identifies an unexpected paradigm in which TEAD1 is essential for postmitotic CM survival by maintaining the expression of nuclear DNA-encoded mitochondrial genes required for ATP synthesis.

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.

Background: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions., Results: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on Enhancer TRAM2 , as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness., Conclusions: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.