RESUMO
Macrophage alternative activation is involved in kidney fibrosis. Previous researches have documented that the transcriptional regulators Yes-associated protein (Yap)/transcriptional coactivator with PDZ-binding motif (Taz) are linked to organ fibrosis. However, limited knowledge exists regarding the function and mechanisms of their downstream molecules in regulating macrophage activation and kidney fibrosis. In this paper, we observed that the Hippo pathway was suppressed in macrophages derived from fibrotic kidneys in mice. Knockout of Taz or Tead1 in macrophages inhibited the alternative activation of macrophages and reduced kidney fibrosis. Additionally, by using bone marrow-derived macrophages (BMDMs), we investigated that knockout of Taz or Tead1 in macrophages impeded both cell proliferation and migration. Moreover, deletion of Tead1 reduces p-Smad3 and Smad3 abundance in macrophages. And chromatin immunoprecipitation (ChIP) assays showed that Tead1 could directly bind to the promoter region of Smad3. Collectively, these results indicate that Tead1 knockout in macrophages could reduce TGFß1-induced phosphorylation Smad3 via transcriptional downregulation of Smad3, thus suppressing macrophage alternative activation and IRI-induced kidney fibrosis.
Assuntos
Proteínas de Ligação a DNA , Fibrose , Ativação de Macrófagos , Macrófagos , Camundongos Knockout , Proteína Smad3 , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Animais , Proteína Smad3/metabolismo , Proteína Smad3/genética , Camundongos , Macrófagos/imunologia , Macrófagos/metabolismo , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Rim/patologia , Rim/metabolismo , Transdução de Sinais , Regulação para Cima , Nefropatias/genética , Nefropatias/patologia , Nefropatias/metabolismo , Nefropatias/imunologia , Via de Sinalização Hippo , Modelos Animais de Doenças , Fator de Crescimento Transformador beta1/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Fosforilação , Proliferação de Células , AciltransferasesRESUMO
Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos , Proteínas de Ligação a DNA , Simulação de Dinâmica Molecular , Proteínas Musculares , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Humanos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dissulfetos/química , Dissulfetos/farmacologia , Ligação Proteica , Sítios de Ligação , Linhagem Celular Tumoral , Desenho Assistido por Computador , Desenho de FármacosRESUMO
Despite the importance of radiation therapy as a non-surgical treatment for non-small cell lung cancer (NSCLC), radiation resistance has always been a concern, due to poor patient response and prognosis. Therefore, it is crucial to uncover novel targets to enhance radiotherapy and investigate the mechanisms underlying radiation resistance. Previously, we demonstrated that NRP1 was connected to radiation resistance in NSCLC cells. In the present study, bioinformatics analysis of constructed radiation-resistant A549 and H1299 cell models revealed that transcription coactivator YAP is a significant factor in cell proliferation and metastasis. However, there has been no evidence linking YAP and NRP1 to date. In this research, we have observed that YAP contributes to radiation resistance in NSCLC cells by stimulating cell proliferation, migration, and invasion. Mechanistically, YAP dephosphorylation after NSCLC cell radiation. YAP acts as a transcription co-activator by binding to the transcription factor TEAD4, facilitating TEAD4 to bind to the NRP1 promoter region and thereby increasing NRP1 expression. NRP1 has been identified as a new target gene for YAP/TEAD4. Notably, when inhibiting YAP binds to TEAD4, it inhibits NRP1 expression, and Rescue experiments show that YAP/TEAD4 influences NRP1 to regulate cell proliferation, metastasis and leading to radiation resistance generation. According to these results, YAP/TEAD4/NRP1 is a significant mechanism for radioresistance and can be utilized as a target for enhancing radiotherapy efficacy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Proteínas de Ligação a DNA , Neoplasias Pulmonares , Neuropilina-1 , Tolerância a Radiação , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Carcinoma Pulmonar de Células não Pequenas/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Movimento Celular , Animais , Células A549 , Camundongos Nus , Ligação Proteica , Transcrição Gênica/efeitos da radiação , CamundongosRESUMO
The Hippo pathway is generally understood to inhibit tumor growth by phosphorylating the transcriptional cofactor YAP to sequester it to the cytoplasm and reduce the formation of YAP-TEAD transcriptional complexes. Aberrant activation of YAP occurs in various cancers. However, we found a tumor-suppressive function of YAP in clear cell renal cell carcinoma (ccRCC). Using cell cultures, xenografts, and patient-derived explant models, we found that the inhibition of upstream Hippo-pathway kinases MST1 and MST2 or expression of a constitutively active YAP mutant impeded ccRCC proliferation and decreased gene expression mediated by the transcription factor NF-κB. Mechanistically, the NF-κB subunit p65 bound to the transcriptional cofactor TEAD to facilitate NF-κB-target gene expression that promoted cell proliferation. However, by competing for TEAD, YAP disrupted its interaction with NF-κB and prompted the dissociation of p65 from target gene promoters, thereby inhibiting NF-κB transcriptional programs. This cross-talk between the Hippo and NF-κB pathways in ccRCC suggests that targeting the Hippo-YAP axis in an atypical manner-that is, by activating YAP-may be a strategy for slowing tumor growth in patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Carcinoma de Células Renais , Proliferação de Células , Neoplasias Renais , Proteínas Serina-Treonina Quinases , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Transdução de Sinais , Fatores de Transcrição de Domínio TEA/metabolismo , NF-kappa B/metabolismo , NF-kappa B/genética , Camundongos Nus , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Serina-Treonina Quinase 3RESUMO
Patients with colorectal cancer (CRC) and diabetes share many risk factors. Despite a strong association between diabetes and CRC being widely studied and confirmed, further genetic research is needed. This study found higher AL049796.1 and TEA domain transcription factor 1 (TEAD1) levels (both mRNA and protein) in CRC tissues of diabetic patients compared with nondiabetics, but no significant difference in miR-200b-3p levels. A positive correlation between AL049796.1 and TEAD1 protein existed regardless of diabetes status, whereas miR-200b-3p was only negatively correlated with TEAD1 protein in nondiabetic CRC tissues. In vitro experiments have shown that high glucose (HG) treatment increased AL049796.1 in CRC cells, and AL049796.1 silencing reduced HG-induced proliferation, migration and invasion, as well as connective tissue growth factor, cysteine-rich angiogenic inducer 61, and epidermal growth factor receptor protein expression. Mechanistic investigations indicated that AL049796.1 could mitigate suppression of miR-200b-3p on TEAD1 posttranscriptionally by acting as a competitive binder. In vivo, subcutaneous CRC tumors in streptozotocin (STZ)-induced mice grew significantly faster; AL049796.1 silencing did not affect the growth of subcutaneous CRC tumors but significantly reduced that of STZ-induced mice. Our study suggests that AL049796.1 independently contributes to the risk of CRC in diabetic patients, highlighting its potential as both a therapeutic target and a novel biomarker for CRC among individuals with diabetes.
Assuntos
Proliferação de Células , Neoplasias Colorretais , Glucose , MicroRNAs , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Humanos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Glucose/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Camundongos , Proliferação de Células/efeitos dos fármacos , Masculino , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Movimento Celular/genética , Movimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Feminino , Pessoa de Meia-Idade , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Progressão da Doença , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Camundongos NusRESUMO
RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Proteínas de Ligação a DNA , Neoplasias Hepáticas , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Ubiquitinação , Proteínas de Sinalização YAP , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Humanos , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Sinalização YAP/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição de Domínio TEA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Progressão da Doença , Camundongos Nus , Movimento Celular/genética , Masculino , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Células HEK293 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Feminino , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked ß-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.
Assuntos
Miofibroblastos , Transdução de Sinais , Miofibroblastos/metabolismo , Animais , Camundongos , Humanos , Fibrose/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Transcrição de Domínio TEA/metabolismo , Masculino , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1 , TEAD1 , AXL , NF2 , CTGF , and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF , or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61 / CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.
Assuntos
Neoplasias Encefálicas , Fator de Crescimento do Tecido Conjuntivo , Proteína Rica em Cisteína 61 , Glioblastoma , Fatores de Transcrição , Humanos , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/genética , Neovascularização Patológica/tratamento farmacológico , Fatores de Transcrição de Domínio TEA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Ferroptose , Neoplasias Pulmonares , Proteínas com Domínio T , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Ferroptose/genética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Animais , Linhagem Celular Tumoral , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Camundongos Nus , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Regulação Neoplásica da Expressão Gênica , Células A549 , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismoRESUMO
In pancreatic ductal adenocarcinomas (PDAC), profound hypoxia plays key roles in regulating cancer cell behavior, including proliferation, migration, and resistance to therapies. The initial part of this research highlights the important role played by long noncoding RNA (lncRNA) MKLN1-AS, which is controlled by hypoxia-inducible factor-1 alpha (HIF-1α), in the progression of PDAC. Human samples of PDAC showed a notable increase in MKLN1-AS expression, which was linked to a worse outcome. Forced expression of MKLN1-AS greatly reduced the inhibitory impact on the growth and spread of PDAC cells caused by HIF-1α depletion. Experiments on mechanisms showed that HIF-1α influences the expression of MKLN1-AS by directly attaching to a hypoxia response element in the promoter region of MKLN1-AS.MKLN1-AS acts as a competitive endogenous RNA (ceRNA) by binding to miR-185-5p, resulting in the regulation of TEAD1 expression and promoting cell proliferation, migration, and tumor growth. TEAD1 subsequently enhances the development of PDAC. Our study results suggest that MKLN1-AS could serve as a promising target for treatment and a valuable indicator for predicting outcomes in PDAC. PDAC is associated with low oxygen levels, and the long non-coding RNA MKLN1-AS interacts with TEAD1 in this context.
Assuntos
Carcinoma Ductal Pancreático , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia , MicroRNAs , Neoplasias Pancreáticas , RNA Longo não Codificante , Fatores de Transcrição de Domínio TEA , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Gastric cancer (GC) exhibits significant heterogeneity and its prognosis remains dismal. Therefore, it is essential to investigate new approaches for diagnosing and treating GC. Desmosome proteins are crucial for the advancement and growth of cancer. Plakophilin-2 (PKP2), a member of the desmosome protein family, frequently exhibits aberrant expression and is strongly associated with many tumor types' progression. In this study, we found upregulation of PKP2 in GC. Further correlation analysis showed a notable association between increased PKP2 expression and both tumor stage and poor prognosis in individuals diagnosed with gastric adenocarcinoma. In addition, our research revealed that the Yes-associated protein1 (YAP1)/TEAD4 complex could stimulate the transcriptional expression of PKP2 in GC. Elevated PKP2 levels facilitate activation of the AKT/mammalian target of rapamycin signaling pathway, thereby promoting the malignant progression of GC. By constructing a mouse model, we ultimately validated the molecular mechanism and function of PKP2 in GC. Taken together, these discoveries suggest that PKP2, as a direct gene target of YAP/TEAD4 regulation, has the potential to be used as an indication of GC progression and prognosis. PKP2 is expected to be a promising therapeutic target for GC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ligação a DNA , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Proteínas Musculares , Placofilinas , Neoplasias Gástricas , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Placofilinas/genética , Placofilinas/metabolismo , Fatores de Transcrição de Domínio TEA/metabolismo , Camundongos , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Prognóstico , Linhagem Celular Tumoral , Masculino , Proliferação de Células , Transdução de Sinais , Feminino , Camundongos Nus , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/genéticaRESUMO
The Hippo pathway controls organ size and homeostasis and is linked to numerous diseases, including cancer. The transcriptional enhanced associate domain (TEAD) family of transcription factors acts as a receptor for downstream effectors, namely yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which binds to various transcription factors and is essential for stimulated gene transcription. YAP/TAZ-TEAD facilitates the upregulation of multiple genes involved in evolutionary cell proliferation and survival. TEAD1-4 overexpression has been observed in different cancers in various tissues, making TEAD an attractive target for drug development. The central drug-accessible pocket of TEAD is crucial because it undergoes a post-translational modification called auto-palmitoylation. Crystal structures of the C-terminal TEAD complex with small molecules are available in the Protein Data Bank, aiding structure-based drug design. In this study, we utilized the fragment molecular orbital (FMO) method, molecular dynamics (MD) simulations, shape-based screening, and molecular mechanics-generalized Born surface area (MM-GBSA) calculations for virtual screening, and we identified a novel non-covalent inhibitor-BC-001-with IC50 = 3.7 µM in a reporter assay. Subsequently, we optimized several analogs of BC-001 and found that the optimized compound BC-011 exhibited an IC50 of 72.43 nM. These findings can be used to design effective TEAD modulators with anticancer therapeutic implications.
Assuntos
Simulação de Dinâmica Molecular , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Sítios de Ligação , Descoberta de Drogas/métodos , Ligação Proteica , Simulação de Acoplamento Molecular , Desenho de FármacosRESUMO
Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology.
Assuntos
Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Linhagem Celular Tumoral , Fatores de Transcrição de Domínio TEA/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proliferação de CélulasRESUMO
Thoracic aortic dissection (TAD) is characterized by extracellular matrix (ECM) dysregulation. Aberrations in the ECM stiffness can lead to changes in cellular functions. However, the mechanism by which ECM softening regulates vascular smooth muscle cell (VSMCs) phenotype switching remains unclear. To understand this mechanism, we cultured VSMCs in a soft extracellular matrix and discovered that the expression of microRNA (miR)-143/145, mediated by activation of the AKT signalling pathway, decreased significantly. Furthermore, overexpression of miR-143/145 reduced BAPN-induced aortic softening, switching the VSMC synthetic phenotype and the incidence of TAD in mice. Additionally, high-throughput sequencing of immunoprecipitated RNA indicated that the TEA domain transcription factor 1 (TEAD1) is a common target gene of miR-143/145, which was subsequently verified using a luciferase reporter assay. TEAD1 is upregulated in soft ECM hydrogels in vitro, whereas the switch to a synthetic phenotype in VSMCs decreases after TEAD1 knockdown. Finally, we verified that miR-143/145 levels are associated with disease severity and prognosis in patients with thoracic aortic dissection. ECM softening, as a result of promoting the VSMCs switch to a synthetic phenotype by downregulating miR-143/145, is an early trigger of TAD and provides a therapeutic target for this fatal disease. miR-143/145 plays a role in the early detection of aortic dissection and its severity and prognosis, which can offer information for future risk stratification of patients with dissection.
Assuntos
Dissecção Aórtica , Matriz Extracelular , MicroRNAs , Músculo Liso Vascular , Miócitos de Músculo Liso , Fenótipo , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Dissecção Aórtica/genética , Dissecção Aórtica/metabolismo , Dissecção Aórtica/patologia , Animais , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Humanos , Camundongos , Masculino , Regulação para Baixo/genética , Fatores de Transcrição de Domínio TEA , Transdução de Sinais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação da Expressão Gênica , Feminino , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of cases are considered YAP1/TEAD dependent. Identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs, which may be necessary for YAP1 protein interactions, has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TEAD complex formation and transcriptional activity. We report the discovery and characterization of a novel YAP1/TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TEAD activity. In vivo, MRK-A showed a favorable tolerability profile in mice and demonstrated pharmacokinetics suitable for twice daily oral dosing in preclinical efficacy studies. Importantly, monotherapeutic targeting of YAP1/TEAD in preclinical models generated regressions in a mesothelioma CDX model; however, rapid resistance to therapy was observed. RNA-sequencing of resistant tumors revealed mRNA expression changes correlated with the resistance state and a marked increase of hepatocyte growth factor (HGF) expression. In vitro, exogenous HGF was able to fully rescue cytostasis induced by MRK-A in mesothelioma cell lines. In addition, co-administration of small molecule inhibitors of the MET receptor tyrosine kinase suppressed the resistance generating effect of HGF on MRK-A induced growth inhibition. In this work, we report the structure and characterization of MRK-A, demonstrating potent and specific inhibition of YAP1/TAZ-TEAD-mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling, including factors resulting in acquired drug resistance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento de Hepatócito , Proteínas Proto-Oncogênicas c-met , Transdução de Sinais , Fatores de Transcrição , Proteínas de Sinalização YAP , Humanos , Fator de Crescimento de Hepatócito/metabolismo , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição de Domínio TEA , Proliferação de Células/efeitos dos fármacosRESUMO
The mouse and human embryo gradually loses totipotency before diversifying into the inner cell mass (ICM, future organism) and trophectoderm (TE, future placenta). The transcription factors TFAP2C and TEAD4 with activated RHOA accelerate embryo polarization. Here we show that these factors also accelerate the loss of totipotency. TFAP2C and TEAD4 paradoxically promote and inhibit Hippo signaling before lineage diversification: they drive expression of multiple Hippo regulators while also promoting apical domain formation, which inactivates Hippo. Each factor activates TE specifiers in bipotent cells, while TFAP2C also activates specifiers of the ICM fate. Asymmetric segregation of the apical domain reconciles the opposing regulation of Hippo signaling into Hippo OFF and the TE fate, or Hippo ON and the ICM fate. We propose that the bistable switch established by TFAP2C and TEAD4 is exploited to trigger robust lineage diversification in the developing embryo.
Assuntos
Proteínas de Ligação a DNA , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-2 , Fatores de Transcrição , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Humanos , Transdução de Sinais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Via de Sinalização Hippo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Desenvolvimento Embrionário/genéticaRESUMO
Microcystins (MCs) are toxic to the central nervous system of mammals. However, the direct toxicity of MCs on mammalian brain cells and the involved molecular mechanisms are not fully elucidated. Here, we incubated primary astrocytes, the major glial cell-type in the brain, with 0-12.5⯵M concentrations of MC-LR for 48â¯h, and the impairment was evaluated. We found that MC-LR caused significant increases in the cell viability at the range of 0.05-1⯵M concentrations with the highest density at 0.1⯵M concentration. Treatment with 0.1⯵M MC-LR induced YAP nuclear translocation and decreased the ratio of p-YAP to YAP. It also decreased mRNA levels of the upstream regulator (AMOT), and enhanced expressions of YAP interacted genes (Egfr, Tead1, and Ctgf) in primary astrocytes. Overexpression of AMOT significantly attenuated the increase of MC-LR-induced astrocyte proliferation and the expression of YAP downstream genes. These results indicate that Hippo signaling contributed to MC-LR-caused astrocyte proliferation. Further, reactive astrogliosis was observed in the mice brain after MC-LR exposure to environmentally relevant concentrations (20 or 100⯵g/L) through drinking water for 16 weeks. Pathological observations revealed that 100⯵g/L MC-LR exposure caused neuronal damages with characteristics of shrunken or vacuolation in the region of the cerebral cortex, striatum and cerebellum. These results were accompanied with increased oxidative stress and inflammatory response. Our data reveal the potential astrocytic mechanisms in MC-induced neurotoxicity and raise an alarm for neurodegenerative disease risk following daily exposure to MC-LR.
Assuntos
Astrócitos , Proliferação de Células , Via de Sinalização Hippo , Toxinas Marinhas , Microcistinas , Transdução de Sinais , Microcistinas/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Via de Sinalização Hippo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Sinalização YAP , Sobrevivência Celular/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Receptores ErbB/metabolismo , Fatores de Transcrição de Domínio TEA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
TEAD transcription factors play a central role in the Hippo signaling pathway. In this study, we focused on transcriptional enhancer factor TEF-3 (TEAD4), exploring its regulation by the deubiquitinase OTU domain-containing protein 6A (OTUD6A). We identified OTUD6A as a TEAD4-interacting deubiquitinase, positively influencing TEAD-driven transcription without altering TEAD4 stability. Structural analyses revealed specific interaction domains: the N-terminal domain of OTUD6A and the YAP-binding domain of TEAD4. Functional assays demonstrated the positive impact of OTUD6A on the transcription of YAP-TEAD target genes. Despite no impact on TEAD4 nuclear localization, OTUD6A selectively modulated nuclear interactions, enhancing YAP-TEAD4 complex formation while suppressing VGLL4 (transcription cofactor vestigial-like protein 4)-TEAD4 interaction. Critically, OTUD6A facilitated YAP-TEAD4 complex binding to target gene promoters. Our study unveils the regulatory landscape of OTUD6A on TEAD4, providing insights into diseases regulated by YAP-TEAD complexes.
Assuntos
Proteínas de Ligação a DNA , Proteínas Musculares , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Células HEK293 , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/química , Transcrição Gênica , Ligação Proteica , Proteínas de Sinalização YAP/metabolismo , Proteínas de Sinalização YAP/genética , Regiões Promotoras GenéticasRESUMO
The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.
Assuntos
Lipoilação , Simulação de Dinâmica Molecular , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Humanos , Aciltransferases/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Regulação Alostérica/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição de Domínio TEA/química , Fatores de Transcrição de Domínio TEA/metabolismo , Transativadores/metabolismo , Transativadores/química , Transativadores/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/química , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/química , Proteínas de Sinalização YAP/metabolismoRESUMO
INTRODUCTION: This study attempted to investigate how proprotein convertase subtilisin/kexin type 9 (PCSK9) influences the stemness of stomach adenocarcinoma (STAD) cells. METHODS: CCK-8 and sphere-formation assays were used to detect cell viability and stemness. qRT-PCR and Western blot were used to detect PCSK9 and TEAD4 expression. The binding relationship was verified by dual-luciferase and chromatin immunoprecipitation assays. The effect of TEAD4 activating PCSK9 on the stemness of STAD cells was detected by bioinformatics, BODIPY 493/503, Oil red O, Western blot, and kits. In vivo experiments verified the role of the TEAD4/PCSK9 axis in tumor formation in nude mice. RESULTS: PCSK9 and TEAD4 were highly expressed in STAD. PCSK9 was enriched in the fatty acid metabolism (FAM) pathway. PCSK9 activated the fatty acid metabolism and promoted the proliferation and stemness of STAD cells. TEAD4 as a transcription factor upstream of PCSK9, cell experiments revealed that knockdown of PCSK9 inhibited STAD cell stemness, whereas further addition of fatty acid inhibitors could attenuate the promoting effect on STAD cell stemness brought by STAD overexpression. Rescue experiments showed overexpressed PCSK9 exerted an inhibitory effect on the stemness of STAD cells brought by TEAD4 knockdown. The hypothesis that TEAD4/PCSK9 axis can promote STAD cell growth was confirmed by in vivo experiments. CONCLUSION: Transcription factor TEAD4 could activate PCSK9 to promote the stemness of STAD cells through FAM. These results added weight to the assumption that TEAD4/PCSK9 axis has the potential to be the therapeutic target that inhibits cancer stem cell in STAD.