RESUMEN
Ferroptosis has attracted extensive interest from cancer researchers due to its substantial potential as a therapeutic target. The role of LATS2, a core component of the Hippo pathway cascade, in ferroptosis initiation in hepatoblastoma (HB) has not yet been investigated. Furthermore, the underlying mechanism of decreased LATS2 expression remains largely unknown. In the present study, we demonstrated decreased LATS2 expression in HB and that LATS2 overexpression inhibits HB cell proliferation by inducing ferroptosis. Increased LATS2 expression reduced glycine and cysteine concentrations via the ATF4/PSAT1 axis. Physical binding between YAP1/ATF4 and the PSAT1 promoter was confirmed through ChIPâqPCR. Moreover, METTL3 was identified as the writer of the LATS2 mRNA m6A modification at a specific site in the 5' UTR. Subsequently, YTHDF2 recognizes the m6A modification site and recruits the CCR4-NOT complex, leading to its degradation by mRNA deadenylation. In summary, N6-methyladenosine modification of LATS2 facilitates its degradation. Reduced LATS2 expression promotes hepatoblastoma progression by inhibiting ferroptosis through the YAP1/ATF4/PSAT1 axis. Targeting LATS2 is a potential strategy for HB therapy.
Asunto(s)
Factor de Transcripción Activador 4 , Adenosina , Ferroptosis , Hepatoblastoma , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Proteínas Señalizadoras YAP , Humanos , Hepatoblastoma/metabolismo , Hepatoblastoma/genética , Hepatoblastoma/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Señalizadoras YAP/metabolismo , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 4/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Ferroptosis/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Línea Celular Tumoral , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Proliferación Celular , Ratones Desnudos , Ratones , Regulación Neoplásica de la Expresión Génica , MetiltransferasasRESUMEN
Although Hippo-YAP/TAZ pathway involvement has been extensively studied in the development of certain cancers, the involvement of this cascade in kidney cancer progression is not well-established and, therefore, will be the focus of this review. Renal cell carcinoma (RCC), the most prevalent kidney tumor subtype, has a poor prognosis and a high mortality rate. Core Hippo signaling inactivation (e.g., LATS kinases) leads to the nuclear translocation of YAP/TAZ where they bind to co-transcriptional factors such as TEAD promoting transcription of genes which initiates various fibrotic and neoplastic diseases. Loss of expression of LATS1/2 kinase and activation of YAP/TAZ correlates with poor survival in RCC patients. Renal-specific ablation of LATS1 in mice leads to the spontaneous development of several subtypes of RCC in a YAP/TAZ-dependent manner. Genetic and pharmacological inactivation of YAP/TAZ reverses the oncogenic potential in LATS1-deficient mice, highlighting the therapeutic benefit of network targeting in RCC. Here, we explore the unique upstream controls and downstream consequences of the Hippo-YAP/TAZ pathway deregulation in renal cancer. This review critically evaluates the current literature on the role of the Hippo pathway in RCC progression and highlights the recent scientific evidence designating YAP/TAZ as novel therapeutic targets against kidney cancer.
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NLRP3 inflammasome activation, essential for cytokine secretion and pyroptosis in response to diverse stimuli, is closely associated with various diseases. Upon stimulation, NLRP3 undergoes subcellular membrane trafficking and conformational rearrangements, preparing itself for inflammasome assembly at the microtubule-organizing center (MTOC). Here, we elucidate an orchestrated mechanism underlying these ordered processes using human and murine cells. Specifically, NLRP3 undergoes palmitoylation at two sites by palmitoyl transferase zDHHC1, facilitating its trafficking between subcellular membranes, including the mitochondria, trans-Golgi network (TGN), and endosome. This dynamic trafficking culminates in the localization of NLRP3 to the MTOC, where LATS1/2, pre-recruited to MTOC during priming, phosphorylates NLRP3 to further facilitate its interaction with NIMA-related kinase 7 (NEK7), ultimately leading to full NLRP3 activation. Consistently, Zdhhc1-deficiency mitigated LPS-induced inflammation and conferred protection against mortality in mice. Altogether, our findings provide valuable insights into the regulation of NLRP3 membrane trafficking and inflammasome activation, governed by palmitoylation and phosphorylation events.
Asunto(s)
Inflamasomas , Lipoilación , Proteína con Dominio Pirina 3 de la Familia NLR , Transporte de Proteínas , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Inflamasomas/metabolismo , Inflamasomas/genética , Animales , Fosforilación , Humanos , Ratones , Células HEK293 , Quinasas Relacionadas con NIMA/metabolismo , Quinasas Relacionadas con NIMA/genética , Aciltransferasas/metabolismo , Aciltransferasas/genética , Centro Organizador de los Microtúbulos/metabolismo , Ratones Endogámicos C57BL , Red trans-Golgi/metabolismo , Ratones Noqueados , Endosomas/metabolismo , Mitocondrias/metabolismoRESUMEN
The degenerative process of follicular atresia in hens naturally commences in granulosa cells, significantly impacting laying hens' reproductive performance. Past studies suggested that granulosa cell autophagy and apoptosis work together to cause follicular atresia. Recent research indicates that miRNA regulates granulosa autophagy and apoptosis, which contributes to the development of follicular atresia. However, the role of miR-302c-3p in follicular atresia and development remains unclear. In this study with the RNA-seq approach, we found that miR-302c-3p expression was significantly decreased in atrophic follicles, suggesting its involvement in the follicular atresia process. Following this, we performed in vitro studies to confirm that miR-302c-3p inhibits autophagy and apoptosis in chicken granulosa cells. Mechanistically, LATS2 is considered as the putative target gene of miR-302c-3p, and it has been demonstrated that LATS2 exerts a positive regulatory role in the modulation of autophagy and apoptosis in chicken granulosa cells. Furthermore, we verified the regulatory function of miR-302c-3p in chicken granulosa cells via the LATS2-YAP signaling pathway. Our results collectively demonstrates that miR-302c-3p targets LATS2 to modulate the YAP signaling pathway, impacting autophagy and apoptosis in granulosa cells leading to follicular atresia.
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Apoptosis , Autofagia , Pollos , Células de la Granulosa , MicroARNs , Animales , Femenino , Células de la Granulosa/fisiología , Células de la Granulosa/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Pollos/genética , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación de la Expresión Génica , Proteínas Señalizadoras YAP/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Atresia Folicular/genética , Atresia Folicular/fisiologíaRESUMEN
The Hippo pathway plays a crucial role in cell proliferation and differentiation during tumorigenesis, tissue homeostasis and early embryogenesis. Scaffold proteins from the ezrin-radixin-moesin (ERM) family, including neurofibromin 2 (NF2; Merlin), regulate the Hippo pathway through cell polarity. However, the mechanisms underlying Hippo pathway regulation via cell polarity in establishing outer cells remain unclear. In this study, we generated artificial Nf2 mutants in the N-terminal FERM domain (L64P) and examined Hippo pathway activity by assessing the subcellular localization of YAP1 in early embryos expressing these mutant mRNAs. The L64P-Nf2 mutant inhibited NF2 localization around the cell membrane, resulting in YAP1 cytoplasmic translocation in the polar cells. L64P-Nf2 expression also disrupted the apical centralization of both large tumor suppressor 2 (LATS2) and ezrin in the polar cells. Furthermore, Lats2 mutants in the FERM binding domain (L83K) inhibited YAP1 nuclear translocation. These findings demonstrate that NF2 subcellular localization mediates cell polarity establishment involving ezrin centralization. This study provides previously unreported insights into how the orchestration of the cell-surface components, including NF2, LATS2 and ezrin, modulates the Hippo pathway during cell polarization.
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Proteínas Adaptadoras Transductoras de Señales , Polaridad Celular , Proteínas del Citoesqueleto , Vía de Señalización Hippo , Neurofibromina 2 , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Proteínas Señalizadoras YAP , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Animales , Ratones , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Transducción de Señal , Embrión de Mamíferos/metabolismo , Mutación/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Transporte de Proteínas , Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genéticaRESUMEN
BACKGROUND: NSCLC is one of the most common causes of death. The hypoxia microenvironment contributes to cancer progression. The purpose was to explore the effects and mechanism of melittin on NSCLC cells in the hypoxic microenvironment. METHODS: NSCLC cell lines (A549 and H1299) were cultured in normoxia or hypoxia conditions with or without melittin treatment. The viability of the cells was detected via MTT assay and the proliferation ability was evaluated by EdU assay. QRT-PCR was performed to evaluate GLUT1, LDHA, HK2, VEGF and LATS2 mRNA levels. Glucose transport was assessed by the 2-NBDG uptake assay. The angiogenesis was determined by the tubule formation assay. The protein expressions of GLUT1, LDHA, HK2, VEGF, LATS2, YAP, p-YAP and HIF-1α were detected via western blotting assay. The tumor formation assay was conducted to examine the roles of melittin and LATS2 in vivo. RESULTS: Melittin inhibited hypoxia-induced cell viability, proliferation, glycolysis and angiogenesis as well as suppressed YAP binding to HIF-1α in NSCLC. Melittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2, ultimately inhibiting cancer progression of NSCLC. Moreover, melittin suppressed tumor growth via up-regulation of LATS2 in vivo. CONCLUSION: Melittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2 to contribute to the development of NSCLC. Therefore, melittin is expected to become a potential prognostic drug for the therapy of NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Pulmonares , Meliteno , Neovascularización Patológica , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Regulación hacia Arriba , Proteínas Señalizadoras YAP , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Glucólisis/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Señalizadoras YAP/metabolismo , Meliteno/farmacología , Meliteno/uso terapéutico , Línea Celular Tumoral , Factores de Transcripción/metabolismo , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fosfoproteínas/metabolismo , AngiogénesisRESUMEN
Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.
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Proteínas Adaptadoras Transductoras de Señales , Diferenciación Celular , Proteínas Serina-Treonina Quinasas , Células de Sertoli , Proteínas Supresoras de Tumor , Proteínas Señalizadoras YAP , Animales , Células de Sertoli/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Masculino , Ratones , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Diferenciación Celular/fisiología , Ratones Noqueados , Transducción de Señal , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Testículo/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Transactivadores/metabolismo , Transactivadores/genéticaRESUMEN
Cell-cell mechanotransduction regulates tissue development and homeostasis. α-catenin, the core component of adherens junctions, functions as a tension sensor and transducer by recruiting vinculin and transducing signals that influence cell behaviors. α-catenin/vinculin complex-mediated mechanotransduction regulates multiple pathways, such as Hippo pathway. However, their associations with the α-catenin-based tension sensors at cell junctions are still not fully addressed. Here, we uncovered the TRIP6/LATS1 complex co-localizes with α-catenin/vinculin at both bicellular junctions (BCJs) and tricellular junctions (TCJs). The localization of TRIP6/LATS1 complex to both TCJs and BCJs requires ROCK1 and α-catenin. Treatment by cytochalasin B, Y-27632 and blebbistatin all impaired the BCJ and TCJ junctional localization of TRIP6/LATS1, indicating that the junctional localization of TRIP6/LATS1 is mechanosensitive. The α-catenin/vinculin/TRIP6/LATS1 complex strongly localized to TCJs and exhibited a discontinuous button-like pattern on BCJs. Additionally, we developed and validated an α-catenin/vinculin BiFC-based mechanosensor that co-localizes with TRIP6/LATS1 at BCJs and TCJs. The mechanosensor exhibited a discontinuous distribution and motile signals at BCJs. Overall, our study revealed that TRIP6 and LATS1 are novel compositions of the tension sensor, together with the core complex of α-catenin/vinculin, at both the BCJs and TCJs.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Vinculina , alfa Catenina , alfa Catenina/metabolismo , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Vinculina/metabolismo , Mecanotransducción Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Uniones Intercelulares/metabolismo , Células HEK293 , Quinasas Asociadas a rho/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Ovarian cancer (OC) is a frequent malignancy of the female reproductive system. Recently, the aberrant expression of numerous lncRNAs has been confirmed as a key factor for cancer development. The regulatory role of PAX8-AS1 in some cancers has been investigated, but its role in OC progression remains unclear. This study focuses on the role and molecular mechanism of PAX8-AS1 in the malignant progression of OC. METHODS: Bioinformatics means were adopted to analyze the expression of PAX8-AS1, microRNA-25-3p, and LATS2 in OC tissues and the binding sites between the three. qRT-PCR was employed to determine the expression of these genes in OC cells. CCK-8, colony formation, scratch healing, and Transwell assays were used to see cell viability, proliferation, migration, and invasion, respectively. Fluorescence in situ Hybridization was performed to probe the subcellular localization of PAX8-AS1. Western blot was applied to evaluate the expression and phosphorylation levels of YAP and TAZ, and an immunofluorescence assay was used to detect the translocation of them. Dual luciferase assay was applied to validate the binding relationship between PAX8-AS1 and microRNA-25-3p, as well as between microRNA-25-3p and LATS2. RESULTS: PAX8-AS1 and LATS2 were lowly expressed. MicroRNA-25-3p was highly expressed in OC. PAX8-AS1 was expressed in cytoplasm and regulated LATS2 expression by sponging microRNA-25-3p. Overexpressing PAX8-AS1 can suppress the malignant behaviors of OC cells, whereas treatment with microRNA-mimic can reverse these results. In addition, the phosphorylation levels of YAP and TAZ increased upon oe-LATS2 treatment, and oe-LATS2 could promote YAP and TAZ translocate from the nucleus to cytoplasm. Rescue experiments demonstrated that sh-PAX8-AS1 fostered malignant progression of OC, which was reversed by simultaneous oe-LATS2. CONCLUSION: In summary, PAX8-AS1/microRNA-25-3p/LATS2 regulated the malignant progression of OC through Hippo signaling, which suggested that PAX8-AS1/microRNA-25-3p/LATS2 axis may be a novel target for OC treatment.
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The cancer epigenome has been studied in cells cultured in two-dimensional (2D) monolayers, but recent studies highlight the impact of the extracellular matrix and the three-dimensional (3D) environment on multiple cellular functions. Here, we report the physical, biochemical, and genomic differences between T47D breast cancer cells cultured in 2D and as 3D spheroids. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, as well as altered expression of ~2000 genes, the majority of which become repressed. Hi-C analysis reveals that cells in 3D are enriched for regions belonging to the B compartment, have decreased chromatin-bound CTCF and increased fusion of topologically associating domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, thereby likely causing the observed TAD fusions. 3D cells show higher chromatin binding of progesterone receptor (PR), leading to an increase in the number of hormone-regulated genes. This effect is in part mediated by LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.
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Neoplasias de la Mama , Factor de Unión a CCCTC , Cromatina , Proteínas Serina-Treonina Quinasas , Humanos , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Cromatina/metabolismo , Cromatina/genética , Femenino , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Vía de Señalización HippoRESUMEN
We tried to elucidate the possible roles of maternal embryonic leucine pull chain kinase (MELK) in lung adenocarcinoma (LUAD) growth and metastasis. Differentially expressed genes in LUAD samples were analysed by the GEPIA database. Clinical tissue samples and cells were collected for MELK, EZH2 and LATS2 expression determination. Co-IP assay was used to verify the interaction between EZH2 and MELK; CHX tracking assay and ubiquitination assay detected the degradation of MELK on EZH2 ubiquitination. ChIP assay detected the enrichment of EZH2 and H3K27me3 on the LATS2 promoter region. LUAD cells were selected for in vitro validation, and the tumorigenic ability of LUAD cells was also observed in a transplantation tumour model of LUAD nude mice. MELK and EZH2 were highly expressed in LUAD samples, while LATS2 was lowly expressed. MELK interacted with EZH2 to inhibit its ubiquitination degradation; EZH2 elevated H3K27me3 modification in the LATS2 promoter to lower LATS2 expression. Silencing MELK or EZH2 or overexpressing LATS2 restrained LUAD cell proliferation and invasion, and facilitated their apoptosis. Silencing MELK or EZH2 or overexpressing LATS2 suppressed tumour formation in nude mice. This study demonstrated that MELK aggravated LUAD by upregulating EZH2 and downregulating LATS2.
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Adenocarcinoma del Pulmón , Proliferación Celular , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica , Histonas , Neoplasias Pulmonares , Ratones Desnudos , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor , Ubiquitinación , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Histonas/metabolismo , Ratones , Proliferación Celular/genética , Metilación , Línea Celular Tumoral , Regiones Promotoras Genéticas/genética , Apoptosis/genética , Femenino , MasculinoRESUMEN
Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s.
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Células Epiteliales Alveolares , Diferenciación Celular , Células Madre Pluripotentes Inducidas , Humanos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Transducción de Señal , Células Cultivadas , Transcriptoma/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
Breast cancer is one of the most common cancers threatening women's health. Our previous study found that silibinin induced the death of MCF-7 and MDA-MB-231 human breast cancer cells. We noticed that silibinin-induced cell damage was accompanied by morphological changes, including the increased cell aspect ratio (cell length/width) and decreased cell area. Besides, the cytoskeleton is also destroyed in cells treated with silibinin. YAP/TAZ, a mechanical signal sensor interacted with extracellular pressure, cell adhesion area and cytoskeleton, is also closely associated with cell survival, proliferation and migration. Thus, the involvement of YAP/TAZ in the cytotoxicity of silibinin in breast cancer cells has attracted our interests. Excitingly, we find that silibinin inhibits the nuclear translocation of YAP/TAZ in MCF-7 and MDA-MB-231 cells, and reduces the mRNA expressions of YAP/TAZ target genes, ACVR1, MnSOD and ANKRD. More importantly, expression of YAP1 gene is negatively correlated with the survival of the patients with breast cancers. Molecular docking analysis reveals high probabilities for binding of silibinin to the proteins in the YAP pathways. DARTS and CETSA results confirm the binding abilities of silibinin to YAP and LATS. Inhibiting YAP pathway either by addition of verteporfin, an inhibitor of YAP/TAZ-TEAD, or by transfection of si-RNAs targeting YAP or TAZ further enhances silibinin-induced cell damage. While enhancing YAP activity by silencing LATS1/2 or overexpressing YAPS127/397A, an active form of YAP, attenuates silibinin-induced cell damage. These findings demonstrate that inhibition of the YAP/TAZ pathway contributes to cytotoxicity of silibinin in breast cancers, shedding lights on YAP/TAZ-targeted cancer therapies.
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Neoplasias de la Mama , Silibina , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Femenino , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Simulación del Acoplamiento Molecular , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Silibina/farmacología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/antagonistas & inhibidores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/efectos de los fármacos , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Verteporfina/farmacología , Proteínas Señalizadoras YAP/antagonistas & inhibidores , Proteínas Señalizadoras YAP/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismoRESUMEN
The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.
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Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Proteínas Señalizadoras YAP , Animales , Humanos , Ratones , Proliferación Celular , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAP/agonistas , Proteínas Señalizadoras YAP/efectos de los fármacos , Proteínas Señalizadoras YAP/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
BACKGROUND: Gastric cancer is a leading cause of cancer-related deaths worldwide. Prognostic assessments are typically based on the tumor-node-metastasis (TNM) staging system, which does not account for the molecular heterogeneity of this disease. LATS2, a tumor suppressor gene involved in the Hippo signaling pathway, has been identified as a potential prognostic biomarker in gastric cancer. AIM: To construct and validate a nomogram model that includes LATS2 expression to predict the survival prognosis of advanced gastric cancer patients following radical surgery, and compare its predictive performance with traditional TNM staging. METHODS: A retrospective analysis of 245 advanced gastric cancer patients from the Fourth Hospital of Hebei Medical University was conducted. The patients were divided into a training group (171 patients) and a validation group (74 patients) to develop and test our prognostic model. The performance of the model was determined using C-indices, receiver operating characteristic curves, calibration plots, and decision curves. RESULTS: The model demonstrated a high predictive accuracy with C-indices of 0.829 in the training set and 0.862 in the validation set. Area under the curve values for three-year and five-year survival prediction were significantly robust, suggesting an excellent discrimination ability. Calibration plots confirmed the high concordance between the predictions and actual survival outcomes. CONCLUSION: We developed a nomogram model incorporating LATS2 expression, which significantly outperformed conventional TNM staging in predicting the prognosis of advanced gastric cancer patients postsurgery. This model may serve as a valuable tool for individualized patient management, allowing for more accurate stratification and improved clinical outcomes. Further validation in larger patient cohorts will be necessary to establish its generalizability and clinical utility.
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The ribose nucleic acid (RNA)-binding motif protein 24 (RBM24) has been recognized as a critical regulatory protein in various types of tumors. However, its specific role in glioblastoma (GBM) has not been thoroughly investigated. The objective of this study is to uncover the role of RBM24 in GBM and understand the underlying mechanism. The expression of RBM24 in GBM was initially analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA). Subsequently, the RBM24 expression levels in clinical samples of GBM were examined, and the survival curves of GBM patients were plotted based on high- and low-expression levels of RBM24 using Kaplan-Meier (KM) plotter. In addition, RBM24 knockdown cell lines and overexpression vectors were created to assess the effects on proliferation, apoptosis, and invasion abilities. Finally, the binding level of RBM24 protein to LATS1 messenger RNA (mRNA) was determined by RNA immunoprecipitation (RIP) assay, and the expression levels of RBM24 and LATS1 were measured through quantitative reverse-transcriptase-polymerase chain reaction (qRT-PCR) and Western blot (WB). Our data revealed a significant decrease in RBM24 mRNA and protein levels in GBM patients, indicating that those with low RBM24 expression had a worse prognosis. Overexpression of RBM24 led to inhibited cell proliferation, reduced invasion, and increased apoptosis in LN229 and U87 cells. In addition, knocking down LATS1 partially reversed the effects of RBM24 on cell proliferation, invasion, and apoptosis in GBM cells. In vivo xenograft model further demonstrated that RBM24 overexpression reduced the growth of subcutaneous tumors in nude mice, accompanied by a decrease in Ki-67 expression and an increase in apoptotic events in tumor tissues. There was also correlation between RBM24 and LATS1 protein expression in the xenograft tumors. RBM24 functions to stabilize LATS1 mRNA, thereby inhibiting the proliferation, suppressing invasion, and promoting apoptosis in GBM cells.
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Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.
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Artefactos , Ganglios Sensoriales , Herpesvirus Humano 1 , Células Receptoras Sensoriales , Análisis de Secuencia de ARN , Análisis de Expresión Génica de una Sola Célula , Latencia del Virus , Animales , Ratones , Muerte Celular , Conjuntos de Datos como Asunto , Ganglios Sensoriales/inmunología , Ganglios Sensoriales/patología , Ganglios Sensoriales/virología , Herpes Simple/inmunología , Herpes Simple/patología , Herpes Simple/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/aislamiento & purificación , MicroARNs/análisis , MicroARNs/genética , Reproducibilidad de los Resultados , ARN Viral/análisis , ARN Viral/genética , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/virologíaRESUMEN
BACKGROUND: LATS1/2 are frequently mutated and down-regulated in endometrial cancer (EC), but the contributions of LATS1/2 in EC progression remains unclear. Impaired antigen presentation due to mutations or downregulation of the major histocompatibility complex class I (MHC-I) has been implicated in tumor immune evasion. Herein, we elucidate the oncogenic role that dysregulation of LATS1/2 in EC leads to immune evasion through the down-regulation of MHC-I. METHODS: The mutation and expression as well as the clinical significance of LATS1/2 in EC was assessed in the TCGA cohort and our sample cohort. CRISPR-Cas9 was used to construct knockout cell lines of LATS1/2 in EC. Differentially expressed genes were analyzed by RNA-seq. The interaction between LATS1/2 and STAT1 was verified using co-immunoprecipitation and GST pull-down assays. Mass spectrometry, in vitro kinase assays, ChIP-qPCR, flow cytometry, immunohistochemistry, immunofluorescence and confocal microscopy were performed to investigate the regulation of LATS1/2 on MHC-I through interaction with and phosphorylate STAT1. The killing effect of activated PBMCs on EC cells were used to monitor anti-tumor activity. RESULTS: Here, we demonstrate that LATS1/2 are frequently mutated and down-regulated in EC. Moreover, LATS1/2 loss was found to be associated with a significant down-regulation of MHC-I, independently of the Hippo-YAP pathway. Instead, LATS1/2 were found to directly interact with and phosphorylate STAT1 at Ser727, a crucial transcription factor for MHC-I upregulation in response to interferon-gamma (IFN-γ) signaling, to promote STAT1 accumulating and moving into the nucleus to enhance the transcriptional activation of IRF1/NLRC5 on MHC-I. Additionally, the loss of LATS1/2 was observed to confer increased resistance of EC cells to immune cell-mediated killing and this resistance could be reversed by over-expression of MHC-I. CONCLUSION: Our findings indicate that dysregulation of LATS1/2 in EC leads to immune evasion through the down-regulation of MHC-I, leading to the suppression of infiltrating activated CD8 + T cells and highlight the importance of LATS1/2 in IFN-γ signaling-mediated tumor immune response, suggesting that LATS1/2 is a promising target for immune checkpoint blockade therapy in EC.
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Neoplasias Endometriales , Escape del Tumor , Femenino , Humanos , Antígenos de Histocompatibilidad Clase I , Presentación de Antígeno , Proteínas Serina-Treonina Quinasas/genética , Neoplasias Endometriales/genética , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype, IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI), a specific LATS inhibitor, or shRNA targeting LATS1/2 significantly reduced clonogenicity, increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore, depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together, our results suggest that LATS could be one of the key factors contributing to the rapid proliferation, reduced apoptosis, and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM.
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Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de Fusión bcr-abl/genética , Proteínas Serina-Treonina Quinasas , Células K562 , Apoptosis , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) cell-intrinsic programmed death 1 (PD-1) promotes tumor progression. However, the mechanisms that regulate its expression are unclear. This study investigated the impact of alpha-fetoprotein (AFP) on HCC cell-intrinsic PD-1 expression. METHODS: The expression of PD-1 and AFP at the gene and protein levels was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB). Proteins interacting with AFP were examined by co-immunoprecipitation (CO-IP). Chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays were used to identify transcription-enhanced association domain 1 (TEAD1) binding to the promoter of PD-1. RESULTS: The expression of HCC cell-intrinsic PD-1 was positively correlated with AFP. Mechanistically, AFP inhibited the phosphorylation of large tumor suppressor 2 (LATS2) and yes-associated protein (YAP). As a result, YAP is transferred to the nucleus and forms a transcriptional complex with TEAD1, promoting PD-1 transcription by binding to its promoter. CONCLUSION: AFP is an upstream regulator of the HCC cell-intrinsic PD-1 and increases PD-1 expression via the LATS2/YAP/TEAD1 axis. GENERAL: Our findings provide insight into the mechanisms of HCC development and offer new ideas for further in-depth studies of HCC.