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In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.
Assuntos
Antioxidantes , Aspergillus nidulans , Antioxidantes/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Peróxido de Hidrogênio/metabolismo , Cisteína/metabolismo , Arabinose , Estresse Oxidativo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Etanol , Carbono , OxirreduçãoRESUMO
Ankyrin repeat and KH domain-containing protein 1, ANKHD1, has been identified as a regulator of signaling pathways and cellular processes of relevance in carcinogenesis. However, the role of ANKHD1 in breast cancer remains unclear. The aim of the present study was to characterize the expression pattern and involvement of ANKHD1 in the malignant phenotype of breast cancer cell lines and to investigate the clinical relevance of ANKHD1 in a breast cancer context. Gene and protein expressions were assessed in the cell lines by quantitative reverse transcription PCR and Western blot analysis, respectively, and ANKHD1 silencing through siRNA transfection was conducted for further in vitro functional assays. The expression of ANKHD1 was identified in non-tumorigenic breast epithelium and breast cancer cell lines, but differences in cellular localization were found among the neoplasia subtypes. ANKHD1 silencing reduced the viability, clonogenicity, and migration of triple-negative breast cancer (TNBC) cells. Bioinformatics analyses demonstrated that patients with triple-negative basal-like 2 and mesenchymal breast cancer subtypes had high ANKHD1 expression associated with poor recurrence-free survival. Therefore, these data indicate that ANKHD1 relevance in breast cancer varies among its subtypes, indicating the importance of ANKHD1 in TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Fenótipo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
OBJECTIVE: Accumulating evidence has been revealed that miR-590 is involved in the progression and carcinogenesis of various cancers. However, the molecular mechanism of miR-590 in non-small-cell lung cancer (NSCLC) remains unclear. METHODS: Quantitative reverse transcription-PCR (qRT-PCR), western blot, MTT, and transwell assay were applied to investigate the functional role of miR-590 in this study. Dual luciferase reporter assay was utilized to investigate the interaction between YAP1 and miR-590 expression. Cells transfected with miR-590 mimic or inhibitor were subjected to western blot to investigate the role of Wnt/ß-catenin signaling in NSCLC modulated by miR-590. RESULTS: MiR-590 was down-regulated in NSCLC tissues and cells. Kaplan-Meier analysis found that the higher expression of miR-590 in NSCLC patients, the more improved survival rate of NSCLC patients. Over-expression of miR-590 inhibited NSCLC cell proliferation, migration, and invasion. Moreover, increasing miR-590 suppressed Yes-associated protein 1 (YAP1) expression and inhibited the Wnt/ß-catenin pathway in NSCLC cells. Furthermore, miR-590 was negatively correlated with YAP1 expression. CONCLUSION: These findings demonstrated that the miR-590/YAP1 axis exerted an important role in the progression of NSCLC, suggesting that miR-590 might be the appealing prognostic marker for NSCLC treatment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/fisiologia , Via de Sinalização Wnt/fisiologia , Proteínas de Sinalização YAP/fisiologia , Progressão da Doença , Humanos , Células Tumorais CultivadasRESUMO
BACKGROUND: Hydrogen peroxide (H2 O2 ) is a key reactive oxygen species (ROS) generated during appendage regeneration among vertebrates. However, its role during tail regeneration in axolotl as redox signaling molecule is unclear. RESULTS: Treatment with exogenous H2 O2 rescues inhibitory effects of apocynin-induced growth suppression in tail blastema cells leading to cell proliferation. H2 O2 also promotes recruitment of immune cells, regulate the activation of AKT kinase and Agr2 expression during blastema formation. Additionally, ROS/H2 O2 regulates the expression and transcriptional activity of Yap1 and its target genes Ctgf and Areg. CONCLUSIONS: These results show that H2 O2 is necessary and sufficient to promote tail regeneration in axolotls. Additionally, Akt signaling and Agr2 were identified as ROS targets, suggesting that ROS/H2 O2 is likely to regulate epimorphic regeneration through these signaling pathways. In addition, ROS/H2 O2 -dependent-Yap1 activity is required during tail regeneration.
Assuntos
Ambystoma mexicanum , Peróxido de Hidrogênio , Animais , Peróxido de Hidrogênio/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regeneração/fisiologia , Cauda/fisiologiaRESUMO
Advances in genomics have led to the identification of twelve relevant molecular subtypes within medulloblastoma (MB). The alpha subtype of Sonic hedgehog-driven MB is resistant to therapy (including smoothened inhibitors) due to activation of genes from the non-canonical SHH pathway, such as MYCN, YAP1, or TP53. Using retrospective cohort microarray data, we found that YAP1 is overexpressed in SHH alpha MB and patients profiled as resistant to SMO inhibitors compared to good responders. Here, we performed YAP1 depletion via CRISPR/Cas9 in two in vitro models of SHH-like MB cells and found that this protein is involved in responsiveness to the SMO inhibitor regarding proliferation, apoptosis, and colony formation. Further, considering the synergic combination of YAP1 depletion with SMO inhibition, we assessed single-cell RNA-seq data from five patients and found that SMO and YAP1 are enriched within cells of SHH MB. Importantly, our data suggest that YAP1 is not only a reliable biomarker for cellular response to SMOi but may indicate prospective testing of combination therapy using YAP1 and SMO inhibitors in preclinical models of SHH MB.
RESUMO
PURPOSE: Increasing evidence suggested that microRNA plays an important role in ovarian cancer. In this study, the role of miR-92 in ovarian cancer was investigated. METHODS: In this study, miR-92 expression in clinical sample was evaluated, role of miR-92 was investigated in vitro, and underlying mechanism was investigated using Chip, co-IP, and western blot. RESULTS: In this study, we show that miR-92 is overexpressed in ovarian cancer tissue compared with normal cancer tissue. Transfection of miR-92 increased proliferation of ovarian cancer cell, and increased migration capacity and colony formation were observed after miR-92 transfection; we found that expression of LATS2 was decreased by miR-92, and this was further confirmed by luciferase assay, which proved that miR-92 is targeting 3' of the endogenous LATS2 gene. Downregulation of LATS2 resulted in increased translocation of YAP1 and upregulation of PD-L1, which subsequently suppressed NK cell function and promoted T cell apoptosis. Moreover, co-transfection of YAP1-targeted shRNA could relieve miR-92-induced immune suppression effect. Mechanically, immunoprecipitation (IP) was used to show that LATS2 interacted with YAP1 and subsequently limited nuclear translocation of YAP1; chromatin immunoprecipitation (ChIP) was used to confirm that YAP1 could bind to enhancer region of PD-L1 to enhance transcription activity of PD-L1. CONCLUSIONS: Our data revealed a novel mechanism which finally resulted in immune suppression in ovarian cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígeno B7-H1/metabolismo , Células Matadoras Naturais/imunologia , MicroRNAs/metabolismo , Neoplasias Ovarianas/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Regulação para Baixo , Elementos Facilitadores Genéticos , Feminino , Inativação Gênica , Humanos , Imunidade Celular , Imunoprecipitação , Células-Tronco Neoplásicas , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno , Transdução de Sinais , Linfócitos T/fisiologia , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Proteínas de Sinalização YAPRESUMO
Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However, the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1), YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP), a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction, in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally, advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly, gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together, our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy.
RESUMO
BACKGROUND & AIMS: Gastric carcinoma is related mostly to CagA+-Helicobacter pylori infection, which disrupts the gastric mucosa turnover and elicits an epithelial-mesenchymal transition (EMT) and preneoplastic transdifferentiation. The tumor suppressor Hippo pathway controls stem cell homeostasis; its core, constituted by the large tumor suppressor 2 (LATS2) kinase and its substrate Yes-associated protein 1 (YAP1), was investigated in this context. METHODS: Hippo, EMT, and intestinal metaplasia marker expression were investigated by transcriptomic and immunostaining analyses in human gastric AGS and MKN74 and nongastric immortalized RPE1 and HMLE epithelial cell lines challenged by H pylori, and on gastric tissues of infected patients and mice. LATS2 and YAP1 were silenced using small interfering RNAs. A transcriptional enhanced associated domain (TEAD) reporter assay was used. Cell proliferation and invasion were evaluated. RESULTS: LATS2 and YAP1 appear co-overexpressed in the infected mucosa, especially in gastritis and intestinal metaplasia. H pylori via CagA stimulates LATS2 and YAP1 in a coordinated biphasic pattern, characterized by an early transient YAP1 nuclear accumulation and stimulated YAP1/TEAD transcription, followed by nuclear LATS2 up-regulation leading to YAP1 phosphorylation and targeting for degradation. LATS2 and YAP1 reciprocally positively regulate each other's expression. Loss-of-function experiments showed that LATS2 restricts H pylori-induced EMT marker expression, invasion, and intestinal metaplasia, supporting a role of LATS2 in maintaining the epithelial phenotype of gastric cells and constraining H pylori-induced preneoplastic changes. CONCLUSIONS: H pylori infection engages a number of signaling cascades that alienate mucosa homeostasis, including the Hippo LATS2/YAP1/TEAD pathway. In the host-pathogen conflict, which generates an inflammatory environment and perturbations of the epithelial turnover and differentiation, Hippo signaling appears as a protective pathway, limiting the loss of gastric epithelial cell identity that precedes gastric carcinoma development.
Assuntos
Transição Epitelial-Mesenquimal/imunologia , Mucosa Gástrica/patologia , Infecções por Helicobacter/patologia , Lesões Pré-Cancerosas/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Ciclo Celular/metabolismo , Feminino , Mucosa Gástrica/microbiologia , Regulação Neoplásica da Expressão Gênica/imunologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Masculino , Metaplasia/genética , Metaplasia/microbiologia , Metaplasia/patologia , Camundongos , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/imunologia , Fatores de Proteção , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAPRESUMO
Preeclampsia (PE) is a complex pregnancy syndrome. Convincing evidence indicates that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of PE. This research mainly investigated the mechanism of family with sequence similarity 99 member A (FAM99A) in PE. The expressions of FAM99A, miR-134-5p, and YAP1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis, migration, and invasion were detected by flow cytometry or transwell assay. The interaction between miR-134-5p and FAM99A or YAP1 was confirmed by dual-luciferase reporter assay. The protein expression of YAP1 was determined by western blot assay. FAM99A and YAP1 were significantly up-regulated, and miR-134-5p was significantly down-regulated in PE tissues (n=30). miR-134-5p was verified as a candidate of FAM99A and YAP1. FAM99A promoted cell metastasis, but reduced apoptosis in HTR8/SVneo cells by regulating miR-134-5p. miR-134-5p down-regulated YAP1 expression to suppress cell metastasis, while it induced apoptosis in HTR8/SVneo cells. FAM99A positively modulated YAP1 expression by sponging miR-134-5p. FAM99A modulated YAP1 to accelerate cell migration and invasion, and inhibited cell apoptosis in PE cells by sponging miR-134-5p. The novel regulatory network may shed light on the pathogenesis of PE.
Assuntos
Humanos , Feminino , Gravidez , Adulto , Pré-Eclâmpsia/genética , RNA Longo não Codificante/genética , Trofoblastos , Movimento Celular/genética , MicroRNAsRESUMO
BACKGROUND: Overexpression of the oncogene yes-associated-protein-1 (YAP1) is associated with increased cell proliferation in human cancers. YAP1 is a potential target of the Wnt/beta-catenin pathway, which plays an important role in adrenocortical tumors (ACT). The role of YAP1 in adrenocortical tumorigenesis has not been assessed. AIMS: To evaluate YAP1 expression in normal adrenals and pediatric ACT and its association with disease outcome. To investigate the interaction between YAP1 and the Wnt/beta-catenin pathway in adrenocortical cells. RESULTS: Strong YAP1 staining was present in fetal adrenals and pediatric ACT but weak in postnatal adrenals. In pediatric ACT, YAP1 mRNA overexpression was associated with death, recurrent/metastatic disease and lower overall survival. The inhibition of the Wnt/beta-catenin pathway increased YAP1 mRNA expression. siYAP1 increased CTNNB1/beta-catenin expression and nuclear staining regardless of DLV2, moreover, it decreased cell growth and impaired cell migration. MATERIALS AND METHODS: We assessed in 42 pediatric ACT samples the YAP1 protein expression by immunohistochemistry and mRNA expression by RT-qPCR and analyzed their association with outcome. As controls, we resort 32 fetal and postnatal normal adrenals for IHC and 10 normal adrenal cortices for RT-qPCR. The interaction between YAP1 and the Wnt/beta-catenin pathway was assessed in NCI-H295 adrenocortical cells by inhibiting the TCF/beta-catenin complex and by knocking down YAP1. CONCLUSION: YAP1 overexpression is a marker of poor prognosis for pediatric patients with ACT. In adrenocortical cells, there is a close crosstalk between YAP1 and Wnt/beta-catenin. These data open the possibility of future molecular therapies targeting Hippo/YAP1 signaling to treat advanced ACT.