RESUMEN
We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.
Asunto(s)
Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas Supresoras de Tumor/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Daño del ADN , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/genética , Humanos , Ligandos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaAsunto(s)
División Celular/genética , Hígado/metabolismo , Proteínas Mad2/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Esfingosina N-Aciltransferasa/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Animales , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas Mad2/genética , Proteínas de la Membrana/genética , Ratones , Esfingosina N-Aciltransferasa/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Cofilina 1/genética , Resistencia a Antineoplásicos/genética , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Proteínas F-Box/genética , Paclitaxel/farmacología , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cofilina 1/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We recently presented Stafia-1 as the first chemical entity that inhibits the transcription factor STAT5a with selectivity over the highly homologous STAT5b. Stafia-1, which was identified from a series of symmetrically substituted m-terphenyl phosphates, binds to the interface between the SH2 domain and the linker domain of STAT5a. Here, we outline a synthetic strategy for the synthesis of asymmetrically substituted m-terphenyl phosphates, which can be tailored to address their asymmetric STAT5a binding site in a more specific manner. The asymmetrically substituted m-terphenyl phosphate with the highest activity against STAT5a was converted to a phosphatase-stable monofluoromethylene phosphonate. The synthetic methodology and activity analysis described here provide first insights into the structure-activity relationships of m-terphenyl phosphates for use as selective STAT5a inhibitors.
Asunto(s)
Compuestos Organofosforados/farmacología , Factor de Transcripción STAT5/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Factor de Transcripción STAT5/metabolismo , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/metabolismoRESUMEN
O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a-c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 µM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1-2 µM), similarly to acridine 8 (GI50 = 0.6 µM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2-0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , ADN/química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Acridinas/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Sitios de Unión/efectos de los fármacos , Bovinos , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanina/química , Guanina/farmacología , Humanos , Estructura Molecular , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.
Asunto(s)
Citocinas/metabolismo , Activación Enzimática/efectos de los fármacos , Malatos/farmacología , Metaloproteinasa 1 de la Matriz/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Cilios/metabolismo , Cilios/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Modelos Biológicos , Estrés Oxidativo/efectos de los fármacos , Material Particulado/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.
Asunto(s)
Carcinoma Hepatocelular/patología , Colina Quinasa/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Regiones Promotoras Genéticas , ARN sin Sentido/genética , Esfingosina N-Aciltransferasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/antagonistas & inhibidores , Factor Nuclear 4 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Pronóstico , Esfingosina N-Aciltransferasa/antagonistas & inhibidores , Esfingosina N-Aciltransferasa/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chloroethylnitrosoureas (CENUs) are an important family of chemotherapies in clinical treatment of cancers, which exert antitumor activity by inducing the formation of DNA interstrand crosslinks (dG-dC ICLs). However, the drug resistance mediated by O6-alkylguanine-DNA alkyltransferase (AGT) and absence of tumor-targeting ability largely decrease the antitumor efficacy of CENUs. In this study, we synthesized an azobenzene-based hypoxia-activated combi-nitrosourea prodrug, AzoBGNU, and evaluated its hypoxic selectivity and antitumor activity. The prodrug was composed of a CENU pharmacophore and an O6-benzylguanine (O6-BG) analog moiety masked by a N,N-dimethyl-4-(phenyldiazenyl)aniline segment as a hypoxia-activated trigger, which was designed to be selectively reduced via azo bond break in hypoxic tumor microenvironment, accompanied with releasing of an O6-BG analog to inhibit AGT and a chloroethylating agent to induce dG-dC ICLs. AzoBGNU exhibited significantly increased cytotoxicity and apoptosis-inducing ability toward DU145 cells under hypoxia compared with normoxia, indicating the hypoxia-responsiveness as expected. Predominant higher cytotoxicity was observed in the cells treated by AzoBGNU than those by traditional CENU chemotherapy ACNU and its combination with O6-BG. The levels of dG-dC ICLs in DU145 cells induced by AzoBGNU was remarkably enhanced under hypoxia, which was approximately 6-fold higher than those in the AzoBGNU-treated groups under normoxia and those in the ACNU-treated groups. The results demonstrated that azobenzene-based combi-nitrosourea prodrug possessed desirable tumor-hypoxia targeting ability and eliminated chemoresistance compared with the conventional CENUs.
Asunto(s)
Antineoplásicos/farmacología , Derivados del Benceno/farmacología , Metilasas de Modificación del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Desarrollo de Medicamentos , Inhibidores Enzimáticos/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Profármacos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Humanos , Masculino , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Esferoides Celulares , Hipoxia Tumoral , Microambiente Tumoral , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mammalian cell cytoskeletal reorganization for efficient directional movement requires tight coordination of actomyosin and microtubule networks. In this study, we show that LRAP35a potentiates microtubule stabilization by promoting CLASP2/EB1 interaction besides its complex formation with MRCK/MYO18A for retrograde actin flow. The alternate regulation of these two networks by LRAP35a is tightly regulated by a series of phosphorylation events that dictated its specificity. Sequential phosphorylation of LRAP35a by Protein Kinase A (PKA) and Glycogen Synthase Kinase-3ß (GSK3ß) initiates the association of LRAP35a with CLASP2, while subsequent binding and further phosphorylation by Casein Kinase 1δ (CK1δ) induce their dissociation, which facilitates LRAP35a/MRCK association in driving lamellar actomyosin flow. Importantly, microtubule dynamics is directly moderated by CK1δ activity on CLASP2 to regulate GSK3ß phosphorylation of the SxIP motifs that blocks EB1 binding, an event countered by LRAP35a interaction and its competition for CK1δ activity. Overall this study reveals an essential role for LRAP35a in coordinating lamellar contractility and microtubule polarization in cell migration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Quinasa Idelta de la Caseína/genética , Línea Celular Tumoral , Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVE: Prostate cancer (PCa) is considered the most serious cancer in the world. Nevertheless, the accuracy of current biomarkers, such as pathological staging, Gleason's score, and serum prostate-specific antigen (PSA) levels, is limited. FOXO1 is a key downstream effector of PTEN and a tumor suppressor in PCA, which has been reported extensively. However, the clinical relevance of FOXO1 in PCa remains unclear. METHODS: In this study, we first detected its expression in four public databases to explore the clinical role of FOXO1. Verification of the knockdown effect of FOXO1 siRNA was performed by real-time PCR analysis. Changes in cell viability were assessed using cell counting kit-8 (CCK-8) assays. In addition, we verified the effect of FOXO1 on the PCa cell cycle using a cell cycle assay. RESULTS: Herein, we found that FOXO1 was significantly downregulated in PCa tissues and was significantly associated with Gleason's score, age, biochemical recurrence (BCR), and lymph node (LN) status, while FOXO1 expression was independent of pathological staging and preoperative PSA levels. The Kaplan-Meier survival analysis showed that PCA patients with high FOXO1 expression were less likely to develop BCR compared with patients with low FOXO1 expression. In terms of function, FOXO1 inhibition significantly promoted the proliferation and cell cycle progression of PCa cells. CONCLUSIONS: In summary, our study suggests that FOXO1 may be one of the prognostic factors that describe the risk of PCa for BCR. These results suggest that FOXO1 may be a therapeutic target for PCa.
Asunto(s)
Proteína Forkhead Box O1/metabolismo , Neoplasias de la Próstata/metabolismo , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Biología Computacional , Regulación hacia Abajo , Proteína Forkhead Box O1/antagonistas & inhibidores , Proteína Forkhead Box O1/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Calicreínas/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , Transcriptoma , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Herein we describe a native mass spectromery protein-peptide model as a competent surrogate for the HOP-HSP90 protein-protein interaction (PPI), application of which led to the qualititive identification of two new peptides capable of in vitro PPI disruption. This proof of concept study offers a viable alternative for PPI inhibitor screening.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Homeodominio/antagonistas & inhibidores , Péptidos/química , Unión Proteica/efectos de los fármacos , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Sitios de Unión , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular , Péptidos/análisis , Prueba de Estudio Conceptual , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor S63845. AML patient samples with a strong response to AC-4-130 and S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirimidinas/uso terapéutico , Factor de Transcripción STAT5/antagonistas & inhibidores , Tiofenos/uso terapéutico , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Dioxigenasas , Humanos , Proteínas Proto-Oncogénicas/genética , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Inhibitor of growth 3 (ING3) is one of five members of the ING tumour suppressor family, characterized by a highly conserved plant homeodomain (PHD) as a reader of the histone mark H3K4me3. ING3 was reported to act as a tumour suppressor in many different cancer types to regulate apoptosis. On the other hand, ING3 levels positively correlate with poor survival prognosis of prostate cancer (PCa) patients. In PCa cells, ING3 acts rather as an androgen receptor (AR) co-activator and harbours oncogenic properties in PCa. Here, we show the identification of a novel ING3 splice variant in both the human PCa cell line LNCaP and in human PCa patient specimen. The novel ING3 splice variant lacks exon 11, ING3∆ex11, which results in deletion of the PHD, providing a unique opportunity to analyse functionally the PHD of ING3 by a natural splice variant. Functionally, overexpression of ING3Δex11 induced morphological changes of LNCaP-derived 3D spheroids with generation of lumen and pore-like structures within spheroids. Since these structures are an indicator of epithelial-mesenchymal transition (EMT), key regulatory factors and markers for EMT were analysed. The data suggest that in contrast to ING3, ING3Δex11 specifically modulates the expression of key EMT-regulating upstream transcription factors and induces the expression of EMT markers, indicating that the PHD of ING3 inhibits EMT. In line with this, ING3 knockdown also induced the expression of EMT markers, confirming the impact of ING3 on EMT regulation. Further, ING3 knockdown induced cellular senescence via a pathway leading to cell cycle arrest, indicating an oncogenic role for ING3 in PCa. Thus, the data suggest that the ING3Δex11 splice variant lacking functional PHD exhibits oncogenic characteristics through triggering EMT in PCa cells.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Neoplasias de la Próstata/genética , Empalme del ARN , Proteínas Supresoras de Tumor/genética , Línea Celular Tumoral , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells.
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Antineoplásicos/farmacología , Aspirina/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Carcinoma Hepatocelular/tratamiento farmacológico , Proteínas de Unión al ADN/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Antineoplásicos/uso terapéutico , Aspirina/uso terapéutico , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Lisina/metabolismo , Fosfopiruvato Hidratasa/química , Fosfopiruvato Hidratasa/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Omics analyses are powerful methods to obtain an integrated view of complex biological processes, disease progression, or therapy efficiency. However, few studies have compared different disease forms and different therapy strategies to define the common molecular signatures representing the most significant implicated pathways. In this study, we used RNA sequencing and mass spectrometry to profile the transcriptomes and proteomes of mouse models for three forms of centronuclear myopathies (CNMs), untreated or treated with either a drug (tamoxifen), antisense oligonucleotides reducing the level of dynamin 2 (DNM2), or following modulation of DNM2 or amphiphysin 2 (BIN1) through genetic crosses. Unsupervised analysis and differential gene and protein expression were performed to retrieve CNM molecular signatures. Longitudinal studies before, at, and after disease onset highlighted potential disease causes and consequences. Main pathways in the common CNM disease signature include muscle contraction, regeneration and inflammation. The common therapy signature revealed novel potential therapeutic targets, including the calcium regulator sarcolipin. We identified several novel biomarkers validated in muscle and/or plasma through RNA quantification, western blotting, and enzyme-linked immunosorbent assay (ELISA) assays, including ANXA2 and IGFBP2. This study validates the concept of using multi-omics approaches to identify molecular signatures common to different disease forms and therapeutic strategies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Miopatías Estructurales Congénitas/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteómica/métodos , Tamoxifeno/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Dinamina II/antagonistas & inhibidores , Humanos , Estudios Longitudinales , Espectrometría de Masas , Ratones , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Análisis de Secuencia de ARN , Proteínas Supresoras de Tumor/antagonistas & inhibidoresRESUMEN
Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases-the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proliferación Celular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Células Ependimogliales/citología , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Células HEK293 , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Down-regulated in renal cell carcinoma 1 (DRR1), a unique stress-induced protein, is highly expressed in the nervous system. This study investigated the roles of DRR1 in the brain by examining its expression pattern at different developmental stages of a rat brain and in cultured primary hippocampal neurons. High expression of DRR1 was observed in all developmental stages of a rat brain and cultured primary hippocampal neurons. We then focused on the role of DRR1 in promoting neurite outgrowth during the early stage of hippocampal neuron development. Results showed that down-regulation of DRR1 suppressed axon outgrowth. Mass spectrometry analysis revealed that tropomodulin-2 (Tmod2) is a novel binding partner of DRR1. Our results showed that both DRR1 and Tmod2 mediate axon formation during the early stage of hippocampal neuron development. Suppression of TMOD2 expression rescued the abnormal axon outgrowth induced by DRR1 knockdown during the early stage of hippocampal neuron development.
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Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Proyección Neuronal/genética , Proyección Neuronal/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células Cultivadas , Regulación hacia Abajo , Femenino , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/metabolismo , Embarazo , Unión Proteica , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Tropomodulina/antagonistas & inhibidores , Tropomodulina/genética , Tropomodulina/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidoresRESUMEN
Deleted in liver cancer 1 (DLC1) is a recognized tumor suppressor gene that negatively regulates Rho family proteins by hydrolyzing the active GTP-bound state to its inactive GDP-bound state. Active Rho proteins play a positive role in tumorigenesis. Numerous in vitro and in vivo experiments have shown that DLC1 is downregulated or inactivated in various solid tumors, which may be due to the following five reasons: genomic deletion, epigenetic modification and ubiquitin-dependent proteasomal degradation may cause DLC1 underexpression; phosphorylation at the post-translation level may cause DLC1 inactivation; and failure to localize at focal adhesions (FAs) may prevent DLC1 from exerting full activity. All of the causes could be attributed to molecular binding. Experimental evidence suggests that direct or indirect targeting of DLC1 is feasible for cancer treatment. Therefore, elucidating the interaction of DLC1 with its binding partners might provide novel targeted therapies for cancer. In this review, we summarized the binding partners of DLC1 at both the gene and protein levels and expounded a variety of anticancer drugs targeting DLC1 to provide information about DLC1 as a cancer diagnostic indicator or therapeutic target.
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Proteínas Activadoras de GTPasa/metabolismo , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Transformación Celular Neoplásica/metabolismo , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/terapia , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
It has been reported that the gut microbiome modulates postoperative cognitive dysfunction (POCD), and that administration of probiotics (VSL#3) may effectively relieve POCD. In this study, we aimed to identify the underlying mechanism of VSL#3 in POCD. A mouse model of POCD was constructed in adult male C57BL/6 mice, which were then treated with VSL#3. VSL#3 exerted a protective role against POCD and resultant neuronal apoptosis. The expression of miR-146a was found to be downregulated in hippocampal tissues of POCD mice, while VSL#3 could restore its expression. Loss- and gain-function approaches were conducted to determine the roles of microRNA (miR)-146a, B-cell translocation gene 2 (BTG2), and Bcl-2-associated X protein (Bax) in post-operative effects on cognitive function and neuronal apoptosis. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured to determine oxidative stress in brain tissue. The dual-luciferase reporter gene assay identified that miR-146a could target BTG2 and negatively regulate its expression. BTG2 knockdown suppressed neuronal apoptosis and contributed to shortened time of latency, prolonged time of mice spent in the target quadrant, and reduced oxidative stress through downregulating Bax expression. Finally, VSL#3 treatment upregulated the expression of miR-146a to block BTG2/Bax axis and consequently inhibited neuronal apoptosis and reduced oxidative stress in POCD mice. Taken together, the study suggested that miR-146a-mediated suppression of BTG2/Bax contributed to the protective role of probiotics treatment against POCD.
Asunto(s)
Proteínas Inmediatas-Precoces/biosíntesis , MicroARNs/biosíntesis , Complicaciones Cognitivas Postoperatorias/dietoterapia , Complicaciones Cognitivas Postoperatorias/metabolismo , Probióticos/administración & dosificación , Proteínas Supresoras de Tumor/biosíntesis , Proteína X Asociada a bcl-2/biosíntesis , Animales , Línea Celular , Expresión Génica , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Complicaciones Cognitivas Postoperatorias/prevención & control , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteína X Asociada a bcl-2/antagonistas & inhibidoresRESUMEN
Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.