RESUMEN
The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.
Asunto(s)
Aeromonas hydrophila/metabolismo , Apoptosis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Enfermedades de los Peces , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas , Riñón/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , Bagres , Enfermedades de los Peces/metabolismo , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/patología , Infecciones por Bacterias Gramnegativas/metabolismo , Infecciones por Bacterias Gramnegativas/patología , Infecciones por Bacterias Gramnegativas/veterinaria , Riñón/patología , Macrófagos/patologíaRESUMEN
MicroRNAs, a class of small non-coding RNAs, are believed to regulate several biological pathways and processes and are implicated in several diseases. They mostly regulate the levels of their target genes at the post transcriptional stage by primarily binding to the 3' UTR. Elevated hepatic levels of miR-107 are a consistent feature associated with several obese and diabetic models. Here, we show that miR-107 post-transcriptionally regulates fatty acid synthase (FASN) by binding to its 3' UTR and reduces its protein levels and the 3'UTR luciferase reporter activity, which are blunted by the miR-107 inhibitor and mutation in the miR-107 binding site in the 3' UTR. Knock-down of endogenous miR-107 levels increased FASN levels in a dose-dependent manner. Overexpression of miR-107 led to significant accumulation of malonyl CoA, accompanied by ER stress induction. All these events were prevented in the presence of the miR-107 inhibitor. While overexpression of FASN could attenuate miR-107 mediated ER stress markers' induction; the ER stress inhibitor, 4-phenyl-butyric acid did not rescue miR-107 induced FASN inhibition. This was followed by increased triglyceride formation and lipid accumulation in the presence of miR-107. These indicate that miR-107 inhibits FASN levels by binding to its 3' UTR and this interaction promotes ER stress induction and malonyl CoA and lipid accumulation in HepG2 cells and primary hepatocytes. Our results suggest that increased levels of miR-107 are critical in promoting lipid accumulation in hepatocytes and this might form the basis of diverse etiologies encountered in a fatty liver.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Ácidos Grasos/biosíntesis , Metabolismo de los Lípidos/genética , MicroARNs/genética , Regiones no Traducidas 3' , Sitios de Unión , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/genética , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Malonil Coenzima A/metabolismo , MicroARNs/metabolismoRESUMEN
PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. EXPERIMENTAL DESIGN: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. RESULTS: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. CONCLUSIONS: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.
Asunto(s)
Neoplasias de la Vaina del Nervio , Neurofibromatosis 1 , Neurofibrosarcoma , Humanos , Ratones , Animales , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromatosis 1/genética , Línea Celular , Apoptosis , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias de la Vaina del Nervio/tratamiento farmacológico , Neoplasias de la Vaina del Nervio/genética , Neoplasias de la Vaina del Nervio/metabolismo , Línea Celular Tumoral , TYK2 Quinasa/genética , TYK2 Quinasa/metabolismo , TYK2 Quinasa/farmacologíaRESUMEN
The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c+CD5+ DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5+ DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5hi T helper and CD8+ T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5+ DCs are an essential component of optimal ICB therapy.
Asunto(s)
Antígenos CD5 , Linfocitos T CD8-positivos , Células Dendríticas , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Melanoma , Linfocitos T Colaboradores-Inductores , Humanos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Células Dendríticas/inmunología , Melanoma/tratamiento farmacológico , Antígenos CD5/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). METHODS: Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. RESULTS: We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to "robust" or "good" microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. CONCLUSIONS: These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.
Asunto(s)
Neoplasias de la Vaina del Nervio , Neurofibromatosis 1 , Neurofibrosarcoma , Humanos , Neurofibrosarcoma/patología , Medicina de Precisión , Neurofibromatosis 1/patología , Neoplasias de la Vaina del Nervio/patología , MutaciónRESUMEN
One of the most common malignancies affecting adults with Neurofibromatosis type 1 (NF1) is the malignant peripheral nerve sheath tumor (MPNST), an aggressive and often fatal sarcoma that commonly arises from benign plexiform neurofibromas. Despite advances in our understanding of MPNST pathobiology, there are few effective therapeutic options, and no investigational agents have proven successful in clinical trials. To further understand the genomic heterogeneity of MPNST, and to generate a preclinical platform that encompasses this heterogeneity, we developed a collection of NF1-MPNST patient-derived xenografts (PDX). These PDX were compared with the primary tumors from which they were derived using copy number analysis, whole exome sequencing, and RNA sequencing. We identified chromosome 8 gain as a recurrent genomic event in MPNST and validated its occurrence by FISH in the PDX and parental tumors, in a validation cohort, and by single-cell sequencing in the PDX. Finally, we show that chromosome 8 gain is associated with inferior overall survival in soft-tissue sarcomas. These data suggest that chromosome 8 gain is a critical event in MPNST pathogenesis and may account for the aggressive nature and poor outcomes in this sarcoma subtype.
Asunto(s)
Cromosomas Humanos Par 8 , Neoplasias de la Vaina del Nervio/genética , Adulto , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neoplasias de la Vaina del Nervio/complicaciones , Neoplasias de la Vaina del Nervio/patología , Neurofibromatosis 1/complicaciones , Análisis de SupervivenciaRESUMEN
DNA methylation is widely known to regulate gene expression in eukaryotes. Here, we unraveled DNA methylation patterns in cultivated chickpea to understand the regulation of gene expression in different organs. We analyzed the methylation pattern in leaf tissue of wild chickpea too, and compared it with cultivated chickpea. Our analysis indicated abundant CG methylation within gene-body and CHH methylation in intergenic regions of the chickpea genome in all the organs examined. Analysis of differentially methylated regions (DMRs) demonstrated a higher number of CG context DMRs in wild chickpea and CHH context DMRs in cultivated chickpea. We observed increased preponderance of hypermethylated DMRs in the promoter regions and hypomethylated DMRs in the genic regions in cultivated chickpea. Genomic location and context of the DMRs correlated well with expression of proximal genes. Our results put forth a positive correlation of promoter hypermethylation with increased transcript abundance via identification of DMR-associated genes involved in flower development in cultivated chickpea. The atypical correlation observed between promoter hypermethylation and increased transcript abundance might be dependent on 24-nt small RNAs and transcription factors binding to the promoter region. This study provides novel insights into DNA methylation patterns in chickpea and their role in regulation of gene expression.
Asunto(s)
Cicer/genética , Genoma de Planta/genética , Sulfitos/química , Metilación de ADN/genética , ADN de Plantas/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Elevated interleukin-1ß (IL-1ß) induces apoptosis in pancreatic ß-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1ß. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca(2+) handling and storage, here we examine the effects of IL-1ß on cellular Ca(2+) movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary ß-cells, IL-1ß alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca(2+) release via IP3R channels and mitochondrial Ca(2+) uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1ß-induced cellular alteration. This is accompanied by IL-1ß-induced apoptosis, which is prevented by JNK1/2 siRNA and the IP3R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca(2+) axis by IL-1ß in apoptotic cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Retículo Endoplásmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , Compuestos Macrocíclicos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Fluorescente , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Modelos Biológicos , Oxazoles/farmacología , Permeabilidad/efectos de los fármacos , Interferencia de ARN , Ratas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The pro-inflammatory cytokine, IL-1ß, is a critical component of the persistent inflammatory milieu that pancreatic cancer cells frequently encounter. Although several studies report diverse mechanisms responsible for this association, yet a comprehensive global analysis of the effect of IL-1ß in these cells is not clearly evident. In this study, we performed whole genome transcriptome analysis of control and IL-1ß treated human pancreatic MIA PaCa-2 cells, validated the most targeted pathway and evaluated the role of JNK therein. 225 Genes were up-regulated and 1215 were down-regulated and these were categorized into biological processes and cellular pathways using the PANTHER classification system. The altered genes categorized into significant biological processes that included those of cell cycle, mitosis, transport and intracellular protein trafficking. The integrin signaling pathway emerged as harboring the maximum number of differentially expressed genes. Two important genes of this pathway, namely vinculin and α5-integrin were validated and both depicted significant inhibition by IL-1ß that was prevented in the presence of JNK siRNA. In a wound healing assay, IL-1ß increased the migratory rate of MIA PaCa-2 and Panc-1 cells that was abrogated by JNK inhibition. Additionally, vinculin and α-integrin siRNAs also increased the migration of these cells along the wound edge. These results suggest that in these pancreatic cancer cells, IL-1ß inhibits components of the integrin signaling pathway in a JNK dependent manner that contributes to their increased migratory potential. Therefore, JNK might be potentially targeted to prevent the migration and invasion of pancreatic cancer cells.
Asunto(s)
Integrina alfa5/metabolismo , Interleucina-1beta/farmacología , MAP Quinasa Quinasa 4/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Línea Celular Tumoral , Regulación hacia Abajo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , MAP Quinasa Quinasa 4/genética , Neoplasias Pancreáticas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Vinculina/antagonistas & inhibidores , Vinculina/metabolismoRESUMEN
MicroRNAs (miRNAs or miRs) are small approximately 22 nucleotide RNA species that are believed to regulate diverse metabolic and physiological processes. In the recent past, several reports have surfaced that demonstrate the role of miRNAs in various biological processes and numerous disease states. For a disease as complex as diabetes, the emergence of miRNAs as key regulators leading to the disease phenotype has added a novel dimension to the area of diabetes research. On the other hand, the liver, a metabolic hub, contributes in a major way towards maintaining normal glucose levels in the body as it can both stimulate and inhibit hepatic glucose output. This equilibrium is frequently disturbed in diabetes and hence, the liver assumes special significance considering the correlation between altered hepatic physiology and diabetes. While the understanding of the mechanisms behind this altered hepatic behavior is not yet completely understood, recent reports on the status and role of miRNAs in the diabetic liver have further added to the complexities of the knowledge of hepatic pathophysiology in diabetes. Here, we bring together the various miRNAs that play a role in the altered hepatic behavior during diabetes.