RESUMEN
Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Nucleares/genética , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , ADN , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Femenino , Inestabilidad Genómica , Mutación de Línea Germinal , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Reparación del ADN por RecombinaciónRESUMEN
BACKGROUND: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold. RESULTS: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses. CONCLUSIONS: Our functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.
Asunto(s)
Canales de Potasio , Animales , Canales de Potasio/metabolismo , Canales de Potasio/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Filogenia , Ratones , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Evolución Molecular , Humanos , Venenos de Artrópodos/químicaRESUMEN
BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1 , Neoplasias de la Mama , Proteína Potenciadora del Homólogo Zeste 2 , Indoles , Inhibidores de Proteínas Quinasas , Piridonas , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Humanos , Indoles/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Mutaciones Letales SintéticasRESUMEN
The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.
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Discapacidades del Desarrollo/genética , Proteínas de Unión al GTP/genética , Progeria/genética , Proteínas de Unión al ARN/genética , Alelos , Consanguinidad , Daño del ADN , Discapacidades del Desarrollo/patología , Genoma Humano , Inestabilidad Genómica , Homocigoto , Humanos , Recién Nacido , Masculino , Mutación , Linaje , Progeria/patología , ARN de Transferencia/genética , Análisis de Secuencia de ARN , Acortamiento del TelómeroRESUMEN
The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.
Asunto(s)
Proteínas de Unión al ADN/genética , Respuesta al Choque Térmico/genética , Proteínas Nucleares/genética , Empalme del ARN , ARN Mensajero/genética , ARN no Traducido/genética , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Exones , Células HeLa , Factores de Transcripción del Choque Térmico , Histona Acetiltransferasas , Chaperonas de Histonas , Calor , Humanos , Intrones , Proteínas Nucleares/metabolismo , Dominios Proteicos , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Fluorouracilo/farmacología , ARN/metabolismo , Ribonucleoproteínas/metabolismo , Estrés Fisiológico , Antimetabolitos Antineoplásicos/metabolismo , Azacitidina/metabolismo , Línea Celular , ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Fluorouracilo/metabolismo , Proteínas de Unión al GTP/análisis , Células HeLa , Humanos , Proteínas de Neoplasias/análisis , Estrés Oxidativo , Receptores de Cinasa C Activada , Receptores de Superficie Celular/análisis , Ribonucleoproteínas/análisis , Tioguanina/metabolismoRESUMEN
Peripheral neuropathy is a common side effect of cancer treatment with paclitaxel. The mechanisms by which paclitaxel is transported into neurons, which are essential for preventing neuropathy, are not well understood. We studied the uptake mechanisms of paclitaxel into neurons using inhibitors for endocytosis, autophagy, organic anion-transporting polypeptide (OATP) drug transporters, and derivatives of paclitaxel. RT-qPCR was used to investigate the expression levels of OATPs in different neuronal tissues and cell lines. OATP transporters were pharmacologically inhibited or modulated by overexpression and CRISPR/Cas9-knock-out to investigate paclitaxel transport in neurons. Through these experiments, we identified OATP1A1 and OATP1B2 as the primary neuronal transporters for paclitaxel. In vitro inhibition of OATP1A1 and OAT1B2 by glycyrrhizic acid attenuated neurotoxicity, while paclitaxel's antineoplastic effects were sustained in cancer cell lines. In vivo, glycyrrhizic acid prevented paclitaxel-induced toxicity and improved behavioral and electrophysiological measures. This study indicates that a set of OATPs are involved in paclitaxel transport into neurons. The inhibition of OATP1A1 and OATP1B2 holds a promising strategy to prevent paclitaxel-induced peripheral neuropathy.
Asunto(s)
Transportadores de Anión Orgánico , Enfermedades del Sistema Nervioso Periférico , Humanos , Paclitaxel/efectos adversos , Ácido Glicirrínico/farmacología , Transportadores de Anión Orgánico/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Membrana , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/prevención & controlRESUMEN
ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Analgésicos Opioides , Colforsina/farmacología , Nocicepción , Células Receptoras Sensoriales , Canales IónicosRESUMEN
Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.
Asunto(s)
Carbunco , Bacillus anthracis , Toxinas Bacterianas , Células Madre Pluripotentes Inducidas , Animales , Carbunco/microbiología , Carbunco/terapia , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Ganglios Espinales/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Nociceptores/metabolismo , Dolor , Receptores de Péptidos/metabolismoRESUMEN
Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteína Quinasa Tipo II Dependiente de AMP Cíclico/metabolismo , Nociceptores/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Venoms are a rich source of highly specific toxins, which allow the identification of novel therapeutic targets. We have now applied high content screening (HCS) microscopy to identify toxins that modulate pain sensitization signaling in primary sensory neurons of rat and elucidated the underlying mechanism. A set of venoms and fractions thereof were analyzed for their ability to activate type II protein kinase A (PKA-II) and extracellular signal-regulated kinases (ERK1/2). We identified MeuNaTxα-1, a sodium channel-selective scorpion α-toxin from Mesobuthus eupeus, which affected both PKA-II and ERK1/2. Recombinant MeuNaTxα-1 showed identical activity to the native toxin on mammalian voltage-gated sodium channels expressed in Xenopus laevis oocytes and induced thermal hyperalgesia in adult mice. The effect of MeuNaTxα-1 on sensory neurons was dose-dependent and tetrodotoxin-sensitive. Application of inhibitors and toxin mutants with altered sodium channel selectivity demonstrated that signaling activation in sensory neurons depends on NaV 1.2 isoform. Accordingly, the toxin was more potent in neurons from newborn rats, where NaV 1.2 is expressed at a higher level. Our results demonstrate that HCS microscopy-based monitoring of intracellular signaling is a novel and powerful tool to identify and characterize venoms and their toxins affecting sensory neurons.
Asunto(s)
Proteína Quinasa Tipo II Dependiente de AMP Cíclico/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Dolor/genética , Canales de Sodio Activados por Voltaje/genética , Animales , Animales Recién Nacidos , Humanos , Hiperalgesia/genética , Hiperalgesia/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Oocitos/efectos de los fármacos , Oocitos/crecimiento & desarrollo , Ratas , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Escorpiones/química , Células Receptoras Sensoriales , Xenopus laevis/crecimiento & desarrolloRESUMEN
Non-coding RNA from pericentromeric satellite repeats are involved in stress-dependent splicing processes, maintenance of heterochromatin, and are required to protect genome stability. Here we show that the long non-coding satellite III RNA (SatIII) generates resistance against the topoisomerase IIa (TOP2A) inhibitor etoposide in lung cancer. Because heat shock conditions (HS) protect cells against the toxicity of etoposide, and SatIII is significantly induced under HS, we hypothesized that the protective effect could be traced back to SatIII. Using genome methylation profiles of patient-derived xenograft mouse models we show that the epigenetic modification of the SatIII DNA locus and the resulting SatIII expression predict chemotherapy resistance. In response to stress, SatIII recruits TOP2A to nuclear stress bodies, which protects TOP2A from a complex formation with etoposide and results in decreased DNA damage after treatment. We show that BRD4 inhibitors reduce the expression of SatIII, restoring etoposide sensitivity.
Asunto(s)
Resistencia a Antineoplásicos/genética , Etopósido/uso terapéutico , ARN Largo no Codificante/fisiología , Animales , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Centrómero/genética , Centrómero/metabolismo , Metilación de ADN/fisiología , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Unión a Poli-ADP-Ribosa/efectos de los fármacos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Largo no Codificante/genética , Factores de Transcripción/antagonistas & inhibidores , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesicswithout eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents.
Asunto(s)
Dolor Crónico , Canal de Sodio Activado por Voltaje NAV1.7 , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Roedores/metabolismo , SumoilaciónRESUMEN
Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.
Asunto(s)
Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Masculino , Ratones Endogámicos C57BL , Microscopía/métodos , Microscopía Fluorescente , Nociceptores/metabolismo , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/inmunologíaRESUMEN
The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent-targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry-based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D -driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. SIGNIFICANCE: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Neoplasias Experimentales/patología , Animales , Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Humanos , Hidroxibenzoatos/farmacología , Ratones , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirazoles/farmacología , Quinazolinas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The α2δ-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ-1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Gabapentina/metabolismo , Trombospondinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Ligandos , Espectrometría de Masas , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismoRESUMEN
Type II isoforms of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA-II) contain a phosphorylatable epitope within the inhibitory domain of RII subunits (pRII) with still unclear function. In vitro, RII phosphorylation occurs in the absence of cAMP, whereas staining of cells with pRII-specific antibodies revealed a cAMP-dependent pattern. In sensory neurons, we found that increased pRII immunoreactivity reflects increased accessibility of the already phosphorylated RII epitope during cAMP-induced opening of the tetrameric RII2:C2 holoenzyme. Accordingly, induction of pRII by cAMP was sensitive to novel inhibitors of dissociation, whereas blocking catalytic activity was ineffective. Also in vitro, cAMP increased the binding of pRII antibodies to RII2:C2 holoenzymes. Identification of an antibody specific for the glycine-rich loop of catalytic subunits facing the pRII-epitope confirmed activity-dependent binding with similar kinetics, proving that the reassociation is rapid and precisely controlled. Mechanistic modeling further supported that RII phosphorylation precedes cAMP binding and controls the inactivation by modulating the reassociation involving the coordinated action of phosphodiesterases and phosphatases.
Asunto(s)
Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Subunidades de Proteína/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos/farmacología , Extractos Celulares , Permeabilidad de la Membrana Celular/efectos de los fármacos , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Isoenzimas/metabolismo , Masculino , Ratones , Modelos Biológicos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Carcinogénesis/patología , Puntos de Control del Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Citoprotección , Daño del ADN , Resistencia a Antineoplásicos , Embrión de Mamíferos/citología , Epitelio/metabolismo , Fibroblastos/metabolismo , Dosificación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , RegeneraciónRESUMEN
BACKGROUND: The sexually dimorphic differentiation of the bipotential gonad into testis or ovary initiates the sexually dimorphic development of mammals and leads to divergent hormone concentrations between the sexes throughout life. However, despite the fact that anatomic and hormonal differences between the sexes are well described, only a few studies have investigated the manifestation of these differences at the transcriptional level in mammalian somatic tissue. OBJECTIVE: This review focuses on basic regulatory mechanisms of sex-specific gene expression and examines recent gene expression profiling studies to outline basic differences between the sexes at the transcriptome level in somatic tissues. METHODS: To identify gene expression profiling studies addressing sexually dimorphic gene expression, the PubMed database was searched using the terms sex and dimorp and gene expression not drosophila not elegans. Abstracts of all identified publications were screened for studies explicitly using microarrays to identify sex differences in somatic tissues of rodents or humans. The search was restricted to English-language articles published in the past 5 years. Reference lists of identified articles as well as microarray databases (Gene Expression Omnibus and ArrayExpress) were also used. RESULTS: The application of microarray technology has enabled the systematic assessment of sex-biased gene expression on the transcriptome level, indicating that the regulatory pathways underlying sexual differentiation give rise to extensive differences in somatic gene expression across organisms. CONCLUSION: Sustainable annotation of sex-biased gene expression provides a key to understanding basic physiological differences between healthy males and females as well as those with diseases.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Caracteres Sexuales , Animales , Femenino , Humanos , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , RatasRESUMEN
Genetic loss of the voltage-gated sodium channel Nav1.7 (Nav1.7-/-) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Nav1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Nav1.7 knockout (Nav1.7-/-) mice, but not those of Nav1.8 knockout (Nav1.8-/-) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT4 receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIß regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the Gαi-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Nav1.7 controls the efficacy and balance of GPCR-mediated pro- and antinociceptive intracellular signaling, such that without Nav1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.