RESUMEN
NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Humanos , Animales , Ratones , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Fosforilación , Proteína Sequestosoma-1/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.
Asunto(s)
Endosomas , Fosfohidrolasa PTEN , Fosfatidilinositoles , Vacuolas , Vacuolas/metabolismo , Vacuolas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/efectos de los fármacos , Humanos , Fosfatidilinositoles/metabolismo , Animales , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Ratones , Morfolinas/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética , Citoplasma/metabolismo , Células HeLa , Aminopiridinas , Compuestos Heterocíclicos con 3 AnillosRESUMEN
Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget's disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteína Sequestosoma-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Mutación Missense , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína Sequestosoma-1/genéticaRESUMEN
Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.
Asunto(s)
Neoplasias Esofágicas , Inmunidad , Factor 1 Regulador del Interferón , Factor de Transcripción STAT1 , Línea Celular/efectos de la radiación , Neoplasias Esofágicas/genética , Humanos , Inmunidad/efectos de la radiación , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 1 Regulador del Interferón/efectos de la radiación , Interferón Tipo I , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/efectos de la radiación , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/efectos de la radiación , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/efectos de la radiaciónRESUMEN
BACKGROUND: Combination therapy based on radiotherapy and immune checkpoint inhibitors (ICIs) was recently reported as effective for various cancers. The radiation-induced immune response (RIIR) is an essential feature in ICI-combined radiotherapy; however, the effects of drugs used concomitantly with RIIR remain unclear. We screened for drugs that can modify RIIR to understand the mutual relationship between radiotherapy and combined drugs in ICI-combined radiotherapy. METHODS: We established a high-throughput system with reporter gene assays for evaluating RIIR, focusing on factors acting downstream of the STING-IRF pathway, which can stimulate cancer cells, T cells, and dendritic cells. We further quantified the effects of 2595 drugs, including those approved by the Food and Drug Administration, on RIIR in vitro. RESULTS: The reporter assay results correlated well with the expression of immune response proteins such as programmed death-ligand 1. This high-throughput system enabled the identification of drugs including cytotoxic agents, molecular-targeted agents, and other agents that activate or suppress RIIR. CONCLUSIONS: Our study provides an encyclopedic catalogue of clinically approved drugs based on their effect on RIIR. In ICIs combined radiotherapy, activation of STING-IFN may improve the therapeutic effect and our result could form a biological basis for further clinical trials combining radiotherapy with ICIs.
Asunto(s)
Anticuerpos Monoclonales , Neoplasias , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inhibidores de Puntos de Control Inmunológico , Inmunidad , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/radioterapia , Preparaciones FarmacéuticasRESUMEN
p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Transducción de Señal , Animales , Autofagia , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismoRESUMEN
Radiotherapy (RT) is an effective treatment option for cancer; however, its efficacy remains less than optimal in locally advanced cancer. Immune checkpoint inhibitor-based therapy, including the administration of anti-PD-L1 antibodies, is a promising approach that works synergistically with RT. Proton beam therapy and carbon-ion therapy are common options for patients with cancer. Proton and carbon ions are reported to induce an immune reaction in cancer cells; however, the underlying mechanisms remain unclear. Here, we aimed to compare the immune responses after irradiation (IR) with X-ray, protons, and carbon ions in an oesophageal cancer cell line and the underlying mechanisms. An oesophageal cancer cell line, KYSE450, was irradiated with 1 fraction/15 GyE (Gy equivalent) of X-ray, proton, or carbon-ion beams, and then, the cells were harvested for RNA sequencing and gene enrichment analysis. We also knocked out STING and STAT1 in the quest for mechanistic insights. RNA sequencing data revealed that gene expression signatures and biological processes were different in KYSE450 irradiated with X-ray, proton, and carbon-ion beams 6-24 h after IR. However, after 3 days, a common gene expression signature was detected, associated with biological pathways involved in innate immune responses. Gene knock-out experiments revealed that the STING-STAT1 axis underlies the immune reactions after IR. X-Ray, proton, and carbon-ion IRs induced similar immune responses, regulated by the STING-STAT1 axis.
Asunto(s)
Carbono , Neoplasias Esofágicas/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Inmunidad/efectos de la radiación , Protones , Transcriptoma/efectos de la radiación , Rayos X , Línea Celular Tumoral , Neoplasias Esofágicas/inmunología , Neoplasias Esofágicas/patología , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/inmunología , Ontología de Genes , Humanos , Inmunidad/genética , Iones , RNA-Seq/métodos , Radiación/clasificación , Transducción de Señal/genética , Transducción de Señal/inmunología , Transducción de Señal/efectos de la radiación , Transcriptoma/inmunologíaRESUMEN
The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62's binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Adenoma/metabolismo , Adenoma/patología , Secuencia de Aminoácidos , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Cristalografía por Rayos X , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Fosforilación , Proteína Sequestosoma-1 , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS (p = 0.049) and cause-specific survival (p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células A549 , Adulto , Anciano , Anciano de 80 o más Años , Animales , Muerte Celular Autofágica/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Doxiciclina/farmacología , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The standard treatment for patients with unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is definitive chemoradiotherapy (CRT) using 5-FU plus cisplatin. However, complete response (CR) rates are low at 11-25%, resulting in 9-10 months of median overall survival (OS). An improved therapeutic efficacy by combining immunotherapy with radiation has been reported in patients with locally advanced non-small cell lung cancer. The results using ESCC cell lines suggest sequential treatment with anti-PD-L1 agents soon after completion of CRT is the most effective combination. METHODS: TENERGY trial is a multicenter, phase II, proof-of-concept study to assess the efficacy and safety of atezolizumab following definitive CRT in patients with locally advanced ESCC. The main inclusion criteria are unresectable locally advanced ESCC without distant metastasis, completion of 60 Gy of radiation plus two concomitant cycles of chemotherapy (cisplatin 70 mg/m2 on day 1 and 5-FU 700 mg/m2 on days 1-4, every 28 days), and adequate organ function. Within 6 weeks after CRT, participants will start taking 1200 mg of atezolizumab every three weeks and continue until 12 months or disease progression. The primary endpoint is the confirmed CR rate by the investigator's assessment. Secondary endpoints include overall response rate, progression-free survival (PFS), OS, adverse events, and confirmed CR rate by central assessment. We will enroll 50 patients (40 with primary locally advanced ESCC and 10 with postoperative locoregionally recurrent ESCC). We will obtain biopsies from the primary site and will collect blood at 3 time points (before CRT, after CRT, and four weeks after the start of atezolizumab) for an exploratory biomarker study. We will analyze the phenotype of immune-competent cells, neoantigens, tumor mutational burden, PD-L1 status, and Human Leukocyte Antigen haplotyping. DISCUSSION: The synergistic efficacies of the sequential combination of CRT and atezolizumab should improve the CR rate, resulting in survival improvement for patients with unresectable locally advanced ESCC. Because CRT is a standard treatment option for patients with early stage to locally advanced ESCC, the sequential combination of CRT and atezolizumab has the potential to change the standard ESCC treatments. TRIAL REGISTRATION: UMIN000034373, 10/04/2018 and EPOC1802.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioradioterapia/mortalidad , Neoplasias Esofágicas/terapia , Carcinoma de Células Escamosas de Esófago/terapia , Terapia Neoadyuvante/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/administración & dosificación , Cisplatino/administración & dosificación , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Fluorouracilo/administración & dosificación , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
BACKGROUND: The number of hemodialysis (HD) patients is increasing worldwide, and they are at a higher risk of cancer than the general population. Because HD patients are more likely to have inflammation, radiotherapy (RT)-induced adverse effects (AEs) are theoretically expected to be worse for HD patients. Since only a few reports have been published on this subject, we aimed to evaluate the tolerability of RT in HD patients. METHODS: We retrospectively analyzed AEs related to RT for HD patients. Our study included patients from three institutions treated between January 2007 and July 2017. The patient eligibility criteria were (i) receipt of maintenance HD 2-3 times per week for end-stage renal disease prior to the start of RT and (ii) pathologically confirmed malignancies. The endpoints were acute and late non-hematologic AEs. RESULTS: The study included 56 patients. The most common histology was head and neck cancer (23%), followed by lung cancer (14%) and prostate cancer (11%). The median radiation dose was 60 (range, 12-93.8) Gy at an equivalent dose in 2-Gy fractions (EQD2). The RT completion rate was 96%. Patients had a median follow-up period after RT of 9.1 (range 0.5-98.1) months. Grade 3 or worse acute and late AEs were noted in 6 (11%) and 3 (7%) patients, respectively. In the acute phase, 2 patients had grade 5 AEs, both of which were infections. CONCLUSION: Our results suggest that RT for HD patients is clinically tolerable. However, some patients can experience severe infections related to treatment.
Asunto(s)
Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias Pulmonares/radioterapia , Neoplasias de la Próstata/radioterapia , Traumatismos por Radiación/etiología , Diálisis Renal , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/fisiopatología , Humanos , Fallo Renal Crónico/terapia , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/fisiopatología , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/fisiopatología , Dosificación Radioterapéutica , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
INTRODUCTION: To investigate enhancement by 5-fluorouracil (5-FU) of the sensitivity of cancer cells to proton beam irradiation and clarify the differences in the responses of the 5-FU-treated cells to proton beam irradiation according to the position of the cells on the spread-out Bragg peak (SOBP). METHODS: OE21 human esophageal squamous cells were irradiated with a 235-MeV proton beam at four different positions on the SOBP. The effects of the irradiation plus 5-FU treatment on the cell survival were assessed by clonogenic assays and determination of the sensitizer enhancement ratio (SER). In addition, DNA double-strand breaks were estimated by measuring phospho-histone H2AX (γH2AX) foci formation in the cells at 0.5 and 24 h after irradiation. RESULTS: The relative biological effectiveness (RBE) of proton beam irradiation against vehicle-control cells tended to increase with an increase in the depth of the cells on the SOBP. On the other hand, the degree of enhancement of the cellular sensitivity to proton beam irradiation by 5-FU was similar across all the positions on the SOBP. Furthermore, a marked increase in the number of residual γH2AX foci at 24 h post-irradiation was observed in the cells at the distal end of the SOBP. CONCLUSIONS: Our data indicated that the degree of enhancement by 5-FU of the sensitivity of OE21 cells to 235-MeV proton beam irradiation did not differ significantly depending on the position of the cells on the SOBP. Furthermore, the degree of increase in the number of γH2AX foci at 24 h after proton beam irradiation with or without 5-FU exposure did not differ significantly according to the position on the SOBP. The effect of 5-FU in enhancing the effect of proton beam irradiation on cancer cells may be constant for all positions on the SOBP.
Asunto(s)
Antimetabolitos Antineoplásicos/efectos adversos , Carcinoma de Células Escamosas/radioterapia , Neoplasias Esofágicas/radioterapia , Fluorouracilo/farmacología , Terapia de Protones/efectos adversos , Traumatismos por Radiación/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Supervivencia Celular , Relación Dosis-Respuesta en la Radiación , Neoplasias Esofágicas/patología , Humanos , Traumatismos por Radiación/etiología , Efectividad Biológica Relativa , Células Tumorales CultivadasRESUMEN
Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and ß-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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Proteínas de Unión al GTP/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células de Purkinje/patología , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina/genética , Alelos , Animales , Conducta Animal , Carboxipeptidasas , Ataxia Cerebelosa/genética , Cerebelo/metabolismo , Cerebelo/patología , Estrés del Retículo Endoplásmico/genética , Exones/genética , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Degeneración Nerviosa/psicología , Fenotipo , Desempeño Psicomotor , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFP(KI/+)). Using these Sqstm1-GFP(KI/+) mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1-GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1-GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFP(KI/+) mouse livers caused accumulation of Sqstm1-GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFP(KI/+) mice are a useful tool for analyzing Sqstm1 in living cells and intact animals.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Autofagia , Regulación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Fagosomas/metabolismo , Estrés Fisiológico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas de Choque Térmico/genética , Humanos , Fallo Hepático/genética , Fallo Hepático/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteína Sequestosoma-1RESUMEN
Obesity, an important risk factor for metabolic syndrome (MetS) and cardiovascular disease, is often complicated by CKD, which further increases cardiovascular risk and causes ESRD. To elucidate the mechanism underlying this relationship, we investigated the role of the endocytic receptor megalin in proximal tubule epithelial cells (PTECs). We studied a high-fat diet (HFD)-induced obesity/MetS model using kidney-specific mosaic megalin knockout (KO) mice. Compared with control littermates fed a normal-fat diet, control littermates fed an HFD for 12 weeks showed autolysosomal dysfunction with autophagy impairment and increased expression of hypertrophy, lipid peroxidation, and senescence markers in PTECs of the S2 segment, peritubular capillary rarefaction with localized interstitial fibrosis, and glomerular hypertrophy with mesangial expansion. These were ameliorated in HFD-fed megalin KO mice, even though these mice had the same levels of obesity, dyslipidemia, and hyperglycemia as HFD-fed control mice. Intravital renal imaging of HFD-fed wild-type mice also demonstrated the accumulation of autofluorescent lipofuscin-like substances in PTECs of the S2 segment, accompanied by focal narrowing of tubular lumens and peritubular capillaries. In cultured PTECs, fatty acid-rich albumin induced the increased expression of genes encoding PDGF-B and monocyte chemoattractant protein-1 via megalin, with large (auto)lysosome formation, compared with fatty acid-depleted albumin. Collectively, the megalin-mediated endocytic handling of glomerular-filtered (lipo)toxic substances appears to be involved primarily in hypertrophic and senescent PTEC injury with autophagy impairment, causing peritubular capillary damage and retrograde glomerular alterations in HFD-induced kidney disease. Megalin could be a therapeutic target for obesity/MetS-related CKD, independently of weight, dyslipidemia, and hyperglycemia modification.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Enfermedades Renales/etiología , Glomérulos Renales/patología , Túbulos Renales Proximales/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Animales , Células Cultivadas , Células Epiteliales , Masculino , Ratones , Ratones NoqueadosRESUMEN
Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Línea Celular Tumoral , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/ultraestructura , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Transcripción/ultraestructuraRESUMEN
The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas del Citoesqueleto/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas del Citoesqueleto/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Immunoblotting , Proteína 1 Asociada A ECH Tipo Kelch , Hígado/metabolismo , Hígado/patología , Hígado/ultraestructura , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Microscopía Inmunoelectrónica , Factor 2 Relacionado con NF-E2/genética , Fagosomas/genética , Fagosomas/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/genética , Proteína Sequestosoma-1 , Factores de Tiempo , Ubiquitina/metabolismoRESUMEN
Autophagy is a unique intracellular protein degradation system accompanied by autophagosome formation. Besides its important role through bulk degradation in supplying nutrients, this system has an ability to degrade certain proteins, organelles, and invading bacteria selectively to maintain cellular homeostasis. In yeasts, Atg8p plays key roles in both autophagosome formation and selective autophagy based on its membrane fusion property and interaction with autophagy adaptors/specific substrates. In contrast to the single Atg8p in yeast, mammals have 6 homologs of Atg8p comprising LC3 and GABARAP families. However, it is not clear these two families have different or similar functions. The aim of this study was to determine the separate roles of LC3 and GABARAP families in basal/constitutive and/or selective autophagy. While the combined knockdown of LC3 and GABARAP families caused a defect in long-lived protein degradation through lysosomes, knockdown of each had no effect on the degradation. Meanwhile, knockdown of LC3B but not GABARAPs resulted in significant accumulation of p62/Sqstm1, one of the selective substrate for autophagy. Our results suggest that while mammalian Atg8 homologs are functionally redundant with regard to autophagosome formation, selective autophagy is regulated by specific Atg8 homologs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Autofagia/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis , Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia , Células Cultivadas , Células HEK293 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiología , Humanos , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/fisiología , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Modelos Moleculares , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Sequestosoma-1 , Electricidad EstáticaRESUMEN
BACKGROUND: To investigate the technical feasibility of RT-PCR and direct sequencing to quantify HPV DNA in the saliva of patients with Human-Papilloma-Virus related oropharyngeal cancer (HPV-OPC), the level of which is known to predict prognosis after treatment. METHODS: Nine patients with locally advanced HPV-OPC treated with definitive radiotherapy with chemotherapy or cetuximab, or radiotherapy alone between April 2016 and September 2017, were enrolled, two of whom also received induction chemotherapy. Saliva was collected before (baseline), during (mid-RT) and after (post-RT) radiotherapy. HPV-16 DNAs (E6 and E7) in saliva were quantified by RT-PCR and sequencing, the latter using a custom cancer panel. Correlations between HPV DNA levels and clinical outcomes were assessed. RESULTS: Compared to the baseline, the relative cycle threshold (Ct) value of E6 and E7 reduced at the point of mid-RT in the majority of the patients (100% and 75% for E6 and E7, respectively). Similarly, the relative Ct value from the baseline to post-RT reduced in 86% and 100% of the patients for E6 and E7, respectively. During the follow-up period, three patients (33%) experienced disease progression. The relative baseline Ct values of these three patients were in the top 4 of all the patients. The sequences of HPV DNA were detected in five (83%) of six samples of the baseline saliva that underwent DNA sequencing, along with several gene mutations, such as TP53,CDKN2A and PIK3CA. CONCLUSIONS: This study demonstrates that, in addition to detection and quantification of HPV DNA by RT-PCR, detection by sequencing of HPV-DNA using a customized cancer panel is technically possible.
Asunto(s)
ADN Viral , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Saliva , Humanos , Neoplasias Orofaríngeas/virología , Neoplasias Orofaríngeas/radioterapia , Saliva/virología , ADN Viral/análisis , Persona de Mediana Edad , Masculino , Femenino , Anciano , Infecciones por Papillomavirus/virología , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/aislamiento & purificaciónRESUMEN
BACKGROUND: We evaluated clinical and dosimetric outcomes of radiotherapy using two anterior oblique portals (AOP), to reduce the dose to the bilateral internal carotid arteries (CAs) and pharyngeal constrictor muscle (PCM) during early-stage glottic cancer (ESGC) treatment. METHODS: We identified patients with ESGC who underwent definitive radiotherapy between June 2014 and May 2020. RESULTS: Among the 66 patients, 32 (48%) underwent radiotherapy using AOP, and the remaining underwent typical radiotherapy using parallel opposed lateral portals (POLP). The median follow-up duration was 53 months. No significant differences were observed in the 5-year local failure (0%/9.4%), progression-free survival (90.6%/90.8%), and overall survival (90.6%/91.0%) rates between the two groups. The grade ≥2 acute mucositis incidence rate was significantly lower in the AOP group (44%/85%). Radiotherapy using AOP maintained an adequate dose coverage to the target while markedly reducing the CAs and PCM doses. CONCLUSION: Radiotherapy with AOP resulted in favorable clinical and dosimetric outcomes.