RESUMO
BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to increase globally with, as of yet, an unmet need for reliable prognostic biomarkers to identify patients at increased risk of metastasis. The aim of the present study was to test the prognostic potential of the combined immunohistochemical expression of the autophagy regulatory biomarkers, AMBRA1 and SQSTM1, to identify high-risk patient subsets. METHODS: A retrospective cohort of 68 formalin-fixed paraffin-embedded primary cSCCs with known 5-year metastatic outcomes were subjected to automated immunohistochemical staining for AMBRA1 and SQSTM1. Digital images of stained slides were annotated to define four regions of interest: the normal and peritumoral epidermis, the tumor mass, and the tumor growth front. H-score analysis was used to semi-quantify AMBRA1 or SQSTM1 expression in each region of interest using Aperio ImageScope software, with receiver operator characteristics and Kaplan-Meier analysis used to assess prognostic potential. RESULTS: The combined loss of expression of AMBRA1 in the tumor growth front and SQSTM1 in the peritumoral epidermis identified patients with poorly differentiated cSCCs at risk of metastasis (*p < 0.05). CONCLUSIONS: Collectively, these proof of concept data suggest loss of the combined expression of AMBRA1 in the cSCC growth front and SQSTM1 in the peritumoral epidermis as a putative prognostic biomarker for poorly differentiated cSCC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Biomarcadores Tumorais , Carcinoma de Células Escamosas , Imuno-Histoquímica , Proteína Sequestossoma-1 , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Masculino , Feminino , Estudos Retrospectivos , Biomarcadores Tumorais/metabolismo , Idoso , Imuno-Histoquímica/métodos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Pessoa de Meia-Idade , Prognóstico , Idoso de 80 Anos ou mais , Estudo de Prova de Conceito , Metástase Neoplásica , AdultoRESUMO
Purpose: Besides regulating paracellular diffusion, claudin-19 modulates the expression of proteins essential for the retinal pigment epithelium (RPE). This study asks how RPE responds when the expression of claudin-19 is reduced. Methods: In stem cell-derived RPE, claudin-19 and sequestosome-1/p62 (SQSTM1) were knocked down with siRNAs. Expression was monitored by quantitative RT-PCR and western blotting. Morphology and function were monitored by immunocytochemistry and transepithelial electrical resistance (TER). Phagocytosis of photoreceptor outer segments (POSs) was followed by fluorescence-activated cell sorting and western blotting. Pharmacology was used to assess the effects of AMP-activated protein kinase (AMPK) and SQSTM1 on phagocytosis. Enzymatic activity was measured using commercial assay kits. Results: Knockdown of claudin-19 reduced the TER without affecting the integrity of the apical junctional complex, as assessed by the distribution of zonula occludens-1 and filamentous actin. AMPK was activated without apparent effect on autophagy. Activation of AMPK alone had little effect on phagocytosis. Without affecting ingestion, knockdown reduced the rate of POS degradation and increased the steady-state levels of LC3B and SQSTM1. Proteasome inhibitors also retarded degradation, as did knockdown of SQSTM1. The expression of metallothioneins and the activity of superoxide dismutase increased. Conclusions: Knockdown of claudin-19 slowed the degradation of internalized POSs. The study questions the role of activated AMPK in phagocytosis and suggests a role for SQSTM1. Further, knockdown was associated with a partial oxidative stress response. The study opens new avenues of experimentation to explore these essential RPE functions.
Assuntos
Claudinas/genética , Regulação da Expressão Gênica , RNA/genética , Doenças Retinianas/genética , Epitélio Pigmentado da Retina/metabolismo , Proteína Sequestossoma-1/genética , Western Blotting , Linhagem Celular , Claudinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Imediatamente Precoces , Imuno-Histoquímica , Microscopia Confocal , Fagocitose , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/patologia , Proteína Sequestossoma-1/biossínteseRESUMO
Alzheimer's disease (AD) is a progressive neurodegeneration characterized by neuron death ending in memory and cognitive decline. A major concern in AD research is the identification of new therapeutics that could prevent or delay the onset of the disorder, with current treatments being effective only in reducing symptoms. In this perspective, the use of engineered probiotics as therapeutic tools for the delivery of molecules to eukaryotic cells is finding application in several disorders. This work introduces a new strategy for AD treatment based on the use of a Lactobacilluslactis strain carrying one plasmid (pExu) that contains a eukaryotic expression cassette encoding the human p62 protein. 3xTg-AD mice orally administered with these bacteria for two months showed an increased expression of endogenous p62 in the brain, with a protein delivery mechanism involving both lymphatic vessels and neural terminations, and positive effects on the major AD hallmarks. Mice showed ameliorated memory, modulation of the ubiquitin-proteasome system and autophagy, reduced levels of amyloid peptides, and diminished neuronal oxidative and inflammatory processes. Globally, we demonstrate that these extremely safe, non-pathogenic and non-invasive bacteria used as delivery vehicles for the p62 protein represent an innovative and realistic therapeutic approach in AD.
Assuntos
Doença de Alzheimer/prevenção & controle , Encéfalo/metabolismo , Terapia Genética , Vetores Genéticos , Lactobacillus/genética , Probióticos , Proteína Sequestossoma-1/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Comportamento Animal , Encéfalo/patologia , Encéfalo/fisiopatologia , Cognição , Modelos Animais de Doenças , Microbioma Gastrointestinal , Mediadores da Inflamação/metabolismo , Lactobacillus/metabolismo , Masculino , Memória , Camundongos Transgênicos , Teste de Campo Aberto , Estresse Oxidativo , Proteína Sequestossoma-1/biossínteseRESUMO
The etiology of preeclampsia (PE), a serious pregnancy complication, remains an enigma. We have demonstrated that proteinopathy, a pathologic feature of neurodegenerative diseases, is a key observation in the placenta and serum from PE patients. We hypothesize that the macroautophagy/autophagy machinery that mediates degradation of aggregated proteins and damaged organelles is impaired in PE. Here, we show that TFEB (transcription factor EB), a master transcriptional regulator of lysosomal biogenesis, and its regulated proteins, LAMP1, LAMP2, and CTSD (cathepsin D), were dysregulated in the placenta from early and late onset PE deliveries. Primary human trophoblasts and immortalized extravillous trophoblasts (EVTs) showed reduced TFEB expression and nuclear translocation as well as lysosomal protein content in response to hypoxia. Hypoxia-exposed trophoblasts also showed decreased PPP3/calcineurin phosphatase activity and increased XPO1/CRM1 (exportin 1), events that inhibit TFEB nuclear translocation. These proteins were also dysregulated in the PE placenta. These results are supported by observed lysosomal ultrastructural defects with decreased number of autolysosomes in hypoxia-treated primary human trophoblasts. Autophagy-deficient human EVTs exhibited poor TFEB nuclear translocation, reduced lysosomal protein expression and function, and increased MTORC1 activity. Sera from PE patients induced these features and protein aggregation in EVTs. Importantly, trophoblast-specific conditional atg7 knockout mice exhibited reduced TFEB expression with increased deposition of protein aggregates in the placenta. These results provide compelling evidence for a regulatory link between accumulation of protein aggregates and TFEB-mediated impaired lysosomal biogenesis and autophagy in the placenta of PE patients. Abbreviation:atg7: autophagy related 7; CTSD: cathepsin D; ER: endoplasmic reticulum; EVTs: extravillous trophoblasts; KRT7: keratin 7; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; mSt: mStrawberry; MTORC1: mechanistic target of rapamycin complex 1; NP: normal pregnancy; NPS: normal pregnancy serum; PE: preeclampsia; PES: preeclampsia serum; p-RPS6KB: phosphorylated ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; XPO1/CRM1: exportin 1.
Assuntos
Autofagia , Hipóxia , Lisossomos/metabolismo , Pré-Eclâmpsia/metabolismo , Proteoma/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Catepsina D/biossíntese , Linhagem Celular , Citoplasma/metabolismo , Feminino , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/biossíntese , Proteínas de Membrana Lisossomal/biossíntese , Camundongos , Camundongos Knockout , Placenta/metabolismo , Gravidez , Prenhez , Proteína Sequestossoma-1/biossíntese , Trofoblastos/metabolismoRESUMO
Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T-antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV-positive tumors. Ectopic expression of MCPyV small T-antigen and truncated large T-antigen (LT), but not the wild-type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV-mediated tumorigenesis. Torin-1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan-caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV-mediated tumorigenesis and potential MCC treatment.
Assuntos
Carcinoma de Célula de Merkel/virologia , Poliomavírus das Células de Merkel/metabolismo , MicroRNAs/biossíntese , Neoplasias Cutâneas/virologia , Antígenos Virais de Tumores/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 7 Relacionada à Autofagia/biossíntese , Proteína 7 Relacionada à Autofagia/genética , Proteína Beclina-1/biossíntese , Proteína Beclina-1/genética , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Humanos , Macrolídeos/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Naftiridinas/farmacologia , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/patologia , Infecções por Polyomavirus/virologia , Processamento Pós-Transcricional do RNA , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologiaRESUMO
Vascular calcification is a complication of diseases and conditions such as chronic kidney disease, diabetes, and aging. Previous studies have demonstrated that high concentrations of inorganic phosphate (Pi) can induce oxidative stress and vascular smooth muscle cell calcification. KEAP1 (Kelch-like ECH-associated protein 1)/NF-E2-related factor 2 (NRF2) signaling has been shown to play important roles in protecting cells from oxidative stress. The current study aims to investigate the possible involvement of the KEAP1/NRF2/P62 -mediated antioxidant pathway in vascular calcification induced by high Pi levels. Exposure of vascular smooth muscle cells (VSMCs) to high Pi concentrations promoted the accumulation of reactive oxygen species (ROS) and the nuclear translocation of NRF2, along with an increase in P62 levels and a decrease in KEAP1 levels. A classic NRF2 activator, tert-butylhydroquinone (tBHQ), significantly decreased ROS levels and calcium deposition in VSMCs by promoting the nuclear translocation of NRF2 and upregulating P62 and KEAP1 expression. In contrast, silencing NRF2 and P62 with siRNAs increased the levels of ROS and calcium deposition in VSMCs. In conclusion, VSMC calcification can be alleviated by the activation of the KEAP1/NRF2/P62 antioxidative pathway, which could have a protective role when it is exogenously activated by tBHQ.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/fisiologia , Fosfatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/fisiologia , Transdução de Sinais/fisiologia , Calcificação Vascular/prevenção & controle , Linhagem Celular , Fluoresceínas/metabolismo , Humanos , Hidroquinonas/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/biossíntese , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Regulação para Cima , Calcificação Vascular/metabolismoRESUMO
Autophagy is a cellular bulk degradation process used as an alternative source of energy and metabolites and implicated in various diseases. Inefficient autophagy in nutrient-deprived cancer cells would be beneficial for cancer therapy making its modulation valuable as a therapeutic strategy for cancer treatment, especially in combination with chemotherapy. Dipyridamole (DIP) is a vasodilator and antithrombotic drug. Its major effects involve the block of nucleoside uptake and phosphodiestesase inhibition, leading to increased levels of intracellular cAMP. Here we report that DIP increases autophagic markers due to autophagic flux blockage, resembling autophagosome maturation and/or closure impairment. Treatment with DIP results in an increased number of autophagosomes and autolysosomes and impairs degradation of SQSTM1/p62. As blockage of autophagic flux decreases the recycling of cellular components, DIP reduced the intracellular ATP levels in cancer cells. Autophagic flux blockage was neither through inhibition of lysosome function nor blockage of nucleoside uptake, but could be prevented by treatment with a PKA inhibitor, suggesting that autophagic flux failure mediated by DIP results from increased intracellular levels of cAMP. Treatment with DIP presented antiproliferative effects in vitro alone and in combination with chemotherapy drugs. Collectively, these data demonstrate that DIP can impair autophagic degradation, by preventing the normal autophagosome maturation, and might be useful in combination anticancer therapy.
Assuntos
Adenocarcinoma/patologia , Autofagia/efeitos dos fármacos , Dipiridamol/farmacologia , Neoplasias da Próstata/patologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Autism spectrum disorders (ASDs) comprise a number of heterogeneous neurodevelopmental diseases. Recent studies suggest that the abnormal transmission of neural signaling pathways is associated with the pathogenesis of autism. The aim of this study was to identify a link between the Notch signaling pathway and the pathogenesis of autism. In this study, we demonstrated that prenatal exposure to valproic acid (VPA) resulted in autistic-like behaviors in offspring rats and that the expression of the Notch signaling pathway-related molecules Notch1, Jagged1, Notch intracellular domain (NICD) and Hes1 increased in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA rats compared to those of controls. However, inhibiting the Notch pathway with (3,5-Difluorophenacetyl)-L-alanyl-S-phenylglycine-2-butyl Ester (Dapt) reduced the overexpression of Notch pathway-related molecules in offspring rats. Notably, Dapt improved autistic-like behaviors in a VPA-exposed rat model of autism. Furthermore, we investigated whether Dapt improved autistic-like behavior in a VPA rat model by regulating autophagy and affecting the morphology of dendritic spines. We found that the expression of the autophagy-related proteins Beclin 1, LC3B and phospho-p62 in the PFC, HC and CB of VPA model rats increased after Notch signal activation and was inhibited by Dapt compared to those of controls. Moreover, postsynaptic density-95 (PSD-95) protein expression also increased significantly compared to that of VPA model rats. The density of dendritic spines decreased in the PFC of VPA rats treated with Dapt compared to that of VPA model rats. Our present results suggest that VPA induces an abnormal activation of the Notch signaling pathway. The inhibition of excessive Notch signaling activation by Dapt can alleviate autistic-like behaviors in VPA rats. Our working model suggests that the Notch signaling pathway participates in the pathogenesis of autism by regulating autophagy and affecting dendritic spine growth. The results of this study may help to elucidate the mechanism underlying autism and provide a potential strategy for treating autism.
Assuntos
Transtorno Autístico/prevenção & controle , Autofagia/efeitos dos fármacos , Espinhas Dendríticas/patologia , Dipeptídeos/farmacologia , Receptor Notch1/metabolismo , Animais , Atrofia/patologia , Transtorno Autístico/induzido quimicamente , Proteína Beclina-1/biossíntese , Comportamento Animal/efeitos dos fármacos , Cerebelo , Modelos Animais de Doenças , Proteína 4 Homóloga a Disks-Large/biossíntese , Feminino , Hipocampo/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Fosforilação , Córtex Pré-Frontal , Gravidez , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Ratos , Proteína Sequestossoma-1/biossíntese , Transdução de Sinais/efeitos dos fármacos , Ácido ValproicoRESUMO
Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.
Assuntos
Autofagia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Lipopolissacarídeos/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Fisostigmina/farmacologia , Estresse Fisiológico , Animais , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/biossíntese , Proteína Beclina-1/metabolismo , Inflamação/genética , Inflamação/patologia , Inflamação/psicologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Microglia/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Sintases/biossíntese , Período Pós-Operatório , Ratos , Ratos Wistar , Proteína Sequestossoma-1/biossínteseRESUMO
OBJECTIVE: Multidrug resistance is the major cause of treatment failure in ovarian cancer. p62 (SQSTM1) is a multifunctional protein involved in multiple cellular processes including proliferation, drug sensitivity and autophagy-associated cancer cell growth. However, the role of p62 in drug resistance remains controversial. METHODS: In this study, we examined p62 expression by immunohistochemistry in a unique ovarian cancer tissue microarray (TMA), which was constructed with paired primary, metastatic, and recurrent tumor tissues. The expression levels of p62 and autophagy related proteins were evaluated in two panels of human cancer cell lines by western blot. Cell viabilities were determined by MTT assay after exposure ovarian cancer cells to different concentrations of paclitaxel alone or in combination with autophagy inhibitors. RESULTS: Both the metastatic and recurrent tumor tissues expressed less p62 than the patient-matched primary tumor. A significant inverse correlation has been found between p62 expression and both the disease-free survival and overall survival. Additionally, multidrug resistant cancer cell lines expressed lower levels of p62 as compared with their parental drug sensitive cell lines. Importantly, inhibition of autophagy enhanced paclitaxel sensitivity in drug resistant ovarian cancer cells. Furthermore, the wound healing assay exhibited that the inhibition of autophagy significantly decreased resistant ovarian cancer cell migration in vitro. CONCLUSION: Our findings highlight the potential of p62 as a new prognostic marker for ovarian cancer patients and p62's associated autophagy pathway may be a promising therapeutic target to prevent metastasis, recurrence and to reverse drug resistance in ovarian cancer.
Assuntos
Neoplasias Ovarianas/metabolismo , Proteína Sequestossoma-1/biossíntese , Adenina/administração & dosagem , Adenina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hidroxicloroquina/administração & dosagem , Imuno-Histoquímica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical behavior. This is the first documented account of ETV6-ALK and SPECC1L-ALK translocations in neoplasms.
Assuntos
Quinase do Linfoma Anaplásico/biossíntese , Histiocitoma Fibroso Benigno/metabolismo , Histiocitoma Fibroso Benigno/patologia , Adolescente , Adulto , Idoso , Quinase do Linfoma Anaplásico/genética , Criança , Estudos de Coortes , Células Epitelioides/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Estudos Retrospectivos , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/metabolismo , Adulto Jovem , Fator de von Willebrand/biossíntese , Fator de von Willebrand/genéticaRESUMO
Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, and is associated with poor prognosis. Autophagy is a programmed cell survival mechanism involved in physiologic processes and various diseases including cancer. However, the relationship between autophagy and cancer is controversial. Several studies have claimed that the expression of autophagy-related proteins, namely microtubule-associated protein light chain3 (LC3) and p62/SQSTM1 (p62), is associated with poor prognosis in OSCC. In this study, we evaluated the expression of the autophagy-related markers LC3A/B and p62 by immunohistochemistry in 71 OSCC patient samples, especially focusing on surgical margins. Results were correlated with clinical characteristics. The expression of LC3A and LC3B was correlated with tumor recurrence and poor overall survival based on multivariate analysis, whereas the expression of p62 was correlated with only tumor recurrence and not prognosis. Thus, we suggest that the expression of autophagy-related markers at the surgical margins might be an indicator of local recurrence and poor prognosis in human OSCC. These results will aid in the development of new therapeutics and diagnostics for OSCC.
Assuntos
Autofagia/fisiologia , Biomarcadores Tumorais/análise , Margens de Excisão , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/biossíntese , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Recidiva Local de Neoplasia/patologia , Prognóstico , Proteína Sequestossoma-1/análise , Proteína Sequestossoma-1/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidadeRESUMO
Inhibition of phosphodiesterase 4 (PDE4) suppressed the inflammatory responses in the brain. However, the underlying mechanisms are poorly understood. Roflupram (ROF) is a novel PDE4 inhibitor. In the present study, we found that ROF enhanced the level of microtubule-associated protein 1 light chain 3 II (LC3-II) and decreased p62 in microglial BV-2 cells. Enhanced fluorescent signals were observed in BV-2 cells treated with ROF by Lysotracker red and acridine orange staining. In addition, immunofluorescence indicated a significant increase in punctate LC3. Moreover, ß amyloid 25-35 (Aß25-35) or lipopolysaccharide (LPS) with ATP was used to activate inflammasome. We found that both LPS plus ATP and Aß25-35 enhanced the conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of mature IL-1ß in BV-2 cells. Interestingly, these effects were blocked by the treatment of ROF. Consistently, knocking down the expression of PDE4B in primary microglial cells led to enhanced level of LC-3 II and decreased activation of inflammasome. What's more, Hoechst staining showed that ROF decreased the apoptosis of neuronal N2a cells in conditioned media from microglia. Our data also showed that ROF dose-dependently enhanced autophagy, reduced the activation of inflammasome and suppressed the production of IL-1ß in mice injected with LPS. These effects were reversed by inhibition of microglial autophagy. These results put together demonstrate that ROF inhibits inflammasome activities and reduces the release of IL-1ß by inducing autophagy. Therefore, ROF could be used as a potential therapeutic compound for the intervention of inflammation-associated diseases in the brain.
Assuntos
Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Derivados de Benzeno/farmacologia , Furanos/farmacologia , Inflamassomos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Peptídeos beta-Amiloides/farmacologia , Animais , Anti-Inflamatórios/química , Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Proteína 7 Relacionada à Autofagia/genética , Derivados de Benzeno/química , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Furanos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Estrutura Molecular , Fragmentos de Peptídeos/farmacologia , Inibidores da Fosfodiesterase 4/química , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genéticaRESUMO
Long-term application of Tamoxifen (TAM) is usually recommended for hormone receptor positive breast cancer patients. Unfortunately, TAM will inevitably increase the incidence of endometrial hyperplasia even endometrial cancer. Despite of substantial investigations, no effective approaches to prevent TAM-induced endometrial carcinogenesis have been acknowledged. In this study, we found that inhibition of Nrf2 could be valuable to prevent TAM-induced endometrial hyperplasia. Upon TAM treatment, the mRNA and protein expression of autophagy adaptor SQSTM1 was specifically increased in endometrial cells but not breast cancer cells. Knocking-down of SQSTM1 expression retarded TAM-promoted growth of endometrial cancer cells. TAM stimulated SQSTM1 transcription specifically in endometrial cells by enhancing phosphorylation and nuclear translocation of Nrf2. Indeed, the expression of Nrf2 and SQSTM1 were positively correlated in primary endometrial tissues. In rats with TAM-induced endometrial hyperplasia, both Nrf2 and SQSTM1 expression were increased. Nrf2 inhibitor brusatol effectively attenuated TAM-induced SQSTM1 upregulation and endometrial hyperplasia. The kinase of Nrf2, PRKCD, was activated by TAM. Once PRKCD was depleted, TAM failed to promote Nrf2 phosphorylation and SQSTM1 expression. In summary, TAM stimulated Nrf2-dependent SQSTM1 transcription to promote endometrial hyperplasia by activating PRKCD. Therefore, blocking PRKCD-Nrf2-SQSTM1 signaling could be useful to prevent TAM-induced endometrial hyperplasia.
Assuntos
Antineoplásicos Hormonais/efeitos adversos , Hiperplasia Endometrial/induzido quimicamente , Fator 2 Relacionado a NF-E2/metabolismo , Proteína Sequestossoma-1/biossíntese , Tamoxifeno/efeitos adversos , Transcrição Gênica , Animais , Antineoplásicos Hormonais/administração & dosagem , Feminino , Humanos , Ratos , Tamoxifeno/administração & dosagemRESUMO
Enhanced macroautophagy/autophagy is recognized as a component of the pathogenesis of smoking-induced airway disease. Based on the knowledge that enhanced autophagy is linked to oxidative stress and the DNA damage response, both of which are linked to smoking, we used microarray analysis of the airway epithelium to identify smoking upregulated genes known to respond to oxidative stress and the DNA damage response. This analysis identified OSGIN1 (oxidative stress induced growth inhibitor 1) as significantly upregulated by smoking, in both the large and small airway epithelium, an observation confirmed by an independent small airway microarray cohort, TaqMan PCR of large and small airway samples and RNA-Seq of small airway samples. High and low OSGIN1 expressors have different autophagy gene expression patterns in vivo. Genome-wide correlation of RNAseq analysis of airway basal/progenitor cells showed a direct correlation of OSGIN1 mRNA levels to multiple classic autophagy genes. In vitro cigarette smoke extract exposure of primary airway basal/progenitor cells was accompanied by a dose-dependent upregulation of OSGIN1 and autophagy induction. Lentivirus-mediated expression of OSGIN1 in human primary basal/progenitor cells induced puncta-like staining of MAP1LC3B and upregulation of MAP1LC3B mRNA and protein and SQSTM1 mRNA expression level in a dose and time-dependent manner. OSGIN1-induction of autophagosome, amphisome and autolysosome formation was confirmed by colocalization of MAP1LC3B with SQSTM1 or CD63 (endosome marker) and LAMP1 (lysosome marker). Both OSGIN1 overexpression and knockdown enhanced the smoking-evoked autophagic response. Together, these observations support the concept that smoking-induced upregulation of OSGIN1 is one link between smoking-induced stress and enhanced-autophagy in the human airway epithelium.
Assuntos
Autofagia , Fumar Cigarros , Proteínas/fisiologia , Mucosa Respiratória/metabolismo , Proteínas Reguladoras de Apoptose , Autofagossomos/ultraestrutura , Autofagia/genética , Células Cultivadas , Humanos , Lisossomos/ultraestrutura , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Estresse Oxidativo , Proteínas/genética , Proteínas/metabolismo , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Regulação para CimaRESUMO
PURPOSE: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.
Assuntos
Autofagia/fisiologia , Diabetes Mellitus Experimental , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , RNA Interferente Pequeno/genética , Retina/metabolismo , Proteína Sequestossoma-1/genética , Animais , Apoptose , Autofagia/efeitos dos fármacos , Western Blotting , Células Cultivadas , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Glucose/farmacologia , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Retina/patologia , Proteína Sequestossoma-1/biossíntese , Edulcorantes/farmacologiaRESUMO
p62/SQSTM1 is a multifunctional signaling hub and autophagy adaptor with many binding partners, which allow it to activate mTORC1-dependent nutrient sensing, NF-κB-mediated inflammatory responses, and the NRF2-activated antioxidant defense. p62 recognizes polyubiquitin chains via its C-terminal domain and binds to LC3 via its LIR motif, thereby promoting the autophagic degradation of ubiquitinated cargos. p62 accumulates in many human liver diseases, including nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC), where it is a component of Mallory-Denk bodies and intracellular hyaline bodies. Chronic p62 elevation contributes to HCC development by preventing oncogene-induced senescence and death of cancer-initiating cells and enhancing their proliferation. In this review, we discuss p62-mediated signaling pathways and their roles in liver pathophysiology, especially NASH and HCC.
Assuntos
Carcinoma Hepatocelular/genética , Inflamação/genética , Neoplasias Hepáticas/genética , Proteína Sequestossoma-1/biossíntese , Carcinoma Hepatocelular/metabolismo , Alimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/metabolismo , Neoplasias Hepáticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , NF-kappa B/genética , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Estresse Oxidativo/genética , Proteína Sequestossoma-1/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Neurônios , Ubiquitinas/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Morte Celular , Humanos , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/psicologia , Mutação/genética , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Aprendizagem Espacial , Ubiquitinas/genéticaRESUMO
Enhanced autophagy has been observed in hypoxic regions of solid tumors. Here we address the hypothesis that autophagy is required for survival of hypoxic cells. We evaluated sensitivity to hypoxia of three human tumor cell lines (MCF7, PC3, and LNCaP) and their autophagy-deficient variants with shRNA knockdown of the genes ATG7 and BECLIN1. Hypoxia-induced cell death was more rapid for autophagy-deficient cells and was increased in the presence of the proton pump inhibitor pantoprazole that inhibits autophagy. Autophagy-deficient cells had a lower rate of oxygen consumption than wild-type cells. In xenografts derived from the three cell lines, autophagy (as determined by increased LC3 and reduced p62/SQSTM1) colocalized with hypoxic regions (identified by EF5). Xenografts derived from autophagy-deficient cells grew more slowly than wild-type tumors. Both LC3 expression and hypoxia were decreased in xenografts generated from single-knockdown cells and absent in double-knockdown tumors. Our results are consistent with the hypothesis that autophagy facilitates the survival of hypoxic cells, although reduced oxygen consumption of autophagy-deficient cells may contribute to lack of hypoxia in tumors derived from them. Because hypoxia is associated with resistance to anticancer therapy, inhibition of autophagy has potential to enhance the effectiveness of cancer treatment.
Assuntos
Autofagia/fisiologia , Hipóxia Celular/fisiologia , Neoplasias/patologia , Consumo de Oxigênio/fisiologia , 2-Piridinilmetilsulfinilbenzimidazóis , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Transplante de Neoplasias , Pantoprazol , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Sequestossoma-1/biossíntese , Transplante HeterólogoRESUMO
Autophagy is a homeostatic process for recycling proteins and organelles that is increasingly being proposed as a therapeutic target for acute and chronic neurodegenerative diseases, including stroke. Confirmation that autophagy is present in the human brain after stroke is imperative before prospective therapies can begin the translational process into clinical trials. Our current study using human post-mortem tissue observed an increase in staining in microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1; also known as p62) and the increased appearance of autophagic vesicles after stroke. These data confirm that alterations in autophagy take place in the human brain after stroke and suggest that targeting autophagic processes after stroke may have clinical significance.