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1.
Nat Commun ; 11(1): 4931, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004795

RESUMO

Testis-restricted melanoma antigen (MAGE) proteins are frequently hijacked in cancer and play a critical role in tumorigenesis. MAGEs assemble with E3 ubiquitin ligases and function as substrate adaptors that direct the ubiquitination of novel targets, including key tumor suppressors. However, how MAGEs recognize their targets is unknown and has impeded the development of MAGE-directed therapeutics. Here, we report the structural basis for substrate recognition by MAGE ubiquitin ligases. Biochemical analysis of the degron motif recognized by MAGE-A11 and the crystal structure of MAGE-A11 bound to the PCF11 substrate uncovered a conserved substrate binding cleft (SBC) in MAGEs. Mutation of the SBC disrupted substrate recognition by MAGEs and blocked MAGE-A11 oncogenic activity. A chemical screen for inhibitors of MAGE-A11:substrate interaction identified 4-Aminoquinolines as potent inhibitors of MAGE-A11 that show selective cytotoxicity. These findings provide important insights into the large family of MAGE ubiquitin ligases and identify approaches for developing cancer-specific therapeutics.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas de Neoplasias/ultraestrutura , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Motivos de Aminoácidos , Aminoquinolinas/farmacologia , Aminoquinolinas/uso terapêutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Estudo de Prova de Conceito , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Domínios Proteicos/genética , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética
2.
Nat Commun ; 11(1): 5079, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033234

RESUMO

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.


Assuntos
Carcinoma Basocelular/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/metabolismo , Ontologia Genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteína Smad3/metabolismo , Transativadores/metabolismo , Regulação para Cima
3.
Nat Commun ; 11(1): 4913, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004813

RESUMO

Reprograming of proline metabolism is critical for tumor growth. Here we show that PINCH-1 is highly expressed in lung adenocarcinoma and promotes proline synthesis through regulation of mitochondrial dynamics. Knockout (KO) of PINCH-1 increases dynamin-related protein 1 (DRP1) expression and mitochondrial fragmentation, which suppresses kindlin-2 mitochondrial translocation and interaction with pyrroline-5-carboxylate reductase 1 (PYCR1), resulting in inhibition of proline synthesis and cell proliferation. Depletion of DRP1 reverses PINCH-1 deficiency-induced defects on mitochondrial dynamics, proline synthesis and cell proliferation. Furthermore, overexpression of PYCR1 in PINCH-1 KO cells restores proline synthesis and cell proliferation, and suppresses DRP1 expression and mitochondrial fragmentation. Finally, ablation of PINCH-1 from lung adenocarcinoma in mouse increases DRP1 expression and inhibits PYCR1 expression, proline synthesis, fibrosis and tumor growth. Our results identify a signaling axis consisting of PINCH-1, DRP1 and PYCR1 that regulates mitochondrial dynamics and proline synthesis, and suggest an attractive strategy for alleviation of tumor growth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma de Pulmão/patologia , Proteínas com Domínio LIM/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Dinaminas/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Proteínas com Domínio LIM/genética , Pulmão/citologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Prolina/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirrolina Carboxilato Redutases/metabolismo , Análise de Sobrevida
4.
Nat Commun ; 11(1): 5077, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033240

RESUMO

Although substantial progress has been made in cancer biology and treatment, clinical outcomes of bladder carcinoma (BC) patients are still not satisfactory. The tumor microenvironment (TME) is a potential target. Here, by single-cell RNA sequencing on 8 BC tumor samples and 3 para tumor samples, we identify 19 different cell types in the BC microenvironment, indicating high intra-tumoral heterogeneity. We find that tumor cells down regulated MHC-II molecules, suggesting that the downregulated immunogenicity of cancer cells may contribute to the formation of an immunosuppressive microenvironment. We also find that monocytes undergo M2 polarization in the tumor region and differentiate. Furthermore, the LAMP3 + DC subgroup may be able to recruit regulatory T cells, potentially taking part in the formation of an immunosuppressive TME. Through correlation analysis using public datasets containing over 3000 BC samples, we identify a role for inflammatory cancer-associated fibroblasts (iCAFs) in tumor progression, which is significantly related to poor prognosis. Additionally, we characterize a regulatory network depending on iCAFs. These results could help elucidate the protumor mechanisms of iCAFs. Our results provide deep insight into cancer immunology and provide an essential resource for drug discovery in the future.


Assuntos
Fibroblastos/patologia , Inflamação/patologia , Análise de Sequência de RNA , Análise de Célula Única , Bexiga Urinária/patologia , Área Sob a Curva , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Polaridade Celular , Proliferação de Células , Citocinas/metabolismo , Variações do Número de Cópias de DNA/genética , Células Dendríticas/metabolismo , Redes Reguladoras de Genes , Humanos , Ligantes , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Monócitos/patologia , Células Mieloides/patologia , Proteínas de Neoplasias/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral , Bexiga Urinária/imunologia
5.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
6.
Nat Commun ; 11(1): 4660, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938908

RESUMO

Intratumor spatial heterogeneity facilitates therapeutic resistance in glioblastoma (GBM). Nonetheless, understanding of GBM heterogeneity is largely limited to the surgically resectable tumor core lesion while the seeds for recurrence reside in the unresectable tumor edge. In this study, stratification of GBM to core and edge demonstrates clinically relevant surgical sequelae. We establish regionally derived models of GBM edge and core that retain their spatial identity in a cell autonomous manner. Upon xenotransplantation, edge-derived cells show a higher capacity for infiltrative growth, while core cells demonstrate core lesions with greater therapy resistance. Investigation of intercellular signaling between these two tumor populations uncovers the paracrine crosstalk from tumor core that promotes malignancy and therapy resistance of edge cells. These phenotypic alterations are initiated by HDAC1 in GBM core cells which subsequently affect edge cells by secreting the soluble form of CD109 protein. Our data reveal the role of intracellular communication between regionally different populations of GBM cells in tumor recurrence.


Assuntos
Antígenos CD/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Histona Desacetilase 1/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Feminino , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Camundongos SCID , Fenilbutiratos/farmacologia , Transdução de Sinais , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS One ; 15(8): e0238466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857809

RESUMO

PURPOSE: Phyllodes tumors (PTs) are biphasic tumors accounting for 0.3-1.5% of all breast tumors. Epithelial membrane proteins (EMPs) have been reported in various malignant tumors but their expression in PTs is unclear. In this study, we aimed to evaluate the expression of EMP1, EMP2, and EMP3 in breast phyllodes tumors (PTs), and to investigate their clinical implications. METHODS: In total, 185 PTs were used for constructing a tissue microarray. Immunohistochemical staining for EMP1, EMP2, and EMP3 was performed, and the results were analyzed along with the clinicopathologic parameters. RESULTS: In total, 185 PTs were included in this study, and comprised 138 benign, 32 borderline, and 15 malignant PTs. In malignant PTs, the epithelial component showed decreased expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032), compared to the benign and borderline PTs. Conversely, stromal component of borderline and malignant PTs showed higher expression of EMP1 (P = 0.027), EMP2 (P = 0.004), and EMP3 (P = 0.032) compared to benign PTs. Expression of EMP1 and EMP3 correlated positively with stromal cellularity and cellular atypia (P < 0.001). In the univariate analysis, stromal EMP3 was associated with shorter disease-free survival (P < 0.001), and shorter overall survival (P = 0.034). CONCLUSION: The expression of EMP1, EMP2, and EMP3 is decreased in the epithelial component and is increased in the stromal component of PT with higher histologic grade. Thus, stromal EMP3 expression may serve as an independent prognostic factor in PT.


Assuntos
Neoplasias da Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Tumor Filoide/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Tumor Filoide/patologia , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Análise Serial de Tecidos
8.
Ecotoxicol Environ Saf ; 202: 110940, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800223

RESUMO

Recent evidence indicates that chronic, low-dose exposure to mixtures of pesticides can cause adverse responses in a variety of cells, tissues and organs, although interactions between pesticides circulating in the blood and cancer cells remain largely unexplored. The aim of this study was to investigate the potential of a mixture of four pesticides to induce multidrug resistance against the chemotherapeutic agents cisplatin, 5-fluorouracil and temozolomide in the human U87 glioblastoma cell line, and to explore the molecular mechanisms underlying this resistance. We found that the repeated administration of the pesticide mixture (containing the insecticides chlorpyrifos-ethyl and deltamethrin, the fungicide metiram, and the herbicide glyphosate) induced a strong drug resistance in U87 cells. The resistance was durable and transferred to subsequent cell generations. In addition, we detected a significant over-expression of the ATP-binding cassette (ABC) membrane transporters P-gp/ABCB1 and BRCP/ABCG2 as well as a glutathione-S-transferase (GST)/M1-type cellular detoxification function, known to have important roles in multidrug resistance, thus providing molecular support for the acquired multidrug resistance phenotype and shedding light on the mechanism of resistance. We further determined that there was lower mortality in the resistant brain tumor cells and that the mitochondrial apoptosis pathway was activated at a lower rate after chemotherapy compared to non-resistant control cells. In addition, multidrug-resistant cells were found to have both higher motility and wound-healing properties, suggesting a greater metastatic potential. Our results suggest that the investigation of P-gp, BRCP and GST/M1 multidrug resistance gene expression and/or protein levels in biopsy specimens of brain tumor patients who were at risk of pesticide exposure could be beneficial in determining chemotherapy dose and prolonging patient survival.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Praguicidas/toxicidade , Testes de Toxicidade Crônica , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia
9.
DNA Cell Biol ; 39(9): 1621-1638, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32758021

RESUMO

Increasing evidence highlights the clinical significance of stromal cells and immune cells in the liver cancer microenvironment. However, reliable prognostic models have not been well established. This study aimed to develop a gene signature for liver cancer based on stromal and immune scores. Using the estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE) algorithm, stromal and immune scores were estimated based on the transcriptome profile of The Cancer Genome Atlas (TCGA) liver cancer cohort. Stromal-/immune-related differentially expressed genes were identified, followed by functional enrichment analysis. The Cox regression model was used to select prognostic genes and construct a gene signature. Its predictive potential was evaluated by receiver operating characteristic (ROC). The correlation between the risk score and immune cell infiltration was analyzed using Tumor Immune Estimation Resource (TIMER). Three hundred sixty-four upregulated and 10 downregulated stromal-/immune-related genes were identified, were mainly enriched in immune-related processes and pathways. Through univariate and multivariate cox survival analysis, a five-gene risk score was constructed, composed of FABP3, HTRA3, OLFML2B, PDZD4 and SLAMF6. Patients with high score indicated a poorer prognosis than those with low risk score. The areas under the ROC curves of overall survival (OS), progression-free interval, 3-, 5-year, OS status were 0.68, 0.57, 0.72, 0.74 and 0.728, indicating its well performance on predicting patients' prognoses. Furthermore, the risk score and the five genes were significantly correlated with immune cell infiltration in the tumor microenvironment. In this study, we proposed a prognostic five-gene signature based on stromal/immune scores in the liver cancer microenvironment.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Hepáticas/genética , Microambiente Tumoral , Idoso , Biomarcadores Tumorais/metabolismo , Proteína 3 Ligante de Ácido Graxo/genética , Proteína 3 Ligante de Ácido Graxo/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Transcriptoma
10.
Prostate ; 80(13): 1071-1086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687633

RESUMO

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.


Assuntos
Colágeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Idoso , Sequência de Aminoácidos , Autoantígenos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/metabolismo , Progressão da Doença , Colágenos Fibrilares/metabolismo , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Colágenos não Fibrilares , Prolina/metabolismo , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
11.
Adv Cancer Res ; 148: 27-67, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32723566

RESUMO

Vascular mimicry is induced by a wide array of genes with functions related to cancer stemness, hypoxia, angiogenesis and autophagy. Vascular mimicry competent (VM-competent) cells that form de novo blood vessels are common in solid tumors facilitating tumor cell survival and metastasis. VM-competent cells display increased levels of vascular mimicry selecting for stem-like cells in an O2-gradient-dependent manner in deeply hypoxic tumor regions, while also aiding in maintaining tumor cell metabolism and stemness. Three of the principal drivers of vascular mimicry are EphA2, Nodal and HIF-1α, however, directly or indirectly many of these molecules affect VE-Cadherin (VE-Cad), which forms gap-junctions to bind angiogenic blood vessels together. During vascular mimicry, the endothelial-like functions of VM-competent cancer stem cells co-opt VE-Cad to bind cancer cells together to create cancer cell-derived blood conducting vessels. This process potentially compensates for the lack of access to blood and nutrient in avascular tumors, simultaneously providing nutrients and enhancing cancer invasion and metastasis. Current evidence also supports that vascular mimicry promotes cancer malignancy and metastasis due to the cooperation of oncogenic signaling molecules driving cancer stemness and autophagy. While a number of currently used cancer therapeutics are effective inhibitors of vascular mimicry, developing a new class of vascular mimicry specific inhibitors could allow for the treatment of angiogenesis-resistant tumors, inhibit cancer metastasis and improve patient survival. In this review, we describe the principal vascular mimicry pathways in addition to emphasizing the roles of hypoxia, autophagy and select proangiogenic oncogenes in this process.


Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/patologia , Animais , Autofagia/fisiologia , Hipóxia Celular/fisiologia , Modelos Animais de Doenças , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia
12.
PLoS Biol ; 18(7): e3000755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644996

RESUMO

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
13.
PLoS One ; 15(7): e0236447, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697788

RESUMO

The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.


Assuntos
Hepacivirus/enzimologia , Hepatite C Crônica/patologia , Interações entre Hospedeiro e Microrganismos , Proteínas de Neoplasias/metabolismo , Proteínas não Estruturais Virais/metabolismo , Biópsia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Hepacivirus/patogenicidade , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Fígado/patologia , Fígado/virologia , Mitocôndrias/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/genética , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Domínios Proteicos/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/genética , Proteínas não Estruturais Virais/antagonistas & inibidores
14.
Mol Cell ; 79(4): 645-659.e9, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32692974

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.


Assuntos
Grânulos Citoplasmáticos/genética , DNA Helicases/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Biossíntese de Proteínas , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , Estresse Fisiológico/genética , Regiões 5' não Traduzidas , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Feminino , Células HCT116 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Espermatogônias/citologia , Espermatogônias/patologia , Testículo/citologia , Testículo/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(28): 16638-16648, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601188

RESUMO

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição NFATC/genética , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Ligação Proteica , Transdução de Sinais , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
16.
Proc Natl Acad Sci U S A ; 117(31): 18600-18607, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32703806

RESUMO

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.


Assuntos
Proteínas Fúngicas , Proteínas de Neoplasias , Piroptose/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Neurospora crassa/metabolismo
17.
Nat Commun ; 11(1): 3563, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678104

RESUMO

Rapidly increasing availability of genomic data and ensuing identification of disease associated mutations allows for an unbiased insight into genetic drivers of disease development. However, determination of molecular mechanisms by which individual genomic changes affect biochemical processes remains a major challenge. Here, we develop a multilayered proteomic workflow to explore how genetic lesions modulate the proteome and are translated into molecular phenotypes. Using this workflow we determine how expression of a panel of disease-associated mutations in the Dyrk2 protein kinase alter the composition, topology and activity of this kinase complex as well as the phosphoproteomic state of the cell. The data show that altered protein-protein interactions caused by the mutations are associated with topological changes and affected phosphorylation of known cancer driver proteins, thus linking Dyrk2 mutations with cancer-related biochemical processes. Overall, we discover multiple mutation-specific functionally relevant changes, thus highlighting the extensive plasticity of molecular responses to genetic lesions.


Assuntos
Neoplasias/genética , Neoplasias/patologia , Proteínas Quinases/genética , Proteômica/métodos , Linhagem Celular , Humanos , Espectrometria de Massas , Complexos Multiproteicos , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Mapas de Interação de Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteoma/metabolismo
18.
PLoS Pathog ; 16(6): e1008514, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479542

RESUMO

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Células Gigantes , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus Oncolíticos/fisiologia , Replicação Viral , Células A549 , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Gigantes/metabolismo , Células Gigantes/virologia , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/virologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética
20.
Life Sci ; 256: 117893, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502539

RESUMO

AIMS: To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF). MATERIALS AND METHODS: Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation. KEY FINDINGS: Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result. SIGNIFICANCE: Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.


Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Pulmão/embriologia , Meliteno/farmacologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/sangue , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiprolina/metabolismo , Proteínas de Neoplasias/sangue , Proteínas Nucleares/sangue , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2C/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitinação/efeitos dos fármacos , Vimentina/metabolismo
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