RESUMO
BACKGROUND: As a class of the opioid receptors, the kappa opioid receptor (KOR) has been verified to be a potential biomarker and therapeutic target for human malignant tumors. However, a thorough understanding of whether KOR affects progression of esophageal squamous cell carcinoma (ESCC) is still lacking. This study focused on exploring the effect of knocking down KOR in ESCC and its underlying mechanism. METHODS: Bioinformatics analysis was used to compare the different expression level of OPRK1 (KOR gene) in tumor and adjacent normal tissues, and predict the relationship between KOR expression and overall survival. RNA-sequence analysis was performed to detect the altered functions and mechanisms after down regulating KOR. The in vitro and in vivo assays were used to detect the effects of down-regulated KOR on cell proliferation, migration and invasion. Substrate gel zymography and 3D cell culture assays were used to find the effect of KOR knockdown on the degradation of extracellular matrix (ECM), and immunefluorescence was performed to detect the altered cytoskeleton. Western blotting and immunohistochemistry were used to explore the underlying mechanism pathway. RESULTS: Bioinformatics analysis revealed that the expression of OPRK1 was lower in tumor tissue than that in adjacent normal tissues, and lowered expression of KOR was associated with poorer overall survival. The in vitro assays demonstrated that down-regulation of KOR enhanced ESCC proliferation, metastasis and invasion. Western blotting revealed that down-regulation of KOR could activate PDK1-AKT signaling pathway, which actively regulated the cancer progression. Down-regulation of KOR enhanced the formation of invadopodia, secretion of matrix metalloproteinase-2 (MMP2) and rearrangement of cytoskeleton, which were positively related with the invasion of ESCC. KOR knockdown enhanced the tumor invasion and elevated the AKT phosphorylation in nude mice. The AKT kinase inhibition could reverse the effect of down-regulation of KOR. CONCLUSION: KOR might act as a tumor suppressor in ESCC and down-regulation of KOR could enhance the ESCC tumor phenotype. Video Abstract.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Regulação para Baixo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Transdução de Sinais/genéticaRESUMO
Filopodia are dynamic membrane extensions generated by F-actin bundling and are involved in cancer cell migration, invasion and metastasis. Fascin is the crucial actin-bundling protein in filopodia, with phosphorylation at fascin serine 39 being well characterized to regulate fascin-mediated actin bundling in filopodia. However, increasing evidence indicates that fascin is phosphorylated at a number of sites. Whether phosphorylation at other sites also regulates fascin function is unknown. In this study, we show that four potential phosphorylation sites in fascin, specifically tyrosine 23, serine 38, serine 39 and serine 274, regulate cell behavior and filopodia formation in esophageal squamous cancer cells. Expression of non-phosphorylatable mutations at each of the four sites promoted anchorage-independent growth, cell motility and filopodia formation, whereas phosphomimetic mutations at each of these sites inhibited these cell behaviors, implying that fascin function in esophageal squamous cancer is regulated by fascin phosphorylation at multiple sites. Furthermore, phosphorylation at S38 and S39 cooperatively regulated cell behavior and filopodia formation, with dual dephosphorylation at both S38 and S39 residues maximally enhancing cell proliferation, migration and filopodia formation, and phosphorylation at any of the two phosphorylatable sites resulting in reduced enhancement. Taken together, our results reveal that phosphorylation at fascin amino acids Y23, S38, S39 and S274, in combination, downregulates the extent of anchorage-independent growth, cell migration and filopodia formation in esophageal squamous cancer cells.
Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Processamento de Proteína Pós-Traducional , Pseudópodes/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/patologia , Esôfago/metabolismo , Esôfago/patologia , Humanos , Proteínas dos Microfilamentos/genética , Mutação , Fosforilação , Pseudópodes/patologia , Pseudópodes/ultraestruturaRESUMO
Lipocalin 2 (LCN2) is a poor prognostic factor in esophageal squamous cell carcinoma (ESCC), however its functional roles and molecular mechanisms of action remain to be clarified. Here, we described the functions and signaling pathways for LCN2 in ESCC. Overexpression of LCN2 in ESCC cells accelerated cell migration and invasion in vitro, and promoted lung metastasis in vivo. Blocking LCN2 expression inhibited its pro-oncogenic effect. Either overexpression of LCN2 or treatment with recombinant human LCN2 protein enhanced the activation of MEK/ERK pathway, which in turn increases endogenous LCN2 to increase MMP-9 activity. The decreased p-cofilin and increased p-ERM induced by pERK1/2 cause the cytoskeleton F-actin rearrangement and alter the behavior of ESCC cells mediated by LCN2. As a consequence, activation of MMP-9 and the rearrangement of F-actin throw light on the mechanisms for LCN2 in ESCC. These results imply that LCN2 promotes the migration and invasion of ESCC cells through a novel positive feedback loop.
Assuntos
Proteínas de Fase Aguda/metabolismo , Carcinoma de Células Escamosas/metabolismo , Movimento Celular , Neoplasias Esofágicas/metabolismo , Lipocalinas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas de Fase Aguda/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Citoesqueleto/genética , Citoesqueleto/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , Lipocalina-2 , Lipocalinas/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
To further our understanding of the pathobiology of esophageal squamous cell carcinoma (ESCC), we previously performed microRNA profiling that revealed downregulation of miR-200b in ESCC. Using quantitative real-time PCR applied to 88 patient samples, we confirmed that ESCC tumors expressed significantly lower levels of miR-200b compared with the respective adjacent benign tissues (P = 0.003). Importantly, downregulation of miR-200b significantly correlated with shortened survival (P = 0.025), lymph node metastasis (P = 0.002) and advanced clinical stage (P = 0.020) in ESCC patients. Quantitative mass spectrometry identified 57 putative miR-200b targets, including Kindlin-2, previously implicated in the regulation of tumor invasiveness and actin cytoskeleton in other cell types. Enforced expression of miR-200b mimic in ESCC cells led to a decrease of Kindlin-2 expression, whereas transfection of miR-200b inhibitor induced Kindlin-2 expression. Furthermore, transfection of miR-200b mimic or knockdown of Kindlin-2 in ESCC cells decreased cell protrusion and focal adhesion (FA) formation, reduced cell spreading and invasiveness/migration. Enforced expression of Kindlin-2 largely abrogated the inhibitory effects of miR-200b on ESCC cell invasiveness. Mechanistic studies revealed that Rho-family guanosine triphosphatases and FA kinase mediated the biological effects of the miR-200b-Kindlin-2 axis in ESCC cells. To conclude, loss of miR-200b, a frequent biochemical defect in ESCC, correlates with aggressive clinical features. The tumor suppressor effects of miR-200b may be due to its suppression of Kindlin-2, a novel target of miR-200b that modulates actin cytoskeleton, FA formation and the migratory/invasiveness properties of ESCC.
Assuntos
Carcinoma de Células Escamosas/patologia , Citoesqueleto/metabolismo , Neoplasias Esofágicas/patologia , Adesões Focais/fisiologia , Proteínas de Membrana/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Mutação/genética , Invasividade Neoplásica , Fosforilação , Prognóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
In contrast to the well-recognized loss of adherens junctions in cancer progression, the role of desmosomal components in cancer development has not been well explored. We previously demonstrated that desmocollin-2 (DSC2), a desmosomal cadherin protein, is reduced in oesophageal squamous cell carcinoma (ESCC), and is associated with enhanced tumour metastasis and poor prognosis. Here, we report that restoration of DSC2 in ESCC cells impeded cell migration and invasion both in vitro and in vivo, whereas siRNA-mediated suppression of DSC2 expression increased cell motility. In E-cadherin-expressing ESCC cells, DSC2 restoration strengthened E-cadherin-mediated adherens junctions and promoted the localization of ß-catenin at these junctions, which indirectly inhibited ß-catenin-dependent transcription. These effects of DSC2 were not present in EC109 cells that lacked E-cadherin expression. ESCC patients with tumours that had reduced E-cadherin and negative DSC2 had poorer clinical outcomes than patients with tumours that lacked either E-cadherin or DSC2, implying that the invasive potential of ESCC cells was restricted by both DSC2 and E-cadherin-dependent junctions. Further studies revealed that DSC2 was a downstream target of miR-25. Enhanced miR-25 promoted ESCC cell invasiveness, whereas restoration of DSC2 abolished these effects. Collectively, our work suggests that miR-25-mediated down-regulation of DSC2 promotes ESCC cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Desmocolinas/metabolismo , Neoplasias Esofágicas/metabolismo , MicroRNAs/metabolismo , Invasividade Neoplásica/genética , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Junções Aderentes/patologia , Adulto , Idoso , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Desmocolinas/genética , Regulação para Baixo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Transfecção , Transplante HeterólogoRESUMO
LOXL2 (lysyl oxidase-like 2), an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC). A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs), following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs) of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.
Assuntos
Processamento Alternativo , Aminoácido Oxirredutases/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Algoritmos , Aminoácido Oxirredutases/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Redes Reguladoras de Genes/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Genéticos , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genéticaRESUMO
Super-enhancers (SEs) comprise large clusters of enhancers that highly enhance gene expression. Long non-coding RNAs (lncRNAs) tend to be dysregulated in cases of stomach adenocarcinoma (STAD) and are vital for balancing tumor immunity. However, whether SE-associated lncRNAs play a role in the immune infiltration of STAD remains unknown. In the present study, we identified SE-associated lncRNAs in the H3K27ac ChIP-seq datasets from 11 tumor tissues and two cell lines. We found that the significantly dysregulated SE-associated lncRNAs were strongly correlated with immune cell infiltration through the application of six algorithms (ImmuncellAI, CIBERSORT, EPIC, quantiSeq, TIMER, and xCELL), as well as immunomodulators and chemokines. We found that the expression of SE-associated lncRNA TM4SF1-AS1 was negatively correlated with the proportion of CD8+ T cells present in STAD. TM4SF1-AS1 suppresses T cell-mediated immune killing function and predicts immune response to anti-PD1 therapy. ChIP-seq, Hi-C and luciferase assay results verified that TM4SF1-AS1 was regulated by its super-enhancer. RNA-seq data showed that TM4SF1-AS1 is involved in immune and cancer-related processes or pathways. In conclusion, SE-associated lncRNAs are involved in the tumor immune microenvironment and act as indicators of clinical outcomes in STAD. This study highlights the importance of SE-associated lncRNAs in the immune regulation of STAD.
RESUMO
Neutrophil gelatinase-associated lipocalin (NGAL) expression has been found to be upregulated in a variety of tumors, but the mechanism of NGAL elevation in gastric carcinoma remains unknown. Here, immunohistochemistry was applied to analyze NGAL expression in gastric carcinoma patients. Reverse transcription PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate NGAL mRNA and protein levels before and after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction. Luciferase reporter assay was carried out to identify the core cis element in NGAL promoter. The binding ability and specificity of transcription factors were analyzed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP), respectively. Results showed that NGAL was overexpressed in gastric tumor tissues. Gastric cancer cells treated with TPA resulted in the transactivation of NGAL promoter and the upregulation of its mRNA and protein levels. We identified the -110 to -79 sequence segment upstream from the transcription initiation site of NGAL as a TPA responsive element (TRE) and confirmed that C/EBPß was able to bind to the -87 to -79 segment. Forced expression of C/EBPß significantly increased the promoter activity of NGAL as well as its mRNA level. These results suggest that NGAL is overexpressed in gastric cancer, the binding of C/EBPß to the TRE of its gene promoter mediates its TPA-induced overexpression in gastric carcinoma cells.
Assuntos
Proteínas de Fase Aguda/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma/metabolismo , Lipocalinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas de Fase Aguda/genética , Sequência de Bases , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/genética , Carcinoma/genética , Carcinoma/patologia , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Humanos , Lipocalina-2 , Lipocalinas/genética , Luciferases/análise , Dados de Sequência Molecular , Plasmídeos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transfecção , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Fascin is overexpressed in esophageal squamous cell [corrected] carcinoma (ESCC) and involved in the proliferation and invasiveness of ESCC cells. In this study, we retrospectively examined the expression of fascin in ESCC samples by immunohistochemistry and revealed that overexpression of fascin was related to poor patient survival. RNAi-mediated knockdown of fascin in ESCC cells significantly inhibited cell proliferation and invasiveness, whereas forced expression of fascin in immortalized esophageal epithelial cells accelerated cell proliferation and invasiveness. To explore the underlying mechanism, cDNA microarray was performed to identify the differential gene expression profiles between a fascin-depleted cell line by RNAi and the corresponding control ESCC cells. Results showed that 296 genes were differentially expressed on fascin depletion. In this study, we focused on two down-regulated genes: CYR61 and CTGF. We found that restored expression of either CYR61 or CTGF led to a recovery of the suppression of cellular proliferation and invasiveness induced by down-regulation of fascin expression; the protein level of CYR61 and CTGF were up-regulated in ESCCs and their expression pattern correlated with fascin overexpression. Finally, analysis of signal transduction revealed that fascin affected the expressions of CYR61 and CTGF through transforming growth factor (TGF)-beta pathway. Taken together, we propose that fascin regulates the proliferation and invasiveness of ESCC cells by modulating the expression of CTGF and CYR61 via TGF-beta pathway.
Assuntos
Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/fisiologia , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Proteína Rica em Cisteína 61/fisiologia , Neoplasias Esofágicas/patologia , Proteínas dos Microfilamentos/fisiologia , Adulto , Idoso , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Proteínas de Transporte/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Proteína Rica em Cisteína 61/genética , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Transdução de Sinais/genética , Análise de Sobrevida , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais CultivadasRESUMO
The overexpression of fascin in human carcinomas is associated with aggressive clinical phenotypes and poor prognosis. However, the molecular mechanism underlying the increased expression of fascin in cancer cells is largely unknown. Here, we identified a Sp1 binding element located at -70 to -60 nts of the FSCN1 promoter and validated that Sp1 specifically bound to this element in esophageal carcinoma cells. Fascin expression was enhanced by Sp1 overexpression and blocked by Sp1 RNAi knockdown. Specific inhibition of ERK1/2 decreased phosphorylation levels of Sp1, and thus suppressed the transcription of the FSCN1, resulting in the down-regulation of fascin. Stimulation with EGF could enhance fascin expression via activating the ERK1/2 pathway and increasing phosphorylation levels of Sp1. These data suggest that FSCN1 transcription may be subjected to the regulation of the EGF/EGFR signaling pathway and can be used as a viable biomarker to predict the efficacy of EGFR inhibitors in cancer therapies.
Assuntos
Carcinoma de Células Escamosas/patologia , Fator de Crescimento Epidérmico/farmacologia , Carcinoma de Células Escamosas/genética , Proteínas de Transporte , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Neoplasias Esofágicas/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas dos Microfilamentos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Heat shock protein (HSP) is a family of highly conserved protein, which exists widely in various organisms and has a variety of important physiological functions. Currently, there is no systematic analysis of HSPs in human glioma. The aim of this study was to investigate the characteristics of HSPs through constructing protein-protein interaction network (PPIN) considering the expression level of HSPs in glioma. After the identification of the differentially expressed HSPs in glioma tissues, a specific PPIN was constructed and found that there were many interactions between the differentially expressed HSPs in glioma. Subcellular localization analysis shows that HSPs and their interacting proteins distribute from the cell membrane to the nucleus in a multilayer structure. By functional enrichment analysis, gene ontology analysis, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, the potential function of HSPs and two meaningful enrichment pathways was revealed. In addition, nine HSPs (DNAJA4, DNAJC6, DNAJC12, HSPA6, HSP90B1, DNAJB1, DNAJB6, DNAJC10, and SERPINH1) are prognostic markers for human brain glioma. These analyses provide a full view of HSPs about their expression, biological process, as well as clinical significance in glioma.
Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Glioma/genética , Proteínas de Choque Térmico/genética , Biomarcadores Tumorais/genética , Biologia Computacional , Glioma/diagnóstico , Glioma/metabolismo , Glioma/patologia , Proteínas de Choque Térmico/metabolismo , Humanos , Espaço Intracelular/metabolismo , Prognóstico , Mapas de Interação de ProteínasRESUMO
PURPOSE: Neutrophil gelatinase-associated lipocalin receptor (NGALR) mRNA level is reduced in isolated chronic myelogenous leukemia blasts but up-regulated in esophageal squamous cell carcinoma (ESCC). The mechanism of NGALR regulation is unknown. Here, we show the expression pattern of NGALR and examine the aberrant methylation of its gene in ESCC and esophageal carcinoma cell lines. EXPERIMENTAL DESIGN: The expression pattern of NGALR was analyzed by immunohistochemistry in 59 ESCCs and compared with noncancerous tissues. The DNA methylation status was investigated by methylation-specific PCR and by bisulfite genomic sequencing in esophageal carcinoma cell lines and surgically resected samples. Methylated cell lines were treated with a methylation inhibitor to restore NGALR expression. RESULTS: The expression of NGALR in ESCC was significantly higher in tumor cell membrane and cytoplasm than in normal esophageal epithelium (P < 0.01). Methylated alleles were detected in three NGALR-nonexpressing cell lines but were not detected in three NGALR-expressing cell lines. Treatment of methylated cell lines with 5-aza-2'-deoxycytidine, a methylation inhibitor, restored NGALR expression. In surgically resected samples, 31 of 77 (40.3%) primary esophageal carcinomas and 46 of 77 (59.7%) paired normal tissues contained methylated NGALR alleles (P < 0.05). CONCLUSIONS: Our results suggest that NGALR hypomethylation contributes to its expression in esophageal carcinomas and that this overexpression may play a role in the pathogenesis of esophageal carcinomas.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Metilação de DNA/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Superfície Celular/biossíntese , Proteínas de Fase Aguda/metabolismo , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Ilhas de CpG , Neoplasias Esofágicas/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Lipocalina-2 , Lipocalinas/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/análise , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
5-lipoxygenase (5-LO) catalyzes the first step of arachidonic acid metabolism to inflammatory mediator leukotrienes. The present study assessed 5-LO expression in esophageal squamous cell carcinoma (ESCC) tissue specimens for associations with clinicopathological and survival data from patients, then explored 5-LO activity in ESCC cells in vitro. 5-LO expression was detected in tissue microarrays containing 297 ESCC samples using immunohistochemistry. Kaplan-Meier curves were used to analyze the survival significance of 5-LO expression and relative risk was evaluated using the multivariate Cox proportional hazards model. Cultured tumor cells were subjected to gene transfection, western blotting, and cell migration and proliferation assays. 5-LO protein was primarily expressed in normal cell cytoplasm and/or membrane, and never in the whole cytoplasm, whereas 5-LO was expressed diffusely in ESCC tissues with nearly homogeneous whole-cytoplasm staining. 5-LO expression was significantly associated with tumor regional lymph node metastasis (P=0.013) and pTNM stage (P=0.004). 5-LO expression was associated with poor overall survival (P=0.029). Multivariate analysis demonstrated that 5-LO overexpression was an independent prognostic factor for ESCC patients (P=0.041). Furthermore, the inhibition of 5-LO expression reduced ESCC cell viability and migration in vitro. These data provide further evidence that the upregulation of 5-LO expression is associated with advanced stages of disease and poor ESCC prognosis, and that 5-LO expression may independently predict overall survival in patients with ESCC. The inhibition of 5-LO expression reduced ESCC malignant behavior in vitro.
RESUMO
Current staging is inadequate for predicting clinical outcome of esophageal squamous cell carcinoma (ESCC). Aberrant expression of LOXL2 and actin-related proteins plays important roles in ESCC. Here, we aimed to develop a novel molecular signature that exceeds the power of the current staging system in predicting ESCC prognosis. We found that LOXL2 colocalized with filamentous actin in ESCC cells, and gene set enrichment analysis (GSEA) showed that LOXL2 is related to the actin cytoskeleton. An ESCC-specific protein-protein interaction (PPI) network involving LOXL2 and actin-related proteins was generated based on genome-wide RNA-seq in 15 paired ESCC samples, and the prognostic significance of 14 core genes was analyzed. Using risk score calculation, a three-gene signature comprising LOXL2, CDH1, and FN1 was derived from transcriptome data of patients with ESCC. The high-risk three-gene signature strongly correlated with poor prognosis in a training cohort of 60 patients (P = 0.003). In mRNA and protein levels, the prognostic values of this signature were further validated in 243 patients from a testing cohort (P = 0.001) and two validation cohorts (P = 0.021, P = 0.007). Furthermore, Cox regression analysis revealed that the signature was an independent prognostic factor. Compared with using the signature or TNM stage alone, the combined model significantly enhanced the accuracy in evaluating ESCC prognosis. In conclusion, our data reveal that the tumor-promoting role of LOXL2 in ESCC is mediated by perturbing the architecture of actin cytoskeleton through its PPIs. We generated a novel three-gene signature (PPI interfaces) that robustly predicts poor clinical outcome in ESCC patients.
Assuntos
Actinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Mapas de Interação de Proteínas , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Biomarcadores Tumorais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Biologia Computacional/métodos , Citoesqueleto , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Estadiamento de Neoplasias , Prognóstico , Mapeamento de Interação de Proteínas , Curva ROC , Reprodutibilidade dos TestesRESUMO
Fascin-1 (FSCN1) is an actin-bundling protein that induces cell membrane protrusions, increases cell motility, and is overexpressed in various human epithelial cancers, including esophageal squamous cell carcinoma (ESCC). We analyzed various protein-protein interactions (PPI) of differentially-expressed genes (DEGs), in fascin knockdown ESCC cells, to explore the role of fascin overexpression. The node-degree distributions indicated these PPI sub-networks to be characterized as scale-free. Subcellular localization analysis revealed DEGs to interact with other proteins directly or indirectly, distributed in multiple layers of extracellular membrane-cytoskeleton/ cytoplasm-nucleus. The functional annotation map revealed hundreds of significant gene ontology (GO) terms, especially those associated with cytoskeleton organization of FSCN1. The Random Walk with Restart algorithm was applied to identify the prioritizations of these DEGs when considering their relationship with FSCN1. These analyses based on PPI network have greatly expanded our comprehension of the mRNA expression profile following fascin knockdown to future examine the roles and mechanisms of fascin action.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Proteínas de Transporte/antagonistas & inibidores , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas dos Microfilamentos/antagonistas & inibidores , RNA Interferente Pequeno/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte/genética , Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas dos Microfilamentos/genética , Mapas de Interação de Proteínas , Células Tumorais CultivadasRESUMO
High-throughput proteomics has successfully identified thousands of proteins as potential therapeutic targets during investigations into mechanisms of drug action. A novel macrolide analog, denoted F806, is a potential antitumor drug. Here, using the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS), we characterize the F806-regulating protein profiles and identify the potential target molecules or pathways of F806 in esophageal squamous cell carcinoma (ESCC) cells. From a total of 1931 quantified proteins, 181 proteins were found to be down-regulated (FDR p-value<0.1, H/L ratio<0.738), and 119 proteins were up-regulated (FDR p-value<0.1, H/L ratio>1.156). Among the down-regulated proteins, we uncovered the over- and under-represented protein clusters in biological process and molecular function respectively by Gene Ontology analysis. Furthermore, down-regulated and up-regulated proteins were significantly enriched in 37 pathways and 60 sub-pathways by bioinformatic analysis (FDR p-value<0.1), while a down-regulated molecule growth factor receptor-bound protein 2 (GRB2) was a prominent node in fourteen cell proliferation-related sub-pathways. We concluded that GRB2 downregulation would be a potential target of F806 in ESCC cells. BIOLOGICAL SIGNIFICANCE: This study used SILAC-based quantitative proteomics screen to systematically characterize molecular changes induced by a novel macrolide analog F806 in esophageal squamous cell carcinoma (ESCC) cells. Followed by bioinformatic analyses, signal pathway networks generated from the quantified proteins, would facilitate future investigation into the further mechanisms of F806 in ESCC cells. Notably, it provided information that growth factor receptor-bound protein 2 (GRB2) would be a prominent node in the F806-targeted cell proliferation network.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/metabolismo , Proteína Adaptadora GRB2/metabolismo , Macrolídeos/farmacologia , Proteínas de Neoplasias/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Proteína Adaptadora GRB2/genética , Humanos , Proteínas de Neoplasias/genética , ProteômicaRESUMO
The paucity of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits the treatment options. This study characterized the therapeutic efficacy and action mechanism of a novel natural macrolide compound F806 in human ESCC xenograft models and cell lines. F806 inhibited growth of ESCC, most importantly, it displayed fewer undesirable side effects on normal tissues in two human ESCC xenograft models. F806 inhibited proliferation of six ESCC cells lines, with the half maximal inhibitory concentration (IC50) ranging from 9.31 to 16.43 µM. Furthermore, F806 induced apoptosis of ESCC cells, contributing to its growth-inhibitory effect. Also, F806 inhibited cell adhesion resulting in anoikis. Mechanistic studies revealed that F806 inhibited the activation of ß1 integrin in part by binding to a novel site Arg610 of ß1 integrin, suppressed focal adhesion formation, decreased cell adhesion to extracellular matrix and eventually triggered apoptosis. We concluded that F806 would potentially be a well-tolerated anticancer drug by targeting ß1 integrin, resulting in anoikis in ESCC cells.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Integrina beta1/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Humanos , Masculino , Camundongos , Camundongos Nus , Oxazóis/farmacologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The lipocalin proteins (lipocalins) are a large family of small proteins characterized by low sequence similarity and highly conserved crystal structures. Lipocalins have been found to play important roles in many human diseases. For this reason, a systemic analysis of the molecular properties of human lipocalins is essential. In this study, human lipocalins were found to contain four structurally conserved regions (SCRs) and could be divided into two subgroups. A human lipocalin protein-protein interaction network (PPIN) was constructed and integrated with their expression data in esophageal carcinoma. Many lipocalins showed obvious co-expression patterns in esophageal carcinoma. Their subcellular distributions also suggested these lipocalins may transfer signals from the extracellular space to the nucleus using the pathway-like paths. These analyses also expanded our knowledge about this human ancient protein family in the background of esophageal carcinoma.
Assuntos
Neoplasias Esofágicas/metabolismo , Lipocalinas/metabolismo , Sequência de Aminoácidos , Expressão Gênica , Humanos , Lipocalinas/química , Lipocalinas/genética , Anotação de Sequência Molecular , Dados de Sequência Molecular , Mapas de Interação de Proteínas , Transporte Proteico , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase (LOX) family, is a copper-dependent enzyme that catalyzes oxidative deamination of lysine residues on protein substrates. LOXL2 was found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous research. We later identified a LOXL2 splicing variant LOXL2-delta72 and we overexpressed LOXL2-delta72 and its wild type counterpart in ESCC cells following microarray analyses. First, the differentially expressed genes (DEGs) of LOXL2 and LOXL2-delta72 compared to empty plasmid were applied to generate protein-protein interaction (PPI) sub-networks. Comparison of these two sub-networks showed hundreds of different proteins. To reveal the potential specific roles of LOXL2- delta72 compared to its wild type, the DEGs of LOXL2-delta72 vs LOXL2 were also applied to construct a PPI sub-network which was annotated by Gene Ontology. The functional annotation map indicated the third PPI sub-network involved hundreds of GO terms, such as "cell cycle arrest", "G1/S transition of mitotic cell cycle", "interphase", "cell-matrix adhesion" and "cell-substrate adhesion", as well as significant "immunity" related terms, such as "innate immune response", "regulation of defense response" and "Toll signaling pathway". These results provide important clues for experimental identification of the specific biological roles and molecular mechanisms of LOXL2-delta72. This study also provided a work flow to test the different roles of a splicing variant with high-throughput data.