RESUMO
Radiotherapy is one of the standard therapies for esophageal squamous cell carcinoma (ESCC), but the efficacy is far from desirable. Large scale genome sequencing reveals PI3Kα is frequently hyper-activated in ESCC. We found that ESCC cells harboring alterations in PI3K pathway were more resistant to radiation and combination of a clinical PI3Kα-selective inhibitor CYH33 and radiation synergistically inhibited cell proliferation in 14 ESCC cell lines. Radiation induced phosphorylation of FOXO1 and Akt, which sensitized ESCC cells to PI3Kα inhibitors. Both S1PR3 and DNA-PK contributed to radiation-induced Akt phosphorylation, which were revealed to be collectively dependent on PI3Kα. By contrast, constitutively active Akt abrogated the synergism between PI3Kα inhibitors and radiation. PI3Kα inhibition enhanced radiation-induced DNA damage, G2/M arrest and apoptosis. Combination of CYH33 and radiation significantly inhibited the growth of xenografts derived from ESCC patients, which was accompanied with abrogation of radiation-induced phosphorylation of Akt and filtration of M2-like macrophages. Taken together, combination of CYH33 and radiation possesses synergism in ESCC, which provides promising rationale to test this combinatorial regimen in ESCC patients.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/radioterapia , Morfolinas/farmacologia , Piperazinas/farmacologia , Pirróis/farmacologia , Radiossensibilizantes/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Técnicas de Cocultura , Dano ao DNA , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Proteína Forkhead Box O1/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Células THP-1 , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metabolic activities are often accompanied by cell-cycle progression, yet known connections between these two processes remain limited. Here, we identified the isocitrate dehydrogenase 3ß (IDH3ß) as a novel substrate of anaphase-promoting complex/cyclosome (APC/C)-CDH1 and an important regulator of the cell cycle. In esophageal squamous cell carcinoma (ESCC), IDH3ß was posttranslationally upregulated in late G1 phase, and overexpression of IDH3ß accelerated G1-S transition, contributing to the promotion of cell proliferation in vitro and in vivo. α-Ketoglutarate (α-KG), a crucial metabolite in tricarboxylic acid (TCA) cycle, was dependent on IDH3ß level and partially accounted for IDH3ß-mediated cell growth. IDH3ß expression increased PFKFB3 protein levels and enhanced glucose uptake, and high expression of IDH3ß correlated with poor survival in patients with ESCC, suggesting a potential application of IDH3ß in prognosis. Overall, our results highlight a new molecular connection between cell-cycle regulation and the TCA cycle in ESCC. SIGNIFICANCE: These findings show that IDH3ß is an APC/C-CDH1 substrate and is expressed in a cell-cycle-dependent manner, highlighting novel molecular cross-talk between the TCA cycle and cell cycle in cancer cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3281/F1.large.jpg.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Ciclo Celular , Proliferação de Células , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/secundário , Isocitrato Desidrogenase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort (n = 241) for five groups of CSC+ HCC tissues, i.e., EpCAM+, CD90+, CD133+, CD44+, and CD24+ HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC+ HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC+ HCC from two independent cohorts (n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC+ HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC+ HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis. SIGNIFICANCE: miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Metilação de DNA , Regulação para Baixo , Redes Reguladoras de Genes , Células HEK293 , Células Hep G2 , Xenoenxertos , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/biossíntese , Células-Tronco Neoplásicas/metabolismo , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/genéticaRESUMO
Small cell lung cancer (SCLC) is a highly aggressive disease and miRNAs may play an important role in modulating SCLC progression. We have previously screened 924 miRNAs and found that miR-886-3P was negatively associated with SCLC survival. In the current study, we further investigated the role of miR-886-3P mimic in regulating SCLC cell phenotypic alteration in vitro and xenograft tumor formation in vivo. We found that transfection of miR-886-3P mimic significantly inhibited SCLC cell proliferation, migration, and colony formation, and induced mesenchymal-epithelial transition (MET) by suppressing TGF-ß1 synthesis in vitro. Furthermore, intra-tumor injection of miR-886-3P mimic lead to necrosis and suppression of tumor invasion to the surrounding tissue in the subcutaneous xenograft tumor, and intra-vein injection of miR-886-3P mimic suppressed xenograft lung cancer growth in vivo. These findings suggested that miR-886-3P functions as a tumor suppressor in SCLC and thus, it might be a potential therapeutic molecule in the treatment of lung cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , MicroRNAs/genética , Interferência de RNA , Carcinoma de Pequenas Células do Pulmão/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Expressão Gênica , Genes Reporter , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Fenótipo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Fator de Crescimento Transformador beta1 , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Pesquisa Biomédica , Conjuntos de Dados como Assunto , China , Tomada de Decisões , HumanosRESUMO
Esophageal squamous cell carcinoma (ESCC) is among the most common malignancies, but little is known about its spatial intratumoral heterogeneity (ITH) and temporal clonal evolutionary processes. To address this, we performed multiregion whole-exome sequencing on 51 tumor regions from 13 ESCC cases and multiregion global methylation profiling for 3 of these 13 cases. We found an average of 35.8% heterogeneous somatic mutations with strong evidence of ITH. Half of the driver mutations located on the branches of tumor phylogenetic trees targeted oncogenes, including PIK3CA, NFE2L2 and MTOR, among others. By contrast, the majority of truncal and clonal driver mutations occurred in tumor-suppressor genes, including TP53, KMT2D and ZNF750, among others. Interestingly, phyloepigenetic trees robustly recapitulated the topological structures of the phylogenetic trees, indicating a possible relationship between genetic and epigenetic alterations. Our integrated investigations of spatial ITH and clonal evolution provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Evolução Clonal/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Mutação/genética , Proteínas de Neoplasias/genética , Carcinoma de Células Escamosas/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Estudos de Coortes , Metilação de DNA , Neoplasias Esofágicas/patologia , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
OBJECTIVES: Anoctamin 1 (ANO1) has been found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous study. Herein we showed the clinical relevance of ANO1 alterations with ESCC and esophageal precancerous lesion progression. RESULTS: ANO1 was detected in 38.1% (109/286) and 25.4% (77/303) of tumors in the two cohorts, but in none of morphologically normal operative margin tissues. ANO1 expression was significantly associated with a shorter overall survival (OS), especially in patients with moderately differentiated and stage IIA tumors. In 499 iodine-unstained biopsies from the endoscopic screening cohort in 2005-2007, all the 72 pathologically normal epithelial mucosa presented negative immunostaining, whereas ANO1 expression was observed in 3/11 tumors and 5/231 intraepithelial lesions. 7/8 ANO1-positive cases had developed unfavorable outcomes revealed by endoscopic follow-up in 2012. Analysis of another independent cohort of 148 intraepithelial lesions further confirmed the correlation between ANO1 expression and progression of precancerous lesions. 3/4 intraepithelial lesions with ANO1 expression had developed ESCC within 4-9 years after the initial endoscopic examination. METHODS: Immunohistochemistry (IHC) was performed to examine ANO1 expression in surgical ESCC specimens and two independent cohorts of esophageal biopsies from endoscopic screening in high-incidence area of ESCC in northern China. Association between ANO1 expression, clinico-pathologic parameters, and the impact on overall survival was analyzed. CONCLUSIONS: Positive ANO1 is a promising biomarker to predict the unfavorable outcome for ESCC patients. More importantly, it can predict disease progression of precancerous lesions.
Assuntos
Anoctamina-1/biossíntese , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/biossíntese , Lesões Pré-Cancerosas/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Progressão da Doença , Neoplasias Esofágicas/diagnóstico , Esôfago/metabolismo , Esôfago/patologia , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/diagnóstico , PrognósticoRESUMO
Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Proteína de Xeroderma Pigmentoso Grupo A/fisiologia , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Cutâneas/etiologia , Raios UltravioletaRESUMO
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common cancer type in China. In this study, we aimed to develop aneuploidy markers for diagnosis and prognosis of ESCC. METHODS: Chromosomal aneuploidies were detected in 493 primary tumors and 61 precancerous lesions by fluorescence in situ hybridization with chromosome enumeration probes (CEP), and cut-off values were set by receiver operating characteristic (ROC) curves. RESULTS: According to the cut-off values, chromosomes 3, 8, 10, 12, 17 and 20 presented frequent gains, with rates of 70.1, 69.7, 58.9, 66.9, 67.5 and 77.2 % in tumors and of 32.1, 26.8, 33.9, 41.2, 44.0 and 42.0 % in precancerous lesions. Loss of chromosome Y was detected in 72.0 % of male patients. An optimal four-probe panel CEP3/12/17/20 was established for detecting ESCC (sensitivity: 86.1 %), and CEP3/10/12/20 for precancerous lesions (sensitivity: 48.0 %). Gain of CEP8 was significantly correlated with lymph node metastasis (LNM) and late stages (P = 0.002 and 0.001), and loss of CEPY with age (P = 0.002, male). Kaplan-Meier survival curves indicated that patients with positive CEP10/17 (pT1 + T2, P = 0.041) and CEP8/17 (stages IIb + III + IV, P = 0.002) had poor overall survival. Combinations of LNM/stage and CEP panels could divide patients into more subgroups, including LNM + CEP3/17, LNM + CEP10/17, LNM + CEP3/10/17, stage + CEP3/17, stage + CEP10/17 and stage + CEP3/10/17 (P = 0.0004, 0.0003, 0.0001, 0.005, 0.001 and 0.0008, respectively). Multivariate Cox regression analysis confirmed that the above combinational models were independent prognostic factors. CONCLUSIONS: Our data suggest that the combinational probe sets may have potential for detection and prognostic prediction of ESCC.
Assuntos
Aneuploidia , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Lesões Pré-Cancerosas/genética , Idoso , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patologia , Aberrações Cromossômicas , Sondas de DNA , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/patologia , PrognósticoRESUMO
DNAJB6 is a member of the heat shock protein 40 (Hsp40) family. We here investigated the clinical correlation and biological role of DNAJB6 overexpression in colorectal cancer (CRC). The expression of DNAJB6 protein was examined in 200 cases of colorectal adenocarcinomas by immunohistochemistry (IHC) technology. Gene transfection and RNA interference were performed to determine the effect of DNAJB6 expression on the invasion of CRC cells and to explore the underlying molecular mechanisms in vitro and in vivo. Overexpression of DNAJB6 was found in 39% (78/200) of the CRC tissues, especially in tumors at pT4 as compared with at pT1-3 (P = 0.02). A Kaplan-Meier survival analysis revealed a correlation between DNAJB6 expression and overall survival (OS) times (P = 0.003). Multivariate analysis confirmed that DNAJB6 overexpression was an independent prognostic factor for CRC (P = 0.002). RNA interference-mediated silencing of the DNAJB6 gene inhibited the invasion of CRC cells in vitro were accompanied by a significant reduction in the protein levels of IQ-domain GTPase-activating protein 1 (IQGAP1) and phosphorylated ERK (pERK). An in vivo assay showed that inhibition of DNAJB6 expression decreased the lung metastases of CRC cells. IHC analysis of serial sections showed that there was a positive correlation between DNAJB6 and IQGAP1 expression in primary CRC tissues (P = 0.013). The data suggest that DNAJB6 plays an important oncogenic role in CRC cell invasion by up-regulating IQGAP1 and activating the ERK signaling pathway and that DNAJB6 may be used as a prognostic marker for CRC.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Choque Térmico HSP40/genética , Sistema de Sinalização das MAP Quinases/genética , Chaperonas Moleculares/genética , Invasividade Neoplásica/genética , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/genética , Proteínas Ativadoras de ras GTPase/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Fosforilação/genética , Prognóstico , Interferência de RNA/fisiologia , Regulação para Cima/genéticaRESUMO
Esophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. In order to identify useful biomarkers for accurately classifying prognostic risks for ESCC patients, we examined the expression of six proteins by immunohistochemistry (IHC) in 590 paraffin-embedded ESCC samples. The candidate proteins include p53, EGFR, c-KIT, TIMP1 and PI3K-p110α reported to be altered in ESCC tissues as well as another important component of PI3K, PI3K-p85α. Of the six proteins tested, p53, EGFR, c-KIT, TIMP1 and PI3K-p85α were detected with high expression in 43.0%, 36.6%, 55.9%, 70.7% and 57.1% of tumors, respectively. Significant associations were found between high expression of PI3K-p85α, EGFR and p53 and poor prognosis (Pâ=â0.00111; 0.00001; 0.00426). Applying these three proteins as an IHC panel could divide patients into different subgroups (P<0.000001). Multivariate cox regression analysis indicated that the three-protein panel was an independent prognostic factor with very high statistical significance (HRâ=â2.090, 95% CI: 1.621-2.696, Pâ=â0.00000001). The data suggest that the three-protein panel of PI3K-p85α, EGFR and p53 is an important candidate biomarker for the prognosis of patients with ESCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/cirurgia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Receptores ErbB/metabolismo , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/cirurgia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-kit/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: We previously revealed that the calreticulin (CRT) gene is a candidate oncogene promoting cell migration and invasion and that neuropilin-1 (NRP1) is a possible effector downstream of CRT in esophageal squamous carcinoma cells. This study aims to explore the mechanisms underlying the migration and invasion of esophageal cancer cells regulated by CRT through NRP1. EXPERIMENTAL DESIGN: Quantitative reverse-transcription polymerase chain reaction, Western blot analysis, chromatin immunoprecipitation, and reporter gene assays were used to investigate the relationship between CRT and NRP1. In vitro and in vivo assays were carried out to evaluate the effects of NRP1 on malignant phenotypes of ESCC cells and tumor metastasis in NOD/SCID mice. Immunohistochemistry was performed to analyze the expression of CRT and NRP1 in esophageal squamous cell carcinomas (ESCC). RESULTS: Knockdown of CRT decreased the expression of NRP1. Inhibition of NRP1 reduced ESCC cell motility in vitro and experimental metastasis in vivo. Ectopic expression of NRP1 rescued the defects of cell migration and invasion in CRT-shRNA cells. CRT depletion inhibited STAT5A phosphorylation at the Y694 site via a CaMKII-independent pathway. Moreover, STAT5A directly regulated NRP1 transcription. Knockdown of CRT or NRP1 led to a downregulation of MMP2, MMP9, and FAK. Notably, positive correlation was found between CRT and NRP1 expression in ESCC tissues (P = 5.87 × 10(-5)). CRT and NRP1 coexpression was significantly associated with lymph node metastasis (P = 0.025). CONCLUSIONS: Our findings suggest that NRP1 is a critical downstream effector of CRT in promoting cell migration and invasion, which might contribute to the metastasis of ESCC.
Assuntos
Calreticulina/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Neuropilina-1/genética , Fator de Transcrição STAT5/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Neoplasias Esofágicas/patologia , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Metástase Linfática , Camundongos , Transcrição GênicaRESUMO
AIM: Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro. METHODS: The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2. RESULTS: In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive. CONCLUSION: Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.
Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Glioma/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: To date, no robust biomarkers have been available in clinical practice that can provide an early diagnostic evaluation of lung cancer. The objective of this study was to identify potential biomarkers for the early detection of lung cancer using bronchial brushing specimens. METHODS: Immunocytochemistry was used to investigate the expression of 35 proteins in 880 bronchial brushing specimens from both outpatients and inpatients who had either lung cancer or benign lung lesions. An optimal panel was identified that had high sensitivity and considerable specificity for detecting lung cancer. Associations between protein expression and clinicopathologic parameters were assessed. RESULTS: Tumor protein 53 (TP53), TP63, Ki67, epidermal growth factor receptor (EGFR), minichromosome maintenance complex component 6 (MCM6), MCM7, uncharacterized proteins KIAA1522 and KIAA0317, and ubiquitin-protein ligase UHR1 (ICBP90) frequently presented high expression in bronchial brushing specimens from patients who had lung cancer compared with patients who had benign lung lesions. A 6-protein panel consisting of TP53, Ki67, MCM6, MCM7, KIAA1522, and KIAA0317 was identified as the best combination, with sensitivity of 81.1% (309 of 381 specimens) for detecting nonsmall cell lung cancer (NSCLC) and 86.8% (145 of 167 specimens) for detecting small cell lung cancer (SCLC) (specificity, 83.3%; 65 of 78 specimens). The combination of cytology and the protein panel significantly improved the sensitivity of bronchial brushing examination for detecting lung cancer (P<.00001), which increased from 49.1% to 81% in early stage NSCLC (stage I and II). In combined analyses, the protein panel was positively associated with patient sex (P=.00033), tumor type (P<.00001), tumor location (P<.00001), and lymph node metastasis (P=.028). CONCLUSIONS: The 6-protein panel is a potential biomarker for the early detection of lung cancer in bronchial brushings.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Brônquios/patologia , Broncoscopia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Citodiagnóstico/métodos , Diagnóstico Diferencial , Diagnóstico Precoce , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Manejo de Espécimes/métodos , Adulto JovemRESUMO
Atypical protein kinase Cι (PKCι) has been identified as an oncoprotein in esophageal squamous cell carcinomas. However, the mechanisms underlying the role of PKCι in this disease remain unclear. In the present work, we found that inhibition of PKCι expression by RNAi induced apoptosis via the down-regulation of ß-catenin in esophageal cancer cells. Furthermore, we found that PKCι regulated ß-catenin in an autophagy dependent way. Since down-regulation of ß-catenin induced by knockdown of PKCι could be rescued by autophagy inhibition; knockdown of PKCι activated autophagy and promoted the recruitment of ß-catenin into autophagosome. These results suggested that PKCι positively regulated ß-catenin through negatively regulated autophagy and depletion of PKCι promoted apoptosis via autophagic degradation of ß-catenin in esophageal cancer cells. These data provide new insights into PKCι signaling in human cancer.
Assuntos
Apoptose/genética , Autofagia/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Isoenzimas/genética , Proteína Quinase C/genética , beta Catenina/genética , Proteína 5 Relacionada à Autofagia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , RNA Interferente Pequeno/genética , beta Catenina/biossíntese , beta Catenina/metabolismoRESUMO
BACKGROUND: Tumor intrinsic chemoradiotherapy resistance is the primary factor in concomitant chemoradiotherapy failure in advanced uterine cervical squamous cell carcinoma. This study aims to identify a set of genes and molecular pathways related to this condition. METHODS: Forty patients with uterine cervical squamous cell carcinoma in International Federation of Gynecology and Obstetrics stage IIb or IIIb, treated with platinum-based concomitant chemoradiotherapy between May 2007 and December 2012, were enrolled in this trial. Patients included chemoradiotherapy resistant (n = 20) and sensitive (n = 20) groups. Total RNA was extracted from fresh tumor tissues obtained by biopsy before treatment and microarray analysis was performed to identify genes differentially expressed between the two groups. RESULTS: Microarray analysis identified 108 genes differentially expressed between concomitant chemoradiotherapy resistant and sensitive patients. Functional pathway cluster analysis of these genes revealed that DNA damage repair, apoptosis, cell cycle, Map kinase signal transduction, anaerobic glycolysis and glutathione metabolism were the most relevant pathways. Platelet-derived growth factor receptor alpha (PDGFRA) and protein kinase A type 1A (PRKAR1A) were significantly upregulated in the chemoradiosensitive group, while lactate dehydrogenase A (LDHA), bcl2 antagonist/killer 1 (BAK1), bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and cyclin-dependent kinase 7 (CDK7) were upregulated in the chemoradiotherapy resistant group. CONCLUSION: We have identified seven genes that are differentially expressed in concomitant chemoradiotherapy resistant and sensitive uterine cervical squamous cell carcinomas, which may represent primary predictors for this condition.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Idoso , Carcinoma de Células Escamosas/radioterapia , Quimiorradioterapia , Feminino , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias do Colo do Útero/radioterapiaRESUMO
PURPOSE: Our aim was to identify frequent genomic aberrations in both esophageal squamous cell carcinoma (ESCC) and esophageal dysplasia and to discover important copy number-driving genes and microRNAs (miRNA) in ESCC. EXPERIMENTAL DESIGN: We conducted array-based comparative genomic hybridization (array CGH) on 59 ESCC resection samples and 16 dysplasia biopsy samples. Expression of genes at 11q13.3 was analyzed by real-time PCR (RT-PCR) and immunohistochemistry (IHC). Integrated analysis was conducted to identify genes or miRNAs with copy number-expression correlations. RESULTS: Array CGH identified 11 amplifications and eight homozygous deletions in ESCC. Integrated analysis of array CGH data with matched gene expression microarray data showed that 90 overexpressed genes and 24 underexpressed genes were consistent with DNA copy number changes, including 12 copy number-driving miRNAs. In esophageal dysplasia, six gains, four losses, 12 amplifications, and four homozygous deletions were detected. Amplifications of 7p11.2 and 11q13.2-11q13.3 (CCND1) and homozygous deletion at 9p21.3 (CDKN2A) were consistent genomic changes in both dysplasia and carcinoma. ANO1 at 11q13.3 was overexpressed at the mRNA and protein levels in tumors, and higher mRNA expression was correlated with the copy number increase. In particular, ANO1 expression was elevated in moderate dysplasia compared with normal esophageal epithelium. IHC revealed that ANO1 overexpression was positively correlated with lymph node metastasis and advanced clinical stage. Knockdown of ANO1 significantly inhibited the proliferation of KYSE30 and KYSE510 cells. CONCLUSION: Copy number aberrations in both esophageal dysplasia and ESCC may be useful as potential biomarkers for early detection. In addition, ANO1 may be a candidate target gene in esophageal tumorigenesis.
Assuntos
Carcinoma de Células Escamosas/genética , Aberrações Cromossômicas , Neoplasias Esofágicas/genética , Esôfago/metabolismo , Esôfago/patologia , Lesões Pré-Cancerosas/genética , Carcinoma de Células Escamosas/diagnóstico , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Neoplasias Esofágicas/diagnóstico , Carcinoma de Células Escamosas do Esôfago , Amplificação de Genes , Deleção de Genes , Expressão Gênica , HumanosRESUMO
OBJECTIVE: To explore the role and mechanism of microRNA185 (miR-185) on proliferation, migration and invasion of esophageal squamous cell carcinoma (ESCC). METHODS: Samples were obtained from 23 ESCC patients undergoing surgery whose were confirmed by pathological diagnosis of esophageal carcinoma from 2002 to 2012,at Department of Thoracic Surgery,Cancer Institute and Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Real-time PCR was used to measure the expression of miR-185. The xCELLigence RTCA MP system and Transwell assay were performed to detect the effect of miR-185 on proliferation, migration and invasion of ESCC respectively. After transfecting of miR-185 mimic into KYSE150, the expression of Six1's downstream gene cyclin A1 was evaluated by real-time PCR. After transfection of miR-185 inhibitor into KYSE30, the expression of E-cadherin, a downstream protein of Six1, was observed under confocal microscope. RESULTS: The expression level of miR-185 was down-regulated in ESCC compared with adjacent normal tissue (0.006 vs 0.039,P = 0.016). After transfection of miR-185 mimic, miR-185 significantly inhibited proliferation, migration and invasion of ESCC.Transwell assay showed, in comparison with the control group, the number of KYSE150 metastatic and invasive cells was respectively decreased(146 ± 15 vs 64 ± 11, 110 ± 12 vs 67 ± 5, both P < 0.05). And the expression level of cyclin A1 decreased. After transfection of miR-185 inhibitor,the expression level of E-cadherin decreased. CONCLUSION: miR-185 may inhibit proliferation, migration and invasion of ESCC through its target gene Six1.