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1.
Proc Natl Acad Sci U S A ; 114(23): E4631-E4640, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533408

RESUMO

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.


Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Edição de RNA , Actinina/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adulto , Idoso , Animais , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Regulação para Baixo , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/secundário , Carcinoma de Células Escamosas do Esôfago , Esôfago/citologia , Esôfago/metabolismo , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Metástase Linfática/genética , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Proteínas de Transporte de Cátions Orgânicos/deficiência , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Risco
2.
Carcinogenesis ; 38(1): 94-104, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27879277

RESUMO

Reprogramming of intracellular metabolism is common in liver cancer cells. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. In our previous study, we reported that a novel oncogene eukaryotic translation initiation factor 5A2 (EIF5A2) promotes tumorigenesis under hypoxic condition. Here, we aim to investigate the role of EIF5A2 in cell metabolic reprogramming during hepatocellular carcinoma (HCC) development. In this study, we reported that the messenger RNA (mRNA) level of EIF5A2 was upregulated in 59 of 105 (56.2%) HCC clinical samples (P = 0.015), and EIF5A2 overexpression was significantly associated with shorter survival time of patients with HCC (P = 0.021). Ectopic expression of EIF5A2 in HCC cell lines significantly promoted cell growth and accelerated glucose utilization and lipogenesis rates. The high rates of glucose uptake and lactate secretion conferred by EIF5A2 revealed an abnormal activity of aerobic glycolysis in HCC cells. Several key enzymes involved in glycolysis including glucose transporter type 1 and 2, hexokinase 2, phosphofructokinase liver type, glyceraldehyde 3-phosphate dehydrogenase, pyruvate kinase M2 isoform, phosphoglycerate mutase 1 and lactate dehydrogenase A were upregulated by overexpression of EIF5A2. Moreover, EIF5A2 showed positive correlations with FASN and ACSS2, two key enzymes involved in the fatty acid de novo biosynthetic pathway, at both protein and mRNA levels in HCC. These results indicated that EIF5A2 may regulate fatty acid de novo biosynthesis by increasing the uptake of acetate. In conclusion, our findings demonstrate that EIF5A2 has a critical role in HCC cell metabolic reprogramming and may serve as a prominent novel therapeutic target for liver cancer treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Glucose/metabolismo , Lipogênese , Neoplasias Hepáticas/metabolismo , Redes e Vias Metabólicas , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Fatores de Iniciação de Peptídeos/genética , Prognóstico , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida , Fator de Iniciação de Tradução Eucariótico 5A
3.
Theranostics ; 8(1): 185-198, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29290801

RESUMO

Background and Aims: Esophageal squamous cell carcinoma (ESCC), a major histologic subtype of esophageal cancer, is increasing in incidence, but the genetic underpinnings of this disease remain unexplored. The aim of this study is to identify the recurrent genetic changes, elucidate their roles and discover new biomarkers for improving clinical management of ESCC. Methods: Western blotting and immunohistochemistry were performed to detect the expression level of RHCG. Bisulfite genomic sequencing (BGS) and methylation-specific PCR (MSP) were used to study the methylation status in the promoter region of RHCG. The tumor-suppressive effect of RHCG was determined by both in-vitro and in-vivo assays. Affymetrix cDNA microarray was used to identify the underlying molecular mechanism. Results:RHCG was frequently downregulated in ESCCs, which was significantly correlated with poor differentiation (P = 0.001), invasion (P = 0.003), lymph node metastasis (P = 0.038) and poorer prognosis (P < 0.001). Demethylation treatment and bisulfite genomic sequencing analyses revealed that the downregulation of RHCG in both ESCC cell lines and clinical samples was associated with its promoter hypermethylation. Functional assays demonstrated that RHCG could inhibit clonogenicity, cell motility, tumor formation and metastasis in mice. Further study revealed that RHCG could stabilize IκB by decreasing its phosphorylation, and subsequently inhibit NF-κB/p65 activation by blocking the nuclear translocation of p65, where it acted as a transcription regulator for the upregulation of MMP1 expression. Conclusions: Our results support the notion that RHCG is a novel tumor suppressor gene that plays an important role in the development and progression of ESCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Neoplasias Esofágicas/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Carcinoma de Células Escamosas/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Metilação de DNA/genética , Metilação de DNA/fisiologia , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Metástase Linfática/genética , Masculino , Metaloproteinase 1 da Matriz/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
4.
Oncotarget ; 8(39): 65957-65968, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-29029485

RESUMO

Frizzled (FZD) proteins are receptors for secreted WNT proteins and play a critical role in the malignant progression of various cancers. However, the role of human FZD family members in esophageal squamous cell carcinoma (ESCC) was rarely investigated. In this study, we found that the FZD7 gene was the most commonly up-regulated FZD member in ESCC cell lines compared with other FZDs. TMA studies further validated that FZD7 protein was up-regulated in 165 of 252 (65.5%) informative ESCC patients and significantly correlated with poor overall survival (P=0.001). Additionally, multivariate Cox regression analysis showed that FZD7 overexpression was an independent prognostic factor for ESCC patients. Ectopic expression of FZD7 could promote ESCC cell metastasis both in vitro and in vivo. Under WNT3A stimulation, FZD7 was able to induce the nuclear translocation of ß-catenin and activate the downstream targets of WNT/ß-catenin signaling, as well as promote epithelial-mesenchymal transition (EMT) potential in ESCC cells. Our study demonstrated for the first time that FZD7 contributes to the malignant progression of ESCC and represents a novel prognostic marker and a potential therapeutic target for ESCC patients.

5.
Cancer Res ; 77(21): 5886-5899, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28883005

RESUMO

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886-99. ©2017 AACR.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas Relacionadas à Folistatina/metabolismo , NF-kappa B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Proteínas Relacionadas à Folistatina/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transplante Heterólogo , Adulto Jovem
6.
Nat Commun ; 7: 13568, 2016 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-27924820

RESUMO

Non-CG methylation has been associated with stemness regulation in embryonic stem cells. By comparing differentially expressed genes affected by non-CG methylation between tumour and corresponding non-tumour tissues in oesophageal squamous cell carcinoma (OSCC), we find that Integrin α7 (ITGA7) is characterized as a potential cancer stem cell (CSC) marker. Clinical data show that a high frequency of ITGA7+ cells in OSCC tissues is significantly associated with poor differentiation, lymph node metastasis and worse prognosis. Functional studies demonstrate that both sorted ITGA7+ cells and ITGA7 overexpressing cells display enhanced stemness features, including elevated expression of stemness-associated genes and epithelial-mesenchymal transition features, as well as increased abilities to self-renew, differentiate and resist chemotherapy. Mechanistic studies find that ITGA7 regulates CSC properties through the activation of the FAK-mediated signalling pathways. As knockdown of ITGA7 can effectively reduce the stemness of OSCC cells, ITGA7 could be a potential therapeutic target in OSCC treatment.


Assuntos
Antígenos CD/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Cadeias alfa de Integrinas/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Cadeias alfa de Integrinas/metabolismo , Metástase Linfática , Camundongos Endogâmicos NOD , Camundongos SCID , Interferência de RNA , Transplante Heterólogo , Carga Tumoral/genética
7.
Nan Fang Yi Ke Da Xue Xue Bao ; 30(10): 2310-3, 2010 Oct.
Artigo em Zh | MEDLINE | ID: mdl-20965834

RESUMO

OBJECTIVE: To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2. METHODS: The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay. RESULTS: The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei. CONCLUSION: The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.


Assuntos
Vetores Genéticos , Células HEK293 , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Expressão Gênica , Humanos , Plasmídeos
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 28(5): 671-4, 2008 May.
Artigo em Zh | MEDLINE | ID: mdl-18504176

RESUMO

OBJECTIVE: To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis. METHODS: Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry. RESULTS: The recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells. CONCLUSION: The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Células Eucarióticas/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética , Animais , Sequência de Bases , Vetores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Transfecção , Proteína Supressora de Tumor p53/metabolismo
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