Effect of Survivin Down-Regulation by Egr1-Survivin shRNA Combined with Radiotherapy on Radiosensitivity of Esophageal Squamous Carcinoma / Efecto de la Regulación Negativa de Survivina por shRNA de Egr1-Survivina Combinado con Radioterapia sobre la Radiosensibilidad del Carcinoma Escamoso de Esófago

SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.

Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.

miR-30e-3p in natural killer cell-derived exosomes inhibits the proliferation and invasion of human esophageal squamous carcinoma cells / 细胞与分子免疫学杂志

Objective To investigate the effects of natural killer (NK)-cell-derived miR-30e-3p-containing exosomes (Exo) on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis and invasion. Methods NK cells were isolated and amplified from the peripheral blood of healthy donors, and NK cell-derived Exo was isolated and identified, which were further co-cultured with NEC cells and were randomly grouped into Exo1 and Exo2 groups. Transmission electron microscopy (TEM) was used to observe the morphology and size of exosomes. Western blot analysis was used to detect the expression levels of exosome markers apoptosis related gene 2- interacting protein X(ALIX), tumor susceptibility gene 101(TSG101), CD81 and calnexin. The NC plasmids, mimics and inhibitors of miR030e-3p were respectively delivered into the NK cells, and the corresponding NK cells-derived Exo were co-cultured with NEC cells, which were divided into NC, Exo, mimic and inhibitor groups. CCK-8 assay was used to evaluate cell proliferation, flow cytometry was conducted to determine cell cycle, annexin V-FITC/PI double staining was employed to detect cell apoptosis, and TranswellTM assay was performed to detect cell invasion abilities. Real-time quantitative PCR was used to detect the expression of miR-23b, miR-422a, miR-133b, miR-124, miR-30e-3p and miR-99a in NCE cells and exosomes. Results The percentages of CD56+CD3+ cells and CD56+CD16+ cells in NK cells were (0.071±0.008)% and (90.6±10.6)%, respectively. Exosome isolated from NK cells ranged from 30 nm to 150 nm, and was positive for ALIX, TSG101 and CD81, while negative for calnexin. NK cell-derived Exos inhibited the proliferation, reduced the proportion of S-phase cells and the number of invaded cells of NEC cells, and promoted the apoptosis and the proportion of G1 phase cells. Overexpression of miR-30E-3p in NK cell-derived exosome inhibited the proliferation and invasion of NEC cells, and blocked cell cycle and promoted apoptosis, while knockdown miR-30e-3p in NK cell-derived exosomes did the opposite. Conclusion miR-30e-3p in NK cell-derived exosomes can inhibit the proliferation and invasion of ESCC cells, block their cell cycle and induce their apoptosis.

Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity / 中华医学杂志(英文版)

BACKGROUND@#Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear.@*METHODS@#To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification.@*RESULTS@#PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs.@*CONCLUSIONS@#As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.

Family history of esophageal cancer modifies the association of serum lipids and malignant esophageal lesions: a nested case-control study from the "Endoscopic Screening for Esophageal Cancer in China" trial / 中华医学杂志(英文版)

BACKGROUND@#The association of lipids and cancer has varied greatly among different cancer types, lipid components and study populations. This study is aimed to investigate the association of serum lipids and the risk of malignant lesions in esophageal squamous epithelium.@*METHODS@#In the "Endoscopic Screening for Esophageal Cancer in China" (ESECC) trial, serum samples were collected and tested for total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol at the time of subject enrollment. Cases were defined as malignant esophageal lesions identified by baseline endoscopic examination or by follow-up to May 31, 2018. Controls were randomly selected using incidence density sampling in the same cohort. Conditional logistic models were applied to identify the association of serum lipids and the risk of malignant esophageal lesions. Effect modification was evaluated by testing interaction terms of the factor under assessment and these serum lipid indicators.@*RESULTS@#No consistent association between serum lipid levels and esophageal malignant lesions were found in a pooled analysis of 211 cases and 2101 controls. For individuals with a family history of esophageal cancer (EC), high TC, and LDL-C were associated with a significantly increased risk of having malignant lesions (odds ratio [OR]High vs. Low TC = 2.22, 95% confidence interval [CI]: 1.14-4.35; ORHigh vs. Low LDL-C = 1.93, 95% CI: 1.01-3.65). However, a negative association was observed in participants without an EC family history (ORHigh vs. Low TC = 0.69, 95% CI: 0.48-0.98, Pinteraction = 0.002; ORHigh vs. Low LDL-C = 0.50, 95% CI: 0.34-0.76, Pinteraction < 0.001).@*CONCLUSIONS@#In this study, we found that the association of serum lipids and malignant esophageal lesions might be modified by EC family history. The stratified analysis would be crucial for population-based studies investigating the association of serum lipids and cancer. The mechanism by which a family history of EC modifies this association warrants further investigation.

Gene mutations of esophageal squamous cell carcinoma based on next-generation sequencing / 中华医学杂志(英文版)

BACKGROUND@#Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers without effective therapy. To explore potential molecular targets in ESCC, we quantified the mutation spectrum and explored the relationship between gene mutation and clinicopathological characteristics and programmed death-ligand 1 (PD-L1) expression.@*METHODS@#Between 2015 and 2019, 29 surgically resected ESCC tissues and adjacent normal tissues from the Fourth Hospital of Hebei Medical University were subjected to targeted next-generation sequencing. The expression levels of PD-L1 were detected by immunohistochemistry. Mutational signatures were extracted from the mutation count matrix by using non-negative matrix factorization. The relationship between detected genomic alterations and clinicopathological characteristics and PD-L1 expression was estimated by Spearman rank correlation analysis.@*RESULTS@#The most frequently mutated gene was TP53 (96.6%, 28/29), followed by NOTCH1 (27.6%, 8/29), EP300 (17.2%, 5/29), and KMT2C (17.2%, 5/29). The most frequently copy number amplified and deleted genes were CCND1/FGF3/FGF4/FGF19 (41.4%, 12/29) and CDKN2A/2B (10.3%, 3/29). By quantifying the contribution of the mutational signatures to the mutation spectrum, we found that the contribution of signature 1, signature 2, signature 10, signature 12, signature 13, and signature 17 was relatively high. Further analysis revealed genetic variants associated with cell cycle, chromatin modification, Notch, and Janus kinase-signal transducer and activator of transcription signaling pathways, which may be key pathways in the development and progression of ESCC. Evaluation of PD-L1 expression in samples showed that 13.8% (4/29) of samples had tumor proportion score ≥1%. 17.2% (5/29) of patients had tumor mutation burden (TMB) above 10 mut/Mb. All samples exhibited microsatellite stability. TMB was significantly associated with lymph node metastasis (r = 0.468, P = 0.010), but not significantly associated with PD-L1 expression (r = 0.246, P = 0.198). There was no significant correlation between PD-L1 expression and detected gene mutations (all P > 0.05).@*CONCLUSION@#Our research initially constructed gene mutation profile related to surgically resected ESCC in high-incidence areas to explore the mechanism underlying ESCC development and potential therapeutic targets.

Human esophageal fibroblast-derived exosomal miR-21 reduced the cisplatin sensitivity to esophageal carcinoma EC9706 cells

The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.

MET overexpression and intratumor heterogeneity in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is among the ten most frequent and deadly cancers, without effective therapies for most patients. More recently, drugs targeting deregulated growth factor signaling receptors have been developed, such as HGF-MET targeted therapy. We assessed MET and HGF genetic alterations and gene and protein expression profiles in ESCC patients from the Brazilian National Cancer Institute and publicly available datasets, as well as the intratumor heterogeneity of the alterations found. Our analyses showed that HGF and MET genetic alterations, both copy number and mutations, are not common in ESCC, affecting 5 and 6% of the cases, respectively. HGF showed a variable mRNA expression profile between datasets, with no alterations (GSE20347), downregulation (GSE45670), and upregulation in ESCC (our dataset and GSE75241). On the other hand, MET was found consistently upregulated in ESCC compared to non-tumor surrounding tissue, with median fold-changes of 5.96 (GSE20347), 3.83 (GSE45670), 6.02 (GSE75241), and 5.0 (our dataset). Among our patients, 84% of the tumors showed at least a two-fold increase in MET expression. This observation was corroborated by protein levels, with 55% of cases exhibiting positivity in 100% of the tumor cells. Intratumor heterogeneity was evaluated in at least four tumor biopsies from five patients and two cases showed a consistent increase in MET expression (at least two-fold) in all tumor samples. Our data suggested that HGF-MET signaling pathway was likely to be overactivated in ESCC, representing a potential therapeutic target, but eligibility for this therapy should consider intratumor heterogeneity.

Long non-coding RNA KCNQ1 overlapping transcript 1 promotes the progression of esophageal squamous cell carcinoma by adsorbing microRNA-133b

OBJECTIVE: The long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) exerts vital regulatory functions in diverse tumors. However, the biological function of KCNQ1OT1 in esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: KCNQ1OT1 expression was detected in ESCC tissues using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, migration, and invasion were detected by the CCK-8 assay, EdU assay, flow cytometry analysis, and Transwell experiments, respectively. Bioinformatics analysis, luciferase reporter experiments, and RNA immunoprecipitation assays were used to predict and validate the regulatory relationships between KCNQ1OT1, microRNA-133b (miR-133b) and epidermal growth factor receptor (EGFR). RESULTS: KCNQ1OT1 expression was remarkably upregulated in ESCC tissues and cell lines. Overexpression of KCNQ1OT1 markedly promoted ESCC cell proliferation, migration, and invasion and enhanced the expression of N-cadherin, MMP-2, and MMP-9, but inhibited apoptosis and E-cadherin expression in ESCC cell lines; KCNQ1OT1 knockdown exerted the opposite effects. KCNQ1OT1 could directly bind to miR-133b and suppress its expression, and miR-133b reversed the effects of KCNQ1OT1 overexpression in ESCC cells. MiR-133b reduced the expression of epidermal growth factor receptor (EGFR); further, KCNQ1OT1 activated the phosphatidylinositol 3-kinase/AKT serine/threonine kinase 1 (PI3K/AKT) signaling pathway by repressing miR-133b repression and indirectly upregulating EGFR. KCNQ1OT1 expression was positively correlated with EGFR mRNA expression and negatively correlated with miR-133b expression. CONCLUSION: KCNQ1OT1 facilitates ESCC progression by sponging miR-133b and activating the EGFR/PI3K/AKT pathway.

Exploration of gene functions for esophageal squamous cell carcinoma using network-based guilt by association principle

Gene networks have been broadly used to predict gene functions based on guilt by association (GBA) principle. Thus, in order to better understand the molecular mechanisms of esophageal squamous cell carcinoma (ESCC), our study was designed to use a network-based GBA method to identify the optimal gene functions for ESCC. To identify genomic bio-signatures for ESCC, microarray data of GSE20347 were first downloaded from a public functional genomics data repository of Gene Expression Omnibus database. Then, differentially expressed genes (DEGs) between ESCC patients and controls were identified using the LIMMA method. Afterwards, construction of differential co-expression network (DCN) was performed relying on DEGs, followed by gene ontology (GO) enrichment analysis based on a known confirmed database and DEGs. Eventually, the optimal gene functions were predicted using GBA algorithm based on the area under the curve (AUC) for each GO term. Overall, 43 DEGs and 67 GO terms were gained for subsequent analysis. GBA predictions demonstrated that 13 GO functions with AUC>0.7 had a good classification ability. Significantly, 6 out of 13 GO terms yielded AUC>0.8, which were determined as the optimal gene functions. Interestingly, there were two GO categories with AUC>0.9, which included cell cycle checkpoint (AUC=0.91648), and mitotic sister chromatid segregation (AUC=0.91597). Our findings highlight the clinical implications of cell cycle checkpoint and mitotic sister chromatid segregation in ESCC progression and provide the molecular foundation for developing therapeutic targets.

Regulação da expressão das DNA metiltransferases pelas proteínas p53 e Sp1 em carcinoma epidermóide de esôfago / [Regulation of DNA methyltransferase expression by proteins p53 and Sp1 in esophageal squamous cell carcinoma]

O câncer de esôfago (CE) é um tumor altamente frequente e letal, correspondendo ao oitavo lugar em incidência e sexto em letalidade, dentro todos os tipos de tumores. O subtipo histopatológico mais frequente de CE é o carcinoma epidermóide de esôfago (CEE) que corresponde a aproximadamente 80% dos tumores de esôfago. O CEE apresenta uma alta frequência de mutações no gene TP53 que respondem pelas alterações genéticas mais frequentes nesse tumor e parecem contribuir significativamente para a carcinogênese esofágica. A proteína p53 exerce um papel central na resposta a sinais de estresse, e já foi descrito que sua associação com SP1 tem um papel importante na regulação de diversos genes, como p21. Resultados obtidos em nosso grupo sugeriram uma associação entre maior expressão da DNMT3B e a presença de mutação no gene supressor de tumor TP53 em CEE. As DNA Metiltransferases (DNMTs) são enzimas que catalisam a transferência do grupamento metil para citosinas em dinucleotideos CpG, sendo esta a modificação epigenética mais estudada em CEE. A associação entre mutações em TP53 e aumento da expressão das DNMTs sugere um papel de p53 na regulação da expressão dessas enzimas e uma interação entre mecanismos genéticos e epigenéticos envolvidos no desenvolvimento desses tumores. Portanto, este projeto teve por objetivo avaliar a regulação da expressão das DNMTs por p53 e SP1 em CEE. Este trabalho demonstrou, pela análise do perfil de expressão das DNMTs e SP1, de acordo com o status mutacional de TP53, em amostras de CEE provenientes de pacientes do INCA e de pacientes cujos dados foram depositados no banco de dados The Cancer Genome Atlas (TCGA), que tanto DNMT1 quanto SP1 encontram-se superexpressos em amostras de CEE que possuem mutação no gene TP53 e que as expressões desses genes se correlacionam positivamente nas amostras mutadas. Além disso, os experimentos in vitro de modulação da expressão de SP1 e/ou p53, seguidos de análise de expressão gênica, de avaliação da ligação dos fatores de transcrição ao promotor de DNMT1 e da investigação da ativação transcricional desse gene, realizados em linhagens celulares derivadas de CEE (que expressam ou não p53 selvagem, ativa), mostraram que SP1 e p53 participam da regulação transcricional de DNMT1, sendo capazes de se ligar ao promotor do gene que codifica esta enzima e ativar a sua transcrição. SP1 é capaz de se ligar ao promotor de DNMT1 mesmo em condições basais da célula, e p53 somente após a indução de sua expressão. Entretanto, especificidades da interação entre SP1 e p53 na regulação transcricional de DNMT1 podem desencadear efeitos distintos. SP1 é capaz de transativar a expressão das DNMT1 independente da presença de p53. No entanto, o aumento da expressão de SP1, em modelos celulares p53 ativo, foi associado à redução da expressão de p53 e das DNMTs. A proteína p53, por sua vez, pode ativar ou reprimir a expressão de DNMT1, e o efeito da interação desta proteína com seus elementos consenso no promotor desse gene é dependente dos níveis disponíveis de p53 nas células. Por fim, nesse estudo foi observado que a diminuição da expressão das DNMTs foi capaz de levar a uma redução dos níveis de metilação global do DNA. Portanto, esses resultados demonstram que a regulação da expressão das DNMTs por p53 é um indicador da interação entre mecanismos genéticos e epigenéticos atuando na carcinogênese esofágica.

Effect of food additive citric acid on the growth of human esophageal carcinoma cell line EC109

Objective: Today, esophageal cancer [EC] has become one of the most common cancer types in China. Therefore, new drug and therapeutic strategies are urgently needed to improve postoperative survival rate of patients with EC. As a food additive, several lines of evidence have shown that citric acid can be served as glycolysis suppressor to inhibit growth of some tumor cells. However, little is known about the effect of this organic acid on the growth of human esophageal carcinoma cell line, EC109

Materials and Methods: In this experimental study, cell proliferation rate was determined using MTT assay. Apoptotic morphological changes were evaluated by fluorescent microscopy using Hoechst 33258 staining. Cell apoptosis rate and mitochondrial membrane potential [MMP] were detected using flow-cytometry. Effect of citric acid on cellular membrane permeability was assessed by measuring lactate dehydrogenase [LDH] activity, using LDH assay kit

Results: Compared to the control group, there was a marked decrease in cells proliferation when the cells were treated with higher citric acid concentrations [800, 1600 micro g/ml]. Typical apoptotic morphology of EC109 cells was observed upon treatment with citric acid, such as chromatin condensation and appearance of apoptotic body. Cell apoptotic indexes were significantly increased [P<0.01] after treatment with citric acid at the concentration of 400-1600 micro g/ml. Extracellular LDH activity and loss of MMP in all of the treated groups were significantly higher than control [P<0.05], in a dose-dependent manner

Conclusion: These results suggest that citric acid prevents EC109 cell growth by inhibiting cell proliferation and inducing apoptosis, which perhaps offers some theoretical guidance for its application in EC treatment

Avaliação das alterações de desmogleína 1 em carcinoma epidermoide de esôfago / [Evaluation of changes in desmoglein 1 in esophageal squamous cell carcinoma]

O câncer de esôfago (CE) é um dos dez tipos de tumores mais frequentes e apresenta uma sobrevida em cinco anos inferior a 20% no Brasil e no mundo. Esta alta letalidade está relacionada ao diagnóstico tardio da doença, o que resulta em um tratamento pouco eficaz. Dentre os diferentes tipos histológicos de CE, o carcinoma epidermóide de esôfago (CEE) corresponde a mais de 80% dos casos de câncer de esôfago no Brasil. Frente a tal panorama, a identificação de potenciais biomarcadores que auxiliem no diagnóstico precoce de CEE é fundamental. Diversos trabalhos têm sido desenvolvidos com o objetivo de elucidar o papel das alterações epigenéticas na iniciação e progressão tumoral, e em estudo do nosso grupo, foi observado que alterações no padrão de metilação do DNA são comuns em CEE. As principais vias celulares afetadas foram as de componentes inflamatórios e adesão/diferenciação celular. Dentro desta última via, destacou-se o gene Desmogleína 1 (DSG1), que codifica uma caderina desmossomal de mesmo nome, já relacionada à diferenciação celular em pele. Desta forma, este trabalho teve como objetivo avaliar a presença de alterações de DSG1 em CEE. Nas análises realizadas em amostras humanas, observamos que os tumores apresentam uma hipermetilação da região promotora e menores níveis de expressão gênica de DSG1 em comparação ao tecido adjacente não tumoral pareado. Além disso, menores níveis de metilação foram observados em indivíduos com idade acima da mediana e tumores moderadamente diferenciados...

Esophageal cancer (EC) is one of the ten most frequent tumors worldwide and in Brazil, showing a five-year overall survival lower than 20%. Such a high lethality is directly associated with a late diagnosis, which results in a poor treatment. Among the different histological subtypes of EC, esophageal squamous cell carcinoma (ESCC) corresponds to more than 80% of the cases. Based on this, the identification of biomarkers that could anticipate ESCC diagnosis is of utmost importance. Several studies have tried to elucidate the role of epigenetic alterations on tumor initiation and progression and our group has already shown that alterations of DNA methylation patterns are common events in ESCC. Among the cellular pathways found to be altered in this study, inflammatory components and cell adhesion and differentiation were the most significant. One of the genes found to be hypermethylated in tumors was Desmoglein 1 (DSG1), which encodes a desmosomal protein and has already been implicated in cellular differentiation in skin keratinocytes. Therefore, the main objective of this study was to evaluate the presence of DSG1 molecular alterations in ESCC. The evaluation of human samples showed that ESCC presents DSG1 hypermethylation and a downregulation of its expression in comparison with the paired non-tumor surrounding tissue. Also, lower DSG1 methylation levels were observed in moderately differentiated tumors and in the tumor tissue of older individuals. DSG1 promoter methylation in tumors was also found to be a prognostic factor in ESCC, with individuals with high levels showing a mean overall survival of 6 months, while patients that presented low methylation levels showed a mean overall survival of 15.5 months...

Expression analysis of miRNA and target mRNAs in esophageal cancer

We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer.

Non-invasive detection of esophageal cancer using genetic changes in circulating cell-free DNA

Cell free DNA [cfDNA] is a genetic biomarker that is present in serum or plasma in high concentration in many types of cancer. Identification of circu-lating cancer related DNA molecules in serum or plasma is a non-invasive tool for early diagnosis and prognosis in many cancer patients. For this review, study selection and data extraction were performed by the authors. Detection of point mutations, microsatellite alterations, DNA hypermethylations and losses of heterozygosity in circulating cell free DNA have been characterized in esophagus cancer. Application of circulating cell free DNA as a biomarker, provide the best opportunity for constructing non-invasive tests for early detection, prognosis and management of cancer patients, after therapy in many types of cancer

A six-nucleotide deletion polymorphism in the casp8 promoter is associated with reduced risk of esophageal and gastric cancers in Kashmir valley.

BACKGROUND: Caspase-8 (CASP8) is a key regulator of apoptosis or programmed cell death, an essential defense mechanism against hyperproliferation and malignancy. To evaluate the role of CASP8 polymorphisms in esophageal (EC) and gastric cancers (GC) in the Kashmir valley, we examined the risk due to -652 6N ins/del polymorphism (rs3834129) in the promoter of CASP8 in a case–control study. MATERIALS AND METHODS: Genotypes of the CASP8 polymorphisms (-652 6N ins/del; rs3834129) were determined for 315 patients (135 EC and 108 GC) and 195 healthy controls by polymerase chain reaction. Data was statistically analyzed using Chi-square test and logistic regression model by using the SPSS software. RESULTS: Carriers for the del allele of rs3834129 single nucleotide polymorphism were associated with decreased risk for both EC (odds ratio [OR] = 0.278; 95% confidence interval [95% CI] = 0.090–0.853; P = 0.025) and GC (OR = 0.397; 95% CI = 0.164–0.962; P = 0.041). Also, in a recessive model, our results showed that CASP8 -652 6N ins/del “del/del” allele was conferring significant low risk for both EC (OR = 0.380; 95% CI = 0.161–0.896; P = 0.027) and GC (OR = 0.293; 95% CI = 0.098–0.879; P = 0.029). However, interaction of CASP8 -652 6N ins/del genotypes with smoking and high consumption of salted tea did not further modulate the risk of EC and GC. CONCLUSIONS: Polymorphism in CASP8 -652 6N ins/del polymorphism modulates the risk of EC and GC in Kashmir valley.

Efeito da p53 sobre a expressão e atividade da enzima de reparo de DNA Timina-DNA Glicosilase / Effect of p53 on the expression and activity of DNA repair enzyme thymine-DNA glycosylase

O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos G.T causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos G.T em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype

Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from G.T mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the G.T mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNA–damage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function

Alterações genéticas e epigenéticas em carcinoma epidermóide de esôfago / Genetic and epigenetic alterations in esophageal squamous cell carcinoma

O câncer de esôfago encontra-se entre os dez tipos de câncer mais incidentes no mundo, sendo o sexto tipo mais mortal. Fatores genéticos, como mutações no gene TP53, e epigenéticos como, por exemplo, a hipermetilação das ilhotas CpG na região promotora de determinados genes, são eventos importantes no desenvolvimento do câncer e podem causar a inativação de genes supressores de tumor. Neste trabalho, avaliamos o perfil de mutação no gene TP53 em 101 pacientes com carcinoma epidermóide de esôfago (CEE) residentes na região sudeste do Brasil. Destes pacientes, 33,7% apresentaram mutações nos éxons 5, 6, 7 e 8 com prevalência nos códons 248, 179 e 220. A metilação das citosinas das ilhotas CpG resulta da atividade de uma família de enzimas, as DNA metiltransferases (DNMTs). A hipermetilação destas ilhotas leva à formação de um complexo de proteínas incluindo proteínas que têm afinidade por CpG metilado (MBDs e MeCP) impedindo que ocorra a transcrição. Neste trabalho nós investigamos, por RT-PCR semi-quantitativo, a expressão das DNMT1, DNMT3A, DNMT3B, MBD1, MBD3, MDB4 e MeCP2 em mucosa esofágica normal. Em seguida, analisamos a expressão das DNMTs, MBDs esofagina, p14ARF, p16INK4a e E-caderina em 17 amostras pareadas, tecidos normal e tumoral, de pacientes com carcinoma epidermóide de esôfago (CEE). Todas as enzimas foram constitutivamente expressas na mucosa esofágica normal. Nos tumores, foi observado um aumento significativo na expressão da DNMT3B (p=0,0038) e da MBD4 (p=0,0197) em relação à mucosa normal adjacente. A expressão dos genes esofagina, p14ARF e p16INK4a, no tecido tumoral, foi ausente ou reduzida em 64,7%, 52,9% e 58,8% das amostras, respectivamente. Apenas 11,7% das amostras de CEE mostraram níveis reduzidos de E-caderina. Quando a correlação entre a expressão da DNMTs com esofagina, p14ARF, p16INK4a e E-caderina, foi analisada pelo teste de Spearman foi observada uma correlação inversamente proporcional entre a expressão de DNMT3B e esofagina...

Esophageal cancer is one of the ten most common malignancies and it is the sixth cause of cancer-related death in the world. Genetic alterations, such as TP53 mutations and epigenetic modifications, such as the hypermethylation of CpG islands, are important events in cancer development and are a common way of inactivating tumor suppressor genes. in this study, we analyzed the spectrum of TP53 mutations in 101 patients with esophageal squamous cell carcinoma (ESCC) living in Southeastern Brazil. Among those patients, 33.7% showed mutations in exons 5, 6, 7 e 8 and these alterations are prevalent in codons 248, 179 and 220. Cytosine methylation is established and maintained by a family of DNA methiltransferase enzymes (DNMTs). Hypermethylation of CpG dinucleotides initiates the formation of a protein complex, including proteins who bind these methylated residues (MBDs and MeCP2), leading to transcriptional repression. We investigated, by RT-PCR, the expression of human DNMT1, DNMT3A, DNMT3B, MBD1, MBD2, MBD3, MBD4 and MeCP2 in normal esophageal mucosa. Then, we analyzed the mRNA expression of these DNMTs, MBDs, esophagin, p14ARF, p16INK4a and E-cacherin in 17 esophageal squamous cell carcinoma (ESCC) samples as well as their adjacent normal epithelial tissues. The expression of esophagin, p14ARF and p16INK4a was absent of reduced in 64.7%, 52.9% and 58.8% of the ESCC samples, respectively. Only 11.7% of the ESCC samples showed reduced levels of E-cadherin mRNA. When the correlation between mRNA expression of the DNMTs and these genes was analyzed by the Spearman rank test we observed that it was inversely correlated for DNMT3B and esophagin (p=0.0112), p14ARF (p=0.0384) and p16INK4a (p=0.0378). The results suggest that DMNT3B overexpression may be involved in the suppression or in the lower expression of p14ARF and p16INK4a seen in esophageal ESCC. Consequently, we selected DNMY3B, MBD4, p14ARF e p16 INK4a to be analyzed by real time PCR...

Análise da expressão gênica de proteínas associadas à coagulação sanguínea na neoplasia maligna de esôfago / Analysis of gene expression of proteins associated with blood coagulation in malignant esophageal neoplasm

Estudos recentes indicam que o fator tecidual (TF) participa no crescimento tumoral, metástase e angiogênese através de uma via independente da coagulação sanguínea. A superexpressão do TF em células tumorais contribui para o estado pró-trombótico em pacientes com câncer. Adicionalmente, uma família de receptores acoplados à proteína G, conhecidos como receptores ativados por proteases (PARs), tem sido associada à biologia do tumor. Estes receptores podem ser ativados por proteases da coagulação como a trombina, o fator VIIa (FVIIa) e o fator FXa (FXa), mediando assim a sinalização celular e podendo levar a um aumento da expressão de IL-8 em vários tipos celulares. O objetivo deste estudo foi analisar a expressão do RNAm de TF, PAR-1, PAR-2 e IL-8 em pacientes com câncer de esôfago. Amostras de tecidos foram obtidas de 35 pacientes submetidos a esofagectomia ou endoscopia em 3 hospitais das regiões Sul e Sudeste do Brasil: Hospital Universitário Pedro Ernesto (HUPE-UERJ), na cidade do Rio de Janeiro, Hospital de Clínicas (HCPA-UFRGS), na cidade de Porto Alegre - Rio Grande do Sul e o Hospital de Clínicas - Gastrocentro (HC-UNICAMP), na cidade de Campinas - São Paulo. Amostras de tecido esofágico tumoral e da mucosa normal adjacente ao tumor, foram obtidas de cada paciente e o diagnóstico foi confirmado através da análise histopatológica do tecido adjacente. O RNA total foi então extraído e analisado por transcrição reversa e reação em cadeia da polimerase (RT-PCR) e por PCR em tempo real (qPCR). Nossos resultados demonstraram um aumento significativo, nas amostras tumorais quando comparadas as amostras normais, da expressão de TF (4,2 +- 5,3, SE=0,9), PAR-1 (6,1 +- 4,7, SE=0,9) e IL-8 (18,2 +- 14,4, SE=3,9), o mesmo porém não foi encontrado para o PAR-2 (1,6 +- 0,8, SE=0,2). Nossos dados sugerem que TF, PAR-1 e IL-8 podem ter um importante papel na biologia dos tumores de esôfago. Na busca por esclarecimentos de como as proteínas analisadas...

A number of studies indicate that Tissue Factor (TF) might participate in tumor growth, metastasis and angiogenesis through a pathway that is independet of blood coagulation. TF overexpression by tumor cells contributes to a pro-thrombotic status in cancer patients. Also, a family of G protein-coupled receptors known as protease-activated receptors (PARs) has been implicated in tumor biology. These receptors may be activated by blood coagulation proteases including thrombin, FVIIa and FXa, thus eliciting cell signalling which might lead to interleukin-8 (IL-8) expression by a variety of cells. The aim of this study was to compare the expression of TF, PAR-1, PAR-2 and IL-8 mRNAs in patients with esophageal cancer. Tissue samples were obtained from 35 patients submitted to esophagectomy or endoscopy in three hospitals from south and southeast regions of Brazil: Hospital Universitário Pedro Esnesto (HUPE-UERJ), located at Rio de Janeiro, Hospital de Clínicas (HCPA-UFRGS), located at Porto Alegre, Rio Grande do Sul, and Hospital de Clínicas - Gastrocentro (HC-UNICAMP), located at Campinas, São Paulo. Tumor samples and the corresponding normal mucosa were obtained from each patient and the diagnosis was confirmed by histopathological analysis of adjacent tissues. Total RNA was extracted and further analyzed by reverse transcriptase (RT)-PCR and Real Time PCR. Our results showed a significant increased expression of TF (4,2 +- 5,3, SE=0,9), PAR-1 (6,1 +- 4,7, SE=0,9) and IL-8 (18,2 +- 14,4, SE=3,9) in tumor samples, but not of PAR-2 (1,6 +- 0,8, SE=0,2). Our data indicate that TF, PAR-1 and IL-8 might play an important role in esophageal cancer. To analyze the role of this proteins in oesophageal cancer patients, we used "in vitro" models with TE-1 cell line. Our results demonstrated that TE-1 cells express TF, PAR-1 and PAR-2 and display potent procoagulant activity. In this context, we will further investigate whether the activation of PAR receptors induces...

Mutations in epidermal growth factor receptor gene in esophageal squamous cell carcinoma patients in Kashmir: a high incidence area of India

Activating mutations in Epidermal Growth Factor Receptor [EGFR] are common in lung adenocarcinoma of never smokers but are rare in other types of cancer. Here we have analysed mutations in exons 19 to 21 of EGFR and in exons 19 and 20 of the EGFR homolog HER2 in 54 cases of Esophageal Squamous Cell Carcinomas [ESCC] from patients recruited in Kashmir, India, a region of high incidence for this cancer. We report the detection of 3 mutations [6%] in the ATP- binding regulatory loops of the tyrosine kinase domain of EGFR [deletion 746-750, p753L, G719D]. No mutation was found in HER2. This is the first report of activating EGFR mutations in ESCC, of the same type as those detected in lung adenocarcinoma of never-smokers. This suggests that a small portion of ESCC patients in this high incidence area may benefit from treatment with EGFR tyrosine kinase inhibitors

[Familial risks of esophageal cancer in the Caspian Littoral of Iran]

In northeastern Iran there is an area of high incidence of esophageal cancer which is populated by residents of Turkmen ancestry. Several environmental risk factors for esophageal cancer have been proposed, but the roles of familial and genetic factors have not been studied extensively in the Turkmen population. We evaluated the importance of familial risk factors for esophageal cancer by performing a case-control study of 167 cases of esophageal squamous cell carcinoma and 200 controls of Turkmen ethnicity. Detailed family pedigrees of the cases and controls were constructed, which documented all cancers in first- and seconddegree relatives. The actuarial risk of cancer was then estimated in 2097 first-degree relatives of cases and 2783 first-degree relatives of the controls. A hazard ratio was constructed, based on a comparison of the two cumulative incidence curves. The risk to age 75 of esophageal cancer in the first-degree relatives of Turkmen patients with esophageal cancer was 34%, versus 14% for the first-degree relatives of the controls [hazard ratio = 2.3; p = 3 x 10[-8]]. 9.6% of the cases reported that their parents were related, versus 2.5% of the controls [odds ratio = 4.1; p -value = 0.006]. Familial factors are important in the etiology of esophageal cancer among the Turkmen residents of Iran. The hazard ratio of 2.3 for cancer among first-degree relatives is consistent with an important contribution of heritable factors. It will be of interest to perform marker studies to establish which genes are responsible