Normal Esophageal Tissues Research Articles

Expression of BRD4 in squamous cell carcinoma and its effects on cell proliferation and invasion ability

Objective: To investigate the expression of BRD4 in squamous cell carcinoma (SCC) tissues and cells, and the effects of its expression on cell proliferation and invasion ability. Methods: Immunohistochemistry was used to detect BRD4 protein expression in SCC tissues and paired normal esophageal squamous epithelial tissues. The expression of BRD4 protein was detected in different SCC cell lines and normal esophageal squamous epithelial cells by Western blot. BRD4 siRNA and control siRNA were used to transfect SCC Eca109 cells, and experiments were divided into three groups: untreated group, control siRNA group and BRD4 siRNA group. Western blot was employed to investigate the expression of BRD4 protein in the three groups of SCC Eca109 cells. CCK-8 kit was utilized to detect cell proliferation ability, and Transwell chamber was used to examine cell invasion ability. Finally, Western blot was used to detect the expression of MMP2 and MMP9 proteins. Results: The positive rate of BRD4 protein expression in SCC tissues was significantly higher than that of normal squamous epithelial tissues. The expression of BRD4 protein in 4 SCC cell lines was higher than that in normal esophageal cell Het-1A. BRD4 siRNA obviously downregulated the expression of BRD4 protein in Eca109 cells, and its downregulation contributed to the suppression of cell proliferation and invasion ability in Eca109 cells (all P<0.05), coupled with the decreases of MMP2 and MMP9 proteins. Conclusion: BRD4 may be closely associated with the proliferation and invasion of SCC, and it thus may be a potential therapeutic target for SCC.

Expression and clinical significance of retinoid X receptor α in esophageal carcinoma.

Esophageal carcinoma (EC) is one of the most aggressive type cancers and dysregulation of retinoid X receptor α (RXRα) involves various tumors. However, the relationship of RXRα with the clinicopathological factors of EC, particularly prognostic characteristics, remains unclear. This present study was to evaluate the effect of RXRα expression in the development of EC. The mRNA and protein expression level of RXRα in EC and normal esophageal tissues using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The subcellular localization was detected by immunohistochemistry (IHC) analysis. The clinicopathological parameters were included age, sex, tumor size, differentiation, TNM stages and lymph node metastasis. Kaplan-Meier method and Cox's regression analyses were performed to evaluate the prognosis of 60 patients with EC. RXRα was elevated in EC tissues comparing with normal esophageal tissues at both mRNA and protein levels. The overexpression level of RXRα was closely associated to the tumor differentiation, TNM stage and lymph node metastasis of patients with EC. In addition, EC patients with RXRα high expression had significantly lower disease-free survival (DFS) and overall survival (OS). Multivariate analysis showed RXRα expression as an independent predictor for the DFS and OS rate of patients with EC. Our results showed that overexpression of RXRα was correlated with unfavorable prognosis, suggesting that RXRα may serve as a potential targeted therapeutic marker in the treatment of EC.

Prognostic significance of the E-box binding zinc finger protein 2 and epithelia-cadherin in esophageal squamous cell carcinoma

Objective To evaluate the expressions of E-box bing zinc finger protein 2 (ZEB2) and epithelia-cadherin (E-cadherin) in esophageal squamous cell carcinoma (ESCC) and their prognostic significance. Methods The expression of ZEB2 and E-cadherin in 218 ESCC and 60 cases of normal esophageal tissue was detected by SP immunohistochemistry. Cox regression model was used to analyze the prognostic factors. Results The expression rate of ZEB2 was 35.3%(77/218) significantly higher in ESCC tissues than those was 18.3% (11/60) in normal tissue (χ2=6.276, P=0.012). The expression of E-cadherin protein was 45.8% (100/218) significantly lower in ESCC tissues than those was 66.7%(40/60) in normal tissue (χ2=8.139, P=0.004). The expressions of ZEB2 and E-cadherin protein were correlated with tumor size, depth of tumor invasion (χ2=4.104, P=0.043), lymph node metastasis (χ2=10.640, P=0.001) and TNM stage (χ2=11.080, P=0.001) in ESCC tissues. ZEB2 was negatively correlated with E-cadherin protein in ESCC tissues (r=-0.440, P=0.000). Multiple-factor analysis showed ZEB2 was the independent prognostic factors for ESCC. Conclusion The positive expression of ZEB2 is associated with epithelial-mesenchymal transformation (EMT) happened and an important factor of poor prognosis in esophageal squamous cell carcinomas. Key words: Esophageal squamous cell carcinoma; E-box bing zinc finger protein 2; Epithelial-mesenchymal transformation; E-cadherin; Prognosis

Expression of programmed death-1 ligand-1 in esophageal squamous cell carcinoma and its effect on radiosensitivity and prognosis

Objective To investigate the relationship of programmed death ligand-1 (PD-L1) with clinicopathological characteristics, radiosensitivity and prognosis of the patients with esophageal squamous cell carcinoma (ESCC). Methods Ninety ESCC patients who received radical radiotherapy diagnosed by ESCC in Affiliated Hospital of North Sichuan Medical College were enrolled. Twenty cases of normal esophageal mucosa were used as the controls. The expression of PD-L1 was detected by using immunohistochemical SP method. Results The expression of PD-L1 protein was not correlated with age, gender, the maximum diameter of tumor, the length of lesion, the local aggressive of tumor, clinical stage and primary tumor volume (all P > 0.05). However, it was statistically correlated with the lymphatic metastasis (χ2= 4.404, P= 0.036). Meanwhile, PD-L1 positive expression was sensitive to radiation (χ2= 4.888, P < 0.05). Single factor analysis showed that the maximum diameter of tumor and radiosensitivity were correlated with progression-free survival (PFS) (χ2= 6.239, P= 0.013; χ2= 6.852, P= 0.008; χ2= 6.312, P= 0.012) and overall survival (OS) (χ2= 8.170, P=0.004; χ2= 4.261, P= 0.039; χ2= 5.003, P= 0.025) of ESCC patients. Multifactor analysis showed that the radiosensitivity and the maximum diameter of tumor affected PFS (OR= 0.512, 95% CI 0.275-0.954, P= 0.035) and OS (OR= 0.507, 95% CI 0.266-0.968, P= 0.039) in ESCC patients, respectively. Conclusions The level of PD-L1 expression is increased significantly in ESCC tissues compared with the normal esophageal mucosa tissues. PD-L1 may be a novel biomarker for predicting metastatic potential and radiosensitivity in ESCC patients, rather than the prognosis predictors of ESCC patients treated by radiotherapy. Key words: Esophageal neoplasms; Programmed death ligand-1; Radiation tolerance; Prognosis

Stromal expression of JNK1 and VDR is associated with the prognosis of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide, and its outcome is poor. The purpose of this study was to determine the association between JNK1 and vitamin D receptor (VDR) expression and the prognosis of ESCC. Immunohistochemical staining was conducted on ESCC tissue microarrays (362 pairs of ESCC and normal esophagus tissues). The epithelial and stromal expression levels of c-jun NH2-terminal kinase 1 (JNK1) and VDR were scored and correlated with the ESCC characteristics. Laser-capture-based quantitative RT-PCR was performed on ESCC tissues. The effects of JNK1 and VDR on ESCC cell proliferation and migration were analyzed in vitro by transient transfection, and protein changes were evaluated by immunoblotting. Both JNK1 and VDR were reduced in ESCC epithelial cells in comparison with the normal esophagus, but the expression of JNK1 and VDR in ESCC stromal tissues, not epithelial cells, was strongly associated with the survival time of ESCC patients. Functional studies showed that increased JNK1 suppressed cancer cell proliferation, mobility, and migration, which were linked to the alterations of VDR and metastasis-associated proteins. JNK1 and VDR act as tumor suppressors, and their stromal expression levels are associated with prognosis in esophageal squamous cell carcinoma.

Selective oestrogen receptor antagonists inhibit oesophageal cancer cell proliferation in vitro

BackgroundOestrogen receptors (ER) have a well-established role to the initiation, progression and regulation of responses to treatment of breast, prostate, and lung cancers. Previous data indicates altered ER expression in oesophageal cancers (OC). However the role of ER subtypes and ER specific inhibitors in the regulation of OC progression remains unclear. This study sought to assess levels of ERα and ERβ in OC. The effects of highly selective ER antagonists on cell proliferation and apoptosis in two OC adenocarcinoma cell lines was also studied.MethodsERα and ERβ expression profiling in paired normal oesophageal mucosa and tumour tissues (n = 34; adenocarcinoma n = 28; squamous cell carcinoma n = 6) was performed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Correlation between levels of ER with the clinico-pathological features for OC was determined. The effect of selective ER antagonists on proliferation of OE33 and OE19 OC cell lines was studied.ResultsERα and ERβ mRNA expression was significantly higher (p < 0.05) in tumour tissues relative to their paired normal mucosa and correlated inversely with survival outcome (p < 0.05). Upregulation of ERα mRNA correlated with higher pathological T-stage (p < 0.05) and lymph node metastasis (p < 0.05) while ERβ mRNA upregulation correlated with positive vascular invasion (p < 0.05). A significant concentration-dependent inhibition of proliferation in OE33 and OE19 cell lines was induced by a highly-selective ERα antagonist (MPP) and an ERβ specific antagonist (PHTPP) (p < 0.05). Moreover, anti-oestrogens induced cell death through stimulation of apoptotic caspase activity.ConclusionThese findings indicate that the ER system is involved in OC progression and thus may provide a novel target for the treatment of OC.

Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference (P=0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P=0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results (P=0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients(P<0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.

Overexpression of Peroxiredoxin 6 (PRDX6) Promotes the Aggressive Phenotypes of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. Peroxiredoxin 6 (PRDX6), a member of peroxidase superfamily, has a function of eliminating the reactive oxygen species (ROS), and participates in development of multiple diseases, including tumors. The purpose of this study was to investigate the expression of PRDX6 in normal and cancerous esophageal tissues and to characterize its role in ESCC progression. We found significantly higher expression of PRDX6 in ESCC tissues than in normal esophageal tissues or tumor-adjacent tissues and that the PRDX6 expression level was positively correlated with the proliferation-related markers. In ESCC cells, PRDX6 distribution was more pronounced in the nucleus region. PRDX6 overexpression by an adenovirus significantly promoted cell proliferation, migration and invasion in TE-1 and Eca-109 cells. Conversely, lentivirus-mediated knock-down of PRDX6 expression significantly reduced cell growth, colony formation and metastasis in ESCC cells. PRDX6 modulated the phosphorylation of Akt and Erk1/2, and the expression of MMP2. We also found that PRDX6 and Erk1/2 pathway were mutually regulated in ESCC cells. In addition, PRDX6 overexpression eliminated radiation-induced ROS and decreased consequent cell apoptosis, indicative of a role in radioresistance. Finally, the role of PRDX6 in promoting tumor growth was further confirmed in nude mice with ESCC xenografts. Taken together, we demonstrated that overexpression of PRDX6 promotes the progression of ESCC through Erk1/2, which provides a potential therapeutic target for human ESCC.

Application of an Advanced Image-Based Virtual Monoenergetic Reconstruction of Dual Source Dual-Energy CT Data at Low keV Increases Image Quality for Esophageal Cancer Patients Imaging

Purpose: To explore the significance of dual-source computed tomography (DECT) virtual monoenergetic reconstructions technology in improving the imaging quality of esophageal cancer patients. Materials and methods: 68 patients with clinically suspected esophageal cancer (all confirmed by pathology) were collected. Routine plain scan was performed with SIEMENS Force dual-energy CT and then dual-phase scans were performed. The venous phase images were respectively subjected to traditional virtual monoenergetic reconstructions (Mono_E) and new generation virtual monoenergetic reconstructions (Mono+). Mono_E 55 keV and Mono+ 55 keV virtual single-energy images were obtained respectively. The signal-to-noise ratio (SNR) of normal esophageal tissue and esophageal cancer lesions, noise and contrast noise ratio (CNR tumor) of normal esophageal tissue and esophageal cancer lesions were compared among 100 kV images, Mono_E images and Mono+ images. At the same time, two imaging physicians read the films and scored the images of each group by using a 5-point scoring method. Results: Mono+ 55 keV images, SNR, SNRtumor, noise and CNRtumor were statistically different from those of 100 kV images and Mono_E images (P Mono+ 55 keV images also had the highest subjective score, with statistical significance (P 55 keV images had the best quality. Conclusion: The new generation of virtual monoenergetic reconstructions post-processing (Mono+) could reduce image noise and improve the contrast between esophageal cancer lesions and normal esophageal tissues, which was of great significance to improve the imaging quality of esophageal cancer patients and improve the early detection rate of esophageal cancer.

Efficacy of Shikonin against Esophageal Cancer Cells and its possible mechanisms in vitro and in vivo.

Increasing evidences indicate that shikonin can suppress the tumor growth. However, the mechanisms remain elusive. In the present study, we investigated the effects and mechanisms of shikonin against esophageal cancer. The expression of hypoxia inducible factor 1α (HIF1α) and pyruvate kinase M2 (PKM2) in esophageal cancer tissues and cells was detected by immunohistochemistry and Western blot. CCK-8 was used to examine the esophageal cancer cell viability. Apoptosis and cell cycle were analyzed by flow cytometry. The expression of EGFR, PI3K, Akt, p-AKT, mTOR, HIF1α and PKM2 was detected by Western blot. EC109/pkm2 was established by lentivirus transducer. Ec109 tumor model was founded to observe the antitumor effect of shikonin in vivo.We found that HIF1α and PKM2 protein expression levels were higher in esophageal cancer tissues and cells than normal esophageal tissues and cells. Shikonin reduced esophageal cancer cells viability and induced cell cycle arrest and apoptosis. Shikonin decreased EGFR, PI3K, p-AKT, HIF1α and PKM2 expression. Overexpression of PKM2 could enhance resistance of esophageal cancer cells to shikonin. In vivo we found that shikonin reduced tumor burden, inducing cell arrest and apoptosis. Taken together, shikonin has a significant antitumor effect in the esophageal cancer by regulating HIF1α/PKM2 signal pathway.

RhoE expression is negatively correlated with epidermal growth factor receptor and its prognosis value in human esophageal squamous cell carcinoma

Objective To investigate the gene expression of RhoE, a small Rho GTP protein in esophageal squamous cell carcinoma (ESCC) tissues and its association with epidermal growth factor receptor (EGFR) expression and patients prognosis. Methods Thirty patietns with confirmed diagnosis of ESCC were included in this study form May 10 2015 to July 10 2016. Real time quantitative PCR was applied to detect the expression of RhoE and EGFR in paired ESCC and corresponding normal tissues. The prognosis of RhoE was further evaluated based on the recorded survival time of the 30 ESCC patients. Results Comparing with the normal esophageal tissues, RhoE was significantly downregulated in ESCC. 63.3% of these ESCC tissues expressed more than 2 fold downregulation of RhoE. Besides, EGFR gene was upregulated in these corresponding ESCC tissues. A significant negative correlation was found between RhoE and EGFR expression (r=-0.550, P=0.032). Kaplan-Meier analysis of association between RhoE expression and overall survival of these ESCC patients revealed that lower RhoE levels indicated poorer prognosis [hazard ratio (HR)=0.210, P=0.038]. Conclusion Down-regulation of RhoE was observed in ESCC tissues, which negatively correlated with EGFR expression and prognosis. All these indicate the vital tumor suppressing roles of RhoE in the development of ESCC. Key words: RhoE; Epidermal growth factor receptor; Esophageal squamous cell carcinoma; Prognosis

Clinical significance and expression of runt-related transcription factor 2 in esophageal squamous cell carcinoma

Objective To evaluate the expression and clinical significance of runt-related transcription factor (RUNX2) in esophageal squamous cell carcinoma (ESCC). Methods We analzyed the RNA-seq data of esophageal carcinoma patients from The Cancer Genome Atlas (TCGA) database. Quantitative real-time PCR and immunohistochemistry were used to detect RUNX2 mRNA and protein expression in 80 ESCC patients’ cancer and adjacent normal esophageal mucosal tissues. The relationship of RUNX2 protein expression and clinicopathological parameters and the association of RUNX2 protein expression with prognosis were analyzed. Results The expression of RUNX2 mRNA in esophageal carcinoma and esophageal squamous cell carcinoma was significantly increased by TCGA database analysis, P=0.000, P=0.002. The RUNX2 mRNA (P=0.000) and protein (16.942, P=0.000) expression was significantly higher in ESCC cancer tissues than in normal tissues. RUNX2 protein expression was significantly associated with the tumor invasion (6.138, P=0.013), differentiation (6.277, P=0.014) and TNM stage (3.903, P=0.048). The overall survival (P=0.048) and progression free survival (P=0.025) in patients with high RUNX2 protein expression were significantly longer than those in the patients with low RUNX2 protein expression. Multivariate analysis showed that the expression of RUNX2 was an independent risk factor of overall survival (P=0.000) and progress free survival (P=0.002). Conclusion RUNX2 might play an important role in the development and progression of ESCC and might be a prognostic factor in ESCC. Key words: Esophageal squamous cell carcinoma; Runt-related transcription factor 2; Prognosis

BMI-1 suppression increases the radiosensitivity of oesophageal carcinoma via the PI3K/Akt signaling pathway.

B-cell‑specific Moloney murine leukaemia virus integration site-1 (BMI-1) contributes to the growth of tumour cells post-irradiation (IR). The aim of the present study was to characterize the effects of BMI-1 on cell viability, radiosensitivity and its mechanisms of action in oesophageal squamous cell cancer (ESCC). Western blotting and immunohistochemistry were employed to evaluate the protein expression of BMI-1 in ESCC cells and specimens, respectively. Additionally, the protein expression levels of BMI-1, H2AK119ub and γH2AX in ESCC cells were detected following different doses of IR and at different times after IR. The protein expression levels of MDC1 and 53BP1 were also measured. Flow cytometry and MTT assays were used to determine cell cycle progression, apoptosis and cell viability. The phosphatidylinositol 3-kinase inhibitor LY294002 and the agonist IGF-1 were employed to suppress or induce the phosphorylation of Akt to determine whether BMI-1 induces radioresistance in ESCC cells via activation of the PI3K/Akt pathway. The expression of BMI-1 was higher in ESCC tissues and cells compared with that in normal oesophageal tissues and cells. In addition, BMI-1 was positively related to tumour size and lymph node metastases and negatively to the overall survival of ESCC patients. IR induced the expression of BMI-1, H2AK119ub and γH2AX in a dose-and time-dependent manner. BMI-1 knockdown lowered the expression of γH2AX, MDC1 and 53BP1, suppressed cell viability and increased radiosensitivity. G2/Mphase arrest was eliminated; this was followed by an increased proportion of cells entering the G0/G1phase after IR and BMI-1 knockdown via the upregulation of P16 and downregulation of cyclinD2 and cyclin-dependent kinase-4. Moreover, BMI-1 knockdown increased cell apoptosis, downregulated MCL-1 and p-Akt and upregulated Bax. Additionally, the inhibitory effect of the downregulation of p-Akt by LY294002 on tumour cell viability was identical to that of BMI-1 knockdown, while the kinase agonist IGF-1 reversed the effects of BMI-1 knockdown on cell viability and radiosensitivity. Taken together, BMI-1 knockdown induces radiosensitivity in ESCC and significantly inhibits cell viability, which may contribute to an increased proportion of cells in the G0/G1phase and cell apoptosis via suppression of the PI3K/Akt signalling pathway.

MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates ESCC tumor growth.

Recently, we have reported that the product of Melanoma Antigens Genes (MAGE) family member MAGE-A11 is an independent poor prognostic marker for esophageal squamous cell carcinoma (ESCC). However, the reason how MAGE-A11 is activated in ESCC progression still remains unclear. In the current study, we demonstrated that DNA methylation and the subsequent histone posttranslational modifications play crucial roles in the regulation of MAGE-A11 in ESCC progression. We found that the methylation rate of TFCP2/ZEB1 binding site on MAGE-A11 promoter in ESCC tissues and cells is higher than the normal esophageal epithelial tissues and cells. Transcription factors TFCP2 and ZEB1 directly bind MAGE-A11 promoter and regulate the endogenous MAGE-A11 expression in a methylation-dependent manner in ESCC cells. Following MAGE-A11 promoter methylation, the methyl-CpG-binding protein MeCP2 was found to bind the methylated MAGE-A11 promoter to mediate histone deactylation by recruiting HDAC1 and HDAC2. Simultaneously, histone inactivation marks including H3K27me3 as well as H3K9me3 were increased, whereas histone activation mark H3K4me3 was decreased. HDAC inhibitor Trichostatin A (TSA) increased DNA methylase inhibitor Decitabine (DAC)-induced MAGE-A11 expression. siRNA-mediated knockdown of histone methltransferase EZH2 or DZNep (a EZH2 inhibitor) treatment increased DAC-induced MAGE-A11 expression. Our results indicate that MAGE-A11 is activated through DNA demethylation, histone acetylation and histone methylation in ESCC, and its activation promotes ESCC tumor growth.

The role of mitochondrial folate enzyme MTHFD1L in esophageal squamous cell carcinoma

Objective: The lack of novel therapeutic targets poses the major challenge to prolong survival and improve the quality of life for esophageal squamous cell carcinoma (ESCC). Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) plays critical roles in folate cycle maintenance. However, little information is available concerning the role of MTHFD1L in cancer cells, and no studies have addressed such issues in esophageal cancer.Materials and method: Surgical cancer and adjacent normal esophageal tissues were obtained from patients with esophagectomy and esophagogastrostomy for ESCC. Western blot, immunohistochemistry and Quantitative RT-PCR were performed to evaluate protein and RNA expression levels of MTHFD1L. Knockdown of MTHFD1L expression was achieved by using short hairpin RNA. The effects of MTHFD1L silencing on ESCC cell proliferation and apoptosis were assessed by the MTT assay, Celigo assays, Annexin V FACS assay and Caspase-3/7 array in vitro.Results: Twenty-three paired cancer and adjacent normal esophageal tissues from patients with ESCC were included in this study. MTHFD1L protein and RNA expression levels were significantly upregulated in ESCC tissue as compared with normal tissue. High expression of MTHFD1 was also detected in two esophageal cancer cell lines (TE-1 and EC109). Knockdown of MTHFD1L expression inhibited the proliferation of TE-1 cells, and the apoptosis was distinctly increased following shMTHFD1L infection.Conclusions: Our preliminary study highlighted for the first time that MTHFD1L might be involved in the development of ESCC, which may provide a new potential tumor-specific therapeutic targeting for anti-folate agents.

Increased copy number of mitochondrial DNA predicts poor prognosis of esophageal squamous cell carcinoma.

Change in mitochondrial DNA (mtDNA) copy number has been reported in esophageal squamous cell carcinoma (ESCC). However, its prognostic implication in ESCC remains largely unknown. Using reverse transcription-quantitative PCR assay, the mtDNA copy number was assessed in a cohort of patients with ESCC (n=141) and normal esophageal tissues (n=45), and the association between variable mtDNA levels and clinical outcomes of patients with ESCC were studied. Data revealed that ESCC patients exhibited an increased mtDNA content compared to control subjects. Furthermore, increased mtDNA content was associated with a significantly increased risk of cancer-associated mortality. This molecular event was associated with poorer survival in patients with ESCC, and was an independent predictor of patient survival. Data demonstrated that increased mtDNA content is a common genetic event in ESCC and may be a predictive factor of poor prognosis for ESCC patients.

Expression of Heat Shock Protein-27 (Hsp27) and P38MAPK in Esophageal Squamous Cell Carcinoma.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a worldwide concern. This study looked at the relationship between the expression of differential proteins and the clinicopathological data and survival rate of ESCC patients to identify potential tumor markers for the growth and metastasis of ESCC.Material/MethodsThis study included 162 patients who underwent surgical excision for management of ESCC. Fresh ESCC tissue and adjacent normal tissue specimens were collected. Protein expressions were detected by western blotting. The expression of Hsp27 and P38MAPK were detected by immunohistochemistry in formalin-fixed paraffin embedded primary tissue specimens.ResultsThe rate of positive Hsp27 and P38MAPK expression in ESCC tissue were higher than in normal esophageal tissue (p<0.05). The expression of P38MAPK was related to the depth of infiltration (p<0.05). The expression of Hsp27 was correlated with lymph node metastasis (p<0.05), but not with age, depth of infiltration, or tumor size. ROC were plotted to estimate the significance of the diagnosis: for Hsp27, AUC=0.735 (p<0.05), for P38MAPK, AUC=0.882 (p<0.05).ConclusionsThe expression of Hsp27 and P38MAPK plays a role in ESCC development. Hsp27 and P38MAPK could be used as prognostic factors in ESCC.

Identification of a key role of widespread epigenetic drift in Barrett\u2019s esophagus and esophageal adenocarcinoma

BackgroundRecent studies have identified age-related changes in DNA methylation patterns in normal and cancer tissues in a process that is called epigenetic drift. However, the evolving patterns, functional consequences, and dynamics of epigenetic drift during carcinogenesis remain largely unexplored. Here we analyze the evolution of epigenetic drift patterns during progression from normal squamous esophagus tissue to Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) using 173 tissue samples from 100 (nonfamilial) BE patients, along with publically available datasets including The Cancer Genome Atlas (TCGA).ResultsOur analysis reveals extensive methylomic drift between normal squamous esophagus and BE tissues in nonprogressed BE patients, with differential drift affecting 4024 (24%) of 16,984 normally hypomethylated cytosine-guanine dinucleotides (CpGs) occurring in CpG islands. The majority (63%) of islands that include drift CpGs are associated with gene promoter regions. Island CpGs that drift have stronger pairwise correlations than static islands, reflecting collective drift consistent with processive DNA methylation maintenance. Individual BE tissues are extremely heterogeneous in their distribution of methylomic drift and encompass unimodal low-drift to bimodal high-drift patterns, reflective of differences in BE tissue age. Further analysis of longitudinally collected biopsy samples from 20 BE patients confirm the time-dependent evolution of these drift patterns. Drift patterns in EAC are similar to those in BE, but frequently exhibit enhanced bimodality and advanced mode drift. To better understand the observed drift patterns, we developed a multicellular stochastic model at the CpG island level. Importantly, we find that nonlinear feedback in the model between mean island methylation and CpG methylation rates is able to explain the widely heterogeneous collective drift patterns. Using matched gene expression and DNA methylation data in EAC from TCGA and other publically available data, we also find that advanced methylomic drift is correlated with significant transcriptional repression of ~ 200 genes in important regulatory and developmental pathways, including several checkpoint and tumor suppressor-like genes.ConclusionsTaken together, our findings suggest that epigenetic drift evolution acts to significantly reduce the expression of developmental genes that may alter tissue characteristics and improve functional adaptation during BE to EAC progression.

Identification and Validation of Fibroblast Growth Factor 12 Gene as a Novel Potential Biomarker in Esophageal Cancer Using Cancer Genomic Datasets.

Esophageal squamous cell carcinoma (ESCC) has a complex, multifactorial etiology in which environmental, geographical, and genetic factors play major roles. It is the second most common cancer among men and the fourth most common among women in India, with a particularly high prevalence in Northeast India. In this study, an integrative in silico [DAVID, NCG5.0, Oncomine, Cancer Cell Line Encyclopedia, and The Cancer Genome Atlas (TCGA)] approach was used to identify the potential biomarkers by using the available three genomic datasets on ESCC from Northeast India followed by its in vitro functional validation. Fibroblast Growth Factor 12 (FGF12) gene was overexpressed in ESCC. The upregulation of FGF12 was also observed on ESCC of TCGA OncoPrint portal, whereas very low expression of FGF12 gene was mapped in normal esophageal tissue on the GTEx database. Silencing of FGF12 showed significant inhibition in activity of tumor cell proliferation, colony formation, and cell migration. The upregulation of FGF12 showed significantly reduced survival in ESCC patients. The protein interaction analysis of FGF12 found the binding with MAPK8IP2 and MAPK13. High expression of FGF12 along with MAPK8IP2, and MAPK13 proteins correlate with poor survival in ESCC patients. Tissue microarray also showed expression of these proteins in patients with ESCC. These results indicate that FGF12 has a potential role in ESCC and suggest that cancer genomic datasets with application of in silico approaches are instrumental for biomarker discovery research broadly and specifically, for the identification of FGF12 as a putative biomarker in ESCC.

Identification of PA28β as a potential novel biomarker in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and serious malignancies in China. However, the exact mechanisms of tumor formation and progression are unclear. As late diagnosis and poor therapeutic efficacy result in lower survival rates, identifying biomarkers for early detection, prognostic evaluation, and recurrence monitoring of ESCC is necessary. Here we analyzed 10 protein expression profiles of ESCC core tissues and paired normal esophageal epithelial tissues using two-dimensional gel electrophoresis. We excised 29 protein spots with two-fold or greater differential expression between cancer and normal tissues and identified them using matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry. The role of PA28β in ESCC cell was confirmed using cell growth, colony formation and soft agar in TE-1 cells pre- and post- PA28β transfection. Compared to their expression in the adjacent normal epithelia, 12 proteins, including transgelin (TAGLN), were upregulated in ESCC tissues; 17 proteins, including proteasome activator 28-beta subunit (PA28β), were downregulated (p < 0.05). Western blotting and immunohistochemistry confirmed that PA28β was significantly underexpressed in ESCC tissues. The functional assays demonstrate that PA28β inhibited cell growth, proliferation and malignancy of TE-1 cells. Among the differentially expressed proteins, PA28β is a potential tumor inhibitor.