Esophageal Squamous Cell Research Articles

Abstract 663: Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin

Abstract Podoplanin is a platelet aggregation-inducing mucin-like sialoglycoprotein that is highly expressed in brain tumors, malignant mesotheliomas, testicular tumors, and lung, esophageal, or head and neck squamous cell carcinomas. Podoplanin has also been reported to be a marker to enrich tumor-initiating cells. We previously established a neutralizing monoclonal antibody (mAb), NZ-1, which inhibits the association between podoplanin and C-type lectin-like receptor-2 (CLEC-2) and blocks the podoplanin-induced platelet aggregation and tumor metastasis. A human chimeric mAb, NZ-8 possesses extremely high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and showed high anti-tumor activities in several xenograft models. However, those mAbs also react with normal cells including lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells throughout the body. Recently, we established the platform to produce a cancer-specific mAb (CasMab) by combining glycobiology and cell engineering technologies. Herein, we report a novel cancer-specific anti-podoplanin mAb, LpMab-2. In Western-blot analyses, LpMab-2 did not react with sialic acid-deficient podoplanin and O-glycan deficient podoplanin, which were expressed in Lec2 and Lec8 cells, respectively. In enzyme-linked immunosorbent assay using several deletion mutants of podoplanin, the epitope of LpMab-2 was shown to be included in 55-88 amino acids of podoplanin, although almost all anti-podoplanin mAbs recognize platelet aggregation-stimulating (PLAG) domain near the N-terminus. Analyses of glycopeptides produced by Edman degradation revealed that O-glycans were attached to glycosylation sites at residues Thr65, Thr66, Thr70, Ser71, Ser74, and Thr76 around LpMab-2 epitope. LpMab-2 did not react with podoplanin-negative cell lines, indicating that not only glycans but also amino acids of podoplanin are necessary for LpMab-2 reaction. In flow cytometric and immunohistochemical analyses, LpMab-2 reacted with tumor cells, but not with lung type I alveolar cells and lymphatic endothelial cells, suggesting that LpMab-2 recognizes the cancer-type conformation of podoplanin. Furthermore, human chimeric LpMab-2 also showed cancer-specific reaction against podoplanin. Taken together, a cancer-specific mAb LpMab-2 should be useful for molecular targeting therapy against podoplanin without side effects such as respiratory or renal failures and lymphatic edema. The CasMab-producing platform could lead to establishment of the side effect-reducing mAbs. Note: This abstract was not presented at the meeting. Citation Format: Yukinari Kato, Mika K. Kaneko. Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2014-663

Abstract 2221: A new promoter of Casp 8 in an esophageal squamous cell carcinoma susceptibility locus

Abstract Our previous Genome wide associationstudy (GWAS) of esophagealsquamous cell carcinoma (ESCC) identified a susceptibility locus on chromosome 2q33.1 encompassing CASP8 gene. Caspase 8 is an essential initiator in cell apoptosis and is associated with many diseases. CASP8 has varieties of transcripts, which might exert distinct functions. However, the transcriptional determinants, which control CASP8 gene expression remain poorly defined. The present study focused on revealing the mechanism behind the GWAS signal and finding clues for the complex regulation of CASP8.We observed a secondary promoter in CASP8 using a series of luciferase reporter assays, which locates 25kb far away from the previously described CASP8 promoter. The ENCODE ChIP-seq data shows the promoter features at the epigenetic level, including high H3K27Ac, H3K4M3 and low CpG methylation around the novel promoter region. Though people have known lots of transcription factors, little research focuses on which factors exert the transcription function for a certain promoteron earth. To explore the transcription factors that contribute to the secondary promoter of CASP8, we developed an effective strategy based on DNA-protein affinity purification and mass spectrometry. PURα was identified from the eluted protein products that the secondary promoter fragment could bind specific. We validated the mass spectrometry result through western blot analysis and immunoprecipitation assay and confirmed that PURα was involved in the transcription machine and might exert the function by forming a complex together with E2F-1 and Pol II. Remarkably, we observed that if knocking down the expression of PURα, the transcription activity decreased significantly. Although the physiologicrole of the CASP8 secondary promoter and its correspondent transcripts remain unclear, the current data suggest that it could explain the complexity of CASP8's transcription in some degree and expand the GWAS results to the function level. We believe these two promoters have thecapacity to play a role in the very different functions of caspase 8 isoforms. Note: This abstract was not presented at the meeting. Citation Format: Zhengwei Lin, Xiaohang Zhao, Yang Xu, Zhimin Guo, Nan Zhao, Yulin Sun. A new promoter of Casp 8 in an esophageal squamous cell carcinoma susceptibility locus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2014-2221

Clinical efficacy of multi-band mucosectomy for high-grade esophageal intraepithelial neoplasia

Objective To investigate the efficacy and safety of multi-band mucosectomy (MBM) for the treatment of high-grade intraepithelial neoplasia.Methods The clinical data of 24 patients with high-grade esophageal intraepithelial neoplasia who were admitted to the Henan Honliv Hospital from April 2011 to October 2012 were retrospectively analyzed.All the 24 patients received MBM,and the operation time,resection of the lesion and complications were observed.All the patients were followed up with gastroscopy at postoperative 1,3,6,12 months.The follow-up was ended in April 2013.Results A total of 26 lesions in 24 patients were resected successfully by MBM (2 patients received MBM twice).The mean operation time was 42 minutes.The mean length of the lesions was 3.1 cm (range,0.8-6.0 cm),and it occupied 3/4 of the circumference of the esophagus to the maximum.The mean number of the band used was 4 (range,1-6).During the operation,bleeding was occurred in 4 patients,and they were cured by hot biopsy forceps.No perforation of the esophagus was detected.The results of post-MBM pathological examination showed that 22 patients were with severe atypical hyperplasia,and 2 were with carcinoma in situ.During the follow-up of 6-24 months,22 patients were cured,and esophageal stricture occurred in 1 patient at post-MBM 1 month,and the symptoms were successfully relieved by endoscopic balloon dilatation.Neoplasia recurrence was observed in 1 patient (2 lesions were resected twice) at post-MBM 3 months,and he received surgical treatment.Histopathological diagnosis showed that he had esophageal squamous cell carcinoma.No stricture or neoplasia was detected by gastroscopy at postoperative month 12.Conclusions MBM is a relatively safe and effective endoscopic technique for the treatment of high-grade esophageal intraepithelial neoplasia.The resection range should not be blindly extended.For patients whose lesions are beyond 3/4 of the circumference of the esophagus in width or with multiple lesions which can not be resected by MBM at one time,MBM should be applied cautiously to avoid esophageal stricture and recurrence. Key words: Esophageal neoplasms; High-grade intraepithelial neoplasia; Multi-band mucosectomy; Treatment

Effects of PLCE1 Gene Silencing by RNA Interference on Cell Cycling and Apoptosis in Esophageal Carcinoma Cells

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase C? gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigate the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.

Progress of cancer research on astrocyte elevated gene-1/Metadherin (Review).

Tumor development is initiated by an accumulation of numerous genetic and epigenetic alterations that promote tumor initiation, invasion and metastasis. Astrocyte elevated gene-1 [AEG-1; also known as Metadherin (MTDH) and Lysine-rich CEACAM1 co-isolated (LYRIC)] has emerged in recent years as a potentially crucial mediator of tumor malignancy, and a key converging point of a complex network of oncogenic signaling pathways. AEG-1/MTDH has a multifunctional role in tumor development that has been found to be involved in the following signaling cascades: i) The Ha-Ras and PI3K/Akt pathways; ii) the nuclear factor-κB signaling pathway; iii) the ERK/mitogen-activated protein kinase and Wnt/β-catenin pathways; and iv) the Aurora-A kinase signaling pathway. Studies have established that AEG-1/MTDH is crucial in tumor progression, including transformation, the evasion of apoptosis, invasion, angiogenesis and metastasis. In addition, recent clinical studies have convincingly associated AEG-1/MTDH with tumor progression and poor prognosis in a number of cancer types, including hepatocellular, esophageal squamous cell, gallbladder and renal cell carcinomas, breast, non-small cell lung, prostate, gastric and colorectal cancers, and glioma, melanoma, neuroblastoma and osteosarcoma. AEG-1/MTDH may be used as a biomarker to identify subgroups of patients who require more intensive treatments and who are likely to benefit from AEG-1/MTDH-targeted therapies. The therapeutic targeting of AEG-1/MTDH may simultaneously block metastasis, suppress tumor growth and enhance the efficacy of chemotherapeutic treatments.

Sa1839 Atonal Homolog 1 (ATOH1) Upregulates Gastric-and Intestinal-Type Protein Expression in Esophageal Squamous Cells, and Increases Proliferation in Barrett's Cells: Potential Roles for ATOH1 in Barrett's Pathogenesis and Carcinogenesis

in response to DCA treatment. 14% of the K14-Cdx2 mice treated with DCA developed two nodules each at the SCJ, whereas none of the K14-Cdx2 mice receiving water alone developed nodules. In contrast, 70% of the IL-1β mice, and an equal number of Cdx2/IL1β mice developed nodular disease at the SCJ after DCA treatment. All DCA treatmentassociated nodules were histologically consistent with metaplasia. However overall the double-transgenic mice had half as many nodules as IL-1β mice. Cdx2 did not affect IL-1β mRNA levels in the double transgenic mice, nor was the inflammation associated with IL1β expression reduced in the Cdx2/IL-1β mice compared to IL-1β mice, as determined by CD45R+ cell infiltration in the esophagi and forestomach. Moreover, cell proliferation rates were not altered by Cdx2 expression, as EdU incorporation was not significantly different. Lastly, the morphologic appearance of the nodules in all three lines, K14-Cdx2, L2-IL-1β, and K14-Cdx2/L2-IL-1β mice was similar, with abundant alcian blue expressing goblet cells detected, along with expression of other markers of intestinalization. Further molecular characterization of the metaplastic nodules by cDNA microarray profiling is underway. Conclusion: Ectopic expression of Cdx2 in the IL-1Beta mouse model for BE reduces the nodular intestinal metaplasia disease burden. These studies raise new questions as to the specific role of inflammation and intestinal transcription factors in the development of Barrett's esophagus in humans.

Mo1660 FOXP3 Inhibits Cell Proliferation of Gastric Cancer Cells Through Modulation of the TGF-β/SMAD Signaling Pathway

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H2O2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immunoprecipitation (ChIP) assay showed that SODDDNAwas detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703.

Mo1658 Acid-Induced Decrease in Cell Apoptosis Is Mediated by Activation of NADPH Oxidase NOX5-S, NF-κB and Sodd in Barrett's Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H2O2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immunoprecipitation (ChIP) assay showed that SODDDNAwas detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703.

Su1972 Differences in Survival Between Patients With Esophageal Adenocarcinoma and Squamous Cell Carcinoma Are Not Due to Differences in Demographic Factors or Tumor Location

Su1972 Differences in Survival Between Patients With Esophageal Adenocarcinoma and Squamous Cell Carcinoma Are Not Due to Differences in Demographic Factors or Tumor Location

The Metalloprotease ADAMTS8 Displays Antitumor Properties through Antagonizing EGFR–MEK–ERK Signaling and Is Silenced in Carcinomas by CpG Methylation

A disintegrins and metalloproteinases with thrombospondin motifs (ADAMTS) family members have been reported dysregulated in various cancers. Through refining a loss of heterozygosity locus at 11q25 by array-CGH, we identified ADAMTS8 as a novel candidate tumor suppressor gene. Although ADAMTS8 downregulation has been reported in several tumors, its biologic function and underlying mechanism remain largely unknown. Here, we found that ADAMTS8 is broadly expressed in normal tissues but frequently downregulated or silenced by promoter methylation in common carcinoma cell lines, including nasopharyngeal, esophageal squamous cell, gastric, and colorectal carcinomas. Pharmacologic or genetic demethylation restored ADAMTS8 expression, indicating that promoter methylation mediates its silencing. Aberrant methylation of ADAMTS8 was also detected in several types of primary tumors but rarely in normal tissues. Further functional studies showed that restoring ADAMTS8 expression suppressed tumor cell clonogenicity through inducing apoptosis. ADAMTS8 as a secreted protease inhibited epidermal growth factor receptor (EGFR) signaling along with decreased levels of phosphorylated MEK and ERK. We further found that ADAMTS8 disrupted actin stress fiber organization and inhibited tumor cell motility. Thus, our data demonstrate that ADAMTS8 metalloprotease acts as a functional tumor suppressor through antagonizing EGFR-MEK-ERK signaling, in addition to its previously reported anti-angiogenesis function, and is frequently methylated in common tumors. This study uncovers the tumor suppressive function of ADAMTS8, one of the ADAMTS family members, and its frequent methylation in certain tumors could be developed as a potential biomarker.

Comparison of the long-term toxicities conferred by two different doses of definitive chemoradiotherapy for stage II/III esophageal squamous cell carcinoma.

96 Background: Definitive chemoradiotherapy is one of the options for stage II/III esophagealsquamous cell carcinoma (ESCC). RTOG9405 recently reported that a higher dose of radiation did not confer any additional survival benefit over the standard dose (50.4 Gy). We comparedthe long-term toxicities conferred by chemoradiation at a dose of 60 Gy and 50.4 Gy for stage II/III ESCC. Methods: Eligibility criteria included clinical stage II/III (non-T4) (UICC-TNM, 6th edition) ESCC, performance status 0-2, and age 20-75 years. The study group comprised 254 patients who received definitive chemoradiotherapy as first-line therapy between January 2000 and August 2010 in our hospital. Group J (n=207) received 2 cycles of cisplatin (40 mg/m2 on days 1 and 8) with fluorouracil infusion (400 mg/m2/day on days 1-5 and 8-12), or 2 cycles of cisplatin(70 mg/m2 on day 1) with fluorouracil infusion (700 mg/m2/day on days 1-4) and concurrent radiotherapy at 60 Gy. Group R (n = 47) received 2 cycles of cisplatin (75 mg/m2 on day 1) with fluorouracil infusion (1000 mg/m2/day on days 1–4) and concurrent radiotherapy at 50.4 Gy. Long-term toxicity was evaluated according to the Common Terminology Criteria for Adverse Event Ver. 3.0. Results: The characteristics of both groups are as follows (J:R group): median age, 64:63; male/female, 178/29:42/5; PS 0/1/2, 90/104/1:33/14/0; stage IIA/IIB/III: 48/58/101:6/20/21. The median follow-up period was more than 60 months for both groups, with 5-year survival rates of 43.6% and 58.6% for the J and R group, respectively. The proportion of patients with grade 3/4 long-term toxicity in each group was as follows (J/R group): pleural effusion, (8.7%/0%; p = 0.036); pericardial effusion, (6.7%/2.1%; p = 0.196); radiation pneumonitis, (2.4%/4.2%); esophagitis, (0.9%/0%); and pericarditis, (2.4%/0%). Grade 3/4 late toxicity was observed more frequently in the J group (15.0%) than in the R group (6.4%) (p = 0.087). Treatment-related death due to pneumonitis was observed in only 1 patient in group J. Conclusions: The RTOG regimen at a dose of 50.4 Gy showed promising results while inducing lower late toxicity rates than when administered at 60 Gy.

Effect of miR-214 on invasive capacity of esophageal squamous carcinoma cell line Eca109

Objective To investigate the effect of miR-214 on the invasive capacity of esophageal squamous carcinoma cell line Eca109,and to explore the possible molecular mechanism.Methods We prepared miR-214 double-stranded mimic and transfected it into Eca109 cells with Lipofectamine 2000.Eca109 cells transfected with nonsense miRNA mimics were taken as control.The expression of mature miR-214 was determined by qPCR.The capacity of cell invasion was determined by Matrigel-coated Transwell assay.E-cadherin protein expression and the percentage of E-cadherin positive cells were examined by Western blotting analysis and flow cytometry,respectively.Results The expression level of mature miR-214 in the miR-214 mimic transfection group was significantly higher than that in the control group 48 h after transfection(P0.01).Eca109 cells transfected with miR-214 mimic showed a significantly lower cell invasive capacity compared to that of cells transfected with control miRNA mimic(P0.05).Moreover,E-cadherin protein expression and the ratio of E-cadherin positive cells in miR-214 mimic transfection group were both significantly lower than those in the control group(P0.05).Conclusion Our data suggest that miR-214 may inhibit the invasive capacity of esophageal squamous carcinoma cells by repressing the epithelial-mesenchymal transition.

Effect of small interfering RNA targeting ΔNp63α gene on the biological characteristics of human esophageal squamous carcinoma cells

Effect of small interfering RNA targeting ΔNp63α gene on the biological characteristics of human esophageal squamous carcinoma cells

Role of autophagy in esophageal squamous cancer cells induced by proteasome inhibitor

Objective To investigate the role of autophagy on proteasome inhibitor MG132 induced-death of esophageal squamous cancer cells.Methods The esophageal squamous cancer EC9706 cells were treated with 20 μmol/L MG132 and/or 5 mmol/L 3-Methyladenine (3-MA).Flow cytometry was used to examine the apoptosis rate of EC9706 cells treated with Annexin V-FITC/propidium iodide (PI) double staining.The change of acidic vesicle organelles (AVO) was detected by using acridine orange (AO) staining under fluorescence microscope.The expression of autophagy-associated protein Beclin-1 and apoptosis-associated protein cysteinyl aspartate-specific protease (Caspase)-9 was detected by using Western blotting.Results Flow cytometry showed that the apoptosis rate of MG132 group,3-MA group and MG132 +3-MA group was (57.37 ± 0.81) ％,(9.43 ± 0.15) ％ and (79.40 ± 0.92) ％ respectively.The autophagic vacuoles were obviously increased in MG132 group,but the phenomenon was inhibited after combined use of 3-MA.The protein average values of Beclin-1 and Caspase-9 in control group,MG132 group,3-MA group and MG132 +3-MA group were 0.57 ±0.01,0.90 ±0.03,0.49 ±0.14 and 0.65 ±0.01,and 0.61 ±0.05,0.88 ±0.02,0.70 ±0.01 and 0.98 ±0.02,respectively.Conclusion MG132 can induce autophagy and apoptosis in esophageal squamous cancer cells,while 3-MA can promote MG132-induced cell killing effect possibly by upregulating the Caspase 9 expression. Key words: MG132; 3-methyladenine; Autophagy; Esophageal squamous carcinoma

Human papillomavirus promotes esophageal squamous cell carcinoma by regulating DNA methylation and expression of HLA‐DQB1

Esophageal cancer (EC) is one of the most prevalent and deadly cancers worldwide. Along with nutrition, smoking and alcohol consumption, human papillomavirus (HPV) infection is one of the major risk factors, which is modulated by host immune response. This study is aimed at elucidating how HPV modifies host immune system in the EC pathogenesis. The HPV and HLA-DQB1 levels in primary esophageal squamous cell (ESC) cancer cells from Han, Khazak and Uygur patients were analyzed by quantitative real-time PCR and immunoblotting. The ability of HPV16 E6/E7 to transform normal primary ESCs was investigated by infecting ESC with pMSCVpuro-carried E6 or E7. The shRNA against HPV16 E6 or E7 was delivered by adenovirus into esophageal squamous cell carcinoma (ESCC) cells with high HPV content. The DNA methylation level of HLA-DQB1 was measured by methylation-specific PCR. The HLA-DQB1 expression level was correlated with the levels of HPV and inversely related to DNA methylation level of HLA-DQB1. Overexpressing HPV16 E6 or E7 alone was enough to transform normal primary ESCs. However, single knockdown of either E6 or E7 in ESC cancer cells did not reduce HLA-DQB1 expression. Oncogenic HPV E6 and E7 genes promoted ESCC pathogenesis by upregulating susceptible HLA-DQB1 via DNA demethylation.

Multiple primary carcinomas including esophageal basaloid squamous cell carcinoma and gastric adenocarcinoma

Synchronous multiple primary carcinomas refers to 2 or more than 2 kinds of different primary malignant tumors develop synchronously or in 6 months.The incidence of synchronous multiple primary carcinoma is low.A patient with esophageal basaloid squamous cell carcinoma (BSCC) and gastric adenocarcinoma was admitted to the First People's Hospital of Lianyungang in October 2011.The main symptom of this patient was dysphagia,and multiple lesions were found in esophagus,cardia and stomach fundus by gastroscopy respectively.On computed tomography image,eminence lesion in esophageal midpiece and wall thickening from esophagus-cardia to stomach fundus were displayed and were both enhanced slightly by enhancement scanning.The esophageal and cardia tumors were resected via left thoracic approach,and postoperative pathological examination revealed esophageal BSCC and moderately differentiated adenocarcinoma of cardia respectively.Comedo necrosis and red basal membrane material were seen under light microscope.The expressions of cytokeratin 5/6 and P63 were positive,the expression of cytokeratin L was weak positive and the expressions of synaptophysin,chromogranin A and CD117 were negative.The patient suffered from pleural effusion and multiple liver metastases after 4 months follow-up and died of liver metastases in May 2012.Multiple primary carcinomas including esophageal BSCC and gastric adenocarcinoma are rarely seen in clinical practice.Their diagnosis and differential diagnosis mainly depend on histological morphology and immunohistochemical method.Surgical resection combined with postoperative radiotherapy and chemotherapy is selectable,but the prognosis is poor. Key words: Esophageal neoplasms; Gastric neoplasms; Multiple primary carcinomas

Comparison of endoscopic therapies and surgical resection in patients with early esophageal cancer: a population-based study

Outcome data comparing endoscopic eradication therapy (EET) and esophagectomy are limited in patients with early esophageal cancer (EC). To compare overall survival and EC-related mortality in patients with early EC treated with EET and esophagectomy. Population-based study. Patients with early EC (stages T0 and T1) were identified from the Surveillance, Epidemiology, and End Results database (1998-2009). Demographics, tumor specific data, and survival were compared. Cox proportional hazards regression models were used to evaluate the association between treatment and EC-specific mortality. EET and esophagectomy. Mid- (2 years) and long- (5 years) term overall survival and EC-specific mortality, outcomes based on histology and stage, treatment patterns, and predictors of cancer-specific mortality. A total of 430 (21%) and 1586 (79%) patients underwent EET and esophagectomy, respectively. There was no difference in the 2-year (EET: 10.5% vs esophagectomy: 12.7%, P = .27).and 5-year (EET: 36.7% vs esophagectomy: 42.8%, P = .16) EC-related mortality rates between the 2 groups. EET patients had higher mortality rates attributed to non-EC causes (5 years: 46.6% vs 20.6%, P < .001). Similar results were noted when comparisons were limited to patients with stage T0 and T1a disease and esophageal adenocarcinoma. There was no difference in EC-specific mortality in the EET compared with the surgery group (hazard ratio 1.4; 95% confidence interval, 0.9-2.03). Variables associated with mortality were older age, year of diagnosis, radiation therapy, higher stage, and esophageal squamous cell carcinoma. Comorbidities and recurrence rates were not available. This population-based study demonstrates comparable mid- and long-term EC-related mortality in patients with early EC undergoing EET and surgical resection.

Effect of PC cell-derived growth factor RNA interference on biological behavior of esophageal squamous carcinoma cells

To investigate the effect of PC cell-derived growth factor (PCDGF) RNA interference on esophageal squamous carcinoma cells Eca-109 in vitro. The PCDGF-shRNA expression vector was transfected into the Eca-109 cells by liposome. After transfection, the mRNA and protein expressions of PCDGF were detected by RT-PCR and Western-blot respectively. Cell Counting Kit-8 (CCK-8) assay and Boyden chamber method were performed to measure the cell proliferation and invasion ability respectively. The expression levels of PCDGF mRNA and protein were both decreased in Eca-109 cells transfected with PCDGF-shRNA expression vector (transfection group). Twenty-four, 48 and 72 h after transfection, the cells proliferation in the transfection group was inhibited, and the inhibition rate was 20.4%, 21.1% and 20.9% respectively. The cell proliferation activity in the transfection group was significantly lower than that in the non-transfection group, liposome group and negative vector group (all P<0.05). The number of cell migration in the non-transfection group,negative vector group, liposome group and transfection group was 118.8±12.0, 100.8±9.0, 114.3±4.7, and 53.5±16.3 respectively. The differences were statistically significant between the transfection group and the other 3 groups (all P<0.05). PCDGF RNA interference can inhibit the proliferation and invasion abilities of esophageal squamous carcinoma cells in vitro. PCDGF gene may be the new target of gene therapy.

Reduction of E-cadherin by human defensin-5 in esophageal squamous cells

Barrett’s esophagus (BE) is metaplastic columnar epithelium converted from normal squamous epithelia in the distal esophagus that is thought to be a precancerous lesion of esophageal adenocarcinoma. BE is attributed to gastroesophageal reflux disease (GERD), and therefore gastric acid or bile acids are thought to be factors that cause epithelial cell damage and inflammation in the gastro-esophageal junction. The decrease of adherent junction molecules, E-cadherin has been reported to be associated with the progression of the Barrett’s carcinoma, but the initiation of BE is not sufficiently understood. BE is characterized by the presence of goblet cells and occasionally Paneth cells are observed at the base of the crypts. The Paneth cells possess dense granules, in which human antimicrobial peptide human defensin-5 (HD-5) are stored and secreted out of the cells. This study determined the roles of HD-5 produced from metaplastic Paneth cells against adjacent to squamous cells in the gastro-esophageal junction. A human squamous cell line Het-1A, was incubated with the synthetic HD-5 peptide as a model of squamous cell in the gastro-esophageal junctions, and alterations of E-cadherin were investigated. Immunocytochemistry, flowcytometry, and Western blotting showed that the expression of E-cadherin protein was decreased. And a partial recovery from the decrease was observed by treatment with a CD10/neprilysin inhibitor (thiorphan). In conclusion, E-cadherin expression in squamous cells was reduced by HD-5 using in vitro experiments. In gastro-esophageal junction, HD-5 produced from metaplastic Paneth cells may therefore accelerate the initiation of BE.

Pattern of lymph node metastasis in limited-stage primary esophageal small-cell carcinoma and its clinical significance: a preliminary study

Objective To study the pattern of lymph node metastasis (LNM) in limited-stage primary esophageal small-cell carcinoma (PESC) and its guiding significance for clinical target volume delineation in radiotherapy.Methods A retrospective analysis was performed on the clinical data of 21 patients with limited-stage PESC who underwent esophagectomy in our hospital from January 2006 to July 2012 to analyze the rate and degree of LNM and distribution of metastatic lymph nodes.Results The mean number of dissected lymph nodes per patient was 27.9.There were 15 patients who had LNM ;8 patients had dispersed distribution of metastatic lymph nodes,and 7 patients had aggregated distribution of metastatic lymph nodes.The LNM rate was 71.4％,and the LNM degree was 17.2％.The Logistic univariate analysis showed that advanced T stage and long PESC lesion were the risk factors for LNM (P =0.004,P =0.044) and that advanced T stage and angiolymphatic invasion were the risk factors for dispersed distribution of metastatic lymph nodes (P =0.007,P =0.005).Conclusions The rate and degree of LNM are higher in PESC than in esophageal squamous cell carcinoma.Among the patients with limited-stage PESC,38％ have dispersed LNM.More research is recommended to evaluate the distribution of metastatic lymph nodes according to T stage and angiolymphatic invasion and investigate the value of prophylactic irradiation to the lymphatic drainage area of PESC. Key words: Esophageal neoplasms; Lymph node metastases; Clinical target volume; Radiotherapy