Esophageal Squamous Carcinoma Cell Lines Research Articles

Enhancing radiosensitivity of TE1, TE8, and TE 11 esophageal squamous carcinoma cell lines by Hdm2-siRNA targeted gene therapy in vitro.

Introduction: Human double minute2 (hdm2) level increases in most human malignancies. Therefore, inhibition of tumor growth and also induction of radiosensitivity may be provided by hdm2 inhibitors. The effects of hdm2-siRNA on hdm2 protein expression, cell apoptosis rate, and radiosensitivity of human esophageal squamous cell carcinoma (ESCC) were studied. Methods: The hdm2 gene was silenced in TE1, TE8, and TE11 ESCC cell lines using 200nM siRNA by liposomal transfection method followed by irradiation with 0.5, 1, 2, 4, and 6 Gy γ-rays in vitro. The gene expression levels were evaluated by real time PCR and Western Blotting methods. MTT, TUNEL, and also colony forming assays were used to compare the radiosensitivity of the cell lines before and after the treatments. Results: Hdm2-siRNA reduced the hdm2 protein as compared to the vehicle control and scrambled groups, and also increased the radiation-induced apoptosis especially in TE11 cells. The related dose reduction factors (DRFs) for the silenced TE1, TE8, and TE11 cells calculated to be 1.20, 1.30, and 2.75, respectively. Conclusion: Increasing radiosensitivity of tumor cells may be provided by silencing the oncogenes.

Transketolase Serves a Poor Prognosticator in Esophageal Cancer by Promoting Cell Invasion via Epithelial-Mesenchymal Transition.

Background: To characterize the potential function and clinical significance of Transketolase (TKT) in esophageal cancer.Methods: High invasive esophageal squamous cell carcinoma (ESCC) cell line CE48T/VGH was used. Cellular functions in response to TKT modulation were examined, including cell growth, migration and invasion. The underlying molecules involved in the TKT regulatory mechanism were determined by western blot and confocal microscopic analysis. Clinically, TKT expressions in 76 ESCC patients were assessed by immunohistochemical (IHC) method, and the association with treatment outcome was determined.Results: TKT silencing inhibited cell migration and invasion but had a minimal effect on cell growth. This TKT silencing also induced the reversion of epithelial-mesenchymal transition (EMT), as evidenced by the spindle to cuboidal morphological change, increased the expression of epithelial markers (γ-catenin), and decreased the levels of mesenchymal markers (fibronectin and N-cadherin). Mechanically, TKT was shown to modulate the EMT through the pERK-Slug/Snail-associated signaling pathway. Clinically, a high level of TKT in the cancer tissues of patients with esophageal squamous cell carcinoma was associated with poor survival (P = 0.042). In the multivariate analysis, a high TKT level was also shown to be an independent unfavorable prognostic factor (Odds ratio: 1.827, 95% confidence interval: 1.045-3.196, P = 0.035).Conclusions: TKT contributes to esophageal cancer by promoting cell invasion via meditating EMT process. Clinically, the over-expression of TKT in ESCC patients predicts poorer survival. TKT inhibition may be a useful strategy to intervene in cancer cell invasion and metastasis, which may lead to better prognosis for ESCC patients.

肉桂酸羽扇烷醇酯的合成及其抗癌活性

以N,N'-二环己基碳二亚胺(DCC)为缩合剂，4-二甲氨基吡啶(DMAP)为催化剂，羽扇烷醇和肉桂酸在二氯甲烷中室温反应24 h，合成了新化合物肉桂酸羽扇烷醇酯，产率为81.6%。研究了肉桂酸羽扇烷醇酯体外对食管鳞癌细胞Eca-109、TE-1和EC-9706增殖的抑制活性。结果表明肉桂酸羽扇烷醇酯对上述3株实验癌细胞的增殖均有良好的抑制活性，其抗癌活性与阿霉素相近。 Lupanol was reacted with cinnamic acid for 24 h in dichloromethane at room temperature to give lupanol cinnamate at 81.6% yield in the presence of N,N'-dicyclohexylcarbodiimide (DDC) as dehydrating agent and 4-dimethylaminopyridine (DMAP) as catalyst. It was evaluated for in vitro antitumor activities against the human esophageal squamous cell carcinoma cell lines Eca- 109, TE-1 and EC-9706. The results showed that lupanol cinnamate had good inhibitory activities against the above three tested cancer cell lines and its anticancer activities were near to those of adriamycin.

As a Novel Prognostic Marker, Cysteine/histidine-rich 1 (CYHR1) is a Therapeutic Target in Patients with Esophageal Squamous Cell Carcinoma.

Cysteine/histidine-rich 1 (CYHR1) was first discovered in a yeast two-hybrid screen with murine galectin-3, and no previous reports have described a relationship between the CYHR1 gene and human cancer. The current study evaluated the role and significance of CYHR1 in esophageal cancer. The human esophageal squamous cell carcinoma (ESCC) cell line TE-8 and the CYHR1 knock-down cell line TE-8/small interfering (si)-CYHR1 were used for in vitro and in vivo assays. For clinical study, ESCC tissues (n=104) were used. Compared with parental cells, TE-8/si-CYHR1 cells had suppressed proliferation and invasion activities. In the in vivo assay, the tumors from TE-8 cells treated with si-CYHR1 had abrogated tumorigenicity. In the clinical study, the expression of CYHR1 mRNA was associated with lymph node metastasis and stage and shown to be an independent prognostic factor. As the findings show, CYHR1 may represent not only a valuable prognostic marker but also a therapeutic target for ESCC patients.

Study on chemotherapeutic sensitizing effect of nimotuzumab on different human esophageal squamous carcinoma cells.

Esophageal cancer is one of the leading causes of mortality worldwide. Although, surgery, radio- and chemotherapy are used to treat the disease, the identification of new drugs is crucial to increase the curative effect. The aim of the present study was to examine the chemotherapeutic sensitizing effect of nimotuzumab (h-R3) and cisplatin cytotoxic drugs cisplatin (DDP) and 5-fluorouracil (5-FU) on esophageal carcinoma cells with two different epidermal growth factor receptor (EGFR) expressions. The expression of EGFR was detected in the human EC1 or EC9706 esophageal squamous cell carcinoma cell line using immunohistochemistry. The inhibitory effect of DDP and 5-FU alone or combined with h-R3 on EC1 or EC9706 cell proliferation was detected using an MTT assay. Flow cytometry and the TUNEL assay were used to determine the effect of single or combined drug treatment on cell apoptosis. The results showed that the expression of EGFR was low in EC1 cells but high in EC9706 cells. The inhibitory effect of the single use of h-R3 on EC1 or EC9706 cell proliferation was decreased. The inhibitory effect between single use of h-R3 alone and combined use of the chemotherapy drugs showed no statistically significant difference (P>0.05) on the EC1 cell growth rate, but showed a statistically significant difference (a=0.05) on EC9706 cell growth rate. The results detected by flow cytometry and TUNEL assay showed that the difference between single use of h-R3 alone and the control group was statistically significant with regard to the EC1 apoptosis rate effect (P<0.05), but not statistically significant for EC9706 (P>0.05). However, statistically significant differences were identified in the apoptotic rate of EC9706 cells between the h-R3 combined chemotherapy group and single chemotherapy group (P<0.05), but not on in the EC1 chemotherapy group (P>0.05). In conclusion, the sensitization effect of h-R3 on chemotherapy drugs is associated with the expression level of EGFR in EC1 or EC9706 cells. The cell killing effect of the combined use of h-R3 with DDP and 5-FU showed no obvious synergistic effect compared to the single-drug group, but only an additive effect.

Sensitivity of esophageal squamous cell carcinoma cells to rapamycin can be improved by siRNA-interfered expression of p70S6K

To explore the differences in sensitivity to rapamycin of five esophageal squamous cell carcinoma cell lines with different differentiation and the changes of sensitivity of the cells after siRNA-interfered expression of p70S6K. Effects of rapamycin on proliferation of ESCC cell lines with different differentiation, EC9706, TE-1, Eca109, KYSE790 and KYSE450 cells, were investigated using cell counting kit 8 (CCK-8) assay, and according to the above results, the EC9706 cells non-sensitive to rapamycin were chosen to be transfected with p70S6K-siRNA. The changes in sensitivity of cells to rapamycin were measured in vitro and in vivo using CCK-8 kit, flow cytometry and tumor formation in nude mice. CCK-8 results showed that all the five cell line cells were sensitive to low concentration of rapamycin (<100 nmol/L), but TE-1 and EC9706 cells, which were with poor differentiation, showed resistance to high concentration of rapamycin. After EC9706 cells were treated with 50, 100, 200, 500 and 1 000 nmol/L rapamycin and p70S6K-siRNA, the proliferation rates of EC9706 cells were (48.67 ± 1.68)%, (15.45 ± 1.54)%, (14.00 ± 0.91)%, (10.97 ± 0.72)% and (2.70 ± 0.32)%, respectively, and were significantly lower than that of cells treated with 50, 100, 200, 500 and 1 000 nmol/L rapamycin and control siRNA [(74.53 ± 1.71)%, (68.27 ± 1.35)%, (71.74 ± 2.44)%, (76.23 ± 1.02)% and (80.21 ± 2.77)%] (P<0.05 for all). The results of flow cytometry showed that the ratios of cells in G1 phase of the p70S6K-siRNA, rapamycin and p70S6K-siRNA+ rapamycin groups were (53.82 ± 1.78)%, (57.87 ± 4.01)% and (73.73 ± 3.68)%, respectively, significantly higher than that in the control group (46.09 ± 2.31)% (P<0.05 for all). The results of tumor formation test in vivo showed that the inhibitory effect of rapamycin on tumor growth was stronger after the cells were transfected with p70S6K-siRNA, and the inhibition rate was 96.5%. ESCC cells with different differentiation have different sensitivity to rapamycin, and p70S6K-siRNA can improve the sensitivity of cells to rapamycin in vitro and in vivo.

Sunitinib modulates the radiosensitivity of esophageal squamous cell carcinoma cells in vitro.

This study aims to explore the radiosensitivity of sunitinib on esophageal cancer cell lines. For in vitro studies, human esophageal squamous cell carcinoma (ESCC) cell lines were treated with sunitinib 24 hours before irradiation. ESCC cell lines were treated with sunitinib with or without radiation. Cell proliferation was detected by Cell Counting Kit 8 assay. Radiosensitization was evaluated by clonogenic survival assay. Cell apoptosis and cell cycle analysis were detected by flow cytometry. Deoxyribonucleic acid (DNA) double-strand breaks were performed by immunocytofluorescence analysis. Western blot analysis was used to determine the effect of sunitinib on radiation induced signal transduction. Sunitinib potently sensitized ESCC cells to radiation with a sensitization enhancement ratio of 1.13-1.72. Furthermore, sunitinib increased radiation induced DNA double-strand breaks, promoted the apoptosis of ESCC cells and induced the G2/M arrest. Radiosensitization was accompanied with enhanced apoptosis and regulated by the intrinsic pathway of apoptosis. Sunitinib sensitized ESCC cells to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in esophageal cancer.

Endostatin combined with radiotherapy suppresses vasculogenic mimicry formation through inhibition of epithelial-mesenchymal transition in esophageal cancer.

The growth of solid tumors requires angiogenesis to provide oxygen and nutrients and to support cell proliferation. The switch from an avascular to a vascular phenotype is typically related to acceleration of tumor growth. Anti-angiogenic therapy is becoming a very promising way for malignant tumors. Meanwhile, malignant tumor cells themselves were able to develop the formation of cell-lined vessels that contribute to tumor neovascularization and supply the nutrients and oxygen, which is called vasculogenic mimicry (VM). However, the molecular mechanism of VM remains unclear. The purpose of this study was to investigate the efficacy of the novel recombinant human endostatin (rh-Endo) protein combined with radiotherapy on human esophageal squamous cell carcinoma (ESCC) cell lines Eca-109 and TE13. Our results showed that rh-Endo combined with radiotherapy significantly inhibited the proliferation, migration, invasion, and VM of human esophageal cancer cells in a dose-dependent manner; however, it has no direct effect on apoptosis of carcinoma cells, which indicated that rh-Endo combined with radiotherapy significantly changed the microenvironment of esophageal carcinoma, and played an important role in preventing distant metastasis. Our findings suggested that rh-Endo inhibited the metastasis of esophageal cancer and the activation of AKT pathway, and the down-regulation of epithelial-mesenchymal transition (EMT) may be associated with such effect of rh-Endo. These results also supported the bright prospect of rh-Endo combined with radiotherapy for clinical applications in the future.

Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method

Cross-contamination during or early after establishment of a new cell line could result in the worldwide spread of a misidentified cell line. Therefore, newly established cell lines need to be authenticated by a reference standard method. This study was conducted to investigate the authenticity of a newly established epithelial cell line of human esophageal squamous cell carcinoma (ESCC) called YM-1 using short tandem repeat (STR) DNA profiling method. Primary human ESCC epithelial cells were cultured from the fresh tumor tissue of an adult female patient. Growth characteristics and epithelial originality of YM-1 cells were studied. Genomic DNA was isolated from YM-1 cells harvested at passage 22 and ESCC donor tumor sample on two different days to prevent probable DNA contamination. STR profiling was performed using AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. To address whether YM-1 cells undergo genetic alteration as the passage number increases, STR profiling was performed again on harvested cells at passage 51. YM-1 cells grew as a monolayer with a population doubling time of 40.66h. Epithelial originality of YM-1 cells was confirmed using ICC/IF staining of cytokeratins AE1/AE3. The STR profile of the ESCC donor tumor sample was the same with YM-1 cells at passage 22. However, STR profile of the donor tumor sample showed an off-ladder (OL) allele in their D7S820 locus. Also, re-profiling of YM-1 cells at passage 51 showed a loss of heterozygosity (LOH) at D18S51 locus. This suggests that long-term culture of cell lines may alter their DNA profile. Comparison of the DNA fingerprinting results in DSMZ, and ATCC STR profiling databases confirmed unique identity of YM-1 cell line. This study provides an easy, fast, and reliable procedure for authentication of newly established cell lines, which helps in preventing the spread of misidentified cells and improving the reproducibility and validity of experiments, consequently.

Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines.

Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol-reactive and readily complex copper. More recently DSF and copper-DSF (Cu-DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti-diabetic drug metformin is anti-proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu-DSF and DSF, with and without metformin, in this present study. We found that Cu-DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin-DSF-induced cytotoxicity since the cell-impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin-treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid-labile compound decreased lysosomal acidification, and DSF-metformin co-treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC.

Abstract 1669: Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines

Abstract Background: Epidermal growth factor receptor (EGFR) is a well-validated oncology target molecule for monoclonal antibody therapies. Sym004 is a novel anti-EGFR antibody mixture that consists of two recombinant IgG1 antibodies against domain III of the EGFR. The primary mechanism of action of Sym004 is thought to be EGFR cross-linking, internalization and degradation of the EGFR from the cell surface. EGFR is highly expressed in the overwhelming majority of esophageal squamous cell carcinoma (ESCC). We, therefore, investigated the effect of Sym004 in human ESCC cell lines in the present study. Method: In this preclinical study, approximately 50 ESCC cell lines and 3 kinds of anti-EGFR antibodies (Sym004, cetuximab, and panitumumab) were used. According to protein expression status and gene mutation status analyzed by Western blotting and next generation sequencing, these ESCC cell lines were classified into several groups. High EGFR-expressing cell lines were used for further investigations. Internalization of anti-EGFR antibodies into ESCC cells and inhibition of the EGFR signaling cascade by anti-EGFR antibodies were studied in in vitro experiments. Growth inhibitions by anti-EGFR antibody treatment were investigated in in vitro and in vivo experiments. The present preclinical studies were conducted at the National Cancer Center East Japan as collaborative studies with Merck Serono. Result: Even though protein expression of EGFR was detected in all ESCC cell lines, a variety of the expression levels were seen among the cell lines consistent with the previous data of clinical specimens. In addition, following gene alterations were found: EGFR amplification, Ras mutation, and PIK3CA mutation, and their frequency was 17% (8/48), 6.3% (3/48) and 8.3% (4/48), respectively. More effective internalization of Sym004 was observed than cetuximab and panitumumab in the high EGFR-expression cell lines. In a growth inhibition assay, more potent anti-proliferative activity of Sym004 was observed than those of other anti-EGFR antibodies in some cell lines. No obvious correlation between EGFR expression and the anti-proliferative activity was observed, although cell lines with higher EGFR expression showed a trend toward higher sensitivity to Sym004. In vivo xenograft studies of Sym004 using human ESCC cell lines are currently underway and will be presented in the annual meeting. Conclusions: These studies showed that Sym004 exhibited antitumor activity in some ESCC cell lines in preclinical settings, and are expected to provide a scientific rationale along with a possible clinical indication of ESCC in clinical setting. Citation Format: Shota Fukuoka, Takashi Kojima, Yoshikatsu Koga, Mayumi Yamauchi, Rie Komatsuzaki, Kazuhiko Aoyagi, Hiroki Sasaki, Masahiro Yasunaga, Yasuhiro Matsumura, Toshio Kuronita, Toshihiko Doi, Atsushi Ohtsu. Preclinical efficacy of Sym004, novel anti-EGFR antibody, in esophageal squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2015-1669

Abstract 2808: Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450

Abstract Esophageal squamous carcinoma (ESCC) may be developed through a progressive sequence from mild to severe dysplasia, carcinoma in situ, and finally, invasive carcinoma. Chemoprevention can block or weaken the influence of development. Recent study has shown luteolin, a bioflavonoid, possesses anti-inflammatory, antioxidant, and anti-proliferative effects, and it might have a preventive effect in this progress. In this study, we focused on the effect of luteolin on cell cycle regulation in human Esophageal Squamous Carcinoma Cell Line EC1 and KYSE450 Cells in vitro and its potential mechanisms. Observations by flow cytometer showed that luteolin inhibited cell cycle progression at G2/M phase in a dose- and time-dependent manner. We also found that luteolin could induce cell apoptosis via decreasing activation of caspase-3 and down-regulation of mitochondrial membrane potential. Western blot results showed the protein expression of cycle related protein CyclinD1 and apoptosis related proteins caspase-3, caspase-9, and Bak were also significantly decreased in luteolin treated cells compared with the non-luteolin treated cells. Our results suggest that the proper use of luteolin might be a practical approach to the prevention of esophageal carcinoma via the inhibition of cell proliferation and other potential high risk regions for this disease. Citation Format: Ping Chen, Tao Hu, Yane Ma, Xiaoyu Chen, Liping Dai, Ningjing Lei, Ziming Dong, Pei Li. Luteolin inhibits cell proliferation and induces cell apoptosis via down-regulation of mitochondrial membrane potential in esophageal carcinoma cells EC1 and KYSE450. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2808. doi:10.1158/1538-7445.AM2015-2808

Abstract 2694: Esophageal cancer exhibits a resistance to the chemical inhibition of IGF-1R with a maintained Ras-MAPK activity

Abstract The type 1 insulin-like growth factor receptor (IGF-1R) and its associated signaling system play a significant role in carcinogenesis and progression of gastrointestinal malignancies, and thus have provoked great interest as a promising target for cancer treatment. The aim of our study was to assess the effects of a novel IGF-1R inhibitor, NVP-AEW541, on the cell proliferation and signal transduction of esophageal squamous cell carcinoma. Five human esophageal squamous carcinoma cell lines (TE-1, TE-4, TE-8, TE-10 and T.Tn) were treated with this inhibitor and the IC50 of NVP-AEW541 for each cell line was more than 1μM, exhibiting that these cells were less sensitive to this compound. The activation of IGF-1R and AKT were dose dependently blocked by NVP-AEW541. However, the activities of the key molecules in Ras-MAPK pathway was not significantly inhibited by NVP-AEW541 without any major mutation of Ras. These results indicate that esophageal squamous cell carcinoma may be resistant to targeting IGF-IR due to its maintenance of Ras-MAPK signaling. Thus, to explore the potential mechanism of resistance will contribute to the therapeutic application for NVP-AEW541. Citation Format: Munenori Takaoka, XiaoHong Bao, Huifang Hao, Naomasa Ishida, Takuya Fukazawa, Tomoki Yamatsuji, Minoru Haisa, Yoshio Naomoto. Esophageal cancer exhibits a resistance to the chemical inhibition of IGF-1R with a maintained Ras-MAPK activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2015-2694

Abstract 173: SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells

Abstract Tumor metastasis is responsible for 90% of cancer-related deaths. A strong link between microRNA dysregulation and human cancers has recently been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear. We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western Blotting using overexpression and shRNA knock-down approaches. Here we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells compared to non-invasive cell lines, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3′-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues. Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas. Citation Format: Rainelli B. Koumangoye, Thomas Andl, Kenneth J. Taubenslag, Steven T. Zilberman, Chase Taylor, Claudia D. Andl. SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2015-173

Abstract 1757: The new molecular target therapy of Hsp90 inhibition in esophageal squamous cell carcinoma

Abstract Heat shock protein 90 (HSP90) has numerous cellular functions, including promotion of proper protein folding, and stabilizes a number of oncogenic proteins, many of which are overexpressed in cancers. The objective of this study is to evaluate an antitumor effect of HSP90 inhibitor, AUY922, in esophageal squamous cell carcinoma(ESCC) cell lines. ESCC cell lines were treated with AUY922 to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. AUY922 inhibited cell proliferation in ESCC cells in a dose- and time-dependent manner. Growth inhibitory effect of AUY922 for ESCC cells was higher than CDDP. With flowcytometry and immunostaining, we showed that AUY922 induced apoptosis in ESCC cells and inhibited chemotaxis of ESCC cells. These antitumor effects of AUY922 were due to inhibition of cell proliferation through suppression of phosphorylation of the client proteins. The HSP90 inhibitor is useful as an antitumor drug in ESCC by inhibiting proliferation of ESCC cells through novel mechanism. Citation Format: Masahiro Yamamura, Tsutomu Nohno, Akira Yamauchi, Naoki Katase, Makoto Okawaki, Akira Sawaki, Hideo Matsumoto, Toshihiro Hirai, Yoshiyuki Yamaguchi. The new molecular target therapy of Hsp90 inhibition in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2015-1757

Abstract 5126: Establishment of an esophageal small cell carcinoma cell line (TYUC-1)

Abstract Background: Small cell carcinoma of the esophagus is rare and its prognosis is extremely poor. However, there is currently no in vitro model of this disease. Methods: After obtaining written informed consent, we obtained a tumor from a 56-year-old female patient with esophageal small cell carcinoma in 2009 and transplanted it into a Severe Combined Immuno Deficiency (SCID) mouse. After successful transplantation, a tumor from the back of this mouse was minced with a sharp scissors without enzymes and was cultured in Ham F12/DMDM supplemented with 5% fetal bovine serum. Results: Culture cells grew as adherent cells during the initial culture phase and gradually grew as floating aggregated cells. We carefully maintained these floating cells and transferred them without enzymes. These cells could passage to another flask with a split ratio of 1/10. We named these cells “TYUC-1”, and passaged them more than 85 times. The population doubling-time of TYUC-1 was approximately 30 hours. The G-band karyotype confirmed that these cells had a human origin. These cells could be transplanted to SCID mice with the same histological features of primary esophageal small cell carcinoma, and were shown to express TrkA, NGF, NSE, FGFRL1, p53, CD56, ALDH1A1, and CD44, but not CD133. A cDNA array also revealed that 9 genes were overexpressed while the expression of 33 genes was significantly lower than that in 28 esophageal squamous cell carcinoma (ESCC) cell lines. Whole exome sequencing revealed that TYUC-1 had TP53, PIK3CA, KMT2D, CACNA1H, NCOR1 and HRAS mutations. Furthermore, TYUC-1 had mutations in 128 genes, whereas 28 ESCC cell lines had no mutations in these genes. Conclusions: We newly established TYUC-1 cells that exhibited neuroendocrine characteristics and expressed several stem cell-related molecules. These cells may be useful for understanding the characteristics of esophageal small cell carcinoma. Citation Format: Yutaka Shimada, Tomoyuki Okumura, Yoshinori Takei, Kazuaki Watanabe, Akira Hirasawa, Arito Yamane, Masahiko Nishiyama, Takuya Nagata, Kazuhiro Tsukada, Kazuharu Shimizu. Establishment of an esophageal small cell carcinoma cell line (TYUC-1). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2015-5126

Abstract 4950: Defective expression of partition-defective 3 (PAR-3) is associated with adverse prognostic factors in esophageal squamous cell carcinoma

Abstract Esophageal cancer is the sixth leading cause of cancer mortality and squamous cell carcinoma is a prevalent histological subtype worldwide. The partition-defective 3 (PAR-3) protein belongs to a partition defective complex that controls polarity, which is mainly composed of PAR-3, PAR-6, and atypical protein kinase C (aPKC). PAR-3 contains one self-oligomerization domain in the N-terminus, three PDZ protein interaction domains and one aPKC-binding domain. Owing to these domains, PAR-3 is able to form a conserved protein complex with PAR-6 and aPKC. In mammalian epithelial cells, PAR complex assembles at tight junctions, and plays a role in controlling apical-basal polarity, asymmetric cell division, and directional cell migration. It is well known that disrupted cell polarity is a feature of epithelial cancers. Therefore, it is believed that PAR proteins may be involved in multiple aspects of oncogenesis and tumor-promoting function. We previously detected a homozygous deletion of PAR-3 gene in esophageal squamous cell carcinoma (ESCC) cell lines by a high-density oligonucleotide microarray approach. (Oncogene 28:2910-2918, 2009). A copy number loss of PAR-3 gene was observed in 15% of primary ESCC tumors. The expression of PAR-3 mRNA was significantly reduced in 70% of ESCC tumors when compared with their nontumorous tissue counterparts. Exogenous expression of PAR-3 gene in ESCC cells lacking this gene (KYSE30 cells) enhanced the recruitment of Zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PAR-3 gene in ESCC cells expressing this gene disrupted ZO-1 localization at cell-cell borders. Although our previous findings suggested that deletion and reduced expression of PAR-3 gene may be a novel mechanism that drives the progression of ESCC, the clinical relevance of PAR-3 in ESCC tumor progression was not known. In the present study, we aimed to investigate the expression pattern of PAR-3 protein in primary ESCC tumors and to clarify any potential relationship between the PAR-3 expression and clinicopathological parameters. Immunohistochemical analyses showed that PAR-3 protein was expressed at the basal layer of the normal squamous epithelium of the esophagus, esophageal glands proper and the duct of esophageal glands. Both normal esophageal cells and ESCC tumor cells showed granular cytoplasmic staining and particularly strong membranous staining of PAR-3 protein. PAR-3 protein expression was lost in 20 (37%) of 74 primary ESCC tumors. Loss of PAR-3 protein expression was significantly associated with invasion depth (P = 0.0065) and lymph node metastasis (P = 0.03). Our results suggest that loss of PAR-3 protein expression may lead to tumor progression and subsequent lymph node metastasis in ESCC. Citation Format: Tomoko Kitaichi, Koichiroh Yasui, Akira Tomie, Yasuyuki Gen, Osamu Dohi, Yuji Naito, Yoshito Itoh. Defective expression of partition-defective 3 (PAR-3) is associated with adverse prognostic factors in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4950. doi:10.1158/1538-7445.AM2015-4950

RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.

Clinical and biological significance of stem-like CD133+CXCR4+ cells in esophageal squamous cell carcinoma

ObjectivesEsophageal squamous cell carcinoma is one of the most frequent malignant tumors. Cancer stem cells are considered to be responsible for tumor growth, metastasis, and recurrence. Cluster of differentiation 133 (CD133) and C-X-C chemokine receptor type 4 (CXCR4) are frequently applied markers for the identification and isolation of cancer stem cells. However, few studies have investigated the coexpression of CD133 and CXCR4 in esophageal squamous cell carcinoma. This study aims to explore the clinical and biological role of stem-like CD133+CXCR4+ cells in esophageal squamous cell carcinoma. MethodsImmunohistochemical staining was performed to detect the expression of CD133 and CXCR4 in esophageal squamous cell carcinoma tissues of patients. Flow cytometry and fluorescence-activated cell sorting were applied to analyze and isolate each subgroup in esophageal squamous cell carcinoma cell line TE-1. The characteristic differences between each subgroup were assayed in vitro. The association between CD133/CXCR4 expression and patients' prognosis was analyzed by Kaplan–Meier and Cox regression. ResultsAmong 154 patient tissues, concomitant high CD133-CXCR4 expression accounts for 20.78% (32/154). In vitro, CXCR4+ cells (CD133+CXCR4+ and CD133−CXCR4+) showed high invasive potential and CD133+CXCR4+ cells showed high proliferative capacity. Clinically, patients with concomitant high CD133-CXCR4 expression had decreased disease-free survival and overall survival (P < .01). ConclusionsEsophageal squamous cell carcinoma cells coexpressing CD133 and CXCR4 possess the characteristics of cancer stem cells. The concomitant high CD133-CXCR4 expression might be a novel marker for predicting the poor prognosis of patients with esophageal squamous cell carcinoma, and CD133 and CXCR4 may serve as potential therapeutic targets.

Overexpression of HSP47 in esophageal squamous cell carcinoma: clinical implications and functional analysis.

Several biomarkers of esophageal squamous cell carcinoma (ESCC) have been explored to improve the prognosis of this disease. One of these, the 47-kDa heat shock protein (HSP47), has been screened as a potential biomarker by genomic profiling and is known to be overexpressed in some malignant diseases. In this study, we explored the role and evaluated the prognostic value of HSP47 expression in ESCC. The function of this protein was analyzed by assaying proliferation, wound healing, and colony formation in an HSP47-knockdown ESCC line. The prognostic implication of HSP47 expression was analyzed by immunohistochemical staining in 157 surgical specimens. HSP47 expression level and other clinical variables were analyzed using multivariate Cox proportional hazards models. Silencing of the HSP47 gene in the ESCC cell line inhibited cell proliferation and colony formation. HSP47 was highly expressed in ESCC tissue samples, compared with normal esophageal tissues. The level of immunohistochemical staining of HSP47 and pathologic stage were significantly correlated with overall and recurrence-free survival, as shown by multivariate analysis (P = 0.014 and 0.044, respectively). We found that overexpression of HSP47 is associated with poor prognosis in patients with ESCC and that this is consistent with the function of HSP47 in terms of increased cell proliferation and colony formation. These results suggest that HSP47 is a potential prognostic biomarker for ESCC and merits further research for novel diagnostic and therapeutic applications.