ESCC Cell Research Articles

Antitumor effect of metformin in esophageal cancer: In vitro study

Recent studies suggest that metformin, which is a member of the biguanide family and commonly used as an oral anti-hyperglycemic agent, may reduce cancer risk and improve prognosis of numerous types of cancer. However, the mechanisms underlying the antitumor effect of metformin on esophageal cancer remain unknown. The goal of the present study was to evaluate the effects of metformin on the proliferation of human ESCC in vitro, and to study changes in the expression profile of microRNAs (miRNAs), since miRNAs have previously been associated with the antitumor effects of metformin in other human cancers. The human ESCC cell lines T.T, KYSE30 and KYSE70 were used to study the effects of metformin on human ESCC in vitro. In addition, we used miRNA array tips to explore the differences between miRNAs in KYSE30 cells with and without metformin treatment. Metformin inhibited the proliferation of T.T, KYSE30 and KYSE70 cells in vitro. Metformin blocked the cell cycle in G0/G1 in vitro. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, as well as decreases in cyclin-dependent kinase (Cdk)4, Cdk6 and phosphorylated retinoblastoma protein (Rb). In addition, the expression of miRNAs was markedly altered with the treatment of metformin in vitro. Metformin inhibited the growth of three ESCC cell lines, and this inhibition may have involved reductions in cyclin D1, Cdk4 and Cdk6.

Rab25 Is a Tumor Suppressor Gene with Antiangiogenic and Anti-Invasive Activities in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is a devastating disease characterized by distinctly high incidences and mortality rates. However, there remains limited understanding of molecular events leading to development and progression of the disease, which are of paramount importance to defining biomarkers for diagnosis, prognosis, and personalized treatment. By high-throughout transcriptome sequence profiling of nontumor and ESCC clinical samples, we identified a subset of significantly differentially expressed genes involved in integrin signaling. The Rab25 gene implicated in endocytic recycling of integrins was the only gene in this group significantly downregulated, and its downregulation was confirmed as a frequent event in a second larger cohort of ESCC tumor specimens by quantitative real-time PCR and immunohistochemical analyses. Reduced expression of Rab25 correlated with decreased overall survival and was also documented in ESCC cell lines compared with pooled normal tissues. Demethylation treatment and bisulfite genomic sequencing analyses revealed that downregulation of Rab25 expression in both ESCC cell lines and clinical samples was associated with promoter hypermethylation. Functional studies using lentiviral-based overexpression and suppression systems lent direct support of Rab25 to function as an important tumor suppressor with both anti-invasive and -angiogenic abilities, through a deregulated FAK-Raf-MEK1/2-ERK signaling pathway. Further characterization of Rab25 may provide a prognostic biomarker for ESCC outcome prediction and a novel therapeutic target in ESCC treatment.

RPN2 expression predicts response to docetaxel in oesophageal squamous cell carcinoma

Background:Neoadjuvant chemotherapy – often using docetaxel in various combinatorial regimens – is a standard treatment choice for advanced oesophageal squamous cell carcinoma (ESCC) in Japan. However, no useful markers exist that predict docetaxel’s effects on ESCC. Ribophorin II (RPN2) silencing, which reduces glycosylation of P-glycoproteins and decreases membrane localisation, promotes docetaxel-dependent apoptosis. We investigated whether RPN2 expression in ESCC biopsy specimens could be a predictive biomarker in docetaxel-based neoadjuvant chemotherapy.Methods:We evaluated RPN2 expression immunohistochemically in biopsy specimens from 79 patients with node-positive ESCC, who received docetaxel-based adjuvant chemotherapy, and compared clinical and pathological responses between the RPN2-positive and RPN2-negative groups. We also studied susceptibility of RPN2-suppressed ESCC cells to docetaxel.Results:The RPN2-negative group had better clinical and pathological responses to docetaxel than the RPN2-positive group. We also found RPN2 suppression to alter docetaxel susceptibility in vitro.Conclusion:Expression of RPN2 in biopsy specimens could be a useful predictive marker for response to docetaxel-based neoadjuvant chemotherapy in ESCC.

P-0004 RNAI-Mediated Silencing of NRF2 Gene Expression in Esophageal Squmous Cell Carcinoma Inhibits Tumor Growth and Increases Efficacy of Chemotherapy

ABSTRACT Introduction NF-E2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. Our previous study found that Nrf2 was over-expression and closely correlated with the lymph node metastasis. Based on our previous results, we investigated the role of Nrf2 in cell proliferation and resistance to anticancer drugs in esophageal squamous cell carcinoma. Methods Human ESCC cell lines Eca-109, Ec-9706, and TE-1 were selected for this study. We first detect the expression and sub-location of Nrf2 by western blot, Real-time PCR and immunofluorescent. Then, the resistance to cisplatin of these three cells was examined by MTT and flow cytometry. Last, effects of Nrf2 on cell growth, cycle, apoptosis, and resistance to cisplatin were determined by inhibition of gene expression of Nrf2 by specific small interfering RNA (Nrf2-siRNA). Results It was found that all of these cells had high expression levels of Nrf2 in mRNA and protein levels, but western bolt and immunofluorescent revealed the distribution of Nrf2 in nucleus or cytoplasm in different cells. Eca-109, with highest nuclear expression of Nrf2, showed higher resistance to cisplatin compared with Ec-9706 and TE-1 cells. Eca-109 was chose for following experiment. By Nrf2-siRNA, the cell proliferation and resistance to cisplatin were both inhibited. Further, the cell apoptosis after treatment with cisplatin was significantly elevated by Nrf2-siRNA, but Knockdown of Nrf2 had no effect on cell cycle. Conclusion These results indicate that Nrf2 is essential for both tumor cell growth and resistance to anticancer drugs in esophageal squamous cell carcinoma. Therefore, Nrf2 is supposed to be a potential target to increase efficacy of chemotherapy.

Overexpression of Aurora-A Enhances Invasion and Matrix Metalloproteinase-2 Expression in Esophageal Squamous Cell Carcinoma Cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.

Abstract 70: Loss of cellular senescence checkpoint functions reveals the oncogene characteristics of Notch1 in squamous cell carcinomas

Abstract Introduction: Notch signaling regulates cell fates that are dependent upon the CSL transcription factor. Both tumor suppressor and oncogenic roles of Notch have been implicated in the pathogenesis of squamous cell carcinomas (SCCs). We investigated the functional consequences of Notch activation in head and neck as well as esophageal SCCs (HNSCC and ESCC). Methods: Primary tumor tissues annotated with known clinical outcomes were analyzed. Human esophageal cells immortalized with telomerase or human papilloma virus E6/E7 genes, their derivatives expressing mutant p53 and ESCC cell lines were stably transduced with ICN1, an active form of Notch1 in a regulatable manner (Tet-On system). Notch was inhibited by dominant negative mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor or γ-secretase inhibitors (GSI). 8xCSL-luciferase reporter was used to assess Notch-mediated transcriptional activity. RNA interference was done targeting either CSL or p16INK4A. Senescence was determined by cell growth inhibition and senescence-associated β-galactosidase assays. Cell growth was tested in soft agar and immunodeficient mice. Gene expression was determined by quantitative RT-PCR, Western blotting and immunohistochemistry (IHC). Results: In primary tumors, the active form of Notch1 (ICN1Val1744) was detected as intense nuclear staining of tumor cells in the invasive fronts in 29% of HNSCC (n=17) and 56% of ESCC (n=171). Nuclear ICN1Val1744 was significantly associated with a poor 5-year survival in postsurgical ESCC patients (n=115). In culture, ICN1 induced cellular senescence through CSL-dependent p16INK4A induction, which was antagonized by DNMAML1 or knockdown of either CSL or p16INK4A. Moreover, ICN1 failed to induce senescence in immortalized cells expressing E6/E7 or ESCC cells with a deleted INK4 locus. However, p53 mutation did not prevent ICN1-induced senescence. In soft agar and xenograft transplantation, ICN1 stimulated colony formation and tumor growth of ESCC cells that negated senescence. Histology revealed an increased number of less-differentiated tumor cells upon ICN1 induction. Moreover, GSI treatment of mice with xenografted ESCC cells resulted in tumor necrosis. Conclusions: These data indicate that Notch activation contributes to disease progression in ESCC. While ICN1 induces senescence, loss of p16INK4A-mediated senescence checkpoint point functions may be required in ICN1-mediated malignant transformation, thus providing a novel mechanistic insight into how Notch signaling may contribute to the pathogenesis of SCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 70. doi:1538-7445.AM2012-70

Abstract 4720: Histone deacetylases as promising drug targets for treatment of esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and the prognosis for advanced esophageal cancer patients is poor in spite of combination therapy including surgery, chemotherapy and radiotherapy. There is urgent need for the development of new therapeutic agents to improve the prognosis of ESCC patients. To find a lead compound effective for the treatment of ESCC, we first screened a set of 285 compounds (SCADS inhibitor kit available at http://scads.jfcr.or.jp/index.html) for their growth inhibitory effect in 21 cell lines of ESCC. Of the 285 compounds tested, 19 compounds showed greater than 80% growth inhibition at 200 nM in at least one cell line. Our attention was drawn to trichostatin A, a histone deacetylase inhibitor (HDACi), because this compound showed antitumor activity in a broad set of ESCC in this initial screening. Also, this class of compound appears to be a promising agent for the treatment of some type of cancer cells. Therefore, we further explored the underlying mechanism for the antitumor effect of HDACi in ESCC. To validate that HDACs are indeed the molecular targets, HDAC1 or HDAC2 expression was knocked down by siRNA, and the effect on cell growth was examined. Knockdown of HDAC1 or HDAC2 expression alone did not show significant cell growth inhibition, but simultaneous knockdown of HDAC1 and HDAC2 expression induced significant cell growth inhibition. We then evaluated the growth inhibitory effect of two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and TrichostatinA (TSA), in our panel of 21 ESCC cell lines. The proliferation of ESCC cells was inhibited by SAHA and TSA in a dose-dependent manner. The IC50 of SAHA and TSA for these cell lines ranged from 0.25 to 6.34 microM and 16.1 to 489nM, respectively. The susceptibilities to SAHA and TSA were correlated. Cell cycle analysis by imaging cytometry showed prominent cell-cycle arrest in G2/M phase in cell lines treated with SAHA. Finally, we sought to determine the gene expression pattern associated with the sensitivities to the HDAC inhibitors. Gene expression profiling revealed that a set of genes related to cell cycle or mitosis was highly expressed in cell lines resistant to the HDAC inhibitors. Hierarchial cluster analysis showed that the cell lines were divided into two groups according to the expression pattern of genes related to cell cycle or mitosis, and significant differences were observed in the susceptibility to HDAC inhibitors between the two groups. One group showed high expression of genes involved in G2/M phase and low susceptibility to HDAC inhibitors, and the other group showed the opposite pattern. These results suggest that HDACs may be promising molecular targets for treatment of ESCC and that the susceptibility to HDAC inhibitors may be predicted by the expression pattern of genes involved in cell cycle, especially in the G2/M phase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4720. doi:1538-7445.AM2012-4720

Abstract 3996: Identification of DEC1-interacting protein DNAJB6 and its role in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ∼14%. Previously, a critical region at 9q33-34 was narrowed down and identified to be associated with tumor-suppressive function in ESCC. Within this region, Deleted in Esophageal Cancer 1 (DEC1), was identified to suppress anchorage-independent growth and tumorigenesis. In this study, we searched for DEC1-interacting partners by yeast two-hybrid screening. DNAJ (Hsp40) homologue subfamily B member 6 (DNAJB6) was identified as a DEC1-interacting protein and its interaction was further confirmed by the GST pull-down assay and co-localization studies. A putative DEC1-interacting sequence was mapped to the C-terminal end of DNAJB6 coding sequence. Using a tissue microarray constructed with tissues from Hong Kong, significantly higher survival in distant metastasis-free (M0) patients was found to be associated with higher DNAJB6 nuclear staining. DNAJB6 is down-regulated in 12 out of 14 (85.7%) ESCC cell lines, when compared to expression in an immortalized normal esophageal epithelial cell line. The two isoforms of DNAJB6 have distinct cellular localizations. DNAJB6a is mainly localized to the nucleus, while DNAJB6b shows diffuse staining in both the nucleus and cytoplasm. Through migration/invasion assay, we showed that expressions of both isoforms were inversely associated with migration/invasion ability of ESCC cell lines. These results suggest DNAJB6 plays a role in ESCC migration/invasion and metastasis. This gene may play a critical role in ESCC metastasis in an isoform-specific manner, which will be the focus of future studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3996. doi:1538-7445.AM2012-3996

Migfilin Regulates Esophageal Cancer Cell Motility through Promoting GSK-3β–Mediated Degradation of β-Catenin

Migfilin, a protein component of focal adhesions, has been implicated in regulation of cell-extracellular matrix adhesion and motility but the underlying mechanisms are not fully elucidated. In this study, we have determined the functions of migfilin in esophageal cancer cells and the mechanisms involved. We show that the expression level of migfilin is negatively associated with clinical metastasis, and enforced expression of migfilin suppressed cell motility through decreased free β-catenin level. Overexpression of migfilin resulted in destabilization of β-catenin in concomitance with reduction of its transcriptional activity. Knockdown of migfilin by siRNA, transfection of a mutant β-catenin at Ser37 which is a critical phosphorylation site of GSK-3β, GSK-3β inhibitor LiCl, or proteasome inhibitor MG132 reversed the migfilin-mediated β-catenin degradation and transcription inhibition. Moreover, migfilin promoted β-catenin degradation by reinforcing the association between β-catenin and GSK-3β. In addition, exogenously expressed β-catenin partially restored migfilin-induced suppression of cell invasion. Collectively, these results suggest that the expression level of migfilin in ESCCs is inversely correlated with clinical metastasis status, and migfilin inhibits ESCC cell invasion at least in part through promoting degradation of β-catenin.

Reelin Is Involved in Transforming Growth Factor-β1-Induced Cell Migration in Esophageal Carcinoma Cells

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice.

Overexpression of DNA damage-induced 45 α gene contributes to esophageal squamous cell cancer by promoter hypomethylation

BackgroundEnvironmental factors-induced dysfunction of esophageal squamous epithelium, including genomic DNA impairment and apoptosis, play an important role in the pathogenesis of esophageal squamous cell cancer. DNA damage-induced 45α (GADD45α) has been found promoting DNA repair and removing methylation marker, Therefore, in this study we will investigate whether GADD45α expression is induced and its mechanism in esophageal squamous cell cancer.MethodsTwo human esophageal squamous cell lines (ESCC), ECA109 and KYSE510 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Lipofectamine 2000 was used to transfect cells. mRNA level of GADD45α was measured by reverse transcription-quantitive PCR (RT-qPCR), protein level of GADD45α was detected by western blot and Immunohistochemistry. Global DNA methylation of tissue sample was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek Group) and promoter methylation was measured by bisulfite sequencing.ResultsGADD45a mRNA and protein levels were increased significantly in tumor tissue than that in adjacent normal tissue. Hypomethylation of global genomic DNA and GADD45α promoter were found in ESCC. The cell sensitivity to Cisplatin DDP was decreased significantly in Eca109 and Kyse510 cells, in which GADD45α expression was down-regulated by RNA interference (RNAi). In addition, silence of GADD45a expression in ESCC cells inhibited proliferation and promoted apoptosis.ConclusionOverexpression of GADD45α gene is due to DNA hypomethylation in ESCC. GADD45α may be a protective factor in DDP chemotherapy for esophageal squamous cell carcinoma.

Overexpression of microRNA-223 regulates the ubiquitin ligase FBXW7 in oesophageal squamous cell carcinoma

Background:F-box and WD repeat domain-containing 7 (FBXW7) is a cell cycle regulatory gene whose protein product ubiquitinates positive cell cycle regulators such as c-Myc, cyclin E, and c-Jun, thereby acting as a tumour-suppressor gene. This study focused on microRNA-223 (miR-223), which is a candidate regulator of FBXW7 mRNA. The aim of this study was to clarify the clinical significance of miR-223 and FBXW7 in oesophageal squamous cell carcinoma (ESCC) patients, and to elucidate the mechanism by which FBXW7 is regulated by miR-223.Methods:The expression levels of miR-223 and the expression of FBXW7 protein was examined using 109 resected specimens to determine the clinicopathological significance. We also investigated the role of miR-223 in the regulation of FBXW7 expression in ESCC cell lines in an in vitro analysis.Results:We found that miR-223 expression was significantly higher in cancerous tissues than in the corresponding normal tissues. There was a significant inverse relationship between the expression levels of miR-223 and FBXW7 protein. Moreover, patients with high miR-223 expression demonstrated a significantly poorer prognosis than those with low expression. On the basis of a series of gain-of-function and loss-of-function studies in vitro, we identified FBXW7 as a functional downstream target of miR-223.Conclusion:Our present study indicates that high expression of miR-223 had a significant adverse impact on the survival of ESCC patients through repression of the function of FBXW7.

Mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) gene expression in esophageal squamous cell carcinoma cell line EC9706

To investigate the mechanism of loss of human esophageal cancer-related gene 4 (ECRG4) expression in esophageal squamous cell carcinoma (ESCC.) PCR-SSCP and DNA sequencing analysis were used to detect the mutation of ECRG4 exons in esophageal cancer and matched adjacent normal tissues of 80 patients. DNA bisulfite-modifying ssPCR sequencing assay was used to examine the methylation status of ECRG4 promoter in human esophageal squamous cell carcinoma EC9706 cells. The re-expression of ECRG4 mRNA was examined by RT-PCR in EC9706 cells, after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. No mutation in the four ECRG4 exons was found in all the ESCC and matched normal adjacent tissues. RT-PCR showed that 11 of 16 CpG islands of ECRG4 promoter were hypermethylated, while ECRG4 mRNA expression level was undetectable in the EC9706 cells. The ECRG4 mRNA was re-expressed after treatment with either demethylation drug 5-aza-2'-deoxycytidine or arsenic trioxide. The epigenetic mechanism of methylation is a reason of loss of ECRG4 gene expression in the ESCC cell line EC9706.

The impact of Metastasis Suppressor-1, MTSS1, on oesophageal squamous cell carcinoma and its clinical significance

BackgroundMetastasis suppressor-1 (MTSS1) has been proposed to function as a cytoskeletal protein with a role in cancer metastasis. Recent studies have demonstrated the clinical significance of MTSS1 in certain type of cancers, yet the clinical relevance of MTSS1 in oesophageal squamous cell carcinoma (ESCC) has not been reported.MethodsIn this study, we assessed the expression levels of MTSS1 in tumours and its matched adjacent non-tumour tissues obtained from 105 ESCC patients. We also used ESCC cells with differing MTSS1 expression and assessed the influence of MTSS1 on ESCC cells.ResultsDown-regulation of MTSS1 expression was observed both in oesophageal tumour tissues and ESCC cancer cell lines. We also reported that MTSS1 expression was associated with tumour grade (p = 0.024), lymph node metastasis (p = 0.010) and overall survival (p = 0.035). Patients with high levels of MTSS1 transcripts had a favorable prognosis in comparison with those who had reduced or absent expression levels. Using over-expression and knockdown approach, we created sublines from ESCC cells and further demonstrated that MTSS1 expression in ESCC cells significantly influenced the aggressiveness of the oesophageal cancer cells, by reducing their cellular migration and in vitro invasiveness.ConclusionMTSS1 serves as a potential prognostic indicator in human ESCC and may be an important target for cancer therapy.

RETRACTED ARTICLE: Slug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice.MethodsWe utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development.ResultsThe EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis.ConclusionsSlug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC.

Abstract 2105: RNA-seq identifies protein tyrosine kinase 6 (PTK6) as a candidate tumor-suppressor gene in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC), the major histologic subtype of esophageal cancer, is one of the most common and deadliest cancers in the world, with survival rates of less than 15%. Like many other solid tumors, the pathogenesis of ESCC is also believed to be a multistep process with accumulation of numerous genetic alterations involving inactivation of tumor-suppressor genes and activation of oncogenes. An understanding of the molecular events that result in the development and progression of the disease may lead to treatments that will increase survival rates of ESCC patients. Recent advances in sequencing technologies offer the opportunity to characterize the cancer genome at unprecedented depth and sensitivity. Large-scale transcriptome sequencing (RNA-Seq) has proved an effective means of precisely quantifying the changing expression levels of each transcript. In this study, we performed integrative RNA-Seq analysis on 14 patient-derived ESCC clinical specimens (4 paired ESCC and their adjacent non-tumorous (NT) counterparts, 1 unpaired NT and 5 unpaired ESCC) and discovered a number of commonly differentially expressed genes in ESCC compared with NT tissues. A total of 526 genes were found to be significantly aberrantly expressed, including 452 genes that were up-regulated and 74 genes that were down-regulated. One candidate tumor suppressor gene, protein tyrosine kinase 6 (PTK6), was chosen for further characterization. By both quantitative real-time PCR and immunohistochemistry analysis, PTK6 was found to be significantly down-regulated in a larger cohort of ESCC tumors compared with NT counterparts. Consistently, expression studies in a panel of esophageal cell lines found PTK6 to be either absent or expressed in low amounts in all eight ESCC cell lines examined compared with two immortalized normal esophageal cell lines, NE1 and NE3. Down-regulation of PTK6 in both ESCC cell lines and clinical samples was found to be significantly associated with promoter hypermethylation, as evident by results obtained from studies involving demethylation treatment with 5-aza-dC, methylation specific PCR as well as bisulfite genomic sequencing. Functional studies in ESCC cell lines, EC109 and KYSE30, with PTK6 stably repressed by lentiviral-based approach stimulated both in vitro and in vivo tumorigenicity ability of the cells including foci formation, colony formation in soft agar as well as tumor formation in nude mice. Taken together, our findings define a function for PTK6 as an important tumor suppressor gene in ESCC development. Additional work on the mechanism by which PTK6 drives ESCC is currently being studied in our laboratory. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2011-2105

Abstract LB-129: Loss of Rab25 expression promotes tumor formation and angiogenesis in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide and is particularly prevalent in some areas of Asian countries. With the advent of next-generation sequencing technologies, it is now possible for us to integrate and analyze large amounts of data at genome-wide scale, with the goal for a better understanding of cellular function and disease development. In order to elucidate the molecular mechanisms that drive tumorigenesis in ESCC, we employed a high-throughput sequencing technology named RNA-Seq (RNA sequencing) to identify dysregulated gene expression in 14 patient-derived ESCC or adjacent non-tumor (NT) clinical specimens of Chinese origin. The expression of Rab25, a family member of Ras-associated binding GTPases, was found to be downregulated in tumour tissues significantly. By both qPCR and immunohistochemistry analysis on a tissue microarray, expression of Rab25 was found to be significantly decreased in a larger cohort of ESCC tumors compared with NT counterparts. Patients with higher levels of Rab25 expression were also significantly associated with a better overall survival. Consistently, absent or downregulated Rab25 expression was also detected in all ESCC cell lines examined as compared to a pooled normal tissue control. Rab25 expression in ESCC cell lines could be restored by demethylation treatment with 5-aza-dC, suggesting Rab25 is silenced via epigenetic alterations. Rab25 functions as an important mediator of vesicle trafficking, regulating cell polarity and distribution of membrane proteins, which are important for the maintenance of normal epithelial lining of the esophagus. We hypothesized that loss of Rab25 expression could lead to dysregulation in the maintenance of this normal epithelium and thus result in tumor development. The tumor suppressive function of Rab25 and the tumorigenic effect of its loss were evaluated by lentiviral-based overexpression and shRNA-knockdown of Rab25 in ESCC cell lines. In vivo studies found Rab25 to suppress tumor formation and angiogenesis in nude mice. Rab25-knockdwon cells gave rise to significantly more and larger tumors as compared to negative control cells. Immunohistochemical staining of resected xenografts revealed an increase in microvessel density and tumor proliferation as shown by higher expressions of endothelial marker CD34 and proliferation marker PCNA, respectively, in Rab25-knockdown cells as compared to negative controls. Similarly, conditioned medium from Rab25-knockdown cells enhanced tube-forming capability of HUVECs, suggesting potential regulation of angiogenic factors by Rab25-mediated pathway. Work on the identification of potential angiogenic factors regulated by Rab25 is now currently underway using a human angiogenesis antibody array. In summary, Rab25 functions as an important tumor suppressor gene in ESCC development by regulating angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2011-LB-129

Inhibition of Oesophageal Squamous Cell Carcinoma Progression by in vivo Targeting of Hyaluronan Synthesis

BackgroundOesophageal cancer is a highly aggressive tumour entity with at present poor prognosis. Therefore, novel treatment options are urgently needed. Hyaluronan (HA) is a polysaccharide present in the matrix of human oesophageal squamous cell carcinoma (ESCC). Importantly, in vitro ESCC cells critically depend on HA synthesis to maintain the proliferative phenotype. The aim of the present study is (1) to study HA-synthase (HAS) expression and regulation in human ESCC, and (2) to translate the in vitro results into a mouse xenograft model of human ESCC to study the effects of systemic versus tumour targeted HAS inhibition on proliferation and distribution of tumour-bound and stromal hyaluronan.MethodsmRNA expression was investigated in human ESCC biopsies by semiquantitative real-time RT PCR. Furthermore, human ESCC were xenografted into NMRI nu/nu mice. The effects on tumour progression and morphology of 4-methylumbelliferone (4-MU), an inhibitor of HA-synthesis, and of lentiviral knock down of HA-synthase 3 (HAS3), the main HAS isoform in the human ESCC tissues and the human ESCC cell line used in this study, were determined. Tumour progression was monitored by calliper measurements and by flat-panel detector volume computed tomography (fpVCT). HA content, cellular composition and proliferation (Ki67) were determined histologically.ResultsmRNA of HAS isoform 3 (HAS3) was upregulated in human ESCC biopsies and HAS3 mRNA was positively correlated to expression of the epidermal growth factor (EGF) receptor. EGF was also proven to be a strong inductor of HAS3 mRNA expression in vitro. During the course of seven weeks, 4-MU inhibited progression of xenograft tumours. Interestingly, remodelling of the tumour into a more differentiated phenotype and inhibition of cell proliferation were observed. Lentiviral knockdown of HAS3 in human ESCC cells prior to xenografting mimicked all effects of 4-MU treatment suggesting that hyaluronan produced by ESCC is accountable for major changes in tumour environment in vivo.ConclusionsSystemic inhibition of HA-synthesis and knockdown of tumour cell HAS3 cause decreased ESCC progression accompanied by tumour stroma remodelling and may therefore be used in novel approaches to ESCC therapy.

MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.MethodsTotal RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.ResultsBased on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.ConclusionsThese results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.

Overexpression of GPR39 contributes to malignant development of human esophageal squamous cell carcinoma

BackgroundBy using cDNA microarray analysis, we identified a G protein-coupled receptor, GPR39, that is significantly up-regulated in ESCC. The aim of this study is to investigate the role of GPR39 in human esophageal cancer development, and to examine the prevalence and clinical significance of GPR39 overexpression in ESCC.MethodsThe mRNA expression level of GPR39 was analyzed in 9 ESCC cell lines and 50 primary ESCC tumors using semi-quantitative RT-PCR. Immunohistochemistry was used to assess GPR39 protein expression in tissue arrays containing 300 primary ESCC cases. In vitro and in vivo studies were done to elucidate the tumorigenic role of GPR39 in ESCC cells.ResultsWe found that GPR39 was frequently overexpressed in primary ESCCs in both mRNA level (27/50, 54%) and protein level (121/207, 58.5%), which was significantly associated with the lymph node metastasis and advanced TNM stage (P < 0.01). Functional studies showed that GPR39 has a strong tumorigenic ability. Introduction of GPR39 gene into ESCC cell line KYSE30 could promote cell proliferation, increase foci formation, colony formation in soft agar, and tumor formation in nude mice. The mechanism by which amplified GPR39 induces tumorigenesis was associated with its role in promoting G1/S transition via up-regulation of cyclin D1 and CDK6. Further study found GPR39 could enhance cell motility and invasiveness by inducing EMT and remodeling cytoskeleton. Moreover, depletion of endogenous GPR39 by siRNA could effectively decrease the oncogenicity of ESCC cells.ConclusionsThe present study suggests that GPR39 plays an important tumorigenic role in the development and progression of ESCC.