Proliferation Of Esophageal Squamous Cell Carcinoma Research Articles

219P - Nestin Promotes Proliferation and Apoptosis and Correlates with Poor Prognosis in Esophageal Squamous Cell Carcinoma

ABSTRACT Aim: Esophageal squamous cell carcinoma (ESCC) is a malignancy of the esophagus that is characterized by its high invasiveness and mortality. Nestin is a member of the class VI family of intermediate filament proteins and we previously demonstrated that the majority of tumor cells in non-small cell lung cancer samples are nestin-positive and showed that nestin expression was positively correlated with the subset of lung cancer patients displaying poor outcomes and high levels of proliferative markers. However, the precise mechanisms of nestin action in the proliferation and metastasis of ESCC require further elucidation. Methods: A total of 93 ESCC samples were randomly selected from our tissue database and we explored the association of the nestin phenotype with malignant proliferation and apoptosis in esophageal squamous cancer cells. Nestin expression was determined in ESCC specimens and cell lines (Eca-109 and TE-1), and correlated with clinicopathological properties, including clinical prognosis and proliferative markers. The association of the nestin phenotype with apoptotic indicators was also analyzed. Results: Nestin was expressed in ESCC specimens and cell lines (Eca-109 and TE-1). ESCC patients with nestin-positive tumors had significantly shorter median survival and progression-free survival times than those with nestin-negative tumors. Positive staining for the proliferation markers Ki67 and PCNA (proliferating cell nuclear antigen) was detected in 56.9% and 60.2% of ESCC specimens, respectively, and was strongly correlated with the nestin phenotype. Notably, expression of cyclin-dependent kinase-5 (CDK5) and P35 was detected in 53.8% and 48.4% of ESCC specimens, respectively, and was strongly associated with the nestin phenotype. Conclusions: Our data demonstrate nestin expression in ESCC specimens and cell lines, and revealed a strong association of the nestin phenotype with poor prognosis in ESCC patients. Furthermore, we showed that nestin plays an important role in the malignant proliferation and supported the tentative conclusion that nestin likely promotes ESCC cell apoptosis. Targeted regulation of nestin may thus have therapeutic applications in the treatment of human esophageal cancer. Disclosure: All authors have declared no conflicts of interest.

Upregulation of SYF2 in esophageal squamous cell carcinoma promotes tumor cell proliferation and predicts poor prognosis.

SYF2, also known as CCNDBP1-interactor or p29, is reported in pre-mRNA splicing and cell cycle progression. However, the role of SYF2 in esophageal squamous cell carcinoma (ESCC) development remains elusive. In the present study, Western blot and immunohistochemistry assays demonstrated that SYF2 was overexpressed in ESCC tumor tissues and cell lines. In addition, immunohistochemistry analysis revealed that SYF2 expression was positively correlated with tumor grade and predicted poor prognosis of ESCC. In vitro studies using serum starvation-refeeding experiment and SYF2-siRNA transfection assay demonstrated that SYF2 expression promoted proliferation of ESCC cells, while SYF2 knockdown led to decreased cell growth rate and colony formation resulted from growth arrest of cell cycle at G0/G1 phase. Furthermore, our results indicated that SYF2 can down-regulate the sensitivity of ESCC cells for cisplatin. Our findings for the first time supported that SYF2 might play an important role in the regulation of ESCC proliferation and would provide a novel therapeutic strategy against human ESCC.

Elevated Orai1 expression mediates tumor-promoting intracellular Ca2+ oscillations in human esophageal squamous cell carcinoma.

Effective treatment as well as prognostic biomarker for malignant esophageal squamous cell carcinoma (ESCC) is urgently needed. The present study was aimed at identifying oncogenic genes involving dysregulated intracellular Ca2+ signaling, which is known to function importantly in cellular proliferation and migration. Tumors from patients with ESCC were found to display elevated expression of Orai1, a store-operated Ca2+ entry (SOCE) channel, and the high expression of Orai1 was associated with poor overall and recurrence-free survival. In contrast to the quiescent nature of non-tumorigenic epithelial cells, human ESCC cells exhibited strikingly hyperactive in intracellular Ca2+ oscillations, which were sensitive to treatments with Orai1 channel blockers and to orai1 silencing. Moreover, pharmacologic inhibition of Orai1 activity or reduction of Orai1 expression suppressed proliferation and migration of ESCC in vitro and slowed tumor formation and growth in in vivo xenografted mice. Combined, these findings provide the first evidence to imply Orai1 as a novel biomarker for ESCC prognostic stratification and also highlight Orai1-mediated Ca2+ signaling pathway as a potential target for treatment of this deadly disease.

Upregulation of the Long Non-coding RNA PlncRNA-1 Promotes Esophageal Squamous Carcinoma Cell Proliferation and Correlates with Advanced Clinical Stage

Recent studies revealed that long noncoding RNAs (lncRNAs) play critical regulatory roles in cancer biology. PlncRNA-1 is one of lncRNAs that is associated with cell apoptosis and proliferation of prostate cancer. This study aimed to assess the potential role of PlncRNA-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression level of PlncRNA-1 in 73 pairs of ESCC and their matched normal tissues. The correlation of PlncRNA-1 with clinicopathological features and clinical stages was also analyzed. Cancer cell proliferation and apoptosis were assessed following knock-down of PlncRNA-1 by MTT, colony formation assay, and flow cytometry. The expression of PlncRNA-1 was significantly higher in human ESCC compared with the adjacent noncancerous tissues (69.8 %, p < 0.05), and the high level of PlncRNA-1 expression was significantly correlated with advanced clinical stage (p < 0.01) and lymph node metastasis (p < 0.05). Furthermore, knockdown of PlncRNA-1 reduced cell proliferation and increased the apoptosis in vitro. PlncRNA-1 plays an important role in ESCC cell proliferation. Overexpression of PlncRNA-1 is correlated with advanced tumor stage and lymph node metastasis, and may serve as a potential prognostic marker and therapeutic target for ESCC.

MicroRNA-27a functions as a tumor suppressor in esophageal squamous cell carcinoma by targeting KRAS

microRNAs (miRNAs) have been suggested to play a vital role in regulating tumor progression and invasion. However, the expression of miR-27a in esophageal squamous cell carcinoma (ESCC) and its effect on the tumorigenesis of ESCC are unclear. In the present study, we found that miR-27a was downregulated in esophageal carcinoma cell lines and ESCC specimens with lymph node metastasis. Furthermore, we demonstrated that miR-27a binds to the 3'-untranslated region (UTR) of KRAS and inhibits the expression of the KRAS protein. miR-27a levels were inversely correlated with levels of KRAS mRNA and protein in ESCC specimens. Both in vitro and in vivo assays revealed that miR-27a attenuated ESCC proliferation, invasion and tumor growth in nude mice. miR-27a exerts its tumor suppressor function through inhibition of the KRAS-related ERK pathways. Our findings suggest, for the first time, that miR-27a suppresses tumorigenesis of ESCC by targeting KRAS.

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.

P-0003 - Rnai-Mediated Silencing of Rap1B Gene Expression in Esophageal Squamous Cell Carcinoma Inhibits Tumor Growth and Invasion

Background: Rap1b is known to play a role in the progression of angiogenesis and migration. Our previous study found that Rap1b was over-expression in esophageal squamous cell carcinoma (ESCC). Based on our previous results, we investigated the role of Nrf2 in cell proliferation and invasion in ESCC.

Abstract 3102: miRNA-mRNA regulatory networks by integrating miRNA and mRNA expression profiling of esophageal squamous cell carcinoma.

Abstract Background: miRNA expression profiling of esophageal squamous cell carcinoma(ESCC) has identified some miRNAs related to the clinical diversity of the disease and this potentially provides novel diagnostic and prognostic tools for ESCC therapy. In order to further understand the associations between miRNA expression and their target mRNA in ESCC, we performed integrating miRNA and mRNA expression profiling in ESCC. Material &amp; Methods: Eight esophageal squamous carcinoma of Stage T2N1MO matched with their corresponding normal tissues was included in the study. Microdissection was performed to assure that more than 70% tumor components were selected for RNA extraction. Microarray technology (Agilent Human miRNA microarray and Agilent whole Human gene expression microarray) was used for miRNA expression and genome wide mRNA profiling. Integrated analysis of miRNAs and their target mRNAs by multi-step bioinformatics methods. Seven important differentiate expressed miRNAs (miR-21, miR-196b, miR-1, miR-29c, miR-125b, miR-195, let-7c) and 4 predicted target mRNAs(CDK-6, VEGFA, IGF1, and FLNC) were selected for future validation in an extended cohort of 50 ESCC by qRT-PCR. The relationship of the miRNA and genes were calculated by their differentiate expression values, and according to the interactions of MicroRNA and genes in Sanger MicroRNA database to create the MicroRNA-Gene-Network by the bioinformatic tools. Results:Compared to esophageal normal tissues, there were 67 differentiately expressed miRNAs and 2992 mRNAs in ESCC. The differentially expressed miRNAs and mRNAs were confirmed by real-time PCR. These differentiated miRNAs and their target genes were associated with many biological processes such as signal transduction, angiogenesis, cell proliferation, cell-cell signaling, response to drug. In the MicroRNA-Gene-Network, the significant pathways were Pathways in cancer, Cell cycle, TGF-beta signaling pathway, p53 signaling pathway, MAPK signaling pathway, mTOR signaling pathway and Jak-STAT signaling pathway. Conclusions: It is the first large-scale study to identify miRNA-mRNA regulatory networks in ESCC by combining expression profiles and bioinformatics analysis demonstrating that miRNA expression is highly associated with the proliferation, prognosis and metastasis of ESCC. Our findings provided a valuable source for future studying the differentially expressed miRNAs and their promising target genes, pathways and processes that may be useful for diagnostic or therapeutic applications in ESCC. Citation Format: Qiao Wei, Kuaile Zhao, Weiwei Yu, Guangqi Qin, Zhuanxu Zhang, Xiang Du, Menghong Sun. miRNA-mRNA regulatory networks by integrating miRNA and mRNA expression profiling of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3102. doi:10.1158/1538-7445.AM2013-3102

SULF1 Inhibits Proliferation and Invasion of Esophageal Squamous Cell Carcinoma Cells by Decreasing Heparin-Binding Growth Factor Signaling

Heparin-binding growth factor signaling is involved in the pathogenesis and development of human cancers. It can be regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPG). SULF1 is a heparin-degrading endosulfatase which can modulate the sulfation of HSPGs. The purpose of this study was to elucidate the role of SULF1 in modulating proliferation and invasion of esophageal squamous cell carcinoma (ESCC) by decreasing heparin-binding growth factor signaling. We restored SULF1 expression in the ESCC cell line KYSE150, and examined the effects of SULF1 expression on the proliferation and invasion of KYSE150 cells. In addition, we investigated the expression of SULF1 in human ESCC tissues and analyzed the correlation of SULF1 expression with clinicopathologic characteristics of ESCC. Our study shows that re-expression of SULF1 in ESCC cell line results in the downregulation of hepatocyte growth factor-mediated activation of MAPK pathways with a resultant decrease in cell invasiveness. Cell proliferation was also inhibited in SULF1-transfected KYSE150 cells. Immunohistochemical assays reveal that SULF1 is expressed in nearly half of the human ESCC tissues but not in normal esophageal epithelial cells. SULF1 expression in human ESCC tissues is negatively correlated with tumor size and tumor invasion. This study identified that SULF1 inhibits proliferation and invasion of ESCC by decreasing heparin-binding growth factor signaling and suggested that SULF1 plays an inhibiting role in the pathogenesis of ESCC.

RETRACTED ARTICLE: HAX-1 Promotes the Chemoresistance, Invasion, and Tumorigenicity of Esophageal Squamous Carcinoma Cells

HAX-1 is an anti-apoptotic factor and regulates the expression of DNA pol β. Interestingly, DNA polymerase pol β is overexpressed in esophageal squamous cell carcinoma (ESCC). However, the functional role of HAX-1 in ESCC remains unclear. To investigate the role of HAX-1 in chemoresistance, invasion, and tumorigenicity of ESCC. Lentivirus-mediated overexpression or knockdown of HAX-1 was employed to establish ESCC EC9706 cell lines that expressed HAX-1 at different levels. The biological behaviors of these engineered cells were characterized in vitro and in vivo using a xenograft nude mice model. In addition, HAX-1 and pol β expression in the tumor tissues was detected by RT-PCR and immunohistochemistry. HAX-1 overexpression promoted cell proliferation and resistance against cisplatin, increased cell invasion and suppressed apoptosis along with increased pol β expression. Conversely, HAX-1 knockdown inhibited the malignant phenotypes of EC9706 cells. The xenograft nude mice model demonstrated that HAX-1 overexpression or depletion led to increased or decreased tumor growth in vivo, respectively. Furthermore, a positive correlation of HAX-1 and pol β expression in the tumor tissues was observed. HAX-1 promotes the proliferation, chemoresistance, invasion, and tumorigenicity of ESCC, and this is correlated with increased poly β expression. HAX-1 may represent a potential target to overcome the resistance and metastasis of ESCC.

Effects of EZH2-siRNA on proliferation and invasion of human esophageal squamous carcinoma cells

Objective To investigate the influence of EZH2 gene down-regulation by RNA interference (RNAi) on the proliferation and invasion of human esophageal squamous carcinoma cell line Ecal09.Methods Two small interfering RNAs (siRNA) targeting EZH2 were transfected into Eca109 cells by using lipofectamine 2000.The proliferation capcity of Eca109 cells was determined by calculating the cell population doubling time,the cell cycle,the cell cloning and the percentage of apoptotic cells.The invasive ability of Eca109 cells was detemined by Transwell assay.Results In vitro experiment showed that Eca109 cells transfected with EZH2-siRNA had significantly longer cell population doubling time [ ( 26.7 ± 0.9) h vs ( 22.6 ± 0.8 ) h,P ＜ 0.01 ],lower percentage of cell cloning [ ( 39.1 ± 2.3 ) ％ vs(65.1±3.0)％,P＜0.01],higher cell apoptotic rate [(9.51±0.19)％ vs (1.11 ±0.08)％,P＜0.01 ],and lower cell invasion rate [ (35.0 ± 0.4) ％ vs (51.8 ± 0.7) ％,P ＜ 0.01 ] than control groups.However,there was no significant diference in the cell cycle at S phase between EZH-2-siRNA group and control group [(32.1 ±0.7)％ vs (31.9 ±0.4)％,P＞0.05].Conclusion EZH2 siRNA can reduce the proliferation and invasion of human esophageal squamous carcinoma cells,which suggests that EZH2 siRNA might be a potential gene therapertic target of human esophageal squamous carcinoma. Key words: Esophageal squamous carcinoma; EZH2; RNA interference; Proliferation; Invasion

Reciprocal Activation Between PLK1 and Stat3 Contributes to Survival and Proliferation of Esophageal Cancer Cells

Aberrant activation of the signal transducer and activator of transcription (Stat)3 and overexpression of polo-like kinase (PLK)1 each have been associated with cancer pathogenesis. The mechanisms and significance of dysregulation of Stat3 and PLK1 in carcinogenesis and cancer progression are unclear. We investigated the relationship between Stat3 and PLK1 and the effects of their dysregulation in esophageal squamous cell carcinoma (ESCC) cells. We used immunoblot, quantitative reverse-transcription polymerase chain reaction, immunochemistry, chromatin immunoprecipitation, mobility shift, and reporter assays to investigate the relationship between Stat3 and PLK1. We used colony formation, fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and xenograft tumor assays to determine the effects of increased activation of Stat3 and PLK1 in proliferation and survival of ESCC cells. Stat3 directly activated transcription of PLK1 in esophageal cancer cells and mouse embryonic fibroblast cell NIH3T3. PLK1 then potentiated the expression of Stat3; β-catenin was involved in PLK1-dependent transcriptional activation of Stat3. This mutual regulation between Stat3 and PLK1 was required for proliferation of esophageal cancer cells and resistance to apoptosis in culture and as tumor xenografts in mice. Furthermore, phosphorylation of Stat3 and overexpression of PLK1 were correlated in a subset of ESCC. Stat3 and PLK1 control each other's transcription in a positive feedback loop that contributes to the development of ESCC. Increased activity of Stat3 and overexpression of PLK1 promote survival and proliferation of ESCC cells in culture and in mice.

The flavonoid Baohuoside-I inhibits cell growth and downregulates survivin and cyclin D1 expression in esophageal carcinoma via β-catenin-dependent signaling

Esophageal cancer is one of the most common malignancies and is associated with a dismal prognosis. Although treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We found that Baohuoside-I, a flavonoid extracted from a Chinese medicinal plant, exhibits anticancer activity. Here, we demonstrated that Baohuoside-I significantly inhibited Eca109 human esophageal squamous carcinoma cell proliferation and induced Eca109 cell apoptosis in vitro and in vivo. The growth inhibitory effect of Baohuoside-I on the Eca109 tumor cell line was examined by MTT assay; the induction of apoptosis was analyzed by flow cytometry. Eca109-luc cells were injected into the subcutaneous tissue of nude mice to establish xenograft tumors. Our results revealed that Baohuoside-I caused a dose- and time-dependent inhibition of cell growth and an induction of apoptosis. Furthermore, Baohuoside-I-treated cells were characterized by decreased expression of the β-catenin gene and protein in the total cell lysates. Thus, the gene and protein expression of the downstream elements survivin and cyclin D1 was downregulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of Baohuoside-I are warranted. Our study provides the first evidence that Baohuoside-I inhibits tumor growth and induces apoptosis by inhibiting β-catenin-dependent signaling pathways. Thus, Baohuoside-I is a potential candidate in ESCC disease therapy.

Inhibition of nuclear factor-κB activity by small interfering RNA in esophageal squamous cell carcinoma cell lines

Chemotherapy with 5-fluorouracil (5-FU) is commonly used in combination therapy for esophageal squamous cell carcinoma (ESCC), but its efficacy is limited in certain patients. Recent studies suggest that constitutive activation of nuclear factor-κB (NF-κB) has a critical role in tumorigenesis and is associated with poor prognosis and resistance to chemoradiation therapy in many types of human cancers. In the present study, we evaluated the effect of small interfering RNA targeting NF-κB (NF-κB siRNA) combined with 5-FU on the proliferation of two cell lines of cultured ESSCs. Immunofluorescence and immunoblot analyses revealed that the NF-κB protein was localized mostly in the cytoplasm of ESCCs. When cultured ESCCs were exposed to tumor necrosis factor-α, NF-κB was transferred to the nucleus and activated. ESCCs with activated NF-κB had poor sensitivity to 5-FU. When cells were transfected with NF-κB siRNA, the levels of NF-κB protein were significantly decreased in the cytoplasm and the nucleus. Transcriptional activity of NF-κB was significantly suppressed in cells treated with 5-FU and NF-κB siRNA compared to cells treated with 5-FU alone. 5-FU consistently suppressed proliferation of ESCCs in a dose-dependent manner, and this effect was significantly enhanced when combined with NF-κB siRNA. These results suggest that combination therapy of 5-FU with NF-κB siRNA may provide a new therapeutic option for ESCC.

S100A4 mediated cell invasion and metastasis of esophageal squamous cell carcinoma via the regulation of MMP-2 and E-cadherin activity

It is well documented that S100A4 is upregulated in a large amount of invasive tumors and plays a pivotal role in tumor invasion and metastasis. However, the precise role and mechanism S100A4 exerts in the invasion and metastasis of esophageal squamous cell carcinoma (ESCC) have not been fully elucidated to date. Our data demonstrated that S100A4 was overexpressed in human ESCC tissues, especially in ESCC with poor differentiation, deep invasion and lymph node metastasis. Subsequently, the knockdown of S100A4 by RNAi in ESCC cell line (EC-1) could reduce cell invasion, metastasis and proliferation ability in vitro. Most importantly, S100A4 regulated MMP-2 positively and E-cadherin negatively in vivo and in vitro to some extent. Our results suggest that S100A4 is an important factor in the invasion, metastasis and proliferation of ESCC and may control invasion and metastasis at least in part through the regulation of MMP-2 and E-cadherin activity. S100A4 may serve as a biomarker for progression of ESCC and a potential molecular target for biotherapy of ESCC.

Abstract 1205: Clinical significance of IL-8 receptor expression in esophageal squamous cell carcinoma

Abstract Introduction: Esophagectomy is one of the most invasive surgery for digestive cancer, and the blood level of inflammatory cytokines such as IL-1, IL-6 and IL-8 arise several hours after surgery or in suffering postoperative complications. It is known that postoperative complications are negative predictors in long-term malignancy outcome. It has been reported that IL-8 receptor, one of the chemokine receptor, is expressed on several carcinomas and IL-8 signaling promotes proliferation of cancer cells, however, it is poorly understood in esophageal squamous cell carcinoma (ESCC). In this study, we demonstrate the significance of IL-8 receptor expression and validate the effect of IL-8 receptor expression and postoperative complications on long-term prognosis in ESCC using resected specimens and cancer cell lines. Materials and Methods: &amp;lt;Immunohistochemical analysis&amp;gt; Eighty-two specimens were sectioned from archived, paraffin-embedded tumor tissues obtained from patients with ESCC underwent esophagectomy and extended lymphadenectomy to have no residual cancer in our institute between 1997 and 2002. These were stained using polyclonal antibody to IL-8 receptor, and correlation between stainability and disease-free survival as well as overall survival was examined. &amp;lt;in vitro&amp;gt; TE4, the human ESCC cell line with the high expression of IL-8 receptor at protein levels and TE11, the one with the low expression were cultured at 1×105cells/mL in medium. Cells were transferred to the 96-well microtitre plates containing diluted IL-8. Plates were incubated for indicated periods and WST assay was performed to evaluate the proliferation of ESCC cells. &amp;lt;in vivo&amp;gt; Four groups of 5 nude mice were injected subcutaneously in the dorsal flank with 3×106 cells (TE4, TE11), and 1 of 2 groups were received intraperitoneal (i.p.) injection of 300ng of IL-8 everyday. Tumor volume was measured twice weekly with a caliper in 2 weeks. Results: &amp;lt;Immunohistochemical analysis&amp;gt; IL-8 receptor was expressed in 33 specimens of 82 (40.2%). The patients with IL-8 receptor expression in primary tumor had shorter disease-free survival compared to those with negative expression. Especially, the patients with IL-8 receptor expression who suffered postoperative complications such as pneumonia and ruptured suture had a poorer prognosis (p=0.013). &amp;lt;in vitro&amp;gt; TE4 cells in IL-8 administered group proliferated significantly more than those in control group (p&amp;lt;0.001) but TE11 showed no significant difference. &amp;lt;in vivo&amp;gt; Tumor growth of TE4 in i.p. group was significantly larger than that in control group (p=0.028). Conclusion: Our results suggest that the expression of IL-8 receptor is associated with the proliferation of ESCC. The patients with IL-8 receptor expression who suffered postoperative complications probably need strict follow-up and aggressive adjuvant therapy. 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 1205. doi:10.1158/1538-7445.AM2011-1205

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma

BackgroundThe esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.AF325503). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC).MethodsIn this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. AF 071882). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells.ResultsWe revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells.ConclusionsECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.

Prognostic Significance of CD151 Expression in Esophageal Squamous Cell Carcinoma with Aggressive Cell Proliferation and Invasiveness

CD151 is a member of the tetraspanins and has recently been reported as a promoter of the malignant progression of cancer. The purpose of this study was to clarify the clinicopathological outcome and prognostic significance of the immunohistochemical expression of CD151 in esophageal squamous cell carcinoma (ESCC). We evaluated the significance of CD151 expression by immunohistochemistry in 138 surgically resected ESCC and the association of CD151 expression with clinicopathological features. Seventy-five (51.7%) ESCC showed a positive expression of CD151, which indicated a significant association with tumor depth (P=0.004), lymph node metastasis (P=0.002), distant metastasis (P=0.025), and lymphatic invasion (P=0.046), as well as the Ki-67 labeling index (P=0.011). The 5-year survival rate of ESCC patients with CD151-positive expression was significantly lower than with CD151-negative expression (positive, 43.1%; negative, 63.8%; P=0.003). Multivariate analysis showed that positive CD151 expression was not an independent factor for poor survival (P=0.096). CD151 expression is associated with tumor proliferation and invasiveness in ESCC.

Abstract 2052: MicroRNA-101 promotes apoptosis and suppresses proliferation in esophageal squamous cell carcinoma by repressing the polycomb group protein EZH2

Abstract Background: MicroRNAs (miRNAs) are ∼22 nucleotide non-coding RNA molecules that regulate gene expression post-transcriptionally. Although aberrant expression of miRNAs has been observed in various types of human cancer, their pathophysiologic role and their relevance to tumorigenesis are still largely unknown. Overexpression of the enhancer of zeste homolog 2 (EZH2) protein, a known repressor of gene transcription, has been reported to be associated with biological malignancy in several types of cancer. Purpose: The aim of this study was (1) to analyze the expression and function of microRNA-101 (miR-101) in esophageal squamous cell carcinoma (ESCC), (2) to elucidate the regulation of the EZH2 expression by miR-101 as its target gene. Methods: miR-101 expression was investigated in 27 matched normal esophageal epitheliums and ESCCs and 12 ESCC cell lines (TE1, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, KYSE30) by TaqMan quantitative real-time-polymerase chain reaction (qRT-PCR). To evaluate the role of miR-101, cell proliferation and apoptosis was analyzed with pre-miR-101-precursor transfected cells. Next, the regulation of EZH2 by miR-101 was analyzed by real-time PCR and western blotting. In addition, to elucidate the role of EZH2 gene in ESCC, we immunohistochemically examined the expression of EZH2 protein in 120 resected ESCC specimens and determined its association with the clinicopathological characteristics and prognosis. Results: In matched 27 resected specimens, the expression of miR-101 was significantly down-regulated in ESCCs compared with normal epitheliums. All 15 ESCC cell lines also showed down-regulation of miR-101. Pre-miR-101-precursor transfected cells promote cellular apoptosis and showed significant reduction in cellular proliferation. Pre-miR-101-precursor transfected cells significantly repressed EZH2 expression in both mRNA level and protein level. In the immunohistochemical analysis of EZH2, 78 (65%) showed positive staining, while 42 (35%) lacked the staining. Positive staining for EZH2 was associated with a poor prognosis significantly. Conclusions: miR-101 promotes apoptosis and suppresses proliferation in ESCC by repressing EZH2 expression as a tumor-suppressive-microRNA. It plays an important role in progression of ESCC and may serve as a novel therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2052.

Comparative proteomic analysis of β-catenin-mediated malignant progression of esophageal squamous cell carcinoma

beta-catenin has emerged as a key regulator of Wnt signaling pathway, which plays an important role in the development and progression of various cancers. Its accumulation in nucleus of the esophagus squamous epithelium might be the crucial step for the carcinogenesis of esophageal squamous cell carcinoma (ESCC). To detect the proteins correlated with beta-catenin function, we used the established cell lines of pGen-3-con (Eca109 cells transfected by control vector) and pGen-3-CTNNB1 (Eca109 cells transfected by beta-catenin siRNA) as cell models for further analysis. Two-dimensional gel electrophoresis technology was performed to separate the proteins of pGen-3-con and pGen-3-CTNNB1 cell lines, respectively. The differential protein spots were analyzed by software analysis, subjected to in-gel digestion, and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Consequently, 13 differentially expressed proteins between the two cell lines were identified, of which 14-3-3sigma, prohibitin, and nm23-H1 were further verified by western blotting and quantitative real-time reverse transcriptase-polymerase chain reaction. Then, the tissue microarray and immunohistochemical analysis were employed to research their relationship in ESCC and their corresponding normal mucosa tissues. The upregulation of prohibitin or the downregulation of 14-3-3sigma and nm23-H1 proteins was significantly associated with the proliferation, invasion depth, and lymph node metastasis of ESCC. There were statistically significant correlations between the expression of beta-catenin and the three proteins. The results presented here might provide potential protein markers to elucidate the mechanism of beta-catenin-mediated biologic characteristics for ESCC.