Li-Yan Xu

Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma: significant correlation with cell differentiation and tumour invasion

Background: Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin family. Recently, an elevated NGAL expression was reported in several types of cancers. However, the characteristics of NGAL expression in oesophageal squamous cell carcinoma (ESCC) are still unknown. Aim: To demonstrate the role of NGAL in ESCC. Methods: NGAL expression in 81 paraffin sections, including ESCC, normal mucosa, simple hyperplasia and dysplasia, and in 73 fresh specimens of ESCC was analysed by immunohistochemistry, western blot and gelatin zymography. Results: On immunohistochemical study, ESCC showed a diverse staining pattern for NGAL. However, only a weak positive signal was present within a restricted cytoplasmic area in the normal oesophageal epithelium. In dysplasia, altered NGAL expression could also be observed. On western blot study, NGAL expression level was found to be significantly higher in ESCC than in normal mucosa (p = 0.030), and to be positively correlated with cell differentiation. However, no significant association was observed between NGAL expression and cell proliferation. In addition, the enzymic activity of the NGAL/matrix metalloproteinase 9 complex was much higher in ESCC than in normal mucosa, and was significantly correlated with the depth of tumour invasion in zymography analysis (p = 0.006). Conclusions: The findings suggest that NGAL is involved in the differentiation pathway and invasive progression of ESCC.

MiR‐142‐3p as a potential prognostic biomarker for esophageal squamous cell carcinoma

microRNAs (miRNAs), small non‐coding RNAs, are always aberrantly expressed in many diseases including human cancers. The aim of this study was to examine and determine the clinical significance of hsa‐miR‐31, hsa‐miR‐142‐3p, hsa‐miR‐338‐3p, and hsa‐miR‐1261 expression in esophageal squamous cell carcinoma (ESCC).

MiRNA profile in esophageal squamous cell carcinoma: Downregulation of miR-143 and miR-145

AIM To investigate the expression profile of miRNA in esophageal squamous cell carcinoma (ESCC). METHODS The expression profile of miRNA in ESCC tissues was analyzed by miRNA microarray. The expression levels of miR-143 and miR-145 in 86 ESCC patients were determined by real-time polymerase chain reaction (PCR) using TaqMan assay. The mobility effect was estimated by wound-healing using esophageal carcinoma cells transfected with miRNA expression plasmids. RESULTS A set of miRNAs was found to be deregulated in the ESCC tissues, and the expression levels of miR-143 and -145 were significantly decreased in most of the ESCC tissues examined. Both miR-143 and miR-145 expression correlated with tumor invasion depth. The transfection of human esophageal carcinoma cells with miR-143 and miR-145 expression plasmids resulted in a greater inhibition of cell mobility, however, the protein level of the previously reported target of miR-145, FSCN1, did not show any significant downregulation. CONCLUSION These findings suggest that the deregulation of miRNAs plays an important role in the progression of ESCC. Both miR-143 and miR-145 might act as anti-oncomirs common to ESCC.

Tumor Initiating Cells in Esophageal Squamous Cell Carcinomas Express High Levels of CD44

BACKGROUND Esophageal Squamous Cell Carcinoma (ESCC) is a major subtype of esophageal cancer causing significant morbility and mortality in Asia. Mechanism of initiation and progression of this disease is unclear. Tumor initiating cells (TICs) are the subpopulation of cells which have the ability to self-renew, as well as, to drive initiation and progression of cancer. Increasing evidence has shown that TICs exist in a variety of tumors. However, the identification and characterization of TICs in esophageal carcinoma has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS to identify TICs in ESCC, ESCC cell lines including two primary cells were used for screening suitable surface marker. Then colony formation assay, drug resistant assay and tumorigenicity assay in immune deficient mice were used to characterize TICs in ESCC. We found that just the CD44 expression correlated with tumorigenicity in ESCC cell lines. And then induced differentiation of ESCC cells by all-trans retinoic acid treatment led to decreased expression of CD44. The FACS isolated cell subpopulations with high CD44 expression showed increased colony formation and drug resistance in vitro, as well as significantly enhanced tumorigenicity in NOD/SICD mice, as compared to the low expressing CD44 ESCC cells. CONCLUSIONS/SIGNIFICANCE our study has discovered a novel TIC surface marker, CD44, which can be utilized to enrich efficiently the TICs in ESCC. These findings will be useful for further studies of these cells and exploring therapeutic approaches.

Fascin Expression in Human Embryonic, Fetal, and Normal Adult Tissue

This study investigates the distribution of fascin in human embryonic, fetal, and normal adult tissues. Tissue microarray technology was used to perform immunohistochemical experiments on human embryos and fetuses at 4–22 weeks of gestation and adult specimens. Fascin was widely expressed in the nervous system. At 4 weeks of gestation, fascin was present in the neural tube. At 8–12 weeks of gestation, homogenous gene expression was seen in cells of the cerebellum and gastrointestinal tract. In later developmental stages and in adults, Purkinje cells of the cerebellum and glandular epithelium of the gastrointestinal tract showed no expression. Fascin was expressed in the cortex and medulla of the adrenal gland at 8–12 weeks of gestation, whereas immunoreactivity decreased from the zona glomerulosa through the zona reticularis and was essentially negative in the adrenal medulla of adults. Significant expression of fascin was seen throughout development in neurons, follicular dendritic cells of lymphoid tissue, basal layer cells of stratified squamous epithelia, mesenchyme, and vascular endothelial cells. Simple columnar epithelia of the biliary duct, colon, ovary, pancreas, and stomach were all negative for fascin expression. These results show that expression of fascin is time specific and highly tissue specific. Parallels between fascin expression in embryogenesis and carcinogenesis are discussed.

Expression and prognostic significance of THBS1, Cyr61 and CTGF in esophageal squamous cell carcinoma

BackgroundThrombospondin1 (THBS1), cystene-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF) are all involved in the transforming growth factor-beta (TGF-β) signal pathway, which plays an important role in the tumorigenesis. The purpose of this study is to explore the expression and prognostic significance of these proteins in esophageal squamous cell carcinoma (ESCC).MethodsWe used immunohistochemistry and western blotting to examine the expression status of THBS1, Cyr61 and CTGF in ESCC. Correlations of THBS1, Cyr61 and CTGF over-expressions with various clinicopathologic factors were also determined by using the Chi-square test or Fishers exact probability test. Survival analysis was assessed by the Kaplan-Meier analysis and the log-rank test. Relative risk was evaluated by the multivariate Cox proportional hazards model.ResultsTHBS1, Cyr61 and CTGF were all over-expressed in ESCC. THBS1 over-expression was significantly associated with TNM stage (P = 0.029) and regional lymph node involvement (P = 0.026). Kaplan-Meier survival analysis showed that over-expression of THBS1, Cyr61 or CTGF was related to poor survival of ESCC patients (P = 0.042, P = 0.020, P = 0.018, respectively). Multivariate Cox analysis demonstrated that Cyr61 and CTGF were independent factors in prognosis of ESCC.ConclusionCyr61, CTGF and THBS1 were all over-expressed in ESCC and might be new molecular markers to predict the prognosis of ESCC patients.

Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells

Ezrin, which crosslinks the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its roles and the underlying mechanism(s) remain unclear. In our study, we first showed that ezrin in ESCC cell is expressed in the nucleus as well as in the cytoplasm and plasma membrane. Then, by using RNAi, we revealed that interference of ezrin expression suppressed the growth, adhesion and invasiveness of ESCC cells. Tumorigenesis experiments revealed that ezrin may directly regulate tumor formation in vivo. To explore the molecular mechanisms through which ezrin contributes to the proliferation and invasiveness of ESCC cells, we used cDNA microarrays to analyze ezrin knockdown cells and the control cells; of 39,000 genes examined, 297 were differentially expressed upon ezrin knockdown, including some proliferation‐ and invasiveness‐related genes such as ATF3, CTGF and CYR61. Furthermore, pathway analysis showed that ezrin knockdown led to decreased activation of the TGF‐β and MAPK pathways, and ezrin‐mediated cell invasiveness alteration was dependent on the activation of these pathways. Finally, immunohistochemical staining on 80 ESCC specimens and 50 normal esophageal mucosae revealed that the expression levels of 3 altered genes involved in the regulation of cell proliferation and tumor metastasis, including CTGF, CYR61 and ATF3, were altered in ESCCs, and their expression pattern correlated with ezrin expression. Taken together, we propose that ezrin might function in the growth and invasiveness of ESCC cells through the MAPK and TGF‐β pathways.

Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells

Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5′-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

Autoantibodies as Potential Biomarkers for the Early Detection of Esophageal Squamous Cell Carcinoma

OBJECTIVES:Esophageal squamous cell carcinoma (ESCC) is one of the most frequent causes of cancer death worldwide and effective diagnosis is needed. We assessed the diagnostic potential of an autoantibody panel that may benefit early diagnosis.METHODS:We analyzed data for patients with ESCC and normal controls in a test cohort and a validation cohort. Autoantibody levels were measured against a panel of six tumor-associated antigens (p53, NY-ESO-1, matrix metalloproteinase-7 (MMP-7), heat shock protein 70 (Hsp70), peroxiredoxin VI (Prx VI), and BMI1 polycomb ring finger oncogene (Bmi-1)) by enzyme-linked immunosorbent assay.RESULTS:We assessed serum autoantibodies in 513 participants: 388 with ESCC and 125 normal controls. The validation cohort comprised 371 participants: 237 with ESCC, and 134 normal controls. Autoantibodies to at least 1 of 6 antigens demonstrated a sensitivity/specificity of 57% (95% confidence interval (CI): 52–62%)/95% (95% CI: 89–98%) and 51% (95% CI: 45–57%)/96% (95% CI: 91–99%) in the test and validation cohorts, respectively. Measurement of the autoantibody panel could differentiate early-stage ESCC patients from normal controls (sensitivity 45% (95% CI: 32–59%) and specificity 95% (95% CI: 89–98%) in the test cohort; 46% (95% CI: 35–58%) and 96% (95% CI: 91–99%) in the validation cohort). In either cohort, no significant differences were seen when patients were subdivided by age, gender, smoking status, size of tumor, site of tumor, depth of tumor invasion, histological grade, lymph node status, TNM stage, or early-stage and late-stage groups.CONCLUSIONS:Measurement of an autoantibody response to multiple tumor-associated antigens in an optimized panel assay, to help discriminate early-stage ESCC patients from normal controls, may aid in early detection of ESCC.

Involvement of CYR61 and CTGF in the fascin-mediated proliferation and invasiveness of esophageal squamous cell carcinomas cells.

Fascin is overexpressed in esophageal squamous cell [corrected] carcinoma (ESCC) and involved in the proliferation and invasiveness of ESCC cells. In this study, we retrospectively examined the expression of fascin in ESCC samples by immunohistochemistry and revealed that overexpression of fascin was related to poor patient survival. RNAi-mediated knockdown of fascin in ESCC cells significantly inhibited cell proliferation and invasiveness, whereas forced expression of fascin in immortalized esophageal epithelial cells accelerated cell proliferation and invasiveness. To explore the underlying mechanism, cDNA microarray was performed to identify the differential gene expression profiles between a fascin-depleted cell line by RNAi and the corresponding control ESCC cells. Results showed that 296 genes were differentially expressed on fascin depletion. In this study, we focused on two down-regulated genes: CYR61 and CTGF. We found that restored expression of either CYR61 or CTGF led to a recovery of the suppression of cellular proliferation and invasiveness induced by down-regulation of fascin expression; the protein level of CYR61 and CTGF were up-regulated in ESCCs and their expression pattern correlated with fascin overexpression. Finally, analysis of signal transduction revealed that fascin affected the expressions of CYR61 and CTGF through transforming growth factor (TGF)-beta pathway. Taken together, we propose that fascin regulates the proliferation and invasiveness of ESCC cells by modulating the expression of CTGF and CYR61 via TGF-beta pathway.