Abstract
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.
MeSH terms
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Adult
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Aged
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Cell Line, Tumor
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Cytoskeletal Proteins / biosynthesis*
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Cytoskeletal Proteins / genetics
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Disease-Free Survival
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Esophageal Neoplasms / genetics
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Esophageal Neoplasms / metabolism*
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Esophageal Neoplasms / mortality
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Female
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Gene Expression Regulation, Neoplastic*
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Humans
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MAP Kinase Kinase 1 / genetics
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MAP Kinase Kinase 1 / metabolism
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MAP Kinase Kinase 2 / genetics
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MAP Kinase Kinase 2 / metabolism
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MAP Kinase Kinase 4 / genetics
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MAP Kinase Kinase 4 / metabolism
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / genetics
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Male
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Microarray Analysis
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Middle Aged
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Mitogen-Activated Protein Kinase 1 / genetics
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / genetics
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Mitogen-Activated Protein Kinase 3 / metabolism
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Mutagenesis, Site-Directed
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Neoplasms, Squamous Cell / genetics
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Neoplasms, Squamous Cell / metabolism*
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Neoplasms, Squamous Cell / mortality
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Phosphorylation / genetics
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Protein Kinase Inhibitors / pharmacology
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Proto-Oncogene Proteins c-fyn / genetics
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Proto-Oncogene Proteins c-fyn / metabolism
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Proto-Oncogene Proteins c-jun / genetics
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Proto-Oncogene Proteins c-jun / metabolism
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Response Elements / genetics
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Retrospective Studies
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Sp1 Transcription Factor / genetics
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Sp1 Transcription Factor / metabolism*
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Survival Rate
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Transcription Factor AP-1 / genetics
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Transcription Factor AP-1 / metabolism*
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p38 Mitogen-Activated Protein Kinases / genetics
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p38 Mitogen-Activated Protein Kinases / metabolism
Substances
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Cytoskeletal Proteins
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Protein Kinase Inhibitors
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Proto-Oncogene Proteins c-jun
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Sp1 Transcription Factor
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Transcription Factor AP-1
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ezrin
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MAP2K2 protein, human
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Proto-Oncogene Proteins c-fyn
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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p38 Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP Kinase Kinase 4
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MAP2K1 protein, human