| Human fascin (fascin-1) gene is located at 7p22. It encodes a 55 kD spherical protein which is associated with the microfilament bundle F-actin and mainly plays a role in cell division, morphological alterations, cell movement and signal transduction pathways.Our group found previously that fascin was an important new oncogene, significantly overexpressed in the development of esophageal cancers, and acting in the proliferation and invasion of cancer cells. However, the regulatory mechanism of its overexpression is still unclear.In the previous studies, we determined that the 5′transcriptional regulatory region of ?74~?41 was the core promoter segment of fascin in esophageal cancer cells. In order to further identify the key transcription activation element of fascin, we first employed the approaches of bioinformatics analysis, site-directed mutagenesis, dual luciferase reporter gene activity assay, electrophoretic mobility shift assay (EMSA), super-EMSA and other experimental techniques, narrowing the key element of fascin to the -70~-60 segment, which can bind to recombinant transcription factor Sp1 detected from the nuclear extracts of esophageal cancer cells.Secondly, we utilized the techniques of gene overexpression, RNA interference, quantitative RT-PCR, western blot and reporter gene activity assay to show that the transcription factor Sp1 not only combined with the -70~-60 section of fascin, but also activated mRNA transcription and protein translation of fascin.In order to identify the upstream kinase that phosphorylated and activated the transcription factor Sp1 in cancer cells, we treated the esophageal cancer cells with eight kinds of specific kinase inhibitors respectively and then detected the expression of fascin and the activity of the reporter gene. The results showed that the MEK1/2 specific inhibitor, not only reduced the phosphorylation levels of both the kinase ERK1/2 and the transcription factor Sp1 significantly, but also inhibited the expression of fascin and the activity of reporter gene in esophageal cancer cells. When we down-regulated the expression level of ERK1/2 using RNAi, the expression level of fascin was effectively decreased. These results strongly suggest that ERK1/2 is an upstream kinase of fascin that directly phosphorylates the transcription factor Sp1, and activates the transcription of fascin in esophageal cancer cells.Finally, to identify the extracellular molecules that activate the signaling pathway of MEK1/2→ERK1/2→Sp1→fascin, we also investigated the impact of epidermal growth factor EGF and transforming growth factor TGF-β1 on fascin expression in esophageal cancer cells respectively. The results show that when treated with EGF (50 ng/ml) for 5 min to 24 h, the mRNA and protein levels of fascin gradually increased. In contrast, TGF-β1 (10 ng/ml) induced no significant effect on fascin expression. Our experimental data from the reporter gene activities, EMSA, western blot and immunoprecipitation supported that, with the stimulation of EGF, the binding capacity of Sp1 with the ?70~?60 core components and the activity of fascin promoter were significantly enhanced, accompanied by significantly increased phosphorylation of ERK1/2 and Sp1. If MEK1/2 specific inhibitors were used to block MEK1/2→ERK1/2→Sp1 signaling pathway before EGF stimulation, EGF could not induce the expression of fascin. These results indicate that EGF mediates the overexpression of fascin by activating the signaling pathway of MEK1/2→ERK1/2→Sp1 in esophageal cancer cells.Summarizing the results above, our study investigated the mechanism of fascin overexpression in esophageal cancer cells and the main conclusions of the study include: 1) the key transcription acitvation elements of fascin is located at the 5′end of ?70~?60 segment; 2) phosphorylated Sp1 is the key transcription activation factor of fascin; 3) fascin overexpression is mainly mediated by MEK1/2→ERK1/2→Sp1 cellular signal transduction pathway in esophageal cancer cells; 4) EGF is the extracellular molecule that activates fascin overexpression in cancer cells. This study helps reveal the mechanism of fascin overexpression and offers the insights for therapeutic interventions in esophageal cancers.
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