Reduction Of AP-2α Results In Up-regulation Of EGF Receptor And An Increase In Proliferation And Invasion Of Human Colon Cancer SW480 Cells In Vitro

ObjectiveTranscription factor AP-2α, a sequence-specific DNA binding transcription factor of 52kD that belongs to AP-2 family, is required for both morphogenesis and cell differentiation. So far, reduction or loss of AP-2αexpression has been detected in several carcinomas including colon cancer, suggesting a tumor-suppressor-like role for AP-2αin colon cancer. This study was to explore the exact mechanism by which reduction or loss of AP-2αcontributes to colon cancer progression.MethodsThe pcDNA3.1(+)-hAP-2αconstruct was created by cloning hAP-2αcDNA into the EcoRI site of pcDNA3.1(+). LipofectamineTM 2000 Reagent was used to mediate the transfection of pcDNA3.1(+)-hAP-2αand pcDNA3.1(+) into SW480 human colon cancer cells. To examine the mRNA and protein level of AP-2αin the cells of each group, reverse transcription-PCR analysis (RT-PCR) and Western Blot analysis were performed. The DNA-binding activity of the exogenous AP-2αprotein in the transfected cells was analyzed by Electrophoretic mobility shift assay (EMSA). Proliferative activities of the cells of each group were evaluated by MTT assay 24,48 or 72 h after the transfection. Anchorage-independent growth assay were performed to detect the effect of AP-2αon the malignant property of SW480 cells. To evaluate the role of AP-2αin SW480 invasion potential in vitro, the method of Transwell invasion assay was performed.A fragment of EGF receptor gene promoter containing the five putative AP-2αbinding sites was obtained from the genomic DNA of SW480 cells. This fragment was successfully cloned into the pMD-18T vector before sequenced. To determine whether AP-2αhas direct interaction with the EGF receptor promoter derived from the genomic DNA of SW480 cells, oligonucleotides containing five putative AP-2αbinding sites were synthesized, and EMSAs were performed. To further assess the effect of the AP-2αprotein on the EGF receptor expression in SW480 colon cancer cells, the mRNA and protein level of endogenous EGF receptor in the AP-2α-transfected cells were detected by the use of RT-PCR and Western Blot analysis respectively. ResultsRT-PCR analysis detected high level of mRNA in the AP-2α-transfected SW480 cells but only residual levels in neo-transfected cells. Expression of AP-2αwas observed in the AP-2α-transfected SW480 cells as compared with the neo-transfected cells. EMSA results revealed high DNA-binding activity of AP-2αin the AP-2α-transfected SW480 cells. MTT results displayed that the overexpression of AP-2αinhibited the proliferation of SW480 cells. The cell growth in soft agar was also inhibited after being transfected with AP-2αgene. Transwell invasion assay showed that AP-2αsignificantly reduced the invasive capacity of SW480 cells as compared with the neo-transfected cells.Sequencing of the fragment of EGF receptor gene promoter region revealed no abnormalities such as deletions, rearrangement, and mutations that are responsible for the aberrant effect of trans-acting factors on the promoter, subsequent perturbation of EGF receptor gene expression. EMSAs in our study showed that AP-2αre-expressed in SW480 colon cancer cells binds directly to the oligonucleotides containing three putative AP-2αbinding sites. Transfection of AP-2αgene into SW480 colon cancer cells down-regulated the endogenous EGF receptor mRNA and expression of protein.ConclusionThe expression of EGF receptor is probably regulated by AP-2αand that forced over-expression of AP-2αsuppresses the growth and invasion potential of human colon cancer cells, possibly through down-regulating EGF receptor gene expression.