Dominant-negative transcription factor AP-2 augments SB-2 melanoma tumor growth in vivo.
We have previously demonstrated that the transition of melanoma to the metastatic phenotype is associated with a loss of expression of the transcription factor AP-2. To further investigate the role of AP-2 in the progression of human melanoma, we attempted to inactivate AP-2 in primary cutaneous SB-2 melanoma cells by using a dominant-negative AP-2, or AP-2B, gene. AP-2B is an alternatively spliced AP-2 variant capable of inhibiting AP-2 trans-activator function. Stable transfection of primary cutaneous melanoma SB-2 cells with the dominant-negative AP-2B gene was confirmed by RT--PCR and Northern blot analyses. Electromobility shift assay using nuclear extracts from these cell lines demonstrated decreased functional binding of AP-2B-transfected cells to the AP-2 consensus binding sequence compared with neo-transfected controls. In addition, CAT activity driven by a construct containing the AP-2 consensus binding sequence was downregulated in the AP-2B transfected cells, indicating AP-2 activity was quenched in the transfected cells. Orthotopic (subcutaneous) injection of the dominant-negative (AP-2B)-transfected cell lines into nude mice increased their tumorigenicity compared to control neo-transfected cells. The AP-2B-transfected cells displayed an increase in MMP-2 expression (by Northern blot) and MMP-2 activity (by zymography), which resulted in an increase in invasiveness through Matrigel-coated filters. The AP-2B-transfected tumors also displayed an increase in MMP-2 expression, microvessel density, and angiogenesis in vivo. These results demonstrate that inactivation of AP-2 contributes to the progression of melanoma, at least partially via deregulation of the MMP-2 gene.
Gershenwald
J E
JE
Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Sumner
W
W
Calderone
T
T
Wang
Z
Z
Huang
S
S
Bar-Eli
M
M
D017398:Alternative Splicing; D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis; D015152:Blotting, Northern; D002465:Cell Movement; D003094:Collagen; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D004338:Drug Combinations; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D007797:Laminin; D008175:Lung Neoplasms / Q000098:blood supply / Q000201:enzymology / Q000556:secondary; D008207:Lymphatic Metastasis; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000235:genetics; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D008546:Melanoma, Experimental / Q000098:blood supply / Q000201:enzymology / Q000473:pathology* / Q000556:secondary; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000473:pathology; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D011509:Proteoglycans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D000951:Antigens, Neoplasm; D003470:Culture Media; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D007797:Laminin; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011509:Proteoglycans; D050656:Transcription Factor AP-2; D014157:Transcription Factors; C059485:matrigel; D003094:Collagen; D020778:Matrix Metalloproteinase 2