Dominant-negative transcription factor AP-2 augments SB-2 melanoma tumor growth in vivo

Oncogene. 2001 Jun 7;20(26):3363-75. doi: 10.1038/sj.onc.1204450.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing
  • Animals
  • Antigens, Neoplasm / analysis
  • Blotting, Northern
  • Cell Movement
  • Collagen
  • Culture Media
  • DNA-Binding Proteins / deficiency
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / physiology*
  • Drug Combinations
  • Genes, Dominant*
  • Genes, Reporter
  • Humans
  • Laminin
  • Lung Neoplasms / blood supply
  • Lung Neoplasms / enzymology
  • Lung Neoplasms / secondary
  • Lymphatic Metastasis
  • Male
  • Matrix Metalloproteinase 2 / biosynthesis
  • Matrix Metalloproteinase 2 / genetics
  • Melanoma / genetics
  • Melanoma / pathology*
  • Melanoma, Experimental / blood supply
  • Melanoma, Experimental / enzymology
  • Melanoma, Experimental / pathology*
  • Melanoma, Experimental / secondary
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Neoplasm Invasiveness
  • Neoplasm Metastasis
  • Neoplasm Transplantation
  • Neovascularization, Pathologic / genetics
  • Neovascularization, Pathologic / pathology
  • Platelet Endothelial Cell Adhesion Molecule-1 / analysis
  • Proteoglycans
  • Reverse Transcriptase Polymerase Chain Reaction
  • Skin Neoplasms / genetics
  • Skin Neoplasms / pathology*
  • Transcription Factor AP-2
  • Transcription Factors / deficiency
  • Transcription Factors / genetics
  • Transcription Factors / physiology*
  • Transfection

Substances

  • Antigens, Neoplasm
  • Culture Media
  • DNA-Binding Proteins
  • Drug Combinations
  • Laminin
  • Platelet Endothelial Cell Adhesion Molecule-1
  • Proteoglycans
  • Transcription Factor AP-2
  • Transcription Factors
  • matrigel
  • Collagen
  • Matrix Metalloproteinase 2