Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells

Carcinogenesis. 2006 Jun;27(6):1134-45. doi: 10.1093/carcin/bgi352. Epub 2006 Feb 12.

Abstract

Metastasis-supporting physiological alterations are regulated by cell signaling molecules, which target signal transduction pathways and gene expression. Osteopontin (OPN) overexpression may represent a key molecular event in cancer metastasis. In this study, using metastatic 4T1 and non-metastatic 4T07 murine mammary cancer cell lines, we demonstrate that 4T1 cells exhibit significantly increased OPN, integrin-linked kinase (ILK), matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (uPA) expression in contrast to 4T07 cells. Blockade of OPN binding to 4T1 cell-surface integrins by the competitive ligand inhibitor, RGD, or a blocking antibody to alphavbeta3 integrin decreases OPN, ILK, MMP-2 and uPA expression. Conversely, exposure of 4T07 cells to exogenous OPN increases ILK, MMP-2 and uPA levels. Further experiments demonstrate that OPN-alphavbeta3 integrin binding in 4T1 with subsequent activation of ILK results in binding of AP-1 to MMP-2 and uPA promoter and increased in vitro promoter activation, as measured by transient transfection assays using MMP-2 and uPA promoter-reporter constructs. AP-1 activity is ablated by co-transfection of DN-ILK or exposure to RGD. Finally, functional correlative assays demonstrate that inhibition of ILK activity or RGD-mediated blockade of alphavbeta3 integrin binding significantly inhibits in vitro invasion, migration and invasion properties of 4T1 cells. In addition, uPA and MMP-2 have overlapping contributions to 4T1 migration and invasion characteristics. However, OPN and ILK activities contribute to 4T1 adhesion activities via mechanisms that are independent of uPA and MMP-2. Our results indicate that binding of an RGD-bearing ligand, such as OPN, to integrin receptors in metastatic 4T1 cells transcriptionally mediates MMP-2, uPA and OPN expression through ILK-dependent AP-1 activity and significantly increases in vitro functional correlates of metastasis. In 4T1 murine mammary cancer cells, we conclude that OPN mediates metastatic behavior, in part, through upregulation of MMP-2 and uPA protein expression.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Epithelial Cells / cytology*
  • Gene Expression Regulation*
  • Gene Expression Regulation, Neoplastic*
  • Mammary Glands, Animal / cytology*
  • Mammary Glands, Animal / metabolism
  • Mammary Neoplasms, Animal / metabolism
  • Matrix Metalloproteinase 2 / biosynthesis*
  • Matrix Metalloproteinase 2 / metabolism
  • Mice
  • Neoplasm Invasiveness
  • Neoplasm Metastasis
  • Osteopontin
  • Protein Serine-Threonine Kinases / physiology*
  • Sialoglycoproteins / metabolism*
  • Urokinase-Type Plasminogen Activator / biosynthesis*

Substances

  • Sialoglycoproteins
  • Spp1 protein, mouse
  • Osteopontin
  • integrin-linked kinase
  • Protein Serine-Threonine Kinases
  • Urokinase-Type Plasminogen Activator
  • Matrix Metalloproteinase 2