Journal Article

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

Zhiyong Mi, Hongtao Guo, Philip Y. Wai, Chengjiang Gao and Paul C. Kuo

in Carcinogenesis

Volume 27, issue 6, pages 1134-1145
Published in print June 2006 | ISSN: 0143-3334
Published online February 2006 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgi352
Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells

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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 αvβ3 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–αvβ3 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 αvβ3 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.

Keywords: AP-1, Activating protein – 1; ChIP, chromatic immunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; DN-ILK, dominant negative integrin linked kinase; EMSA, electrophoretic mobility shift assays; ILK, integrin-linked kinase; MMP-2, matrix mettalloproteinase-2; OPN, osteopontin; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RGD, Arginine-Glycine-Aspartic acid; RGE, Arginine Glycine-Glutamic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; uPA, urokinase-type plasminogen activator; WI-ILK, Wild type integrin linked kinase

Journal Article.  9315 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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