Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells.
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.
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Zhiyong
Z
Mi
Hongtao
H
Guo
Philip Y
PY
Wai
Chengjiang
C
Gao
Paul C
PC
Kuo
D000818:Animals; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology*; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000378:metabolism; D015674:Mammary Neoplasms, Animal / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis* / Q000378:metabolism; D051379:Mice; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D053495:Osteopontin; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012795:Sialoglycoproteins / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis*; D000099200:Scaffold Protein ILK
D020778:Matrix Metalloproteinase 2; D053495:Osteopontin; D017346:Protein Serine-Threonine Kinases; D012795:Sialoglycoproteins; D014568:Urokinase-Type Plasminogen Activator; D000099200:Scaffold Protein ILK; C506896:Spp1 protein, mouse