2695

Growth factor-induced synthesis of several proteases and their co-expressed inhibitors modulates the highly interdependent processes of cell migration and matrix proteolysis during tumor stromal invasion and the associated angiogenic response. Transcription of the gene encoding plasminogen activator inhibitor type-1 (PAI-1), a serine protease inhibitor expressed by angiogenic elements as well as tumor cells and an important regulator of barrier proteolysis and stromal invasion, is controlled by pro-migratory growth factors including members of the TGF-β family. Transplantation of squamous cell carcinoma in wild-type (PAI-1+/+) and PAI-1-null mice, moreover, indicated that PAI-1 expression is required for tumor invasion and the angiogenic response making this SERPIN an important target for cancer therapy. Comparison of the migratory properties of epidermal keratinocytes and endothelial cells isolated from PAI-1-null and wild-type mice, moreover, indicated that PAI-1 was essential for basal as well as TGF-β-stimulated invasion for both cell types. Migration through complex stromal “equivalent” 3-D barriers was inhibited in wild-type cells by transfection of a PAI-1 antisense construct. The motile phenotype by PAI-1-null cells and wild-type PAI-1 antisense transfectants could be rescued by addition of active PAI-1 protein although optimal migration was clearly dependent upon the concentration of PAI-1 used. This requirement for PAI-1 synthesis and the TGF-β1-dependent invasive phenotype necessitated an analysis of TGF-β1 signaling pathways leading to PAI-1 transcription. TGF-β1-induced PAI-1 expression in keratinocytes and endothelial cells was ERK-dependent. Both ERK activation and PAI-1 transcription were effectively attenuated by the MEK inhibitor PD98059 at concentrations that ablated TGF-β1-stimulated 2-D planar cell motility and stromal invasion. TGF-β1-dependent ERK phosphorylation and PAI-1 transcription were similarly decreased by preincubation with the src family kinase inhibitor PP1 prior to growth factor addition. Consistent with the pharmacologic results, a dominant-negative pp60c-src construct effectively suppressed TGF-β1-stimulated PAI-1 mRNA/protein synthesis in transfectant cell lines. Dominant-negative pp60c-src expression also attenuated TGF-β1-induced ERK activation suggesting that pp60c-src was upstream of ERK in this TGF-β1-initiated pathway. These data indicate that pp60c-src and MEK are critical elements in the TGF-β1-activated signaling cascade leading to PAI-1 transcription. This study has significantly increased the number of potential targets in the PAI-1 transcriptional control network that may be clinically useful in management of tumor metastasis and the associated angiogenic response. Supported by NIH grant GM-57242.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]