Up-regulation of urokinase-type plasminogen activator by insulin-like growth factor-I depends upon phosphatidylinositol-3 kinase and mitogen-activated protein kinase kinase

Elevated levels of urokinase plasminogen activator-1 (uPA) and the insulin- like growth factor-1 receptor (IGF-IR) are associated with breast cancer re currence and decreased survival. It is possible that activation of IGF-IR a nd elevations in uPA are mechanistically linked, Our laboratory recently sh owed that insulin-like growth factor-I (IGF-I) induces uPA protein and mRNA in the breast cancer cell line MDA-MB-231. We also found that IGF-IR and u PA were commonly overexpressed in primary breast cancers. In this study, we investigated the signal transduction pathway through which IGF-I regulates uPA, Phosphatidylinositol 3-kinase, mitogen-activated protein kinase kinas e, and p70 kinase were inhibited with LY294002, PD98059, and rapamycin, res pectively. Induction of uPA protein by IGF-I was partially inhibited by LY2 94002 (60% inhibition) or PD98059 (30% inhibition) but not by rapamycin, Th e production of uPA protein induced by IGF-I was blocked up to 90% by the t yrosine kinase inhibitor herbimycin A, Furthermore, herbimycin A suppressed the phosphorylation of AKT and Erk1/2. Next, we tested the impact of the s ignal transduction inhibitors on uPA gene expression, Both LY294002 and PD9 8059 were required to completely inhibit uPA mRNA expression, whereas each drug alone resulted in approximately 50% reduction in uPA expression. Next, using a minimal uPb-luciferase promoter construct containing the binding s ites for the AP-I and Ets transcription factors, we observed that IGF-I sti mulated the uPA promoter via these sites. Furthermore, both Ly294002 and PD 98059 were necessary to block IGF-I-stimulated uPA-Luc activity. In summary , we conclude that IGF-I requires both phosphatidylinositol 3-kinase and mi togen-activated protein kinase kinase-dependent pathways to optimally induc e uPA expression. These findings suggest that the development of drugs targ eting these pathways may benefit breast cancer patients at a high risk of r ecurrence, such as those who have primary tumors overexpressing IGF-IR and uPA.