Transforming growth factor beta-1 and amphiregulin act in synergy to increase the production of urokinase-type plasminogen activator in transformed breast epithelial cells

Amphireguline (AR) is an epidermal growth factor (EGF)-related peptide that seems to play an important role in breast cancer progression. We have demonstrated recently that suppression of AR expression in transformed breast epithelial cells considerably reduced both size and neovascularization of tumors developed in nude mice. We show that the reduction of AR expression allowed to an important decrease of the levels of urokinase-type plasminogen activator (uPA) and transforming growth factor-beta1 (TGFbeta1). According to these data, exogenous AR (10(-10) M-10(-8) M) stimulated the production of uPA and TGFbeta1 in AR antisense-transfected A2-15 and A2-P17F25 cells. The addition of 2 x 10(-10) M TGFbeta1 into culture medium increased the level of uPA produced by AR-expressing parental cells but not by A2-15 and A2-P17F25 cell clones. Whereas AR alone stimulated uPA production to 200% of control, combined AR and TGFbeta1 treatment increased protease level in A2-15 and A2-P17F25 cells to 500-600% of control, demonstrating a synergism between TGFbeta1 and AR. This was accompanied by an important augmentation of the number of tumoral cells that invaded matrigel in vitro. The synergistic induction of uPA protein resulted of an early and transient augmentation of steady state mRNA level and was blocked in the presence of the MAP kinase kinase inhibitor PD098059, strongly suggesting that synergistic effect of AR and TGFbeta1 on uPA expression required MAPK pathway. This data demonstrates concerted action between AR and TGFbeta1 that may have profound effect on protease production and consequently on breast cancer progression.