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Regulation by Epidermal Growth Factor of Human Squamous Cell Carcinoma Plasminogen Activator-mediated Proteolysis of Extracellular Matrix^1,2,

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510

The interaction of epidermal growth factor (EGF) with specific cell surface receptors initiates biochemical events in target cells which result in cellular proliferation and differentiation. In this report the regulation of extracellular-associated plasminogen activator (PA) production by EGF in human squamous cell carcinomas and its influence on tumor cell-mediated degradation of extracellular matrix (ECM) is described. The studies utilized the vulvar carcinoma cell line A431, which possesses an unusually large number of EGF receptors (EGF-Rs), and two A431 EGF-R expression variants (A5 and A7), which contain up to 20-fold fewer cell surface EGF-Rs. EGF enhanced the production of urokinase (u) PA activity by two- to threefold in A431 tumor cells, in a concentration-dependent manner, following a 24-h treatment, as determined by substrate hydrolysis assays, while no changes in tissue-type PA occurred. In contrast, A5 and A7 tumor cells failed to demonstrate such a response. Time course studies of the EGF-mediated induction of uPA activity in A431 tumor cells indicated that within 8 h after exposure to EGF, a twofold increase above basal untreated control levels was observed using the substrate hydrolysis assay. EGF increased the steady state levels of uPA mRNA threefold in A431 tumor cells following a 24-h treatment, while in contrast, no such response was observed in EGF-R variant tumor cells. In accord with an EGF enhancement of uPA mRNA levels in A431 tumor cells, a similar increase of two- to threefold in the de novo synthesis of [³⁵S]methionine-radiolabeled uPA was observed by immunoprecipitation following EGF treatment, while no measurable increase was observed in the EGF-R tumor variants. A431 tumor cells progressively degraded [³H]glucosamine-radiolabeled bovine corneal subendothelial ECM in the presence of EGF, resulting in 8.7-, 4.3-, and 1.7-fold increases above untreated control values, after a 48-h exposure to 100, 10, and 1 ng/ml of EGF, respectively. In contrast, A5 and A7 tumor cells did not demonstrate an increase in ECM degradation in the presence of EGF, even though these tumor cells possessed the ability to degrade ECM in the absence of the growth factor. The observed increase in ECM degradation mediated by EGF in A431 tumor cells was dependent upon the presence of plasminogen and could be inhibited by an anticatalytic uPA monoclonal antibody. The findings suggest that EGF may be involved in the regulation of the proteolytically active phenotype associated with certain tumor cells and thereby may be a potential chemotherapeutic target for limiting the invasion and metastasis of squamous cell carcinoma.

¹ Portions of this work were presented at the Annual Meeting of the American Association for Cancer Research, New Orleans, May, 1988.

² This research was supported in part by USPHS Grant CA-02817 from the National Cancer Institute and by American Cancer Society Grant PF-3142.

³ American Cancer Society Postdoctoral Fellow.

⁴ To whom reprint requests should be addressed, at Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

Received 8/ 3/88. Revised 12/20/89. Revised 3/20/89. Accepted 3/22/89.