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Inhibition of Urokinase Activity Reduces Primary Tumor Growth and Metastasis Formation in a Murine Lung Carcinoma Model

Ingrid Henneke¹, Susanne Greschus^2,, Rajkumar Savai¹, Martina Korfei¹, Philipp Markart¹, Poornima Mahavadi¹, Ralph T. Schermuly^1,4, Malgorzata Wygrecka¹, Jörg Stürzebecher^3,, Werner Seeger¹, Andreas Günther^1,5,* and Clemens Ruppert^1,*

¹ University of Giessen Lung Center (UGLC), Department of Internal Medicine, Medical Clinic II; ² Department of Neuroradiology, Justus-Liebig-University, Giessen; ³ Friedrich-Schiller University, Center for Vascular Biology and Medicine, Erfurt; ⁴ Max-Planck-Institute for Heart and Lung Research, Department of Lung Development and Remodeling, Bad Nauheim; and ⁵ Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany

Correspondence and requests for reprints should be addressed to Andreas Günther, M.D., Universty of Giessen Lung Center (UGLC), Dept. of Internal Medicine, Medical Clinic II, Klinikstr. 36, 35392 Giessen, Germany. E-mail: Andreas.Guenther{at}uglc.de

Rationale: Lung cancer is the most common malignancy in humans. Urokinase (uPA) plays a crucial role in carcinogenesis by facilitating tumor cell invasion and metastasis.

Objectives: We investigated the effect of the highly specific urokinase inhibitor CJ-463 (benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide) on tumor growth, metastasis formation, and tumor vascularization in the murine Lewis lung carcinoma (LLC) and a human small lung cancer model.

Methods: A quantity of 3 x 10⁶ LLC cells were subcutaneously injected into the right flank of C57Bl6/N mice, uPA knock out, and uPA receptor knockout mice. Seven days later mice were randomized to receive intraperitoneally either saline (control group), CJ-463 (10 and 100 mg/kg, twice a day), or its ineffective stereoisomer (10 mg/kg, twice a day). Tumor volume was measured every second day and metastasis formation was monitored by volumetric-computed tomography. Twelve days after onset of treatment mice were killed and tumors were prepared for histologic examination.

Measurements and Main Results: Treatment with CJ-463 resulted in a significant inhibition of primary tumor growth, with the highest efficacy seen in the 100 mg/kg group. In addition, histological analysis of the lung revealed a significant reduction in lung micrometastasis in the 100 mg/kg group. Similarly, a reduced seeding of tumor cells into the lung after intravenous injection of LLC cells was observed in inhibitor-treated mice. In these mice, treatment with CJ-463 appeared not to significantly alter the relative extent of tumor vascularization. In vitro, proliferation of LLC cells remained unchanged upon inhibitor treatment. CJ-463 was found to similarly reduce tumor growth in uPA receptor knockout mice, but was ineffective in uPA knockout mice.

Conclusions: Our results suggest that synthetic low-molecular-weight uPA-inhibitors offer as novel agents for treatment of lung cancer.

Key Words: SCLC • NSCLC • plasminogen activator • angiogenesis • active site inhibitor

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Prognosis of lung cancer is still poor and there is an unmet clinical need for new treatment modalities and for studies assessing new strategies and compounds for anticancer therapy. The urokinase (uPA) plasminogen activator system plays a crucial role in carcinogenesis by facilitating tumor cell invasion and metastasis and might be a potential target for new treatment options. What This Study Adds to the Field Here we show that inhibition of uPA activity using the highly specific low-molecular-weight uPA inhibitor CJ-463 not only reduces metastasis formation but also results in a significant reduction of the primary tumor growth in both an orthotopic mouse model of Lewis Lung carcinoma and of the human small cell lung cancer model in SCID mice. The beneficial effect was shown to be mediated via inhibition of stromal uPA. Thus, the uPA system may be a suitable target for anticancer strategies, and highly specific low-molecular-weight uPA inhibitors could lead to an improvement in cancer therapy.