Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor, in Human Cancer Cells with Acquired Resistance to Antiepidermal Growth Factor Receptor Therapy Fortunato Ciardiello, 1 Roberto Bianco, 2 Roberta Caputo, 2 Rosa Caputo, 2 Vincenzo Damiano, 2 Teresa Troiani, 2 Davide Melisi, 2 Ferdinando De Vita, 1 Sabino De Placido, 2 A. Raffaele Bianco, 2 and Giampaolo Tortora 2 1 Cattedra di Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F Magrassi e A Lanzara,” Seconda Universita ` degli Studi di Napoli, Naples, Italy, and 2 Cattedra di Oncologia Medica, Dipartimento di Endocrinologia e Oncologia Molecolare e Clinica, Universita ` degli Studi di Napoli Federico II, Naples, Italy ABSTRACT Purpose: The epidermal growth factor receptor (EGFR) autocrine signaling pathway is involved in cancer development and progression. EGFR inhibitors such as C225 (cetuximab), a chimeric human-mouse anti-EGFR monoclonal antibody, and ZD1839 (gefitinib), a small mol- ecule EGFR-selective tyrosine kinase inhibitor, are in ad- vanced clinical development. The potential emergence of cancer cell resistance in EGFR-expressing cancers treated with EGFR inhibitors could determine lack of activity of these drugs in some cancer patients. Vascular endothelial growth factor (VEGF) is secreted by cancer cells and plays a key role in the regulation of tumor-induced endothelial cell proliferation and permeability. ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR tyrosine kinase. Experimental Design: The antitumor activity of ZD1839, C225, and ZD6474 was tested in athymic mice bearing human GEO colon cancer xenografts. GEO cell lines resistant to EGFR inhibitors were established from GEO xenografts growing in mice treated chronically with ZD1839 or C225. Expression of EGFR was evaluated by flow cytometry. Expression of various proteins involved in intracellular cell signaling was assessed by Western blotting. Tumor growth data were evaluated for statistical signifi- cance using the Student’s t test. All Ps were two-sided. Results: Although chronic administration of optimal doses of C225 or ZD1839 efficiently blocked GEO tumor growth in the majority of mice, tumors slowly started to grow within 80 –90 days, despite continuous treatment. In contrast, continuous treatment of mice bearing established GEO xenografts with ZD6474 resulted in efficient tumor growth inhibition for the entire duration of dosing (up to 150 days). ZD6474 activity was also determined in mice pre- treated with ZD1839 or C225. When GEO growth was apparent after 4 weeks of treatment with EGFR inhibitors, mice were either re-treated with EGFR inhibitors or treated with ZD6474. GEO tumor growth was blocked only in mice treated with ZD6474, whereas tumor progression was ob- served in mice re-treated with C225 or ZD1839. GEO tu- mors growing during treatment with C225 or with ZD1839 were established as cell lines (GEO-C225-RES and GEO- ZD1839-RES, respectively). Cell membrane-associated EGFR expression was only slightly reduced in these cell lines compared with parental GEO cells. Western blotting re- vealed no major change in the expression of the EGFR ligand transforming growth factor  of bcl-2, bcl-xL, p53, p27, MDM-2, akt, activated phospho-akt, or mitogen-acti- vated protein kinase. However, both GEO-C225-RES and GEO-ZD1839-RES cells exhibited a 5–10-fold increase in activated phospho-mitogen-activated protein kinase and in the expression of cyclooxygenase-2 and of VEGF compared with GEO cells. GEO-C225-RES and GEO-ZD1839-RES growth as xenografts in nude mice was not significantly affected by treatment with either C225 or ZD1839 but was efficiently inhibited by ZD6474. Conclusions: Long-term treatment of GEO xenografts with selective EGFR inhibitors results in the development of EGFR inhibitor-resistant cancer cells. Growth of EGFR inhibitor-resistant tumors can be inhibited by ZD6474. These data indicate that inhibition of VEGF signaling has potential as an anticancer strategy, even in tumors that are resistant to EGF inhibitors. INTRODUCTION Growth factors regulate cancer development through sev- eral mechanisms. These include uncontrolled cell growth caused Received 07/15/03; accepted 10/13/03. Grant support: Grants from the Associazione Italiana per la Ricerca sul Cancro and Consiglio Nazionale delle Ricerche-Ministero dell’ Istruzi- one, Universita `, Ricerca 449/97-99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Rosa Caputo is the recipient of a postdoctoral fellowship from the Fondazione Italiana per la Ricerca sul Cancro. Requests for reprints: Fortunato Ciardiello, Cattedra di Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi e A. Lanzara,” Seconda Universita ` degli Studi di Napoli, Via S. Pansini, 5-80131 Naples, Italy. Phone: 39-081-5666713; Fax: 39-081-5666728 or 39-081-2203147; E-mail: fortunatociardiello@yahoo.com or fortunato.ciardiello@unina2.it. 784 Vol. 10, 784 –793, January 15, 2004 Clinical Cancer Research Downloaded from http://aacrjournals.org/clincancerres/article-pdf/10/2/784/1955435/zdf00204000784.pdf by guest on 14 June 2022