Cancer Therapy : Preclinical ERBB 1 and ERBB 2 Have Distinct Functions in Tumor Cell Invasion and Intravasation

Purpose: The epidermal growth factor receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis. Clinical studies using ERBB inhibitors have focused on tumor growth effects, but ERBBs can contribute to malignancy independent of their effects on tumor growth. Our studies were designed to evaluate the effect of ERBB inhibition on tumor cell motility and intravasation in vivo using clinically relevant small-molecule inhibitors. Experimental Design: Using in vivo mouse models of breast cancer, we test the effects of ERBB1 and ERBB2 inhibitors AC480 and lapatinib, ERBB1 inhibitor gefitinib, and ERBB2 inhibitor AG825 on in vivo tumor cell invasive properties in mammary fat pad tumors. Results: ERBB1 and ERBB2 inhibition rapidly (within 3 h) inhibits both tumor cell motility and intravasation. Using gefitinib, ERBB1 inhibition rapidly inhibits tumor cell motility and invasion but not intravasation, whereas ERBB2 inhibition by AG825 rapidly blocks intravasation. Conclusions: ERBB1 and ERBB2 inhibition can rapidly block tumor cell invasive properties. In addition, we differentiate for the first time the contributions of ERBB1 and ERBB2 to the key metastatic properties of in vivo tumor cell invasion and intravasation. These experiments temporally and molecularly separate two key stages in tumor cell entry into blood vessels: invasion and intravasation. These results indicate that ERBB inhibition should be considered for blocking other tumor cell malignant properties besides growth. Metastatic spread is complex, requiring stromal invasion, intravasation (entry of cells into the vasculature), arrest at a metastatic site, and growth of a metastasis. The development of therapies that target specific steps in the cascade is growing, with the current therapeutic armamentarium focused on inhibiting growth (1). The epidermal growth factor (EGF) receptor (ERBB1) and related family member HER-2/neu (ERBB2) are often overexpressed in aggressive breast cancers and their overexpression is correlated with poor prognosis (2–4). In addition to their well-characterized contributions to cell proliferation and survival, ERBB1 and ERBB2 also contribute to other characteristics of aggressive tumors such as local invasion and intravasation potentially independent of their effects on growth (5–7). Important for the optimization of anti-ERBB treatments in cancer is a clear in vivo identification of the specific tumor properties that are dependent on ERBB1 and ERBB2. The interpretation of studies that use stable, long-term alteration of ERBB1 or ERBB2 expression is limited by the time (weeks to months) required to produce a tumor or metastasis. During that time, the altered ERBB expression can cause dramatic changes in gene expression within the tumor cells, which may in turn induce changes in the surrounding tumor stroma. The availability of drugs targeted to ERBBs that rapidly act to inhibit ERBB activity provides a novel opportunity to examine cellular processes that are more directly dependent on ERBB activity. In this article, we make use of ERBB-targeted drugs to rapidly inhibit ERBB function to dissect the contributions of ERBB1 and ERBB2 to invasion and intravasation at the primary tumor site. We find that ERBB1 is important for local stromal invasion, whereas ERBB2 is more directly important for intravasation. Authors’ Affiliations: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York; Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; and Departments of Medicine and Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee Received 8/19/08; revised 1/20/09; accepted 1/27/09; published OnlineFirst 5/19/09. Grant support: Department of Defense grants BC061403 (D. Kedrin), CA100324 (J. Wyckoff and J.E. Segall), CA77522 (J.E. Segall), and CA80195 (C.L. Arteaga) and Novartis Research Foundation (N.E. Hynes). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Jeffrey E. Segall, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-678-1109; Fax: 718-678-1019; E-mail: segall@aecom.yu. edu. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2163 3733 Clin Cancer Res 2009;15(11) June 1, 2009 www.aacrjournals. org Research. on July 17, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals. org Downloaded from Materials and Methods Cell culture. MTLn3 cells expressing GFP and human ERBB1 were generated (MTLn3E) and propagated as described previously (6). Leibowitz L-15 medium supplemented with 0.3% bovine serum albumin was used as serum-free starvation medium. MDA-MB-231-4173 cells (in vivo selected lung metastatic MDA-MB-231 cells) generously provided by Joan Massague (8) were transduced with a GFP-expressing lentivirus and GFP-expressing transductants selected by fluorescenceactivated cell sorting. MDA-MB-231 cells were cultured in DMEM high-glucose supplemented with 10% fetal bovine serum. 1R, 5R, and control (pBabe) vectors for down-regulation of surface ERBB1 and ERBB2 expression, respectively, were used as described previously (9). Inhibitors. Gefitinib (Iressa), lapatinib (GW572016), and AC480 [also described as BMS-599626 (Ambit Biosciences)] were kindly provided by AstraZeneca, GlaxoSmithKline, and Bristol Myers Squibb, respectively. AG825 was purchased from Tocris. Tumor formation and drug treatment. One million MTLn3E or MDAMB-231 cells were injected under the second nipple from the rear of 4to 6-week-old severe combined immunodeficient mice. For polyoma middle T tumors, mice carrying the polyoma middle T oncogene under the control of the MMTV promoter and expressing GFP in the mammary gland (10) were used. For all tumors, analysis was done when tumor diameters were between 1.5 and 2 cm (∼35-40 days for MTLn3E or 5057 days for MDA-MB-231). Mice were treated with carrier alone (0.5% hydroxypropylmethylcellulose, 0.1% Tween 80 for gefitinib or 50% propylene glycol for AC480 and lapatinib) or carrier containing the inhibitor (100 mg/kg). AG825 treatment was administered via intraperitoneal injection in 10% DMSO at 20 mg/kg. To test the effects of drug treatment on cell viability, cells were seeded at low density on 10 cm plates and allowed to attach. To mimic 3 h treatment by oral gavage, the medium was changed to one containing 10 μmol/L drug or DMSO control for 3 h and then replaced with fresh medium. Cells were allowed to grow and form colonies for several days and the number of colonies was counted. In vivo imaging. For a detailed protocol, see ref. 11 (Unit 19.7). Mice were treated with carrier or drug 3 h before the start of the imaging session. Multiple fields were imaged for each animal and the numbers of moving cells per field were counted and compared. For each field, a 30 min z-stack time-lapse series was collected and analyzed. In vivo invasion and intravasation. MTLn3E tumor-bearing mice were treated via oral gavage with the appropriate carrier compound or drug for 3 h before beginning of the needle collection assay. The in vivo invasion and intravasation assays were done as described pre-