Tumor-Selective Response to Antibody-Mediated Targeting Of
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Volume 10 Number 11 November 2008 pp. 1259–1267 1259 www.neoplasia.com † Tumor-Selective Response to Charles N. Landen*, Tae-Jin Kim , Yvonne G. Lin*, William M. Merritt*, Aparna A. Kamat*, Liz Y. Han*, Whitney A. Spannuth*, Alpa M. Nick*, Antibody-Mediated Targeting of ‡ 1 Nicholas B. Jennnings*, Michael S. Kinch , α β § ,¶ v 3 Integrin in Ovarian Cancer David Tice and Anil K. Sood* *Department of Gynecologic Oncology, The University of Texas M.D. Anderson Cancer Center, 1155 Herman Pressler Boulevard, Unit 1362, Houston, TX 77030, USA; †Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Cheil General Hospital, Kwandong University College of Medicine, Seoul, South Korea; ‡Functional Genetics, 708 Quince Orchard Rd, Gaithersburg, MD 20878, USA; §MedImmune, Inc., 1 MedImmune Way, Gaithersburg, MD 20878, USA; ¶Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 173, Houston, TX 77030, USA Abstract The αvβ3 integrin is expressed on proliferating endothelial cells and some cancer cells, but its expression on ovar- ian cancer cells and its potential as a therapeutic target are unknown. In this study, expression of the αvβ3 integrin on ovarian cancer cell lines and murine endothelial cells was tested, and the effect of a fully humanized mono- clonal antibody against αvβ3, Abegrin (etaracizumab), on cell invasion, viability, tumor growth, and the Akt pathway were examined in vitro and in vivo. We found that etaracizumab recognizes αvβ3 on the ovarian cancer cell lines SKOV3ip1, HeyA8, and A2780ip2 (at low levels) but not on murine endothelial cells. Etaracizumab treatment de- creased ovarian cancer proliferation and invasion. In vivo, tumor-bearing mice treated with etaracizumab alone gave variable results. There was no effect on A2780ip2 growth, but a 36% to 49% tumor weight reduction in the SKOV3ip1 and HeyA8 models was found (P < .05). However, combined etaracizumab and paclitaxel was superior to paclitaxel in the SKOV3ip1 and A2780ip2 models (by 51-73%, P < .001) but not in the HeyA8 model. Treatment with etaracizumab was then noted to decrease p-Akt and p-mTOR in SKOV3ip1, but not in HeyA8, which is Akt-independent. Tumors resected after therapy showed that etaracizumab treatment reduced the proliferating cell nuclear antigen index but not microvessel density. This study identifies tumor cell αvβ3 integrin as an attractive target and defines the Akt pathway as a predictor of response to function-blocking antibody. Neoplasia (2008) 10, 1259–1267 Abbreviations: ECM, extracellular matrix; MICS, Membrane Invasion Culture System; IP, intraperitoneal; eta, etaracizumab Address all correspondence to: Anil K. Sood, MD, Professor, Departments of Gynecologic Oncology and Cancer Biology, The University of Texas M.D. Anderson Cancer Center, 1155 Herman Pressler, CPB6.3244, Unit 1362, Houston, TX 77230-1439. E-mail: [email protected] 1Funding: In part by the Reproductive Scientist Development Program through the Ovarian Cancer Research Fund and the National Institutes of Health (K12 HD00849, to C.N.L.); the Department of Defense (W81XWH-04-1-0227); the University of Texas MD Anderson Cancer Center Specialized Program of Research Excellence in ovarian cancer (CA083639); National Institutes of Health grants CA10929801 and CA11079301; Program Project Development Grant from the Ovarian Cancer Research Fund, Inc.; and the Marcus Foundation (to A.K.S.). Received 3 July 2008; Revised 14 August 2008; Accepted 19 August 2008 Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00 DOI 10.1593/neo.08740 1260 Targeting αvβ3 in Ovarian Cancer Landen et al. Neoplasia Vol. 10, No. 11, 2008 Introduction mors grown in the mouse intraperitoneal (IP) cavity after IP injection Integrins are a family of cell surface receptors that are primarily re- of A2780, through two passes. Endothelial cells isolated from the sponsible for exchanging information between cells and the sur- mesenteryorovaryoftheimmortomousewereakindgiftfrom rounding extracellular matrix (ECM) [1]. They are heterodimers Dr. Robert Langley [28] and maintained in DMEM with 10% composed of 1 of 10 α subunits and 1 of 8 β subunits, and each FBS. These cells proliferate indefinitely by means of SV40 transfor- subtype has specificity for different ECM proteins. In response to mation at 33°C. However, when switched to 37°C, SV40 expression binding components of the ECM, such as collagen, fibronectin, or is turned off, and cells proliferate for only a few more cycles [28]. vitronectin, signals are generated within the cell that can affect When using these cells, they were kept at 37°C for 48 hours before growth, migration, invasion differentiation, and survival [2,3]. As any experiments were performed, to allow elimination of SV40 ex- more is learned about the importance of a tumor cell’s microenviron- pression. All in vitro experiments were conducted at 60% to 80% ment to survival and invasive potential, integrins are seen to play an confluence, unless otherwise specified. For vitronectin-coating ex- important role in tumor biology and may serve as useful targets to periments, 20 μg/ml vitronectin (Chemicon, Temecula, CA) in tumor therapy. PBS (or PBS alone) was added to culture vessels and incubated at α β The v 3 integrin [4] is preferentially expressed on developing, 37°C overnight. Afterwards, vitronectin/PBS was removed and re- rather than mature vasculature, and is considered the most important placed with 1% bovine serum albumin in PBS for 1 hour at 37°C. integrin for angiogenesis [5]. Its primary ligand is vitronectin, but it This was then removed immediately before plating cells for an experi- also interacts with fibrinogen, fibronectin, and thrombospondin ment. For in vivo injection, cells were trypsinized and centrifuged α β [6,7]. Furthermore, associations have been found between v 3 at 1000 rpm for 7 minutes at 4°C, washed twice, and reconstituted and matrix metalloproteinase 2, platelet-derived growth factor, insu- in Hank’s balanced salt solution (Gibco, Carlsbad, CA) at a concen- lin, and vascular endothelial growth factor receptor 2 (VEGFR-2) tration of 5 × 106 cells/ml for 200-μl IP injections of 1 × 106 cells. [8–11]. In a self-promoting loop, VEGF, one of the more potent stim- α β ulators of angiogenesis [12,13], up-regulates v 3 expression and Flow Cytometry increases affinity for its ligands [14], which in turn interacts with Cells growing in monolayer culture at 60% to 80% confluence VEGFR-2 to further amplify VEGF [15]. Administration of a mouse were trypsinized with EDTA and washed in PBS. Cells were re- α β 6 μ monoclonal antibody against v 3 (LM609) was shown to disrupt constituted to equal 5 × 10 cell/ml, and 200 l was incubated with μ α β tumor-induced angiogenesis on chick chorioallantoic membrane 1 g/ml anti- v 3 antibody (LM609; Upstate, San Francisco, CA) (CAM) [5], and in subsequent studies, disrupt tumor-associated vas- with gentle rotation at 4°C for 30 minutes. Cells were spun at culature and induce tumor regression without significant adverse 2000 rpm for 5 minutes, washed twice with PBS, and reincubated effects on established, mature blood vessels. Subsequent studies of with antimouse IgG-FITC (Upstate) at 4°C for 30 minutes. Cells the LM609 antibody showed tumor growth inhibition in preclini- were washed with PBS and reconstituted in 1 ml of PBS for imme- cal mouse models of melanoma [16,17] and breast cancer [18], diate reading with an EPICS XL-MCL flow cytometer (Beckman and synergy with immunotherapy in neuroblastoma [19]. Coulter Inc., Miami, FL). α β More recently, v 3 expression has been demonstrated on meta- static human melanoma, breast, prostate, and glioblastoma tumor Immunoprecipitation and Western Blot α β cells, where its expression contributes to malignant phenotype. A fully Immunoblot detection of v and 3 integrin subunits was per- α β humanized antibody targeted to v 3 has demonstrated encouraging formed using a modified immunoprecipitation technique that not α β activity (etaracizumab, Abegrin; MedImmune, Inc., Gaithersburg, only allowed detection of both v and 3 together in a single sample α β MD) [20]. The v 3 integrin has been studied in ovarian cancer, with but also allowed detection of other integrin subunits associated with α β targeting by either antibodies or small molecule inhibitors shown to either v or 3. Cells in monolayer culture were labeled with biotin, inhibit migration, adhesion, motility, angiogenesis, and proliferation and cell lysates were prepared by washing cells with PBS followed by – α in vitro [11,21 24]. The v subunit has been found in malignant ef- incubation in modified RIPA lysis buffer (50 mM Tris, 150 mM fusions and solid tumors from ovarian cancer patients [25]. However, NaCl, 1% Triton, 0.5% deoxycholate plus 25 μg/ml leupeptin, α β μ the biologic significance of v 3 targeting is not fully understood. The 10 g/ml aprotinin, 2 mM EDTA, and 1 mM sodium orthovana- α β aim of this study was to determine the effects of v 3 on ovarian can- date; Sigma Chemical Co., St. Louis, MO) for 10 minutes at 4°C. cer cell line invasion, proliferation, vascularization, and tumor growth Cells were scraped from plates and centrifuged at 13,000 rpm for in an in vivo orthotopic model of advanced ovarian cancer. Examining 20 minutes at 4°C, and the supernatant was stored at −80°C. Protein several cell lines in vivo, we have also identified molecular correlates of concentrations were determined using a BCA Protein Assay Reagent α β anti- v 3 therapy. These findings support the use of etaracizumab as kit (Pierce Biotechnology, Rockford, IL), and samples were separated α β μ therapy for ovarian cancer and demonstrate the benefit of tailoring for v or 3 testing. Protein (300 g) was immunoprecipitated with ’ α β therapy based on an individuals tumor biology.