Selective Blockade of Matrix Metalloprotease-14 with a Monoclonal Antibody Abrogates Invasion, Angiogenesis, and Tumor Growth in Ovarian Cancer

Published OnlineFirst February 19, 2013; DOI: 10.1158/0008-5472.CAN-12-1426

Cancer Microenvironment and Immunology Research

Rajani Kaimal1, Raid Aljumaily1,2, Sarah L. Tressel1, Rutika V. Pradhan1, Lidija Covic1,2,5, Athan Kuliopulos1,2,5, Corrine Zarwan6, Young B. Kim3, Sheida Shariﬁ4,7, and Anika Agarwal1,2

Abstract Most patients with ovarian cancer are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease (MMP)-14 on ovarian cancer cells stimulates a tumor–stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efﬁcacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor–stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring Matrigel plugs. These effects on angiogenesis correlated with downregulation of several important angiogenic factors. Furthermore, mice with ovarian cancer tumors treated with anti–MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared with immunoglobulin G (IgG)-treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14–dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer. Cancer Res; 73(8); 2457–67. 2013 AACR.

Introduction Angiogenic and inflammatory markers such as interleukin a Ovarian cancer remains the fifth leading cause of death in (IL)-8, IL-6, growth-related oncogene (GRO)- , monocyte women and the most fatal gynecologic cancer, accounting for chemoattractant protein (MCP)-1, VEGF, and matrix metal- more than 15,000 deaths in the United States annually (1). The loprotease (MMP)-1 are highly upregulated in epithelial – vast majority of patients with ovarian cancer are diagnosed in ovarian cancers (4 7). These markers are postulated to play fl late stages and experience recurrences and relapses due to a pivotal role in tumor growth, in ammation, angiogenesis, drug resistance despite initial response to surgical debulking and metastasis and are associated with poor survival (8, 9). fi and standard first-line chemotherapy (2). In addition, there are Angiogenesis inhibitors, as exempli ed by the VEGF-anti- no established and effective treatments of platinum refractory body Avastin, have recently emerged as potential treatments ovarian cancer. Therefore, identification of novel targeted of recurrent ovarian and primary peritoneal cancer (10). But, therapies that block critical signaling pathways are important anti-VEGF therapy has not improved overall survival (11) for improving survival in ovarian cancer (3). and one of the reasons is that decreased tumor size has been associated with increased invasiveness and propensity to metastasize (12). MMPs are zinc-dependant endopeptidases that play a key Authors' Affiliations: 1Molecular Oncology Research Institute; Depart- role in cancer cell invasion, metastases, and tumor angio- 2 3 4 ments of Medicine, Obstetrics and Gynecology, and Pathology, Tufts genesis (13, 14). Abundant evidence has indicted MMPs Medical Center; 5Departments of Biochemistry and Genetics, Tufts Uni- versity Medical School, Boston; 6Department of Hematology and Oncol- including MMP-1, -2, -9, and -14 as critical mediators of ogy, Lahey Clinic Medical Center, Burlington; and 7Mt Auburn Hospital, invasion, blood vessel penetration, and metastasis of many Cambridge, Massachusetts solid tumors including ovarian cancer (4, 13, 15–18). MMP- Note: Supplementary data for this article are available at Cancer Research 14, (MT1-MMP), is a transmembrane collagenase, which is Online (http://cancerres.aacrjournals.org/). not detected in normal ovarian surface epithelium but is R. Kaimal and R. Aljumaily contributed equally to this work. widely expressed on ovarian carcinomas of all histologic Corresponding Author: Anika Agarwal, Tufts Medical Center, 800 types (18). High expression of epithelial MMP-14 by immu- Washington Street, Boston, MA 02111. Phone 617-636-4522; Fax 617- nohistochemistry is associated with poorer prognosis and 636-7855; E-mail: [email protected] shorter disease survival in patients with ovarian cancer (19). doi: 10.1158/0008-5472.CAN-12-1426 MMP-14 is critical to the acquisition of the invasive (20) and 2013 American Association for Cancer Research. metastatic phenotype of breast, prostate, melanoma, renal,

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and ovarian carcinomas (21–23). MMP-14 promotes a col- and MMP-9 have been described (4) and were obtained from lagen-invasive phenotype (24) in ovarian carcinoma, which Dharmacon. triggers peritoneal dissemination of metastatic ovarian tumors (23). MMP-14 is known to activate gelatinase Cell culture MMP-2 (25) that has been implicated in the mesothelial Ovarian cancer cell lines OVCAR-4, SKOV-3, and OVCAR-3 attachment and metastatic spread of ovarian cancer cells were obtained from the National Cancer Institute and were (20, 26). We have recently shown that ovarian cancer cell grown in RPMI with 10% FBS. Cells were serum-starved MMP-14 leads to activation of proMMP-1 (4) via MMP-9, overnight in 0.25% bovine serum albumin (BSA) for migration which culminates in chemokine production, angiogenesis and invasion assays. Human umbilical vein endothelial cells (4, 16, 27), and ovarian tumor growth (4, 27). Endothelial cell (HUVEC) were bought from Cambrex (Lonza) and cultured in MMP-14 has been implicated in angiogenesis (28), however, EBM2 medium with Bullet kit and 10% FBS. HUVEC cells were the mechanism of cancer cell produced MMP-14 in stimu- serum-starved for 1 hour before tube formation. Gelatinase lating angiogenesis in the ovarian tumor microenvironment activity was assayed from conditioned media by measuring the is unknown. cleavage of fluorescein-conjugated DQ gelatin (Molecular A causal relationship between MMP expression and cancer Probes). Gelatinase assays contained 10 mg DQ gelatin in 50 progression led to the evaluation of MMP inhibitors in several mmol/L Tris–HCl, pH 7.6, 150 mmol/L NaCl, 5 mmol/L CaCl2, fi large clinical trials but met with limited ef cacy and treatment and 0.2 mmol/L NaN3 and cleavage was monitored at 538 nm failure (29, 30). These small-molecule MMP inhibitors elicited using a fluorescence microplate reader with excitation at 485 debilitating musculoskeletal toxicities that precluded ade- nm at 37C. Gelatinase activity was expressed as percentage of quate dosing and poor patient compliance (31, 32). Toxicity the control group. Experiments were repeated 3 times in was assumed to be due to the broad-spectrum nature of these triplicates. compounds, which led to off-target inhibition of protective MMPs such as MMP-8 and -12 leading to worse clinical out- Detection of MMP-14 in patient samples comes (29). Moreover, most of the patients recruited to the All patient blood samples were collected in full compliance trials were in late stages (stage III–IV) of cancer, which could with Tufts and Lahey Institutional Review Board guidelines. potentially miss the positive therapeutic benefits of blocking Blood was collected from women presenting at Tufts Medical MMP pathway at earlier stages in cancer (33). Center (Boston, MA) or Lahey Clinic (Burlington, MA) with In this study, we used a monoclonal antibody to inhibit benign or malignant pelvic masses, before surgery. Blood was MMP-14 on the ovarian carcinoma cells. MMP-14 inhibition processed to collect serum that was stored at 80C till further led to suppression of both migratory and invasive propensity use. Several serum samples were also obtained from Gynecol- of ovarian carcinoma cells. MMP-14 antibody suppressed ogy Oncology Group Tissue bank. Discarded patient fluids ovarian cancer cell-stimulated angiogenesis in vitro and in were collected from patients undergoing a peritoneal and mice. Further, MMP-14 antibody treatment led to a marked pulmonary tap at Tufts Medical Center. Serum samples were regression in ovarian tumor growth and tumor blood vessel diluted 10-fold and MMP-14 levels were quantified using ELISA formation in a mouse xenograft model indicating that MMP-14 (USCN E92056Hu). Groups were compared with one-way may be an attractive target for antiangiogenic therapy in ANOVA followed by t test. , P < 0.05. Patient fluids and serum ovarian cancer and perhaps other solid tumors. Furthermore, samples were normalized using Bradford assay. Two microliter in a peritoneal model of aggressive ovarian cancer, combina- of normalized samples (2.2 mg/mL) were mixed with citrate tion intraperitoneal therapy of MMP-14 antibody and doce- buffer, EDTA, and 5 sample buffer in a 10 mL volume, heated taxol inhibited invasion through the mouse diaphragm and for 40 minutes at 37C, and immunoblotted with monoclonal thoracic metastasis. We believe, blockade of MMP-14 as mono- MMP-14 antibody (Ab; MAB 918). therapy or as combinatorial therapy along with conventional chemotherapy has direct clinical relevance for adjuvant and Flow cytometry metronomic treatment regimens in ovarian cancer. Flow cytometry was conducted as described before (4) using a mouse monoclonal antibody specifictoMMP-14 Materials and Methods (R&D Systems MAB9181). Reagents MMP-14 monoclonal antibodies were obtained from Cal- Migration and invasion biochem (EMD Biosciences), R&D (MAB 9181, 918), and Milli- Chemotactic migration and invasion was conducted using pore (MAB 3319). Isotypic immunoglobulin G (IgG) control an 8 mm Transwell apparatus (Corning), as described previ- was purchased from R&D System and fluorescein isothiocya- ously (16). Of note, 50,000 serum-starved ovarian cancer cells nate (FITC)–conjugated rabbit anti-mouse from Invitrogen. were treated with 10 mg/mL of antibody and placed in the top MMP-7 (Ab-2), MMP-8 (Ab-1), and TIMP-1 (Ab-1) mouse chamber of the Transwell in 200 mL of 0.25% BSA in RPMI. The monoclonal antibodies were obtained from Oncogene. Human cells were migrated toward stromal matrix (fibroblast condi- MMP-14 ELISA was obtained from USCN (E92056Hu; Uscn Life tioned media) or malignant ovarian ascites, as previously Science, Inc.) and human Quantikine MMP-9 and VEGF ELISA described (16) for a period of 5 hours. For chemoinvasion, from R&D Systems and used as per manufacturer's protocol. wells were coated with of 20 mg of reconstituted matrix RNA interference reagents (RNAi) against Luciferase, MMP-14, (Matrigel; BD Biosciences; ref. 4) and cells were migrated

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similarly for 20 hours. For collagen invasion assays, Transwell injections of 2.5 mg/kg of either IgG or MMP-14 antibody were coated with 30 mg of rat tail high concentration type I twice a week with concomitant docetaxel (5 mg/kg at day 21). collagen (BD Biosciences) and cells were migrated for 40 hours. At the end of 35 days, tumors and organs were excised from Migrated and invaded cells on the lower surface of the Trans- euthanized mice, fixed in 10% formalin/PBS, and imbedded in well were fixed, stained, and counted and data were compared paraffin. Tissue sections (5 mm) from omentum, diaphragm, with the respective IgG controls. One-way ANOVA followed by and thoracic organs were prepared and stained with hema- Student t test was used to compare the effect of treatment with toxylin and eosin and examined in a blinded manner by 2 the control. , P < 0.05; , P < 0.005. pathologists.

Tube formation assay Results MatTek plates were chilled to 4C and coated with 100 mL Serum MMP-14 is increased in patients with invasive of Matrigel per well. Freshly passaged HUVEC cells (35,000; ovarian cancer P2-5) in EBM2 media with 0.25% BSA were plated on We have previously shown that MMP-14 initiates the acti- Matrigel-coated MatTek plates and stimulated with condi- vation of a MMP cascade in the ovarian tumor microenviron- tioned media from OVCAR-4 or SKOV-3 cells treated with ment that leads to angiogenesis and ovarian tumor growth (4). either MMP-14 antibody, IgG or RNAi. Endothelial tube High expression of epithelial MMP-14 by immunohistochem- formation was observed after 18 hours and photographed istry was associated with poor outcome in patients with under phase contrast microscopy using an inverted Olympus ovarian cancer (19). Therefore, we investigated whether microscope. Digital images were used to count total tubes MMP-14 could be detected as a tumor biomarker in the serum per 5 40 fields and quantified for tube length and branch- and malignant ascites of patients with ovarian carcinoma. ing complexity using NIH ImageJ software. Measures were These patients are known to produce copious amounts of expressed in number of branch points and actual tubal peritoneal ascites and we explored whether we could detect length. , P < 0.05; , P < 0.005. MMP-14 or a cleaved part of MMP-14 in malignant ovarian ascites. We collected fluids from 14 patients with malignant RNA interference and benign disease (malignant ovarian ascites, n ¼ 5; malig- Cells were transfected with Luci, MMP-14, or MMP-9 nant ovarian cyst, n ¼ 1; malignant pleural effusions from RNAi using oligofectamine as previously described (4). Thirty patients with ovarian cancer, n ¼ 4; benign ascites, n ¼ 3; and hours after recovery, the cells were either used for RNA benign ovarian cyst, n ¼ 1) and found that the levels of MMP-14 preparation or serum-starved for conditioned media. were more than 3-fold increased in the malignant fluids of patients with ovarian cancer compared with benign fluids (Fig. Matrigel plugs and ovarian cancer xenograft models 1A). Because MMP-14 was increased in the malignant ovarian All animal experiments were carried out in full compliance ascites, we further explored whether it could be detected in the with Tufts Medical Center Institutional Animal Care and Use serum of patients with malignant ovarian cancer. In a cohort of Committee. Female NCR Nu/Nu mice (5–7 weeks) were pur- 92 patients comprising healthy females (n ¼ 34), those with chased from Taconic farms. Mice were injected with 300 mL benign ovarian tumors (n ¼ 32) and malignant ovarian cancer Matrigel plugs of equal parts conditioned media and Matrigel (serous epithelial cancer, stages II–IV; n ¼ 26), we found that in their flanks subcutaneously. Animals were subsequently serum MMP-14 was significantly increased in patients with treated 24 hours later with either MMP-14 antibody, IgG, or malignant ovarian carcinoma (Fig. 1A). To our knowledge, Avastin (2.5 mg/kg twice weekly) in a 100 mL volume. At the end this is the firstreportofincreasedMMP-14intheserumand of 7 days, mice were euthanized and plugs were excised and malignant ascites of patients with ovarian carcinoma and fixed in 10% formalin/PBS and stained with CD31 (Chemicon, has important implications not only as a biomarker of the MAB 1398Z) for confocal microscopy, as previously described disease but also as a tumor marker for monitoring response (27). For ovarian xenograft experiments, mice were injected to therapy. We also confirmed the presence of MMP-14 in subcutaneously with 3.5 million OVCAR-4 cells in their flanks. these samples of malignant ovarian ascites, malignant pleu- Mice were randomized into 2 treatment groups and 24 hours ral effusion, and the serum of patients with ovarian carci- later injected with 2.5 mg/kg of IgG or MMP-14 antibody, noma using immunoblot analysis (Fig. 1B and C). Immuno- intraperitoneally (i.p.), twice weekly till day 26. Tumor sizes blotting for MMP-14 showed several fragments of MMP-14 were measured twice weekly initially and every other day after exodomain as previously described (34). Our data correlate day 26 for the duration of the experiment. Tumor volume was with the increased expression of MMP-14 found by immu- calculated using the formula: V ¼ length diameter2/2. Mice nohistochemistry in metastatic ovarian lesions as compared were euthanized on day 35 and tumors and tissues were with primary ovarian tumors (23) and suggests an important collected. Tumors were stained for endothelial marker CD31 role for MMP-14 in ovarian cancer progression. We also (760-4378; Ventana Medical Systems, automated as per man- tested aggressive OVCAR-4, SKOV-3, or OVCAR-3 ovarian ufacturer's protocol) and evaluated by a pathologist in a cancer cells and found a robust expression of MMP-14 by blinded manner. flow cytometry (Fig. 1D). This finding reiterates the fact that For the Peritoneal Ovarian Tumor Model, mice were injected MMP-14 can be detected as a tumor biomarker in malignant into the peritoneal cavity with 1.5 million OVCAR-4 cells. ovarian cancer and emphasizes the importance of this MMP Animals were treated 24 hours later with intraperitoneal in ovarian carcinogenesis.

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A 26 * Figure 1. MMP-14 is a biomarker of 22 malignant ovarian cancer. A, serum samples were collected from females with malignant 18 n ¼ Patient serum ovarian cancer ( 26), females with benign ovarian masses Patient fluids 14 (n ¼ 32), and normal females (n ¼ 34) and MMP-14 cellular 10 exodomain levels were measured * * by ELISA. Various fluids were MMP-14 (ng/mL) collected from 14 patients with 6 benign or malignant disease. 4 MMP-14 was measured by ELISA Normal Benign Malignant n ¼ Ov tumor Ov tumor in malignant ovarian ascites ( 5), malignant ovarian cyst (n ¼ 1), Serum 34 32 26 pleural effusion of patients with Patient fluids 4 10 ovarian cancer (n ¼ 4), benign ascites (n ¼ 3), and benign ovarian B Patient fluid samples C Patient serum samples cyst (n ¼ 1). Columns, mean; bars, SE; , P < 0.05. B and C, gels C showing cleaved MMP-14 BO MPE BA MA MA Norm Ben Malig Malig 72 kD fragments in benign and malignant patient fluids. Patient serum and 56 kD fluids (BOC, benign ovarian cyst; 43 kD MPE, malignant pleural effusion; BA. benign ascites; MA, malignant 34 kD ascites) were diluted, mixed with citrate buffer, and immunoblotted 26 kD with MAB 918 in 1:1,000 dilution. The cleaved MMP-14 fragment D OVCAR-4 OVCAR-3 SKOV-3 seen at 27 kDa is most representative of the ELISA data 100 100 100 shown earlier. D, surface expression of MMP-14 was 80 80 80 measured in ovarian cancer cell lines OVCAR-4, OVCAR-3, and 60 60 60 SKOV-3 by flow cytometry using a MMP-14 monoclonal antibody. 40 40

40 Counts

Counts Counts The dotted line represents secondary alone and the solid line 20 20 20 represents primary plus secondary antibody. 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10

Monoclonal MMP-14 antibody inhibits migration and cancer cells, respectively, compared with IgG controls (Fig. 2A). invasion of ovarian cancer cells We also used several other relevant monoclonal antibodies as MMP-14, when overexpressed, promotes cancer cell migra- controls. MMP-7 is a matrilysin important for inducing ovarian tion and invasion (24, 35) critical to the acquisition of the cancer invasion (39), MMP-8 is tumor collagenase such as metastatic phenotype in ovarian cancer (23). MMP-14 silenc- MMP-14, which is known to play a protective role in cancers ing suppresses migration and invasion of tumor cells in vitro (40) but is not expressed in our ovarian cancer cells (Supple- (36) and pharmacologic inhibition of MMPs, including MMP- mentary Fig. S1A) and TIMP-1 (tissue inhibitor of metallopro- 14, using broad-spectrum MMP inhibitors such as prinimostat teinase-1) inhibits most MMPs except membrane-tethered inhibits tumor progression (37). However, as broad-spectrum MMPs such as MMP-14, -16, and -25 (41). Unlike MMP-14 MMP inhibitors have not been successful in clinical trials (38), antibody, antibodies against MMP-7, -8, and TIMP-1 did not we investigated whether selective blockade of MMP-14 using a suppress migration of either cell line (Fig. 2A), suggesting that monoclonal antibody could inhibit the migratory ability of the migratory ability of ovarian cancer cells was dependent ovarian carcinoma cells. We treated OVCAR-4 and OVCAR-3 on MMP-14. Anti-VEGF therapy is currently being used as cells with monoclonal MMP-14 antibody or corresponding IgG first-line therapy in ovarian cancer (10) along with standard and allowed them to migrate toward fibroblast conditioned chemotherapeutic regimens but has not yet improved media (Fig. 2A) or malignant ovarian ascites (Fig. 2B). Treat- overall survival perhaps due to increased local invasion and ment with MMP-14 antibody significantly decreased migration metastasis (12). We therefore tested whether antibodies by 46% to 69% of OVCAR-3 (P < 0.05) and OVCAR-4 (P < 0.005) against VEGF (Avastin) would inhibit migration of ovarian

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Ovarian carcinoma cells OVCAR-4 A B Migration to Migration OVCAR-3 ovarian ascites Figure 2. Blockade of MMP-14 120 inhibits migration and invasion of 120 ovarian cancer cells. A, OVCAR-4 and OVCAR-3 cells were treated with 100 100 10 mg/mL of either IgG or MMP-14 antibody, Avastin (VEGF antibody), 80 TIMP-1 antibody, MMP-7 antibody, 80 or MMP-8 antibody and migrated 60 ﬁ toward stromal matrix ( broblast Migration (%) 60 conditioned media) for 5 hours. B, Migration (%) 40 OVCAR-4 and OVCAR-3 cells were treated with MMP-14 or luciferase 20 40 Luci MMP-14 RNAi, serum starved, and migrated Ab: IgG MMP-14 VEGF MMP-7 TIMP-1 MMP-8 RNAi: toward malignant ovarian ascites for 5 hours. C, Transwells were coated with 20 mg of Matrigel. OVCAR-4 and C Matrigel invasion D Collagen invasion OVCAR-3 cells were treated with either IgG, MMP-14, Avastin (VEGF antibody), MMP-7, or TIMP-1 antibody and migrated toward stromal matrix for 20 hours. D, 3 Transwells were coated with type I rat tail collagen, and cells were migrated toward stromal matrix for 40 hours. Columns, mean; bars, SE; , P < 0.05; , P < 0.005. Invaded cells x10 Chemoinvasion (%)

Ab: IgG MMP-14 VEGF MMP-7 TIMP-1 Ab: IgG MMP-14

cancer cells. Anti-VEGF antibody had no effect on the migra- tuted type I collagen matrices, which is highly dependent on tion of OVCAR-4 cells, whereas they had a nonsignificant MMP-14 activity (42). We migrated MMP-14 antibody-treated decrease on the migration of OVCAR-3 cells (Fig. 2A). OVCAR-4 and OVCAR-3 cells through a type I collagen matrix In the peritoneal cavity, malignant ovarian ascites serves as a and found that MMP-14 antibody significantly inhibited more rich chemoattractant for the migratory ovarian carcinoma than 60% of the invasive propensity of OVCAR-4 (P < 0.005) and cells. To examine the role of MMP-14 in migration toward OVCAR-3 cells compared with respective IgG-treated controls ascites, we treated OVCAR-4 or OVCAR-3 cells with MMP-14 (P < 0.05; Fig. 2D). These data suggest that selectively blocking RNAi (36) and migrated them toward malignant ovarian MMP-14 may be physiologically relevant for inhibiting the ascites. MMP-14 silencing of OVCAR-4 and OVCAR-3 cells led migratory and invasive potential of ovarian carcinoma cells. to a 60% to 80% decrease in MMP-14 levels at the mRNA level (Supplementary Fig. S1B) and 80% to 95% decrease by flow Blockade of cancer cell MMP-14 inhibits endothelial cytometry (Supplementary Fig. S1C and S1D, respectively), angiogenesis which was confirmed by immunoblotting (Supplementary Fig. Endothelial cell MMP-14 is known to be important for blood S1E and S1F). MMP-14 RNAi-treated OVCAR-4 and OVCAR-3 vessel formation (43) because MMP-14 knockout mice display cells showed decreased migration toward ascites compared skeletal defects due to impaired vascularization of the cartilage with the corresponding luciferase-treated cells (Fig. 2B), con- and endothelial cells from these mice show decreased fibro- sistent with the MMP-14 antibody data, suggesting that MMP- blast growth factor (FGF2)-induced capillary tube formation 14 is important for the movement of ovarian cancer cells in the (44). We postulated that the cancer cell MMP-14 was regulating peritoneal cavity. the release of angiogenic factors in the ovarian tumor micro- We further explored the effect of MMP-14 antibody on environment and stimulating blood vessel formation. To test ovarian cancer cell invasion. MMP-14 antibody-treated cells the role of cancer cell MMP-14 in cancer cell–stimulated showed a more than 50% reduction in the invasion of OVCAR-4 angiogenesis, we treated OVCAR-4 or SKOV-3 cells overnight and a 40% reduction in the invasion of OVCAR-3 cells through with either MMP-14–blocking antibody or IgG (10 mg/mL), reconstituted basement membrane (Matrigel) compared with collected serum-starved conditioned media, and stimulated corresponding IgG controls. Antibodies against VEGF (Avas- HUVECs on Matrigel-coated plates. HUVECs stimulated with tin), MMP-7, and TIMP-1 had no significant effect on Matrigel conditioned media from MMP-14 antibody-treated OVCAR-4 invasion (Fig. 2C). We also tested invasion through reconsti- or SKOV-3 cells showed 60% to 83% reduction in branching

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complexity and 50% or more reduction in tubal length com- tumor microenvironment that promoted endothelial tube pared with those stimulated with IgG-treated conditioned formation and branching complexity. media (Fig. 3A and B). Furthermore, to ensure that these Furthermore, we characterized the MMP-14 antibodies used effects were a function of blockade of MMP-14 on the cancer in this study (Calbiochem and R&D Systems) and found that cell, we also collected serum-starved conditioned media from they blocked the conversion of proMMP-2 to active MMP-2 MMP-14 and Luci RNAi-treated OVCAR-4 cells (Supplemen- (Supplementary Fig. S2B). Furthermore, MMP-14 antibody tary Fig. S1C and S1E) and stimulated tube formation. MMP-14 (R&D Systems) was speciﬁc in recognizing MMP-14 protein gene silencing of the ovarian cancer cells completely blocked and did not cross-react with other MMPs such as MMP-3, -7, carcinoma cell–stimulated angiogenesis (Fig. 3C) conﬁrming and -8 (Supplementary Fig. S2C). that endothelial tube formation was at least partly regulated through cancer cell MMP-14. We conducted a mitochondrial Cancer cell MMP-14 regulates angiogenesis in the activity assay (MTT) to rule out that MMP-14 antibody treat- ovarian tumor microenvironment ment affected the viability of treated cells. MMP-14 antibody- Cancer cell MMP-14 is known to convert proMMP-2 to and IgG-treated endothelial and cancer cells did not show any MMP-2 (25). We have previously shown that MMP-14 on the differences in viability after 72 hours of treatment (Fig. 3D). ovarian cancer cells regulates activation of proMMP-1 to This suggests that the cancer cell MMP-14 was regulating the MMP-1, which upregulates chemokine production and ovarian production or release of angiogenic factors in the ovarian cancer angiogenesis (4). Therefore, we speculated that cancer

Figure 3. Blockade of MMP-14 disrupts tumor–stromal communication and angiogenesis. A and B, ovarian cancer cell– stimulated angiogenesis is B inhibited by MMP-14 antibody. OVCAR-4 and SKOV-3 cells were treated overnight with either MMP- 14 antibody or IgG (10 mg/mL) in serum-starved conditioned media. Ovarian cancer cell conditioned media was collected 18 hours later and used to stimulate HUVECs on Matrigel-coated MatTEK plates. C, OVCAR-4 ovarian cancer cells were treated with RNAi against C MMP-14 and luciferase, and serum-starved conditioned media was collected to stimulate HUVEC cells. Results from A–C were quantiﬁed using NIH ImageJ. Tubal length and branch points were compared between the treatment groups using a t test. , P < 0.01. D, HUVECs, OVCAR-4, and SKOV- 3 ovarian cancer cells were treated with either 10 mg/mL of IgG or MMP-14 antibody and allowed to grow for 72 hours. Mitochondrial D activity for all the cells was assessed after 72 hours and expressed as a percentage of IgG controls.

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cell MMP-14 was regulating angiogenesis by modulating the MMP-14 gene suppression significantly reduced gelatin activ- activity of other MMPs or angiogenic factors (45) in the ovarian ity (Fig. 4B), part of which can be attributed to MMP-9 activity tumor microenvironment. Having seen the unequivocal effects as OVCAR-4 cells lack MMP-2 (Supplementary Fig S2A and of MMP-14 antibody and MMP-14 RNAi on in vitro cancer cell– S2B). stimulated angiogenesis, we explored the angiogenic factors We further tested whether MMP-14 blockade also had an being regulated through MMP-14. It is well established that effect on VEGF levels. MMP-14 knockdown also significantly MMP-9 and VEGF are important for tumor angiogenesis (46), decreased VEGF A (165) levels in serum-starved OVCAR-4 but it is not well known that MMP-14 regulates MMP-9 (47). We conditioned media (Fig. 4C) establishing the importance of hypothesized that MMP-14 was suppressing blood vessel MMP-14 in ovarian tumor angiogenesis. To ensure that these formation by downregulating production or release of effects were a function of decreased MMP-9, we also collected MMP-9 or VEGF, or both of these factors. We tested MMP- serum-starved conditioned media from OVCAR-4 cells that 14 antibody-treated OVCAR-4 cell conditioned media and had been treated with MMP-9 and Luci RNAi (Fig. 4D and confirmed the decrease in MMP-9 levels by ELISA (Fig. 4A). Supplementary Fig. S3A) and stimulated tube formation. Because active MMP-9 is required for the release of VEGF from MMP-9 gene silencing of the ovarian cancer cell showed more the cancer cells and for promoting angiogenesis (46), we than 75% decrease in tubal length and branching complexity further tested whether MMP-14 inhibition decreased MMP-9 (Fig. 4D). This suggested that at least a part of the MMP-14– activity by measuring in situ gelatinase (largely MMP-2 and -9) regulated ovarian cancer angiogenesis was mediated through activity from RNAi-treated OVCAR-4 cell conditioned media. suppression of MMP-9 (Fig. 4A and B and Supplementary Fig.

A OVCAR-4 CM B OVCAR-4 CM C OVCAR-4 CM

ELISA ELISA

Figure 4. MMP-14 regulates ovarian cancer angiogenesis 100 100 100 through MMP-9 and VEGF 80 ** pathways. A, OVCAR-4 cells were * 80 80 treated with MMP-14 antibody or 60 MMP-14 RNAi along with respective MMP-9 (%) VEGF-A (%) controls. Serum-starved 40 activity (%) 60 60 DQ Gelatinase conditioned media was collected to Ab: IgG MMP-14 RNAi: Luci MMP-14 RNAi: Luci MMP-14 assess levels of MMP-9 by ELISA (A), gelatin cleavage activity using DQ OVCAR4 RNAi CM OVCAR-4 CM gelatinase assay (B), and VEGF by 4 ELISA (C). D, OVCAR-4 cells were D Luci MMP-9 treated with either MMP-9 or 5 s 80 luciferase RNAi and conditioned 4 media was used to stimulate 3 40 endothelial tube formation. Results 2 ** were quantiﬁed as tube length and 1 **

Tube Iength x 10 branch points. , P < 0.05; 0 Branch point 0 , P < 0.005. E, Matrigel plugs of RNAi: Luci MMP-9 Luci MMP-9 300 mL were injected into the flanks of female NCR nu/nu mice MMP-9 92 k Da subcutaneously. The plugs RNai Luci MMP-9 consisted of equal volumes of OVCAR-4 conditioned media and Matrigel along with HUVECs. On day E CD31 staining in matrigel plugs 2, mice were treated with either MMP-14 (n ¼ 8) or corresponding 2,500 IgG antibody (n ¼ 8) or Avastin (n ¼ 7) in a dose of 2.5 mg/kg i.p., twice a week for 7 days. At the end of 7 days, 2,000 * * mice were euthanized and the fi Matrigel plugs were excised, xed, 1,500 and stained for CD31. Figure shows CD31-stained microscopic sections of Matrigel plugs and quantification 1,000 CD31 staining of CD31 counts from the three CD31 staining treatment groups. Columns, mean; 500 bars, SE; , P < 0.05. Ab: IgG Ab: IgG MMP-14 Avastin MMP-14Avastin

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S3B) especially as MMP-14 antibody added directly on the ovarian cancer angiogenesis and tumor growth and could HUVECs did not significantly suppress tube formation (Sup- potentially be a novel target for antiangiogenic therapy in plementary Fig. S3C). ovarian cancer. Having seen the importance of cancer cell MMP-14 in ovarian cancer angiogenesis and the inhibitory effects of Intraperitoneal combination of MMP-14 antibody plus MMP-14 antibody on in vitro tube formation, we tested wheth- docetaxol inhibits invasion and metastasis of ovarian er this would be true for in vivo angiogenesis. We subcutane- cancer in nude mice ously injected Matrigel plugs into the flanks of female nude Patients whose cancer has spread throughout the peritoneal mice. The plugs consisted of equal volumes of OVCAR-4 cavity have a poor survival rate, thus inhibition of peritoneal conditioned media and Matrigel along with HUVECs. The dissemination and metastatic progression is critical for the mice were then treated on day 2 with either MMP-14 antibody successful treatment of ovarian cancer. Therefore, we assessed or corresponding IgG antibody or Avastin (VEGF) twice a week whether inhibition of MMP-14 using a monoclonal antibody in at 2.5 mg/kg i.p. At the end of 7 days, Matrigel plugs were combination with docetaxel was able to slow progression of excised, fixed overnight, and stained with CD31, a marker for peritoneal ovarian cancer in mice. We have previously devel- endothelial cells (27). Both MMP-14 and VEGF antibody-trea- oped a histologic staging system that used the diaphragm as a ted mice showed a marked decrease in vascularization of marker of the progressive invasion and metastasis from the Matrigel plugs as compared with the IgG-treated controls (Fig. peritoneal cavity/omentum to the thoracic organs (4). We 4E). The Matrigel plug experiment not only established the injected 1.5 million aggressive OVCAR-4 ovarian cancer cells efficacy of the MMP-14 antibody in vivo, but also the adequate into the peritoneal cavity of mice. Starting on day 2, mice dose required for inhibition of blood vessels suggesting that received either IgG or MMP-14 antibody (2.5 mg/kg i.p., twice blocking MMP-14 may be a therapeutic strategy to inhibit a week) and 2 doses of docetaxel (5 mg/kg) i.p. on day 21 and ovarian cancer angiogenesis. 28 until the end of the experimental period. At the 35-day time point, all of the IgG-treated mice had complete penetra- MMP-14 antibody inhibits ovarian tumor growth and tion through the diaphragm and metastasis to the lungs and angiogenesis mediastinum, confirming that the OVCAR-4 cells were Small-molecule MMP inhibitors have not been successful in extremely aggressive and metastatic in vivo (Fig. 5D and clinical trials due to their broad-spectrum effects. As shown Supplementary Fig. S4A). In comparison, mice treated with earlier, monoclonal MMP-14 antibody had striking effects on in MMP-14 antibody had a marked decrease in metastatic pro- vivo ovarian cancer angiogenesis and did not bind nonspecif- gression (P ¼ 0.01) and the cancer remained confined to the ically to other MMPs such as MMP-8 (Supplementary Fig. S2C); peritoneal surface of the diaphragm in 75% of the mice (Fig. 5D therefore, we further tested whether these effects could be and Supplementary Fig S4A). Although we did not see a translated into decreased tumor growth. We injected 3.5 significant reduction in ascites volume between the two treat- million OVCAR-4 ovarian carcinoma cells subcutaneously into ment groups, the ascites in the MMP-14–treated mice was the flanks of nude mice. Mice were randomized 24 hours later straw colored as opposed to the bloody ascites in the IgG- into 2 treatment groups and treated with either MMP-14 treated mice (Supplementary Fig. S4B). MMP-14 antibody antibody or corresponding IgG (2.5 mg/kg, twice per week, conferred significant (P ¼ 0.01) protection against invasion i.p.). MMP-14 antibody–treated mice showed a significant into the diaphragm and thoracic metastases suggesting that reduction in tumor growth (P < 0.05) from day 26 onward MMP-14–targeted therapy maybe clinically relevant for inhi- (Fig. 5A). As antibodies usually have long lasting effects, we biting progression of advanced ovarian cancer. stopped treatment in both groups at day 27 and let the tumors grow for another week. After halting treatment, the IgG-treated Discussion mice had exponential tumor growth, whereas the MMP-14 MMP-14: a new antiangiogenic molecular therapeutic antibody-treated mice continued to show a significant reduc- target in human ovarian cancer tion (P < 0.01) in tumor growth compared with the IgG-treated Ovarian cancers critically depend on blood vessels for their mice. Furthermore, within the MMP-14 antibody treatment expansive growth and belong to the group of highly angiogenic group there was no significant increase in tumor size between tumors that respond to even monotherapy with antiangiogenic the day 28 and 35 time point showing the long lasting effect of drugs (48). Bevacizumab, a monoclonal antibody against the MMP-14 antibody treatment on tumor growth. Mice were VEGF, has been increasingly used upfront for therapy in euthanized and tumors collected for CD31 immunohistochem- ovarian cancer (10). Despite early benefits seen with these istry on day 35. MMP-14-treated mice showed a marked anti-VEGF pathway targeting drugs, the clinical benefits in reduction in blood vessels (P < 0.005) both at the tumor edge terms of overall survival have been modest and associated with and the tumor center compared with the IgG-treated controls enhanced local invasion and metastatic progression (12). (Fig. 5B and C). We correlated decreased angiogenesis with Therefore, it is possible that therapies that target multiple, reduced tumor growth in MMP-14 antibody-treated tumors distinct angiogenic pathways are superior to those targeting compared with their IgG-treated counterparts. Furthermore, singular angiogenic pathways. We believe MMP-14 could be we examined the livers of mice in both treatment groups and such a target in ovarian cancer. found no obvious signs of toxicity (data not shown). These MMP-14 has been associated with poor overall survival findings suggest that MMP-14 is an important regulator of in breast cancer (22), and in this study, we found MMP-14

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MMP-14 Antibody Inhibits Ovarian Tumor Progression

Figure 5. MMP-14 antibody inhibits tumor growth, angiogenesis, A B OVCAR-4 ovarian invasion, and metastasis in mice. carcinoma xenografts CD31-stained tumor sections A, OVCAR-4 ovarian cancer cells (3.5 million) were injected into the 800 Tumor edge Tumor center flanks of nude mice and the mice ) were treated with either IgG or MMP- 3 14 antibody i.p., 24 hours after 600 IgG implantation. Treatment was MMP14 IgG stopped from day 27 onward but 400 tumors were monitored and measured by calipers every other * day until day 35 (, P < 0.05). B and C, 200 * * * tumors were stained with CD31 * antibody and blood vessels counted Tumor growth (mm 0 in sections from each tumor. Intense 12 16 20 24 28 32 36 staining of individual endothelial cells was scored as a blood vessel in Days from tumor implantation a blinded manner from paraffin- embedded tissue sections. Vascular Treatment stopped MMP-14 density was determined by counting 5to9fields (magnification, 160) from at least 5 tumors in each group. CD31-stained blood vessels are CD shown by black arrows in tumor H&E-stained sections from treatment groups edge and center. Columns, mean; Lung Diaphragm bars, SE; , P < 0.05; , P < 0.005. D, female NCR Nu/Nu mice were Tumor CD31 injected i.p. with 1.5 million OVCAR- 4.5 T 4 cells and randomized into T n ¼ treatment groups of IgG ( 5) or 4.0 MMP-14 antibody (n ¼ 4, IgG 2.5 mg/kg/day, twice a week, i.p.), 3.5 starting on day 2. Both groups received docetaxel (5 mg/kg/week, 3.0 i.p.) starting on day 21 and a repeat dose after 7 days. Mice were 2.5 euthanized and diaphragm, peritoneal, and thoracic organs were 2.0 harvested, stained with hematoxylin **

Blood vessels per field 1.5 and eosin, and analyzed by 2 independent pathologists blinded

1.0 MMP-14 to the treatment groups. IgG MMP-14 Representative sections from lung and diaphragm are shown and tumor areas are marked by arrows. to be increased in the serum of patients with malignant of the disease, remission and potentially, response to anti- ovarian cancer compared with normal women and those MMP-14–targeted therapy. with benign ovarian masses confirming its importance for Recently, there have been several reports of proinvasive ovarian carcinogeneis. MMP-14 is a 66-kDa membrane- consequences of antiangiogenic tumor therapy (50) that led tethered MMP and unlike other MMPs it is not known to the authors to suggest that a combination of different treat- be secreted into the tumor milieu. However, we postulated ment regimens, which include anti-invasive therapy, may be that other MMPs and serine proteases present in the more effective in blocking tumor progression. MMP-14, which ovarian tumor microenvironment might cleave the extra- is expressed on the invadopodia of cancer cells (20), could be cellular domain of MMP-14 (49) and allow for its detection such a therapeutic target. However targeting MMPs has been in patient with ovarian cancer fluids and serum. We con- challenging and like other systemic therapy broad-spectrum firmed the increased presence of cleaved MMP-14 in malig- MMP inhibitors failed to produce enduring efficacy in terms of nant ovarian ascites, pleural effusions from patients with either tumor shrinkage or long-term survival benefit (30, 32). In ovarian cancer, and serum of these patients using ELISA this study, we used commercially available monoclonal anti- and immunoblot analysis using a MMP-14 monoclonal body against MMP-14 to significantly impair the migratory and antibody. To our knowledge, this is the first study to report invasive ability of MMP-14 expressing ovarian cancer cells. the detection of MMP-14 in the serum and malignant fluids This is especially relevant for the spread of ovarian cancer cells ofpatientswithovariancancer.DetectionofMMP-14inthe within the rich milieu of the peritoneal cavity (23). These serum is important for monitoring relapse and recurrence results show that antimigratory and anti-invasive property of

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Kaimal et al.

the MMP-14 antibody could be a significant advantage over the advanced ovarian cancer and implications for adjuvant ther- VEGF antibody for long-term antiangiogenic therapy. apeutic regimens not only for ovarian cancer but also for other Using a monoclonal antibody or RNAi against ovarian solid tumors. cancer cell MMP-14, we disrupted tumor–stromal communication that normally stimulated in vitro and in vivo angiogen- Disclosure of Potential Conflicts of Interest fl esis. In vivo, MMP-14 antibody was equivalent to Avastin in No potential con icts of interest were disclosed. blocking blood vessel growth within Matrigel plugs in mice. We fi Authors' Contributions con rmed that these effects were not due to inhibition of Conception and design: R. Aljumaily, A. Kuliopulos, C. Zarwan, A. Agarwal cellular proliferation but due to production of angiogenic Development of methodology: R. Aljumaily, S. Sharifi, A. Agarwal factors. Cancer cell MMP-14 not only regulated MMP-9 and Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Kaimal, S.L. Tressel, R.V. Pradhan, A. Kuliopulos, C. VEGF production and but also MMP-9 activity, which is Zarwan, Y.B. Kim, A. Agarwal important for VEGF release (46). Although OVCAR-4 cells do Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Kaimal, R. Aljumaily, S. Sharifi, A. Agarwal not express MMP-1, we have previously shown that MMP-14 on Writing, review, and/or revision of the manuscript: R. Aljumaily, L. Covic, A. the ovarian cancer cell, along with MMP-9 activates stromal Kuliopulos, Y.B. Kim, A. Agarwal proMMP-1 to MMP-1 (4), which is known to be important for Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): R.V. Pradhan tumor angiogenesis (9, 16) directly and through the upregula- Study supervision: A. Agarwal tion of several chemokines (27). Furthermore, mice bearing OVCAR-4 ovarian cancer tumors Acknowledgments when treated with MMP-14 antibody as monotherapy showed The authors thank all the authors whose important contributions could not be cited because of reference limits. The authors also thank Ms. Susan Turner, a marked regression in tumor growth. Even a week after the Drs. Katie Wakeley and John Yang for their help in collecting the patient samples, treatment had been stopped, there was no significant growth in Goli Javid for help with the histopathologic analysis, Ms. Namrata Nammi for the MMP-14 antibody-treated tumors, whereas the IgG-treated technical assistance, Dr. Katie O'Callaghan for reading the manuscript, and Prof. Phil Hinds for helpful suggestions. cohort continued to grow exponentially. The MMP-14 antibody-treated tumors showed a marked reduction in CD31- Grant Support stained blood vessels compared with the IgG controls provid- This work was funded by Liz Tilberis award from Ovarian Cancer Research ing proof-of-concept for targeting MMP-14 for antiangiogenic Foundation (to A. Agarwal), NIH T32 CA009429 training grant (to R. Aljumaily), Beauchamp Memorial Fund (to C. Zarwan), and NIH HL064701 (to A. therapy in ovarian cancer. In addition, in an aggressive, ortho- Kuliopulos). topic mouse model of ovarian cancer, MMP-14 antibody along The costs of publication of this article were defrayed in part by the payment of with docetaxel, inhibited invasion through the diaphragm and page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. thoracic metastasis. We believe that MMP-14 blockade as monotherapy or as combinatorial therapy with standard-of- Received April 19, 2012; revised January 11, 2013; accepted February 7, 2013; care drugs may have direct clinical relevance for patients with published OnlineFirst February 19, 2013.

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Selective Blockade of Matrix Metalloprotease-14 with a Monoclonal Antibody Abrogates Invasion, Angiogenesis, and Tumor Growth in Ovarian Cancer

Rajani Kaimal, Raid Aljumaily, Sarah L. Tressel, et al.

Cancer Res 2013;73:2457-2467. Published OnlineFirst February 19, 2013.

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