Abituzumab Targeting of Αv-Class Integrins Inhibits Prostate Cancer Progression

Published OnlineFirst March 17, 2017; DOI: 10.1158/1541-7786.MCR-16-0447

Oncogenes and Tumor Suppressors Molecular Cancer Research Abituzumab Targeting of aV-Class Integrins Inhibits Prostate Cancer Progression Yuan Jiang1,2, Jinlu Dai1, Zhi Yao2, Greg Shelley1, and Evan T. Keller1,3

Abstract

Integrins that contain an integrin aV subunit contribute to viability, cell-cycle, and caspase-3/7 activity. DI17E6 inhibited multiple functions that promote cancer progression. The goal of migration and invasion of prostate cancer cells. In addition, this study was to determine whether abituzumab (DI17E6, EMD DI7E6 decreased phosphorylation of FAK, Akt, and ERK. These 525797), a humanized monoclonal antibody (mAb) against results indicate that inhibition of integrin aV with DI17E6 integrin aV impacts, prostate cancer progression. To evaluate inhibits several prometastatic phenotypes of prostate cancer this, prostate cancer cells were treated with DI17E6 and its effects cells and therefore provide a rationale for further evaluation of on proliferation, apoptosis, cell-cycle, adhesion, detachment, DI17E6 for diminishing prostate cancer progression. migration, invasion and phosphorylation of downstream targets, including FAK, Akt, and ERK, were determined. DI17E6 Implications: This work identiﬁes that therapeutic targeting promoted detachment and inhibited adhesion of prostate can- of integrins containing an aV integrin unit inhibits cancer pro- cer cells to several extracellular matrix (ECM) proteins and cells gression and thus may be of clinical beneﬁt. Mol Cancer Res; 1–9. found in the bone microenvironment but had no impact on cell 2017 AACR.

Introduction without cross-reacting with other members of the integrin family (7). Results from a phase I trial in patients with progressive Prostate cancer is a major cause of cancer-related deaths (1, 2). castration-resistant prostate cancer with bone metastases after Although multiple therapies have been used to target prostate chemotherapy showed DI17E6 to be well-tolerated with potential cancer; once it has advanced, most therapies eventually fail antitumor activity (6). However, the mechanisms through which resulting in the patient's death. Development of additional ther- targeting aV integrins could provide an antitumor effect in pros- apeutic strategies is critical to increase survival rates of those tate cancer have not been defined. To explore these mechanisms, afflicted with prostate cancer. we evaluated DI17E6 on prostate cancer cell lines. Integrins are cell surface molecules that mediate cell-to-cell and cell-to-extracellular matrix adhesion and play important roles in Materials and Methods multiple cellular activities including cell mobility, cell viability, cell signaling network, and maintaining the prostate cancer stem Cells and cell culture pool (3). Through their ability to modulate cell adhesion and Human prostate cancer cell lines DU145, LNCaP, and PC3 were migration, integrins can promote cancer metastasis (4); therefore, obtained from the ATCC and cultured in RPMI-1640 (Invitrogen integrins may serve as therapeutic targets. Co.) supplemented with 10% FBS and 1% penicillin/streptomy- Modulation of aV-based integrins has significant effects on cin (Life Technologies, Inc.). The C4-2B cell line, which is an metastasis in preclinical in vivo prostate cancer models (5) leading LNCaP subline, and ARCaP (kindly provided by Dr. Leland to testing an integrin aV inhibitor, abituzumab (DI17E6, EMD Chung, Cedars Sinai) were maintained in T medium [80% DMEM 525797), in clinical prostate cancer (6). DI17E6 recognizes the (Life Technologies, Inc.), 20% F12 (Invitrogen), 100 units/L extracellular domains of the integrin aV chain and inhibits ligand penicillin G, 100 mg/mL streptomycin, 5 mg/mL insulin, 13.6 binding to all aV heterodimers (aVb1, aVb3, aVb5, aVb6, aVb8) pg/mL triiodothyronine, 5 mg/mL transferrin, 0.25 mg/mL biotin, and 25 mg/mL adenine] supplemented with 10% FBS. VCaP cells (kindly provided by Dr. Kenneth Pienta, University of Michigan, Ann Arbor, MI) were maintained in DMEM (Life Technologies, 1Department of Urology, University of Michigan, Ann Arbor, Michigan. 2Depart- 3 Inc.) with 10% FBS and 1% penicillin/streptomycin (Life Tech- ment of Immunology, Tianjin Medical University, Tianjin, China. Biointerfaces nologies, Inc.). The human fetal osteoblast line (hfOB1.19) was Institute, University of Michigan, Ann Arbor, Michigan. obtained from ATCC and was maintained in DMEM with F12 Note: Supplementary data for this article are available at Molecular Cancer (DMEM þ F12, Invitrogen) with 10% FBS and 1% penicillin/ Research Online (http://mcr.aacrjournals.org/). streptomycin (Life Technologies, Inc.). HS-5 and Saos2 cells were Corresponding Author: Evan T. Keller, Department of Urology, University of obtained from the ATCC and were maintained in a-MEM (Gibco) Michigan, NCRC B14 RM116, 2800 Plymouth Road, Ann Arbor, MI 48109. Phone: with 10% FBS and 1% penicillin/streptomycin (Life Technolo- 734-615-0280; Fax: 734-936-9220; E-mail: [email protected] gies, Inc.). Primary cultures of neonatal foreskin-derived human doi: 10.1158/1541-7786.MCR-16-0447 dermal microvascular endothelial cells (HDMEC) were obtained 2017 American Association for Cancer Research. commercially (Lonza). For cell culture, endothelial cell basal

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medium (EBM; Cambrex) was used and supplemented with 10% For cell-to-cell detachment assay, HDMECs were grown in 48- FBS (Life Technologies), EGF (10 ng/mL; BD Biosciences), hydro- well plate at 5 104 per well for 24 hours. Prostate cancer cells cortisone (1 mg/mL; Sigma-Aldrich), and 1% penicillin/strepto- were labeled with Qtracker 565 Cell Labeling Kit for 60 minutes mycin (Life Technologies, Inc.). All cultures were maintained at and washed twice with completed media and washed once with 37 C, 5% CO2, and 100% humidity. The identities of cell lines FBS-free medium. Prostate cancer cells were then resuspended in were confirmed using short tandem repeat fingerprinting. 250-mL serum-free media and added into the plates coated with HDMECs and allowed to incubate for 12 hours. The cells were Antibodies then treated with DI7E6 (0, 0.01, 0.1, 1, 10, 30, 100 mg/mL) for 60 a DI1E76 (Merck KGaA) is an anti-integrin V chain antibody minutes at which time media were removed and wells were gently a (7). Anti-integrin V antibody [EPR16800] (ab179475; Abcam) is washed 2 times with PBS. The cell fluorescence was measured a rabbit monoclonal antibody. using a plate reader at 405 to 565 nm. Cell proliferation Cell adhesion assay LNCaP, C4-2B, ARCaP, VCaP, PC3, and DU145 cells were For cell substratum adhesion assay, prostate cancer cells were grown in 96-well plates. Cells were then treated with DI17E6 m m treated with DI17E6 (0.01, 0.1, 1, 10, 30, 100 g/mL) or vehicle (0.1, 1, 10, 100 g/mL) or vehicle for 24, 48, and 72 hours. Cell 5 for 24 hours, and then 250 mL cell suspension containing 5 10 proliferation reagent WST-1 was added and incubated at 37 C and cells/well in serum-free media were added to fibronectin/vitro- 5% CO for 4 hours. Absorbance was then measured at 440 nm 2 nectin/osteopontin/collagen I–coated plates and incubated for 60 with a plate reader. Data are presented as mean SD from minutes. The media were then removed and plate gently washed triplicate determinations. with PBS. Then, 0.2 mL of 96% ethanol was added into each well Caspase-3/7 assay and plates incubated at room temperature for 10 minutes. Then, LNCaP, C4-2B, ARCaP, VCaP, PC3, and DU145 were cultured 0.5 mL 0.1% crystal violet was added into each well, and plates in white-walled 96-well plates at a concentration of 1 104 per 50 were incubated at room temperature for 30 minutes. Plates were mL per well. Cells were treated with 50 mL DI176E (0.1, 1, 10, and then gently washed with deionized water. Extraction solution 100 mg/mL final concentrations) or vehicle or etoposide (100 (0.05 mL 0.2% Triton X in deionized water) was then added, and nmol/L final concentration; Sigma) as a positive control and were optical density was measured at 570 nm in a plate reader. – allowed to grow for 24 hours at which point 100 mL of Apo-ONE For cell cell adhesion assay, hFOB, Saos2, or HS-5 cells were 4 Caspase-3/7 Reagent (Promega) was added to each well and the grown in 48-well plate at 5 10 per well for 24 hours. Prostate cells were incubated for extended periods (>4 hours). Absorbance cancer cells were labeled with Qtracker 565 Cell Labeling Kit for was determined at 499 and 521 nm using a plate reader (Multi- 60 minutes and washed twice with completed media and washed Mode Microplate Reader, SpectraMax M5, Molecular Devices once with FBS-free medium. Prostate cancer cells were then m MDS Analytical Technologies). resuspended in 250 L serum-free media and added into the plates coated with target cells. The cells were then treated with Cell-cycle analysis DI7E6 (0, 0.01, 0.1, 1, 10, 30, 100 mg/mL) for 60 minutes at which LNCaP, C4-2B, ARCaP, VCaP, PC3, and DU145 were cultured time media were removed and wells were gently washed 2 times in 100-mm dishes at a concentration of 1 105 per dish in 10 mL. with PBS. The media and PBS wash were combined, and fluores- Cells were treated with DI176E (100 mg/mL) or vehicle for 72 cence was measured and reported as relative to vehicle. hours. Cells were then collected and fixed with ice-cold 70% ethanol for overnight. After that, cell-cycle analysis was performed Scratch wound-healing assay using flow cytometry as previously described (8). Briefly cells were Prostate cancer cells (LNCaP, C4-2B, ARCaP, PC3, VCaP, and centrifuged and washed with DPBS twice for removing ethanol DU145) were seeded into 24-well plates. After cells reached 70% fl and then 0.8 mL of DNAse-free RNAse was added into each tube to 80% con uence, a 1-mL pipette tip was used to scratch across and incubated at 37C for 10 minutes. Staining was carried out by the center of the well and another straight line, perpendicular to fi adding 500 mL of propidium iodide (50 mg/mL) to each tube. Cells the rst line. After scratching, the wells were gently washed twice were then subjected to flow cytometry (BD Biosciences), and with medium to remove the detached cells, and cells were replen- ModFit LT 4.1 software (Verity Software House) was used for ished with fresh medium. The cells were treated with vehicle or m determining cell-cycle parameters. DI7E6 (0.01, 0.1, 1, 10, 30, 100 g/mL). Images were captured every 6 hours. The gap distance was quantitatively measured. Cell detachment assay Ninety-six-well plates were coated with fibronectin/vitronec- Invasion assay tin/osteopontin (OPN)/collagen I. LNCaP, C4-2B, ARCaP, and Modified Boyden chambers (BD Biocoat Matrigel chambers) VCaP were seeded at the concentration of 3 104 into wells of 96- were used to assess invasion in vitro. Prostate cancer cell lines well plates in FBS-free media for 12 hours; PC3 and DU145 were (PC3, DU145, C4-2B, LNCaP, ARCaP, and VCaP cells) were seeded at the concentration of 1 104 into wells of 96-well plates seeded in the chamber inserts at density of 5 104 cells in 0.5 in FBS-free media for 12 hours in 100 mL and then treated with mL and treated with vehicle or 100 mg/mL DI17E6 in a CO2 DI17E6 (0, 0.01, 0.1, 1, 10, 30, 100 mg/mL) for 24 hours. WST-1 incubator at 37 C for 22 hours. The chamber inserts were then was added after gently washing off detached cells with PBS 3 stained using the Diff-Quick Staining Kit (Dade-Behring) accord- times, and plates were incubated at 37C for 4 hours. Absorbance ing to the manufacturer's instructions. Finally, the membranes was then measured at 440 nm using a plate reader. Prostate cancer were separated with a sterile scalpel and observed using a light cell detachment was determined by subtraction of the number of microscope and quantified by counting the numbers of cells that attached cells from the number of control unexposed DI17E6. penetrated through the membrane in 5 random microscopic

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A C 24 h 48 h Vehicle DI17E6 0.8 2.0 (µg/mL) 0.6 LNCap 1.5 LNCap ARCap C4-2B 0.4 C4-2B 1.0 ARCap DU145 PC3 PC3 Number 0.2 DU145 0.5 DU145 Number OD Value VCap OD Value VCap 0.0 0.0 Channels (FL3-HPI LIN) Channels (FL3-HPI LIN) 200 40 60 80 100 200 40 60 80 100

2.5 72 h VCaP Number 2.0 LNCap Number 1.5 C4-2B ARCap Channels (FL3-HPI LIN) Channels (FL3-HPI LIN) 1.0 PC3 0.5 DU145 OD Value 0.0 VCap LNCaP

200 40 60 80 100 Number Number

DI17E6 (µg/mL) Channels (FL3-HPI LIN) Channels (FL3-HPI LIN)

B ARCaP Number Number DU145 ARCaP Channels (FL3-HPI LIN) Channels (FL3-HPI LIN) 200 200 C4-2B 150 150 Number Number

100 100 Channels (FL3-HPI LIN) Channels (FL3-HPI LIN)

(% vehicle) 50 (% vehicle) 50 PC3 0 0 Number Number Caspase-3/7 activity 1 Caspase-3/7 activity 1 0.1 10 100 0.1 10 100 Vehicle Vehicle Etoposide Etoposide Channels (FL3-HPI LIN) Channels (FL3-HPI LIN) PC3 LNCaP 200 200 DU145 PC3 60 80 Control 150 150 60 40 DI176E 100 100 40 (µg/mL) 20 20 50 50 0

Percentage (%) Percentage (%) Percentage 0 (% vehicle)

(% vehicle) G0–G1 S G2–M G –G S G –M 0 0 0 1 2 0.1 1 10 0.1 1 10 100 Caspase-3/7 activity 100 C4-2B LNCaP Caspase-3/7 activity Vehicle Vehicle Etoposide Etoposide 80 80 C4-2B VCaP 60 60 200 200 40 40 20 20 150 150 0 Percentage (%) Percentage Percentage (%) Percentage 0 G –G S G –M G –G S G –M 100 100 0 1 2 0 1 2 ARCaP VCaP 50 50 60 80 (% vehicle) (% vehicle) 0 60 0 40 0.1 1 10 0.1 1 10 100 Caspase-3/7 activity 100 40 Caspase-3/7 activity Vehicle Vehicle Etoposide Etoposide 20 20

µ 0 (%) Percentage 0 DI176E ( g/mL) (%) Percentage G0–G1 S G2–M G0–G1 S G2–M

Figure 1. DI17E6 does not impact prostate cancer cell viability, proliferation, or apoptosis. Prostate cancer cell lines (LNCaP, C4-2B, ARCaP, PC3, DU145, and VCaP cells) were treated with vehicle and indicated dose of DI17E6. Viability (A), caspase-3/7 activity (B), and cell cycle (C) of prostate cancer cells were determined. The experiment was repeated 3 times and reported as mean SD%.

fields (at 200 magnification) per filter. Invasive ability was Numerical data are expressed as mean SD. Statistical analysis defined as the proportion of cells that penetrated through the was performed by analysis of one-way ANOVA and/or the Student matrix-coated membrane divided by the number of cells that t test for independent analysis. P < 0.05 was considered statisti- migrated through the uncoated membrane (baseline migration). cally significant. Results Western blot assay Prostate cancer cell lines (PC3, DU145, C4-2B, LNCaP, ARCaP, DI17E6 has no direct effect on overall prostate cancer cell and VCaP cells) were treated with vehicle or 100 mg/mL DI17E6 growth for 24 hours. Then, cells were lysed with cell lysis buffer and To determine whether DI17E6 modulated cell growth para- separated on 12% SDS-PAGE, transferred to polyvinylidene meters, we quantified its impact on cell viability, cell-cycle dis- difluoride membranes (Millipore), and then blocked with 10% tribution, and apoptosis in 6 different prostate cancer cell lines. defatted milk for 1 hour at 37C and incubated with antibodies DI17E6 had no impact on cell viability at various doses up to 100 (see Supplemental Table 1) overnight at 4C. The immunoreac- mg/mL and up to 72 hours' time (Fig. 1A). Similarly, DI17E6 had tive bands were visualized using an enhanced chemiluminescence no impact on apoptotic rate at various doses up to 100 mg/mL, (ECL) system (Pierce) and GADPH was internal control. although etoposide, a known inducer of apoptosis, increased apoptosis of the prostate cancer cells (Fig. 1B). In addition, Statistical analyses DI17E6 had no impact on cell-cycle distribution (Fig. 1C). Taken All in vitro experiments were performed at least 3 times with together, these results indicate that DI17E6 does not directly similar results. Results from representative assays are shown. modulate tumor growth activity.

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A B Detachment assay (vitronectin-coated plates) Detachment assay (osteopontin-coated plates) DU145 (OPN) PC3 (OPN) DU145 (vitronectin) PC3 (vitronectin) P < 0.0001 P = 0.0005 40 P < 0.0001 30 40 P < 0.0001 30 P = 0.0009 P < 0.0001 P = 0.0007 P < 0.0001 30 P = 0.0005 30 P = 0.0005 P = 0.0005 20 P < 0.0001 20 P = 0.0012 20 P = 0.0002 20 P < 0.0001 P = 0.0005 10 P = 0.0369 10 10 10 0 0 0 0 1 1 0.1 10 0.1 10 1 1 Detached cell number (%) cell number Detached 100 Detached cell number (%) cell number Detached 100 10 10 0.01 0.01 0.01 0.1 100 0.01 0.1 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) LNCaP (vitronectin) C4-2B (vitronectin) C4-2B (OPN) LNCaP (OPN) 40 P < 0.0001 40 P < 0.0001 P = 0.0014 P < 0.0001 P < 0.0001 40 40 P < 0.0001 P < 0.0001 P = 0.0015 30 30 P < 0.0001 30 P = 0.0215 30 P = 0.0005 P < 0.0001 P = 0.0014 P = 0.0044 20 P = 0.0143 20 P = 0.0210 20 P = 0.0457 20 P = 0.0004 10 10 10 10 P = 0.0014 0 0 0 0 1 0.1 10 100 1 10 0.01 (%) cell number Detached 0.1 1 1 Detached cell number (%) cell number Detached 100 0.01 0.1 10 100 0.1 10 Vehicle DI17E6 (µg/mL) Vehicle µ 0.01 0.01 100 DI17E6 ( g/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) ARCaP (vitronectin) VCaP (vitronectin) 40 P < 0.0001 30 P < 0.0001 ARCaP (OPN) VCaP (OPN) 30 P = 0.0004 20 P = 0.0017 40 P = 0.0052 40 P = 0.0011 P = 0.0102 P = 0.0031 20 30 P = 0.0071 30 P = 0.0078 10 P = 0.0022 P = 0.0016 10 20 P = 0.0017 20 P = 0.0022 0 0 10 10 1 1 0.1 10 0.1 10 0 0 100 (%) cell number Detached 0.01 100 Detached cell number (%) cell number Detached 0.01 Vehicle µ Vehicle µ DI17E6 ( g/mL) DI17E6 ( g/mL) 1 10 1 10

0.1 (%) cell number Detached (%) cell number Detached (%) cell number Detached 0.1 Detached cell number (%) cell number Detached (%) cell number Detached 0.01 (%) cell number Detached 100 0.01 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) CD Detachment assay (collagen I-coated plates) Detachment assay (fibronectin-coated plates)

DU145 (fibronectin) PC3 (fibronectin) DU145 (Collagen I) PC3 (Collagen I) 30 40 P = 0.0001 4 4 P = 0.0051 30 P = 0.0002 20 P = 0.0409 P = 0.0019 2 2 P = 0.0407 20 10 P = 0.0352 10 0 0 0 0 –2 –2

Detached cell number (%) cell number Detached 1 1

0.1 10 (%) cell number Detached 0.1 10 Detached cell number (%) cell number Detached 1 1 0.01 100 0.01 100 0.1 10 100 0.1 10 100 0.01 (%) cell number Detached 0.01 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL)

C4-2B (Fibronection) LNCaP (Fibronectin) C4-2B (Collagen I) LNCaP (Collagen I) 40 40 P < 0.0001 4 P = 0.0005 4 30 P < 0.0001 30 P = 0.0019 2 20 P < 0.0001 20 P = 0.0003 2 P < 0.0001 10 10 0 0 0 0 –2 –2

1 (%) cell number Detached 1 1 10 1 10 0.1 10 0.1 10 (%) cell number Detached 0.1 100 0.1 100 100 100 0.01 (%) cell number Detached 0.01 Detached cell number (%) cell number Detached 0.01 0.01 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL)

ARCaP (Fibronectin) VCaP (Fibronectin) ARCaP (Collagen I) VCaP (Collagen I) 30 P = 0.0234 30 P = 0.0002 4 4 P = 0.0217 20 20 P = 0.0005 P = 0.0237 2 2 10 10 0 0 0 0 –2 –2 1 1 1 1 (%) cell number Detached 10 Detached cell number (%) cell number Detached 10 Detached cell number (%) cell number Detached 10 0.1 Detached cell number (%) cell number Detached 10 100 0.1 0.01 0.1 100 0.01 0.1 100 0.01 0.01 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL)

Figure 2. DI17E6 promotes detachment of prostate cancer cells from vitronectin, ﬁbronectin, and osteopontin. Prostate cancer cell lines (LNCaP, C4-2B, ARCaP, PC3, DU145, and VCaP cells) were seeded on 96-well plates coated with vitronectin (A), ﬁbronectin (B), osteopontin (C), and collagen I (D) and incubated for 12 hours. Then, cells were treated with vehicle and the indicated dose of DI17E6. After 24 hours, the plates were gently washed, and the cells that detached were then counted. The experiment was repeated 3 times and reported as mean SD%.

DI7E6 inhibits adhesion of prostate cancer to multiple We also explored the effect of DI17E6 on the adhesion of prostate extracellular matrix proteins but not collagen I cancer cells to the various matrices in which we incubated the cells To determine whether DI7E6 could reverse established cell-to- with DI17E6 simultaneously upon introducing them to the extracellular matrix adhesion, we incubated prostate cancer cells various coated cell culture plates. DI17E6 inhibited adhesion to in cell culture plates coated with various extracellular matrix vitronectin, osteopontin, and fibronectin (between 20% and proteins including vitronectin, osteopontin, fibronectin, and col- 40% at 100 mg/mL for the various cell lines and matrices) but not lagen I and then added DI17E6 to determine its impact on collagen I (Fig. 3). detachment. DI17E6 promoted detachment of prostate cancer cells from vitronectin (up to approximately 20% cells detached at DI17E6 inhibits motility and invasion of prostate cancer cells 100 mg/mL), osteopontin (up to approximately 10% cells Integrins play a major role in motility and invasive ability. detached at 100 mg/mL), and fibronectin (up to approximately Thus, we explored the effect of DI17E6 on the motility and 10% cells detached at 100 mg/mL) but not from collagen I (Fig. 2). invasion of prostate cancer cells. DI17E6 (100 mg/mL) reduced

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ABAdhesion assay Adhesion assay (vitronectin coated) (fibronectin coated) PC3 PC3 100 DU145 100 DU145 LNCaP 80 LNCaP 80 C4-2B C4-2B 60 60 ARCaP ARCaP OD value VCaP 40 VCaP 0204060 80 100 0 20406080100 (ratio vs. vehicle) (ratio vs. (ratio vs. vehicle) (ratio vs. DI17E6 (µg/mL) Relative OD value DI17E6 (µg/mL) DU145 (Fibronectin) PC3 (Fibronectin) DI17E6 (µg/mL) DU145 (Vitronectin) PC3 (Vitronectin) 100 100 µ 100 100 Vehicle 0.01 0.1 1 10 30 100 BSA DI17E6 ( g/mL) 80 80 80 Vehicle 0.01 0.1 1 10 30 100 BSA 80 60 DU145 60 60 60 40 40 0 20406080100 DU145 40 40

(ratio vs. vehicle) (ratio vs. 0 20406080100 (ratio vs. vehicle) (ratio vs. Relative OD value µ Relative OD value 0 20406080100 0 20406080100

DI17E6 ( g/mL) µ vehicle) (ratio vs. Relative OD value DI17E6 ( g/mL) µ vehicle) (ratio vs. PC3 DI17E6 ( g/mL) Relative OD value DI17E6 (µg/mL) C4-2B (Fibronectin) LNCaP (Fibronectin) PC3 C4-2B (Vitronectin) C4-2B 100 100 LNCaP (Vitronectin) 100 80 80 C4-2B 100 80 LNCaP 60 60 80 60 40 40 LNCaP 60 ARCaP 020406080100 0 20406080100 40 (ratio vs. vehicle) (ratio vs. 40 (ratio vs. vehicle) (ratio vs. Relative OD value Relative OD value DI17E6 (µg/mL) µ DI17E6 ( g/mL) ARCaP 0 20406080100 0 20406080100 (ratio vs. vehicle) (ratio vs. Relative OD value

µ vehicle) (ratio vs. DI17E6 ( g/mL) Relative OD value DI17E6 (µg/mL) VCaP ARCaP (Fibronectin) VCaP (Fibronectin) VCaP 100 100 ARCaP (Vitronectin) VCaP (Vitronectin) 100 100 Fibronectin-coated plate 80 80 Vitronectin-coated plate 60 60 80 80 40 40 60 60 0 20406080100 0 20406080100 (ratio vs. vehicle) (ratio vs. Relative OD value (ratio vs. vehicle) (ratio vs. 40 40 µ Relative OD value µ DI17E6 ( g/mL) DI17E6 ( g/mL) 020406080100 020406080100 µ (ratio vs. vehicle) (ratio vs. (ratio vs. vehicle) (ratio vs. µ Relative OD value DI17E6 ( g/mL) CDRelative OD value DI17E6 ( g/mL) Adhesion assay (OPN coated) Adhesion assay PC3 (collagen-I coated) 100 DU145 120 LNCaP PC3 80 C4-2B DU145 60 ARCaP 100 LNCaP VCaP C4-2B 40 ARCaP 0 20406080100 µ 80 VCaP µ vehicle) (ratio vs. DI17E6 ( g/mL) DI17E6 ( g/mL) Relative OD value DI17E6 (µg/mL) 0 20406080100

Vehicle 0.01 0.1 1 10 30 100 BSA vehicle) (ratio vs.

Relative OD value µ Vehicle 0.01 0.1 1 10 30 100 BSA DU145 (OPN) PC3 (OPN) DI17E6 ( g/mL) DU145 (Collagen I) PC3 (Collagen I) DU145 100 100 DU145 120 120 80 80 100 100 60 60 PC3 PC3 80 80 40 0 20406080100 40 0 20406080100 60 60

(ratio vs. vehicle) (ratio vs. µ µ (ratio vs. vehicle) (ratio vs. Relative OD value 0 20406080100 0 20406080100 C4-2B DI17E6 ( g/mL) Relative OD value DI17E6 ( g/mL) C4-2B

(ratio vs. vehicle) (ratio vs. µ (ratio vs. vehicle) (ratio vs. µ Relative OD value DI17E6 ( g/mL) Relative OD value DI17E6 ( g/mL) C4-2B (OPN) LNCaP (OPN) C4-2B (Collagen I) LNCaP (Collagen I) LNCaP 100 100 LNCaP 120 120 80 80 100 100 60 ARCaP 60 ARCaP 80 80 40 40 0 20406080100 0 20406080100 60 60

(ratio vs. vehicle) (ratio vs. µ 0 20406080100 0 20406080100 (ratio vs. vehicle) (ratio vs. Relative OD value µ VCaP DI17E6 ( g/mL) Relative OD value DI17E6 ( g/mL) µ µ (ratio vs. vehicle) (ratio vs. DI17E6 ( g/mL) vehicle) (ratio vs.

Relative OD value DI17E6 ( g/mL)

VCaP Relative OD value Osteopontin-coated plate ARCaP (OPN) VCaP (OPN) ARCaP (Collagen I) VCaP (Collagen I) 100 100 Collagen I-coated plate 120 120 80 80 100 100 60 60 80 80 40 40 0 20406080100 0 20406080100 60 60 0 20406080100 0 20406080100

(ratio vs. vehicle) (ratio vs. µ (ratio vs. vehicle) (ratio vs. Relative OD value DI17E6 ( g/mL) Relative OD value µ µ

DI17E6 ( g/mL) vehicle) (ratio vs. (ratio vs. vehicle) (ratio vs. DI17E6 ( g/mL)

µ Relative OD value Relative OD value DI17E6 ( g/mL)

Figure 3. DI17E6 inhibits adhesion of prostate cancer cells on vitronectin, fibronectin, and osteopontin. Prostate cancer cell lines (LNCaP, C4-2B, ARCaP, PC3, DU145, and VCaP cells) were seeded on the 96-well plates precoated with fibronectin (A), vitronectin (B), osteopontin (C), and collagen I (D) and then the cells were treated with vehicle or indicated dose of DI17E6. After 24 hours, the plates were gently washed and the cells that remained attached were stained with crystal violet. The level of crystal violet was measured. The experiment was repeated 3 times and reported as mean SD%. cell motility at 12 and 18 hours postinitiation of the assay, detachment of prostate cancer cells from to a known expressor of whereas no inhibition was noted at lower doses (Fig. 4A). DI17E6 aV integrins, HDMECs (Fig. 5). inhibited motility by approximately 30% to 40% compared with that of vehicle control. In addition, DI17E6 inhibited invasive Effect of DI17E6 on the expression of tumor-related genes in ability by approximately 25% to 30% compared with vehicle (Fig. prostate cancer cell lines 4B). To determine whether the effect was specific to DI17E6 or Upon engagement of integrins, intracellular signaling is applied to general inhibition of integrin aV, we assessed the activated. To determine whether DI17E6 blocked integrin- ability of an alternative anti-aV antibody, EPR16800, to impact mediated cell signaling, prostate cancer cells were plated in prostate cancer cell migration and invasion. It was able to inhibit collagen-coated plates with the addition of DI17E6 or vehicle. both migration and invasion similar to that seen using DI17E6 DI17E6 inhibited FAK phosphorylation starting at 12 and 6 (Supplementary Fig. S1). hours in C4-2B and LNCaP, respectively; AKT phosphorylation starting at 3 and 2 hours in C4-2B and LNCaP, respectively; and DI17E6 inhibits the ability of prostate cancer cells to adhere to ERK phosphorylation at starting at 1 and 1.5 hours in C4-2B osteoblast and bone stromal cell lines and LNCaP, respectively (Fig. 6). DI1E76 was associated with Bone metastasis occurs in most men with advanced prostate decreased expression of Integrin aV in both cell lines. Similar cancer. Integrins may mediate bone metastasis through their results were found using an alternative anti-aVantibody, ability to allow adherence to cells in the bone microenvironment. EPR16800 (Supplementary Fig. S2). Accordingly, we evaluated whether DI17E6 impacted the ability of prostate cancer cells to bind to osteoblast and bone marrow stroma cell lines. Furthermore, DI17E6 inhibited prostate cancer Discussion cell adhesion to human osteoblast cell lines hFOB and Saos2 and As prostate cancer advances, the majority of men will develop a bone marrow stromal cell line, HS-5 (Fig. 5). DI17E6 promoted bone metastases and its associated mortality. Because of the

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Jiang et al.

DU145 A DU145 PC3 150 P = 0.0454 (two-way ANOVA) 0 h 6 h 12 h 18 h 0 h 6 h 12 h 18 h 100 P = 0.0012 Vehicle P = 0.0243 50 (% of T0) (% of

0.1 µg/mL width Wound 0 1 1 1 1 0.1 10 100 0.1 10 100 0.1 10 100 0.1 10 100 Vehicle Vehicle Vehicle Vehicle 0 h 1 µg/mL PC3 6 h 150 12 h P = 0.0192 (two-way ANOVA) 10 µg/mL 18 h 100 P = 0.0061 100 µg/mL P = 0.0280

(% of T0) (% of 50 Wound width Wound 0 1 1 1 1 0.1 10 100 0.1 10 100 0.1 10 100 0.1 10 100 B Vehicle Vehicle Vehicle Vehicle DI17E6 (µg/mL) Migration Invasion Vehicle DI17E6Vehicle DI17E6 (100 µg/mL) (100 µg/mL)

DU145

150 C4-2B

100 LNCaP Vehicle DI17E6 50 (100 µg/mL) Invasion rate Invasion PC3 Vehicle) (% of 0 PC3 DU145 C4-2B LNCaP ARCaP VCaP VCaP

ARCaP

Figure 4. DI17E6 inhibits prostate cancer cell motility and invasiveness. A, Scratch wound assays were performed on prostate cancer cells (LNCaP, C4-2B, ARCaP, PC3, DU145, and VCaP cells) grown to 90% conﬂuency followed by treatment with vehicle and or the indicated dose of DI17E6. The wound gap was measured every 6 hours. B, Modiﬁed Boyden chamber assay was performed with prostate cancer cell lines (LNCaP, C4-2B, ARCaP, PC3, DU145, and VCaP cells) without Matrigel (motility) and with Matrigel (invasion). Cells were treated with vehicle or the indicated dose of DI17E6. After 24 hours, cells that had invaded through the Matrigel-coated membrane were stained and counted. The experiment was repeated 3 times and reported as mean SD%.

prevalence of bone metastases and its impending lethality, DI17E6 has been demonstrated to block growth of several many efforts have been put forth toward defining mechanisms tumors using in vivo models (e.g., melanoma, renal, and colorectal and therapeutic targets for bone metastasis. This work has led cancers) independently of antiangiogenic activity (6, 12). We to identification of a variety of mediators that may play role in demonstrated that DI17E6 suppressed aV integrin expression; the process of bone metastasis including integrins. Well-rec- however, it did not impact prostate cancer cell growth, cell-cycle ognized to play roles in multiple aspects of biology and distribution, or apoptotic rate. These results in combination with pathology, integrins are a family of proteins that function in the previous reports of inhibition of tumor growth in vivo suggest cell–cell and cell–matrix adhesion. Integrins are composed of that inhibition of aV integrins does not directly impact tumor a-andb-subunits and various combinations of the a-and cells but rather mediates their activity indirectly. Possible mechan- b-subunits result in formation of integrins with various spe- isms include altering angiogenesis or immune cell infiltration into cificities for ligands. tumors. One integrin subunit, aV, composes the a-subunit for at least 5 The ability of cells to detach and attach has been shown to different integrins. Inhibition of aV has been shown to reduce be important for tumor metastasis and its potential inhibition tumor growth and metastasis of a variety of tumors (9). Inhibition (13, 14). Our finding that DI17E6 promotes detachment and of activated aV integrins reduced cell survival, induced cell–cycle inhibits attachment of prostate cancer cell lines in vitro suggests blockade, and sensitized tumor to combination therapies includ- that it would impact the establishment and progression of metas- ing radio- and chemotherapy (10, 11). These findings provided tasis. The potential of migration and invasion is also an important the rationale to design a clinically viable method to target aV. component of metastasis (15–17). Our observation that DI17E6 Accordingly, a humanized monoclonal antibody that targets the reduces migration and invasion of prostate cancer cell lines aV subunit of integrins, DI17E6, was designed. suggests that it could suppress invasion of clinical prostate cancer.

OF6 Mol Cancer Res; 2017 Molecular Cancer Research

aV-Class Antibody Inhibits Prostate Cancer Progression

Adhesion assay (hFOB-coated plate) A PC3 µ 100 DU145 DI17E6 ( g/mL) LNCaP Vehicle 0.01 0.1 1 10 30 100 80 C4-2B ARCaP 60 VCaP

DU145 565 Qtracker 0 20 40 60 10080 (ratio vs. vehicle) (ratio vs.

relative fluorescence DI17E6 (µg/mL)

PC3 DU145 PC3 100 100 80 80 60 60 Qtracker 565 Qtracker Qtracker 565 Qtracker

(ratio vs. vehicle) (ratio vs. 0 20 40 60 80 100 C4-2B vehicle) (ratio vs. 0 20 40 60 80 100 relative fluorescence relative fluorescence DI17E6 (µg/mL) DI17E6 (µg/mL) LNCaP C4-2B 100 100 LNCaP 80 80 60 60 Qtracker 565 Qtracker Qtracker 565 Qtracker

(ratio vs. vehicle) (ratio vs. 0 20 40 60 80 100 (ratio vs. vehicle) (ratio vs. 0 20406080 100 relative fluorescence ARCaP relative fluorescence DI17E6 (µg/mL) DI17E6 (µg/mL) ARCaP VCaP 100 100 VCaP 80 80 60 60 Qtracker 565 Qtracker Qtracker 565 Qtracker (ratio vs. vehicle) (ratio vs. 0 20406080 100 vehicle) (ratio vs. 0 20406080 100 relative fluorescence DI17E6 (µg/mL) relative fluorescence DI17E6 (µg/mL) B DI17E6 (µg/mL) Vehicle 0.01 0.1 1 10 30 100 Detachment assay (HDMEC coated plate) DU145 PC3 DU145 30 P < 0.0001 30 P = 0.0009 P = 0.0014 P < 0.0001 20 P = 0.0331 20 P = 0.0123 P = 0.0171 P = 0.0021 10 P = 0.0390 10 Qtracker 565 Qtracker PC3 565 Qtracker 0 0 (ratio vs. vehicle) (ratio vs. (ratio vs. vehicle) (ratio vs. 1 10 30 1 10 30 relative fluorescence relative fluorescence 0.01 0.1 100 0.01 0.1 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) C4-2B LNCaP C4-2B 30 P = 0.0009 30 P < 0.0001 P = 0.0025 P < 0.0001 20 P = 0.0066 20 P = 0.0004 P = 0.0135 P = 0.0007 10 P = 0.0109 10 P = 0.0121

LNCaP 565 Qtracker 0 565 Qtracker 0 (ratio vs. vehicle) (ratio vs. 1 10 30 vehicle) (ratio vs. 1 10 30 relative fluorescence 0.01 0.1 100 relative fluorescence 0.01 0.1 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) ARCaP VCaP ARCaP P = 0.0006 P = 0.0002 30 P < 0.0001 30 P = 0.0012 P = 0.0162 P < 0.0001 20 P = 0.0420 20 P = 0.0047 P = 0.0004 10 10 Qtracker 565 Qtracker VCaP 0 565 Qtracker 0 (ratio vs. vehicle) (ratio vs. 1 10 30 vehicle) (ratio vs. 1 10 30 relative fluorescence 0.01 0.1 100 relative fluorescence 0.01 0.1 100 Vehicle DI17E6 (µg/mL) Vehicle DI17E6 (µg/mL) C Adhesion to HS-5 Adhesion to Saos2 P = 0.0421 P = 0.0238 150 P = 0.0198 150 P = 0.0153 P = 0.0007 P = 0.0474 P = 0.0284 P = 0.0133 P = 0.006 DU145 100 100 PC3

50 50 Qtracker 565 Qtracker Qtracker 565 Qtracker (ratio vs. vehicle) (ratio vs. 0 vehicle) (ratio vs.

relative fluorescence 1 1 0

relative fluorescence 1 1 0.1 10 30 0.1 10 30 10 30 10 30 100 100 0.1 100 0.1 100 Vehicle Vehicle Vehicle Vehicle DI17E6 (µg/mL) DI17E6 (µg/mL)

Figure 5. DI17E6 inhibits adhesion of prostate cancer cells onto hFOB, HDMEC, HS-5, and Saos5 cells. A, Prostate cancer cell lines were stained with Qtracker 565 and seeded on plates precoated with hFOB and then treated with the indicated dose of DI17E6 for 1 hour, at which time the plates were gently washed and the fluorescence of the remaining cells was measured. , P < 0.05 versus vehicle. B, Prostate cancer cell lines were stained with Qtracker 565 and seeded on plates precoated with HDMECs, incubated for 12 hours, and then treated with the indicated dose of DI17E6 for 24 hours, at which time the plates were gently washed and the cells in the wash fluid were collected and their fluorescence measured. C, Prostate cancer cell lines (Du145 and PC3) were stained with Qtracker 565 and seeded on plates precoated with HS-5 or Saos2 and then treated with the indicated dose of DI17E6 for 24 hours, at which time the plates were gently washed and the fluorescence of the remaining cells was measured. These experiments were repeated 3 times and reported as mean SD%.

Engagement of integrins activates multiple signaling path- aspects of tumor progression including growth and invasion ways that are associated with protumorigenic activities, includ- (21, 22). ing invasion and metastasis (18, 19). Our observation that In summary, the ability of DI17E6 to target integrin aV pro- DI17E6 suppressed activation of multiple integrin-activated vides the potential to suppress prostate cancer metastasis through signaling pathways suggests that inhibition of signaling could a variety of mechanisms including inhibition of cell–cell and cell– contribute to the anti-migration and invasive activity of extracellular matrix interactions, cellular invasion, and cell sig- DI17E6. For example, FAK is well-recognized to promote naling (23, 24). These ﬁndings provide the rationale to perform motility and invasion (20). In addition, ERK affects multiple future studies to provide in vivo validation and further insight into

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Jiang et al.

C4-2B

DI17E6 2.5 P = 0.0089 (one-way ANOVA) 2.0 P = 0.0134 1.5 Vehicle15 min 30 min 1 h 1.5 h 2 h 3 h 12 h 1.0

Integrin alpha V (p-FAK/FAK) 0.5

Image ratio density 0.0 Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 12 h

p-FAK 2.5 P = 0.0012 (one-way ANOVA) 2.0 1.5 P = 0.0494 FAK 1.0 0.5 (p-FAK/FAK) 0.0

p-Akt Image ratio density Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 12 h

2.5 Akt P = 0.0003 (one-way ANOVA) 2.0 P = 0.0310 p-ERK 1.5 P = 0.0167 1.0 0.5 ERK (p-FAK/FAK) 0.0 Image ratio density Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 12 h GAPDH

LNCaP P < 0.0001 (one-way ANOVA) 2.5 DI17E6 2.0 P = 0.0102 P = 0.0083 1.5 P = 0.0430 1.0

Vehicle15 min30 min 1 h 1.5 h 2 h 3 h 6 h 12 h 24 h (p-FAK/FAK) 0.5

Image ratio density Image 0.0 Integrin alpha V Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 6 h 12 h 24 h P < 0.0001 (one-way ANOVA) p-FAK 2.5 2.0 P = 0.0126 P = 0.0018 FAK 1.5 P = 0.0002 1.0

p-Akt (p-Akt/Akt) 0.5 0.0 Image ratio density Image Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 6 h 12 h 24 h Akt P < 0.0001 (one-way ANOVA) 2.5 P = 0.0030 p-ERK 2.0 P = 0.0069 P = 0.0034 1.5 P = 0.0117 ERK 1.0 0.5 (p-ERK/ERK) GAPDH 0.0 Image ratio density Image Vehicle 15 min 30 min 1 h 1.5 h 2 h 3 h 6 h 12 h 24 h

Figure 6. DI17E6 inhibits expression of aV integrin and phosphorylation of FAK, Akt, and ERK in LNCaP and C4-2B cells. LNCaP and C4-2B cells treated with vehicle and 100 mg/mL DI17E6 for the indicated times and then total protein was harvested and subjected to Western blot analysis; aV integrin, phospho-FAK, FAK, phospho-AKT, Akt, phospho-ERK, and ERK. Densitometry was performed and presented as the ratio of phosphorylated to total protein.

integrin control of prostate cancer progression and to pursue Development of methodology: J. Dai inhibition of integrin aV as a therapeutic target for prostate cancer. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Dai, G. Shelley ﬂ Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Disclosure of Potential Con icts of Interest computational analysis): Y. Jiang, Z. Yao, E.T. Keller ﬂ No potential con icts of interest were disclosed. Writing, review, and/or revision of the manuscript: E.T. Keller Administrative, technical, or material support (i.e., reporting or organizing Authors' Contributions data, constructing databases): G. Shelley Conception and design: Y. Jiang, E.T. Keller Study supervision: E.T. Keller

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aV-Class Antibody Inhibits Prostate Cancer Progression

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This work was supported, in part, by the National Cancer Institute Grant P01 this fact. CA093900. The costs of publication of this article were defrayed in part by the Received December 6, 2016; revised January 17, 2017; accepted March 10, payment of page charges. This article must therefore be hereby marked 2017; published OnlineFirst March 17, 2017.

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www.aacrjournals.org Mol Cancer Res; 2017 OF9

Abituzumab Targeting of αV-Class Integrins Inhibits Prostate Cancer Progression

Yuan Jiang, Jinlu Dai, Zhi Yao, et al.

Mol Cancer Res Published OnlineFirst March 17, 2017.

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