ORIGINAL STUDY
The Novel IJB Kinase A Inhibitor, IMD-0560, Has Potent Therapeutic Efficacy in Ovarian Cancer Xenograft Model Mice Ikuko Sawada, MD,* Kae Hashimoto, MD, PhD,* Kenjiro Sawada, MD, PhD,* Yasuto Kinose, MD, PhD,* Koji Nakamura, MD,* Aska Toda, MD,* Erika Nakatsuka, MD,* Akihiko Yoshimura, MD,* Seiji Mabuchi, MD, PhD,* Tomoyuki Fujikawa, PhD,Þ Akiko Itai, PhD,Þ and Tadashi Kimura, MD, PhD*
Objective: Aberrant activation of nuclear factor-kappa A (NF-JB) signaling has been correlated with poor outcome among patients with ovarian cancer. Although the therapeutic potential of NF-JB pathway disruption in cancers has been extensively studied, most classical NF-JB inhibitors are poorly selective, exhibit off-target effects, and have failed to be applied in clinical use. IMD-0560, N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2hydroxybenzamide, is a novel low-molecular-weight compound that selectively inhibits the IJB kinase complex and works as an inhibitor of NF-JB signaling. The aim of this study was to assess the therapeutic potential of IMD-0560 against ovarian cancer in vitro and in vivo. Methods: NF-JB activity (phosphorylation) was determined in 9 ovarian cancer cell lines and the inhibitory effect of IMD-0560 on NF-JB activation was analyzed by Western blotting. Cell viability, cell cycle, vascular endothelial growth factor (VEGF) expression, and angiogenesis were assessed in vitro to evaluate the effect of IMD-0560 on ovarian cancer cells. In vivo efficacy of IMD-0560 was also investigated using an ovarian cancer xenograft mouse model. Results: The NF-JB signaling pathway was constitutively activated in 8 of 9 ovarian cancer cell lines. IMD-0560 inhibited NF-JB activation and suppressed ovarian cancer cell proliferation by inducing G1 phase arrest. IMD-0560 decreased VEGF secretion from cancer cells and inhibited the tube formation of human umbilical vein endothelial cells. IMD-0560 significantly inhibited peritoneal metastasis and prolonged the survival in an ovarian cancer xenograft mice model. Immunohistochemical staining of excised tumors revealed that IMD-0560 suppressed VEGF expression, tumor angiogenesis, and cancer cell proliferation. Conclusions: IMD-0560 showed promising therapeutic efficacy against ovarian cancer xenograft mice by inducing cell cycle arrest and suppressing VEGF production from cancer cells. IMD-0560 may be a potential future option in regimens for the treatment of ovarian cancer. Key Words: Angiogenesis, IKKA inhibitor, NF-JB, Ovarian cancer
*Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka; and †Institute of Medicinal Molecular Design, Inc, Bunkyo-ku, Tokyo, Japan. Address correspondence and reprint requests to Kae Hashimoto, MD, PhD, Department of Obstetrics and Gynecology, Copyright * 2016 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000668 International Journal of Gynecological Cancer
Osaka University Graduate School of Medicine, 2-2, Yamadaoka Suita, Osaka 5650871, Japan. E-mail: [email protected]. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (2390100 to K.H.; 24592515, 26670725, and 26293360 to K.S.; and 24249080 to T.K.) The authors declare no conflicts of interest.
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Received October 20, 2015. Accepted for publication November 9, 2015. (Int J Gynecol Cancer 2016;00: 00Y00)
cancer has the highest mortality rate of all gyO varian necologic malignancies and is the fifth leading cause of
cancer-related death among women.1 In recent years, surgical resection followed by chemotherapy has resulted in clinical response in 50% to 80% of patients with stage III and IV disease. However, the relapse rates for patients with ovarian cancer are extremely high,2 and the prognosis of ovarian cancer has not changed drastically since the late 1990s. Hence, there is a clear need for new chemotherapeutic or targeted agents for ovarian cancer treatment. Nuclear factor-kappa B (NF-JB) is a ubiquitous transcription factor that regulates the expression of various genes associated with immune responses, the cell cycle, and apoptosis.3,4 Dysregulation of NF-JB plays an important role in many disease processes, including inflammatory and autoimmune diseases, viral infection, and cancers.5 NF-JB is activated in certain cancers, including breast, colon, lung, and ovarian cancers,6 and is associated with cell proliferation,7 suppression of apoptosis,8 angiogenesis,9,10 metastasis,11 and chemoresistance.12,13 NF-JB activation increases the aggressiveness of ovarian cancer cell lines14 and NF-JB overexpression has a positive correlation with poor outcome among patients with ovarian cancer.15 On the basis of these findings, we speculated that targeting NF-JB signaling could be a potential therapeutic option for the treatment of ovarian cancer. NF-JB is a dimer of Rel family member proteins, and the most prevalent activated form is composed of p50 or p52 and p65. NF-JB exists in the cytoplasm in an inactive form associated with its regulatory protein, inhibitor of NF-JB (IJB). NF-JB activation requires phosphorylation of IJB by the IJB kinase (IKK) complex. Phosphorylated IJB is then degraded by the proteasome, which releases the NF-JB dimer, allowing it to translocate to the nucleus where it binds to promoters and enhancers, leading to gene transcription. As NF-JB has various physiological functions, unselective and complete inhibition would likely lead to several serious adverse effects.16 Therefore, it has been practically difficult to use laboratory NF-JB, inhibitors in the clinic. Although many NF-JB inhibitory drugs have been identified, none have been successfully translated into clinical medicine as specific NF-JB inhibitors.17 During the past decade, many small-molecule inhibitors have been synthesized and evaluated for potential therapeutic activity and antineoplastic strategies. Some have shown promising antitumor activity in preclinical trials and are used clinically for cancer treatment.18 IMD-0560, N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2-hydroxybenzamide, is a newly synthesized low-molecular-weight compound that selectively inhibits IKKA in the IKK complex.19 IMD-0560 blocks IJB> phosphorylation and prevents NF-JB p65 nuclear translocation. Importantly, the prodrug of IMD-0560 is under assessment
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in a clinical trial for inflammation-related cardiovascular diseases and rheumatoid arthritis.20 In this study, we assessed the potential of the lowmolecular-weight IKK inhibitor, IMD-0560, as a novel therapeutic option for the treatment of ovarian cancer.
MATERIALS AND METHODS Reagents and Cell Lines IMD-0560 was provided by the Institute of Medical Molecular Design Inc (Tokyo, Japan). A 10-mM stock solution was prepared in dimethyl sulfoxide and stored at j20-C until use. The human ovarian cancer cell line, SKOV3ip1, was gifted by Dr Ernst Lengyel (University of Chicago, Chicago, IL) and HeyA8 cells were gifted by Dr Anil K Sood (MD Anderson Cancer Center, Houston, TX) in 2007. Caov-3, SKOV-3, OVCAR-3, and TOV-21G cell lines were purchased from the American Type Culture Collection (Manassas, VA), and the A2780 cell line was purchased from the European Collection of Cell Cultures (Salisbury, UK). Human umbilical vein endothelial cells (HUVECs) were collected as previously reported.21 Rat anti-mouse CD31 (M-20) and anti-vascular endothelial growth factor (VEGF) antibodies (A-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ki-67 antibody (RM-9160), Alexa Fluor 488Ylabeled goat anti-rabbit IgG, Lipofectamine 2000, and Opti-MEM I Reduced Serum Media were from Thermo Fisher Scientific (Waltham, MA). AntiYphospho-NF-JB p65 (Ser536) and antiYNF-JB p65 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Growth factorYreduced basement membrane proteins (Matrigel) were purchased from BD Biosciences (Bedford, MA). The CyQUANT cell proliferation assay kit was purchased from Molecular Probes (Eugene, OR). The pGL4-phVEGFA plasmid bearing the human VEGF-A promoter sequence (position: 3853 5157 of human VEGF sequence; NCBI Reference Sequence: NG008732) with firefly luciferase was obtained from RIKEN BioResource Center (Tsukuba, Japan). Passive Lysis Buffer and the Luciferase Reporter Assay System were purchased from Promega (Madison, WI).
Animal Procedures and Treatment All animal procedures were conducted in accordance with institutional and national guidelines. Female BALB/c nude mice (aged 4Y5 weeks) were purchased from CLEA Japan Inc (Tokyo, Japan). SKOV3ip1 cells (1 106) were suspended as single cells in phosphate-buffered saline and injected intraperitoneally into the mice. One week after inoculation, mice showed tumor dissemination with multiple lesions on the peritoneal surface, omentum, surface of the * 2016 IGCS and ESGO
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liver, and small bowel mesentery. Therefore, treatment was initiated 1 week after tumor inoculation. The mice were administered intraperitoneally with either 0.5 mL 0.5% carboxymethylcellulose sodium salt (CMC-Na; Wako, Osaka, Japan) or IMD-0560 (10 mg/kg per day) daily. The dosage levels were determined based on previously reported dosages.22 Mice were assessed daily for general health and the development of ascites. Treatment was continued until mice became moribund or until killed. Mice were killed at 35 days after the treatment; the number of metastases in each mouse was counted, mice were dissected carefully, and the tumors were removed and weighed.
incubated with the indicated antibodies and the proteins were visualized with Western Lightning Plus ECL (PerkinElmer Life Science, Waltham, MA).
Tube Formation Assay Human umbilical vein endothelial cells were collected as previously reported.23 Human umbilical vein endothelial cells were seeded onto 96-well plates coated with growth factorYreduced Matrigel (3 104 cells/well) and incubated in conditioned medium or DMEM supplemented with 10% fetal bovine serum. After 12 hours, tube formation was visualized under an inverted microscope (400), and the images were analyzed.
Immunohistochemical Study Tumor tissue was fixed in formalin and embedded in paraffin. Following antigen retrieval, the sections were stained with human VEGF (1:40), mouse CD-31 antibody (1:300), and Ki-67 antibodies (1:300) for 1 hour at room temperature. After washing with Tris-buffered saline/Tween 20, sections were stained using N-Histofine Simple Stain MAX PO(R) (Nichirei Biosciences, Tokyo, Japan) and then counterstained with Carazzi hematoxylin.
Immunofluorescent Analysis
Cells (5 103 cells) were plated on 8-well chamber slides and cultured under serum-free condition overnight. After pretreatment with 10 KM of IMD-0560 for 12 hours, cells were fixed with 10% formalin and stained with rabbit anti-human NF-JB p65 (1:200) followed by incubation with Alexa Fluor 488Ylabeled goat anti-rabbit IgG (1:200). The samples were observed using an FV1000-D Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).
Cell Proliferation Assay
SKOV3ip1 (3 103 cells/well) and HeyA8 cells (1 103 cells/well) were seeded in 96-well plates. At the indicated time points, cells were washed with serum-free medium and frozen at j70-C until analysis. Cell proliferation was measured using the CyQUANT Cell Proliferation Assay Kit as according to the manufacturer’s instructions.
Cell-Cycle Analysis
Cells (3 105/well) were seeded in 6-well culture plates and treated for 24 hours with IMD-0560 in growth medium. Cells were harvested by brief trypsinization, washed, fixed in 70% ethanol, and kept at j70-C until use. The samples were stained with propidium iodide with RNase and analyzed by flow cytometry on a FACScan cytometer (BD Biosciences, Rockville, MD) using CellQuest (BD Biosciences) software.
Luciferase Activity Assay
SKOV3ip1 cells (1 105) were seeded in 24-well plates. After replacement of culture media with Opti-MEM I Reduced Serum Media, cells were transfected with 1.0 Kg of pGL4-phVEGFA luciferase vector using Lipofectamine 2000. The next day, cells were treated for 12 hours with IMD0560. Both cell number and cell viability did not differ between control and IMD-0560Ytreated group. Cells were lysed with Passive Lysis Buffer and luciferase activity was determined using the Luciferase Reporter Assay System.
Statistical Analysis All statistical analyses were performed with Statcel version 3 (OMS-Publishing Inc, Saitama, Japan). Survival estimates were computed using the KaplanYMeier method, and comparisons between groups were analyzed using the log-rank test. The level of significance was set at P G 0.05.
RESULTS NF-JB Signaling Pathway Was Constitutively Activated in Ovarian Cancer Cell Lines We first examined NF-JB activity in various ovarian cancer cell lines by Western blotting. Primary cultured ovarian surface epithelium was collected from a normal ovary during benign gynecological surgeries at Osaka University Hospital and used as a control. Although ovarian surface epithelium did not express phosphorylated NF-JB, 8 of 9 ovarian cancer cell lines, except Caov-3, showed phosphorylation of NF-JB without stimulation, implying that NF-JB is constitutively activated in ovarian cancer cell lines (Fig. 1A).
Western Blot Analysis
Constitutive Activation of NF-JB in Ovarian Cancer Cells Were Inhibited by IMD-0560
Cells were cultured for 24 hours under serum-free conditions followed by incubation for 24 hours in growth medium with IMD-0560 and lysed in ice-cold lysis buffer (Cell Signaling Technology). Samples were resolved by SDSpolyacrylamide gel electrophoresis and transferred to Hybond-P membranes (Millipore, Bedford, MA). The membranes were
IKK serves as a relevant molecular target because most of signaling pathways mediated by NF-JB involve IKK. To assess the effect of the selective IKKA inhibitor IMD-0560 on ovarian cancer cells, cells were treated with IMD-0560 and NF-JB phosphorylation was analyzed by Western blotting. We used SKOV3ip1 and HeyA8 ovarian cancer cell lines
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FIGURE 1. IMD-0560 suppresses aberrant activation of NF-JB in ovarian cancer cell lines. A, Western blot analysis. Cell lysates from ovarian cancer cells including normal ovarian surface epithelium (OSE) were resolved by SDS-PAGE and immunoblotted with a phosphorylated NF-JB p65 antibody. A-Actin was used as a loading control. Blots are representative of 3 experiments. B, Effect of IMD-0560 on NF-JB phosphorylation. SKOV3ip1 cells were incubated with the indicated dose of IMD-0560 for 24 hours and subjected to Western blotting. C, NF-JB nuclear translocation was examined by immunocytochemistry. NF-JB p65 was stained with Alexa Fluor 488 (green). NF-JB p65 was predominantly located in the nucleus in approximately half of the SKOV3ip1 cells (top panel), IMD-0560 treatment inhibited nuclear translocation of NF-JB p65 (bottom panel). Bars represent 50 Km. because these cell lines exhibit constitutive NF-JB activation and showed tumorigenic potential in an immunodeficient mouse peritoneal cavity.24 IMD-0560 at 10 KM inhibited phosphorylation of NF-JB p65 in both cell lines (Fig. 1B). We next examined the localization of NF-JB p65 by immunofluorescence analysis. NF-JB p65 was predominantly located in the nucleus in approximately half of the untreated control SKOV3ip1 cells. Treatment with 10 KM IMD-0560 inhibited
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the nuclear translocation of NF-JB, and p65 remained in the cytoplasm in almost all cells (Fig. 1C).
IMD-0560 Suppressed Ovarian Cancer Cell Proliferation We next evaluated the effect of IMD-0560 on the proliferation of ovarian cancer cells. SKOV3ip1 and HayA8 cells were incubated with increasing concentrations of IMD-0560 * 2016 IGCS and ESGO
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FIGURE 2. IMD-0560 inhibits cancer cell proliferation by inducing cell-cycle arrest. A, SKOV3ip1 and HeyA8 cells were incubated with increasing concentration of IMD-0560 for the indicated times. Data represent mean T SD compared with control (dimethyl sulfoxide treatment). *P G 0.05, **P G 0.01, ***P G 0.001. B, Cell-cycle analysis of SKOV3ip1 cells treated with IMD-0560. DNA histograms of SKOV3ip1 cells collected after 24 hours of treatment and stained with propidium iodide. C, The percentages of cells in cell-cycle phases after treatment with IMD-0560. Lower table shows the mean T SE of percentage of cells in G0/G1, G2/M, and S phases of the cell cycle. for 24, 48, and 72 hours. As shown in Fig. 2A, IMD-0560 suppressed cell proliferation of both cell lines in a dosedependent manner. The inhibitory effect of IMD-0560 showed significant differences at concentrations above 1 KM for SKOV3ip1 cells and above 10 KM for HeyA8 cells. To more closely examine IMD-0560 inhibition of ovarian cancer cell proliferation, we next performed cell-cycle analysis of IMD0560Ytreated SKOV3ip1 cells. Upon treatment of SKOV3ip1 cells with 10 KM IMD-0560, we observed an increased of G0/G1 phase cells by 15.1% whereas S phase cells decreased by 5.2%, indicating that IMD-0560 induced G0/G1 phase arrest of SKOV3ip1 ovarian cancer cells (P = 0.004) (Figs. 2B, C).
IMD-0560 Suppressed VEGF Production From Ovarian Cancer Cells and Inhibited In Vitro Tube Formation of Endothelial Cells Angiogenesis has been shown to be an important contributor to ovarian carcinogenesis and progression, and previous studies have shown that NF-JB signaling regulates VEGF expression.25 Therefore, we examined VEGF production in ovarian cancer cells treated with IMD-0560 by Western blotting. Results showed that 10 KM IMD-0560 decreased VEGF expression in SKOV3ip1 cells (Fig. 3A). Furthermore, luciferase assays with a luciferase reporter controlled by VEGF-A
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10% feral bovine serum as control for 12 hours, and the formed tube lengths were measured. IMD-0560 significantly reduced tube formation by 39.2% T 25.7% at 1 KM (P = 0.05) and 87.5% T 16.7% at 10 KM (P = 0.002) (Fig. 3C), suggesting that IMD-0560 may suppress in vitro angiogenesis in ovarian cancer cells through the inhibition of VEGF production.
IMD-0560 Inhibited Peritoneal Metastasis and Prolonged the Survival of Ovarian Cancer Xenograft Mice
FIGURE 3. IMD-0560 inhibits angiogenesis through the inhibition of VEGF production from ovarian cancer cells. A, SKOV3ip1 cells were cultured with IMD-0560 for 24 hours and VEGF expression was analyzed by Western blotting. A-Actin were used as a loading control. B, Luciferase assay. pGL4-hVEGFA firefly luciferase vector was transfected into SKOV3ip1 cells treated with IMD-0560 as indicated. Data represent mean T SD. C, Effect of IMD-0560 on tube formation by HUVECs cultured on Matrigel. HUVECs were seeded with conditioned medium containing IMD-0560Ytreated SKOV3ip1 cells. Representative endothelial capillary-like tubes are shown. While SKOV3ip1 culture media treated with IMD-0560 significantly reduced tube formation. *P G 0.05, **P G 0.01, ***P G 0.001. promoter showed that VEGF-A transcriptional activity was significantly inhibited by concentrations of IMD-0560 above 1 KM (Fig. 3B). In vitro tube formation assay was performed using primary cultured HUVECs. Human umbilical vein endothelial cells were seeded with conditioned medium containing IMD0560Ytreated SKOVip1 cells or DMEM supplemented with
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Finally, we evaluated the therapeutic efficacy of IMD0560 in an ovarian cancer xenograft mouse model. The daily treatment of IMD-0560 significantly prolonged the survival of mice. The median survival value of mice in the IMD-0560 treatment group was 65 days compared with 51 days for the control group (P = 0.028) (Fig. 4A). At 35 days after treatment, both tumor weight and the number of peritoneal implants were significantly reduced in mice treated with IMD-0560 compared with control mice (tumor weight: IMD-0560, 0.310 T 0.226 vs control, 0.861 T 0.307 g, P G 0.001; number of peritoneal implants: 24.2 T 18.3 vs 81.5 T 31.5, respectively, P G 0.001) (Figs. 4B, C). To examine the mechanism by which IMD-0560 affects cancer cells in vivo, we next performed immunohistochemical analyses. Peritoneal tumors were stained with the cell proliferation marker Ki-67 (Fig. 4D). The number of Ki-67Ypositive cells was significantly decreased by IMD-0560 treatment (14.26 T 5.80 vs control, 34.6 T 6.79/200 field; P = 0.03) (Fig. 4E). Human VEGF expression was inhibited in tumors treated with IMD-0560 compared with those of the control (Fig. 4D). Accordingly, the number of intratumoral vessels, as visualized by anti-mouse CD31 staining (Fig. 4D), was significantly inhibited by IMD-0560 treatment (14.26 T 5.62 vs control, 30.46 T 3.89/200 field; P = 0.03) (Fig. 4F). Collectively, these findings indicated that IMD-0560 inhibits ovarian cancer progression by inhibiting cancer cell proliferation and decreases angiogenesis through suppression of VEGF production in ovarian cancer cells.
DISCUSSION The present study demonstrates that the novel selective IKKA inhibitor, IMD-0560, suppressed ovarian cancer progression and extended survival of ovarian cancer xenograft mice. Activation of NF-JB is associated with up-regulation of cell survival signaling molecules and regulation of cell cycleYrelated molecules.5,25 IMD-0560 inhibited NF-JB activation through IJB phosphorylation and led to cell-cycle arrest of ovarian cancer cell lines in vitro and suppressed peritoneal metastases in vivo. Another significant effect of IMD-0560 is antiangiogenic activity. IMD-0560 inhibited VEGF-A promoter activity in vitro and suppressed VEGF production in cancer cells, which led to the inhibition of tumor angiogenesis in vivo. Angiogenesis is an important contributor to ovarian carcinogenesis and progression. Angiogenesis often requires the release of angiogenic growth factors from tumor cells or inflammatory cells, such as macrophages and neutrophils. NF-JB regulates the expression of such growth factors and * 2016 IGCS and ESGO
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FIGURE 4. Effect of IMD-0560 in a xenograft ovarian cancer mouse model. A, IMD-0560 prolonged the survival of ovarian cancer model mice. Ovarian cancer model mice were generated as described in Materials and Methods. IMD-0560 was administered IP 10 mg/kg everyday from 1 week after implantation. KaplanYMeier survival curves were calculated. Median survival of IMD-0560Ytreated mice was 51 days compared with 65 days in control mice (P = 0.028). B and C, IMD-0560 decreased tumor weight and the number of tumor deposit. Mice were killed at 35 days after initiation of treatment. Tumor weight (B) and numbers of metastatic sites (C) were significantly decreased by IMD-0560 treatment. D, Tissue sections were immunostained with anti-VEGF, CD31, and Ki-67 antibodies. Representative histological stains are shown. Bars represent 10 Km. E, IMD-0560 treatment significantly decreased CD31-positive cells (P = 0.03). F, IMD-0560 treatment significantly decreased the number of vessels (P = 0.03). *P G 0.05. cytokines, including VEGF, tumor necrosis factor, and monocyte chemoattractant protein.8,26 Among these factors, VEGF is considered a key regulator of angiogenesis.27 Bevacizumab, a VEGF-AYblocking monoclonal antibody, is active against advanced ovarian cancer as shown by several large randomized
studies.28,29 However, despite promising early preclinical data, the clinical benefits of bevacizumab with respect to progressionfree survival are few, with progression-free survival of usually a few months; the benefits in overall survival are more limited30,31 or absent.32,33 Bevacizumab targets only VEGF-dependent
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angiogenesis; however, whereas VEGF is a key driver of cancer angiogenesis, numerous other angiogenic factors have been identified. Therefore, even if the VEGF pathway is successfully blocked by bevacizumab, other angiogenic factors might be eventually up-regulated leading to tumor resistance to bevacizumab. Following the success of bevacizumab, new antiangiogenesis molecules have been developed and several studies on these molecules have been conducted or are underway.34 Thus, the antitumor effect of targeting a single growth factor would most likely be very transient. In that regard, an NF-JB inhibitor can target not only VEGF but also various other proangiogenic factors (eg, tumor necrosis factor, monocyte chemoattractant protein-1, and interleukin-8). Thus far, IMD-0560 can be considered as a potential treatment option for future antiangiogenic therapy. The therapeutic potential of IKKA-dependent NF-JB pathway disruption in cancers has been extensively studied, and a number of NF-JB inhibitors have been developed.35,36 However, most classical NF-JB inhibitors are poorly selective and show off-target effects; therefore, few have progressed to phase III clinical trials.37 The proteasome inhibitor, bortezomib, is the only clinically available NF-JB inhibitor.37 However, proteasome inhibition may affect other signaling pathways. Notably, IMD-0560 specifically inhibits IKKA, which induces the inhibition of NF-JB activation only in inflammatory conditions. Additionally, the prodrug of IMD-0560 is already under assessment in a clinical trial for cardiovascular diseases and rheumatoid arthritis.19,20 The current study has some limitations. We did not assess combination therapy. Because it is difficult to show the overall survival benefit of monotherapy with most small-molecule inhibitors, it is essential to consider combination therapy with existing or novel therapies for clinical application. Thus, further investigation of combination therapy is required for future clinical application. In summary, our finding shows that IMD-0560 treatment has promising therapeutic efficacy in an ovarian cancer xenograft mouse model. IMD-0560 not only induced cellcycle arrest but also suppressed VEGF production in ovarian cancer cells. On the basis of the increasing importance of antiangiogenic agents for ovarian cancer treatment, IMD0560 looks promising and may be valuable in future regimens for the treatment of ovarian cancer.
REFERENCES 1. Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9Y29. 2. Matsuo K, Lin YG, Roman LD, et al. Overcoming platinum resistance in ovarian carcinoma. Expert Opin Investig Drugs. 2010;19:1339Y1354. 3. Dixit V, Mak TW. NF-kappaB signaling. Many roads lead to Madrid. Cell. 2002;111:615Y619. 4. Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol. 2009;27:693Y733. 5. Aggarwal BB. Nuclear factor-kappaB: the enemy within. Cancer Cell. 2004;6:203Y208.
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6. Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov. 2009;8:33Y40. 7. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest. 2001;107:241Y246. 8. Webster GA, Perkins ND. Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol. 1999;19:3485Y3495. 9. Tsujii M, Kawano S, Tsuji S, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell. 1998;93:705Y716. 10. Huang S, DeGuzman A, Bucana CD, et al. Nuclear factor-kappaB activity correlates with growth, angiogenesis, and metastasis of human melanoma cells in nude mice. Clin Cancer Res. 2000;6:2573Y2581. 11. Andela VB, Schwarz EM, Puzas JE, et al. Tumor metastasis and the reciprocal regulation of prometastatic and antimetastatic factors by nuclear factor kappaB. Cancer Res. 2000;60:6557Y6562. 12. Cusack JC Jr, Liu R, Houston M, et al. Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-kappaB inhibition. Cancer Res. 2001;61:3535Y3540. 13. Mabuchi S, Ohmichi M, Nishio Y, et al. Inhibition of NFkappaB increases the efficacy of cisplatin in in vitro and in vivo ovarian cancer models. J Biol Chem. 2004;279:23477Y23485. 14. Hernandez L, Hsu SC, Davidson B, et al. Activation of NF-kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian cancer. Cancer Res. 2010;70:4005Y4014. 15. Annunziata CM, Stavnes HT, Kleinberg L, et al. Nuclear factor kappaB transcription factors are coexpressed and convey a poor outcome in ovarian cancer. Cancer. 2010;116:3276Y3284. 16. Niederberger E, Geisslinger G. The IKK-kappaB pathway: a source for novel molecular drug targets in pain therapy? FASEB J. 2008;22:3432Y3442. 17. Horie R. Molecularly-targeted strategy and NF-JB in lymphoid malignancies. J Clin Exp Hematop. 2013;53:185Y195. 18. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer. 2009;9:28Y39. 19. Suzuki J, Ogawa M, Muto S, et al. Novel IJB kinase inhibitors for treatment of nuclear factor-JB-related diseases. Expert Opin Investig Drugs. 2011;20:395Y405. 20. Young ER. IKKA as a therapeutic intervention point diseases related to inflammation. In: Levin JI, Laufer S, eds. Anti-inflammatory Drug Discovery. RSC Publishing; 2012;255Y296. 21. Sawada K, Morishige K, Mabuchi S, et al. In vitro and in vivo assays to analyze the contribution of Rho kinase in angiogenesis. Methods Enzymol. 2008;439:395Y412. 22. Wakatsuki S, Suzuki J, Ogawa M, et al. A novel IKK inhibitor suppresses heart failure and chronic remodeling after myocardial ischemia via MMP alteration. Expert Opin Ther Targets. 2008;12:1469Y1476. 23. Yin L, Morishige K, Takahashi T, et al. Fasudil inhibits vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Mol Cancer Ther. 2007;6:1517Y1525. 24. Kinose Y, Sawada K, Makino H, et al. IKKA regulates VEGF expression and is a potential therapeutic target for ovarian cancer as an antiangiogenic treatment. Mol Cancer Ther. 2015;14:909Y919. 25. Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF-kappaBYdependent: studies using * 2016 IGCS and ESGO
Copyright © 2016 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
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26. 27.
28.
29.
30.
31.
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adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2. J Cell Sci. 2003;116:665Y674. Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431Y436. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol. 2001;280:1358Y1366. Burger RA, Sill MW, Monk BJ, et al. Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group Study. J Clin Oncol. 2007;25:5165Y5171. Cannistra SA, Matulonis UA, Penson RT, et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J Clin Oncol. 2007;25:5180Y5186. Perren TJ, Swart AM, Pfisterer J, et al. A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med. 2011;365:2484Y2496. Burger RA, Brady MF, Bookman MA, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. N Engl J Med. 2011;365:2473Y2483.
32. Aghajanian C, Blank SV, Goff BA, et al. OCEANS: a randomized, double-blind, placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer. J Clin Oncol. 2012;30:2039Y2045. 33. Liu FW, Cripe J, Tewari KS. Anti-angiogenesis therapy in gynecologic malignancies. Oncology (Williston Park). 2015;29:350Y360. 34. Klempner SJ, Myers AP, Mills GB, et al. Clinical investigation of receptor and non-receptor tyrosine kinase inhibitors for the treatment of epithelial ovarian cancer. Expert Opin Pharmacother. 2013;14:2171Y2182. 35. Burke JR, Pattoli MA, Gregor KR, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem. 2003;278:1450Y1456. 36. Sanda T, Iida S, Ogura H, et al. Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor. Clin Cancer Res. 2005;11:1974Y1982. 37. San Miguel JF, Schlag R, Khuageva NK, et al. Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med. 2008;359:906Y917.
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The Novel IJB Kinase A Inhibitor, IMD-0560, Has Potent Therapeutic Efficacy in Ovarian Cancer Xenograft Model Mice Ikuko Sawada, MD,* Kae Hashimoto, MD, PhD,* Kenjiro Sawada, MD, PhD,* Yasuto Kinose, MD, PhD,* Koji Nakamura, MD,* Aska Toda, MD,* Erika Nakatsuka, MD,* Akihiko Yoshimura, MD,* Seiji Mabuchi, MD, PhD,* Tomoyuki Fujikawa, PhD,Þ Akiko Itai, PhD,Þ and Tadashi Kimura, MD, PhD*
Objective: Aberrant activation of nuclear factor-kappa A (NF-JB) signaling has been correlated with poor outcome among patients with ovarian cancer. Although the therapeutic potential of NF-JB pathway disruption in cancers has been extensively studied, most classical NF-JB inhibitors are poorly selective, exhibit off-target effects, and have failed to be applied in clinical use. IMD-0560, N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2hydroxybenzamide, is a novel low-molecular-weight compound that selectively inhibits the IJB kinase complex and works as an inhibitor of NF-JB signaling. The aim of this study was to assess the therapeutic potential of IMD-0560 against ovarian cancer in vitro and in vivo. Methods: NF-JB activity (phosphorylation) was determined in 9 ovarian cancer cell lines and the inhibitory effect of IMD-0560 on NF-JB activation was analyzed by Western blotting. Cell viability, cell cycle, vascular endothelial growth factor (VEGF) expression, and angiogenesis were assessed in vitro to evaluate the effect of IMD-0560 on ovarian cancer cells. In vivo efficacy of IMD-0560 was also investigated using an ovarian cancer xenograft mouse model. Results: The NF-JB signaling pathway was constitutively activated in 8 of 9 ovarian cancer cell lines. IMD-0560 inhibited NF-JB activation and suppressed ovarian cancer cell proliferation by inducing G1 phase arrest. IMD-0560 decreased VEGF secretion from cancer cells and inhibited the tube formation of human umbilical vein endothelial cells. IMD-0560 significantly inhibited peritoneal metastasis and prolonged the survival in an ovarian cancer xenograft mice model. Immunohistochemical staining of excised tumors revealed that IMD-0560 suppressed VEGF expression, tumor angiogenesis, and cancer cell proliferation. Conclusions: IMD-0560 showed promising therapeutic efficacy against ovarian cancer xenograft mice by inducing cell cycle arrest and suppressing VEGF production from cancer cells. IMD-0560 may be a potential future option in regimens for the treatment of ovarian cancer. Key Words: Angiogenesis, IKKA inhibitor, NF-JB, Ovarian cancer
*Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka; and †Institute of Medicinal Molecular Design, Inc, Bunkyo-ku, Tokyo, Japan. Address correspondence and reprint requests to Kae Hashimoto, MD, PhD, Department of Obstetrics and Gynecology, Copyright * 2016 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0000000000000668 International Journal of Gynecological Cancer
Osaka University Graduate School of Medicine, 2-2, Yamadaoka Suita, Osaka 5650871, Japan. E-mail: [email protected]. This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (2390100 to K.H.; 24592515, 26670725, and 26293360 to K.S.; and 24249080 to T.K.) The authors declare no conflicts of interest.
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Received October 20, 2015. Accepted for publication November 9, 2015. (Int J Gynecol Cancer 2016;00: 00Y00)
cancer has the highest mortality rate of all gyO varian necologic malignancies and is the fifth leading cause of
cancer-related death among women.1 In recent years, surgical resection followed by chemotherapy has resulted in clinical response in 50% to 80% of patients with stage III and IV disease. However, the relapse rates for patients with ovarian cancer are extremely high,2 and the prognosis of ovarian cancer has not changed drastically since the late 1990s. Hence, there is a clear need for new chemotherapeutic or targeted agents for ovarian cancer treatment. Nuclear factor-kappa B (NF-JB) is a ubiquitous transcription factor that regulates the expression of various genes associated with immune responses, the cell cycle, and apoptosis.3,4 Dysregulation of NF-JB plays an important role in many disease processes, including inflammatory and autoimmune diseases, viral infection, and cancers.5 NF-JB is activated in certain cancers, including breast, colon, lung, and ovarian cancers,6 and is associated with cell proliferation,7 suppression of apoptosis,8 angiogenesis,9,10 metastasis,11 and chemoresistance.12,13 NF-JB activation increases the aggressiveness of ovarian cancer cell lines14 and NF-JB overexpression has a positive correlation with poor outcome among patients with ovarian cancer.15 On the basis of these findings, we speculated that targeting NF-JB signaling could be a potential therapeutic option for the treatment of ovarian cancer. NF-JB is a dimer of Rel family member proteins, and the most prevalent activated form is composed of p50 or p52 and p65. NF-JB exists in the cytoplasm in an inactive form associated with its regulatory protein, inhibitor of NF-JB (IJB). NF-JB activation requires phosphorylation of IJB by the IJB kinase (IKK) complex. Phosphorylated IJB is then degraded by the proteasome, which releases the NF-JB dimer, allowing it to translocate to the nucleus where it binds to promoters and enhancers, leading to gene transcription. As NF-JB has various physiological functions, unselective and complete inhibition would likely lead to several serious adverse effects.16 Therefore, it has been practically difficult to use laboratory NF-JB, inhibitors in the clinic. Although many NF-JB inhibitory drugs have been identified, none have been successfully translated into clinical medicine as specific NF-JB inhibitors.17 During the past decade, many small-molecule inhibitors have been synthesized and evaluated for potential therapeutic activity and antineoplastic strategies. Some have shown promising antitumor activity in preclinical trials and are used clinically for cancer treatment.18 IMD-0560, N-[2,5-bis (trifluoromethyl) phenyl]-5-bromo-2-hydroxybenzamide, is a newly synthesized low-molecular-weight compound that selectively inhibits IKKA in the IKK complex.19 IMD-0560 blocks IJB> phosphorylation and prevents NF-JB p65 nuclear translocation. Importantly, the prodrug of IMD-0560 is under assessment
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in a clinical trial for inflammation-related cardiovascular diseases and rheumatoid arthritis.20 In this study, we assessed the potential of the lowmolecular-weight IKK inhibitor, IMD-0560, as a novel therapeutic option for the treatment of ovarian cancer.
MATERIALS AND METHODS Reagents and Cell Lines IMD-0560 was provided by the Institute of Medical Molecular Design Inc (Tokyo, Japan). A 10-mM stock solution was prepared in dimethyl sulfoxide and stored at j20-C until use. The human ovarian cancer cell line, SKOV3ip1, was gifted by Dr Ernst Lengyel (University of Chicago, Chicago, IL) and HeyA8 cells were gifted by Dr Anil K Sood (MD Anderson Cancer Center, Houston, TX) in 2007. Caov-3, SKOV-3, OVCAR-3, and TOV-21G cell lines were purchased from the American Type Culture Collection (Manassas, VA), and the A2780 cell line was purchased from the European Collection of Cell Cultures (Salisbury, UK). Human umbilical vein endothelial cells (HUVECs) were collected as previously reported.21 Rat anti-mouse CD31 (M-20) and anti-vascular endothelial growth factor (VEGF) antibodies (A-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ki-67 antibody (RM-9160), Alexa Fluor 488Ylabeled goat anti-rabbit IgG, Lipofectamine 2000, and Opti-MEM I Reduced Serum Media were from Thermo Fisher Scientific (Waltham, MA). AntiYphospho-NF-JB p65 (Ser536) and antiYNF-JB p65 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Growth factorYreduced basement membrane proteins (Matrigel) were purchased from BD Biosciences (Bedford, MA). The CyQUANT cell proliferation assay kit was purchased from Molecular Probes (Eugene, OR). The pGL4-phVEGFA plasmid bearing the human VEGF-A promoter sequence (position: 3853 5157 of human VEGF sequence; NCBI Reference Sequence: NG008732) with firefly luciferase was obtained from RIKEN BioResource Center (Tsukuba, Japan). Passive Lysis Buffer and the Luciferase Reporter Assay System were purchased from Promega (Madison, WI).
Animal Procedures and Treatment All animal procedures were conducted in accordance with institutional and national guidelines. Female BALB/c nude mice (aged 4Y5 weeks) were purchased from CLEA Japan Inc (Tokyo, Japan). SKOV3ip1 cells (1 106) were suspended as single cells in phosphate-buffered saline and injected intraperitoneally into the mice. One week after inoculation, mice showed tumor dissemination with multiple lesions on the peritoneal surface, omentum, surface of the * 2016 IGCS and ESGO
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liver, and small bowel mesentery. Therefore, treatment was initiated 1 week after tumor inoculation. The mice were administered intraperitoneally with either 0.5 mL 0.5% carboxymethylcellulose sodium salt (CMC-Na; Wako, Osaka, Japan) or IMD-0560 (10 mg/kg per day) daily. The dosage levels were determined based on previously reported dosages.22 Mice were assessed daily for general health and the development of ascites. Treatment was continued until mice became moribund or until killed. Mice were killed at 35 days after the treatment; the number of metastases in each mouse was counted, mice were dissected carefully, and the tumors were removed and weighed.
incubated with the indicated antibodies and the proteins were visualized with Western Lightning Plus ECL (PerkinElmer Life Science, Waltham, MA).
Tube Formation Assay Human umbilical vein endothelial cells were collected as previously reported.23 Human umbilical vein endothelial cells were seeded onto 96-well plates coated with growth factorYreduced Matrigel (3 104 cells/well) and incubated in conditioned medium or DMEM supplemented with 10% fetal bovine serum. After 12 hours, tube formation was visualized under an inverted microscope (400), and the images were analyzed.
Immunohistochemical Study Tumor tissue was fixed in formalin and embedded in paraffin. Following antigen retrieval, the sections were stained with human VEGF (1:40), mouse CD-31 antibody (1:300), and Ki-67 antibodies (1:300) for 1 hour at room temperature. After washing with Tris-buffered saline/Tween 20, sections were stained using N-Histofine Simple Stain MAX PO(R) (Nichirei Biosciences, Tokyo, Japan) and then counterstained with Carazzi hematoxylin.
Immunofluorescent Analysis
Cells (5 103 cells) were plated on 8-well chamber slides and cultured under serum-free condition overnight. After pretreatment with 10 KM of IMD-0560 for 12 hours, cells were fixed with 10% formalin and stained with rabbit anti-human NF-JB p65 (1:200) followed by incubation with Alexa Fluor 488Ylabeled goat anti-rabbit IgG (1:200). The samples were observed using an FV1000-D Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).
Cell Proliferation Assay
SKOV3ip1 (3 103 cells/well) and HeyA8 cells (1 103 cells/well) were seeded in 96-well plates. At the indicated time points, cells were washed with serum-free medium and frozen at j70-C until analysis. Cell proliferation was measured using the CyQUANT Cell Proliferation Assay Kit as according to the manufacturer’s instructions.
Cell-Cycle Analysis
Cells (3 105/well) were seeded in 6-well culture plates and treated for 24 hours with IMD-0560 in growth medium. Cells were harvested by brief trypsinization, washed, fixed in 70% ethanol, and kept at j70-C until use. The samples were stained with propidium iodide with RNase and analyzed by flow cytometry on a FACScan cytometer (BD Biosciences, Rockville, MD) using CellQuest (BD Biosciences) software.
Luciferase Activity Assay
SKOV3ip1 cells (1 105) were seeded in 24-well plates. After replacement of culture media with Opti-MEM I Reduced Serum Media, cells were transfected with 1.0 Kg of pGL4-phVEGFA luciferase vector using Lipofectamine 2000. The next day, cells were treated for 12 hours with IMD0560. Both cell number and cell viability did not differ between control and IMD-0560Ytreated group. Cells were lysed with Passive Lysis Buffer and luciferase activity was determined using the Luciferase Reporter Assay System.
Statistical Analysis All statistical analyses were performed with Statcel version 3 (OMS-Publishing Inc, Saitama, Japan). Survival estimates were computed using the KaplanYMeier method, and comparisons between groups were analyzed using the log-rank test. The level of significance was set at P G 0.05.
RESULTS NF-JB Signaling Pathway Was Constitutively Activated in Ovarian Cancer Cell Lines We first examined NF-JB activity in various ovarian cancer cell lines by Western blotting. Primary cultured ovarian surface epithelium was collected from a normal ovary during benign gynecological surgeries at Osaka University Hospital and used as a control. Although ovarian surface epithelium did not express phosphorylated NF-JB, 8 of 9 ovarian cancer cell lines, except Caov-3, showed phosphorylation of NF-JB without stimulation, implying that NF-JB is constitutively activated in ovarian cancer cell lines (Fig. 1A).
Western Blot Analysis
Constitutive Activation of NF-JB in Ovarian Cancer Cells Were Inhibited by IMD-0560
Cells were cultured for 24 hours under serum-free conditions followed by incubation for 24 hours in growth medium with IMD-0560 and lysed in ice-cold lysis buffer (Cell Signaling Technology). Samples were resolved by SDSpolyacrylamide gel electrophoresis and transferred to Hybond-P membranes (Millipore, Bedford, MA). The membranes were
IKK serves as a relevant molecular target because most of signaling pathways mediated by NF-JB involve IKK. To assess the effect of the selective IKKA inhibitor IMD-0560 on ovarian cancer cells, cells were treated with IMD-0560 and NF-JB phosphorylation was analyzed by Western blotting. We used SKOV3ip1 and HeyA8 ovarian cancer cell lines
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FIGURE 1. IMD-0560 suppresses aberrant activation of NF-JB in ovarian cancer cell lines. A, Western blot analysis. Cell lysates from ovarian cancer cells including normal ovarian surface epithelium (OSE) were resolved by SDS-PAGE and immunoblotted with a phosphorylated NF-JB p65 antibody. A-Actin was used as a loading control. Blots are representative of 3 experiments. B, Effect of IMD-0560 on NF-JB phosphorylation. SKOV3ip1 cells were incubated with the indicated dose of IMD-0560 for 24 hours and subjected to Western blotting. C, NF-JB nuclear translocation was examined by immunocytochemistry. NF-JB p65 was stained with Alexa Fluor 488 (green). NF-JB p65 was predominantly located in the nucleus in approximately half of the SKOV3ip1 cells (top panel), IMD-0560 treatment inhibited nuclear translocation of NF-JB p65 (bottom panel). Bars represent 50 Km. because these cell lines exhibit constitutive NF-JB activation and showed tumorigenic potential in an immunodeficient mouse peritoneal cavity.24 IMD-0560 at 10 KM inhibited phosphorylation of NF-JB p65 in both cell lines (Fig. 1B). We next examined the localization of NF-JB p65 by immunofluorescence analysis. NF-JB p65 was predominantly located in the nucleus in approximately half of the untreated control SKOV3ip1 cells. Treatment with 10 KM IMD-0560 inhibited
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the nuclear translocation of NF-JB, and p65 remained in the cytoplasm in almost all cells (Fig. 1C).
IMD-0560 Suppressed Ovarian Cancer Cell Proliferation We next evaluated the effect of IMD-0560 on the proliferation of ovarian cancer cells. SKOV3ip1 and HayA8 cells were incubated with increasing concentrations of IMD-0560 * 2016 IGCS and ESGO
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FIGURE 2. IMD-0560 inhibits cancer cell proliferation by inducing cell-cycle arrest. A, SKOV3ip1 and HeyA8 cells were incubated with increasing concentration of IMD-0560 for the indicated times. Data represent mean T SD compared with control (dimethyl sulfoxide treatment). *P G 0.05, **P G 0.01, ***P G 0.001. B, Cell-cycle analysis of SKOV3ip1 cells treated with IMD-0560. DNA histograms of SKOV3ip1 cells collected after 24 hours of treatment and stained with propidium iodide. C, The percentages of cells in cell-cycle phases after treatment with IMD-0560. Lower table shows the mean T SE of percentage of cells in G0/G1, G2/M, and S phases of the cell cycle. for 24, 48, and 72 hours. As shown in Fig. 2A, IMD-0560 suppressed cell proliferation of both cell lines in a dosedependent manner. The inhibitory effect of IMD-0560 showed significant differences at concentrations above 1 KM for SKOV3ip1 cells and above 10 KM for HeyA8 cells. To more closely examine IMD-0560 inhibition of ovarian cancer cell proliferation, we next performed cell-cycle analysis of IMD0560Ytreated SKOV3ip1 cells. Upon treatment of SKOV3ip1 cells with 10 KM IMD-0560, we observed an increased of G0/G1 phase cells by 15.1% whereas S phase cells decreased by 5.2%, indicating that IMD-0560 induced G0/G1 phase arrest of SKOV3ip1 ovarian cancer cells (P = 0.004) (Figs. 2B, C).
IMD-0560 Suppressed VEGF Production From Ovarian Cancer Cells and Inhibited In Vitro Tube Formation of Endothelial Cells Angiogenesis has been shown to be an important contributor to ovarian carcinogenesis and progression, and previous studies have shown that NF-JB signaling regulates VEGF expression.25 Therefore, we examined VEGF production in ovarian cancer cells treated with IMD-0560 by Western blotting. Results showed that 10 KM IMD-0560 decreased VEGF expression in SKOV3ip1 cells (Fig. 3A). Furthermore, luciferase assays with a luciferase reporter controlled by VEGF-A
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10% feral bovine serum as control for 12 hours, and the formed tube lengths were measured. IMD-0560 significantly reduced tube formation by 39.2% T 25.7% at 1 KM (P = 0.05) and 87.5% T 16.7% at 10 KM (P = 0.002) (Fig. 3C), suggesting that IMD-0560 may suppress in vitro angiogenesis in ovarian cancer cells through the inhibition of VEGF production.
IMD-0560 Inhibited Peritoneal Metastasis and Prolonged the Survival of Ovarian Cancer Xenograft Mice
FIGURE 3. IMD-0560 inhibits angiogenesis through the inhibition of VEGF production from ovarian cancer cells. A, SKOV3ip1 cells were cultured with IMD-0560 for 24 hours and VEGF expression was analyzed by Western blotting. A-Actin were used as a loading control. B, Luciferase assay. pGL4-hVEGFA firefly luciferase vector was transfected into SKOV3ip1 cells treated with IMD-0560 as indicated. Data represent mean T SD. C, Effect of IMD-0560 on tube formation by HUVECs cultured on Matrigel. HUVECs were seeded with conditioned medium containing IMD-0560Ytreated SKOV3ip1 cells. Representative endothelial capillary-like tubes are shown. While SKOV3ip1 culture media treated with IMD-0560 significantly reduced tube formation. *P G 0.05, **P G 0.01, ***P G 0.001. promoter showed that VEGF-A transcriptional activity was significantly inhibited by concentrations of IMD-0560 above 1 KM (Fig. 3B). In vitro tube formation assay was performed using primary cultured HUVECs. Human umbilical vein endothelial cells were seeded with conditioned medium containing IMD0560Ytreated SKOVip1 cells or DMEM supplemented with
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Finally, we evaluated the therapeutic efficacy of IMD0560 in an ovarian cancer xenograft mouse model. The daily treatment of IMD-0560 significantly prolonged the survival of mice. The median survival value of mice in the IMD-0560 treatment group was 65 days compared with 51 days for the control group (P = 0.028) (Fig. 4A). At 35 days after treatment, both tumor weight and the number of peritoneal implants were significantly reduced in mice treated with IMD-0560 compared with control mice (tumor weight: IMD-0560, 0.310 T 0.226 vs control, 0.861 T 0.307 g, P G 0.001; number of peritoneal implants: 24.2 T 18.3 vs 81.5 T 31.5, respectively, P G 0.001) (Figs. 4B, C). To examine the mechanism by which IMD-0560 affects cancer cells in vivo, we next performed immunohistochemical analyses. Peritoneal tumors were stained with the cell proliferation marker Ki-67 (Fig. 4D). The number of Ki-67Ypositive cells was significantly decreased by IMD-0560 treatment (14.26 T 5.80 vs control, 34.6 T 6.79/200 field; P = 0.03) (Fig. 4E). Human VEGF expression was inhibited in tumors treated with IMD-0560 compared with those of the control (Fig. 4D). Accordingly, the number of intratumoral vessels, as visualized by anti-mouse CD31 staining (Fig. 4D), was significantly inhibited by IMD-0560 treatment (14.26 T 5.62 vs control, 30.46 T 3.89/200 field; P = 0.03) (Fig. 4F). Collectively, these findings indicated that IMD-0560 inhibits ovarian cancer progression by inhibiting cancer cell proliferation and decreases angiogenesis through suppression of VEGF production in ovarian cancer cells.
DISCUSSION The present study demonstrates that the novel selective IKKA inhibitor, IMD-0560, suppressed ovarian cancer progression and extended survival of ovarian cancer xenograft mice. Activation of NF-JB is associated with up-regulation of cell survival signaling molecules and regulation of cell cycleYrelated molecules.5,25 IMD-0560 inhibited NF-JB activation through IJB phosphorylation and led to cell-cycle arrest of ovarian cancer cell lines in vitro and suppressed peritoneal metastases in vivo. Another significant effect of IMD-0560 is antiangiogenic activity. IMD-0560 inhibited VEGF-A promoter activity in vitro and suppressed VEGF production in cancer cells, which led to the inhibition of tumor angiogenesis in vivo. Angiogenesis is an important contributor to ovarian carcinogenesis and progression. Angiogenesis often requires the release of angiogenic growth factors from tumor cells or inflammatory cells, such as macrophages and neutrophils. NF-JB regulates the expression of such growth factors and * 2016 IGCS and ESGO
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FIGURE 4. Effect of IMD-0560 in a xenograft ovarian cancer mouse model. A, IMD-0560 prolonged the survival of ovarian cancer model mice. Ovarian cancer model mice were generated as described in Materials and Methods. IMD-0560 was administered IP 10 mg/kg everyday from 1 week after implantation. KaplanYMeier survival curves were calculated. Median survival of IMD-0560Ytreated mice was 51 days compared with 65 days in control mice (P = 0.028). B and C, IMD-0560 decreased tumor weight and the number of tumor deposit. Mice were killed at 35 days after initiation of treatment. Tumor weight (B) and numbers of metastatic sites (C) were significantly decreased by IMD-0560 treatment. D, Tissue sections were immunostained with anti-VEGF, CD31, and Ki-67 antibodies. Representative histological stains are shown. Bars represent 10 Km. E, IMD-0560 treatment significantly decreased CD31-positive cells (P = 0.03). F, IMD-0560 treatment significantly decreased the number of vessels (P = 0.03). *P G 0.05. cytokines, including VEGF, tumor necrosis factor, and monocyte chemoattractant protein.8,26 Among these factors, VEGF is considered a key regulator of angiogenesis.27 Bevacizumab, a VEGF-AYblocking monoclonal antibody, is active against advanced ovarian cancer as shown by several large randomized
studies.28,29 However, despite promising early preclinical data, the clinical benefits of bevacizumab with respect to progressionfree survival are few, with progression-free survival of usually a few months; the benefits in overall survival are more limited30,31 or absent.32,33 Bevacizumab targets only VEGF-dependent
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angiogenesis; however, whereas VEGF is a key driver of cancer angiogenesis, numerous other angiogenic factors have been identified. Therefore, even if the VEGF pathway is successfully blocked by bevacizumab, other angiogenic factors might be eventually up-regulated leading to tumor resistance to bevacizumab. Following the success of bevacizumab, new antiangiogenesis molecules have been developed and several studies on these molecules have been conducted or are underway.34 Thus, the antitumor effect of targeting a single growth factor would most likely be very transient. In that regard, an NF-JB inhibitor can target not only VEGF but also various other proangiogenic factors (eg, tumor necrosis factor, monocyte chemoattractant protein-1, and interleukin-8). Thus far, IMD-0560 can be considered as a potential treatment option for future antiangiogenic therapy. The therapeutic potential of IKKA-dependent NF-JB pathway disruption in cancers has been extensively studied, and a number of NF-JB inhibitors have been developed.35,36 However, most classical NF-JB inhibitors are poorly selective and show off-target effects; therefore, few have progressed to phase III clinical trials.37 The proteasome inhibitor, bortezomib, is the only clinically available NF-JB inhibitor.37 However, proteasome inhibition may affect other signaling pathways. Notably, IMD-0560 specifically inhibits IKKA, which induces the inhibition of NF-JB activation only in inflammatory conditions. Additionally, the prodrug of IMD-0560 is already under assessment in a clinical trial for cardiovascular diseases and rheumatoid arthritis.19,20 The current study has some limitations. We did not assess combination therapy. Because it is difficult to show the overall survival benefit of monotherapy with most small-molecule inhibitors, it is essential to consider combination therapy with existing or novel therapies for clinical application. Thus, further investigation of combination therapy is required for future clinical application. In summary, our finding shows that IMD-0560 treatment has promising therapeutic efficacy in an ovarian cancer xenograft mouse model. IMD-0560 not only induced cellcycle arrest but also suppressed VEGF production in ovarian cancer cells. On the basis of the increasing importance of antiangiogenic agents for ovarian cancer treatment, IMD0560 looks promising and may be valuable in future regimens for the treatment of ovarian cancer.
REFERENCES 1. Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9Y29. 2. Matsuo K, Lin YG, Roman LD, et al. Overcoming platinum resistance in ovarian carcinoma. Expert Opin Investig Drugs. 2010;19:1339Y1354. 3. Dixit V, Mak TW. NF-kappaB signaling. Many roads lead to Madrid. Cell. 2002;111:615Y619. 4. Vallabhapurapu S, Karin M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol. 2009;27:693Y733. 5. Aggarwal BB. Nuclear factor-kappaB: the enemy within. Cancer Cell. 2004;6:203Y208.
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6. Baud V, Karin M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov. 2009;8:33Y40. 7. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappaB. J Clin Invest. 2001;107:241Y246. 8. Webster GA, Perkins ND. Transcriptional cross talk between NF-kappaB and p53. Mol Cell Biol. 1999;19:3485Y3495. 9. Tsujii M, Kawano S, Tsuji S, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell. 1998;93:705Y716. 10. Huang S, DeGuzman A, Bucana CD, et al. Nuclear factor-kappaB activity correlates with growth, angiogenesis, and metastasis of human melanoma cells in nude mice. Clin Cancer Res. 2000;6:2573Y2581. 11. Andela VB, Schwarz EM, Puzas JE, et al. Tumor metastasis and the reciprocal regulation of prometastatic and antimetastatic factors by nuclear factor kappaB. Cancer Res. 2000;60:6557Y6562. 12. Cusack JC Jr, Liu R, Houston M, et al. Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: implications for systemic nuclear factor-kappaB inhibition. Cancer Res. 2001;61:3535Y3540. 13. Mabuchi S, Ohmichi M, Nishio Y, et al. Inhibition of NFkappaB increases the efficacy of cisplatin in in vitro and in vivo ovarian cancer models. J Biol Chem. 2004;279:23477Y23485. 14. Hernandez L, Hsu SC, Davidson B, et al. Activation of NF-kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian cancer. Cancer Res. 2010;70:4005Y4014. 15. Annunziata CM, Stavnes HT, Kleinberg L, et al. Nuclear factor kappaB transcription factors are coexpressed and convey a poor outcome in ovarian cancer. Cancer. 2010;116:3276Y3284. 16. Niederberger E, Geisslinger G. The IKK-kappaB pathway: a source for novel molecular drug targets in pain therapy? FASEB J. 2008;22:3432Y3442. 17. Horie R. Molecularly-targeted strategy and NF-JB in lymphoid malignancies. J Clin Exp Hematop. 2013;53:185Y195. 18. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer. 2009;9:28Y39. 19. Suzuki J, Ogawa M, Muto S, et al. Novel IJB kinase inhibitors for treatment of nuclear factor-JB-related diseases. Expert Opin Investig Drugs. 2011;20:395Y405. 20. Young ER. IKKA as a therapeutic intervention point diseases related to inflammation. In: Levin JI, Laufer S, eds. Anti-inflammatory Drug Discovery. RSC Publishing; 2012;255Y296. 21. Sawada K, Morishige K, Mabuchi S, et al. In vitro and in vivo assays to analyze the contribution of Rho kinase in angiogenesis. Methods Enzymol. 2008;439:395Y412. 22. Wakatsuki S, Suzuki J, Ogawa M, et al. A novel IKK inhibitor suppresses heart failure and chronic remodeling after myocardial ischemia via MMP alteration. Expert Opin Ther Targets. 2008;12:1469Y1476. 23. Yin L, Morishige K, Takahashi T, et al. Fasudil inhibits vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. Mol Cancer Ther. 2007;6:1517Y1525. 24. Kinose Y, Sawada K, Makino H, et al. IKKA regulates VEGF expression and is a potential therapeutic target for ovarian cancer as an antiangiogenic treatment. Mol Cancer Ther. 2015;14:909Y919. 25. Kiriakidis S, Andreakos E, Monaco C, et al. VEGF expression in human macrophages is NF-kappaBYdependent: studies using * 2016 IGCS and ESGO
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International Journal of Gynecological Cancer
26. 27.
28.
29.
30.
31.
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adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2. J Cell Sci. 2003;116:665Y674. Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431Y436. Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol. 2001;280:1358Y1366. Burger RA, Sill MW, Monk BJ, et al. Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group Study. J Clin Oncol. 2007;25:5165Y5171. Cannistra SA, Matulonis UA, Penson RT, et al. Phase II study of bevacizumab in patients with platinum-resistant ovarian cancer or peritoneal serous cancer. J Clin Oncol. 2007;25:5180Y5186. Perren TJ, Swart AM, Pfisterer J, et al. A phase 3 trial of bevacizumab in ovarian cancer. N Engl J Med. 2011;365:2484Y2496. Burger RA, Brady MF, Bookman MA, et al. Incorporation of bevacizumab in the primary treatment of ovarian cancer. N Engl J Med. 2011;365:2473Y2483.
32. Aghajanian C, Blank SV, Goff BA, et al. OCEANS: a randomized, double-blind, placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer. J Clin Oncol. 2012;30:2039Y2045. 33. Liu FW, Cripe J, Tewari KS. Anti-angiogenesis therapy in gynecologic malignancies. Oncology (Williston Park). 2015;29:350Y360. 34. Klempner SJ, Myers AP, Mills GB, et al. Clinical investigation of receptor and non-receptor tyrosine kinase inhibitors for the treatment of epithelial ovarian cancer. Expert Opin Pharmacother. 2013;14:2171Y2182. 35. Burke JR, Pattoli MA, Gregor KR, et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J Biol Chem. 2003;278:1450Y1456. 36. Sanda T, Iida S, Ogura H, et al. Growth inhibition of multiple myeloma cells by a novel IkappaB kinase inhibitor. Clin Cancer Res. 2005;11:1974Y1982. 37. San Miguel JF, Schlag R, Khuageva NK, et al. Bortezomib plus melphalan and prednisone for initial treatment of multiple myeloma. N Engl J Med. 2008;359:906Y917.
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