Novel Proteasome Inhibitor PS-341 Inhibits Activation of Nuclear Factor-kB, Cell Survival, Tumor Growth, and Angiogenesis in Squamous Cell Carcinoma

We have shown that activation of nuclear factor-kB (NF-kB) promotes cell survival and expression of cytokines such as growth-regulated oncogene-a, which can modulate angiogenesis, growth, and metastasis of squamous cell carcinoma (SCC). Activation of NF-kB and cytoprotective genes in cancer may result from signal-induced phosphorylation and proteasome-dependent degradation of inhibitorkB. In this study, we examined the effects of the novel proteasome inhibitor PS-341 on activation of NF-kB and cell survival, growth, and angiogenesis in murine and human SCC cell lines. PS-341 inhibited activation of NF-kB DNA binding and functional reporter activity at concentrations between 10 and 10 M. Cytotoxicity was observed at 10 M in four murine and two human SCC lines, and followed early cleavage of poly(ADP-ribose) polymerase, a marker of caspase-mediated apoptosis. In vivo, PS-341 inhibited growth of murine and human SCC in mice at doses of 1–2 mg/kg given three times weekly, and dose-limiting toxicity was encountered at 2 mg/kg. Tumor growth inhibition was associated with a marked decrease in vessel density. PS-341 inhibited expression of the proangiogenic cytokines growthregulated oncogene-a and vascular endothelial growth factor by SCC in the range at which PS-341 inhibits NF-kB. We conclude that PS-341 inhibits activation of NF-kB pathway components related to cell survival, tumor growth, and angiogenesis in SCC. INTRODUCTION Constitutive activation of NF-kB has been implicated in the development and progression of a number of human malignancies, including head and neck cancer (1), pancreatic cancer (2), colon cancer (3), breast cancer (4, 5), T-cell leukemia (6), and Hodgkin’s lymphoma (7). We reported that the constitutive activation of NF-kB in SCCHN promotes the expression of cytokines with proinflammatory and proangiogenic activities (1). Activation of NF-kB was found to be correlated with development of a metastatic phenotype (8) and with autonomous overexpression of an NF-kB-dependent chemokine, called GRO-a (KC), which promotes tumor growth and the development of metastases in vivo (9). Further evidence for the significance of NF-kB activation in SCCHN comes from experiments in which transfection of a dominant-negative inhibitor of NF-kB into SCCHN cells resulted in a 70–90% reduction in cell viability (10). Thus, NF-kB activation appears to be important for the survival of SCCHN cells, as well as the conferment of a more aggressive tumor phenotype. These observations indicate that NF-kB may be an important target for therapy. The prototypical mechanism of NF-kB activation depends on the signal-induced phosphorylation and ubiquitination of an inhibitory protein called IkB-a, which is subsequently degraded by the proteasome (11–14). A novel proteasome inhibitor, PS-341 (Millennium Pharmaceuticals, Inc.) has recently been described to be a potent inhibitor of NF-kB activation. This compound is a dipeptidyl boronic acid analogue that selectively inhibits the chymotryptic activity of the 20S proteasome. It has excellent bioavailability and stability and has been shown to have in vivo antitumor activity in a human prostate carcinoma xenograft model (15) and to inhibit both tumor growth and the development of metastases in an animal model of lung carcinoma (16). In this study, we examined the effect of PS-341 on multiple murine and human SCC cell lines in vitro and in vivo. Exposure to the compound was found to inhibit activation of NF-kB and expression of the NF-kB-dependent proangiogenic chemokine GRO-a (KC) and VEGF in SCC. Significant effects of PS-341 on cell survival, tumor growth, and angiogenesis were observed. MATERIALS AND METHODS Cells Lines. The PAM 212 cell line is a spontaneously transformed cell line derived from neonatal BALB/c keratinoReceived 11/3/00; revised 1/12/01; accepted 2/7/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Institute on Deafness and Other Communication Disorders (NIDCD) intramural research project Z01-DC-00016. 2 To whom requests for reprints should be addressed, at Tumor Biology Section, Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, 10 Center Drive, Building 10, Room 5D55, Bethesda, MD 20892. 3 The abbreviations used are: NF-kB, nuclear factor-kB; SCCHN, squamous cell carcinoma of the head and neck; GRO-a (KC), growthregulated oncogene-a; IkB, inhibitor of nuclear factor-kB; VEGF, vascular endothelial growth factor; EMSA, electrophoretic mobility shift analysis; PARP, poly(ADP-ribose) polymerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1419 Vol. 7, 1419–1428 May 2001 Clinical Cancer Research Research. on September 22, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from cytes in vitro that forms SCCs in vivo and was provided by Dr. Stuart Yuspa of the NCI (Bethesda, MD; Ref. 17). The PAMLY2 cell line is a metastatic variant of the PAM 212 line isolated from lymph node metastases that formed after s.c. inoculation of PAM 212 tumor fragments in BALB/c mice. Details of the development and characterization of the PAMLY2 variant have been described previously (18). PAM 212 and PAM-LY2 cells exhibit constitutive activation of NF-kB at low and high levels with respect to each other, respectively (8). The B4B8 and B7E3 cell lines were derived by exposure of murine oral mucosa cells to carcinogens by Dr. Fred Hendler at the University of Louisville and Dr. Giovanna Thomas in our laboratory (19). The human SCCHN cell lines UM-SCC-9 and -11B were obtained from the well-characterized UM-SCC series maintained by Dr. T. E. Carey at the University of Michigan (Ann Arbor, MI; Ref. 20). UM-SCC-9 and -11B have constitutive activation of NF-kB at low and high levels with respect to each other (1). All of the cell lines were maintained in Eagle’s Minimal Essential Medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. SCC Tumor Model. BALB/c and BALB/c SCID mice were obtained from the NCI, Frederick Cancer Research and Development Center, Animal Production Area. The mice used were between 4 and 6 weeks of age, male, weight #20 g, and housed in a specific pathogen-free animal facility. Animal care was provided under an NIH Animal Care and Use Committee approved protocol (no. 894-99), in accordance with NIH guidelines. In vivo experiments with PAM-LY2 cells were conducted by injecting 5 3 10 cells s.c. over the flanks of immunocompetent syngeneic BALB/c mice. In vivo experiments with UMSCC-11B cells were conducted by injecting 1 3 10 cells s.c. over the flanks of immunodeficient BALB/c SCID mice. Tumors were measured by independent animal care personnel in a blinded fashion. Inhibitor PS-341. The synthesis and purification of PS341 was described previously by Adams et al. (21). For in vitro studies, PS-341 was reconstituted in DMSO at a concentration of 10 M. Appropriate dilutions were performed such that the concentration of DMSO never exceeded 0.001%. For in vivo studies, PS-341 was carried in a vehicle containing 10% DMSO in PBS. Solutions were sterilized by filtration through a 45 mm syringe filter. PS-341 was administered using sterile technique by i.p. injections three times/week on a Monday/Wednesday/ Friday schedule. For PAM-LY2 tumor-bearing mice, initiation of treatment occurred 6 days after inoculation with tumor cells and prior to the development of palpable tumors. For UM-SCC11B tumor-bearing mice, initiation of treatment occurred after the development of palpable tumors: at least 0.02 cm for the experiment in which mice received PS-341 at a dose of 1.0 mg/kg and at least 0.25 cm for the experiment in which mice received a dose of 2.0 mg/kg. Nuclear and Cell Extracts. PAM-LY2 cells (5 3 10) were grown either in medium without PS-341 or in medium containing 1 3 10, 1 3 10, 1 3 10, or 1 3 10 M PS-341 for 12 h. The cells were harvested by scraping in PBS and resuspended in lysis buffer [10 mM Tris-HCl (pH 8.0), 60 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, and protease inhibitor cocktail]. After 5 min on ice, the nuclei were pelleted by centrifugation and separated from the cytoplasmic extract. The nuclei were resuspended in nuclear extract buffer [20 mM Tris-HCl (pH 8.0), 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM DTT] and incubated at 4°C for 10 min. Nuclear debris was removed from the extract by high-speed centrifugation. Protein concentrations were determined using the BCA protein assay method (Protein Assay Kit; Pierce Chemical Co., Rockford, IL). EMSA. Double-stranded DNA oligonucleotides for NF-kB and ornithine carbamyl transferase-1 were purchased from Promega (Madison, WI). The consensus sequences (underlined) were: NF-kB, 59-AGTTGAGGGGACTTTCCCAGGC-39 and ornithine carbamyl transferase-1: 59-TGTCGAATGCAAATCACTAGAA39. Oligonucleotides were labeled using T4 polynucleotide kinase and [g-P]ATP (6000 Ci/mmol; Amersham, Arlington Heights, IL). EMSA was performed as described previously (1). Briefly, nuclear extracts (5 mg) were added to binding buffer [100 mM HEPES-KOH (pH 7.9), 25 mM MgCl2, 300 mM KCl, 5 mM DTT, 0.5% NP-40, and 50% glycerol] containing poly(dI-dC). Competition for oligonucleotide binding was accomplished by the addition of unlabeled oligonucleotide prior to addition of P-labeled probes. Nuclear extract proteins were incubated at 20°C for 25 min. Binding complexes were resolved on 5% polyacrylamide gels in 0.253 Tris-borate-EDTA buffer at 20°C and visualized by autoradiography. Cell Transfection and Reporter Gene Assays. The IgkB-Luc plasmid, containing two copies of the NF-kB-binding site upstream of the minimal promoter fused with the luciferase gene, was described previously (22) and was a kind gift of Dr. U. Siebenlist (National Institute of Allergy and Infectious Disease, NIH). The pCMV-LacZ construct was made by Dr. Giovana Thomas in our laboratory and consists of a LacZ gene inserted between the CMV promoter and BGH poly(A) signal sequence in pcDNA3 (Invitrogen, Carlsbad, CA). PAM-LY2 cells (2 3 10/well) were transfected for 5 h at 37°C in 6-well culture plates with 2 mg of pIgk-luc and 0.1 mg of pCMVLacZ DNA plus 16 ml of Lipofectamine (Life Technologies, Gaithersburg, MD) in Optimem medium. Following transfection, the cells were grown in Eagle’s Minimal Essential Medium plus 10% fetal bovine serum and exposed to PS-341 at either 10 or 10 M for 12 h or left untreated. The cells were harvested, and reporter gene activities were assayed using the Dual-Light Luciferase and b-Galactosidase Reporter Gene Assay System (Tropix, Bedford, MA), and chemiluminescence was measured by a Monolight 2010 luminometer (Analytical Luminescence Lab, San Diego, CA). The relative light units were calculated as follows: RLU 5 RLU from Luciferase RLU from b-Galactosidase where RLU is relative light unit(s). Measurement of Cell Proliferation and Viability. Cells (2 3 10/well) were grown in 6-well culture plates and exposed to PS-341 at either 10 or 10 M or left untreated. At the 4-, 12-, 24-, and 72-h time points, cells were harvested by trypsinization, stained with trypan blue dye, and visually counted. The percentage of viability was determined by the number of nonstained cells divided by the total number of cells counted. 1420 Proteasome Inhibitor PS-341 and SCC Research. on September 22, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Isolation of Whole Cell Lysates and Assay for PARP Cleavage. PAM-LY2 cells were grown to 60–90% confluency in sterile 100 3 20 mm polystyrene cell culture dishes. Cells were exposed to either medium without PS-341 or medium containing 1 3 10 M PS-341. At 1, 2, 3, 6, 9, and 12 h following exposure to PS-341, the cells were rinsed once with ice-cold PBS, scraped, and lysed in 250 ml of lysis buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 0.2 mM Na3VO4, and 0.2 mM phenylmethylsulfonyl fluoride], and incubated on ice for 15 min. The lysates were passed three times through a 23-gauge needle, centrifuged at 14,000 rpm for 5 min at 4°C, and transferred to a fresh microcentrifuge tube. Total protein concentrations were determined using the BCA protein assay method (Protein Assay Kit; Pierce). Each sample (40 mg total protein) was gently vortexed with loading buffer and boiled at 100°C for 5 min. The samples were pulse centrifuged and electrophoresed through 10% Tris-Glycine precast gels (Novex, San Diego, CA) at 120 V. Proteins were transferred to nitrocellulose (Bio-Rad, Hercules, CA) for 90 min at 20 V, using the Novex gel blot module. The nitrocellulose blot was subsequently stained using Ponceau-S (Sigma Chemical Co., St. Louis, MO) to determine transfer efficiency. Blots were blocked with 5% nonfat powdered milk in Tris-buffered saline-Tween overnight at 4°C and incubated for 2 h at room temperature with a rabbit polyclonal IgG to the COOH terminus of PARP that recognizes the fulllength and 89-kDa cleaved fragment (Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA). The blot was washed with three times with Tris-buffered saline-Tween and incubated with horseradish peroxidase-conjugated goat antirabbit IgG at a 1:2000 dilution (Bio-Rad) for 1 h at room temperature. Chemiluminescent detection of horseradish peroxidase was accomplished using the SuperSignal Substrate method (Pierce) followed by exposure to X-OMAT AR film (Eastman Kodak Co.,