Background
Ovarian cancer is the second most common gynecologic malignancy in the United States. However, it is the deadliest of all gynecologic cancers; of the 28,080 expected deaths from gynecologic malignancies annually, about 50% or 14,030 will be from ovarian cancer [
1]. Most patients respond well to the initial treatment with surgical de-bulking and combination chemotherapy [
2]. The disease eventually recurs in a large number of patients leading to death mainly due to the lack of effective treatments against drug resistant disease [
3]. There is a clear need to identify novel therapeutic agents that target critical drug resistance pathways to improve survival in ovarian cancer.
Apoptosis or programmed cell death is a cell suicide mechanism which plays a critical role in the development and homeostasis in vertebrates and invertebrates [
4]. Inhibition of apoptosis can prevent cancer cell death and promote the development of drug resistance in various malignancies [
5]. The inhibitor of apoptosis proteins (IAPs) are among the principal molecules that contribute to this phenomenon [
6]. The anti-apoptotic activity of IAPs can be overcome by second mitochondria-derived activator of caspases (SMAC), a mitochondrial protein which is released into the cytoplasm in response to apoptotic stimuli [
7]. A number of compounds that mimic the function of the SMAC proteins have been described recently with striking pro-apoptotic activity reported in vitro and in vivo [
8‐
11].
We have previously described the development and potential diagnostic and therapeutic application of sigma-2 ligands for various types of malignancies [
12‐
15], and shown that sigma-2 receptor ligands bind to the PGRMC1 protein complex [
16]. Sigma-2 ligands are especially suitable for purposes of diagnostic imaging and therapeutic targeting of solid tumors due to their high selectivity for tumor cells in vivo [
17,
18]. Furthermore, by virtue of their rapid internalization and binding to the sigma-2 receptor [
19], these ligands represent excellent candidates for selective delivery of anticancer drugs into the tumor cells [
14]. Taking advantage of these two unique properties of sigma-2 ligands, we have generated dual-domain therapeutics, wherein sigma-2 ligands additionally function as targeting domains for a cancer-selective delivery of effector molecules such as pro-apoptotic peptides into the tumor cells (12). One such compound is SW IV-134, which is a conjugate of the sigma-2 ligand SW43 and a small molecule SMAC mimetic SW IV-52. In our current report, we describe in detail the in vitro characterization of SW IV-134 and explore its effectiveness in preclinical models of ovarian cancer.
Discussion
Sigma-2 receptors are preferentially expressed in proliferating tumor cells [
27], and are therefore considered potential targets for a selective delivery of therapeutics into the cancer cells. Insights from the current understanding of the cell death pathways have led to the identification of several candidate proteins that could be targeted for the eradication of cancer cells. One such class of prominent cell survival factors are the inhibitors of apoptosis proteins (IAPs). IAPs are frequently overexpressed in many types of human malignancies, making them attractive targets for therapeutic intervention [
28]. In the current work we have shown the feasibility of conjugating a small molecule SMAC mimetic to a sigma-2 ligand and demonstrated the effectiveness of the conjugate in treatment of ovarian cancer using in vitro and in vivo studies.
Our results indicate that SW IV-134 potentiates the sigma-2 ligand related cell death in different ovarian cancer cell lines (Figure
2). Furthermore, the cell death induced by SW IV-134 was significantly greater than that observed with a combination of SW43 and SW IV-52 (Figure
2). This is likely due to the more efficient delivery of the small molecule SMAC mimetic into the tumor cells following conjugation to the sigma-2 ligand SW43. The SMAC mimetic in turn enhances the cytotoxicity of the compound by negating the activity of two anti-apoptotic proteins, cIAP-1 and cIAP-2 (Figure
4A), thus establishing an intracellular environment that is conducive for NF-қB activation. The loss of cIAPs with SW IV-134 treatment is likely secondary to the SMAC mimetic-induced auto-ubiquitination and proteasomal degradation, and has been shown by others [
29].
The NF-қB transcription factors [such as NF-қB1 (p105/p50), NF-қB2 (p100/p52), RelA(p65), ReLB, and c-ReL] are usually retained in the cytoplasm of un-stimulated cells by IқB (Inhibitor of қB) proteins [
30]. In the canonical NF-қB pathway, phosphorylation of IқB by IKK2 (IқB Kinase) leads to its proteasomal degradation, allowing nuclear translocation of the transcription factors [
31,
32]. It has been shown that phosphorylation of NF-қB p65 is critical for the canonical NF-қB signaling [
33]. On the other hand, NF-қB-inducing-kinase (NIK) is one of the key regulators of the non-canonical NF-қB pathway. NIK phosphorylates IKKα, resulting in the phosphorylation of p100, which leads to the ubiquitination and partial proteasomal degradation of p100 to its mature p52 form [
34,
35]. Treatment with SW IV-134 led to phosphorylation of NF-қB p65 as well as accumulation of NIK (Figure
4A), suggesting its role in activation of both canonical and non-canonical NF-қB signaling pathways. The activation of NF-қB pathways, in turn, led to the transcriptional upregulation of TNFα mRNA (Figure
5A), directly corresponding with an increase in TNFα protein (Figure
5B) and subsequent potentiation of cell death via activation of the extrinsic pathway of apoptosis, similar to what has been described by other investigators [
26].
SW IV-134 reduced the tumor burden of the intraperitoneal xenografts and significantly improved survival in mice (Figure
6). A frequent criticism of many xenograft models is the location of tumor formation and its resemblance to human disease [
36]. Because most ovarian cancers in humans are diagnosed at late stages, we believe that our intraperitoneal tumor model resembles more closely the advanced-stage human disease than a localized orthotopic model. In this widely metastatic intraperitoneal model, SW IV-134 demonstrated modest effectiveness, extending median survival by 10 days. Furthermore, the treatment was well tolerated with minimal adverse effects on the animals as evidenced by blood testing and histopathological analysis of various organs and tissues harvested at the end of treatment. Treatment with sigma-2 ligands has repeatedly been shown to induce apoptosis with limited off-site toxicities. This is because cancer selectivity is a key feature of our sigma-2 based platform concept, and has been demonstrated in numerous previous studies [
12,
15,
19,
21]. In one of these studies it was demonstrated that nude mice bearing either mouse 66 (mammary carcinoma) or MDA-MB-435 cells (human melanoma) injected with the fluorescently-labeled sigma-2 ligand SW120 was taken up selectively by proliferating tumor cells and not by the generally quiescent peripheral blood mononuclear cells [
21]. Similar results were seen in vitro, where sigma-2 ligands were preferentially taken up by transformed cancer cells and not by the immortalized normal human HPDE cells (data not shown). Furthermore, new clinical data have recently become available that once again highlight the tendency of our drugs to localize primarily to solid tumors with negligible accumulation in the surrounding, healthy tissue [
37]. These results suggest that SW IV-134 may have considerable potential for the treatment of ovarian cancers with a favorable therapeutic window.
Anti-tumor efficacy of SMAC mimetics has been reported in a variety of tumor-types by other investigators [
20,
38]. Furthermore, SMAC mimetics have been shown to exhibit considerable synergism in combination with a wide variety of therapeutic agents ranging from conventional therapy to TRAIL and other biologic agents [
9‐
11,
39]. Because SW IV-134 induced apoptosis targets the same mechanistic pathways as the SMAC mimetics, it can also be potentially combined with different chemotherapeutic agents to overcome resistance to therapy-induced apoptosis. We believe that the selective delivery of SMAC mimetics into the tumor cells by sigma-2 ligands (as achieved with SW IV-134) will likely enhance its on-target effects while keeping the off-target effects to a minimum. We thus envision the unique properties of SW IV-134 highly advantageous when used alone but especially in combination with existing and/or novel chemotherapeutics. As a result, we are currently in the process of exploring the tumoricidal effects of SW IV-134 in combination with different chemotherapeutic agents, to identify the most efficacious treatment regimen which could be moved forward for further clinical investigations.
Conclusions
In summary, we have described the effectiveness and potential application of SW IV-134, a novel conjugate of sigma-2 ligand and a small molecule SMAC peptidomimetic, in the treatment of ovarian cancer. We believe this approach is particularly meritorious considering the cancer selectivity of sigma-2 ligands and the important contribution of IAPs to both de-novo and acquired treatment resistance, and therefore deserves serious consideration for future clinical development.
Materials and methods
Cell lines and reagents
SKOV3 cells were obtained from Dr. Robert Mach (Washington University School of Medicine, St. Louis, MO), Hey A8 and Hey A8 MDR cells from Dr. Anil Sood [
40] (M.D. Anderson Cancer Center, Houston, TX), and OVCAR3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). SKOV3 cells were labeled with a eYFP/luciferase reporter fusion protein by retroviral infection to generate SKOV3-Luc cells (G. Garg and D. Spitzer, unpublished data). Protein expression was confirmed by flow cytometry and in vitro luciferin conversion. SW43 [
19] and SW IV-52 [
41] were synthesized as previously reported. Synthesis of SW IV-134 is described in detail in the Additional file
7: Supplementary methods. MG-132 was purchased from Calbiochem (Billerica, MA), Z-VAD-FMK from Enzo Life Sciences (Ann Arbor, MI), and anti-TNFα antibody was purchased from R&D systems (Minneapolis, MN).
Receptor binding assays
The sigma-1 and sigma-2 receptor binding affinities of SW IV-134 were determined as previously described [
42]. Briefly, guinea pig brain (sigma-1 assay) or rat liver (sigma-2 assay) membrane homogenates (~300 μg protein) were diluted with 50 mM Tris-HCl, pH 8.0 and incubated with the radioligand {~5 nM [
3H](+)-pentazocine (34.9 Ci/mmol; sigma-1 assay) or ~5 nM [3H](+)-DTG (58.1 Ci/mmol; sigma-2 assay)} and SW IV-134 with concentrations ranging from 0.1 nM to 10 μM in a total volume of 150 μL in 96-well plates at 25°C. After incubating for 120 minutes, the reactions were terminated by the addition of 150 μL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a 96-channel transfer pipette (Fisher Scientific, Pittsburg, PA), and the samples harvested and filtered rapidly into a 96-well fiberglass filter plate (Millipore, Billerica, MA) that had been presoaked with 100 μL of 50 mM Tris-HCl, pH 8.0 for 1 hour. Each filter was washed three times with 200 μL of ice-cold wash buffer, and the bound radioactivity quantified using a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, MA). Nonspecific binding was determined in the presence of 10 μM cold haloperidol.
Blocking studies
SKOV3 cells (3 × 104/well) were seeded into 6 cm plates for 24 hours before treatment. The cells were incubated with 5, 10, 50, and 100 μM doses of SW IV-134 for 30 minutes at 37°C. After this step, 10 nM SW120 was added to the cell culture medium containing SW IV-134. After 30 minute incubation at 37°C, cells were washed twice with phosphate buffered saline (PBS) and harvested with 0.05% trypsin EDTA (Life Technologies, Grand Island, NY). The cells were centrifuged at 1000 × g for 5 minutes and pellets washed twice with PBS. Internalization of SW120 was determined by flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA).
Annexin V binding
SKOV3 cells were treated with different concentrations of SW IV-134 (3 μM, 6 μM, and 10 μM) for 16 hours. Staining was performed using the Annexin V Apoptosis Detection Kit (BioLegend, San Diego, CA) according to the manufacturer’s instructions. The apoptosis rate was determined by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA).
Western blot analysis
Cells were lysed in radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM sodium chloride, 1.0 mM EDTA, 1% Nonidet P40, and 0.25% SDS (pH 7.0)], supplemented with complete protease inhibitor cocktail (Roche, Mannheim) and phosphatase inhibitor cocktail 1 (Sigma Chemical Co., St. Louis, MO). The protein concentration was determined using a BioRad Dc protein assay kit (Bio-Rad Laboratories, Hercules, CA). Lysates containing 30 μg of protein were run on a 12% polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA). The PVDF membrane was incubated with 5% nonfat dry milk for 1 h at room temperature, then overnight with a primary antibody at 4°C, and finally with the secondary antibody, horse-radish peroxidase-conjugated IgG, and signal visualized using the super Signal West Pico Chemiluminiscent Substrate assay kit (Pierce Biotechnology, Rockford, IL). The primary antibodies against cIAP1, cIAP2, XIAP, NIK, NF-қb (p65), phospho- NF-қB (p65), caspase3, caspase 8, and caspase 9 (all caspase-detecting antibodies react with both their precursors and cleaved [activated] forms) were purchased from Cell Signalling Technology (Danvers, MA, USA), whereas actin antibody was from Santa Cruz Biotechnology (Dallas, TX). The secondary antibodies against mouse, rabbit, and goat were also purchased from Santa Cruz Biotechnology (Dallas, TX).
Quantitative RT-PCR
SKOV3 cells were treated with SW IV-134 (1 μM, 3 μM) or vehicle-control for 4 hours, 12 hours, and 24 hours. At the end of each treatment, cells were harvested and total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY) and RT-PCR performed as per the protocol described before [
43]. Briefly, one microgram of RNA from each sample was reverse transcribed to cDNA with Retroscript (Ambion, Austin, TX). Resulting cDNA was diluted to an equivalent of 10 ng/μL of input RNA. Primer/probe sets for TNFα and GAPDH were purchased from Applied Biosystems (Grand Island, NY). Each reaction consisted cDNA, TaqMan Master Mix (Applied Biosystems), and primer/probe set in a total of 10 μL, following the manufacturer’s standard protocol. For each transcript/sample, triplicate reactions were done in an ABI7500FAST Sequence Detection System. TNFα data were normalized for expression with GAPDH, and results were expressed as fold change over untreated controls.
ELISA
SKOV3 cells were treated with SW IV-134 (1 μM and 3 μM). Cell culture supernatants were collected at 4, 12, and 24 hours after treatment. Medium was collected, floating cells were removed by centrifugation at 1200 × g for 5 minutes, and samples were frozen at -20°C until analysis. TNFα ELISA was performed using a quantitative high sensitivity sandwich immunoassay from eBioscience (San Diego, CA) as per the manufacturer’s instructions.
Analysis of cell death
Cells (1 × 104) were seeded into 96 well plates. Treatment as described in the figure legends was initiated the following day. Cell viability was determined 18 hours after treatment using CellTiter-Glo Luminiscent Viability Assay (Promega, Madison, WI). Data were recorded with a SpectraMax Gemini microplate spectrofluorometer, Molecular Devices (Silicon Valley, CA).
Caspase activation assays
Caspase 3, 8 and 9 activities were measured using Caspase-Glo® Assay Systems according to the manufacturer’s instructions (Promega, Madison, WI). Briefly, the assay systems are based on caspase-specific substrates, which are activated by cleavage, resulting in caspase specific luminescence signals. HeyA8 cells were plated at a density of 1 × 104 in white 96 well, clear bottom plates for 24 hours before treatment. Cells were treated with different concentrations of SW IV-134. Caspase 3, 8 and 9 assays were performed by adding 100 μl of caspase reagents to each well. The contents were mixed using a plate shaker for 30 seconds and incubated at room temperature for 90 minutes. Luminescence signal was measured using a multi-mode microplate reader (BioTek, Winooski, VT).
Animal studies
All studies were performed in accordance with an animal protocol approved by the Washington University Institutional Animal Care Facility. Female severe combined immunodeficient mice (SCID) were purchased from Taconic Farms (Hudson, NY) at age 6 weeks. SKOV3-Luc cells (5 × 10
6) were inoculated intraperitoneally and mice were randomized 7 days later into one of the three treatment groups according to their baseline luciferase activity employing bioluminescence imaging (BLI). For bioluminescence imaging, mice were injected intraperitoneally with 150 μg/g D-luciferin (Biosynth, Naperville, IL) in PBS, anesthetized with 2.5% isoflurane, and imaged with a charge-coupled device (CCD) camera-based bioluminescence imaging system (IVIS 100; Caliper, Hopkinton, MA; exposure time 300 seconds, binning 16, field of view 12, f/stop 1, open filter). Signal was displayed as radiance (photons/sec/cm
2/sr) [
44].
In vivo experiments were conducted with 15 mice per treatment group. Treatment involved daily intraperitoneal injections with 90 μL of SW43 (7.5 mM stock), SW IV-134 (7.5 mM stock), or vehicle-only (25% cremophor in H2O) for 3 weeks. Tumor burden was monitored by bioluminescence imaging once a week during the treatment period. At the end of the treatment, 5 mice were randomly removed per treatment group and submitted for necropsy to the Washington University Department of Comparative Medicine. Blood samples were collected by intracardiac withdrawal prior to necropsy for chemistry and hematology work-up. In addition, various key organs were harvested for gross and histopathological examination. The remaining 10 mice from each treatment group were followed over time to examine overall survival. For survival studies, actual death or poor physical condition/large tumor size meeting criteria for euthanasia, constituted a death event.
Statistical analyses
Statistical analyses and data plotting were performed using GraphPad Prism software version 5 (San Diego, CA). Results were expressed as mean ± SEM of at least 3 biological replicates. One-way ANOVA was used to analyze the differences in cell killing assays (Titer-Glow), TNFα quantification assays (RT-PCR and ELISA) and for measuring tumor sizes. The Kaplan-Meier survival curve was plotted and the difference between the groups was compared with a Log-rank test. P values < 0.05 were considered significant for all analyses.
Acknowledgements
We thank Dr. Premal H. Thaker from the Division of Gynecologic Oncology for obtaining cell lines from MD Anderson Cancer Center; Dr. Kimberly Trinkaus from the Division of Bio-statistics for statistical analysis; Suellen Greco from the Division of Comparative Medicine for performing necropsy and review of pathologic specimens; Dr. Sriraj Pillai, Jesse Gibbs, and Stacey Plambeck-Suess from the Department of Surgery for technical assistance. This work was funded by grants of the National Institute of Health 5R01CA16376402 (W.G. Hawkins), P50 CA094056 (D. Piwnica-Worms), The Staenberg and The Eberle family donation to the Department of Gynecologic Oncology (M.A. Powell and D.G. Mutch), the Mallinckrodt Institute of Radiology (R.H. Mach), and the National Cancer Institute P30 CA91842 to the Siteman Cancer Center.
Competing interest
Robert Mach and William Hawkins own patent rights for the sigma-2 related drugs. All other authors declare no conflict of interest.
Authors’ contributions
Conception and design: GG, WGH, DS. Development of methodology: GG, SV, CZ. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): GG, LC, CH. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): GG, WGH, DS. Writing, review, and/or revision of the manuscript: GG, YH, DP-W, MAP, DGM, RHM, WGH, DS. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): SV, CZ, LC, CH, YH. Study supervision: WGH, DS. All authors read and approved the final manuscript.