The Targeted SMAC Mimetic SW IV-134 is a strong enhancer of standard chemotherapy in pancreatic cancer
© The Author(s). 2016
Received: 27 August 2016
Accepted: 5 December 2016
Published: 17 January 2017
Pancreatic cancer is a lethal malignancy that frequently acquires resistance to conventional chemotherapies often associated with overexpression of inhibitors of apoptosis proteins (IAPs). We have recently described a novel means to deliver second mitochondria-derived activator of caspases (SMAC) mimetics selectively to cancer cells employing the sigma-2 ligand/receptor interaction. The intrinsic death pathway agonist SMAC offers an excellent opportunity to counteract the anti-apoptotic activity of IAPs. SMAC mimetics have been used to sensitize several cancer types to chemotherapeutic agents but cancer-selective delivery and appropriate cellular localization have not yet been considered. In our current study, we tested the ability of the sigma-2/SMAC drug conjugate SW IV-134 to sensitize pancreatic cancer cells to gemcitabine.
Using the targeted SMAC mimetic SW IV-134, inhibition of the X-linked inhibitor of apoptosis proteins (XIAP) was induced pharmacologically and its impact on cell viability was studied alone and in combination with gemcitabine. Pathway analyses were performed by assessing caspase activation, PARP cleavage and membrane blebbing (Annexin-V), key components of apoptotic cell death. Single-agent treatment regimens were compared with combination therapy in a preclinical mouse model of pancreatic cancer.
The sensitizing effect of XIAP interference toward gemcitabine was confirmed via pharmacological intervention using our recently designed, targeted SMAC mimetic SW IV-134 across a wide range of commonly used pancreatic cancer cell lines at concentrations where the individual drugs showed only minimal activity. On a mechanistic level, we identified involvement of key components of the apoptosis machinery during cell death execution. Furthermore, combination therapy proved superior in decreasing the tumor burden and extending the lives of the animals in a preclinical mouse model of pancreatic cancer.
We believe that the strong sensitizing capacity of SW IV-134 in combination with clinically relevant doses of gemcitabine represents a promising treatment option that warrants clinical evaluation.
KeywordsSigma-2 receptors Sigma-2/SMAC drug conjugate Gemcitabine Combination therapy Pancreatic cancer
Pancreatic cancer is one of the most aggressive malignancies with an estimated 53,070 new cases and 41,780 deaths in the United States in 2016, with a 5-year survival rate of only 8% (Ref. ). Surgical intervention remains the only potential cure and even after surgery, the 5-year survival rate is only 10 - 25 percent due to the high rates of local recurrence and metastases . Gemcitabine is a recommended treatment for pancreatic cancer and is used to treat both localized and metastatic disease. Gemcitabine can also be used in combination with radiation therapy . Unfortunately, the benefits of gemcitabine therapy and other chemotherapies are rather limited. For example, gemcitabine chemotherapy only modestly improved overall survival to 6.8 months in patients with stage IV disease . The response rate by Response Evaluation Criteria in Solid Tumors (RECIST criteria) for first line gemcitabine is only 9.4% . A comparison between a combination of multiple active components such as FOLFIRINOX (5-FU, Irinotecan, Leucovorin and Oxaliplatin), versus a mix of Gemzar and albumin-complexed paclitaxel (Abraxane), did show similar disease control but both regimens were associated with substantial off-side toxicities . Outcomes like these highlight the urgent need to develop more effective treatment options for patients with pancreatic cancer.
Pancreatic cancers use several mechanisms to evade apoptosis as they acquire resistance to conventional chemotherapy . The inhibitor of apoptosis proteins (IAP) frequently contribute to drug resistance via blockade of caspase activation . More specifically, the X-linked inhibitor of apoptosis proteins (XIAP) is a well-characterized member of the IAP family. It contains baculovirus IAP repeat (BIR) domains, of which BIR-2 is involved in blocking caspases-3/7 while BIR-3 interferes with activation of caspase-9 [8–10]. High intracellular XIAP levels have been attributed to chemoresistance in many pancreatic cancer cell lines as well as primary tumors . Second mitochondria-derived activator of caspases (SMAC) is a mitochondrial protein that is released into the cytosol when cells are exposed to stress, and amplifies the apoptotic pathway by inhibiting IAP activity . SMAC competitively binds to the caspase-binding domains of XIAP, resulting in their activation [12, 13]. Several SMAC mimetics have been described as potential therapeutics for cancer therapy and as sensitizers for traditional chemotherapeutics [14–17].
We have previously shown that sigma-2 receptors are overexpressed in human pancreatic cancer cells . We have also demonstrated that sigma-2 ligands can enter and deliver additional drug cargos into pancreatic cancer cells . We have recently described the novel drug conjugate SW IV-134, composed of the SMAC mimetic SW IV-52 and the sigma-2 ligand SW43 . This potent cancer drug selectively targets the cancer cells via binding to the overexpressed sigma-2 receptor and induces cell death by delivering the SMAC mimetic SW IV-52 . SW IV-134 has a high cytotoxic activity in the low micromolar range on pancreatic cancer cells in vitro and in mouse models of pancreatic cancer .
A key limitation of conventional chemotherapy is the toxicity to normal tissues due to a lack of selective cancer cell delivery. The purpose of this study was to evaluate the therapeutic potential of combining a targeted SMAC mimetic (SW IV-134) with gemcitabine in an effort to improve the efficacy of the non-targeted chemotherapeutic.
The synthesis of SW IV-134 was performed in our laboratory and has been previously described . Gemcitabine (Gemzar®) was purchased from Eli Lilly (Indianapolis, IN).
PANC-1, CFPAC-1, BxPC-3, AsPC-1, and MIA PaCa-2 were obtained from American Type Culture Collection (ATCC, Manassas, VA). CFPAC-1 was cultured in Iscove’s modified medium with 4 mM L-glutamine, 1.5 g/L Sodium bicarbonate, and 10% fetal bovine serum (FBS). MIA PaCa-2 was cultured in Dulbecco's Modified Eagle's medium with 10% FBS and 2.5% horse serum. BxPC-3 and AsPC-1 were cultured in RPMI- 1640 medium with 10% FBS. Antibiotics, penicillin (100 μg/mL) and streptomycin (100 μg/mL) were added to the media. Cells were maintained in a humidified incubator at 37 °C with 5% CO2.
Evaluation of cytotoxicity in vitro
Cells were plated at a density of 1 × 104/well in 96-well plates for 24 hours prior to treatment. SW IV-134 was dissolved in DMSO and diluted in culture medium to achieve a final concentration of 1 μM (the DMSO concentration was always kept below 1% and had thus no impact on the experimental results). Gemcitabine was dissolved in PBS to achieve a concentration of 0.5 μM. Cells were treated with the SW IV-134, gemcitabine, and combination of both drugs that contained 1 μM of SW IV-134 and 0.5 μM of gemcitabine. Cell viability was determined 3 - 4 days after treatment using CellTiter-Glo Luminescent Viability Assay (Promega, Madison, WI). Luminescence signal was measured using a multi-mode microplate reader (Bio-Tek, Winooski, VT). All assays were performed in triplicates.
Lentivirus mediated XIAP knockdown
The lentiviral constructs, pLKO.1 for XIAP (sh-1, TRCN0000003785; sh-2, TRCN0000003787) and Luciferase (sh-Luc) were obtained from Washington University genome center. shRNA constructs were transfected into HEK293T cells together with the lentiviral packaging plasmids VSVG (envelope) and ∆8.9 (gag, pol), using Fugene 6 (Roche, Indianapolis, IN). Viral supernatants were collected at 48 and 72 hours and added to PANC-1 and MIA PaCa-2 cells in the presence of protamine sulfate (10 μg/mL). Infected cells were selected with Puromycin (2 μg/mL). The drug selection process was monitored via GFP fluorescence (as part of the lentiviral vectors) and was in the range of 90% positive cells.
Cells were lysed in radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM sodium chloride, 1 mM EDTA, 1% Nonidet P40, and 0.25% SDS (pH 7.0)]. The buffer was supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentration was measured by BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL). Samples containing equal amounts of protein were run on NuPAGE Bis-Tris 4 - 12% gradient gels and then transferred onto PVDF membranes (Life Technologies, Grand Island, NY). The membranes were incubated in blocking buffer (5% dry milk) for 1 hour, followed by addition of the respective primary antibodies at 4 °C overnight. The following day, membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour. SuperSignal West Dura Substrate (Thermo Fisher Scientific Rockford, IL) was used to detect the secondary antibodies. Primary and secondary antibodies for capase-3, cleaved caspase-3, Poly ADP-ribose polymerases (PARP), cleaved PARP, and XIAP were purchased from Cell Signaling Technology (Danvers, MA). Primary and secondary actin antibodies were purchased from Santa Cruz (Dallas, TX). Antibody dilutions were made according to the manufacturer’s instructions.
In vitro evaluation of apoptosis (Annexin-V staining)
AsPC-1 cells were seeded in 6-well plates at a density of 5 × 105/well for 24 hours. Cells were treated for 48 hours with 0.5 μM of SW IV-134, 0.5 μM of gemcitabine, equimolar concentration of both drugs, and DMSO as a control. Apoptosis was detected using Annexin-V FITC Kit (Biolegend, San Diego, CA). Propidium iodide was added to differentiate early apoptotic cells from necrotic and late stage apoptotic cells. Cells were prepared according to the manufacturer's instructions and analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
In vitro caspase activation assays
Caspase-3, 8 and 9 activities were measured in PANC-1 and AsPC-1 cells using Caspase-Glo Assay Systems (Promega, Madison, WI) according to the manufacturer’s instructions. This assay is based on luminogenic caspase substrates which are cleaved by activated caspases resulting in generation of a luminescence signal. Cells were seeded at a density of 1 × 104 in 96-well plates for 24 hours. Then, they were treated for 48 hours with 1 μM of SW IV-134, 0.5 μM of gemcitabine, combination of the two drugs, and DMSO as a control. The contents of the plate were mixed using an orbital shaker for 30 seconds and incubated at room temperature for 90 minutes. Luminescence signal was measured using a multi-mode microplate reader (Bio-Tek, Winooski, VT).
In vivo assessment of tumor growth, survival, and toxicity
Immunocompromised female nude mice (6 weeks old, Harlan Laboratories, Indianapolis, IN) were injected in the right flank with 200 μL single cell suspension of 1 × 106 AsPC-1 cells in RPMI medium. Treatment started when tumors reached approximately 5 mm in diameter. Mice were randomized into four groups (n = 14). The groups received daily i.p. injections with 100 μL of vehicle (25% cremophor in H2O), weekly gemcitabine (3 mg), daily SW IV-134 (750 nmoles) with and without weekly gemcitabine (3 mg). Tumors were measured every other day with a digital caliper and the volumes were calculated using the equation V = d1 (d2)2/2, (V = volume, d1 = larger diameter, d2 = smaller diameter). Following conclusion of treatment at day 21, five mice from each group were sent to the Division of Comparative Medicine at our institution for toxicity evaluation. Blood was collected for complete blood count (CBC) and biochemical analysis (AST, ALT, BUN, total bilirubin, and Cr). Organs were examined grossly and histologically. Mice were euthanized when tumors reached a diameter of 2 cm or ulcerated. Animal euthanasia was performed using a carbon-dioxide (CO2) chamber. Mice were placed in the CO2 chamber (≤10 mice at a time) and 100% CO2 was introduced at a slow rate, replacing 30% of the chamber volume in 1 minute. Mice were exposed to CO2 for five minutes followed by a two minute dwell period. Animal studies were carried out in accordance with the Washington University Division of Comparative Medicine guidelines for care and use of laboratory animals. The protocol was approved by the Animal Studies Committee of Washington University (protocol 20130073).
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 viability and caspase activity assays. Unpaired two tailed t-tests were used to evaluate the difference in CBC, biochemistry analyses, viability of knock-down cells, and to confirm the difference in subgroups. Two-way ANOVA was used to analyze the difference in tumor sizes. Kaplan-Meier survival analysis was used and the difference between the groups was compared with a log-rank test. A p-value < 0.05 was considered significant for all analyses.
Low XIAP expression sensitizes pancreatic cancer cells to gemcitabine
SW IV-134 sensitizes pancreatic cancer to gemcitabine
SW IV-134 augments gemcitabine-induced activation of both the intrinsic and the extrinsic pathways of apoptosis
For further assessment of apoptosis involvement during combination treatment, AsPC-1 cells were exposed to SW IV-134 and gemcitabine alone, an equimolar mix of the drugs and DMSO (control). The cells were subsequently assessed for membrane blebbing (Annexin-V) and PI uptake. The combination of both drugs resulted in the highest degree of early apoptosis with 34% of cells being Annexin-V+/PI– (Fig. 3b). Only gemcitabine single-agent treatment resulted in a somewhat elevated level of early apoptosis (5.5%), while none of the other groups showed clear evidence of treatment effects (DMSO and SW IV-134 with 0.9% and 1.3%, respectively). These data further suggest that low-dose combination therapy mediates strong cytotoxicity in an apoptosis-dependent fashion.
SW IV-134 enhances gemcitabine therapy in a preclinical xenograft model of pancreatic cancer
Pancreatic cancer has a poor prognosis and novel therapeutic approaches are desperately needed. Gemcitabine has been combined safely with several other chemotherapeutics in efforts to improve outcomes but success has been very modest. For example, combination of gemcitabine with erlotinib or capecitabine mildly improved overall survival by a few weeks [30, 31]. Recently, an intensive multi-drug combination has shown more significant gains in survival. The regimen includes doses of 5-FU, oxaliplatin, irinotecan, and leucovorin (FOLFIRINOX). Overall survival improved from 6.8 months to 11.1 months when gemcitabine was added but at the cost of significant toxicity. FOLFIRINOX was associated with substantially more adverse effects, including febrile neutropenia, thrombocytopenia, diarrhea, and sensory neuropathy . As a consequence, this treatment option is only suitable for the healthiest of patients suffering from pancreatic cancer.
Evading apoptosis is an important mechanism of acquired or preexisting chemoresistance in pancreatic cancer [32, 33]. Restoring the ability to undergo programmed cell death is therefore an attractive strategy to enhance treatment efficiencies . IAPs belong to a family of proteins frequently involved in resistance of pancreatic cancer to chemotherapeutics [28, 35]. XIAP is the most prominent and potent member of this family and its transcriptional down regulation or pharmacologic blockade using SMAC mimetics has been shown to sensitize pancreatic cancer cells to gemcitabine [11, 28]. Interestingly, depletion of cIAP-1 and cIAP-2 alone was not sufficient to sensitize pancreatic cancer cells to gemcitabine . In agreement to these earlier studies, we confirmed that decreased XIAP levels substantially improved sensitivity to gemcitabine, even in the context of residual XIAP expression (Fig. 1). We therefore believe that this up-regulated cellular survival pathway represents an attractive target in patients with pancreatic cancer. In order to exploit XIAP as a putative pancreatic cancer target most efficiently, delivery of its antagonist, the SMAC peptidomimetic, needs to be rendered cancer selective, since a non-selective mimetic would increase the risks for systemic toxicities. By linking a SMAC mimetic to the sigma-2 ligand SV119, we created a targeted therapeutic capable of delivering its payload directly into the cancer cells [20, 22]. It is important to note that SMAC mimetics, including our targeted SW IV-134, induce cancer cell death via complex mechanisms, currently only incompletely elucidated. While XIAP is functionally blocked, cIAP-1/2 is rapidly degraded, which leads to NIK-dependent TNFα production and augmented target cell killing by inducing apoptosis [8, 20, 22]. Even though SW IV-134 targets the same pathways as SMAC itself, we found it to be far more effective than the unconjugated SMAC mimetic . The reason(s) for its enhanced activity profile is not completely understood and constitutes an active research area but we believe it is likely caused by an enhanced and cancer-selective uptake/internalization mechanism, as we have recently shown for the targeted delivery of an erastin analog to treat pancreatic cancer in vitro and in vivo .
This is the first description of using SW IV-134 in combination with a standard chemotherapeutic, gemcitabine, in PDAC. However, it might achieve even better treatment outcomes when combined with non-standard experimental therapies, such as targeted TRAIL biologics . In order to show the potential synergy between SW IV-134 and gemcitabine, we reduced the doses of the individual drugs. We are currently in the process of designing preclinical models of PDAC in order to identify the maximum tolerated dose in vivo. At the reduced doses, combination of SW IV-134 with gemcitabine significantly slowed tumor growth and increased in the median overall survival of our animals. Apart from a decrease in the WBC count, commonly seen following gemcitabine chemotherapy , and mild peritonitis at the site of injection, no toxicities were observed.
Sigma-2 receptors are up-regulated in many cancer types [39, 40] and SMAC mimetics have been shown to sensitize many types of cancer to a wide array of chemotherapeutic agents [17, 41, 42]. As a result, we believe that SW IV-134 could potentially be used to sensitize several additional sigma-2-expressing malignancies, including head and neck tumors, breast cancer, lymphomas , and ovarian cancer  to different chemotherapeutics. As such, the combination therapy approach employed here may have implications beyond the treatment of patients with pancreatic adenocarcinoma.
In this study, we described the ability of the sigma-2 receptor-targeted SMAC mimetic SW IV-134 to act as a sensitizing agent for chemotherapy in a model of pancreatic cancer. This approach merits further investigation, since this cancer has been recalcitrant to most standard therapies. This work is significant because sigma-2 receptors are highly expressed in many types of cancer. As a result, we believe that the targeted combination strategy will likely work for additional malignancies. Since many chemotherapeutics, including SW IV-134, activate the intrinsic arm of the apoptotic pathway, it may work well to accentuate the effects of a variety of therapeutics that trigger complementing effector arms of programmed cell death, such as the TRAIL-induced extrinsic death pathway.
Blood urea nitrogen
Cellular inhibitor of apoptosis proteins-1
Inhibitor of apoptosis proteins
Poly ADP-ribose polymerase
Response Evaluation Criteria in Solid Tumors
Second mitochondria-derived activator of caspases
X-linked inhibitor of apoptosis proteins
The authors thank Mary Hornick for technical assistance (Department of Surgery, Washington University).
This work was funded by the National Institute of Health R01 grant (US NIH 5R01CA16376402) (W.G. Hawkins) and the Barnes-Jewish Hospital Auxiliary Cancer Frontier Fund - 3795 (W.G Hawkins and D. Spitzer). Statistical services were provided by the Biostatistics Core at the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO. The Siteman Cancer Center is supported in part by NCI Cancer Center Support Grant #P30 CA091842. Toxicology and pathology studies were supported by the Digestive Diseases Research Core Center at Washington University School of Medicine in St. Louis, MO. Salary support for YH was provided by the Washington University Surgical Oncology Training Grant (T32 CA009621).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files. Please contact the corresponding author for requests of materials described in this study.
YMH performed the experiments, analyzed the data and wrote the manuscript. SW synthesized and characterized the sigma-2-based drugs. NVS designed XIAP-specific shRNA constructs and verified XIAP knockdown by Western blot analysis. PSB performed in vitro killing assays. SPG, RHM, DS, and WGH were involved in study supervision and critical review of the manuscript. All authors have read and approve the final version of the manuscript.
Robert Mach and William Hawkins own patent rights for the sigma-2 related drugs. All other authors declare no conflicts of interest.
Consent for publication
Ethics approval and consent to participate
Animal studies were carried out in accordance with the Washington University Division of Comparative Medicine guidelines for care and use of laboratory animals (Protocol #20160058).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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