Ovarian cancer is the most lethal of the gynecologic malignancies and this is due in large part to the resistance of recurrent ovarian cancer cells to standard chemotherapeutic strategies. Resistance to apoptotic cell death is a fundamental characteristic of cancer cells, and a primary cause of treatment failure. A critical step in apoptosis is the activation of cysteine-dependent aspartate-specific proteases, the caspases. Once activated, the caspases are responsible for DNA fragmentation and cleavage of numerous critical proteins. Because of the pathological consequences of deregulated apoptosis, the activation of caspases is under direct and stringent regulation by the inhibitors of apoptosis [IAPs; reviewed in Ref.1].
X-chromosome-linked IAP (XIAP), the most potent member of the IAP family, binds directly to and blocks the activity of caspase-3, -7 and -9, as well as interferes with the BAX/cytochrome c cell death pathway.2, 3 It is overexpressed in a variety of cancers, including ovarian,4–6 and there is accumulating evidence that XIAP is a major contributor to chemoresistance in ovarian cancer cells. Specifically, downregulation of XIAP expression in ovarian cancer cells increased the sensitivity of the cells to cisplatin- and docetaxel-induced cell death.7–9 Studies on the efficacy of antisense oligonucleotides against XIAP (AS XIAP) in vivo show that AS XIAP treatment significantly impeded tumor growth or caused tumor regression in xenograft models representing several types of cancer (prostate, lung, colon and breast) when administered either as a single agent or in combination with clinically-relevant chemotherapy.10, 11 The therapeutic effect of antisense knockdown of XIAP in ovarian tumor models has not yet been evaluated.
Herein, we report that downregulation of XIAP expression using antisense oligonucleotide AEG35156 significantly increases cell death of ovarian cancer cells in vitro, in the absence or presence of wild-type p53. Furthermore, we extended these findings to the in vivo setting and demonstrate for the first time that AS XIAP treatment prolongs animal survival and reduces tumor viability in an ovarian cancer xenograft model.
AS, antisense; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IAP, inhibitor of apoptosis; IP, intraperitoneally; PARP, poly-ADP ribose polymerase; RIAP, rat IAP; SEM, standard error of the mean; TBST, Tris-buffered saline with tween; XIAP, X-linked inhibitor of apoptosis.
Material and methods
The following cell lines were used in this study: A2780-s (cisplatin-sensitive parental line) and A2780-cp (a cisplatin-resistant cell line established by stepwise exposure of A2780-s cells to increasing doses of cisplatin) from Dr. Molepo (Ottawa, ON, Canada) and ES-2 human ovarian carcinoma cells from Dr. J. Bell (Ottawa, ON, Canada). For maintenance, all 3 cell lines were cultured in DMEM plus 2.5% fetal calf serum, 7.5% donor bovine serum and 1% nonessential amino acids. Transfection of the cells with oligonucleotides was facilitated by Oligofectamine in Opti-MEM (Gibco BRL, Burlington, ON, Canada) according to manufacturer's instructions. Specifically, the antisense oligonucleotides against XIAP (AEG35156, AS XIAP; Aegera Therapeutics, Montreal, PQ, Canada) were mixed-backbone, fully phosphorothioated 19-mers with 4 2′O-methyl RNA residues at both the 5′ and 3′ ends and a central core of 11 DNA residues. The scrambled control (AEG35187) had the same base composition and thus the same molecular weight as the parent compound.
Western blot analyses
To confirm successful knockdown of XIAP expression and to analyze Caspase 3 and poly-ADP ribose polymerase (PARP) cleavage characteristic of apoptotic cell death, protein lysates were extracted in RIPA buffer according to the manufacturer's instructions (Upstate Cell Signalling Solutions, Waltham, MA). Denatured protein samples (50 μg) were separated on NuPAGE precast gels (Invitrogen Life Technologies, Burlington, ON, Canada) and transferred to nitrocellulose membranes. Membranes were blocked in 5% skim milk in TBS/T (180 mM NaCl, 10 mM Tris, 0.05% Tween 20) for 1 hr at room temperature prior to incubation with the primary antibody overnight at 4°C. Specifically, a mouse monoclonal antibody to GAPDH (Abcam, Cambridge, UK) was used at a dilution of 1:2,000, a rabbit polyclonal antibody to RIAP3 [Aegera Therapeutics12] was diluted 1:500 and mouse monoclonal antibodies to Caspase 3 or PARP (Cell Signaling Technology, Danvers, MA) were diluted 1:1,000. Membranes were blotted according to standard protocols, with the following horseradish peroxidase-conjugated IgG secondary antibodies: anti-mouse (KPL, Gaithersburg, MD; 1:5,000); and anti-rabbit (Cedarlane Laboratories, Hornby, ON, Canada; 1:5,000). Visualization of protein bands was performed using an enhanced chemiluminescence detection system (LumiGLO; KPL) and Kodak X-Omat Blue XB-1 film (Rochester, NY).
Equal numbers of cells were plated in 6 wells of a 12-well plate on Day 1 and allowed to adhere overnight. The first transfection was conducted on Day 2. In the morning of Day 3, all cells from each well were replated in wells of a 6-well plate and allowed to adhere for ∼6 hr. A second transfection was conducted in the afternoon of Day 3, based on a previous report of efficient knock-down with this strategy.10 On Day 4, adherent cell numbers were determined by Coulter counter.
DNA sequence analysis of p53
Following trypsinization, A2780-s and A2780-cp cells (∼1 × 106) were resuspended in DNA extraction buffer (250 μl; 10 mM Tris/0.1 M EDTA/0.5% SDS/20 μg/ml proteinase K) and incubated at 58°C overnight. DNA was precipitated by a standard protocol using saturated sodium chloride and isopropanol. The DNA pellet was washed once with 75% ethanol and resuspended to a final concentration of 100 ng/μl in Tris-EDTA (50 mM, pH 6.8).
PCR for p53 was performed as follows: the 50 μl PCR reaction contained 1 μl DNA (at 100 ng/μl), 20 pmol of each primer (p53U: 5′ CTCTTCCTGCAGTACTCCC-CTGC 3′; p53D: 5′ GCCCCAGCTGCTCACCATCGCTA 3′), 100 μM dNTPs, 1.5 mM MgCl2, 1× Advantage PCR Buffer and 5 U AdvanTaq DNA polymerase (BD Biosciences, Palo Alto, CA). The protocol had an initial 3 min denaturation at 94°C, followed by 35 cycles of 94°C for 30 s, 66°C for 45 s and 72°C for 90 s, and a final elongation at 72°C for 7 min. PCR products were separated by electrophoresis on a 1.0% agarose gel and stained with ethidium bromide for visualization. As only a single band was visualized following PCR amplification, the PCR products were sent directly for sequencing by the Ottawa Health Research Institute Ontario Genomics Innovation Centre (Ottawa, ON, Canada).
All animal experiments were performed according to the Guidelines for the Care and Use of Animals established by the Canadian Council on Animal Care. Female CD-1 nu/nu mice (Charles River Laboratories; Wilmington, MA) aged 6–8 weeks were housed with free access to food and water. A2780-cp cells (1 × 107) were resuspended in 500 μl of phosphate-buffered saline and injected with a 25-gauge needle intraperitoneally (IP) into at least 8 mice per treatment group per experiment on Day 0. IP antisense oligonucleotide treatment (10 mg/kg or 25 mg/kg) began on Day 7 and continued for 6 weeks (5 days on, 2 days off, as previously described10). Disease progression was monitored based on overall health until a predetermined endpoint was reached. Survival time reflects the time required for the animals to reach any endpoint(s), including weight gain/loss exceeding 20%, anorexia and/or diarrhea.
Histological analysis of tumors
Tumor samples were fixed in 10% buffered formalin (VWR, Mississauga, ON, Canada) for 6–24 hr, paraffin-embedded and sectioned at 5 μm for hematoxylin and eosin (H&E) staining according to standard protocols. Slides were analyzed by microscopy (Zeiss Axioskop2; Northern Eclipse Imaging Software Version 6.0, Empix Imaging, Mississauga, ON, Canada) or scanned at 600 dpi, 3× magnification with an Epson Perfection 2450 Photo Scanner, and the resulting images were analyzed for the quantity of hematoxylin-positive pixels (indicative of live cells) relative to total number of pixels in the tumor using Adobe Photoshop Software as previously described.13
All cell count values are from at least 3 independent experiments in triplicate and are expressed as the mean ± standard error of the mean (SEM). The probabilities of significant differences upon comparison of the 2 groups (scrambled control vs. AS XIAP-treated) were determined by 2-tailed paired Student's t-tests. Kaplan-Meyer survival curves were plotted using GraphPad Prism software (version 4.0; GraphPad, San Diego, CA) and were compared using a log-rank test. In the analysis of the tumor sections for the proportion consisting of viable cells, one outlier determined by the Quartile/Fourth Spread method was removed from the control group. Significance for all comparisons was inferred at p < 0.05.
AS XIAP transfection decreased XIAP protein expression in ovarian cancer cell lines
Three ovarian cancer cell lines were transfected with mixed-backbone, fully phosphorothioated 19-mer antisense oligonucleotides against XIAP, or a scrambled control of the same base composition, and analyzed by western blot for XIAP expression. Our results show that expression was substantially decreased following transfection with AS XIAP (Fig. 1).
Downregulation of XIAP decreased ovarian cancer cell survival, independent of p53 status
To determine whether downregulation of XIAP expression would affect the survival of A2780-cp, A2780-s or ES-2 cells, equal numbers of cells were transfected with control or AS XIAP oligonucleotides on 2 consecutive days, and adherent cell number was determined by Coulter counter 24 hr following the second transfection. Cell number was significantly decreased by AS XIAP transfection. Specifically A2780-cp, A2780-s and ES-2 cell numbers were reduced relative to cells transfected with scrambled oligonucleotides by 43.3 ± 2.0%, 63.3 ± 7.6% and 21.4 ± 3.6%, respectively (p < 0.05; Fig. 2a). For all 3 cell lines, the number of cells originally plated far exceeded the number of adherent cells following AS XIAP transfection, which suggested that the reduction in cell number was attributable to apoptosis, as opposed to decreased cell cycle progression.14 This was confirmed by western blot analysis for Caspase 3 and PARP: 17 and 19 kDa Caspase 3 cleavage products became evident, and full-length PARP was noticeably reduced relative to GAPDH, in the AS XIAP- transfected population compared to the transfected control cells (Fig. 2b).
On the basis of previous reports that impaired cell survival from XIAP downregulation relies upon wild-type p53,9, 15 we sought to confirm that the p53 mutation previously reported for the A2780-cp cells was present in the population of cells used in this study.16 A single base pair missense mutation (G to T) resulting in a valine to phenylalanine amino acid substitution was confirmed at codon 172 by PCR amplification and sequencing (data not shown).
AS XIAP treatment prolonged the survival of A2780-cp xenograft mouse models
CD-1 nude mice were injected IP with 1 × 107 A2780-cp cells on Day 0, and treatment with AS XIAP or scrambled control oligonucleotides commenced on Day 7, when animals had established tumors, as determined by necropsy of a subset of animals (data not shown). Animals were treated IP with 10 or 25 mg/kg of AS XIAP or 25 mg/kg control oligonucleotide, reconstituted in saline, on a 5 days on/2 days off schedule for up to 6 weeks, as described.10 Kaplan-Meyer survival curves generated from a compilation of 3 identical experiments demonstrate a significant improvement in median survival time in animals treated with 10 mg/kg AS XIAP, relative to controls (51 vs. 81 days; p = 0.036; Fig. 3). The same trend was observed for the mice treated with the 25 mg/kg dose (51 vs. 67; p = 0.069). The number of mice in each arm of the survival curve is Control, n = 20; 10 mg/kg AS XIAP, n = 10; 25 mg/kg AS XIAP, n = 28.
AS XIAP treatment increased intratumoral cell death
Our observation that AS XIAP was able to improve animal survival time was substantiated by histological evaluation of the tumors. To compare the proportion of the tumors consisting of viable cells, digital images of tumor sections from control (n = 12) and 25 mg/kg/day AS XIAP-treated mice (n = 14) were analyzed using hematoxylin and eosin staining. The hematoxylin-positive areas were deemed to represent regions containing viable cells, whereas the regions staining positive for eosin were considered to contain no or very few viable cells. The proportion of hematoxylin-positive (viable) pixels in the image relative to the total number of pixels stained by either stain (Figs. 4a and 4b) revealed that AS XIAP-treated tumors had significantly less area occupied by viable cells than the control tumors (54.99 ± 4.1 vs. 68.03 ± 3.8, p = 0.031; Fig. 4c).
This study has found that downregulation of XIAP expression using antisense oligonucleotides significantly increased cell death of ovarian cancer cells in vitro. Furthermore, we extended this finding to the in vivo setting and demonstrated for the first time in an ovarian cancer xenograft model that AS XIAP treatment prolonged animal survival and reduced tumor viability. This is the first indication that decreasing XIAP expression can impair ovarian cancer cell survival, in the absence or presence of wild-type p53.
XIAP is a major player in counteracting normal apoptosis in many chemoresistant malignancies and based on this, we hypothesized that downregulation of its expression would negatively impact the survival of A2780-cp, A2780-s and ES-2 ovarian cancer cells. We have demonstrated that downregulation of XIAP is sufficient to induce apoptosis in ovarian cancer cells, which is consistent with reports in lung, prostate and colon cancer cells.10, 17 AEG35156 is not thought to cross-react with and consequently decrease expression of other IAP family members, since HIAP2/cIAP1 transcript levels are unaffected by treatment,10 but perhaps only comprehensive gene expression array data18 would allow us to conclude definitively that the cell death we have observed is attributable to XIAP downregulation alone. Manipulation of other proapoptotic proteins that indirectly decrease the cellular XIAP content, such as Smac19 and XAF1,6 has also been shown to induce apoptosis without additional insult, which supports our findings. However, in contrast to what we have observed, it has been reported that a decrease in XIAP expression alone does not cause cell death in some cancer cell types, but does increase their susceptibility to chemotherapy-induced death.7–9, 20 We propose that the delicate balance of pro- and anti-apoptotic molecules may differ with cell type and/or the environment to which the cells are exposed. Comparable decreases in XIAP content may be refractory in one cell type, but tip the balance beyond the apoptotic threshold in another. In addition, different transfection conditions may impart variable degrees of stress to cells that affect their susceptibility to apoptosis resulting from downregulated XIAP.
It has previously been reported that, in ovarian cancer cells, impaired cell survival arising from XIAP downregulation occurs in p53 wild-type cells, but not in p53 mutated cells.9 However, we observed a significant decrease in cell survival following AS XIAP transfection not only in the A2780-s cell line containing wild-type p53, but in A2780-cp cells, which have been reported to contain a p53 mutation.21 As previously discussed, the balance of pro- and antiapoptotic signals is delicate and may vary between cells. One main difference between the studies that may explain the dissimilar outcome is the method used to decrease XIAP. Both the adenoviral strategy employed by Sasaki et al. (2000) and antisense oligonucleotides have the potential to have nonsequence specific effects,18, 22 and although this was controlled for (i.e., control oligonucleotides with the same base composition as the AS XIAP compound), studies of this kind need to be interpreted cautiously as there is a possibility that the delivery method may be imparting an effect. Also, the degree of knockdown achieved in the mutant-p53 cell lines relative to wild-type cells in the Sasaki et al. study may have been less, and insufficient to induce apoptosis.
The decrease in cell survival that we observed in vitro motivated an in vivo evaluation of AS XIAP in xenograft mouse models. It has previously been reported in lung cancer mouse models that decreasing XIAP content induces apoptosis and enhances sensitivity to chemotherapy,23, 24 but the therapeutic efficacy in invivo ovarian cancer models has not yet been evaluated. We chose to test this strategy in a xenograft model using A2780-cp cells, chosen for their chemoresistance and mutant-p53 status, which may have hindered their ability to apoptose in response to XIAP downregulation, but represents a disease that is more difficult to treat and that more reasonably reflects the patient population in whom this new therapeutic approach will be tested. Cells were injected IP in an effort to accurately represent the human disease.25 Intraperitoneal AS XIAP treatment (10 mg/kg/day) of mice xenografted IP with A2780-cp cells prolonged the survival time of the animals, which is consistent with the effects of AEG35156 in p53-mutant models of breast and lung cancer.11 Interestingly, the lower 10 mg/kg/day dose reached statistical significance in lengthening survival time compared to control oligonucleotide treated mice; however, the 25 mg/kg/day dose, which showed a trend for efficacy, did not reach statistical significance. As the survival curves of the 2 doses are not statistically different from each other, one interpretation could be that there was simply no additional benefit from the higher dose. Alternatively, this could be taken as evidence that the higher dose was less effective, suggesting that there is either a greater amount of nonspecific toxicity associated with the higher dose that may have compromised the efficacy, or that cells are triggered to compensate when the knockdown of XIAP expression is sufficiently large.
Our in vivo findings that AEG35156 improves the survival of A2780-cp xenografted mice were substantiated by histological evaluation of the tumors. Our primary objective was to evaluate the effect of decreased XIAP expression on animal survival, and since the majority of mice survived beyond the final treatment, it was not possible for us to evaluate the tumors for apoptotic cells by more conventional strategies, such as TUNEL, as they would have long been eliminated. However, analysis of H&E sections of tumors revealed significantly less “cellularity” in the cross-sectional area of tumors in AS XIAP-treated mice, which indicates an increase in intratumoral cell death.
Although XIAP is ubiquitously expressed in all tissues, the downregulation of XIAP should not, in principle, impact normal cells and tissues unless they are subjected to insults that trigger caspase activation and the initiation of the apoptotic process. Based on body weight and gross pathology at the time of necropsy, we did not observe any toxicity in mice that received up to 25 mg/kg/day of AS XIAP for up to 100 days (data not shown). Moreover, formal GLP preclinical toxicology studies conducted in rat and monkey show that AEG35156 can be administered via the intravenous route with an acceptable safety profile for clinical evaluation.11 That XIAP-deficient mice are viable26 further suggests that normal cells can accommodate this change to their “apoptotic balance,” which is thought to be more tenuous in cancer cells. Phase I clinical trials of AEG35156 AS XIAP therapy will further assess the safety of this therapeutic approach.
Improvement of ovarian cancer survival statistics will rely in part upon the development of novel therapeutics targeting molecules that are responsible for or contribute to tumor formation and chemoresistance. Not only are there a myriad of potential molecular targets in need of validation, there are numerous strategies for altering their expression and/or activity. Modulation of gene expression by antisense oligonucleotides is a valuable therapeutic approach since it is both specific and applicable to any gene.27, 28 Future research will clarify the most successful means for XIAP downregulation, be it antisense oligonucleotides,11, 23 adenovirus,9 RNA interference20 or small molecule inhibitors,29 but this study has validated XIAP as a promising molecular target for ovarian cancer therapy. AEG35156 is currently being evaluated in humans in several Phase I clinical trials in the UK, Canada, and the US as a single agent and in combination with other anticancer agents.
The authors thank Ms. Gabriele Cherton-Horvat, Dr. Herman Cheung, Dr. Katherine Clark-Knowles, Ms. Olga Collins, Ms. Colleen Crane and Ms. Elizabeth Macdonald for their excellent assistance with the experiments. This research was supported by Aegera Therapeutics Inc., the National Cancer Institute of Canada with funds from the Terry Fox Foundation (BCV), and the Betty Irene West Doctoral Research Scholarship from the Canadian Institutes of Health Research (TJS). EL and JD are both employees of Aegera Therapeutics Inc., which is developing the compound AEG35156 for therapeutic application.