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XIAP is not required for human tumor cell survival in the absence of an exogenous death signal
© Sensintaffar et al; licensee BioMed Central Ltd. 2010
Received: 12 June 2009
Accepted: 12 January 2010
Published: 12 January 2010
The X-linked Inhibitor of Apoptosis (XIAP) has attracted much attention as a cancer drug target. It is the only member of the IAP family that can directly inhibit caspase activity in vitro, and it can regulate apoptosis and other biological processes through its C-terminal E3 ubiquitin ligase RING domain. However, there is controversy regarding XIAP's role in regulating tumor cell proliferation and survival under normal growth conditions in vitro.
We utilized siRNA to systematically knock down XIAP in ten human tumor cell lines and then monitored both XIAP protein levels and cell viability over time. To examine the role of XIAP in the intrinsic versus extrinsic cell death pathways, we compared the viability of XIAP depleted cells treated either with a variety of mechanistically distinct, intrinsic pathway inducing agents, or the canonical inducer of the extrinsic pathway, TNF-related apoptosis-inducing ligand (TRAIL).
XIAP knockdown had no effect on the viability of six cell lines, whereas the effect in the other four was modest and transient. XIAP knockdown only sensitized tumor cells to TRAIL and not the mitochondrial pathway inducing agents.
These data indicate that XIAP has a more central role in regulating death receptor mediated apoptosis than it does the intrinsic pathway mediated cell death.
An underlying feature of all human cancer is uncontrolled cell proliferation. However, for a tumor to increase in cell mass and malignant potential, the increase in replication rate must be accompanied by suppression of apoptosis . While tumor cells can subvert many apoptotic regulators, the anti-apoptotic IAP family is thought to have a central role in this process.
There are eight IAPs in humans. All IAPs contain multiple functional domains that potentially modulate many biological processes, including apoptosis. For instance, IAPs have a role in cell-cycle regulation through mitotic spindle formation, ubiquitination of target proteins, and modulation of several signal transduction pathways . Elevated IAP protein levels are common in many tumor types, and a wealth of data supports their role in suppressing cell death, although the exact mechanisms by which different IAPs mediate this effect remains unclear [3, 4]
XIAP is the most thoroughly characterized of this family, and is the only member that can directly inhibit the proteolytic activity of caspases in vitro (reviewed in Eckelmen ). Caspase inhibition is mediated through an 80 amino acid motif, the Baculovirus IAP Repeat domain (BIR), common to all IAPs. By contrast, cIAPs can also directly interact with caspases, but largely to target caspase degradation through the ubiquitin ligase activity of the C-terminal RING domain . Importantly, XIAP inhibits caspases at both the initiation phase (caspase-9) and the execution phase (caspases-3 and 7) of apoptosis . In light of these activities, XIAP inhibition through small molecules or antisense has received considerable pharmaceutical industry focus, and multiple agents have progressed to clinical trials .
A hallmark of apoptotic cell death is the presence of proteolytically cleaved, catalytically active caspases. Viable cells of many well-studied cancer cell lines have been reported to exhibit high steady-state levels of activated caspases in the absence of other markers of cell death . The resistance of these cells to apoptosis is thought to be mediated, at least in part, by XIAP. If XIAP function is essential for survival of these cancer cells, then its inhibition by pharmacological or genetic targeting should increase the rate of apoptosis, without the requirement of additional exogenous signals. XIAP loss of function has been studied extensively using various genetic tools including germ line deletion , somatic cell deletion , and both transient and stable mRNA knockdown. The results have varied widely; in some reports XIAP knockdown alone resulted in decreased viability, while other studies demonstrated no effect. Mice harboring XIAP null alleles are viable and do not exhibit any overt defects in developmental or homeostatic apoptosis, aside from a slight delay in mammary alveolar development [11, 12]. These same XIAP null mice crossed to the TRAMP mouse model of prostate cancer did not result in an alteration in tumor progression, suggesting that XIAP is not a critical regulator of tumor apoptosis in this context . However, loss of XIAP function can sensitize some cell lines in vitro to apoptosis mediated by activation of either the extrinsic, caspase 8 dependent pathway, using exogenous ligands such as TRAIL [10, 14] and TNFα , or chemotherapeutic agents, which largely activate the intrinsic, caspase 9-dependent pathway [16–18]
Some of the different outcomes in XIAP depleted cells may be attributable to varying functional dependence on XIAP. On the other hand, there are conflicting reports even in the same cell line. In MCF-7 cells, Hu et. al.,  reported that siRNA-mediated knockdown of XIAP had no effect on cell viability in the absence of an exogenous apoptotic stimulus. By contrast, Zhang et. al.  reported a 70% decrease in MCF-7 viability within 60 hr after transient siRNA-mediated loss of XIAP. Also, Lima et. al.  reported an approximately 50% decrease in viability in MCF-7 cells, 96 hr post transfection with XIAP-targeted siRNA. In another example, the effect of XIAP depletion in NCI-H460 cells ranged from approximately 20%  to 55% reduced viability . The reported differences in phenotype upon XIAP knockdown for a given tumor cell line could be a function of degree and or duration of knockdown, the methodology for quantifying viability, or a more subtle parameter such as cell-culture conditions.
We present a systematic study of siRNA mediated knockdown of XIAP in human tumor cell lines of diverse tissue origin, including cell lines used in previous reports. In addition to assessing the effect of XIAP knockdown under normal growth conditions, we also explored whether loss of XIAP sensitizes tumor cells to either intrinsic or extrinsic inducers of cell death. Interestingly, loss of XIAP function sensitizes human tumor cell lines to TRAIL, but not inducers of the intrinsic death pathway.
DU-145, HCT-116, MCF7, PC-3 and SW-620 were from the Division of Cancer Treatment and Diagnosis, National Cancer Institute (Frederick, Maryland). A-375, BxPC-3, LS 174T, and T24 were from American Type Culture Collection (Manassas, VA). PATU-I cells were from Dr. David Hockenberry at the Fred Hutchinson Cancer Research Center (Seattle, WA). All cell lines were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) at 37° with humidified air containing 5% CO2.
Cells were lysed in modified radioimmunoprecipitation buffer (mRIPA; 10 mM Tris, 150 mM NaCl, 1% (v/v) NP-40, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, pH 7.4) containing cOmplete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) 48 hr after transfection. Total protein of the clarified lysates was quantitated by Lowry Assay (Biorad DC protein assay). NuPAGE® LDS Sample Buffer and Sample Reducing Agent were added to the lysates and heated to 85°C for 10 mins. 20 μg of total cell protein was separated in NuPAGE 4-12% Bis Tris Gel and transferred to a nitrocellulose membrane using an iBlot® Dry Blotting System (Invitrogen). Membranes were incubated in Blocking Buffer (LI-COR, Lincoln, NE) for 30 minutes at room temperature, followed by 60 minute incubations with mouse monoclonal antibodies against XIAP (BD Transduction Laboratories, Cat. No. 610716), Hsp90 (BD Transduction Laboratories, Cat. No. 610418), or PLK1 (Cell Signaling Technology Cat. No. 208G4). Following incubation with an IRDye® Conjugated Goat Anti Mouse IgG (LI-COR), protein bands were quantified using an Odyssey® Infrared Imaging System. Graphing of data to determine XIAP levels was performed using Graphpad PRISM® software. Percent XIAP levels were calculated as follows:
% XIAP = (fluorescence XIAP band of sample-bkgrd/fluorescence Hsp90 band of sample-bkgrd) ÷ (fluorescence XIAP band of untreated cells-bkgrd/fluorescence Hsp90 of untreated cells-bkgrd)
After transfection, cells were immediately diluted in RPMI 1640 supplemented with 10% fetal bovine serum and added to clear bottom white wall 96 well plates at a concentration of 2500 cells/well in a 100 μL volume. 100 μL of ATPlite™ 1 step reagent was added to each well and the luminescence was measured using a Spectramax®L microplate luminometer. Data was acquired using SOFTMAX®Pro software. Graphing and statistical analysis was performed using Graphpad PRISM® software. For experiments to determine TRAIL sensitivity, soluble human recombinant KillerTRAIL™ (Alexis Biochemicals, San Diego) was added at 32 hr after transfection; cells were incubated for an additional 24 hr prior to addition of ATPlite™.
Efficient siRNA-mediated knockdown of XIAP protein levels
Human tumor cell lines.
Effect of XIAP protein knockdown on tumor cell viability
XIAP depleted cells are sensitized to TRAIL but not intrinsic pathway inducing agents
Sensitivity of XIAP depleted HCT-116, SW-620 and PC-3 tumor cells to various mechanistically distinct anti-cancer agents.
Here we report that transient, siRNA-mediated depletion of XIAP alone does not significantly decrease human tumor cell viability. We interpret the results to mean that XIAP does not have an essential role in growth and survival of tumor cell lines under normal, optimized growth conditions in vitro. This conclusion is consistent with a lack of effect on developmental apoptosis in mice harboring a germ line XIAP mutation and in transformed mouse embryo fibroblasts derived from these XIAP knockout mice . Similar results were obtained with human colorectal cancer cells in which the XIAP locus was deleted via homologous recombination . However, in these studies and others using transient or stable XIAP knockdown, loss of XIAP function sensitized the cells to TRAIL induced apoptosis. Our study is a more expansive survey, and supports the idea that XIAP has a critical role in negatively regulating death receptor mediated apoptosis across a wide array of tumor cell lines derived from diverse tissue types. Surprisingly, similar enhancement of apoptosis was not observed with multiple mechanistically distinct chemotherapeutics or the proteasome inhibitor bortezomib, or the HDAC inhibitor SAHA. All of these agents are thought to induce apoptosis predominantly (but perhaps not exclusively) through the mitochondrial pathway, involving cytochrome c and SMAC release and subsequent activation of caspase-9 by the apoptosome. Our data strongly suggest that XIAP has a more central role in inhibiting the extrinsic caspase-8 mediated death pathway than the intrinsic, caspase-9 dependent pathway. One potential explanation for the difference between extrinsic versus intrinsic death inducers is that the latter cause a release of SMAC, an endogenous inhibitor of XIAP. In wild-type cells, the caspase inhibitory activity of XIAP may be neutralized by SMAC following a robust intrinsic death pathway signal, essentially mimicking XIAP depletion. Therefore, no further increase in apoptotic response would be expected in XIAP siRNA treated cells. Support of this hypothesis comes from the elegant studies of the Prehn group, where loss of XIAP function in staurosporine treated HeLa cells did not accelerate substrate cleavage after detection of mitochondrial outer membrane permeabilization .
In contrast to our studies, Ras/E1A transformed MEFs derived from XIAP KO mice exhibited an increased sensitivity to the apoptosis inducing effects of etoposide compared to their wild-type counterparts . It is possible that Ras/E1A transformed MEFs are under different apoptotic pressures than the human cancer cells used in our study, resulting in XIAP having a more central role in suppressing intrinsic pathway mediated cell death. Testing the effects of other mechanistically distinct inducers of the intrinsic cell death pathway in Ras/E1A transformed MEFs should help clarify this and determine if the observed effects in MEFs are specific to etoposide.
Yang et al  reported that several cell lines, including a subset of those used in this study (BxPC-3, MCF-7, and SW-620) exhibited high basal levels of activated caspase-3 and -8 activity in the absence of other markers of apoptosis. It was argued that these cells were non-apoptotic via a compensatory increase in XIAP expression, which neutralized the caspase activity. Within the same study, over-expression of XIAP-associated factor 1 (XAF-1) in MCF-10A and MDA-MB-231 resulted in an increase in apoptosis. However, the biological activities of XAF-1 are complex and not yet fully elucidated, and thus it is difficult to ascertain whether this increase in cell death is solely mediated by XIAP. The more definitive XIAP knockdown experiments were not performed. If viable tumor cells such as BxPC3 and SW620 do in fact have activated caspases, our data suggests that these "death enzymes" are unlikely to be directly inhibited by XIAP, but rather by some other mechanism. Alternatively, in the context of XIAP knockdown the level of active caspases is still below a threshold necessary to induce cell death. Since 100% knockdown is never achieved with siRNA, the residual XIAP protein in the siRNA treated cells may be sufficient to inhibit the activated caspases present in these cells.
Several authors have reported that functional p53 is required for XIAP depletion to result in cell death. Tong and colleagues  found that the p53 positive MKN-45 gastric carcinoma cell line exhibited an elevated apoptotic rate following XIAP depletion, while the p53 mutant cell line MKN-28 was unaffected. Mohapatra and colleagues  reported that XIAP depletion did not result in increased apoptosis in p53 wild type LNCaP or p53 deficient PC-3 prostate cancer cells although over-expression of p53 in both cell lines resulted in apoptosis following XIAP depletion. Our studies included cell lines that harbor wild-type and mutant (loss-of-function) p53, however, there was no obvious correlation between response to XIAP knockdown and p53 status.
Recently, multiple reports indicated that tumor cell death induced by multiple, chemically distinct SMAC mimetics was in fact dependent on the proteasomal degradation of multiple members of the IAP family and subsequent induction of TNFα production and caspase-8 mediated death . Treated cells that did not have detectable levels of TNFα did not undergo apoptosis nor did TNFα-positive cells that were simultaneously treated with a TNFα blocking antibody. These results lend support to our conclusions from the knockdown experiments that under normal growth conditions in vitro, most tumor cells have not sufficiently engaged an apoptotic pathway such that their survival is dependent on XIAP. Some other death signal is needed (e.g. TNFα production or exogenous TRAIL), which, together with XIAP antagonism results in enhanced apoptosis. One outstanding question is whether the anti-tumor activity of the SMAC mimetics in vivo is also dependent on engagement of the TNFα pathway. It is possible that the associated stresses of in vivo tumor growth (e.g. hypoxia) generate a death signal (activated caspases) that is sufficient to render the tumor cells sensitive to inhibition of XIAP solely via the disruption of the caspase 9/XIAP interaction. In support of this notion, multiple reports have shown that stable shRNA or antisense knockdown of XIAP resulted in decreased tumor cell growth, as subcutaneous xenografts in vivo, but not as culture mono-layers, in vitro [14, 18, 32]. In vivo studies with inducible shRNAs that target XIAP in both nascent and established tumors may help resolve this issue, and should provide further insight for validation of XIAP as a cancer drug target.
Our work is consistent with others and predicts that agents that simply disrupt the caspase-3/9-XIAP interaction may hold limited therapeutic promise as monotherapy and that their utility will be likely found in the combination setting, in particular with therapies that engage the extrinsic death receptor pathway. Ultimate validation of XIAP as a cancer drug target will come from the clinical development of both the SMAC mimetics and the anti-sense based XIAP cancer therapies, both of which have recently entered Phase I clinical trials.
The authors would like to acknowledge the entire Apoptos Team for support and Dr. David Hockenbery (Seattle, WA) for the PATU-I cells.
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