Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis (original) (raw)
Identification of mitochondrial survivin. Because of its distribution among multiple subcellular compartments (16), we investigated whether survivin could also localize to mitochondria, an organelle with a central role in cell death pathways (4). A pool of survivin was found in purified cytosolic extracts of various tumor cell lines (Figure 1A), in agreement with previous observations (16). In addition, a pool of survivin was present in isolated mitochondrial fractions of tumor cells and colocalized with the reactivity of the mitochondrial proteins Smac and Cox-4, but not cytoplasmic Aurora A (Figure 1A). Next, we asked whether mitochondrial localization of survivin was a feature of transformed cells or was also observed in normal tissues. For these experiments, we prepared extracts from normal tissues known to express endogenous survivin and analyzed its distribution in cytosolic or mitochondrial fractions. Cytosolic extracts isolated from normal testis, liver, and spleen contained endogenous survivin, by immunoblotting (Figure 1B). In contrast, survivin was not detected in isolated mitochondrial fractions of the same tissues (Figure 1B). In immunoelectron microscopy experiments, analysis of MCF-7 cell pellets with a nonimmune IgG revealed negligible accumulation of gold particles (Figure 1C). In contrast, analysis of whole cell pellets (Figure 1D) or isolated mitochondrial fractions (Figure 1E) revealed staining for survivin in close proximity to the mitochondrial membrane(s), and colocalization with the reactivity of an antibody to Smac (Figure 1E). Quantification of 19 individual electron microscopy fields containing an average of 2.9 mitochondria each revealed that 94% of mitochondria examined exhibited dual localization of survivin and Smac, with comparable accumulation of gold particles per each marker (Table 1).
Mitochondrial localization of survivin. (A) Survivin localization in tumor cell lines. Cytosolic (left panel) or mitochondrial (right panel) extracts from the indicated tumor cell lines were analyzed by immunoblotting. MW, molecular weight. (B) Subcellular fractionation of normal tissues. Mitochondrial (M) or cytosolic (C) fractions extracted from normal testis, spleen, and liver or unfractionated HeLa cell extracts were analyzed by immunoblotting. Cox-4 was used as a mitochondrial marker. (C–E) Immunoelectron microscopy. Mitochondrial pellets (C and E) isolated from MCF-7 cells or whole MCF-7 cell extracts (D) were stained with nonimmune IgG (C) or an antibody to survivin (D) followed by colloidal gold-conjugated secondary IgG. Isolated mitochondrial pellets (E) were simultaneously stained with antibodies to survivin (12 nm diameter gold particles) and Smac (6 nm diameter gold particles). Magnification, ×53,000 (C and D), ×104,000 (E).
Quantification of survivin-Smac mitochondrial colocalization by electron microscopy
We next used a biochemical approach to map more precisely the submitochondrial topography of survivin. Treatment of mitochondrial pellets with proteinase K resulted in concentration-dependent loss of Bcl-2 (Figure 2A), which localizes to the outer mitochondrial membrane (3). In contrast, proteinase K treatment did not reduce survivin levels in mitochondria (Figure 2A). Conversely, proteinase K digested both Bcl-2 and survivin in isolated cytosolic extracts, confirming comparable susceptibility of both proteins to proteolysis (Figure 2A). We next permeabilized the outer mitochondrial membrane with digitonin (17) and looked for potential changes in survivin distribution. Digitonin treatment resulted in concentration-dependent relocalization of survivin from mitochondrial pellets into supernatants (Figure 2B). This was also associated with release of Smac (18) from mitochondrial pellets into supernatants, whereas the localization of nonextractable mitochondrial Hsp70 was unaffected by digitonin treatment (Figure 2B). Collectively, these data demonstrate that a novel pool of survivin localizes to mitochondria in tumor cells but not in normal tissues and is compartmentalized in the intermitochondrial membrane space.
Topography of mitochondrial survivin. (A) Sensitivity to proteinase K. Mitochondrial or cytosolic fractions were treated with the indicated concentrations of proteinase K and analyzed by immunoblotting. Lower panel: Quantification of Bcl-2 or survivin proteolysis by proteinase K treatment. (B) Permeabilization of mitochondrial membrane. Mitochondrial fractions were treated with the indicated increasing concentrations of digitonin, and pellets (P) or supernatants (S) were analyzed by immunoblotting. mt-Hsp70, mitochondrial Hsp70.
Stress-response modulation of mitochondrial survivin. We next asked whether cellular stress stimuli influenced the expression and/or localization of the mitochondrial pool of survivin. Exposure of HeLa cells to hypoxia (∼1% O2) caused a dramatic upregulation of the IAP family protein cIAP2 (Figure 3A), in agreement with previous observations (19). Hypoxia also increased survivin levels approximately threefold, by immunoblotting (Figure 3A). Analysis of isolated subcellular fractions demonstrated that hypoxia-induced upregulation of survivin occurred exclusively in the mitochondrial pool with minimal changes in cytosolic survivin levels (Figure 3B). To begin to determine the mechanism(s) of survivin modulation by hypoxia, we examined changes in protein levels in cycloheximide block experiments. Cycloheximide treatment resulted in time-dependent turnover of endogenous survivin levels in HeLa cells, by immunoblotting (Figure 3C). Conversely, hypoxic HeLa cells exhibited stabilization of survivin levels over a 6-hour time interval, as compared with normoxic cultures (Figure 3C). In control experiments, no significant decrease of cell viability was observed 6 hours after hypoxia (data not shown). Finally, treatment of MCF-7 cells with nonapoptotic concentrations of the DNA-damaging agent adriamycin also resulted in selective expansion of mitochondrial survivin, with no significant changes in cytosolic survivin levels (Figure 3D).
Modulation of mitochondrial survivin during the cellular stress response. (A) Regulation of survivin by hypoxia. HeLa cells were exposed to hypoxia, and analyzed by immunoblotting followed by densitometry. N, normoxic cultures; H, hypoxic cultures. (B) Subcellular fractionation. Cytosolic or mitochondrial fractions from HeLa cells were exposed to hypoxia, and analyzed by immunoblotting. (C) Cycloheximide block. Normoxic or hypoxic HeLa cells were treated with cycloheximide, and analyzed by immunoblotting at the indicated time intervals. Lower panel: β-Actin–normalized densitometric quantification of differential survivin stability in control versus hypoxic conditions. (D) Modulation by DNA damage. Untreated (None) or MCF-7 cells treated with nonapoptotic concentrations of adriamycin (Adriam) were fractionated in cytosolic and mitochondrial fractions, and analyzed by immunoblotting.
Loss of mitochondrial survivin influences cell death. To begin to probe the functional role of mitochondrial survivin, we studied a rat insulinoma cell line, INS-1, in which survivin was found almost exclusively in the cytosol, but not in purified mitochondrial fractions (Figure 4A). When transduced with a replication-deficient adenovirus (pAd) encoding WT survivin (pAd-Survivin), INS-1 cells exhibited increased survivin expression in the cytosol, but not in mitochondria (Figure 4A). Conversely, MCF-7 cells expressed endogenous survivin in cytosolic and mitochondrial fractions, and both pools were coordinately increased by transduction with pAd-Survivin (Figure 4A). Transduction of MCF-7 cells with pAd-Survivin inhibited staurosporine-induced cell death by 65–70% (Figure 4B). In contrast, pAd-Survivin did not reverse apoptosis in INS-1 cells (Figure 4B). In control experiments, both INS-1 and MCF-7 cells expressed comparable levels of transduced survivin (Figure 4B, inset).
Participation of mitochondrial survivin in cell death. (A) Defective mitochondrial import of survivin in INS-1 cells. INS-1 (upper panel) or MCF-7 (lower panel) cells were left untreated (None) or transduced with pAd-Survivin, and isolated mitochondrial or cytosolic fractions were analyzed by immunoblotting. (B) Cytoprotection. INS-1 or MCF-7 cells were transduced with pAd-GFP or pAd-Survivin, treated with staurosporine, and analyzed for hypodiploid DNA content. Data are the mean ± SEM of 3 independent experiments. Inset: Immunoblotting of survivin in transduced INS-1 or MCF-7 cells at the indicated time intervals. (C) Differential sensitivity to apoptosis. INS-1 or MCF-7 cells were treated with suboptimal concentrations of staurosporine (STS) or UVB and analyzed for nuclear morphology of apoptosis by DAPI staining. Data are the mean ± SD of 2 independent experiments. (D) RNAi knock-down. INS-1 or MCF-7 cells were transfected with dsRNA oligonucleotides to survivin (S4) or control (VIII), and analyzed by immunoblotting. (E) Cell cycle analysis. INS-1 or MCF-7 cells transfected with control or survivin dsRNA oligonucleotides were analyzed for cell cycle distribution by flow cytometry. The percentage of cells with hypodiploid (Sub-G1) DNA content is indicated.
To determine whether the lack of mitochondrial survivin resulted in increased sensitivity to apoptosis, we challenged INS-1 or MCF-7 cells with suboptimal doses of cell death stimuli. Concentrations of staurosporine (0.1 μM) or UVB (50 joules per square meter [J/m2]) that did not reduce the viability of MCF-7 cells resulted in extensive apoptosis of INS-1 cells (Figure 4C). In reciprocal experiments, we used RNA interference (RNAi) to acutely knock down survivin levels in INS-1 or MCF-7 cells and analyzed apoptosis and cell cycle progression (20). A survivin-derived double-stranded RNA (dsRNA) oligonucleotide (S4) ablated survivin levels in cytosolic and nuclear fractions of both MCF-7 and INS-1 cells, whereas a control dsRNA sequence (VIII) was without effect (Figure 4D and data not shown) (20). In control experiments, no phosphorylation of eIF2α was observed in RNAi-treated cells (Figure 4D). RNAi ablation of survivin in MCF-7 cells resulted in a dual phenotype of apoptosis and mitotic defects with polyploidy, by DNA content analysis and flow cytometry (Figure 4E), in agreement with previous observations (20). Although loss of survivin in INS-1 cells also caused mitotic defects predominantly seen as a G2/M block, this was not associated with significantly enhanced cell death, as compared with death of cells transfected with control dsRNA (VIII) (Figure 4E).
Mitochondrial survivin inhibits apoptosis. To further investigate a cytoprotective function of mitochondrial survivin, we engineered INS-1 cells to stably express WT survivin (Surv) or mitochondrially targeted survivin (MT-S), survivin targeted to mitochondria by the cytochrome c mitochondrial import sequence and containing GFP as a marker (INS-1/MT-S). By fluorescence microscopy, INS-1/MT-S cells exhibited perinuclear GFP expression consistent with mitochondrial localization (see below). Staurosporine treatment of parental INS-1 cells, or INS-1 cells stably transfected with mitochondrially targeted GFP (INS-1/MT-GFP), caused caspase-dependent cell death by multiparametric DEVDase activity (Figure 5A). Stable expression of Surv in INS-1 cells was unable to counteract staurosporine-induced apoptosis (Figure 5A). In contrast, mitochondrial targeting of survivin in INS-1/MT-S cells inhibited staurosporine-induced apoptosis and increased 5- to 16-fold the percentage of viable cells, by multiparametric flow cytometry (Figure 5A).
Mitochondrial targeting of survivin in INS-1 cells inhibits apoptosis. (A) Cytoprotection in stable transfectants. Parental INS-1 cells or INS-1 cells stably transfected with Surv, MT-GFP, or MT-S were treated with staurosporine and analyzed for caspase-3 and caspase-7 activity (DEVDase activity, green fluorescence) and plasma membrane integrity (propidium iodide, red fluorescence), by multiparametric flow cytometry. The percentage of cells in each quadrant is indicated. (B) Caspase-3 cleavage. INS-1 cells expressing MT-GFP or MT-S were treated with staurosporine and analyzed at the indicated time intervals by immunoblotting. The position of active caspase-3 bands of 17 and 19 kDa is shown. (C) Caspase-9 cleavage. The experimental conditions were as in B except that samples were analyzed for generation of 37-kDa active caspase-9 fragments by immunoblotting. (D) Inhibition of caspase-dependent apoptosis. Parental INS-1 cells or INS-1 cells stably expressing MT-GFP or MT-S were treated with the indicated concentrations of staurosporine in the presence or absence of zVAD-fmk, and scored for nuclear morphology of apoptosis by DAPI staining. Data are the mean ± SEM of 3 independent experiments.
We next tested the effect of mitochondrially targeted survivin on proteolytic processing of effector and initiator caspases. Exposure of INS-1/MT-GFP cells to staurosporine resulted in time-dependent proteolytic cleavage of proform caspase-3 and caspase-9 to generate active fragments of 17 kDa and 19 kDa (Figure 5B), and 37 kDa (Figure 5C), respectively. Conversely, expression of mitochondrial survivin in stably transfected INS-1 (INS-1/MT-S) cells suppressed the generation of active caspase-3 (Figure 5B) and caspase-9 (Figure 5C) at the same time intervals examined. To further distinguish between caspase-dependent and -independent cell death modulated by mitochondrial survivin, we exposed differentially transfected INS-1 cells to staurosporine in the presence or absence of a broad-spectrum caspase inhibitor, z-Val-Ala-Asp (OMe)-fmk (zVAD-fmk). INS-1/MT-S cells were protected against apoptosis over a wide range of staurosporine concentrations, in a reaction that was not further enhanced by zVAD-fmk (Figure 5D). Conversely, staurosporine-induced apoptosis in INS-1 or INS-1/MT-GFP cells was inhibited by 50–75% by zVAD-fmk (Figure 5D), suggesting that mitochondrial survivin selectively protected against caspase-dependent cell death.
We next generated pAds encoding either MT-GFP (pAd-MTGFP) or hemagglutinin-tagged (HA) MT-S (pAd-MTS). Transduction of INS-1 cells with pAd-MTS resulted in selective accumulation of survivin in mitochondrial, but not cytosolic, extracts (Figure 6A). This was associated with strong inhibition of apoptosis induced by UVB, serum deprivation, or staurosporine, by hypodiploid DNA content and flow cytometry (Figure 6B). In contrast, transduction of INS-1 cells with pAd-MTGFP did not reverse apoptosis induced by the various cell death stimuli (Figure 6B). We next asked whether selective expansion of mitochondrial survivin was sufficient to inhibit apoptosis. Transduction of MCF-7 cells with pAd-Survivin resulted in increased expansion of both cytosolic and mitochondrial survivin, and inhibition of staurosporine-induced apoptosis (Figure 6C). By comparison, pAd-MTS resulted solely in expansion of mitochondrial, not cytosolic, survivin levels and was equally as effective as pAd-Survivin at suppressing staurosporine-induced apoptosis (Figure 6C). In control experiments, transduction of MCF-7 cells with pAd-MTGFP did not reduce staurosporine-mediated cell death (Figure 6C).
Adenoviral targeting of survivin to mitochondria inhibits apoptosis. (A) Subcellular localization. INS-1 cells were infected with pAd-MTS, and isolated mitochondrial or cytosolic fractions were analyzed by immunoblotting. The position of endogenous or transduced (HA-MTS) survivin is indicated. (B) Cytoprotection by adenoviral transduction. INS-1 cells transduced with pAd-MTGFP or pAd-MTS were treated with UVB, exposed to serum starvation, or treated with staurosporine, and analyzed for DNA content by flow cytometry. Samples labeled as INS-1 were untreated cultures. The percentage of cells with hypodiploid (apoptotic) DNA content is indicated for each condition tested. (C) Mitochondrial survivin is sufficient for cytoprotection. MCF-7 cells were transduced with pAd-MTGFP, pAd-Survivin, or pAd-MTS, treated with staurosporine, and analyzed for hypodiploid DNA content and immunoblotting of whole cell extracts (W), mitochondrial, or cytosolic fractions. The percentages of cells with hypodiploid (apoptotic) DNA content were 47% (nontransduced cultures, data not shown), 40% (pAd-MTGFP), 25% (pAd-Survivin), and 29% (pAd-MTS).
Release of mitochondrial survivin during apoptosis antagonizes caspase-9 generation. To begin to identify a mechanistic role of mitochondrial survivin in cytoprotection, we first monitored changes in permeability transition, i.e., the release of mitochondrial apoptogenic proteins following cell death stimulation (5). Stable expression of mitochondrially targeted survivin in INS-1/MT-S cells did not reduce the cytoplasmic accumulation of cytochrome c in response to staurosporine, as compared with cultures expressing mitochondrially targeted GFP (Figure 7A). Similar results were obtained with analysis of Smac in differentially transfected INS-1 cells following induction of apoptosis (data not shown). In contrast, treatment of INS-1/MT-S with staurosporine resulted in redistribution of the GFP signal from a punctate, perinuclear pattern observed in resting cells to a diffuse cytoplasmic localization (Figure 7B), suggestive of release of mitochondrial survivin. Fluorescence analysis of 264 individual stably transfected INS-1/MT-S cells revealed that staurosporine treatment resulted in translocation of the GFP signal from mitochondria to the cytosol in 93.3% ± 1.1% of the cell population examined. To further quantify the dynamics of mitochondrial release of survivin during apoptosis, single-cell fluorescence-analysis experiments were carried out. Determination of fluorescence intensity in individual areas corresponding to mitochondrial (Figure 7C, yellow squares) or cytosolic (Figure 7C, gray circles) regions demonstrated that staurosporine caused loss of mitochondrial GFP signal (1,475 ± 30.3 versus 980 ± 19, P < 0.0001, n = 16), which was associated with a parallel increase in cytosolic fluorescence (628 ± 21 versus 887 ± 14.4, P < 0.0001, n = 14).
Mitochondrial release of survivin during apoptosis. (A) Effect on cytochrome c release. INS-1 cells stably transfected with MT-GFP or MT-S were treated with staurosporine, harvested at the indicated time intervals, and analyzed by immunoblotting. (B) Fluorescence microscopy. INS-1/MT-S cells were treated with staurosporine and analyzed for cytosolic redistribution of GFP. (C) Single-cell analysis. Areas corresponding to mitochondria (yellow squares) or cytosol (gray circles) were quantitatively analyzed for changes in fluorescence distribution before (None) or after staurosporine treatment (left panel). Right panel: Quantification of single-cell analysis. Changes in fluorescence intensity in individual mitochondrial or cytosolic areas were analyzed in the presence or absence of staurosporine treatment. Data represent an average of 16 individual determinations for mitochondrial areas and 14 individual determinations for cytosolic areas. (D) Time course of mitochondrial release of survivin during cell death. INS-1/MT-S cells were treated with staurosporine, and isolated mitochondrial or cytosolic fractions were analyzed at the indicated time intervals by immunoblotting. Right panel: Densitometric quantification of time-dependent depletion of mitochondrial survivin in response to staurosporine.
We next studied the time course of release of mitochondrial survivin during cell death in isolated cytosolic or mitochondrial fractions. In these experiments, staurosporine induced a rapid depletion of mitochondrial survivin, which was reduced by about 70% 4 hours after induction of apoptosis and was completely discharged by 12–18 hours (Figure 7D). This coincided with increased accumulation of survivin in isolated cytosolic extracts at the same time intervals (Figure 7D) and thus agrees with the fluorescence analysis reported above.
To determine the requirements of cytoprotection by mitochondrial survivin, we studied modulation of caspase-9 cleavage in transduced MCF-7 cells. Staurosporine treatment of MCF-7 cells transduced with pAd-MTGFP resulted in time-dependent cleavage of 46-kDa proform caspase-9 and generation of an active, 37-kDa caspase-9 fragment (Figure 8A). In contrast, transduction with pAd-MTS both delayed and attenuated the generation of active caspase-9 in staurosporine-treated MCF-7 cells (Figure 8A). Because survivin can associate with mitochondrial Smac (21), we asked whether cytoprotection by mitochondrial survivin involved binding and sequestration of Smac, which would be expected to relieve its inhibitory function on XIAP suppression of caspases (18). A specific interaction between survivin and Smac was demonstrated in vitro and in vivo (data not shown), in agreement with published data (21). However, transduction of MCF-7 cells with pAd-MTS did not affect the amount of Smac associated with XIAP during staurosporine-induced apoptosis, by coimmunoprecipitation and immunoblotting (Figure 8B). In control experiments, immune complexes precipitated by nonimmune IgG did not contain XIAP or Smac (Figure 8B).
Mechanisms of cytoprotection by mitochondrial survivin. (A) Inhibition of caspase-9 processing. MCF-7 cells transduced with pAd-MTGFP or pAd-MTS were treated with staurosporine and analyzed for caspase-9 processing at the indicated time intervals, by immunoblotting. The position of approximately 37-kDa active caspase-9 is indicated. Lower panel: Quantification of caspase-9 generation by densitometry. (B) XIAP-Smac interaction. Control cultures or MCF-7 cells transduced with pAd-MTS were treated with staurosporine and immunoprecipitated with an antibody to Smac or control IgG, and pellets and supernatants were analyzed by immunoblotting with antibodies to XIAP or Smac.
Role of mitochondrial survivin in tumorigenesis. To determine whether the distinct subcellular pools of survivin were differentially involved in cellular transformation, we studied the ability of differentially transfected INS-1 cells to form colonies in soft agar, i.e., anchorage-independent cell growth. INS-1 cells expressing MT-GFP exhibited a limited degree of colony formation in soft agar over a 2-week interval (Figure 9, A and B). Expression of MT-S in INS-1 cells resulted in an approximately threefold increase in colony formation, as compared with expression of MT-GFP in INS-1 cells (Figure 9, A and B). In contrast, expression of cytosolic survivin that cannot be transported to mitochondria (INS-1/Surv) nearly completely abolished colony formation in soft agar (Figure 9, A and B).
Mitochondrial survivin promotes tumorigenicity. (A) Colony formation in soft agar. Differentially transfected INS-1 cells were plated in semisolid medium and scored for colony formation by phase-contrast microscopy. (B) Quantification of colony formation in soft agar. The experimental conditions were as in A. Data are the mean ± SD of a representative experiment of at least 2 independent determinations. ***P = 0.0001. (C) Kinetics of tumor growth. Stably transfected INS-1 cells were injected subcutaneously in the flank of CB17 SCID/beige mice, and tumor volume was determined at the indicated time intervals. Statistical analysis compared growth of INS-1/Surv versus INS-1/MT-S tumors at day 28 (P = 0.024), day 35 (P = 0.0069), and day 45 (P = 0.015). *P < 0.05; **P < 0.01. (D) Histology. Tissue sections from the indicated INS-1 tumors were stained by H&E, Ki67 reactivity as a measure of cell proliferation, and TUNEL as a measure of apoptosis, in vivo. Magnification, ×400. (E) Mitotic index in vivo. The number of Ki67-positive (proliferating) cells was counted in 7 independent fields, each containing an average of 400 cells. #P = 0.031. (F) Apoptotic index in vivo. The number of TUNEL-positive (apoptotic) cells was counted in 7 independent fields, each containing an average of 400 cells.
We next injected differentially transfected INS-1 cells in immunocompromised mice and studied the kinetics of tumor formation. Tumors formed by INS-1/MT-GFP cells exhibited delayed onset and a flattened growth rate that did not significantly change during the last 3 weeks of examination (Figure 9C). Conversely, INS-1 cells expressing mitochondrially targeted survivin (INS-1/MT-S) gave rise to solid tumors characterized by early onset and steady exponential growth rate (Figure 9C). Stable expression of non-mitochondrially-targeted cytosolic survivin in INS-1 cells was associated with strong inhibition of tumor growth in immunocompromised animals (Figure 9C); this was similar to the findings regarding formation of colonies in soft agar (Figure 9, A and B). Histologically, tumors formed by INS-1 cells had neuroendocrine appearance and exhibited an extensive network of vascular sinusoids filled with blood (Figure 9D). Tumor cell proliferation as detected by Ki67 labeling (Figure 9D) was indistinguishable in tumors formed by INS-1 cells expressing mitochondrially targeted GFP or non-mitochondrially-targeted, cytosolic survivin (INS-1/Surv) (Figure 9E). Conversely, INS-1/MT-S tumors revealed a higher mitotic index (P = 0.031) as compared with INS-1/Surv tumors (Figure 9E). Quantification of internucleosomal DNA fragmentation by TUNEL staining (Figure 9D) revealed that expression of mitochondrially targeted survivin in INS-1/MT-S tumors resulted in nearly complete suppression of apoptosis in vivo (P = 0.0001), as compared with that in INS-1/Surv or INS-1/MT-GFP tumors (Figure 9F). When compared with INS-1/MT-GFP tumors, tumors expressing non-mitochondrially-targeted, INS-1/Surv resulted in a considerably increased (P = 0.0003) apoptotic index (Figure 9F), which may explain the reduction in colony formation (Figure 9, A and B) and the slower growth rate of these tumors in vivo (Figure 9C).