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A study into the potential role of Survivin localization in resistance to drug-induced apoptosis

A study into the potential role of Survivin localization in resistance to drug-induced apoptosis Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn012 ......................................................................................................................................................................................................................................... Research article A study into the potential role of Survivin localization in resistance to drug-induced apoptosis Helen Angell* Faculty of Life Sciences, University of Manchester, Manchester, UK. * Corresponding author: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel: þ44 0161 275 5626. Email: cstreuli@ manchester.ac.uk Supervisor: Dr Fiona M. Foster and Prof. Charles H. Streuli*, University of Manchester, Manchester, UK. ........................................................................................................................................................................................................................................ The aim of this study was to test the hypothesis that diverting the cytoplasmic subcellular localization of the anti-apoptotic form of Survivin to the nucleus would sensitize cancer cells to chemotherapeutics. Apoptosis is a morphologically and biochemically distinctive form of cell death, critical in the maintenance of tissue homeostasis. Caspases are a family of cysteine proteases that have a vital role in the implementation of apoptosis, and their activity is regulated by Inhibitors of Apoptosis Proteins. Recent studies indicate that one such inhibitor, Survivin, may have dual functions that are specific to its cellular location, including suppression of apoptotis (cytosolic) and regulation of cell division (nuclear). Since both apoptosis and proliferation are altered in cancer, identifying whether these roles for Survivin are dependent on its subcellular localization will inform future approaches to treat chemotherapeutically resistant tumour cells. After initially confirming the specificity of several Survivin antibodies, the distribution of Survivin was examined by immunofluor- escence microscopy and sub-cellular fractionation in breast cancer cell lines. The threshold of drug-induced apoptosis was compared in cells over-expressing either wildtype Survivin or a form of Survivin unable to exit the nucleus due to a mutation in its nuclear export sequence. Endogenous Survivin localized to both nucleus and cytoplasm of breast cancer cell lines. Over-expressed Survivin had an anti-apoptotic, protective function. In contrast, cells expressing Survivin with the mutated nuclear export sequence had a lower apoptotic threshold to chemotherapeutic drugs. These results demonstrate for the first time that Survivin is localized to both the nucleus and cytoplasm of breast cancer cell lines. Importantly, the sensitivity of cells to chemotherapeutic drugs was increased when Survivin’s local- ization was restricted to the nucleus, consistent with cytoplasmic Survivin having the anti-apoptotic role. Since clinical studies have shown that nuclear Survivin is a positive prognostic factor in breast cancer patients, the data suggest that strategies to alter Survivin distribution may be useful in the fight against cancer. Key words: apoptosis, Survivin, subcellular localization, breast cancer. ........................................................................................................................................................................................................................................ 1, 3 and intrinsic pathways. Crucial regulation of apoptosis is Introduction mediated by the inhibitor of apoptosis protein (IAP) family. Apoptosis is the complex process of regulated cell death, IAPs function as intrinsic regulators of the caspase cascade, 5, 6 essential for maintenance of tissue homeostasis and embryo- where they inhibit active caspases. IAPs are distinguished nic development. Numerous signalling pathways are involved by containing one or more baculoviral IAP repeat (BIR) 7–11 in triggering apoptosis and determining the fate of a cell. domains, essential for inhibitory function. Mis-regulated Apoptosis is orchestrated by the caspases, a family of cysteine- apoptosis and promotion of cell survival can lead to cancer. 1, 2 dependent death proteases, activated in apoptotic cells. IAPs are therefore becoming increasingly targeted within The caspase family can be subdivided into initiator caspases oncology research. and effector caspases. Initiator caspases mediate cellular sig- One member of the IAP family, Survivin, binds and inhi- nalling to activate downstream effector caspases that then bits caspase-9 thereby contributing to its anti-apoptotic func- 7, 13 execute apoptosis via a ‘caspase cascade’ within the extrinsic tion. In addition, Survivin can inhibit Smac/DIABLO ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 85 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... function, while conflicting literature debates whether it can supplemented with 10% v/v Foetal Bovine Serum 15 – 17 bind to other caspases. Survivin also binds and stabil- (Invitrogen), 1% v/v Penicillin/Streptomycin, 1% v/v gluta- izes other IAPs, e.g. XIAP, promoting their anti-apoptotic mine. MCF10A cells were cultured in DMEM:Hams F-12 effect. 1:1 mix supplemented with 5% v/v horse serum In addition to its anti-apoptotic function, Survivin has a (Invitrogen), 10 mg/ml insulin, 500 ng/ml hydrocortisone, second role within the cell cycle, as a regulator of cell prolifer- and 20 ng/ml EGF, 1% v/v Penicillin/Streptomycin, 1% ation. Current evidence suggests that Survivin is a chromo- v/v glutamine. somal passenger protein, involved in orchestrating the 19 – 22 Sub-cellular fractionation chromosomal passenger complex (CPC). Since Survivin is found in both the cytoplasm and the nucleus of cells, Cells were gently scraped in 1 ml of hypotonic buffer; there has been controversy as to whether Survivin’s functions 10 mM Tris – Cl pH 7.5, 10 mM NaCl, 1.5 mM MgCl , can be separated according to localization, and if this can then 1:100 PIC (Protease Inhibitor Cocktail, Calbiochemw), and 23 – 25 be used as a prognostic indicator for cancer patients. The transferred to ice for 5 min in Dounce homogenizer. Cells prevailing opinion is that cytosolic localization appears to were homogenized and NaCl was then added back to enable the IAP function of Survivin, whereas nuclear localiz- regain the correct physiological concentration (i.e. ation facilitates cell division. Survivin is transported from 150 mM). Samples were centrifuged at 100 000g for the nucleus to the cytosol by nuclear export receptor, Crm1, 30 min at 48C (TLA 110 rotor, 55 000 rpm). The super- which is regulated by a leucine-rich nuclear export signal natant was collected, forming the cytosolic fraction. The 26, 27 (NES). Influencing Survivin’s nuclear export as a strategy remaining pellet was re-suspended in 150 ml of CHAPS to antagonize its proliferative activity has been demonstrated buffer (1% CHAPS, 0.75 M aminocarproic acid, 1:100 using Leptomycin B, a Crm1-specific inhibitor. In addition, PIC) and stored on ice for 5 min, then spun at 100 000g mutations in the Survivin NES region result in the accumu- for 30 min (48C) and the supernatant collected, forming lation of nuclear Survivin, unable to exit the nucleus. For the CHAPS soluble (enriched membrane fraction). The example, Survivin-L98A causes a lower apoptotic threshold pellet was then finally re-suspended, forming the CHAPS 27, 28 to X-irradiation than wild type Survinin. Apredominant insoluble (enriched nuclear fraction), in 2% SDS in 5 mM nuclear localization could also influence a cell’s susceptibility Tris – Cl pH 6.8 and boiled for 10 min until fully to apoptosis by increased transcription of pro-apoptotic genes. re-suspended. The protein concentration for each sample Survivin is highly expressed in most cancers, where it may of cell fraction was calculated using the BCA assay (Pierce). have an anti-apoptotic function. We suggest that tumour cell Immunoblotting resistance to chemotherapeutic agents may be mediated by increased nuclear export of Survivin. Survivin localization Twenty micrograms of protein was run on a 15% resolving is of considerable clinical relevance, as nuclear localization gel, transferred onto nitrocellulose membrane (Whatman) is a favourable prognostic factor in breast cancer patients before being treated with block buffer (5% v/v BSA, 0.1% while a cytoplasmic localization is unfavourable. v/v NaN , 0.1% v/v Tween 20). Primary antibodies for The current study aimed to investigate further the sub- protein detection were: 1/1000 rabbit anti-Apaf-1 (Stressgen cellular localization of Survivin, how this localization links Bioreagents); 1/1000 rabbit anti-Histone H3 and 1/1000 with Survivin function, and whether Survivin localization mouse anti-Survivin (Cell Signalling Technology); 1/1000 might affect apoptotic susceptibility in response to chemo- mouse anti-HSP 70 (Affinity Bioreagents); 1/150 rabbit therapeutic agents. It is hypothesized that cytoplasmic anti-X-IAP (Cell Signalling Technology). IR-dye-conjugated Survivin contributes to IAP function while the nuclear secondary antibodies (1/10 000 diluted in 50% Odyssey form results in a non-protective role for apoptosis. Block Buffer, 50% 1 PBS) were used for detection. TM Sub-cellular fractionation will determine Survivin localiz- Membranes were read on an Odyssey LI-COR , and band ation in breast cancer cell lines. Survivin over-expression staining intensities were obtained from the gel using studies will investigate if increased cytoplasmic Survivin Odyssey V1.1 software. results in a protective anti-apoptotic function, and whether Over-expression of Survivin constructs Survivin restricted to the nucleus, using Survivin containing a mutation within the NES (Survivin-L98A-GFP), has a 293T cells were plated at 0.810 cells per well of a 12-well lower threshold to apoptotic stimuli. plate, and the following day transfection was carried out with QIAGEN QIAprepw-purified pcDNA3.1 vectors Materials and methods (Invitrogen) encoding Survivin-GFP, Survivin-L98A-GFP or TM GFP alone, using Lipofectamine (4 h). Forty-eight hours Tissue culture following transfection, microscopic examination on a Zeiss MDA-MB-231, MDA-MB-468, BT20, and 293T cells were Axiovert 40CFL showed a 30% transfection efficiency. cultured in Dulbecco’s Modified Eagles Medium (DMEM) A sample of cells were lysed (in sample buffer containing ......................................................................................................................................................................................................................................... 86 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 5% v/vNa VO ,1%v/v NaF, 1PIC) and separated on a higher level in the three breast cancer cell lines than in 3 4 15% gel to confirm transfection levels. Localization of MCF10A cells. While XIAP was largely cytoplasmic, the Survivin constructs was also confirmed by confocal distribution of Survivin was more diverse, being present in microscopy. Transfected cells were stained with the nuclear both cytosolic and nuclear fractions. These results indicate marker, DAPI, and visualized on a Leica SP2 inverted micro- first that IAPs may be upregulated in cancer cells, and scope. Single sections through the mid-point of the cell are second that Survivin, but not XIAP, is not only present presented. within the cytoplasm but is also nuclear. Survivin protects against Etoposide-induced apoptosis Drug treatment in 293T cells Twenty-four hours post-transfection, cells were drug treated In order to determine whether the sub-cellular localization of overnight with 100 mM Etoposide (Calbiochem), 1 mM Survivin influences the sensitivity of cells to chemotherapeu- Melphalan (Sigma, St Louis) or 1 mM Staurosporine tics, the ability of a mutant form of Survivin, unable to exit (Calbiochem). Etoposide and Melphalan are both DNA the nucleus, was compared with the ability of wildtype damaging agents, currently in clinical use. Staurosporine is Survivin to protect cells from drugs known to induce apop- a kinase inhibitor, which acts to induce cell stress and apop- tosis, and which are currently in clinical use. tosis. Drug concentrations were chosen to induce maximal Initially 293T cells were transfected with plasmids expres- apoptosis in the cell lines used, over 24 h, without resulting sing a GFP control, Survivin-GFP and Survivin-L98A-GFP. in complete cellular loss, based on previous concentration- To confirm equivalent levels of transfection, cell lysates dependence studies performed in the lab (unpublished) and were extracted and probed for the presence of Survivin and elsewhere. Cells were detached and cytospun onto slides, GFP (Fig. 2A). In the Survivin-GFP (both WT and mutant) fixed in 4% formaldehyde (10 min) and permeabilized in expressing cells, the Survivin (red) and GFP bands (green) 0.2% Triton-X100 (5 min). Cells were treated with 10% overlap to give a yellow merged image. Confocal microscopy calf serum in PBS before staining with 1/500 mouse confirmed that wildtype Survivin-GFP was present within the anti-Ki67 primary antibody (BD Transduction cytosol, whereas Survivin-L98A-GFP was limited to the Laboratories), Alexa 594 goat anti-mouse secondary anti- nucleus (Fig. 2B). Previous studies using tagged survivin body (1:500) and DAPI. Slides were visualized on an have shown that it behaves exactly the same as over- Axioplan2 Imaging microscope and images analysed using 28, 37, 38 expressed survivin constructs. OpenLab software. GFP, Survivin-GFP and Survivin-L98A-GFP transfected cells were then treated with Etoposide, Melphalan or Statistical analysis Staurosporine for 24 h, and the GFP-expressing cells were A Student’s two-tailed t-test was used. examined for either apoptosis or proliferation. Nuclear DAPI stain was used to identify the distinct morphological Results features associated with apoptotic cells including nuclear fragmentation and the presence of apoptotic bodies. Cells Endogenous Survivin localization in breast cancer undergoing proliferation were analysed by Ki67 staining. cell lines The percentage of apoptotic or proliferating cells was calcu- In order to investigate the sub-cellular localization of lated and expressed as a percentage of the total number of endogenous Survivin in breast cancer cells, a panel of three transfected cells (Fig. 2C and D). cancer lines (MDA-MB-231; MDA-MB-468; BT20) was Transfection with the GFP-construct did not result in an compared with a normal line (MCF10A). Cells were sepa- increase in basal apoptosis rates, and transfection with rated into enriched fractions of cytoplasm (soluble), mem- Survivin-GFP and Survivin-L98A-GFP caused 1.5% and branes (CHAPS soluble) and nuclei (CHAPS insoluble) 6.5%, respectively, of the cells to apoptose spontaneously. (Fig. 1). To confirm accurate fractionation of samples, However, in each case, the percentage of cells undergoing immunoblots were probed with antibodies to (i) anti- apoptosis increased significantly (P, 0.05) in the drug- Apaf-1 antibody, a cytoplasmic marker which migrates at treated cells in comparison to the untreated controls 33 – 35 130 kDa; (ii) Heat Shock Protein 70 (HSP70), a (Fig. 2C, white bars). Moreover, and importantly, the cells 70 kDa protein that localizes to the mitochondria and was expressing Survivin-GFP showed a significant protection used as a membrane and mitochondrial marker and against Etoposide-induced apoptosis compared with the (iii) anti-Histone H3 antibody, a 17 kDa nuclear marker. GFP control cells. This resistance to apoptosis was not In addition, anti-Survivin and anti-XIAP antibodies were afforded by over-expression of the nuclear-restricted used to probe for IAP levels and location (Fig. 1). Notably, Survivin-L98A-GFP. Interestingly, neither Survivin nor both Survivin, which migrates at 16.5 kDa, and XIAP, Survivin-L98A conferred significant resistance to cells migrating at 50 kDa, were expressed at considerably treated with Melphalan or Staurosporine. ......................................................................................................................................................................................................................................... 87 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. Sub-cellular location of Survivin and XIAP in breast cancer cell lines. Twenty-microgram fractions of MDA-MB-231, MDA-MB-468, BT20 and MCF10A were separated by SDS–PAGE and examined by immunoblotting with antibodies to Apaf1, a cytoplasmic control; HSP70, a membrane marker; Histone H3, a nuclear marker, as well as the IAPs, Survivin and XIAP. Data presented are representative of three different experiments. In contrast to the apoptosis results, all the drugs inhibited In each of the cell lines tested, XIAP localized to the cyto- proliferation, though there was no significant additional plasm, correlating with the central function of XIAP, which 10, 42, 43 effect with either WT or L98A Survivin (Fig. 2D). is to buffer the caspase cascade. In contrast, Survivin Interestingly, there was a significant inhibition of basal pro- localized to both the nucleus and the cytoplasm in each of liferation (i.e. no drug treatment) in the cells transfected the cancer lines. These data correlate with a study of tissue with either Survivin-GFP or Survivin-L98A-GFP. from breast cancer patients including atypical hyperplasias Together these experiments demonstrate a protective role and malignant lymph tissue, where Survivin was most fre- for certain forms of Survivin on Etoposide-induced apoptosis quently present either only in the cytoplasm (31.3%) or in in breast cancer cells. the cytoplasm and nucleus (22.5%), but was restricted to the nucleus in 11.3% of cases. A separate statistical analy- sis showed that cytoplasmic Survivin expression correlates Discussion with tumour stage and histological grade, and with a lower recurrence-free patient survival rate. The main discovery arising from this study is that over- expressed Survivin can protect breast cancer cells against apoptosis induced by some chemotherapeutic drugs, but Over-expression of exogenous Survivin only if Survivin is present within the cytosol. Since The decrease in the percentage of cells undergoing Survivin was found to be upregulated in three independent Etoposide-induced apoptosis in cells over-expressing cancer lines, we speculate that it may have an apoptosis- Survivin-GFP is consistent with the protective role of suppressing function that contributes to disease progression. Survivin within the apoptotic pathway. This supports pre- vious literature showing a protective IAP function of over- Endogenous Survivin localization by sub-cellular expressed Survivin in cells treated with various apoptotic 46, 47 fractionation stimuli. However, we found that over-expressed Both XIAP and Survivin are up-regulated in the three cancer Survivin did not prevent Melphalan or STS-induced apopto- cell lines, MDA-MB-231, MDA-MB-468 and BT20, com- sis. The differential response to Etoposide and the other two pared with the normal breast cell line, MCF10a. This is con- agents might reflect differences in the caspase activation. For sistent with the general observation that IAPs are example, in HeLa cells, Etoposide induced greater activation up-regulated in cancer and are part of the mechanism of of caspase 9 than STS, while STS induced greater activation 41 32 apoptosis resistance displayed by cancer cells. of caspases 8 and 3. Survivin has previously been shown to ......................................................................................................................................................................................................................................... 88 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Survivin protects against Etoposide-induced apoptosis. (A) 293T cells were transfected with Survivin-GFP, Survivin-L98A-GFP and GFP alone. Whole cell lysates were separated on a 15% gel, with (a) Rainbow marker, (b) un-transfected control, (c) GFP control, (d) transfected Survivin-GFP and (e) transfected Survivin-L98A-GFP. The gel was probed with anti-Survivin (chemifluorescence at 700 nm) and anti-GFP antibody (chemifluorescence at 800 nm). Data are representative of n ¼ 2. (B) 293T cells transfected with Survivin-GFP (top panel), Survivin-L98A-GFP (middle panel) or GFP (bottom panel) were stained with DAPI (left column), and examined for Survivin localization (middle column and merged picture in right column) by confocal microscopy. (C and D) 293T cells were transfected with GFP alone, Survivin-GFP (SUR-GFP) or Survivin-L98A-GFP (SUR-NES-GFP). Cells were treated for 24 h with drugs as indicated, detached and cytospun onto slides, then fixed, permeabilized and stained with antibodies to Ki67. DAPI was used as a nuclear marker and transfected cells located by their GFP-tag. (C) Cells with condensed, apoptotic nuclei were scored in the GFP-positive population of the transfected cells. (D) Similarly, GFP-positive cells were scored for Ki67 staining. Results are expressed as percentages, taken from an average of 60 samples from three independent experiments. Error bars depict standard error of the mean. Asterisks represent significant difference (P, 0.05) com- pared with GFP-only transfected cells for each drug treatment. ......................................................................................................................................................................................................................................... 89 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 6. Deveraux QL, Roy N, Stennicke HR et al. (1998) IAPs block apoptotic events inhibit caspase 9, whereas its effects on caspase 3 are indirect induced by caspase-8 and cytochrome c by direct inhibition of distinct cas- and mediated through interactions with other IAPs. It is pases. EMBO J 17: 2215–2223. therefore possible that at least in breast epithelia, an over- 7. Ambrosini G, Adida C, Altieri DC (1997) A novel anti-apoptosis gene, survivin, expressed IAP only suppresses specific drug-induced expressed in cancer and lymphoma. Nat Med 3: 917–921. caspase pathways. 8. LaCasse EC, Baird S, Korneluk RG et al. (1998) The inhibitors of apoptosis In comparison to the results with Survivin-GFP, no protec- (IAPs) and their emerging role in cancer. Oncogene 17: 3247–3259. tive effect to therapeutic agents was seen in samples trans- 9. Borden KL, Freemont PS (1996) The RING finger domain: a recent example of fected with Survivin-L98A-GFP, where the over-expressed a sequence-structure family. Curr Opin Struct Biol 6: 395–401. protein is restricted to the nucleus. This is consistent with 10. Rothe M, Pan MG, Henzel WJ et al. 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A study into the potential role of Survivin localization in resistance to drug-induced apoptosis

Angell, Helen
Bioscience Horizons , Volume 1 (2) – Jun 22, 2008
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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn012 ......................................................................................................................................................................................................................................... Research article A study into the potential role of Survivin localization in resistance to drug-induced apoptosis Helen Angell* Faculty of Life Sciences, University of Manchester, Manchester, UK. * Corresponding author: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Tel: þ44 0161 275 5626. Email: cstreuli@ manchester.ac.uk Supervisor: Dr Fiona M. Foster and Prof. Charles H. Streuli*, University of Manchester, Manchester, UK. ........................................................................................................................................................................................................................................ The aim of this study was to test the hypothesis that diverting the cytoplasmic subcellular localization of the anti-apoptotic form of Survivin to the nucleus would sensitize cancer cells to chemotherapeutics. Apoptosis is a morphologically and biochemically distinctive form of cell death, critical in the maintenance of tissue homeostasis. Caspases are a family of cysteine proteases that have a vital role in the implementation of apoptosis, and their activity is regulated by Inhibitors of Apoptosis Proteins. Recent studies indicate that one such inhibitor, Survivin, may have dual functions that are specific to its cellular location, including suppression of apoptotis (cytosolic) and regulation of cell division (nuclear). Since both apoptosis and proliferation are altered in cancer, identifying whether these roles for Survivin are dependent on its subcellular localization will inform future approaches to treat chemotherapeutically resistant tumour cells. After initially confirming the specificity of several Survivin antibodies, the distribution of Survivin was examined by immunofluor- escence microscopy and sub-cellular fractionation in breast cancer cell lines. The threshold of drug-induced apoptosis was compared in cells over-expressing either wildtype Survivin or a form of Survivin unable to exit the nucleus due to a mutation in its nuclear export sequence. Endogenous Survivin localized to both nucleus and cytoplasm of breast cancer cell lines. Over-expressed Survivin had an anti-apoptotic, protective function. In contrast, cells expressing Survivin with the mutated nuclear export sequence had a lower apoptotic threshold to chemotherapeutic drugs. These results demonstrate for the first time that Survivin is localized to both the nucleus and cytoplasm of breast cancer cell lines. Importantly, the sensitivity of cells to chemotherapeutic drugs was increased when Survivin’s local- ization was restricted to the nucleus, consistent with cytoplasmic Survivin having the anti-apoptotic role. Since clinical studies have shown that nuclear Survivin is a positive prognostic factor in breast cancer patients, the data suggest that strategies to alter Survivin distribution may be useful in the fight against cancer. Key words: apoptosis, Survivin, subcellular localization, breast cancer. ........................................................................................................................................................................................................................................ 1, 3 and intrinsic pathways. Crucial regulation of apoptosis is Introduction mediated by the inhibitor of apoptosis protein (IAP) family. Apoptosis is the complex process of regulated cell death, IAPs function as intrinsic regulators of the caspase cascade, 5, 6 essential for maintenance of tissue homeostasis and embryo- where they inhibit active caspases. IAPs are distinguished nic development. Numerous signalling pathways are involved by containing one or more baculoviral IAP repeat (BIR) 7–11 in triggering apoptosis and determining the fate of a cell. domains, essential for inhibitory function. Mis-regulated Apoptosis is orchestrated by the caspases, a family of cysteine- apoptosis and promotion of cell survival can lead to cancer. 1, 2 dependent death proteases, activated in apoptotic cells. IAPs are therefore becoming increasingly targeted within The caspase family can be subdivided into initiator caspases oncology research. and effector caspases. Initiator caspases mediate cellular sig- One member of the IAP family, Survivin, binds and inhi- nalling to activate downstream effector caspases that then bits caspase-9 thereby contributing to its anti-apoptotic func- 7, 13 execute apoptosis via a ‘caspase cascade’ within the extrinsic tion. In addition, Survivin can inhibit Smac/DIABLO ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 85 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... function, while conflicting literature debates whether it can supplemented with 10% v/v Foetal Bovine Serum 15 – 17 bind to other caspases. Survivin also binds and stabil- (Invitrogen), 1% v/v Penicillin/Streptomycin, 1% v/v gluta- izes other IAPs, e.g. XIAP, promoting their anti-apoptotic mine. MCF10A cells were cultured in DMEM:Hams F-12 effect. 1:1 mix supplemented with 5% v/v horse serum In addition to its anti-apoptotic function, Survivin has a (Invitrogen), 10 mg/ml insulin, 500 ng/ml hydrocortisone, second role within the cell cycle, as a regulator of cell prolifer- and 20 ng/ml EGF, 1% v/v Penicillin/Streptomycin, 1% ation. Current evidence suggests that Survivin is a chromo- v/v glutamine. somal passenger protein, involved in orchestrating the 19 – 22 Sub-cellular fractionation chromosomal passenger complex (CPC). Since Survivin is found in both the cytoplasm and the nucleus of cells, Cells were gently scraped in 1 ml of hypotonic buffer; there has been controversy as to whether Survivin’s functions 10 mM Tris – Cl pH 7.5, 10 mM NaCl, 1.5 mM MgCl , can be separated according to localization, and if this can then 1:100 PIC (Protease Inhibitor Cocktail, Calbiochemw), and 23 – 25 be used as a prognostic indicator for cancer patients. The transferred to ice for 5 min in Dounce homogenizer. Cells prevailing opinion is that cytosolic localization appears to were homogenized and NaCl was then added back to enable the IAP function of Survivin, whereas nuclear localiz- regain the correct physiological concentration (i.e. ation facilitates cell division. Survivin is transported from 150 mM). Samples were centrifuged at 100 000g for the nucleus to the cytosol by nuclear export receptor, Crm1, 30 min at 48C (TLA 110 rotor, 55 000 rpm). The super- which is regulated by a leucine-rich nuclear export signal natant was collected, forming the cytosolic fraction. The 26, 27 (NES). Influencing Survivin’s nuclear export as a strategy remaining pellet was re-suspended in 150 ml of CHAPS to antagonize its proliferative activity has been demonstrated buffer (1% CHAPS, 0.75 M aminocarproic acid, 1:100 using Leptomycin B, a Crm1-specific inhibitor. In addition, PIC) and stored on ice for 5 min, then spun at 100 000g mutations in the Survivin NES region result in the accumu- for 30 min (48C) and the supernatant collected, forming lation of nuclear Survivin, unable to exit the nucleus. For the CHAPS soluble (enriched membrane fraction). The example, Survivin-L98A causes a lower apoptotic threshold pellet was then finally re-suspended, forming the CHAPS 27, 28 to X-irradiation than wild type Survinin. Apredominant insoluble (enriched nuclear fraction), in 2% SDS in 5 mM nuclear localization could also influence a cell’s susceptibility Tris – Cl pH 6.8 and boiled for 10 min until fully to apoptosis by increased transcription of pro-apoptotic genes. re-suspended. The protein concentration for each sample Survivin is highly expressed in most cancers, where it may of cell fraction was calculated using the BCA assay (Pierce). have an anti-apoptotic function. We suggest that tumour cell Immunoblotting resistance to chemotherapeutic agents may be mediated by increased nuclear export of Survivin. Survivin localization Twenty micrograms of protein was run on a 15% resolving is of considerable clinical relevance, as nuclear localization gel, transferred onto nitrocellulose membrane (Whatman) is a favourable prognostic factor in breast cancer patients before being treated with block buffer (5% v/v BSA, 0.1% while a cytoplasmic localization is unfavourable. v/v NaN , 0.1% v/v Tween 20). Primary antibodies for The current study aimed to investigate further the sub- protein detection were: 1/1000 rabbit anti-Apaf-1 (Stressgen cellular localization of Survivin, how this localization links Bioreagents); 1/1000 rabbit anti-Histone H3 and 1/1000 with Survivin function, and whether Survivin localization mouse anti-Survivin (Cell Signalling Technology); 1/1000 might affect apoptotic susceptibility in response to chemo- mouse anti-HSP 70 (Affinity Bioreagents); 1/150 rabbit therapeutic agents. It is hypothesized that cytoplasmic anti-X-IAP (Cell Signalling Technology). IR-dye-conjugated Survivin contributes to IAP function while the nuclear secondary antibodies (1/10 000 diluted in 50% Odyssey form results in a non-protective role for apoptosis. Block Buffer, 50% 1 PBS) were used for detection. TM Sub-cellular fractionation will determine Survivin localiz- Membranes were read on an Odyssey LI-COR , and band ation in breast cancer cell lines. Survivin over-expression staining intensities were obtained from the gel using studies will investigate if increased cytoplasmic Survivin Odyssey V1.1 software. results in a protective anti-apoptotic function, and whether Over-expression of Survivin constructs Survivin restricted to the nucleus, using Survivin containing a mutation within the NES (Survivin-L98A-GFP), has a 293T cells were plated at 0.810 cells per well of a 12-well lower threshold to apoptotic stimuli. plate, and the following day transfection was carried out with QIAGEN QIAprepw-purified pcDNA3.1 vectors Materials and methods (Invitrogen) encoding Survivin-GFP, Survivin-L98A-GFP or TM GFP alone, using Lipofectamine (4 h). Forty-eight hours Tissue culture following transfection, microscopic examination on a Zeiss MDA-MB-231, MDA-MB-468, BT20, and 293T cells were Axiovert 40CFL showed a 30% transfection efficiency. cultured in Dulbecco’s Modified Eagles Medium (DMEM) A sample of cells were lysed (in sample buffer containing ......................................................................................................................................................................................................................................... 86 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... 5% v/vNa VO ,1%v/v NaF, 1PIC) and separated on a higher level in the three breast cancer cell lines than in 3 4 15% gel to confirm transfection levels. Localization of MCF10A cells. While XIAP was largely cytoplasmic, the Survivin constructs was also confirmed by confocal distribution of Survivin was more diverse, being present in microscopy. Transfected cells were stained with the nuclear both cytosolic and nuclear fractions. These results indicate marker, DAPI, and visualized on a Leica SP2 inverted micro- first that IAPs may be upregulated in cancer cells, and scope. Single sections through the mid-point of the cell are second that Survivin, but not XIAP, is not only present presented. within the cytoplasm but is also nuclear. Survivin protects against Etoposide-induced apoptosis Drug treatment in 293T cells Twenty-four hours post-transfection, cells were drug treated In order to determine whether the sub-cellular localization of overnight with 100 mM Etoposide (Calbiochem), 1 mM Survivin influences the sensitivity of cells to chemotherapeu- Melphalan (Sigma, St Louis) or 1 mM Staurosporine tics, the ability of a mutant form of Survivin, unable to exit (Calbiochem). Etoposide and Melphalan are both DNA the nucleus, was compared with the ability of wildtype damaging agents, currently in clinical use. Staurosporine is Survivin to protect cells from drugs known to induce apop- a kinase inhibitor, which acts to induce cell stress and apop- tosis, and which are currently in clinical use. tosis. Drug concentrations were chosen to induce maximal Initially 293T cells were transfected with plasmids expres- apoptosis in the cell lines used, over 24 h, without resulting sing a GFP control, Survivin-GFP and Survivin-L98A-GFP. in complete cellular loss, based on previous concentration- To confirm equivalent levels of transfection, cell lysates dependence studies performed in the lab (unpublished) and were extracted and probed for the presence of Survivin and elsewhere. Cells were detached and cytospun onto slides, GFP (Fig. 2A). In the Survivin-GFP (both WT and mutant) fixed in 4% formaldehyde (10 min) and permeabilized in expressing cells, the Survivin (red) and GFP bands (green) 0.2% Triton-X100 (5 min). Cells were treated with 10% overlap to give a yellow merged image. Confocal microscopy calf serum in PBS before staining with 1/500 mouse confirmed that wildtype Survivin-GFP was present within the anti-Ki67 primary antibody (BD Transduction cytosol, whereas Survivin-L98A-GFP was limited to the Laboratories), Alexa 594 goat anti-mouse secondary anti- nucleus (Fig. 2B). Previous studies using tagged survivin body (1:500) and DAPI. Slides were visualized on an have shown that it behaves exactly the same as over- Axioplan2 Imaging microscope and images analysed using 28, 37, 38 expressed survivin constructs. OpenLab software. GFP, Survivin-GFP and Survivin-L98A-GFP transfected cells were then treated with Etoposide, Melphalan or Statistical analysis Staurosporine for 24 h, and the GFP-expressing cells were A Student’s two-tailed t-test was used. examined for either apoptosis or proliferation. Nuclear DAPI stain was used to identify the distinct morphological Results features associated with apoptotic cells including nuclear fragmentation and the presence of apoptotic bodies. Cells Endogenous Survivin localization in breast cancer undergoing proliferation were analysed by Ki67 staining. cell lines The percentage of apoptotic or proliferating cells was calcu- In order to investigate the sub-cellular localization of lated and expressed as a percentage of the total number of endogenous Survivin in breast cancer cells, a panel of three transfected cells (Fig. 2C and D). cancer lines (MDA-MB-231; MDA-MB-468; BT20) was Transfection with the GFP-construct did not result in an compared with a normal line (MCF10A). Cells were sepa- increase in basal apoptosis rates, and transfection with rated into enriched fractions of cytoplasm (soluble), mem- Survivin-GFP and Survivin-L98A-GFP caused 1.5% and branes (CHAPS soluble) and nuclei (CHAPS insoluble) 6.5%, respectively, of the cells to apoptose spontaneously. (Fig. 1). To confirm accurate fractionation of samples, However, in each case, the percentage of cells undergoing immunoblots were probed with antibodies to (i) anti- apoptosis increased significantly (P, 0.05) in the drug- Apaf-1 antibody, a cytoplasmic marker which migrates at treated cells in comparison to the untreated controls 33 – 35 130 kDa; (ii) Heat Shock Protein 70 (HSP70), a (Fig. 2C, white bars). Moreover, and importantly, the cells 70 kDa protein that localizes to the mitochondria and was expressing Survivin-GFP showed a significant protection used as a membrane and mitochondrial marker and against Etoposide-induced apoptosis compared with the (iii) anti-Histone H3 antibody, a 17 kDa nuclear marker. GFP control cells. This resistance to apoptosis was not In addition, anti-Survivin and anti-XIAP antibodies were afforded by over-expression of the nuclear-restricted used to probe for IAP levels and location (Fig. 1). Notably, Survivin-L98A-GFP. Interestingly, neither Survivin nor both Survivin, which migrates at 16.5 kDa, and XIAP, Survivin-L98A conferred significant resistance to cells migrating at 50 kDa, were expressed at considerably treated with Melphalan or Staurosporine. ......................................................................................................................................................................................................................................... 87 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. Sub-cellular location of Survivin and XIAP in breast cancer cell lines. Twenty-microgram fractions of MDA-MB-231, MDA-MB-468, BT20 and MCF10A were separated by SDS–PAGE and examined by immunoblotting with antibodies to Apaf1, a cytoplasmic control; HSP70, a membrane marker; Histone H3, a nuclear marker, as well as the IAPs, Survivin and XIAP. Data presented are representative of three different experiments. In contrast to the apoptosis results, all the drugs inhibited In each of the cell lines tested, XIAP localized to the cyto- proliferation, though there was no significant additional plasm, correlating with the central function of XIAP, which 10, 42, 43 effect with either WT or L98A Survivin (Fig. 2D). is to buffer the caspase cascade. In contrast, Survivin Interestingly, there was a significant inhibition of basal pro- localized to both the nucleus and the cytoplasm in each of liferation (i.e. no drug treatment) in the cells transfected the cancer lines. These data correlate with a study of tissue with either Survivin-GFP or Survivin-L98A-GFP. from breast cancer patients including atypical hyperplasias Together these experiments demonstrate a protective role and malignant lymph tissue, where Survivin was most fre- for certain forms of Survivin on Etoposide-induced apoptosis quently present either only in the cytoplasm (31.3%) or in in breast cancer cells. the cytoplasm and nucleus (22.5%), but was restricted to the nucleus in 11.3% of cases. A separate statistical analy- sis showed that cytoplasmic Survivin expression correlates Discussion with tumour stage and histological grade, and with a lower recurrence-free patient survival rate. The main discovery arising from this study is that over- expressed Survivin can protect breast cancer cells against apoptosis induced by some chemotherapeutic drugs, but Over-expression of exogenous Survivin only if Survivin is present within the cytosol. Since The decrease in the percentage of cells undergoing Survivin was found to be upregulated in three independent Etoposide-induced apoptosis in cells over-expressing cancer lines, we speculate that it may have an apoptosis- Survivin-GFP is consistent with the protective role of suppressing function that contributes to disease progression. Survivin within the apoptotic pathway. This supports pre- vious literature showing a protective IAP function of over- Endogenous Survivin localization by sub-cellular expressed Survivin in cells treated with various apoptotic 46, 47 fractionation stimuli. However, we found that over-expressed Both XIAP and Survivin are up-regulated in the three cancer Survivin did not prevent Melphalan or STS-induced apopto- cell lines, MDA-MB-231, MDA-MB-468 and BT20, com- sis. The differential response to Etoposide and the other two pared with the normal breast cell line, MCF10a. This is con- agents might reflect differences in the caspase activation. For sistent with the general observation that IAPs are example, in HeLa cells, Etoposide induced greater activation up-regulated in cancer and are part of the mechanism of of caspase 9 than STS, while STS induced greater activation 41 32 apoptosis resistance displayed by cancer cells. of caspases 8 and 3. Survivin has previously been shown to ......................................................................................................................................................................................................................................... 88 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Figure 2. Survivin protects against Etoposide-induced apoptosis. (A) 293T cells were transfected with Survivin-GFP, Survivin-L98A-GFP and GFP alone. Whole cell lysates were separated on a 15% gel, with (a) Rainbow marker, (b) un-transfected control, (c) GFP control, (d) transfected Survivin-GFP and (e) transfected Survivin-L98A-GFP. The gel was probed with anti-Survivin (chemifluorescence at 700 nm) and anti-GFP antibody (chemifluorescence at 800 nm). Data are representative of n ¼ 2. (B) 293T cells transfected with Survivin-GFP (top panel), Survivin-L98A-GFP (middle panel) or GFP (bottom panel) were stained with DAPI (left column), and examined for Survivin localization (middle column and merged picture in right column) by confocal microscopy. (C and D) 293T cells were transfected with GFP alone, Survivin-GFP (SUR-GFP) or Survivin-L98A-GFP (SUR-NES-GFP). Cells were treated for 24 h with drugs as indicated, detached and cytospun onto slides, then fixed, permeabilized and stained with antibodies to Ki67. DAPI was used as a nuclear marker and transfected cells located by their GFP-tag. (C) Cells with condensed, apoptotic nuclei were scored in the GFP-positive population of the transfected cells. (D) Similarly, GFP-positive cells were scored for Ki67 staining. Results are expressed as percentages, taken from an average of 60 samples from three independent experiments. Error bars depict standard error of the mean. Asterisks represent significant difference (P, 0.05) com- pared with GFP-only transfected cells for each drug treatment. ......................................................................................................................................................................................................................................... 89 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 6. Deveraux QL, Roy N, Stennicke HR et al. (1998) IAPs block apoptotic events inhibit caspase 9, whereas its effects on caspase 3 are indirect induced by caspase-8 and cytochrome c by direct inhibition of distinct cas- and mediated through interactions with other IAPs. It is pases. 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Journal

Bioscience Horizons – Oxford University Press

Published: Jun 22, 2008

Keywords: Key words apoptosis Survivin subcellular localization breast cancer

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