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CA125 (MUC16) tumor antigen selectively modulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis
Gynecologic Oncology 115 (2009) 407–413
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
CA125 (MUC16) tumor antigen selectively modulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis Marianne Boivin, Denis Lane, Alain Piché, Claudine Rancourt ⁎ Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Canada J1H 5N1
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Article history: Received 12 March 2009 Available online 10 September 2009 Keywords: Ovarian cancer CA125/MUC16 Drug resistance Genotoxic drugs
a b s t r a c t Objective. Little is known about the biological functions of CA125/MUC16 tumor antigen. Here, we examined the role of CA125/MUC16 in regulating the sensitivity of epithelial ovarian carcinoma (EOC) cells to different drugs. Methods. An endoplasmic reticulum targeted single-chain antibody (scFv) was used to down-regulate cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells and the C-terminal domain (CTD) of MUC16 was ectopically expressed in CA125-negative SKOV3 cells. Sensitivity to genotoxic agents and to inhibitors of microtubule depolymerization was examined in NIH:OVCAR3 and SKOV3 cell sublines. Cell viability was determined by XTT assay, apoptosis by propidium iodide staining and caspase activation by Western blot and fluorogenic assay. Results. Down-regulation of cell surface MUC16 decreases cisplatin IC50 by 5-fold in NIH:OVCAR3 cells but does not affect paclitaxel IC50. We found that the sensitivity to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide was also increased by down-regulation of MUC16. Caspase-9 and caspase-3 activation also significantly augmented in cisplatin-treated NIH:OVCAR3 cells expressing the anti-MUC16 scFv. Ectopic expression of MUC16 CTD has the opposite effect. Cisplatin sensitivity and caspases activation are decreased by the ectopic expression of MUC16 CTD in SKOV3 cells. Conclusions. CA125/MUC16 selectively modulates the sensitivity of EOC cells to genotoxic agents. The MUC16 CTD appears to be sufficient to promote cisplatin resistance. © 2009 Elsevier Inc. All rights reserved.
Introduction Epithelial ovarian carcinoma (EOC) is the fifth most common cancer among women. The high mortality rate makes ovarian cancer the leading cause of death among gynecologic cancers. Over 70% of women with EOC present with late stage disease and dissemination of tumor implants throughout the peritoneal cavity [1]. Despite the standard therapy with surgical cytoreduction and the combination of cisplatin and paclitaxel, the treatment efficacy is significantly limited by the frequent development of drug resistance [2]. Novel therapeutic targets are urgently needed to improve ovarian cancer treatment efficacy. The human CA125 tumor antigen is a mucin-like transmembrane glycoprotein that shares many characteristics of the membranebound mucin proteins [3-6]. Its proposed structure comprises an Nterminal domain of more than 22,000 amino acid residues that is presumably heavily glycosylated, a central domain containing up to 60 glycosylated repeat sequences constituting the tandem repeats characteristic of mucins and a C-terminal domain composed of a ⁎ Corresponding author. Département de Microbiologie et Infectiologie, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. Fax: +1 819 564 5392. E-mail address: [email protected] (C. Rancourt). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.08.007
unique region, a potential proteolytic cleavage site, a transmembrane domain and a short cytoplasmic tail with possible sites of phosphorylation [5,6]. CA125 is found in the majority of EOC but is not usually detectable in normal ovary tissues [7]. There is a strong correlation between rising and falling levels of serum CA125 with progression and regression of the disease [8,9] making a useful clinical marker of diseases. Levels of CA125 in serum however have not been clearly associated with drug resistance. Because altered expression of mucins has been associated with carcinomas [10], increased invasiveness [11], enhanced tumor cell growth and metastasis [12,13], and modulation of cell adhesion [14], it is plausible to assume that CA125 may exert a number of functions that parallel those of mucins in ovarian cancer. In the present study, we examined the functional role of CA125/ MUC16 in modulating the sensitivity of EOC cells to different chemotherapeutic drugs. Materials and methods Reagents and cell culture The NIH:OVCAR3 and SKOV3 human ovarian cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA).
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NIH:OVCAR3 cells were grown in RPMI 1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20% heat-inactivated FBS (Wisent), 2 mM L-glutamine (Wisent), 100 units/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml insulin, and maintained at 37 °C in a humidified 5% CO2 incubator. The SKOV3 cell line was maintained at 37 °C in a humidified incubator containing 5% CO2 in DMEM/F12 (Wisent) supplemented with 10% heat-inactivated FBS and antibiotics. Cisplatin was obtained from Faulding, etoposide and
paclitaxel from Bristol-Myers Squibb, doxorubicin from Pfizer, cyclophosphamide from Baxter and vinorelbin from Pierre Fabre Pharma. Anti-caspase-9, anti-caspase-3, and anti-PARP antibodies were purchased from Cell Signaling (Beverly, MA). Anti-tubulin was from Sigma. HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from GE Healthcare and Cell Signaling respectively. Phenazine methosulfate, propidium iodide and antitubulin antibody were obtained from Sigma.
Fig. 1. MUC16 scFvs down-regulate cell surface expression of CA125 in NIH:OVCAR3 cells. NIH:OVCAR3 cells were transfected to stably expressed anti-MUC16 scFvs (1:9#7 and 1:9#9) or a control scFv (Ctrl scFv). (A) Cells were incubated with anti-c-myc or isotypic IgG antibodies and analyzed for anti-MUC16 scFvs expression by immunofluorescence. (B) Cells were incubated with anti-CA125 M11 mouse monoclonal antibody or an isotypic IgG antibody and analyzed for immunofluorescence by flow cytometry. (C) Nonpermeabilized cells were incubated with anti-CA125 M11 and propidium iodine, and analyzed for CA125 expression by immunofluorescence. (D) Conditioned media from stable transfectants 1:9#7 and 1:9#9, control scFv (Ctrl scFv) or parental NIH:OVCAR-3 cells were collected when cell confluence had reached 80–90%. Samples were quantitatively analyzed for CA125 content by an ELISA-based method using the ADVIA Centaur CA125 II detection assay. Results are shown as a mean ± SEM of 3 independent experiments performed in triplicate.
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The hybridoma cell line VK-8, which expresses a monoclonal antibody against the extracellular domain of CA125 tumor antigen was kindly provided by K.O. Lloyd (Sloan-Kettering Memorial Cancer Center, New York, NY) [16]. The scFv DNA fragments were derived from the VK-8 hybridoma cell line using the Amersham Biosciences mouse scFv module (Amersham Biosciences, Baie d'Urfé, Québec, Canada) according to the manufacturer's instructions. Briefly, the variable heavy chain (VH) and light chain (VL) were amplified from the cDNA by PCR (Expand high fidelity PCR, Roche Diagnostics, Laval Canada) using mouse variable region primers provided by Amersham Biosciences (sequences unavailable). The VH and VL DNA fragments were linked together by PCR overlap extension using a (Gly4Ser)3 linker sequence to generate 750 bp scFv constructs with flanking SfiI and NotI sites. The scFv
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DNA fragments were inserted into SfiI/NotI sites of the prokaryotic expression vector pCANTAB5E (Amersham Pharmacia Biotech, Piscataway, NJ). After selection with 1500 U/ml of purified CA125 (Meridian Life Science Inc., Saco, MN) for binding in vitro, the anti-CA125 scFv DNA fragments were subcloned into the SfiI/ NotI sites of the previously described pSTCF.KDEL eukaryotic vector upstream and in frame with a c-myc tag and the KDEL sequence which enable targeting to the lumen of the endoplasmic reticulum (ER). The scFv DNA fragments were subcloned into the pLTR.KDEL retroviral plasmid in SfiI/NotI sites after insertion of a SfiI/NotI containing polylinker at XhoI site of pLTR.KDEL to generate the pLTR.KDEL-ER-1:9 plasmid. This plasmid was transfected into NIH: OVCAR3 cells to generate the stable NIH:OVCAR3 clones expressing pLTR.KDEL-ER-1:9#9, pLTR.KDEL-ER-1:9#7 (two independent
Fig. 2. Effect of CA125 cell surface down-regulation on drug sensitivity in NIH:OVCAR3 cell line. (A) Cells were incubated with increasing concentrations of cisplatin or paclitaxel. Cell viability was determined by XTT assay after 96 h. (B) Cells were treated with fixed concentrations of the indicated drug and cell viability was determined. All experiments were performed in triplicate and are representative of three independent experiments. Values represent the mean ± SEM. ⁎Statistically significant with p b 0.01.
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clones isolated) and a control pLTR.KDEL-ER-ctrl scFv. The pLTR. KDEL-ER-ctrl scFv contains a scFv that does not bind CA125 by ELISA whereas scFvs 1:9 bind CA125 with high affinity. Clones were selected in complete media (as for NIH:OVCAR3) containing 1.5 μg/ml blasticidin. Independent colonies were picked, expanded and maintained subsequently in 0.5–1.0 μg/ml blasticidin-containing media. SKOV3 cells were transfected with pSTCFcyto (empty vector) or pSTCFcyto-MUC16-CTD in the presence of FuGENE 6 (Roche Applied Science, Indianapolis, IN). MUC16-CTD cDNA (FLJ14303, accession number: AK024365) contains a IgK leader sequence, the unique 229 a.a. extracellular domain, the transmembrane and cytosolic domains of CA125 (MUC16) tagged at the C-terminal with His6 peptide, which is separated by a small linker with a c-myc peptide tag. Primers for PCR amplification of MUC16CTD were as follow: 5′-ATGCGGCCCAGCCGGCCATACACCCTGCTGAGGGAC-3′ (sense) and 5′-ATAGTTTAGCGGCCGCATGATGATGATGATGATGACCACCGCTTTGCAGATCCTCCAGGTCT-3′ (antisense). SKOV3 stable transfectants were selected in 500 μg/ml zeocin (Invitrogen, Carlsbad, CA). Pooled colonies were expanded to generate the SKOV3-EV (empty vector) and SKOV3-CTD (containing MUC16-CTD) cell lines.
were separated by 12% SDS-PAGE gels, proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech Inc.) by electroblotting, and immunoblot analysis was performed as previously described [15]. All primary antibodies were incubated overnight at 4 °C. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc.). Statistical analysis The data are presented as the means ± SEM from three independent experiments. Statistical comparisons between groups were performed using the unpaired Student's t-test. Statistical significance was indicated by p b 0.05.
Cytotoxicity assays Cell viability was determined by XTT assay. Briefly, cells were plated at 20,000 cells/well in 96-well plates. The next day, cells (confluence 60–70%) were treated with increasing concentration of cisplatin or paclitaxel incubated for 72 h. For other drugs, cells were treated with a fixed dose (as indicated) and incubated for 72 h to 96 h depending on the drug. At the termination of the experiment, the culture media was removed and fresh media containing phenazine methosulfate and XTT (Molecular Probes) was added for 30 min. The absorbance of each well was determined using a microplate reader at 450 nm. Pilot experiments verified that the cell densities encountered in these experiments were within the linear portion of the XTT assay. The percentage of cell survival was defined as the relative absorbance of untreated versus treated cells. All assays were performed in triplicate and repeated three times. Apoptosis assays Caspase-3 fluorogenic protease assay was performed according the manufacturer's protocol (R&D Systems, Minneapolis, MN). In brief, 3 × 106 cells were lysed in 250 μl of cell lysis buffer, and total cell lysates were incubated with 50 μM of DEVD-AFC substrate for 1 h. Caspase-3 activity was measured on a Versa Fluor fluorometer (BioRad, Hercules, CA). Protein concentration of the lysates was measured with Bio-Rad protein assay kit according to the manufacturer's recommendations. To determine the sub-G0 DNA content, floating and adherent cells were harvested, washed with PBS and fixed with cold ethanol overnight. Cell pellets were resuspended, washed with PBS/1 % BSA, filtered on nylon mesh membrane (40 μ mesh) to remove cell aggregates and counted. Cells were then incubated with propidium iodide (final concentration 50 μg/ml in 38 mM sodium citrate pH 7.0) and with boiled RNase A (1 mg/ml) 30 min at 37 °C. Cells were analyzed on a FACSCAN flow cytometer (Becton Dickinson). Immunoblot analysis Cells were lysed in Nonidet P-40 isotonic lysis buffer (283 mM Kcl, 10 mM MgCl2, 50 mM HEPES, pH 7.2, 4 mM EGTA, 0.6% Nonidet P-40 with freshly added protease inhibitors (1 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 2 μg/ml aprotinin, 0.7 μg/ml pepstatin, and 0.5 μg/ml leupeptin) (Sigma) and proteins were quantities by a Bradford assay. Equal amount of whole cell extracts
Fig. 3. Down-regulation of cell surface CA125 potentiates cisplatin-induced apoptosis and caspase activation. (A) Cells were treated with cisplatin (1,5 μM) and the percentage of apoptosis was determined by measuring the percent of sub-G1 cells after nuclear propidium iodide staining 48 h after adding drugs to the medium. The experiments shown are means ± SEM of two individual experiments done in duplicates. (B) Western blot analysis of whole-cell lysates from cells treated with cisplatin (1,5 μM) for 48 h probed with anti-caspase-9, anti-caspase-3, anti-PARP and anti-tubulin (to ensure equal loading). (C) Lysates from cells treated with cisplatin (1,5 μM) were assayed for caspase-3 activity by monitoring the fluorescence produced by hydrolysis of caspase-3 substrate DEVD-AFC. Results are expressed as fold increase relative to untreated cells (n = 3). The relative fluorescence unit (RFU) of caspase-3 activity was normalized for the protein content of each extract. ⁎Statistically significant with p b 0.001.
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Results Down-regulation of cell surface CA125 selectively sensitizes OVCAR3 cells to genotoxic drugs CA125 positive OVCAR3 cells were transfected to stably expressed anti-MUC16 scFvs or a control scFv. Two independent clones (1:9#7 and 1:9#9) were selected. Immunofluorescence analysis with anti-cmyc demonstrated expression of the c-myc tagged anti-MUC16 scFvs in a pattern that suggested ER localization (Fig. 1A). Indeed, colocalization of the MUC16 scFv anti-calreticulin, an ER-resident protein, supported targeting of MUC16 scFv to the ER (data not shown). The anti-MUC16 scFv was derived from the hybridoma cell line VK-8 [16], which expresses a monoclonal antibody against the extracellular domain of CA125 tumor antigen. The entrapment of CA125 mediated by intracellular expression of CA125-specific scFvs targeted within the secretion pathway was expected to prevent localization of CA125 at the cell surface. Indeed, the stable expression of the anti-MUC16 scFv induces a 10- to 100-fold decrease of CA125 expression at the cell surface as shown by flow cytometry (Fig. 1B). Immunofluorescence studies with anti-CA125 antibodies in nonpermeabilized cells, confirmed the down-regulation of cell surface CA125 in 1:9#7 and 1:9#9 sublines (Fig. 1C). Because CA125 tumor antigen is usually released in conditioned media of cultured cells expressing this antigen, we also determined whether the shedding of CA125 was affected in our stable transfectants. Conditioned media from confluent cell cultures was collected and quantitatively analyzed using an ELISA-based method. Fig. 1D shows that the parental cells and the control scFv (Ctrl scFv) transfectant both shed almost similar
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amounts of CA125 in the culture media. However, the 1.9#7 and 1:9#9 transfectants showed a drastic reduction of CA125 release (p b 0.05). Altogether, these data suggest that entrapment of the CA125 protein prevents its cell surface localization and leads to its degradation within the ER. Of note, FACS analysis and microarray analysis have shown that MUC1 expression is not altered by the down-regulation of MUC16 expression (data not shown). Cisplatin and paclitaxel are two drugs used in first-line therapy for the treatment of EOC. Cisplatin is a genotoxic drug that produces DNA damage and paclitaxel is an inhibitor of microtubule depolarization. Although their mechanism of action differs, both drugs ultimately induce the apoptotic cascade leading to cell death. We thus assessed the effect of MUC16 down-regulation at the cell surface on in vitro sensitivity of NIH:OVCAR3 cells. The parental NIH:OVCAR3 cell line and cells stably expressing the anti-MUC16 scFv (1:9#7 and 1:9#9) or an irrelevant MUC16 non-binding scFv (Ctrl scFv) were exposed to increasing concentrations of cisplatin and paclitaxel for 48 h. The fold differences were calculated at the point where 50% cytotoxicity was observed for the controls versus cells expressing anti-MUC16 scFvs. Cisplatin cytotoxicity was approximately 5-fold greater when MUC16 cell surface expression was down-regulated whereas the paclitaxel sensitivity remained unchanged (Fig. 2A). Next, we examined the sensitivity of NIH:OVCAR3 cells expressing the control scFv or the anti-MUC16 scFvs to a number of genotoxic drugs versus drugs that affect microtubule assembly. Drug cytotoxicity was significantly (p b 0.001) enhanced in anti-MUC16 scFv expressing cells for all genotoxic agents tested (cyclophosphamide, doxorubicine, etoposide) but not for taxol and vinorelbine, which affect microtubule assembly (Fig. 2B).
Fig. 4. Effect of ectopic expression of MUC16-CTD on cisplatin and paclitaxel sensitivity in SKOV3 cells. (A) Lysates were subjected to immunoblot analysis with anti-c-myc and antitubulin. (B) Cells were incubated with anti-c-myc and propidium iodine to stain nuclei, and expression of MUC16-CTD was analyzed by immunofluorescence. (C) Cells were treated with cisplatin (CDDP) (1,5 μM) or paclitaxel (10 nM) for 96 h and cell viability was determined by XTT assay. All experiments were performed in triplicate and are representative of three independent experiments. Values represent the mean ± SEM. ⁎Statistically significant with p b 0,01.
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Down-regulation of cell surface CA125 increases cisplatin-mediated apoptosis and caspases activation Treatment of tumor cells with DNA-damaging agents such as cisplatin is associated with activation of the intrinsic apoptotic pathway. We examined the effect of MUC16 cell surface downregulation on cisplatin-induced apoptosis. Cells were treated with cisplatin (1,5 μM), harvested 96 h later, and analyzed for sub-G1 DNA content. After cisplatin treatment, the percentage of apoptotic cells in the clones expressing the anti-MUC16 scFv increased to 35–40% whereas control populations (parental NIH:OVCAR3 and Ctrl scFv) only increased to 10% (Fig. 3A). We next assessed caspase activity in response to MUC16 down-regulation and cisplatin treatment. Caspase-9 and caspase-3 activation, as monitored by cleavage of proisoforms, was enhanced by cisplatin in cells expressing the antiMUC16 scFv (1:9#7 and 1:9#9) as compared to cells expressing the control scFv (Ctrl scFv) (Fig. 3B). Down-regulation of MUC16 cell surface expression by itself did not alter the expression levels of procaspase-9, pro-caspase-3 and PARP in either control cells, clone 1:9#7 or clone 1:9#9 (data not shown). Similarly, the basal levels of the apoptotic regulatory proteins Bcl-2, Bcl-XL, Bax and XIAP were not affected by down-regulation of MUC16. Because the antibody we used does not properly detect the active fragments of caspase-3, caspase-3 activity was measured using a fluorogenic assay. Caspase-3 activity was 3-fold lower in the NIH:OVCAR3 cells expressing the anti-MUC16 scFv compared to the control cells (Fig. 3C). Next, we examined the effect of MUC16 down-regulation with taxol on caspase-3 activity in the NIH:OVCAR3 cell populations. In contrast to cisplatin, the combination of paclitaxel and MUC16 down-regulation did not affect the levels of caspase-3 activity when compared to control NIH: OVCAR3 cells (data not shown). Thus, down-regulation of MUC16 alone does not increase apoptosis and caspases activation, however down-regulation of MUC16 enhances the cisplatin- but not taxolmediated apoptosis and caspases activation. MUC16 C-terminal domain selectively decreases the sensitivity to cisplatin To further confirm the selectivity of MUC16 on cisplatin sensitivity, the CA125-negative SKOV3 cell line was transfected to stably express an empty vector (SKOV3-EV) or the C-terminal domain of CA125 (MUC16-CTD) comprising a small extracellular domain of CA125, its transmembrane domain and a His6- and c-myc tagged cytoplasmic tail (SKOV3-CTD). The MUC16-CTD protein was detectable in SKOV3CTD but not in SKOV3-EV and SKOV3 cells as shown by immunoblot with anti-c-myc antibody (Fig. 4A). MUC16-CTD protein showed a pattern consistent with a diffuse distribution at the cell membrane by immunofluorescence (Fig. 4B). SKOV3 cells expressing the empty vector or the MUC16-CTD were treated with cisplatin or paclitaxel. Treatment with cisplatin significantly increased cell viability of MUC16-CTD-expressing SKOV3 cells when compared to SKOV3 or empty vector-expressing cells (Fig. 4C). In contrast, there was no difference in cell viability in the different SKOV3 cell population treated with paclitaxel. These findings further support the fact that MUC16 selectively affect the sensitivity of ovarian cancer cells to genotoxic agents. MUC16 C-terminal domain attenuates cisplatin-apoptosis in SKOV3 cells We examined the effect of MUC16-CTD exogenous expression on cisplatin-induced caspases activation in SKOV3 cells. We first assessed the basal expression levels of pro-caspase-9, pro-caspase3, PARP, Bcl-2, Bcl-XL, Bax and XIAP to ensure that their expression was not altered by MUC16 CTD. Western blot analysis showed that expression of the proteins was similar in parental SKOV3, empty vector-expressing SKOV3 and MUC16-CTD-expressing SKOV3 cells
Fig. 5. Ectopic expression of MUC16-CTD attenuates cisplatin-induced caspase activation in SKOV3 cells. (A) Western blot analysis of whole-cell lysates from cells treated with cisplatin (1,5 μM) for 48 h probed with anti-caspase-9, anti-caspase-3, anti-PARP and anti-tubulin (to ensure equal loading). (B) Lysates from cells treated with cisplatin (1,5 μM) were assayed for caspase-3 activity by monitoring the fluorescence produced by hydrolysis of caspase-3 substrate DEVD-AFC. Results are expressed as fold increase relative to untreated cells (n = 3). The relative fluorescence unit (RFU) of caspase-3 activity was normalized for the protein content of each extract. ⁎Statistically significant with p b 0.01.
(data not shown). As shown in Fig. 5A, following cisplatin treatment, the levels of pro-caspase-9, pro-caspase-3 and full length PARP were higher in MUC16-CTD-expressing SKOV3 indicating decreased caspases activation in these cells compared to control cells. Consistent with these results, caspase-3 activity was also reduced in MUC16-CTD-expressing SKOV3 cells following cisplatin treatment indicating that expression of MUC16-CTD attenuates cisplatinmediated caspase activation. Discussion The efficacy of treatment for EOC is frequently impaired by the rapid appearance to resistance to chemotherapeutic drugs. Therefore, the identification of proteins that may impact tumor cell drug sensitivity is of importance. The major findings from our study are that MUC16 modulates the sensitivity of ovarian cancer cells to genotoxic agents but not to drugs that affect microtubules. To strongly support this conclusion, we used two approaches. We down-regulated MUC16 in epithelial ovarian carcinoma cells that endogenously expressed this protein and we stably express MUC16CTD in MUC16-negative ovarian cancer cells. Indeed, we showed that scFv-mediated down-regulation of cell surface MUC16 promotes cisplatin-induced apoptosis and caspase activation in NIH:OVCAR3 cells. These findings were not restricted to cisplatin since down-
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regulation of cell surface MUC16 also sensitizes to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide. Conversely, ectopic expression of MUC16 C-terminal domain decreases cisplatin-mediated caspase activation in SKOV3 cells. However, the down-regulation of cell surface MUC16 or the ectopic expression of MUC16-CTD has no effect on paclitaxel sensitivity. Although CA125/MUC16 is a well-established marker for ovarian cancer progression [8], little is known about its biological functions. These findings therefore establish a novel role for CA125/MUC16 in EOC cells. Our data also suggest that the MUC16-CTD is sufficient to promote cisplatin resistance in SKOV3 cells. The findings that downregulation of CA125/MUC16 cell surface expression or ectopic expression of MUC16-CTD do not display alteration of Bax, Bcl-2, Bcl-XL or XIAP indicate that modulation of MUC16 affects genotoxic drug-induced apoptosis by other mechanisms. CA125 is a large membrane-bound mucin overexpressed in most human epithelial ovarian cancer [7]. The C-terminal domain of the protein is composed of a unique region, a potential proteolytic cleavage site, a transmembrane domain and a short cytoplasmic tail which appears to be sufficient to attenuate cisplatin-induced caspases activation. The cytoplasmic tails of membrane-bound mucins such as MUC1 have been shown to be involved in signaling events that contribute to resistance to drug-induced apoptosis. Although the cytoplasmic tails of MUC16 and MUC1 do not share sequence homology [17], it is not entirely unexpected that the MUC16-CTD can promote cisplatin resistance. The cytoplasmic tail of MUC16 harbors several potential phosphorylation sites that may serve to activate different signaling cascades including proteins that regulate the apoptotic cascade. Proteins that interact with MUC16 cytoplasmic tail, however, remain to be identified. Interestingly, the MUC1 oncoprotein has been shown to activate FOXO3a transcription factor and knockdown of MUC1 inactivates FOXO3A thereby increasing the sensitivity of cells to oxidative stress [18]. The phosphorylation of FOXO3a blocks its translocation to the nucleus and consequently FOXO3a-mediated transactivation of gene transcription [19]. Importantly, FOXO3a contributes to DNA repair in response to genotoxic stress by activating Gadd45a expression [20]. In the presence of irreparable damage, FOXO3a contributes to an apoptotic response. It is conceivable that MUC16 knockdown, like MUC1 knockdown, could inactivate FOXO3a, consequently decreasing DNA repair, increasing DNA damage induced by genotoxic drugs and selectively sensitizing tumor cells to these drugs. In support of this hypothesis, we found that MUC16 cell surface down-regulation decreases FOXO3a expression levels in NIH:OVCAR3 cells and decreases FOXO3a nuclear localization (unpublished data). These results could explain, at least in part, the fact that MUC16 selectively modulates the sensitivity to genotoxic drugs in EOC cells. It is interesting that MUC1 overexpression confers resistance to genotoxic drugs [21,22] and that, conversely, knocking down of endogenous MUC1 sensitizes cells to drug-induced apoptosis [21]. These observations are in concert with our findings with MUC16. They further support its implication in the modulation of genotoxic drug sensitivity. Overexpression of MUC16 may therefore confer a selective advantage to EOC cells under conditions of genotoxic stress. Downregulating MUC16 may represent an alternative approach for sensitizing EOC cells to genotoxic therapeutic modalities such as platinum-based therapies.
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Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by an internal grant from Université de Sherbrooke (A.P. and C.R.) and by the National Cancer Institute with funds from the Canadian Cancer Society to C.R. (#011225 and #014263).
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
CA125 (MUC16) tumor antigen selectively modulates the sensitivity of ovarian cancer cells to genotoxic drug-induced apoptosis Marianne Boivin, Denis Lane, Alain Piché, Claudine Rancourt ⁎ Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Canada J1H 5N1
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Article history: Received 12 March 2009 Available online 10 September 2009 Keywords: Ovarian cancer CA125/MUC16 Drug resistance Genotoxic drugs
a b s t r a c t Objective. Little is known about the biological functions of CA125/MUC16 tumor antigen. Here, we examined the role of CA125/MUC16 in regulating the sensitivity of epithelial ovarian carcinoma (EOC) cells to different drugs. Methods. An endoplasmic reticulum targeted single-chain antibody (scFv) was used to down-regulate cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells and the C-terminal domain (CTD) of MUC16 was ectopically expressed in CA125-negative SKOV3 cells. Sensitivity to genotoxic agents and to inhibitors of microtubule depolymerization was examined in NIH:OVCAR3 and SKOV3 cell sublines. Cell viability was determined by XTT assay, apoptosis by propidium iodide staining and caspase activation by Western blot and fluorogenic assay. Results. Down-regulation of cell surface MUC16 decreases cisplatin IC50 by 5-fold in NIH:OVCAR3 cells but does not affect paclitaxel IC50. We found that the sensitivity to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide was also increased by down-regulation of MUC16. Caspase-9 and caspase-3 activation also significantly augmented in cisplatin-treated NIH:OVCAR3 cells expressing the anti-MUC16 scFv. Ectopic expression of MUC16 CTD has the opposite effect. Cisplatin sensitivity and caspases activation are decreased by the ectopic expression of MUC16 CTD in SKOV3 cells. Conclusions. CA125/MUC16 selectively modulates the sensitivity of EOC cells to genotoxic agents. The MUC16 CTD appears to be sufficient to promote cisplatin resistance. © 2009 Elsevier Inc. All rights reserved.
Introduction Epithelial ovarian carcinoma (EOC) is the fifth most common cancer among women. The high mortality rate makes ovarian cancer the leading cause of death among gynecologic cancers. Over 70% of women with EOC present with late stage disease and dissemination of tumor implants throughout the peritoneal cavity [1]. Despite the standard therapy with surgical cytoreduction and the combination of cisplatin and paclitaxel, the treatment efficacy is significantly limited by the frequent development of drug resistance [2]. Novel therapeutic targets are urgently needed to improve ovarian cancer treatment efficacy. The human CA125 tumor antigen is a mucin-like transmembrane glycoprotein that shares many characteristics of the membranebound mucin proteins [3-6]. Its proposed structure comprises an Nterminal domain of more than 22,000 amino acid residues that is presumably heavily glycosylated, a central domain containing up to 60 glycosylated repeat sequences constituting the tandem repeats characteristic of mucins and a C-terminal domain composed of a ⁎ Corresponding author. Département de Microbiologie et Infectiologie, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Québec, Canada J1H 5N4. Fax: +1 819 564 5392. E-mail address: [email protected] (C. Rancourt). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.08.007
unique region, a potential proteolytic cleavage site, a transmembrane domain and a short cytoplasmic tail with possible sites of phosphorylation [5,6]. CA125 is found in the majority of EOC but is not usually detectable in normal ovary tissues [7]. There is a strong correlation between rising and falling levels of serum CA125 with progression and regression of the disease [8,9] making a useful clinical marker of diseases. Levels of CA125 in serum however have not been clearly associated with drug resistance. Because altered expression of mucins has been associated with carcinomas [10], increased invasiveness [11], enhanced tumor cell growth and metastasis [12,13], and modulation of cell adhesion [14], it is plausible to assume that CA125 may exert a number of functions that parallel those of mucins in ovarian cancer. In the present study, we examined the functional role of CA125/ MUC16 in modulating the sensitivity of EOC cells to different chemotherapeutic drugs. Materials and methods Reagents and cell culture The NIH:OVCAR3 and SKOV3 human ovarian cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA).
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NIH:OVCAR3 cells were grown in RPMI 1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20% heat-inactivated FBS (Wisent), 2 mM L-glutamine (Wisent), 100 units/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml insulin, and maintained at 37 °C in a humidified 5% CO2 incubator. The SKOV3 cell line was maintained at 37 °C in a humidified incubator containing 5% CO2 in DMEM/F12 (Wisent) supplemented with 10% heat-inactivated FBS and antibiotics. Cisplatin was obtained from Faulding, etoposide and
paclitaxel from Bristol-Myers Squibb, doxorubicin from Pfizer, cyclophosphamide from Baxter and vinorelbin from Pierre Fabre Pharma. Anti-caspase-9, anti-caspase-3, and anti-PARP antibodies were purchased from Cell Signaling (Beverly, MA). Anti-tubulin was from Sigma. HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from GE Healthcare and Cell Signaling respectively. Phenazine methosulfate, propidium iodide and antitubulin antibody were obtained from Sigma.
Fig. 1. MUC16 scFvs down-regulate cell surface expression of CA125 in NIH:OVCAR3 cells. NIH:OVCAR3 cells were transfected to stably expressed anti-MUC16 scFvs (1:9#7 and 1:9#9) or a control scFv (Ctrl scFv). (A) Cells were incubated with anti-c-myc or isotypic IgG antibodies and analyzed for anti-MUC16 scFvs expression by immunofluorescence. (B) Cells were incubated with anti-CA125 M11 mouse monoclonal antibody or an isotypic IgG antibody and analyzed for immunofluorescence by flow cytometry. (C) Nonpermeabilized cells were incubated with anti-CA125 M11 and propidium iodine, and analyzed for CA125 expression by immunofluorescence. (D) Conditioned media from stable transfectants 1:9#7 and 1:9#9, control scFv (Ctrl scFv) or parental NIH:OVCAR-3 cells were collected when cell confluence had reached 80–90%. Samples were quantitatively analyzed for CA125 content by an ELISA-based method using the ADVIA Centaur CA125 II detection assay. Results are shown as a mean ± SEM of 3 independent experiments performed in triplicate.
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The hybridoma cell line VK-8, which expresses a monoclonal antibody against the extracellular domain of CA125 tumor antigen was kindly provided by K.O. Lloyd (Sloan-Kettering Memorial Cancer Center, New York, NY) [16]. The scFv DNA fragments were derived from the VK-8 hybridoma cell line using the Amersham Biosciences mouse scFv module (Amersham Biosciences, Baie d'Urfé, Québec, Canada) according to the manufacturer's instructions. Briefly, the variable heavy chain (VH) and light chain (VL) were amplified from the cDNA by PCR (Expand high fidelity PCR, Roche Diagnostics, Laval Canada) using mouse variable region primers provided by Amersham Biosciences (sequences unavailable). The VH and VL DNA fragments were linked together by PCR overlap extension using a (Gly4Ser)3 linker sequence to generate 750 bp scFv constructs with flanking SfiI and NotI sites. The scFv
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DNA fragments were inserted into SfiI/NotI sites of the prokaryotic expression vector pCANTAB5E (Amersham Pharmacia Biotech, Piscataway, NJ). After selection with 1500 U/ml of purified CA125 (Meridian Life Science Inc., Saco, MN) for binding in vitro, the anti-CA125 scFv DNA fragments were subcloned into the SfiI/ NotI sites of the previously described pSTCF.KDEL eukaryotic vector upstream and in frame with a c-myc tag and the KDEL sequence which enable targeting to the lumen of the endoplasmic reticulum (ER). The scFv DNA fragments were subcloned into the pLTR.KDEL retroviral plasmid in SfiI/NotI sites after insertion of a SfiI/NotI containing polylinker at XhoI site of pLTR.KDEL to generate the pLTR.KDEL-ER-1:9 plasmid. This plasmid was transfected into NIH: OVCAR3 cells to generate the stable NIH:OVCAR3 clones expressing pLTR.KDEL-ER-1:9#9, pLTR.KDEL-ER-1:9#7 (two independent
Fig. 2. Effect of CA125 cell surface down-regulation on drug sensitivity in NIH:OVCAR3 cell line. (A) Cells were incubated with increasing concentrations of cisplatin or paclitaxel. Cell viability was determined by XTT assay after 96 h. (B) Cells were treated with fixed concentrations of the indicated drug and cell viability was determined. All experiments were performed in triplicate and are representative of three independent experiments. Values represent the mean ± SEM. ⁎Statistically significant with p b 0.01.
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clones isolated) and a control pLTR.KDEL-ER-ctrl scFv. The pLTR. KDEL-ER-ctrl scFv contains a scFv that does not bind CA125 by ELISA whereas scFvs 1:9 bind CA125 with high affinity. Clones were selected in complete media (as for NIH:OVCAR3) containing 1.5 μg/ml blasticidin. Independent colonies were picked, expanded and maintained subsequently in 0.5–1.0 μg/ml blasticidin-containing media. SKOV3 cells were transfected with pSTCFcyto (empty vector) or pSTCFcyto-MUC16-CTD in the presence of FuGENE 6 (Roche Applied Science, Indianapolis, IN). MUC16-CTD cDNA (FLJ14303, accession number: AK024365) contains a IgK leader sequence, the unique 229 a.a. extracellular domain, the transmembrane and cytosolic domains of CA125 (MUC16) tagged at the C-terminal with His6 peptide, which is separated by a small linker with a c-myc peptide tag. Primers for PCR amplification of MUC16CTD were as follow: 5′-ATGCGGCCCAGCCGGCCATACACCCTGCTGAGGGAC-3′ (sense) and 5′-ATAGTTTAGCGGCCGCATGATGATGATGATGATGACCACCGCTTTGCAGATCCTCCAGGTCT-3′ (antisense). SKOV3 stable transfectants were selected in 500 μg/ml zeocin (Invitrogen, Carlsbad, CA). Pooled colonies were expanded to generate the SKOV3-EV (empty vector) and SKOV3-CTD (containing MUC16-CTD) cell lines.
were separated by 12% SDS-PAGE gels, proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech Inc.) by electroblotting, and immunoblot analysis was performed as previously described [15]. All primary antibodies were incubated overnight at 4 °C. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc.). Statistical analysis The data are presented as the means ± SEM from three independent experiments. Statistical comparisons between groups were performed using the unpaired Student's t-test. Statistical significance was indicated by p b 0.05.
Cytotoxicity assays Cell viability was determined by XTT assay. Briefly, cells were plated at 20,000 cells/well in 96-well plates. The next day, cells (confluence 60–70%) were treated with increasing concentration of cisplatin or paclitaxel incubated for 72 h. For other drugs, cells were treated with a fixed dose (as indicated) and incubated for 72 h to 96 h depending on the drug. At the termination of the experiment, the culture media was removed and fresh media containing phenazine methosulfate and XTT (Molecular Probes) was added for 30 min. The absorbance of each well was determined using a microplate reader at 450 nm. Pilot experiments verified that the cell densities encountered in these experiments were within the linear portion of the XTT assay. The percentage of cell survival was defined as the relative absorbance of untreated versus treated cells. All assays were performed in triplicate and repeated three times. Apoptosis assays Caspase-3 fluorogenic protease assay was performed according the manufacturer's protocol (R&D Systems, Minneapolis, MN). In brief, 3 × 106 cells were lysed in 250 μl of cell lysis buffer, and total cell lysates were incubated with 50 μM of DEVD-AFC substrate for 1 h. Caspase-3 activity was measured on a Versa Fluor fluorometer (BioRad, Hercules, CA). Protein concentration of the lysates was measured with Bio-Rad protein assay kit according to the manufacturer's recommendations. To determine the sub-G0 DNA content, floating and adherent cells were harvested, washed with PBS and fixed with cold ethanol overnight. Cell pellets were resuspended, washed with PBS/1 % BSA, filtered on nylon mesh membrane (40 μ mesh) to remove cell aggregates and counted. Cells were then incubated with propidium iodide (final concentration 50 μg/ml in 38 mM sodium citrate pH 7.0) and with boiled RNase A (1 mg/ml) 30 min at 37 °C. Cells were analyzed on a FACSCAN flow cytometer (Becton Dickinson). Immunoblot analysis Cells were lysed in Nonidet P-40 isotonic lysis buffer (283 mM Kcl, 10 mM MgCl2, 50 mM HEPES, pH 7.2, 4 mM EGTA, 0.6% Nonidet P-40 with freshly added protease inhibitors (1 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 2 μg/ml aprotinin, 0.7 μg/ml pepstatin, and 0.5 μg/ml leupeptin) (Sigma) and proteins were quantities by a Bradford assay. Equal amount of whole cell extracts
Fig. 3. Down-regulation of cell surface CA125 potentiates cisplatin-induced apoptosis and caspase activation. (A) Cells were treated with cisplatin (1,5 μM) and the percentage of apoptosis was determined by measuring the percent of sub-G1 cells after nuclear propidium iodide staining 48 h after adding drugs to the medium. The experiments shown are means ± SEM of two individual experiments done in duplicates. (B) Western blot analysis of whole-cell lysates from cells treated with cisplatin (1,5 μM) for 48 h probed with anti-caspase-9, anti-caspase-3, anti-PARP and anti-tubulin (to ensure equal loading). (C) Lysates from cells treated with cisplatin (1,5 μM) were assayed for caspase-3 activity by monitoring the fluorescence produced by hydrolysis of caspase-3 substrate DEVD-AFC. Results are expressed as fold increase relative to untreated cells (n = 3). The relative fluorescence unit (RFU) of caspase-3 activity was normalized for the protein content of each extract. ⁎Statistically significant with p b 0.001.
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Results Down-regulation of cell surface CA125 selectively sensitizes OVCAR3 cells to genotoxic drugs CA125 positive OVCAR3 cells were transfected to stably expressed anti-MUC16 scFvs or a control scFv. Two independent clones (1:9#7 and 1:9#9) were selected. Immunofluorescence analysis with anti-cmyc demonstrated expression of the c-myc tagged anti-MUC16 scFvs in a pattern that suggested ER localization (Fig. 1A). Indeed, colocalization of the MUC16 scFv anti-calreticulin, an ER-resident protein, supported targeting of MUC16 scFv to the ER (data not shown). The anti-MUC16 scFv was derived from the hybridoma cell line VK-8 [16], which expresses a monoclonal antibody against the extracellular domain of CA125 tumor antigen. The entrapment of CA125 mediated by intracellular expression of CA125-specific scFvs targeted within the secretion pathway was expected to prevent localization of CA125 at the cell surface. Indeed, the stable expression of the anti-MUC16 scFv induces a 10- to 100-fold decrease of CA125 expression at the cell surface as shown by flow cytometry (Fig. 1B). Immunofluorescence studies with anti-CA125 antibodies in nonpermeabilized cells, confirmed the down-regulation of cell surface CA125 in 1:9#7 and 1:9#9 sublines (Fig. 1C). Because CA125 tumor antigen is usually released in conditioned media of cultured cells expressing this antigen, we also determined whether the shedding of CA125 was affected in our stable transfectants. Conditioned media from confluent cell cultures was collected and quantitatively analyzed using an ELISA-based method. Fig. 1D shows that the parental cells and the control scFv (Ctrl scFv) transfectant both shed almost similar
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amounts of CA125 in the culture media. However, the 1.9#7 and 1:9#9 transfectants showed a drastic reduction of CA125 release (p b 0.05). Altogether, these data suggest that entrapment of the CA125 protein prevents its cell surface localization and leads to its degradation within the ER. Of note, FACS analysis and microarray analysis have shown that MUC1 expression is not altered by the down-regulation of MUC16 expression (data not shown). Cisplatin and paclitaxel are two drugs used in first-line therapy for the treatment of EOC. Cisplatin is a genotoxic drug that produces DNA damage and paclitaxel is an inhibitor of microtubule depolarization. Although their mechanism of action differs, both drugs ultimately induce the apoptotic cascade leading to cell death. We thus assessed the effect of MUC16 down-regulation at the cell surface on in vitro sensitivity of NIH:OVCAR3 cells. The parental NIH:OVCAR3 cell line and cells stably expressing the anti-MUC16 scFv (1:9#7 and 1:9#9) or an irrelevant MUC16 non-binding scFv (Ctrl scFv) were exposed to increasing concentrations of cisplatin and paclitaxel for 48 h. The fold differences were calculated at the point where 50% cytotoxicity was observed for the controls versus cells expressing anti-MUC16 scFvs. Cisplatin cytotoxicity was approximately 5-fold greater when MUC16 cell surface expression was down-regulated whereas the paclitaxel sensitivity remained unchanged (Fig. 2A). Next, we examined the sensitivity of NIH:OVCAR3 cells expressing the control scFv or the anti-MUC16 scFvs to a number of genotoxic drugs versus drugs that affect microtubule assembly. Drug cytotoxicity was significantly (p b 0.001) enhanced in anti-MUC16 scFv expressing cells for all genotoxic agents tested (cyclophosphamide, doxorubicine, etoposide) but not for taxol and vinorelbine, which affect microtubule assembly (Fig. 2B).
Fig. 4. Effect of ectopic expression of MUC16-CTD on cisplatin and paclitaxel sensitivity in SKOV3 cells. (A) Lysates were subjected to immunoblot analysis with anti-c-myc and antitubulin. (B) Cells were incubated with anti-c-myc and propidium iodine to stain nuclei, and expression of MUC16-CTD was analyzed by immunofluorescence. (C) Cells were treated with cisplatin (CDDP) (1,5 μM) or paclitaxel (10 nM) for 96 h and cell viability was determined by XTT assay. All experiments were performed in triplicate and are representative of three independent experiments. Values represent the mean ± SEM. ⁎Statistically significant with p b 0,01.
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Down-regulation of cell surface CA125 increases cisplatin-mediated apoptosis and caspases activation Treatment of tumor cells with DNA-damaging agents such as cisplatin is associated with activation of the intrinsic apoptotic pathway. We examined the effect of MUC16 cell surface downregulation on cisplatin-induced apoptosis. Cells were treated with cisplatin (1,5 μM), harvested 96 h later, and analyzed for sub-G1 DNA content. After cisplatin treatment, the percentage of apoptotic cells in the clones expressing the anti-MUC16 scFv increased to 35–40% whereas control populations (parental NIH:OVCAR3 and Ctrl scFv) only increased to 10% (Fig. 3A). We next assessed caspase activity in response to MUC16 down-regulation and cisplatin treatment. Caspase-9 and caspase-3 activation, as monitored by cleavage of proisoforms, was enhanced by cisplatin in cells expressing the antiMUC16 scFv (1:9#7 and 1:9#9) as compared to cells expressing the control scFv (Ctrl scFv) (Fig. 3B). Down-regulation of MUC16 cell surface expression by itself did not alter the expression levels of procaspase-9, pro-caspase-3 and PARP in either control cells, clone 1:9#7 or clone 1:9#9 (data not shown). Similarly, the basal levels of the apoptotic regulatory proteins Bcl-2, Bcl-XL, Bax and XIAP were not affected by down-regulation of MUC16. Because the antibody we used does not properly detect the active fragments of caspase-3, caspase-3 activity was measured using a fluorogenic assay. Caspase-3 activity was 3-fold lower in the NIH:OVCAR3 cells expressing the anti-MUC16 scFv compared to the control cells (Fig. 3C). Next, we examined the effect of MUC16 down-regulation with taxol on caspase-3 activity in the NIH:OVCAR3 cell populations. In contrast to cisplatin, the combination of paclitaxel and MUC16 down-regulation did not affect the levels of caspase-3 activity when compared to control NIH: OVCAR3 cells (data not shown). Thus, down-regulation of MUC16 alone does not increase apoptosis and caspases activation, however down-regulation of MUC16 enhances the cisplatin- but not taxolmediated apoptosis and caspases activation. MUC16 C-terminal domain selectively decreases the sensitivity to cisplatin To further confirm the selectivity of MUC16 on cisplatin sensitivity, the CA125-negative SKOV3 cell line was transfected to stably express an empty vector (SKOV3-EV) or the C-terminal domain of CA125 (MUC16-CTD) comprising a small extracellular domain of CA125, its transmembrane domain and a His6- and c-myc tagged cytoplasmic tail (SKOV3-CTD). The MUC16-CTD protein was detectable in SKOV3CTD but not in SKOV3-EV and SKOV3 cells as shown by immunoblot with anti-c-myc antibody (Fig. 4A). MUC16-CTD protein showed a pattern consistent with a diffuse distribution at the cell membrane by immunofluorescence (Fig. 4B). SKOV3 cells expressing the empty vector or the MUC16-CTD were treated with cisplatin or paclitaxel. Treatment with cisplatin significantly increased cell viability of MUC16-CTD-expressing SKOV3 cells when compared to SKOV3 or empty vector-expressing cells (Fig. 4C). In contrast, there was no difference in cell viability in the different SKOV3 cell population treated with paclitaxel. These findings further support the fact that MUC16 selectively affect the sensitivity of ovarian cancer cells to genotoxic agents. MUC16 C-terminal domain attenuates cisplatin-apoptosis in SKOV3 cells We examined the effect of MUC16-CTD exogenous expression on cisplatin-induced caspases activation in SKOV3 cells. We first assessed the basal expression levels of pro-caspase-9, pro-caspase3, PARP, Bcl-2, Bcl-XL, Bax and XIAP to ensure that their expression was not altered by MUC16 CTD. Western blot analysis showed that expression of the proteins was similar in parental SKOV3, empty vector-expressing SKOV3 and MUC16-CTD-expressing SKOV3 cells
Fig. 5. Ectopic expression of MUC16-CTD attenuates cisplatin-induced caspase activation in SKOV3 cells. (A) Western blot analysis of whole-cell lysates from cells treated with cisplatin (1,5 μM) for 48 h probed with anti-caspase-9, anti-caspase-3, anti-PARP and anti-tubulin (to ensure equal loading). (B) Lysates from cells treated with cisplatin (1,5 μM) were assayed for caspase-3 activity by monitoring the fluorescence produced by hydrolysis of caspase-3 substrate DEVD-AFC. Results are expressed as fold increase relative to untreated cells (n = 3). The relative fluorescence unit (RFU) of caspase-3 activity was normalized for the protein content of each extract. ⁎Statistically significant with p b 0.01.
(data not shown). As shown in Fig. 5A, following cisplatin treatment, the levels of pro-caspase-9, pro-caspase-3 and full length PARP were higher in MUC16-CTD-expressing SKOV3 indicating decreased caspases activation in these cells compared to control cells. Consistent with these results, caspase-3 activity was also reduced in MUC16-CTD-expressing SKOV3 cells following cisplatin treatment indicating that expression of MUC16-CTD attenuates cisplatinmediated caspase activation. Discussion The efficacy of treatment for EOC is frequently impaired by the rapid appearance to resistance to chemotherapeutic drugs. Therefore, the identification of proteins that may impact tumor cell drug sensitivity is of importance. The major findings from our study are that MUC16 modulates the sensitivity of ovarian cancer cells to genotoxic agents but not to drugs that affect microtubules. To strongly support this conclusion, we used two approaches. We down-regulated MUC16 in epithelial ovarian carcinoma cells that endogenously expressed this protein and we stably express MUC16CTD in MUC16-negative ovarian cancer cells. Indeed, we showed that scFv-mediated down-regulation of cell surface MUC16 promotes cisplatin-induced apoptosis and caspase activation in NIH:OVCAR3 cells. These findings were not restricted to cisplatin since down-
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regulation of cell surface MUC16 also sensitizes to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide. Conversely, ectopic expression of MUC16 C-terminal domain decreases cisplatin-mediated caspase activation in SKOV3 cells. However, the down-regulation of cell surface MUC16 or the ectopic expression of MUC16-CTD has no effect on paclitaxel sensitivity. Although CA125/MUC16 is a well-established marker for ovarian cancer progression [8], little is known about its biological functions. These findings therefore establish a novel role for CA125/MUC16 in EOC cells. Our data also suggest that the MUC16-CTD is sufficient to promote cisplatin resistance in SKOV3 cells. The findings that downregulation of CA125/MUC16 cell surface expression or ectopic expression of MUC16-CTD do not display alteration of Bax, Bcl-2, Bcl-XL or XIAP indicate that modulation of MUC16 affects genotoxic drug-induced apoptosis by other mechanisms. CA125 is a large membrane-bound mucin overexpressed in most human epithelial ovarian cancer [7]. The C-terminal domain of the protein is composed of a unique region, a potential proteolytic cleavage site, a transmembrane domain and a short cytoplasmic tail which appears to be sufficient to attenuate cisplatin-induced caspases activation. The cytoplasmic tails of membrane-bound mucins such as MUC1 have been shown to be involved in signaling events that contribute to resistance to drug-induced apoptosis. Although the cytoplasmic tails of MUC16 and MUC1 do not share sequence homology [17], it is not entirely unexpected that the MUC16-CTD can promote cisplatin resistance. The cytoplasmic tail of MUC16 harbors several potential phosphorylation sites that may serve to activate different signaling cascades including proteins that regulate the apoptotic cascade. Proteins that interact with MUC16 cytoplasmic tail, however, remain to be identified. Interestingly, the MUC1 oncoprotein has been shown to activate FOXO3a transcription factor and knockdown of MUC1 inactivates FOXO3A thereby increasing the sensitivity of cells to oxidative stress [18]. The phosphorylation of FOXO3a blocks its translocation to the nucleus and consequently FOXO3a-mediated transactivation of gene transcription [19]. Importantly, FOXO3a contributes to DNA repair in response to genotoxic stress by activating Gadd45a expression [20]. In the presence of irreparable damage, FOXO3a contributes to an apoptotic response. It is conceivable that MUC16 knockdown, like MUC1 knockdown, could inactivate FOXO3a, consequently decreasing DNA repair, increasing DNA damage induced by genotoxic drugs and selectively sensitizing tumor cells to these drugs. In support of this hypothesis, we found that MUC16 cell surface down-regulation decreases FOXO3a expression levels in NIH:OVCAR3 cells and decreases FOXO3a nuclear localization (unpublished data). These results could explain, at least in part, the fact that MUC16 selectively modulates the sensitivity to genotoxic drugs in EOC cells. It is interesting that MUC1 overexpression confers resistance to genotoxic drugs [21,22] and that, conversely, knocking down of endogenous MUC1 sensitizes cells to drug-induced apoptosis [21]. These observations are in concert with our findings with MUC16. They further support its implication in the modulation of genotoxic drug sensitivity. Overexpression of MUC16 may therefore confer a selective advantage to EOC cells under conditions of genotoxic stress. Downregulating MUC16 may represent an alternative approach for sensitizing EOC cells to genotoxic therapeutic modalities such as platinum-based therapies.
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Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by an internal grant from Université de Sherbrooke (A.P. and C.R.) and by the National Cancer Institute with funds from the Canadian Cancer Society to C.R. (#011225 and #014263).
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