Inhibition of Phosphorylation of a Forkhead Transcription Factor ...

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Endocrinology 145(4):2014 –2022 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-1199

Inhibition of Phosphorylation of a Forkhead Transcription Factor Sensitizes Human Ovarian Cancer Cells to Cisplatin EMI ARIMOTO-ISHIDA, MASAHIDE OHMICHI, SEIJI MABUCHI, TOSHIFUMI TAKAHASHI, CHIKA OHSHIMA, JUN HAYAKAWA, AKIKO KIMURA, KAZUHIRO TAKAHASHI, YUKIHIRO NISHIO, MASAHIRO SAKATA, HIROHISA KURACHI, KEIICHI TASAKA, AND YUJI MURATA Department of Obstetrics and Gynecology (E.A.-I., M.O., S.M., J.H., A.K., Y.N., M.S., K.Tas., Y.M.), Osaka University Medical School, Osaka 565-0871, Japan; Department of Obstetrics and Gynecology (M.O., T.T., K.Tak., H.K.) and Division of Nursing (C.O.), Yamagata University, School of Medicine, Yamagata 990-9585, Japan The Forkhead family transcription factor FKHRL1 is an inducer of apoptosis in its unphosphorylated form and was recently reported to be a substrate of Akt kinase. We studied the roles of FKHRL1 in both cisplatin-resistant Caov-3 (a papillary adenocarcinoma cell line) and cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of Caov-3 cells but not A2780 cells with cisplatin transiently stimulated the phosphorylation of FKHRL1. Transfection experiments revealed that a kinase inactive-mutant of Akt or a triple mutant (TM) of FKHRL1, in which all three of the putative Akt phosphorylation sites were converted to alanine, was unable to phosphorylate the FKHRL1 protein in cells treated with cisplatin. Because the phosphorylated form of FKHRL1 is known to be localized in the cytoplasm, we examined whether cisplatininduced phosphorylation of FKHRL1 might have an effect on the subcellular distribution of FKHRL1. Cisplatin induced the

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HE MAJORITY OF patients with ovarian cancer require treatment with cytotoxic chemotherapy. It is now well established that platinum agents (cisplatin or carboplatin) are the most important drugs to be included in first-line regimens. More recently, randomized trials have confirmed the benefit of the addition of taxanes to platinum-containing regimens. Despite the high response rate to first-line chemotherapy, the majority of patients with advanced ovarian cancer relapse and become candidates for further chemotherapy, which can palliate symptoms and improve survival even in recurrent disease. Therefore, clarifying the mechanism of resistance to chemotherapeutic drugs is very important. Eukaryotes have a complex yet conserved response to DNA damage, including changes in cell cycle kinetics (1) and transcriptional induction of multiple genes (2). We recently demonstrated that both the ERK and c-Jun N-terminal kinase (JNK) cascades are independently involved in maintaining the cell viability after cisplatin treatment and may share a crucial downstream step such as the formation of an active Abbreviations: GSK, Glycogen synthase kinase; GST, glutathioneS-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; PI-3K, phosphatidylinositol 3-kinase; Ser, serine; TM, triple mutant. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

localization of FKHRL1 in the cytoplasm in Caov-3 cells but not in A2790 cells. Moreover, cisplatin induced the association of 14 –3-3 protein with phosphorylated-FKHRL1 in Caov-3 cells but not in A2790 cells. Because the unphosphorylated form of FKHRL1 binds the Fas ligand promoter, thereby inducing apoptosis, we further examined the effect of the phosphorylation status of FKHRL1 on the activity of the Fas ligand promoter in the presence of cisplatin. Transfection with the kinase-inactive mutant of Akt or TM of FKHRL1 induced the activity of the Fas ligand promoter in Caov-3 cells. Moreover, exogenous expression of TM of FKHRL1 in Caov-3 cells decreased the cell viability after treatment with cisplatin. Our findings suggest that cisplatin causes the phosphorylation of FKHRL1 via a phosphatidylinositol 3-kinase/Akt cascade, and inhibition of this cascade sensitizes ovarian cancer cells to cisplatin. (Endocrinology 145: 2014 –2022, 2004)

c-Jun/c-Fos complex (3). Moreover, we showed that both ERK-dependent BAD phosphorylation at serine (Ser)-112 and phosphatidylinositol 3-kinase (PI-3K)-Akt-dependent BAD phosphorylation at Ser-136 seem to be also involved in the repair of cisplatin treatment (4). Akt phosphorylates and inactivates components of the apoptotic machinery, including BAD and caspase-9, in a transcription-independent manner. The question arises: are there any transcription-dependent mechanisms in the enhancement of cell survival by the PI-3K-Akt cascade induced by cisplatin? It was recently reported that Akt promotes cell survival by phosphorylating a Forkhead transcription factor (5). Three members of the mammalian Forkhead family of transcription factors termed FKHRL1, FKHR, and AFX have been isolated thus far (6 –9). This subfamily of transcription factors possess sequence homology to DAF16, which is a transcription factor and a target of the Akt cascade expressed in Caenorhabditis elegans (9, 10). It has been shown that the phosphorylation of FKHRL1 by Akt inhibits the stimulatory effect of FKHRL1 on the transcription of genes that encode death-activating proteins such as the Fas ligand, leading to the survival of target cells (5). Indeed, it was reported that the Fas ligand promoter contains within its regulatory region three Forkhead-responsive elements that bind FKHRL1 (5). It was reported that the expression of Fas ligand correlates with cisplatin-induced apoptosis (11). More recently, a Forkhead transcription factor

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Arimoto-Ishida et al. • Effects of Cisplatin on Forkhead Phosphorylation

was reported to be involved in chemotherapeutic druginduced apoptosis (12) and regulate the resistance of cells to stress by inducing DNA repair (13). These considerations led us to examine whether the phosphorylation status of FKHRL1 is involved in the sensitivity of cells to cisplatin. In the present study, we show that cisplatin induces the phosphorylation of FKHRL1, the cytoplasmic localization of FKHRL1 via the PI-3K/Akt cascade, and the association of 14 –3-3 protein with phosphorylatedFKHRL1 in cisplatin-resistant Caov-3 cells but not in cisplatin-sensitive A2780 cells. Moreover, expression of a triple mutant (TM) of FKHRL1 in which all three of the putative Akt phosphorylation sites were converted to alanine in Caov-3 cells induced the activity of the Fas ligand promoter and decreased the cell viability after treatment with cisplatin. Materials and Methods Materials Wortmannin was purchased from Sigma Chemical Co. (St. Louis, MO). LY294002 was purchased from Calbiochem (La Jolla, CA). Geneticin was purchased from Life Technologies (Grand Island, NY). Enhanced chemiluminescence Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Antibodies against phospho-FKHRL1 (Thr32), phospho-FKHRL1 (Ser253), and total FKHRL1 were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). An Akt kinase assay kit including glycogen synthase kinase (GSK)-3 fusion protein and a phospho-specific GSK3␣/␤ antibody was obtained from New England Biolabs (Beverly, MA). A Cell Titer 96 cell proliferation assay kit was obtained from Promega (Madison, WI). Hoechst 33258 was obtained from Molecular Probes (Eugene, OR).

Cell cultures Human ovarian papillary adenocarcinoma cell line Caov-3 was obtained from American Type Culture Collection (Manassas, VA). The human ovarian cancer A2780 cell line derived from a patient before treatment and the cisplatin-resistant cell line 2780CP were kindly provided by Dr. T. Tsuruo (Institute of Molecular and Cellular Biosciences, Tokyo, Japan) and Drs. R. F. Ozols and T. C. Hamilton (National Cancer Institute, Bethesda, MD) (14). The cells were cultured at 37 C in DMEM with 10% fetal bovine serum in a water-saturated atmosphere of 95% O2 and 5% CO2.

Constructs The plasmid encoding the hemagglutinin (HA)-tagged form of kinase-dead Akt (DN-Akt) (15), the plasmids encoding the HA-tagged wild-type FKHRL1 (WT-HA-FKHRL1) and triple mutant of FKHRL1 (5) in which all three of the putative Akt phosphorylation sites (T32, S253, and S315) were converted to alanine (TM-HA-FKHRL1), and FHRE-Luc plasmid in which the Fas ligand promoter containing the three FKHRL1 binding sites was inserted 5⬘ of a basal promoter controlling the expression of the luciferase gene were kind gifts from Dr. M. E. Greenberg and Dr. S. R. Datta (Harvard Medical School, Boston, MA). The glutathione-S-transferase (GST)-14-3-3␪ construct was a kind gift from Dr. Takashi Tsuruo (Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan) (16).

Clone selection Caov-3 and A2780 cells were transfected for 12 h in 6-well tissue culture plates with 2 ␮g of the empty vector (pECE), WT-HA-FKHRL1, or TM-HA-FKHRL1 and the neomycin resistance gene using Lipofectamine plus (Invitrogen Corp., Carlsbad, CA) (17). Clonal selection was performed by adding geneticin to the medium at 200 ␮g/ml final concentration 2 d after the transfection. After 3 wk, several clones were isolated using cloning rings. Selected clones were then maintained in

Endocrinology, April 2004, 145(4):2014 –2022 2015

medium supplemented with geneticin (100 ␮g/ml), and only lowpassage cells (P ⬍ 10) were used for the experiments described here.

Assay of Akt kinase activity Cells were incubated in the absence of serum for 16 h and then treated with various materials. They were then washed twice with PBS and lysed in ice-cold lysis buffer [20 mm Tris (pH 7.4), 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 2.5 mm sodium pyrophosphate, 1 mm ␤-glycerol phosphate, 1 mm sodium orthovanadate, 1 ␮g/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride]. The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Two hundred fifty micrograms of protein from the lysate samples were incubated with gentle rocking at 4 C overnight with immobilized Akt antibody cross-linked to agarose hydrazide beads. After Akt was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were washed twice with lysis buffer and twice with kinase assay buffer [25 mm Tris (pH 7.5), 10 mm MgCl2, 5 mm ␤-glycerol phosphate, 0.1 mm sodium orthovanadate, and 2 mm dithiothreitol] and then resuspended in 40 ␮l of kinase assay buffer containing 200 ␮m ATP and 1 ␮g GSK-3␣ fusion protein. The kinase reaction was allowed to proceed at 30 C for 30 min and stopped by the addition of Laemmli sodium dodecyl sulfate sample buffer (18). Reaction products were resolved by 10% SDS-PAGE followed by Western blotting with anti-phospho-GSK-3␣ antibody. For analysis of the total amount of Akt, 250 ␮g of protein from the lysate samples was resolved by 10% SDS-PAGE, followed by Western blotting with anti-Akt antibody.

Phosphorylation of Forkhead protein Cells cultured in 100-mm dishes were transfected with 4 ␮g WTHA-FKHRL1 or TM-HA-FKHRL1 with or without DN-Akt using Lipofectamine plus (Life Technologies). At 24 h after transfection, serumdeprived cells were treated with cisplatin for the various times. They were then washed twice with PBS and lysed in ice-cold HNTG buffer [50 mm HEPES (pH 7.5), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EDTA, 10 mm sodium pyrophosphate, 100 ␮m sodium orthovanadate, 100 mm NaF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride] (19). The lysate samples were immunoprecipitated with anti-HA antibody. Immune complexes were precipitated with protein A Sepharose, and the isolated proteins were analyzed by SDS-PAGE. Transfer to nitrocellulose, Western blotting with a mixture of anti-phospho-FKHRL1 (Thr32) and anti-phosphoFKHRL1 (Ser 253) antibodies or anti-FKHRL1 antibody, and washings were performed as described elsewhere (20). For analysis of the effect of expression of various forms of FKHRL1 on FKHRL1 expression or phosphorylation, empty vector (pECE)-, WTHA-FKHRL1-, and TM-HA-FKHRL1 expressing Caov-3 cells grown in 100-mm dishes were treated with 1 ␮m cisplatin for 3 h. The lysate samples were resolved by SDS-PAGE, followed by Western blotting with anti-HA, or the lysate samples were immunoprecipitated with anti-HA antibody and immune complexes were resolved by SDS-PAGE, followed by Western blotting with phospho-FKHRL1 antibody, as described above.

Fluorescence microscopy Cells were grown on glass cover slips in 6-well dishes. The cells were transfected with WT-HA-FKHRL1 plasmid for 24 h. Then the cells were incubated with or without 1000 ␮m cisplatin for 3 h in the absence or presence of 50 ␮m LY294002. The cells were fixed with 10% formalin for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked with 3% BSA for 1 h. Anti-HA antibody and Alexa Fluor secondary antibodies were used at 2 ␮g/␮l in blocking solution. Cells were conterstained with 10 mmol/liter Hoechst 33258 to visualize the nucleus. Samples were mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA). The cells were examined using fluorescence microscopy.

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Endocrinology, April 2004, 145(4):2014 –2022

GST-14-3-3␪ pull-down assay Caov-3 cells were transfected with WT-HA-FKHRL1 for 24 h using Lipofectamine plus according to the manufacturer’s protocol. Cells were treated with 1000 ␮m cisplatin and then harvested. Total lysates from the Caov-3 cells were prepared with HNTG lysis buffer. Portions of the lysates were preincubated with GST-beads and the resulting supernatants were incubated with GST-14-3-3␪ fusion protein prepared as described previously (16). The beads were washed with the lysis buffer and the eluted proteins were subjected to SDS-PAGE and transferred to nitrocellulose membranes. The specific signals were detected by Western blot analysis with anti-phospho-FKHRL1 antibody as the primary antibody.


medium with fresh medium. The number of surviving cells was determined 5 d later by determination of the A590 nm of the dissolved formazan product after addition of 3-[4,5,dimethylthiazol-2-yl]5-[3-carboxymethoxy-phenyl]2-[4-sulfophenyl]2H-tetrazolium (inner salt) for 1 h as described by the manufacturer (Promega). All experiments were carried out in quadruplicate and the viability was expressed as the ratio of the number of viable cells with cisplatin treatment to that without treatment.

Statistics Statistical analysis was performed using one-way ANOVA followed by Fisher’s least significant difference test, and P ⬍ 0.05 was considered significant. Data are expressed as the mean ⫾ se

Luciferase assay FHRE-Luc reporter plasmid was transiently transfected into cells for 24 h using Lipofectamine plus according to the manufacturer’s protocol. Cells were harvested and subjected to luciferase assays using the luciferase assay system (Promega). A plasmid expressing the bacterial ␤galactosidase gene was also cotransfected in each experiment to serve as an internal control for transfection efficiency as described elsewhere (21).

Cytotoxicity Cell viability (3) was assessed by the addition of cisplatin for 1 h 1 d after seeding test cells into 96-well plates followed by replacement of the

Results Phosphorylation of FKHRL1 in an Akt-dependent manner

To evaluate whether Akt is activated by only the DNAdamaging cisplatin isomer in Caov-3 human ovarian cancer cells, which are resistant to cisplatin, the cultured cells were exposed to 1000 ␮m cisplatin or 1000 ␮m transplatin for 3 h (Fig. 1A). It is known that cisplatin but not transplatin forms covalent cross-links between the N7 position of adjacent guanine or adenine-guanine residues (22). Akt activity was

FIG. 1. Cisplatin induces the phosphorylation of FKHRL1 in an Akt-dependent manner. Cells were grown in 100-mm dishes. A, Caov-3 cells were pretreated with or without 100 nM wortmannin for 15 min, followed by treatment with 1000 ␮M cisplatin for 3 h (lanes 2 and 3) or 1000 ␮M transplatin for 3 h (lane 4). Akt activity (middle panel) and the total amount of Akt (lower panel) were analyzed as described in Materials and Methods. Relative densitometric units of the phospho-GSK3␣ bands are shown in the upper panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ⫾ SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P ⬍ 0.01. The positions of molecular weight markers are noted on the left. B, Caov-3 cells were transfected with WT-HA-FKHRL1, WT-HA-FKHRL1⫹DN-Akt, or TM-HA-FKHRL1. C, A2780 cells were transfected with WT-HA-FKHRL1. After 72 h, cells were treated with 1000 ␮M cisplatin for the indicated times. Phosphorylation of FKHRL1 (middle panel) and the total amount of FKHRL1 (lower panel) were analyzed as described in Materials and Methods. Relative densitometric units of the phospho-FKHRL1 bands are shown in the upper panel, with the density of the control bands set arbitrarily at 1.0. Values shown represent the mean ⫾ SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P ⬍ 0.01. The positions of molecular weight markers are noted on the left.


analyzed by monitoring the phosphorylation of GSK3␣, which is a substrate of Akt. Treatment of cells with transplatin had no apparent effect on Akt activation (Fig. 1A, lane 4), whereas cisplatin clearly induced Akt activation (Fig. 1A, lane 2). Because Akt is an effector of survival signaling downstream from PI-3K, we next determined whether stimulation of cells with cisplatin could increase the activity of Akt through a PI-3K-dependent mechanism. Cells were stimulated with cisplatin in the presence of wortmannin, a PI-3K inhibitor, and the kinase activity of Akt was assayed. The induction of Akt activity by cisplatin was inhibited by wortmannin (Fig. 1A, lane 3). These results confirm our previous data (4) that only the DNA-damaging cisplatin isomer activates Akt activity through a PI-3K-dependent mechanism. It was recently reported that Akt promotes cell survival by phosphorylating a Forkhead transcription factor (5). Therefore, we next examined the effect of cisplatin on the phosphorylation of FKHRL1. HA epitope-tagged wild-type FKHRL1 (WT-HA-FKHRL1) was transfected into Caov-3 cells (Fig. 1B, lanes 1– 4). Cultured cells were exposed to 1000 ␮m cisplatin for the indicated times. Stimulation of the phosphorylation of FKHRL1 by cisplatin in Caov-3 cells was detected at 1 h, reached a peak at 3 h, and declined thereafter (Fig. 1B, lanes 1– 4). Because FKHRL1 is known to be a substrate of Akt, we examined whether cisplatin induces phosphorylation of FKHRL1 via the Akt cascade. Cisplatin failed to stimulate the phosphorylation of FKHRL1 protein in cells cotransfected with a kinase-inactive mutant of Akt and WT-


HA-FLHRL1 (Fig. 1B, lane 5). In addition, in cells expressing a TM of FKHRL1 in which all three of the putative Akt phosphorylation sites (T32, S253, and S315) were converted to alanine, there was no detectable stimulation of phosphorylation by cisplatin (Fig. 1B, lane 6). These results indicate that cisplatin induced the phosphorylation of FKHRL1 through an Akt-dependent mechanism. Moreover, we examined the phosphorylation status of FKHRL1 in A2780 cells, which are sensitive to cisplatin. Cisplatin did not induce the phosphorylation of FKHRL1 in A2780 cells (Fig. 1C), suggesting the possibility that the phosphorylation status of FKHRL1 might be involved in the sensitivity of cells to cisplatin. Cisplatin-induced localization of FKHRL1 in the cytoplasm

Whereas the unphosphorylated form of FKHRL1 is known to be localized within the nucleus, the phosphorylated form of FKHRL1 produced by the PI-3K/Akt cascade is known to be excluded from the nucleus and detected in the cytoplasm (5). Therefore, we examined the effect of cisplatin on the subcellular distribution of FKHRL1. We transfected WT-HAFKHRL1 into cells and examined the FKHRL1 subcellular distribution. When Caov-3 cells were treated with cisplatin, wild-type FKHRL1 was largely excluded from the nucleus and was detected in the cytoplasm (Fig. 2B, left panel). By contrast, when the endogenous PI-3K/Akt cascade was inhibited by treatment with LY294002, the cisplatin-induced

FIG. 2. Cisplatin-induced localization of FKHRL1 within the cytoplasm is Akt dependent. Caov-3 cells (left panel) and A2780 cells (right panel) were transfected with WT-HA-FKHRL1. After transfection, the cells were incubated with 1000 ␮M cisplatin for 3 h in the absence (B) or presence of 1 ␮M LY249002 (C). Cells were fixed and subjected to indirect immunofluorescence staining. The expression of HA is indicated by green coloration. Nuclei were stained with Hoechst 33258. Bar, 10 ␮M.

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localization of FKHRL1 in the cytoplasm was abolished (Fig. 2C, left panel). On the other hand, wild-type FKHRL1 was not excluded from the nucleus in A2780 cells (Fig. 2B, right panel), in which cisplatin did not induce the phosphorylation of FKHRL1 (Fig. 1C). These results indicate that cisplatininduced cytoplasmic localization of FKHRL1 through an Akt-dependent mechanism in Caov-3 cells, but not in A2780 cells, and the localization of FKHRL1 may influence the sensitivity of cells to cisplatin. Cisplatin induces the association of 14-3-3 protein with phosphorylated-FKHRL1

It has been reported that phosphorylation of Forkhead family transcription factors triggers their nuclear exit and binding to 14-3-3 proteins and inhibits transactivation in mammalian cells (5). Therefore, we examined whether cisplatin induces the association of 14-3-3 protein with phosphorylated-FKHRL1. Caov-3 cells (Fig. 3A) or A2780 cells (Fig. 3B) were transfected with WT-HA-FKHRL1 and then treated with cisplatin for the indicated times and used to prepare cell lysates that were incubated with GST-14-3-3 fusion protein immobilized on glutathione-Sepharose, followed by Western blotting with anti-phospho-FKHRL1 antibody. Although cisplatin did not induce the association of 14-3-3 protein with phosphorylated-FKHRL1 in A2780 cells (Fig. 3B), cisplatin induced the association of 14-3-3 protein with phosphorylated-FKHRL1 in Caov-3 cells, with a peak at 1 h, and declined thereafter (Fig. 3A).


Cisplatin-induced activity of the Fas ligand promoter

It was reported that the Fas ligand promoter contains within its regulatory region three Forkhead-responsive elements that bind FKHRL1 (5). We further examined the effect of the phosphorylation status of FKHRL1 on the activity of the Fas ligand promoter. We confirmed that the cisplatininduced phosphorylation of FKHRL1 was inhibited by transfection with either the kinase-inactive mutant of Akt (Fig. 1B, lane 5) or TM of FKHRL1 (Fig. 1B, lane 6). Using a construct in which 2.5 kb of the Fas ligand promoter was fused to the luciferase reporter gene, we found that transfection with the kinase-inactive mutant of Akt (Fig. 4A) or TM of FKHRL1 (Fig. 4B) induced the activity of the Fas ligand promoter in the presence of cisplatin but not in the absence of cisplatin in both Caov-3 cells (Fig. 4, top panel) and 2780CP cells (Fig. 4, middle panel), which are known to be resistant to cisplatin (14), whereas transfection with WT-HA-FKHRL1 had no effect on the activity of the Fas ligand promoter in either the absence or presence of cisplatin in both Caov-3 cells (Fig. 4, top panel) and 2780CP cells (Fig. 4, middle panel). On the other hand, transfection with the kinase-inactive mutant of Akt (Fig. 4A) or the TM of FKHRL1 (Fig. 4B) had no effect on the activity of the Fas ligand promoter in either the absence or presence of cisplatin in A2780 cells (Fig. 4, bottom panel). Thus, these results indicate that cisplatin induced the activity of the Fas ligand promoter when FKHRL1 was in the unphosphorylated form in cisplatin-resistant cells but not in cisplatinsensitive cells. TM of FKHRL1 sensitizes Caov-3 cells to cisplatin

FIG. 3. Cisplatin induces the association of 14-3-3 protein with phosphorylated-FKHRL1. Caov-3 (A) or A2780 (B) cells were transfected with WT-HA-FKHRL1. After transfection, cells were incubated with 1000 ␮M cisplatin for the indicated times. The GST 14-3-3␪ fusion protein was immobilized on glutathione-Sepharose and incubated with the cell lysates. After extensive washing, the bound fraction was analyzed by Western blotting with anti-phospho FKHRL1 antibody. Equal loading of each GST fusion protein was confirmed by Coomassie blue staining of the SDS-PAGE gel (data not shown). The positions of molecular weight markers are noted on the left. Significant differences are indicated by asterisks. **, P ⬍ 0.01.

To determine whether the phosphorylation of FKHRL1 is necessary for cell survival signaling after cisplatin-induced DNA damage, the effect of cisplatin treatment on the viability of a cell clone expressing the triple mutant of FKHRL1 (TM-HA-FKHRL1) was compared with that of an emptyvector-expressing control line (pECE) in both Caov-3 and A2780 cells. We first confirmed the overexpression of ectopically expressed FKHRL1 protein products (Fig. 5A) and the negative effects of the expression of TM-HA-FKHRL1 on the phosphorylation of FKHRL1 (Fig. 5B). Although both the cell clones expressing the wild-type FKHRL1 (WT-HAFKHRL1) and TM-HA-FKHRL1 ectopically expressed FKHRL1 protein products in both Caov-3 (Fig. 5A, upper panel) and A2780 cells (Fig. 5A, lower panel), cisplatin induced the phosphorylation of FKHRL1 in cells expressing WT-HAFKHRL1 but not in cells expressing TM-HA-FKHRL1 in Caov-3 cells (Fig. 5B, upper panel), whereas cisplatin did not induce the phosphorylation of FKHRL1 in cells, even in A2780 cells expressing WT-HA-FKHRL1 (Fig. 5B, lower panel), confirming the data of Fig. 1C. The viability of the control Caov-3 cells was not affected by increasing concentrations of cisplatin of more than 100 ␮m, as we reported previously (3, 4). Further titrations revealed IC50 values of 400 and 379 ␮m for parental and empty vector-expressing Caov-3 cells, respectively (Table 1). In contrast, TM-HAFKHRL1-expressing Caov-3 cells exhibited an IC50 as low as 97 ␮m, indicating more than 3.9-fold greater sensitivity to cisplatin than the empty vector-expressing control line (Fig. 5C and Table 1). WT-HA-FKHRL1 did not affect the sensi-



FIG. 4. Cisplatin induced the activity of the Fas ligand promoter when FKHRL1 was in the unphosphorylated form. Caov-3 (top panel), 2780CP (middle panel), and A2780 (bottom panel) cells were cotransfected with luciferase reporter plasmids containing 2.5 kb of the Fas ligand promoter and cytomegalovirus-6 or DN-Akt (A) or pECE, TMFKHRL1, or WT-HA-FKHRL1 (B). After transfection, the cells were incubated with or without 1000 ␮M cisplatin. Twenty-four hours later, cell pellets were collected and used to prepare lysates that were subjected to luciferase assays. The transcriptional activity was normalized with respect to that in cells treated with empty vector taken as 1.0. Values shown represent the mean ⫾ SE from at least three separate experiments. Significant differences are indicated by asterisks. **, P ⬍ 0.01.

tivity to cisplatin, compared with that of the control line (data not shown). Thus, the sensitization to cisplatin observed in TM-HA-FKHRL1-expressing Caov-3 cells appeared to be due to interference with the phosphorylation of FKHRL1. On the other hand, the IC50 values of parental and empty vectorexpressing A2780 cells were 84 and 86 ␮m, respectively (Table 1), as we reported previously (3, 4). TM-HA-FKHRL1expressing A2780 cells exhibited an IC50 as low as 88 ␮m, indicating no greater sensitivity to cisplatin than the empty vector-expressing control line (Fig. 5D and Table 1), probably due to the inability of cisplatin to induce the phosphorylation of FKHRL1 in A2780 cells (Figs. 1C and 5B). Discussion

Resistance to cisplatin is a multifactorial phenomenon, the elements of which may be divided into three general categories: 1) reduced accumulation of cisplatin, 2) elevated levels of glutathione and metallothionein, and 3) increased DNA damage tolerance or repair (23–26). Because cisplatin acts by forming DNA-DNA cross-links (both intrastrand and interstrand) and DNA-protein crosslinks, resulting in DNA damage, the repair of the affected DNA is clearly an important mechanism of resistance to

cisplatin (27). Although the mechanism of this DNA repair is not completely clear, the intracellular signaling that modulates apoptosis may be involved in DNA repair and may be an appropriate target for strategies to overcome the resistance to chemotherapeutic DNA-damaging drugs. The sensitivity of cells to chemotherapeutic druginduced apoptosis appears to depend on the balance between proapoptotic and antiapoptotic signals. Therefore, it is possible that antiapoptotic signals such as the PI-3KAkt survival cascade are involved in the sensitivity to chemotherapeutic drugs. We reported that Akt inactivation sensitizes human ovarian cancer cells to cisplatin (4) and paclitaxel (17), suggesting that Akt inactivation could be used as a hallmark in examining the sensitivity of cells to some chemotherapeutic drugs. Forkhead family transcription factors, such as FKHR, FKHRL1, and AFX, are reported to be nuclear substrates of Akt, and phosphorylation by Akt inhibits the stimulatory effect of Forkhead family proteins on the transcription of genes that encode death-activating proteins, as in the case of other Akt substrates such as BAD (15) and Raf-1 (28). Studies of the factors regulating the phosphorylation of Forkhead have been limited to serum, IGF-1/insulin, epithelial growth

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FIG. 5. Interference with phosphorylation of FKHRL1 sensitizes Caov-3 cells to cisplatin. A and B, Empty vector (pECE)-, WT-HA-FKHRL1-, or TM-HA-FKHRL1-expressing Caov-3 (upper panel) and A2780 (lower panel) cells were grown in 100-mm dishes. For analysis of the level of FKHRL1 (A), the lysate samples were analyzed by electrophoresis on 8% sodium dodecyl sulfate-polyacrylamide gels, followed by Western blotting with anti-HA antibody. For analysis of the effects of the expression of TM-HA-FKHRL1 on FKHRL1 phosphorylation (B), the cells were incubated with 1000 ␮M cisplatin for 3 h. The lysate samples were immunoprecipitated with anti-HA antibody and immune complexes were resolved by 8% SDS-PAGE, followed by Western blotting with anti-phospho-FKHRL1 antibody. The positions of molecular weight markers are noted on the left. Cell viability was assessed in empty vector (pECE)- or TM-HA-FKHRL1-expressing Caov-3 (C) and A2780 (D) cells after treatment with the indicated concentrations of cisplatin as described in Materials and Methods. Values shown represent the mean ⫾ SE from at least three separate experiments. Significant differences between pECE and TM-FKHRL1 are indicated by asterisks. *, P ⬍ 0.01. TABLE 1. Effect of TM-FKHRL1 on cisplatin-induced cytotoxicity in Caov-3 cells or A2780 cells Control, IC50 (␮M)a,c

Caov-3 A2780

400 84

Empty vector (pECE)b

TM-FKHRL1

IC50

Sensitizationd

IC50

Sensitizatione

379 86

1.1 0.98

97 88

3.9 0.98

a Control cells were analyzed in parallel with equal concentrations of cisplatin in the range 0 –1 mM in quadruplicate. b Empty vector-expressing cells were analyzed in parallel with equal concentrations of cisplatin in the range 0 –1 mM in quadruplicate. c IC50 values were determined by direct titration of viability with cisplatin as described in Materials and Methods. d Sensitization is defined as the ratio of the IC50 value for the parental cells to the IC50 value for the empty vector-expressing cells. e Sensitization is defined as the ratio of the IC50 value for the empty vector-expressing cells to the IC50 value for the TM-FKHRL1expressing cells.

factor (29), estrogen (30), or constitutively active Akt. We have provided new evidence that cisplatin-induced DNA damage induces the phosphorylation of FKHRL1 through a PI-3K/Akt-dependent mechanism. We reported that cisplatin-induced DNA damage differentially activates the JNK and ERK cascades (3) and that cisplatin-induced DNA damage also induces the phosphorylation of both BAD Ser-112 via an ERK cascade and BAD

Ser-136 via a PI-3K-protein kinase B/Akt cascade (4) in both cisplatin-resistant cells and -sensitive cells, suggesting that these cascades seem to be necessary for maintaining cell viability after the genotoxic stress of cisplatin. However, cisplatin-induced DNA damage induced the phosphorylation of FKHRL1 in cisplatin-resistant cells, but not -sensitive cells (Fig. 1). Thus, it is possible that the ability of cisplatin to regulate FKHRL1 phosphorylation (Fig. 1) and binding to its target genes, such as Fas ligand, through regulating its exclusion from the nucleus (Fig. 2) and association with 14-3-3 protein (Fig. 3), regulates the apoptosis. Thus, the phosphorylation status of FKHRL1 might be a more important and sensitive characteristic factor than the phosphorylation status of Akt itself in the mechanism of resistance to cisplatin, suggesting that the phosphorylation status of FKHRL1 after cisplatin treatment is a hallmark of sensitivity of the cells to cisplatin. However, in addition to Forkhead family transcription factors, multiple signaling molecules, such as BAD, caspase-9, GSK3, and nuclear factor ␬B, have been identified as Akt substrates, and therefore it remains unknown which downstream pathway(s) that issue from Akt are important and sensitive factor(s) in the mechanism of resistance to cisplatin. Further experiments will be necessary to address this question. The 14-3-3 proteins are a family of proteins reported to bind Forkhead transcription factors in a phosphorylation-


dependent manner and thereby sequester Forkhead transcription factors in the cytoplasm so that they are unable to regulate their target genes in the nucleus (5). Because the 14-3-3 proteins bind Forkhead transcription factors in a phosphorylation-dependent manner, both cisplatin-induced phosphorylation of FKHRL1 (Fig. 1B) and association of 143-3 protein with phosphorylated-FKHRL1 (Fig. 3A) are transient phenomena. However, cisplatin induces the association of 14-3-3 protein with phosphorylated-FKHRL1 (Fig. 3A) with a different time frame than it induces the phosphorylation of FKHRL1 (Fig. 1B). Although the exact reason for the difference of these time frames is unknown, there is a possibility that 14-3-3 protein might be tightly associated with the initial step of phosphorylation of FKHRL1, followed by maintenance of its function. Accordingly, cisplatin-induced localization of FKHRL1 in the cytoplasm was detected from 1 h (data not shown). Because inhibition of a PI-3K-Akt-BAD cascade sensitizes ovarian cancer cells to cisplatin (4), inhibition of the PI-3KAkt-FKHRL1 cascade by expression of the TM of FKHRL1, in which all three of the putative Akt phosphorylation sites (T32, S253, and S315) were converted to alanine, sensitizes ovarian cancer cells to cisplatin (Fig. 5). Because PI-3K inhibitors such as wortmannin and LY294002 block the phosphorylation of Akt, and consequently inhibit the phosphorylation of both BAD and FKHRL1, the combined use of cisplatin with PI-3K inhibitors is a new anticancer strategy. Moreover, we reported that inhibition of either of ERK or JNK cascades differentially sensitizes ovarian cancer cells to cisplatin (3). The first-line chemotherapy of ovarian cancer is the combined use of platinum agents and taxanes. Although we (17) and other groups (31) reported that both PI-3K inhibitors and MAPK kinase inhibitors also sensitize ovarian cancer cells to paclitaxel, the effect of JNK inhibitors on the sensitivity to paclitaxel has remained obscure and we are currently investigating this question. In addition, it was reported that both Akt (32) and ERK (33) cascades are involved in the invasion and metastasis of ovarian cancer cells. Overall, these findings render the combined use of cisplatin and paclitaxel with PI-3K inhibitors and/or with MAPK kinase inhibitors a promising new strategy for cancer chemotherapy that should improve the response rate and expand the usefulness of cisplatin and paclitaxel in the treatment of resistant tumors with metastatic lesions that affect a large percentage of cancer patients. Acknowledgments We are grateful to Dr. Michael E. Greenberg and Dr. Sandeep Robert Datta (Children’s Hospital and Department of Neurobiology, Harvard Medical School, Boston, MA) for the gift of DN-Akt, WT-HA-FKHRL1, TM-HA-FKHRL1, and FHRE-Luc plasmid, and Dr. Takashi Tsuruo (Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan) for providing GST-14-3-3␪ constructs. We are also grateful to Ms. Ayako Okamura and Ms. Tomoko Iwaki for technical and secretarial assistance. Received September 10, 2003. Accepted December 24, 2003. Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 56-0871, Japan. E-mail: [email protected].


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