SIRT1-Mediated FoxO1 Deacetylation Is Essential for Multidrug

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SIRT1-Mediated FoxO1 Deacetylation Is Essential for Multidrug Resistance-Associated Protein 2 Expression in Tamoxifen-Resistant Breast Cancer Cells Hoo-Kyun Choi,† Kyoung Bin Cho,‡ Nguyen Thi Thuy Phuong,†,‡ Chang Yeob Han,‡ Hyo-Kyung Han,§ Tran Thi Hien,† Hong Seok Choi,† and Keon Wook Kang*,‡ †

BK21 Project Team, College of Pharmacy, Chosun University, Gwangju 501-759, South Korea College of Pharmacy, Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, South Korea § College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 410-820, South Korea ‡

ABSTRACT: Our previous studies have shown that multidrug resistance protein 2 (MRP2) is overexpressed in tamoxifen-resistant MCF-7 breast cancer cells (TAMR-MCF-7 cells) and forkhead boxcontaining protein, O subfamily1 (FoxO1), functions as a key regulator of multidrug resistance 1 (MDR1) gene transcription. This study aimed to investigate the role of FoxO1 in regulating MRP2 gene expression in TAMR-MCF-7 cells. The proximal promoter region of the human MRP2 gene contains four putative FoxO binding sites, and MRP2 gene transcription was stimulated by FoxO1 overexpression in MCF-7 cells. Subcellular fractionation and immunoblot analyses revealed that basal MRP2 expression and nuclear levels of FoxO1 were enhanced in TAMRMCF-7 cells compared to MCF-7 cells and the enhanced MRP2 gene transcription was suppressed by FoxO1 siRNA. Because nuclear localization of FoxO1 is regulated by SIRT1 deacetylase, we were further interested in whether SIRT1 is involved in MRP2 expression. Overexpression of SIRT1 with FoxO1 potentiated the gene transcriptional activity of MRP2, and the basal activity and expression of SIRT1 was increased in TAMR-MCF-7 cells. In addition, SIRT1 inhibition reduced both the nuclear FoxO1 levels and MRP2 expression and enhanced cytotoxic effects of paclitaxel and doxorubicin in TAMR-MCF-7 cells. These results suggest that FoxO1 activation via SIRT1-mediated deacetylation is closely related with up-regulation of MRP2 in TAMR-MCF-7 cells. KEYWORDS: MRP2, SIRT1, FoxO1, tamoxifen resistant breast cancer

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antiestrogen therapy.6 We previously showed that overexpression of MRP2 occurs in TAM-resistant MCF-7 breast cancer cells (TAMR-MCF-7 cells) and suggested that the upregulation of ABC transporters plays a role in the additional acquisition of resistance to chemotherapy.7 In mammals, Forkhead bOX-containing proteins, which includes the O subfamily (FoxO) of transcription factors, consists of four isoforms.8 Transcriptional activity of FoxO factors is regulated by a shuttling system running between the nucleus and the cytoplasm, and this can be regulated by phosphorylation-dependent ubiquitination and acetylation, which affect their DNA binding activity and, consequently, control of target gene expression.9−11 FoxO proteins play a key role in regulating diverse cellular processes such as differentiation and proliferation, and their expression is frequently dysregulated in cancers.9,12,13 We recently showed that FoxO1 is consistently increased in adriamycin-resistant breast cancer

INTRODUCTION Multidrug resistance (MDR) is defined as concurrent resistance to structurally different anticancer agents. One of the most important mechanisms for MDR is the active efflux of chemotherapeutic agents through the increased expression of drug transporters.1 ATP-binding cassette (ABC) transporters are transmembrane proteins that are involved in the pumping out of various substrates across the plasma membrane. Among them, multidrug resistance-associated proteins (MRPs, ABCC subfamily) and multidrug resistance1 (MDR1, p-glycoprotein) interact with a broad range of substrates; hence their overexpression is considered to confer resistance to chemotherapeutic agents by preventing the accumulation of cytotoxic agents inside cancer cells, but its molecular mechanism has not been fully understood.2−4 Tamoxifen (TAM), a nonsteroidal antiestrogen and an orally active selective estrogen receptor modulator, has been approved for the chemoprevention of breast cancer and is the most widely used antiestrogen in estrogen receptor-positive breast cancer patients.5 Although most patients are initially responsive, the acquisition of resistance to TAM is the main problem of © XXXX American Chemical Society

Received: September 28, 2012 Accepted: June 3, 2013

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Figure 1. Role of FoxO1 in MRP2 gene transcription. (A) Putative binding sites to transcription factors in the proximal promoter region of human MRP2 gene. The asterisks (*, **) indicate putative FoxO binding sites based on highly conserved sequence of Forkhead protein. (B) MRP2 induction by FoxO1 overexpression. MCF-7 cells were transiently transfected with pCMV5 (0.3 μg), pCMV5-FoxO1 (0.1 and 0.3 μg), or pCMV5FoxO3 (0.1 and 0.3 μg) plasmid and incubated for 24 h. Relative densitometric scanning results were shown in top of the representative blot. (C) Transactivation of MRP2 gene by FoxO1. Reporter activities of p2635-MRP2-Luc in MCF-7 cells transiently transfected with pCMV5-FoxO1 (3−30 ng) or pCMV5 vector (30 ng). Data represent means ± SD with three different samples (significant versus the pCMV5-transfected group, *p < 0.05, **p < 0.01).

they consequently accumulate in the nucleus and affect transactivation activity of target genes.19 In the present study, we investigated a possible role for FoxO1 in MRP2 gene expression in TAMR-MCF-7 cells. Moreover, based on the hypothesis that SIRT1-dependent FoxO1 activity is important for the expression and regulation of ABC transporters, we

cells and that FoxO1 plays an essential role in the expression of the MDR1 gene.14 Silent information regulator two ortholog 1 (SIRT1) is a member of the sirtuin deacetylase family.15,16 Several transcription factors such as p53 and nuclear factor-κB (NF-κB) have been reported to be substrates of SIRT1.17,18 FoxO transcription factors are also deacetylated by the enzyme, and B

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Figure 2. continued

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Figure 2. FoxO1-dependent MRP2 induction in TAMR-MCF-7 cells. (A) Immunoblot analysis of MRP2. Left: A representative immunoblot shows MRP2 protein in both MCF-7 and TAMR-MCF-7 cells serum-deprived for 24 h. Right: Relative changes in MRP2 expression were assessed by scanning densitometry. Data represent the means ± SD of four separate experiments (significant versus MCF-7 cells, **p < 0.01). (B) Immunoblot analysis of FoxO1, FoxO3, FoxO4, and FoxO6 in the total cell lysates, nuclear fractions, or cytoplasmic fractions. (C) Reporter activities of deletion mutants of human MRP2 promoters in MCF-7 and TAMR-MCF-7 cells. Each cell type was transiently transfected with p2635-MRP2-Luc, p491MRP2-Luc, p392-MRP2-Luc, or p245-MRP2-Luc plasmid. Dual luciferase reporter assays were performed on the lysed cells transfected with each pMRP2-Luc plasmid 18 h after transfection. Data represents means ± SD with six different samples (significant versus MCF-7 cells, **p < 0.01). (D) Gel shift analysis of the FoxO binding in MRP2 promoter. Nuclear extracts were prepared from overnight serum-deprived MCF-7 and TAMR-MCF7 cells. All lanes contained 10 μg of nuclear extracts and 5 ng of the labeled FoxO binding region (−442to −422 bp) of MRP2 promoter. Competition studies were carried out by adding a 20-fold excess of unlabeled FoxO1 oligonucleotide to the nuclear extracts of TAMR-MCF-7 cells. Immunodepletion was performed using FoxO1 antibody. (E) Inhibition of MRP2 protein and mRNA expression by FoxO1 suppression. MRP2 and FoxO1 protein levels were determined by immunoblottings in TAMR-MCF-7 cells transfected with FoxO1 siRNA (60 pmol) or control siRNA (60 pmol) (left panel). MRP2 mRNA levels were determined by real-time PCR in TAMR-MCF-7 cells transfected with FoxO1 siRNA (60 pmol) or control siRNA (60 pmol) (right panel). Data represent the means ± SD with three separate experiments (significant versus the control siRNAtransfected MCF-7 cells, **p < 0.01; significant versus the control siRNA-transfected TAMR-MCF-7 cells, ##p < 0.01). (F) Inhibition of MRP2 gene transcriptional activity by FoxO1 suppression. MCF-7 and TAMR-MCF-7 cells were cotransfected with p2635-MRP2-Luc in combination with FoxO1 siRNA (20 pmol) or control siRNA (20 pmol). Data represent the means ± SD with six separate samples (significant versus the control siRNA-transfected MCF-7 cells, **p < 0.01; significant versus the control siRNA-transfected TAMR-MCF-7 cells, ##p < 0.01).

containing the human MRP2 promoter region (−2635 bp, four putative FoxO binding sites) and p491-MRP2-Luc (three putative FoxO binding sites) were kindly provided by Dr. T. Uchiumi (Kyushu University, Japan),20 and p392-MRP2-Luc (1 putative FoxO binding site) and p245-MRP2-Luc (no putative FoxO binding sites) was generated by ligating PCR-amplified MRP2 promoter regions with a pGL2-luciferase vector (Promega, Madison, WI). The FoxO1 forkhead response element (FHRE) containing a minimal reporter plasmid, pCMV5-FoxO1, and pCMV5-FoxO3 overexpression plasmid was supplied from Addgene Inco. (Cambridge, MA). SIRT1constitutive active plasmid (SIRT1-CA) was donated from Dr. K. Y. Lee (Chonnam National University, Gwangju, Korea).

further investigated a possible role for SIRT1 activation in the up-regulation of MRP2 in TAM-resistant breast cancer cells.

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MATERIALS AND METHODS Materials. FoxO1 and FoxO3a specific antibodies, SIRT1 antibody, horseradish peroxidase-conjugated antirabbit, and antimouse IgGs were purchased from Cell Signaling Technology (Beverly, MA). Anti-MRP2 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Most of the reagents used for molecular studies were obtained from Sigma (St. Louis, MO). Small inhibitory RNA (siRNA) targeting human FoxO1 was acquired from Ambion (Austin, TX). A Fluor de Lys SIRT1 activity kit was purchased from Biomol (Plymouth Meeting, PA). The p2635-MRP2-Luc reporter plasmid D

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Cell Culture and Establishment of TAMR-MCF-7 Cells. MCF-7 cells were cultured at 37 °C in 5% CO2/95% air in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/ mL streptomycin. TAMR-MCF-7 cells were established using methods previously reported.7 Immunoblot Analysis and Immunocytochemistry. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblot analyses were performed, as described previously.7 For the siRNA transfection, 70% confluent cells cultured in 6-well plates were transfected with 60 pmol of control or FoxO1 siRNA (Ambion, Austin, TX) using Hilymax reagent (Dojindo Molecular Technologies, Gaithersburg, MD). MRP2 immunocytochemistry was performed following the standard protocol. TAMR-MCF-7 cells were grown on Lab-TEK chamber slides (Nalge Nunc International Corporation, Rochester, NY), and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) was used to verify the location of nuclei. Reporter Gene Assay. Promoter activity was determined using a dual-luciferase reporter assay system (Promega, Madison, WI). Briefly, cells (3 × 105 cells/well) were replated in 12-well plates overnight and transiently transfected with each reporter plasmid/phRL-SV plasmid (hRenilla luciferase expression for normalization) (Promega, Madison, WI) using Hilymax reagent (Dojindo Molecular Technologies, Gaithersburg, MD). Cells were then incubated in culture medium without serum for 18 h, and firefly and hRenilla luciferase activities in the cell lysates were measured using a luminometer (LB941, Berthold Technologies, Bad Wildbad, Germany). Gel Shift Analysis. A double stranded putative FoxO binding site oligonucleotide in human MRP2 gene promoter was used for gel shift analysis after end-labeling the probe with [γ-32P]ATP using T4 polynucleotide kinase. The sequence of the consensus oligonucleotide was 5′-TTTGATGAAACAAGTAAAGA-3′. Gel shift analysis was performed as described previously.20 Binding specificity was determined by using competition experiments, which were carried out by adding a 20-fold excess of an unlabeled oligonucleotide. For immunoinhibition assays, 2 μg of FoxO1 antibody was added to reaction mixtures. Samples were loaded onto 4% polyacrylamide gels at 100 V, and removed gels were dried and autoradiographed. Determination of SIRT1 Deacetylase Enzyme Activity. The Fluor de Lys fluorescence assay was used to assess SIRT1 enzyme activity in cell extracts. The Fluor de Lys fluorescence assay was done as indicated in the BioMol product sheets.15 Statistical Analysis. One-way analysis-of-variance (ANOVA) was used to determine whether there was a significant difference among treatment groups. When treatment was found to have a significant effect, the Newman-Keuls test was used to compare multiple group means.

suggested FoxO1 as a master regulator of ABC transporter expression.14 As shown in Figure 1A, we found four putative FoxO binding sites in the human MRP2 promoter region. To determine whether FoxO binding to the putative binding sites is related to MRP2 gene transcription, we measured the effects of FoxO1 and FoxO3 overexpression on MRP2 expression using MCF-7 cell lines. Expression levels of MRP2 were increased in MCF-7 cells transfected with FoxO1 overexpression vector (pCMV5-FoxO1) (Figure 1B). In contrast, MRP2 protein levels were rarely elevated by FoxO3 overexpression plasmid (pCMV5-FoxO3), and the induction intensity was much weaker than that induced by FoxO1 overexpression. We next tested whether FoxO1 introduction stimulates MRP2 promoter activity. In MCF-7 cells, the human p2635MRP2-Luc reporter activity was significantly increased by the presence of a FoxO1 overexpressing plasmid (Figure 1C). These results demonstrate that FoxO1 functions as an active transcription factor in the expression of MRP2 as well as MDR1. FoxO1 Is Essential for the Induction of MRP2 in TAMResistant Breast Cancer Cells. We found sustained MRP2 up-regulation in TAM-resistant breast cancer cells, which could be associated with the acquisition of additional chemoresistance.7 We confirmed that basal MRP2 levels were higher in TAMR-MCF-7 cells than in control MCF-7 cells (Figure 2A). Because nuclear localized FoxO only acts as a functional transcription factor, nuclear FoxO1 and FoxO3 as well as those in total cell lysates were also compared in the two cell types. In comparison to control MCF-7 cells, total expression and nuclear levels of FoxO1, but not those of FoxO3, were sharply increased in TAMR-MCF-7 cells (Figure 2B). When we determined nuclear levels of FoxO4 and FoxO6, nuclear FoxO6 was also up-regulated in TAMR-MCF-7 cells (Figure 2B). To assess involvement of the putative FoxO binding sites in MRP2 gene transcription in TAMR-MCF-7 cells, deletion mutant constructs (p2635-MRP2-Luc containing four FoxO binding sites, p491-MRP2-Luc containing three FoxO binding sites, p392-MRP2-Luc containing 1 FoxO binding site, and p245MRP2-Luc containing no FoxO binding site) were used for reporter gene analyses. As shown in Figure 2C, the relative luciferase activity in TAMR-MCF-7 cells was gradually decreased by the deletion of FoxO binding sites, and the reporter activity in cells transfected with p245-MRP2-Luc was only 3.3% of the inducible luciferase activity that was observed with the p2635-MRP2-Luc plasmid. This promoter deletion experiment demonstrates that the four FoxO binding sites in the human MRP2 promoter are functional. The relative reporter activity in 245-MRP2-transfected TAMR-MCF-7 cells was significantly higher than that in MCF-7 cells, which suggest that other transcription factor binding sites in proximal promoter region (within −245 bp) of MRP2 gene may play a role in TAMR-MCF-7 cells. Electrophoretic mobility shift assays were performed to determine FoxO1 DNA binding activity using nuclear extracts prepared from MCF-7 and TAMR-MCF-7 cells. In TAMRMCF-7 cells, DNA binding activity in putative FoxO binding site found in human MRP2 gene promoter was increased compared to control MCF-7 cells (Figure 2D). Specificity of protein binding to the FoxO binding site was confirmed by complete reversal of DNA binding activity in unlabeled oligonucleotide-tretaed sample (Figure 2D). Moreover, antiFoxO1 antibody reduced the band intensity, suggesting that

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RESULTS Role of FoxO1 in the MRP2 Gene Transcription. There are many transcription factor binding sites in the human MRP2 promoter region.21 Activator protein-1 (AP-1), hepatocyte nuclear factor-3β, specific protein-1, and CCAAT/enhancerbinding protein β (C/EBPβ) are examples of transcription factors that bind to the MRP2 promoter (Figure 1A). We showed that the FoxO1 binding site in the MDR1 gene promoter is essential for basal expression of MDR1 in adriamycin-resistant cancer cells (MCF-7/ADR cells), and we E

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Figure 3. SIRT1 activation in TAMR-MCF-7 cells. (A) Akt phosphorylation in MCF-7 and TAMR-MCF-7 cells. A representative immunoblot shows phospho-Akt protein in both MCF-7 and TAMR-MCF-7 cells serum-deprived for 24 h (upper). Effect of LY294002 (PI3K inhibitor) on Akt phosphorylation in TAMR-MCF-7 cells was also determined (lower). (B) FoxO1 phosphorylation (Ser 319) in MCF-7 and TAMR-MCF-7 cells. A representative immunoblot shows phospho-FoxO1 protein in both MCF-7 and TAMR-MCF-7 cells serum-deprived for 24 h (upper). Effect of LY294002 (PI3K inhibitor) on FoxO1 phosphorylation in TAMR-MCF-7 cells was also determined (lower). (C) Protein expression (upper panel) and activities (lower panel) of SIRT1 in MCF-7 and TAMR-MCF-7 cells (upper, left panel). SIRT1 activities were measured by a Fluor de Lys fluorescence assay. Data represent means ± SD with three different samples (significant versus the control MCF-7 cells, **p < 0.01). (D) Effects of PI3-kinase inhibitor and SIRT1 inhibitor on the expression of FoxO1 and MRP2 in TAMR-MCF-7 cells. Expression of FoxO1 and MRP2 (left panel) was measured in TAMR-MCF-7 cells treated with PI3-kinase inhibitor (LY; LY294002 20 μM) and SIRT inhibitor (NAM; nicotinamide 1 mM) for 24 h. MRP2 gene transcription was also assessed by determining p2635-MRP2-Luc reporter activity (right panel). Data represent means ± SD with four different samples (significant versus the control MCF-7 cells, **p < 0.01; significant versus the untreated TAMR-MCF-7 cells, ##p < 0.01). (E) Effects of amurensin G (10 μM) on the expression of FoxO1 and MRP2 in TAMR-MCF-7 cells.

FoxO1 via its deacetylation and subsequently increases the transcriptional activity of FoxO1.24 Among several kinases regulating FoxO proteins, PI3Kcontrolled Akt directly phosphorylates FoxO1 and subsequently causes ubiquitination-dependent degradation, which leads to inhibition of FoxO1 transcriptional activity.10 Levels of phosphorylated Akt were higher in TAMR-MCF-7 cells than in MCF-7 cells, which is consistent with our previous result (Figure 3A).7 We further determined the levels of Ser 319 phosphorylation of FoxO1. Ser 319 phosphorylation of FoxO1 has been believed to export signal from the nucleus.25 As shown in Figure 3B, Ser 319 phosphorylated FoxO1 was increased in TAMR-MCF-7 cells versus control MCF-7 cells. Moreover, LY294002, a PI3K inhibitor, suppressed the phosphorylation of Akt and FoxO1 in TAMR-MCF-7 cells (Figure 3A and B), which show that PI3K activation in TAMR-MCF-7 cells consistently phosphorylates FoxO1. Because Akt activation decreases the transactivator function of FoxO1, this result seems to conflict with the sustained activation of FoxO1 in TAMR-MCF-7 cells shown in Figure 2A. However, our data, showing that PI3K inhibitor did not affect nuclear FoxO1 expression in TAMR-MCF-7 cells (Figure 3D, left), clearly demonstrate that the sustained PI3K/Akt activation in TAMRMCF-7 cells seems to be unrelated with the enhanced levels of nuclear FoxO1. Instead, SIRT1 expression was enhanced in

FoxO DNA binding activity mainly depends on FoxO1 form (Figure 2D). To confirm the role of FoxO1, Western blot analysis was done for TAMR-MCF-7 cells transfected with siRNA that specifically silences FoxO1. When FoxO1 expression was inhibited by FoxO1 siRNA, the elevation of MRP2 levels in TAMR-MCF-7 cells was prevented (Figure 2E, left). Moreover, real-time PCR analysis showed that the enhanced MRP2 mRNA expression in TAMR-MCF-7 cells was significantly inhibited by FoxO1 siRNA transfection (Figure 2E, right). Reporter gene assays with p2635-MRP2-Luc were next done in MCF-7 and TAMR-MCF-7 cells cotransfected with FoxO1 siRNA. MRP2 reporter activity was significantly diminished by FoxO1 inactivation (Figure 2F). These data suggest that FoxO1 is a critical regulator of human MRP2 gene transcriptional activity as well as MDR1 gene expression. Involvement of SIRT1 in FoxO1-Mediated MRP2 Expression in TAM-Resistant Breast Cancer Cells. FoxO proteins can be controlled by two different mechanisms: phosphorylation and acetylation. Multiple kinase pathways including phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (ERK), and p38 kinase have been shown to regulate FoxO via phosphorylation.10,22,23 In addition, it has been reported that SIRT1 causes nuclear translocation of F

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Figure 4. Requirements of SIRT1 to induce FoxO1-mediated MRP2 expression in TAMR-MCF-7 cells. (A) Transactivation of MRP2 gene by SIRT1 overexpression. Reporter activity of p2635-MRP2-Luc (left panel) and nuclear FoxO1 levels (right panel) were measured in MCF-7 cells transiently transfected with SIRT1-constitutive active plasmid (SIRT1-CA, 30−300 ng or 300 ng) or pCMV5 vector (300 ng). Data represents means ± SD with three different samples (significant versus the pCMV5-transfected group, *p < 0.05; **p < 0.01). (B) The additive effects of FoxO1 and SIRT1. MCF-7 cells were cotransfected with p2635-MRP2-Luc reporter and pCMV5-FoxO1 (30 ng) in the presence or absence of SIRT1-CA (300 ng), and then the luciferase activities were measured 18 h after transfection. Data represent the means ± SD of three different samples (significant versus the pCMV5-transfected MCF-7 cells, **p < 0.01; significant versus the FoxO1-trasnfected MCF-7 cells, ##p < 0.01). (C) Reversion of SIRT1 induced MRP2 gene transactivation by FoxO1 suppression. p2635-MRP2-Luc reporter activities were measured in MCF-7 cells cotransfected with pCMV5 (300 ng) or SIRT1-CA (300 ng) in combination with control siRNA (20 pmol) or FoxO1 siRNA (20 pmol) (left panel). Data represents means ± SD with three different samples (significant versus the control siRNA and pCMV5-transfected MCF-7 cells, **p < 0.01; significant versus the control siRNA and SIRT1-CA-transfected MCF-7 cells, #p < 0.05). Protein expression of MRP2 was detected after cotransfection of MCF-7 cells with SIRT-CA and FoxO1 siRNA (right panel). MCF-7 cells were cotransfected with pCMV5 (300 ng) or SIRT1-CA (300 ng) in combination with control siRNA (60 pmol) or FoxO1 siRNA (60 pmol), and then total cell lysates were obtained 24 h after transfection. G

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Figure 5. Effect of SIRT inhibitor on (A) paclitaxel- or (B) doxorubicin-induced cell proliferation inhibition in TAMR-MCF-7 cells. TAMR-MCF-7 cells were plated at 96-well plate and preincubated with or without 1 mM nicotinamide (NAM) for 12 h. The cells were further incubated in the presence or absence of paclitaxel (1−30 nM) or doxorubicin (1−30 μM) for 36 h in the serum-containing condition. Data represents means ± SD with eight different samples (significant versus the untreated control, **p < 0.01; significant versus NAM-untreated control group, ##p < 0.01). (C) MRP2 immunostaining in TAMR-MCF-7 cells. Green fluorescence (Alexa Fluor 488) represents MRP2 localization.

also accelerated in TAMR-MCF-7 cells, but SIRT1-mediated deacetylation of FoxO1 may overcome it. We then determined whether SIRT1 activity is essential for the FoxO1-dependent MRP2 expression. According to results for nuclear FoxO1 determination (Figure 3D, left), nicotinamide, a representative SIRT inhibitor, had an inhibitory effect on FoxO1. Nicotinamide also substantially reduced expression levels of MRP2 protein (Figure 3D, left) and p2635-MRP2-Luc

TAMR-MCF-7 cells compared to control MCF-7 cells (Figure 3C, upper). Moreover, TAMR-MCF-7 cells showed higher basal SIRT1 activity than MCF-7 cells (Figure 3C, lower). These data strongly support the possibility that FoxO1 activity in TAMR-MCF-7 cells can be regulated by SIRT1 instead of by the PI3K/Akt pathway. PI3K/Akt-dependent FoxO1 phosphorylation and subsequent FoxO1 export from nucleus could be H

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DISCUSSION Multidrug resistance has been considered a serious problem in the treatment of many cancers including breast cancer.31 MDR1 and MRP2 are the overt proteins that drive this severe resistance.32 Among ABC transporters, MRP2 protein is reported to be overexpressed in malignant neoplastic tissues.33 In addition to transporting endogenous conjugates, MRP2 pumps out cancer chemotherapeutics such as vinblastine, vincristine, and methotrexate. Therefore, this protein appears to contribute to drug resistance in mammalian cells.33,34 However, the molecular mechanisms by which MRP2 is increased are largely unknown. Although it has been suggested that C/EBPβ binding region in the human MRP2 promoter is essential for the transactivation of the MRP2 gene,20 we concluded that C/ EBPβ is probably not responsible for MRP2 gene activation in TAMR-MCF-7 cells.7 FoxO may play an important role in cell growth, proliferation, differentiation, longevity, metabolism, and tumor development.9,12,13 In the present study we showed that there was a close correlation between MRP2 and FoxO1 transcription factor. MRP2 protein levels were elevated in FoxO1 overexpressing MCF-7 human breast cancer cells. Remarkably enhanced FoxO1 levels were observed in TAMR-MCF-7 cells, which have canonically overexpressed MRP2 proteins. Although FoxO1 overexpression marginally increased the reporter activity of MRP2 gene promoter in MCF-7 cells, the increase intensity was concentration-dependent and significant. Moreover, FoxO1 knockdown experiments showed the potent inhibitory effects of FoxO1 siRNA on gene transcription and protein expression of MRP2 in TAMR-MCF-7 cells. Thus, FoxO1 overexpression alone may not be enough for the MRP2 gene transcription in normal breast cancer cells. However, FoxO1 activation may act as a crucial factor for MRP2 gene transcription in TAM-resistant breast cancer cells. Hisaeda et al. showed that compared with p1659-MRP2, reporter activity decreased when p491-MRP2 was assayed in HepG2 cells, leading to the conclusion that a putative positive regulator is localized in the −1659/−491 bp region.35 In contrast, Tanaka et al. previously showed that promoter reporter activity decreased in HepG2 cells when p1659MRP2 was assayed, compared with p491 MRP2, and concluded that there was a putative silent regulatory element in the −1659/−491 region.21 The conclusions of these two studies are exactly contrary to each other. To resolve this conflict, more detailed investigation is needed. Among such complex elements, FoxO1 proved to be a positive regulator of MRP2. Although a FoxO1 binding region is truncated in p491-MRP2, the reporter still reserves three putative binding sites for FoxO1, and p491-MRP2 was also shown to play a role in increasing MRP2 luciferase activity in TAMR-MCF-7 cells compared to MCF-7 cells. Likewise, three FoxO1 binding sitestruncated p392-MRP2 reporter activity was enhanced in TAMR-MCF-7 cells. In contrast, promoter activity in TAMRMCF-7 cells transfected with p245-MRP2 containing no putative FoxO1 binding sites showed only minimal activity. These results suggest the possibility that a FoxO1 binding in the human MRP2 promoter region is critical for gene transcription. Further study was done by introducing a FoxO1 specific siRNA. Western blots and reporter gene analyses revealed that inhibition of FoxO1 expression by siRNA restrained MRP2 protein expression as well as MRP2 reporter activity in TAMR-MCF-7 cells.

reporter activity (Figure 3D, right) in TAMR-MCF-7 cells. We have previously shown that amurensin G acts as a potent SIRT1 inhibitor.26 Pretreatment of TAMR-MCF-7 cells with 10 μM amurensin G inhibited both nuclear FoxO1 expression and MRP2 expression in TAMR-MCF-7 cells (Figure 3E). In our previous PI3K inhibition also reduced basal MRP2 levels in TAMR-MCF-7 cells, but nuclear FoxO1 levels were not affected (Figure 3D, left). In our previous study, we showed that PI3K/Akt activation up-regulates MRP2 protein in a pregnane X receptor (PXR) dependent manner.7 Hence, MRP2 expression in TAMR-MCF-7 cells may be controlled by two distinct mechanisms: SIRT1-dependent FoxO1 activation and PI3K/Akt-dependent PXR activation. Additive Effects of SIRT1 on FoxO1-Dependent MRP2 Overexpression. We then assessed the effect of SIRT1 overexpression on MRP2 gene transcription in MCF-7 cells. Reporter activity of p2635-MRP2-Luc was increased as expected following addition of SIRT1 overexpression vector (Figure 4A, left). SIRT1 overexpression also enhanced nuclear level of FoxO1 in MCF-7 cells (Figure 4A, right), which implies that SIRT1 is closely connected with FoxO1 activity and the subsequent MRP2 gene transcription. To confirm whether SIRT1 overexpression potentiates FoxO1-mediated MRP2 gene transcription, MCF-7 cells were cotransfected with a constitutively active SIRT1 plasmid with or without a FoxO1 overexpression plasmid. SIRT1 overexpression in the presence of FoxO1 additively enhanced MRP2 promoter activity, whereas each one alone also showed MRP2 increasing effects (Figure 4B). Furthermore, both SIRT1-induced MRP2 gene transactivation and MRP2 protein expression were reversed by FoxO1 siRNA (Figure 4C). These data support the idea that SIRT1 acts as a critical regulator of MRP2 expression via a FoxO1 dependent mechanism. Potentiation of Cytotoxic Effects of Paclitaxel and Doxorubicin by SIRT Inhibition in TAMR-MCF-7 Cells. MRP2 is one of the most important transporters for the efflux of various chemotherapeutic agents such as paclitaxel and methotrexate.27 Because we have previously reported that MDR1 expression is extremely low in TAMR-MCF-7 cells,7 MRP2 may function as a main efflux pump for paclitaxel and doxorubicin in TAMR-MCF-7 cells. To test the functional role of SIRT1-mediated MRP2 expression, we determined the effect of nicotinamide on the paclitaxel- and doxorubicin-induced cell death. As shown in Figure 5A and B, 1 mM nicotinamide significantly enhanced the cell growth inhibitory effects of paclitaxel (3 and 10 nM) and doxorubicin (1−30 μM) in TAMR-MCF-7 cells. The results suggest that SIRT1-mediated MRP2 expression is crucial for the responsiveness of paclitaxel and doxorubicin in TAM-resistant breast cancer cells. Evers et al. have shown that MRP2 is primarily localized in polarized cells and its expression is difficult to achieve in nonpolarized cells such as MCF-7 cells.28 Although MCF-7 cells is a typical nonpolarized cell, we and other groups already showed that TAMR-MCF-7 cells acquired several characteristics of fibroblast-like mesenchymal cell type through epithelial mesenchymal transition (EMT).29,30 The cobblestone-like morphology of MCF-7 cells at confluence was changed in TAMR-MCF-7 cells to a spindle-like fibroblastic morphology. Immunocytochemistry revealed that MRP2 was mainly localized in a plasma membrane region in TAMR-MCF-7 cells (Figure 5C). I

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elevation of MRP2 transcription was completely reversed by FoxO1 suppression. These data demonstrate that the increased SIRT1 activity is a key mechanism responsible for MRP2 upregulation in TAMR-MCF-7 cells. PI3K/Akt-dependent Ser 319 phosphorylation of FoxO1 is known as an export signal from nucleus.25 However, we found that PI3K inhibition did not change the nuclear levels of FoxO1 in TAMR-MCF-7 cells. As mentioned in the Results section, SIRT1-mediated deacetylation of FoxO1 may overcome it. This result raises the possibility that SIRT1-mediated FoxO1 deacetylation may overcome the effect of PI3K/Akt-dependent FoxO1 phosphorylation. In addition, Fukunaga et al. suggested that association of Ran with FoxO1 contributes to the movement of FoxO1 from the nucleus after Akt-mediated phosphorylations.40 Thus, exporting assembly may be dys-regulated in TAMR-MCF-7 cells even after consistent activation of PI3K/Akt pathway. In summary, this study showed that FoxO1 positively regulates MRP2 and SIRT1 activation is an upstream regulator of FoxO1 in the sustained up-regulation of MRP2 in TAMresistant breast cancer cells. MRP2 is one of the most important transporters for the efflux of various chemotherapeutic agents, and its expression is up-regulated in TAM-resistant breast cancer cells. Our findings are worthy of attention because the SIRT1/FoxO1 pathway could be a new therapeutic target for overcoming the acquisition of additional chemoresistance in TAM-resistant breast cancer. In fact, SIRT inhibition sensitizes paclitaxel- or doxorubicin-mediated cytotoxic effects in TAMresistant breast cancer cells.

Here, we also found that the nuclear level of FoxO6 was enhanced in TAMR-MCF-7 cells (Figure 2B). Hence, it seems to be possible that FoxO6 plays a role in the enhanced MRP2 gene transcription in TAMR-MCF-7 cells. However, FoxO1 antibody efficiently reduced the enhanced FoxO DNA binding activity in TAMR-MCF-7 cells, whereas the band intensity in anti-FoxO1 IgG-treated sample was comparable to that in control MCF-7 cells (Figure 2D, lane 8). In comparison to FoxO1 and FoxO3, the physiological role of FoxO6 has not been covered in cancer biology. The potential involvement of FoxO6 in the regulation of ABC transporter genes could be tested in future study. The activity of FoxO is controlled by post-translational modifications, including phosphorylation/ubiquitination and acetylation.10 It is a target through which PI3K/Akt can induce tumorigenicity through serine/threonine phosphorylation and nuclear exclusion. In TAMR-MCF-7 cells, not only SIRT1 but phosphorylated Akt levels are increased. Akt activation phosphorylates FoxO1 and subsequently incapacitates its transcriptional activity through ubiquitination and proteasomal degradation, which may have an influence on MRP2 expression. However, though the basal activity of PI3K/Akt is very high in TAMR-MCF-7 cells and consequently Ser 319 of FoxO1 is phosphorylated, the cells retain highly expressed FoxO1 and MRP2. Our previous data can be used as a clue to resolve this contradiction. The PI3K pathway plays a key role in controlling pregnane X receptor (PXR) activity, and PXR is thought to be an important transcription factor for MRP2 induction.7 When a PI3K/Akt specific inhibitor, LY294002, was added to TAMRMCF-7 cells, PXR reporter activities and MRP2 protein levels were significantly inhibited, implying a role for PI3K/Akt in PXR-mediated MRP2 expression in TAMR-MCF-7 cells.7 This action is unlikely to be dependent on FoxO1 action because PI3K inhibition did not change nuclear FoxO1 expression in the present study. SIRT1, a member of the sirtuin family, is known to be a regulator of lifespan extension. Frescas et al. suggested that deacetylation of FoxO by SIRT1 increases its nuclear retention time and thus increases transcriptional activity.24 SIRT1 acts as one of the critical regulators of FoxO transcription in response to cellular stress via its role as an inducer of NAD-dependent deacetylation.36 Coactivator p300 directly acetylates FoxO1 and stimulates FoxO1-induced transcription.37 In contrast, Motta et al. demonstrated an inhibitory effect of SIRT1 on p300mediated activation of FoxO3a.16 Although there are conflicting opinions about the role of SIRT1 in FoxO1 induced transcriptional regulation, recent studies have suggested SIRT1 as an assistor of FoxO activation. SIRT1-mediated deacetylation results in nuclear retention of FoxO and thereby prolongs FoxO-dependent transcription of subnuclear stressregulating genes. Through this mechanism, SIRT1 is thought to be able to promote cellular survival and increase lifespan.38 A recent study also demonstrated that FoxO3a acetylation was decreased in response to cisplatin treatment and the acetylation intensity was lower in cisplatin-resistant cancer cells.39 In this study, we found that the basal expression and activity of SIRT1 were significantly enhanced in TAMR-MCF-7 cells and that the SIRT1 inhibitor nicotinamide reduced the protein level and promoter activity of FoxO1 and MRP2 in TAMR-MCF-7 cells. Moreover, when SIRT1 was cotransfected with FoxO1 in MCF-7 cells, MRP2 transcriptional activities were significantly increased, while each one alone also contributes to the induction of MRP2 transactivation. Vice versa, SIRT1-mediated

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The first two authors (H.K.C. and K.B.C.) equally contributed to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation (NRF) funded by the Korean government (MSIP) [No. 2012-053532 (Bio & Medical Technology Development Program) and 2007-0056817].

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REFERENCES

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