ANTICANCER RESEARCH 34: 4127-4134 (2014)

Metformin Inhibits Tumor Cell Migration via Down-regulation of MMP9 in Tamoxifen-resistant Breast Cancer Cells

SOON YOUNG JANG1, AEREE KIM1,2, JIN KYOUNG KIM1,2, CHUNGYEL KIM1,2, YOUL-HEE CHO3, JONG-HO KIM4, CHUL HWAN KIM1 and JI-YUN LEE1

1Department of Pathology, College of Medicine, Korea University, Seoul, South Korea; 2Department of Pathology, Korea University Guro Hospital, Seoul, South Korea; 3Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, South Korea; 4Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, South Korea

Abstract. Metformin is the most widely used anti-diabetic responsible for the degradation of type-IV , which is a drug in the world. Recent evidence indicates that metformin major component of the basement membrane. These two could potentially inhibit tumorigenesis. In the present study, MMPs are known to be associated with tumor migration and we found that metformin inhibited cell migration and invasion in several types of cancers (1). Regulation of MMP9 invasion of phorbol 12-myristate 13-acetate-induced MCF-7 is important for several biological processes, including and tamoxifen-resistant MCF-7 breast cancer cells. This , inflammatory response, and (5). inhibition was correlated with the modulation of matrix The elevated expression of MMP9 occurs in response to -9 (MMP9) via the suppression of its inflammatory and oncogenic signals and is involved in several expression and proteolytic activity. These results indicate that pathological processes such as , tumor-induced metformin leads to the suppression of migration and invasion angiogenesis, lupus, and asthma (5, 6). The expression of through regulation of MMP9 and it may have potential as an MMP9 can be induced at the transcriptional level in response anticancer drug for therapy in human breast cancer, to different agents, such as growth factors, interleukins, tumor especially of chemoresistant cancer cells. necrosis factor (TNF)-α and phorbol 12-myristate 13-acetate (PMA). Modulation of the MMP9 is achieved via a 2.2-kb Cancer cell invasion and metastasis represent a series of events upstream regulatory sequence containing binding sites for in the tumor microenvironment. One such event is the activator protein 1 (AP1), nuclear factor kappa-light-chain- proteolytic degradation of components (1, enhancer of activated B cells (NFκB), and specificity protein 1 2). Certain , such as serine proteases, cysteine (SP1) (7, 8). Two AP1 binding sites (proximal and distal) have proteases, and matrix (MMPs), contribute been shown to contribute to the transcriptional induction in to invasion and metastasis. MMPs can be divided into four sub- response to several stimuli (9). However, all DNA binding classes: , , stromelysin, and membrane- sites, namely AP1, NFκB, and SP1, are required for full associated MMPs (3). Recent studies have described MMPs as activation of MMP9 (10). critical regulators of the tumor microenvironment, as well as The oral anti-diabetic drug metformin belongs to the being involved in tumor progression, metastasis, invasion, and family of bi-guanides and is the most widely used anti- inflammation (4). Human MMPs, such as gelatinase-A hyperglycemic drug in the world. Metformin has been shown (MMP2) and gelatinase-B (MMP9), are key that are to suppress the energy-sensitive AMP-activated protein kinase/mammalian target of rapamycin signaling pathway, which leads to reduced protein synthesis and cell proliferation. Recent retrospective epidemiological studies Correspondence to: Ji-Yun Lee, Ph.D., 5-1 Anam-Dong, have shown that metformin treatment is associated with Seoungbuk-Gu,Seoul, 136-705 South Korea. Tel: +82 29206141, reduced risk of various types of cancer, such as prostate, Fax: +82 29533130, e-mail: [email protected] and Chul Hwan breast, lung, and pancreatic cancer (11-13). In addition, Kim, MD, Ph.D., 5-1 Anam-Dong, Seoungbuk-Gu, Seoul, 136-705 several groups have reported the inhibitory effect of South Korea,Tel: +82 29205991, Fax: +82 29533130, e-mail: [email protected] metformin on melanoma cell proliferation through the induction of autophagic cell death (14, 15). These results Key Words: Metformin, MMP9, tamoxifen resistance, invasion, suggest that metformin might potentially be used as an migration. anticancer drug for different types of cancer.

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Tamoxifen resistance presents a serious challenge to the treatment of patients with breast cancer. In spite of an initial Western blotting. Cell extracts were prepared using RIPA buffer (1× response to such therapies, many patients will eventually phosphate buffered saline (PBS), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS containing 100 μl of 10 mg/ml show tumor relapse and disease progression (16, 17). phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride Previous studies have shown that acquisition of tamoxifen (PMSF), and one tablet of the complete mini inhibitors) resistance in breast cancer cells is indicative of an increased after washing cells twice with 1×PBS. The protein lysates were metastatic ability, including invasion and migration. resolved by SDS–PAGE and transferred using nitrocellulose In the present study, we investigated whether metformin membranes (Whatman PROTEAN® BA83, 0.2 μm). The exerts its effects through inhibition of MMP9 in MCF-7 cells membranes were incubated with buffer containing 0.1% Tween 20 as well as tamoxifen-resistant TamR-MCF-7 breast cancer cells. and 5% skim milk and were then exposed to the desired primary antibody. After treatment with a suitable secondary antibody, the immunoreactive bands were visualized using the standard enhanced Materials and Methods chemiluminescence (Pierce, Rockford, IL, USA) method.

Materials and reagents. Metformin was purchased from Sigma (St. Reverse transcription-PCR (RT-PCR). Total RNA was extracted from Louis, MO, USA), and antibodies against MMP2 and MMP9 were the treated cells, and cDNA was synthesized from 1 μg of total RNA purchased from Cell Signaling (Boston, MA, USA). Antibodies using a High Capacity cDNA Synthesis Kit (Applied Biosystems, against proliferating cell nuclear antigen, p65, c-JUN, and c-FOS were Foster city, CA, USA) for the RT-PCR analysis. The following PCR purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). primers were used: MMP9 sense: 5’- TTTGACAGCGAC PMA was purchased from Calbiochem (San Diego, CA, USA). MMP9 AAGAAGTGG-3’, antisense: 5’- TCCCATC CTTGAACAAATACA- ELISA kits were purchased from R&D Systems (Abingdon, UK). 3’; MMP2 sense: 5’- CATTCCGCTTCCA GGGCACAT-3’, antisense: 5’- GCTCCTGAATGCCCTTGAT GTCA-3’; Glyceraldehyde 3- Cell lines and culture. The human breast cancer cell lines MCF-7 phosphate dehydrogenase (GAPDH) sense: 5’-GCCATCGTCA were obtained from America Type Culture Collection (Mannassa, CCAACTGGGAC-3’, antisense: 5’-CGATTTCCCGCTCGGCCG VA, USA) and Tamoxifen-resistant MCF-7 were gifts from Dr. A. TGG-3’. The PCR products were analyzed using agarose gel Kim, Korea University Guro Hospital. MCF-7 cells were grown in electrophoresis and visualized after treatment with ethidium bromide. RPMI 1640 medium supplemented with 10% fetal bovine serum. Stable TamR-MCF-7 cells were maintained in phenol red-free RPMI Luciferase reporter assay. MCF-7 and TamR-MCF-7 cells were medium supplemented with 10% charcoal-stripped and steroid- transiently co-transfected with 0.5 μg MMP9-, NFκB-, and AP1- depleted fetal bovine serum (FBS). luciferase plasmids and 0.5 μg pSV-β-galactosidase reporter vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) transfection Transwell invasion assays. The invasiveness of tumor cells was reagent. The MMP9- and NF-κB-luciferase plasmids were kindly assessed by an invasion assay in Transwell chambers consisting of provided by Dr. I. Shin (Hanyang University, South Korea) and the AP1- a Transwell membrane (8 μm pore size, 6.5 mm diameter) (Corning luciferase plasmid was kindly provided by Dr. S. Jang (University of Life Science, Corning, NY, USA) coated with Matrigel (100 μg/ml, Ulsan College of Medicine, South Korea). After transfection for 24 h, 100 μl/well). Cells were seeded onto the upper wells in the presence the cells were treated with metformin for the described period, 24 h. Cell of different concentrations (5 mM to 100 mM) of metformin. The extracts were prepared for the luciferase assays. The luciferase activity bottom chambers of the Transwell were filled with cell growth was normalized by β-galactosidase activity. medium. Cells were fixed, stained, and counted under a light microscope after 24 h incubation. Electrophoretic mobility shift assay (EMSA). The oligonucleotides were labeled with (γ-32P) ATP and incubated with nuclear extracts In-gel zymography. MMP activities were assayed as described for 30 min using gel shift assay kit (Promega, Madison, WI, USA). elsewhere (18). Briefly, 5×105 cells in a six-well plate were cultured in The DNA–protein complexes were resolved on non-denaturing and serum-free medium for 16 to 24 h, and the conditioned medium thus non-reducing 6% acrylamide gels. The probes used for EMSA were produced was separated on a sodium dodecyl sulfate –polyacrylamide as follows: for AP1, 5’-CGC TTG ATG ACT CAG CCG GAA-3’; electrophoresis gel (by SDS–PAGE) containing 1 mg/ml gelatin. The and for NFκB, 5’-AGT TGA GGG GAC TTT CCC GAA C-3’. gel was washed with buffer I (Tris–HCl; pH 7.5) and 2.5% Triton X- 100), incubated overnight in buffer II (150 mM NaCl, 5 mM CaCl2, ELISA assay. Cells were cultured at 5×105 cells per well in 6-well and 50 mM Tris–HCl; pH 7.6) at 37˚C, and stained with Coomassie plates. At approximately 80% confluence, the cells were stimulated by blue. Clear bands indicated where MMPs had degraded the gelatin. metformin (5 mM to 10 mM) in the absence and presence of 100 nM PMA. The supernatants were collected after 24 h, and the level of Cell migration assay. Cell migration was assessed using a wound- MMP9 in the cultured media were determined using an ELISA kit healing assay. Cells were seeded at 2×104 cells per 6-well plate. (R&D Systems, Abingdon, UK). After scraping the cell monolayer with a sterile micropipette tip, the wells were washed with serum-free medium and incubated several Statistical analysis. The results are represented as the times with different concentrations (5 mM to 100 mM) of mean±standard error (SE), and the statistical comparisons between metformin. The first image of each scratch was acquired at time groups were performed using one-way ANOVA followed by zero. Each scratch was examined and captured at the same location, Student’s t-test. A value of p≤0.05 was considered statistically and the healed area was measured after 24 h. significant.

4128 Jang et al: Metformin Inhibits Breast Cancer Cell Migration

Figure 1. Suppression of migration and invasion by metformin in MCF-7 and tamoxifen-resistant TamR-MCF7 human breast cancer cells. A: Cultures of MCF-7 (upper panel) and TamR-MCF-7 (lower panel) cells were ‘wounded’ by scratching with a pipette tip and incubated with or without 100 nM PMA and the indicated concentrations of metformin. Representative images of wound healing were taken at the time of the scratch and 24 h later. B: Invasion was determined using a Transwell assay after treatment of MCF-7 (upper panel) and TamR-MCF-7 (lower panel) cells with the indicated concentrations of metformin, with or without 100 nM PMA for 24 h. Representative photomicrographs show membrane-associated cells were stained with eosin. The data represent the average of three independent experiments±SD. Statistically significant difference (p≤0.05) from *PMA treatment only, #from the control group.

Results PMA-induced migration and invasion in a somewhat dose- dependent manner (Figure 1A and B, upper panel). In Metformin suppresses migration and invasion in both MCF-7 addition, we confirmed the anti-invasive effects of metformin and TamR-MCF-7 cells. Since TamR-MCF-7 cells display on TamR-MCF-7 cells. Metformin inhibited the migration and enhanced invasive capacity (19), we first examined the invasion of TamR-MCF-7 cells in a manner similar to its inhibitory effects of metformin on the invasive potency of effects on MCF-7 cells (Figure 1A and B, lower panel). These MCF-7 cells and TamR-MCF-7 cells using in vitro migration results indicate that metformin effectively inhibits migration and invasion assays. MCF-7 cells treated with PMA, of which and invasion of MCF-7 and TamR-MCF-7 cells. the effects on cancer cell migration have been previously described (20), presented a 2- to 3-fold increase in migration Metformin reduces expression and secretion of MMP9. It is and invasion. However, treatment with metformin inhibited known that MMP9 and MMP2 are critical for invasion and

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Figure 2. The reduction of MMP9 expression and secretion by metformin in MCF-7 and tamoxifen-resistant TamR-MCF7 human breast cancer cells. A: MMP2 and MMP9 protein and mRNA levels in MCF-7 and TamR-MCF-7 cells were evaluated using western blot analysis and semi-quantitative RT-PCR. Expression levels of β-actin and GAPDH were included as internal control. The asterisk represents an unspecified band. B: MCF-7 (upper panel) and TamR-MCF-7 (lower panel) cells in serum-free medium were treated with the indicated concentrations of metformin with or without 100 nM PMA for 24 h. The cell-conditioned medium was collected after treatment and assayed using gelatin zymography to analyze the activity of MMP9. Assays were performed several times. C: MMP9 secreted by MCF-7 cells in the conditioned media was quantified using ELISA. Data are the means±SD of triplicate experiments. **Statistically significant difference from PMA treatment only (p≤0.001).

migration in several cancer cell lines (21). Therefore, we zymography was performed in PMA-treated MCF-7 cells examined the effect of metformin on the levels of both and TamR-MCF-7 cells. The results showed that metformin MMP2 and MMP9 in MCF-7 and TamR-MCF-7 cells. As reduced MMP9 activity in both cell lines (Figure 2B). To shown in Figure 2A, metformin reduced MMP9 expression confirm the inhibitory effects of metformin on MMP9, an in both PMA-treated MCF-7 cells and TamR-MCF-7 cells in ELISA assay was performed to estimate MMP9 secretion. It a dose-dependent manner. However, the expression of MMP2 was shown that a 24-h treatment of metformin significantly was not significantly affected by PMA nor by metformin inhibited the secretion of MMP9 from PMA-treated MCF-7 treatment in either cell line (Figure 2A). To further test the cells (Figure 2C). These results indicate that metformin effect of metformin on MMP activity, which is related to the inhibits cell migration and invasion through down-regulation invasion and metastasis of human cancer, gelatin of MMP9 in both MCF-7 and TamR-MCF7 cells.

4130 Jang et al: Metformin Inhibits Breast Cancer Cell Migration

Figure 3. The inhibition of MMP9 promoter activity by metformin in MCF-7 and tamoxifen-resistant TamR-MCF-7 human breast cancer cells. The MCF-7 and Tam-MCF-7 cells were transfected with MMP9-Luc (0.5 μg) (A), NFκB-Luc (0.5 μg) (B), and AP1-Luc (0.5 μg) (C) reporter plasmids. After 24 h, cells were treated with the indicated concentrations of metformin for 24 h (for MCF-7 cells in the presence of 100 nM PMA) and harvested for luciferase assays. The luciferase activity was normalized to that of β-galactosidase. Data are the means±SD of triplicate experiments and denote a statistically significant difference at *p≤0.05 and **p≤0.001 from PMA treatment only, and at #p≤0.05 and ##p≤0.001 from the control.

Metformin inhibits transcription of MMP9. To investigate the Luc activity in MCF-7 cells (Figure 3B, upper panel) but molecular mechanisms underlying the inhibitory effects of significantly reduced NFκB-Luc activity in TamR-MCF-7 metformin on MMP9 expression, we performed a promoter cells at concentrations ranging from 25 to 75 mM (Figure assay in MCF-7 and TamR-MCF-7 cells. We used a 3B, lower panel). However, AP1-Luc activity was reduced luciferase reporter plasmid assay containing the minimal by metformin in a dose-dependent manner in both MCF-7 response elements, NFκB and AP1, located in the -710 bp and TamR-MCF-7 cells (Figure 3C). These results indicate region upstream of the transcription start site of the human that the AP1 transcription factor contributed to the inhibition MMP9 gene. After MCF-7 cells were transfected with of MMP9 by metformin in MCF-7 and TamR-MCF7 cells. MMP9-Luc, the promoter activity in response to PMA, as In addition, TamR-MCF-7 cells may employ another well as to PMA with metformin, were examined. MMP9-Luc signaling pathway for the regulation of MMP9. activity showed an 8-fold increase in response to PMA and 75% repression of this response on treatment with both PMA Metformin inhibits specific transcription factor binding to and 10 mM metformin (Figure 3A, upper panel). the MMP9 promoter region. To determine whether Additionally, metformin treatment of TamR-MCF7 cells metformin leads to the repression of MMP9 transcription by inhibited MMP9-Luc activity in a dose-dependent manner inhibiting the binding of transcription factors such as AP1 or (Figure 3A, lower panel). This suggests that metformin can NFκB to the proximal promoter region, we performed inhibit MMP9 expression at the transcriptional level. To EMSA. Nuclear extracts from MCF-7 cells were prepared confirm this observation, luciferase reporter plasmids and used to detect AP1 binding to the proximal AP1 containing tandem repeats of the AP1- or NFκB-binding sites sequence within the MMP9 promoter. As shown in Figure were used to study the effects of metformin on the promoter 4A, metformin significantly reduced PMA-induced AP1 activity. Treatment with metformin had no effect on NFκB- DNA-binding activity in MCF-7 cells. Nuclear extracts from

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Figure 4. The effects of metformin on the DNA-binding activities and expression in MCF-7 and tamoxifen-resistant TamR-MCF-7 human breast cancer cells. A: MCF-7 cells were treated with metformin for 24 h in the presence of 100 nM PMA, and nuclear extracts (5 μg) were prepared. The nuclear extracts were incubated with radiolabeled oligonucleotides containing the AP1 motif in the MMP9 promoter. Nuclear extracts (5 μg) from TamR-MCF-7 cells treated with metformin for 24 h were mixed with radioactive oligonucleotides containing the AP1 and NFκB motifs in the MMP9 promoter. Bound complexes were analyzed by EMSA. B: The nuclear fraction was extracted from MCF-7 cells treated with metformin and 100 nM PMA for 24 h, and from TamR-MCF-7 cells treated with metformin for 24 h. The nuclear levels of AP1 (c-FOS and c-JUN) and NFκB (p65) subunits were determined by western blot analysis using subunit-specific antibodies. PCNA expression was included as an internal control.

TamR-MCF-7 cells treated with different concentrations of AP1 and/or NFκB transcription factors. To identify the metformin for 24 h were isolated and analyzed for AP1 and subunit of the AP1 transcription factor that is regulated by NFκB DNA-binding activities. Metformin reduced the NFκB metformin, we examined the expression of c-FOS and c-JUN as well as AP1 DNA-binding activities in a dose-dependent through nuclear fractionation. Our data show that metformin manner (Figure 4A). These data were consistent with the reduced PMA-induced c-JUN expression but had little effect promoter analysis (Figure 3), suggesting that metformin on the expression of c-FOS or p65 in MCF-7 cells (Figure inhibits MMP9 expression through DNA binding activity of 4B). Furthermore, metformin markedly inhibited p65 and c-

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FOS expression in TamR-MCF-7 cells but had no effect on factors related in the regulation of MMP9 in the expression of c-JUN (Figure 4B). These results suggest TamR-MCF-7 cells. Finally, we evaluated the suppression of that the mechanism of metformin inhibition of MMP9 is DNA-binding activity by metformin from the MMP9 based on the DNA binding activity of different transcription promoter in both MCF-7 and TamR-MCF-7 cells. factors in MCF-7 (AP1 only) and TamR-MCF-7 cells (AP1 Importantly, we found that treatment with metformin and NFκB). dramatically reduces DNA-binding activity of the transcription factors AP1/NFκB in both cell lines. Our results Discussion demonstrate that metformin suppression of AP1 or NFκB is an effective strategy for blocking MMP9 induction. Thus, the TamR-MCF-7 cells express increased levels of epidermal regulation of AP1 and NFκB, which is downstream of growth factor receptor (EGFR), have enhanced motility, and several signaling pathways such as Focal Adhesion Kinase, a fibroblast-like phenotype (19, 22). The progression of phosphoinositide 3-kinase/protein kinase B, and Mitogen- tamoxifen-resistant tumors is one of the challenges activated protein kinases, is essentially involved in the encountered in endocrine therapy of breast cancer. Several cascade of tumor invasion and metastasis (27). studies have supported the hypothesis that tamoxifen In conclusion, we demonstrate, as far as we are aware for resistance occurs via augmentation of altered signaling the first time, that metformin inhibits MCF-7 and TamR- pathways. Specifically, EGFR, EGFR2/HER2, and insulin- MCF-7 breast cancer cell migration and invasion through like growth factor-1 receptor signaling pathways are MMP9 regulation. This finding brings new clues to the elevated, and the activity of kinases, such as extracellular understanding of the differences inaction of metformin regulated kinase 1/2, p38, and protein kinase B, is also between MCF-7 and TamR-MCF-7 cells. Metformin increased (23). treatment might be considered in preventing the invasion and MMP9 is a 92-kDa type-IV collagenase that belongs to metastasis of human malignant tumors, such as drug-resistant the gelatinase group of human MMPs. The expression of breast cancer. MMP9 is regulated by diverse growth factors, cytokines, and xenobiotics, such as PMA (24), and is related to the invasion, Declaration of Interest metastasis, and angiogenesis of various types of cancer cell lines. Therefore, developing drugs that inhibit MMP9 could None. be useful in human cancer therapy. Metformin, which reduces blood glucose levels and Acknowledgements enhances insulin sensitivity, is the most commonly This work was supported by grant K1133691 from Korea prescribed oral drug in patients with type II diabetes. University, and by the Basic Science Research Program Metformin treatment has growth-inhibitory effects involving (2014R1A2A2A01003566) from the National Research Foundation the activation of AMP-activated kinase and serine/threonine of Korea (NRF) grant, funded by the Ministry of Education, Science protein kinases, which serve as energy sensors in all and Technology (MEST), Republic of Korea. eukaryotic cell types (25, 26). Recently, several studies have shown that metformin can inhibit cell proliferation in several References cancer cell lines including MCF-7 cells (25). In the present study, we observed that metformin was 1 Chambers AF and Matrisian LM: Changing views of the role of effective in inhibiting in vitro invasion through down- matrix metalloproteinases in metastasis. J Natl Cancer Inst regulation of MMP9 in both MCF-7 and TamR-MCF-7 cells. 89(17): 1260-1270, 1997. Metformin suppressed the PMA-induced activity of MMP9 2 Woodhouse EC, Chuaqui RF and Liotta LA: General mechanisms in MCF-7 cells, whereas the activity of MMP2 was not of metastasis. Cancer 80(8 Suppl): 1529-1537, 1997. 3 Yan C and Boyd DD: Regulation of significantly affected. Similar data were also found for gene expression. J Cell Physiol 211(1): 19-26, 2007. TamR-MCF7 cells. To determine the underlying mechanism, 4 Kessenbrock K, Plaks V and Werb Z: Matrix metalloproteinases: we performed several experiments including gelatin regulators of the tumor microenvironment. Cell 141(1): 52-67, zymography, real time (RT)-PCR, western blot analysis, and 2010. luciferase reporter gene assay. Based on the above data, we 5 Galewska Z et al: Gelatinase matrix metalloproteinase (MMP)- found that the inhibitory effect of metformin on MMP9 2 and MMP-9 of the umbilical cord blood in preeclampsia. Clin activity was due to its effects on transcriptional regulation. Chem Lab Med 46(4): 517-22, 2008. 6 Ram M, Sherer Y and Shoenfeld Y: Matrix metalloproteinase-9 In addition, we observed that the major target of metformin and autoimmune diseases. J Clin Immunol 26(4): 299-307, 2006. in the regulation of MMP9 was AP-1 transcription factor, 7 Himelstein BP et al: Transcriptional activation of the matrix whereas NFκB had little effect in MCF-7 cells. However, metalloproteinase-9 gene in an H-ras and v-myc transformed rat metformin modulated both AP1 and NFκB, key transcription embryo cell line. Oncogene 14(16): 1995-1998, 1997.

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