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Cancer Research 68, 4201, June 1, 2008. doi: 10.1158/0008-5472.CAN-07-2570
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Mel-18 Negatively Regulates INK4a/ARF-Independent Cell Cycle Progression via Akt Inactivation in Breast Cancer

Jeong-Yeon Lee1, Ki-Seok Jang1, Dong-Hui Shin1, Mi-Yun Oh1, Hyun-Jun Kim1, Yongseok Kim2 and Gu Kong1

Departments of 1 Pathology and 2 Biochemistry, College of Medicine, Hanyang University, Seoul, Republic of Korea

Requests for reprints: Gu Kong, Department of Pathology, College of Medicine, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea. Phone: 82-2-2290-8251; Fax: 82-2-2295-1091; E-mail: gkong{at}hanyang.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G1 arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G1-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27Kip1 phosphorylation at Thr157, but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser473 was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27Kip1 was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3β phosphorylation, β-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18->Akt->G1 phase regulators. [Cancer Res 2008;68(11):4201–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Polycomb group (PcG) proteins are highly conserved factors that act as transcriptional repressors. They were originally identified in Drosophila and play a crucial role in maintaining repression of homeotic genes during development (1). PcG proteins control many diverse biological processes, including cellular differentiation, proliferation, and tumorigenesis (2, 3). They form multimeric protein complexes and epigenetically regulate many target genes through chromatin modification (2). These proteins are classified into two distinct groups: one is the polycomb repressive complex 2 group, which is thought to initiate repression by methylating histone H3 lysine 9 and 27, and another is the polycomb repressive complex 1 group, which is considered to be involved in maintenance of repression (2, 3).

Mel-18, also known as polycomb group ring finger protein, is a constituent of polycomb repressive complex 1. Mel-18 is highly homologous to Bmi-1 by sharing 57% amino acid identity and a zinc finger named RING at its NH2 terminus. Wiederschain et al. (4) have recently reported that Bmi-1 and Mel-18 in HeLa cells compose nearly identical core proteins by sharing many constituents, such as Ring1, Ring2, RYBP, HPH1, HPH2, HPH3, CBX4, and CBX8. Despite their structural similarities, many studies report differences in their roles in cancer cell growth and survival. Unlike Bmi-1, which is overexpressed in various human tumors and generally accepted as a proto-oncogene (4–7), Mel-18 is shown to be either oncogenic or tumor suppressive, depending on the cancer system. Mel-18 was originally cloned from B16 mouse melanoma cells and their transcripts were shown to be highly expressed in many tumor cells including human melanoma and Hodgkin's lymphomas (8, 9). Mel-18 and Bmi-1 are highly expressed in DAOY medulloblastoma cells and small hairpin RNA–mediated knockdown of either one or both of them in DAOY cells inhibits cancer cell growth in vitro and suppresses the growth of human medulloblastoma xenografts in vivo (4). However, markedly enhanced tumorigenicity has been shown in nude mice injected with NIH 3T3 cells overexpressing antisense mel-18, but not with sense construct (10). We have also reported that Mel-18 directly interacts with cyclin D2 and knockdown of Mel-18 by antisense construct in T-47D breast cancer cells enhances cell proliferation (11). Recently, Guo et al. (12) reported an inverse correlation between Mel-18 and Bmi-1 expression in breast cancer cell lines and primary human breast tumors. Furthermore, they have shown Mel-18 as a tumor suppressor in MCF7 breast cancer cells and suggested repression of Bmi-1 expression and down-regulation of Akt activity as its mechanism. Therefore, the functional role of Mel-18 should be carefully interpreted within the context of a cancer system.

Akt signaling plays a crucial role in many biological processes including cell proliferation and survival. Its aberrant activation contributes to tumor progression in many types of human cancer including breast cancer (13). Akt promotes cell proliferation through its many downstream effectors. Particularly, Akt positively regulates G1 to S transition in cell cycle progression through regulation of D-type cyclins and cyclin-dependent kinase (Cdk) inhibitors, such as p27Kip1, at the transcriptional level. Akt controls the canonical Wnt pathway (14, 15). Akt-dependent phosphorylation of the Forkhead family transcription factors leads to their nuclear exclusion and, thus, inhibition of FoxO factor–mediated gene expression including p27Kip1 (16, 17) and up-regulation of cyclin D1 (18). Moreover, Akt regulates the stability and subcellular localization of G1 regulators. Akt-dependent inactivation of glycogen synthase kinase-3β (GSK-3β) blocks cyclin D1 phosphorylation at Thr286, which protects cyclin D1 from proteolytic degradation (19). Phosphorylation of p27Kip1 at Thr157 by Akt causes cytoplasmic retention, which blocks binding to nuclear Cdk2/cyclin E (20–22). Similarly, p21Cip1/WAF1 is retained in the cytoplasm on phosphorylation at Thr145 by Akt (23).

Previously, we have reported that loss of Mel-18 expression accelerates cell proliferation of T-47D human breast cancer cells (11). However, its exact mechanism remains undefined. To elucidate the growth regulatory mechanism of Mel-18, we investigated the effect of Mel-18 on cell cycle in breast cancer cells. In this study, we found that Mel-18 attenuated cell growth and caused G1 arrest by down-regulation of cyclin D1 expression and p27Kip1 phosphorylation at Thr157 via inactivation of the Akt signaling pathway. Our data suggest that Mel-18 controls cell cycle as a negative regulator of Akt and may have a possibility of being the tumor suppressor gene in human breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples from patients. Fresh frozen normal and tumor tissues were obtained from 40 patients of invasive ductal carcinoma who underwent surgery between 2002 and 2005 at Hanyang University Hospital (Seoul, Korea).

Cell lines, kinase inhibitors, and antibodies. T-47D cells were maintained in RPMI 1640 (Welgene) supplemented with 10% fetal bovine serum (FBS; Welgene) and SK-BR-3 cells were maintained in DMEM (Welgene) containing 10% FBS at 37°C in a humidified 5% CO2 incubator. The phosphatidylinositol 3-kinase inhibitor LY294002 and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (MEK) inhibitor PD98059 were from Sigma. For immunoblottings and immunoprecipitations, the following antibodies were used: antibodies against Akt, Ser473 phospho-Akt, GSK-3β, Ser9 phospho-GSK-3β, phospho-MAPK, and p15INK4b (Cell Signaling Technology); cyclin D1, cyclin E, p27Kip1, Cdk2, Cdk4, Cdk6, Mel-18, Bmi-1, and Sp1 (Santa Cruz Biotechnologies); Thr157 phospho-p27Kip1 (R&D Systems); total MAPK (Erk42/44; Zymed Laboratories); total β-catenin (Upstate Biotechnology); {alpha}-tubulin (Calbiochem); p27Kip1 and β-catenin (BD Transduction Laboratories); Cy5-conjugated antimouse IgG (Jackson Immunoresearch); and β-actin and phycoerythrin-conjugated antirabbit IgG (Sigma).

Plasmids, transfection, and retroviral infection. Full-length Mel-18 cDNA in pOTB7 vector was purchased (clone MGC-10336; American Type Culture Collection). The retroviral vector pCL-IRES3-EGFP was a generous gift from Chang-Hwan Park (Hanyang University, Seoul, Korea). The p27Kip1{Delta}NLS mutant construct (24) was kindly provided by Incheol Shin (Hanyang University, Seoul, Korea). Mel-18 cDNA in pOTB7 was cut with BamHI and XhoI restriction enzymes, blunt-ended with Klenow, and subcloned into XhoI-cut and blunt-ended pCL-IRES3-EGFP vector. Subcloned Mel-18 cDNA clones in the forward and reverse orientations were used as expression and antisense constructs, respectively. H29D cells were transfected with each plasmid using Lipofectamine (Invitrogen). After 48-h transfection, the medium containing retroviruses was harvested, filtered through 0.45-µm filters, and transferred to target cells with 6 µg/mL polybrene (Sigma) or stored in aliquots at –70°C for later use. The infected cells were transiently used in experiments or selected with 10 µg/mL blasticidin S (Invitrogen) for 6 d, maintained with the medium containing 2 µg/mL blasticidin S, and used as stable cells.

Cell proliferation assay. The CellTiter 96 nonradioactive cell proliferation assay kit (Promega) was used for measurement of cell proliferation. As described by the manufacturer, cells were seeded onto 96-well plates and incubated with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye solution at 37°C for 4 h, and stopped by addition of solubilization/stop solution. The formazan product was measured at 570-nm absorbance using a microplate reader (Bio-Rad).

Cell cycle analysis. Cells were trypsinized, washed once with ice-cold PBS, and fixed with 75% ethanol at –20°C for 2 h. After washing twice with PBS, cells were stained with 10 µg/mL propidium iodide (Sigma) containing 1 mg/mL RNase at 37°C for 20 min in the dark and analyzed with FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

Reverse transcription-PCR. Total RNA was isolated with Trizol (Invitrogen) and reverse transcription-PCR (RT-PCR) was done with Access RT-PCR system (Promega) according to the instructions of the manufacturers. The cDNA amplification was carried out using the GeneAmp PCR system (Perkin-Elmer) as follows: 94°C for 5 min, 22 to 28 cycles of 94°C for 1 min, 54°C to 58°C for 1 min, 72°C for 1.5 min, and 72°C for 10 min. The following specific primers were used for PCR reaction: cyclin D1, 5'-AGCCATGGAACACCAGCTC-3' and 5'-GCACCTCCAGCATCCAGGT-3'; cyclin E, 5'-ATACAGACCCACAGAGACAG-3' and 5'-TGCCATCCACAGAAATACTT-3'; p27Kip1, 5'-CAAACGTGCGAGTGTCTAACG-3' and 5'-GCAGGTCGCTTCCTTATTCCT-3'; p15INK4b, 5'-GGTAAGAAAATAAAGTCGTT-3' and 5'-GGAATGCGCGAGGAGAACAA-3'; mel-18, 5'-GTACTTCATCGACGCCACCACTATC-3' and 5'-CTCGTCCTCGTACAGAACCTCCA-3'; and β-actin, 5'-CCAGAGCAAGAGAGGTATCC-3' and 5'-CTGTGGTGGTGAAGCTGTAG-3'.

Immunoblotting and immunoprecipitation. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and immunoblotting was carried out as described (25). For immunoprecipitation, 1 µg of Cdk4 and Cdk2 antibodies was chemically cross-linked with 20 mmol/L dimethyl pimelimidate (Sigma) in 0.2 mol/L sodium tetraborate (pH 9.0) to 20 µL of protein A agarose (Upstate Biotechnology). Cell lysates (1 mg) and the cross-linked antibodies were immunoprecipitated as described (25).

In vitro kinase assay. Total cell lysates were immunoprecipitated with anti-Cdk4 or anti-Cdk2 antibodies at 4°C for 16 h. The immunoprecipitant was washed thrice with RIPA buffer, once with kinase buffer [50 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2, 2.5 mmol/L EGTA, 1 mmol/L DTT], and reacted in kinase buffer containing 20 µmol/L of ATP, 10 µCi of [{gamma}-32P]ATP (6,000 Ci/mmol; Amersham Life Science), and 5 µg of histone H1 (Sigma), a Cdk2 substrate, or 2 µg of glutathione S-transferase-pRb (Santa Cruz Biotechnologies), a Cdk4 substrate, at 30°C for 30 min. The reaction was stopped by adding 10 µL of 5x SDS sample buffer, and the mixture was heated at 95°C for 5 min. The radioactive kinase activity was visualized by SDS-PAGE (15% gel) and autoradiography.

Cell fractionation. Cell fractionation was carried out using NE-PER Nuclear and cytoplasmic extraction reagents (Pierce) as described by the manufacturer. Briefly, 2 x 106 cells were pelleted and lysed in 200 µL of ice-cold CER I buffer and 11 µL of ice-cold CER II buffer. After centrifugation at 4°C for 5 min, the supernatant (cytoplasmic extract) was collected and the pellet was resuspended in 50 µL of ice-cold NER buffer and vortexed for 15 s every 10 min, for a total of 50 min. After centrifugation at 4°C for 10 min, the supernatant was collected as nuclear extract. The extracts were subjected to immunoblotting or stored at –80°C until use.

Immunofluorescence staining. Cells (1 x 105 per chamber) were seeded onto a four-chamber slide glass (Lab-Tek II Chamber Slide System, Nalge Nunc International) for 16 h, washed with PBS, fixed with 4% paraformaldehyde for 30 min, and permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 3% bovine serum albumin for 1 h, cells were stained with appropriate primary antibodies diluted 1:200 at 4°C for 16 h. Phycoerythrin-conjugated antirabbit IgG (1:200) and Cy5-conjugated antimouse IgG (1:200) were used as secondary antibodies. To visualize the nucleus, 4'6-diamidino-2-phenylindole (DAPI) staining was also done as previously described (26). Immunofluorescence was detected by fluorescence microscopy (Nikon).

Reporter assay. Cells were seeded at 1 x 105 per well in a 12-well plate, and then transfected with 1 µg of pTOP-FLASH, a T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter, or pFOP-FLASH, a TOP mutant form (27), and β-gal cDNAs using FuGENE6 reagent (Roche Diagnostics Corp.). After incubation for 24 h, the cells were infected with retrovirus encoding empty vector or Mel-18 for 48 h. Luciferase activity, expressed as relative light units, was measured using Luciferase Assay system (Promega) and MicroLumat Plus LB96V luminometer (Berthold Technologies) and normalized by β-galactosidase activity.

Statistical analysis. The Western blot and RT-PCR band densities were determined using AlphaEase FC Software (AlphaInnotech, Inc.) and statistical analysis was done with Fisher's exact test or Student's t test using SPSS software (SPSS version 11.0).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Loss of Mel-18 correlates with phosphorylated Akt in primary human breast cancer. Increased Akt activity plays a key role in cancer progression and it is frequently activated in various human cancers including breast cancer (28). To investigate whether Mel-18 expression and Akt activity have correlation in human primary breast cancer, we tested Mel-18 expression and Akt phosphorylation at Ser473, a recognition site for Akt activation, in pairs of normal and tumor breast tissues from individual breast cancer patients by immunoblotting. Mel-18 was expressed in both normal and tumor tissues. However, 32 of 40 tumors showed significantly reduced Mel-18 expression compared with normal tissues (Fig. 1, top ). Furthermore, we found that the tumor cells, which have lower expression of Mel-18 compared with normal tissue, showed enhanced Akt phosphorylation (Fig. 1, bottom). Akt phosphorylation was increased in 24 of 40 tumor tissues but total Akt expression remained unchanged. Our result shows an inverse correlation between Mel-18 and Akt activity in human clinical samples, suggesting that loss of Mel-18 may play an important role in breast cancer progression.


Figure 1
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  		Figure 1. The relationship between Mel-18 expression and Akt phosphorylation at Ser473 in human primary breast tumors. Cell lysates (50 µg of protein) of normal breast tissue (N) and breast tumor tissue (T) pairs from individual breast cancer patients were subjected to immunoblotting with Mel-18, phospho-Akt, and total Akt antibodies (top). The densities of the immunoblotting bands were measured, and the expression levels of phospho-Akt in 40 of breast cancer patients who have low Mel-18 expression (normal > tumor) or high expression (normal ≤ tumor) were measured and statistically analyzed with Fisher's exact test (bottom).

 
Mel-18 attenuates cell growth by G1 arrest in human breast cancer. To examine the role of Mel-18 in human breast cancer cells, we first measured Mel-18 protein and mRNA levels in various human breast cancer cell lines by Western blot analysis and RT-PCR, respectively. In estrogen receptor–positive T-47D and MCF7 cell lines, Mel-18 was moderately or highly expressed, whereas in estrogen receptor–negative SK-BR-3, MDA-MB-231, and MDA-MB-435 cell lines, low expression was seen (Fig. 2A ). Next, the role of Mel-18 in breast cancer cell proliferation was examined in two breast cancer cell lines, SK-BR-3 and T-47D cells, where endogenous Mel-18 expression levels are low and high, respectively. To overexpress or suppress Mel-18 expression in low- or high-expressing cells, we generated retroviral Mel-18 and Mel-18 antisense constructs, respectively. SK-BR-3 cells, which have low expression of Mel-18, were infected with retrovirus encoding Mel-18 cDNA and selected with blasticidin S. Mel-18 overexpression in the stable cells was confirmed by Western blot analysis (Fig. 2B, top left) and the effect of Mel-18 on cell growth was determined by MTT assay. There was an inverse relationship between Bmi-1 and Mel-18 on Mel-18 overexpression or suppression (Fig. 2B), which is consistent with a previous report (12). As shown in Fig. 2B (bottom left), cell growth was attenuated in Mel-18–overexpressing SK-BR-3 cell lines (P < 0.05, Student's t test). Because of the difficulty in long-term maintenance of the stable cells, attributed to the growth inhibitory effect of Mel-18, and comparable efficiency in Mel-18 transduction, all further experiments in this article were done after transient retroviral transduction. SK-BR-3 cells were retrovirally infected with Mel-18 for 24 hours and cell cycle was analyzed by flow cytometry after 72 hours. As seen in Fig. 2B (right), the percentage of G1 phase cells was increased and S phase was slightly reduced in the Mel-18–overexpressing cells (P < 0.05, Student's t test). Furthermore, we blocked Mel-18 expression in T-47D cells using a retrovirus encoding a Mel-18 antisense construct. As shown in Fig. 2C, Mel-18 expression was effectively suppressed by the Mel-18 antisense construct as assessed by Western blot analysis (top left). In nonsynchronized cells, cell growth was not affected by expression of Mel-18 antisense (data not shown). However, when cells were restimulated with 10% FBS after serum starvation for 48 hours, cell growth was enhanced and the proportion of S phase cells was increased by antisense construct–mediated knockdown of Mel-18 expression as assessed by MTT assay (Fig. 2C, bottom left) and cell cycle analysis, respectively (Fig. 2C, right; P < 0.05, Student's t test). Taken together, these results indicate that Mel-18 attenuates growth of breast cancer cells through G1 arrest.


Figure 2
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  		Figure 2. Effect of Mel-18 on cell proliferation and cell cycle in human breast cancer cell lines. A, Mel-18 expression was quantified in various breast cancer cell lines. Cell lysates (50 µg) and total RNA of human breast cancer cell lines were subjected to Western blot analysis and RT-PCR, respectively. β-Actin was used as a loading control. ER, estrogen receptor. B, SK-BR-3 cells were infected with retrovirus encoding empty vector (Con) or Mel-18 cDNA (Mel-18) and selected with antibiotics as described in Materials and Methods. Two weeks after selection, which is equivalent of 16 d after viral infection, Mel-18 overexpression was confirmed by immunoblotting (top left). These cells were serum starved for 24 h for synchronization and subjected to MTT assay for up to 5 d (bottom left, day 0 set as time of release with complete medium with 10% FBS). After transient transduction of Mel-18 cDNA by retroviral infection for 24 h, the cell cycle was analyzed by flow cytometry after 72 h and the percentage of each phase (G1-M) was indicated on the right. C, T-47D cells were infected with retrovirus expressing empty vector (Con) or Mel-18 antisense construct (MelAS). At 24 h posttransduction, cells were serum starved for 48 h, restimulated with 10% FBS–containing medium for 24 h, and then subjected to immunoblotting (top left). Identically transduced, starved, and released cells were subjected to MTT assay for up to 5 d (bottom left; day 0 set as the time of release with complete medium). Cell cycle analysis was done with cells treated identically as day 5 of MTT assay (right). All of data were done in triplicate and statistically analyzed with Student's t test. The results are shown as mean ± SD. *, P < 0.05.

 
Mel-18–mediated G1 arrest occurs by down-regulation of Cdk4 and Cdk2 activities via changes in cyclin D1, cyclin E, and p27Kip1, but not INK4a/ARF family genes. Because we observed changes in G1 to S transition by Mel-18 overexpression or suppression (Fig. 2), we examined whether Mel-18 affected Cdk4 and Cdk2 activities. In Mel-18–overexpressing SK-BR-3 cells, Cdk4 kinase activity was decreased whereas Cdk2 kinase activity was not changed (P = 0.001, Student's t test; Fig. 3A, left ). On knockdown of Mel-18 expression in T-47D cells, both Cdk4 and Cdk2 activities were augmented (P = 0.034 and P = 0.038, respectively, Student's t test; Fig. 3A, right). Total amounts of Cdk4 and Cdk2 proteins were unchanged in both cell lines (Fig. 3A, Input). We next explored the effect of Mel-18 on the expression of G1 phase regulators, cyclins and Cdk inhibitors, which could affect Cdk4 and Cdk2 activities. In Mel-18–infected SK-BR-3 cells, cyclin D1 expression was down-regulated whereas cyclin E expression was unchanged (P < 0.001, Student's t test; Fig. 3B, left). On the other hand, both cyclin D1 and cyclin E expressions were up-regulated by Mel-18 blockade in T-47D cells (P = 0.002 and P = 0.045, respectively, Student's t test; Fig. 3B, right). In addition, total amount of p27Kip1 remained unchanged, but phosphorylation of p27Kip1 at Thr157, which is involved in cytoplasmic localization, was significantly reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells (P < 0.001 and P = 0.019, respectively, Student's t test; Fig. 3B). Identical expression patterns were seen at the mRNA level when assessed by RT-PCR (cyclin D1 in SK-BR-3, P = 0.023; in T-47D, P = 0.023, Student's t test; Fig. 3C). Because Mel-18 is known as a regulator of INK4a/ARF family (29–31), we also investigated p16INK4a, p15INK4b, and p14ARF expressions in these cell lines. Although p15INK4b was expressed in both cell lines, its expression remained unchanged by overexpression or suppression of Mel-18 (Fig. 3B and C). The protein level of p16INK4a was not detected in both SK-BR-3 and T-47D cells (data not shown). Both protein and mRNA levels of p14ARF were very weak in T-47D cells and its expression was unchanged in both SK-BR-3 and T-47D cell lines (data not shown). Next, we examined whether the changes seen with total protein were reflected in the association with G1 phase Cdks. As shown in Fig. 3D (left), on Mel-18 overexpression in SK-BR-3, the amount of Cdk4-associated cyclin D1 was decreased (P < 0.001, Student's t test). Moreover, the amount of Cdk4-associated p27Kip1 was decreased whereas Cdk2-associated p27Kip1 was increased as determined by coimmunoprecipitaion, suggesting that p27Kip1 was shifted from Cdk4 to Cdk2 in Mel-18–overexpressing SK-BR-3 cells (P < 0.001 and P = 0.019, respectively, Student's t test). The amount of Cdk2-associated cyclin E remained unchanged. In T-47D cells, coimmunoprecipitation showed the increase in the amounts of both Cdk4-associated cyclin D1 and Cdk2-associated cyclin E (P = 0.023 and P = 0.016, respectively, Student's t test; Fig. 3D, right). The amounts of both Cdk2- and Cdk4-associated p27Kip1 were unchanged (Fig. 3D, right). Therefore, these results indicate that Mel-18–mediated G1 arrest is attributed to changes in G1 cyclin expression and p27Kip1 phosphorylation, but not in INK4a/ARF expressions, in human breast cancer cell lines.


Figure 3
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  		Figure 3. Effect of Mel-18 on G1 regulators in human breast cancer cell lines. Cell lysates of SK-BR-3 cells, retrovirally transduced to express empty vector (Con) or Mel-18, were prepared at 72 h after transient infection for 24 h. Cell lysates of T-47D cells, infected with empty vector (Con) or Mel-18 antisense (Mel AS), were made 5 d after release from synchronization as in Fig. 2C. A, in vitro kinase assays of immunoprecipitated Cdks (IP: Cdk4 and IP: Cdk2) from Mel-18–overexpressing SK-BR-3 cells (left) and Mel-18–suppressed T-47D cells (right) were done with appropriate substrates as indicated. Input, immunoblots of whole-cell lysates with antibodies indicated on the right. GST, glutathione S-transferase. B and C, transiently transduced SK-BR-3 and T-47D were subjected to immunoblotting and RT-PCR to examine the expressions of G1 regulators. D, the identical cell lysates in A were immunoprecipitated with anti-Cdk2 or Cdk4 antibody and the amounts of associated cyclin D1, cyclin E, and p27Kip1 were quantified by immunoblotting. Representative of three independent experiments with similar results. Each blot density was quantified. * and **, P < 0.05, statistically significant increase and decrease, respectively (Student's t test).

 
Mel-18–induced G1 arrest is mediated by the Akt signaling pathway. To understand the molecular mechanism involved in the Mel-18–mediated regulation of cyclin D1 expression and p27Kip1 phosphorylation, we investigated the upstream signaling pathways of cell cycle regulators. Because we saw an inverse correlation between Mel-18 and Akt phosphorylation in primary breast cancer (Fig. 1) and both cyclin D1 expression and p27Kip1 phosphorylation at Thr157 can be regulated by Akt (32), we investigated the Akt signaling pathways in Mel-18– or Mel-18 antisense–infected breast cancer cell lines. In SK-BR-3 cells, Akt phosphorylation at Ser473, a recognition site for Akt activation, was significantly diminished by Mel-18 overexpression as assessed by immunoblotting (P < 0.001, Student's t test; Fig. 4A ). Consistent with these results, knockdown of Mel-18 expression augmented Akt phosphorylation at Ser473 (P = 0.001, Student's t test; Fig. 4B). Total amount of Akt was unchanged in both cells. Because cyclin D1 expression can also be regulated by the MAPK signaling pathway (33), we also investigated this pathway in Mel-18– or Mel-18 antisense–transduced breast cancer cell lines. Phosphorylation and total amount of Erk1/2 were unchanged in both cell lines (Fig. 4A and B). To confirm that the changes of cell cycle regulators were Akt-dependent effects in Mel-18 antisense–infected T-47D cells, we treated the cells with 40 µmol/L of LY294002, a phosphatidylinositol 3-kinase inhibitor, and/or 50 µmol/L of PD98059, a MEK inhibitor. As shown in Fig. 4C, the effect of Mel-18 antisense on cyclin D1 expression and p27Kip1 phosphorylation was abrogated when cells were treated with LY294002 (lane 3 versus lane 2), but not with PD98059 (lane 4 versus lane 2; P = 0.008 and P = 0.009, respectively, Student's t test). Interestingly, endogenous expression of Mel-18 was up-regulated by LY294002 treatment (lane 3 versus lane 2). Likewise, the phosphatidylinositol 3-kinase inhibitor wortmannin also increased Mel-18 expression (data not shown). Collectively, these results show that the changes seen in cyclin D1 expression and p27Kip1 phosphorylation by Mel-18 are mediated by the Akt signaling pathway but not by the Erk pathway.


Figure 4
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  		Figure 4. Mel-18 regulates cyclin D1 expression and p27Kip1 phosphorylation via the Akt signaling pathway but not the Erk pathway. A and B, transiently transduced SK-BR-3 and T-47D, prepared as in Fig. 3, were subjected to immunoblotting to examine the changes in Akt and Erk expression and phosphorylation. The Western blot densities were measured and statistically analyzed with Student's t test. * and **, P < 0.05, statistically significant increase and decrease, respectively. C, four days after release from synchronization, T-47D cells were treated with 40 µmol/L LY294002 and/or 50 µmol/L PD98059 for 24 h, and subjected to immunoblotting with antibodies as indicated. Representative of three independent experiments with similar results. *, P < 0.05, statistically significant increase compared with lane 1; {dagger} and {dagger}{dagger}, P < 0.05, statistically significant increase and decrease compared with lane 2, respectively.

 
Mel-18 blocks Akt-mediated cytoplasmic localization of p27Kip1. Direct phosphorylation of p27Kip1 at Thr157 by Akt is implicated in inhibition of its nuclear import (20–22). Because we found that Mel-18 down-regulated p27Kip1 phosphorylation at Thr157 (Fig. 3A), the effect of Mel-18 on subcellular localization of p27Kip1 was examined. SK-BR-3 cells were infected with retroviral vector encoding each construct (control, Mel-18, p27Kip1{Delta}NLS mutant) or treated with 40 µmol/L LY294002. The overexpression of Mel-18 and p27Kip1{Delta}NLS mutant and the Akt inhibition effect of LY294002 were confirmed by Western blot analysis (Fig. 5A ). We found that cytoplasmic p27Kip1 expression was reduced and nuclear p27Kip1 expression was enhanced by Mel-18 as determined by cytoplasmic and nuclear fractionations and immunoblotting (Fig. 5B). To confirm these data, we also carried out immunofluorescence staining in these cells. Consistent with the above result, cytoplasmic p27Kip1 was reduced in Mel-18–overexpressing SK-BR-3 cells (P < 0.05, Student's t test; Fig. 5C). Therefore, these results suggest that Mel-18 affects Akt-mediated p27Kip1 localization in human breast cancer.


Figure 5
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  		Figure 5. Inhibition of cytoplasmic localization of p27Kip1 by Mel-18 overexpression in SK-BR-3 cells. SK-BR-3 cells were transiently infected with retroviral empty vector (Control), Mel-18, and p27Kip1{Delta}NLS (p27{Delta}NLS), as in Fig. 3, or treated with 40 µmol/L LY294002 for 24 h, as in Fig. 4C. A, each cell lysate was subjected to immunoblotting with indicated antibodies. B, cells were fractionated and the amounts of nuclear and cytoplasmic p27Kip1 were estimated by immunoblotting. p27Kip1{Delta}NLS mutant construct and LY294002 treatment were used as positive controls for cytoplasmic p27Kip1 and nuclear p27Kip1, respectively. {alpha}-Tubulin and Sp1 were tested as controls of cytoplasmic and nuclear fractionations, respectively. C, cells were costained with anti-p27Kip1 antibody coupled with Cy5-conjugated antimouse immunoglobulin (IgG; red) and anti–Mel-18 antibody coupled with phycoerythrin-conjugated antirabbit IgG (white). The efficiency of retroviral infection was determined by observation of green fluorescent protein (GFP) expression (green). To visualize the nucleus, DAPI staining was also done (blue). The ratio of the cytoplasmic and nuclear p27Kip1 expression was measured by counting 1,000 cells each in three independent experiments and the result was shown as percentage of total cells counted ± SD (bottom). *, P < 0.05 (Student's t test).

 
Mel-18 inhibits the Akt-mediated Wnt signaling pathway. Because cyclin D1 expression was affected by Mel-18 at both protein and transcript levels (Fig. 3B and C) and the Akt signaling pathway was implicated (Fig. 4C), we tested whether the Akt-mediated Wnt signaling pathway was involved in the regulation of cyclin D1 expression. In Mel-18–infected SK-BR-3 cells, GSK-3β phosphorylation at Ser9 was diminished whereas total GSK-3β expression remained unchanged as determined by immunoblotting (Fig. 6A, left ). Likewise, enhanced GSK-3β phosphorylation at Ser9 was seen in Mel-18 antisense–infected T-47D cells (Fig. 6A, right). However, total β-catenin expression remained unchanged in both cell lines (Fig. 6A). Because inhibition of GSK-3β phosphorylation implicates reduced nuclear β-catenin (15), we next investigated the subcellular localization of β-catenin on Mel-18 overexpression in SK-BR-3 cells. As shown in Fig. 6B, cytoplasmic β-catenin expression was increased and nuclear β-catenin expression was decreased by Mel-18 overexpression as assessed by biochemical fractionation of nuclear and cytoplasmic proteins and immunoblotting. Consistent with this result, enhanced localization of β-catenin in the cytoplasm was seen in Mel-18–overexpressing cells as determined by immunofluorescence staining (P < 0.05, Student's t test; Fig. 6C). Furthermore, the reduction of nuclear β-catenin by Mel-18 in SK-BR-3 cells also reduced TCF/LEF promoter activity, as assessed by reporter assays with β-catenin–dependent TCF/LEF promoter (TOP-FLASH) and its mutant (FOP-FLASH; P = 0.01, Student's t test; Fig. 6D). Taken together, Mel-18 effect on cyclin D1 transcription is mediated by the Akt-dependent canonical Wnt signaling pathway.


Figure 6
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  		Figure 6. Mel-18 inhibits the Akt-mediated canonical Wnt pathway. A, transiently transduced SK-BR-3 and T-47D cells, as in Fig. 3, were subjected to immunoblotting with indicated antibodies. B, Mel-18–infected SK-BR-3 cells were fractionated and the amounts of nuclear and cytoplasmic β-catenin expression were estimated by Western blot analysis. {alpha}-Tubulin and Sp1 were tested as controls of cytoplasmic and nuclear fractions, respectively. C, cells were stained with β-catenin antibody coupled with Cy5-conjugated antimouse IgG (red) and Mel-18 antibody as in Fig. 5C. To observe the nucleus, cells were also subjected to DAPI staining (blue; left). Right, ratio of the cytoplasmic and nuclear β-catenin expression quantified by counting 1,000 cells each in three independent experiments and shown as percentage of total cells counted; bars, SD. D, the effect of Mel-18 overexpression on TCF/LEF promoter activity was determined by reporter assay as described in Materials and Methods. Columns, mean of triplicate transfections; bars, SD. TOP, reporter assay with TOP-FLASH construct; FOP, reporter assay with FOP-FLASH construct; RLU, relative light units. *, P < 0.05 (Student's t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report that Mel-18 regulates cell cycle progression by down-regulation of the Akt signaling pathway in human breast cancer. In primary breast tumors, the expression of Mel-18 was inversely correlated with phosphorylation of Akt at Ser473, which is phosphorylated in activated Akt. Mel-18 expression was also correlated with the percentage of the G1 phase population by regulating the activities of Cdk4 and Cdk2 in T-47D and SK-BR-3 breast cancer cell lines. Those Cdk activities were affected by changes in cyclin D1 expression and p27Kip1 phosphorylation, but not in INK4a/ARF gene family. Cyclin D1 expression was regulated by the Akt-mediated Wnt signaling pathway, and cytoplasmic retention of p27Kip1 was reduced by inhibition of Akt-dependent phosphorylation at Thr157. Taken together, we show a linear relationship between Mel-18, Akt, and G1 phase regulators.

The oncogenic properties of several PcG proteins such as Bmi-1, RNF2, and CBX7 are, at least in part, attributed to repression of Cdkn2a locus, the tumor-suppressive locus encoding INK4a and ARF proteins (2). They can control G1-S transition in cell cycle progression and senescence via targeting INK4a/ARF locus (29, 34, 35). Mel-18 has also been reported as an inhibitor of INK4a/ARF genes (29, 30). However, a recent report shows that Mel-18 accelerates cellular senescence through down-regulation of Bmi-1 and up-regulation of its target genes, p16INK4a and p14ARF, in human fibroblast (31). As we previously showed that Mel-18 interacts with cyclin D2 and inhibits the proliferative activity of cyclin D2 in breast cancer (11), we have further examined the role of Mel-18 in cell cycle progression. Here, we suggest that Mel-18 negatively regulates cell cycle of human breast cancer in a INK4a/ARF–independent manner. The INK4a/ARF locus is frequently silenced in human primary breast tumors (36–38). The expression levels of these genes were also very weak or undetectable in human breast cancer cell lines including T-47D (data not shown). Although their expression patterns were undetectable or remained unchanged, Mel-18 could affect the G1-S transition through changes in Cdk4 and Cdk2 activities via regulation of other G1 phase regulators, particularly cyclin D1 and p27Kip1 (Fig. 3).

At the molecular level, we show here that Mel-18–induced G1 arrest via changes in cyclin D1 expression and p27Kip1 phosphorylation at Thr157 is mediated by Akt signaling. We found that Mel-18 down-regulated cyclin D1 transcription via the Akt-mediated Wnt signaling pathway (Figs. 3B–C and 6). Moreover, reduced p27Kip1 phosphorylation at Thr157 by Mel-18 led to inhibition of Akt-mediated cytoplasmic localization of p27Kip1 (Figs. 3B–C and 5). These changes in Akt-mediated G1 regulators may explain how Mel-18 regulated cell cycle progression of breast cancer cells, which have hypermethylated INK4a/ARF genes, in an INK4a/ARF–independent manner. Recently, Guo et al. (12) reported the novel relationship between Mel-18 and Akt. Although our results partially overlap with their results in showing the relationship between Mel-18 and Akt, the significance of this study is that our focus is different from them. They suggest that Bmi-1 enhances Akt activity and Mel-18–mediated Bmi-1 down-regulation leads to inhibition its activity. Although, we did not look into the role of Bmi-1 in our system, we also show the relationship between Mel-18 expression and Akt activation both in vitro and in vivo. We found an inverse correlation between Mel-18 expression and Akt phosphorylation at Ser473 in human primary breast cancer (Fig. 1). This result is consistent with two previous reports, which showed decreased Mel-18 mRNA in human breast cancer cell lines (39) and low expression of Mel-18 in primary breast cancer (12). Although Guo et al. (12) first reported the relationship between Mel-18 and Akt activity, they have seen it in the in vitro cultured cells. We also show Mel-18–mediated Akt activity in other two breast cancer cell lines, SK-BR-3 and T-47D, by either overexpression or knockdown of Mel-18. Taken together, we established a linear relationship between Mel-18->Akt->G1 phase regulators, which is compatible with the Mel-18->Bmi-1->Akt model of Guo et al., yet an extension of their model. Moreover, our results may be another evidence of the tumor-suppressive effect of Mel-18 in human breast cancer.

Although we observed Mel-18–mediated Akt signaling, the exact regulatory mechanism of Akt activity by Mel-18 remains to be elucidated. Because Mel-18 is reported as a transcriptional repressor by binding of specific DNA sequence (10), and Mel-18 is dominantly expressed in the nucleus (11) whereas Akt is expressed in the cytosol, Mel-18 may regulate Akt activity indirectly by repressing transcription of certain positive regulators of Akt, such as phosphatidylinositol 3-kinase or phosphoinositide-dependent kinases, by binding to its target sequences rather than by directly binding to Akt and inhibiting its activity.

Collectively, we propose that Mel-18 acts as a negative regulator of cell cycle in G1-S transition through regulation of Akt-mediated cyclin D1 expression and p27Kip1 phosphorylation at Thr157 in INK4a/ARF–silenced human breast cancer, and it may suggest the greater possibility of tumor-suppressive activity of Mel-18 in these cancer cells.


   Acknowledgments
 
Grant support: Korea Science and Engineering Foundation grant 2007-04369 funded by the Korean government (Ministry of Science and Technology).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. S.J. Hwang (Hanyang University, Seoul, Korea) for the technical support for immunofluorescence staining and Dr. H.K. Chung (Hanyang University, Seoul, Korea) for critical comments and reading of the manuscript.

Received 7/ 7/07. Revised 12/23/07. Accepted 2/28/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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