MAGE-A4 Interacts with the Liver Oncoprotein Gankyrin and Suppresses Its Tumorigenic Activity*

Toshikazu NagaoDagger , Hiroaki HigashitsujiDagger , Kohsuke NonoguchiDagger , Toshiharu SakuraiDagger , Simon Dawson§, R. John Mayer§, Katsuhiko ItohDagger , and Jun FujitaDagger

From the Dagger  Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan and the § Laboratory of Intracellular Proteolysis, School of Biomedical Sciences, University of Nottingham Medical School, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom

Received for publication, June 19, 2002, and in revised form, January 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gankyrin (gann ankyrin repeat protein, also known as PSMD10 and p28) is an oncoprotein, the expression of which is increased (1, 2) in hepatocellular carcinomas (HCCs).1 Gankyrin consists of six ankyrin repeats and a 38-amino acid N-terminal extension and binds to the retinoblastoma tumor suppressor protein (Rb), the S6 ATPase subunit of the 26 S proteasome (PSMC4, RPT3, TBP7), and cyclin-dependent kinase 4 (Cdk4) (1, 3, 4). Overexpression of gankyrin increases both the phosphorylation and degradation of Rb in vivo and oncogenically transforms NIH/3T3 cells. Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16INK4A and p18INK4C (4). In a rodent model of hepatocarcinogenesis, gankyrin is overexpressed from the earliest stage of tumor development (5). These findings suggest that gankyrin is a major player in cell cycle control and tumorigenesis in HCCs.

The MAGE (melanoma antigen) genes were initially identified because they encode tumor antigens that can be recognized by cytolytic T lymphocytes derived from the blood lymphocytes of cancer patients (6). The MAGE gene family is composed of more than 25 genes in humans and are classified as type I MAGE genes (including MAGE-A, MAGE-B, and MAGE-C genes) and type II MAGE genes, which include those that reside outside of the MAGE-A, MAGE-B, and MAGE-C genomic clusters (7, 8). The MAGE-A subfamily comprises 12 genes (MAGE-A1 to MAGE-A12), and is expressed in various types of tumors but not in normal adult tissues, except for testis and placenta. The MAGE-A antigens are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. Despite the isolation of growing numbers of MAGE genes, their function in normal tissues remains mostly unknown.

To further characterize the molecular mechanism underlying the oncogenic activity of gankyrin and facilitate development of a therapeutic agent against HCCs, we have used a yeast two-hybrid screen to identify further gankyrin interactions. We report here that MAGE-A4 binds to human gankyrin and suppresses its oncogenicity.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Yeast Two-hybrid Assay-- A full-length human gankyrin cDNA was cloned into pAS2-1 vector (BD Biosciences) and co-transformed into Y190 yeast cells with a placenta or a U-2 OS cDNA library in pACT2 vector (BD Biosciences). Yeast clones containing interacting proteins were identified by growth on media lacking tryptophan, leucine, and histidine, followed by assaying for beta -galactosidase activity. The interaction was confirmed by co-transformation into Y190 cells with pAS2-1-gankyrin and their growth on the selection medium with 25 mM 3-aminotriazole (3AT). For analysis of interactions, full-length and various mutant cDNAs of gankyrin (GenBankTM accession number D83197) and MAGE-A4 (GenBankTM accession number U10687) were generated by the polymerase chain reaction and cloned into a pAS2-1 or pACT2 vector, respectively. cDNAs corresponding to the C-terminal regions of MAGE-A1 (amino acids 203-309, GenBankTM accession number NM_004988), MAGE-A2 (amino acids 210-314, GenBankTM accession number NM_005361), and MAGE-A12 (amino acids 210-314, GenBankTM accession number XM_010079) were also cloned into pACT2.

Plasmids-- FLAG-tagged MAGE-A4, HA-tagged MAGE-A4, and HA-tagged gankyrin cDNAs were cloned into the eukaryotic expression vectors pMKit-neo, pMKit-hygro, and pCMV4. pEGFP-C1 vector (BD Biosciences) was used to express EGFP-S6 and EGFP-MAGE-A4 fusion proteins. For conditional expression of FLAG-MAGE-A4, we used the tetracycline-regulated system as described (9). The pMACS4-IRES vector (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was used to express MAGE-A4 or C-terminal 107 or 55 amino acids of MAGE-A4 with truncated human CD4. The CD4-expressing transfectants were enriched by using the MACSelect 4 system (Miltenyi Biotec GmbH).

Cell Culture-- Mouse NIH/3T3 embryonal fibroblasts and its derivatives, monkey COS-7 kidney cells, human U-2 OS osteosarcoma cells, Huh-7 hepatoma cells, PLC/PRF/5 hepatoma cells, and HEK293 human embryonal kidney cells and its derivatives were cultured in Dulbecco's modified Eagle medium supplemented with 10% calf or fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2 in air. Transfection was performed by the calcium phosphate method. pl16-GK-6 cells were generated from NIH/3T3 cells so that they constitutively expressed HA-tagged human gankyrin and the tetracycline activator, rtTA (9). In the presence of 2 µg/ml doxycycline (Dox) in culture medium, they expressed FLAG-tagged MAGE-A4. GK-S25 cells were derived from NIH/3T3 cells and constitutively expressed gankyrin. To assess the anchorage dependence of growth in vitro, 5 × 102 cells were plated in 0.3% agarose on top of 0.6% agarose in the presence or absence of Dox in a 35-mm dish. Four weeks later, the number of colonies (>20 cells) was counted microscopically. Statistical differences between sample means were calculated by analysis of variance, followed by an unpaired Student's t test.

Western Blot Analysis and Immunoprecipitation-- Western blot analysis and immunoprecipitation were performed as described previously (1, 9). Antibodies used were mouse monoclonal anti-HA antibody (Roche Diagnostics), anti-FLAG antibody (Sigma), anti-GFP antibody (BD Biosciences), anti-actin antibody (Chemicon International, Inc., Temecula, CA), rabbit polyclonal anti-gankyrin antibody (Santa Cruz Biotechnology), anti-MAGE antibody (FL-309; Santa Cruz Biotechnology), horseradish peroxidase-conjugated goat anti-mouse antibody (DAKO, Kyoto, Japan), and anti-rabbit antibody (DAKO). To analyze interaction between endogenous MAGE-A4 and endogenous gankyrin, U-2 OS cell lysates were immunoprecipitated using rabbit anti-MAGE antibody (FL-309) immobilized to the protein G support (Seize X mammalian immunoprecipitation kit; Pierce). The antibody is broadly reactive with all MAGE family members according to the manufacturer. The immobilized antibody was incubated consecutively with four aliquots of cell lysates (total of 1 ml), the first aliquot at 4 °C overnight and the others at 20 °C sequentially for 1 h. The bound proteins were eluted with the elution buffer supplied in the kit. The elution was repeated three times, and each fraction was analyzed by Western blotting using rabbit polyclonal anti-gankyrin antibody (Santa Cruz Biotechnology.). Interaction of transiently expressed exogenous MAGE-A4 or its mutant with exogenous gankyrin was examined by co-transfecting COS-7 or U-2 OS cells with plasmids expressing FLAG-tagged MAGE-A4, FLAG-tagged C-terminal 107 amino acids of MAGE-A4, HA-tagged full-length gankyrin, FLAG alone, or HA alone in various combinations. Interaction of stably transfected gankyrin with MAGE-A4 was examined by using pl16-GK-6 cells, which were derived from mouse NIH/3T3 cells, constitutively expressed HA-tagged human gankyrin, and inducibly expressed FLAG-tagged MAGE-A4 in the presence of Dox. To analyze the effects of MAGE-A4 on binding of gankyrin to Rb, S6, and Cdk4, U-2 OS cells and 293T cells were co-transfected with plasmids expressing FLAG-gankyrin, HA-tagged Rb, EGFP-S6 fusion protein, HA-tagged Cdk4, HA-tagged MAGE-A4, EGFP-MAGE-A4 fusion protein, EGFP alone, or FLAG alone in various combinations.

Immunofluorescence Staining-- Immunofluorescence staining was performed essentially as described (9, 10). COS-7 cells were replated on chamber slides after transfection, fixed with phosphate-buffered saline containing 4% paraformaldehyde for 30 min, and then rendered permeable with phosphate-buffered saline containing 0.2% Triton X-100 for 30 min at room temperature. After blocking nonspecific antibody-binding sites with bovine serum albumin, the cells were incubated with mouse monoclonal anti-FLAG antibody (Sigma) and rabbit polyclonal anti-HA antibody ( Berkeley Antibody, Richmond, CA). Then the bound antibodies were reacted with FITC-linked anti-mouse and TRITC-linked anti-rabbit IgGs (Amersham Biosciences) and observed under a confocal laser microscope (Olympus, Tokyo, Japan).

Tumorigenicity Assay in Mice-- Twenty-four female BALB/c Slc-nu/nu athymic mice (4 weeks old) were injected subcutaneously with pl16-GK-6 cells or GK-S25 cells (8 × 106 cells each) and divided into two groups. Twelve were given Dox (2 mg/ml) in the drinking water ad libitum. Tumor size was calculated by measuring the length, width, and thickness with calipers.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of MAGE-A4 as a Gankyrin-interacting Protein-- We performed yeast two-hybrid assays to search for proteins capable of physically interacting with human gankyrin. Using full-length gankyrin as a bait, we identified 18 clones of 3.5 × 106 yeast clones transformed with human placenta or U-2 OS cell cDNA libraries. Each clone proliferated on media containing the histidine inhibitor 3AT and was positive for beta -galactosidase staining (Fig. 1A, and data not shown). DNA sequencing analysis of the rescued plasmids revealed that two of them encoded the C-terminal 107 amino acids of MAGE-A4. Consistent with our previous findings (3), the remaining 16 clones encoded different C-terminal sequences of the S6 ATPase subunit of the 26 S proteasome. MAGE-A4 did not interact with the GAL4 DNA binding domain alone (Fig. 1A), indicating that it interacted with gankyrin.


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Fig. 1.   Identification of MAGE-A4 as a gankyrin-binding protein. A, interaction in yeasts. Yeast cells were co-transformed with the human full-length gankyrin cDNA, p53 cDNA, or none (Gal4-BD) in pAS2-1 vector in combination with the isolated partial MAGE-A4 cDNA, S6 cDNA, SV40 T-antigen cDNA, or none (Gal4-AD) in pACT2 vector as indicated. They were then plated onto media lacking tryptophan and leucine (SD/-W -L) and media lacking tryptophan, leucine, and histidine and supplemented with 3AT (SD/-W-L-H+3AT). B, interaction of isolated clones in COS-7 cells. Lysates were prepared after co-transfection with vectors expressing HA-tagged gankyrin, FLAG-tagged C-terminal 107 amino acids of MAGE-A4 (FLAG-MAGEDelta N), FLAG alone, and HA alone as indicated. The lysates and those immunoprecipitated with antibodies to FLAG or HA were analyzed by Western blotting using the indicated antibodies. Arrowheads indicate mobilities of specific bands. C, interaction of full-length MAGE-A4 in COS-7 cells. Lysates were prepared after co-transfection with vectors expressing HA-tagged gankyrin, FLAG-tagged full-length MAGE-A4, FLAG alone, and HA alone as indicated, and analyzed as described for panel B. D, co-localization of gankyrin and MAGE-A4. COS-7 cells co-transfected with HA-gankyrin and FLAG-MAGE-A4 were incubated with rabbit anti-HA and mouse anti-FLAG antibodies, visualized with TRITC-linked anti-rabbit and FITC-linked anti-mouse IgGs, and observed under confocal microscope. Note cytoplasmic co-localization (yellow) of gankyrin (red) and MAGE-A4 (green).

To confirm that gankyrin interacts with the isolated MAGE-A4 fragment in mammalian cells, COS-7 cells were co-transfected with plasmid DNAs expressing HA-tagged human gankyrin and FLAG-tagged C-terminal 107 amino acids of MAGE-A4. When cell lysates were immunoprecipitated with an anti-FLAG antibody, HA-gankyrin was detected in them but not in precipitates from cells co-transfected with parental FLAG vector and HA-gankyrin (Fig. 1B, left panels). Reciprocally, truncated MAGE-A4 was detected in the anti-HA immunoprecipitates from cells co-transfected with plasmids expressing HA-gankyrin and FLAG-tagged truncated MAGE-A4 (Fig. 1B, right panels).

We next examined whether full-length MAGE-A4 interacts with gankyrin in COS-7 cells. As shown in Fig. 1C, HA-tagged gankyrin was immunoprecipitated with anti-FLAG antibody in lysates from cells co-transfected and expressing FLAG-tagged full-length MAGE-A4 and HA-tagged gankyrin. Reciprocally, FLAG-tagged MAGE-A4 was immunoprecipitated with an anti-HA antibody. Similar interactions were observed in U-2 OS cells as well (data not shown). Furthermore, in the COS-7 cells expressing FLAG-MAGE-A4 and HA-gankyrin, double immunofluorescence staining showed that both proteins were co-localized in the cytoplasm (Fig. 1D).

COS-7 cells are well known to vastly over-express transfected proteins, which makes it a problem to conclude that physiologically relevant associations occur between such expressed proteins. Therefore, we generated an NIH/3T3-derived clone (pl16-GK-6) in which HA-gankyrin was stably overexpressed and FLAG-MAGE-A4 was inducibly expressed in the presence of Dox. As shown in Fig. 2A, Dox increased the level of FLAG-MAGE-A4 but not that of HA-gankyrin in pl16-GK-6 cells. Only in the presence of Dox was FLAG-MAGE-A4 co-immunoprecipitated with HA-gankyrin by the anti-HA antibody and HA-gankyrin co-immunoprecipitated with FLAG-MAGE-A4 by anti-FLAG antibody (Fig. 2A, bottom two panels). About 40% of total MAGE-A4 and 5% of total gankyrin present in pl16-GK-6 cells after transfection were estimated to be in a complex (data not shown). Western blot analysis demonstrated that the level of stably overexpressed gankyrin in pl16-GK-6 cells was less than those observed in human U-2 OS osteosarcoma and Huh-7 hepatoma cells (Fig. 2B, top panels). The level of MAGE-A4 in the presence of Dox was probably less than that in U-2 OS osteosarcoma and PLC/PRF/5 hepatoma cells (Fig. 2B, bottom panels). Further confirmation is necessary for this observation, because the anti-MAGE antibody used was reactive with MAGE proteins other than MAGE-A4. We next examined the interaction between endogenous gankyrin and MAGE-A4 in U-2 OS cells from which we isolated the original truncated MAGE-A4 cDNA clones. As shown in Fig. 2C, the endogenous gankyrin was co-immunoprecipitated with MAGE proteins by anti-MAGE antibody. Taken together, these results strongly suggest that gankyrin and MAGE-A4 interacts in human cancer cells.


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Fig. 2.   Interaction of MAGE-A4 with gankyrin in mammalian cells. A, interaction of inducibly overexpressed MAGE-A4 with stably overexpressed human gankyrin in mouse cells. pl16-GK-6 cells were cultured in the presence (+) or absence (-) of Dox, and lysates were prepared from them. 5% of the input lysates (top two panels) and one-quarter of the immune complexes precipitated by anti-HA antibody or anti-FLAG antibody (bottom two panels) were analyzed by Western blotting using anti-HA antibody or anti-FLAG antibody as indicated. Arrowheads indicate mobilities of the specific bands. B, levels of overexpressed HA-gankyrin and FLAG-MAGE-A4 in pl16-GK-6 cells in the presence of Dox compared with those in human cancer cells. Cell lysates were prepared from pl16-GK-6 cells, U-2 OS cells, Huh-7 cells, and PLC cells as indicated and analyzed by Western blotting using anti-gankyrin antibody (top left panel), anti-HA antibody (top right panel), anti-MAGE antibody (bottom left panel), and anti-FLAG antibody (bottom right panel). Mobilities of HA-gankyrin, endogenous gankyrin, FLAG-MAGE-A4, and endogenous MAGE proteins are indicated on the left. C, interaction of endogenous MAGE proteins with endogenous gankyrin. Lysates from U-2 OS cells were immunoprecipitated with anti-MAGE antibody immobilized to the protein G support. 2% of input, eluate fractions 1, 2, and 3, and flow-through were analyzed by Western blotting using anti-gankyrin antibody. The mobility of endogenous gankyrin is indicated on the right.

Because gankyrin binds to Rb, the S6 subunit of the 26 S proteasome, and Cdk4 (1, 3, 4), the effects of overexpression of MAGE-A4 on the binding of gankyrin to these proteins were examined. When U-2 OS cells were co-transfected with plasmids expressing FLAG-tagged gankyrin and HA-tagged Rb, HA-Rb was immunoprecipitated with anti-FLAG antibody as expected (Fig. 3A). Overexpression of EGFP-MAGE-A4 fusion protein by co-transfection did not affect the amount of immunoprecipitated HA-Rb. When U-2 OS cells were co-transfected with plasmids expressing FLAG-tagged gankyrin and EGFP-S6 fusion protein, EGFP-S6 was immunoprecipitated with anti-FLAG antibody as expected (Fig. 3B). Overexpression of HA-tagged MAGE-A4 by co-transfection did not affect the amount of immunoprecipitated EGFP-S6. Similarly, no effect of EGFP-MAGE-A4 was observed on the binding of gankyrin to Cdk4 (data not shown). Similar results were obtained with 293T cells (data not shown).


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Fig. 3.   Effects of MAGE-A4 on binding of Rb and S6 to gankyrin. A, no effect on binding of Rb to gankyrin. U-2 OS cells were co-transfected with plasmids expressing FLAG-gankyrin and HA-Rb together with increasing amount of plasmids expressing the EGFP-MAGE-A4 fusion protein as indicated. Total amount of plasmids was adjusted to 5 µg/60-mm dish with empty vectors expressing FLAG alone or EGFP alone. 48 h after transfection, lysates were prepared from them. One-quarter of immune complexes precipitated by anti-FLAG antibody (top panel) and 10% of the input lysates (bottom four panels) were analyzed by Western blotting using antibodies as indicated. B, no effect on binding of S6 to gankyrin. U-2 OS cells were co-transfected with plasmids expressing FLAG-gankyrin and EGFP-S6 fusion protein together with increasing amount of plasmids expressing the HA-MAGE-A4 fusion protein as indicated. Total amount of plasmids was adjusted to 5 µg/60-mm dish with empty vectors expressing FLAG alone or HA alone. 48 h later, lysates were prepared from them. One-quarter of immune complexes precipitated by anti-FLAG antibody (top panel) and 10% of the input lysates (bottom four panels) were analyzed by Western blotting using antibodies as indicated.

Specific Binding of C-terminal Portion of MAGE-A4 to Full-length Gankyrin-- To characterize the interacting domains of gankyrin and MAGE-A4, we made various deletion mutants and analyzed their interactions in the yeast two-hybrid system. We demonstrated previously that full-length is necessary for the interaction with Rb (1). Similarly, no deletion mutant of gankyrin interacted with MAGE-A4, indicating that all ankyrin repeats and the N-terminal extension are necessary for the binding (Fig. 4A, and data not shown).


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Fig. 4.   Specific interaction of C-terminal part of MAGE-A4 with gankyrin. A, interaction between full-length MAGE-A4 and various gankyrin deletion mutants analyzed by the yeast two-hybrid assay. Top row, full-length gankyrin. Locations of ankyrin repeats are shown as ANK1 to ANK6. Plus (+) and minus (-) signs indicate the presence and absence of the interaction with MAGE-A4, respectively. B, interaction between full-length gankyrin and various MAGE-A4 deletion mutants analyzed by the yeast two-hybrid assay. Top row, full-length MAGE-A4. Striped box, MAGE homology domain. The numbers on top of the boxes indicate the amino acid positions, and the numbers inside the boxes indicate the length of each mutant. Plus (+) and minus (-) signs indicate the presence and absence of the interaction with gankyrin, respectively. C, comparison of the C-terminal amino acid sequence of MAGE-A4 to those of MAGE-A1, MAGE-A2, and MAGE-A12. Amino acid residues identical to MAGE-A4 are highlighted, and the regions that give rise to the MAGE-A4 peptides that are presented on the cell surface (13, 14) are indicated by lines. D, interaction between full-length gankyrin and C-terminal regions of various MAGE-A proteins shown in panel C analyzed by the yeast two-hybrid assay. Yeast cells were co-transformed with the indicated plasmids and plated onto media lacking tryptophan and leucine (SD/-W-L) and media lacking tryptophan, leucine, and histidine and supplemented with 3AT (SD/-W-L-H+3AT). Note that only the C-terminal potion of MAGE-A4 interacted with gankyrin.

The MAGE-A4 clone originally isolated by the two-hybrid screen contained only the C-terminal 107 amino acids (position 211 to 317, Fig. 1, A and B, and Fig. 4B). Further N-terminal truncation up to residue 226 did not prevent the interaction with gankyrin. By contrast, a truncation of 10 amino acids from the C terminus of MAGE-A4 abolished its binding to gankyrin (Fig. 4B). The interaction was specific to MAGE-A4, because the corresponding regions of other MAGE proteins, although structurally quite similar to MAGE-A4, did not interact with gankyrin (Fig. 4, C and D). These results indicate that the C-terminal region of MAGE-A4 containing the HLA-A2-presented peptides (11, 12) (GVYDGREHTV and YLEYRQVPV) specifically interacts with gankyrin.

Effects of MAGE-A4 on Tumorigenic Activity of Gankyrin-- To investigate the biological effects of the interaction of gankyrin and MAGE-A4, we used the NIH/3T3-derived pl16-GK-6 cells in which gankyrin was stably overexpressed and MAGE-A4 expression could be induced with Dox (Fig. 2A). These gankyrin-transformed cells formed colonies in soft agar (Fig. 5A). When the cells were cultured in the presence of Dox, the numbers of colonies was decreased to 65% of those in the absence of Dox (Fig. 5B). When the mouse NIH/3T3-derived GK-S25 cells stably overexpressing gankyrin were transfected with plasmids expressing the C-terminal 55 amino acids of MAGE-A4 that did not bind to gankyrin (Fig. 4B), no decrease in the number of colonies were observed (Fig. 5C). By contrast, the C-terminal 107 amino acids of the MAGE-A4 that binds to gankyrin inhibited the colony formation, suggesting that the ability to bind to gankyrin correlates with the ability to suppress anchorage-independent growth in vitro.


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Fig. 5.   Effects of MAGE-A4 and its mutants on anchorage-independent growth of gankyrin-transformed cells in vitro. A and B, effects of MAGE-A4 on colony formation in soft agar of pl16-GK-6 cells stably overexpressing gankyrin. Dox induces the expression of FLAG-MAGE-A4 in them. pl16-GK-6 cells were cultured in soft agar in the absence (-) or presence (+) of Dox, photographed (A), and the numbers of colonies were counted under a microscope (B). Shown are mean ± S.E. of triplicates. *, p < 0.01 versus Dox (-) group. C, effects of mutant MAGE-A4 on colony formation in soft agar of GK-S25 cells overexpressing gankyrin. GK-S25 cells were transfected with bicistronic vectors expressing CD4 in combination with full-length MAGE-A4 (FL), C-terminal 107 amino acids of MAGE-A4 (107aa), C-terminal 55 amino acids of MAGE-A4 (55aa) or no protein (mock). CD4-expressing cells were enriched and analyzed for colony formation. Results are mean ± S.E. of triplicates. *, p < 0.05; **, p < 0.01 versus mock group.

The anchorage-independent phenotype in cell culture has been closely correlated with the ability of cells to form tumors in animals (13). We therefore evaluated the effect of MAGE-A4 on tumor formation of gankyrin-transformed cells in athymic nude mice. After being subcutaneously inoculated with pl16-GK-6 cells, the animals were divided into two groups, one of which was administered Dox in drinking water. In the absence of Dox, all mice developed tumors 5 weeks after the inoculation of pl16-GK-6 cells (Fig. 6). By contrast, the tumors appeared later and grew slower in mice given Dox. Dox by itself showed no suppressive effects on tumor formation by GK-S25 cells (data not shown). Taken together, these results demonstrate that MAGE-A4 directly binds to gankyrin and suppresses its tumorigenic activity.


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Fig. 6.   Effects of MAGE-A4 on anchorage-independent growth of gankyrin-transformed cells in vivo. nu/nu mice were subcutaneously injected with pl16-GK-6 cells stably overexpressing gankyrin. Then, they were divided into two groups, one of which was given Dox in drinking water. A, photos of mice given Dox (+) or vehicle alone (-) for 6 weeks. Arrowhead indicates the tumor. B, tumor volumes (mean ± S.E., n = 6 each) in mice given Dox () or vehicle alone (black-square).*, p < 0.05 versus Dox (-) group.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rb and p53 play critical roles in transducing a variety of growth inhibitory signals to the cell cycle control machinery via distinct mechanisms (14-16). The concurrent inactivation of these two pathways occurs frequently in human cancers and argues that unscheduled entry into the cell cycle and escape from cell cycle arrest/apoptosis are two critical events that a cell cycle requires to become cancerous. Gankyrin destabilizes Rb and is commonly overexpressed in HCCs (1, 2). Gankyrin therefore plays an important role in hepatocarcinogenesis and is a promising target for therapeutic drug development. Here, we demonstrated that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. The interaction was identified by the yeast two-hybrid assay and reproduced in mammalian cells. Because endogenous gankyrin is associated with endogenous MAGE protein(s) in human cancer cells in which MAGE-A4 transcripts were detected, it is highly likely that the interaction is physiologically relevant. MAGE-A4 is a member of the MAGE family, a large group of proteins that contain a well conserved ~200 amino acid region known as the MAGE homology domain (7, 8). Although its general function is unknown, the MAGE homology domain of necdin binds to E2F-1 (17). In the present study, the C terminus of MAGE-A4, in addition to the C-terminal half of MAGE homology domain, was found to be necessary for MAGE-A4 to bind to gankyrin. This binding was specific for MAGE-A4, because similar amino acid sequences from other MAGE-A family members did not bind to gankyrin.

Despite their normally restricted physiological expression, the MAGE genes are expressed in a wide variety of cancer cells. In the case of HCC, more than 60% of the carcinomas express MAGE-A1 and/or MAGE-A3 transcripts, which have been regarded as tumor-specific markers (18, 19). Expression of other MAGE family members, including MAGE-A4, has also been detected in HCCs (20). When overexpressed in the cytosol of cancer cells, MAGE proteins are proteolytically processed, transported to the endoplasmic reticulum, and then presented on the cell surface as antigenic major histocompatibility complex-associated peptides (8). Thus, attention has been focused on the potential of MAGE as a target for cancer immunotherapy. The C-terminal region of MAGE-A4 that binds to gankyrin contains decapeptides presented by HLA-A2 (11, 12). Whether binding of gankyrin affects degradation of MAGE-A4 and/or the presentation of antigenic peptide remain to be investigated.

The physiological roles played by the MAGE gene family are unknown with a few exceptions (8). Necdin has an important role in development and/or maintenance of discrete cells in the nervous system. Overexpression of necdin causes cell cycle arrest through mechanisms that may involve physical interactions with E2F-1 or p53 (17). NRAGE (MAGE-D1), which binds to several proteins including the p75 neurotrophin receptor and inhibitor of apoptosis proteins (IAP), blocks cell cycle progression and enhances apoptosis (21, 22). Magphinin (MAGE-D4) is suggested as regulating cell proliferation during gametogenesis, and its ectopic expression suppresses cell proliferation (23). In contrast to NRAGE, necdin, and magphinin, which belong to the type II MAGE genes, MAGE-A4 did not affect the cell cycle progression, proliferation rate, nor apoptosis by itself. 2 However, MAGE-A4 showed anti-tumorigenic effects on gankyrin-overexpressing cells. Because this effect was observed in vitro as well as in vivo in athymic nude mice, it is cell autonomous and not mediated by T cells.

In mammals, the execution of G1 Cdks, namely Cdk4, Cdk6, and Cdk2, is essential for onset of the S phase (24). A major target for the G1 Cdks is Rb. Phosphorylation of Rb leads to activation of the E2F-DP1 transcriptional factor complex that controls the expression of genes essential for onset of the S phase (14). Although there appears to be cell type- and genotype-specific differences in the control of anchorage-dependent growth, a recent study has demonstrated that the G1 Cdks and Cdc6 constitute major cell cycle targets for the regulation of the G1-S transition by anchorage and oncogenic stimulation (25). Gankyrin binds to Cdk4, evades inhibition by p16INK4A (3, 4), increases phosphorylation and degradation of Rb, and transforms cells to grow in an anchorage-independent manner (1). Because the suppressive effect of MAGE-A4 on anchorage-independent growth correlated with its binding to gankyrin, it is possibly mediated by gankyrin and/or dependent on association with gankyrin. However, we were unable to detect effects of MAGE-A4 on the degradation of Rb.3 MAGE-A4 did not reduce the binding of gankyrin to Rb, S6, or Cdk4. Molecules involved in the anti-tumorigenic activity of MAGE-A4 are yet to be elucidated. Recently, continued activity of a specific oncogene has been found to be necessary to maintain the cancer phenotype in some cancer cells, which suggests a possibility that suppression of gankyrin alone has a therapeutic effect in HCCs (26). Further clarification of the mechanisms underlying the effects of MAGE-A4 on gankyrin and anchorage-independent growth will facilitate development of novel therapeutics against HCCs and also shed light on normal physiological functions of MAGE-A proteins.

    ACKNOWLEDGEMENT

We thank Dr. Manabu Sugai for helpful suggestions.

    FOOTNOTES

* This work was partially supported by Grants-in-aid from the Ministry of Science, Culture, Sports, and Education of Japan, the Yasuda Anti-Cancer Foundation, and the Smoking Research Foundation of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-75-751-3751; Fax: 81-75-751-3750; E-mail: jfujita@virus.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M206104200

2 T. Nagao and J. Fujita, unpublished observation.

3 T. Nagao, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: HCC, hepatocellular carcinoma; Rb, retinoblastoma tumor suppressor protein; Cdk, cyclin-dependent kinase; MAGE, melanoma antigen; 3AT, 3-aminotriazole; HA, hemagglutinin; Dox, doxycycline; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Higashitsuji, H., Itoh, K., Nagao, T., Dawson, S., Nonoguchi, K., Kido, T., Mayer, R. J., Arii, S., and Fujita, J. (2000) Nat. Med. 6, 96-99[CrossRef][Medline] [Order article via Infotrieve]
2. Fu, X. Y., Wang, H. Y., Tan, L., Liu, S. Q., Cao, H. F., and Wu, M. C. (2002) World J. Gastroenterol. 8, 638-643[Medline] [Order article via Infotrieve]
3. Dawson, D., Apcher, S., Mee, M., Higashitsuji, H., Baker, R., Uhle, S., Dubiel, W., Fujita, J., and Mayer, R. J. (2002) J. Biol. Chem. 277, 10893-10902[Abstract/Free Full Text]
4. Li, J., and Tsai, M.-D. (2002) Biochemistry 41, 3977-3983[CrossRef][Medline] [Order article via Infotrieve]
5. Park, T. J., Kim, H. S., Byun, K. H., Jang, J. J., Lee, Y. S., and Lim, I. K. (2001) Mol. Carcinog. 30, 138-150[CrossRef][Medline] [Order article via Infotrieve]
6. van der Bruggen, P., Traversari, C., Chomoz, P., Lurquin, C., De Plaen, E., van den Eynde, B., Knuth, A., and Boon, T. (1991) Science 254, 1643-1647[Medline] [Order article via Infotrieve]
7. Chomez, P., De Backer, O., Bertrand, M., De Plaen, E., Boon, T., and Lucas, S. (2001) Cancer Res. 61, 5544-5551[Abstract/Free Full Text]
8. Baker, P. A., and Salehi, A. (2002) J. Neurosci. Res. 67, 705-712[CrossRef][Medline] [Order article via Infotrieve]
9. Higashitsuji, H., Higashitsuji, H., Nagao, T., Nonoguchi, K., Fujii, S., Itoh, K., and Fujita, J. (2002) Cancer Cell 2, 335-346[CrossRef][Medline] [Order article via Infotrieve]
10. Hopwood, D. (1998) in Cell Biology, A Laboratory Handbook (Celis, J. E., ed), 2nd Ed. , pp. 221-231, Academic Press, San Diego, CA
11. Duffour, M.-T., Chaux, P., Lurquin, C., Cornelis, G., Boon, T., and van der Bruggen, P. (1999) Eur. J. Immunol. 29, 3329-3337[CrossRef][Medline] [Order article via Infotrieve]
12. Graff-Dubois, S., Faure, O., Gross, D.-A., Alves, P., Scardino, A., Chouaib, S., Lemonnier, F. A., and Kosmatopoulos, K. (2002) J. Immunol. 169, 575-580[Abstract/Free Full Text]
13. Shin, S.-I., Freedman, V. H., Risser, R., and Pollack, R. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4435-4439[Abstract]
14. Vogelstein, B., Lane, D., and Levine, A. J. (2000) Nature 408, 307-310[CrossRef][Medline] [Order article via Infotrieve]
15. Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348[CrossRef][Medline] [Order article via Infotrieve]
16. Hahn, W. C., and Weinberg, R. A. (2002) Nat. Rev. Cancer 2, 331-341[CrossRef][Medline] [Order article via Infotrieve]
17. Taniura, H., Matsumoto, K., and Yoshikawa, K. (1999) J. Biol. Chem. 274, 16242-16248[Abstract/Free Full Text]
18. Tahara, K., Mori, M., Sadanaga, N., Sakamoto, Y., Kitano, S., and Makuuchi, M. (1999) Cancer 85, 1234-1240[CrossRef][Medline] [Order article via Infotrieve]
19. Mou, D. C., Cai, S. L., Peng, J. R., Wang, Y., Chen, H. S., Pang, X. W., Leng, X. S., and Chen, W. F. (2002) Br. J. Cancer 86, 110-116[CrossRef][Medline] [Order article via Infotrieve]
20. Chen, C. H., Huang, G. T., Lee, H. S., Yang, P. M., Yan, M. D., Chen, D. S., and Sheu, J. C. (1999) Liver 19, 110-114[Medline] [Order article via Infotrieve]
21. Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288[Medline] [Order article via Infotrieve]
22. Jordan, B. W., Dinev, V., LeMellay, V., Troppmair, J., Gotz, R., Wixler, L., Sendtner, M., Ludwig, S., and Rapp, U. R. (2001) J. Biol. Chem. 276, 39985-39989[Abstract/Free Full Text]
23. Saburi, S., Nadano, D., Akama, T. O., Hirama, K., Yamanouchi, K., Naito, K., Tojo, H., Tachi, C., and Fukuda, M. N. (2001) J. Biol. Chem. 276, 49378-49389[Abstract/Free Full Text]
24. Weinberg, R. A. (1995) Cell 81, 323-330[Medline] [Order article via Infotrieve]
25. Jinno, S., Yageta, M., Nagata, A., and Okayama, H. (2002) Oncogene 21, 1777-1784[CrossRef][Medline] [Order article via Infotrieve]
26. Weinstein, I. B. (2002) Science 297, 63-64[Free Full Text]


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