Although X chromosome transfer experiments indicated that tumor suppressor genes are present on the X chromosome, they have not been previously identified. In this report, we show that the ETS transcription factor MEF (ELF4), which is located on chromosome Xq26.1, possesses tumor suppressive capability. MEF expression was up-regulated by 5-azacytidine in some cancer cell lines. MEF overexpression induced morphological changes, such as the conversion of normally loose cell-cell contacts to strong interactions similar to those seen in the presence of matrix metalloproteinase (MMP) inhibitor BB94. In the colony formation assay, A549 cells, but not MEF-overexpressing cells, formed colonies in soft agar culture. Furthermore, MEF-overexpressing cells s.c. injected in the nude mice did not grow, whereas the control cells did. The A549 tumors were poorly differentiated, whereas the MEF-overexpressing tumors were well differentiated. By immunostaining with CD31, a marker on vascular endothelial cells, we found that tumor angiogenesis was significantly suppressed in the tumors formed from MEF-overexpressing cells. In addition, the conditioned media from A549 cell cultures stimulated the migration of human umbilical vein endothelial cells, whereas conditioned media from MEF-overexpressing cell cultures had less of an effect. By gelatin zymography, Western blotting analysis, and immunohistochemistry, we found that the expression levels of MMP-9 and MMP-2 were significantly reduced in MEF-overexpressing tumors. Immunohistochemical analyses showed that interleukin (IL)-8 expression was reduced in the MEF-overexpressing tumors in nude mice. Furthermore, IL-8 mRNA expression in vitro was significantly down-regulated in MEF-overexpressing cells, compared with A549 cells. MEF suppressed the transcription and promoter activities of the genes encoding MMP-9 and IL-8, whereas ETS-2 up-regulated these activities. Therefore, we propose that MEF is a candidate tumor suppressor gene on the X chromosome with activities that are opposite to those of ETS-2.

It is generally accepted that tumorigenesis is a multistep process that involves a series of genetic and epigenetic alterations, such as the activation of dominantly acting oncogenes and the inactivation of tumor suppressor genes. Sequential mutations in key growth-regulatory genes of somatic cells and their progeny represent “multiple-hits” according to a broader interpretation of the original Knudson “two-hits” theory (1). If a tumor suppressor gene is localized on the X chromosome, one hit is sufficient to induce tumorigenesis because the other allele on the X chromosome is inactivated by epigenetic modification. Therefore, the identification of tumor suppressor genes on the X chromosome has important ramifications for cancer therapy. It has been suggested that a senescence gene (or genes), which is putatively located on the Chinese hamster Xq chromosome, is regulated by DNA methylation and that escape from senescence and loss of tumor suppressor gene activity can occur via epigenetic mechanisms (2). Furthermore, the presence of genes that cause senescence-like cell growth arrest in rodent and human tumor cells has been demonstrated through X chromosome transfer experiments (2). In addition, it has recently been reported that X chromosome methylation is unstable in aberrant crypt foci (precancerous lesions) in the colon and that this may be an early event in colon carcinogenesis (3). The treatment of Chinese hamster embryo cells with Ni induced a high recovery of transformants that exhibited nonrandom deletions of the heterochromatic Xq chromosome (4). Recently, allelic LOH3 was located at Xq25–26.1 in ovarian and breast carcinomas (5, 6). Although the tissue inhibitor of the metalloproteinase-1 gene, which is a putative tumor suppressor gene, is located on the human X chromosome Xq11 (7), the suppressor gene, which is regulated by epigenetic modifications to the X chromosome, remains to be precisely defined.

The ETS transcription factor family plays a key role in cell growth, death, and differentiation (8). In addition to their importance in normal cellular control and based on the predominance of target genes, ETS factors have also been implicated in several malignant and genetic disorders. For example, the human ETS genes, Fli-1, TEL, and ERG are located at the translocation breakpoints of several leukemias and solid tumors, and they form chimeric proteins that are believed to be responsible for tumorigenesis (9). In addition, ETS factors are overexpressed [e.g., ETS-2 in prostate and breast cancer (10, 11)] or lost [e.g., hPSE in prostate cancer (12)] during cancer development. Among ETS factors, ETS-2 is known to be an important mediator of cellular transformation because dominant negative constructs of ETS-2 can block transformation by Ras or Her2 (10, 13), and because mammary tumors are suppressed by mating with heterozygous mice that carry a targeted mutation in ETS-2 (14).

Some ETS factors have repressor activities (e.g., ERF, YAN, TEL, and NET) that allow them to compete directly with other ETS factors for ETS-binding sites (15). In addition, interactions with other proteins can block the ability of ETS factors to activate transcription (15). The potencies of individual ETS proteins depend on their particular promoter and cell targets. For example, Fli-1 can function as an activator of the tenascin-C promoter but acts as a repressor of the collagen promoter (16). On the other hand, ETS-1 activates both of these promoters (16, 17). Unique promoter interactions have also been demonstrated for ETS-2 (or ETS-1) and Erg on the MMP-1 and MMP-3 promoters. Although Erg appears to act as an activator of the MMP-1 promoter and an inhibitor of the stimulation of the MMP-3 promoter by ETS-2, ETS-2 stimulates both of these promoters (18). Thus, interplay between ETS factors may serve as a molecular switch between gene repression and activation.

We reported previously (19) that myeloid elf-1-like factor (MEF or ELF4), which is an ETS transcription factor, activated lysozyme transcription in the human non-small cell lung carcinoma cell line A549. MEF, which was at one time referred to as ELFR (20), is located on chromosome Xq26.1, which is the location identified for LOH in ovarian and breast carcinoma (5, 6). In this report, we show that MEF is down-regulated by methylation in some cancer cells and that MEF suppresses tumorigenesis by inhibition of tumor growth and angiogenesis and by blocking the invasiveness of A549 cells. We also show that MEF suppresses the transcription and promoter activities of the genes encoding MMP-9 and IL-8, whereas ETS-2 up-regulates these activities. Therefore, we propose that MEF is a candidate tumor suppressor gene on the X chromosome that acts as a negative regulator of ETS-2.

Cell Lines, Transfection Protocols, and Luciferase Measurements.

The A549, HeLa, Caco-2, NCI-H292, and HEK293 cell lines were obtained from the American Type Culture Collection. A549, Caco-2, and HEK293 cells were grown in DMEM supplemented with 10% FBS (Hyclone, Logan, UT), HeLa cells were grown in MEM supplemented with 10% FBS, and NCI-H292 cells were grown in RPMI 1640 supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For methylation analysis, A549, HEK293 and Caco-2 cells were cultured in the presence of 5-AC (Nacalai tesque). 5-AC (1.0 or 5.0 μm) was added to the cultures for 3 days.

Transient transfections were performed with transfectam (Promega, Madison, WI) according to the manufacturer’s recommendations. Specifically, 2 μl of transfectam reagent and 1.5 μg of total DNA in DMEM were incubated for 10 min before the mixture was applied to subconfluent cells on 24-well plates. Cotransfection experiments were performed with 0.5 μg of the reporter plasmid and 1 μg of MEF and/or ETS2 plasmids. The empty vector (pCB6) was added to ensure a constant amount of input DNA. Cotransfection with the pRL-CMV vector (10 ng of DNA in each sample), which expresses Renilla luciferase (Promega), was used to verify that the differences in expression of the firefly luciferase reporter gene were not due to variations in transfection efficiency. Cells were incubated for 2 h without serum, at which time additional medium containing serum was added. The medium was removed 40–48 h after transfection, and cells were harvested. Luciferase activity was measured using a Dual-Luciferase Reporter Assay (Promega) and a luminometer (Lumat LB9507; EG&G Berthold). The absolute light emission generated by the luciferase enzyme reaction was determined. Relative luciferase activity was plotted and expressed as the fold induction of activity generated by experimental treatments relative to the basal luciferase activity (expressed from the empty vector). The results are shown as the means ± SE (n = 4).

The expression constructs were introduced into the human lung adenocarcinoma cell line A549 (RCB0098) by electroporation (19) to generate stably transfected clones. Briefly, approximately 1 × 106 cells were transfected with 100 μg of a plasmid (pCB6) that carried MEF cDNA and was linearized with ApaLI. Electroporation was performed using an ECM 600 apparatus (BTX Inc.) at 500 V and 1350 μF. The transformed cells were cultured in 6-well plates at 1 × 105 cells/well. When the culture reached 50% confluence, 1 mg/ml G418 sulfate (Calbiochem) was added. G418-resistant clones were picked 1 week later and analyzed individually or as pools of several hundred clones.

Plasmids.

The MMP-9 and IL-8 promoters were prepared by PCR using human genomic DNA as the template. The MMP-9 promoter was amplified using the primers 5′-CTAGAGGCTTACTGTCCCCTTTACTG-3 and 5-CAGAGGCTCATGGTGAGGGCAGAG-3′ and Pyrobest (Takara) for 30 PCR cycles (30 s at 94°C, 30 s at 58°C, and 30 s at 72°C). A second, nested PCR was performed under the same conditions with primers 5′-CCCTGAAGATTCAGCCTGCGG-3′ and 5′-GGTGAGGGCAGAGG-TGTCTG-3′. Amplification of the IL-8 promoter region was performed using primers 5′-GATTGGCTGGCTTATCTTCACC-3′ and 5′-TTGTCCTAGAAGCTTGTGTGCTCTGCTGTC-3′ and Pyrobest for 30 PCR cycles (30 s at 94°C, 30 s at 54°C, and 30 s at 72°C). The PCR products were ligated into the PCR2.1 vector using an Original TA Cloning Kit (Invitrogen, Carlsbad, CA). The inserted DNA sequences were verified and cloned into the KpnI-XhoI and XhoI-HindIII sites of pGL2 basic vector.

RT-PCR.

Total RNA was extracted from 1 × 106 A549, HeLa, Caco-2, NCI-H292, and HEK293 cells using Isogen (Nippongene). RT-PCR experiments were performed with a RNA PCR kit (AMV version 2.1; Takara) according to the manufacturer’s instructions. The reverse transcription reaction was carried out at 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min. PCR was performed for 20–40 cycles (for IL-8, 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C; for GAPDH and MEF, 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C) The following primer pairs were used: (a) for IL-8, 5′-ATGACTTCCAAGCTGGCCGTGCT-3′ and 5′-TCTCAGCCCTCTTCAAAAACTTCTC-3′; and (b) for MEF, 5′-GGAAGACCCCTCTGTGTTCCCAGCTG-3′ and 5′-CAGTCTTCTTGGCTCTTTCCTCTCTGG-3′.

F-actin Staining.

For F-actin staining, the cells were grown on glass coverslips, fixed in 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS. The cells were incubated with rhodamine phalloidin (Molecular Probes, Eugene, OR) for 30 min at room temperature.

Matrigel Invasion Assay.

A549 cells and HUVECs were resuspended in FBS-free DMEM supplemented with 0.1% BSA, and the cultures were incubated at 37°C for 30 min. Invasion assays were carried out in 24-well Matrigel invasion chambers (BD Biocoat Matrigel, Franklin Lakes, NJ). The lower chambers were filled with serum-free medium or conditioned medium from cultures of A549 or clone71 cells, and the upper chambers were seeded with 2.5 × 105 A549 cells or 5.0 × 104 HUVECs. After a 24-h incubation, the number of cells that had passed through the filter into the lower chamber was counted and expressed as a percentage relative to the controls.

In Vitro Soft Agar and in Vivo Growth Assays.

For the in vitro growth assay, 1 × 104 cells were plated on a 24-well plate with or without 10 μm BB94. The cells were counted using the WST assay every 24 h for 3 or 4 days. For the soft agar assays, 1 ml of 1.0% agarose was diluted with 1 ml of 2× DMEM. A 2-ml volume of bottom agar was plated in a 6-well plate. The cells (1.0 × 104) were resuspended in 1 ml of 3× DMEM and mixed with 2 ml of 0.5% agarose. Finally, 3 ml of the mixture were overlaid on a bottom agar layer. For the in vivo growth assays, a single cell suspension of 1.0 × 106 cells in PBS was injected s.c. into four nude mice. The mice were sacrificed after 2 months, and the tumor volume (length × width2/2) and tumor weight were measured.

Immunohistochemical Staining.

Tumor sections from nude mice were cut using a LEICA cryostat. The sections were fixed in 4% paraformaldehyde in PBS. H&E staining was performed by standard methods. Tumor sections were stained with a 1:100 dilution of rat antihuman CD31 (MEC13.3; PharMingen, San Diego, CA), a 1:150 dilution of goat antihuman MMP-9 (Santa Cruz Biotechnology, Santa Cruz, CA), a 1:150 dilution of sheep anti-lysozyme, or a 1:150 dilution of rabbit anti-IL-8 (Santa Cruz Biotechnology) antibody. Incubation with primary antibody was carried out at 4°C overnight. Incubation with a 1:100 dilution of TRITC antigoat and antirabbit secondary antibodies and a 1:100 dilution of biotinylated antirat secondary antibody (Vector Laboratories, Burlingame, CA) was carried out for 45 min at room temperature. The biotinylated secondary antibody was revealed with 3,3′-diaminobenzidine (Dojindo) according to the manufacturer’s instructions.

Gelatin Zymography.

Tumor nodules were homogenized in lysis buffer [20 mm Tris-HCl (pH 7.4), 0.1 m NaCl, and 0.5% Triton X-100]. The soluble and insoluble extracts were separated by centrifugation and stored at −20°C. This lysate was mixed with SDS sample buffer [0.185 m Tris-HCl (pH 6.8), 30% glycerol, and 6% SDS] and analyzed by gelatin zymography on 10% SDS-polyacrylamide gels containing 0.5 mg/ml gelatin. After electrophoresis, SDS was removed from the gels by soaking twice for 20 min in 2.5% Triton X-100 and washed for 20 min in rinse buffer [50 mm Tris-HCl (pH 7.4) and 0.1 m NaCl]. The gels were incubated for 20 h at 37°C in gel incubation buffer (50 mm Tris-HCl, 10 mm CaCl2, and 0.02% NaN3), stained in 0.5% Coomassie Blue, and destained in a solution of 5% methanol and 7.5% acetic acid.

Western Blot Analysis.

Tumor nodules were homogenized in lysis buffer, and soluble and insoluble extracts were separated by centrifugation and stored at −20°C. This lysate was mixed with SDS sample buffer, and the samples were electrophoresed on 10% SDS-polyacrylamide gels. For protein analysis using cell lines, cells were washed twice and scraped with cold PBS, pelleted for 10 min at 4°C, and lysed using radioimmunoprecipitation assay buffer [150 mm NaCl, 1% NP40, 0.15% sodium deoxycholate, 0.1% SDS, 50 mm Tris (pH 8.0) and a protease inhibitor mixture (Sigma)] with 1 h of rotation at 4°C. Proteins were quantitated by the BCA assay. Protein samples (80 μg) were electrophoresed on 10% SDS-PAGE gel. The samples were subsequently electroblotted onto a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was blocked with a solution of PBST [PBS containing 0.05% (v/v) Tween 20] and 5% nonfat milk. After three washes in PBST, the membrane was incubated for 1 h in a 1:1,000 dilution of a primary antibody against MMP-9 (Santa Cruz Biotechnology). The secondary antibody used was mouse antigoat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a dilution of 1:10,000. As for CD31, a 1:100 dilution of a primary antibody against CD31 (BD Pharmingen, San Diego, CA) and a secondary antibody rabbit antirat IgG (Vector Laboratories) were used. A 1:1,000 dilution of Ets-2 antibody (Santa Cruz Biotechnology) and a 1:500 dilution of anti-MEF polyclonal antibodies (a kind gift from TransGenic Inc., Kumamoto, Japan) were used to detect Ets-2 and MEF proteins, respectively. Goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted to 1:10,000 was used as a secondary antibody. The blots were detected with the enhanced chemiluminescence detection kit (Amersham).

Regulation of MEF Expression in Normal and Cancer Cell Lines.

We analyzed the expression of MEF mRNA in human cancer cell lines and non-cancer cell lines by semiquantitative RT-PCR (Fig. 1,A). MEF was highly expressed in HEK293 cells, which are derived from normal human kidney cells. However, MEF expression was undetectable or low in cancer cell lines compared with HEK293 cells when the data were normalized for the GAPDH mRNA levels. By Western blotting, we determined that MEF protein expression level in cancer cell lines was lower compared with the level in HEK293 cells (Fig. 1,A). We next examined the effect of the demethylating agent 5-AC on MEF expression in some cancer cell lines. As shown in Fig. 1,B, 5-AC increased MEF expression but not GAPDH expression in A549 cells and Caco-2 cells. Consistent with this data, the protein levels of MEF were higher in the 5-AC-treated cells compared with the controls (Fig. 1 B). Therefore, these findings indicate that methylation of the MEF gene is involved in reducing the expression of MEF in A549 cells and Caco-2 cells and that treatment with 5-AC induces the expression of MEF to a level comparable with that of normal cells, especially in the case of Caco-2 cells.

Effect of MEF Overexpression in A549 Cells.

It was shown that DNA methyltransferase-1 was highly expressed in A549 cells and that antisense molecules directed against DNA methyltransferase-1 mRNA inhibited cell growth and activated p21 transcription in A549 cells (21, 22). Assuming that MEF is a tumor suppressor gene, MEF overexpression may suppress tumorigenesis in A549 cells that express MEF at low levels. Therefore, we produced stable transfectants of A549 to determine the in vitro characteristics of A549 cells and their MEF-overexpressing counterparts. As we reported previously, functional MEF is expressed in the stable transfectant of A549 (19). MEF protein was highly expressed in the MEF stable transfectant of A549 compared with the parental cell line (Fig. 1,A, bottom panel, lanes 1 and 2). MEF overexpression induced morphological changes, such as the conversion of normally loose cell-cell contacts to strong interactions (Fig. 2,A). Other stable transfectants also showed similar morphological changes (data not shown). We also observed that F-actin was localized at the cell-cell borders in MEF-overexpressing cells (Fig. 2,A), suggesting that cell motility is decreased and that the formation of functional adherent junctions is increased by MEF overexpression. When A549 cells were cultured in the presence of 10 μm 5-AC, morphological changes were induced that were similar to those seen in the MEF-overexpressing cells (data not shown). Because it has been reported that epithelial-to-mesenchymal transitions were induced by an E-cadherin deficiency or by the activation of MMPs (23), we also determined the expression levels of MMPs and E-cadherin. MMP-9 expression was decreased in MEF-overexpressing cells, as indicated by Western blotting analysis and gelatin zymography (Fig. 2,B), whereas the E-cadherin mRNA levels were unchanged in these cells (data not shown). To mimic the reduced activity of MMP-9 in MEF-overexpressing cells, we treated A549 cells with the MMP inhibitor BB94. As shown in Fig. 2,A, BB94 (10 μm) induced morphological changes and F-actin localization patterns at cell-cell borders that were typical of MEF-overexpressing cells. Normally, these morphological changes are indicative of reduced invasiveness. Therefore, we performed Matrigel invasion assays to determine the invasion activities of A549 cells and MEF-overexpressing cells. A549 cells showed potent invasiveness in this assay, whereas MEF-overexpressing cells and BB94-treated cells showed low levels of invasiveness (Fig. 2,C). Because BB94 has been reported to suppress the growth of cancer cells by blocking the processing and/or release of various growth factors that are induced by MMPs (24, 25), we examined whether MEF overexpression and BB94 (10 μm) affected the in vitro growth of A549 cells (Fig. 2,D). The growth of MEF-overexpressing cells in serum-free conditions was suppressed compared with A549 cells, probably due to MMP inhibition, because BB94 showed similar effect (Fig. 2 D). Although MEF slowed the growth of A549 cells, there were no signs of apoptosis as determined by morphology or by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay (data not shown). These findings imply that MEF affects the morphology, invasiveness, and growth of cells, possibly by inhibiting the expression of MMP-9 and other MMPs.

We also determined whether MEF suppressed the cellular transformation and tumorigenic activity of A549 cells in vitro and in vivo. Using a colony formation assay in soft agar culture, we found that A549 cells, but not MEF-overexpressing cells, formed colonies (Fig. 3,A). This suggests that MEF suppresses anchorage-independent cell growth, which is a marker of tumor malignancy. To test this hypothesis, we injected s.c. A549 cells and MEF-overexpressing cells into nude mice and examined in vivo tumorigenic activity (Fig. 3,B). Two months after injection, tumors consisting of A549 cells grew to about 2 cm in diameter in the nude mice, whereas the tumors formed from MEF-overexpressing cells had diameters of less than 0.4 cm. H&E staining showed that the A549 tumors were poorly differentiated. In contrast, the tumors formed from MEF-overexpressing cells contained well-differentiated glands with numerous central lumens (Fig. 3,C). Previously, we showed that MEF up-regulated the expression of lysozyme, which is a marker of gland serous cell differentiation, in A549 cells (19). In this regard, high-level expression of lysozyme was seen exclusively in the central lumens of MEF-overexpressing tumors (Fig. 3 C). Therefore, the induction of epithelial cell differentiation by MEF may be related to the suppression of tumorigenicity in A549 cells in vivo.

To further determine whether MEF affected tumor angiogenesis, we analyzed the vascularization of nude mice tumors by immunostaining with CD31, which is a marker on vascular endothelial cells. The number of medium- and large-sized vessels in MEF-overexpressing tumors was significantly reduced (to 75% and 31%, respectively) compared with the controls (Fig. 4,A). The difference in expression of CD31 between the tumors was confirmed by Western blot analysis using CD31 antibodies (Fig. 4,B). In addition, we determined whether MEF overexpression in A549 cells affected the migration of vascular endothelial cells. We found that the conditioned media from A549 cell cultures stimulated the migration of HUVECs, whereas conditioned media from MEF-overexpressing cell cultures had less of an effect (Fig. 4,C). In the positive control, basic fibroblast growth factor increased HUVEC migration (Fig. 4 C).

It has been reported that blockage of IGF-I signaling induced glandular differentiation in A549 tumors (26). Various growth factors are found within the stromal matrix that are bound to various proteins and to the extracellular matrix (25). It has been reported that MMPs are involved not only in tumor invasion but also in the processing and release of various growth factors, such as vascular endothelial growth factor and IGF-I (24, 25). Because MMPs are necessary for the mobilization of growth factors, they are indirectly involved in tumor growth and angiogenesis. To examine the factors involved in the tumor-suppressive effects of MEF, we first focused on the expression and activities of MMPs, especially because the expression of vascular endothelial growth factor and IGF-I in A549 cells was not affected by MEF overexpression (data not shown). Using gelatin zymography, we found that the expression levels of MMP-9 and MMP-2 were significantly reduced in MEF-overexpressing tumors (Fig. 5,A). The gelatin zymography result on MMP-9 was verified by Western blotting analysis and immunohistochemistry (Fig. 5, A and B, respectively). MEF inhibited MMP-2 expression, as shown in Fig. 5 A. Although it appears that the MMP-2 promoter, which lacks the ets consensus motif, is not directly regulated by ETS transcription factors, the observed inhibition of MMP-2 may be related to the suppression of tumor malignancy. This hypothesis is based on the finding that the suppression of MMP-2 alone inhibited the transition from the prevascular to vascular stage during tumor development and consequently inhibited tumor growth.

Angiogenesis is induced by the proliferation and migration of blood endothelial cells, which are stimulated by angiogenic factors; in the case of A549 cells, IL-8 is a potent angiogenic factor (27). A recent report suggested that PEA3 is involved in tumor angiogenesis mediated by the induction of IL-8 (28). We suggest that MEF suppresses tumor angiogenesis by inhibiting IL-8 expression. Immunohistochemical analyses showed that IL-8 expression was reduced in MEF-overexpressing tumors in nude mice (Fig. 5,B). Furthermore, IL-8 mRNA expression in vitro was significantly reduced in MEF-overexpressing cells, compared with A549 cells (Fig. 5 C). These results indicate that MEF inhibits tumor angiogenesis and that this inhibition is mediated, at least in part, by the suppression of IL-8 transcription.

Effects of MEF on MMP-9 and IL-8 Transcription.

To examine whether MEF directly suppresses the transcription of MMP-9 and IL-8, we performed luciferase assays using reporter constructs that contained the MMP-9 promoter (+18 to –643) and IL-8 promoter (+22 to –431). In A549 cells, high constitutive luciferase activities were observed (Fig. 6,A). However, MEF dramatically decreased the activities of these promoters. Additionally, the activities of MMP-9 and IL-8 were down-regulated in the A549 cells stably expressing MEF (Fig. 6,A). This down-regulation is clearly related to MEF expression levels as shown in the protein blots (Fig. 6,A, right panel). In contrast, among all of the tested ETS transcription factors (ELF-1, ETS-1, PEA3, and ESE-1; data not shown), only ETS-2 increased the promoter activities of the MMP-9 and IL-8 promoters, and antisense ETS-2 mRNA decreased the activities of these promoters (Fig. 6,B). Furthermore, the endogenous ETS-2 expression in A549 cells was down-regulated when antisense ETS-2 was transfected (Fig. 6,B, right panel). We predicted that MEF competed with ETS-2 on the ets-binding sites of these promoters because MEF, unlike PU-1 (29), does not have a repressor domain to interact with HDAC and Sin3A, which have deacetylase activities, and because MEF did not affect the endogenous expression of ETS-2 (Fig. 6,C). Based on the findings shown in Fig. 1,B, we investigated whether demethylation of the MEF gene regulated the promoter activity of IL-8. Thus, we examined the effect of 5-AC (1 μm) on the activity of the promoter. As shown in Fig. 6 D, the activity of the IL-8 promoter in A549 cells was suppressed by treatment with 5-AC to an extent similar to that of transient expression of MEF. Therefore, we propose that MEF is a tumor suppressor gene that is down-regulated by methylation in cancer cells.

The data presented here show that MEF suppresses the tumorigenesis of human non-small cell lung carcinoma A549 cells both in vitro and in vivo, most likely due to the inhibition of MMPs and IL-8. MEF may compete with ETS-2 for binding to the ets-binding sites on the promoters of the MMP and IL-8 genes. MEF expression in some cancer cells was down-regulated by methylation of the MEF gene. However, additional methylation experiments remain to be performed to verify this. In the growth assay, the reduced colony size in soft agar culture and reduced tumor size in nude mice are due, in part, to the reduced growth rate of MEF-expressing A549 cells. However, because the reduced growth rate of MEF-expressing A549 cells was smaller relative to the reduced rate of colony size and tumor size, other mechanisms such as the inhibition of angiogenesis in the case of the reduced tumor growth need to be considered. We propose that MEF may be a novel tumor suppressor gene that is localized on the X chromosome. This is further supported by the clinical finding that LOH is found at Xq25–26.1 in ovarian and breast carcinomas (5, 6). A recent study suggested that transcriptional regulation by MEF was restricted to the G1 phase of the cell cycle (30). It is reasonable to assume that MEF is inactivated during the S phase because MEF inhibits tumor growth.

It has been suggested that several ETS transcription factors up-regulate the transcription of positive regulators, such as MMPs and IL-8, in cancer cells (8, 28). In addition, ETS factors that are activated by oncogenic factors control tumor progression. Although many researchers have focused on how the balance of extracellular factors controls cellular transformation, the mechanism by which the balance of intracellular factors (such as transcription factors) has not been studied extensively. We studied ETS transcription factors to find out whether transcription factor levels controlled cellular transformation. Many of the genes associated with cellular transformation contain adjacent binding sites for ETS- and AP-1 family transcription factors, and these elements mediate transcriptional activation in a wide range of activated oncogenes (9). Several ETS transcription factors are known to be downstream targets of the Ras-Raf-MEK signaling pathway (31). The oncogenic stimulation of this pathway activates gene transcription of ETS-1 and ETS-2 by phosphorylation of threonine residues in the pointed domain (32, 33). Our present findings that ETS-2 activates tumor malignant factors, such as MMP-9 and IL-8, in A549 cells are consistent with studies indicating that ETS-2 mediates cellular transformation (13, 14). Thus, it appears that the inhibition of ETS-2 could be a good strategy for cancer therapy.

Recently, it was suggested that some ETS transcription factors suppress the transcription of MMPs and HER2 (15). Therefore, it is possible that tumor progression is controlled by the balance between positive and negative ETS factors. The present findings suggest that the balance between MEF and ETS-2 in cancer cells plays a critical role in determining tumor malignancy. It has been suggested that several ETS transcription factors are phosphorylated by Ras signaling and enter the nucleus, where they activate the transcription of oncogenes. In contrast, ESE-3, which is an ETS transcription factor, was recently shown to be a nuclear protein that suppresses transcription depending on Ras signaling (34). Therefore, we propose that nuclear ETS factors, such as ESE-3, may be negative regulators that work against cytoplasmic ETS factors to reestablish cellular homeostasis. We recently determined the intracellular localization of MEF using a green fluorescence protein-MEF fusion protein under serum and nonserum conditions and in the presence of dominant-negative Ras and MEK. A549 cells have a mutation in K-Ras (35), which results in activation of the Ras-MEK-mitogen-activated protein kinase pathway. We found that MEF was constitutively localized in the nucleus under all conditions, whereas ETS-2 was localized in the cytoplasm and nucleus under nonstimulatory conditions (data not shown). If our hypothesis is valid, the balance between different ETS factors may control cellular transformation. In other words, MEF and ESE-3 may act as tumor suppressors in opposition to oncogenic ETS transcription factors, such as ETS-2. Previous works indicated that ETS activators could reverse transformed phenotypes in tumor cell lines and that the DNA binding domain (the ETS domain) is essential for this reversion (36, 37, 38). In light of these reports, we cannot discount the possibility that MEF acts as a dominant negative Ets factor. Within the context of the system used here, ETS-2, which was highly expressed endogenously in A549 cells (Fig. 6, B and C), may act as an activator of ETS-dependent transcription, and MEF would bind to ETS binding sites to broadly inhibit ETS-dependent gene expression in lung carcinoma cells.

We reported previously that MEF activated the lysozyme promoter in A549 cells (19). However, the present study indicated that MEF suppressed the promoter activities of the MMP-9 and IL-8 genes. Thus, MEF-mediated transcriptional regulation is dependent on the promoter and cellular contexts. Similarly, Fli-1 can function as an activator of the tenascin-C promoter (16) while acting as a repressor of the collagen promoter (39). In any case, interplay between ETS factors that depends on target promoter context may serve as a molecular switch between gene repression and activation.

The present finding may contribute not only to cancer therapies but also to cancer diagnostics that are based on the detection of LOH, single nucleotide polymorphisms, and epigenetic modifications to the MEF gene. Projects are under way to investigate these potential applications. From a clinical viewpoint, the reactivation of tumor suppressor genes by demethylation, especially by 5-AC, represents a promising target for tumor therapy. The MEF gene on the X chromosome may be a potential target for epigenetic modification.

Fig. 1.

Expression and reactivation of MEF induced by 5-AC in various cancer cell lines. A, top panel, mRNA expression of MEF was analyzed in human cell lines. HEK293, normal kidney; A549, lung adenocarcinoma; Caco-2, colon carcinoma; NCI-H292, human bronchial epithelial cells; HeLa, uterus cancer cells. A, bottom panel, protein blots to detect MEF expression in different cell lines. The second lane is A549 stably expressing MEF. Nonspecific bands are indicated (NS) to show equal loading. B, top panel, effect of demethylating agent 5-AC on MEF mRNA expression in cell lines. B, bottom panel, MEF protein expression in A549 and Caco-2 cells in the presence or absence of 5-AC.

Fig. 1.

Expression and reactivation of MEF induced by 5-AC in various cancer cell lines. A, top panel, mRNA expression of MEF was analyzed in human cell lines. HEK293, normal kidney; A549, lung adenocarcinoma; Caco-2, colon carcinoma; NCI-H292, human bronchial epithelial cells; HeLa, uterus cancer cells. A, bottom panel, protein blots to detect MEF expression in different cell lines. The second lane is A549 stably expressing MEF. Nonspecific bands are indicated (NS) to show equal loading. B, top panel, effect of demethylating agent 5-AC on MEF mRNA expression in cell lines. B, bottom panel, MEF protein expression in A549 and Caco-2 cells in the presence or absence of 5-AC.

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Fig. 2.

Characteristics of MEF-overexpressing cell line. A, morphological features (a–c) and F-actin staining (d−f) of untransfected A549 cells (a and d), MEF-overexpressing A549 cells (b and e), and BB94 (10 μm)-treated A549 cells (c and f). B, gelatin zymography of MMP-9 and Western blotting using an anti-MMP-9 antibody to detect MMP-9 content in conditioned media from A549 and MEF-overexpressing cells. To show equal loading of samples, unknown bands at approximately Mr 70,000 were excised from the gel stained with Coomassie Brilliant Blue (right panel). C, Matrigel invasion assay. The upper chambers were seeded with 2.5 × 105 A549, MEF-overexpressing A549, or BB94-treated A549 cells. After a 24-h incubation, the number of cells that had passed through the filter into the lower chamber was counted, and the number was expressed as a percentage relative to the control value. D, cell growth rate. The growth rates of MEF-overexpressing A549 and BB94-treated A549 cells are expressed as a percentage relative to the growth rate of A549.

Fig. 2.

Characteristics of MEF-overexpressing cell line. A, morphological features (a–c) and F-actin staining (d−f) of untransfected A549 cells (a and d), MEF-overexpressing A549 cells (b and e), and BB94 (10 μm)-treated A549 cells (c and f). B, gelatin zymography of MMP-9 and Western blotting using an anti-MMP-9 antibody to detect MMP-9 content in conditioned media from A549 and MEF-overexpressing cells. To show equal loading of samples, unknown bands at approximately Mr 70,000 were excised from the gel stained with Coomassie Brilliant Blue (right panel). C, Matrigel invasion assay. The upper chambers were seeded with 2.5 × 105 A549, MEF-overexpressing A549, or BB94-treated A549 cells. After a 24-h incubation, the number of cells that had passed through the filter into the lower chamber was counted, and the number was expressed as a percentage relative to the control value. D, cell growth rate. The growth rates of MEF-overexpressing A549 and BB94-treated A549 cells are expressed as a percentage relative to the growth rate of A549.

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Fig. 3.

Tumor-suppressive effect of MEF. A, colony formation assay in soft agar showed that the MEF stable transfectant had a reduced growth rate in comparison with A549 cells. B, tumor formation in nude mice. A single-cell suspension of 1.0 × 106 cells in PBS was injected s.c. into four nude mice. The mice were sacrificed after 2 months, and tumor volumes were measured (bar graph). Tumor volume = length × width2/2. Arrows indicate formed tumors. C, histological features and immunostaining for lysozyme in tumor sections.

Fig. 3.

Tumor-suppressive effect of MEF. A, colony formation assay in soft agar showed that the MEF stable transfectant had a reduced growth rate in comparison with A549 cells. B, tumor formation in nude mice. A single-cell suspension of 1.0 × 106 cells in PBS was injected s.c. into four nude mice. The mice were sacrificed after 2 months, and tumor volumes were measured (bar graph). Tumor volume = length × width2/2. Arrows indicate formed tumors. C, histological features and immunostaining for lysozyme in tumor sections.

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Fig. 4.

MEF suppresses tumor angiogenesis. A, analysis of the vascularization of tumors formed from A549 or MEF-overexpressing A549 cells in nude mice by immunostaining with CD31. B, CD31 expression in tumors was examined by Western blotting. The right panel shows unknown bands of about Mr 70,000 excised from the gel stained with Coomassie Brilliant Blue to determine equal loading of samples. C, HUVEC migration assay. Conditioned media from A549 cell cultures and media from MEF-overexpressing A549 cell cultures were compared for their ability to promote migration (bottom panel). The top panels shows negative (top left panel) and positive (top right panel) controls. The graph on the right shows the quantitation of migration activity of HUVECs.

Fig. 4.

MEF suppresses tumor angiogenesis. A, analysis of the vascularization of tumors formed from A549 or MEF-overexpressing A549 cells in nude mice by immunostaining with CD31. B, CD31 expression in tumors was examined by Western blotting. The right panel shows unknown bands of about Mr 70,000 excised from the gel stained with Coomassie Brilliant Blue to determine equal loading of samples. C, HUVEC migration assay. Conditioned media from A549 cell cultures and media from MEF-overexpressing A549 cell cultures were compared for their ability to promote migration (bottom panel). The top panels shows negative (top left panel) and positive (top right panel) controls. The graph on the right shows the quantitation of migration activity of HUVECs.

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Fig. 5.

Expression levels of MMP-9 and IL-8 are reduced by overexpression of MEF. A, MMP-9 expression in tumors formed from A549 and MEF-overexpressing A549 cells was examined by gelatin zymography and Western blotting using antibody raised against MMP-9. As a loading control, the gel was stained with Coomassie Brilliant Blue, and unknown bands at about Mr 70,000 were excised to show equal loading (bottom panel). Bottom panel shows the loading control (Coomassie Brilliant Blue staining). B, immunostaining of MMP-9 and IL-8 in nude mice tumors formed from A549 and MEF stable clone. C, RT-PCR of IL-8 in A549 cells and MEF-expressing clone.

Fig. 5.

Expression levels of MMP-9 and IL-8 are reduced by overexpression of MEF. A, MMP-9 expression in tumors formed from A549 and MEF-overexpressing A549 cells was examined by gelatin zymography and Western blotting using antibody raised against MMP-9. As a loading control, the gel was stained with Coomassie Brilliant Blue, and unknown bands at about Mr 70,000 were excised to show equal loading (bottom panel). Bottom panel shows the loading control (Coomassie Brilliant Blue staining). B, immunostaining of MMP-9 and IL-8 in nude mice tumors formed from A549 and MEF stable clone. C, RT-PCR of IL-8 in A549 cells and MEF-expressing clone.

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Fig. 6.

Reciprocal effects of MEF and ETS-2 on MMP-9 and IL-8 promoter activities. A, MMP-9 and IL-8 promoter activities are suppressed by transient expression of MEF as well as in a stable transfectant. Left panel, Western blot showing MEF expression in untransfected A549 cells, transiently transfected MEF (3 μg), and stable cell clone. The nonspecific band is indicated (NS) to show equal loading. B, ETS-2 activates the MMP-9 and IL-8 promoters, and antisense ETS-2 suppresses both of these promoters. Left panel, Western blot of ETS-2 in the absence and presence of antisense ETS-2. ETS-2 expression was down-regulated by antisense ETS-2. C, MEF does not affect the expression of ETS-2. Bottom panel shows nonspecific bands excised from the same blot to show equal loading. D, reactivation of MEF by 5-AC may suppress IL-8 promoter activities in A549.

Fig. 6.

Reciprocal effects of MEF and ETS-2 on MMP-9 and IL-8 promoter activities. A, MMP-9 and IL-8 promoter activities are suppressed by transient expression of MEF as well as in a stable transfectant. Left panel, Western blot showing MEF expression in untransfected A549 cells, transiently transfected MEF (3 μg), and stable cell clone. The nonspecific band is indicated (NS) to show equal loading. B, ETS-2 activates the MMP-9 and IL-8 promoters, and antisense ETS-2 suppresses both of these promoters. Left panel, Western blot of ETS-2 in the absence and presence of antisense ETS-2. ETS-2 expression was down-regulated by antisense ETS-2. C, MEF does not affect the expression of ETS-2. Bottom panel shows nonspecific bands excised from the same blot to show equal loading. D, reactivation of MEF by 5-AC may suppress IL-8 promoter activities in A549.

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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.

1

Supported in part by grants in aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and by grants from Kowa Life Science Foundation, Japan.

3

The abbreviations used are: LOH, loss of heterozygosity; MMP, matrix metalloproteinase; IL, interleukin; RT-PCR, reverse transcription-PCR; 5-AC, 5-azacytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; FBS, fetal bovine serum; IGF, insulin-like growth factor; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase.

We thank Dr. W. G. Stetler-Stevenson, (NIH) for BB94 and Drs. H. Saya and M. Nakao (Kumamoto University) for valuable discussion. Anti-MEF polyclonal antibodies were a kind gift from TransGenic Inc.

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