Overexpression of Activating Transcription Factor-2 Is Required for Tumor Growth and Progression in Mouse Skin Tumors

INTRODUCTION

The mammalian activator protein (AP)-1 transcription factor is composed of homodimers and heterodimers of basic region-leucine zipper proteins, which belong to the Jun, Fos, and the closely related activating transcription factor (ATF) subfamily (1) , including (among others) ATF-2 and ATF-a. The c-Jun/Fos dimers preferentially bind to the 7-bp consensus sequence TGAGTCA, a phorbol ester and growth factor-inducible element (2 , 3) , whereas c-Jun/ATF dimers recognize 8-bp motifs, i.e., the jun2 12-O-tetradecanoylphorbol-13-acetate–responsive element (TRE) TTACCTCA sequence (4 , 5) . The trans-activating capacity of ATF-2 can be regulated in response to stress stimuli through phosphorylation by the c-Jun NH₂-terminal kinase (JNK)/stress-activated protein kinase group of the mitogen-activated protein kinase (MAPK) family (6) and by p38 MAPK (7) at Thr⁶⁹ and Thr⁷¹ residues. The regulation of ATF-2 phosphorylation by growth factors was found to be performed by a two-step mechanism: The extracellular signal-regulated kinase (ERK) pathway induces phosphorylation of ATF-2 Thr⁷¹, whereas subsequent ATF-2 Thr⁶⁹ requires the p38 pathway (8) .

The biological role of ATF-2 remains largely undefined. It has been implicated in various processes such as central nervous system and skeleton development (9 , 10) , tissue regeneration and proliferation (11) , and immune and adaptive responses to deleterious cellular stresses (12 , 13) . ATF-2 is involved in the transcriptional regulation of c-jun and a number of cell cycle genes, such as cyclin A, cyclin D1, and ATF3 (14, 15, 16) . Moreover, it has been shown to mediate a transcriptional response to the transforming adenovirus protein E1A (17) .

The data concerning the involvement of ATF-2 in carcinogenesis are rather limited. In human late-stage melanoma cells, ATF-2 enhances cell resistance to ultraviolet (UV) radiation-induced apoptosis (18) . Furthermore, ATF-2 mRNA levels were higher in human tumors of the gastrointestinal tract than in adjacent normal counterparts (11) . Moreover, the human chromosome locus 2q32, which includes ATF-2, is a region of frequent losses and alterations in various tumors (19, 20, 21, 22) ; however, alterations of the ATF-2 gene were found to be infrequent in human neuroblastomas and lung and breast carcinomas (23) , suggesting that ATF-2 is not a major tumor suppressor gene in humans. Recently, ATF-2 was reported to play an important role in the development of diffuse breast cancers in ATF-2 heterozygous mice (ATF-2^+/−; ref. 23 ), whereas it was demonstrated to promote proliferation and tumor formation in chicken embryo fibroblasts only in cooperation with v-Jun (24) . These findings further complicate the picture of the involvement ATF-2 in oncogenesis. Finally, the implication of ATF-2 in human or mouse skin carcinogenesis has not yet been addressed in detail.

Recently, we demonstrated (25) increased levels of ATF-2 and other AP-1 components at different stages of carcinogenesis in mouse skin cell lines derived from tumors induced by chemical mutagens. The multistage mouse skin carcinogenesis system, which was used in our study, has been developed after chemical treatment of mouse epidermis with 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate. It comprises a series of cell lines isolated from different stages of mouse skin tumor progression (25, 26, 27, 28) , including the highly anaplastic, invasive spindle cell lines A5 and CarB, which exhibit metastatic tumor growth in vivo.

In the present study, we carried out a series of in vitro and in vivo experiments using A5 and CarB cells stably transfected with a dominant negative form of ATF-2 (dnATF-2; ref. 29 ) to examine the specific role of ATF-2 in mouse oncogenicity. Our findings showed that ATF-2 is required for mouse skin tumor growth and progression, possibly by up-regulating the expression of the vital cell cycle genes c-jun, ATF-3, cyclin A, and cyclin D1 and affecting the composition and activity of the AP-1 complex. To the best of our knowledge, such findings have not been reported previously.

MATERIALS AND METHODS

Chemical Carcinogenesis Protocols.

For two-stage chemical carcinogenesis, the backs of ten 8-week–old mice were shaved and treated with a single application of 7,12-dimethylbenz(a)anthracene [25 μg of 7,12-dimethylbenz(a)anthracene in 200 μL of acetone) followed by biweekly application of 12-O-tetradecanoylphorbol-13-acetate (200 μL of a 10⁻⁴ mol/L solution of 12-O-tetradecanoylphorbol-13-acetate in acetone) for 20 weeks. Mice were visually examined weekly and sacrificed when individual tumors reached a diameter of 1 to 1.5 cm or at termination of the experiments. Tumors from sacrificed animals were snap-frozen in liquid nitrogen, fixed in 4% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin or used for immunohistochemical staining.

Immunohistochemistry.

For immunohistochemical staining, a modification of the immunoglobulin enzyme bridge technique [avidin-biotin-peroxidase (ABC) method] reported by Hsu et al. (30) was used. Paraffin-embedded tissue sections of 4 μm were dried for 30 minutes at 58°C before deparaffinization in xylene and rehydration by sequential incubation in EtOH/water solutions. The sections were treated with 3% hydrogen peroxide in methanol for 30 minutes at room temperature, in darkness, to quench endogenous peroxidase activity. After rinsing in water and PBS, sections were blocked with serum (diluted 1:5 in PBS) from a nonimmunized animal for 40 minutes to reduce nonspecific binding. Subsequently, the sections were incubated with a primary polyclonal antibody against ATF-2 (diluted 1:40 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA) or c-jun (diluted 1:50 in PBS; Santa Cruz Biotechnology) in a moist chamber at 4°C overnight. After washing in PBS, biotinylated antirabbit immunoglobulin (Dako Corp., Carpinteria, CA) at a dilution of 1:200 was added for 45 minutes, followed by a washing step and incubation with ABC reagent (streptABComplex/HRP; Dako Corp.) for 45 minutes. The peroxidase reaction was developed with 0.02% 3,3′-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in PBS containing 0.06% hydrogen peroxide for 1 minute. Finally, sections were rinsed with water and counterstained with Harris’ hematoxylin.

Cells and Culture Conditions.

The A5 and CarB mouse skin spindle cell lines as well as the derived transfected cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were grown at 37°C in a humidified atmosphere with 5% CO₂ and expanded to 75-cm² cell culture flasks.

Preparation of Dominant Negative ATF-2 Transfectants.

The A5 and CarB cell lines were transfected with the ATF-2d18d12 dominant negative ATF-2 mutant plasmid (29) or with CMV-neo control vector. Both plasmids contained a gene conferring resistance to the antibiotic Geneticin.

A5 and CarB cells were seeded at a confluence of 20% per 10-cm culture dish in DMEM supplemented with 10% FBS. After 24 hours, the cells were transfected with 5 μg of plasmid by the calcium phosphate method (31) . The cells were incubated with precipitated DNA for 18 hours, washed twice in PBS, and incubated for 24 hours in fresh DMEM supplemented with 10% FBS. Forty eight hours after transfection, the cells were diluted 10-fold and incubated with 800 μg/mL Geneticin (Sigma) for 2 weeks. Individual Geneticin-resistant colonies were finally isolated with cylinder trypsinization and grown in DMEM supplemented with 10% FBS.

Preparation of Total Cell Lysates.

Cells were allowed to grow exponentially in DMEM supplemented with 10% FBS in 10-cm dishes. For preparation of total cell lysates, cells were washed twice in ice-cold PBS and lysed in lysis buffer [20 mmol/L Tris (pH 7.6), 0.5% Triton X-100, 250 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 10 μg/mL pefabloc, 2 mmol/L sodium orthovanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L dithiothreitol]. Alternatively, fresh tissue from mouse skin tumors was homogenized in lysis buffer using a Teflon glass homogenizer and subsequently filtered. Lysates were incubated on ice for 30 minutes and then centrifuged at 10,000 rpm at 4°C for 10 minutes. The supernatant was aliquoted and stored at −70°C. Protein estimations were performed by the Bradford method (32) .

To induce hyperosmolality stress, cells were seeded on 10-cm culture dishes and allowed to grow in DMEM supplemented with 10% FBS until they reached 70% confluence. They were subsequently grown in DMEM supplemented with 0.5% FBS for 24 hours and then induced with 150 mmol/L NaCl. Total cell lysates were prepared at 0 and 30 minutes after induction. Alternatively, cells were exposed to three 200 J/m² doses of UV-B at 30-minute intervals and harvested 1 hour after the last dose (33) .

Protein (Western Blot) Analysis.

Forty micrograms of total cell proteins were electrophoresed on a 10% SDS-polyacrylamide gel or a 7.5% SDS-polyacrylamide gel (when the phospho-ATF-2–specific antiserum was used after NaCl induction, or after exposure of cells to UV-B). Reducing agent was added in a 1:10 ratio. Proteins were then transferred to nitrocellulose membranes, which were blocked for 2 hours at room temperature, in either 5% bovine serum albumin (BSA) with Tris-buffered saline (TBS)-0.1% Tween 20 and 0.5 μmol/L sodium orthovanadate (when phospho-specific antisera were used) or 5% nonfat dry milk with TBS-0.1% Tween 20 (for the rest of specific antisera). The blots were subsequently incubated for 3 hours at room temperature with the corresponding primary antibodies.

The primary antibodies used were as follows. Anti–ATF-2, anti–c-Jun, anti–c-JunB, anti–Fra-2, anti–c-Fos, anti-ERK2, anti-p38, anti–phospho-ERK1/2, cyclin A, cyclin D1, and ATF-3 were purchased from Santa Cruz Biotechnology. Anti-JunD and anti–Fra-1 were kindly provided by Drs. D. Lallemand and M. Yaniv (Pasteur Institute, Paris, France; ref. 34 ). Anti-JNK1/2 was kindly provided by Dr. D. Gillespie (Beatson Institute, Glasgow, Scotland, United Kingdom; ref. 35 ). Anti–phospho-JNK1/2 and anti–phospho-ATF-2 Thr^69/71 were purchased from New England Biolabs (Beverley, MA). Primary antibodies were diluted 1:1,000 in 3% BSA or nonfat dry milk with TBS-0.1% Tween 20. After incubation, the membranes were washed twice in TBS-0.1% Tween 20 for 15 minutes.

Horseradish peroxidase-conjugated secondary antibody (diluted 1:5,000 in 1% BSA or nonfat dry milk with TBS-0.1% Tween 20) was then added for 1 hour at room temperature, and after washing twice, detection of protein levels was carried out using an enhanced chemiluminescence system (Pierce, Rockford, IL).

Electrophoretic Mobility Shift Assay.

Annealed oligonucleotides for the collagenase TRE (5′-AGCTTGATTGAGTCAGCCGGATC-3′ and 5′-GATCCGGCTGACTCATCAAG CT-3′; collagenase promoter position −73 to −65) and the jun2TRE motif (5′-TACACAGGATGTCCATATTAGGACA-3′ and 5′-TGTCCTAATATGGACAT CCTGTGTA-3′; the c-jun promoter position −194 to −179) were end-labeled with [γ-³²P]ATP using T4 polynucleotide kinase, and the reaction products were purified on an 8% polyacrylamide gel (25) .

DNA binding reactions were carried out by mixing 2,000 cpm of [γ-³²P]ATP–labeled oligonucleotide with 10 μg of total cell protein in binding buffer [50 mmol/L HEPES (pH 8.0), 500 mmol/L NaCl, 0.5 mol/L phenylmethylsulfonyl fluoride, 0.5 mg/mL BSA, 20% glycerol, and 1 mmol/L EDTA] plus 1 mmol/L dithiothreitol and 150 μg/mL poly(deoxyinosinic-deoxycytidylic acid) (Sigma). The reaction mixture was left at room temperature for 30 minutes, and the samples were subsequently subjected to electrophoresis on a 6% polyacrylamide gel at 150 V for 90 minutes, dried, and visualized by autoradiography. Control experiments were carried out using 100-fold molar excess of the unlabeled oligonucleotides collTRE cold and H-ras-p53 (36) . For the supershift assays, the reaction mixture was incubated with anti–c-jun or anti—ATF-2 for 30 minutes or with anti-hemagglutinin (HA) tag antibody (Santa Cruz Biotechnology) for 3 hours at 4°C.

Plasmids, Transfections, and Luciferase Reporter Assay.

A luciferase construct carrying the collTRE sequence (5XcollTRE-tata-Luc) or the jun2TRE motif (5Xjun2TRE-tata-Luc) and a control vector carrying no AP-1 binding site (tata-pG13Luc) were used for transfection experiments. For the construction of pcGNATF2T69+T71, vector pcGN (37) was linearized with XbaI and BamHI, and ATF2T69+T71 was amplified by polymerase chain reaction from pCMVATF2T69+T71. In pCMVATF2T69+T71, ATF-2 has been mutated at positions 69 and 71 from threonines into alanines (29) . Polymerase chain reaction amplification of ATF2T69+T71 was carried out using primers that add XbaI and BamHI restriction sites to the mutant ATF2T69+T71 gene. Primers (upstream, 5′-GAGCTCTAGAGGC-atgagtgatgacaaaccc-3′; downstream, 5′-GAGGGATCCTCA-tcaacttcctgagggctg-_3′) keep ATF2T69+T71 in-frame with the HA tag. Restriction with XbaI and BamHI was followed by ligation into linearized pCGN. Restriction sites are indicated in bold.

Cells were transfected by the calcium phosphate method (31) with 20 μg per 75-cm² flask of plasmid DNA containing 2.5 μg of pSG5-lacZ (control for the efficiency of transfection), 2.5 μg of reporters, and completed with pSG5. Cells at 30% confluence were incubated with precipitated DNA for 18 hours, washed twice with PBS, incubated with DMEM supplemented with 10% FBS for 24 hours, scraped, and subjected to three freeze/thaw cycles in 1× cell lysis reagent (Promega, Madison, WI). The lysate was analyzed for luciferase activity according to the manufacturer’s instructions (Promega) and normalized for transfection efficiency with β-galactosidase activity. The assay was repeated three times for each cell line investigated.

Growth Rate Assays.

Growth rate was estimated by three different methods. Approximately 5 × 10⁴ cells were plated on 5-cm culture dishes and allowed to grow in DMEM supplemented with 10% FBS. The growth rates of parental and transfected cell lines were compared 0, 2, 4, 6, and 8 days after plating of cells. The cells were methanol-fixed on the culture dish for 5 minutes and stained with 0.5% crystal violet solution for 5 minutes. After removal of the excess dye under running tap water, the dishes were air dried and destained with 33% acetic acid, and the absorbance was measured at 595 nm. The assay was repeated three times for each cell line investigated.

The rapid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay for cell proliferation was performed in a 96-well flat-bottomed tissue culture. One thousand cells for each clone were seeded; 6 hours after plating, the standard medium was removed, and 100 μl of culture medium with 0.5% of FBS were applied. The determinations were carried out at time points of 0, 2, 4, 6, and 8 days as follows: 0.01 mL of MTT (Sigma Chemical Co., Poole, United Kingdom) in PBS (5 mg/mL; filter sterilized) was added to each well. Samples were incubated at 37°C for 4 hours, the medium was removed, and 0.1 mL isopropyl alcohol/0.04 N HCl was added to each well. After thorough mixing to dissolve the dark blue crystals, the plates were left for a few minutes at room temperature and then placed on a Titertek Flow MicroELIZA reader, in which absorbance was recorded at a wavelength of 540 nm. Plates were read within 1 hour of adding the acid isopropanol solution. The assay was repeated three times for each cell line investigated.

[³H]Thymidine incorporation assay was carried out as follows. Approximately 5 × 10⁴ cells were plated on 5-cm culture dishes and allowed to grow in DMEM supplemented with 10% FBS. After 96 hours, serum was removed (0.5% fetal bovine serum), and the following day, the starvation medium was replaced by standard medium, in which [³H]thymidine (0.5 μCi; obtained from ICN) was added. Cells were collected at 6, 12, 18, and 24 hours after stimulation; DNA was extracted; and radioactivity was determined by liquid scintillation. All experiments were repeated three times.

Anchorage-Independent Growth.

Approximately 5 × 10³ cells were plated on 5-cm culture dishes onto a 3-mL basal layer of 0.3% agar in DMEM supplemented with 10% FBS, over a solidified cushion of 2 mL of 0.6% agar in DMEM supplemented with 10% FBS. Cells were allowed to grow for 2 weeks, and 0.5 mL of fresh DMEM supplemented with 10% FBS was added into the wells every 2 days. Individual macroscopic colonies were finally counted. The assay was repeated three times for each cell line investigated (38) . We used the immortalized mouse skin cell line C50 as negative control (39) and the A5 and CarB cell lines, which exhibit metastatic growth in vivo, as positive controls.

In vivo Tumorigenicity Studies.

Parental A5 and CarB cells, control vector-transfected A5-V and CarB-V cells, and dnATF-2–transfected A5-1, A5-2, CarB-T7, and CarB-T8 cells were harvested, washed, and resuspended in PBS. Approximately 10⁶ cells were injected subcutaneously at two sites in the abdominal region of 9-week–old BALB/c severe combined immunodeficient (SCID) mice (The Jackson Laboratory, Bar Harbor, ME). Tumor growth was measured two to three times weekly, and animals were killed when tumors reached a diameter of approximately 1.5 cm or at the end of the observation period. All experiments were performed at least twice, and samples from the skin tumors and the lungs and liver were obtained. Tissues for histologic examination were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde overnight, dehydrated, and embedded in paraffin by standard methods. Sections (5 μm) were stained with hematoxylin and eosin. Immunohistochemical expression of keratins was subsequently assessed with a specific wide spectrum screening polyclonal anti–pan-keratin antibody (Dako Corp.).

Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling Assay.

Double-stranded DNA breaks were detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) according to the method of Gavrieli et al. (40) . Briefly, 5-μm paraffin sections were mounted on poly-l-lysine–coated slides, dewaxed, rehydrated, and incubated for 30 minutes with 0.3% hydrogen peroxide to quench the endogenous peroxidase activity. Pretreatment was carried out by incubating the sections with proteinase K (Sigma; 20 μg/mL) for 15 minutes at 37°C. The labeling step was performed with terminal deoxynucleotidyl transferase (15 units per slide; New England Biolabs) for 1 hour at 37°C in 25 mmol/L Tris-Cl (pH 7.2), 200 mmol/L potassium cacodylate, 0.25 mmol/L CoCl₂, 25 mg/mL bovine serum albumin, and 24 μmol/L biotin-dATP (Life Technologies, Inc., Carlsbad, CA). The reaction was stopped by rinsing the sections in 20 mmol/L EDTA. The next stage comprised a 30-minute incubation in StreptAB Complex solution (Dako Corp). For color development, we used 3,3′-diaminobenzidine tetrahydrochloride as chromogen and hematoxylin as counterstain. We used tissue sections incubated with DNase I before treatment with terminal deoxynucleotidyl transferase as positive controls and sections incubated in terminal deoxynucleotidyl transferase buffer without the presence of the enzyme as negative controls. Cells were considered to undergo apoptosis when nuclear staining, without cytoplasmic background, was observed. Apoptotic index was estimated as the percentage of apoptotic cells in 10 high power fields (number of cells counted, 900–1,000).

RESULTS

Increased Levels of ATF-2 and c-Jun Were Observed in Spindle Cell Carcinomas of Mouse Skin.

We first studied the in vivo status of ATF-2 and c-Jun in early (papillomas) and advanced stages (spindle tumors) of the mouse skin carcinogenesis. Immunohistochemical expression of ATF-2 and c-Jun was assessed in serial sections from chemical-induced skin tumors from 10 mice. In the papillomas examined, only a faint nuclear staining for ATF-2 (Fig. 1A) ⇓ and c-Jun (Fig. 1B) ⇓ was observed, whereas the spindle cell carcinomas exhibited a strong nuclear staining for ATF-2 (Fig. 1A) ⇓ and c-Jun (Fig. 1B) ⇓ . Western blotting analysis of ATF-2 and c-Jun in representative chemical-induced skin tumors (Fig. 1C) ⇓ showed increased levels of total and phospho–ATF-2 and c-Jun expression in spindle tumors, compared with the papillomas examined. These data confirm our previous in vitro results showing enhanced levels of ATF-2 and c-Jun in the metastatic spindle cell lines of the mouse skin carcinogenesis model (25) . Therefore, we continued our experiments by using the A5 and CarB spindle cell lines, which, with regard to their ATF-2 and c-Jun status, seem to be an in vitro analog of the chemical-induced mouse skin tumors.

Transfected A5 and CarB Cell Lines Stably Expressed Dominant Negative ATF-2.

To study the effect of ATF-2 on the phenotype of the spindle cell lines, we stably introduced in A5 and CarB cells a dominant negative form of ATF-2 (dnATF-2) whose MAPK phosphorylation sites Thr⁶⁹ and Thr⁷¹ have been replaced by alanines (29) . Moreover, this dnATF-2 also lacks amino acids 1 to 18.

Western blot analysis showed that four independently obtained cell lines (A5-1, A5-2, CarB-T7, and CarB-T8) stably expressed dnATF-2 because expression of this shorter version of ATF-2 was >5-fold higher than that of endogenous ATF-2 in these cell lines (Fig. 2A and B) ⇓ .

Overexpression of Dominant Negative ATF-2 Has a Negative Effect on the Phosphorylation of Endogenous ATF-2.

To examine whether the overexpressed dnATF-2 in stable transfectants was functional, we determined the levels of Thr⁶⁹ + Thr⁷¹ phosphorylated wild-type ATF-2 by Western blot, using a phospho-specific antibody. The presence of dnATF-2 was found to reduce the phosphorylation state of full-length ATF-2 in exponentially growing A5 and CarB stable clones (Fig. 2A and B) ⇓ . Moreover, induction of hyperosmolality stress (Fig. 2C) ⇓ or exposure to UV-B (Fig. 2D) ⇓ , which are both known to activate JNK or p38 by inducing their phosphorylation, did not result in increased phosphorylation of ATF-2 in dnATF-2–transfected A5 and CarB cells under serum starvation conditions. The reduced phosphorylation levels of endogenous ATF-2 in the dnATF-2–transfected A5 and CarB cell lines was not due to inadequate activation of JNK1/2 or p38 MAPK molecules because the total levels, as well as the amount of the phosphorylated forms of these kinases, did not differ between dnATF-2–transfected A5 or CarB cells and their respective parental cells (Fig. 2C and D) ⇓ .

Dominant Negative ATF-2 Affects the Composition and Activity of the AP-1 Complex.

To investigate whether overexpression of the dnATF-2 affects the composition and activity of AP-1 in spindle cells, we first carried out Western blot analysis using specific antisera against Jun and Fos subfamily members. As presented in Fig. 3 ⇓ , the amount of JunB, JunD, Fra-1, Fra-2, and c-Fos was not affected by dnATF-2 overexpression. The expression levels of total and p-c-Jun, however, were decreased in dnATF-2–transfected A5 (Fig. 3A) ⇓ and CarB cells (Fig. 3B) ⇓ . This finding suggests that overexpression of dnATF-2 in A5 and CarB cells specifically inhibits the expression of c-Jun through its inhibitory effects on the c-jun promoter (29) .

Electromobility shift assays demonstrated that the decrease in c-Jun expression in dnATF-2–transfected A5 and CarB cells (Fig. 3A and B) ⇓ was accompanied by a strong reduction in AP-1 binding activity on the Jun/Fos binding site of the collagenase promoter (collTRE; Fig. 4A and B ⇓ ). To confirm the specificity of the AP-1/DNA complex obtained in the electromobility shift assays, we carried out competition experiments using cold collTRE in excess, which led to the disappearance of the AP-1/DNA complex. On the contrary, in competition experiments using the nonspecific cold H-ras-p53 oligonucleotide, we did not observe any decrease in the AP-1/DNA complex. Supershift assays with specific antisera against c-Jun protein revealed that its participation decreased in the dnATF-2–transfected CarB cells (Fig. 4B) ⇓ .

Consistent with the above-mentioned finding was the fact that a luciferase reporter construct driven by five copies of the collTRE showed reduced transcriptional activity in the dnATF-2–transfected A5 cells (Fig. 4C) ⇓ , as well as in dnATF-2–transfected CarB cells (Fig. 4D) ⇓ . These results indicate that overexpression of dnATF-2 in A5 and CarB cells inhibits Jun/Fos activity by reducing the expression of c-Jun.

Electromobility shift assays on the distal Jun/ATF-2 and ATF-2/ATF-2 binding site of the c-jun promoter (jun2TRE; ref. 5 ) showed a slight increase in Jun and/or ATF-2 binding activity in dnATF-2–transfected A5 and CarB cells (Fig. 5A and B) ⇓ . Supershift assays with specific antisera against c-Jun and ATF-2 proteins revealed that in the dnATF-2–transfected CarB cells, the participation of the ATF-2 protein was increased, whereas the participation of the c-Jun protein was decreased (Fig. 5B) ⇓ . Using a HA-tagged dnATF-2 stably transfected A5 cell line, we demonstrated with electrophoretic mobility shift assay analysis that dnATF-2 indeed participated in AP-1 binding to jun2TRE (Fig. 5C) ⇓ . Although increased binding was observed, a luciferase reporter construct driven by five copies of the jun2TRE showed reduced transcriptional activity in the dnATF-2–transfected A5 and CarB cells (Fig. 5D and E) ⇓ . These findings suggest that the AP-1 complexes binding to Jun/ATF-2–dependent promoters (such as the c-jun promoter) consist of inactive dnATF-2 molecules, rather than active Jun/ATF-2 dimers, resulting in reduced c-jun promoter activity.

Overexpression of Dominant Negative ATF-2 Altered, In vitro, the Characteristics of the A5 and CarB Transfected Cells.

To examine, in vitro, the effect of dnATF-2 on the biological characteristics of the dnATF-2–transfected spindle cells, we evaluated the growth rate of parental and transfected cells, as well as their ability to grow on soft agar. Our first interesting observation, as depicted in Fig. 6A ⇓ , was that the dnATF-2–transfected A5 and CarB cell lines had lost their mesenchymal/spindle morphology and acquired an epithelial-like phenotype. The cells were enlarged and flattened and had become more rounded and more refractive to light. On the other hand, A5 and CarB cells transfected with the control vector (A5-V and CarB-V cells) demonstrated the same spindle characteristics as the parental cells.

The dnATF-2–transfected cells had a significantly slower growth rate than the parental and control vector-expressing A5 and CarB cells (Fig. 6B–D) ⇓ . Furthermore, parental cells reached confluence at day 4, but they continued their exponential growth. On the contrary, dnATF-2–transfected cells reached confluence at day 6, and growth was arrested. The striking differences between the growth rate of parental and dnATF-2–transfected cells were verified by three different methods of cell growth assessment, including trypan blue (Fig. 6B) ⇓ , MTT (Fig. 6C) ⇓ , and thymidine incorporation (Fig. 6D) ⇓ . In addition, when we overexpressed dnATF-2 in squamous B9 and PDV cells, we noticed a decrease in cellular proliferation, with other phenotypic properties remaining unaffected (data not shown).

As presented in Fig. 7A and B ⇓ and in Table 1 ⇓ , anchorage-independent growth was reduced in the dnATF-2 transfectants compared with the A5 and CarB parental cell lines, producing fewer and smaller colonies. On the contrary, the colony-forming efficiency did not differ between control vector-transfected and A5 and CarB parental cell lines. As a negative control, we used the immortalized mouse skin cell line C50 (39) , which produced no colonies on soft agar.

Overexpression of Dominant Negative ATF-2 Altered, In vivo, the Properties of the A5 and CarB Transfected Cells.

We subsequently assessed the in vivo effects of dnATF-2 by injecting parental control vector-transfected and dnATF-2–transfected cells subcutaneously into BALB/c SCID mice. We examined their tumorigenicity and latency period of tumor onset. We observed that the ratio of the number of positive sites to the total number of injected sites was reduced in mice that received injection with the stable transfectants, compared with mice that received injection with parental or control vector-transfected cells (Table 2) ⇓ . Moreover, the latency period of tumor onset was markedly prolonged in mice injected with the stable transfectants (Table 2) ⇓ .

More specifically, a number of mice were sacrificed according to the tumor onset period (Table 2) ⇓ . Mice that received injection with parental or control vector-transfected cells had developed tumors of >1.5 cm in diameter (Fig. 8A) ⇓ , which were resected for histologic examination. Liver and lung samples were also obtained, although no metastases were observed macroscopically. The histologic examination demonstrated that the subcutaneous tumors developed from CarB or control vector-transfected cells had a spindle, fibroblastoid morphology and invaded the abdominal wall (Fig. 8B, i and ii) ⇓ . Microscopically, no metastases were observed in the liver and lungs. Mice that received injection with dnATF-2–transfected cells had developed tumors with a diameter of 0.2 to 0.3 cm (Fig. 8A) ⇓ . The histologic features of these tumors were altered in comparison with the phenotype of tumors developed from parental or control vector-transfected cells because they had acquired an epithelial-like appearance with a more rounded cellular shape (Fig. 8B, iv) ⇓ . Invasion of the abdominal wall was observed several weeks later, in comparison with mice that received injection with parental or control vector-transfected cells. No macroscopic or microscopic metastases were observed in liver or lung samples. Immunohistochemical analysis of tumors resected from mice that received injection with CarB, control vector-transfected CarB, or dnATF-2–transfected CarB cells showed that all tumors were negative for the epithelial marker keratin (Fig. 8B, iii) ⇓ . On the contrary, all tumors resected from mice that received injection with A5, control vector-transfected A5, or dnATF-2–transfected A5 cells showed that all tumors were positive for the epithelial marker keratin (data not shown). TUNEL assay analysis has shown no difference in the apoptotic index between tumors derived after injection of parental and dnATF-2–transfected cells (data not shown).

Overexpression of Dominant Negative ATF-2 Results in Decreased Expression of ATF-2 Target Genes.

Finally, we examined the expression levels of c-Jun/ATF-2–dependent genes, which are involved in oncogenic transformation (14, 15, 16) . Western blot analysis showed decreased levels of c-Jun, ATF-3, cyclin D1, and cyclin A proteins in tumors from mice that received injection with dnATF-2–transfected A5 and CarB cells (Fig. 9A) ⇓ , compared with control vector-transfected and parental A5 and CarB cells. Analogous results were obtained by Western blotting of dnATF-2– and control vector-transfected A5 and CarB cells (Fig. 9B) ⇓ . According to these results, dnATF-2 most probably suppresses the transformed phenotype of the spindle cells and the fibroblastoid morphology of the tumors derived from them through down-regulation of c-Jun/ATF-2–dependent genes.

DISCUSSION

The aim of our present study was to thoroughly examine the involvement of ATF-2 in mouse skin carcinogenesis. This report is a continuation of our previous work showing elevated levels of the phosphorylated forms of c-Jun, Fra-1, Fra-2, and ATF-2 accompanied by increased AP-1 complex DNA binding and transactivation activity in the metastatic spindle cell lines A5 and CarB (25) . In addition, constitutive AP-1 binding and transactivation have been shown in malignant but not in benign mouse epidermal cells (41) .

In our current investigation, we initially evaluated the expression levels of ATF-2 and c-Jun in chemical-induced tumors of the mouse skin. Significantly higher levels of ATF-2 and c-Jun were demonstrated in spindle cell carcinomas, compared with the papillomas studied (Fig. 1A and B) ⇓ . Furthermore, Western blotting analysis of ATF-2 and c-Jun in representative chemical-induced skin tumors showed increased levels of ATF-2 and c-Jun expression in spindle tumors (Fig. 1C) ⇓ . These findings confirm our previous in vitro results (25) and indicate that, with regard to their ATF-2 and c-Jun status, A5 and CarB spindle cell lines seem to be an in vitro analog of the chemical-induced mouse skin tumors. The potential role of ATF-2 in the progression of mouse skin tumors was assessed by examining, at the molecular and cellular level, the in vitro and in vivo biological properties of A5 and CarB spindle mouse skin cell lines transfected with dnATF-2. This dnATF-2 carried alanine substitutions at Thr⁶⁹ and Thr⁷¹ phosphorylation sites, whereas its transactivation and DNA-binding domains remained intact. The two mutated phosphorylation sites are located in the transactivation domain of ATF-2 but are physically separated from the kinase-binding subdomain (12 , 42) . Thus, dnATF-2 is still able to bind activated kinase molecules, although it cannot be phosphorylated by them.

Overexpression of dnATF-2 in A5 and CarB cells had a negative effect on the phosphorylation of the endogenous ATF-2 by JNK1/2 or p38 MAPK (Fig. 2) ⇓ , implying that the abundant exogenous dnATF-2 molecules compete with wild-type ATF-2 for active MAPKs. Furthermore, we observed that dnATF-2 affected the composition and activity of AP-1 because the levels of c-Jun were decreased in dnATF-2–transfected A5 and CarB cells (Fig. 3) ⇓ . This finding suggests that dnATF-2 inhibits c-Jun expression through a direct effect on its promoter (29) . Decreased c-Jun expression was accompanied by a strong reduction in AP-1 binding and transactivation activity on the Jun/Fos binding site of the collagenase promoter, a well-defined AP-1 target gene (refs. 2 and 43 ; Fig. 4 ⇓ ), as well as by a slight increase in Jun and/or ATF-2 binding activity on the distal Jun/ATF-2 and ATF-2/ATF-2 binding site of the c-jun promoter (jun2TRE) in dnATF-2–transfected cells (Fig. 5) ⇓ . In addition, a luciferase reporter construct driven by five copies of collTRE or jun2TRE showed reduced transcriptional activity in the dnATF-2–transfected A5 and CarB cells. Overexpression of dnATF-2 therefore restrains Jun/Fos transcriptional activity on the collTRE oligonucleotide, as well as the Jun/ATF-2 and ATF-2/ATF-2 transcriptional activity on the jun2TRE oligonucleotide, through reduction of c-Jun levels (29 , 44) . A possible explanation for the mechanism is the following: c-jun is a target gene of ATF-2 (5) and is positively autoregulated by its product (45) . The c-jun promoter contains the jun2TRE consensus, which is preferentially targeted by ATF-2 homodimers or c-Jun/ATF-2 heterodimers (5) . In parental cells, phospho–c-Jun and phospho–ATF-2 bind to the jun2TRE site and activate the transcription of c-jun. In the dnATF-2 transfectants, however, there are only small amounts of phospho–c-Jun and phospho–ATF-2 molecules. The jun2TRE site is therefore targeted mostly by inactive dnATF-2 molecules, as shown by electrophoretic mobility shift assay (Fig. 5B and C) ⇓ , which are able to dimerize and bind to their consensus DNA motifs. The c-jun transcription is therefore suppressed, and through the autoregulating mechanism, even fewer c-Jun molecules are produced (Fig. 5) ⇓ . These results suggest that dnATF-2 could squelch either the c-Jun protein or other Jun or Fos proteins off of the TREs via its leucine zipper domain. Also, it is possible that the dominant negative ATF-2 heterodimerizes with wild-type AP-1 proteins, binds the TRE, and inhibits transactivation because of either the deletion or point mutations. This effect on AP-1 activity most likely influences the function of a variety of AP-1 targets with a role in cell cycle progression (46, 47, 48, 49) and cell–cell and cell–matrix interactions (50, 51, 52) and is probably responsible for the altered biological properties of the stable transfectants.

Our in vitro data provided evidence that the observed biochemical changes were accompanied by altered biological behavior of dnATF-2–transfected cells. In particular, we demonstrated that the stable transfectants had a slower growth rate than the parental cells (Fig. 6B–D) ⇓ and lost their spindle cell morphology and acquired an epithelial-like phenotype (Fig. 6A) ⇓ . Furthermore, the anchorage-independent growth assay, which is a stringent determinant of cell transformation capacity (38) , revealed that the dnATF-2–transfected spindle cell lines acquired a less aggressive phenotype because they formed fewer and smaller colonies (Fig. 7 ⇓ ; Table 1 ⇓ ).

Because epithelial malignantly transformed cells do not always form colonies in soft agar (53) , we further strengthened our in vitro data by testing the tumorigenicity and latency period of tumor onset after injection of parental and transfected cells into BALB/c SCID mice. The ratio of the number of positive sites to the total number of injected sites was lower in mice that received injection with dnATF-2–transfected cells than in the control group (mice injected with the parental or control vector-transfected cells). Furthermore, the tumor onset latency period and the time of abdominal wall invasion were markedly prolonged (Fig. 8A ⇓ ; Table 2 ⇓ ). This finding could be corelated with the slower growth rate exhibited by the dnATF-2 stable transfectants in vitro. TUNEL assay analysis has shown no difference in the apoptotic index between tumors derived after injection of parental and dnATF-2 cells. Interestingly, we also observed altered histologic features in the tumors developed from mice that received injection with stable transfectants. These tumors had acquired an epithelial-like morphology with a more rounded cellular shape, which resembled the phenotype of poorly differentiated epithelial tumors rather than the spindle-fibroblastoid morphology of the control group tumors (Fig. 8B) ⇓ . Immunohistochemical staining for keratin was negative in tumors developed from both CarB parental cells and CarB-derived transfected cells. Keratin is a well known epithelial marker. As shown previously (54) , CarB cells do not express keratin. If tumors developed from dnATF-2–transfected CarB cells expressed keratin, we would suggest that the introduction of dnATF-2 in these cells results in the reversion of the mesenchymal, spindle phenotype to an epithelial morphology. Nevertheless, the fact that this was not the case with our dnATF-2–transfected CarB cells does not exclude the possibility that ATF-2 overexpression and enhanced levels of phosphorylated ATF-2 actually have a role in epithelial to mesenchymal transition because A5 parental cells are characterized by mesenchymal properties, although they express keratin (54) . The only known apparent discrepancy between the genetic profiles of A5 and CarB cells (26 , 54) is the imbalance between the H-ras mutant/wild-type allele ratio because CarB cells do not harbor any wild-type H-ras alleles. Oncogenic ras proteins regulate AP-1 activity through the JNK and ERK pathways (55 , 56) . Our present data, therefore, suggest that a factor(s) in the CarB cells cooperates with the constantly activated ras/MAPK/ATF-2 pathway to disrupt the keratin network and completely suppresses epithelial differentiation. A recent study has actually demonstrated that mutant H-ras must cooperate with the dominant activated transcription factor Smad2 to induce epithelial to mesenchymal transition, characterized by replacement of the epithelial intermediate filament cytokeratins by the mesenchymal filaments vimentin and α-smooth muscle actin (57) . Nevertheless, our findings underscore the significance of ATF-2 phosphorylation and overexpression as a key regulatory event in mouse skin tumor growth and progression.

The altered biological behavior of dnATF-2–transfected cells could be related to the inhibition of c-Jun expression observed in those cells because previous studies have shown that stable expression of a c-jun deletion mutant in two mouse epidermal cell lines blocks tumor formation in nude mice (58) . It could also be the consequence of down-regulation of a series of AP-1 targets implicated in cell cycle control and progression. We therefore examined the expression levels of the c-Jun/ATF-2–dependent genes cyclin D1, cyclin A, and ATF-3 (14, 15, 16) . Our analysis showed significantly decreased levels of cyclin D1, cyclin A, and ATF-3 proteins in tumors derived after injection of stable clones in mice, as well as in stably transfected A5 and CarB cells, supporting the role of ATF-2 in the regulation of these genes (Fig. 9) ⇓ . Cyclins D1 and A are pivotal cell cycle regulators. They function by binding and activating the cyclin-dependent kinases (CDKs) CDK4/6 and CDK2, respectively, which in turn phosphorylate the pRb protein, thus facilitating a series of events that lead to the progression through G₁ and S phase (59) . Both cyclins D1 and A are overexpressed and associated with adverse prognosis in a variety of human tumors (60, 61, 62) . The role of ATF-3 in carcinogenesis has not been yet clarified. The ATF-3 transcription factor is an immediate early response protein to various stress signals (63) , which may repress transcription by forming homodimers or activate transcription by forming heterodimers with jun proteins, depending on the cellular context (64) . Our study is one of the first reports suggesting a role of ATF-3 in the process of carcinogenesis and is in agreement with the data reported by Ishiguro et al. (65) , implicating ATF-3 in metastasis through modulation of cell adhesion and invasion.

In conclusion, our data support for the first time a nodal role of ATF-2 in the progression of carcinogenesis in the mouse skin and provide evidence that this effect is mediated through regulation of AP-1 family members and their target genes involved in cell cycle promotion. Based on these findings, ATF-2 could be considered, in the future, as a putative molecular target for antineoplastic therapy. Our approach, which resulted in the suppression of aggressive mouse skin malignant phenotypes, may be the first indication toward this direction.