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Molecular Cancer Research 3:237-249 (2005)
© 2005 American Association for Cancer Research

Signaling and Regulation

Fos-Related Antigen 1 Modulates Malignant Features of Glioma Cells

Waldemar Debinski and Denise M. Gibo

Brain Tumor Center of Excellence, Comprehensive Cancer Center, Departments of Neurosurgery, Radiation Oncology, and Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina

Requests for reprints: Waldemar Debinski, Brain Tumor Center of Excellence, Comprehensive Cancer Center, Departments of Neurosurgery, Radiation Oncology, and Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. Phone: 336-716-9712; Fax: 336-713-7639. E-mail: debinski{at}wfubmc.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Malignant gliomas, and high-grade gliomas (HGG) in particular, are nonmetastasizing but locally infiltrating, hypervascularized brain tumors of poor prognosis. We found previously that a c-fos-inducible vascular endothelial growth factor D is ubiquitously up-regulated in HGG grade IV, glioblastoma multiforme, and that glioblastoma multiforme overexpress Fos-related antigen 1 (Fra-1) rather than the c-Fos. We have thus become interested in the role Fra-1 may play in malignant glioma progression/maintenance, because Fra-1 has the capacity to modulate transcription of a variety of target genes. In this work, we have analyzed the biological effects of ectopic Fra-1 expression or Fra-1 knockdown in malignant glioma cells. Ectopic Fra-1 induced prominent phenotypic changes in all three malignant glioma cell lines examined: H4, U-87 MG, and A-172 MG. These changes were reflected in cells becoming more elongated with larger number of cellular processes. Furthermore, Fra-1 transgene caused H4 cells, which do not form tumor xenografts, to regain tumorigenic capacity. The genotype of these cells changed too, because 50 of 1,056 genes examined became either up-regulated or down-regulated. Conversely, Fra-1 knockdown altered prominently the morphology, anchorage-independent growth, tumorigenic potential, and Fra-1 effector expression, such as vascular endothelial growth factor D, in HGG cells. For example, cells transfected with antisense fra-1 showed shorter cellular processes than the control cells that did not grow in agar, and their tumorigenic potential was significantly diminished. Thus, Fra-1 may likely play an important role in the maintenance/progression of malignant gliomas and potentially represents a new target for therapeutic interventions.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Malignant gliomas are brain tumors of astroglial cell origin and a variety of their forms are highly progressive, morphologically heterogeneous malignancies that are resistant to current therapy (1). High-grade gliomas (HGG) are a prevalent type of rapidly progressing brain tumors of dismal prognosis (2). In search for glioblastoma multiforme (GBM; HGG grade IV)–specific markers/therapeutics targets, we uncovered previously a receptor for interleukin (IL)-13, IL-13R{alpha}2, whose gene is located on chromosome X (3-6). IL-13R{alpha}2 is overexpressed in a vast majority of patients with HGG and is a molecularly defined target for therapeutic deliveries to HGG (7-9). We subsequently documented that a X-linked, mammalian member of vascular endothelial growth factor (VEGF) family, VEGF-D (10, 11), is a ubiquitous angiogenic factor in GBM (12). Notably, GBM is characterized by a well-developed and abnormal neovascularization and is in fact one of the most highly vascularized human tumors (1, 13, 14). Of interest, VEGF-D had been isolated originally as a c-fos oncogene, a member of the activating protein-1 (AP-1) transcription factor family, inducible mitogen (11). In expectation that AP-1 factors may take part in up-regulating VEGF-D in GBM, we uncovered that a Fos-related antigen 1 (Fra-1) rather than c-Fos is involved in up-regulation of VEGF-D in the diseased cells (12).

The role of AP-1 factors, especially that of c-Fos and c-Jun, in oncogenesis has been studied extensively (e.g., refs. 15-17). However, more recent hypotheses relate to a possible central role AP-1 may play in tumor invasiveness and progression and thus in determining dismal prognosis of many cancers (18, 19). The finding of Fra-1 being highly up-regulated in GBM directed our attention to the role of AP-1 transcription factors in up-regulating gene expression in GBM. For example, other proteins, whose genes possess AP-1 binding sites and have been widely implicated in the invasiveness and neovascularization of GBM, such as urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, gelatinase B (matrix metalloproteinase-9), tissue inhibitor of metalloproteinase-1, and fibroblast growth factor-2 (FGF-2), are prominently up-regulated in GBM (e.g., refs. 20-27). This is consistent with an unopposed, activated AP-1 axis in GBM and an up-regulation of gene expression in a transcription factor–specific manner (18, 19). The presence of up-regulated Fra-1, which is also an AP-1 controlled factor, in several aggressive cancers, including HGG, further supports a role for AP-1 in tumor progression/maintenance (27-32).

We therefore proposed a hypothetical model in which Fra-1, being under the positive control of AP-1 activity, is continuously up-regulated in transformed cells (12). This sustained up-regulation may come from two complementary pathways: (a) abundance of epidermal growth factor (EGF), leukemia inhibitory factor, oncostatin M, FGF-2, and others in GBM, which produce AP-1 stimulatory signals, and (b) accumulation of the Fra-1 protein in cancer cells. These lead to a selection of Fra-1 as a mediator of mechanisms important for the oncogenic characteristics of glioma cells, because c-Fos was found not to be stably activated in glioma cells (12), and Fra-1 is known to overtake c-Fos-dependent functions (33). Other cancers may use the phenomenon of Fra-1 substituting for c-Fos (31), because Fra-1 is up-regulated, for example, in a vast majority of colorectal cancer cases (30). Interestingly, VEGF-D is also overexpressed in >70% of colorectal cancer, representing an independent prognostic marker of survival (34). Moreover, VEGF-D that seems to be under Fra-1 control is a prognostic factor in other aggressive cancers, such as breast and ovary (35, 36).

Recent findings strongly support the biological importance of Fra-1. The fra-1(–/–) genotype in a mouse knockout model was lethal due to the lack of proper vascularization of the placenta (37) and we suspect that VEGF-D is one of the missing angiogenic factors in fra-1-deprived mice. This notion is based on our data demonstrating that fra-1 transgene induced VEGF-D expression in otherwise Fra-1/VEGF-D-negative cancer cells (12). It is noteworthy that an insufficient expression of urokinase-type plasminogen activator and matrix metalloproteinase-9 has been suggested to be responsible at least in part for the lethal deficiency in vascularization of the placenta in fra-1(–/–) mice fetuses (37).

However, Fra-1 as well as Fra-2 lack the AP-1 transactivation potential that distinguishes these factors from other AP-1 family members (32). All members of the Fos family contain highly conserved basic leucine zipper domains, which enable heterodimerization with Jun proteins, the complexes of which bind to 12-O-tetradecanoylphorbol-13-acetate–responsive elements (32, 38). The NH2- and COOH-terminal domains of the Fos proteins are much less homologous to the NH2- and COOH-terminal domains of other AP-1 factors, unlike their basic leucine zipper domains. The COOH-terminal regions of c-Fos and FosB harbor transactivation function, which are absent in Fra-1 and Fra-2 proteins. There is also another transactivation region in the NH2-terminal domains of c-Fos and FosB proteins, which similar to the COOH-terminal domains is completely absent in Fra-1 and Fra-2 proteins (32). Nevertheless, Fra-1 exerts potent biological effects on its own (39) and they were mapped to the NH2-terminal end of the protein (40, 41). The transactivation potential of Fra-1 is, however, low as revealed in transfection assays in different types of cells (40, 42). It has been recently documented that under these low transactivation potential conditions Fra-1 nevertheless controlled cell motility in at least three different model systems (42-44).

A limited number of studies on the pathobiological importance of Fra-1 have been conducted thus far on epithelial cells or epithelioid carcinomas. We documented that Fra-1 is overexpressed in brain tumors of astroglial origin, malignant gliomas (12). Thus, in the present work, we attempted to explore whether Fra-1 may be important for malignant behavior of glioma cells by examining phenotypic, growth, and tumorigenic characteristics of malignant glioma cells with ectopic Fra-1 or Fra-1 silenced.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Fra-1 Expression Increases Prominently during AP-1 Stimulation of GBM Cells
We have continued testing our hypothesis of AP-1 stimulatory factors up-regulating Fra-1 in astrocytoma cells (12). Thus, we treated SNB-19 and A-172 MG GBM cells with GBM-relevant stimulatory factors, such as EGF and FGF-2, as well as IL-4 as a negative control, which is primarily a STAT-6 inducer (45). We examined the presence of immunoreactive Fra-1 protein using Western blots on cell lysates. Twenty-four hours of stimulation with either EGF or FGF-2 resulted in a large, more than a 10-fold, increase in an immunoreactive Fra-1 in both SNB-19 and A-172 MG cells (Fig. 1A), suggestive of Fra-1 being either overly produced and/or accumulated in those cells. This was not observed using IL-4.



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  		FIGURE 1. A. Immunoblots for Fra-1 in SNB-19 and A-172 MG GBM cells that were mock treated (Control) or treated with EGF, FGF-2, or IL-4 for 24 hours. B. Immunoblot for c-Fos in SNB-19 cells treated with EGF, FGF-2, or IL-4 for 24 hours and in A-431 epidermoid carcinoma cells treated with EGF for 24 hours. C. Northern blot of fra-1 mRNA in SNB-19 GBM cells treated with 20 ng/mL EGF for 1, 4, 6, and 24 hours. D. Analysis of fra-1 mRNA levels in SNB-19 GBM cells. Histogram of fra-1 gene expression as determined by Northern blot (C) was normalized for the internal control (actin).

 
In contrast to a large increase in Fra-1 in GBM cells in response to EGF and FGF-2, the levels of c-Fos remained below the detection limit independently of the stimulant (EGF or FGF-2) and GBM cell line analyzed. c-Fos immunoreactivity was not seen in SNB-19 cells stimulated with either EGF or FGF-2 (Fig. 1B) and a similar result was obtained in A-172 MG cells (data not shown); only nonspecific immunoreactive bands were observed in these GBM cells. However, the A-431 epidermoid adenocarcinoma cells treated with EGF for 24 hours showed specific c-Fos immunoreactive band of ~53 kDa, as expected (Fig. 1B). Of interest, the expression of Fra-1 can be similarly up-regulated in A-431 cells using either EGF or FGF-2 (data not shown). Thus, c-Fos is silent during prolonged AP-1 stimulation of GBM cells, similar to what was observed in some other malignancies (32), which is in sharp contrast to the ability of Fra-1 to accumulate in response to growth factor/mitogen stimulation.

Fra-1 Accumulation in AP-1-Activated Cells Is Discordant with the Levels of Its Message
We next monitored the gene expression profile for fra-1 in GBM cells treated with EGF. The analysis of SNB-19 cells stimulated with the growth factor (two separate experiments) revealed that fra-1 mRNA was higher in cells treated with EGF compared with nontreated cells at 1, 4, 6, and 24 hours of stimulation [1.1-, 1.3-, 1.1-, 1.9-, and 1.5-fold increase of normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message, respectively] as determined by Northern blot (Fig. 1C and D). The magnitude of change in mRNA was, however, much lower than that seen with the gene product.

Ectopic fra-1 Causes Prominent Phenotypic Changes in H4 Glioma Cells
In further search for a direct link between Fra-1 and the behavior of glioma cells, we have cloned a human fra-1 gene from the G48 GBM cells into a pcDNA3.1 eukaryotic cell expression vector. H4 malignant glioma cells were stably transfected with the construct, because we have found the levels of both Fra-1 and VEGF-D low in this glioma cell line, and H4 cells are completely nontumorigenic when injected into nude mice. First, we examined pooled transfectants of H4 cells for their morphology. The mock-transfected cells retained the features of parental cells (Fig. 2A). However, we found that H4[fra-1](+) cells became prominently elongated when compared with their controls and these cells formed readily noticeable multiple processes (Fig. 2B). The phalloidin staining further emphasized the profound changes in the transfected cells architecture (Fig. 2B, inset) when compared with mock-treated cells (Fig. 2A, inset).



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  		FIGURE 2. General morphology (crystal violet) of mock-transfected or fra-1-transfected H4 cells (A and B, respectively) and their actin/nuclear (phalloidin/DAPI) staining (A and B, insets).

 
Immunoreactive Fra-1 Is Readily Detected in H4 Cells Transfected with fra-1 Transgene
We found that H4 cells with fra-1 transgene express the protein at higher levels than the mock-transfected and parental counterparts, with clone 10 being a higher expressor of Fra-1 than clone 4 (Fig. 3A). Fra-1 was also readily detected in the pooled transfectant H4[fra-1](+) cells and the levels of the immunoreactive protein corresponded to the ones seen in the isolated clones: higher than in clone 4 and lower than in clone 10. As seen in Fig. 3B, the message for endogenous fra-1 was detected at low levels using Northern blot analysis, whereas H4[fra-1](+) clones 4 and 10 had elevated levels of the message for fra-1 transgene; the size of the transgene transcript was slightly lower from the endogenous one (Fig. 3B). Microscopic examination revealed readily noticeable differences between the parental, mock-transfected and ectopic Fra-1-expressing H4 cells, and these differences were similar to the ones shown in Fig. 2.



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  		FIGURE 3. A. Western blot immunoreactivity for Fra-1 in H4 cells. Parental, mock-transfected clones 4 and 10 of H4[fra-1](+), pooled mock-transfected, and pooled H4[fra-1](+) cells are shown. The immunoreactivity was developed using chemiluminescence. B. Gene expression analysis of fra-1 in H4 cells. Northern blot of fra-1 message was done in parental, mock-transfected, and clones 4 and 10 of H4[fra-1](+) cells. Human GAPDH was used as reference. C. Actin/nuclear staining (phalloidin/DAPI) of H4 cells: I, parental; II, mock-transfected; III, H4[fra-1](+)clone 4; and IV, H4[fra-1](+)clone 10.

 
H4[fra-1](+) Cells Become Tumorigenic
H4 cells do not form tumors in nude mice at all. It was surprising to observe that H4[fra-1](+) cells, unlike parental or mock-transfected H4 cells, produced tumors at an 80% of tumor take in immunocompromised mice. We harvested and preserved some of these tumors and stained for Fra-1 (Fig. 4). We found Fra-1 to be expressed in H4[fra-1](+) tumors, indicating that the transgene was still present in situ. Of interest, Fra-1 localized to both nuclei (Fig. 4A) and cytoplasm (Fig. 4B) of tumor cells.



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  		FIGURE 4. A. Immunostaining for Fra-1 in paraffin-embedded sections of H4[fra-1](+) tumor. Arrows (yellow) point to the cells nuclei stained for Fra-1. B. Same as A, but the arrows (white) point to the cell cytoplasm stained for Fra-1.

 
Transcriptionally Active AP-1 Complexes in Malignant Gliomas
There are few data on the involvement of specific AP-1 factors in forming transcriptionally active complexes in brain tumors. The results of DNA binding by productive AP-1 complexes formed in SNB-19 GBM cells and H4 cells are shown in Fig. 5A. We found that Fra-1 and Fra-2 as well as JunB and JunD, but not c-Fos of FosB, participated in the most effective binding of AP-1 complexes to the DNA in SNB-19 and H4 cells under normal culture conditions. It is noteworthy that although the immunoreactive Fra-1 is relatively low in H4 cells it could evidently produce productive complexes to bind AP-1 sites (Fig. 5A). This is similar to what we have observed with Fra-2 whose immunoreactivity is relatively low (data not shown) but whose participation in forming the AP-1 complexes is readily detected (Fig. 5). Next, we measured AP-1 activity in H4 cells from the fra-1 transfection experiments. The most striking difference was seen in H4[fra-1](+) clone 10 when compared with clone 4 and control cells (Fig. 5B). These cells exhibited a prominent, almost a 2-fold, increase in Fra-1 participation in forming active DNA binding complexes and this was not seen with Fra-2. Furthermore, there was an increase in the c-Jun and JunB involvement in the binding to AP-1 sites.



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  		FIGURE 5. A. AP-1 activity profiling in SNB-19 and H4 cells under baseline conditions. Nuclear extracts were assayed for the c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD activity using the TransAm AP-1 family kit. WI-38 is the assay's positive control. B. AP-1 activity profiling in H4 cells transfected with fra-1 transgene. Nuclear extracts were assayed for the c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD activity as in A.

 
The Genotype of H4 Cells Changes with the Introduction of Ectopic Fra-1
Prominent phenotypic and malignant behavior [i.e., tumorigenicity changes occurring in H4[fra-1](+) cells] prompted us to seek at least preliminarily for the cellular mediators/targets of the Fra-1 transgene actions. Thus, we did a targeted cDNA microarray analysis (1,056 genes) focusing on a subset of genes related to the cytokines, their receptors, and oncogenes/tumor suppressor genes (Table 1). Our experiments revealed that 18 different genes were overexpressed in H4[fra-1](+) clones 4 and 10 when compared with their controls (Table 1). The most significant changes took place for insulin-like growth factor binding proteins 3 and 6, JunB, RBBP4/RbAp48, PLK, TNFRSF1A, and GRO{gamma}. Noteworthy are increases in VEGF, furin, and MAPK3. Expression of several genes diminished as well. Among them were N-cadherin, granulocyte macrophage colony-stimulating factor, and insulin-like growth factor binding protein 1.


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  		Table 1. Up-Regulated Genes in the H4[fra-1](+) Cells Compared with Parental Cells

 
Transient Silencing of Ectopic Fra-1 Reverses Morphologic Changes in H4[fra-1](+) Cells
Ectopic Fra-1 changed the phenotype of H4 cells (Figs. 2 and 3). We transiently silenced fra-1 in H4[fra-1](+) cells clone 10 using small interfering RNA (siRNA) approach. We observed that fra-1 siRNA, but not nonsense nucleotides, produced >75% decrease in immunoreactive Fra-1 (Fig. 6A). Under these conditions, a substantial reversal of the morphologic features associated with ectopic Fra-1 in H4 cells (i.e., their ability to form long processes and assuming more elongated cell shape) was evident (Fig. 6B).



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  		FIGURE 6. A. Western blot immunoreactivity for Fra-1 in H4[fra-1](+) cells in which silencing of fra-1 was achieved with siRNA. Mock-, nonsense-, and fra-1 siRNA-transfected H4[fra-1](+) cells clone 10 are shown. B. Phase-contrast microscopy of H4[fra-1](+) cells in which fra-1 was silenced using siRNA.

 
fra-1 Transgenes Alter Immunoreactive Fra-1, Morphology, Anchorage-Independent Growth, and Tumorigenicity in U-87 MG GBM Cells
Ectopic Fra-1 caused glioma cells to change morphology and genotype and to produce tumors in nude mice in one malignant glioma cell line (Figs. 2-4). Next, we asked whether Fra-1 is linked to the tumorigenic potential of malignant glioma cells in which Fra-1 is elevated and which forms tumors in nude mice. We thus transfected the U-87 MG GBM cells with fra-1 in both sense (+) and antisense (–) orientation. Immunoreactive Fra-1 in Western blot decreased to almost undetectable levels in U-87[fra-1](–) cells or increased in U-87[fra-1](+) cells (Fig. 7A). Furthermore, the message for fra-1(–) was detected in the U-87 MG cells transfected with an antisense fra-1 by Northern blot, but it was absent in the control cells (Fig. 7B). The staining revealed profound morphologic differences between the parental, mock-transfected and ectopic Fra-1-expressing U-87 cells. The U-87[fra-1](–) clones became larger, more rounded, and with fewer and/or shorter process than the controls (Fig. 7C). The opposite was seen in U-87[fra-1](+) clones, because the cells became more elongated and started to form longer processes that spanned even more than one magnifying field (Fig. 7C).



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  		FIGURE 7. A. Immunoblots for Fra-1 in U-87 MG cells. The mock-transfected, antisense, and sense fra-1 gene transfected clones are shown. B. Gene expression analysis of an antisense fra-1 in U-87 MG cells. Northern blot of fra-1 antisense message was done in both mock-transfected and U-87[fra-1](–) clones. Human GAPDH was used as reference. C. General morphology/architecture of the U-87 MG cells: I, parental; II, mock-transfected; III, U-87[fra-1](+); IV, U-87[fra-1](–) cells as revealed by phalloidin/DAPI staining.

 
We next determined whether supplanting or silencing fra-1 in the U-87 MG cells had any effect on their anchorage-independent growth. We noticed that the U-87 MG cells formed colonies in soft agar rather poorly and the mock-transfected cells exhibited a similar or slightly lower potential to expand in agar (Fig. 8A). However, the U-87[fra-1](–) cells lost an ability to grow in agar completely, whereas the U-87[fra-1](+) cells started to form multiple colonies much more so than the control cells (Fig. 8A). We also monitored the tumorigenicity of U-87[fra-1](+) and U-87[fra-1](–) cells implanted into nu/nu mice. We found that the U-87[fra-1](–) tumors were on average one tenth of the size of tumors that grew from the parental U-87 MG cells, whereas the mock-transfected cells formed tumors efficiently although somewhat smaller than that of parental cells by day 30 of the experiment (Fig. 8B). By day 22, there were 80% U-87[fra-1](–) tumors formed (8 of 10), with an average size 20 mm3, whereas the size of U-87[fra-1](+) tumors (10 of 10) was 135 mm3. On the other hand, the U-87[fra-1](+) cells produced tumors as efficiently as the U-87, if not better (Fig. 8B).



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  		FIGURE 8. A. Anchorage-independent growth of U-87 MG cells transfected with fra-1 in sense and antisense orientation. B. Tumor-forming abilities of the U-87 MG cells transfected with fra-1 in sense and antisense orientation in nu/nu mice. Vertical bars correspond to the SE, which can be smaller than the data point marks. The number of animals was 10 per group. C. VEGF-D staining of the U-87 MG tumors growing in nude mice.

 
We harvested the U-87, U-87[fra-1](–), and U-87[fra-1](+) tumors when grown to larger sizes and the respective sections were stained for VEGF-D (12). The U-87 MG tumors were highly vascularized with the cytosolic VEGF-D readily detected in astrocytoma cell bodies as well as cell processes (Fig. 8C). The U-87[fra-1](–) tumors were, however, poorly vascular and the immunoreactive VEGF-D was close to the detection limit of the assay in these tumors (Fig. 8C). Furthermore, the U-87[fra-1](+) tumors showed gross morphology similar to the control tumors and VEGF-D was also readily detected (data not shown). However, VEGF-D in the U-87[fra-1](+) tumors was associated more with the glioma cell processes rather than cell bodies seen in the parental U-87 MG tumors (Fig. 8C). The shift in the pattern of VEGF-D localization dependent on the Fra-1 status in U-87 MG tumors was unexpected and is most likely related to the changes in morphology of these cells (i.e., cell elongation and formation of long cellular processes; Fig. 7C).

fra-1 Transgenes Alter Immunoreactive Fra-1, Morphology, and Anchorage-Independent Growth in A-172 MG Cells
To examine whether other HGG cells of different characteristics than the U-87 MG cells would be similarly affected by the varying levels of intracellular Fra-1, we examined the A-172 MG GBM cells. The A-172 MG cells exhibit lower levels of Fra-1 under baseline conditions than the U-87 MG cells but considerably higher than that in H4 cells. Fra-1 did not change significantly in mock-transfected cells when compared with parental cells but decreased to almost undetectable levels in A-172[fra-1](–) cells and increased prominently in A-172[fra-1](+) cells (Fig. 9A). Furthermore, the morphologic differences between parental, mock-transfected and ectopic Fra-1-expressing A-172 MG cells could be characterized as very similar to that observed with the two other glioma cells studied (Fig. 9B). We also stained the A-172 cells for VEGF-D and the A-172[fra-1](–) cells showed a visible drop in the levels of VEGF-D immunoreactivity when compared with parental and mock-transfected cells (data not shown). However, the immunoreactive VEGF-D in A-172[fra-1](+) cells was higher than in all other A-172 cells examined.



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  		FIGURE 9. A. Western blot immunoreactivity for Fra-1 in A-172 MG cells. The immunoreactivity was developed using chemiluminescence. B. Phalloidin/DAPI staining of the U-87 MG cells transfected with fra-1 in sense and antisense orientation. C. Anchorage-independent growth of the A-172 MG cells transfected with fra-1 in sense and antisense orientation. Ten random low-power fields in duplicate with minimum of 10 cells per colony were counted.

 
We next analyzed anchorage-independent growth of the A-172 MG cells. These cells, surprisingly, had little capacity to form colonies in agar. The mock-transfected cells were not different, with the exception that a few colonies were in fact formed (Fig. 9D). The A-172[fra-1](–) cells did not grow well in agar either (Fig. 9). However, the A-172[fra-1](+) cells showed a completely different picture in that they formed multiple colonies in agar in sharp contrast to all other A-172 cells used in this assay (Fig. 9).


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this work, we have documented an ability of Fra-1, an AP-1 transcription factor that is deprived of transactivating potential, to modulate malignant characteristics of brain tumor cells originating from malignant gliomas. Firstly, Fra-1 is frequently up-regulated as well as accumulates stably to even higher levels in response to an AP-1 activation in malignant glioma cells. Secondly, the introduction of ectopic Fra-1 causes prominent phenotypic changes and alters the malignant characteristics of glioma cells independently of the background levels of this transcription factor. This phenomenon takes place in all three malignant glioma cell lines studied: H4, U-87 MG, and A-172 MG. Thirdly, Fra-1 transgene is sufficient to confer tumorigenicity in otherwise nontumorigenic cells expressing low levels of endogenous Fra-1, H4 cells. The ectopic Fra-1 also induces prominent changes to the genotype of H4 cells. Fourthly, silencing fra-1 by its antisense transgene alters the morphology, anchorage-independent growth, and tumor formation in vivo of glioma cells. The alterations seen with the antisense fra-1 were opposite to the ones associated with the ectopic Fra-1. In general, a characteristic pattern of biological effects occurs in response to manipulation with Fra-1 in malignant glioma cell lines.

Fra-1 is either up-regulated in HGG cells or its levels are increased to a large extent by the treatment with growth factors and/or mitogens, AP-1 activators, such as EGF, or FGF-2. Our results show for the first time an ability of glioma cells to raise dramatically the levels of immunoreactive Fra-1 in response to AP-1 stimulatory factors. Considering the observed discordance between the mRNA and the protein levels in glioma cells treated with AP-1 activators, we suggest that the stability of the Fra-1 protein is likely enhanced in HGG cells. Previously, it was found that the COOH-terminal deletions of Fra-1 increased prominently both its accumulation and its biological activity. This was documented in a mouse breast carcinoma model when the mRNA for fra-1 changed disproportionately less than the protein levels (40). Hypothetically, a higher stability of Fra-1 in glioma cells may be pertinent to one of the possible mechanisms for Fra-1 to exert prominent biological actions in these cancer cells.

Malignant glioma cells exhibiting low basal levels of Fra-1, H4 cells, responded with prominent phenotypic changes to the ectopic transcription factor. Of interest, very similar, if not identical, phenotypic changes have been reported in low-grade mouse mammary adenocarcinoma CSML0 cells supplanted with Fra-1 (40, 43). Whether this is reflective of a general biological function of Fra-1 remains to be seen. However, the fact that two other malignant glioma cell lines, U-87 MG and A-172 MG, showed similar phenotypic changes in response to the fra-1 transgene suggests the existence of Fra-1-dependent mechanism of cellular morphology control. Furthermore, antisense fra-1 strategy resulted in phenotypic changes that are opposite to the ones observed with ectopic Fra-1.

Although Fra-1 lacks the transactivation potential, it has a proven function of regulating expression of a subset of genes, some already known (e.g., ref. 43). Interestingly, the biological function of Fra-1 is confined to its NH2-terminal end, because the removal of amino acids 1 to 94 inactivates Fra-1 (41). Moreover, previous studies have shown that the removal of the entire COOH-terminal domain increased the osteoclastogenic activity of Fra-1 (41). It is therefore plausible that the experimental COOH-terminal deletions in Fra-1 activate the protein due to the enhancement of its stability. Pathobiologically, the COOH terminus of Fra-1 may undergo post-translational modifications and/or interact with other partnering proteins mimicked by the COOH-terminal deletional experiments; AP-1 factors do function through tight protein-protein interaction mechanisms (46, 47).

Fra-1 is very effective in changing the tumorigenic potential of malignant glioma cells. We have found that H4 cells regained an ability to form xenografts in nude mice when transfected with a Fra-1 transgene unlike their controls. To the best of our knowledge, such an effect of Fra-1 has not been reported yet. Furthermore, antisense fra-1 significantly altered the potential of highly tumorigenic cells, the U-87 MG cells, to grow tumors in immunocompromised mice. Thus, Fra-1 plays a prominent role in tumorigenicity of glioma cells in immunocompromised mice.

Ectopic Fra-1 induced multiple changes in the genotype of malignant glioma cells. One of the most intriguing is the fact that junB is being highly up-regulated in H4[fra-1](+) cells (Table 1). This is of interest because the lack of junB has been documented to be the only one to produce identical abnormality to fra-1 knockout in mice and which are different from all other individual AP-1 factor knockout effects (13). Thus, in view of our results, the question arises whether fra-1 may directly regulate junB and whether the cancer-related effects of Fra-1, such as the ones presented in this work, can be mimicked by JunB. These possibilities are currently under investigation. In addition, an up-regulation of furin may be also of pathobiological significance, as this protease has been recently linked to the control of the invasive properties of glioma cells (48).

Recent data are indicative of other cancers, such as the breast, head and neck, thyroid, and skin cancers (28-32), using AP-1 members to regulate specific subsets of genes and self-sustainable loops of activation of the factors, such as Fra-1, which are important in more advanced stages of disease. An unopposed, activated AP-1 axis, including Fra-1 functions, may thus be a hallmark of advanced cancers (12) and Fra-1 may be an attractive target for new rational molecular anticancer therapies.

We have documented a direct involvement of Fra-1 in the modulation of several malignant features of glioma cells. With this new knowledge, it will be of interest to examine in detail the role of Fra-1 in glioma cells migration, motility, and invasiveness. The motility of other cells has been already shown to be under the Fra-1 control and our pilot studies are pointing in the same direction. Another important aspect of Fra-1 biological function to explore is to verify whether there is a direct dependence of JunB on Fra-1 and/or whether the two represent at least in part pathobiologically redundant mechanisms. Moreover, the mechanism of Fra-1 biological activity is not known and the search for its interactive partner(s) in mediating this activity in malignant gliomas is under way.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Lines
GBM cell lines A-172 MG, U-87 MG, SNB-19, and malignant glioma H4 cells were obtained from the American Type Culture Collection (Manassas, VA). The GBM A-172 MG and H4 malignant glioma cells were grown in DMEM with 10% FCS (Life Technologies, Rockville, MD). The U-87 MG cells were grown in Earle's MEM, 10% FCS, 0.1 mmol/L nonessential amino acids, 2 mmol/L glutamine (Life Technologies), and 100 µg/mL sodium pyruvate. G48a human explant cells were grown in RPMI 1640 (Life Technologies) 10% FCS, 100 µg/mL sodium pyruvate, 100 µg/mL L-cysteine (Life Technologies), 20 µg/mL L-proline (Sigma, St. Louis, MO), 1x HT supplement, consisting of 0.1 µmol/L sodium hypoxanthine and 0.016 µmol/L thymidine, 5 units/mL penicillin G, and 5 units/mL streptomycin sulfate (Life Technologies). G48a GBM cells were isolated in our laboratory and their karyotype is as follows: 49~67, X?Y, +X, +add(1)(p11), +2, +add(4)(q35), +add(4)(p11), +5, +5, –6, –7, +8, +8 add(9)(q34), add(9)(q34), +add(9)(q34), +10, +10, +dup(11)(q25p15), +12, +16, +16, –18, –19, add(19)(q13.4), and +mar1[cp2].

Transfection Experiments
Cells were plated in six-well dishes and allowed to grow until 60% to 70% confluent. Then, the cells were rinsed with PBS and the medium was replaced with Opti-MEM (Invitrogen, Carlsbad, CA). DNA (2 µg; fra-1 sense, fra-1 antisense, or pcDNA vector control) was transfected using LipofectAMINE 2000 (Invitrogen). After 24 hours, the Opti-MEM was replaced with appropriate growth medium containing 20% fetal bovine serum. Twenty-four hours later, the cells were split into 100 cm2 Petri dishes and geneticin (800 µg/mL) was added to select clones. First, pooled transfectants were monitored and harvested, and individual clones were isolated (at least 10 per transfected cells) and maintained in appropriate growth medium containing 200 µg/mL geneticin.

siRNA Transfections
Cells were plated in six-well dishes at 5 x 104 cells per well. The cells are allowed to grow until 60% to 70% confluent. The wells were rinsed with PBS and the medium was replaced with Opti-MEM. siRNA (100 pmol/L, Dharmacon, Lafayette, CO) was transfected using LipofectAMINE 2000. Morphology was monitored and protein lysates were collected at 2, 3, and 5 days following transfection. Target sequence for siRNA was 5'-CACCAUGAGUGGCAGUCAG-3' and the control nonsense siRNA was G/C content matched.

Western Blots
Cell lysates were collected from subconfluent cultures. Cells were washed in PBS and lysed in radioimmunoprecipitation assay buffer (PBS, 1% Igepal CA-630, ICN Biomedicals, Inc., Costa Mesa, CA), 0.5% sodium deoxycholate (Fisher Scientific, Fairlawn, NJ), and 0.1% SDS containing mammalian protease inhibitor cocktail (Sigma). Lysates were passed through a 21-gauge needle to shear the DNA. Phenylmethylsulfonyl fluoride (1 mmol/L, Sigma) was added to the lysates and incubated on ice for 30 to 60 minutes. The nonsolubilized debris was pelleted at 10,000 x g for 10 minutes. The supernatant was collected, aliquoted, and stored at –80°C until use. For AP-1 activation experiments, cells were allowed to grow to 70% to 90% confluence. Cells were washed in PBS twice and serum-free medium was added and left for 24 hours. EGF, FGF-2, or IL-4 (20 ng/mL) in PBS/0.1% bovine serum albumin was added and the controls contained PBS/0.1% bovine serum albumin.

Proteins (40 µg) were loaded on 12% SDS-PAGE gel. Proteins were transferred to polyvinylidene difluoride membrane (Pierce, Rockford, IL) and blocked overnight with 5% blotto (5% dry milk, PBS/0.05% Tween 20) at 4°C. Membranes were incubated with primary antibody diluted in 5% blotto for 1 hour at room temperature while shaking. Primary antibodies include Fra-1 (1:100) and c-Fos (1:100) from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ß-actin (1:50,000) antibody (Sigma) was also used as a loading control. Following the three washes of 5 minutes each in PBS/0.05% Tween 20, membranes were incubated in secondary antibody in 5% blotto for 1 hour at room temperature while shaking. Secondary antibody conjugated with horseradish peroxidase (goat anti-mouse IgG or goat anti-rabbit IgG) was diluted at 1:10,000 in 5% blotto. Membranes were washed with PBS for three changes of 5 minutes each. Detection was done using the SuperSignal West Pico chemiluminescent substrate (Pierce). Membranes were exposed to autoradiographic film X-OMAT AR (Eastman Kodak Co., Rochester, NY) for up to 1 hour. Films were scanned at 600 dpi and images were compiled in Paint Shop Pro version 6.0.

Immunostaining
Cells were plated at 104 and grown overnight on glass slides in their respective medium. After 24 hours, slides were washed in PBS and fixed for 2 minutes in cold acetone and washed in PBS twice left to air dry and stored at –80°C until ready to use or assayed right after washes. Primary antibodies include rabbit polyclonal Fra-1 (1:100, Santa Cruz Biotechnology) and VEGF-D (7.5 µg/mL). Slides were then washed in three changes of PBS and blocked for 30 minutes with 10% normal goat serum in PBS at room temperature. Primary antibodies were diluted in 1.5% normal goat serum/PBS and incubated at room temperature for 2 hours (Fra-1 and VEGF-D). Slides were washed in PBS for three changes at room temperature for 5 minutes each. Secondary antibodies, goat anti-rabbit rhodamine (1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or goat anti-mouse Oregon green (1:200, Molecular Probes, Eugene, OR), were diluted in 1.5% normal goat serum/PBS and incubated in the dark at room temperature for 1 hour. Hoechst no. 33258 nuclear counterstain [4',6-diamidino-2-phenylindole (DAPI), 1 ng/mL, Sigma] was added to the secondary antibodies and was used to counterstain the cells. Double labeling was used with the experiments. Slides were washed in three changes of PBS for 5 minutes each and then mounted with Gel-Mount (Biomeda Corp., Foster City, CA). Pictures were taken at x40 magnification.

Phalloidin staining was done as follows: cells were plated on glass slides and allowed to grow for 2 to 3 days. The slides were washed twice in PBS, fixed for 5 minutes with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 for 1 minute. Then, the slides were rinsed in PBS and then stained with 1:200 Alexa Fluor 488 phalloidin (Molecular Probes) at room temperature for 1 hour. The cells were counterstained with DAPI, washed with PBS, and coverslipped for analysis. The crystal violet staining was done on cells plated on either glass slides or six-well dishes and cultured for 2 to 3 days. The cells were washed twice with PBS and fixed for 5 minutes in 10% acetic acid/10% methanol. Then, the cells were stained with 0.4% crystal violet for 5 minutes followed by two rinses in distilled water. Slides were air dried and the coverslips were mounted with Permount. For the crystal violet staining, the pictures were taken at x20 magnification.

Treatment and Isolation of RNA
Cells were plated in 100 mm dishes. After 24 hours of serum starvation, EGF (20 ng/mL) or vehicle alone was added. RNA was isolated using the acid-guanidium isothiocyanate-phenol-chloroform method (49) at 0.5, 1, 4, 6, and 24 hours after treatment. Polyadenylate [poly(A)+] RNA was further isolated using the Oligotex mRNA kit (Qiagen, Inc., Valencia, CA). Total poly(A)+ RNA (2 µg) was electrophoresed on a 1% agarose formaldehyde gel, transferred to 0.45 µm magna charge nylon membrane (MSI, Westborough, MA), and UV cross-linked (Stratagene, La Jolla, CA).

fra-1 Riboprobe Construction
Poly(A)+ RNA was isolated from GBM explant cells, G48a (a GBM cell line isolated in our laboratory), as described above. Following first-strand synthesis with oligo(dT) primers and Superscript (Invitrogen), full-length fra-1 was amplified with pfu Turbo polymerase (Stratagene) and the following primers: forward 5'-TAGCTAGAATTCATGTTCCGAGAGTTCGGG-3' and reverse 5'-ATAAGTGAATTCTCACAAAGCGAGGAGGGT-3'.The resulting fragment was ligated into pcDNA 3.1 vector (Invitrogen) in both forward and antisense orientation. The constructs were sequenced using an ABI 377 automated sequencer (PE, Norwalk, CT). fra-1 antisense and sense were labeled with 32P using T7-Riboprobe System (Promega Corp., Madison, WI).

Northern Blot Analyses
The membrane was prehybridized overnight at 60°C in a solution consisting of 50% formamide, 50 mmol/L sodium phosphate, 5x SSC (0.6 mol/L NaCl/0.6 mol/L sodium citrate), 1 mm EDTA, 2.5x Denhardt's solution, 10 µg/mL sheared salmon sperm DNA, and 1% SDS. Membrane was subsequently hybridized overnight at 60°C in the same solution with the addition of [32P]fra-1 riboprobe. The membrane was washed with 0.1x SSC/0.1% SDS thrice for 30 minutes each at 60°C. The membrane was exposed to autoradiographic film X-OMAT AR and placed at –80°C for up to 3 days. The membrane was subsequently stripped and reprobed with GAPDH as described previously (4, 8).

cDNA Arrays
Atlas Oncogene/Tumor Suppressor and Cytokine Arrays were purchased from R&D Systems and poly(A)+ RNA (1 µg) was labeled with [{alpha}-33P]dATP according to the manufacturer. Membranes were prehybridized overnight at 68°C in ExpressHyb (Clontech Laboratories, Inc. Palo Alto, CA) containing 0.1 mg/mL sheared salmon sperm DNA. Labeled cDNA probe was denatured and added to the prehybridization solution and the membranes were hybridized overnight at 68°C. Membranes were then washed twice in 2x SSC/1% SDS for 20 minutes followed by two washes in 0.1% SSC/0.5% SDS at 68°C. The membranes were exposed to autoradiographic film for up to 10 days at –70°C. Films were scanned on a transparency scanner at 88 x 88 pixels and the densities were acquired using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Duplicate spots were averaged and normalized to the GAPDH values on the same films. The arrays contain cDNA-specific fragments for oncogenes, such as c-fos, junB, and c-myc. Housekeeping genes included ubiquitin, liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and phospholipase. RNA used for the cDNA microarray assays was isolated from subconfluent cultures of GBM cells using the acid-guanidium isothiocyanate-phenol-chloroform method (49). Poly(A)+ RNA was further isolated using the Oligotex mRNA kit. Normal human brain poly(A)+ RNA was purchased from Clontech Laboratories.

Preparation of Nuclear Extracts for AP-1 Activity Measurement in Cultured Cells
Confluent cell layers were washed with ice-cold PBS/phosphate inhibitor buffer. The cells were scraped into PBS/phosphate inhibitor buffer with a cell lifter. The cells were pelleted in 15 mL tubes at 300 x g for 5 minutes at 4°C and the pellet was resuspended in 1 mL ice-cold hypotonic buffer. The cells stayed on ice for 15 minutes. Then, 10% NP40 (0.5% final) was added and cells were vortexed vigorously for 10 seconds. The homogenate were microcentrifuged for 30 seconds at 4°C. The supernatant was saved for other uses. The nuclear pellet was resuspended in Complete lysis buffer and rocked gently on ice for 30 minutes on a shaking platform. The nuclear suspension was then centrifuged for 10 minutes at 14,000 x g at 4°C and the supernatant was saved as a nuclear extract, aliquoted, protein concentration determined, and stored at –80°C until used.

We employed TransAm AP-1 family transcription assay ELISA-based kit (Active Motif, Carlsbad, CA). The assay uses 96-well plates to which oligonucleotide containing a 12-O-tetradecanoylphorbol-13-acetate–responsive element (5'-TGAGTCA-3') is immobilized. AP-1 dimers contained in nuclear extracts bind specifically to this oligonucleotide and are detected by an antibody directed against c-Fos, Fra-1, etc. Secondary antibody is horseradish peroxidase conjugated and the readout of the assay is colorimetric and quantified by spectrophotometry. Briefly, binding buffer (30 µL, according to the manufacturer) was added to each well. Then, cell extract (5 µg) was diluted in 20 µL complete lysis buffer and added to wells. Control nuclear extract (WI-38) was also added to wells; no antibody-containing wells served as a blank. The plates were incubated for 1 hour at room temperature with mild agitation. Then, the wells were washed thrice with washing buffer. After wash, one of the diluted AP-1 primary antibodies was added (100 µL/well), such as Fra-1, c-Jun, etc., to each well being used. The plates were incubated for 1 hour at room temperature and then washed again thrice in washing buffer. Horseradish peroxidase–conjugated antibody (100 µL) was then added to all wells and another 1-hour incubation at room temperature ensued. The plates were then washed four times with washing buffer. Developing solution was added from 2 to 20 minutes at room temperature in the dark to develop color. The stop solution was added when medium became medium blue; the solution then turns yellow. The absorbance was read at 450 nm within 5 minutes.

Transient Silencing of fra-1 with siRNA
We attempted to down-regulate ectopic Fra-1 in H4 cells by transient transfection with silencing RNA duplexes (siRNA). The siRNA sequence that already successfully targeted human fra-1 in cancer cells is 5'-CACCAUGAGUGGCAGUCAG-3' (44). Those sequences were Blast searched against expressed sequence tag libraries to ensure the specificity of the siRNA molecule. Desalted, deprotected, and duplexed synthetic oligonucleotides were made by Dharmacon. Control duplex oligonucleotides were a siRNA nonsense duplex. siRNAs were transfected using the LipofectAMINE 2000 reagent. Transient transfections were carried out on 50% to 60% confluent cells plated 2 days before the experiment in Opti-MEM without antibiotics using the siRNA fra-1 duplex and corresponding controls in duplicates at least. For one well of a six-well plate, 100 pmol siRNA duplex were added. The effect of silencing was assayed 2, 3, and 5 days after transfection. The transfection efficiency was evaluated by Western blotting of Fra-1 and general cell morphology and compared with our stably transfected and their control cell lines.

Soft Agar Assay
Base layers consisting of 0.5% Noble agar (Becton Dickinson, Sparks, MD) in medium were prepared in six-well plates and allowed to harden. Cells were trypsinized, washed, and counted before plating. Cells (1 x 105) were added to prewarmed medium containing 0.35% Noble agar and layered over the base. Plates were allowed to harden at room temperature for several minutes and then placed in the 37°C incubator. Plates were regularly inspected for anchorage-independent growth. Cells were plated in triplicates and the experiment with each cell line was done at least twice.

In vivo Studies
Parental, mock-transfected fra-1(–) clone 33 (U-87 MG) and parental, mock-transfected fra-1(+) clones 4 and 10 (H4) and clone 34 (U-87 MG) glioma cells were cultured and harvested in a log phase of growth for tumor implantation in nu/nu mice. H4 (6 x 106) or U-87 MG (10 x 106) cells were injected s.c. into the flank of 8- to 10-week-old nu/nu mice in a volume of 100 µL. A total of 5 (H4 cells) or 10 (U-87 MG cells) mice were injected per group. Tumors were measured every 2 to 3 days according to a standard formula (3). The experiment with each cell line, H4 and U-87 MG, was done twice.

Karyotyping
The karyotype of G48a cells was done in a blinded fashion by clinical cytogeneticists at the Cancer Genetics Laboratory, Genetics & IVF Institute (Fairfax, VA).


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Becky Slagle-Webb for excellent technical assistance in this project, Marc G. Achen and Steven A. Stacker (Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Australia) for the generous gift of VEGF-D antibody, and Dr. Costas Koumenis for comments on the article.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
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.

Received 1/ 5/05; revised 3/ 8/05; accepted 3/16/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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J. Wykosky, D. M. Gibo, C. Stanton, and W. Debinski
Interleukin-13 Receptor {alpha}2, EphA2, and Fos-Related Antigen 1 as Molecular Denominators of High-Grade Astrocytomas and Specific Targets for Combinatorial Therapy
Clin. Cancer Res., January 1, 2008; 14(1): 199 - 208.
[Abstract] [Full Text] [PDF]

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 Molecular Cancer TherapeuticsHome page
J. Wykosky, D. M. Gibo, and W. Debinski
A novel, potent, and specific ephrinA1-based cytotoxin against EphA2 receptor expressing tumor cells
Mol. Cancer Ther., December 1, 2007; 6(12): 3208 - 3218.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
P. Adiseshaiah, D. J. Lindner, D. V. Kalvakolanu, and S. P. Reddy
FRA-1 Proto-Oncogene Induces Lung Epithelial Cell Invasion and Anchorage-Independent Growth In vitro, but Is Insufficient to Promote Tumor Growth In vivo
Cancer Res., July 1, 2007; 67(13): 6204 - 6211.
[Abstract] [Full Text]