Loss of Net as repressor leads to constitutive increased c-fos transcription in cervical cancer cells

In the present study we have unravelled the mechanism underlying deregulated c-fos expression in cervical carcinoma cells. Tumorigenic cells express relatively low levels of net and high levels of c-fos . Re-introduction of Net, in either a transient or a stable fashion, suppresses c-fos promoter directed transcription and triggers the disappearance of the c-Fos protein from the AP-1 complex. In contrast, non-tumorigenic HPV-positive cells express relatively high levels of net and low levels of c-fos . Within these cells, Net can be detected bound to the endogenous c-fos promoter and decreasing Net levels with siRNA increases c-fos expression. These results support the notion that deregulation of Net, and in turn of c-fos expression, is an important event in the multi-step progression to cervical cancer, the second leading cancer in women worldwide. These data suggest that the loss of Net leads to increased c-fos transcription in malignant HPV-positive cells.


INTRODUCTION
Development of cervical cancer is a multi-step process that is initiated by infection with "high risk" human papillomaviruses (HPVs). The viral oncoproteins E6 and E7 are indispensable for proliferation of cervical carcinoma cells. They cause unlimited growth, which eventually leads to accumulation of cell damage, chromosomal instability, aneuploidy and loss of tumor suppressor genes. However, expression of E6/E7 in primary human keratinocytes merely results in immortalization without further progression to tumorigenicity, showing that further events are important (1). Moreover, HPV-induced cancer can be considered as a recessive genetic trait that can be complemented by somatic cell hybridization with primary human fibroblasts or keratinocytes. A genetically well defined in vitro model is provided by fusion of HPV18-positive HeLa cells with primary human fibroblasts, which results in hybrids that still express HPV18 E6/E7, but are non-tumorigenic in nude mice (2).
Long-term in vitro cultivation generates segregants with morphological features of tumorigenic cells (3).
The transcription factor AP-1, which is composed of heterodimers between Jun and Fos family members plays a crucial role in various cellular processes such as enhanced proliferation, differentiation and neoplastic transformation (4). In the case of HPV-positive cells, there is substantial evidence that AP-1 composition determines their in vivo behaviour in nude mice, their sensitivity against growth-inhibitory cytokines, as well as their ability to express certain chemokines that are necessary to maintain immunological surveillance of persisting HPV infections (5). While in asynchronous growing primary human fibroblasts,

Cell extracts and Western blot analysis
Nuclear extracts for EMSA and Western blot analysis were prepared as described previously (35).  µg of c-Fos antibody (sc-52 X, Santa Cruz Biotechnology) was added and further incubated for 1 h at 4°C. The complexes were resolved with 5.5% non-denaturing polyacrylamide gels (29:1 cross-linking ratio). The gels were dried and exposed to X-ray films (Amersham) or analysed using a Phosphor-Imager Storm 820 and the Image-Quant software (Molecular Dynamics, Amersham).

ChIP assay
The ChIP Assay Kit (#17-295) from Upstate Cell Signaling Solutions (Lake Placid, NY, USA) was used according to the manufacturer's instructions. Subconfluent cells were crosslinked with 1% formaldehyde for 10 min at 37°C, washed and scraped in ice-cold PBS containing proteinase inhibitor cocktail Complete (Roche Diagnostics, Penzberg, Germany).
After lysis of 1 x 10 6 cells, the DNA was sheared into 200-1000 bp fragments by sonification

HPV-positive cell lines
To

Serum inducibility of Fos expression in non-malignant and malignant cells
To determine whether increased mRNA and protein levels in tumorigenic cells remain constitutive after serum depletion, cells were starved for 24 hours one day after seeding.
Expression was monitored by Western blot and RT-PCR analyses ( Fig

The SRE mainly contributes to an increased c-fos promoter activity in tumorigenic cells
To test whether c-fos is differentially regulated at the level of transcriptional initiation, transient transfection assays were performed. Comparing the relative luciferase activity, where the absolute counts for the 444 hybrids were arbitrarily set as 1, the c-fos promoter was found to be 2 to 4-fold more active in malignant than in non-tumorigenic cells (Fig. 3). In another set of experiments, we analyzed the SRE and the CRE, known to be critical in spatial and temporal gene response (42,14). While CRE-controlled reporter plasmids did not reveal any notable differences, SRE-driven luciferase constructs showed a 5-fold higher activity in HeLa cells and an about 7-fold higher activity in tumorigenic segregants when compared with

Expression and phosphorylation levels of the SRE-activator proteins Elk-1 and SRF
In response to stimulation, c-fos SRE is activated by phosphorylation of TCFs such as Elk-1 via the Ras-ERK pathway (44). In addition, c-fos can be also induced via Rho-actin signalling, targeting SRF in a TCF-independent manner (27,28). To investigate the potential contribution of these two signalling routes, we examined the basal phosphorylation levels of both Elk-1 and SRF. Treatment with TNF-α again served as positive control for a temporal defined MAPK activation. Western blot analyses (Fig. 5A) revealed similar basal levels of Elk-1 and SRF phosphorylation in tumorigenic and non-tumorigenic cells, which could be further stimulated by TNF-α. In addition, inhibition of Rho-acting signalling by Latrunculin B (28) did not revealed any effect of c-fos expression in CGL3 cells (Fig. 5B), also excluding this pathway being responsible for enhanced c-fos transcription. Taken together, these data argue against the concept that increased SRE-activity was due to constitutive hyperphosphorylation of Elk-1 or SRF.

Diminished Net expression in tumorigenic cells: Loss of repressor function at the c-fos promoter
Because none of the classical activators were found to be involved in differential c-fos  (Fig. 6A). Moreover, as depicted in Fig. 6B, we also found an inverse correlation of Net and c-fos transcription in other cervical carcinoma cells. Both HPV16-positive SiHa as well as HPV18-positive SW756 cells exhibited low levels of Net, while c-fos transcription was highly up-regulated.
Exceptional for cervical carcinoma cells, c-fos was weakly transcribed in HPV16-positive CaSki cells (8). Notably, examining these cells by RT-PCR and Western blot analysis, a high amount of Net could be discerned (Fig. 6B). Since it has been postulated that Net significantly contributes to the repression of basal c-fos SRE-activity (22), these data may provide the first direct mechanistic link between its diminished expression and increased c-fos transcription in a highly clinical relevant form of human cancer.

In vivo binding of Net to the c-fos SRE: Loss of repressor function in tumorigenic cells
In order to demonstrate that Net is differentially recruited at the c-fos SRE nucleoprotein complex in vivo, chromatin-immunopreciptiation assays (ChIPs) were carried out (Fig. 7).
Formaldehyde cross-linked protein-DNA complexes were immunoprecipitated using two different polyclonal antibodies against Net. Co-precipitated DNA fragments were amplified using specific primers surrounding the area of the c-fos SRE. The specificity of the ChIP reaction was confirmed using a randomly selected DNA sequence of the c-fos upstream region (position -600) and for a coding stretch within the GAPDH gene. Equal amounts of sheared genomic DNA were used as template. SRF was found to be constitutively associated with the SRE in both cell lines. In contrast, Net binding at the c-fos SRE occurred exclusively in 444 cells, but was absent in CGL3. These data suggest that the loss of Net leads to

Exogenous Net suppresses c-fos in tumorigenic cells while Net siRNA induces c-fos transcription in the non-malignant counterparts
To prove a causal relationship between the lack of repressor function at the SRE and increased c-fos transcription, we first examined the effect of ectopically expressed Net in transient transfection assays. As shown for CGL3 cells, Net expression inhibited SRE and cfos promoter activity, but had no effect on the CRE (Fig. 8A). showed distinct net expression levels which inversely correlated with endogenous amount of c-fos mRNA and protein (Fig. 8B). In addition, supershift EMSA demonstrated that the amount of c-Fos associated with AP-1 was reduced (Fig. 8C). To further corroborate the repressor function, non-malignant cells were transfected with siRNA directed against Net. As shown in Fig. 8D, in the same way as endogenous Net mRNA and protein was diminished, cfos transcription increased. Delivery of a non-specific siRNA showed had no effect on both genes. These results provide strong evidence that constitutive expression of c-fos transcription in tumorigenic cells is due to the loss of Net repressor activity.

DISCUSSION
The dimeric transcription factor AP-1 acts as a junction point for many regulatory mechanisms associated with proliferation, apoptosis and tumorigenicity (4). AP-1 becomes activated upon a whole variety of extra/intracellular stimuli including growth factors, cytokines, tumor promoters and oncogenes (46). The decision, however, which target genes finally turns out to be induced, is apparently depending on AP-1 composition (47). For HPVpositive human cells, AP-1 is part of an intracellular surveillance network determining not only the in vivo phenotype but also the sensitivity/response against growth-inhibitory cytokines and chemokines (1,7,8).
Raising the question how c-fos is deregulated in HPV18-positive cervical carcinoma cells, we took advantage of a model system where fusion of malignant and normal cells resulted in somatic hybrids that are non-tumorigenic in nude mice (2). We found that only malignant cells transcribed high levels of c-fos under asynchronized growth conditions, while there was almost no detectable signal when non-tumorigenic cells were examined (Fig. 1).
Although the intracellular availability of c-Fos can be also regulated in a post-translational manner (48), the amount of protein in Western blot closely matched with the steady state level of the corresponding mRNA (Fig. 1). Moreover, since actinomycin D treatment (49) also did not reveal any differences of the mRNA half-life (data not shown), c-fos expression was evidently controlled at the level of initiation of transcription. Although both viral oncoproteins can stimulate the c-fos promoter in rodent cells (50), transcriptional regulation of c-fos in the human system was actually independent of E6/E7 expression. This supports previous data demonstrating that the induction of particular cytokines/chemokines was determined by the in vivo phenotype, rather then by the expression of the viral oncoproteins per se (35,51).
We found that non-tumorigenic hybrids behave similar to normal cycling cells (IMR-90), where both the extent and the temporal range of c-fos expression were tightly controlled (12,13). Such a negative regulatory loop is obviously missing in tumorigenic cells, where cfos remained expressed both under asynchronous growth conditions and during serum starvation (Fig. 2). Remarkably, although not followed up in molecular terms at the time, an earlier study has already provided initial clues for a constitutive re-expression of higher c-fos levels after transition of non-tumorigenic HPV-positive somatic hybrids towards malignancy (52). Accordingly, ectopic expression of c-fos in 444 hybrids provoked rapid tumor formation by shifting the AP-1 composition from Jun/Fra-1 to Jun/c-Fos. Hence, a potential linkage between c-fos deregulation and E6/E7 expression during human tumor progression has been predicted (7).
In fact, inappropriate transcriptional control of c-fos in malignant cells could be further substantiated in transient transfections (Fig. 3). Using either the entire c-fos promoter or individual cis-regulatory elements, luciferase reporter activity was consistently higher in malignant cells than in their non-malignant counterparts. This property was apparently mediated by the SRE, known to be the major target sequence responsive to extracellular signal transduction (53).
Signal transduction is controlled both by the strength and duration of MAPK activation (54). Since increased MAPK activity is normally considered as a hallmark for cancer cells (55), we reasoned that sustained activation of this pathway might account for enhanced SRE activity. However, neither JNK nor p38 MAPK was affected in its basal activity (Fig. 4). Only ERK1/2 showed constitutive phosphorylation in non-tumorigenic hybrids ("444"), arguing for a stronger functionality of the upstream kinases MEK1 and 2 (56). Elevated ERK1/2 activity, however, could not be responsible for constitutive c-fos expression, because treatment with the MEK1/2 inhibitor U0126 (55) abrogates ERK1/2 phosphorylation without any consequences on the steady state level of the c-fos specific mRNA (Fig. 4B). Notably, even though enhanced ERK1/2 activity has been described for many other human malignancies as the result of Ha-ras proto-oncogene activation (57), HeLa cells as well as primary tumor 20 specimens directly obtained from cervical cancer patients apparently lack mutated H-ras allele expression (58).
Although c-fos expression was maintained at an elevated level through a mechanism independent of increased MAPK activity, constitutive SRE-activity could be either due to deficient antagonistic phosphatase function (59,60) or by stimuli-independent phosphorylation by other kinases (61). Alternatively, SRE-activity could be also regulated by a pathway involving the Rho family GTPases (62). These options, however, could be excluded, because both the steady-state and phosphorylation levels of potential target proteins such as Elk1 and SRF (54) were the same (Fig. 5).
In the absence of active MAPK signalling, the TCF family members Net and to a lesser significantly reduced endogenous c-fos transcription (Fig. 8B), whereby c-Fos was diminished from the AP-1 transcription complex (Fig. 8C). However, the amount of ectopically expressed net was apparently not sufficient to completely suppress c-fos in CGL3 cells (Fig. 8B). Hence, we currently cannot answer the question, whether Net is acting as tumor suppressor, since its