HPV16E6-Dependent c-Fos Expression Contributes to AP-1 Complex Formation in SiHa Cells

To date, the major role of HPV16E6 in cancer has been considered to be its ability to inhibit the p53 tumor-suppressor protein, thereby thwarting p53-mediated cytotoxic responses to cellular stress signals. Here, we show that HPV16E6-dependent c-fos oncogenic protein expression contributes to AP-1 complex formation under oxidative stress in SiHa cells (HPV16-positive squamous cell carcinoma of the cervix). In addition, we examined the role of HPV16E6 in TGF-α-induced c-fos expression and found that the c-fos protein expression induced by TGF-α is HPV16E6 dependent. Thus, our results provide the first evidence that HPV16E6 contributes to AP-1 complex formation after both ligand-dependent and independent EGFR activation, suggesting a new therapeutic approach to the treatment of HPV-associated tumors.


Introduction
HPV (human papillomavirus) infection is a hallmark of uterine cervical cancer and is thought to be important, if not causal, for some types of tumorigenesis [1][2][3]. Two HPVencoded proteins, E6 and E7, inactivate and inhibit the expression of the p53 and RB tumor suppressor proteins, respectively. Nevertheless, HPV infection is not sufficient for tumorigenesis, because HPV infection is also present in healthy individuals [4], and the overexpression of E6/E7 alone is capable of immortalizing primary human epithelial cells but does not induce tumor cell transformation [5]. Thus, understanding the conditions that stimulate HPVinfected cells is crucial for the development of effective treatments for HPV-associated tumors.
The transcription factor AP-1, which is composed of heterodimers of members of the c-Jun and c-Fos families, regulates various cellular processes such as enhanced proliferation, apoptosis, and tumor metastasis [6]. In particular, among the various members of the AP-1 family, c-fos acts as a tumor promoter, and c-fos upregulation causes cellular transformation that is characterized by colony formation in soft-agar and tumor formation in nude mice [7,8]. In addition, when c-fos binds to c-jun, it increases the gene expression of cyclinD1 and contributes to the potentiation of malignancy [9][10][11].
In cervical cancer, the AP-1 complex formed during tumor development consists of a c-fos/c-jun heterodimer [9,[12][13][14], and c-fos/c-jun formation is also implicated in HPV-induced esophageal tumor development [15]. In fact, various c-fos target genes are reported to be expressed at higher levels in cervical cancer cells with comparison to normal cervical epithelial cells [16]. While c-fos showed very low expression in normal samples and moderate expression in cervical premalignant lesions, the tumor tissues showed very strong expression [13]. It is, therefore, reasonable to assume that c-fos regulation plays a fundamental role in HPV-induced tumor development. However, the extracellular conditions in which c-fos expression and AP-1 complex formation are induced in HPV-infected cells are unknown.

Mediators of Inflammation
A shift in the composition of AP-1 from fra-1/c-jun to c-fos/c-jun heterodimers occurs during HPV-infected tumor development [7,14]. The same shift in AP-1 composition also takes place under oxidative stress, such as after UV-B exposure [10]. As transient transfection experiments showed that HPV16E6 induces the transcription of the c-fos promoter [17], we hypothesized that under oxidative stress HPV16E6 function might contribute to c-fos expression and c-fos/c-jun heterodimer formation.
There are at least 15 cancer-associated HPV types, and of these, HPV16 is found in more than 50% of HPV-positive cancer tissues [1,2]. Here, we used SiHa cells (HPV16positive squamous cell carcinoma of the cervix) and showed that H 2 O 2 and TGF-α, which are promoters of the multistep carcinogenic process [18,19], induced c-fos/c-jun heterodimer formation in SiHa cells, whereas o-phenanthroline inhibited H 2 O 2 -induced c-fos upregulation. H 2 O 2 and TGFα-induced c-fos upregulation was impaired in SiHa cells transfected with siRNA HPV16E6. This result further implicates H 2 O 2 and TGF-α signaling in the pathogenesis of HPVinduced tumor development.

SDS-PAGE and Western
Blotting. The cell cultures (80-100% confluent) were washed twice with ice-cold phosphate buffered saline and lysed in 1 × Laemmli sample buffer supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Roche). The cells were scraped and homogenized at 4 • C with a syringe and a thin needle, and the cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% resolving gel). The resultant proteins were electrophoretically transferred onto nitrocellulose membranes (Pall Corporation), before being incubated for 2 h at room temperature with blocking buffer containing 4% nonfat dried milk in TBS (20 mM Tris (pH 7.6) and 150 mM NaCl). They were then washed with TBS-T (0.1% Tween-20) and incubated overnight at 4 • C in TBS buffer containing 4% BSA (Wako) and appropriate antibodies. The antibodies used in the experiment were anti-c-fos (Santa Cruz, sc-52, 1 : 500) and anti-c-jun (Cell Signaling, L70B11, 1 : 500), and the secondary antibodies were Donkey Antirabbit IgG/HRP (Amersham NA934, 1 : 5000) and Goat Antimouse IgG/HRP (Chemicon, AP124P 1 : 5000).
The protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare Bio-Sciences). The signals from the membrane were detected and imaged using a LAS-4000 imager (Fujifilm Corporation). The membranes were then stripped (30 min at 56 • C) in stripping buffer containing 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) and reprobed with β-actin or tubulin as a loading control. All Western blots were performed at least three times for each experiment. To quantify the Western blot data, densitometric analysis of ECL-exposed blots was performed using the NIH ImageJ software (version 1.45f).

CoIP Assay.
The antibodies used for the Western blot analysis were c-Fos, c-Jun, and CBP/β. For the c-Fos coIP assay, SiHa cells were lysed in a modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% NP-40) and a protease inhibitor mixture (Roche). CoIP was performed using 2 μg of the c-Fos antibody or the respective isotype control.
2.6. RT-PCR. Total RNA were extracted from the harvested cells using Isogen (Nippon Gene). After the removal of genomic DNA, approximately 1 μg of RNA was used to generate cDNA using Reverse Transcriptase M-MLV (RNase H-) (TaKaRa) and random hexamers (Fermentas). The primer sequences used were as follows: 5 -CGGAATTCATGCACCAAAAGCGAAC-3 (sense) and 5 -CCCAAGCTTACAGCTGGGTTTCTCT-3 (antisense) for HPV16E6 [21] and 5 -CAGGGCTGCTTTTAACTCTG-3 (sense) and 5 -GATGATCTTGAGGCTGTTGTC-3 (antisense) for GAPDH. The PCR reactions for HPV16E6 were run for 30 cycles. GAPDH amplification products were used as a loading control. All amplification products were separated in 2% agarose gels. All RT-PCR were performed at least three times for each experiment.

Statistical
Analysis. Data are expressed as the mean ± s.e.m. The paired-sample t-test was performed for statistical comparisons. For all tests, a P value of less than 0.05 was considered to be significant.

o-phenanthroline Inhibits H 2 O 2 -Induced c-Fos Expression.
The upregulation of c-fos has been observed after H 2 O 2 treatment in both epithelial and endothelial cells [19,22], which raises the possibility that SiHa cells might respond to H 2 O 2 in a similar manner. To test this, we analyzed c-fos protein expression after H 2 O 2 treatment in SiHa cells. Western blotting analysis revealed aberrantly high levels of c-fos protein expression after H 2 O 2 treatment (Figure 1(a)).
In the H 2 O 2 -treated SiHa cells, the expression of the AP-1 family member c-jun was also upregulated (Figure 1(a)). o-phenanthroline is an iron chelator [23] that protects cells from cancer progression by inhibiting the Fenton reaction,  (Figure 1(b)).

Characterization of c-Fos/c-jun Heterodimers after Ligand -Dependent and Independent EGFR Activation.
The upregulation of c-fos observed in epithelial cells after H 2 O 2 treatment is mainly mediated through EGFR activation [18]. We, therefore, examined c-fos protein expression in SiHa cells that had been treated with the EGFR ligand TGF-α. Western blotting analysis revealed aberrantly high levels of c-fos protein expression after TGF-α treatment (Figure 2(a)). We then investigated c-fos/c-jun heterodimer formation in SiHa cells. We stimulated SiHa cells with H 2 O 2 or TGF-α for 4 hours, immunoprecipitated c-fos, and determined its partner AP-1 family member by Western blotting (Figure 2(b)).
Interactions between c-fos and c-jun were observed after both H 2 O 2 and TGF-α stimulation (Figure 2(b): the IP with isotype control did not show any specific band).

HPV16E6 Mediates H 2 O 2 and TGF-α-Induced c-Fos Expression.
We next tested whether HPV16E6 is necessary for the c-fos expression induced by H 2 O 2 or TGF-α treatment.
To directly address the role of HPV16E6 protein in c-fos expression, we transfected SiHa cells with siRNA HPV16E6 (Figure 3(a)). A scrambled nonspecific siRNA duplex was used as a negative control. We observed siRNA HPV16E6dependent reductions in H 2 O 2 and TGF-α induced c-fos expression (Figures 3(b) and 3(c)). Similar results that siRNA HPV16E6-dependent reduction in c-fos expression induced by H 2 O 2 treatment were obtained in another cervical cancer cell line, Caski cell (Figure 3(d)). These data suggest that HPV16E6 is necessary for H 2 O 2 and TGF-α induced-c-fos expression.

Discussion
Our findings provide new insights into the mechanism of c-fos/c-jun heterodimer formation during the development of cervical cancer. First, our findings show that c-fos/cjun heterodimer formation results from both H 2 O 2 and Mediators of Inflammation TGF-α stimulation. The current view is that in cervical cancer c-fos/c-jun heterodimer formation results from the inefficient expression of the ternary complex factor Net [25], but the evidence we have uncovered for HPV16E6 dependent c-fos expression after H 2 O 2 or TGF-α stimulation broadens the scope of the mechanisms of c-fos/c-jun heterodimer formation in extracellular conditions such as oxidative stress. For example, the observed shift in AP-1 composition from fra-1/c-jun to c-fos/c-jun heterodimers in HaCaT keratinocytes after UV-B exposure [10] suggests that after being induced by extracellular and environmental stimuli, c-fos expression and c-fos/c-jun heterodimer formation contribute to the pathological process of HPV positive skin cancer. Therefore, studies of c-fos expression, HPV infection, and skin tumor development are necessary. Second, our data indicate that HPV16E6 is necessary and sufficient for c-fos/c-jun heterodimer formation to occur in SiHa cells under both H 2 O 2 and TGF-α stimulation. In the presence of transition metals such as iron and copper, H 2 O 2 can be oxidized into extremely reactive and toxic HO• via the Fenton reaction. The hydroxyl radical (HO•) then activates EGFR [26]. Our data show that H 2 O 2 and TGF-α are stimulators of c-fos/c-jun heterodimer formation and confirm that the iron chelator is an inhibitor of c-fos expression that acts by inhibiting the Fenton reaction. The negative effect of o-phenanthroline on HPV16E6 mediated c-fos expression might explain why iron chelation inhibits growth and induces the apoptosis of HPV-positive carcinoma cells [27]. Tumorigenic cervical cells express higher levels of intracellular iron [28], which creates an environment that favors HPV16E6-mediated c-fos/c-jun heterodimer formation in HPV-infected cells. This implies that iron is necessary for c-fos/c-jun heterodimer formation. The precise triggers of H 2 O 2 expression around cervical epithelial cells are unknown, but it has been proposed that chronic inflammation triggers the accumulation of macrophages in the vicinity of the cervix, and that these cells subsequently produce the antimicrobial molecule H 2 O 2 .
Finally, our data indicate that cervical cancer, the hallmark of which is HPV infection, is an EGFR-sensitive disease regardless of ligand-dependent or ligand-independent stimulation. EGFR mAb therapies are effective for treating squamous cell carcinoma of the head and neck and are also being evaluated in other HPV-associated tumors [29]. One of the best-known mechanisms of EGFR mAb therapy resistance is ligand-independent EGFR activation [30]. This was observed in studies of squamous carcinoma cells (T-Hep3) that naturally overexpress the uPA receptor, one of which found that EGFR was activated by the uPA receptor in a ligand-independent fashion, resulting in resistance to EGFR mAb [31]. Therapies such as the use of o-phenanthroline, or alternative approaches such as the use of small molecule EGFR tyrosine kinase inhibitors that block the intracellular tyrosine kinase domain, could hold great potential for the treatment of head and neck cancer.