FGF8b oncogene mediates proliferation and invasion of Epstein–Barr virus-associated nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b upregulation

Abstract

The fibroblast growth factor 8b (FGF8b) oncogene is known to be primarily involved in the tumorigenesis and progression of hormone-related cancers. Its role in other epithelial cancers has not been investigated, except for esophageal cancer, in which FGF8b overexpression was mainly found in tumor biopsies of male patients. These observations were consistent with previous findings in these cancer types that the male sex-hormone androgen is responsible for FGF8b expression. Nasopharyngeal carcinoma (NPC) is a highly metastatic cancer of head and neck commonly found in Asia. It is etiologically associated with Epstein–Barr Virus (EBV) infection, inflammatory tumor microenvironment and relatively higher male predominance. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated non-hormone-related cancer of the head and neck, NPC. More importantly, overexpression of FGF8b mRNA and protein was detected in a large majority of NPC tumors from both male and female genders, in addition to multiple NPC cell lines. We hypothesized that FGF8b overexpression may contribute to NPC tumorigenesis. Using EBV-associated NPC cell lines, we demonstrated that specific knockdown of FGF8b by small interfering RNA inhibited cell proliferation, migration and invasion, whereas exogenous FGF8b stimulated these multiple phenotypes. Further mechanistic investigation revealed that in addition to NF-κB signaling (a major inflammatory signaling pathway known to be activated in NPC), an important EBV oncoprotein, the latent membrane protein 1 (LMP1), was found to be a direct inducer of FGF8b overexpression in NPC cells, whereas androgen (testosterone) has minimal effect on FGF8b expression in EBV-associated NPC cells. In summary, our study has identified LMP1 as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells. This novel mechanism of viral-mediated FGF8 upregulation may implicate a new role of oncoviruses in human carcinogenesis.

Introduction

Fibroblast growth factor 8b (FGF8b) is an oncogene found to be predominantly overexpressed in hormone-related cancers, namely prostate and breast cancers (Tanaka et al., 1998; Mattila and Harkonen, 2007). The potent oncogenic activity of FGF8b has been demonstrated in vivo as FGF8b-overexpressing murine fibroblasts causes transformation and rapid tumor formation in nude mice (MacArthur et al., 1995a). Such a remarkable transforming ability of FGF8b was further demonstrated in transgenic mouse models overexpressing FGF8b. Transgenic mice overexpressing FGF8b (under the control of the mouse mammary tumor virus promoter, MMTV) frequently developed mammary tumors with lung metastases, salivary gland tumors, invasive lobular adenocarcinomas, as well as ductal hyperplasia (Daphna-Iken et al., 1998). Similarly, tissue-specific overexpression of FGF8b in the prostate epithelium of transgenic mice resulted in the development of prostate neoplasia, stromal hyperplasia, prostate adenocarcinoma with lymph node metastasis (Song et al., 2002; Zhong et al., 2006). Consistent with these findings, it has been demonstrated that FGF8b overexpression in both prostate and breast cancer cells can promote tumor cell growth and formation in nude mice (Song et al., 2000; Ruohola et al., 2001), whereas specific downregulation of FGF8b by antisense RNA inhibited the in vivo tumorigenicity of prostate cancer cells (Rudra-Ganguly et al., 1998). These cumulative evidences establish the role of FGF8b in carcinogenesis of hormone-related cancers (Tanaka et al., 1998; Marsh et al., 1999; Gnanapragasam et al., 2003).

FGF8b is a member of a large family of FGF8 (previously known as the androgen-induced growth factor, AIGF). FGF8 is known to be essential for embryogenesis (Crossley and Martin, 1995), in particular, for the development/organogenesis of the craniofacial, pharyngeal, brain, cardiac, kidney, urogenital organs and limbs (Heikinheimo et al., 1994; Crossley et al., 1996; Mattila and Harkonen, 2007). Investigation on the involvement of FGF8b in hormone-related cancers was indeed based on the initial discovery of FGF8 (androgen-induced growth factor) in an androgen-dependent mouse mammary carcinoma cell line (Tanaka et al., 1992) and detection of FGF8 overexpression in human breast and prostate cancers (Tanaka et al., 1995; Leung et al., 1996; Gnanapragasam et al., 2002). Further genomic and functional characterization of the FGF8 family demonstrated that the FGF8b isoform (one of the four human FGF8 isoforms: FGF8a, b, e and f (Gemel et al., 1996)) possesses the greatest mitogenic and, more importantly, the greatest transforming activity than the other isoforms, both in vitro and in vivo (MacArthur et al., 1995a, 1995b; Blunt et al., 1997; Song et al., 2000; Olsen et al., 2006). It is believed that FGF8b is one of the most important FGF8 isoforms in human carcinogenesis (Marsh et al., 1999; Gnanapragasam et al., 2003), although the exact physiological function of each isoform is yet-to-be fully revealed.

Owing to the original discovery of FGF8 in an androgen-dependent system (Tanaka et al., 1992), as well as cumulative findings that FGF8b knockin mice developed prostate and breast cancers, investigations on the role of FGF8b in human oncogenesis have primarily been focused on hormone-related cancers. Clinical studies revealed that FGF8b overexpression was found in the majority of prostate cancer (40–80%) (Dorkin et al., 1999; Gnanapragasam et al., 2002, 2003; Tanaka et al., 2002; Zhong et al., 2006) and breast cancer (70–100%) (Marsh et al., 1999). Moreover, its overexpression was clinically associated with disease progression (mainly staging and grading), poor prognosis, poor survival (Dorkin et al., 1999; Valve et al., 2001), and potentially metastasis in prostate cancer (Gnanapragasam et al., 2003; Valta et al., 2008). Although the molecular mechanisms for FGF8b overexpression in prostate and breast cancers are not fully understood, the human sex hormone androgen (Tanaka et al., 1992) is known to play a key role, even in the case of breast cancer (Castellano et al., 2010 Park et al., 2010) (in addition to prostate cancer (Gnanapragasam et al., 2002)). Similar findings in esophageal cancer, which has recently found to be androgen-related (Yamashita et al., 1989; Awan et al., 2007; Thaver et al., 2009), also revealed that FGF8b overexpression is linked to androgen signaling (Tanaka et al., 2001; Awan et al., 2007). With the limited number of cancer types being investigated thus far, it is unclear whether FGF8b is involved in the carcinogenesis of other human malignancies, especially in those hormone-unrelated cancers.

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer commonly found in Asia with a high incidence rate of 15–50/100 000 persons/year (comparable with that of pancreatic cancer in the US) (Chan et al., 2002; Spano et al., 2003; Anderson et al., 2006). In endemic regions, NPC is characterized by its strong etiologic association with Epstein–Barr virus infection (EBV, a known oncogenic virus), high metastatic potential (Skinner et al., 1991), highly inflammatory tumor microenvironment with heavy lymphocyte infiltration (Lo and Huang, 2002), as well as a male predominance of 3:1 (Bhattacharyya, 2004). It is believed that NPC occurs as a result of complex interplay of local factors in the nasopharynx, EBV and the inflammatory tumor microenvironment. The molecular mechanisms underlying its highly invasive and metastatic nature, as well as the reported male predominance are not fully understood. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated non-hormone-related cancer of the head and neck, NPC. More importantly, overexpression of FGF8b mRNA and protein was detected in a large majority of NPC tumors from both male and female genders. We hypothesized that FGF8b overexpression contributed to NPC tumorigenesis. In addition to the identification of NF-κB-mediated FGF8b upregulation in EBV-associated NPC cells, this mechanistic investigation has also identified latent membrane protein 1 (LMP1) as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells, which may reveal a new role of oncoviruses in human carcinogenesis.

Results

FGF8b overexpression in NPC biopsies and cell lines

To investigate the possible involvement of FGF8b in NPC tumorigenesis, we first examined the expression of FGF8b mRNA in 26 NPC tumor biopsies (18 from males and 8 from females) by reverse transcriptase–PCR (RT–PCR). Normal human colon epithelial tissues (as well as a normal esophageal epithelial control cell line, Het-1A; ATCC, Manassas, VA, USA) were included for comparison. Figure 1A showed that normal control tissues expressed very low basal levels of FGF8b mRNA, whereas 21/26 (81%) NPC tumors showed elevated FGF8b mRNA expression (1.5-fold higher expression versus controls), ranging from 1.51- to 6.28-fold higher expression versus control tissues. Statistical analysis demonstrated a significant overexpression of FGF8b mRNA versus normal controls (P=0.0048**, Mann–Whitney test) (Figure 1B). Interestingly, FGF8b mRNA overexpression was found in a very high percentage of NPC tumors from both genders: 14/18 tumors in male NPC (78%) and 7/8 tumors in female patients (88%) (Figure 1A). This finding in NPC was distinct from previous reports in esophageal cancer, in which FGF8b overexpression was mainly found in tumor biopsies of male patients (absent in female esophageal adenocarcinoma in one report; Tanaka et al., 2001, and detected only in two female cases in another report; Awan et al., 2007). Indeed, immunohistochemical staining also confirmed FGF8b protein overexpression in NPC tumors from both genders, as indicated by intense FGF8b-positivity (Figure 1C). Immunostaining for human FGF8b protein was optimized and performed using an automated Bond-max immunostaining platform (Mount Waverly, Victoria, Australia; see Materials and methods). Specificity of FGF8b staining was confirmed with positive control tissues (normal breast epithelium) as a previous study demonstrated that ‘FGF8b-positive staining was exclusively found in normal breast epithelium (and breast cancer cells), but absent in stromal cells’ (Tanaka et al., 2002). An additional normal control tissue (normal colon epithelial tissue) was also included for FGF8b expression in normal tissues. Consistent with the previous findings, our results showed that only normal breast epithelium, but not the adjacent stromal tissue, was distinctly stained positive for FGF8b (Figure 1C). Moreover, the normal colon epithelium also showed low level of FGF8b expression, which is consistent with our finding using RT–PCR (Figure 1A). Similar finding was observed in normal lung epithelium (data not shown). In an additional cohort of 40 NPC tumors from 24 male and 16 female patients, intense cytoplasmic FGF8b staining was observed in all NPC tumor tissues and their corresponding adjacent normal nasopharyngeal epithelial cells (Figure 1C), when compared with the normal control tissues. The median cytoplasmic FGF8b (cFGF8b) immunohistochemistry (IHC) scores for NPC tumor cells and adjacent normal cells were 204 and 264, respectively (with no statistically significant differences between the IHC scores, P=0.3813, Wilcoxon signed-rank test) (Supplementary Figure 1). As shown in Figure 1C, intense cytoplasmic FGF8b staining was observed in both male and female NPC biopsies, and no statistically significant difference was found between male and female cases (median cFGF8b IHC scores for male and female were 208.5 and 194.5, respectively; P=0.4314, Mann–Whitney test). This result, together with our RT–PCR findings (Figure 1A), did indicate an overall upregulation of FGF8b protein in both NPC tumor cells and the immediate epithelial tissues around the tumors. This interesting phenomenon was not observed with previous findings in esophageal cancer (Tanaka et al., 2001) nor prostate cancer (Valta et al., 2008), where FGF8b expression was exclusively found in tumor cells. As NPC is well known to have a highly inflammatory tumor microenvironment with viral infection (thus named lymphoepithelioma because of heavy lymphocyte infiltration), it is likely that such an inflammatory microenvironment plays a key role in such a wide-spread upregulation of FGF8b in NPC.

Figure 1
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FGF8b overexpression in NPC tumor biopsies and NPC cell lines. (A) Elevated expression of FGF8b mRNA in NPC tumor biopsies from both genders (Upper Panel). RT–PCR assays demonstrated that FGF8b mRNA was overexpressed in 14/18 (78%) male and 7/8 (88%) female NPC biopsies tested. RT–PCR for GAPDH was included as the internal control. Lower Panel shows the graphical representation of the relative expression level of the FGF8b mRNA in comparison with the normal control tissues (obtained from adjacent normal colon epithelial tissue of colon cancer patients). All mRNA expression data were normalized to the respective GAPDH expression (by densitometry). (B) Scatter plot showing the expression of FGF8b mRNA in NPC tumor biopsies and normal controls (P=0.0048**). FGF8b mRNA expression levels were shown as mean±s.e.m. Normal controls were included for comparison (two adjacent normal colon epithelial tissues of colon cancer patients—indicated by black triangles, normal esophageal total RNA from Applied Biosystems/Ambion—indicated by gray triangle, as well as normal esophageal epithelial cell line, Het-1A from ATCC—indicated by open triangles for two independent measurements). FGF8b mRNA expression levels in NPC tumors were indicated by filled circles. Statistical analysis was performed using Mann–Whitney test (a non-parametric statistical analysis comparing two unpaired groups) with GraphPad Prism software version 4.0 (PRISM4) (GraphPad Software Inc.). Results with P<0.05 were considered as statistically significant. (C) Overexpression of FGF8b protein was detected in both male- and female-undifferentiated NPC tumors by immunohistochemical staining. Paraffin-embedded specimens of NPC tumors were immunostained for FGF8b with goat anti-human FGF-8b antibody at 1:30 dilution (detailed in Materials and methods). Inserts showing FGF8b immunostaining in a: normal breast ductal cells indicated by black arrows, normal stromal tissues of the breast indicated by white arrows; b: normal colon epithelial cells indicated by black arrows; c: no antibody negative control (Tris-buffered saline) for NPC tumor was shown; d–e: two male NPC tumors (T); f: normal (N) epithelial cells from NPC male patient; g–h: two female NPC tumors (T); i: normal (N) epithelial cells from NPC female patient. All stained slides were evaluated under light microscope at × 400 magnification. Pictures were taken at × 400 magnification. (D) Overexpression of FGF8b mRNA (Upper Panel) and FGF8b protein (Lower Panel) in NPC cell lines. FGF8b mRNA expression was determined in seven NPC cell lines. The relative expression of FGF8b mRNA in NPC cell lines versus that of Het1A was shown as bar graph below. Western blotting results showed that FGF8b protein was expressed at a much higher level than the control cell line, Het-1A (Lower Panel). Actin was shown as loading control. Similar results were obtained in three independent experiments.

Consistent with the observed FGF8b overexpression in NPC tumor biopsies, FGF8b mRNA and protein expression was also found to be elevated in a panel of seven NPC cell lines tested when compared with the control Het-1A cells (Figure 1D) (C666-1 was from undifferentiated NPC origin; HONE-1, HONE-1-EBV, HONE-1-LMP1(B95.8) and HONE-1-LMP1(2117) were from poorly differentiated NPC; whereas HK1 and HK1-EBV were derived from well-differentiated NPC). Among all NPC cell lines tested, the poorly differentiated NPC cell line, HONE-1-LMP1(B95.8), expressed the highest level of FGF8b protein (Figure 1D), whereas the control Het-1A cells only expressed a barely detectable level of FGF8b protein. Cumulative results showed that stable expression of LMP1(B95.8) was able to induce FGF8b upregulation to 8-fold when compared with the parental HONE-1 cells (Supplementary Figure 2), suggesting the potential involvement of LMP1 in FGF8b upregulation in NPC.

FGF8b siRNA inhibits NPC cell proliferation, migration and invasion

To examine the functional involvement of FGF8b overexpression in NPC tumorigenesis, we determined the effects of specific knockdown of FGF8b by small interfering RNA (siRNA) in NPC cells. Two highly transfectable EBV-associated NPC cell lines were chosen: HONE-1-LMP1(B95.8) (poorly differentiated NPC origin with the highest level of FGF8b protein expression) and HONE-1-EBV (representative of EBV-genome-positive NPC cells). HK1 and HK1-EBV were not transfectable. Figure 2a showed that transient transfection with FGF8b siRNA (300 pmol) inhibited cellular proliferation of HONE-1-EBV and HONE-1-LMP1(B95.8) by 45.6 and 44.2% at 72 h, respectively, when compared with control siRNA transfection. Moreover, functional knockdown of FGF8b also resulted in reduction of cellular migration and invasion abilities of these NPC cells. The transwell migration abilities of HONE-1-EBV and HONE-1-LMP1(B95.8) were reduced by 34.4 and 50.9%, respectively (Figure 2b). Similarly, the matrigel invasion abilities of HONE-1-EBV and HONE-1-LMP1(B95.8) were reduced by 34.0 and 40.8%, respectively, upon FGF8b siRNA transfection (Figure 2c). These inhibitory activities of FGF8b knockdown was also observed in C666-1, an undifferentiated NPC cell line with lower transfectability. As shown in Figure 2d, siRNA-mediated knockdown of FGF8b was accompanied by growth inhibition (32% versus control) and inhibition of migration and invasion of 71 and 78%, respectively. This demonstrated that FGF8b was functionally involved in NPC cell proliferation, migration and invasion.

Figure 2
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Transient transfection of FGF8b siRNA inhibited growth (a), migration (b) and invasion (c) of EBV-associated NPC cell lines. (a) Two highly transfectable EBV-associated NPC cell lines, HONE-1-EBV or HONE-1-LMP1(B95.8) were transfected with 300 pmol of control siRNA or FGF8b siRNA for 72 h. Cell growth inhibition was assessed by MTT assay (n=3 in each experiment). Similar results were obtained in three independent experiments. Day 2 transfectants of FGF8b siRNA and control siRNA (300 pmol) were used to assess the transwell migration (b) and matrigel invasion (c) abilities of HONE-1-EBV or HONE-1-LMP1(B95.8) cells at 24 h. For both the transwell migration and matrigel invasion assays, NPC cells (7 × 104 cells/chamber in serum-free medium) contained in the upper chamber were immersed in complete medium (RPMI containing 10% fetal bovine serum) in a 24-well culture plate for 24 h at 37 °C in a 5% CO2 incubator. After 24 h, migrated cells through the transwell migration chamber (b) or invaded cells through the matrigel invasion chamber (c) were fixed and stained with 1% Toluidine Blue O (Sigma-Aldrich) in 1% Borax (USB, Cleveland, OH, USA). Average number of migrated or invaded cells (n=4–10 fields/chamber) was counted under light microscope ( × 200 magnification). Similar results were obtained in three independent experiments. (d) Transient transfection of FGF8b siRNA inhibited growth, cellular migration and invasion of C666-1 cells. C666-1 cells (with medium transfectability) were transfected with 600 pmol of FGF8b siRNA or control siRNA. Specific knockdown of FGF8b protein was verified by western blotting. Effects of FGF8b knockdown on growth, migration and invasion of C666-1 cells (1 × 105 cells) were assessed as above. Similar results were observed in three independent experiments.

Exogenous FGF8b promotes NPC cell growth, migration and invasion with upregulation of mesenchymal markers

Consistent with our FGF8b siRNA results, exogenous FGF8b was able to induce NPC cell growth, migration and invasion in vitro. The effect of exogenous FGF8b stimulation was assessed in HONE-1-EBV and HONE-1-LMP1(B95.8) cells. Figure 3a showed that exogenous human FGF8b (50 ng/ml) was able to stimulate proliferation of both NPC cell lines by 50% at 48 h. In addition, exogenous FGF8b also promoted cellular invasion of HONE-1-LMP1(B95.8) and HONE-1-EBV by 40.1 and 35.3% (at 24 h), respectively (Figure 3b). Similarly, migration abilities of these cells were also enhanced moderately by FGF8b stimulation (37.8 and 21.2% increase for HONE-1-LMP1(B95.8) and HONE-1-EBV, respectively) (Figure 3c). This FGF8b-induced invasion of NPC cells was consistent with the observed upregulation of mesenchymal markers, vimentin and N-cadherin by FGF8b treatment at 24 h (Figure 3d). In another EBV-positive NPC cell line of undifferentiated origin, C666-1, FGF8b also stimulated marked induction of cellular invasion by 115.5%. This further confirmed the important role of FGF8b in NPC invasion (Supplementary Figure 3).

Figure 3
figure3

Exogenous FGF8b stimulated proliferation (a), invasiveness (b, c) of NPC cells with upregulation of mesenchymal markers (d). (a) FGF8b-induced NPC cell proliferation: HONE-1-EBV or HONE-1-LMP1(B95.8) cells (7 × 104 cells) were treated with 50 ng/ml FGF8b (in serum-free medium) or vehicle control for 48 h. Cell proliferation was measured by MTT assay (n=3). Similar results were observed in three independent experiments. (b, c) Exogenous FGF8b-induced cellular invasion of EBV-associated NPC cells through matrigel invasion chambers (b), as well as cellular migration through transwell migration chambers (c). Both cell lines were allowed to migrate through the transwell or invade through the matrigel invasion chambers for 24 h with either 50 ng/ml FGF8b or vehicle control placed in the lower chamber. The average number of invaded (b) or migrated (c) cells/field was presented as mean±s.e.m. (n=10 fields). Similar results were obtained in three independent experiments. (d) FGF8b induced upregulation of mesenchymal markers, vimentin and N-cadherin. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were incubated with vehicle or 50 ng/ml of FGF8b (in serum-free medium) for 24 h. Western blot analysis demonstrated an induction of vimentin and N-cadherin expression by FGF8b versus vehicle control. Actin was shown as loading control. Similar results were obtained in three independent experiments.

FGF8b activates MAPK in NPC cells

The key mitogenic pathway, mitogen-activated protein kinase (MAPK), is known to be the major downstream signaling pathway of FGF8b (Ishibe et al., 2005). Here, we examined whether FGF8b signals through MAPK in the context of EBV influence. Using these two EBV-associated NPC cell lines, HONE-1-EBV and HONE-1-LMP(B95.8), we found that exogenous FGF8b stimulation resulted in activation of MAPK (induction of phospho-p44/42 MAPK) as early as 10 and 30 min upon stimulation (Figure 4A), indicating an immediate activation of MAPK by FGF8b in EBV-associated NPC cells. The involvement of MAPK activation was further confirmed with FGF8b siRNA, where FGF8b siRNA (versus control siRNA) transient transfection resulted in specific downregulation of phospho-p44/42 MAPK in both HONE-1-EBV and HONE-1-LMP1(B95.8) (Figure 4B). Although another major growth-related signaling pathway, AKT was not significantly altered in these EBV-associated NPC cell lines upon FGF8b siRNA or exogenous FGF8b stimulation (data not shown).

Figure 4
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(a) Activation of p44/42 MAPK pathway in NPC cells by FGF8b. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were serum starved for 48 h, then exposed to FGF8b (50 ng/ml in serum-free medium) or vehicle for 10, 30 and 240 min, respectively. Western blot analysis showed that p44/42 MAPK was phosphorylated as early as 10 min upon FGF8b stimulation. Actin was shown as loading control. Similar results were obtained in three independent experiments. (b) Specific knockdown of FGF8b by siRNA inhibited p44/42 MAPK phosphorylation in NPC cells. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were transfected with control siRNA or FGF8b siRNA (300 pmol) for 72 h. Western blot analysis demonstrated a specific downregulation of pi-p44/42 MAPK by FGF8b siRNA versus control siRNA. Similar results were obtained in three independent experiments.

NF-κB is an upstream regulator of FGF8b expression in EBV-associated NPC cells, but not androgen

Previous studies in hormone-related cancers (prostate and breast cancers), as well as esophageal cancer demonstrated that FGF8b gene expression was regulated by androgen/testosterone (Tanaka et al., 1992, 2001; Gnanapragasam et al., 2002). Given the relatively higher expression levels of FGF8b mRNA in male NPC tumors (Figure 1A), we examined if androgen (in the form of testosterone) was responsible for FGF8b regulation in EBV-associated NPC cells. As shown in Figure 5A, exogenous stimulation of both HONE-1-EBV and HONE-1-LMP1(B95.8) cells with testosterone up to 1000 ng/ml for 48 h did not result in significant induction of FGF8b mRNA, FGF8b protein nor its downstream p44/p42 MAPK pathway. Our results suggested that FGF8b may not be directly regulated by androgen in NPC, which seemed to be consistent with our observation that FGF8b overexpression was detected in both male and female NPC tumors (Figure 1A). Thus, factors other than androgen may be responsible for the regulation of FGF8b overexpression in NPC cells.

Figure 5
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(a) Testosterone did not significantly alter the expression of FGF8b at both the mRNA and protein levels. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were serum starved for 48 h and then subjected to vehicle or testosterone treatment (300 or 1000 ng/ml) for 48 h. Both cellular mRNA and protein were collected for the detection of FGF8b expression by RT–PCR (Upper Panel) and western blot analysis (Lower Panel), respectively. Similar results were observed in three independent experiments. (b) Involvement of NF-κB signaling in FGF8b upregulation in NPC cells as specific inhibition of NF-κB by BAY 11-7082 downregulated FGF8b protein expression in EBV-associated NPC cells (Upper Panel). HONE-1-EBV or HONE-1-LMP1(B95.8) cells were incubated with vehicle (DMSO) or BAY 11-7082 (7 μg/ml) for 24 and 48 h. Western blot analysis showed that BAY 11-7082 inhibited FGF8b protein expression in both cell lines. Actin was shown as loading control. Similar results were obtained in three independent experiments. Lower Panel showing the effect of specific knockdown of NF-κB subunits p50 and p65 (by siRNA) on FGF8b protein expression in NPC cells. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were transfected with 300 pmol of NF-κB p50 siRNA, p65 siRNA or control siRNA. Western blotting showing the expression of FGF8b, NF-κB p50 and p65 subunits on day 3 after transfection. Similar results were observed in three independent experiments. (c) TNF-α-induced expression of FGF8b was inhibited by BAY 11-7082. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were cotreated with TNF-α (60 ng/ml) and BAY 11-7082 (7 μg/ml) or vehicle control for 24 or 48 h. By western blot analysis, a prominent induction of FGF8b was observed in HONE-1-EBV, which was almost completely abolished by BAY 11-7082 at 24 h. Similar result was observed in HONE-1-LMP1(B95.8) at a later time point at 48 h. Actin was shown as loading control. Similar results were obtained in three independent experiments. (d) LMP1-induced FGF8b expression in NPC cells. HONE-1 cells (2 × 105 cells/well) were transfected with 4 μg of control vector (pcDNA3.1/Hygro(+)) or LMP1(B95.8)-expression plasmid for 48 h. Induction of FGF8b protein and activation of p44/42 MAPK were detected by western blotting. Actin was shown as loading control. Similar results were obtained in three independent experiments.

NPC is characterized by its inflammatory pathology and the demonstrated involvement of a major inflammatory signaling pathway, the NF-κB pathway (Thornburg et al., 2003; Lo et al., 2006; Shi et al., 2006; Chou et al., 2008; Wong et al., 2009). A recent study in a hormone-independent prostate cancer cell line indicated that regulation of FGF8 expression (though not for FGF8b in particular) required NF-κB (Armstrong et al., 2006). This prompted us to investigate whether this major inflammatory signaling pathway in NPC was responsible for FGF8b regulation in EBV-associated NPC cells. Using a specific inhibitor of NF-κB, BAY 11-7082, we found that the expression of FGF8b protein was markedly reduced in HONE-1-EBV and HONE-1-LMP1(B95.8) cells at 24 h (versus dimethyl sulfoxide control), and such inhibition on FGF8b expression was found to persist even up to 48 h (Figure 5b, upper panel). Our data indicated the potential involvement of NF-κB in FGF8b regulation in EBV-associated NPC cells. Moreover, transient transfection of both NPC cell lines with siRNAs against human NF-κB p65 and p50 subunits were found to result in marked downregulation of FGF8b (Figure 5b, lower panel), confirming a direct role of this inflammatory signaling pathway, NF-κB, in FGF8b regulation in NPC cells (consistent with previous finding in prostate cancer cells) (Armstrong et al., 2006).

To further confirm that NF-κB is an upstream regulator of FGF8b expression in EBV-associated NPC cells, a well-known NF-κB stimulus, TNF-α, was employed (TNF-α is also known to be overexpressed in NPC patients) (Kuo et al., 1994; Cui et al., 2001). Both EBV-associated NPC cell lines were serum starved for 48 h and then subjected to TNF-α stimulation (60 ng/ml) either in the presence or absence of BAY 11-7082 (7 μg/ml) to demonstrate the specificity of NF-κB-dependent regulation of FGF8b expression in NPC cells. Figure 5c showed that TNF-α stimulation induced FGF8b expression in HONE-1-EBV as early as 24 h. In the HONE-1-LMP1(B95.8) cell line, which harbors a much higher basal/endogenous level of FGF8b than HONE-1-EBV (Figure 1D), TNF-α stimulation was found to induce FGF8b upregulation more prominently at a later time point at 48 h. Similarly, BAY 11-7082 was able to completely abrogate the expression of both basal and TNF-α-induced FGF8b protein in HONE-1-EBV at 24 h and in HONE-1-LMP1(B95.8) at 48 h, respectively. Our finding demonstrated that NF-κB is a key regulator for FGF8b expression in NPC cells.

LMP1 oncoprotein directly induces FGF8b upregulation in EBV-associated NPC cells

The EBV oncoprotein, LMP1, is a key player in NPC carcinogenesis (Zheng et al., 2007). Prompted by the finding that the poorly differentiated LMP1-expressing stable NPC cell line, HONE-1-LMP1(B95.8), did express FGF8b at a much higher level than its LMP-1-negative parental cell line, HONE-1 (Figure 1D and Supplementary Figure 2), we examined whether this viral oncoprotein could directly regulate the expression of this important cellular oncoprotein, FGF8b. The parental HONE-1 cells, which do not express the LMP1 oncoprotein, expressed a relatively lower level of FGF8b protein (Supplementary Figure 4 and Figure 1D). However, when the LMP1 gene was introduced into the parental HONE-1 cells by transient transfection, a marked induction of FGF8b upregulation was detected (Figure 5d), which demonstrate a direct role of LMP1 in FGF8b induction. Moreover, this novel LMP1/FGF8b axis was found to be associated with prominent activation of p44/42 MAPK. Furthermore, such an induction was found to be LMP1 specific, as the introduction of another important EBV protein, LMP2A, did not result in FGF8b upregulation upon transient transfection (data not shown). Taken together the results from the stable cell line and transient transfection (Figures 1d and 5d), we demonstrated for the first time that a viral oncoprotein of EBV origin, namely LMP1, was a potent inducer of FGF8b protein. This is the first viral protein identified to be a direct inducer of FGF8b in human cancer.

Discussion

FGF8b overexpression has been shown to be primarily associated with hormone-related cancers in human, mainly prostate and breast cancers. Its role in other epithelial cancers has not been investigated, except for esophageal cancer, where FGF8b overexpression was mainly found in tumor biopsies of male patients. Here, we demonstrated for the first time that FGF8b was overexpressed in a high percentage of tumor biopsies and cell lines of NPC, a well-known EBV-associated head and neck cancer. Interestingly, FGF8b overexpression was detected in high percentage of NPC tumor biopsies from both male and female genders. Functional studies demonstrated its involvement in NPC cell proliferation and invasion. Androgen (testosterone) was found to have minimal effect on FGF8b expression in EBV-associated NPC cells. In addition to the identification of NF-κB-mediated FGF8b upregulation in EBV-associated NPC cells (NF-κB is a major inflammatory signaling pathway known to be activated in NPC), the current study has also identified the EBV LMP1 as the first viral oncogene capable of directly inducing FGF8b (an important cellular oncogene) expression in human cancer cells, which may reveal a new role of oncoviruses in human carcinogenesis.

The cellular mechanisms of FGF8b upregulation in human cancers are not fully understood. Androgen signaling is believed to be an important regulatory mechanism of FGF8b upregulation in prostate cancer (Gnanapragasam et al., 2002), as well as breast cancer (Tanaka et al., 1998), and, more recently, in esophageal cancer (Tanaka et al., 2001; Awan et al., 2007). Of particular interest was the unanticipated clinical association of androgen receptor expression and FGF8b expression in breast cancer (but not with estrogen nor progesterone receptor status) (Tanaka et al., 1995, 1998). Esophageal cancer (which has a male to female ratio of 7:1) (Blot et al., 1993) has recently been shown to be clinically associated with androgen dependence (as indicated by elevated serum testosterone, overexpression of androgen receptor, as well as FGF8b overexpression) (Awan et al., 2007). Although NPC has a relatively higher predominance for male (male:female ratio of 3:1) (Bhattacharyya, 2004), we demonstrated for the first time that the EBV oncoprotein, LMP1, as well as NF-κB are direct inducers of FGF8b expression in NPC cells, whereas androgen may not be a crucial factor for FGF8b overexpression in this EBV-associated cancer. Our findings that both viral and inflammatory component (NF-κB) can directly induce FGF8b upregulation in human cancer cells may imply the potential involvement of FGF8b in a wider spectrum of human cancers, especially cancers of EBV or inflammatory origin.

LMP1 is one of the most oncogenic proteins encoded by the EBV genome. Our study has identified LMP1 as the first viral oncogene capable of inducing FGF8b upregulation in human cancer. This novel LMP1/FGF8b induction mechanism may represent a new role of EBV in human oncogenesis. A potential role of viral infection in FGF8 regulation (not for FGF8b in particular) has been previously implicated in a transgenic mouse system; however, it was mainly described as a viral gene insertion mechanism. MacArthur et al. (1995c) demonstrated in a mouse mammary tumor virus-infected Wnt-1 transgenic mouse system that mouse mammary tumor virus was able to activate murine FGF8 expression by proviral insertion into the mouse genome at the FGF8 locus, thus activating FGF8 transcription (MacArthur et al., 1995c). Although this viral-mediated genomic insertion mechanism for FGF8b gene upregulation has not been confirmed in human breast cancer system, it is unlikely that similar viral insertion mechanism (by EBV) would be responsible for FGF8b overexpression in NPC, as EBV integration into the human genome is uncommon in NPC (Ohshima et al., 1998). Nevertheless, LMP1 has not been indicated in the EBV integration process in NPC, thus far. Another possible mechanism of LMP1-induced FGF8b upregulation in NPC would be via the NF-κB pathway as LMP1 is known to induce the activation of this key inflammatory transcriptional factor in NPC (Tsao et al., 2002). This is consistent with our finding that NF-κB inhibitor could suppress FGF8b expression, whereas the common activator of NF-κB, TNF-α, stimulated FGF8b expression in EBV-associated NPC cells lines. Indeed, promoter analysis by us and others indicated that several putative NF-κB-binding sites are located in the promoter region of the human FGF8b gene (data not shown) (Armstrong et al., 2006). Additional mechanisms may also contribute to this previously undescribed regulation of FGF8b by LMP1, as LMP1 is known to activate various signaling pathways involved in gene transcription/regulation, including STATs, p53, Id-1 and so on (Liao et al., 2001; Tsao et al., 2002; Dawson et al., 2003; Lui et al., 2009b), which may require further in-depth investigations.

Cumulative evidences demonstrated that this head and neck cancer, which originates from the epithelium of the nasopharynx, requires a complex interplay of cellular factors in the nasopharynx, EBV and the inflammatory tumor microenvironment. It is interesting to note that results from the current study revealed that both viral and inflammatory factors are involved in the upregulation of FGF8b, as both are known to be important factors involved in NPC pathogenesis. Moreover, FGF8 (although studies have not dissected in details which FGF8 isoforms are in particular involved) is known to be crucially involved and developmentally regulated in the organogenesis of the head and neck region, including the craniofacial and nasopharynx (Heikinheimo et al., 1994; Tucker et al., 1999). It is likely that dysregulation of FGF8 expression (possibly FGF8b) is involved in NPC pathogenesis, as possibly contributed by both viral and inflammatory factors. Findings from this study may provide important information for the complex model of NPC pathogenesis.

Our finding that NF-κB signaling is involved in FGF8b upregulation in NPC has several important implications in NPC. As NF-κB stimulation by TNF-α increased FGF8b expression in NPC cells, it is possible that TNF-α (especially TNF-α is known to be elevated in NPC patients) or other NF-κB-activating cytokines or cellular factors (for example, EGF) may mediate FGF8b overexpression in NPC in situ. It is likely that these inflammatory signals or NF-κB-activating cytokines or growth factors may induce a general inflammatory tumor microenvironment in which the immediate adjacent epithelial cells around the tumors are also exposed to FGF8b-inducing signals. A recent study by Song et al. (2000) demonstrated that coculturing of prostate cancer cells and prostate stromal cells induced cellular proliferation of the prostate stromal cells, indicating a complex interaction of cancer cells with stromal tissue (Song et al., 2000). It is unclear whether such a tumor–stromal interaction exists in NPC or not. Results from this study encourage further investigation in NPC on the existence of such a tumor–stromal interaction and the actual pathological contribution of various NF-κB-activating factors to FGF8b upregulation in NPC.

NPC is not a hormone-related cancer. Our data demonstrated that FGF8b overexpression occurs at a high rate in NPC patients of both genders, which is rather different from two independent studies in esophageal cancer where FGF8b overexpression is mainly found in male, but completely absent in female patients (Tanaka et al., 2001), and only two cases of female esophageal cancers (Awan et al., 2007) (and of course, only male patients in prostate cancer cases). Although such a distinct gender-specific pattern of FGF8b expression was not observed in NPC, nevertheless, there seemed to be a relatively higher level of FGF8b expression in NPC tumors from male than female patients. We cannot exclude at the moment the possibility that male NPC patients may have other predisposing factors linked to elevated FGF8b expression. Therefore, further investigations on gender inclination, clinical association with prognosis, disease progression and metastasis in larger cohort studies in NPC are warranted.

In summary, we have identified the functional involvement of FGF8b in NPC tumorigenesis, as well as a novel viral mechanism of LMP1/FGF8b upregulation, in addition to an inflammation-related mechanism (via NF-κB) for FGF8b upregulation in NPC. These may facilitate the development of therapeutic approach for FGF8b targeting in NPC.

Materials and methods

See Supplementary data for Materials and methods.

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Acknowledgements

Financial support by research fund, Clinical Oncology, CUHK (VWYL). We thank Drs Q Tao and GT Chung for experimental assistance.

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Correspondence to V W Y Lui or A T C Chan.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Oncogene website

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Lui, V., Yau, D., Cheung, C. et al. FGF8b oncogene mediates proliferation and invasion of Epstein–Barr virus-associated nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b upregulation. Oncogene 30, 1518–1530 (2011) doi:10.1038/onc.2010.529

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Keywords

  • FGF8b
  • NPC
  • LMP1
  • NF-κB

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