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(2011) 30, 1518–1530 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE FGF8b oncogene mediates proliferation and of Epstein–Barr -associated nasopharyngeal cells: implication for viral-mediated FGF8b upregulation

VWY Lui1,7, DM-S Yau1,7, CS-F Cheung1, SCC Wong1, AK-C Chan2, Q Zhou1, EY-L Wong1, CPY Lau1, EKY Lam1, EP Hui1, B Hong1, CWC Hui1, AS-K Chan1, PKS Ng3, Y-K Ng4, K-W Lo5, CM Tsang6, SKW Tsui3, S-W Tsao6 and ATC Chan1

1State Key Laboratory of in South , Sir YK Pao Center for , Department of Clinical Oncology, Chinese University of Hong Kong, Hong Kong; 2Department of Pathology, Queen Elizabeth Hospital, Hong Kong; 3School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong; 4Department of Surgery, Chinese University of Hong Kong, Hong Kong; 5Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Hong Kong and 6Department of Anatomy, University of Hong Kong, Hong Kong

The fibroblast growth factor 8b (FGF8b) oncogene is identified LMP1 as the first viral oncogene capable of known to be primarily involved in the tumorigenesis and directly inducing FGF8b (an important cellular oncogene) progression of hormone-related . Its role in other expression in human cancer cells. This novel mechanism of epithelial cancers has not been investigated, except for viral-mediated FGF8 upregulation may implicate a new esophageal cancer, in which FGF8b overexpression was role of in human . mainly found in tumor of male patients. These Oncogene (2011) 30, 1518–1530; doi:10.1038/onc.2010.529; observations were consistent with previous findings in published online 29 November 2010 these cancer types that the male sex-hormone androgen is responsible for FGF8b expression. Nasopharyngeal Keywords: FGF8b; NPC; LMP1; NF-kB carcinoma (NPC) is a highly metastatic cancer of head and neck commonly found in . It is etiologically associated with Epstein–Barr Virus (EBV) , inflammatory tumor microenvironment and relatively Introduction higher male predominance. Here, we reported for the first time that FGF8b is overexpressed in this EBV-associated Fibroblast growth factor 8b (FGF8b) is an oncogene non-hormone-related cancer of the head and neck, NPC. found to be predominantly overexpressed in hormone- More importantly, overexpression of FGF8b mRNA and related cancers, namely prostate and breast cancers protein was detected in a large majority of NPC tumors (Tanaka et al., 1998; Mattila and Harkonen, 2007). The from both male and female genders, in addition to multiple potent oncogenic activity of FGF8b has been demon- NPC cell lines. We hypothesized that FGF8b over- strated in vivo as FGF8b-overexpressing murine fibro- expression may contribute to NPC tumorigenesis. Using blasts causes transformation and rapid tumor formation EBV-associated NPC cell lines, we demonstrated that in nude mice (MacArthur et al., 1995a). Such a specific knockdown of FGF8b by small interfering RNA remarkable transforming ability of FGF8b was further inhibited cell proliferation, migration and invasion, demonstrated in transgenic mouse models overexpres- whereas exogenous FGF8b stimulated these multiple sing FGF8b. Transgenic mice overexpressing FGF8b phenotypes. Further mechanistic investigation revealed (under the control of the mouse mammary tumor virus that in addition to NF-jB signaling (a major inflamma- promoter, MMTV) frequently developed mammary tory signaling pathway known to be activated in NPC), an tumors with lung metastases, salivary gland tumors, important EBV oncoprotein, the latent membrane protein invasive lobular , as well as ductal 1 (LMP1), was found to be a direct inducer of FGF8b (Daphna-Iken et al., 1998). Similarly, tissue- overexpression in NPC cells, whereas androgen (testos- specific overexpression of FGF8b in the prostate terone) has minimal effect on FGF8b expression in EBV- of transgenic mice resulted in the develop- associated NPC cells. In summary, our study has ment of prostate neoplasia, stromal hyperplasia, pros- tate with lymph node (Song et al., 2002; Zhong et al., 2006). Consistent with these Correspondence: Dr ATC Chan or Dr VWY Lui, Department of findings, it has been demonstrated that FGF8b over- Clinical Oncology, The Chinese University of Hong Kong, Prince of expression in both prostate and breast cancer cells can Wales Hospital, Sha Tin, Hong Kong. promote tumor cell growth and formation in nude mice E-mails: [email protected] or [email protected] 7Denotes cofirst authorship (Song et al., 2000; Ruohola et al., 2001), whereas specific Received 11 March 2010; revised 15 October 2010; accepted 16 October downregulation of FGF8b by antisense RNA inhibited 2010; published online 29 November 2010 the in vivo tumorigenicity of prostate cancer cells FGF8b upregulation in NPC VWY Lui et al 1519 (Rudra-Ganguly et al., 1998). These cumulative evi- involved in the carcinogenesis of other human malig- dences establish the role of FGF8b in carcinogenesis of nancies, especially in those hormone-unrelated cancers. hormone-related cancers (Tanaka et al., 1998; Marsh Nasopharyngeal carcinoma (NPC) is a highly invasive et al., 1999; Gnanapragasam et al., 2003). and metastatic commonly found in FGF8b is a member of a large family of FGF8 Asia with a high incidence rate of 15–50/100 000 persons/ (previously known as the androgen-induced growth year (comparable with that of pancreatic cancer in the factor, AIGF). FGF8 is known to be essential for US) (Chan et al., 2002; Spano et al., 2003; Anderson embryogenesis (Crossley and Martin, 1995), in particu- et al., 2006). In endemic regions, NPC is characterized by lar, for the development/organogenesis of the cranio- its strong etiologic association with Epstein–Barr virus facial, pharyngeal, brain, cardiac, kidney, urogenital infection (EBV, a known oncogenic virus), high meta- organs and limbs (Heikinheimo et al., 1994; Crossley static potential (Skinner et al., 1991), highly inflamma- et al., 1996; Mattila and Harkonen, 2007). Investigation tory tumor microenvironment with heavy lymphocyte on the involvement of FGF8b in hormone-related infiltration (Lo and Huang, 2002), as well as a male cancers was indeed based on the initial discovery of predominance of 3:1 (Bhattacharyya, 2004). It is believed FGF8 (androgen-induced growth factor) in an andro- that NPC occurs as a result of complex interplay of local gen-dependent mouse mammary carcinoma cell line factors in the nasopharynx, EBV and the inflammatory (Tanaka et al., 1992) and detection of FGF8 over- tumor microenvironment. The molecular mechanisms expression in human breast and prostate cancers underlying its highly invasive and metastatic nature, as (Tanaka et al., 1995; Leung et al., 1996; Gnanapraga- well as the reported male predominance are not fully sam et al., 2002). Further genomic and functional understood. Here, we reported for the first time that characterization of the FGF8 family demonstrated that FGF8b is overexpressed in this EBV-associated non- the FGF8b isoform (one of the four human FGF8 hormone-related cancer of the head and neck, NPC. isoforms: FGF8a, b, e and f (Gemel et al., 1996)) More importantly, overexpression of FGF8b mRNA possesses the greatest mitogenic and, more importantly, and protein was detected in a large majority of NPC the greatest transforming activity than the other iso- tumors from both male and female genders. We forms, both in vitro and in vivo (MacArthur et al., 1995a, hypothesized that FGF8b overexpression contributed 1995b; Blunt et al., 1997; Song et al., 2000; Olsen et al., to NPC tumorigenesis. In addition to the identification 2006). It is believed that FGF8b is one of the most of NF-kB-mediated FGF8b upregulation in EBV- important FGF8 isoforms in human carcinogenesis associated NPC cells, this mechanistic investigation has (Marsh et al., 1999; Gnanapragasam et al., 2003), also identified latent membrane protein 1 (LMP1) as the although the exact physiological function of each first viral oncogene capable of directly inducing FGF8b isoform is yet-to-be fully revealed. (an important cellular oncogene) expression in human Owing to the original discovery of FGF8 in an cancer cells, which may reveal a new role of oncoviruses androgen-dependent system (Tanaka et al., 1992), as in human carcinogenesis. well as cumulative findings that FGF8b knockin mice developed prostate and breast cancers, investigations on the role of FGF8b in human oncogenesis have primarily Results been focused on hormone-related cancers. Clinical studies revealed that FGF8b overexpression was found FGF8b overexpression in NPC biopsies and cell lines in the majority of prostate cancer (40–80%) (Dorkin To investigate the possible involvement of FGF8b in et al., 1999; Gnanapragasam et al., 2002, 2003; Tanaka NPC tumorigenesis, we first examined the expression of et al., 2002; Zhong et al., 2006) and breast cancer (B70– FGF8b mRNA in 26 NPC tumor biopsies (18 from 100%) (Marsh et al., 1999). Moreover, its overexpres- males and 8 from females) by reverse transcriptase–PCR sion was clinically associated with disease progression (RT–PCR). Normal human colon epithelial tissues (as (mainly staging and ), poor prognosis, poor well as a normal esophageal epithelial control cell line, survival (Dorkin et al., 1999; Valve et al., 2001), and Het-1A; ATCC, Manassas, VA, USA) were included for potentially metastasis in prostate cancer (Gnanapraga- comparison. Figure 1A showed that normal control sam et al., 2003; Valta et al., 2008). Although the tissues expressed very low basal levels of FGF8b mRNA, molecular mechanisms for FGF8b overexpression in whereas 21/26 (B81%) NPC tumors showed elevated prostate and breast cancers are not fully understood, the FGF8b mRNA expression (X1.5-fold higher expression human sex hormone androgen (Tanaka et al., 1992) is versus controls), ranging from 1.51- to 6.28-fold higher known to play a key role, even in the case of breast expression versus control tissues. Statistical analysis cancer (Castellano et al., 2010 Park et al., 2010) (in demonstrated a significant overexpression of FGF8b addition to prostate cancer (Gnanapragasam et al., mRNA versus normal controls (P ¼ 0.0048**, Mann– 2002)). Similar findings in esophageal cancer, which has Whitney test) (Figure 1B). Interestingly, FGF8b mRNA recently found to be androgen-related (Yamashita et al., overexpression was found in a very high percentage of 1989; Awan et al., 2007; Thaver et al., 2009), also NPC tumors from both genders: 14/18 tumors in male revealed that FGF8b overexpression is linked to NPC (78%) and 7/8 tumors in female patients (88%) androgen signaling (Tanaka et al., 2001; Awan et al., (Figure 1A). This finding in NPC was distinct from 2007). With the limited number of cancer types being previous reports in esophageal cancer, in which FGF8b investigated thus far, it is unclear whether FGF8b is overexpression was mainly found in tumor biopsies of

Oncogene FGF8b upregulation in NPC VWY Lui et al 1520 male patients (absent in female esophageal adenocarci- confirmed with positive control tissues (normal breast noma in one report; Tanaka et al., 2001, and detected epithelium) as a previous study demonstrated that only in two female cases in another report; Awan et al., ‘FGF8b-positive staining was exclusively found in 2007). Indeed, immunohistochemical staining also con- normal breast epithelium (and breast cancer cells), firmed FGF8b protein overexpression in NPC tumors but absent in stromal cells’ (Tanaka et al., 2002). An from both genders, as indicated by intense FGF8b- additional normal control tissue (normal colon epithe- positivity (Figure 1C). Immunostaining for human lial tissue) was also included for FGF8b expression in FGF8b protein was optimized and performed using normal tissues. Consistent with the previous findings, an automated Bond-max immunostaining platform our results showed that only normal breast epithelium, (Mount Waverly, Victoria, Australia; see Materials but not the adjacent stromal tissue, was distinctly and methods). Specificity of FGF8b staining was stained positive for FGF8b (Figure 1C). Moreover, the

Figure 1 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 ¼ 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 Po0.05 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.

Oncogene FGF8b upregulation in NPC VWY Lui et al 1521

Figure 1 Continued. normal colon epithelium also showed low level of prostate cancer (Valta et al., 2008), where FGF8b FGF8b expression, which is consistent with our finding expression was exclusively found in tumor cells. As NPC using RT–PCR (Figure 1A). Similar finding was is well known to have a highly inflammatory tumor observed in normal lung epithelium (data not shown). microenvironment with viral infection (thus named In an additional cohort of 40 NPC tumors from 24 male because of heavy lymphocyte infil- and 16 female patients, intense cytoplasmic FGF8b tration), it is likely that such an inflammatory micro- staining was observed in all NPC tumor tissues and their environment plays a key role in such a wide-spread corresponding adjacent normal nasopharyngeal epithe- upregulation of FGF8b in NPC. lial cells (Figure 1C), when compared with the normal Consistent with the observed FGF8b overexpression control tissues. The median cytoplasmic FGF8b in NPC tumor biopsies, FGF8b mRNA and protein (cFGF8b) immunohistochemistry (IHC) scores for expression was also found to be elevated in a panel of NPC tumor cells and adjacent normal cells were 204 seven NPC cell lines tested when compared with the and 264, respectively (with no statistically significant control Het-1A cells (Figure 1D) (C666-1 was from differences between the IHC scores, P ¼ 0.3813, Wilcox- undifferentiated NPC origin; HONE-1, HONE-1-EBV, on signed-rank test) (Supplementary Figure 1). As HONE-1-LMP1(B95.8) and HONE-1-LMP1(2117) shown in Figure 1C, intense cytoplasmic FGF8b were from poorly differentiated NPC; whereas HK1 staining was observed in both male and female NPC and HK1-EBV were derived from well-differentiated biopsies, and no statistically significant difference NPC). Among all NPC cell lines tested, the poorly was found between male and female cases (median differentiated NPC cell line, HONE-1-LMP1(B95.8), cFGF8b IHC scores for male and female were 208.5 and expressed the highest level of FGF8b protein 194.5, respectively; P ¼ 0.4314, Mann–Whitney test). (Figure 1D), whereas the control Het-1A cells only This result, together with our RT–PCR findings expressed a barely detectable level of FGF8b protein. (Figure 1A), did indicate an overall upregulation of Cumulative results showed that stable expression of FGF8b protein in both NPC tumor cells and the LMP1(B95.8) was able to induce FGF8b upregulation immediate epithelial tissues around the tumors. This to B8-fold when compared with the parental HONE-1 interesting phenomenon was not observed with previous cells (Supplementary Figure 2), suggesting the potential findings in esophageal cancer (Tanaka et al., 2001) nor involvement of LMP1 in FGF8b upregulation in NPC.

Oncogene FGF8b upregulation in NPC VWY Lui et al 1522 FGF8b siRNA inhibits NPC cell proliferation, migration transfectable EBV-associated NPC cell lines were and invasion chosen: HONE-1-LMP1(B95.8) (poorly differentiated To examine the functional involvement of FGF8b NPC origin with the highest level of FGF8b protein overexpression in NPC tumorigenesis, we determined expression) and HONE-1-EBV (representative of EBV- the effects of specific knockdown of FGF8b by small genome-positive NPC cells). HK1 and HK1-EBV interfering RNA (siRNA) in NPC cells. Two highly were not transfectable. Figure 2a showed that transient

Figure 2 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 ¼ 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 Â 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 1Cina5%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 ¼ 4–10 fields/chamber) was counted under light microscope ( Â 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 Â 105 cells) were assessed as above. Similar results were observed in three independent experiments.

Oncogene FGF8b upregulation in NPC VWY Lui et al 1523

Figure 2 Continued. transfection with FGF8b siRNA (300 pmol) inhibited invasion of HONE-1-LMP1(B95.8) and HONE-1-EBV cellular proliferation of HONE-1-EBV and HONE-1- by 40.1 and 35.3% (at 24 h), respectively (Figure 3b). LMP1(B95.8) by 45.6 and 44.2% at 72 h, respectively, Similarly, migration abilities of these cells were also when compared with control siRNA transfection. enhanced moderately by FGF8b stimulation (37.8 and Moreover, functional knockdown of FGF8b also 21.2% increase for HONE-1-LMP1(B95.8) and HONE- resulted in reduction of cellular migration and invasion 1-EBV, respectively) (Figure 3c). This FGF8b-induced abilities of these NPC cells. The transwell migration invasion of NPC cells was consistent with the observed abilities of HONE-1-EBV and HONE-1-LMP1(B95.8) upregulation of mesenchymal markers, vimentin and were reduced by 34.4 and 50.9%, respectively N-cadherin by FGF8b treatment at 24 h (Figure 3d). In (Figure 2b). Similarly, the matrigel invasion abilities another EBV-positive NPC cell line of undifferentiated of HONE-1-EBV and HONE-1-LMP1(B95.8) were origin, C666-1, FGF8b also stimulated marked induc- reduced by 34.0 and 40.8%, respectively, upon FGF8b tion of cellular invasion by 115.5%. This further siRNA transfection (Figure 2c). These inhibitory confirmed the important role of FGF8b in NPC activities of FGF8b knockdown was also observed in invasion (Supplementary Figure 3). C666-1, an undifferentiated NPC cell line with lower transfectability. As shown in Figure 2d, siRNA- mediated knockdown of FGF8b was accompanied by FGF8b activates MAPK in NPC cells growth inhibition (32% versus control) and inhibition The key mitogenic pathway, mitogen-activated protein of migration and invasion of 71 and 78%, respectively. kinase (MAPK), is known to be the major downstream This demonstrated that FGF8b was functionally signaling pathway of FGF8b (Ishibe et al., 2005). Here, involved in NPC cell proliferation, migration and we examined whether FGF8b signals through MAPK invasion. 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 Exogenous FGF8b promotes NPC cell growth, migration FGF8b stimulation resulted in activation of MAPK and invasion with upregulation of mesenchymal markers (induction of phospho-p44/42 MAPK) as early as 10 Consistent with our FGF8b siRNA results, exogenous and 30 min upon stimulation (Figure 4A), indicating an FGF8b was able to induce NPC cell growth, migration immediate activation of MAPK by FGF8b in EBV- and invasion in vitro. The effect of exogenous FGF8b associated NPC cells. The involvement of MAPK stimulation was assessed in HONE-1-EBV and HONE- activation was further confirmed with FGF8b siRNA, 1-LMP1(B95.8) cells. Figure 3a showed that exogenous where FGF8b siRNA (versus control siRNA) transient human FGF8b (50 ng/ml) was able to stimulate transfection resulted in specific downregulation of proliferation of both NPC cell lines by B50% at 48 h. phospho-p44/42 MAPK in both HONE-1-EBV and In addition, exogenous FGF8b also promoted cellular HONE-1-LMP1(B95.8) (Figure 4B). Although another

Oncogene FGF8b upregulation in NPC VWY Lui et al 1524

Figure 3 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 Â 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 ¼ 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 ¼ 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.

Oncogene FGF8b upregulation in NPC VWY Lui et al 1525

Figure 4 (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.

major growth-related signaling pathway, AKT was NPC is characterized by its inflammatory pathology not significantly altered in these EBV-associated NPC and the demonstrated involvement of a major inflam- cell lines upon FGF8b siRNA or exogenous FGF8b matory signaling pathway, the NF-kB pathway (Thorn- stimulation (data not shown). burg et al., 2003; Lo et al., 2006; Shi et al., 2006; Chou et al., 2008; Wong et al., 2009). A recent study in a NF-kB is an upstream regulator of FGF8b expression hormone-independent prostate line indicated in EBV-associated NPC cells, but not androgen that regulation of FGF8 expression (though not for Previous studies in hormone-related cancers (prostate FGF8b in particular) required NF-kB (Armstrong et al., and breast cancers), as well as esophageal cancer 2006). This prompted us to investigate whether this demonstrated that FGF8b expression was regulated major inflammatory signaling pathway in NPC was by androgen/testosterone (Tanaka et al., 1992, 2001; responsible for FGF8b regulation in EBV-associated Gnanapragasam et al., 2002). Given the relatively higher NPC cells. Using a specific inhibitor of NF-kB, BAY 11- expression levels of FGF8b mRNA in male NPC tumors 7082, we found that the expression of FGF8b protein (Figure 1A), we examined if androgen (in the form of was markedly reduced in HONE-1-EBV and HONE-1- testosterone) was responsible for FGF8b regulation in LMP1(B95.8) cells at 24 h (versus dimethyl sulfoxide EBV-associated NPC cells. As shown in Figure 5A, control), and such inhibition on FGF8b expression was exogenous stimulation of both HONE-1-EBV and found to persist even up to 48 h (Figure 5b, upper HONE-1-LMP1(B95.8) cells with testosterone up to panel). Our data indicated the potential involvement of 1000 ng/ml for 48 h did not result in significant induction NF-kB in FGF8b regulation in EBV-associated NPC of FGF8b mRNA, FGF8b protein nor its downstream cells. Moreover, transient transfection of both NPC cell p44/p42 MAPK pathway. Our results suggested that lines with siRNAs against human NF-kB p65 and p50 FGF8b may not be directly regulated by androgen in subunits were found to result in marked downregulation NPC, which seemed to be consistent with our observa- of FGF8b (Figure 5b, lower panel), confirming a direct tion that FGF8b overexpression was detected in both role of this inflammatory signaling pathway, NF-kB, male and female NPC tumors (Figure 1A). Thus, factors in FGF8b regulation in NPC cells (consistent with other than androgen may be responsible for the previous finding in prostate cancer cells) (Armstrong regulation of FGF8b overexpression in NPC cells. et al., 2006).

Oncogene FGF8b upregulation in NPC VWY Lui et al 1526

Figure 5 (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-kB signaling in FGF8b upregulation in NPC cells as specific inhibition of NF-kB 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 mg/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-kB 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-kBp50siRNA,p65 siRNA or control siRNA. Western blotting showing the expression of FGF8b, NF-kB p50 and p65 subunits on day 3 after transfection. Similar results were observed in three independent experiments. (c)TNF-a-induced expression of FGF8b was inhibited by BAY 11-7082. HONE-1-EBV or HONE-1-LMP1(B95.8) cells were cotreated with TNF-a (60 ng/ml) and BAY 11-7082 (7 mg/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 Â 105 cells/well) were transfected with 4 mgof control vector (pcDNA3.1/Hygro( þ )) 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.

Oncogene FGF8b upregulation in NPC VWY Lui et al 1527

Figure 5 Continued.

To further confirm that NF-kB is an upstream induction was found to be LMP1 specific, as the regulator of FGF8b expression in EBV-associated introduction of another important EBV protein, NPC cells, a well-known NF-kB stimulus, TNF-a, was LMP2A, did not result in FGF8b upregulation upon employed (TNF-a is also known to be overexpressed in transient transfection (data not shown). Taken together NPC patients) (Kuo et al., 1994; Cui et al., 2001). the results from the stable cell line and transient Both EBV-associated NPC cell lines were serum starved transfection (Figures 1d and 5d), we demonstrated for for 48 h and then subjected to TNF-a stimulation the first time that a viral oncoprotein of EBV origin, (60 ng/ml) either in the presence or absence of BAY namely LMP1, was a potent inducer of FGF8b protein. 11-7082 (7 mg/ml) to demonstrate the specificity of NF- This is the first viral protein identified to be a direct kB-dependent regulation of FGF8b expression in NPC inducer of FGF8b in human cancer. cells. Figure 5c showed that TNF-a 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 Discussion HONE-1-EBV (Figure 1D), TNF-a stimulation was found to induce FGF8b upregulation more prominently FGF8b overexpression has been shown to be primarily at a later time point at 48 h. Similarly, BAY 11-7082 was associated with hormone-related cancers in human, able to completely abrogate the expression of both basal mainly prostate and breast cancers. Its role in other and TNF-a-induced FGF8b protein in HONE-1-EBV epithelial cancers has not been investigated, except for at 24 h and in HONE-1-LMP1(B95.8) at 48 h, respec- esophageal cancer, where FGF8b overexpression was tively. Our finding demonstrated that NF-kB is a key mainly found in tumor biopsies of male patients. Here, regulator for FGF8b expression in NPC cells. 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 LMP1 oncoprotein directly induces FGF8b upregulation head and neck cancer. Interestingly, FGF8b overexpres- in EBV-associated NPC cells sion was detected in high percentage of NPC tumor The EBV oncoprotein, LMP1, is a key player in NPC biopsies from both male and female genders. Functional carcinogenesis (Zheng et al., 2007). Prompted by the studies demonstrated its involvement in NPC cell finding that the poorly differentiated LMP1-expressing proliferation and invasion. Androgen (testosterone) stable NPC cell line, HONE-1-LMP1(B95.8), did was found to have minimal effect on FGF8b expression express FGF8b at a much higher level than its LMP-1- in EBV-associated NPC cells. In addition to the negative parental cell line, HONE-1 (Figure 1D and identification of NF-kB-mediated FGF8b upregulation Supplementary Figure 2), we examined whether this in EBV-associated NPC cells (NF-kB is a major viral oncoprotein could directly regulate the expression inflammatory signaling pathway known to be activated of this important cellular oncoprotein, FGF8b. The in NPC), the current study has also identified the EBV parental HONE-1 cells, which do not express the LMP1 LMP1 as the first viral oncogene capable of directly oncoprotein, expressed a relatively lower level of FGF8b inducing FGF8b (an important cellular oncogene) protein (Supplementary Figure 4 and Figure 1D). expression in human cancer cells, which may reveal a However, when the LMP1 gene was introduced into new role of oncoviruses in human carcinogenesis. the parental HONE-1 cells by transient transfection, a The cellular mechanisms of FGF8b upregulation in marked induction of FGF8b upregulation was detected human cancers are not fully understood. Androgen (Figure 5d), which demonstrate a direct role of LMP1 in signaling is believed to be an important regulatory FGF8b induction. Moreover, this novel LMP1/FGF8b mechanism of FGF8b upregulation in prostate cancer axis was found to be associated with prominent (Gnanapragasam et al., 2002), as well as breast cancer activation of p44/42 MAPK. Furthermore, such an (Tanaka et al., 1998), and, more recently, in esophageal

Oncogene FGF8b upregulation in NPC VWY Lui et al 1528 cancer (Tanaka et al., 2001; Awan et al., 2007). transcription/regulation, including STATs, p53, Id-1 Of particular interest was the unanticipated clinical and so on (Liao et al., 2001; Tsao et al., 2002; Dawson association of androgen receptor expression and FGF8b et al., 2003; Lui et al., 2009b), which may require further expression in breast cancer (but not with estrogen nor in-depth investigations. progesterone receptor status) (Tanaka et al., 1995, Cumulative evidences demonstrated that this head 1998). Esophageal cancer (which has a male to female and neck cancer, which originates from the epithelium ratio of 7:1) (Blot et al., 1993) has recently been shown of the nasopharynx, requires a complex interplay of to be clinically associated with androgen dependence (as cellular factors in the nasopharynx, EBV and the indicated by elevated serum testosterone, overexpression inflammatory tumor microenvironment. It is interesting of androgen receptor, as well as FGF8b overexpression) to note that results from the current study revealed that (Awan et al., 2007). Although NPC has a relatively both viral and inflammatory factors are involved in the higher predominance for male (male:female ratio of 3:1) upregulation of FGF8b, as both are known to be (Bhattacharyya, 2004), we demonstrated for the first important factors involved in NPC pathogenesis. More- time that the EBV oncoprotein, LMP1, as well as NF- over, FGF8 (although studies have not dissected in kB are direct inducers of FGF8b expression in NPC details which FGF8 isoforms are in particular involved) cells, whereas androgen may not be a crucial factor for is known to be crucially involved and developmentally FGF8b overexpression in this EBV-associated cancer. regulated in the organogenesis of the head and neck Our findings that both viral and inflammatory compo- region, including the craniofacial and nasopharynx nent (NF-kB) can directly induce FGF8b upregulation (Heikinheimo et al., 1994; Tucker et al., 1999). It is likely in human cancer cells may imply the potential involve- that dysregulation of FGF8 expression (possibly FGF8b) ment of FGF8b in a wider spectrum of human cancers, is involved in NPC pathogenesis, as possibly contributed especially cancers of EBV or inflammatory origin. by both viral and inflammatory factors. Findings from LMP1 is one of the most oncogenic proteins encoded this study may provide important information for the by the EBV genome. Our study has identified LMP1 as complex model of NPC pathogenesis. the first viral oncogene capable of inducing FGF8b Our finding that NF-kB signaling is involved in upregulation in human cancer. This novel LMP1/ FGF8b upregulation in NPC has several important FGF8b induction mechanism may represent a new role implications in NPC. As NF-kB stimulation by TNF-a of EBV in human oncogenesis. A potential role of viral increased FGF8b expression in NPC cells, it is possible infection in FGF8 regulation (not for FGF8b in that TNF-a (especially TNF-a is known to be elevated particular) has been previously implicated in a trans- in NPC patients) or other NF-kB-activating genic mouse system; however, it was mainly described as or cellular factors (for example, EGF) may mediate a viral gene insertion mechanism. MacArthur et al. FGF8b overexpression in NPC in situ. It is likely that (1995c) demonstrated in a mouse mammary tumor these inflammatory signals or NF-kB-activating cyto- virus-infected Wnt-1 transgenic mouse system that kines or growth factors may induce a general inflam- mouse mammary tumor virus was able to activate matory tumor microenvironment in which the murine FGF8 expression by proviral insertion into the immediate adjacent epithelial cells around the tumors mouse genome at the FGF8 locus, thus activating FGF8 are also exposed to FGF8b-inducing signals. A recent transcription (MacArthur et al., 1995c). Although this study by Song et al. (2000) demonstrated that cocultur- viral-mediated genomic insertion mechanism for FGF8b ing of prostate cancer cells and prostate stromal cells gene upregulation has not been confirmed in human induced cellular proliferation of the prostate stromal breast cancer system, it is unlikely that similar viral cells, indicating a complex interaction of cancer cells insertion mechanism (by EBV) would be responsible for with stromal tissue (Song et al., 2000). It is unclear FGF8b overexpression in NPC, as EBV integration into whether such a tumor–stromal interaction exists in NPC the is uncommon in NPC (Ohshima or not. Results from this study encourage further et al., 1998). Nevertheless, LMP1 has not been indicated investigation in NPC on the existence of such a in the EBV integration process in NPC, thus far. tumor–stromal interaction and the actual pathological Another possible mechanism of LMP1-induced FGF8b contribution of various NF-kB-activating factors to upregulation in NPC would be via the NF-kB pathway FGF8b upregulation in NPC. as LMP1 is known to induce the activation of this key NPC is not a hormone-related cancer. Our data inflammatory transcriptional factor in NPC (Tsao et al., demonstrated that FGF8b overexpression occurs at a 2002). This is consistent with our finding that NF-kB high rate in NPC patients of both genders, which is inhibitor could suppress FGF8b expression, whereas rather different from two independent studies in the common activator of NF-kB, TNF-a, stimulated esophageal cancer where FGF8b overexpression is FGF8b expression in EBV-associated NPC cells lines. mainly found in male, but completely absent in female Indeed, promoter analysis by us and others indicated patients (Tanaka et al., 2001), and only two cases of that several putative NF-kB-binding sites are located in female esophageal cancers (Awan et al., 2007) (and of the promoter region of the human FGF8b gene (data not course, only male patients in prostate cancer cases). shown) (Armstrong et al., 2006). Additional mechan- Although such a distinct gender-specific pattern of isms may also contribute to this previously undescribed FGF8b expression was not observed in NPC, never- regulation of FGF8b by LMP1, as LMP1 is known to theless, there seemed to be a relatively higher level of activate various signaling pathways involved in gene FGF8b expression in NPC tumors from male than

Oncogene FGF8b upregulation in NPC VWY Lui et al 1529 female patients. We cannot exclude at the moment the Materials and methods possibility that male NPC patients may have other predisposing factors linked to elevated FGF8b expres- See Supplementary data for Materials and methods. sion. Therefore, further investigations on gender in- clination, clinical association with prognosis, disease progression and metastasis in larger cohort studies in Conflict of interest NPC are warranted. In summary, we have identified the functional The authors declare no conflict of interest. involvement of FGF8b in NPC tumorigenesis, as well as a novel viral mechanism of LMP1/FGF8b upregula- Acknowledgements tion, in addition to an inflammation-related mechanism (via NF-kB) for FGF8b upregulation in NPC. These Financial support by research fund, Clinical Oncology, may facilitate the development of therapeutic approach CUHK (VWYL). We thank Drs Q Tao and GT Chung for for FGF8b targeting in NPC. experimental assistance.

References

Anderson KE, Mack T, Silverman DT. (2006). Cancer of the Dorkin TJ, Robinson MC, Marsh C, Bjartell A, Neal DE, Leung HY. pancreas. In: Schottenfeld D, Fraumeni JF Jr (eds) Cancer (1999). FGF8 over-expression in prostate cancer is associated with and Prevention. Oxford University Press: New York, decreased patient survival and persists in androgen independent pp 721–762. disease. Oncogene 18: 2755–2761. Armstrong K, Robson CN, Leung HY. (2006). NF-kappaB activation Gemel J, Gorry M, Ehrlich GD, MacArthur CA. (1996). Structure and upregulates fibroblast growth factor 8 expression in prostate cancer sequence of human FGF8. Genomics 35: 253–257. cells. Prostate 66: 1223–1234. Gnanapragasam VJ, Robinson MC, Marsh C, Robson CN, Hamdy Awan AK, Iftikhar SY, Morris TM, Clarke PA, Grabowska AM, FC, Leung HY. (2003). FGF8 isoform b expression in human Waraich N et al. (2007). Androgen receptors may act in a paracrine prostate cancer. Br J Cancer 88: 1432–1438. manner to regulate oesophageal adenocarcinoma growth. Eur J Gnanapragasam VJ, Robson CN, Neal DE, Leung HY. (2002). Surg Oncol 33: 561–568. Regulation of FGF8 expression by the androgen receptor in human Bhattacharyya N. (2004). The impact of race on survival in prostate cancer. Oncogene 21: 5069–5080. nasopharyngeal carcinoma: a matched analysis. Am J Otolaryngol Heikinheimo M, Lawshe A, Shackleford GM, Wilson DB, MacArthur 25: 94–97. CA. (1994). FGF-8 expression in the post-gastrulation mouse Blot WJ, Devesa SS, Fraumeni Jr JF. (1993). Continuing climb in rates suggests roles in the development of the face, limbs and central of esophageal adenocarcinoma: an update. Jama 270: 1320. nervous system. Mech Dev 48: 129–138. Blunt AG, Lawshe A, Cunningham ML, Seto ML, Ornitz DM, Hong B, Lui VW, Hui EP, Ng MH, Cheng SH, Sung FL et al. (2009). MacArthur CA. (1997). Overlapping expression and redundant Hypoxia-targeting by tirapazamine (TPZ) induces preferential activation of mesenchymal fibroblast growth factor (FGF) receptors growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 by alternatively spliced FGF-8 ligands. J Biol Chem 272: 3733–3738. activation. Invest New Drugs (e-pub ahead of print 16 December Castellano I, Allia E, Accortanzo V, Vandone AM, Chiusa L, 2009). Arisio R et al. (2010). Androgen receptor expression is a significant Ishibe T, Nakayama T, Okamoto T, Aoyama T, Nishijo K, Shibata prognostic factor in estrogen receptor positive breast cancers. Breast KR et al. (2005). Disruption of fibroblast growth factor signal Cancer Res Treat 124: 607–617. pathway inhibits the growth of synovial : potential Chan AT, Teo PM, Johnson PJ. (2002). Nasopharyngeal carcinoma. application of signal inhibitors to molecular target therapy. Clin Ann Oncol 13: 1007–1015. Cancer Res 11: 2702–2712. Chou J, Lin YC, Kim J, You L, Xu Z, He B et al. (2008). Kuo WR, Yu HS, Chang KL, Juan KH, Jan YS, Yu CL. (1994). Nasopharyngeal carcinoma—review of the molecular mechanisms Increased production of tumor necrosis factor-alpha and release of of tumorigenesis. Head Neck 30: 946–963. soluble CD4 and CD8 molecules, but decreased responsiveness to Crossley PH, Martin GR. (1995). The mouse Fgf8 gene encodes a phytohemagglutinin in patients with nasopharyngeal carcinoma. family of polypeptides and is expressed in regions that direct J Formos Med Assoc 93: 569–575. outgrowth and patterning in the developing embryo. Development Leung HY, Dickson C, Robson CN, Neal DE. (1996). Over-expression 121: 439–451. of fibroblast growth factor-8 in human prostate cancer. Oncogene Crossley PH, Martinez S, Martin GR. (1996). Midbrain development 12: 1833–1835. induced by FGF8 in the chick embryo. Nature 380: 66–68. Liao W, Tang M, Yin L. (2001). [EBV latent membrane protein 1 Cui D, Zhao Y, Deng Y, Luo G, Su M, Chen X. (2001). [Clinical induces p53 expression via NF-kappa B in nasopharyngeal observation of TNF-alpha content in nasopharyngeal secretion of carcinoma]. Zhonghua Zhong Liu Za Zhi 23: 199–201. patients with nasopharyngeal carcinoma]. Lin Chuang Er Bi Yan Lo AK, Lo KW, Tsao SW, Wong HL, Hui JW, To KF et al. (2006). Hou Ke Za Zhi 15: 202–203. Epstein-Barr virus infection alters cellular signal cascades in human Daphna-Iken D, Shankar DB, Lawshe A, Ornitz DM, Shackleford nasopharyngeal epithelial cells. Neoplasia 8: 173–180. GM, MacArthur CA. (1998). MMTV-Fgf8 transgenic mice develop Lo KW, Huang DP. (2002). Genetic and epigenetic changes in mammary and salivary gland neoplasia and ovarian stromal nasopharyngeal carcinoma. Semin Cancer Biol 12: 451–462. hyperplasia. Oncogene 17: 2711–2717. Lui VW, Wong EY, Ho Y, Hong B, Wong SC, Tao Q et al. (2009a). Dawson CW, Tramountanis G, Eliopoulos AG, Young LS. (2003). STAT3 activation contributes directly to Epstein-Barr virus- Epstein-Barr virus latent membrane protein 1 (LMP1) activates the mediated invasiveness of nasopharyngeal cancer cells in vitro. phosphatidylinositol 3-kinase/Akt pathway to promote cell Int J Cancer 125: 1884–1893. survival and induce actin filament remodeling. J Biol Chem 278: Lui VW, Yau DM, Wong EY, Ng YK, Lau CP, Ho Y et al. (2009b). 3694–3704. Cucurbitacin I elicits anoikis sensitization, inhibits cellular invasion

Oncogene FGF8b upregulation in NPC VWY Lui et al 1530 and in vivo tumor formation ability of nasopharyngeal carcinoma Tanaka A, Kamiakito T, Takayashiki N, Sakurai S, Saito K. (2002). cells. Carcinogenesis 30: 2085–2094. Fibroblast growth factor 8 expression in breast carcinoma: MacArthur CA, Lawshe A, Shankar DB, Heikinheimo M, Shackle- associations with androgen receptor and prostate-specific antigen ford GM. (1995a). FGF-8 isoforms differ in NIH3T3 cell expressions. Virchows Arch 441: 380–384. transforming potential. Cell Growth Differ 6: 817–825. Tanaka A, Miyamoto K, Matsuo H, Matsumoto K, Yoshida H. MacArthur CA, Lawshe A, Xu J, Santos-Ocampo S, Heikinheimo M, (1995). Human androgen-induced growth factor in prostrate and Chellaiah AT et al. (1995b). FGF-8 isoforms activate receptor splice breast cancer cells: its molecular cloning and growth properties. forms that are expressed in mesenchymal regions of mouse FEBS Lett 363: 226–230. development. Development 121: 3603–3613. Tanaka A, Miyamoto K, Minamino N, Takeda M, Sato B, Matsuo H MacArthur CA, Shankar DB, Shackleford GM. (1995c). Fgf-8, et al. (1992). Cloning and characterization of an androgen-induced activated by proviral insertion, cooperates with the Wnt-1 transgene growth factor essential for the androgen-dependent growth of in murine mammary tumorigenesis. J Virol 69: 2501–2507. mouse mammary carcinoma cells. Proc Natl Acad Sci USA 89: Marsh SK, Bansal GS, Zammit C, Barnard R, Coope R, Roberts- 8928–8932. Clarke D et al. (1999). Increased expression of fibroblast growth Tanaka S, Ueo H, Mafune K, Mori M, Wands JR, Sugimachi K. factor 8 in human breast cancer. Oncogene 18: 1053–1060. (2001). A novel isoform of human fibroblast growth factor 8 is Mattila MM, Harkonen PL. (2007). Role of fibroblast growth factor 8 induced by androgens and associated with progression of esopha- in growth and progression of hormonal cancer. Growth geal carcinoma. Dig Dis Sci 46: 1016–1021. Factor Rev 18: 257–266. Thaver V, Lottering ML, van Papendorp D, Joubert A. (2009). In vitro Ohshima K, Suzumiya J, Kanda M, Kato A, Kikuchi M. (1998). effects of 2-methoxyestradiol on cell numbers, morphology, cell Integrated and episomal forms of Epstein-Barr virus (EBV) in EBV cycle progression, and apoptosis induction in oesophageal carcino- associated disease. Cancer Lett 122: 43–50. ma cells. Cell Biochem Funct 27: 205–210. Olsen SK, Li JY, Bromleigh C, Eliseenkova AV, Ibrahimi OA, Lao Z Thornburg NJ, Pathmanathan R, Raab-Traub N. (2003). Activation et al. (2006). Structural basis by which alternative splicing of nuclear factor-kappaB p50 homodimer/Bcl-3 complexes in modulates the organizer activity of FGF8 in the brain. Dev nasopharyngeal carcinoma. Cancer Res 63: 8293–8301. 20: 185–198. Tsao SW, Tramoutanis G, Dawson CW, Lo AK, Huang DP. (2002). Park S, Koo J, Park HS, Kim JH, Choi SY, Lee JH et al. (2010). The significance of LMP1 expression in nasopharyngeal carcinoma. Expression of androgen receptors in primary breast cancer. Ann Semin Cancer Biol 12: 473–487. Oncol 21: 488–492. Epub 2009 Nov 3. Tucker AS, Yamada G, Grigoriou M, Pachnis V, Sharpe PT. (1999). Rudra-Ganguly N, Zheng J, Hoang AT, Roy-Burman P. (1998). Fgf-8 determines rostral-caudal polarity in the first branchial arch. Downregulation of human FGF8 activity by antisense constructs in Development 126: 51–61. murine fibroblastic and human prostatic carcinoma cell systems. Valta MP, Tuomela J, Bjartell A, Valve E, Vaananen HK, Harkonen Oncogene 16: 1487–1492. P. (2008). FGF-8 is involved in bone metastasis of prostate cancer. Ruohola JK, Viitanen TP, Valve EM, Seppanen JA, Loponen NT, Int J Cancer 123: 22–31. Keskitalo JJ et al. (2001). Enhanced invasion and tumor growth of Valve EM, Nevalainen MT, Nurmi MJ, Laato MK, Martikainen PM, fibroblast growth factor 8b-overexpressing MCF-7 human breast Harkonen PL. (2001). Increased expression of FGF-8 isoforms and cancer cells. Cancer Res 61: 4229–4237. FGF receptors in human premalignant prostatic intraepithelial Shi W, Bastianutto C, Li A, Perez-Ordonez B, Ng R, Chow KY et al. neoplasia lesions and prostate cancer. Lab Invest 81: 815–826. (2006). Multiple dysregulated pathways in nasopharyngeal carcinoma Wong JH, Lui VW, Umezawa K, Ho Y, Wong EY, Ng MH et al. revealed by gene expression profiling. Int J Cancer 119: 2467–2475. (2009). A small molecule inhibitor of NF-kappaB, dehydroxymethy- Skinner DW, Van Hasselt CA, Tsao SY. (1991). Nasopharyngeal lepoxyquinomicin (DHMEQ), suppresses growth and invasion of carcinoma: modes of presentation. Ann Otol Rhinol Laryngol 100: nasopharyngeal carcinoma (NPC) cells. Cancer Lett 287: 23–32. 544–551. Wong SC, Chan JK, Lee KC, Hsiao WL. (2001). Differential Song Z, Powell WC, Kasahara N, van Bokhoven A, Miller GJ, Roy- expression of p16/p21/p27 and cyclin D1/D3, and their relationships Burman P. (2000). The effect of fibroblast growth factor 8, isoform to cell proliferation, apoptosis, and tumour progression in invasive b, on the biology of prostate carcinoma cells and their interaction ductal carcinoma of the breast. J Pathol 194: 35–42. with stromal cells. Cancer Res 60: 6730–6736. Wong SC, Lo SF, Lee KC, Yam JW, Chan JK, Wendy Hsiao WL. Song Z, Wu X, Powell WC, Cardiff RD, Cohen MB, Tin RT et al. (2002). Expression of frizzled- related protein and Wnt-signalling (2002). Fibroblast growth factor 8 isoform B overexpression in molecules in invasive breast tumours. J Pathol 196: 145–153. prostate epithelium: a new mouse model for prostatic intraepithelial Yamashita Y, Hirai T, Mukaida H, Kawano K, Toge T, Niimoto M neoplasia. Cancer Res 62: 5096–5105. et al. (1989). Detection of androgen receptors in human esophageal Spano JP, Busson P, Atlan D, Bourhis J, Pignon JP, Esteban C et al. cancer. Jpn J Surg 19: 195–202. (2003). Nasopharyngeal : an update. Eur J Cancer 39: Zheng H, Li LL, Hu DS, Deng XY, Cao Y. (2007). Role of 2121–2135. Epstein-Barr virus encoded latent membrane protein 1 in the Tanaka A, Furuya A, Yamasaki M, Hanai N, Kuriki K, Kamiakito T carcinogenesis of nasopharyngeal carcinoma. Cell Mol Immunol 4: et al. (1998). High frequency of fibroblast growth factor (FGF) 8 185–196. expression in clinical prostate cancers and breast tissues, immuno- Zhong C, Saribekyan G, Liao CP, Cohen MB, Roy-Burman P. (2006). histochemically demonstrated by a newly established neutralizing Cooperation between FGF8b overexpression and PTEN deficiency monoclonal antibody against FGF 8. Cancer Res 58: 2053–2056. in prostate tumorigenesis. Cancer Res 66: 2188–2194.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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