Introduction
Nasopharyngeal carcinoma (NPC) is a common malignant disease in Southern China where its annual incidence rate is more than 20 cases per 100,000 populations. Men are twice as likely to develop NPC as women. The incidence generally increases from ages 20 to 50 [
1]. Because the primary anatomical site of tumor growth is located in a cryptic area and the disease is usually asymptomatic at early stages, the NPC patients tend to present at a more advanced stage with higher metastatic potential. Therefore, it is of great interest to find effective biomarkers for early diagnosis of as well as therapeutic targets for this malignancy.
The tumorigenesis and metastasis of NPC are complex and continuous processes that involve a cascade of oncogene activations [
1,
2]. Latent membrane protein 1 (
LMP1), a protein encoded by Epstein-Barr virus (EBV), is considered as a major oncogenic protein promoting the development of NPC.
LMP1-positive cells have greater mobility, leading to higher metastatic potential [
3] and faster disease progression [
4]. Protein
XBP-1 induces
LMP1 expression, and knockdown of
XBP-1 blocks up-regulation of
LMP1 in NPC cells. Furthermore,
XBP-1 significantly correlates with
LMP1 expression in NPC tumor biopsies, suggesting that
XBP-1 can promote virus-associated cancer in a unique way by driving expression of a viral oncogene [
5].
Survivin is an inhibitor of apoptosis and promoter of cell proliferation, and it is usually absent in normal adult tissues but present in numerous tumors [
6]. In NPC,
survivin has been constantly found to be over-expressed [
7]. Inhibiting survivin expression can decrease NPC cell viability as well as increase tumor radiosensitivity [
8].
In a previous study, we investigated differentially expressed genes between non-cancerous nasopharyngeal tissues and NPC tissues using cDNA microarray. One of the genes showed markedly increased expression in NPC was eukaryotic translation initiation factor (
EIF4G1) [
7].
EIF4G1 participates in protein translation by serving as a scaffold for several other initiation factors [
9]. In addition, recent studies have found that EIF4G1 possesses tumorigenesis activities including promoting angiogenesis [
10], inducing malignant transformation [
11], and inhibiting apoptosis [
12]. In a variety of cancers, including NPC, lung cancer, and breast cancer, the expression levels of
EIF4G1 are significantly up-regulated [
13‐
16].
In the present study, we investigated the role of EIF4G1 in the pathogenesis of NPC, and examined its association with clinicopathologic features, including the survival of patients with NPC. We identified the effects of EIF4G1 on cell growth, invasion, and in vivo tumorigenesis, confirmed the increased expression levels of EIF4G1 in primary NPC tissues and cell lines, and found an inverse correlation between the levels of EIF4G1 and clinical outcome of patients with NPC.
Discussion
It is well known that the majority of NPC deaths result from tumor metastases rather than from primary tumors. However, the molecular mechanisms underlying the regulation of tumor cell invasion and metastasis of NPC remain incompletely understood.
EIF4G1, a member of the translational initiation factor family, is recognized as the central organizing protein in the recruitment of mRNA during translational initiation. Gradually accumulated evidences indicate that increased
EIF4G1 expression may play an oncogenic role and promote the process of tumorigenesis. For instance, over-expression of
EIF4G1 has been found in inflammatory breast cancer (IBC) and promotes the formation of IBC tumor emboli by enhancing translation of IRES-containing
p120 mRNA, which facilitated tumor cell survival [
15]. In addition, frequent amplification of
EIF4G1 on chromosome 3q27.1 and over-expression of
EI4G1 mRNA was also displayed in lung cancer and hypopharyngeal cancer [
13,
14], which suggest the involvement of
EIF4G1 in tumorigenesis [
11,
20]. Yet, it remains largely unknown about the role of
EIF4G1 in the pathogenesis of NPC.
In the previous microarray study, we found that the levels of
EIF4G1 mRNA in NPC were increased compared to noncancerous nasopharyngeal tissues [
7]. In this report, we further presented the proof that
EIF4G1 was over-expressed at both mRNA and protein levels in NPC tissues and cell lines. Furthermore, we found that
EIF4G1 over-expression was significantly associated with T classification, lymph node involvement, and clinical stages of NPC patients. Our results also indicated that
EIF4G1 protein expression was inversely correlated with patients' overall survival time. NPC patients with higher expression levels of
EIF4G1 protein had shorter survival time, and the protein levels of
EIF4G1 were an independent prognostic factor. These results suggested the clinical significance of
EIF4G1 as a biomarker for NPC prognosis.
To understand the biological functions of EIF4G1, we employed the loss-of-function approach, that is, by knocking down the expression level of endogenous EIF4G1. To achieve this purpose, we chose to use NPC 5-8F cells, a cell line with high tumorigenic and metastatic ability. The 5-8F cells exhibit the highest expression levels of endogenous EIF4G1 in all the six NPC cell lines examined, and thus represent an ideal model for our study. Using this system, we identified the roles of EIF4G1 in promoting cell proliferation, cell cycle, migration, and invasion; we further found that EIF4G1 could enhance tumorigenesis in vivo. These results strongly suggest an oncogenic role of EIF4G1 in NPC development.
EIF4G1 may be used as a useful molecular marker for NPC and an indicator for tumor progression and prognosis. In combination with other biomarkers of NPC,
EIF4G1 would be useful for novel therapeutic strategies. Assembly of the
EIF4E/EIF4G complex plays a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the
4EBP1, a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (
EIF4E), which compete with
EIF4G1 for binding to
eIF4E and have tumor-suppressor activity[
21]. To pharmacologically mimic 4E-BP function Moerke et al developed the most potent small-molecule compound 4EGI-1 for disrupting
EIF4E/EIF4G association, which inhibited cap-dependent translation but not initiation factor independent translation. While 4EGI-1 displaces
EIF4G from
EIF4E, it effectively enhances
4EBP1, a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (
EIF-4E), association with
EIF4E both in vitro and in cells, and suppressed cell proliferation [
22,
23]. Additionally,
EIF4G1 has been reported to implicate in another signal pathway. For example,
EIF4A/EIF4G complex has been shown to be involved in the key regulation of translation initiation. Tumor suppressor
PDCD4[
24,
25] could compete with
EIF4A for binding to
EIF4G1 and thus inhibit the initialization of translation [
26]. Although
PDCD4 is known to be a key regulator that controls tumor growth and invasion [
27], the role of
PDCD4 in NPC progression remains unclear. Recently, in a microarray study, we observed a significant down-expression of
PDCD4 in NPC samples [
7]. We therefore speculated that
EIF4G1 may play a role in NPC by inhibiting the functions of
PDCD4. In the present study, we found that the reduction of endogenous
EIF4G1 protein expression increased the expression levels of
PDCD4. This result supports the potential involvement of
EIF4G1 in regulating
PDCD4 expression.
In summary, our results provided the first evidence that EIF4G1 may be involved in the development of NPC. In addition, we also demonstrated that EIF4G1 could serve as a biomarker for the prognosis of NPC. Further works are needed to investigate the mechanisms and pathways of NPC pathogenesis mediated by EIF4G1.
Materials and methods
Cell Culture and Sample Collection
NPC cell lines 5-8F, 6-10B, CNE2, HONE1, HNE1, and SUNE1 were maintained in RPMI 1640 culture medium supplemented with 10% newborn calf serum (NBCS) (PAA Laboratories, Austria). NP69, an immortalized human nasopharyngeal epithelial cell line, was grown in defined-KSFM medium supplemented with EGF (Invitrogen, Carlsbad, CA). All of the cell lines were incubated in a humidified chamber with 5% CO2 at 37°C. Seven primary NPC tissues, 5 noncancerous nasopharyngeal tissues, 132 paraffin-embedded NPC specimens (107 cases with prognosis information), and 53 noncancerous paraffin-embedded nasopharyngeal specimens were obtained from People's Hospital of Zhongshan City, China, at the time of diagnosis before any therapy. An informed consent was obtained from each patient. All freshly collected samples were immediately stored in liquid nitrogen.
Real-time PCR
Real-time PCR was performed to measure the expression of
EIF4G1 mRNA in NPC cell lines, tumor tissues, or normal nasopharyngeal tissues using SYBR Premix Ex Taq (Takara, Japan) as described previously [
28]. The primers used were: forward 5'-TTGTGGATGATGGTGGCT-3' and reverse 5'-TTATCTGTGCTTTCTGTGGGT-3'.
GAPDH was used as an internal control and the primers were: forward 5'-GCACCGTCAAGGCTGAGAAC-3' and reverse 5'-TGGTGAAGACGCCAGTGGA-3'. Each measurement was repeated three times.
Immunohistochemistry
Paraffin sections (3 μm) from 132 NPC and 53 nasopharyngeal samples were deparaffinized in 100% xylene and re-hydrated in descending ethanol series according to standard protocols [
7]. Heat-induced antigen retrieval was performed in 10 mM citrate buffer for 2 min at 100°C. The endogenous peroxidase activity and non-specific antigens were blocked with peroxidase blocking reagent containing 3% hydrogen peroxide and serum. The slides were incubated with rabbit anti-human
EIF4G1 antibody (1:50) (Cell Signaling Technology, CA, USA) for 1 h at 37°C. After washing, the sections were incubated with biotin-labeled goat anti-rabbit IgG antibody for 10 min at room temperature followed by incubation with streptavidin-conjugated horseradish peroxidase (HRP) (Maixin Inc, China). The peroxidase reaction was developed with 3,3-diaminobenzidine chromogen solution in DAB buffer substrate. The slides were reviewed and scored independently by two pathologists blinded to the clinical parameters. The staining intensity was scored as described [
29,
30]. The extent of the staining, defined as the percentage of positive staining cells in relation to the whole field, was scored on a scale of 0 to 3 as 0: < 10%; 1: 10-25%; 2: 26-75%; and 3: ≥ 76%. The sum of the staining-intensity and staining-extent scores (0-6) was used as the final staining score for
EIF4G1. For statistical analysis, a final staining score of 0, 1~2, 3~4, and 5~6 were respectively considered to be negative, low, medium, and high expression levels.
Establishment of NPC 5-8F cell line with stably expressing shRNA-EIF4G1
The construction of lentiviral vector of shRNA targeting
EIF4G1 and establishment of NPC 5-8F cell line stably expressing shRNA had been described previously [
17,
18,
31]. In brief, we selected one sequence for targeting the
EIF4G1 gene using the BLOCK-It RNAi Designer (Invitrogen, Carlsbad, CA). The preparation of lentiviral vectors expressing human
EIF4G1 short hairpin RNA (shRNA) was performed using the BLOCK-It Lentiviral RNAi Expression System (Invitrogen, Carlsbad, CA), following the manufacturer's instruction and replication-incompetent lentivirus was produced by cotransfection of the pLenti6/
EIF4G1 expression vector and ViraPower packaging mix (Invitrogen) containing an optimized mixture of three packaging plasmids: pLP1, pLP2, and pLP/VSVG into 293FT cells. NPC 5-8F cells were infected with lentiviral particles containing specific or negative control vectors and selected for stable integrants by culturing a complete medium containing blasticidin. After 15 days of selection, there were no viable cells in mock wells and discrete blasticidin resistance colonies. The total RNA of these cell clones was isolated, and the levels of
EIF4G1 mRNA were measured using real-time PCR as described above.
Western blot Analysis
Cells were lysed in RIPA Buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 5 mM DTT, 2% SDS), and protein concentration was determined using BCA assay (Beyotime Inc, China). Total protein (30 μg) was resolved using a 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The primary antibodies used were rabbit polyclonal anti-EIF4G1 antibody(1:500), anti-ACTB antibody(1:400) (both from Santa Cruz Biotechnology, CA, USA) and PDCD4(1:500, Abcam Inc., Cambridge, MA). An HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Zhongshan Inc, China).
Cell Proliferation and Cell Cycle Analyses
Cell proliferation was analyzed using MTT assay (Sigma, St. Louis, USA). Briefly, 1 × 103 cells were seeded into a 96-well plate with quadruplicate repeat for each condition. After 72 h of incubation, MTT reagent was added to each well and incubated for 4 h. The formazan crystals formed by viable cells were then solubilized in DMSO and measured at 490 nm for the absorbance (A) values. Each experiment was performed in triplicates.
To evaluate cell cycle distribution, cells were seeded on 10 cm-diameter plates in RPMI 1640 culture medium containing 10% NBCS. After 48 h of incubation, a total of 1 × 106 cells were harvested, rinsed with cold PBS, and fixed with 70% ice-cold ethanol for 48 h at 4°C. Fixed cells were rinsed with cold PBS followed by incubation with PBS containing 10 μg/mL propidium iodide and 0.5 mg/mL RNase A for 15 min at 37°C. The DNA content of labeled cells was acquired using FACS Caliber cytometry (BD Biosciences). Each experiment was performed in triplicates.
In vitro Cell Migration and Invasion Assays
Cells growing in the log phase were treated with trypsin and re-suspended as single-cell solution. A total of 1 × 105 cells were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning Inc., Corning, NY). In the lower chamber, 600 μl of RPMI 1640 with 10% NBCS was added as chemoattractant. After the cells were incubated for 12 h, the insert was washed with PBS, and cells on the top surface of the insert were removed by a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa, and counted under a microscope in five predetermined fields (×200). For the Matrigel invasion assay, the procedure was similar with the cell migration assay, except that the transwell membrane was precoated with ECMatrix™ and the cells were incubated for 14 h. All assays were independently repeated at least three times.
About 100 cells were added to each well of a 6-well culture plate, and each cell group contained 2 wells. After 2 weeks of incubation, cells were washed twice with PBS and stained with Giemsa solution. The number of colonies containing ≥ 50 cells was counted under a microscope. The colony formation efficiency was calculated as: efficiency = (number of colonies/number of cells inoculated) × 100%. Each experiment was performed in triplicates.
Growth of Tumor Xenografts in Nude Mice
All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals under assurance number A3873-1. Nude mice of 4 to 6 weeks old (n = 6) were purchased from the Animal Center, Southern Medical University. A total of 1 × 106 cells growing in log phase were re-suspended in serum-free RPMI 1640 medium and injected subcutaneously into the nude mice. To minimize inter-individual variation, cells containing control vector and cells containing shRNA-EIF4G1 vector were injected symmetrically to the either flank of the same mice. After 25 d, mice were sacrificed and tumors isolated and weighted. EIF4G1 protein levels in the xenograft tumors were examined using immunohistochemistry assay.
Statistical analysis
All data were analyzed for statistical significance using SPSS 13.0 software. The Mann-Whitney U test was applied to the analysis of relationship between EIF4G1 expression levels and clinicopathologic characteristics. Survival analysis was performed using Kaplan-Meier method (the log-rank test). Multivariate Cox proportional hazards method was used for analyzing the relationship between the variables and patient's survival time. One-way ANOVA was used to determine the differences between groups for all in vitro and in vivo analyses. A P value of less than 0.05 was considered statistically significant.
Authors' contributions
LT, ZL, and WF carried out the molecular and cell biology studies. WF and ZL participated in the design of the study and performed the statistical analysis, collected, analyzed, and interpreted data and wrote the manuscript. XL interpreted data. GX revised the manuscript. XH, QJ, YH and SX collected and analyzed data. WF, KY supervised all the work. All authors have read and approved the final manuscript.