FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway

To measure the expression of the FLJ10540 and osteopontin transcripts in NPC tissues, fresh tissues were obtained from patients with NPC biopsy specimens. Controls included fresh normal nasopharyngeal mucosal tissues from other patients with biopsies for other non-neoplastic diseases. These materials were histologically confirmed by frozen sections before quantitative RT-PCR and western blotting assays. Available paraffin-embedded tissue blocks were retrieved from 63 NPC patients. Clinicopathological information for each subject, including gender, age, tumor-(T) stage, nodal-(N) status, TNM stage, and disease-specific survival (DSS), was obtained retrospectively from clinical records and pathology reports. NPC patients received local head and neck examinations before treatment, along with staging examinations, including whole body bone scans, abdominal ultrasonography, computed tomography, and/or magnetic resonance imaging. Using the 2002 American Joint Committee on Cancer staging system, 19 patients were classified as T1, 25 as T2, four as T3, and 15 as T4. Twenty-eight patients were classified as N0, 13 as N1, 18 as N2, and four as N3. Twelve patients were determined to be at stage I, 20 at stage II, 11 at stage III, and 20 at stage IV. The method of radiotherapy for NPC was, in general, uniform throughout this period. All patients were regularly monitored after receiving radiotherapy and/or chemotherapy until death or their last appointment, according to the intervals and protocols of follow-up as detailed in a previous study [3]. Survival data were either obtained from the cancer registry of our hospital or collected from the patients’ attending physicians. Locoregional failure was determined by pathological diagnosis or progressive deterioration, as shown on consecutive imaging studies. To identify distant metastases, patients underwent chest radiography yearly and bone scans or abdominal ultrasonography whenever indicated. All study participants provided informed consent, and study design was approved by the Medical Ethics and Human Clinical Trial Committee of Chang Gung Memorial Hospital.

Tissue samples were frozen in liquid nitrogen and stored at −80°C prior to RNA extraction. The RNA extraction, and quantitative RT-PCR assays were performed as described previously [4]. For Q-RT-PCR, FLJ10540 and osteopontin Taq-Man probes (ABI) were used. Data were represented as mean ± s.d. To analyze the distribution of normal and tumor areas, we used the Wilcoxon signed rank test between two groups for statistical analysis. A P-value of less than 0.05 was considered statistically significant. GAPDH was used as an internal control for comparison and normalization of the data. Assays were performed in triplicate using an Applied Biosystems Model 7500-Fast instrument.

For tissue protein extraction, frozen samples were homogenized in RIPA lysis buffer (50 mm Tris–HCl, pH 7.5, 150 mm NaCl, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% SDS). The western blotting assay was performed as described previously [4]. The antibodies used in this study included polyclonal antibodies against FLJ10540 (generated by us) [23], HA (3F10, Roche Biochemicals, Indianapolis, IN, USA), and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The proteins were investigated using X-ray films.

Normal and tumor NPC tissue samples were selected by a pathologist based on diagnosis and microscopic morphology. Immunohistochemical staining was performed as described previously [4, 23, 24]. After antigen retrieval, the sections were incubated with diluted anti-FLJ10540 antibody (polyclonal; generated by us; 1:200; polyclonal; Abnova, Taiwan 1:100), and anti-osteopontin (polyclonal; Abnova, Taiwan 1:100; polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50 at room temperature for 1 hour, followed by washing with PBS. Horseradish peroxidase/Fab polymer conjugate (PicTure™-Plus kit; Zymed, South San Francisco, CA, USA) was then applied to the sections for 30 min followed by washing with PBS. Finally, the sections were incubated with diaminobenzidine for 5 min to develop the signals. A negative control was run simultaneously by omitting the primary antibody. The reactivity level of the immunostained tissues was evaluated independently by two pathologists who were blind to the subjects’ clinical information. Between 15 and 20 high-power fields were viewed. Criteria were developed for quantitating the immunoreactivities of the osteopontin staining in both the normal and tumor sections using a score range of 0 to +3, where 0 indicated no positive cell staining, +1 less than 5% positive cell staining, +2 5-50% positive cell staining, and +3 more than 50% positive cell staining. Similarly, the stain intensity was graded as +0, +1, +2, or +3 as previously described [25]. The quantitating of the immunoreactivities of the FLJ10540 staining followed the protocol of osteopontin. High-expressions of FLJ10540 and osteopontin were defined as +2 or higher for both scoring methods.

NPC-TW01 and Hone-1 cell lines derived from primary nasopharyngeal tumors of untreated NPC patients were used for functional assays [26‐28]. All cell culture-related reagents were purchased from Gibco-BRL (Grand Island, NY, USA). TW01 cells were grown in DMEM, however the Hone1 cells were grown in RPMI containing 10% FBS and 100 U/ml penicillin and streptomycin (Gibco-BRL). HA-vector (pcDNA3.1), and HA-FLJ10540 were transiently transfected into cancer cells using Lipofectamine (Invitrogen) according to the manufacturer’s instructions. TW01 and Hone1 cells stably expressing FLJ10540 were selected with 400 μg/ml G418 (Calbiochem Novabiochem, San Diego, CA, USA). The cells were then harvested and analyzed for exogenous FLJ10540 expression by Western blotting. 5’-upstream fragments of FLJ10540 gene was amplified from human genomic DNA and verified by sequencing. The PCR fragments were cloned into firefly luciferase reporter vector pGL3-Basic (Promega) NheI and HindIII sites which were designed into the forward and the reverse primers, respectively. pRL-TK (Promega), a Renilla luciferase internal control vector, was used for normalization of firefly luciferase signals. For co-transfection experiments, TW01 cells were co-transfected with 100 ng firefly luciferase reporter plasmids (pGL3-Basic or pGL3-FLJ10540) and 10 ng of pRL-TK Renilla luciferase internal control plasmid. After 24h, the cells were incubated with serum-free medium for 18h after osteopontin was added. The Luciferase activity was measured using Dual Glo™ Luciferase Assay System (Promega), One double-stranded synthetic RNA oligomers (5’-GGGAGAAATTGCACACTTAtt-3’; Ambion) deduced from human FLJ10540 and one negative control siRNA (#4611G; Ambion) were used in the siRNA experiments. The specific protein levels were determined by Western blotting analysis with specific antibodies.

The viability of sub-confluent cells was analyzed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The assay was performed in 96-well plates seeded with 4000–6000 cells/well in 200 μl. After 24 hours, 100 μl of culture medium containing 25 μl MTT solutions was added (1 g MTT (Sigma M5655) dissolved in 200 mL D-PBS). It was stored for 4 hours at 37°C, and then 100 μl DMSO buffer was added and left protected from light for 10 min. Readout was performed using a microplate reader (Labsystems Multiscan MCC/340) at 540 nm. Absorbance at 692 nm was used as the reference.

Migration and invasion assays were conducted with TW01-/Hone1-vehicle, TW01-/Hone1-FLJ10540, TW01-negative, and TW01-siFLJ10540 stable clones using 24-well Transwell chambers (8-μm pore size polycarbonate membrane; Costar). The migration and invasion assays were performed as described previously [4, 23, 24]. Briefly, cell migration and invasion were evaluated by counting the number of TW01-/Hone1-vehicle, TW01-/Hone1-FLJ10540, TW01-negative, and TW01-siFLJ10540 stable clone cells that had migrated or invaded by 200X phase-contrast microscopy on three independent membranes, then normalized against the vehicle cells to determine the relative ratio.

Several clinicopathological factors were evaluated, including gender, age, T stage, N status, and TNM stage. Fisher’s exact test was used to evaluate the correlation between the clinicopathological variables and the expressions of FLJ10540 and osteopontin. A p value of less than 0.05 was considered to indicate statistical significance in all analyses. Clinicopathological variables and the expression of FLJ10540 and osteopontin were taken into account for the analysis of survival, based on the Kaplan-Meier method; statistical significance, defined as a p value of less than 0.05, was assessed by log-rank test. To determine the effect of particular prognostic factors on survival, a multivariate analysis was performed according to Cox’s regression model.

A significant challenge in the post-genomic era is determining how to prioritize differentially expressed and poorly characterized targets in cancer-related microarray profiling studies. This study aimed to identify and characterize novel oncogenes that are only conserved in vertebrates, but not in invertebrates, based on the hypothesis that these novel genes may possess unique function in human cancer development. We used a bioinformatics approach to search for potentially important regulatory genes involved in growth control of human NPC. The publicly accessible microarray data has been deposited in NCBIs Gene Expression Omnibus (GEO) but can be accessed through the GEO Series accession number GSE12452 [29]. According to the microarray data of 31 primary NPC specimens, 592 out of 54,675 differentially expressed transcripts between normal and tumor areas were clustered by the ANOVA (p<0.05) based on three-fold differences and tested using the Benjamini and Hochberg false discovery rate correlation (p<0.05). These molecular portraits, containing 193 up-regulated and 399 down-regulated transcripts, could successfully distinguish the patients’ normal and tumor areas by hierarchical clustering ( Additional file 1: Figure S1). As a result, we focused on the poorly characterized gene, FLJ10540. FLJ10540 exhibited up-regulated expression patterns in NPC specimens in microarray database (Figure 1A). To validate the gene expression profile data for FLJ10540, Q-RT–PCR was performed on 12 NPC-tumor and five normal tissue samples. Figure 1B shows that overexpression of FLJ10540 was observable in our NPC patient samples. This result raises the possibility that upregulation of FLJ10540 may lead to the abnormalities found in human NPC.

Using the concept of syn-expression, genes belonging to the same pathway often show the same regulatory profiles under a variety of biological situations [30]. Hence, we employed the same publicly accessible microarray dataset, as described earlier, to gain insight into the functional concordance of co-expressed genes. To test this hypothesis, we first examined whether the mRNA expression profile of FLJ10540 correlated with any ligand in the publicly accessible microarray database of NPC. Of these co-expressed genes, the top candidate was osteopontin, which was highly positively correlated with the mRNA expression level of FLJ10540 in NPC cancer patients (r=0.72 p<0.001). This raises the possibility that these two molecules are functionally linked or in the same pathway.

The same panels of 12 NPC-tumor and five normal tissues were also applied to examine the mRNA expression level of osteopontin by Q-RT-PCR. As shown in Figure 1B, the mRNA expression levels of osteopontin were increased in the tumor samples as compared to those in normal tissue samples. The expressions of FLJ10540 and osteopontin in another batch of freshly frozen tumor and normal samples were further verified by western blotting. Our data indicated that the western blot results were consistent with the observed mRNA expressions of FLJ10540 and osteopontin in NPC specimens (Figure 1C). Taken together, these results suggest that there is a significant positively correlation between FLJ10540 and osteopontin in human NPC.

To investigate the clinical implications of FLJ10540 and osteopontin in NPC, immunohistochemical analysis was performed to determine the expressions and distributions of FLJ10540 and osteopontin in 63 human NPC samples and 10 normal tissues. The representative results of FLJ10540 and osteopontin immunostaining in NPC are shown in Figure 2A. First, FLJ10540 and osteopontin revealed very weak but detectable staining in the epithelium (Figure 2A, a and f). In the tumor samples, FLJ10540 and osteopontin expressions were positively correlated with tumor stage and node stage of the tumor cells (Figure 2A, b-e and g-j). However, strong stainings were not observed in tumor tissues when anti-FLJ10540 and anti-osteopontin antibodies were pre-incubated with protein corresponding to a portion of FLJ10540 and osteopontin, respectively (data not shown). Third, FLJ10540 and osteopontin were largely localized in the cytoplasm of the tumor sample cells as well as in normal tissues. To further confirm our immunohistochemisty results, anti-FLJ10540 and anti-osteopontin antibodies from Abnova produced almost the same results. In addition, χ² test was done to assess the association between FLJ10540 and osteopontin. As shown in Table 1, FLJ10540 again was significantly associated with osteopontin. Taken together, these results suggest that patients with a high FLJ10540 score had a significantly higher osteopontin score in human NPC.

As shown in Table 2, patients with stage III-IV tumors, a higher rate of lymph node metastasis, and TNM stages III-IV had significantly higher expressions of FLJ10540 or osteopontin compared with patients with stage I-II tumors (p< 0. 001), with no or a lower rate of lymph node metastasis (p=0. 001 and p= 0. 007), and TNM stages I/II (p< 0. 001). No significant differences between high and low levels of FLJ10540 and osteopontin were observed in terms of the patients’ age, gender, or histological type at the time of diagnosis. These findings suggest that both FLJ10540 and osteopontin expressions correlate with higher tumor progression from an early stage to advanced stage.

A DSS analysis using the Kaplan-Meier methods revealed that the prognosis of patients with high tumor expressions of FLJ10540 and osteopontin were significantly poorer than that of patients with low tumor expressions of FLJ10540 and osteopontin (p< 0. 001) (Figure 2B). Univariate analysis showed that DSS in this series was significantly associated with several clinico-pathological factors, including T (p< 0. 001), N (p= 0. 009), and TNM stage (p= 0. 001) (Table 3). Clinical prognosis, however, was not associated with age, gender or histological type. For multivariate analysis, however, T stage (p< 0. 001; hazard ratio = 5.091) remained significant independent prognostics factor of DSS for NPC patients. These results clearly indicate that the 5-year survival and clinical prognosis of NPC are associated with FLJ10540 and osteopontin expressions in NPC patients.

As shown in the Figure 3A, the mRNA and protein expression levels of FLJ10540 were increased in an osteopontin dose-dependent manner in TW01 cells by Q-RT-PCR and Western blotting. Similar results were also obtained in the Hone1 cells (Figure 3B). Next, to investigate the role of osteopontin in regulating FLJ10540 transcription, we sought to determine whether FLJ10540 promoter activity could be regulated by osteopontin stimulation. We transfected the FLJ10540 promoter-luciferase constructs into TW01 cells following osteopontin stimulation in a dose-dependent manner. The data showed that a significant activation of the FLJ10540 promoter was detected under osteopontin stimulation (Figure 3C). These results illustrated that FLJ10540 is one of the downstream targets of the osteopontin signaling pathway in human NPC cancer cells.

In Figure 2A, FLJ10540 expression is significantly higher in the NPC patients with a late tumor stage. This raises the possibility that FLJ10540 expression may promote tumor growth in NPC. Two different cell types were employed to address this question. First, TW01 and Hone1 cells stably expressing HA-tagged FLJ10540 were established (Figure 4A and B, left panel). FLJ10540-TW01 stable transfectants or vehicle control cells were seeded in 96-well plates to test whether they could grow in low serum medium. The results showed that FLJ10540-TW01 stable transfectants could promote cell growth in low-serum media compared to the vehicle control (Figure 4A, right panel). Similar results were also observed in FLJ10540-Hone1cells (Figure 4B, right panel). To determine whether osteopontin contributes to FLJ10540-mediated cell growth, FLJ10540-NPC transfectants, and vehicle control in the presence of osteopontin stimulation were seeded in 96-well plates to test cell proliferation. The results showed that FLJ10540-NPC stable transfectants in the presence of osteopontin grew faster than the vehicle control with osteopontin stimulation (Figure 4A and B, right panel). Furthermore, we also found that stimulated FLJ10540 transfectants with osteopontin was also growth faster than FLJ10540 stable cells alone. Taken together, these results suggest that FLJ10540 transfectants with or without osteopontin stimulation may decrease the requirement for serum, a characteristic of transformed cells.

We used a siRNA approach to inhibit endogenous expression of FLJ10540, and assayed for the growth rate of NPC cells. First, we transfected a chemically synthesized FLJ10540 siRNAs (siFLJ10540) and a negative control into TW01 and Hone1 for 24 hours, followed by Western blot analysis using antibodies against FLJ10540. The results showed a dramatic reduction in the FLJ10540 protein level with FLJ10540 siRNA (Figure 5A and B, left panel). Both TW01 and Hone1 siFLJ10540-transfected and negative control were determined the growth rates by MTT assay. The results demonstrated that the proliferation rate of FLJ10540 siRNA transfected cells was much slower than the cells transfected with negative siRNA (Figure 5A and B, right panel). Using the same panel, we also asked whether osteopontin encourages cell growth in depleted-FLJ10540 NPC cells. The result showed that FLJ10540-depleted NPC cells had a slight increased growth curve under osteopontin stimulation, compared to siFLJ10540 alone (Figure 5A and B, right panel). Take together these results strongly supports the idea that FLJ10540 plays a role in the cell proliferation of NPC cells.

FLJ10540 expression was higher in the NPC patients with lymph node metastasis (N+) than those that without lymph node metastasis (N-) (Figure 2A). We hypothesized that FLJ10540 may participate in the progression of metastasis in cells. FLJ10540-TW01 stable cells and vehicle control were seeded on a Transwell chamber to perform a motility assay for migration and invasion. Quantitatively speaking, the migration ability of FLJ10540-TW01 stable transfectants was approximately 4.8-5.5-fold higher than that of the vehicle control (Figure 6A, left panel). We next performed an invasion assay using a Matrigel-coated barrier filter. After 24 hours of incubation in the Matrigel-coated Transwell chamber, the TW01-FLJ10540 transfectants had invaded the basement membrane material and passed through the pores to reach the underside of the filter. Similarly, the TW01-FLJ10540 expressing cells showed a 6.8-7.8-fold higher invasive ability than the vehicle control cells (Figure 6A, right panel). The overexpressed FLJ10540 stable transfectants in the presence of osteopontin stimulation were analyzed the cell motility in a Transwell chamber. Again, FLJ10540 transfectants with osteopontin stimulation had a higher migration and invasion abilities than that the FLJ10540 alone or vehicle control (Figure 6A). Conversely, the motility of the NPC cells was strongly inhibited, while endogenous FLJ10540 was depleted by siRNA (Figure 6B). However, compared with siFLJ10540-NPC cells, the migratory and invasive abilities of siFLJ10540-NPC cells were slightly increased after cell-treated with osteopontin (Figure 6B). Taken together, these results strongly suggest that FLJ10540 is required for proper osteopontin-dependent signaling, and that it contributes to cell migration and invasion in NPC.

We next asked whether the involvement of CD44 on the biological activities of osteopontin could be attributed to the modulation of FLJ10540 expression induced by osteopontin in NPC cancer cells. The CD44 receptors were blocked using neutralizing antibodies, and analyzed FLJ10540 protein expression in TW01 cells upon osteopontin stimulation. The results demonstrated that upregulation of FLJ10540 in the presence of osteopontin was inhibited by the simultaneous addition of CD44 antibodies in a dose-dependent manner (Figure 7A). Similar results were also observed in Hone1 cells (data not shown). To confirm the involvement of CD44 in mediating FLJ10540 function upon osteopontin stimulation, we treated FLJ10540-TW01 and vehicle cells with anti-CD44 blocking antibody or isotypes IgG antibody in the presence of osteopontin and monitored cell growth and motility. Anti-CD44 antibodies resulted in a significant inhibition of cell growth and motility by FLJ10540 stable cells in the presence of osteopontin stimulation, compared to FLJ10540 stable cells treated with isotype IgG antibody (Figure 7B, and C). Taken together, these results indicate that FLJ10540 is required for proper osteopontin/CD44-dependent signaling and that it contributes to cell proliferation and metastasis.