FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway
© Chen et al.; licensee BioMed Central Ltd. 2012
Received: 27 December 2011
Accepted: 2 May 2012
Published: 16 May 2012
Nasopharyngeal carcinoma (NPC) is well-known for its highly metastatic characteristics, but little is known of its molecular mechanisms. New biomarkers that predict clinical outcome, in particular the ability of the primary tumor to develop metastatic tumors are urgently needed. The aim of this study is to investigate the role of FLJ10540 in human NPC development.
A bioinformatics approach was used to explore the potentially important regulatory genes involved in the growth/metastasis control of NPC. FLJ10540 was chosen for this study. Two co-expression strategies from NPC microarray were employed to identify the relationship between FLJ10540 and osteopontin. Quantitative-RT-PCR, immunoblotting, and immunohistochemistry analysis were used to investigate the mRNA and protein expression profiles of FLJ10540 and osteopontin in the normal and NPC tissues to confirm microarray results. TW01 and Hone1 NPC cells with overexpression FLJ10540 or siRNA to repress endogenous FLJ10540 were generated by stable transfection to further elucidate the molecular mechanisms of FLJ10540-elicited cell growth and metastasis under osteopontin stimulation.
We found that osteopontin expression exhibited a positive correlation with FLJ10540 in NPC microarray. We also demonstrated comprehensively that FLJ10540 and osteopontin were not only overexpressed in NPC specimens, but also significantly correlated with advanced tumor and lymph node-metastasis stages, and had a poor 5-year survival rate, respectively. Stimulation of NPC parental cells with osteopontin results in an increase in FLJ10540 mRNA and protein expressions. Functionally, FLJ10540 transfectant alone, or stimulated with osteopontin, exhibited fast growth and increased metastasis as compared to vehicle control with or without osteopontin stimulation. Conversely, knockdown of FLJ10540 by siRNA results in the suppression of NPC cell growth and motility. Treatment with anti-CD44 antibodies in NPC parental cells not only resulted in a decrease of FLJ10540 protein, but also affected the abilities of FLJ10540-elicited cell growth and motility in osteopontin stimulated-NPC cells.
These findings suggest that FLJ10540 may be critical regulator of disease progression in NPC, and the underlying mechanism may involve in the osteopontin/CD44 pathway.
Nasopharyngeal carcinoma (NPC), the most common head and neck cancer, is a disease in which malignant cells form in the epithelium of the nasopharyngeal cavity. There is a high incidence of NPC in southern China and Southeast Asia, where the annual incidence is more than 20–30 cases per 100,000 people , and this pattern persists in those who have emigrated. The etiology of NPC seems to follow a multistep process, in which EBV infection, ethnic background, consumption of food and alcohol, and environmental carcinogens all seem to play a role . Advances in diagnostic imaging, radiation therapy, and concurrent chemoradiotherapy have achieved better locoregional control, however final treatment outcomes are not satisfactory [3, 4].
The process of metastasis which is considered a late event in tumorigenesis is very complex. Due to the rapid growth behavior of NPC, it has a high tendency to invade adjacent regions and metastasize to regional lymph nodes and distant organs [5–7]. More than 60% of NPC patients are first diagnosed with metastasis, leading to a high rate of treatment failure . The prognosis of NPC depends primarily on clinical TNM staging and tumor size . However, NPC patients with the same clinical stage often have different clinical courses, suggesting that TNM staging and tumor size are insufficient to accurately predict prognosis . Thus it is important to search for new therapeutic targets and a better understanding of the mechanisms involved in the spread of NPC.
Osteopontin, also known as SPP-1, was first detected in 1979 from malignantly transformed epithelial cell lines and T cells . Human osteopontin is composed of 314 amino acid residues containing two crucial functional domains, the calcium-binding domain and arginine-glycine-aspartic acid (RGD) sequences . It is a multifunctional cytokine which mediates in bone resorption, atherosclerosis, angiogenesis, wound repair, immune function, tissue injuries, and disease formation [12, 13]. Osteopontin expression has been associated with cancer markers for many years . Strong osteopontin expression in tumor cells has been shown in a variety of cancer types and is associated with poor prognosis and metastasis including gastric, ovarian, lung, breast, prostate, liver, sarcomas, colon, renal, and head and neck cancers [15–18]. However, the precise role of osteopontin in tumor development and promoting cancer metastasis in NPC is largely unknown.
FLJ10540 is also known by other names, including CEP55 , and C10orf3 . FLJ10540 is overexpressed in human colon cancer , hepatocellular carcinoma (HCC) , lung cancer  and oral cavity squamous cell carcinoma (OCSCC) , suggesting that it may function as an oncogenic characteristics in tumor development. However, there is little information regarding the expression and significance of FLJ10540 with tumor histological grade, stage, and patient outcome in NPC patients. In addition, the precise roles of FLJ10540 in tumor growth and metastasis in NPC are large unknown. The purpose of the present study was to examine the expression levels of FLJ10540 in NPC specimens, and to correlate the results with clinicopathologic variable survival and to investigate the functional role of FLJ10540 in human NPC development.
Patients and tumor samples
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 . 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.
RNA extraction, and quantitative RT-PCR (Q-RT-PCR)
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 . 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 . The antibodies used in this study included polyclonal antibodies against FLJ10540 (generated by us) , 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 . 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.
Cell culture, establishment of stable clones, gene silencing using siRNA, promoter plasmids, and luciferase assays
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.
Cell viability assay
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
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.
Microarray analysis, data analysis and clustering algorithm
The microarray data of NPC (GEO Series accession number is GSE12452) has been deposited in NCBIs Gene Expression Omnibus (GEO) and were analyzed using GeneSpring® 7.3.1 software (Silicon Genetics, Redwood City, CA). The microarray data was normalized by Robust Multichip Average (RMA) algorithm to produce and normalize the Affymetrix expression signals for the transcripts based on corresponding probe pairs of oligonucleotides. One-way analysis of variance (ANOVA) was used to build an explicit model about the sources of variances that affect the measurements. The values of p< 0.05 were considered as statistically significant.
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.
Elevated FLJ10540 expression in NPC tissues
Using the concept of syn-expression to uncover osteopontin, as the upstream regulator of FLJ10540 in NPC specimens
Using the concept of syn-expression, genes belonging to the same pathway often show the same regulatory profiles under a variety of biological situations . 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.
χ²test was done to assess the association between FLJ10540 and osteopontin
Prognostic value of FLJ10540 and osteopontin expression levels in NPC
Comparisons of FLJ10540 and osteopontin expressions between the subgroups of various clincopathological parameters
WHO type II
WHO type III
Disease-specific survival by Cox regression analyses between the subgroups of various clincopathological parameters
No of events
HR (95% CI a )
WHO type II
WHO type III
Primary tumor (T)
Nodal status (N)
Osteopontin triggered FLJ10540 expression in NPC cancer cells
FLJ10540 transfectants alone or stimulated with osteopontin could promote cell growth in NPC
Knockdown of endogenous FLJ10540 by siRNA suppressed NPC cell growth
FLJ10540 and osteopontin synergistically induced the migration and invasion of NPC cancer cells
Osteopontin-mediated FLJ10540 expression promoted cell proliferation and motility via CD44
Our data illustrates that the up-regulation of FLJ10540 and osteopontin expressions are associated with a poor prognosis in NPC patients. It is worth noting that prognosis was not dependent on histologic type, but rather dependent on the expressions of FLJ10540 and osteopontin. FLJ10540 and osteopontin positively correlated with a poor prognosis in NPC patients, especially with T and N stages respectively. Because it is difficult to predict the prognosis of such patients, investigating FLJ10540 and osteopontin stainings together in NPC cancer cells may be helpful in determining the therapeutic strategy. Functionally, Ectopic expression of FLJ10540 enhanced cell growth, migration and invasion. Conversely, siRNA-mediated repression of FLJ10540 suppressed cell growth and motility in NPC. Moreover, blocked CD44 receptors by using CD44-specific antibodies, not only decreased the protein expression of FLJ10540 under osteopontin stimulation, but also suppressed FLJ10540-elicited NPC cell growth and metastasis, supporting the participation of FLJ10540 in the osteopontin/CD44 pathway. These findings suggest that FLJ10540 is not only a good prognostic indicator of NPC, but also a candidate for a molecular targeted therapy.
FLJ10540 overexpression is disclosed in a number of human cancers and cell lines, and has been correlated with tumor progression as well as decreased survival in patients with HCC, lung and oral cancer[21–23]. This suggests that FLJ10540 plays an important role in tumor formation. This is the first time we have shown that FLJ10540-NPC stable transfectants promoted cell growth at low serum levels. Conversely, suppressed endogenous FLJ10540 by FLJ10540-mediated siRNA inhibited cell growth, suggesting that FLJ10540 displays a characteristic associated with oncogenes in NPC. Furthermore, stimulation of FLJ10540-NPC stable cells by adding osteopontin could encourage cell growth faster than FLJ10540 alone or the vehicle control. Taken together, we suggest that FLJ10540, mediated by osteopontin stimulation, may play a significant role in tumor progression in NPC.
The metastatic spread of cancer cells is composed of multiple sequential steps such as dys-regulation of intercellular adhesion, degradation of the extracellular matrix, and increasing cell motility . To the best of our knowledge, this is the first report to demonstrate that an increased FLJ10540 expression was not only positively correlated with clinical advanced N stage, but that is also promoted NPC cell migration and invasion in vitro. Moreover, the migratory and invasive abilities were abolished with knockdown endogenous FLJ10540 by siRNA in NPC cells. Importantly, FLJ10540-NPC transfectants stimulated by osteopontin enhanced cancer cell migration and invasion. Our results suggest that one mechanism by which FLJ10540 may promote tumor cell migration and invasion is through osteopontin regulation in NPC.
We re-arranged a gene expression microarray dataset and employed the concept of syn-expression to identify that FLJ10540 is one of the downstream targets of osteopontin in NPC. Recently, it has been reported that osteopontin serves as a serum marker for some human tumor types . Furthermore, an increased expression of osteopontin has been shown to correlate with disease outcome and patient survival. Osteopontin is able to engage receptors, including CD44, and thus may stimulate diverse signaling pathways and influence cellular events that in turn, favor tumorigenesis . Using osteopontin-null mutant mice as a model system, osteopontin was demonstrated to have a role in the growth or survival of cancer cells . However, the specific mechanisms by which osteopontin functions to promote malignant cell behavior have not been completely elucidated. In the present study, it is crucial to demonstrate that the mRNA and protein expression levels of FLJ10540 are positively correlated with osteopontin in NPC specimens. In addition, the expression level of FLJ10540 was upregulated in an osteopontin dose-dependent manner in NPC cell lines. The mechanisms responsible for the overexpression of FLJ10540 in malignant cancer cells are still not clear. Here, our results demonstrated that one of the important upstream regulators of FLJ10540 may be regulated by a cytokine, osteopontin, in NPC cancer cells. Furthermore, blockade of CD44 receptor caused significant inhibitions of FLJ10540-induced cell growth, migration and invasion in the presence of osteopontin stimulation in NPC cells. In this regard, our results show that CD44 provides an important link for understanding the role of osteoponint/FLJ10540 in cell proliferation and metastasis.
FLJ10540 was regulated by osteopontin and plays an important role in NPC because increased FLJ10540 protein levels are observed to associate with advanced stage of NPC. In addition, FLJ10540 seems to contribute to tumor aggressiveness because high FLJ10540 protein levels are related to reduce patient survival. If the independence from established prognostic factors is demonstrated in larger clinical study, FLJ10540 genetic alterations in NPC may eventually be able to guide therapeutic strategies.
This study was funded by grants obtained by Dr. Chung-Feng Hwang and by Dr. Chang-Han Chen from the Kaohsiung Chang Gung Memorial Hospital Taiwan (grant numbers: CMRPG890461 and CMRPG890462, and CMRPG890921, respectively) and from the National Science Council [NSC 98-2314-B-182A-046-MY3, (NMRPG886051; NMRPG886052) and NSC 100-2320-B-182A-001 (NMRPG8A0011)]. We thank the Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, (CLRPG871342) for providing the instruments used for this study and Tissue Bank, Kaohsiung Chang Gung Memorial Hospital for providing the study materials (CLRPG870463, CMRPG870461). We also thank the technical supports provided by Core Facilities for High Throughput Experimental Analysis of Institute of Systems Biology and Bioinformatics, College of Science, National Central University. The Core Facilities for High Throughput Experimental Analysis are supported by the Aim of Top University Project from the Ministry of Education.
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