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

Chen, Chang-Han; Shiu, Li-Yen; Su, Li-Jen; Huang, Chi-Ying F; Huang, Shun-Chen; Huang, Chao-Cheng; Yin, Yu-Fang; Wang, Wei-Sheng; Tsai, Hsin-Ting; Fang, Fu-Min; Chuang, Wan-Chu; Kang, Hong-Chang; Hwang, Chung-Feng

doi:10.1186/1479-5876-10-93

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

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Published: 16 May 2012

Volume 10, article number 93, (2012)
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FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway

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Chang-Han Chen^1,2,3,
Li-Yen Shiu⁴,
Li-Jen Su⁵,
Chi-Ying F Huang⁶,
Shun-Chen Huang⁷,
Chao-Cheng Huang^3,7,
Yu-Fang Yin^2,3,
Wei-Sheng Wang^2,3,
Hsin-Ting Tsai^2,3,
Fu-Min Fang^3,8,
Wan-Chu Chuang^2,3,
Hong-Chang Kang^2,3 &
…
Chung-Feng Hwang^2,3,9

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Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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Background

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 [Full size image

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 [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.

Table 1 χ²test was done to assess the association between FLJ10540 and osteopontin

Full size table

Prognostic value of FLJ10540 and osteopontin expression levels in 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.

Table 2 Comparisons of FLJ10540 and osteopontin expressions between the subgroups of various clincopathological parameters

Full size table

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.

Table 3 Disease-specific survival by Cox regression analyses between the subgroups of various clincopathological parameters

Full size table

Osteopontin triggered FLJ10540 expression in NPC cancer cells

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.

FLJ10540 transfectants alone or stimulated with osteopontin could promote cell growth in NPC

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.

Knockdown of endogenous FLJ10540 by siRNA suppressed NPC cell growth

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 and osteopontin synergistically induced the migration and invasion of NPC cancer 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.

Osteopontin-mediated FLJ10540 expression promoted cell proliferation and motility via CD44

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.

Discussion

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 [31]. 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 [32]. 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 [33]. Using osteopontin-null mutant mice as a model system, osteopontin was demonstrated to have a role in the growth or survival of cancer cells [34]. 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.

Conclusions

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.

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Acknowledgements

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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Authors and Affiliations

Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Chang-Han Chen
Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Chang-Han Chen, Yu-Fang Yin, Wei-Sheng Wang, Hsin-Ting Tsai, Wan-Chu Chuang, Hong-Chang Kang & Chung-Feng Hwang
Kaohsiung Chang Gung Head and Neck Oncology Group, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan
Chang-Han Chen, Chao-Cheng Huang, Yu-Fang Yin, Wei-Sheng Wang, Hsin-Ting Tsai, Fu-Min Fang, Wan-Chu Chuang, Hong-Chang Kang & Chung-Feng Hwang
The Department of Medical Research and Development, Show Chwan Memorial Hospital and Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan
Li-Yen Shiu
Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan
Li-Jen Su
Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
Chi-Ying F Huang
Department of Pathology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Shun-Chen Huang & Chao-Cheng Huang
Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan
Fu-Min Fang
Department of Otolaryngology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, No. 123, Ta-Pei Road, Niao Sung District, Kaohsiung, 833, Taiwan
Chung-Feng Hwang

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Chang-Han Chen
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Li-Yen Shiu
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Li-Jen Su
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Chi-Ying F Huang
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Shun-Chen Huang
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Chao-Cheng Huang
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Yu-Fang Yin
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Wei-Sheng Wang
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Hsin-Ting Tsai
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Fu-Min Fang
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Wan-Chu Chuang
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Hong-Chang Kang
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Chung-Feng Hwang
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Correspondence to Chung-Feng Hwang.

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The authors declare that they have no competing interests.

Authors' contributions

CHC, LYS and CFH: collected the clinical data of patients, conceived the study design, carried out and coordinated immunohistochemical examinations of tumor specimens, and drafted the manuscript. LJS: NPC microarray analysis and interpretation. CYH: conceived the study design. SCH and CCH: participated in the interpretation of data and conducted immunohistochemistry analysis. WSW, YFY, HTT and WCC: performed the IHC staining and biochemical experiments. FMF: performed statistical data analysis. All authors have read and approved the final version of the manuscript.

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Additional file 1: The up-regulated and down-regualted genes involved in the development of human NPC. (PDF 175 KB)

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Chen, CH., Shiu, LY., Su, LJ. et al. FLJ10540 is associated with tumor progression in nasopharyngeal carcinomas and contributes to nasopharyngeal cell proliferation, and metastasis via osteopontin/CD44 pathway. J Transl Med 10, 93 (2012). https://doi.org/10.1186/1479-5876-10-93

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Received: 27 December 2011
Accepted: 02 May 2012
Published: 16 May 2012
DOI: https://doi.org/10.1186/1479-5876-10-93

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

Abstract

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Background

Using the concept of syn-expression to uncover osteopontin, as the upstream regulator of FLJ10540 in NPC specimens

Prognostic value of FLJ10540 and osteopontin expression levels in NPC

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

Discussion

Conclusions

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