Journal of Cancer Research and Therapeutics Medknow Publications on behalf of the Association of Radiation Oncologists of India (AROI) ISSN: 0973-1482 EISSN: 1998-4138 Vol. 7, Num. 2, 2011, pp. 138-142

Journal of Cancer Research and Therapeutics, Vol. 7, No. 2, April-June, 2011, pp. 138-142

Original Article

Osteopontin expression in nasopharyngeal carcinoma: Its relevance to the clinical stage of the disease

Hong-Han Wang¹, Xing-Wei Wang¹, Can-E Tang²

¹ Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central-South University, Changsha City, Hunan Province, China
² Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central-South University, Changsha City, Hunan Province, China
Correspondence Address: Xing-Wei Wang, Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central-South University, Changsha City, Hunan Province, China, wang-xingwei@126.com

Code Number: cr11033

PMID: 21768699
DOI: 10.4103/0973-1482.82926

Abstract

Keywords: Metastasis, nasopharyngeal carcinoma, osteopontin, pathogenesis, progression

Introduction

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China, where an annual incidence reaches up to 15-50 cases per 100,000. Likely etiological factors of NPC include Epstein-Barr virus (EBV), ethnic background, genetic susceptibility, consumption of sodium and salted foods, environmental carcinogens and so on. Over 90% of NPCs are poorly differentiated or undifferentiated squamous cell carcinoma and radiotherapy is the main choice for the treatment. Currently, with the improvement of early diagnosis and radiotherapy regime, the five-year overall survival rate of NPC patients is about 55%. Many factors may affect its prognosis, including the clinical stages, presence of keratinization, lymph node metastasis, and genetic factors. ^[1]

Osteopontin (OPN), also known as SPP-1, is a secreted glycophosphoprotein. It is a multifunctional cytokine, which mediates cell adhesion, migration, and Type I immunity through interacting with several cell surface receptors, ^[2] including αvβ3 integrin and CD44. It has been reported that OPN is overexpressed in a variety of human tumors and is associated with the tumorgenesis, development, and metastasis. ^{[3],[4],[5],[6],[7]} There is evidence that targeting OPN significantly improves the therapeutic potency in some malignancies. ^[8],[9] Other studies have shown that OPN expression levels in the liver, breast, stomach, and uterine cancers are related to patient survival and clinical stages, ^{[10],[11],[12],[13]} suggesting that OPN is a potential prognosis biomarker for these types of cancer. ^{[5],[14],[15],[16]} However, the expression and clinical relevance of OPN in NPC tissue samples are still unclear.

Materials and Methods

All tissue samples were from preliminary diagnosed patients who visited the Department of Otolaryngology-Head and Neck Surgery at Xiangya Hospital of Central-South University for biopsy, from March to December 2009. All patients and volunteers were informed. Each tissue sample was split into two parts. One part was immersed into formalin solution, embedded in paraffin, and routinely stained with hematoxylin and eosin and the other part was stored in liquid nitrogen immediately after resection. After careful screening, we obtained 67 NPC and 21 NNET samples which were used for IHC. Corresponding frozen tissue samples were used to extract total RNA. After assessing the quality of mRNA, we got 44 NPC and 15 NNET samples, which were used for qRT-PCR. In addition, 12 paired NPC and PCT samples were used for WB. All NPC samples in the study were differentiated squamous cell carcinoma. The detailed clinical data for patient sex, age, T stage, N stage, clinical stages are listed in [Table - 1]. Tumor size and situation of regional lymph node metastasis were clinically measured by magnetic resonance imaging (MRI) or computed tomography (CT) scan. Tumor stages were evaluated according to Union for International Cancer Control (UICC, 2003).

Approximately 100 mg frozen tissue per sample was taken to isolate total RNA using trizol reagent (Invitrogen). The mRNA quality was evaluated by the OD260/280 ratio and samples were only used when the ratio was 1.8-2.0. Reverse transcription (RT) was performed using Reverse Transcriptase XL (AMV) kit (TaKaRa Biotechnology Co. Ltd.) according to the manufacturer′s instructions. The PCR program (30 cycles) consisted of denaturation at 94°C for 30 sec, annealing at 52°C for 30 sec, then extension at 72°C for 45 sec, and extension at 72°C for 5 min. The predicted 424 bp PCR product was amplified. OPN primer sequences were: forward; 5′-CAACTCCTCGCTTTCCAT-3′; reverse; 5′-GCTAAACCCTGACCCATCT-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the internal control. GAPDH primer sequences were: forward; 5′-GTCAGTGGTGGACCTGACCT-3′; reverse; 5′-TGAGGAGGGGAGATTCAGTG-3′. The PCR products were examined on 1.5 agarose gels with ethidium bromide staining. A FluorChem FC2 imaging system (Cell Biosciences) was used to calculate the ratio of OPN/GAPDH.

Paraffin sections (4 μm) were deparaffinized routinely in xylene and rehydrated through a series of graded ethanol to Tris-buffered saline (TBS) buffer. Antigen retrieval was performed in a constant high-temperature water bath (99°C) with the sections in citrate buffer (pH 6.0) for 15 min. The sections were incubated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase and were washed with TBS. Then they were incubated with goat serum for 30 min to inhibit nonspecific binding followed by incubation with anti-osteopotin mouse monoclonal antibody (Santa Cruz Biotechnology Inc., dilution at 1:100) overnight at 4°C. On the following day, the sections were subsequently incubated with biotin-conjugated goat anti-mouse immunoglobulin G antibody for 30 min at room temperature and incubated with streptavidin-biotin peroxidase solution for 10 min followed by incubation with freshly prepared DAB solution (Maixin-Bio Inc.) for 3 min. All of the sections were counterstained with Mayer′s hematoxylin for 30 sec, washed in running water, dehydrated and mounted with neutral gum. A section of thyroid papillary carcinoma which was previously proved to be OPN-positive was used as positive control. For a negative control, the primary antibody was replaced with phosphate buffered saline. Staining was assessed from 5 to 10 high-power fields at ×400 magnification. Expression of OPN was determined by assessing the percentage of stained tumor cells and the staining intensity. The percentage of positive cells was rated as follows: 1-10% positive cells (1+), 11-49% positive cells (2+); 50-74% positive cells (3+); and > 75% positive cells (4+). Staining intensity was scored as negative (0), weak (1+), moderate (2+), and intensive (3+). Scores for expression and scores for percentage of positive cells were multiplied to calculate an immune-reactive score (IRS) ranging from 0 to 12, with 0-1 as (-), 2-3 as (+), 4-6 as (++), and > 6 as (+++). All of the sections were examined and scored independently by two investigators.

Total protein was extracted from each frozen tissue sample and protein concentration was determined by BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Twenty-five μg of total protein was loaded onto 10% sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. β-actin was used as the loading control. The membranes were then blocked with 5% skimmed milk and 0.1% Tween 20 for 1 h, followed by incubation with the OPN primary antibody (1:400, Santa Cruz Biotechnology Inc.) overnight at 4°C. The membranes were subsequently incubated for 1 h at room temperature with the mouse secondary antibody (1:2000, Maixin-Bio Inc.), and protein was detected using the enhanced chemiluminescent (ECL) reagent (Beyotime Institute of Biotechnology). Quantitative analysis was carried out using FluorChem FC2 image analysis system (Cell Biosciences).

All of the statistical analyses were carried out using SPSS 17.0 for Windows (SPSS Inc., CA.). The quantitative data was presented as mean ± standard deviation (x ± SD). Significance in the numerical data between the two groups was evaluated using t-test or Fisher′s exact probability test. The significance in OPN expression between NPC and NNET was evaluated using Wilcoxon rank sum test. Results were considered statistically significant when P < 0.05.

Results

The expression of OPN mRNA in 44 NPC tissue samples and 15 NNET samples by qRT-PCR has been shown in [Figure - 1]. The expression level of OPN mRNA in NPC samples was significantly higher than in NNET samples [Table - 2] (P = 0.000). OPN protein expression in 67 cases of NPC samples and 21 cases of NNET samples was examined by IHC. OPN was mainly localized to the cytoplasm of tumor cells. The staining intensity and the percentage of positive cells were heterogeneous in some sections. The representative staining was scored [Figure - 2]. There were 59 cases which showed strong or moderate positive OPN staining in 67 NPC samples (88.1%) and only six cases out of 15 NNET samples (28.6%) displayed weak positive OPN staining [Table - 3]. The expression levels of OPN in NPC and NNET were significantly different (P = 0.001).

To compare OPN protein expression in NPC and its corresponding PCT samples, 12 paired NPC and PCT samples were analyzed by WB [Figure - 3]. This analysis found that OPN expression levels in NPC were much higher than in PCT, and the differences between them were significant [Table - 4] (P = 0.000).

Comparing OPN mRNA and protein expression levels with various clinicopathological parameters of NPC, we found that OPN mRNA and protein expression were not significantly associated with patient age (P = 0.579, P = 0.554, respectively) and sex (P = 0.962, P = 0.594, respectively). However, it was noted that there was a significant correlation between OPN mRNA and protein expression and NPC tumor stage (T stage) (P = 0.031, P = 0.011, respectively), regional lymph nodal metastasis (N stage) (P = 0.039, P = 0.008, respectively), and clinical stages (P = 0.015, P = 0.001, respectively) [Table - 5] and [Table - 6].

Discussion

The existence of OPN was first discovered in bone mineralization of the extracellular matrix, and later it was found in various tissues and cells. ^[17] OPN protein contains a highly conserved arginine-glycine-aspartic acid (RGD) domain, which interacts with integrin receptors αvβ3, αvβ5 and mediates cell adhesion and migration. ^[18],[19] In addition to integrin, thrombin (Thrombin) and matrix metalloproteinase (MMP) also interact with OPN via its RGD domain. The non-RGD-binding sites in the carboxy-terminal of OPN can interact with cell surface receptors CD44v (CD44′s variant) and also play an important role in cell adhesion signaling. ^[3]

The interactions of OPN with various cell surface receptors induce the activation of various signal transduction pathways, resulting in changes in the expression of series of genes. ^[20],[21] It has been shown that inhibition of αv-integrins by a cyclic RGD-peptide results in significant reduction of functional vessel density, retardation of tumor growth and metastasis in vivo. ^{[22],[23],[24],[25],[26]}

It has been reported that OPN is involved in tumorgenesis, metastasis and progression of several different types of human tumors, including pancreatic, renal, endometrial, esophageal, head and neck carcinomas. ^[10] A certain study found that OPN levels in NPC patients′ plasma were significantly higher than in healthy individuals. In addition, high OPN levels have been shown in patients with advanced cancer and this was related to nodal metastasis. This suggested that OPN may have a potential role in the pathogenesis and nodal metastasis of NPC. ^[27]

In the present study, three different methods (qRT-PCR, IHC, and WB) were used for the investigations. We found that OPN/GAPDH ratios in 44 cases of NPC and 15 cases of NNET samples were 0.80±0.24 and 0.32±0.18 respectively; 88.1% (59/67) of NPC samples showed strong or moderate positive OPN staining. Only 28.6% of NNET, by comparison, displayed a weak positive OPN staining (6/15). As a result of WB, OPN/β-actin ratios in 12 paired NPC and PCT samples were 0.83±0.16 and 0.51±0.21 respectively. These results clearly showed that OPN expression is much higher in NPC than in NNET and PCT. The analysis of the relationship between OPN mRNA and NPC T, N and clinical stages showed the ratios for OPN/GAPDH in all the stages and the ratios are as follows: In T stage, OPN/GAPDH ratio was 0.73±0.27 for T1/T2 and 0.89±0.16 for T3/T4, in N stage, OPN/GAPDH ratio was 0.66±0.31 for N0 and 0.84±0.20 for N1/N2/N3 and finally OPN/GAPDH ratio was 0.63±0.31 for I/II and 0.88±0.15 for III/IV. In the same way, the positive rates of OPN staining were 77.8% (28/36) and 100% (31/31) in T1/T2 and T3/T4, 64.3% (9/14) and 94.3% (50/53) in N0 and N1/N2/N3, and 66.7% (14/21) and 97.8% (45/46) in I/II and III/IV. The analysis indicates that OPN expression is significantly correlated with NPC T, N and clinical stages.

In summary, OPN overexpression is strongly related to more aggressive behavior of NPC. It suggests that OPN might be associated with the pathogenesis, metastasis and progression of NPC. However, our study did not reveal further associations of OPN in NPC and if there is any mediation by interacting with integerin and other adhesive molecules, including MMP, thrombin, CD44 and so on. More investigations are required for this.

Acknowledgment

The authors would like to thank Drs Robert J. Rounbehler and Jun-li Luo for their invaluable guidance and help.

References

Copyright 2011 - Journal of Cancer Research and Therapeutics

The following images related to this document are available:

Photo images