Upregulation of caveolin-1 and CD147 expression in nasopharyngeal carcinoma enhanced tumor cell migration and correlated with poor prognosis of the patients

Authors

  • Zi-Ming Du,

    1. State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China
    2. Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China
    3. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
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  • Chun-Fang Hu,

    1. Institute for Cancer Studies, University of Birminghan, Birminghan, England, United Kingdom
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  • Qiong Shao,

    1. Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China
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  • Ma-Yan Huang,

    1. Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China
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  • Chang-Wei Kou,

    1. Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China
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  • Xiao-Feng Zhu,

    1. State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China
    2. Department of Experiment Research, Sun Yat-Sen University Cancer Center, Guangzhou, China
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  • Yi-Xin Zeng,

    1. State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China
    2. Department of Experiment Research, Sun Yat-Sen University Cancer Center, Guangzhou, China
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  • Jian-Yong Shao

    Corresponding author
    1. State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, Guangzhou, China
    2. Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China
    3. Department of Experiment Research, Sun Yat-Sen University Cancer Center, Guangzhou, China
    • Department of Pathology, Sun Yat-Sen University Cancer Center, 651 Dong Feng Road East, Guangzhou 510060, People's Republic of China
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    • Fax: +86-20-87343391.


Abstract

Expression of caveolin-1 (Cav-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) and their prognostic significance were analyzed in archive NPC samples. Cav-1 and CD147 were overexpressed in 49.48% (96/194) and 59.39% (117/197) of NPC, respectively. Both Cav-1 and CD147 expression levels correlated significantly with metastasis (p = 0.025 and 0.017, respectively) and a lower 5-year survival rate (p = 0.02 and 0.0009, respectively). In addition, Cav-1 expression levels correlated significantly with local recurrence (p = 0.038). Multivariate Cox regression analysis indicated that combination of high Cav-1 and CD147 expression was a significant, independent prognosis predictor in patients with NPC (HR = 2.135; p = 0.006). Functional studies revealed that overexpression of Cav-1 promoted secretion of MMP-3 and MMP-11 (active) proteins, as well as an increase in the migratory ability of CNE1 and CNE2 cells, while siRNA-mediated silencing of Cav-1 or CD147 led to reduced levels of MMP-3 and MMP-11(active) secretion, and reduced migration capacity of CNE1 and CNE2 cells. We observed a positive correlation between Cav-1 and CD147 expression in NPC (ρ = 0.330, p = 0.000), CD147 protein levels were upregulated in Cav-1 overexpressing CNE1 and CNE2 cells, whereas siRNA-mediated silencing of Cav-1 led to the downregulation of CD147 expression. Our results indicate that Cav-1 and CD147 overexpression predict poor NPC prognosis and enhanced tumor cell migration, which is associated with MMP-3 and MMP-11 (active) secretion. © 2009 UICC

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in certain areas of Southern China, Southeast Asia and North Africa.1, 2 NPC has a dominant clinicopathological behavior of locoregional recurrence and metastasis, which differs from other types of head and neck cancers.3 Although NPC is radiosensitive, the 5-year survival rate remains between 50 and 60%. Regional lymph node and distant metastasis and locoregional recurrence are the 2 major reasons resulted in failure of treatment for this cancer.4, 5 Currently, an evaluation of NPC prognosis is primarily based on the clinical TNM staging; however, patients with NPC with the same clinical stage often undergo different clinical courses, suggesting that the TNM staging is insufficient for precisely predicting the prognosis of this disease. Therefore, it is important to identify molecular biomarkers, which will help clinicians identify patients with NPC with elevated risk, improve prognosis prediction and facilitate therapeutic intervention.

Caveolin-1 (Cav-1) is a major structural component of caveolae, which are involved in several cellular functions, including vesicle trafficking, cholesterol homeostasis and signal transduction.6, 7 Reduced Cav-1 expression has been reported in ovarian cancer,8 breast cancer9 and lung cancer.10 On the other hand, Cav-1 overexpression has been observed in bladder cancer,11 prostate cancer12 and esophageal squamous cell carcinoma (ESCC).13 Furthermore, recent evidence suggests a central role for Cav-1 in the regulation of cellular invasion and metastasis.14, 15

Extracellular matrix metalloproteinase inducer (EMMPRIN), also named CD147, is a glycoprotein that belongs to the immunoglobulin superfamily.16 CD147 is composed of 2 extracellular Ig domains: a single transmembrane domain and a short cytoplasmic domain. The first Ig domain of CD147 is required for counter receptor binding activity, which is involved in matrix metalloproteinases (MMPs) induction and oligomerization; and the second Ig domain of CD147 is known to associate with Cav-1.17 CD147 is enriched on the plasma membrane of tumor cells and triggers the production or release of MMPs in surrounding mesenchymal cells and tumor cells.18, 19 Several recent studies have found that the overexpression of CD147 is correlated with poor prognosis in human cancers, including ESCC,20 breast carcinoma,21 serous ovarian carcinoma22 and gastric carcinoma.23

To date, the expression patterns of Cav-1 and CD147 and their clinical significance in NPC have not been determined. The aim of our study was to investigate the expression patterns of Cav-1 and CD147 and their clinicopathological implications in NPC progression, and to determine potential underlying molecular mechanisms that may lead to an increased understanding of NPC.

Material and methods

Patients and clinical tissue samples

For this retrospective study, we used archival formalin-fixed, paraffin-embedded specimens from 232 primary patients with NPC (174 males and 58 females, aged from 14 to 86 years; median, 46 years) who underwent radical radiotherapy and were recruited from 1992 to 2002 at the Sun Yat-Sen University Cancer Center (Guangzhou, China). All the NPC samples in our study were obtained before treatment with standard curative radiotherapy with or without chemotherapy. Sixty-six patients were diagnosed with nonkeratinized differentiated (WHO types II) and 166 were diagnosed with undifferentiated carcinoma (WHO types III). The disease stages of all patients were classified or reclassified according to the 1992 NPC staging system of China.4, 24, 25 The staging system is characterized according to the following model: T, primary tumor: T1, limited to the nasopharynx; T2, involvement of the nasal cavity, oropharynx, soft palatine, anterior cervical vertebrae soft tissue and parapharyngeal space extension before the SO line (the SO line is between the styloid process and the midpoint on the posterior edge of the great occipital foramen); T3, extension over the SO line, involvement of the anterior or posterior cranial nerves alone, the base of the skull, the pterygoprocess zone and the pterygopalatine fossa; T4, involvement of both anterior and posterior cranial nerves, parabasal sinus, cavernous sinus, orbit, infratemporal fossa and direct invasion of the first or second cervical vertebrae; N, regional lymph node involvement: N0, no enlarged lymph nodes; N1, greatest dimension of upper neck lymph node <4 cm, movable; N2, lower neck lymph node or greatest lymph node dimension between 4 and 7 cm; N3, supraclavicular lymph node, lymph node greatest dimension >7 cm, fixed, or skin infiltration (the border between the upper neck and the lower neck is the inferior margin of the cricoid cartilage); M, distant metastasis: M0, absence of distant metastasis; M1, presence of distant metastasis; staging: Stage I, T1N0M0; Stage II, T2N0-N1M0, T0-T2N1M0; Stage III, T3N0-N2M0, T0-T3N2M0; Stage IVa, T4N0-N3M0, T0-T4N3M0; Stage IVb, M1. Among 232 primary patients with NPC in our study, 6 patients were classified as stage I, 53 patients as stage II, 105 patients as stage III and 68 patients as stage IV.

Tissue microarray construction

Paraffin-embedded specimens from the 232 primary patients with NPC were used to construct the tissue microarray (TMA), and the procedures for the TMA construction were as described previously.26

Immunohistochemistry

Primary antibodies against Cav-1 (1:500 dilution; sc-894, Santa Cruz, USA) and CD147 (1:100 dilution; ZA-0455, ZYMED, USA) were used in our study. Briefly, tissue sections were dewaxed, incubated with hydrogen peroxide for 10 min, incubated in retrieval buffer solution for antigen recovery, blocked with normal serum for 10 min and incubated with a primary antibody for 60 min, followed by detection using a Catalyzed Signal Amplification Kit (DAKO, USA); signal was visualized using diaminobenzidine. Nonimmune goat or rabbit serum was substituted for the primary antibody as a negative control. The immunohistochemistry results were evaluated and scored independently by 2 pathologists without the knowledge of the clinicopathological outcomes of the patients. A semiquantitative estimation was made, as described previously,27 using a composite score obtained by multiplying the values of the staining intensity and the relative abundance of Cav-1 and CD147 positive cells. The intensity was graded as 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive; equivalent to endothelial cell Cav-1 staining). The abundance of the positive cells was graded from 0 to 4 (0, <5% positive cells; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%). A composite score greater than or equal to the mean value was considered as high expression, and composite scores less than the mean value were considered as low expression.

Cell culture

The human NPC cell lines CNE1 and CNE2 were cultured in RPMI 1640 medium (GIBCO, USA) containing 10% fetal calf serum (FCS). The human NPC cell line C666-1 was cultured in RPMI 1640 medium (GIBCO, USA) containing 15% FCS. The immortalized nasopharyngeal epithelial cell line NP69 was cultured in keratinocyte serum-free medium (Invitrogen) supplemented with 5% FCS, 25 μg/ml bovine pituitary extract and 0.2 ng/ml recombinant epidermal growth factor, as suggested by the manufacturer. All the cell lines were grown in a humidified incubator at 37°C with 5% CO2.

Real-time PCR assays

Total RNA was extracted from NPC cell lines of CNE1, CNE2, C666-1 and an immortalized nasopharyngeal epithelial cell line NP69, as well as 2 NPC tumors and paired normal biopsies using TRIzol reagent (Invitrogen). After reverse transcription of the total RNA, the first-strand cDNA was then used as templates for detection of Cav-1 and CD147 expression by quantitative real-time PCR (QT-PCR) with the SYBR Green I chemistry (ABI, USA). GAPDH was used as internal control. The primers were Cav-1 (Forward: AAG CAC AGC CCA GGG AAA C and Reverse: GGC AGA CAG CAA GCG GTA A); CD147 (Forward: CCA GAA TGA CAA AGG CAA GAA CG and Reverse: TGG CTT CAG ACA GGC AGG ACA CG); GAPDH (Forward: CCA CCC ATG GCA AAT TCC ATG GCA and Reverse: TCT AGA CGG CAG GTC AGG TCC ACC). Thermal cycle conditions were as follows: 1 cycle of preincubation at 95°C for 5 min (segment 1); 40 cycles of amplification at 95°C for 30 sec, 55°C (Cav-1) or 61°C (CD147) or 57°C (GAPDH) 45 sec and 72°C for 45 sec (segment 2); and melting temperature curve analysis at 95°C for 30 sec, 60°C for 30 sec and 95°C for 30 sec (segment 3). The relative expression level was determined as 2−ΔΔCt. Data are presented as the expression level relative to the calibrator (NP69 cells), with the standard error of the mean of triplicate measures for each test sample.

siRNA transfection

Before transfection, 2 × 105 cells per well were plated into 6-well plates and grown for 1 day in antibiotic-free medium containing 10% FCS. When the cells were 40 to 60% confluent, they were transfected with an siRNA reagent designed to specifically target Cav-1 (sc-29941, Santa Cruz, USA), CD147 (CGG CCA UGC UGG UCU GCA A) or with a nonspecific negative control siRNA (UUC UCC GAA CGU GUC ACG U) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. Transfected cells were grown at 37°C for 6 hr, followed by incubation with complete medium for an additional 42 hr. When transient transfection was performed for 48 hr, cells were harvested and followed by the further assay.

Plasmid transfection

Briefly, 4 × 105 cells per well were plated into 6-well plates and grown for 1 day in antibiotic-free medium containing 10% FCS before transfection. When the cells were 85 to 90% confluent, they were transfected with a Cav-1 expressing vector (pCI-neo-Cav-1, kindly donated by Professor Jian-Wen Chen from Chinese Academy of Sciences), or a CD147 expressing vector (pCI-neo-CD147) or a corresponding empty pCI-neo vector (control), all transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. The transfected cells were grown at 37°C for 6 hr, followed by incubation in complete medium. The cells were grown for 24 hr before further assays were conducted.

Immunoblot analysis

Cells were harvested and lysed with RIPA buffer (Upstate, USA). Equal amounts of denatured protein sample were separated by SDS-PAGE and were then transferred electrophoretically to PVDF membranes (Pall, USA) for immunoblot analysis. Antibodies used for immunoblot analysis were against Cav-1 (1:1,000 dilution, sc-894, Santa Cruz, USA), CD147 (1:200 dilution, sc-21746, Santa Cruz, USA), MMP-3 (1:1,000 dilution, sc-21732, Santa Cruz, USA) and MMP-11 (1:500 dilution, MS-1035, NeoMARKERS, USA); an anti-GAPDH antibody (1:3,000 dilution, sc-32233, Santa Cruz, USA) was used as loading control. All protein bands were detected using an enhanced chemiluminescent (ECL) Western blot Kit (Cell Signaling Technology, USA).

To determine the protein levels secreted into the cell medium, the same number of cells were plated and transfected, followed by incubation in 2 ml serum-free RPMI 1640 medium for 48 hr. Conditioned media protein samples harvested from 4 duplicate wells were pooled (8 ml) and concentrated to 200 μl using a Vivaspin 20 column with a 10 kDa exclusion limit (Sartorius, Germany). Then, 10 μl of each concentrated conditioned media protein sample was subjected to immunoblot analysis.

Transwell migration assay

Transwell migration assays were performed using 24-well transwell units (Corning, USA) with a 8-μm pore size polycarbonate filter coated with 100 μl of 1% Matrigel™ (BD Biosciences, USA) in PBS to form a continuous thin layer. Cells were harvested and resuspended in serum-free RPMI 1640 medium at a concentration of 1 × 106 cells/ml. Cell suspensions of 120 μl were placed onto the upper chamber, and 600 μl RPMI 1640 with 10% FCS was added to the bottom chamber. Cells were grown overnight at 37°C in a 5% CO2 incubator. After overnight incubation, the top cells were removed and the bottom cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI; 5 μg/ml) to visualize nuclei. The number of migrating cells in 5 fields at a 200× magnification was countered under a fluorescence microscope, and the mean cell number for each chamber was calculated. Triplicate samples were acquired and the data were presented as the average cell number of 15 fields.

Statistical analysis

Data was analyzed using SPSS12.0 software. The association between Cav-1 and CD147 expression, and clinicopathological parameters were assessed using a Chi-Square test or Correlations test. Kaplan-Meier analysis and log-rank tests were used to assess the survival rate and to compare the difference in survival curves. Cox regression analysis was performed to assess the significance of multiple predictor of survival. Results of the transwell cell migration assay were presented as mean ± SD, and Student's t-test was used to determine the differences in multiple comparisons. It was considered as significant differences when p < 0.05.

Results

Expression of Cav-1 and CD147 and their associations with clinicopathological parameters in NPC

To determine Cav-1 and CD147 expression in NPC, quantitative real-time PCR (QRT-PCR) was performed to evaluate the expression levels of Cav-1 and CD147 transcripts in NPC cell lines of CNE1, CNE2 and C666-1, an immortalized primary nasopharyngeal epithelial cell line of NP69 cells, as well as in 2 NPC biopsies and the paired normal tissues. Compared to NP69 cells, overexpression of Cav-1 and CD147 mRNA was observed in NPC cell lines of CNE1, CNE2 and C666-1. Meanwhile, the expression level of Cav-1 mRNA, especially CD147 mRNA were also significantly upregulated in NPC tumor tissues, compared to their normal counterparts (Figs. 1a and 1b). Western blot analysis also revealed the overexpression of Cav-1 and CD147 protein in NPC cell lines of CNE1, CNE2 and C666-1, compared to NP69 cells (Fig. 1c). Haematoxylin and Eosin (H&E) staining of NPC tumor was shown in Figure 1d. An immunohistochemistry staining revealed the strong expression of Cav-1 predominantly in cytoplasm of NPC tumor cells (hollow arrows), while medium expression in paired normal epithelium (black arrows) (Fig. 1e). In addition, strong expression of CD147 was observed predominantly in plasma membrane of NPC tumor cells (hollow arrows), while weak expression in paired normal tissues (black arrows) (Fig. 1f).

Figure 1.

Expression of Cav-1 and CD147 in NPC cell lines and clinical samples. QRT-PCR detection showed that NPC cell lines of CNE1, CNE2 and C666, as well as NPC tumors (NPC1 and NPC2) presented higher level of mRNA expression of Cav-1 (a) and CD147 (b) than that in immortalized primary nasopharyngeal epithelial cell line of NP69 cells and normal nasopharynx epithelium (NPNE1 and NPNE2). (c) Western blot analysis showed that the expression of Cav-1 and CD147 protein levels in NPC cell lines of CNE1, CNE2, C666-1 were higher than that in NP69 cells. (d) H&E staining of NPC tumor cells (×400). (e) Cav-1 (IHC, ×400) staining showed strong expression of Cav-1 predominantly in cytoplasm of NPC tumor cells (hollow arrows), while medium expression of Cav-1 in paired normal epithelium (black arrows). (f) (IHC, ×400) Strong CD147 staining was observed predominantly in plasma membrane of NPC tumor cells (hollow arrows), while weak expression of Cav-1 was observed in paired normal tissues (black arrows).

For immunohistochemistry staining of Cav-1, in total, 194 cases were evaluated. Overexpression of Cav-1 was observed in 96 cases (49.48%) of NPC, which was associated significantly with local recurrence (p = 0.038) and metastasis (p = 0.025) of patients with NPC. No significant association between Cav-1 expression and age, gender and TNM stage of the patients was observed in our study (Table I). For CD147 staining, in the informative 197 cases, overexpression of CD147 was observed in 117 cases (59.39%) of NPC, which was associated significantly with metastasis of the disease (p = 0.017). No significant association between CD147 expression and age, gender, clinical staging and recurrence of the disease was observed in our study (Table I). In total, there were 157 qualified cases for both Cav-1 and CD147 immunostaining evaluation. Among the 75 Cav-1 overexpression cases, 74.67% (56/75) also showed CD147 overexpression. In contrast, in the 82 Cav-1 low expression cases, only 40.24% (33/82) showed CD147 overexpression. A significant positive correlation was observed between Cav-1 and CD147 expression (ρ = 0.330, p = 0.000).

Table I. Correlation Between Cav-1 and CD147 Expression and Clinicopathological Parameters of NPC
ParametersCases (n = 194)Cav-1 expressionp ValueCases (n = 197)CD147 expressionp Value
Low expression (n = 98)High expression (n = 96)Low expression (n = 80)High expression (n = 117)
Age        
 <468746410.5549440540.596
 ≥461075255 1034063 
Gender        
 Male14469750.21914857910.298
 Female502921 492326 
WHO classification        
 II5430240.3835024260.218
 III1406872 1475691 
T stage        
 T1+T27837410.4828032480.886
 T3+T41166155 1174869 
N stage        
 N05026240.8075223290.535
 N1+N2+N31447272 1455788 
TNM stage        
 I+II4922270.3634818300.614
 III+IV1457669 1496287 
Recurrence        
 Yes4818300.0385019310.664
 No1468066 1476186 
Metastasis        
 Yes247170.025265210.017
 No1709179 1717596 

Combined Cav-1 and CD147 overexpression predicts poor survival of the patients with NPC

When the patient cohort was stratified according to tumor expression of Cav-1, the 5-year overall survival rates in patients with low Cav-1 expression (n = 98) and high Cav-1 expression (n = 96) were 62.42 and 50.34%, respectively; there was a significant difference between the 2 groups (p = 0.02, Fig. 2a). With respect to CD147 expression, the 5-year overall survival rates in patients with NPC with low CD147 expression (n = 80) and high CD147 expression (n = 117) were 66.91 and 46.08%, respectively; there was a significant difference between the 2 groups (p = 0.0009, Fig. 2b). When the patient cohort was stratified according to expression of both Cav-1 and CD147, the 5-year overall survival rates were 68.32, 55.85 and 45.17%, in patients that showed low expression of both Cav-1 and CD147 (n = 49), high expression of Cav-1 or CD147 (n = 52) and high expression of both Cav-1 and CD147 (n = 56), and there was significant difference between both Cav-1 and CD147 high and low expression groups (p = 0.004, Fig. 2c). It was noteworthy that patients who had tumor with high expression of both Cav-1 and CD147 had a worse prognosis than that of in patients with Cav-1 and/or CD147 low expression groups (p = 0.001, Fig. 2c).

Figure 2.

Kaplan-Meier curves of overall NPC patient survival. (a) The 5-year overall survival rates were 62.42 and 50.34% in patients seen with low levels of Cav-1 expression (n = 98) and high levels of Cav-1 expression (n = 96), respectively; there was a significant difference in the overall survival rate between the 2 groups (p = 0.02). (b) The 5-year overall survival rates were 66.91 and 46.08% in patients with NPC seen with low levels of CD147 expression (n = 80) and high levels of CD147 expression (n = 117), respectively; there was a significant difference in the overall survival rate between the 2 groups (p = 0.0009). (c) The 5-year overall survival rates were 68.32, 55.85 and 45.17%, in patients seen with low levels of both Cav-1 and CD147 expression (n = 49), high expression of Cav-1 or CD147 (n = 52) and high levels of both Cav-1 and CD147 expression (n = 56), respectively; there were significant differences between both Cav-1 and CD147 high and low expression groups (p = 0.004), and Cav-1 and/or CD147 low expression groups (p = 0.001).

Univariate analyses showed CD147 alone [Hazard ratio (95% CI), 2.463(1.368–4.436); p = 0.003], combined Cav-1 and CD147 expression [Hazard ratio (95% CI), 2.335(1.363–4.000); p = 0.002], gender [Hazard ratio (95% CI), 0.384(0.186–0.791); p = 0.009] and metastasis [Hazard ratio (95% CI), 2.046(1.021–4.097); p = 0.043] were prognostic predictor of overall survival in patients with NPC (Table II). Multivariate Cox regression analysis indicated that neither Cav-1 alone nor CD147 alone was informative independent prognostic factors in this group of patients with NPC (Cav-1 alone: p = 0.664; CD147 alone: p = 0.083). However, the combination of high Cav-1 and CD147 expression had significant, independent predictive value of survival in patients with NPC [Hazard ratio (95% CI), 2.135(1.242–3.672); p = 0.006]. In addition, gender was also an independent prognosis predictors in patients with NPC [Hazard ratio (95% CI), 0.427(0.206–0.882); p = 0.022] (Table II).

Table II. Univariate and Multivariate Cox Regression Analyses of the Relationships between Clinicopathological Outcomes of NPC
VariableSubsetHazard ratio (95% CI)p
Univariate analysis (n = 157)
 Cav-1 expression aloneLow vs. High1.701 (0.984–2.940)0.057
 CD147 expression aloneLow vs. High2.463 (1.368–4.436)0.003
 Cav-1 and CD147 expressionCav-1 and/or CD147 low vs. Cav-1 and CD147 high2.335 (1.363–4.000)0.002
 Age<46 vs. ≥461.363 (0.790–2.352)0.266
 GenderMale vs. Female0.384(0.186–0.791)0.009
 Clinical stageI, II vs. III, IV1.579 (0.826–3.019)0.167
 T-stageT1+T2 vs. T3+T41.440 (0.827–2.509)0.198
 N-stageN0 vs. N1+N2+N30.862 (0.474–1.565)0.625
 RecurrenceNo vs. Yes1.354 (0.771–2.377)0.292
 MetastasisNo vs. Yes2.046 (1.021–4.097)0.043
Multivariate analysis (n = 157)
 Cav-1 expression aloneLow vs. High 0.664
 CD147 expression aloneLow vs. High 0.083
 Cav-1 and CD147 expressionCav-1 and/or CD147 low vs. Cav-1 and CD147 high2.135 (1.242–3.672)0.006
 Age<46 vs. ≥46 0.369
 GenderMale vs. Female0.427 (0.206–0.882)0.022
 Clinical stageI, II vs. III, IV 0.129
 T-stageT1+T2 vs. T3+T4 0.196
 N-stageN0 vs. N1+N2+N3 0.84
 RecurrenceNo vs. Yes 0.659
 MetastasisNo vs. Yes 0.054

Cav-1 and CD147 increase the migration ability of CNE1 and CNE2 cells

CNE1 and CNE2 cells transfected with Cav-1 siRNA (50 nM), CD147 siRNA (50 nM) or control siRNA (50 nM) were used in transwell migration assays at 48 hr posttransfection. The average number of the migrated cells was 21.8 ± 4.3 and 19.6 ± 4.3, respectively, in Cav-1 siRNA and CD147 siRNA transfected CNE1 cells, which were significantly lower than that (35.2 ± 7.5) of with siRNA control treated CNE1 cells (p < 0.05). Similarly, the average number of the migrated cells was 58.4 ± 10.2 and 25.1 ± 7.8, respectively, in Cav-1 siRNA and CD147 siRNA treated CNE2 cells, which was significantly lower than the number (160.5 ± 18.3) of siRNA control treated CNE2 cells (p < 0.05) (Figs. 3a and 3c). In contrast, the average number of migrated cells of which were transfected with a vector of pCI-neo-Cav-1 (CNE1, 15.3 ± 4.1; CNE2, 73.8 ± 14.1) or pCI-neo-CD147 (CNE1, 30.2 ± 7.1; CNE2, 114.6 ± 16.6), were significantly higher that of which was transfected with the control vector in CNE1 (5.4 ± 0.9) and CNE2 (57.2 ± 10.1) cells, respectively. There was statistically difference (p < 0.05) (Figs. 3b and 3d).

Figure 3.

Both Cav-1 and CD147 alter CNE1 and CNE2 cell migration. (a) CNE1 and CNE2 cells were transfected with a Cav-1 siRNA (50 nM), with a CD147 siRNA (50 nM) or with a control siRNA (50 nM), and transwell migration assays were carried out at 48 hr posttransfection. The cells passed through the filter and attached to the lower side of the filter as determined by DAPI staining (×200). (b) CNE1 and CNE2 cells transfected with a vector to overexpress Cav-1 (pCI-neo-Cav-1), or with a vector to overexpress CD147 (pCI-neo-CD147) or with the vector alone (pCI-neo, control) were used in transwell migration assays at 24 hr posttransfection. The cells passed through the filter and attached to the lower side of the filter (DAPI staining, ×200). (c) The average number of Cav-1 siRNA-transfected (21.8 ± 4.3) and CD147 siRNA-transfected (19.6 ± 4.3) migrating CNE1 cells was significantly lower than the number of control siRNA-transfected migrating CNE1 cells (35.2 ± 7.5) (p < 0.05). The average number of Cav-1 siRNA-transfected (58.4 ± 10.2) and CD147 siRNA-transfected (25.1 ± 7.8) migrating CNE2 cells was significantly lower than the number of control siRNA-transfected CNE2 cells (160.5 ± 18.3) (p < 0.05). (d) The average number of migrated cells that transfected with a vector of pCI-neo-Cav-1 (CNE1, 15.3 ± 4.1; CNE2, 73.8 ± 14.1) or pCI-neo-CD147 (CNE1, 30.2 ± 7.1; CNE2, 114.6 ± 16.6) were significantly higher than that of cells which was transfected with the control vector in CNE1 (5.4 ± 0.9) and CNE2 (57.2 ± 10.1) cells, respectively. There were statistically significances (p < 0.05).

Cav-1 and CD147 induce the secretion of MMP-3 and MMP-11 (active) in CNE1 and CNE2 cells

Immunoblot analysis of secreted MMPs protein in the cell culture supernatant showed that MMP-3 and MMP-11 (active) secretion of CNE1 and CNE2 cells was regulated by Cav-1 and CD147. In CNE1 cells transfected with the vector pCI-neo-Cav-1, in which 4.4-fold Cav-1 overexpressed, it has been observed a 1.7-fold and 1.9-fold increase in the secretion of MMP-3 and MMP-11 (active) in the cell culture supernatant, respectively. Similarly, a 5.1-fold overexpression of Cav-1 in CNE2 cells was accompanied by a 1.9-fold and 1.5-fold increase in the secretion of MMP-3 and MMP-11 (active) in the cell culture supernatant, respectively (Figs. 4a and 4b). On the other hand, CNE1 and CNE2 cells that showed reduced 74 and 59% of Cav-1 expression by siRNA mediated inhibition, were observed a 37 and 62%, as well as 15 and 59% decrease in the secretion of MMP-3 and MMP-11 (active) in the cell culture supernatant, respectively (Figs. 4c and 4d). After transfection with a CD147 specific siRNA, the CNE1 cells of which CD147 expression decreased by 75%, accompanied by a 34 and 60% reduction in the secretion of MMP-3 and MMP-11 (active) in the cell culture supernatant. Similarly, siRNA-mediated inhibition of CD147 by 65% in CNE2 cells was accompanied by a 100 and 45% reduction in MMP-3 and MMP-11 (active) secretion in the cell culture supernatant (Figs. 4e and 4f). However, the expression of MMP-3 and MMP-11 (active) in cell lysate was not altered in CNE1 cells transfected with the Cav-1 overexpression vector (pCI-neo-Cav-1), or with the siRNA to target Cav-1 and CD147 expression (Fig. 4g).

Figure 4.

Both Cav-1 and CD147 regulate MMP-3 and MMP-11 (active) secretion in CNE1 and CNE2 cells. (a) Immunoblot analysis of secreted MMP-3 and MMP-11 (active) in media from CNE1 and CNE2 cells transfected with a vector to overexpress Cav-1 (pCI-neo-Cav-1) or with the vector alone as a negative control (pCI-neo). (b) Densitometry analysis revealed that CNE1 cells transfected with pCI-neo-Cav-1 show a 4.4-fold increase in Cav-1 protein expression, a 1.7-fold increase in MMP-3 secretion and a 1.9-fold increase in MMP-11 (active) secretion. CNE2 cells transfected with pCI-neo-Cav-1 show a 5.1-fold increase in Cav-1 protein expression, a 1.9-fold increase in MMP-3 secretion and a 1.5-fold increase in MMP-11 (active) secretion. (c) Immunoblot analysis to examine MMP-3 and MMP-11 (active) secretion in CNE1 and CNE2 cells transfected with an a Cav-1 siRNA (50 nM) or with a control siRNA (50 nM). (d) Densitometry analysis showed that CNE1 cells transfected with the Cav-1 siRNA exhibit a 74% reduction in Cav-1 protein expression, and a 37 and 62% reduction in MMP-3 and MMP-11 (active) secretion, respectively. CNE2 cells transfected with the Cav-1 siRNA show a 59% reduction in Cav-1 expression, and a 15 and 59% reduction in MMP-3 and MMP-11 (active) secretion, respectively. (e) Immunoblot analysis to examine MMP-3 and MMP-11 (active) secretion in CNE1 and CNE2 cells transfected with a CD147 siRNA (50 nM) or with a control siRNA (50 nM). (f) Densitometry analysis showed that CNE1 cells transfected with the CD147 siRNA exhibit a 75% reduction in CD147 expression, and a 34 and 60% reduction in MMP-3 and MMP-11 (active) secretion, respectively. CNE2 cells transfected with the CD147 siRNA exhibit a 65% reduction in CD147 expression, and a 100 and 45% reduction in MMP-3 and MMP-11 (active) secretion, respectively. (g) Western blot analysis showed the expression of MMP-3 and MMP-11 (active) in cell lysate was not altered in CNE1 cells after transfected with the Cav-1 overexpression vector (pCI-neo-Cav-1), or with the siRNA to target Cav-1 and CD147 expression.

To investigate whether CD147 is a mediator of Cav-1 regulation of MMP-3 and MMP-11 (active) secretion in NPC cells, CNE1 cells was cotransfected with pCI-neo-Cav-1 and CD147 siRNA, then immunoblot analysis of secreted MMP-3 and MMP-11 (active) was taken in the cell culture supernatant (Fig. 5a). Compared to CNE1 cells transfected with pCI-neo-Cav-1, CNE1 cells co-transfected with pCI-neo-Cav-1 and CD147 siRNA (100nM) showed a 20 and 27% reduction in MMP-3 and MMP-11 (active) secretion, respectively, meanwhile CNE1 cells transfected with CD147 siRNA showed a 33 and 25% reduction in MMP-3 and MMP-11 (active) secretion, respectively (Fig. 5b).

Figure 5.

Cav-1 regulates MMP-3 and MMP-11 (active) secretion in CNE1 cells partially through CD147. (a) Immunoblot analysis of secreted MMP-3 and MMP-11 (active) in media from CNE1 cells transfected with pCI-neo-Cav-1/CD147 siRNA (100 nM), pCI-neo-Cav-1 or CD147 siRNA (100 nM). (b) Densitometry analysis revealed that, compared to CNE1 cells transfected with pCI-neo-Cav-1, CNE1 cells cotransfected with pCI-neo-Cav-1 and CD147 siRNA (100 nM) showed a 20 and 27% reduction in MMP-3 and MMP-11 (active) secretion, respectively, meanwhile CNE1 cells transfected with CD147 siRNA showed a 33 and 25% reduction in MMP-3 and MMP-11 (active) secretion, respectively.

Cav-1 regulates CD147 expression in CNE1 and CNE2 cells

To verify whether Cav-1 regulates CD147 expression in NPC cell, we transiently transfected CNE1 and CNE2 cells with a vector to overexpress Cav-1 [pCI-neo-Cav-1] and a Cav-1-specific siRNA. Immunoblot analysis indicated that in CNE1 and CNE2 cells transfected with a Cav-1-specific siRNA (50 nM) at 48 hr posttransfection (The siRNA-mediated inhibition of Cav-1 expression in CNE1 and CNE2 NPC cell lines was shown in Fig. S1), the Cav-1 protein expression was decreased in 74 and 60%, respectively; meanwhile a 45 and 40% reduction of total CD147 expression was observed (Figs. 6a and 6b). On the other hand, in CNE1 and CNE2 cells transfected with the vector pCI-neo-Cav-1, Cav-1 protein expression increased in 4.4-fold and 2.6-fold, respectively; consistently, a 2.8-fold and 1.5-fold overexpression of CD147 protein was observed in CNE1 and CNE2 cells transfected with pCI-neo-Cav-1 (Figs. 6c and 6d).

Figure 6.

Cav-1 regulates CD147 expression in CNE1 and CNE2 cells. (a) Immunoblot analysis of Cav-1 and CD147 protein expression in CNE1 and CNE2 cells transfected with a specific siRNA to target Cav-1 (50 nM) or with a negative control siRNA (50 nM). Cells were analyzed at 48 hr posttransfection, and GAPDH was used as a loading control. (b) Densitometry analysis showed that transfection of CNE1 cells with a Cav-1 siRNA (50 nM) led to a 74% reduction in Cav-1 protein levels, and a 45% reduction in CD147 protein levels. Transfection of CNE2 with the Cav-1 siRNA led to a 60% reduction in Cav-1 protein levels, and a 40% reduction in CD147 protein levels. (c) Immunoblot analysis to determine Cav-1 and CD147 protein expression levels in CNE1 and CNE2 cells transfected with a vector to overexpress Cav-1 (pCI-neo-Cav-1) or with the empty vector as a negative control (pCI-neo, control). Cells were collected and analyzed at 24 hr posttransfection; GAPDH was used as loading control. (d) Densitometry analysis showed that CNE1 cells transfected with a vector to overexpress Cav-1 (pCI-neo-Cav-1) exhibited a 4.4-fold and 2.8-fold increase in Cav-1 and CD147 protein expression, respectively. CNE2 cells overexpressing Cav-1 exhibited a 2.6-fold and 1.5-fold increase in Cav-1 and CD147 protein expression, respectively.

Discussion

Cav-1 overexpression was reported previously to be correlated with the metastasis of several malignancies, including prostate cancer,12 ESCC,13 breast cancer28 and lung cancer.29 Cav-1 overexpression in the tumor cells has also been reported to be correlated with poor prognosis of the patients with ESCC,13 renal clear cell carcinoma,30 prostate cancer,31 lung cancer32 and pancreatic ductal adenocarcinoma.33 In the present study, we revealed that Cav-1 overexpression in NPC tumor cells was significantly correlated with recurrence (p = 0.038) and metastasis (p = 0.025) of the disease, and with poor prognosis of patients with NPC. Overexpression of CD147 in the tumor cells has been reported to be correlated with metastasis of breast cancer,21 and oral squamous cell carcinoma34; and with poor prognosis of patients with ESCC,20 breast cancer,21 serous ovarian cancer22 and gastric cancer.23 We found that CD147 overexpression in NPC tumor cells was significantly correlated with metastasis (p = 0.017) of the disease and poor prognosis of patients with NPC in present study. In addition, we found that patients with NPC with overexpression of both Cav-1 and CD147 in tumor cells had a significantly worse prognosis and a significantly lower 5-year overall survival rate relative to patients with NPC with low expression of Cav-1 and / or CD147 (p = 0.001). To our knowledge, this is the first report of Cav-1 and CD147 overexpression and their significance with respect to the metastasis and prognosis of NPC. Transwell migration assays further revealed that loss of Cav-1 and CD147 expression inhibited CNE1 and CNE2 cell migration ability, whereas Cav-1 and CD147 overexpression promoted cell migration ability. Taken together, our results are consistent with previous studies of Cav-1 and CD147 expression in other malignancies, and indicate that these 2 genes may play a key role in the invasion and metastasis of NPC and correlate with poor prognosis of patients with NPC.

Growing evidence suggests a close association between Cav-1, CD147 and the expression or secretion of MMPs. Overexpression of Cav-1 in HEK293 cells decreased MMP-1 secretion in a coculture assay with primary human fibroblasts,35 and overexpression of Cav-1 in metastatic mammary tumor cells could inhibit MMP-2 and MMP-9 secretion, although the expression of MMP-2 and MMP-9 in whole cell lysates was not altered,9 In contrast, Cav-1 can induce MMP-11 secretion and the invasive potential of a murine hepatocarcinoma cell line.36 CD147 is a tumor cell-derived MMP inducer that is expressed on the tumor cell surface and triggers the production or release of MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP and MT2-MMP in the surrounding mesenchymal cells and tumor cells.18, 19, 37–39 In the present study, we found that the suppression of Cav-1 and CD147 expression led to decreased MMP-3 and MMP-11 (active) secretion in CNE1 and CNE2 cells, whereas overexpression of Cav-1 in CNE1 and CNE2 cells promoted MMP-3 and MMP-11 (active) secretion. Furthermore, suppression of CD147 expression in Cav-1 overexpressing CNE1 cells could also lead to decreased MMP-3 and MMP-11 (active) secretion. Taken together, these results indicate that Cav-1 and CD147 overexpression in NPC tumors can promote tumor cell migration by stimulating MMP-3 and MMP-11 (active) secretion in NPC tumor cells, and Cav-1 regulates MMP-3 and MMP-11 (active) secretion partially, maybe directly, through CD147 in NPC tumor cells.

To date, the relationship between Cav-1 and CD147 genes is controversial. It was reported that the overexpression of Cav-1 in HEK293 cells decreased CD147 clustering and the HG-CD147/LG-CD147 ratio, whereas downregulation of Cav-1 increased the HG-CD147/LG-CD147 ratio in human rhabdomyosarcoma cells.17, 35 On the contrary, it was demonstrated that Cav-1 can upregulate CD147 glycosylation and the invasive capability of a murine hepatocarcinoma cell line.36 The precise role of Cav-1 in carcinogenesis remains far from being elucidated. Cav-1 was reported to play a critical role in the modulation of cell cycle progression in vivo.40 The expression of Cav-1 in human breast cancer cells enhances matrix-independent cell survival that is mediated by upregulation of IGF-I receptor expression.41 In our study, data showed that siRNA-mediated inhibition of Cav-1 expression in the human NPC cell lines led to significant downregulation of CD147 protein expression. On the other hand, Cav-1 overexpression led to significant upregulation in CD147 protein expression. Cav-1 expression was positively correlated with CD147 expression in NPC tumor cells (ρ = 0.330, p = 0.000). The results of our study differ from those earlier reports; and this difference may be due to the different culture or genera of the cell lines and different forms of Cav-1. These results indicate that Cav-1 regulates the expression of CD147 in NPC cell lines, and one of the roles of Cav-1 in NPC carcinogenesis may be partly meditated by the upregulation of CD147 expression. The precise mechanism which Cav-1 regulates the expression of CD147 in NPC cell lines was deserved to further study.

In conclusion, the results of our study suggest that overexpression of Cav-1 partly leads to CD147 upregulation in NPC, both of which can enhance tumor cell migration by inducing MMP-3 and MMP-11 (active) secretion in NPC cells, and is associated with tumor recurrence, metastasis, as well as poor prognosis of the patients with NPC. Collectively, the results of our study suggest that Cav-1 and CD147 may represent new potential therapeutic targets relevant to NPC prognosis.

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