High expression of Talin-1 is associated with poor prognosis in patients with nasopharyngeal carcinoma

Human NPC cell lines (CNE-1, CNE-2, SUNE-1, C666-1, HNE1 and HONE1) were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum. The immortalized nasopharyngeal epithelial cell line (NP69) was cultured in keratinocyte/serum-free medium (Invitrogen) supplemented with bovine pituitary extract, as previously described [12]. All cell lines were incubated at 37°C in a 5% CO₂ incubator.

Eight freshly-frozen NPC specimens and six normal nasopharyngeal epithelium samples were obtained from Sun Yat-sen University Cancer Center. In addition, a total of 233 pretreatment paraffin-embedded NPC specimens were collected from our hospital between January 2003 and February 2006; none of the 233 NPC patients had received radiotherapy or chemotherapy before biopsy. The clinical characteristics of all patients were recorded (Table 1). All patients were treated with conventional two-dimensional radiotherapy, and patients with stage III-IV disease also received platinum-based concurrent chemotherapy [21,22]. The median follow-up time for the entire cohort was 63.2 months (range, 5.2-91.87). The following end points (time to the first defining event) were assessed: 5-year overall survival (OS) and distant metastasis-free survival (DMFS). This study was approved by the Institutional Ethical Review Board of Sun Yat-sen University Cancer Center, and written informed consent was obtained from each patient.

Total RNA was extracted from the cell lines using TRIzol reagent (Life Technologies, Grand Island, NY, USA). The extracted RNA (2 μg) was reverse transcribed using random primers (Promega, Madison, WI, USA) and M-MLV reverse transcriptase (Promega) for 60 min at 37°C and then 15 min at 70°C, and the product was stored at -20°C until use. Quantitative RT-PCR was performed on the PRISM 7900HT sequence detection system (Applied Biosystems, Carlsbad, CA, USA) in 15 μL reactions containing 0.5 μL of reverse transcription product, 7.5 μL of Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen), 1.5 μL of each PCR forward primer and reverse primer (final concentration, 2.5 μM), and 5.5 μL of ddH₂O. The reactions were pre-incubated at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s and annealing/extension at 60°C for 1 min, then ramped from 60°C to 95°C to obtain a melting curve. The following PCR primers were used: Talin-1 forward, 5′-CTGGAGGCAACCACAGAAC-3′ and Talin-1 reverse, 5′-GTGGCTCTGGGGAACAGA-3′; glyceraldehyde-3-phosphatedehydrogenase (GAPDH) forward, 5′-CTCCTCCTGTTCGACAGTCAGC-3′ and GAPDH reverse, 5′-CCCAATACGACCAAATCCGTT -3′. All of the assays were performed in triplicate, and reactions containing either no template or no reverse transcriptase were used as negative controls. GAPDH was used as the normalization control. Agarose gel electrophoresis was performed to examine the mRNA expression level of Talin-1. PCR amplications were performed using the GoTaq Green Master Mix kit (Promega, USA) and PCR cycling conditions were as follows: 95°C for 2 min, followed by 27 cycles of 95°C for 30 s, 53°C for 30 s and 72°C for 8 s, then extension at 72°C for 5 min (Biorad, S1000 Thermal Cycler, USA). PCR products were checked by 1.5% agarose gel electrophoresis.

Total proteins were extracted using sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% 2-β-mercaptoethanol), and the protein concentration was quantified using the Pierce® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Total proteins were separated on 6% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed-milk powder in Tris buffered saline with tween-20 (TBS-T), incubated with mouse monoclonal anti-Talin-1 (clone 1A11) antibody (1:200; Abnova, Taipei, Taiwan), then incubated with anti-mouse IgG secondary antibody (1:5,000; Sigma, St. Louis, MO, USA). The bands were detected by enhanced chemiluminescence; α-tubulin antibody (1:5,000; Epitomics) was used as a loading control.

The expression of Talin-1 in the sections from the 233 paraffin-embedded NPC specimens was examined by immunohistochemical staining, as described previously [23]. Briefly, the tissue blocks were cut into 5 μm-thick sections, followed by deparaffinization and rehydration. The sections were microwaved for antigen retrieval in EDTA buffer, and 3% hydrogen peroxide was used to quench endogenous peroxidase activity. Non-specific binding was blocked using 1% bovine serum albumin. The sections were then incubated overnight at 4°C with mouse monoclonal anti-Talin-1 (clone 1A11) antibody (1:100; Abnova); normal goat serum was used as a negative control. After rinsing, the sections were incubated with biotinylated secondary antibody bound to a streptavidin-horseradish peroxidase complex. The bound antibody was visualized by adding 3,3-diaminobenzidine, and then the sections were counterstained with hematoxylin, dehydrated and mounted.

The sections were scored independently by two pathologists, and any disagreements were resolved by consensus. Both the proportion of positively-stained tumor cells and the intensity of staining were assessed [23,24]. The proportion of positive tumor cells was scored as follows: 1 (<10%), 2 (10-35%), 3 (35-70%), and 4 (>70%). The intensity of staining was graded on a scale from 0 to 3 as follows: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow brown), and 3 (strong staining, brown). The staining index was calculated as the product of the staining intensity score and the proportion of positive tumor cells; using this method, staining index scores of 0, 1, 2, 3, 4, 6, 8, 9 or 12 were possible. The optimal cutoff value for high and low expression was selected based on a receiver operating characteristic (ROC) curve analysis, as previously described [9,25-27]. The best cutoff score of Talin-1 was 5. Therefore, a staining index score of < 5 was used to define NPC with low Talin-1 expression and a staining index score of > 5 was designated as high Talin-1 expression.

For the wound healing assay, transfected CNE-2 or SUNE-1 cells were seeded into 6-well plates. After serum starvation in serum-free media for 24 h, an artificial wound was created on the confluent cell monolayer using a standard 200 μl plastic pipette tip. Cells migrated into the scratch area as single cells from the confluent sides; the width of the scratch gap was viewed under an inverted microscope and photographed at 0 h and 24 h. Three replicate wells from a six-well plate were used for this experiment.

Invasion assays were performed in Transwell chambers (Corning, Steuben County, New York, USA) coated with Matrigel (BD Biosciences) on the upper surface of membrane with 8 μm-pore size. In brief, transfected CNE-2 or SUNE-1 cells were harvested, suspended in serum-free medium and 1 × 10⁵ transfected cells were plated into the upper chamber for the invasion assays, and media supplemented with 10% FBS was placed into the lower chamber. After incubation for 24 h, the cells that had invaded through the membrane to the lower surface were fixed, stained and counted under an inverted microscope. Five random fields of view were analyzed for each chamber; three independent experiments were conducted for each assay.

The Chi-square test or Fisher’s exact test were used to analyze the relationships between Talin-1 protein expression and clinical characteristics. Talin-1 mRNA expression levels in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed using the Student’s t-test. For functional analyses, data were presented as mean ± standard deviation and Student’s t-tests were used to determine the significance of the differences between two groups. The Kaplan-Meier method was used to estimate OS and DMFS, and the differences were compared using the log-rank test. A Cox proportional hazards regression analysis with backward stepwise selection was used to identify independent predictive factors for OS and DMFS and calculate hazard ratio (HR) values. The Talin-1 expression level, age, sex, clinical stage, WHO type, VCA-IgA and EA-IgA were included as covariates. All statistical analyses were performed using SPSS 16.0 software (SPSS, Chicago, IL, USA); two-tailed P-values < 0.05 were considered significant.