Patients with NSCLC who underwent radical resection of their primary cancer at the Department of Radiation Oncology, Jinan Military General Hospital (Jinan, China) were used in the present study. Patients were excluded from the study if they had previously undergone radiotherapy or chemotherapy for cancer treatment. Two groups were included in the present study. The first group included 42 fresh NSCLC tumor samples, which were immediately frozen and stored in liquid nitrogen for protein and RNA extraction following surgical resection. In addition to the first group, 240 NSCLC tissue specimens, including 139 adenocarcinomas and 101 squamous cell carcinomas, obtained from the Department of Radiation Oncology, Jinan Military General Hospital between January 2005 and January 2008, were used. All specimens were histologically analyzed and classified using the World Health Organization classification system. Detailed clinical, pathological and survival data were available. Written informed consent was obtained from all patients for the use of their tissues. Furthermore, the present study was approved by the Institutional Review Board at Jinan Military General Hospital. Patient follow-up was performed at three-month intervals. The median follow-up period was 48 months (range, 9–66 months) for all patients. Overall survival was defined as the period from the time of surgery to mortality.

The total RNA from frozen fresh samples was extracted using a TRIzol^® extraction kit (Invitrogen Life Technologies, Carslbad, CA, USA) and reverse transcribed in a 25 μl reaction volume using Taqman^® reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The complementary (c)DNA was diluted and quantified using qPCR analysis using SYBR^® Green I. The primer sequences used for the qPCR analysis were as follows: AP-4 forward, 5′-GAGGGCTCTGTAGCCTTGC-3′ and reverse, 5′-GAATCCCGCGTTGATGCTCT-3′; GAPDH forward, 5′-ACAACTTTGGTATCGTGG-3′ and reverse,5′-GCCATCACGCCACAGTTTC-3′. Data were analyzed using the ΔΔCt method and normalized using GAPDH expression.

Frozen tumor tissues were prepared by washing twice in cold phosphate-buffered saline (PBS). Approximately 20 mg tissue from each fresh sample was homogenized in 0.5 ml ice-cold cell lysis buffer (Roche Applied Science, Penzberg, Germany) containing fresh protease and phosphate inhibitors. Lysates were then centrifuged at 12,000 × g in a microcentrifuge at 4°C for 20 min and the resulting supernatants were used as tissue extracts. The extracted proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked using Tris-buffered saline (TBS) containing 5% non-fat dried milk and then probed with primary antibodies in PBS containing 5% bovine serum. The following primary antibodies were used: Rabbit anti-AP-4 (Millipore Corp., Billerica, MA, USA) and mouse anti-GAPDH (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactive bands were detected and quantified using an imaging system (Invitrogen Life Tecnologies).

The antibodies used for western blot analysis were also used for immunohistochemical staining. Formalin-fixed and paraffin-embedded tissue sections (5-μm thick) were deparaffinized, hydrated and heated in a steamer for 10 min for antigen retrieval. Peroxidase activity was blocked using 3% H₂O₂ in methanol at room temperature for 10 min, followed by incubation in 10% bovine serum albumin in TBS-Tween 20 for 30 min. Slides were then incubated with primary antibodies against AP-4 at a 1:100 dilution for 60 min at room temperature. Subsequent to washing with PBS, the slides were incubated with biotin-labeled secondary antibodies for 30 min. The samples were then incubated with streptavidin-peroxidase at a 1:40 dilution for 30 min. Samples were stained with 0.05% 3′,3-diaminobenzidine tetrahydrochloride prepared in 0.05 mol/l TBS (pH 7.6) containing 0.02% H₂O₂, then counterstained with hematoxylin. Formalin-fixed and paraffin-embedded lung tissues with normal bronchial epithelia were used as a positive control. Tissue samples which were not incubated with the primary antibodies were used as a negative control. Immunohistochemical staining was quantified by two independent pathologists.

Staining was quantified using a scoring method based on the intensity and proportion of the immunohistochemically stained cells. The proportion of positively stained tumor cells was determined semi-quantitatively and each sample was scored as follows: 0, <1%; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The staining intensity of the positively stained tumor cells was scored as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The immunoreactive score of each tumor was calculated by the sum of the two parameters. The immunohistochemical staining was ultimately graded as either negative (total score, 0–1) or positive (total score, 2–7). All stained sections were assessed by two independent pathologists without knowledge of the clinicopathological features.

Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Independent sample Student’s t-tests and χ² tests were used to analyze the continuous and categorical variables, respectively. Survival probability was assessed using the Kaplan-Meier estimator. The log-rank test was used for the comparison of patient survival. The Cox proportional hazards model was used to calculate the effect of AP-4 expression on patient survival, with adjustments made for clinical and histopathological parameters, including age, gender and smoking status. P<0.05 was considered to indicate a statistically significant difference.

AP-4 expression was assessed using qPCR analysis in 42 fresh NSCLC samples and matched adjacent non-cancerous lung tissues. AP-4 mRNA expression was found to be significantly higher in the NSCLC samples compared with the adjacent noncancerous tissues (P<0.0001; Fig. 1). Furthermore, western blot analysis revealed that AP-4 protein expression was significantly increased in the 42 fresh NSCLC samples compared with the matched adjacent noncancerous tissues, as quantified using densitometry (P<0.0001; Fig. 2).

Immunohistochemical staining revealed that AP-4 was expressed in the nuclei of the cells in the NSCLC tissues. The rate of AP-4 expression was significantly higher in the NSCLC tissue samples (48.3%; 116/240) compared with the adjacent noncancerous lung tissues (5.8%; 14/240; P<0.01; Fig. 3). The correlation between AP-4 expression and clinicopathological features is shown in Table I. AP-4 expression was found to be significantly associated with the tumor, nodes and metastasis (TNM) stage.

Kaplan-Meier survival estimates revealed that overall survival was significantly lower in patients with positive AP-4 expression than in those with negative AP-4 expression (P=0.0026; Fig. 4). Furthermore, Cox proportional hazard multivariate analysis was used to analyze the correlation between AP-4 expression in NSCLC tissues and other features, including patient gender and smoking history, as well as tumor histology, size, differentiation, metastasis status and TNM stage. Positive AP-4 expression (hazard ratio, 2.543; 95% confidence interval, 1.18–5.016; P=0.016) was found to be an independent prognostic indicator in patients with NSCLC, in addition to lymph node status and distant metastasis (Table II).