RETRACTED ARTICLE: Psoriasin (S100A7) is a novel biomarker for lung squamous cell carcinoma in humans

NCI-H520 cells were from American Type Culture Collection. LC-2/ad cells were from RIKEN BRC. Cells were maintained in a humidified atmosphere with 5% carbon dioxide using Ham’s F-12 media (Cellgro, Manassas, Va) or Roswell Park Memorial Institute Media 1640 (Gibco, Grand Island, NY), respectively, with 10% fetal bovine serum (FBS). Tumor necrosis factor-alpha (TNF-α) was used to induce NF-κB activation. It was dissolved in phosphate buffered saline (PBS) and used at a concentration of 20 ng/mL. Antibodies for phosphorylated and total NF-κB and GAPDH were obtained from Cell Signaling Technology, Inc (Beverly, Mass). Antibody for S100A7 was obtained from Abcam (Cambridge, Mass).

Surgically resected lung squamous cell carcinomas (SCC) tissues (n = 240) and specimens of normal lung adjacent to the tumor tissues (n = 60) were obtained from People’s hospital of Linyi, Shandong. Follow-up information was available for 143 cases. None of the patients had been treated with any preoperative chemotherapy or radiotherapy before surgery. After surgical resection, all patients received standard therapies according to the 2004 NCCN Clinical Practice Guidelines in Oncology for NSCLC. After cancer recurrence, the treatment was determined by individual physicians caring for their patients. Written informed consent was obtained from each patient, and institutional review board approval of this study was obtained from People’s Hospital of Linyi. The histological classification of these tumors was based on the WHO criteria [13]. Pathologic staging was done according to the tumor-node metastasis system for lung cancer staging [14].

Four-micron-thick TMA sections were used in all analyses. The sections were deparaffinized with xylene and rehydrated through graded alcohol incubations into water. They were then processed following the optimal staining protocol, as described above. In short, heat-induced epitope retrieval was performed using a Decloakig Chamber (DAKO, Kyoto, Japan), in which tissues were heated to 125°C then cooled to 90°C in Tris/EDTA buffer at pH 9 (Target Retrieval Solution; DAKO). After heat-induced epitope retrieval treatment, endogenous peroxidases were blocked with Peroxidase-Blocking Solution (DAKO) for 5 min. The sections were then incubated with anti-S100A4 antibody (1:50 dilution) for 60 min, followed by incubation with EnVision + Dual Link system, according to the manufacturer’s recommendations (DAKO). The reaction products were visualized with DAB+ (DAKO). All procedures were carried out at room temperature.

Semiquantitative evaluation of the sections was performed by 2 pathologists in a blinded manner. The negative control for immunostaining was performed without primary antibody. Both the fraction and intensity of the immunostained tumor cells were considered. The fraction score was calculated as the average of 5 randomly selected high-power fields assessed by light microscopy as follows: 0, no stained target cells (tumor, bronchial or alveolar cells); 1,20% of cells stained; 2, 20,50% of cells stained; and 3, over 50% of cells stained. The intensity score was defined as follows: 0, no appreciable staining of the target cells; 1, barely detectable staining in the cytoplasm and/or nucleus of the target cells; 2, readily apparent brown staining; and 3, dark brown staining. The fraction and intensity scores were multiplied to give a total score ranging from 0 to 9. Scores between 2 and 9 were regarded as positive expression. Immunohistochemical assays were also performed on histological sections of paraffin-embedded xenograft tumors.

Staining of the proliferative marker Ki-67 was performed to assess the growth of the xenograft tumors. The Ki-67 antibody (diluted 1:100) was obtained from Thermo Fisher Scientific, Rockford, USA. The Ki-67 labeling index (Ki-67 LI) was calculated according to the following formula: Ki-67 LI = number of Ki-67 positive cells/total number of tumor cells counted × 100%.

pUSE vectors containing non-targeted shRNA and 4 distinct shRNA sequences targeting S100A7 were purchased from Sigma-Aldrich. Cells were transfected with the appropriate expression construct, or control empty vector (pUSE), using Lipofect-AMINE 2000 (Invitrogen, Carlsbad, CA) in accordance with the manufacturer’s protocol. Stable clones were selected by continuous treatment with G418 (Life Technologies, 0.8 mg/mL) over 4 weeks.

Total RNA was prepared from cultured cells using an RNeasy® Mini Kit (Qiagen, Maryland, USA), according to the manufacturer's protocol. cDNA was synthesized using SuperScript™II RNase H-Reverse Transcriptase (Invitrogen™, Sweden), as described by the manufacturer. The relative expression of S100A7 was analyzed using real-time PCR and the LightCycler instrument (Roche Applied Science, Sweden), together with the Light Cycler Fast Start DNA Master SYBR Green I kit (Roche Applied Science, Sweden), according to the manufacturer’s instructions. Primers for S100A7 [5] and β-actin [15] were as previously described. Two μl of cDNA were added to 18 μl of PCR Master Mixture (including 3 mM of MgCl2, 0.5 Mm of primers (S100A7 and β-actin) and 2 μl of LightCycler ™Fast Start Master SYBR Green). The protocol consisted of a 10-min denaturation step at 95°C and 40–45 cycles of amplification at 95°C for 10 seconds, 58°C (S100A7) and 60°C (β-actin) for five seconds and 72°C for eight seconds (β-actin) and 10 seconds (S100A7) respectively. After amplification, melting curves were obtained to verify the specificity of the amplification reaction and the products were also run on a 2% agarose gel. Quantitative analysis was performed with the LightCycler software version 3.5 (Roche Applied Science, Sweden), which estimates the crossing point (CP) for each sample. The CP values determined for psoriasin were normalized to β-actin (endogenous control).

Cells were plated on 6-well plates at a density of 3 × 10⁶ in full media for 24 hours, then serum-starved (0.5% FBS) for 24 hours, then treated. Cells were stimulated with or without TNF-α (20 ng/mL) for 4 hours, then lysed with Laemelli buffer under reducing conditions. Time trials with TNF-α identified sustained changes in NF-κB activation at this time point (data not shown). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis techniques and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in 1 × PBS, 0.1% Tween-20 (Sigma-Aldrich). Primary antibodies were dissolved in 5% bovine serum albumin (Sigma-Aldrich), 1 × PBS, 0.1% Tween-20. Secondary antibodies were prepared in 5% nonfat milk in 1 × PBS, 0.1% Tween-20. The protein of interest was developed using Pierce ECL Chemiluminescent (Thermo Fisher Scientific, Inc, Rockford, Ill). Densitometric analysis was performed using ImageJ Software (National Institutes of Health, Bethesda, Md).

The cells were seeded onto 96-well plates at 4,000 per well in culture medium (100 μL). After culturing for 72 hr, cell numbers were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described previously [16]. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (10 μL; 5 mg/mL) was added to each well and incubated for 4 hours at 37°C at 72 hr. The reaction was stopped by adding 100 μL of 0.04 N HCl in isopropanol to each well, with vigorous mixing to solubilize colored crystals produced by the reaction. The absorbance at 570 nm to absorbance at 630 nm as reference wave was measured by a multi well scanning spectrophotometer. Each data point is the average of six determinations and each experiment was repeated thrice.

All xenograft experiments were performed under a protocol approved by the People’s Hospital of Linyi, Animal Care and Use Committee. Male nude mice aged 6 weeks were purchased from Shandong University and quarantined for 2 weeks. At age 8 weeks the mice were injected into their left flanks with 1 × 10⁶ cells in 100 μL PBS. Flank tumors were measured every 2 to 3 days using digital calipers and volume was calculated by multiplying height × length × width. Xenograft tumors were removed en bloc and a portion from each tumor was preserved in 4% formalin in PBS and RNA later. Immunohistochemical staining of tumor tissue homogenate was used to examine S100A7 as described above. Tumors preserved in formalin were paraffin embedded; cut onto slides; and stained with hematoxylin and eosin, and for Ki-67. Histology was reviewed by a pathologist blinded to the study.

SPSS 11.0 for Windows (SPSS Inc., Chicago, Ill, USA) was used for all data analysis. The immunohistochemical results and their associations with clinical characteristics were analyzed using the chi-square test. Spearman’s rank test was used to analyze the correlation between protein phenotypes. The Kaplan-Meier method was used to analyze univariate survival, and comparisons of the survival distributions among groups were performed using the log-rank test. The prognostic significance of S100A7 expression with respect to other pathological variables was assessed using multivariate Cox regression analysis. The quantitative data are expressed as the mean ± S.D. Student t test was performed for 2 group comparisons, and analysis of variance test was performed when comparing 3 or more groups. All P values were 2-tailed, and values of P < 0.05 were considered statistically significant.