Expression of Nuclear Factor- B and Its Clinical Significance in Nonsmall-Cell Lung Cancer

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1 Expression of Nuclear Factor- B and Its Clinical Significance in Nonsmall-Cell Lung Cancer Zhenfa Zhang, MD, Jianqun Ma, MD, Ni Li, PhD, Nan Sun, MD, and Changli Wang, BM Department of Lung Cancer, Affiliated Cancer Hospital, Tian Jin Medical University, Tian Jin; Department of Thoracic Surgery, Affiliated Tumor Hospital, Ha Er Bin Medical University, Ha Er Bin; Department of Epidemiology, China Medical University, Shen Yang; and Department of Thoracic Surgery, Tumor Hospital of Liao Ning Province, Shen Yang, China Background. It has been known that transcription factor nuclear factor (NF)- B plays an important role in cell proliferation and oncogenesis. The aims of this study were to evaluate expression levels of NF- B in nonsmallcell lung cancer (NSCLC) and to elucidate its clinical significance and prognostic value for patients with NSCLC. Methods. Using 45 tumor tissue specimens from 45 patients with NSCLC who underwent surgery, we investigated the expression of NF- B using Western blotting analysis. Apoptotic rate of NSCLC cells with different expression of NF- B was determined by TUNEL (terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end-labeling) assay. Paraffin-embedded tissue blocks from 71 consecutive patients with NSCLC were obtained for immunohistochemical staining. Results. The expression level of NF- B in poorly or moderately differentiated lung cancer cells was higher than that in well-differentiated ones (p 0.001). The apoptotic rate was lower for lung cancer cells with higher NF- B expression than for those with lower NF- B expression (p ). Furthermore, expression of NF- B was correlated with caspase-3, cyclooxygenase-2, and p53 expression in lung cancer cells that were examined. Most NSCLC cells showed nuclear staining pattern and the nuclear positive rate was 67.6% (48 of 71 specimens). Immunohistochemical NF- B expression in patients with NSCLC was an independent prognostic factor for overall survival. Conclusions. The results of this study suggest that expression of NF- B may correlate with lung cancer differentiation. Overexpression of NF- B inhibits tumor cell apoptosis and indicates an unfavorable prognosis for overall survival in some patients with NSCLC. (Ann Thorac Surg 2006;82:243 8) 2006 by The Society of Thoracic Surgeons The nuclear factor (NF)- B transcription factor has a central role in several cellular processes, including proliferation, cell adhesion, apoptosis, inflammatory response, and regulation of the immune response. Nuclear factor- B regulates genes that encode cytokines and chemokines, angiogenesis, and a lot of antiapoptotic proteins. Recently, NF- B has been implicated in the biology of cancer. Numerous cancers have been shown to display constitutive activation of NF- B. Cancers that display evidence of NF- B activation include multiple myeloma, lymphoma, breast cancer, pancreatic cancer, prostate cancer, head and neck squamous cell carcinoma, colon cancer, and others [1]. Although considerable information about NF- B and its function has been accumulated, its behavior in nonsmall-cell lung cancer (NSCLC) cells has not been clarified. The aims of this study were to investigate expression of NF- B and its influence on apoptosis of lung cancer cells; and to elucidate its clinical significance in NSCLC. Accepted for publication Jan 10, Address correspondence to Dr Wang, Department of Lung Cancer, Affiliated Cancer Hospital, Tian Jin Medical University, Huan Hu Xi St, Tian Jin City, China ; wangchangli1973@yahoo.com. Material and Methods Patients Forty-five tumor tissue specimens were sampled from 45 patients with NSCLC who underwent surgery between October and December All patients were required to sign a written informed consent. Patients who received induction therapy were excluded from the study. Samples were histologically confirmed to have been obtained from tumor masses. A portion of each sample was frozen immediately after surgical resection and stored at 80 C until use. The clinicopathologic features of the specimens were assessed according to the World Health Organization classification system [2] and the tumor, lymph node, metastasis (TNM) staging system [3]. Locations and sizes of tumors were determined during operation. This patient group was used to investigate expression of NF- B by Western blotting, and the apoptotic rate of lung cancer cells by TUNEL (terminal deoxynucleotidyl- transferasemediated dutp-biotin nick end-labeling) assay. To determine the correlation between expression of NF- B and 5-year survival of lung cancer patients, we performed a retrospective immunohistochemical experiment using resected tissue blocks from patients who had undergone surgical resection 5 years before. From July to October 1999, 71 consecutive patients who underwent 2006 by The Society of Thoracic Surgeons /06/$32.00 Published by Elsevier Inc doi: /j.athoracsur

2 244 ZHANG ET AL Ann Thorac Surg ROLE OF NF- B IN NSCLC 2006;82:243 8 surgery for NSCLC in the thoracic department of First Affiliated Hospital of China Medical University were included in the study. The surgical procedure was a potentially curative complete resection in combination with hilar and mediastinal lymphadenectomy. Patients with typical carcinoid tumor and patients who had preoperative neoadjuvant chemotherapy or radiation therapy or who had palliative surgery were excluded from the study. Follow-up information was obtained from hospital case records or from a questionnaire completed by the local chest physician or general practitioner, or from death certificates. Our study was approved by the Clinical Trials Review Board of First Affiliated Hospital of China Medical University and by the principle investigator of every participating center. Western Blotting In brief, 100 mg frozen tissue was homogenized, and extracts were centrifuged at 10,000g for 5 minutes at 4 C to remove debris. Then, 10 L supernatant was heated in a boiling water bath for 4 minutes and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrotransfer of proteins from the gel to a nitrocellulose membrane was performed for 30 minutes using a semidry transfer system (BioRad, Hercules, CA). The membrane was then soaked in 5% defatted dry milk diluted with phosphate-buffered saline containing 0.02% Tween20 to reduce nonspecific protein binding. Next, the membrane was incubated with 50 L anti NF- B antibody (NF- B p50, rabbit IgG; Santa Cruz Biotechnology, Inc, Santa Cruz, CA) for 45 minutes at room temperature and then treated with a suitable second antibody (goat anti-rabbit IgG; Santa Cruz Biotechnology, Inc) for 30 minutes at room temperature. Enhanced chemiluminescence analysis was performed according to the manufacture s instructions, and the image was developed in a film in a darkroom. The same membrane was subsequently probed for the detection of -actin, caspase-3, p53, and cyclooxygenase-2 (COX-2). TUNEL Staining Apoptotic cells were detected by TUNEL staining using a commercially available kit (purchased from Roche Applied Science, Shang Hai, China). Cells were processed according to the manufacture s recommended protocol. Immunohistochemistry Paraffin tissue blocks were sectioned (thickness, 4 m), and immunohistochemical studies were performed on these paraffin sections using the avidin-biotinylated peroxidase complex method. In brief, sections were deparaffinized and treated with the buffer for antigen restorationin in accordance with the manufacturer s recommendation for each primary antibody. Next, sections were incubated in 0.3% H 2 O 2 in methanol for 20 minutes to quench endogenous peroxidase activity, and then treated with diluted normal blocking serum. The NF- B antibody (NF- B p50, rabbit IgG) was diluted in the antibody diluent and applied to the sections for 1 hour at room temperature. Incubation with a secondary antibody (goat anti-rabbit IgG) solution was Fig 1. Expression of nuclear factor (NF)- B in nonsmall-cell lung cancer by Western blotting. Lung cancer tissue samples were analyzed by probing with an anti NF- B polyclonal antibody. The blots were subsequently probed with anti-actin antibody as a loading control. The expression level of NF- B in each band was semiquantified by calculating the ratio of NF- B to -actin. performed for 1 hour at room temperature, after which incubation with an avidin-biotin-peroxidase complex for 30 minutes was performed. Peroxidase activity was visualized using diaminobenzidine tetrahydrochloride as the substrate, and sections were counterstained with hematoxylin. We counted 800 of the cancer cells. All immunostained sections were examined by at least two pathologists. Positive staining was defined as the presence of NF- B immunoreactivity in at least 10% of cancer cells [4]. Data Analysis Statistical analyses were performed using Student s t test to compare individual means. Correlation between NF- B and caspase-3, COX-2, and p53 was determined by bivariate correlation analysis. Survival rates were calculated by the Kaplan-Meier method using the date of operation as the starting point and the date of death or last follow-up as the endpoint; patients who died of other causes without evidence of recurrent disease or who were unavailable for follow-up were censored either at the time of death or at the last follow-up. Comparisons were made with log-rank test, and the Cox proportional hazards ratio model was used to investigate the simultaneous effect of multiple predictors on survival. All tests of significance were two-sided, and differences were considered statistically significant at p values less than The SPSS software (version 10.0; SPSS, Chicago, Illinois) was used for the analysis. Results Correlation With Clinical Characteristics The Western blotting products from NF- B protein were semiquantified using -actin as an intrinsic control. The density of each band was separately converted to a numeric value by Bandleader software. The ratio of NF- Bto -actin in each case was calculated. The average NF- B/ -actin value was (Fig 1). There was no significant correlation between NF- B and patient age, sex, tumor staging, and pathologic type. Expression level of NF- B was higher in poorly and moderately differentiated cancer cells than in welldifferentiated ones (t 3.640, p 0.001; Table 1).

3 Ann Thorac Surg ZHANG ET AL 2006;82:243 8 ROLE OF NF- B IN NSCLC 245 Table 1. Expression of Nuclear Factor- B and Clinical Characteristics of Nonsmall-Cell Lung Cancer Factor n Mean SE t p Value Sex Male Female Age (years) Stage I II Size (cm) Location Central Peripheral Differentiation Well Moderate/poor Histology Squamous cell Others Correlation With Apoptosis A TUNEL assay was performed to determine the influence of NF- B on apoptosis of lung cancer cells. Cells were determined to be TUNEL positive by counting a total of at least 1,000 neoplastic nuclei in 10 randomly chosen fields at 400-fold magnification. Positive signals were defined as presence of a distinct brown color nuclear staining of neoplastic cells. The apoptotic index was calculated by dividing the number of TUNEL positive cells by the total number of cells in 10 random fields. The Student t test was used to test the statistical significance using the percentage of apoptosis obtained from the data in the two groups. Our experiments showed that the apoptotic rate( ) in lung cancer cells with higher NF- B expression ( , n 31) was less than that ( ) in ones with lower NF- B expression (p , data not shown), indicating that NF- B negatively affects apoptosis of lung cancer cells. Correlation With Caspase-3, p53, and COX-2 The NF- B signaling pathway and the transcription factors that it activates have emerged as critical regulators of the apoptotic response, depending on the interplay within a complex yet precisely orchestrated network of proteins [5]. In the current study, we examined a number of biomarkers of apoptosis and searched for correlations with NF- B expression. Western blotting was used to determine expressions of NF- B, caspase-3, p53, and COX-2. Bivariate correlation analysis showed significant correlation between NF- B and caspase-3, p53, and COX-2; and the Pearson correlation coefficients were 0.789, 0.825, and 0.705, respectively (all p 0.000; Fig 2). Predicting Prognosis To evaluate whether nuclear NF- B expression by immunohistochemistry (data not shown) has prognostic significance in lung cancer, we analyzed the survival of patients using the Kaplan-Meier method. Univariate analysis revealed that histologic type, NF- B expression, lymph node involvement, tumor differentiation, and staging were significant prognostic factors (p , p , p , p , p , respectively; Table 2). Patients with NF- B positive expression had a survival significantly shorter than those who were NF- B negative. The overall survival curve for the patients with different expression of NF- B is shown in Figure 3. Table 3 summarizes the multivariate analysis of prognostic value of prognostic factors, which were determined by univariate analysis (p 0.05), in overall survival in 71 patients. In this study, a significant value in overall survival was observed for staging (p 0.000), tumor differentiation (p 0.007), lymph node involvement (p 0.034), and NF- B expression (p 0.018). Pathologic type was not an independent prognostic factor (p 0.057). Comment The transcription factor NF- B has been implicated in the control of cell proliferation and oncogenesis [6]. Nuclear factor- B transcription factors bind to DNA as heterodimers or homodimers that are composed of five possible subunits in mouse and human (RelA/p65, c-rel, RelB, p50, and p52). These proteins mediate DNA binding, dimerization, and interactions with inhibitory factors known as inhibitor B (I- B) proteins [7]. Degradation of the I- B proteins results in the liberation of NF- B, allowing nuclear translocation and binding to cognate DNA motifs in the regulatory regions of a host of target genes. As a consequence, transcription of these genes, which are involved in immune and inflammatory responses and regulation of apoptosis, as well as in cell growth control, is induced [8]. The transcription factor NF- B is activated in a range of human cancers and is thought to promote tumorigenesis, mainly due to its ability to protect transformed cells from apoptosis [9]. Rahman and colleagues [10] demonstrated that the inactivation of NF- B activity plays important roles in mediating I3C (indole-3 carbinol) induced apoptosis in breast cancer cells. Exposure of NF- B in human colon carcinoma tumor cell line HCT-116 cells to betalapachone resulted in growth inhibition and induction of apoptosis in a dose-dependent manner. After betalapachone treatment, the nuclear protein levels of NF- B and the activity of NF- B DNA binding were markedly decreased. Beta-lapachone treatment also resulted in inhibition of the transcriptional activity of NF- B luciferase reporter plasmid, suggesting that betalapachone induced apoptosis may be partly regulated through the inactivation of NF- B [11]. Denlinger and coworkers [12] suggested that the majority of NSCLC

4 246 ZHANG ET AL Ann Thorac Surg ROLE OF NF- B IN NSCLC 2006;82:243 8 Table 2. Univariate Analysis Showed Correlation Between Nuclear Factor- B and Other Factors and Prognosis of Patients With Nonsmall-Cell Lung Cancer Factors n Overall 5-Year Survival p Value Sex Male Female Age (years) Histology Squamous cell Others Location Central Peripheral Differentiation Good Moderate/poor Stage I II Necrosis factor- B Positive Negative Fig 2. Scatter graph for correlation between nuclear factor (NF)- B and caspase-3, p53, and COX-2. (A) Expression of NF- B and caspase-3. The linear regression equation was y x (p 0.000), and the correlation coefficient was (p 0.000). (B) Expression of NF- B and p53. The linear regression equation was y x (p 0.000), and the correlation coefficient was (p 0.000). (C) Expression of NF- B and COX-2. The linear regression equation was y x (p 0.000), and the correlation coefficient was (p 0.000). Fig 3. This chart illustrates the correlation between expression of nuclear factor (NF)- B and overall survival among patients with nonsmall-cell lung cancer. Survival distributions were calculated with the Kaplan-Meier method and were compared in a log-rank analysis ( : censored; p ). Nuclear factor- B is expressed as ratio to actin expression. Nuclear factor- B(-): median survival time, 52 months; 95% confidence interval: 48 to 55. Nuclear factor- B( ): median survival time, 20 months; 95% confidence interval: 17 to 23.

5 Ann Thorac Surg ZHANG ET AL 2006;82:243 8 ROLE OF NF- B IN NSCLC 247 Table 3. Correlation Between Nuclear Factor- B and Other Factors and Prognosis of Patients With Nonsmall-Cell Lung Cancer by Cox Proportional Hazards Ratio Model Factors OR 95% CI p Value Stage I II Differentiation Good Moderate/poor Lymph node Negative Positive Nuclear factor- B Positive Negative CI confidence interval; OR odds ratio. exhibits dysregulated antiapoptotic pathways involving the transcription factor NF- B. In the current study, we also found that expression of NF- B had a significant association with apoptosis in NSCLC cells, indicating that its function in development of NSCLC may be caused by its ability to inhibit apoptosis in lung cancer cells. To further elucidate its antiapoptotic role in lung cancer, we analyzed its correlation with biomarkers known to be involved in apoptosis: caspase-3, p53, and COX-2. Nonsmall-cell lung cancer cells exhibit intrinsic apoptosis resistance, while caspase-3 occupies a pivotal position in the final common pathway of apoptosis. Increasing evidence suggested that this protease was constitutively inhibited in NSCLC [13]. A subunit of NF- B, p65/rela, is cleaved at Asp(97) by caspase-3 during apoptosis. Caspase-3 cleaved p65 loses transcriptional activity and potentiates naphthoquinone (NA)- induced apoptosis, in contrast to an uncleavable mutant of p65, which protects the cell from apoptosis [14]. Our data also showed a negative correlation between NF- B and caspase-3, indicating that NF- B protection against apoptosis may be accompanied by inhibition of caspase-3 in NSCLC. The p53 gene is the most frequently mutated gene in human cancers. Loss of functional p53 leads to impaired responses of cancer cells to apoptosis induction and to poor prognosis in patients with certain types of cancer [15]. The p53-responsive element was predicted to be a binding site for NF-kappa B [16]. Cyclooxygenase-2 overexpression is seen in many malignancies including lung cancer. In NSCLC, COX-2 is overexpressed in most adenocarcinomas and squamous cell carcinomas. Elevated tumor COX-2 levels have been implicated in angiogenesis, tumor invasion, resistance to apoptosis, and suppression of antitumor immunity [17]. Cyclooxygenase-2 has an NF- B binding site on its promoter [18], and the inhibition of COX-2 expression correlated with the suppression of NF- B activity in the lung cancer cells [19]. These previous reports were consistent with our results that expression of NF- B was correlated with p53 and COX-2 in NSCLC, suggesting that many genes involved in development of lung cancer were related with the function of NF- B as a transcription factor. Our data showed that positive nuclear staining for NF- B (67.6%) by immunohistochemistry was consistent with overexpression ( ) of NF- B by Western blotting (68.9%; 31 of 45). The current trial suggested that nuclear staining for NF- B, tumor differentiation, lymph node status, and pathologic stage all showed a significant correlation with overall 5-year survival. To know whether NF- B was an independent prognostic factor, we performed multivariate analysis. Cox proportional hazard models indicated that nuclear NF- B staining was an independent predicator and portended a 2.65-fold (1/0.377) increase in the 5-year risk of death. In addition, we have demonstrated a link between nuclear localization of NF- B and tumor differentiation. Tumor staging and nodal status are important factors associated with prognosis of lung cancer patients, but we did not find clear evidence that expression of NF- B had a correlation with tumor staging and nodal status. Our results indicate that prognosis of lung cancer is very complicated and may be affected by various kinds of factors. Furthermore, the difference of the two methodology, Western blotting and immunohistochemistry, may have an influence on the result of this experiment. Our results may augment our ability to predict prognosis by providing additional prognostic information independently and in addition to the current clinical factors used to predict recurrence and metastasis. These findings also may allow physicians to tailor therapeutic regiments better for their patients. By predicting those patients who have a poor prognosis, we may be able to determine better which patients require more aggressive local treatments and which of those systemic treatments. Nuclear factor- B may represent an attractive therapeutic target in a select patient group, although that remains to be determined through further studies. References 1. Veiby OP, Read MA. 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