Abstract
Lung cancer is the leading cause of cancer-related deaths worldwide. Although our knowledge on the pathobiology of the disease has increased in the last decades, the prognosis of lung cancer patients has hardly changed. Many signaling pathways are implicated in lung carcinogenesis, but the role of the alternative pathway of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in lung cancer pathogenesis and progression has not been investigated. The aim of our study was to investigate the role of this pathway in non-small cell lung cancer (NSCLC) patients. NF-κB2 and RelB protein expression was retrospectively assessed by immunohistochemistry in tissue samples from 109 NSCLC patients. RelB and NF-κB2 protein levels differed between tumors and adjacent nonneoplastic lung parenchyma. Cytoplasmic immunoreactivity of NF-κB2 and RelB was correlated with tumor stage (p = 0.03 and p = 0.016, respectively). In addition, cytoplasmic NF-κB2 levels were related to tumor grade (p = 0.046). Expression of RelB in the cytoplasm was tumor histologic type-specific, with squamous cell carcinomas having the highest protein levels. Nuclear expression of RelB and NF-κB2 differed between tumor and nonneoplastic tissues, possibly indicating activation of the alternative pathway of NF-κB in cancer cells. Moreover, lymph node metastasis was related to nuclear NF-κB2 expression in tumor cells. The deregulation of the alternative NF-κB pathway in NSCLC could play a role in the development and progression of the disease.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Primary tumors of the lung were rare until the beginning of the twentienth century [1, 2]. At present, lung cancer is the first cause of cancer-related deaths in both sexes (30 % in males, 26 % in females) in the USA and worldwide [3, 4]. Non-small cell lung cancer (NSCLC) and small cell lung cancer are the two major clinical lung cancer subtypes. The majority (85 %) of lung carcinomas are NSCLC (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) [4]. The current therapeutic approaches, which include surgical resection, platinum-based doublet chemotherapy, radiotherapy, and targeted therapies, rarely cure the disease, and the overall survival rate remains low at approximately 15 % [5].
In the last decades, there has been an expansion of knowledge relating to the pathobiology of malignant epithelial lung tumors, but the prognosis of the disease has little changed. Although a wide spectrum of genes and proteins has been studied in human lung cancer, it remains unclear whether the alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway can promote tumorigenesis or change the tumor progression. At present, it is well documented that NF-κB has a pleiotropic function by binding to discrete DNA sequences in promoters and enhancers.
The family of NF-κB transcriptional regulators consists of seven members p105/p50 (NF-κB1), p100/p52 (NF-κB2), p65 (RelA), RelB, c-Rel, which are encoded by five genes (NF-κB1, NF-κB2, RELA, RELB, and c-REL) [6, 7]. NF-κB1 and NF-κB2 encode the precursor proteins p105 and p100, which are proteasomaly cleaved to give rise to the functional molecules p50 and p52, respectively [7, 8]. The NF-κB family gives rise to two major signaling pathways, the classical and the alternative. In the alternative pathway, two of the central players are p100/p52 and RelB. As only the Rel family members carry a carboxy-terminal transcriptional activating domain, which is a positive regulator of gene expression, a heterodimer between p100/p52 and RelB must form in order to bind to promoters and enhancers [8, 9].
Until recently, the role of NF-κBs and their signaling pathways has been thought to be limited to coordinating the innate and adaptive immune responses. Their role in cancer development has not been elucidated, although it is now documented that NF-κB is a major factor linking inflammation and carcinogenesis [10, 11]. Moreover, NF-κB is a central player in virus-associated carcinogenesis [12]. In many types of cancer, not only in hematological but also in solid malignancies, e.g., melanoma, breast, prostate, ovarian, pancreatic, colon, head and neck, renal, bladder, liver, astrocytoma, glioblastoma, and thyroid, NF-κB family members exhibit elevated activity [13, 14]. In lung cancer, only p50 and p65 have been studied, while little is known about the role of the alternative pathway of NF-κB in lung carcinogenesis and the progression of the disease [15–17].
Methods
Tissue specimens
This study was carried out according to the principles and after the approval of the Committee on Research and Ethics and the Scientific Committee of the University Hospital of Patras, Greece. The study comprised 109 formalin-fixed paraffin-embedded (FFPE) lung tissue specimens of invasive NSCLCs including adjacent nonneoplastic lung parenchyma, retrieved from the archives of the Laboratory of Pathology of the University Hospital of Patras. All cases were surgically managed at the University Hospital of Patras Medical School between 2005 and 2008.
Clinicopathological information was obtained from the pathology reports and is given in Table 1. Sixty (n = 60) samples were squamous cell carcinomas, 38 (n = 38) were adenocarcinomas, and 8 (n = 8) were large cell carcinomas. The majority of patients were men (90.9 %) with a mean age of 67 years (range, 46–81 years). The pathological stage was defined according to the primary pathology reports. All stages were represented with the majority of samples equally distributed between stages I, II, and III. The tumor grade was (2 %) grade 1, (49 %) grade 2, and (43 %) grade 3, respectively. Lymph node status was known for 102 patients. Fifty percent of them had lymph node metastatic disease. First- and second-year survival rates were available in 101 of the 109 patients. Moreover, the regional relapse status in the 2-year observational period was known for 29 patients (11 relapsed).
Immunohistochemical analysis
Four-micrometer sections from FFPE tissue specimens were deparaffinised in xylene and rehydrated in a series of graded alcohols. The sections were then pre-treated in a microwave oven, and peroxidase activity was blocked with 3 % hydrogen peroxide for 20 min, followed by incubation with an appropriate protein blocking solution. Primary antibodies used were mouse monoclonal anti-NF-κΒ2 (dilution 1:500, clone: C-4, sc-7386, Santa Cruz) and rabbit polyclonal antibody against ReB (dilution 1:100, clone: C-19, sc-226, Santa Cruz). Detection was performed using the Envision detection kit (DAKO) according to the manufacturer’s instructions. Diaminobenzidine was used as the chromogen for visualization. Sections were counterstained with Harris’ hematoxylin solution, dehydrated, and mounted. To test for specificity, the procedure was repeated in consecutive sections substituting the anti-NF-κΒ2 and anti-RelB antibodies with protein blocking solution.
Evaluation of immunohistochemistry
All slides were assessed by one pathologist (H.P.) and one investigator (F.D.) independently and blinded to the case. The histological type and tumor grade were confirmed according to the 2004 WHO classification of lung tumors [18]. Cases with staining in >10 % of cells were considered positive. Immunohistochemical reactivity was graded on a scale of 0–3 according to the intensity of the staining, and the percentage of immunopositive cells as follows: 0, no staining or <10 % positive cells; 1, weak staining in >10 % of cells or moderate staining in 10–70 % of cells; 2, moderate staining in >70 % of cells or strong staining in 10–70 % of cells; and 3, strong staining in >70 % of cells. NFkB2 and RelB expression in cancer cells was categorized in three groups (high vs medium vs low) using as a cutoff point the 33rd and 66th percentiles [19]. The intensity and distribution of the NF-κB2 and RelB signal were the parameters used to estimate NF-κB2 and RelB expression. The total score for each slide was the sum of the intensity and distribution (between 0 and 6). Per tissue section, the more representative areas were selected using low-power fields (magnification ×10). The accurate quantification was performed on high-power fields (magnification ×40). Microphotographs were obtained using a Nikon DXM 1200C digital camera mounted on a Nikon Eclipse 80i microscope and ACT-1C software (Nikon Instruments Inc., Melville, NY, USA).
Statistical analysis
Statistical analysis was performed with the Statistical Package for Social Sciences version 17 (SPSS, Chicago, IL, USA). Possible associations between NF-κB and RelB protein expression with the clinicopathological parameters of the tumors were evaluated with the Kruskal–Wallis or the Mann–Whitney tests for ordinal variables and χ 2 test for nominal variables. Spearman’s correlations were used to assess associations between variables. Survival rates were estimated using the Kaplan–Meier method and then compared with the log rank test. Cox regression analysis was used to assess the role of the two NF-κB molecules as prognostic factors. For all comparisons, statistical significance was defined as p < 0.05.
Results
RelB and NF-κB2 protein levels differ between NSCLC and control tissue
The association of NF-κB2 and RelB expression with the clinicopathological characteristics is listed in Tables 2 and 3, respectively. As shown in Fig. 1a–f, immunoreactivity of NF-κB2 and RelB in epithelial cells was cytoplasmic and nuclear in all NSCLC histological subtypes. Cytoplasmic expression of NF-κB2 and RelB was observed in 96.3 and 81.7 % of the tumor specimens, respectively, in contrast to 10 % of tumor-adjacent nonneoplastic tissue (Fig. 2a–b). Moreover, expression levels were higher in tumor cells than in nonneoplastic tissue (p < 0.001 for both molecules). Regarding nuclear staining, it was noted exclusively in tumor specimens. Approximately half (48.6 %) of the tumor specimens were positive for RelB and 15.7 % for NF-κB2. However, concurrent expression of these molecules was detected only in 12.1 % of cases.
NF-κB2 and RelB expression is correlated with the tumor stage
Cytoplasmic NF-κB2 expression in tumor cells was related to disease stage (p = 0.03). More specifically, NF-κB2 levels were higher in stages II and III and lower in stages I and IV, with the metastatic stage having the lowest score. Likewise, cytoplasmic RelB levels were lower at stage IV compared to stages I to III (p = 0.016). In contrast, nuclear NF-κB2 (p52) and RelB expression was similar across stages (p = 0.178 and p = 0.10, respectively).
Regional lymph node infiltration is associated with NF-κB2 nuclear expression levels
Lymph node metastasis was found to be related to NF-κB2 expression in the nucleus of primary site tumor cells. That is, the nuclear NF-κB2 protein levels were higher in tumor cells of patients without lymph node metastasis (p = 0.044). However, no association was detected between NF-κB2 cytoplasmic expression and lymph node positivity. RelB expression, whether cytoplasmic or nuclear, was independent of lymph node metastatic frequency.
NF-κB2 cytoplasmic expression is correlated with tumor grade
Using the two-tier grading system, we found that NF-κB2 levels in the cytoplasm were related to the grade of the tumor. Low-grade tumors had higher NF-κB2 levels compared to high-grade tumors (p = 0.046). However, p52 in the nucleus was unrelated to grade. Moreover, RelB protein levels, independently of subcellular location, remained unchanged across grades.
RelB cytoplasmic expression is higher in squamous cell carcinoma
RelB cytoplasmic expression differed between NSCLC types. In particular, RelB expression in the cytoplasm of squamous cell carcinomas was higher than in the other NSCLC histological types (p = 0.008). Moreover, expression levels were lower in adenocarcinomas than large cell carcinomas, although the difference was not significant. In addition, we observed a trend of higher nuclear RelB expression in the squamous cell subtype compared to other subtypes (p = 0.066), although no differences were observed between adenocarcinomas and large cell carcinomas (p = 1.00). Furthermore, NF-κB2 levels, independently of localization, did not differ between tumor types.
Correlation of protein levels between RelB and NF-κB2
NF-κB2 and RelB levels (cytoplasmic as well as nuclear) were positively correlated (p = 0.029 and p = 0.004, respectively). Additionally, RelB cytoplasmic and nuclear protein levels were correlated (p < 0.001), although no such relation was noted for NF-κB2.
NF-κB2 in the cytoplasm is overexpressed in right lung tumors
In right lung tumors, cytoplasmic NF-κB2 levels were higher compared to left lung tumors. The difference was significant (p = 0.032) when cytoplasmic expression was divided into three groups (low vs intermediate vs high). NF-κB2 nuclear expression and RelB nuclear or cytoplasmic expression were unrelated to primary site.
No correlation was found with age, sex, smoking, maximum tumor diameter, relapse rate, and overall survival
Patients’ age was unrelated to the expression levels of NF-κB2 and RelB. Patients of both age groups, over and under the age of 65 (the median of our cohort), had the same NF-κB2 and RelB protein levels. Likewise, smoking, as estimated using the pack-years (median exposure of 80 pack-years), had no impact on the expression pattern of NF-κB2 and RelB. In addition, no significant correlation was found with maximum tumor diameter. Tumors with maximum diameter bigger or smaller than 4.5 cm, which was the median of our group, had similar levels of NF-κB2 and RelB. Furthermore, neither NF-κB2 nor RelB protein expression correlated with the regional disease recurrence and overall survival.
Discussion
NF-κB is a functionally pleiotropic factor implicated in the pathobiology of a variety of diseases [20]. In the last years, experimental evidence implicates the deregulation of the non-canonical NF-κB pathway in solid tumors, but its role in lung cancer pathogenesis and progression remains unknown [21].
In the present study, the immunohistochemical analysis of NF-κB2 and RelB proteins in NSCLC tissue samples revealed a statistically different expression pattern compared to adjacent nonneoplastic lung parenchyma. Cytoplasmic expression of both proteins was noted more frequently in neoplastic cells compared to adjacent nonneoplastic epithelium, while nuclear expression of both molecules was restricted to tumor tissue. These findings may reflect the activation of the alternative pathway of NF-κB in NSCLC patients. This is consistent with the observation of Lukashev et al. [22] that the TNF receptor family member, lymphotoxin-β receptor, whose activation results in the activation of the alternative pathway of NF-κB, is overexpressed in 87 to 96 % of a wide range of solid tumors, including lung cancer. Similarly, 51.9 and 58.9 % of NSCLC patients were positive for another activator of the alternative NF-κB pathway, CD40 and its ligand, CD154, respectively. Moreover, CD40 expression in tumors is correlated with a poor prognosis since the activation of CD40 in cancer cells boosts the malignant potential of NCSLC [23, 24].
Furthermore, NF-κB2 and RelB seem to be expressed in the cytoplasm of cancer cells in a stage-dependent manner. NF-κB2 and RelB expression is lowest in patients with metastatic disease. However, previous studies in NK/T lymphomas, prostate, and esophageal tumors reported no relation of NF-κB2 and RelB expression with stage [25–27]. It is possible that the role of these molecules differs between different tumor types. Although our results should be treated with caution because of the small number of stage IV patients, they may reflect the different roles of NF-κB2 and RelB in the different stages of lung cancer progression. Additionally, it is documented that the final effect of NF-κB function may depend on the duration of NF-κB activation and the cellular context, and it can be either tumor suppressive or tumor promoting [28, 29]. This possible dual role of NF-κB during tumor progression needs to be clarified by further studies in NSCLC.
Moreover, supportive to the stage-dependent expression of NF-κB subunits is our finding that nuclear NF-κB (p52) levels are associated with lymph node metastasis. The reduced protein levels of NF-κB in the nucleus of cancer cells seem to be related to lymph node infiltration. In agreement with our findings, the nuclear localization of p65 (another member of the NF-κB family) in primary prostate tumors is a valuable marker for the prediction of lymph node metastasis [30]. It is possible that the activation of the alternative pathway of NF-κB and the subsequent nuclear translocation of NF-κB (p52) may hamper metastasis of the cancer cell. Perhaps the repeal of this brake, in a particular moment of the tumor’s physical history, increases the lymph node metastatic potential. In addition, the higher levels of NF-κB2 in the cytoplasm were associated with low-grade tumors. This indicates that NF-κB2 in the cytoplasm may be correlated with an impediment of the dedifferentiation process. In prostate cancer, no relationship was noted between p52 nuclear expression and grade, although RelB was associated with tumor grade [25]. One intriguing point of our finding is that only the cytoplasmic NF-κB2 expression is correlated with tumor grade and not the nuclear.
In this study, for the first time, we found that RelB cytoplasmic expression is higher in squamous cell carcinomas compared to adenocarcinomas and large cell carcinomas. The difference between the histological groups may reflect the different microenvironments of these carcinomas that influence the activation of the NF-κB pathways in a cell type-specific manner. In agreement with our findings is the different molecular biology of the three NSCLC histological types [4].
NF-κB2 and RelB, the pivotal players of the alternative NF-κB pathway, are thought to have an active transcriptional activity when they are localized nuclearly. In our study, both were located mainly in the cytoplasm posing the question of whether cytoplasmic NF-κB2 and RelB maintain a biological function. Moreover, it would be of interest to investigate whether this cellular lodging reflects an escape mechanism of the malignant cell, which may trap p52 and RelB in the cytoplasm being benefitted more from their presence in the cytoplasm rather than of their absence from the nucleus.
References
Miller YE (2005) Pathogenesis of lung cancer: 100 year report. Am J Respir Cell Mol Biol 33(3):216–223
Aggarwal BB (2004) Nuclear factor-kappa-B: the enemy within. Cancer Cell 6(3):203–208
Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ (2009) Cancer statistics, 2009. CA Cancer J Clin 59(4):225–249
Herbst RS, Heymach JV, Lippman SM (2008) Lung cancer. N Engl J Med 359(13):1367–1380
Sato M, Shames DS, Gazdar AF, Minna JD (2007) A translational view of the molecular pathogenesis of lung cancer. J Thorac Oncol 2(4):327–343
May MJ, Ghosh S (1997) Rel/NF-kappa B and I kappa B proteins: an overview. Semin Cancer Biol 8(2):63–73. doi:10.1006/scbi.1997.0057
Chen LF, Greene WC (2004) Shaping the nuclear action of NF-kappaB. Nat Rev Mol Cell Biol 5(5):392–401. doi:10.1038/nrm1368 nrm1368
Perkins ND (2007) Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8(1):49–62. doi:10.1038/nrm2083
Hayden MS, Ghosh S (2008) Shared principles in NF-kappaB signaling. Cell 132(3):344–362. doi:10.1016/j.cell.2008.01.020
Karin M (2006) Nuclear factor-kappa B in cancer development and progression. Nature 441(7092):431–436. doi:10.1038/Nature04870
Grivennikov SI, Greten FR, Karin M (2010) Immunity, inflammation, and cancer. Cell 140(6):883–899. doi:10.1016/j.cell.2010.01.025
Okamoto T, Sanda T, Asamitsu K (2007) NF-kappa B signaling and carcinogenesis. Curr Pharm Des 13(5):447–462
Pacifico F, Leonardi A (2006) NF-kappa B in solid tumors. Biochem Pharmacol 72(9):1142–1152. doi:10.1016/j.bcp. 2006.07.032
Rayet B, Gelinas C (1999) Aberrant rel/nfkb genes and activity in human cancer. Oncogene 18(49):6938–6947
Tang X, Liu D, Shishodia S, Ozburn N, Behrens C, Lee JJ, Hong WK, Aggarwal BB, Wistuba II (2006) Nuclear factor-kappaB (NF-kappaB) is frequently expressed in lung cancer and preneoplastic lesions. Cancer 107(11):2637–2646. doi:10.1002/cncr.22315
Zhang Z, Ma J, Li N, Sun N, Wang C (2006) Expression of nuclear factor-kappaB and its clinical significance in nonsmall-cell lung cancer. Ann Thorac Surg 82(1):243–248. doi:10.1016/j.athoracsur.2006.01.049
Zhang D, Jin X, Wang F, Wang S, Deng C, Gao Z, Guo C (2007) Combined prognostic value of both RelA and IkappaB-alpha expression in human non-small cell lung cancer. Ann Surg Oncol 14(12):3581–3592. doi:10.1245/s10434-007-9560-z
Travis WD, World Health Organization, International Agency for Research on Cancer, International Association for the Study of Lung Cancer, International Academy of Pathology (2004) Pathology and genetics of tumours of the lung, pleura, thymus, and heart. IARC Press, Lyon
Taylor C (2006) Quantifiable internal reference standards for immunohistochemistry: the measurement of quantity by weight. Appl Immunohistochem Mol Morphol 14:253–259
Wong ET, Tergaonkar V (2009) Roles of NF-kappaB in health and disease: mechanisms and therapeutic potential. Clin Sci (Lond) 116(6):451–465. doi:10.1042/CS20080502
Dejardin E (2006) The alternative NF-kappa B pathway from biochemistry to biology: pitfalls and promises for future drug development. Biochem Pharmacol 72(9):1161–1179. doi:10.1016/j.bcp. 2006.08.007
Lukashev M, LePage D, Wilson C, Bailly V, Garber E, Lukashin A, Ngam-ek A, Zeng W, Allaire N, Perrin S, Xu X, Szeliga K, Wortham K, Kelly R, Bottiglio C, Ding J, Griffith L, Heaney G, Silverio E, Yang W, Jarpe M, Fawell S, Reff M, Carmillo A, Miatkowski K, Amatucci J, Crowell T, Prentice H, Meier W, Violette SM, Mackay F, Yang D, Hoffman R, Browning JL (2006) Targeting the lymphotoxin-beta receptor with agonist antibodies as a potential cancer therapy. Cancer Res 66(19):9617–9624. doi:10.1158/0008-5472.CAN-06-0217
Ishikawa K, Miyamoto M, Yoshioka T, Kato T, Kaji M, Ohbuchi T, Hirano S, Itoh T, Dosaka-Akita H, Kondo S (2008) Up-regulation of CD40 with juxtacrine activity in human nonsmall lung cancer cells correlates with poor prognosis. Cancer 113(3):530–541. doi:10.1002/cncr.23618
Loskog AS, Eliopoulos AG (2009) The Janus faces of CD40 in cancer. Semin Immunol 21(5):301–307. doi:10.1016/j.smim.2009.07.001
Lessard L, Begin LR, Gleave ME, Mes-Masson AM, Saad F (2005) Nuclear localisation of nuclear factor-kappaB transcription factors in prostate cancer: an immunohistochemical study. Br J Cancer 93(9):1019–1023. doi:10.1038/sj.bjc.6602796
Kang MR, Kim MS, Kim SS, Ahn CH, Yoo NJ, Lee SH (2009) NF-kappaB signalling proteins p50/p105, p52/p100, RelA, and IKKepsilon are over-expressed in oesophageal squamous cell carcinomas. Pathology 41(7):622–625. doi:10.3109/00313020903257756
Liu X, Wang B, Ma X, Guo Y (2009) NF-kappaB activation through the alternative pathway correlates with chemoresistance and poor survival in extranodal NK/T-cell lymphoma, nasal type. Jpn J Clin Oncol 39(7):418–424. doi:10.1093/jjco/hyp037
Chen F, Castranova V (2007) Nuclear factor-kappaB, an unappreciated tumor suppressor. Cancer Res 67(23):11093–11098
Chen F, Beezhold K, Castranova V (2008) Tumor promoting or tumor suppressing of NF-kappa B, a matter of cell context dependency. Int Rev Immunol 27(4):183–204. doi:10.1080/08830180802130327
Lessard L, Karakiewicz PI, Bellon-Gagnon P, Alam-Fahmy M, Ismail HA, Mes-Masson AM, Saad F (2006) Nuclear localization of nuclear factor-kappaB p65 in primary prostate tumors is highly predictive of pelvic lymph node metastases. Clin Cancer Res 12(19):5741–5745. doi:10.1158/1078-0432.CCR-06-0330
Acknowledgments
This research has been co-financed by the European Union (European Social Fund–ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)–Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.
Conflict of interest
We declare that we have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Dimitrakopoulos, FI.D., Antonacopoulou, A.G., Kottorou, A. et al. NSCLC and the alternative pathway of NF-κB: uncovering an unknown relation. Virchows Arch 460, 515–523 (2012). https://doi.org/10.1007/s00428-012-1230-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00428-012-1230-2