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Introduction

Despite recent improvements in diagnosis, lung cancer is a major cause of mortality from malignant diseases, due to its high incidence, malignant behavior and the lack of major advancements in treatment strategy (1). Lung cancer was the leading indication for respiratory surgery (42.2%) in 1998 in Japan (2). More than 15,000 patients underwent surgery at Japanese institutions in 1998 (2). The clinical behavior of lung cancer is largely associated with its stage. The cure of the disease by surgery is only achieved in cases presenting with an early stage of lung cancer (3).

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor belonging to the cap ‘n’ collar subfamily of the basic-leucine zipper (bZIP) family of transcription factors, which plays a significant role in adaptive responses to oxidative stress (4). At the expression level, NRF2 is expressed widely in various human tissues (5), including lung cancer (6). It has been indicated that the overexpression of NRF2 in premalignant cells may enable the cancer cells to survive in an oxidizing tumor environment. Subsequently, the cancer cells alter their metabolic processes, mitochondrial dysfunction and activation of oncogenic signals. It has been shown that patients with lung tumors containing the NRF2 gene (NFE2L2) mutation display a poorer prognosis than patients with non-mutant tumors (7,8). It has been reported that mutations of the NRF2 gene have been associated with primary lung cancer (6–9). NRF2 gene somatic mutation is more common in lung squamous cell carcinomas (7). Recently, NRF2 overexpression was investigated in lung cancer (10–12), and the NRF protein has also been shown to be highly expressed in squamous cell carcinomas of the lung (6).

Although we have previously reported the status of the NRF2 gene (NFE2L2) mutations in lung cancers (7), the association of NRF2 gene copy number status and Japanese lung cancer has not been previously reported. Therefore, in the present study, in order to determine the NRF2 gene copy number status in Japanese lung squamous cell carcinomas, we investigated the copy number by real-time polymerase chain reaction (real-time-PCR) amplifications. The findings were compared to the clinicopathological features of lung squamous cell carcinomas.

Patients and methods

Patients

The study group included 90 lung squamous cell carcinoma patients who had undergone surgery at the Department of Surgery, Nagoya City University Hospital, Nagoya, Japan. All tumor samples were immediately frozen and stored at −80°C until assayed.

The clinical and pathological characteristics of the 90 lung squamous cell carcinoma patients were as follows: 43 cases at stage I, 20 at stage II and 27 at stage III. The mean age was 66.7 years (range, 36–85). Among the 90 lung cancer patients, 35 had lymph node metastasis, 83 were male and 16 had NRF2 gene mutations. The samples from these patients had previously been sequenced for the NRF2 gene (NFE2L2) (7).

PCR assays for NRF2

Genomic DNA was extracted from lung cancer tissues using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. DNA concentration was determined by a NanoDrop spectrophotometer (NanoDrop Technologies, Inc. Rockland, DE, USA) and adjusted to a concentration of 2.5 ng/ml. We then used 5 μl of each DNA for PCR assays. NRF2 copy number was analyzed by quantitative real-time PCR, performed using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) by using the QuantiTect SYBR-Green kit (Qiagen Inc., Valencia, CA, USA) (13–15). NRF2 primers used for amplification were: 5′GGTTTCTTCGGCTACGTTT-3′ and 5′TAACTCAGGAAT GGATAATAGCTC-3′. Total DNA content was estimated by assaying Line-1 elements for each sample using the primers, 5′-AAAGCCGCTCAACTACATGG-3′ and 5′-TGCTTTG AATGCGTCCCAGAG-3′. The cycling conditions were as follows: initial denaturation at 95°C for 15 min, followed by 40 cycles at 94°C for 15 sec, 56°C for 30 sec and 72°C for 34 sec. Copy numbers >3 were considered as increased, according to previous reports (13,16,17).

Statistical analysis

Statistical analyses were performed using the Mann-Whitney U-test for unpaired samples and Wilcoxon’s signed rank test for paired samples. Linear relationships between variables were determined by means of a simple linear regression. Correlation co-efficients were determined by rank correlation using the Spearman’s test and χ2 test. The overall survival of the lung cancer patients was examined by the Kaplan-Meier method, and differences were examined by the Log-rank test. All analyses were performed using the Stat-View software package (Abacus Concepts Inc., Berkeley, CA, USA), and a p-value <0.05 was considered to indicate a statistically significant difference.

Results

NRF2 gene mutation in Japanese lung cancer

We investigated the NRF2 gene (NFE2L2) mutation status in the N-terminal domain by direct sequencing as previously described (7). In total, 13 out of 109 squamous cell carcinoma patients had NRF2 gene mutations. In addition, we analyzed 39 squamous cell carcinoma patients and 3 had NRF2 gene mutations. These 3 were known mutations (T80A, W24C, R34Q). In total, 16 out of 148 (10.8%) had NRF2 gene mutations (Fig. 1). All of the mutations were found in squamous cell carcinomas. The NRF2 gene mutations were clustered on exon 2 and resulted in amino acid changes in either the DLG or the ETGE motif of the regulatory Neh2 domain (7).

Figure 1 Results from the direct sequencing of the NRF2 gene. Upper panel, 101 G to A transversion resulted in 34 arginine (Arg) to proline (Pro) conversion (R34P mutation). Lower panel, 238 A to G conversion resulted in thyreonine (Thy) to alanine (Ala) conversion (T80A mutation).

NRF2 gene status in Japanese lung cancer patients

Using the primer sets for the NRF2 gene, from 90 lung cancer patients, 7 patients had more than 3 copies of the NRF2 gene and the clinicopathological background is shown in Table I. NRF2 gene copy number status significantly correlated with gene mutations (mutant, 31.25% vs. wild-type, 2.7%; p=0.0017). However, the copy number status did not correlate with smoking status (Brinkman index <400 vs. ≥400; p=0.4443), pathological stage (stage I vs. stage II and III, p=0.4375), lymph node metastasis (positive vs. negative, p=0.4242) or age (<65 vs. ≥65, p=0.9999). NRF2 gene copy number significantly correlated with gene mutations (mutant, 2.478±0.668 vs. wild-type, 1.917±0.737; p=0.0048). However, the mean NRF2 copy number did not correlate with smoking status (Brinkman index <400, 2.239±0.499 vs. ≥400, 1.998±0.593; p=0.3741), lymph node metastasis (negative, 2.074±0.567 vs. positive, 1.926±0.980; p=0.1695), age (≥65, 2.083±0.782 vs. <65, 1.96±0.732; p=0.1237) or pathological stage.

Table I Clinicopathological data of the 90 lung squamous cell carcinoma patients.

Table I

Clinicopathological data of the 90 lung squamous cell carcinoma patients.

	NRF2 gene status
Factors	Number of patients	Copy number	P-value
Mean age (years)	66.7±8.4	90
Pathological stage			NS
I	43 (47.8%)	2.035±0.490
II	20 (22.2%)	2.165±1.033
III	27 (30.0%)	1.877±0.859
Lymph node metastasis			0.1695
N0	55 (61.1%)	2.074±0.567
N+	35 (38.9%)	1.926±0.980
BI status			0.3741
<400	7 (7.8%)	2.239±0.499
≥400	83 (92.2%)	1.998±0.593
Differentiation
Well	27 (30.0%)	1.955±0.817	0.8671
Moderate/poor or other	63 (70.0%)	2.042±0.729
NRF2 mutations			0.0048
Mutant	16 (17.8%)	2.478±0.668
Wild-type	74 (82.2%)	1.917±0.737
Age, years			0.1237
≥65	41 (45.6%)	2.083±0.782
<65	49 (54.4%)	1.960±0.732
Gender			0.1545
Male	3 (92.2%)	1.984±0.751
Female	7 (7.8%)	2.404±0.720

* NS, not significant. N0, lymph node metastasis-negative; N+, lymph node metastasis-positive; BI, Brinkman index.

The overall survival of the 90 lung squamous cell carcinoma patients, with follow-up through to December 31, 2010, was studied in reference to the NRF2 gene status. The survival of the patient with an increased copy number of the NRF2 gene (n=7) and the patient with a normal copy number of NRF2 (n=83) was not significantly different (Log-rank test, p=0.6582).

Discussion

In our analysis, an increased NRF2 gene copy number was found in 7.8% of Japanese lung squamous cell carcinomas. The NRF2 gene statuses correlated with NRF2 mutations, indicating that the mutations were activating mutations.

NRF2 (NFE2L2) is a master transcriptional activator of genes encoding many cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents (18–20). A previous report showed that the RNAi-mediated silencing of NRF2 gene expression in non-small cell lung cancer inhibited tumor growth (21). A NRF2 gene promoter polymorphism has been identified and has been suggested to correlate with carcinogenesis (22). The association of NRF2 mutation and increased copy number in lung squamous cell carcinomas suggests a role of NRF2 in tumorigenesis. The constitutive expression of NRF2 may provide a survival advantage to invasive and metastatic cancer cells, by adaptation to the microenvironment and evolution of chemoresistance in cancer cells under hypoxic conditions (23,24). A previous study showed that the degree of cisplatin (CDDP)-induced DNA crosslinking and the number of cells undergoing apoptosis were increased significantly in A549 cells transfected with NRF2-siRNA (25). The expression of multidrug resistance-associated proteins, the drug efflux proteins, has also been shown to be significantly reduced in NRF2-silenced A549 cells (25). Another study also showed that the inhibition of NRF2 function restored CDDP sensitivity in human ovarian cancer cells (26).

However, in our analysis, more than half of NRF2 gene mutations did not have an increased NRF2 gene copy number. The prognosis analysis between NRF2 gene copy number statuses was not significantly different, which suggests that another mechanism may exist for the activation of NRF2 mutations in cancer. As previously reported, in vitro, wild-type NRF2 was efficiency polyubiqitinated while mutant NRF2 proteins were only weakly polyubiquitinated after treatment with MG132 (8). Wild-type NRF2 protein decreased rapidly, whereas mutant NRF2 proteins were degraded more slowly, having half-lives of approximately twice that of wild-type (8). In addition, mutant NRF2 proteins were significantly more active than wild-type NRF2 by analyzing luciferase activity (8). In addition, the prognostic value of NRF2 expression in lung cancers was controversial. Merikallio et al showed that NRF2 protein expression was correlated with poor prognosis using multi-variate analysis (10). Kim et al showed that high NRF2 expression was not correlated with poor prognosis in stage I lung cancers (27). Interaction between other genes, such as Keap1, would be also important for control of NRF2 expression in NSCLC (28).

Acknowledgements

The authors would like to thank Mrs. Miki Mochizuki for her excellent technical assistance. This study was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS) (Nos. 23659674, 24592097 and 21591820) and a grant for cancer research of the Program for developing the supporting system for upgrading education and research (2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References