Genetic polymorphism in the NRF2 gene as a prognosis marker for cancer chemotherapy

NF-E2-related factor 2 (NRF2) is a transcription factor that controls the expression of a variety of antioxidant and detoxification genes. Accumulating evidence strongly suggests that NRF2 mediates cancer cell proliferation and drug resistance, as well. Single nucleotide polymorphism (SNP) -617C > A in the anti-oxidant response element-like loci of the human NRF2 gene play a pivotal role in the positive feedback loop of transcriptional activation of the NRF2 gene. Since the SNP (-617A) reportedly decreases the binding affinity to the transcription factors of NRF2/small multiple alignment format (MafK), the homozygous -617A/A allele may attenuate the positive feedback loop of transcriptional activation of the NRF2 gene and reduce the NRF2 protein level. As the consequence, cancer cells are considered to become more sensitive to therapy and less aggressive than cancer cells harboring the -617C (WT) allele. Indeed, Japanese lung cancer patients carrying SNP homozygous alleles (c. -617A/A) exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021). The genetic polymorphism in the human NRF2 gene is considered as one of prognosis markers for cancer therapy.


INTRODUCTION HISTORICAL BACKGROUND
In the field of cancer chemotherapy, it has been well documented that glutathione (GSH) plays a pivotal role in conferring cancer cells resistance to anti-tumor drugs, such as cisplatin and alkylating agents. Several lines of evidence suggest that certain multi-drug efflux pumps encoded by ATP-binding cassette (ABC) transporter genes are up-regulated by oxidative stress and/or chemotherapeutic agents to contribute to multi-drug resistance of cancer cells. About two decades ago, Ishikawa and Kuo first reported that many cytotoxic agents induced the expression of both γglutamylcysteine synthetase (γ-GCS) and ABCC1 (MRP1) genes Kuo et al., 1996Kuo et al., , 1998Gomi et al., 1997;Yamane et al., 1998). Coordinated up-regulation of both γ-GCS and ABCC1 genes was found in human malignant tissues. Among 32 cases of human colorectal cancer biopsies, 78% of the cases exhibited co-elevated expression of ABCC1 and γ-GCS genes in tumor samples as compared with their corresponding adjacent naïve normal samples (Kuo et al., 1996). At that time, it was speculated that a common transcriptional regulator might exist for the coordinated expression of both γ-GCS and ABCC1 genes .

TRANSCRIPTION FACTOR NRF2 AS A MASTER SWITCH IN GENE EXPRESSION
During the past two decades, evidence has accumulated to show that one transcription factor named NF-E2-related factor 2 (NRF2) is a common redox regulator to control cellular adaptation/protection to external stimuli by inducing antioxidant and detoxification genes (Motohashi and Yamamoto, 2004;Kobayashi and Yamamoto, 2006;Nguyen et al., 2009). In fact, NRF2 is a major player in the transcriptional upregulation of many target genes in phase II drug metabolizing enzymes and certain phase III ABC transporters (ABCC2, ABCC3, and ABCG2; Adachi et al., 2007). The 5 -flanking region of many of phase II xenobiotic detoxifying genes (e.g., γ-GCS) contains an antioxidant response element (ARE). NRF2 directly binds to the ARE sequence in those target genes (Shen and Kong, 2009;Singh et al., 2010). Furthermore, it has recently been reported NRF2 mediates cancer cell proliferation and drug resistance (Lau et al., 2008;Hayes and McMahon, 2009;Homma et al., 2009;Taguchi et al., 2011;Sporn and Liby, 2012;Yamadori et al., 2012;Shelton and Jaiswal, 2013).

FIGURE 2 | Kaplan-Meier plots showing the overall survival of patients harboring the WT homozygote (−617C/C), WT/SNP heterozygote (−617C/A), or SNP homozygote (−617A/A) in the NRF2 gene (A) and genotyping of NRF2 SNP −617C > A by the rapid SNP-detection method (B, upper panels) and DNA sequence analysis (B, lower panels). Data from Okano et al. (2013).
was not known at that time. Three years later, Marzec et al. (2007) reported the impact of those SNPs on the regulation of NRF2 gene expression. In fact, the −617C > A SNP significantly affected basal NRF2 protein levels in vitro (Marzec et al., 2007). Moreover, the SNP −617C > A was found to be associated with a higher risk of oxidant-induced acute lung injury in humans (Marzec et al., 2007). These findings suggest that the SNP (−617C > A) in the ARE-like loci of the human NRF2 gene is important for self-induction of the NRF2 gene (Okano et al., 2013); refer to schematic illustrations in Figure 1.

SNP (-617C > A) IN THE NRF2 GENE AS A BIOMARKER FOR PROGNOSIS OF LUNG CANCER
NF-E2-related factor 2 plays a pivotal role in protecting normal cells from external toxic challenges and oxidative stress, whereas it can modulate the cancer phenotype (Figure 1A) www.frontiersin.org and also endow cancer cells resistance to anticancer drugs ( Figure 1B). NRF2 activation appears to be associated with the emergence of cancer resistance to various anticancer drugs by transcriptionally activating a battery of self-defense genes. Indeed, NRF2 can induction the expression of γ-GCS and ABCC1 genes involved cancer cell resistance to cisplatin and alkylating agents Adachi et al., 2007). In addition, ABCG2 is known to mediate the efflux of gefitinib (Iressa) from cancer cells (Saito et al., 2006), and its expression is regulated by NRF2 (Singh et al., 2010) and the EGFR-tyrosine kinase cascade (Meyer zu Schwabedissen et al., 2006;Huang et al., 2011; Figure 1B).
Single nucleotide polymorphism −617C > A could affect the positive feedback loop of transcriptional activation of the NRF2 gene, and thereby it can regulate the NRF2 protein level. It is proposed that the homozygote −617A/A significantly attenuates the positive feedback loop of transcriptional activation of the NRF2 gene. Interestingly, Asians, including Japanese, have higher frequencies of the −617A allele as compared with African-Americans and Caucasians (Okano et al., 2013). As demonstrated in Figure 2A, Japanese lung cancer patients carrying SNP homozygous alleles (c. −617A/A) exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021). This SNP is considered as a new biomarker for prognosis of lung cancer in Japanese population, and a hypothetical molecular mechanism has been proposed (Okano et al., 2013).

SOMATIC MUTATIONS IN NRF2 AND KEAP1 GENES
The genetic polymorphisms are the "intrinsic" mechanism, whereas the mutations are the "acquired" mechanism in cancer cells. Hitherto, several mutations in the NRF2 and KEAP1 genes have been reported in carcinomas of the lung (Sporn and Liby, 2012), liver (Yoo et al., 2012), stomach (Yoo et al., 2012), and breast (Sjöblom et al., 2006). Abnormalities in NRF2 activity were correlated with poor prognosis in terms of either recurrence-free or overall 5-year survival. Increased expression of NRF2 protein and decreased expression of KEAP1 protein were often observed as common abnormalities in non-small cell lung cancer (NSCLC), being associated with poor prognosis (Solis et al., 2010).

FUTURE PERSPECTIVES
Identification and validation of biomarkers for personalized cancer therapy is one of the challenges in cancer management. To practically realize personalized medicine, development of costeffective methods is required. Furthermore, genetic information in each patient's record should be timely provided for individualized cancer treatment. In this regards, we have recently developed a rapid isothermal method to detect genetic polymorphisms in the NRF2 gene and correlated the genotyping data with the survival of patients who had primary lung cancer (Okano et al., 2013). By means of the new method (Ishikawa and Hayashizaki, 2013), we could detect the SNP −617C > A in the NRF2 gene within 30 to 45 min without DNA isolation and PCR amplification ( Figure 2B). Such genotyping methods would provide a simple and practical tool for personalized cancer therapy and assessment of prognosis.

ACKNOWLEDGMENTS
The author's study was supported by a research fund of Japan Science and Technology Agency (JST) project named "Development of the world's fastest SNP detection system." This means, except for the following governmental research fund, the author had no financial relationships with third parties.