Frequent epigenetics inactivation of KEAP1 gene in non-small cell lung cancer

The KEAP1/Nrf2 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumor cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1 expression. We performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 47 non-small cell lung cancer tissues and normal specimens. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time. Methylation at the KEAP1 promoter region was detected in 22 out of the 47 NSCLCs (47%) and in none of the normal tissues analyzed. Somatic mutations were detected in 7 out of the 47 tumors (15%) and loss of heterozygosity (LOH) in 10 out of the 47 (21%) of the cases. Overall, we found at least one molecular alteration in 57% of the cases. Approximately one third of the tumors had two alterations and this feature was associated with higher risk of disease progression in univariate COX regression analysis (HR = 3.62; 95% CI 1.24–10.65, p = 0.02). This result was confirmed by Kaplan-Meier analysis, which demonstrated an association between worst outcome and KEAP1 double alterations (p = 0.01, Log rank test). Our results further suggest that deregulation of the NRF2/KEAP1 system could play a pivotal role in the cancerogenesis of NSCLC. In addition identifying patients with KEAP1 genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

[1]  M. McMahon,et al.  NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. , 2009, Trends in biochemical sciences.

[2]  M. Copetti,et al.  Abstract 65: Regulation of KEAP1 expression by promoter methylation in malignant gliomas and association with patient's outcome , 2011 .

[3]  P. Nioi,et al.  A mutation of Keap1 found in breast cancer impairs its ability to repress Nrf2 activity. , 2007, Biochemical and biophysical research communications.

[4]  A. Kong,et al.  Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters. , 2009, Biopharmaceutics & drug disposition.

[5]  Donna D. Zhang.  Mechanistic Studies of the Nrf2-Keap1 Signaling Pathway , 2006, Drug metabolism reviews.

[6]  S. Yokoyama,et al.  Structural analysis of the complex of Keap1 with a prothymosin alpha peptide. , 2008, Acta crystallographica. Section F, Structural biology and crystallization communications.

[7]  R. Bordoni.  Consensus conference: multimodality management of early- and intermediate-stage non-small cell lung cancer. , 2008, The oncologist.

[8]  H. Hansen,et al.  Lung cancer. , 1990, Cancer chemotherapy and biological response modifiers.

[9]  Warren C. Lathe,et al.  Prediction of deleterious human alleles. , 2001, Human molecular genetics.

[10]  M. Kwak,et al.  Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. McMahon,et al.  Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds. , 2009, Carcinogenesis.

[12]  J. Herman,et al.  Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer. , 1999, Journal of the National Cancer Institute.

[13]  D. Seripa,et al.  Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors , 2004, Clinical Cancer Research.

[14]  E Gabrielson,et al.  Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Rui Wang,et al.  Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues. , 2008, Biochemical and biophysical research communications.

[16]  Volume CT for diagnosis of nodules found in lung-cancer screening. , 2009, The New England journal of medicine.

[17]  T. Kensler,et al.  Nrf2 as a target for cancer chemoprevention. , 2005, Mutation research.

[18]  Yi-feng Lin,et al.  The Protective Role of Nrf2 in Streptozotocin-Induced Diabetic Nephropathy , 2010, Diabetes.

[19]  Irina Petrache,et al.  Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. , 2004, The Journal of clinical investigation.

[20]  S. Hirohashi,et al.  Genetic alteration of Keap1 confers constitutive Nrf2 activation and resistance to chemotherapy in gallbladder cancer. , 2008, Gastroenterology.

[21]  Yue Xiong,et al.  BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase , 2005, Molecular and Cellular Biology.

[22]  David Haussler,et al.  Improved splice site detection in Genie , 1997, RECOMB '97.

[23]  C. Moon,et al.  Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer , 2004, Clinical Cancer Research.

[24]  Steven Henikoff,et al.  SIFT: predicting amino acid changes that affect protein function , 2003, Nucleic Acids Res..

[25]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

[26]  R. Lothe,et al.  Evaluation of loss of heterozygosity/allelic imbalance scoring in tumor DNA. , 2001, Cancer genetics and cytogenetics.

[27]  M. Sonobe,et al.  Mutations in Keap1 are a potential prognostic factor in resected non‐small cell lung cancer , 2010, Journal of surgical oncology.

[28]  Bengt Bergman,et al.  Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. , 2004, The New England journal of medicine.

[29]  S. Hirohashi,et al.  Loss of Keap1 function activates Nrf2 and provides advantages for lung cancer cell growth. , 2008, Cancer research.

[30]  Shyam Biswal,et al.  Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. , 2007, Annual review of pharmacology and toxicology.

[31]  Jinhua Wang,et al.  ESEfinder: a web resource to identify exonic splicing enhancers , 2003, Nucleic Acids Res..

[32]  S. Goodman,et al.  RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy. , 2008, Cancer research.

[33]  L. Zipper,et al.  The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm* , 2002, The Journal of Biological Chemistry.

[34]  Se-Jin Kim,et al.  Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin. , 2008, Lung cancer.

[35]  Masayuki Yamamoto,et al.  Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2 , 2004, Molecular and Cellular Biology.

[36]  Deborah Schrag,et al.  Annual report to the nation on the status of cancer, 1975-2002, featuring population-based trends in cancer treatment. , 2005, Journal of the National Cancer Institute.

[37]  Tsutomu Ohta,et al.  Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. , 2006, Molecular cell.

[38]  J. Nevins,et al.  Complex Transcriptional Regulatory Mechanisms Control Expression of the E2F3 Locus , 2000, Molecular and Cellular Biology.

[39]  Stephen G Swisher,et al.  Nrf2 and Keap1 Abnormalities in Non–Small Cell Lung Carcinoma and Association with Clinicopathologic Features , 2010, Clinical Cancer Research.

[40]  M. Kwak,et al.  Acquisition of doxorubicin resistance in ovarian carcinoma cells accompanies activation of the NRF2 pathway. , 2009, Free radical biology & medicine.

[41]  R. Treisman,et al.  The POZ domain: a conserved protein-protein interaction motif. , 1994, Genes & development.

[42]  M. Sporn,et al.  Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-imidazolide. , 2009, European journal of pharmacology.

[43]  T. Guilarte,et al.  In vivo modulation of the Parkinsonian phenotype by Nrf2. , 2006, Neurotoxicology.

[44]  D. Sidransky,et al.  Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. , 2002, Cancer research.

[45]  M. Kwak,et al.  Role of the Nrf2-antioxidant system in cytotoxicity mediated by anticancer cisplatin: implication to cancer cell resistance. , 2008, Cancer letters.

[46]  Geoffrey J. Barton,et al.  The Jalview Java alignment editor , 2004, Bioinform..

[47]  J. Herman,et al.  Predicting lung cancer by detecting aberrant promoter methylation in sputum. , 2000, Cancer research.