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

Abstract

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.

The Keap1–Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases

“…Methylation in the promoter region of Keap1 affects its expression and hinders the ability to bind to the Nrf2. Therefore, this leads to the expression of Nrf2 (Wang et al 2008;Muscarella et al 2011). Conversely, DNA methylation by DNA methyltransferases (DNMTs) appears to downregulate the Nrf2 expression indirectly (Khor et al 2014;Yu et al 2010;Rajabi et al 2016;Tagde et al 2016b).…”

AberrantKeap1methylation in breast cancer and association with clinicopathological features

“…The highest frequency of mutations was found in gallbladder cancer (30.7%), ovarian clear cell carcinoma (29%) and NSCLC (19%). [6][7][8][13][14][15][16][17][18][19][20] Lower frequencies of mutations were demonstrated in endometrioid endometrial tumors (8.5%), non-clear cell ovarian cancer (8%), hepatocellular carcinoma (8.9%), colorectal cancer (7.8%), biliary duct carcinomas (5%), breast cancer (2%), prostate cancer (1.3%) and gastric cancer (1%). 13,17,21,22 However, immunohistochemical studies in lung cancer, endometrial tumors, renal and breast cancer demonstrated a high frequency of KEAP1 downregulation and/or Nrf2 overexpression in these tumor types, suggesting that the deregulation of KEAP1 may play a role in carcinogenesis beside the presence of genomic alterations.…”

“…Aberrant methylation of the KEAP1 promoter was found in approximately 50% of NSCLC and in 70% of malignant gliomas. 18,26 The presence of two genetic or epigenetic abnormalities was associated with worst progression free survival in patients affected by NSCLC, 18 whereas concomitant methylation of MGMT and KEAP1 genes was able to better predict response to treatment with radiotherapy and temozolomide in malignant gliomas. 26 Hanada et al 27 recently reported aberrant promoter methylation in 53% of colorectal cancer without finding correlation with patients' survival.…”

“…DNA obtained from the 20 invasive breast tumors was analyzed by fluorescence based direct sequencing for mutations in exons 4-7. 18 Amplification reactions were performed in a final reaction volume of 25 μL containing 100 ng of genomic DNA template, 0.25 nM dNTPs, 20 pmol of each primers, 1U HotMaster Taq polymerase (Eppendorf AG), in 1X PCR Reaction Buffer. PCR cycling conditions consisted of an initial denaturation step at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, annealing for 30 sec, extension at 72°C for 30 sec, and ending with a final elongation step at 72°C for 7 min.…”

“…We determined KEAP1 methylation levels in normal tissues and tumor specimens by using a primers/ probe set designed to amplify a CpG region showing the highest frequency of methylation and correlating with KEAP1 mRNA expression. 18,26,28 Methylation analysis was performed on DNA obtained from 6 normal breast tissues from reductive mammoplasty (NB), 14 pre-invasive lesions (PreINV), 102 invasive tumors (INV) along with 38 paired normal breast tissues distant from tumor (NBDT). Of the pre-invasive lesions, 12 were synchronous (9 DCIS and 3 ADH) to invasive cancer and 2 were only DCIS.…”