Epigenetic Scanning of KEAP1 CpG Sites Uncovers New Molecular-Driven Patterns in Lung Adeno and Squamous Cell Carcinomas

Abstract

Background: The KEAP1/NRF2 (Kelch-like ECH-associated protein 1/nuclear factor erythroid 2–related factor 2) pathway modulates detoxification processes and participates in the resistance of solid tumors to therapy. Scientific evidence about the presence of genetic and epigenetic abnormalities of the KEAP1 gene was firstly reported in non-small-cell lung cancer (NSCLC) and then described in other tumors. At present, the prognostic role of aberrant methylation at cytosine-guanine dinucleotide (CpG) sites of the KEAP1 gene promoter is debated in NSCLC, and its correlation with transcriptional changes and protein levels remains to be defined in large sample cohorts. Methods: We evaluated and compared multiple KEAP1 omics data (methylation, transcript, and protein expression levels) from The Cancer Genome Atlas (TCGA) to explore the role of CpGs located in different portions of KEAP1 and the correlation between methylation, transcription, and protein levels. Data from two subsets of lung adenocarcinoma (LUAD, n = 617) and lung squamous cell carcinoma (LUSC, n = 571) cohorts of NSCLC patients with different disease stages were evaluated. Results: We found that the methylation levels of many KEAP1 CpGs at various promoter and intragenic locations showed a significant inverse correlation with the transcript levels. Interestingly, these results were limited to the KRAS wild-type LUSC and LUAD cohorts, whereas in LUAD the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in the EGFR mutated subpopulation. Conclusions: These results support the idea that the prognostic role of KEAP1 CpG sites warrants more in-depth investigation and that the impact of their changes in methylation levels may differ among specific NSCLC histologies and molecular backgrounds. Moreover, the observed impact of epigenetic silencing on KEAP1 expression in specific KRAS and EGFR settings may suggest a potential role of KEAP1 methylation as a predictive marker for NSCLC patients for whom anti-EGFR treatments are considered.

Keap1/Nrf2 Signaling Pathway

“…Therefore, investigations of the protective effects of Keap1/Nrf2 signaling that focus on specific tissues or organs are of great interest. Such studies have traditionally focused on tissues with detoxification functions such as the liver [ 6 , 7 ] but have also expanded to diverse tissues, including the kidney [ 8 , 9 ], skin [ 10 ], lung [ 11 ], colon [ 12 ], heart [ 13 ], nervous system [ 14 ], adipose tissue [ 15 ], pancreas [ 16 ] and thyroid [ 17 , 18 ], as evidenced by the respective tissue/organ-specific studies that are outlined below and comprise the majority of studies in the present Special Issue. Lastly, research on Nrf2 has expanded to encompass not only basic but also translational and clinical studies.…”

“…In the lung, the Keap1/Nrf2 pathway has a central role not only in maintaining homeostasis and protecting against various diseases [ 29 ] but also in the aberrant activation of Keap1/Nrf2 signaling due to mutations or epigenetic modifications, which has been implicated as a driver event in lung carcinomas [ 30 , 31 ]. The study by Fabrizio et al expanded on this knowledge by analyzing -omics data from a public repository corresponding to more than 1000 patients with non-small cell lung carcinoma (NSCLC) [ 11 ]. They report that the inverse correlation between the methylation levels of many CpG islands at various promoter and intragenic locations of the human KEAP1 gene and the transcript levels were limited to patients with Kirsten rat sarcoma virus (KRAS) wild-type lung squamous cell carcinoma (LUSC) or lung adenocarcinoma (LUAD), whereas in LUAD, the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in patients with epidermal growth factor receptor (EGFR)-mutated tumors [ 11 ].…”

“…The study by Fabrizio et al expanded on this knowledge by analyzing -omics data from a public repository corresponding to more than 1000 patients with non-small cell lung carcinoma (NSCLC) [ 11 ]. They report that the inverse correlation between the methylation levels of many CpG islands at various promoter and intragenic locations of the human KEAP1 gene and the transcript levels were limited to patients with Kirsten rat sarcoma virus (KRAS) wild-type lung squamous cell carcinoma (LUSC) or lung adenocarcinoma (LUAD), whereas in LUAD, the effect of the epigenetic silencing of KEAP1 on its transcription was also observed in patients with epidermal growth factor receptor (EGFR)-mutated tumors [ 11 ]. These results suggest that the prognostic role of such epigenetic alterations may differ among specific NSCLC histologies and molecular genetic backgrounds.…”

The Renaissance of KRAS Targeting in Advanced Non-Small-Cell Lung Cancer: New Opportunities Following Old Failures

“…In conclusion, the study has revealed that the epigenetic regulation of KEAP1 expression has different features in KRAS and EGFR settings of NSCLC, suggesting KEAP1 methylation as a predictive marker for response to anti-EGFR agents in oncogene addicted disease. Moreover, the correlation between epigenetic features and histotype underlies an interplay with lung cancer pathogenesis and development ( 141 ). Despite the many obstacles and its intrinsic complexity, the intriguing scenario of epigenetic targeting has led to many efforts trying to find the weak spot of KRAS-mutant tumors, although in this regard there is still a long way to go.…”

NRF2 Regulation by Noncoding RNAs in Cancers: The Present Knowledge and the Way Forward

“…Aberrant methylation at the promoter island of the KEAP1 gene has been widely reported as one of the most important mechanisms of KEAP1 silencing in solid tumors, such as glioma, breast, prostate, colorectal, thyroid cancers, clear renal cell carcinoma and lung cancer and was linked to tumor development, chemoresistance and mortality risk [14]. More recently, in NSCLC Kirsten rat sarcoma viral oncogene homolog (KRAS) wild-type subpopulations, a novel and significant correlation was observed among promoter and in intragenic exon 3 cytosine-guanine dinucleotide island (CpG-I) methylation and the transcription levels of KEAP1, NFE2L2 and many ARE genes [46,47].…”