Loss-of-function mutations in KEAP1 drive lung cancer progression via KEAP1/NRF2 pathway activation

Abstract

Background and purpose: Targeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations. Methods: Lung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines. Results: The expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385. Conclusion: Somatic mutations of KEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.

KEAP1 Is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer

“…KEAP1 binds to nuclear factor erythroid 2-related factor 2 (NRF2), a key regulator of cellular response to oxidative stress, in the cytoplasm. Under normal physiological conditions, KEAP1 forms a complex with NRF2 and CUL3 (Cullin-3), which facilitates the ubiquitination and subsequent proteasomal degradation of NRF2 [ 9 , 10 , 11 ]. In response to oxidative stress, KEAP1 cysteine residues are oxidized, leading to conformational changes that cause release of NRF2, allowing for NRF2 to translocate to the nucleus, and regulate the transcriptional activation of antioxidant response genes, including heme oxygenase-1 ( HO-1 ) and NAD(P)H quinone dehydrogenase 1 ( NQO-1 ) [ 10 ].…”

“…Mutations in this pathway occur in approximately 30% of NSCLC and are of considerable clinical interest. In NSCLC cell lines, overexpression of wild-type, but not inactivating mutant, KEAP1 results in reduced colony formation in soft agar, decreased cell migration, and reduced growth of tumors in subcutaneous xenografts [ 9 ]. Additionally, overexpressing wild-type KEAP1 reduces the expression of NRF2 protein and the expression of transcriptional targets of NRF2 including HO-1 and NQO-1 [ 9 ].…”

“…In NSCLC cell lines, overexpression of wild-type, but not inactivating mutant, KEAP1 results in reduced colony formation in soft agar, decreased cell migration, and reduced growth of tumors in subcutaneous xenografts [ 9 ]. Additionally, overexpressing wild-type KEAP1 reduces the expression of NRF2 protein and the expression of transcriptional targets of NRF2 including HO-1 and NQO-1 [ 9 ]. Genetically engineered mouse models have also demonstrated that NRF2 activation or deletion of Keap1 accelerates Kras G12D driven lung tumorigenesis [ 12 , 13 , 14 ].…”

Predictive biomarkers of anti-PD-1/PD-L1 therapy in NSCLC

“…Lung cancer patients with MET exon 14 mutations responded modestly to single-agent or combination immune checkpoint inhibitors (objective response rate: 17%, 95% CI: 6%-36%) [ 55 ], and didn’t seem to benefit from immunotherapy [ 56 ]. Kelch-like ECH-associated protein 1 ( KEAP1 ) somatic mutations promoted tumorigenesis and reduced therapeutic sensitivity by activating the KEAP1 /nuclear factor erythroid-2-related factor-2 ( NFE2L2 ) stress response pathway [ 57 – 60 ]. NFE2L2 / KEAP1 mutations were associated with high TMB and PD-L1 expression, and the efficacy of immunotherapy was better in patients with NFE2L2 / KEAP1 mutations than other treatments (median OS: 22.52 months vs. 12.89 months, p = 0.0034) [ 61 ].…”

Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals

“…However, inactivating mutations in KEAP1 that lead to constitutive NRF2 activation [5,6] can provide a growth advantage in some cancer cells. The yin/yang aspect of NRF2 signaling was first elaborated by Wang et al, reporting that knockdown of NRF2 using siRNA, or stable overexpression of KEAP1, sensitized human cancer cells to chemotherapy [7].…”