Loss-of-function mutations in KEAP1 drive lung squamous cell carcinoma progression via KEAP1/NRF2 pathway activation

doi:10.21203/rs.2.18320/v1

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Loss-of-function mutations in KEAP1 drive lung squamous cell carcinoma progression via KEAP1/NRF2 pathway activation

https://doi.org/10.21203/rs.2.18320/v1

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published 23 Jun, 2020

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Background and Purpose Targeted therapy has led to dramatic change in the treatment of lung adenocarcinoma, but lung squamous cell carcinoma(LSCC) lacks targeted therapy options. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in LSCC. In this study, 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. Inhibited proliferation was more apparent in the A549 cell line trasfected with the R320Q KEAP1 mutant than the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.

Conclusion Somatic mutations in KEAP1 identified from patients with lung carcinoma 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.

Cell Communication and Signaling

Cancer Biology

KEAP1/NRF2

somatic mutation

NRF2 inhibitor

lung squamous cell carcinoma

targeted therapy

Lung cancer is a leading cause of cancer-related death, with a 5-year overall survival rate less than 15%³⁴, a significantly lower survival rate than that of most epithelial malignancies. Lung cancer is divided into small cell lung cancer and non-smallcell lung cancer (NSCLC). The proportion of the NSCLC type is more than 85%, mainly including lung adenocarcinoma and lung squamous cell carcinoma^{6, 16}.Lung squamous cell carcinoma (LSCC) accounts for approximately 30% of all lung cancers, and the mortality rate is extremely high⁹. Because most lung cancer patients are already in the advanced stage of disease at the time of diagnosis, they have lost the opportunity for surgical treatment, and the prognosis of LSCC has not obviously improved. The finding of high-frequency mutations in epidermal growth factor receptor (EGFR) kinase has led to a dramatic change in the treatment of patients with lung adenocarcinoma^{23, 26}. More recent data have indicated that targeting mutations in BRAF,AKT1,ERBB2, and PIK3CA as well as fusions that involve receptor tyrosine kinase genes ALK, ROS1, and RET may also be successful^{8, 18}. Unfortunately, the activating mutations in EGFR and ALK fusions are limited in lung adenocarcinoma andare not present in LSCC²⁹, and targeted agents developed for these activating mutations are largely ineffective against LSCC.

Currently, approximately 29 possible pathogenic genes for LSCC have been identified and are widely accepted^{1, 19, 21}. However, therapeutic drugstargeting these driver genes are lacking. Interestingly, a search of the TCGA database revealed that approximately 30% of LSCCs undergo recurrent mutations in KEAP1 and NFE2L2(alsonamed as NRF2)^{1, 19}. In our previous study, we identified that KEAP1and NRF2 mutations are recurrent in Chinese patients with LSCC, with a 5.8% frequency for KEAP1 and a 27.9% frequency for KEAP1/NRF2 mutations. However, mutations in KEAP1/NRF2 in Chinese patients with lung adenocarcinoma are rarely found, which is consistent with reports from Takahashi T³⁹. Interestingly, KEAP1 and NRF2 mutations show mutual repulsion in Chinese patients with LSCC¹. KEAP1 and NRF2 are the two key genes that regulate the oxidative stress pathway. At homeostasis, NRF2 is bound by the adapter protein KEAP1, which recruits the CUL3 ubiquitin ligase, leading to the proteasomal degradation of NRF2²⁰. Oxidative stress acts on KEAP1, causing its conformation to change and dissociate from NRF2, thereby losing the ability to mediate NRF2 degradation^{32, 38} and leading to NRF2 activation. Abnormal activation of NRF2 increases glutathione and related detoxification enzymes and decreases oxidative stress. Thus, chemotherapy drugs inactivate rapid metabolism in cells, significantly reducing the efficacy of anti-tumor drugs^{13, 33, 35}. More recently, the data have shown that loss of function of KEAP1 promotes KRAS-driven lung cancer and results in the dependence on glutaminolysis³¹.

Therefore, we aimed to test whether mutations in KEAP1, identified in our previous study, accelerate the development of LSCC, and whether a NRF2 inhibitor can be used as a targeted therapeutic drug in patients with lung cancer carrying KEAP1/NRF2 mutations.

Cell culture,reagents, and nude mice

The NCI-H1299,A549, H838, H460,H1299, 95D, and SPCA1 human lung cancer cell lines and HEK293T cells were obtained from American Type Culture Collection (Manassas, VA, USA). H1299, H838, H460, H292, 95D, and SPCA1 cells were maintained in RPMI 1640 medium(Gibco, Grand Island, NY, USA).A549 cells were cultured in F-12K(Gibco) supplemented with 10% fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere containing 5% CO₂. Twelve 4–6-week-old male BALB/c nude mice were purchased and reared from the Shanghai Ninth People's Hospital Central Laboratory Animal Law.

Plasmids, site-directed mutagenesis, and stable transfection

Mutations were conducted using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and were validated by sequencing; the primer sequences for mutagenesis are shown in (Supplementary Table 1). A retrovirus-mediated infection system was used.to construct A549 and H460 cells stably over-expressing 3FLAG-tagged KEAP1(WT or mutant). For PMSCV production, DNA encoding 3FLAG-tagged KEAP1 was inserted into the multi-cloning site of the pMSCV vector. Each PMSCV vector was co-transfected with gag-pol and VSVG using Lipofectamine 2000(Invitrogen,Waltham, MA, USA) in 293T cells. The virus was collected 2 days later and was transfected into A549 and H460 cells. The infected cells were selected with 1 µg/mL (A549) or 0.5 µg/mL (H460) of puromycin for 3–4 weeks.

Western blot and immunoprecipitation analyses

The antibodies used in our study were as follows: anti-FLAG M2 monoclonal(Sigma, St. Louis, MO, USA),anti-NRF2(Abcam,Cambridge, United Kingdom),anti-β-actin(Cell Signaling,Danvers, MA, USA), anti-HO-1(Cell Signaling),anti-LaminB1(Cell Signaling),anti-Lamin A/C(Cell Signaling).The Nuclear and Cytoplasmic Protein Extraction kit was obtained from (Thermo Fisher Scientific,Waltham, MA, USA).For immunoprecipitation, cell lysates were cleared by centrifugation and were incubated with FLAG resin (Sigma) before washing with lysis buffer, followed by overnight incubation at 4 °C.After washing three times by 1 × phosphate-buffered saline (PBS), the precipitates were analyzed by immunoblotting.

Real-time quantitative PCR

Total RNA was prepared from cells using Trizol reagent(Invitrogen),and tumors using Animal Total RNA Isolation kit(Sangon Biotech,Shanghai, China) and reverse transcription were performed using the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio,Shiga, Japan).The sequences for each primer are listed in Supplementary Table 2.

ROS measurement

After the cells were washed with PBS twice, the cells were incubated with 1 µM CM-H2DCF-DA (Nanjing Jiancheng, Nanjing, China) in culture conditions for 30 min in the dark and then were trypsinized and collected. The ROS levels were examined by flow cytometry.

Colony-formation assay

Exponentially growing cells were counted,diluted, and seeded in triplicate at 800–1,000 cells/well in 6-well plates.To assess clonogenic survival following drug exposure, the cell cultures were incubated in complete growth medium at 37 °C for 11–14 days.Colonies were fixed with precooled methanol and then were stained with 0.5% (w/v) crystal violet for 30 min at room temperature, followed by washing with PBS, photographing and counting. Only colonies with more than 50 cells were counted³⁶.

Wound-healing assay

Cell motility was determined by measuring the movement of cells close to an artificial wound. Cells were wounded with a 200-µL pipette tip, washed with PBS, and incubated in F12-K or RPMI 1640 medium without FBS. The distances removed by cells were monitored by microscopy at the indicated time points. The scratched area was analyzed using ImageJ.

Tumor xenograft model

Twelve BALB/c nude mice (4–6 weeks old, male) were randomly assigned to two groups(WT or mutant). We infected A549-KEAP1-WT or A549-KEAP1-R320W cells (1 × 10⁸) subcutaneously into the right flank of BALB/c nude mice and measured the tumor dimensions by calipers every 3–4 days. The tumor volumes were calculated using the formulalength [(mm) × width(mm)²]/2¹⁷. All experimental protocols conducted on mice were performed in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Shanghai Jiaotong University Animal Care and Use Committee.

Statistical analysis

All experiments were performed in quadruplicate and were repeated at least three times with similar results unless otherwise indicated.All statistical analyses were performed using unpaired two-tailed Student’s t-test and the mean ± standard error of the mean.A P-value of 0.05 or less was considered statistically significant.These analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA) or GraphpadPrism 7 (GraphpadSoftware, San Diego, CA, USA).

The KEAP1/NRF2 pathway was activated in lung cancer cell lines with KEAP1 mutations

To explore the effect of KEAP1 loss on activation of the KEAP1/NRF2 pathway,we collected three NSCLC cell lines with KEAP1 mutations (A549, NCI-H460, and NCI-H838) and four NSCLC cell lines without KEAP1/NRF2 mutations (NCI-H1299, NCI-H292, 95D, and SPCA1). As expected, Sanger sequencing revealed that the A549, H460, and H838 cell lines carried homozygous point mutations at D236H, G333C, and E444* in KEAP1, respectively; however, H1299, NCI-H292, 95D, and SPCA1 cell lines did not carry mutations in neither KEAP1 nor NRF2(Fig. 1A). Next, we found that the mRNA expression of downstream target genes of the KEAP1/NRF2 pathway, such as GCLC, GCLM, TXN, TXNRD, HO1, NQO1, GSR, and G6PD, which encode detoxifying enzymes and antioxidant proteins, were significantly higher in KEAP1 mutant cell lines than in wild-type (WT) lung cancer cells by real-time polymerase chain reaction (PCR), but the mRNA expression of NRF2 showed no significant difference between lung cancer cell lines with and without KEAP1 mutation (Fig. 1B). Given that NRF2 protein translocates into the nucleus to activate the transcription of downstream target genesin the KEAP1/NRF2 pathway, we further examined the protein expression of nuclear NRF2 and the downstream target HO-1 in these tumor cells by western blot analysis. As expected, function loss of KEAP1 significantly increased nuclear NRF2 levels and HO-1 levels incytoplasm (Fig. 1C). Thus, KEAP1 loss decreased the degradation of NRF2to activate the KEAP1/NRF2 pathway.

Activation of the KEAP1/NRF2 pathway decreases intracellular reactive oxygen species (ROS) levels through upregulating the expression of detoxifying enzymes and antioxidant genes. Thus, we checked whether KEAP1 loss could decrease ROS production in lung cancer cell lines. As shown in Fig. 1D, ROS levels were significantly decreased in lung cancer cells with KEAP1 mutation. Collectively, these findings demonstrate that KEAP1 loss can enhance the ability of lung cancer cells to resist oxidative stress.

Mutations in KEAP1 identified in Chinese patients with lung cancer promoted tumorigenesis via activation of the KEAP1/NRF2 pathwayin lung cancer cells

In our previous study, we identified five nonsynonymous mutations in KEAP1 from five patients with LSCC. However, the role of these KEAP1 mutations in tumorigenesis is unclear. The function these five nonsynonymous mutations in KEAP1 were predicted by Polyhen2_HDIV and SIFT. The results showed that these five nonsynonymous mutations in KEAP1 were harmful, affecting protein function (Table 1).

To verify whether these five somatic mutations in KEAP1 influence the function of KEAP1, WT and five KEAP1 mutants were stably transfected with retroviral vectors into A549/H460 lung cancer cell lines that carry loss-of-function mutations in KEAP1. As expected, nuclear NRF2 protein levels and expression of theNRF2 target gene HO-1 were significantly decreased after A549 or H460 lung cancer cell lines were stably transfected with WT KEAP1 (Fig. 2A). However, the levels of nuclear NRF2 protein and expression of NRF2 target gene HO-1 showed no significant difference after A549 or H460 lung cancer cell lines were stably transfected with the five KEAP1 mutants (Fig. 2A). Compared with A549 or H460 lung cancer cell linesstably transfected with empty vector, mRNA levels of NRF2 and its target genes HO-1, GCLC, and FTH1 were significantly decreased after the cell lines were stably transfected with WT KEAP1 (Fig. 2B). However, mRNA levels of NRF2and its target genes HO-1, GCLC, and FTH1 were significantly increased in the A549 or H460 lung cancer cell linesstably transfected with the five KEAP1 mutants (Fig. 2B).Together, these data suggested that these five nonsynonymous mutations in KEAP1, derived from Chinese patients with LSCC, were loss-of-function mutations that upregulated the expression of detoxifying enzymes and antioxidant genes.

KEAP1 is located at 19p13.2 and its protein has three major domains: an N-terminal broad complex, tramtrack, and the bric-a-brac (BTB) domain; a central intervening region (IVR); and a series of six C-terminal Kelch repeats ². The Kelch repeats of KEAP1 bind the NEH2 domain of NRF2¹⁵, whereas the IVR and BTB domains are required for the redox-sensitive regulation of NRF2 through a series of reactive cysteines present throughout this region^{20, 40}. In our present study, we found somatic mutations at R320Q, R413L, and D479H in the Kelch repeat domains of KEAP1, a somatic mutation at R234W in the IVR domain, and a somatic mutation at F174L in the BTB domain of KEAP1 (Fig. 2C). The binding of KEAP1 mutants to NRF2 was detected in coimmunoprecipitation experiments. Interestingly, mutants at R413L and D479H in the Kelch repeat domain of KEAP1 did not bind to NRF2(Fig. 2C). However, compared with WT KEAP1, binding of the mutants at F174L in the BTB domain and at R234W in the IVR domain of NRF2 was significantly increased (Fig. 2C). Unexpectedly, binding of NRF2 to the KEAP1 mutants at R320Q in the Kelch repeat domainwas not affected (Fig. 2C).

To uncover whether these five somatic mutations in KEAP1 influence the biological behavior of lung cancer cells, cell proliferation and migration were detected by colony-formation and scratch experiments. Compared with A549/H460 lung cancer cell lines stably transfected with retroviral empty vector, the colony formation and migration of A549/H460 lung cancer cell lines stably transfected with WT KEAP1 were significantly decreased (Fig. 2D, E). However, after being stably transfected with KEAP1 mutants, the colony formation and migration of A549/H460 lung cancer cell lines significantly increased (Fig. 2D, E). These data suggest that the newly found somatic mutations in KEAP1 promote tumor cell activity by activating antioxidant stress signaling pathways.

The somatic mutation at R320Q in KEAP1accelerated tumor growth in vivo

To further examine the effect of the somatic mutations in KEAP1 on the growth of lung cancer cell lines in vivo, the A549 cell lines stably transfected with WT KEAP1 or the R320Q mutant of KEAP1 were grafted subcutaneously into 4- to 5-week-old nude mice. After the cancer cell lines were grafted subcutaneously into nude mice, tumor sizes weremeasured using Vernier caliperseach day for 4 days. After 45 days of subcutaneous engraftment, tumors were peeled from the subcutis of the nude mice. The tumor sizes of the A549 cell line stably transfected with the R320Q KEAP1 mutant were significantly larger than those of the A549 cell line stably transfected with WT KEAP1(Fig.3A). Additionally, the tumor growth of theA549 cell line stably transfected with the R320Q KEAP1 mutant was strongly accelerated compared with that of the A549 cell line stably transfected with WT KEAP1, as measured by the change intumor volume(Fig.3B). Consistent with the in vitro results, the KEAP1 mutant showed significantly accelerated tumor growth in vivo. These results indicate that KEAP1 likely is a novel tumor driver gene for LSCC.

Next, we examined the expression of oxidative stress-related genes in the grafted tumor tissues from the nude mice. Compared with the expression of NRF2 in the nucleus and its target protein HO-1 in the cytoplasm in tumor tissues from the A549 cell line transfected with WT KEAP1, the expression levels of NRF2 and its target protein HO-1 were significantly increased in the tumor tissues from the A549 cell line transfected with the R320Q KEAP1 mutant (Fig. 3D). The mRNA levels of NRF2 and its target genes HMOX1, HO-1, GCLC, and NQO1 in the grafted tumor tissues from the A549 cell line transfected with the R320Q KEAP1 mutant were remarkably increased compared with that in grafted tumor tissues from the A549 cell line transfected with WT KEAP1(Fig.3C).

The NRF2 inhibitor ML385 inhibited proliferation of lung cancer cells carrying KEAP1 mutations

Increased cellular oxidative stress levels by small-molecule compounds to enhance cytotoxicity have been identified as a viable cancer treatment strategy¹¹. High levels of ROS not only inhibit cancer cell proliferation but also trigger apoptosis. A subset of NRF2 inhibitors has been reported to inhibit the proliferation of cancer cells by down-regulating the expression of NRF2, resulting in elevated levels of intracellular ROS and increased cytotoxicity. However, it is unknown whether NRF2 inhibitors have different effects on these LSCCs with or without KEAP1 somatic mutations. Thus, we selected an effective NRF2 inhibitor, ML385, which specifically and directly interacts with NRF2 protein, blocks NRF2 transcriptional activity, and enhances the efficacy of carboplatin and other chemotherapeutic drugs in lung cancer cells³⁷. The lung cancer cell line A549 transfected with the R320Q KEAP1 mutant (KEAP1-R320Q mutant) or with WT KEAP1(KEAP1-WT) and H1299 lung cancer cells, which carry both WT KEAP1 and NRF2, were selected and treated with ML385. The number of formed colonies was decreased in all three groups in a dose-dependent manner with increased ML385 treatment (Fig. 4A). Interestingly, when the lung cancer cell lines were treated with ML385 at a low dose, such as 0.25 or 0.5 µM/L, the proliferation of theA549 lung cancer cell line transfected with the R320Q KEAP1 mutant showed more significant inhibition than that of A549 cells transfected with WT KEAP1 or that of H1299 lung cancer cells (Fig. 4A, B). The proliferation of lung cancer cell lines showed no significant difference between A549 cells transfected with WT KEAP1 and H1299 lung cancer cells without KEAP1 and NFR2 mutation (Fig. 4A, B). These results suggest that lung cancer cell lines with KEAP1 mutations may have higher sensitivity to ML385 treatment.

To further detect whether the effect of ML385 on lung cancer cell proliferation is mediated by inhibiting the KEAP1/NRF2 pathway, the expression of NRF2 and its target genes HO-1, GCLC and NQO1 in lung cancer cell lines treated with ML385 were investigated by western blot and real-time PCR. As shown in Fig.4C, the expression levels of HO-1 and NRF2 protein in A549 lung cancer cells transfected with F174L,R234W, R320Q, and R413L KEAP1 mutants were significantly inhibited by ML385. However, the expression levels of HO-1 and NRF2 protein showed no significant difference between A549 transfected with WT KEAP1 treated with and without ML385(Fig. 4C). Although mRNA expression levels of NRF2 and its target gene NQO1 in A549 cells transfected with WT KEAP1 were significantly inhibited by ML385, the mRNA expression levels of the NRF2 target genes GCLC and HO-1 were not influenced by ML385(Fig. 4D). Notably, the mRNA expression levels of NRF2 and its target genes HO-1, GCLC, and NQO1 in A549 cells transfected with F174L, R234W, R320Q, and R413L KEAP1 mutants were dramatically decreased after treatment with ML385(Fig. 4D).

Recently, high-frequency somatic mutations of KEAP1 and NRF2 in the oxidative stress response pathway have been identified in patients with NSCLC by large-scale genomic studies³. Previous studies have revealed that NRF2 is involved in cancer development, especially lung cancer^{12, 14, 24, 27}. The loss-of-function mutation of KEAP1 promoted the tumorigenesis of Kras- and Pten-driven lung cancer cell lines in mice^{4, 31}. Moreover, the KEAP1/NRF2 pathway synergized with TP53 deletion mutation could induce LSCC and radiation resistance¹⁷. However, no evidence that KEAP1 or NRF2 mutations identified in lung cancer patients are involved in tumorgenesis has been reported. In the present study, we found that the ability of the antioxidative stress response mediated by activation of the KEAP1/NRF2 pathway is higher in lung cancer cell lines with KEAP1 mutation(A549, NCI-H460, NCI-H838) than in lung cancer cell lines without KEAP1 mutation(NCI-H1299, NCI-H292, 95D, and SPCA1). Interestingly, compared with the lung cancer cell lines A549 and H460, which carry KEAP1 mutation, after stable transfection with WT KEAP1,the mRNA levels of NRF2 and its target genes are significantly increased in A549 and H460 lung cancer cell lines transfected with KEAP1 mutants. Moreover, colony formation and migration were increased inA549 and H460 lung cancer cell lines transfected with KEAP1mutants. Similarly, the grafted subcutaneous tumor sizes in nude mice were significantly larger in A549 cells transfected with the R320Q KEAP1 mutant than those inA549 cells transfected with WT KEAP1. These data suggest that the somatic mutations of KEAP1 identified in Chinese patients with LSCC likely promote the development of lung cancer through activation of the antioxidative stress response in the KEAP1/NRF2 pathway.

Although genomic analysis identified some high-frequency gene mutations from LSCC, such as TP53, PI3KCA, and SOX2, no clear operationable targets for the treatment of LSCC have been found thus far^{1, 10, 19}. Practically, molecular targeted therapy improves the survival of patients with lung adenocarcinoma, but no effective targeted drugs have been identifiedin clinical trials for LSCC²⁸. Convenient treatment of LSCC remainsto be platinum-based chemotherapy. In recent years, immune checkpoint inhibitors, such as the PD-L1 monoclonal antibody, have been developed to treat NSCLC and bring certain benefitsfor the treatment of LSCC⁵. However, the overall effects of therapy for LSCC remain grim, revealing the immediate need for an effective treatment. In the present study, we found that the loss-of-function mutation in KEAP1 promotes the development of lung cancer mediated by activation of the KEAP1/NRF2 pathway. Thus, it is tempting to presume that inhibitors of the KEAP1/NRF2 pathway will likely treat LSCC patients carrying KEAP1/NRF2 mutations. To date, some inhibitors of the KEAP1/NRF2 pathway have been identified, such as NRF2 inhibitors that inhibit this pathway: ML385³⁷, Brucea chinensis³⁰, clonazepa propionate⁷, luteolin, all-trans retinoic acid, and flavonoid molecular compounds ^{22, 25}. The NRF2 inhibitor ML385 blocks activation of the pathway by inhibiting NRF2 expression. We further explored the effect of ML385 on the development of lung cancercell lines with or without KEAP1 mutations. Compared with A549 lung cancer cell lines trasfected with WT KEAP1, proliferation of the A549 lung cancer cell line trasfected with the R320Q KEAP1 mutant was dramatically inhibited by ML385. These preliminary data suggest that ML385 inhibits the proliferation of lung cancer cells with KEAP1 mutations by blocking the KEAP1/NRF2 antioxidant stress response pathway. It will provide a new option for therapy targeted to the KEAP1/NRF2 pathway in patients with LSCC carrying the KEAP1 mutation.

In summary, high-frequency mutationin KEAP1 has been identified in Chinese patients with LSCC. The somatic nonsynonymous mutations in KEAP1 derived from patients with lung cancer likely promote tumorigenesis via activation of the KEAP1/NRF2 antioxidant stress response pathway. Notably, NRF2 inhibitor ML385 may inhibit the proliferation of tumor cells with KEAP1 mutation by enhancing the oxidative stress level of lung cancer cells.

NRF2/ NFE2L2------- a nuclear factor erythroid 2-related factor 2

NSCLC---------- non-smallcell lung cancer

LSCC---------- Lung squamous cell carcinoma

EGFR--------- epidermal growth factor receptor

WT------- wild-type

Polyhen2_HDIV---------http://genetics.bwh.harvard.edu/pph2/

SIFT-------http://sift.jcvi.org/

ROS------- reactive oxygen species

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81772460 and 81472177).

Author information

Affiliations

1.Yangpu Hospital, Tongji Universtiy

2.Shanghai Jiaotong University

3.Shanghai University of Traditional Chinese Medicine

# Shanghai Changzheng Hospotal

* These authors contributed equally to this work.

**Correspondence author , addressed at: [email protected].

Gong meiling:[email protected];

Li yan : [email protected];

Ye Xiaoping: [email protected] ;

Zhang linlin : [email protected];

Wang zhifang: [email protected]

Xu xiaowen: [email protected]

Shen yejing: [email protected]

Contributions

ML-G, Y-L and LL-Z were major contributor in writing the manuscript. CX-Z was Corresponding Author.All authors read and approved the final manuscript.

corresponding author

Correspondence to CX-Z

Availability of data and materials

Not applicable.

Ethics declarations

Ethics approval and consent to participate

Experiments with Nude mouse tumor formation experiment were carried out under the ethical approval of Research Ethics Committee of the Shanghai Ninth People's Hospital Central Laboratory Animal Law.

Consent for publication

Not applicable

Availability of supporting data

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Loss-of-function mutations in KEAP1 drive lung squamous cell carcinoma progression via KEAP1/NRF2 pathway activation

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