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

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.

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 high 9 . 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 LSCC 29 , 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   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 counted 36 .
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 × 10 8 ) 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 ]/2 17 . 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 Pvalue 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).
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 8 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 9 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.  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 colonyformation 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).

KEAP1 mutations
Increased cellular oxidative stress levels by small-molecule compounds to enhance cytotoxicity have been identified as a viable cancer treatment strategy 11 . 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 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 dosedependent 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 13 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).   Quantitative analysis of the mean fluorescence intensity using unpaired twotailed Student's t-test. The results are expressed as mean ± standard error of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. The scratch wound-healing assay showed that the migration of A549/H460 cells stably transfected withmutant KEAP1 was faster at 0h,24h,48h, and 72h than that of A549/H460 cells transfected withWTKEAP1. Scratch area quantitative analysis by Image J software.Each assay was repeated at least three times. Mean± standard error of the mean (SEM)are reported (* P < 0.05; **, P < 0.01; ***, P < 0.001). Mean±SEM are reported (* P < 0.05, ** P < 0.01, *** P < 0.001).