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. 2021 Dec;10(23):8673-8692.
doi: 10.1002/cam4.4338. Epub 2021 Oct 6.

Integrated analysis of patients with KEAP1/NFE2L2/CUL3 mutations in lung adenocarcinomas

Xing Jin  1 , Yuansheng Zheng  1 , Zhencong Chen  1 , Fei Wang  2 , Guoshu Bi  1 , Ming Li  1 , Jiaqi Liang  1 , Qihai Sui  1 , Yunyi Bian  1 , Zhengyang Hu  1 , Yulei Qiao  1 , Songtao Xu  1   3
Affiliations

Integrated analysis of patients with KEAP1/NFE2L2/CUL3 mutations in lung adenocarcinomas

Xing Jin et al. Cancer Med. 2021 Dec.

Abstract

Objectives: To explore the clinical features, molecular characteristics, and immune landscape of lung adenocarcinoma patients with KEAP1/NFE2L2/CUL3 mutations.

Methods: The multi-omics data from the GDC-TCGA LUAD project of The Cancer Genome Atlas (TCGA) database were downloaded from the Xena browser. The estimate of the immune infiltration was implemented by using the GSVA analysis and CIBERSORT. The status of KEAP1/NFE2L2/CUL3 mutation in 50 LUAD samples of our department was detected by using Sanger sequencing, following the relative expression level of differentially expressed genes (DEGs), miRNAs (DEmiRNAs), and lncRNAs (DElncRNAs) was validated by IHC and real-time quantitative polymerase chain reaction (RT-qPCR).

Results: The Kaplan-Meier and multivariable Cox regression analyses demonstrated that KEAP1/NFE2L2/CUL3 mutations had independent prognostic value for OS and PFS in LUAD patients. The differential analysis detected 207 upregulated genes (like GSR/UGT1A6) and 447 downregulated genes (such as PIGR). GO, KEGG, and GSEA analyses demonstrated that DEGs were enriched in glutamate metabolism and the immune response. The constructed ceRNA network shows the linkage of differential lncRNAs and mRNAs. Three hundred and nine somatic mutations were detected, alterations in immune infiltration DNA methylations and stemness scores were also founded between the two groups. Eight mutated LUAD patients were detected by Sanger DNA sequencing in 50 surgical patients. GSR and UGT1A6 were validated to express higher in the Mut group, whereas the expression of PIGR was restrained. Furthermore, the IHC staining conducted on paraffin-embedded tissue emphasizes the consistency of our result.

Conclusion: This research implemented the comprehensive analysis of KEAP1/NFE2L2/CUL3 somatic mutations in the LUAD patients. Compared with the wild type of LUAD patients, the Mut group shows a large difference in clinical features, RNA sequence, DNA methylation, and immune infiltrations, indicating complex mechanism oncogenesis and also reveals potential therapeutic targets.

Keywords: cullin 3 (CUL3); kelch-like ECH-associated protein 1 (KEAP1); lung adenocarcinoma (LUAD); mutation; nuclear factor erythroid 2-like 2 (NFE2L2).

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Conflict of interest statement

The authors declare no competing interests in this work.

Figures

FIGURE 1
FIGURE 1
Flowchart diagram: a flow chart of the whole study design and analysis
FIGURE 2
FIGURE 2
Survival analysis and nomogram. Survival curves and forest plots of overall survival and progression‐free survival in LUAD patients with or without the KEAP1/NFE2L2/CUL3 mutations. Kaplan–Meier survival curves show significant differences between the Mut and the Wild groups in overall survival (OS) (A) and progression‐free survival (B). The forest plots manifested that the KEAP1/NFE2L2/CUL3 mutations are a risk factor for LUAD patients in overall survival (OS) (C) Nomogram of the overall survival in LUAD patients (D). 1‐year and 3‐year internal calibration plots of the overall survival nomogram (E)
FIGURE 3
FIGURE 3
Genetic mutations related to Mut and Wild groups. Waterfall plot of (A) KEAP1/NFE2L2/CUL3 Mut and (B) Wild group summarizing the somatic alterations and copy number variations. Different colors annotated the type of alterations. (C) The top eight differential mutations in the Mut and the Wild groups and their distributions. p value indicated. (D) A violin plot is presenting the tumor mutation burden in the two groups. The differences between the two groups were compared through the Wilcoxon test. p values indicated
FIGURE 4
FIGURE 4
Differential and function enrichment analysis of DEmRNAs and ceRNA network. (A) Differential expressed genes between the KEAP1/NFE2L2/CUL3 Mut and the Wild groups were shown in a volcano plot. (B) Cytoscape's plugin “MCODE” found the hub genes in DEGs and established the PPI network. The node's size represents the degree of the gene, and the width of the line indicates the combined score. (C) Dot plot of GO‐BP and (D) KEGG pathway analysis to the DEGs (top 10). (E) Heatmap of the top 40 DEmRNAs and the phenotype of the two groups.
FIGURE 5
FIGURE 5
DEmiRNAs, DElncRNAs, and the ceRNA network analyses. (A) DEmiRNAs and (B) DElncRNAs between the Mut and Wild groups were presented in the volcano plots. (C) Dot plot of KEGG pathway analysis of the downstream mRNAs regulated by LINC00473 (D) Upset plot of hallmark enrichment analysis of the linc00473’s modified mRNAs. (E) The ceRNA network of the DElncRNA–DEmiRNA–DEmRNA demonstrates the cascade regulation relationship in the KEAP1/NFE2L2/CUL3 mutant LUAD patients
FIGURE 6
FIGURE 6
Immune landscape, methylation differences, and stemness indices of the KEAP1/NFE2L2/CUL3 Mut and Wild groups. (A) Comparison of each immune cell fraction between Mut and Wild groups, (B) relative expression level of molecules participated in innate immunity (left) and MHC‐I/II antigen‐presenting procedure (right), (C) relative expression level of immune coinhibitors (left) and costimulators (right), (D) the differential genes related to the immune checkpoint. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. (E) Heatmap of DMPs between the KEAP1/NFE2L2/CUL3 Mut and Wild groups (adjusted p value <0.05, |Deltabeta| >0.2); (F) mRNAsi and (G) mDNAsi differences of the two groups in the LUAD patients were displayed in the violin plots. p values indicated
FIGURE 7
FIGURE 7
Validation of expression levels in our own cohort and the external validation of survival analysis. (A) The expression level of GSR, PIGR, UGT1A6, miR‐205‐5p, miR‐193b‐3p, RP11‐499O7.7, CTD‐2139B15.5, and LINC00473. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. (B) The IHC staining of three DEGs in Mut and Wild KEAP1 LUAD tissue samples. Quantification of percent positive regions (right) was performed using the IHC profiler plugin for ImageJ. Data are presented as the mean ± SD. (C) Kaplan–Meier survival curves between the Mut and the Wild groups in OS, RFS, and PFS
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