Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells

Abstract

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.

Keywords: Apoptosis; KEAP1; Lung cancer; NFκB; TNFα; TRAF2.

Fig. 1

KEAP1 mutations from TCGA lung adenocarcinoma cohort classified as clusters 1–3 show differential expression of genes in oncogenic pathways. (A) Lollipop map of hotspot mutations in KEAP1 protein. (B) Clustering of KEAP1 mutations into three clusters by PCA according to their mRNA expression assessed by RNA-seq. (C) NRF2 activity score calculated in clusters 1–3. (D) Gene Set Enrichment Analysis (GSEA) from RNA-seq data between clusters 1 and 2. Positive and negative enrichment score (p < 0.05) are shown in red and blue colors, respectively. (E) Analysis of differential expression of genes between clusters 1 and 2. Data represents mean values from n = 7–9 patient samples from each KEAP1 mutant group.

Fig. 2

Molecular modeling and simulations of KEAP1 wt and mutants R320Q and R470C. (A) Full model of dimeric Keap1 containing BTB and Kelch domains. At the middle, pairwise interactions for native Keap1 for mutated residues R470 (left) and R320 (right) taken from AlphaFold model after full protein preparation. (B) 2D interaction charts including rough estimations for hydrogen bond and ionic interaction energies according to MOE software. (C) Interactions of native (left: green) and R320Q mutant (right: yellow) of KEAP1 with surrounding amino acids. (D) Interactions of native (left: green) and R470C mutant (right: yellow) of KEAP1 with surrounding amino acids. (E) Snapshot after 1 ms of simulation shows that in native form H311 acts as linger between mainchain carbonyl (N489) and carboxylic acid sidechain of E493. (F) Snapshot shows interactions of R470C mutant at the beginning of the simulation and (G) shows interactions with D422 and E493 at the end of 1 ms simulation.

Fig. 3

Identification of protein interaction partners of wild type KEAP1 and R320Q and R470C mutants. (A) Overexpression of HA-tagged GFP (mock), KEAP1 wild type and its R320Q and R470C mutants in Hek-TREx-293T cells. NRF2 protein expression was analyzed, with Actin as a loading control. (B) Top hits of affinity purification and mass spectrometry (AP-MS) analysis from the KEAP1 overexpressing cells. (C) Interaction partners from pathway enrichment analysis in KEAP1 mutants R320Q and R470C, as fold change to wt KEAP1. (D) Pathway enrichment analysis from AP-MS results by Ingenuity Pathway Analysis (IPA). Heatmap is based on the Z-score. (E) Co-immunoprecipation of Myc-tagged TRAF2 and HA-tagged Keap1 (F) Input controls. (G) Quantification of results from Co-IP. Data represents mean ± s.e.m from n = 3 (*p < 0.05, One way ANOVA, Tukey's test).

Fig. 4

KEAP1 mutants R320Q and R470C interact with TRAF2 and RIPK1 complex. (A) Schematic illustration of TNF-TNFR1 complex proteins, consisting of TRAF2, BIRC-family of proteins, RIPK1 and TRADD. Anti-apoptotic NFκB signaling or Caspase 8-mediated apoptotic signaling is mediated by RIPK1 and its status of phosphorylation. TRAF2 is degraded and removed from RIPK1 complex to initiate apoptosis signaling. (B) A549 cells with HA-tagged KEAP1 were co-immunoprecipitated with Flag-RIPK1 or (C) Myc-TRAF2. (D) and (E) Corresponding input lysates are shown. (F) Quantification of results from Flag IP and (G) Myc IP. Data represents mean ± s.e.m from n = 3 (**p < 0.01, One way ANOVA, Tukey's test).

Fig. 5

KEAP1 mutants R320Q and R470C activate NFκB transcriptional activity by stabilizing TRAF2 protein. (A) Immunoblots with respective antibodies in A549-LV cells. (B) Luciferase assay of NFκB reporter in A549-LV cells, showing NFκB transcriptional activity in KEAP1 R320Q and R470C cells with or without TNFα treatment. (C) NFκB transcriptional activity in A549-LV cells with QNZ and TRAF2 knockdown by siRNAs. (D) TRAF2 stability was studied in Hek-293T cells co-transfected with HA-KEAP1 (wt and mutants) and Myc-TRAF2 plasmids along with cycloheximide treatment for 0, 2 and 8 h. (E) Immunoblots with respective antibodies showing S11-phosphorylation of TRAF2 in A549-LV cells, transfected with Myc-TRAF2. Representative blots from three independent experiments are shown here. Data represents mean ± s.e.m from n = 3 (*p < 0.05, **p < 0.01, One way ANOVA, Tukey's test). CHX = cycloheximide.

Fig. 6

TRAF2-NFκB activation by KEAP1 R320Q and R470C leads to reduced apoptotic activity. (A) Nuclear staining of cleaved Caspase 3/7 and propidium iodide in A549-LV cells with and without TNFα treatment. Data shows representative images from n = 15 from three independent experiments. Scale bar = 100 μm. (B) Immunoblots showing full-length and cleaved PARP in A549-LV cells treated with TNFα. (C) Caspase 3/7 staining of A549-LV cells treated with THP1 CM and (D) M1 CM and QNZ. (E) Caspase 3/7 staining of A549-LV cells co-treated with TNFα and QNZ. (F) Caspase 3/7 staining of A549-LV cells with TNFα and TRAF2 knockdown. Data represents mean ± s.e.m from n = 3 (*p < 0.05, **p < 0.01, One way ANOVA, Tukey's test).

Fig. 7

TNFα treatment in A549-LV cells show differential expression of XIAP and other key apoptotic regulators. (A) A549-LV cells treated with TNFα show differentially expressed protein markers in an apoptosis array. (B) Quantification of results from the apoptosis array. Heatmap represents average values from two dots in the array, with samples stained from three independent replicates. (C) Luciferase assay of NFκB reporter in A549-LV cells with XIAP knockdown. (D) Caspase 3/7 staining in A549-LV cells with XIAP knockdown. Data represents mean ± s.e.m from n = 3 (*p < 0.05, One way ANOVA, Tukey's test). (E) In our hypothetical scenario, TNFα stimulated TNFR1 forms a protein complex including RIPK1, BIRC2 and 3, TRADD and TRAF2, which leads to NFκB transcriptional activity. KEAP1 mutants, R320Q and R470C (left: shown as red colored box) stabilize TRAF2 from proteasomal degradation, thereby increasing NFκB activation. Wt KEAP1 (right: shown as blue colored box) does not inhibit TRAF2 degradation thereby leading to apoptosis. XIAP, an inhibitor of TRAF2 degradation is enriched in lung cancer cells with KEAP1 R320Q and R470C mutations and contributes to anti-apoptotic signaling. Activation of TRAF2-NFκB signaling leads to increased production of cytoprotective genes, resulting in the survival of lung cancer cells against the stress induced by immune cells and chemotherapeutics. Image created with BioRender.com.

Supplementary Fig. 1

Differential expression of genes between KEAP1 mutants in cluster 1 and 2 from TCGA lung adenocarcinoma cohort. Log2 fold change and significantly upregulated and downregulated genes in cluster 1, when compared to cluster 2 KEAP1 mutant samples are shown highlighted in red color. NS = not significant. Threshold lines indicate the following: FDR-value = -log10(0.05) ∼1.3; LogFC = 1.5.

Supplementary Fig. 2

Characterization of A549-LV cells. (A) qRT-PCR analysis of KEAP1, (B) NQO1, (C) GCLM, (D) HMOX1, (E) TNXRD1 in A549-LV cells. Data represents mean ± s.e.m from n = 3 (*p < 0.05, **p < 0.01, One way ANOVA, Tukey's test). (F) Immunoprecipitation of HA-tagged KEAP1 from A549-LV cells and immunoblotting against endogenous TRAF2. Representative immunoblots from three independent repeats show respective antibodies staining from immunoprecipitation and corresponding lysate samples.

Supplementary Fig. 3

TRAF2 knockdown and qRT-PCR analysis of IL23A and BIRC2 in A549-LV cells. (A) qRT-PCR analysis of TRAF2 knockdown by siRNAs in A549 cells. (B) qRT-PCR analysis of IL23A and (C) BIRC2 genes in A549-LV cells with TNFα and QNZ treatments. Data represents mean ± s.e.m from n = 3 (*p < 0.05, **p < 0.01, One way ANOVA, Tukey's test).

Supplementary Fig. 4

Cytokine secretion in A549-LV cells. (A) Cytokine array analysis of extracellular media collected from A549-LV cells. Differentially expressed cytokines are highlighted with colored boxes. (B) Quantification of differentially expressed cytokines between different A549-LV cell lines. Data obtained from media pooled samples from three independent repeats.

Supplementary Fig. 5

S11-phosphorylation of Myc-TRAF2 in Hek-293T cells. Hek-293T cells co-transfected with Myc-TRAF2 and HA-tagged GFP (mock), KEAP1 wt and its mutants R320Q and R470C. Immunoblots with respective antibodies are shown. Data represents blots from three independent repeats.

Supplementary Fig. 6

Caspase 3/7 staining of A549-LV cells. (A) A549 mock cells treated with cisplatin, staurosporine and TNFα show Caspase 3/7 and propidium iodide staining. (B) A549 mock cells without Caspase 3/7 and propidium iodide staining, showing nuclei with DRAQ5 (in white color) staining. Scale bar = 100 μm. (C) Caspase 3/7 staining of A549-LV cells with cisplatin and staurosporine, and QNZ co-treatment. Data represents mean ± s.e.m from n = 3 (*p < 0.05, **p < 0.01, One way ANOVA, Tukey's test).

Supplementary Fig. 7

XIAP knockdown with siRNAs. qRT-PCR analysis of XIAP gene expression in A549 cells treated with siXIAP-1 and siXIAP-2. Data represents mean ± s.e.m from n = 3 **p < 0.01, One way ANOVA, Tukey's test).