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. 2022 Oct 25;10(5):1727–1730. doi: 10.1016/j.gendis.2022.10.009

Figure 1.

Figure 1

NRF2 participates in the suppressive tumor immune microenvironment of KRAS/KEAP1 co-mutant non-small cell lung cancer by inhibiting the STING pathway. (A) The mutation characters of cell lines used in this study were identified through CCLE database. (B) The protein expression of NRF2 were determined by Western blot. (C) Nuclear and cytoplasm isolation assay was performed to detect NRF2 localization in both KK- and K-type NSCLC cell lines. (D) The expression of NRF2 was negatively correlated with CD8+ T cell abundance based on TISIDB database in lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). (E) NRF2 was upregulated in K-type cells and knocked down in KK-type cells. CD3+/CD8+ T cell recruitment was evaluated by the chemotaxis assay; cells that migrated to the lower chamber were stained and recognized by a flow cytometer, and analyzed by Flowjo software. (F) ELISA was performed to determine the secretion levels of CCL5 and CXCL10 after upregulating NRF2 in K-type cells and downregulating NRF2 in KK-type cells. (G) The protein expression of STING in LUAD and normal tissues were confirmed by immunohistochemistry results from the HPA database. Scale bar = 50 μm. (H) The expression of STING in both KK- and K-type cells were determined by Western blot. (I) Changes of key protein levels in STING pathway were tested by Western blot after up- or down-regulating NRF2 in K- and KK-type cells, respectively. (J) After knockdown of NRF2 and addition of H151 in A549 and H460 cells, the secretion levels of CCL5 and CXCL10 were tested by ELISA. (K) CD3+/CD8+ T cell recruitment was evaluated by the chemotaxis assay; cells that migrated to the lower chamber were stained and recognized by a flow cytometer, and analyzed by Flowjo software. (L) The expression of BRCA1 in both KK- and K-type cells were determined by Western blot. (M) Changes of BRCA1 and DNA damage marker γ-H2AX levels were tested by Western blot after up- or down-regulating NRF2 in K- and KK-type cells, respectively. (N) Binding sites between the BRCA1 promoter sequence and NRF2 were predicted by JASPAR database. (O) Luciferase activities following transfection with NRF2 expression vector and pGL3-Basic or BRCA1 vector were detected by dual luciferase reporter assay. pRL-TK vectors were transfected as the internal control. (P) Changes of BRCA1, NRF2 and key protein levels in STING pathway were tested by Western blot after down-regulating BRCA1 in A549 and H460 cells. (Q) Schematic representation of the potential mechanisms of “hot” and “cold” tumors in K- and KK-type NSCLC. (Left) In K-type cells, exogenous risks lead to DNA damage, increase the cytoplasmic double-stranded DNA (dsDNA), and activate the STING pathway, resulting in the recruitment of CD8+ T cells by promoting the chemokines CCL5 and CXCL10, leading to a “hot tumor”; (Right) In KK-type cells, when KEAP1 mutation (KEAP1 MUT) occurred, NRF2 translocated into the nucleus and bond to the ARE sequence in the promoter region of BRCA1 gene, to promote BRCA1 transcription and expression, and DNA damage repair, resulting in STING pathway inactivation, chemokines synthesis and secretion reduction, and CD8+ T cell recruitment inhibition, leading to a suppressive TIME (cold tumor). EV, empty vector. NC, negative control. si-NRF2, small interfering RNA that targets NRF2. si-B, small interfering RNA that targets BRCA1. Data are shown as mean ± standard deviation of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not significant.