Article Figures & Data
Figures
- Figure 1.
LKB1 modulates STING expression in KRAS-mutant lung cancer. A, Venn diagram showing differentially expressed genes between KL and KP in TCGA and CCLE. Top-ranked gene signatures derived from differentially expressed genes are represented. B, Schematic of dsRNA and dsDNA sensing pathways that induce type 1 IFN. C, Relative RPKM values of genes related to these pathways in KL and KP cells from CCLE. D, Representative STING IHC images of primary KL and KP NSCLC samples (top plot). Insets highlight tumor cell STING expression. STING intensity in cancer cells was scored in a blinded manner (bottom plot) on a scale of 0 to 3. IHC0, no staining; IHC1, low staining; IHC2, moderate staining; and IHC3, high staining. E, Immunoblot (IB) of the indicated proteins in KL (red) or KP (blue) cells (left). KL cell lines with an asterisk contain p53 mutation. STING expression was quantified by ImageJ and normalized to β-actin (right). F, qRT-PCR of STING in KL or KP cells. Each point represents one cell line. G, IB of the indicated proteins in KP cells transduced with scramble or LKB1 single-guide RNA (sgRNA). H and I, IB of the indicated proteins in KL cells transduced with the indicated vectors. P values were calculated by unpaired two-tailed Student t test (C, E, and F) or Fisher exact test (D). *, P < 0.05; **, P < 0.01.
- Figure 2.
Hyperactivation of DNMT1 and EZH2 suppresses STING expression in KL cells. A, qRT-PCR of STING in KL cells transduced with GFP or LKB1. B, IB of the indicated proteins in KL cells. C, Dot plot heat maps showing DNA methylation levels in the STING promoter between KL and KP cells in CCLE (red = hypermethylation). The location of each position is as follows: P1, 5:138861649; P2, 5:138861807; P3, 5:138862442; and P4, 5:138862470. D and E, Levels of DNA methylation (D) or DNMT1 binding (E) within the 5′ UTR of STING (see Methods) normalized to input in KL cells. F, qRT-PCR of STING in KL cells treated with 100 nmol/L DAC, 5 μmol/L GSK126, 5 μmol/L KDM5-C70, or 500 nmol/L UNC0638 for 7 days. G and H, IB of the indicated proteins (G) or qRT-PCR of STING (H) in KL cells transduced with GFP or LKB1, and treated ± 100 nmol/L DAC for 7 days. I and J, H3K27me3 levels at 5′ UTR of STING normalized to the input in KL cells transduced with GFP or LKB1 (I) or treated with 5 μmol/L GSK126 for 7 days (J; n = 4 replicates from two independent experiments). K, IB of the indicated proteins in KL cells treated with 100 nmol/L DAC and/or 5 μmol/L GSK126 for 7 days. L, Measurement of SAM in A549 cells treated with 100 nmol/L DAC and/or 5 μmol/L GSK126 for 3 days (n = 4 replicates, representative of two independent experiments). P values were calculated by one-way (E, F, H, and L), two-way (A) ANOVA followed by Tukey post hoc test, or unpaired two-tailed Student t test (D, I, and J). *, P < 0.05; **, P < 0.01.
- Figure 3.
Defective sDNA sensing and impaired T-cell chemotaxis due to LKB1 inactivation. A, IB of the indicated proteins in KL cells transduced with GFP or LKB1, and treated ±1 μg/mL poly (dA:dT) for 4 hours. B, ELISA of human IFNβ, CXCL10, or CCL5 levels in conditioned medium (CM) derived from KL cells transduced with GFP or LKB1, and treated ±1 μg/mL poly (dA:dT) for 24 hours. C, IB of the indicated proteins in A549 cells transduced with GFP or LKB1, pretreated ±100 nmol/L DAC for 7 days, and treated ± 1 μg/mL poly (dA:dT) for 4 hours. D, ELISA of human CXCL10 levels in CM derived from A549 cells transduced with GFP or LKB1, pretreated ±100 nmol/L DAC for 7 days, and treated ±1 μg/mL poly (dA:dT) for 24 hours. E, Quantification of Jurkat CXCR3 infiltration into H1355 tumor spheroids (see Supplementary Fig. S3I and S3J). Values were normalized to each control. F and G, PD-L1 expression in H1944 cells transduced with NanoLuc or LKB1 (F), or A549 cells transduced with NanoLuc or LKB1, pretreated ±100 nmol/L DAC for 7 days (G), and treated ±125 ng/mL poly (dA:dT) for 12 hours. MFI, mean fluorescence intensity. ΔMFI, (dAdT-Ctr)/Ctr. Data are representative of three independent experiments. H, I, and J, IHC images and analysis from primary LKB1-negative; STING IHC 0/1 (n = 12) and LKB1-positive; STING IHC 2/3 NSCLC (n = 22) samples (see Fig. 1D). Red arrows highlight stained CD8+ T cells in both tumor epithelium (red) and stroma (green; H). PathAI (see Methods) was used to quantify CD3+/CD4+/CD8+ T-cell infiltration (I) and tumor PD-L1 expression (J). P values were calculated by two-way (B, D, and E) ANOVA followed by Tukey post hoc test, or unpaired two-tailed Student t test (F, G, I, and J). *, P < 0.05; **, P < 0.01.
- Figure 4.
STING is poorly tolerated by KRAS-LKB1–mutant cells due to the pathologic accumulation of cytoplasmic dsDNA. A and B, IB of the indicated proteins (left) and quantification (right) of pTBK1 induction (A) or qRT-PCR of CXCL10 (B) in KL (red) or KP (blue) cells 7 days following STING overexpression (n = 4 replicates from two independent experiments). C, CXCL10 ELISA in conditioned medium (CM) derived from KL cells 7 days following STING overexpression ± 1 μmol/L TBK1 inhibitor compound 1 for 72 hours. D and E, Relative cell number 9 days following STING overexpression ±1 μmol/L ruxo or 1 μmol/L TBK1 inhibitor Compound 1 (n > 3, biological replicates) following STING overexpression (n = 4, biological replicates). F, IB of the indicated proteins 7 days following STING reexpression ± 1 μmol/L ruxo treatment. G, H2009, H1944, or H2122 cells stained with PicoGreen, Mitotracker, and Hoechst (left). Scale bars, 3 μm. Signal intensity at each region of interest (ROI) was quantified by ImageJ (right). H, qPCR of mtDNA in cytoplasmic fraction (n = 6, biological replicates). I, Parental H2122, H2122 Rho 0, or H2122 cells transduced with scramble or POLG sgRNA stained with PicoGreen, Mitotracker, and Hoechst (left). Scale bars, 3 μm. Signal intensity at each ROI was quantified by ImageJ (right). J, Heat map of cytokine profiles in CM 7 days following STING reexpression. CMs were collected 72 hours after medium change. Scores, ratio of log2 fold change following STING reexpression relative to parental H2122 (left) or H2122 transduced with scramble sgRNA (right). K, IB of the indicated proteins 7 days following STING reexpression. L, Relative cell number 9 days following STING overexpression (n = 3, biological replicates). Values of STING-overexpressing cells were normalized to each control. P values were calculated by one-way (A,G,H, and I) or two-way (B,C,D,E, and L) ANOVA followed by Tukey post hoc test. *, P < 0.05; **, P < 0.01.
Additional Files
Supplementary Data
- Supplementary Figures and Table legends - The supplementary file shows Supplementary figure S1-S4 and their legends, and the legends for Supplementary table S1-S4.
- Table S1 - Table S1 shows list of TCGA IDs of KP and KL lung adenocarcinoma patients related with figure 1.
- Table S2 - Table S2 shows list of differentially expressed genes between KL and KP in TCGA and CCLE related with figure 1.
- Table S3 - Table S3 shows list of KRAS WT; LKB1 WT and KRAS WT; LKB1 Mut lung adenocarcinoma cell lines related with supplementary figure S1.
- Table S4 - Table S4 shows primers for qRT-PCR, ChIP-qPCR, construction of sgRNA-expressing vectors and mycoplasma monitoring.
Volume 9, Issue 1
