Promotion of liquid-to-solid phase transition of cGAS by Baicalein suppresses lung tumorigenesis

Abstract

Kras and p53 mutation are among the most common gene mutations in lung cancer, which has both the highest incidence and mortality rate among cancers. 1 Kras/p53 mutation also causes mitochondrial dysfunction, which has been implicated to promote the inflammation-to-cancer transition. 2 We established a lung adenocarcinoma model by using conditional alleles of Kras LSL-G12D /p53 flox/flox in mice 3 to evaluate the effect of Baicalein (5,6,7-trihydroxyflavone), a principal component of Scutellaria baicalensis in traditional Chinese medicine, 4 on the initiation and progression of lung cancer. Cre-mediated expression of Kras G12D and deletion of p53 caused obvious tumor lesions in the lung, which were strongly inhibited by the administration of Baicalein (Fig. 1a, b and Supplementary Fig. 1a, b ), indicating that Baicalein is highly potent in inhibiting the progression of primary lung cancer. Fig. 1 Baicalein promotes the liquid-to-solid phase transition of cGAS to suppress lung tumorigenesis. a Hematoxylin and eosin (H&E) staining of the section of lung tissue from mice of indicated groups. A lung adenocarcinoma (LUAD) model using conditional alleles of Kras LSL-G12D ; p53 flox/flox in mice (KP mice) was established. The mice were divided into three groups, one group was treated with adenovirus expressing Cre recombinase and fed with a normal diet (Cre), and the other group was also treated with adenovirus expressing Cre recombinase and mixed with Baicalein in a normal diet (Cre+Baicalein). The third group was treated with control adenovirus and fed with a normal diet (Cre) (Blank). Scale bars left: 200 μm. The red frame indicates the magnified area, which is shown on the right side. b The analysis of the number of tumors formed in one lobe of the left lung in mice of the indicated groups. The symbol indicates one mouse from n = 6 mice per group. The data shown are representative of n = 3 independent experiments. Data were expressed as the mean ± SD and one-way ANOVA followed by Dunnett’s post hoc test was used for the statistical analysis. c Gene set enrichment analysis (GSEA) for oxidative phosphorylation pathways correlated with the differentially expressed genes (DEGs). d Heatmap of DEGs in the pathway of cytosolic DNA-sensing. e qRT-PCR measurement of mtDNA in MEF cells treated as indicated. DNA was extracted from digitonin extracts of MEF cells generated from LSL-Kras G12D/WT ;p53 flox/flox mice that has been stably transduced with HBAD-Cre (Cre) left untreated or treated with Baicalein (60 μM) for 24 h. Cytosolic mtDNA was quantitated via qRT-PCR using a mitochondrial D-loop primer set. Normalization was performed as described in the Methods. f Oxygen consumption rate (OCR) in untreated MEF cells (Control) or MEF cells that has been stably transduced with an empty vector (Vehicle) or HBAD-Cre (Cre) in the absence of the presence of Baicalein (60 μM) for 24 h. n = 3. g , h Representative confocal microscopy images showing the presence of dsDNA in MEF cells that has been stably transduced with HBAD-Cre (Cre) in the absence of the presence of Baicalein (60 μM) for 24 h. Cells were stained with MitoTracker Red (red), Picogreen (green), and DAPI (blue), visualized by confocal microscopy. Scale bars = 10 μm. The quantification of the cytosolic Picogreen signal is shown in ( d ). i qRT-PCR measurement of Ifnb1 transcripts in the lung of mice as in (Fig. 1a ). j – l qRT-PCR measurement of transcripts of Ifnb1 ( a ), Cxcl10 ( b ), and Ccl5 ( c ) in untreated MEF cells generated from LSL-Kras G12D/WT ;p53 flox/flox mice (Blank), and MEF cells that has been stably transduced with an empty vector (Vehicle) or an HBAD-Cre (Cre) in the absence or presence of Baicalein at indicated concentrations for 12 h. n = 3. m Immunoblotting of indicated protein in untreated MEF cells generated from LSL-Kras G12D/WT ;p53 flox/flox mice (Blank), and MEF cells that has been stably transduced with an empty vector (Vehicle) or an HBAD-Cre (Cre) in the absence or presence of Baicalein at indicated concentrations for 12 h. n Surface Plasmon Resonance (SPR) assay showing the binding of Baicalein with cGAS (Kd = 3.02 μM). o An ELISA assay of the production of cGAMP by purified SUMO-cGAS in the absence or presence of ISD left untreated or treated with increasing dose of Baicalein. p FRAP assay of mEGFP-cGAS in Hela cells. The intensity was normalized with the pre-bleached as 100%. Graphical data were mean ± SD. Statistical analyses were done using unpaired Student’s t -test ( d , f ) or one-way ANOVA followed by Dunnett’s post hoc test ( a ). * p < 0.05; ** p < 0.01 Full size image

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