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, 114 (23), E4612-E4620

cGAS Is Essential for Cellular Senescence

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cGAS Is Essential for Cellular Senescence

Hui Yang et al. Proc Natl Acad Sci U S A.

Abstract

Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.

Keywords: DNA damage; DNA sensing; cGAS; cancer; senescence.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
cGAS deletion accelerates spontaneous immortalization of MEFs. (A) Growth curve of MEFs derived from the embryos of two pairs of WT and cGas−/− mice during the course of 20 serial passages according to a modified 3T3 protocol. (B) Similar to A except that MEFs were from littermate WT and cGas-/ embryos in the same cGas+/− mother that was crossed to a cGas+/− male.
Fig. S1.
Fig. S1.
cGas but not Sting deletion accelerates spontaneous immortalization of MEFs. (A) Growth curve of MEFs from the embryos of WT, cGas−/−, and Stinggt/gt mice. (B) Western blotting of cell lysates from indicated MEF cells with cGAS- or STING-specific antibodies.
Fig. 2.
Fig. 2.
cGAS deletion abrogates the senescence phenotypes of MEFs during spontaneous immortalization. (A) WT and cGas-/ MEFs at indicated passages were analyzed by SA-β-Gal staining. (B) Quantification of the β-Gal positive cells shown in A. (CE) qRT-PCR analyses of the indicated SASP genes. Error bars indicate SEs of triplicate measurements. All results in this and other figures are representative of at least two independent experiments.
Fig. 3.
Fig. 3.
cGAS deletion abrogates the senescence phenotypes induced by etoposide. (A and B) WT and cGas−/− MEFs were treated with the indicated concentrations of etoposide (Eto) for 24 h followed by 5 d of culturing in normal media before cells were stained by SA-β-Gal (A) and quantified (B). (CE) Aliquots of the MEF cells treated with Eto were analyzed for the expression of SASP genes by qRT-PCR. p21 is a cell cycle-dependent kinase inhibitor and a senescence marker. (FK) Similar to AE, except that WT and cGas−/− BJ cells were used. Error bars indicate SEs of triplicate measurements. Analyses of additional SASP genes are shown in Fig. S2 H–M. ND, not detectable.
Fig. S2.
Fig. S2.
cGAS deletion abrogates etoposide-induced senescence phenotypes of BJ cells. (A) Targeted mutation of exon 2 of human cGAS gene in BJ cells using TALEN technology. (B) Sequence verification of disruption of both alleles of the cGas gene in a BJ clone. (C) Western blotting of extracts of the cGAS−/− BJ cell clone with a human cGAS antibody. (DG) cGas−/− BJ cells were stimulated with HT-DNA, poly[I:C] or cGAMP as indicated, then total RNA was isolated for qRT-PCR. (HM) WT and cGAS−/− BJ cells were treated with the indicated concentrations of etoposide (Eto) for 24 h, followed by culturing in normal media for 5 d. Total cellular RNA was prepared from the cells to measure SASP gene expression by qRT-PCR. ND, not detectable.
Fig. S3.
Fig. S3.
cGAS is essential for etoposide-induced senescence of B16F10 cells. (A) Sequence verification of disruption of the cGAS gene in a B16F10 clone generated by CRISPR. (B) Western blotting of l from lysates of WT and cGas−/− B16F10 cells. (C and D) WT and cGas−/− B16F10 cells were stimulated by HT-DNA transfection, then the induction of IFNβ and CXCL10 RNA was measured by qRT-PCR. (E and F) WT and cGas−/− B16F10 cells were stimulated by transfection with poly[I:C] and the induction of IFNβ and CXCL10 RNA was measured by qRT-PCR. (G and H) WT and cGas−/− B16F10 cells were treated with the indicated concentrations of etoposide for 24 h before growing in normal culture media for 5 d. Cells were stained with β-Gal (G) and the results quantified (H). (IM) Total cellular RNA was isolated for qRT-PCR analyses of SASP genes.
Fig. 4.
Fig. 4.
cGAS deletion abrogates the senescence phenotypes induced by ionizing radiation. (A and B) WT and cGas−/− MEFs were irradiated with 3 Gy X-ray and then cultured in normal media for the indicated days before cells were stained with SA-β-Gal (A) and quantified (B). (CE) Aliquots of the MEF cells were analyzed for expression of the indicated SASP genes. (FK) Similar to AE, except that BJ cells were used. Error bars indicate SEs of triplicate measurements. Analyses of additional SASP genes are shown in Fig. S4 CF. ND, not detectable.
Fig. S4.
Fig. S4.
cGAS deletion abrogates the senescence phenotypes induced by IR. (A and B) WT and cGas−/− B16F10 cells were irradiated with the indicated IR dosages. At 7 d after radiation, cells were stained with β-Gal (A) and the blue cells quantified (B). (CG) WT and cGas−/− BJ cells were irradiated as described in Fig. 4, and the expression of the indicated SASP genes was measured by qRT-PCR. ND, not detectable.
Fig. 5.
Fig. 5.
cGAS enters the nucleus in proliferating cells and associates with damaged DNA in the cytosol in response to DNA damage. (A) MEF cells stably expressing GFP-cGAS were grown at low (8 × 104 per well of a 12-well plate) or high (3 × 105 per well of a 12-well plate) densities as indicated. The cells were cultured in the presence of EdU for 12 h and then EdU on the DNA was labeled and detected by fluorescence microscopy. Highlighted area indicates Edu-negative cells that contained GFP-cGAS predominantly in the cytoplasm. (B) GFP-cGAS MEF cells were treated with etoposide or IR and then stained with DAPI or antibodies specific for dsDNA or γ-H2AX. Zoomed cells highlight colocalization of cGAS with DNA and γ-H2AX in the cytoplasmic DNA foci.
Fig. S5.
Fig. S5.
cGAS, but not STING, associates with the chromatin during mitosis. (A) WT MEF cells were seeded at low (2.5 × 104 per well of a 12-well plate) or high (1 × 105 per well of a 12-well plate) densities. The cells were cultured for 2 d before staining with a STING antibody followed by immunofluorescence microscopy. (B) Immunofluorescent staining of STING in a dividing cell.
Fig. 6.
Fig. 6.
cGAS expression levels positively correlate with better survival of human lung adenocarcinoma patients. Kaplan–Meier curves were generated from the public microarray databases of human patients using online software (kmplot.com/analysis/). Groups of patients with high (top one-third) expression levels of the indicated genes cGAS (A), STING (B), MDA5 (C), and MAVS (D), were compared with those with low expression levels (bottom one-third). The Affymetrix microarray ID for each gene is shown in Materials and Methods. Statistical significance was determined using the log-rank test. HR, hazard ratio.
Fig. S6.
Fig. S6.
Kaplan–Meier analysis of gene expression microarray and survival data of human lung cancer patients. Data analysis of lung adenocarcinoma (A and B) and squamous cell carcinoma (CH) was carried out online at kmplot.com/analysis/. For comparisons between groups of high and low expression, the high expression group is defined as those having the top one-third expression values and the low expression group represents those with the bottom one-third expression values. The Affymetrix microarray ID for each gene is shown in Materials and Methods. Statistical significance was determined using the log-rank test. HR, hazard ratio.

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