cGAS–STING cytosolic DNA sensing pathway is suppressed by JAK2-STAT3 in tumor cells

www.nature.com/scientificreports OPEN cGAS–STING cytosolic DNA sensing pathway is suppressed by JAK2‑STAT3 in tumor cells Manuel Adrian Suter1, Nikki Y. Tan1, Chung Hwee Thiam1, Muznah Khatoo1, Paul A. MacAry1, Veronique Angeli1, Stephan Gasser1,2 & Y. L. Zhang1* Deficiencies in DNA repair and DNA degrading nucleases lead to accumulation of cytosolic DNA. cGAS is a critical DNA sensor for the detection of cytosolic DNA and subsequent activation of the STING signaling pathway. Here, we show that the cGAS-STING pathway was unresponsive to STING agonists and failed to induce type I interferon (IFN) expression in many tested human tumor cells including DU145 prostate cancer cells. Inhibition of IL-6 or the downstream JAK2/STAT3 signaling restored responsiveness to STING agonists in DU145 cells. STING activity in murine TRAMP-C2 prostate cancer cells was critical for tumor rejection and immune cell infiltration. Endogenous STING agonists including double-stranded DNA and RNA:DNA hybrids present in TRAMP-C2 cells contribute to tumor rejection, but tumor growth was further suppressed by administration of cGAMP. Intratumoral co-injections of IL-6 significantly reduced the anti-tumor effects of cGAMP. In summary, STING in tumor cells contributes to tumor rejection in prostate cancer cells, but its functions are frequently suppressed in tumor cells in part via JAK2 and STAT3 pathways. Cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, catalyzes the synthesis of cyclic dinucleotide cGAMP, which binds and activates Stimulator of interferon (IFN) genes (STING), an endoplasmic reticulum-located adaptor molecule1, 2. STING plays a crucial role in mediating inflammation by inducing type I IFN production through recruitment of TANK binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3)3. Type I IFNs induce the transcription of many anti-viral genes and activate important components of the innate and adaptive immune system including natural killer cells and T cells4. Mb21d1 and Tmem173, the genes encoding cGAS and STING respectively, are expressed by many cell types and t issues5. cGAS and STING deficiency in various cancer cell lines results in abolished or declined level of type I IFNs in response to exogenous cytosolic DNA, which may contribute to non-inflamed cancer m icroenvironment6. Early evidence that STING agonists can exert anti-tumor activity was provided by preclinical mouse models using flavone acetic acids and its derivate 5,6-dimethyllxanthenone-4-acetic acid (DMXAA)7. Recent studies showed that DMXAA selectively binds mouse, but not human STING, which was suggested to have contributed to its poor performance in phase III t rials8. The discovery of bacterial cyclic dinucleotides (CDN) and cGAMP allowed the design of STING agonists based on the structure of human S TING9. Treatment with such novel STING agonists upregulate the expression of pro-inflammatory cytokines and type I IFNs in human and mouse dendritic cells (DCs)10. Also, intra-tumoral injection of STING agonists led to complete tumor regressions and protective T-cell responses in several mouse tumor m odels7, 11. Based on these encouraging preclinical data, STING agonists have entered clinical development. Although the role of STING and STING agonists in immune cells has been well studied, the role of STING in non-immune cells including tumor cells remains poorly understood. We have previously shown that cytosolic DNA in cancer cells induces and maintains low levels of type I IFNs and induce an anti-cancer T-cell response in mouse prostate tumor cells in a STING-dependent manner12. Consistently, recent evidence suggests that STING expression in B16 melanoma cells contributes to the activation of immune cells and tumor retardation13, suggesting that tumoral STING may play an important role in anti-cancer immunity. Dysregulated DNA repair processes and expression of nucleases lead to accumulation of cytosolic DNA in tumor cells12, 14, 15. Here we show that many tested human cancer cells failed to respond to exogenous STING agonists or double-stranded (ds) DNA. Our data suggest that the cGAMP unresponsiveness of many human tumors is caused by impaired STING activity, but not by dysfunction of pathways downstream of STING. Reduction of 1 Department of Microbiology, Immunology Programme, National University of Singapore, Singapore 117456, Singapore. 2NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117597, Singapore. *email: Scientific Reports | (2021) 11:7243 | https://doi.org/10.1038/s41598-021-86644-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ cytosolic DNA levels or cGAS deficiency did not restore cGAMP responsiveness of these cancer cells indicating that unresponsiveness was not caused by overstimulation of the pathway. In line with previous studies, we found that responsiveness of tumor cells to STING agonists could be restored by chemical inhibition of IL-6 in DU145 cells or JAK2/STAT3 in all tested cells16, 17. STING activity in cancer cells is functionally important as STING expression in prostate TRAMP-C2 cancer cells contributed to their rejection and mediated immune infiltration of the tumor. STING activity was partially mediated by endogenous cGAS agonists including double-stranded DNA and RNA:DNA hybrids present in the cytosol of tumor cells. However, tumor rejection was further boosted by intratumoral injection of cGAMP suggesting that endogenous cGAS agonists fail to fully activate STING in TRAMP-C2 cells. Supporting the finding that JAK2/STAT3 suppresses STING in tumor cells, co-administration of IL-6, a JAK2/STAT3 activator, impaired the anti-tumor effects of cGAMP. In summary, our data show that STING activity in tumor cells contributes to anti-cancer responses, but is often repressed in human cancer cells. Restoration of STING activity by for example blocking JAK2/STAT3 pathways may increase the efficacy of cancer immunotherapies in particular therapies using STING agonists. Results STING signaling is defective in the majority of tested human cancer cell lines. The STING signaling pathway plays a critical role in tumor suppression and immune s urveillance1. Immune selection of STING expressing cancer cells may lead to loss of STING activity in some tumor cells. In support of this possibility, a recent study found that a majority of human colorectal cancer cells are defective in STING-dependent signaling pathways18. In addition, STING was found to be epigenetically silenced in KRAS-LKB1–mutant lung cancers, which may facilitate immune e scape19. To examine STING activity in different human cancer cells, we first analyzed IRF3 nuclear localization in several human cancer cells upon activation of STING. IRF3 transcriptional activity correlates with nuclear IRF3 translocation, but not with minimal post-translational modifications20. cGAMP and ISD induced nuclear translocation of endogenous IRF3 and expre (...truncated)