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

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.

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 cells 16,17 . 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 surveillance 1 . 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 pathways 18 . In addition, STING was found to be epigenetically silenced in KRAS-LKB1-mutant lung cancers, which may facilitate immune escape 19 . 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 modifications 20 . cGAMP and ISD induced nuclear translocation of endogenous IRF3 and expression of IRF3 target genes in TRAMP-C2 and THP-1 leukemia cells ( Fig. 1A and B). In contrast, endogenous nuclear IRF3 levels or expression of IRF3 target genes did not increase in human DU145 prostatic carcinoma cells, A549 lung carcinoma cells, HeLa cervix carcinoma cells, and HCT116 colorectal carcinoma cells in response to ISD or cGAMP ( Fig. 1A and B). Similar findings were observed in cells transduced with a retrovirus encoding an IRF3-GFP fusion protein (Fig. S1A). However, it is worth noting that the steady-state levels of nuclear IRF3-GFP in ISD and cGAMP unresponsive cells were significantly higher when compared to responsive cells. No nuclear localization of the activation-defective IRF3-GFP mutant A7 (IRF3A7-GFP) was observed in all tested cancer cells upon treatments (Fig. S1B). The inability of the unresponsive cancer cells to respond to STING agonists was unlikely due to mutations in the Tmem173 or Mb21d1 genes as nonsynonymous substitutions are not present in either gene in DU145, A549, HeLa and HCT116 cells 21,22 . The average transcript intensity z-scores for Tmem173 and Mb21d1 were within the range found in other cancer cells (n = 60) including ISD/cGAMP responsive cells. While Tmem173 transcript levels were somewhat lower in A549 cells (z = − 1.73) and Mb21d1 transcript levels were decreased in HCT116 cells (z = − 1.28), no significant difference in STING/cGAS protein levels was observed in either cell line when compared to other tested cells ( Fig. 1C and S2). Furthermore, the average transcript intensity z scores for TBK1 and IKKe, two kinases activated by STING, were similar in the analyzed cell lines when compared to other cells (n = 60). The IKKe transcript (z = − 0.9) and protein levels were slightly reduced in HCT116 cells ( Fig. 1C and S2) 21,22 . Finally, ENPP1, which degrades cGAMP was not amplified in any of the tested cells and no gain-offunction mutations were found (Data not shown) [21][22][23] .
To gain insights into the mechanisms contributing to the inability of these human cancer cells to respond to STING agonists, we treated the different cancer cells with the Toll-like receptor (TLR) 3 agonist Poly(I:C). Similar to cGAMP, Poly(I:C) activates IRF3 through the serine/threonine kinases TBK1 or IKKe 24 . However, unlike the STING-dependent activation of TBK1/IKKe by cGAMP, TLR3 signals require the adaptor TRIF 3 . The TLR3 agonist Poly(I:C) induced the expression of the IRF3 target genes IFNB and CXCL10 in all tested cancer cell lines suggesting that defects upstream of TBK1/IKKe render the cancer cells unresponsive to STING agonists (Fig. 1D). The data also demonstrate that the lower levels of IKKe in HCT116 cells are unlikely to explain their inability to respond to STING agonists. Hence, the inability of some human tumor cells to respond to STING agonist is likely due to the dysfunction of STING activity in these cells.
Cytosolic DNA does not contribute to STING dysfunction in cancer cells. Activation of the cytosolic DNA sensor cGAS was found to trigger negative feedback pathways leading to suppression of STING activity 25 . Cytosolic dsDNA and RNA:DNA hybrids were reported to be the major substrates of cGAS 2,26 . To evaluate whether these DNA species in the cytosol contribute to constitutive cGAS activation and the induction of STING unresponsiveness, we first labelled cGAMP-responsive and unresponsive cancer cell lines for dsDNA and RNA:DNA hybrids in the cytosol. Both dsDNA and RNA:DNA hybrids recognized by the S9.6 antibody were present in the cytosol of all tested tumor cells ( Fig. 2A). To investigate if cGAS binds cytosolic DNA in tumor cells, we first co-labelled tumor cells for cGAS and different cytosolic DNA species. Cytosolic dsDNA and RNA:DNA hybrids partially co-localized with cGAS in all tested tumor cells (Figs. S3 and S4).To demonstrate that cGAS physically binds to dsDNA and RNA:DNA hybrids in tumor cells, cytosolic dsDNA and RNA:DNA hybrids were immunoprecipitated in A549 cells. Immunoblot analysis showed that cGAS co-immunoprecipitated with dsDNA and to a lesser degree with RNA:DNA hybrids ( Fig. 2B and Fig. S8A). Treatment of the tumor cell lysate with DNase or RNase H abrogated the binding of cGAS to dsDNA or RNA:DNA hybrids, respectively. In summary, our data show that cGAS binds to cytosolic dsDNA and to a lesser degree RNA:DNA hybrids in cancer cells, which may result in the activation of cGAS.   S5). As expected, the levels of dsDNA decreased in the cytosol of TREX1-transduced cells (Fig. 2C), while cytosolic RNA:DNA hybrids levels were lower in cells overexpressing RNASEH1 (Fig. 2D). The lower levels of cytosolic DNA in TREX1-or RNASEH1transduced A549 and HCT116 cells resulted in decreased transcript levels of IFNA (p < 0.05), IFNB (p < 0.02), and CXCL10 (p < 0.03) (Fig. 2E). However, overexpression of TREX1 or RNASEH1 in A549 and HCT116 cells did not render cells responsive to STING agonists (Fig. 2F), suggesting that cGAMP unresponsiveness of A549 and HCT116 cells was not a result of cytosolic DNA-mediated suppression of STING activity. cGAS does not contribute to STING dysfunction. cGAS was functional in TRAMP-C2 and A549 cells as cGAMP was detected in untreated cells and cGAMP levels significantly increased upon transfection of ISD (Fig. S6A). To analyse if cGAS activity contributes to the dysfunction of STING in cGAMP unresponsive cancer cells, we inhibited cGAS expression by cGAS-specific siRNAs in DU145 and TRAMP-C2 cells or by a cGAS-specific RNA-guided Cas9 nuclease in A549, HeLa, and HCT116 cells 27 . Using different approaches, we confirmed that cGAS expression levels were significantly reduced (p < 0.0001) in cGAS siRNA-transfected TRAMP-C2 and DU145 cells and that CRISPR-induced frame-shift mutations resulted in cGAS-deficient A549, HeLa, and HCT116 cells (Figs. 3A and B, S6B and C, and S8B). IFNA, IFNB and CXCL10 transcript levels were decreased in TRAMP-C2 and DU145 cells transfected with cGAS siRNAs and cGAS-deficient A549, HeLa, and HCT116 cells (Fig. 3C) demonstrating that sensing of cytosolic DNA by cGAS contributes to the constitutive expression of type I IFNs in tumor cells. However, cGAS deficiency in cGAMP unresponsive cancer cells did not enable cells to respond to STING agonists ( Fig. 3D) showing that STING dysfunction is not maintained by cGAS overstimulation in the tested tumor cells.

The JAK2/STAT3 pathway contributes to sting dysfunction in tumor cells. Recent reports
showed that IL-6 and JAK2/STAT3 pathway negatively regulates STING activity in THP-1 cells 16 and fibroblastic MRC-5 cells 17 . To explore the role of JAK2/STAT3 in the suppression of type I IFN expression, cells were treated with the JAK2/STAT3 inhibitor WP1066 at doses 3 times above published IC 50 (Fig. 4A) 28 . Strikingly, inhibition of JAK2/STAT3 activity increased the expression of IFNA or IFNB transcripts in all tested cell lines. To investigate if cytosolic DNA activates the JAK2/STAT3 pathway via IL-6, we blocked IL-6 signals by using neutralizing antibodies against IL-6. Blocking of IL-6 rendered DU145 cells responsive to cGAMP (Fig. 4B). Blocking of IL-6 correlated with reduced numbers of phosphorylated STAT3 foci in the nucleus of DU145 cells (Fig. 4C). These data suggested that STAT3 is activated by IL-6 in DU145 cells but not the other tested cells. In summary, our data show that activation of the JAK2/STAT3 pathway, which can be activated by diverse signals including IL-6 29 , abrogates the response of cells to endogenous type I IFN inducers such as cGAMP.

STING function in tumor cells contributes to control of tumor growth and inflammation of the tumor microenvironment.
Recent reports have shown significant anti-cancer effects of STING agonists in several mouse cancer models and various small-molecule STING agonists are currently being evaluated in clinical trials 7,30 . To address if STING activity in tumor cells contributes to the anti-cancer responses, we challenged C57BL/6 mice with STING-sufficient or STING-deficient TRAMP-C2 cells. As expected, STINGdeficient TRAMP-C2 cells no longer upregulated type I IFNs levels in response to the STING agonists cGAMP or the cGAS agonist IFN stimulatory DNA (ISD) (Fig. 5A and Fig. S8C). Tumor cells that lacked STING grew significantly faster when compared to STING expressing TRAMP-C2 cells (p < 0.001; Fig. 5B). Intratumoral injection of cGAMP 14 days after administration of TRAMP-C2 cells further slowed the growth rate of STING     Fig. 5B). Strikingly, intratumoral administration of cGAMP had no effect on the growth rate of TRAMP-C2 tumors that were deficient in STING ( Fig. 5B and C) suggesting that activation of STING in TRAMP-C2 cells is critical for anti-cancer responses of cGAMP in this model.   www.nature.com/scientificreports/ STING agonists were shown to mediate tumor infiltration of lymphocytes by inducing the production of pro-inflammatory cytokines and chemokines 31 . Tumor-infiltrating lymphocytes are associated with suppressed tumor growth and favorable prognostic value in patients with different types of cancers 32,33 . To determine the role of tumoral STING expression in mediating immune cell infiltration of tumors, we stained STING CTRL and STING CRISPR TRAMP-C2 tumors for the presence of different immune cells upon administration of cGAMP. Fewer immune cells expressing the pan-leukocyte marker CD45 were present in STING-deficient TRAMP-C2 tumors when compared to tumors expressing STING (p < 0.029; Fig. 5D). Treatment of tumors with cGAMP increased the presence of CD45 + immune cells in the tumor microenvironment in a STING-dependent manner (p < 0.021; Fig. 5D and Fig. S7A). STING-sufficient TRAMP-C2 tumors contained higher numbers of CD11c + dendritic cells (DCs), which play an important role in anti-cancer immune responses (p < 0.03; Fig. 5D and Fig. S7A) 34 . Consistent with the conclusion that STING activation attracts DCs to the tumor microenvironment, intratumoral injection of cGAMP increased the number of CD11c + DCs in the tumor (p < 0.02; Fig. 5D and Fig. S7A). CD11c + DCs in tumors did not co-express CD68, a marker for monocytes and macrophages (Fig. S7B). To investigate if STING activation also attracts cytotoxic T cells, tumors were stained for the presence of CD8α + T cells. CD8α + T cells were only rarely observed in the tumor microenvironment of STING-sufficient or -deficient TRAMP-C2 tumors (Figs. 5D and S7A). Intratumoral injection of cGAMP notably increased the number of CD8α + T cells in STING-sufficient tumors, but not in STING-deficient tumors (p < 0.05; Fig. 5D). In summary, our data support the conclusion that activation of STING expression in TRAMP-C2 cells contributes to anti-cancer immunity by attracting DCs and T cells.
Our in vitro data suggest that the IL-6 and JAK2/STAT3 pathways suppress STING function in cancer cells. Addition of exogenous IL-6 to TRAMP-C2 cells inhibited their ability to induce the expression of type I IFNs in response to cGAMP (Fig. 5E). To test the ability of IL-6 to suppress the anti-cancer effects of cGAMP in vivo, IL-6 was co-administrated with cGAMP in the TRAMP-C2 prostate cancer model. Co-injection of mouse IL-6 significantly decreased the anti-tumor effects of cGAMP, while IL-6 on its own had no impact on tumor growth, demonstrating that tumor growth promoting effects of the co-injection depended on cGAMP (Fig. 5F). In summary, our data suggest that signals leading to STAT3 activation may counter the effects of STING agonists. Combination of STAT3 small molecule antagonists that are currently being developed or IL-6 inhibitors might be interesting combination partners for STING agonists in future clinical trials.

Discussion
It was suggested that STING is activated by tumor-derived DNA upon engulfment of necrotic tumor cells by DCs 35 . The subsequent production of type I IFNs and other factors may facilitate cross-presentation of tumor antigens by DCs and activation of tumor-specific T cells. Here we show that the cytosolic DNA accumulates in tumor cells and activates the cGAS/STING pathway. Recognition of cytosolic DNA by this intracellular DNA sensor pathway slows the formation of TRAMP-C2 prostate cancer cells consistent with findings in the B16 melanoma model 13 . The anti-cancer effects of cytosolic DNA may in part be mediated by STING-dependent signals that induce tumor infiltration of immune cells. Interestingly, very few CD8 + T cells were present in TRAMP-C2 tumor tissue suggesting that endogenous STING activity in TRAMP-C2 tumors is not sufficient to attract T cells possibly due to an immunosuppressive microenvironment in TRAMP-C2 tumors, which prevents infiltration of T cells 36,37 .
The anti-cancer effects of STING have led to the evaluation of therapeutic potential of STING agonists, which were effective in several mouse tumor models 7 . Encouragingly, injection of cGAMP was able to enhance T cell infiltration of TRAMP-C2 tumors. While STING activation in antigen-presenting cells (APCs) plays an important role in the anti-cancer effects of STING agonists 38 , STING activation by cGAMP in TRAMP-C2 cells was critical for CD8 + T-cell infiltration into the tumor microenvironment. Analogous to stimulation of  www.nature.com/scientificreports/ STING in APCs, cGAMP induced the expression of type I IFNs, pro-inflammatory cytokines and chemokines in TRAMP-C2 cells. The T cell attracting factors released by TRAMP-C2 in response to cGAMP are currently not known, but possibly include chemokines such as CCL5 and CXCL10. Hence, our data indicate that the ability of STING agonists to convert an "excluded infiltrate phenotype" into an "inflamed" tumor depends on STING expression in tumor cells and APCs 39 . Immune cell infiltration of human tumors has been associated with responses to checkpoint inhibitors and favorable clinical outcome in many different tumor types including prostate cancer 40 . High densities of memory CD8 + T cells correlate with longer disease-free survival and overall survival. In agreement, infiltration of CD8 + T cell and other immune cell in TRAMP-C2 tumors negatively correlated with tumor growth consistent with the conclusion that STING activity in tumor cells is paramount to mount effective anti-cancer immune responses. We previously found that repair of genomic DNA contributes to the accumulation of cytosolic DNA in prostate cancer cells 12 . Here we show that cytosolic DNA present in many tumor cells, but not normal healthy cells, activates the cGAS/STING pathway. In accordance, cGAS deficiency or overexpression of RNASEH1 or the nuclease TREX1 reduced the expression of type I IFN and other IRF3-target genes. However, transfection of exogenous DNA or cGAMP stimulation failed to upregulate the production of type I IFNs in many of tested tumors suggesting that cGAS-STING pathway is suppressed in these tumor cells in line with previous reports 18,19 . Here we found that unresponsive cells were able to respond to stimuli that activate pathways downstream of STING indicating that the suppression acts at the level of STING and potentially also cGAS. The suppression was not a consequence of cGAS or STING mutations or significant differences in the expression of cGAS or STING. It is also unlikely that cytosolic DNA or constitutive cGAS signals were maintaining the suppression as lowering of cytosolic DNA levels, cGAS deficiency or blocking of type I IFNs (data not shown) failed to restore cGAMP responsiveness of STING. In the prostate cancer cells DU145, blocking of autocrine IL-6 restored the ability of cells to induce type IFN expression in response to cGAMP. In accordance, addition of exogenous IL-6 suppressed cGAMP-induced type I IFN expression and anti-tumor responses. It is possible that other IL-6-like signals that activate the JAK2/STAT3 pathway are mediating some of the suppression in other cancer cells as treatment of cGAMP-unresponsive cancer cells with the JAK2/STAT3 inhibitor WP1066 upregulated type I IFN expression in the tested cancer cells.
In summary, we found that many human cancer cell lines fail to respond to STING agonists, which can be rescued by JAK2/STAT3 inhibitors in some cases. Recent reports also found evidence for STING silencing in human cancer samples 19 . Future studies will be needed to investigate the role of the JAK2/STAT3 pathway in silencing STING function in human cancers. Such studies will be of particular importance as STING agonists and STAT3 inhibitors are currently being developed as new anti-cancer therapeutics. IL-6 antagonists such as Tocilizumab and Sarilumab, are approved for rheumatoid arthritis. Our data show that patient stratification based on STING activity in tumors should be considered when recruiting patients for clinical trials. Furthermore, combination of STING agonists with IL-6 antagonists or STAT3 inhibitors may increase the efficacy of STING agonists. Overall, a better understanding of the molecular mechanisms contributing to the unresponsiveness of tumor cells to STING agonists may open new approaches for cancer treatment.

Methods
Study in this article was approved by the Ethics Committee Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore (NUS) in accordance with the guideline R14-0204. The study on animals was carried out in compliance with the ARRIVE guidelines.
Metamorph was used for the quantification of mean fluorescence intensities (MFI) and determination of percentage of positive immune cells (Metamorph NX, Molecular Devices, USA).
Western blotting. Immunoblots were performed as previously described 26 . Real-time PCR. RNA extraction and reverse transcription were previously described 12 . The PCR conditions were used according to TaqMan Gene Expression Assays (Thermo Fisher). Triplicates were performed for the PCR reaction using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Singapore). Finally, gene-specific values were normalized to the GAPDH or HPRT levels. Samples prepared without RNA served as negative controls.
ELISA. For cGAMP measurements, 1 × 10 6 TRAMP-C2 and A549 cells were grown in a culture dish and treated with 4 μg/ml ISD or water as control for 18 h. Cells were collected and lysed using M-PER mammalian protein extraction reagent. After lysed cells were spun down, supernatant was collected and cGAMP levels were measured using the 2′3′-cGAMP ELISA kit according to manufacturer's instructions (Cayman Chemical, USA). Statistical analysis. Data distribution was determined by Shapiro-Wilk normality test. P values were determined using Student's t-tests, ANOVA or Pearson correlation coefficient analysis as appropriate (Prism 7d, Graphpad). *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.