cGAS-STING Signaling Pathway Research Articles

Emodin Attenuates Acetaminophen-Induced Hepatotoxicity via the cGAS-STING Pathway.

Emodin is a natural bioactive compound from traditional Chinese herbs that exerts anti-inflammatory, antioxidant, anticancer, hepatoprotective, and neuroprotective effects. However, the protective effects of emodin in acetaminophen (APAP)-induced hepatotoxicity are not clear. The present study examined the effects of emodin on APAP-induced hepatotoxicity and investigated the potential molecular mechanisms. C57BL/6 mice were pretreated with emodin (15 and 30mg/kg) for 5 consecutive days and then given APAP (300mg/kg) to establish an APAP-induced liver injury model. Mice were sacrificed to detect the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB) and the liver tissue levels of glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD). Histological assessment, Western blotting, and ELISA were performed. Emodin pretreatment significantly reduced the levels of ALT, AST, and ALP; increased the levels of ALB; alleviated hepatocellular damage and apoptosis; attenuated the exhaustion of GSH and SOD and the accumulation of MDA; and increased the expression of antioxidative enzymes, including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H quinone dehydrogenase 1 (NQO1). Emodin also inhibited the expression of NLRP3 and reduced the levels of pro-inflammatory factors, including interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α). Emodin inhibited interferon (IFN)-α, cyclic GMP-AMP synthase (cGAS), and its downstream signaling effector stimulator of interferon genes (STING) expression to protect the liver against APAP-induced inflammatory responses and apoptosis. These results suggest that emodin protected hepatocytes from APAP-induced liver injury via the upregulation of the Nrf2-mediated antioxidative stress pathway, the inhibition of the NLRP3 inflammasome, and the downregulation of the cGAS-STING signaling pathway.

Activation of STING Pathway Contributed to Cisplatin-Induced Cardiac Dysfunction via Promoting the Activation of TNF-α-AP-1 Signal Pathway.

Cardiovascular complications are a well-documented limitation of conventional cancer chemotherapy. As a notable side effect of cisplatin, cardiotoxicity represents a major obstacle to the treatment of cancer. Recently, it has been reported that cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) signaling pathway was associated with the occurrence and development of cardiovascular diseases. However, the effect of STING on cardiac damage caused by cisplatin remains unclear. In this study, cisplatin was shown to activate the cGAS-STING signaling pathway, and deficiency of STING attenuated cisplatin-induced cardiotoxicity in vivo and in vitro. Mechanistically, the STING-TNF-α-AP-1 axis contributed to cisplatin-induced cardiotoxicity by triggering cardiomyocyte apoptosis. In conclusion, our results indicated that STING might be a critical regulator of cisplatin-induced cardiotoxicity and be considered as a potential therapeutic target for preventing the progression of chemotherapy-associated cardiovascular complications.

Intrinsic STING expression inversely associates with survival in ovarian cancer patients

<h3>Objectives:</h3> The DNA sensing cGAS-STING signaling pathway plays a dual role of anti-tumor immunity and tumorigenesis. Chemoradiotherapy and PARPi induce DNA damage leading to activation of the cGAS-STING pathway, robust anti-tumor responses and improved clinical outcomes. Chronic activation of STING signaling creates an immunosuppressive TME and promotes metastases. Studies show that ovarian cancer cells have defective STING signaling. Current therapeutic strategies are focused on harnessing the beneficial effects of PARPi, IC blockade and STING agonists. However, the role for intrinsic ovarian tumor cGAS-STING levels in treatment responses and patient survival is unknown. The goal of this study is to determine the effect of tumor-specific cGAS-STING expression on survival in ovarian cancer patients. <h3>Methods:</h3> Using the Roswell Park Cancer Registry (RPCR), TMAs were generated from 488 patients treated for ovarian, fallopian tube or primary peritoneal cancer from 1991-2012. TMAs were stained for cGAS, STING and CD8 by IHC. cGAS and STING were scored based on the staining intensity (0-3) and the percent of positive cells (0-3) with a maximum combined score of 6 (0=no expression, 6=strong staining in >50% of cells). Additionally, the OS, PFS and cGAS-STING expression in 558 ovarian cancer patients from TCGA were analyzed. Clinico-pathologic data (age, demographics, histopathology, stage, tumor grade, treatment response, recurrence and survival) were retrieved from the electronic health records. <h3>Results:</h3> TCGA ovarian cancer patients with tumors expressing cGAShighSTINGhigh compared to cGASlowSTINGlow had OS of 38.4 months vs. 31.18 months (p=0.05). There was a significant positive correlation between gene and protein expression for cGAS (r=0.39, p=0.01) and STING (r=0.62, p<0.0001). Of the 488 patients in the RPCR, 95.3% were Caucasian with a median age at diagnosis of 63 years (range 20-93) and a median follow up of 44 months (range 0-279). The majority were diagnosed at an advanced surgical stage (68% and 14% at stage III and IV respectively), serous histology in 72% and high grade in 90.6% of cases. 72% received optimal and 27% had suboptimal debulking surgery. All patients received adjuvant platinum and taxane chemotherapy and the initial recurrence rate was 52%. In addition, 42.2% were platinum sensitive, 30% were refractory and 18% were platinum resistant. During the first 60 months of follow up, there was a trend toward improved OS and PFS in patients with cGASlowSTINGlow compared to cGAShighSTINGhigh tumors. Independently, cGAS did not associate with OS or PFS. Interestingly, STING high tumors correlated positively with CD8+ TILs (r=0.05, p=0.0002) but associated with decreased PFS (p=0.016). <h3>Conclusions:</h3> TCGA dataset revealed cGAShighSTINGhigh expressing tumors correlated with improved OS. By contrast, high grade ovarian tumors with intrinsically high levels of STING correlate with increased CD8+ TILs and decreased PFS.

Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.

Targeting STING attenuates ROS induced intervertebral disc degeneration

DNA damage induced by ROS is considered one of the main causes of nucleus pulposus (NP) cells degeneration during the progression of intervertebral disc degeneration (IVDD). cGAS-STING pathway acts as DNA-sensing mechanism for monitoring DNA damage. Recent studies have proved that cGAS-STING contributes to the development of various diseases by inducing inflammation, senescence, and apoptosis. This work explored the role of STING, the main effector of cGAS-STING signaling pathway, in NP degeneration. Immunohistochemistry was conducted to measure STING protein levels in the nucleus pulposus tissues from human and puncture-induced IVDD rat models. TBHP induces degeneration of nucleus pulposus cells in vitro. For in vivo experiments, lv-NC or lv-STING were injected into the central intervertebral disc space. The degeneration level of IVDD was assessed by MRI, X-ray, HE, and Safranin O staining. We found that the expression of STING was upregulated in human and rat degenerated NP tissue as well as in TBHP-treated NP cells. Overexpression of STING promoted the degradation of extracellular matrix; it also promoted apoptosis and senescence of TBHP-treated and untreated NP cells. Knock-down of STING significantly reversed these effects. Mechanistically, STING activated IRF3, whereas blockage of IRF3 attenuated STING-induced apoptosis, senescence and ECM degradation. In vivo experiments revealed that STING knock-down alleviated puncture-induced IVDD development. STING promotes IVDD progress via IRF3, while suppression of STING may be a promising treatment for IVDD.

Activation of the cGAS-STING signaling pathway in adenomyosis patients.

ObjectiveAdenomyosis is characterized by the presence of endometrium or endometrium‐like glands and stroma within the myometrium. In this study, we aimed to investigate whether the cGAS–STING pathway was activated and correlated with clinical outcomes in adenomyosis patients.Materials and MethodsTwenty patients diagnosed with adenomyosis and 10 patients diagnosed with cervical intraepithelial neoplasia grade 3 (CIN‐3) but no adenomyosis were enrolled in this study. Specimens were collected during surgery from August 2017 to December 2017 at Third Xiangya Hospital. The messenger RNA (mRNA) and protein levels of key cGAS–STING pathway factors in uterine tissue were detected by real‐time reverse‐transcription polymerase chain reaction and immunohistochemistry, respectively. The correlations of gene expression and clinical outcomes, including dysmenorrhea and uterine volume, were analyzed.ResultsThe cGAS, STING, TANK‐binding kinase 1 (TBK‐1), interferon‐α (IFN‐α), IFN‐β, and tumor necrosis factor‐α (TNF‐α) mRNA and protein levels in the ectopic endometrial tissue from adenomyosis patients were significantly higher compared with that from the controls in endometrium (p < .05). cGAS and STING gene expression were correlated with TBK‐1, IFN‐β, and TNF‐α expression (p < .05). Importantly, TBK‐1 and TNF‐α expression were correlated with the clinical outcome of dysmenorrhea (p < .05).ConclusionOur study reveals that the cGAS–STING pathway is activated in adenomyosis patients and its activation is subsequently correlated with clinical outcomes, which suggests that the cGAS–STING pathway may contribute to adenomyosis pathogenesis.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) contributes to the pathogenesis of coronavirus disease 2019 (COVID‐19). The strategy used by SARS‐CoV‐2 to evade antiviral immunity needs further investigation. Here, we reported that SARS‐CoV‐2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS‐CoV‐2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS‐CoV‐2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA‐sensing pathways of RIG‐I/MDA5‐MAVS signaling, including RIG‐I, MDA‐5, MAVS, TBK1, and IKKε, rather than IRF3‐5D, which is the active form of IRF3. SARS‐CoV‐2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA‐sensing pathway of TLR3‐TRIF signaling and the adaptor protein of the cytosolic DNA‐sensing pathway of cGAS–STING signaling, respectively. A mechanistic analysis revealed that the SARS‐CoV‐2 ORF9b protein interacted with RIG‐I, MDA‐5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS‐CoV‐2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS‐CoV‐2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS‐CoV‐2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID‐19.

Pulmonary inflammatory and fibrogenic response induced by graphitized multi-walled carbon nanotube involved in cGAS-STING signaling pathway

Graphitized multi-walled carbon nanotubes (GMWCNTs) are a new type of nanomaterial. Recently, their production and application in biological medicine have grown rapidly. However, GMWCNTs may cause adverse health effects, including the common occupational disease of pulmonary fibrosis. Pulmonary fibrosis is a serious progressive disease that often leads to lung failure, high mortality, and disability, and there is no effective therapy currently available. Therefore, identifying new biomarkers of the disease is important to better understand the disease mechanisms and explore new therapeutic strategies. In this study, 40 μg of GMWCNTs was used to treat mice in vivo by pharyngeal aspiration, and different genes were screened by transcriptome sequencing. Activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) signal pathway had an important effect on the development of pulmonary inflammation and fibrosis. GMWCNTs were then administered to the mice with a STING inhibitor (C-176). Inhibition of STING effectively decreased pulmonary inflammation and fibrosis in mice induced by GMWCNTs. Collectively, activation of the cGAS-STING signaling pathway is involved in GMWCNT-induced pulmonary inflammation and fibrosis in mice.

CGAS knockdown promotes microglial M2 polarization to alleviate neuroinflammation by inhibiting cGAS-STING signaling pathway in cerebral ischemic stroke

Inflammation plays a pivotal role in promoting the pathophysiology of ischemic stroke (IS). Microglia is the major immunocompetent cells involved in different neuropathologies. The activation of cyclic GMP-AMP synthase (cGAS) and its downstream signaling protein-stimulator of interferon genes (STING) is increasingly recognized as a crucial determinant of neuropathophysiology. However, the mechanisms underlying cGAS-STING signaling regulating inflammatory response during IS remains to be elucidated. In this study, HT22 cells was used to establish an oxygen-glucose deprivation (OGD) cell model in vitro, and then this cell culture supernatant containing OGD-induced DAMPs (OIDs) was employed to stimulate BV2 microglia. Furthermore, a middle cerebral artery occlusion (MCAO) mouse model was established. Cells and MCAO mice were treated with si-cGAS or si-NC lentivirus. The expression levels of STING, cGAS and p-IRF3 in BV2 cells or MCAO mouse brain; the microglial M1/M2 polarization of BV2 microglia or isolated microglial cells from MCAO mouse brain; the contents of iNOS, TNF-α, TGF-β and IL-10 in the culture medium of BV2 cells or in murine brain homogenates, were all detected. In addition, the severity of cerebral infarction with or without the knockdown of cGAS in a MCAO mouse model was also determined by TTC staining. Results showed that OGD-induced DAMPs strongly activated cGAS-STING pathway and triggered microglia polarization in BV2 cells, reflecting as the accumulation of a plethora of pro-inflammatory factors in activated microglia. However, these effects could be inhibited by cGAS knockdown. In the MCAO mouse model, the inhibition of cGAS-STING pathway resulted from cGAS knockdown could effectively diminish cell apoptosis in mouse brain stimulated by MIDs (MCAO-induced DAMPs), reduced the area ratio of cerebral infarction and ultimately improved the injured nerve function during IS. Taken together, our elucidation of underlying mechanisms involved in the microglial inflammatory response, triggered by cGAS-STING signaling, highlights this pathway as a potential therapeutic target in IS.

Immune Regulation of the cGAS-STING Signaling Pathway in the Tumor Microenvironment and Its Clinical Application.

As a DNA receptor in the cytoplasm, cyclic GMP-AMP synthase (cGAS) contributes to the recognition of abnormal DNA in the cytoplasm and contributes to the stimulator of interferon genes (STING) signaling pathway. cGAS could mediate the expression of interferon-related genes, inflammatory-related factors, and downstream chemokines, thus initiating the immune response. The STING protein is a key effector downstream of the DNA receptor pathway. It is widely expressed across cell types such as immune cells, tumor cells, and stromal cells and plays a role in signal transduction for cytoplasmic DNA sensing and immunity. STING agonists, as novel agonists, are used in preclinical research and in the treatment of various tumors via clinical trials and have displayed attractive application prospects. Studying the cGAS-STING signaling pathway will deepen our understanding of tumor immunity and provide a basis for the research and development of antitumor drugs.

The Trinity of cGAS, TLR9, and ALRs Guardians of the Cellular Galaxy Against Host-Derived Self-DNA.

The immune system has evolved to protect the host from the pathogens and allergens surrounding their environment. The immune system develops in such a way to recognize self and non-self and develops self-tolerance against self-proteins, nucleic acids, and other larger molecules. However, the broken immunological self-tolerance leads to the development of autoimmune or autoinflammatory diseases. Pattern-recognition receptors (PRRs) are expressed by immunological cells on their cell membrane and in the cytosol. Different Toll-like receptors (TLRs), Nod-like receptors (NLRs) and absent in melanoma-2 (AIM-2)-like receptors (ALRs) forming inflammasomes in the cytosol, RIG (retinoic acid-inducible gene)-1-like receptors (RLRs), and C-type lectin receptors (CLRs) are some of the PRRs. The DNA-sensing receptor cyclic GMP–AMP synthase (cGAS) is another PRR present in the cytosol and the nucleus. The present review describes the role of ALRs (AIM2), TLR9, and cGAS in recognizing the host cell DNA as a potent damage/danger-associated molecular pattern (DAMP), which moves out to the cytosol from its housing organelles (nucleus and mitochondria). The introduction opens with the concept that the immune system has evolved to recognize pathogens, the idea of horror autotoxicus, and its failure due to the emergence of autoimmune diseases (ADs), and the discovery of PRRs revolutionizing immunology. The second section describes the cGAS-STING signaling pathway mediated cytosolic self-DNA recognition, its evolution, characteristics of self-DNAs activating it, and its role in different inflammatory conditions. The third section describes the role of TLR9 in recognizing self-DNA in the endolysosomes during infections depending on the self-DNA characteristics and various inflammatory diseases. The fourth section discusses about AIM2 (an ALR), which also binds cytosolic self-DNA (with 80–300 base pairs or bp) that inhibits cGAS-STING-dependent type 1 IFN generation but induces inflammation and pyroptosis during different inflammatory conditions. Hence, this trinity of PRRs has evolved to recognize self-DNA as a potential DAMP and comes into action to guard the cellular galaxy. However, their dysregulation proves dangerous to the host and leads to several inflammatory conditions, including sterile-inflammatory conditions autoinflammatory and ADs.

The cGAS-STING signaling pathway contributes to the inflammatory response and autophagy in Aspergillus fumigatus keratitis

Fungal keratitis is a serious corneal infection, which can lead to significant visual impairment and blindness. The cGAS-STING signaling pathway has emerged as a key player in innate immunity by sensing of invading pathogens. However, the role of the cGAS-STING pathway in Aspergillus fumigatus (A. fumigatus) keratitis is still unknown. In this study, we showed that the cGAS-STING signaling pathway was activated in human corneal epithelial cells (HCECs) and in mouse corneas infected with A. fumigatus. Knockdown of cGAS reduced A. fumigatus-induced production of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IFN-β. However, reconstruction of cGAS activity restored the inflammatory response in HCECs infected with A. fumigatus. A specific cGAS inhibitor, RU.521, could also significantly inhibit A. fumigatus-induced inflammatory cytokine expression. In addition, we found that cGAS was indispensable for the autophagy flux evoked by A. fumigatus infection. Moreover, inhibition of cGAS using siRNA or RU.521 alleviated the severity of A. fumigatus keratitis in the mouse cornea. Therefore, the cGAS-STING signaling pathway contributes to the progression of A. fumigatus keratitis and targeting this pathway may provide therapeutic potential.

Role of cGAS-STING signaling pathway in colon cancer

Role of cGAS-STING signaling pathway in colon cancer

Activation of the cGAS-STING Signaling Pathway Is Associated with Glomerular Diseases

Activation of the cGAS-STING Signaling Pathway Is Associated with Glomerular Diseases

Crosstalk between cGAS-STING signaling and cell death.

Cytosolic nucleic acid sensors have a critical role in detecting endogenous nucleic acids to initiate innate immune responses during microbial infections and/or cell death. Several seminal studies over the past decade have delineated the conserved mechanism of cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) andthe downstream signaling adapter stimulator of interferon genes (STING) in mediating innate immune signaling pathways as a host defense mechanism. Besides the predominant role in microbial infections and inflammatory diseases, there is an increased attention on alternative functional responses of cGAS-STING-mediated signaling. Here we review the complexity of interactions between the cGAS-STING signaling and cell death pathways. A better understanding of molecular mechanisms of this interplay is important with regard to the development of new therapeutics targeting cGAS-STING signaling in cancer, infectious, and chronic inflammatory diseases.

Abstract 3348: Exogenous DNA upregulates DUOX2 expression in pancreatic cancer via activation of the cGAS-STING signaling pathway

Abstract Dual oxidase 2 (DUOX2) is a transmembrane protein that produces hydrogen peroxide in the extracellular environment, mediates innate immunity at mucosal surfaces, contributes to chronic inflammation-related tissue injury and angiogenesis, and can promote the initiation of epithelial-derived cancers. In pancreatic adenocarcinomas (PDAC), the composition of the tumor microbiome significantly affects patient outcomes. However, it remains unclear how microbes in the tumor microbiome affect PDAC on a cellular level. In recent years, the cGAS-STING signaling pathway has been identified as a crucial mediator of anti-viral defense by recognizing the presence of DNA in the cytosol and subsequently producing Type I interferons (IFNs) as well as a host of other anti-viral genes. Using Western blot and qPCR, we show that the introduction of exogenous DNA into the cytosol significantly upregulates DUOX2 expression in human PDAC cells after activating cGAS-STING signaling. Expression of DUOX2 occurs most strongly 2 days post-transfection of DNA, while cGAS-STING signaling is activated within hours. Treatment of BxPC-3 cells with exogenous cyclic GMP-AMP (cGAMP) for 24 hrs increases DUOX2 expression in a concentration-dependent manner, suggesting the necessity of cGAS activity for DUOX2 induction. Furthermore, Western analysis of phospho-NF-κB p65 in BxPC-3 cells demonstrates activation of NF-κB that mirrors the time-dependent induction of DUOX2. In CFPAC-1 cells, another PDAC line, Jak-Stat signaling is activated along with the components of cGAS-STING. These data suggest that DNA-induced DUOX2 upregulation in PDAC cells may be mediated in a cell context-specific fashion, beyond the canonical components of the cGAS-STING pathway. Upregulation of DUOX2 by exogenous DNA is not observed in colon cancer cell lines. In summary, these data suggest that extracellular DNA could contribute to a DUOX2-induced, H2O2-mediated pro-inflammatory milieu that specifically contributes to the pathogenesis of PDAC, in part through activation of the cGAS-STING pathway. Citation Format: Stephen L. Wang, Yongzhong Wu, Smitha Antony, Agnes Juhasz, Jennifer Meitzler, Goujian Jiang, Iris Dahan, Jiamo Lu, Krishnendu Roy, James Doroshow. Exogenous DNA upregulates DUOX2 expression in pancreatic cancer via activation of the cGAS-STING signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3348.

Irradiation induces cancer lung metastasis through activation of the cGAS\u2013STING\u2013CCL5 pathway in mesenchymal stromal cells

Emerging evidence indicates that mesenchymal stromal cells (MSCs) have an important role in cancer metastasis. Although tumor microenvironment, which includes MSCs and immune cells, can be altered by ionizing radiation (IR), whether irradiation can promote metastasis through MSCs remains unclear. Using the lung colonization model of transplanted 4T1 breast cancer cells, we found an increased lung metastasis in mice exposed to ionizing radiation, even when the thorax was shielded during whole-body irradiation. This radiation-induced lung metastasis can be replicated using irradiated MSCs. cGAS–STING signaling pathway was found to be activated in MSCs, accompanied by upregulation of type I interferon-related genes, including chemokine CCL5. Disruption of cGAS–STING signaling in MSCs abolished their pro-metastatic effect. Deletion of CCL5 in MSCs also abrogated the pro-metastatic effect endowed by IR. Furthermore, we showed that the lung pro-metastatic effect of irradiated MSCs required the presence of macrophages. Our results indicate that radiation-induced alterations in distant mesenchymal stromal cells facilitate cancer metastasis.

Potential Role of cGAS‐STING Pathway in the Induction of Diabetic Kidney Disease

The role of the cyclic GMP–AMP synthase (cGAS) / Stimulator of interferon genes (STING) / interferon‐β (INFβ) axis in innate immunity has been explored during the last decade. Recently, it has been revealed that the activity of this pathway could be a double‐edged sword; besides protection against invading organisms, its overactivity has been associated with pathological conditions e.g., Aicardi‐Goutieres syndrome (AGS) syndrome. It was also recently reported that activation of the STING‐cGAS pathway had been associated with renal inflammation and fibrosis. However, the impact of the cGAS‐STING pathway on diabetic kidney disease (DKD) has not been studied before. Therefore, the goal of this study was to assess the contribution of the cGAS‐STING‐INFβ pathway towards the development of DKD. We used two established DKD models that develop advanced diabetic kidney lesions ‐ eNOSdb/db mice and type 2 diabetic nephropathy (T2DN) rat. Western blot analysis was used to estimate the expression and/or activity level of different molecules involved in the cGAS‐STING pathway in the kidney tissue from 28 wks old eNOSdb/db mice and corresponding controls. RT‐qPCR was also used to test the transcriptional level of interferon genes, which are the downstream target of this pathway. Comparison between male and female in T2DN rats were used to define changes in the cGAS‐STING signaling pathway during the progression of DKD. As was previously reported, female rats exhibit significant attenuation in progression of diabetes and renal damage in this model. Western blot analysis showed increased expression and/or activity of specific proteins involved in the cGAS‐STING pathway. RT‐qPCR confirmed the Western blot results and showed a significant elevation of the checked INFβ genes. Overall, the activity of cGAS‐STING signaling was higher in males, which is consistent with the protective phenotype of T2DN female rats. In addition, immunohistochemistry (IHC) staining for STING molecule in described rodent models and human renal samples was performed. The results indicate the significant elevation of STING in the renal tissue of both rodents and humans with type 2 diabetes. Therefore, our data revealed that cGAS‐STING pathway is markedly activated in diabetes, and we speculate that inhibiting this pathway would ameliorate the deleterious outcome of the pathologic processes in DKD.

Inhibition of cGAS-Mediated Interferon Response Facilitates Transgene Expression.

SummaryDNA transfection is often the bottleneck of research and gene therapy practices. To explore the mechanism regulating transgene expression, we investigated the role of the cGAS-STING signaling pathway, which induces type-I interferons in response to DNA. We confirmed that deletion of cGAS enhances transgene expression at the protein level by ~2- to 3-fold. This enhancement is inversely correlated with the expression of interferons and interferon stimulated genes (ISGs), which suppress expression of transfected genes at the mRNA level. Mechanistically, DNA transfection activates the cGAS-STING pathway and induces the expression of the OAS family proteins, leading to the activation of RNaseL and degradation of mRNA derived from transgenes. Administration of chemical inhibitors that block cGAS-mediated signaling cascades improves the expression of transgenes by ~1.5- to 3-fold in multiple cell lines and primary cells, including T cells. These data suggest that targeting the cGAS-STING pathway can improve transgene expression, and this strategy may be applied to gene therapy.

Relative Contributions of the cGAS-STING and TLR3 Signaling Pathways to Attenuation of Herpes Simplex Virus 1 Replication

The innate immune response is crucial for defense against viral infections. Cells recognize virus infection through pattern recognition receptors and induce type I interferons as well as proinflammatory cytokines to orchestrate an innate immune response. Herpes simplex virus 1 (HSV-1) triggers both the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and Toll-like receptor 3 (TLR3) pathways. It is well known that TLR3 uses the adaptor protein Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing beta interferon (TRIF) for signaling, but we recently reported that STING signaling also requires TRIF. Because STING directly binds to TRIF, we identified the STING-interacting domain of TRIF and generated STING-noninteracting mutants of human and mouse TRIFs. The mutant TRIFs were unable to support STING signaling, although they were fully functional in the TLR3 pathway. These mutants were used to assess the relative contributions of the TLR3 and STING pathways to the attenuation of HSV-1 replication in mouse and human cell lines. For this purpose, the mouse L929 and NB41A3 cell lines and the human HT1080 and HeLa-M cell lines, in which both the TLR3 and the STING pathways are operational, were used. The TRIF gene was disrupted in these lines by CRISPR/Cas9, before reconstituting them with mutant and wild-type TRIF expression vectors. Infection of the reconstituted cells with HSV-1 revealed that both the cGAS-STING and the TLR3 signaling pathways are required for the attenuation of virus replication, but their relative contributions in attenuating HSV-1 replication were found to be different in mouse versus human cell lines. Thus, our study suggests that the relative contributions of the cGAS-STING and the TLR3 pathways in the attenuation of viral infection may be species specific.IMPORTANCE The magnitude of fatal infections caused by all different viruses in human and animal populations justifies a better understanding of the host innate immune response process that attenuates virus replication. In particular, the relative contributions of different signaling pathways which are responsible for the generation of the innate immune response are still largely unknown. In this study, we used STING-noninteracting TRIF mutants to decipher the relative contributions of the TLR3 and cGAS-STING signaling pathways to the attenuation of HSV-1 infection. We show that the relative contributions of the two pathways to the attenuation of viral infection are different in mouse versus human cell lines. Together, our results provide new insights into the relative contributions of two different signaling pathways in the attenuation of viral infection and may lead to the development of new antiviral strategies aimed at blocking viral infection at very early stages.