Human Cancer Cells Sense Cytosolic Nucleic Acids Through the RIG-I–MAVS Pathway and cGAS–STING Pathway

Pattern recognition receptors (PRRs) are germline-encoded host sensors of the innate immune system. Some human cancer cells have been reported to express PRRs. However, nucleic acid sensors in human cancers have not been studied in detail. Therefore, we systematically analyzed the expression, molecular cascade, and functions of TLR3, RIG-I, MDA5, LGP2, cGAS, and STING in human cancer cells. TLR3, TRIF, RIG-I, MDA5, LGP2, and MAVS were expressed in 22 cell lines. The majority of cell lines responded to only RIG-I ligands 5′-ppp-dsRNA, Poly(I:C)-HMW, Poly(I:C)-LMW, and/or Poly(dA:dT), as revealed by IRF3 phosphorylation and IFN-β secretion. IFN-β secretion was inhibited by RIG-I and MAVS knockdown. cGAS and STING were co-expressed in 10 of 22 cell lines, but IFN-β secretion was not induced by STING ligands ISD, HSV60, VACV70, Poly(dG:dC), and 3′3′-cGAMP in cGAS and STING intact cell lines. Further experiments revealed that the cGAS–STING pathway was activated, as revealed by TBK1 and IRF3 phosphorylation and IFN-β and ISG mRNA expression. These results suggest that human epithelial cancer cells respond to cytosolic RNA through the RIG-I–MAVS pathway but only sense cytosolic DNA through the cGAS–STING pathway. These findings are relevant for cancer immunotherapy approaches based on targeting nucleic acid receptors.


INTRODUCTION
Pattern recognition receptors (PRRs) are germline-encoded host sensors of the innate immune system that detect pathogen-associated molecular patterns and self-tissue damage-associated molecular patterns. A major type of PRRs is dedicated to sensing nucleic acids, including DNA and RNA. There are two classes of nucleic acid sensors: those that sense nucleic acids in endosomes, such as TLR3, TLR7, TLR8, and TLR9, and those that sense nucleic acids in the cytosol, such as RIG-I-like receptors (RLRs) and cGAS/STING (Hoffmann and Akira, 2013;Cui et al., 2015;Sparrer and Gack, 2015).
Extensive studies on nucleic acid sensors have focused on their biology and molecular pathways in myeloid cells and antigen-presenting cells (Akira et al., 2006;Gilliet et al., 2008). Upon infection, PRRs recognize nucleic acids and then recruit adaptors and trigger the phosphorylation of IRF3/7, leading to the production of IFN-β and expression of interferonstimulated genes (Goubau et al., 2013). TLR3, TLR7 and TLR8, and TLR9 recognize dsRNA, ssRNA, and unmethylated CpG DNA, respectively (Kawai and Akira, 2011). Following nucleic acid binding, TLRs undergo conformational changes and recruit adaptor proteins TRIF for TLR3 and MyD88 for TLR7/8/9, leading to IRF3/7 phosphorylation and IFN-β secretion (Akira et al., 2006). RLRs comprise three members: RIG-I (DDX58), MDA5 (IFIH1), and laboratory of genetics and physiology 2 (LGP2; DHX58) (Yoneyama et al., 2015). These RLRs share a DExD/H-box RNA helicase domain and a C-terminal domain, both of which are required for dsRNA detection, while RIG-I and MDA5, but not LGP2, have an N-terminal caspase recruitment domain for interaction with MAVS and downstream signaling (Kawai et al., 2005;Meylan et al., 2005;Seth et al., 2005;Xu et al., 2005;Yoneyama et al., 2005). DNA can also be transcribed into 5 -triphosphate-containing small dsRNA by RNA polymerase III for RLR binding to initiate IFN-β secretion (Ablasser et al., 2009;Chiu et al., 2009). cGAS is a cytosolic DNA sensor that catalyzes the synthesis of cGAMP (Li et al., 2013). cGAMP binds to STING and mediates the activation of TBK1 and IRF3 to initiate IFN-β secretion (Wu et al., 2013). These findings have translated into the development of new adjuvants for the next generation of vaccines and new immunotherapies for cancer that can reverse anti-PD-1/anti-PD-L1 resistance by converting "cold tumors" into "hot tumors" (Fu et al., 2015).
Certain human cancer cells have been reported to express PRRs and respond to cytosolic nucleic acids to produce type I IFNs. TLR3 is expressed in the intestinal epithelium and hepatocytes and senses extracellular Poly(I:C) Broquet et al., 2011). It is also expressed in human lung epithelial cells to recognize influenza A virus and respiratory syncytial virus (Groskreutz et al., 2006;Le Goffic et al., 2007). Meanwhile, RIG-I and MDA5 are expressed in hepatocytes, intestinal epithelial cells, lung epithelial cells, primary human astrocytes, and glioblastoma, and they respond to cytosolic Poly(I:C) and viruses Hirata et al., 2007;Wang et al., 2009;Furr et al., 2010;Broquet et al., 2011;Glas et al., 2013;Sugimoto et al., 2014). Upon delivery of Poly(I:C) or viruses into the cytosol, RIG-I or MDA5 expression is significantly increased to initiate the innate immune response. Both cGAS and STING have been found to be expressed in 54.4% and 45.5% of human melanoma and colorectal cancer cell lines, respectively; and cells defective for the cGAS-STING pathway are sensitive to oncolytic DNA virus (Xia et al., 2016a,b). However, there have been no systematic studies on all known nucleic acid sensors in human cancers.
In this study, we systematically analyzed the expression, molecular cascade, and function of the endosomal RNA sensor TLR3; cytosolic RNA sensors RIG-I, MDA5, and LGP2; and cytosolic DNA sensors cGAS and STING in 22 human epithelial cancer cell lines to obtain useful insights into targeting nucleic acid receptors for cancer immunotherapy.
ELISA IFN-β secretion was assessed using an ELISA kit (PBL Interferon Source) according to the manufacturer's instructions.
GAPDH was used as a reference gene to normalize the amounts of cDNA. The relative expression was calculated using the 2 (− Ct) method.

Confocal Microscopy
Cells were cultured and transfected with rhodamine-labeled Poly(dA:dT) for 3 h. Images were captured with an Olympus confocal microscope at the Institute of Immunology, the First Hospital of Jilin University. Image deconvolution was carried out with ImageJ (National Institutes of Health).

RNA Sequencing
Total RNA was extracted using the EasyPure RNA kit (TransGen, Beijing, China) according to the manufacturer's instructions. Approximately 1,000 ng of RNA was used for library preparation and subsequent sequencing on an Illumina HiSeq 4000 platform. Reads were aligned to the reference genome (GRCh38.p13) by TopHat2 and HISAT2 software. Differentially expressed genes were analyzed by DEGseq software, and heatmap was generated by GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States).

Statistical Analysis
Statistical differences were determined by using the two-tailed Student's t-test with GraphPad Prism 7 (GraphPad Software, San Diego, CA, United States); p-values less than 0.05 were considered statistically significant.

Expression of Major Molecules in the TLR3-TRIF, RLR-MAVS, and cGAS-STING Pathways in Human Epithelial Cancer Cell Lines
The TLR3-TRIF, RLR-MAVS, and cGAS-STING pathways are the most important signaling pathways in immune cells in the defense against invading pathogens. To investigate their roles in human epithelial cancer cells, we collected 22 human epithelial cancer cell lines derived from nine cancer types, including breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioma, human hepatocellular cancer, lung cancer, human ovarian cancer, and human pancreatic cancer (Supplementary Table 1) and detected the major molecules in the TLR3-TRIF, RLR-MAVS, and cGAS-STING pathways in these cells, including TLR3, TRIF, RIG-I, MDA5, LGP2, MAVS, cGAS, and STING. The results showed that TLR3, TRIF, RIG-I, MDA5, LGP2, and MAVS, which were reported to be expressed in the A549 cell line, were expressed in all these cells lines at both the mRNA and protein levels (Figures 1A,B,D), indicating that the TLR-TRIF and RLR-MAVS pathways are constitutively intact in all cancer cells. Interestingly, cGAS was expressed in 13 of the 22 cell lines, and STING was expressed in 16 of the 22. cGAS and STING were co-expressed in 10 of the 22 (45.5%) cell lines at both the mRNA and protein levels (Figures 1C,D). cGAS and STING expression in HeLa cells served as the positive control.  Table 2. To ascertain the capability of these nucleic acids in the cytosol to induce IFN-β, we transfected the cell lines with rhodamine-labeled Poly(dA:dT) and photographed them using a fluorescence confocal microscope. We found that rhodamine-labeled Poly(dA:dT) could be delivered into the cytosol via Lipofectamine 2,000 in all the cell lines (Supplementary Figures 1A-V). However, there was no rhodamine-labeled Poly(dA:dT) in the cytosol of PANC-1 cells when they were cocultured with rhodamine-labeled Poly(dA:dT) (Supplementary Figure 1W).
Thus, the RLR-MAVS pathway is functional in most human epithelial cancer cells, but the cGAS-STING pathway is inactive.
Human Epithelial Cancer Cells Sense Cytosolic RNA via RIG-I, but Not TLR3 or MDA5 To demonstrate the mechanisms through which cancer cells sense cytosolic Poly(I:C) and Poly(dA:dT), we first examined the activation and expression of PKR, TLR3, RIG-I, MDA5, LGP2, and DHX29 stimulated by Poly(I:C) and Poly(dA:dT) in PANC-1 cells. We found that these receptors were constitutively expressed in PANC-1 cells and that the phosphorylation of PKR and the expression of RIG-I, MDA5, and LGP2 were significantly increased upon Poly(I:C) and Poly(dA:dT) transfection in PANC-1 cells ( Figure 3A).
It is reported that Poly(dA:dT) is transcribed into dsRNA by RNA polymerase III, which is then recognized by RIG-I (Ablasser et al., 2009;Chiu et al., 2009). To investigate the role of RNA polymerase III in the RIG-I signaling pathway in human cancer cells, PANC-1 and A549 cells were treated with the RNA polymerase III inhibitor ML-60218, and the expression of IFN-β induced by Poly(dA:dT) was determined. ML-60218 treatment had no effect on the Poly(dA:dT)-induced IFN-β expression in human cancer cells (Supplementary  Figures 3A,B) but significantly inhibited this expression in the 293T cells (Supplementary Figure 3C), indicating that RNA polymerase III is not involved in the RIG-I signaling pathway in human cancer cells.
Therefore, RIG-I, rather than TLR3 or MDA5, is required for sensing cytosolic nucleic acids in human cancer cells.

Cytosolic Poly(I:C) and Poly(dA:dT) Activate the MAVS-IRF3 Pathway in Human Epithelial Cancer Cells
Invading pathogens are recognized by PRRs, after which adaptors are recruited for the activation of downstream signaling pathways, leading to the secretion of type I IFN. To determine which adaptor is necessary for RIG-I to activate downstream signaling pathways in cancer cells, we studied the expression of TRIF, MAVS, and STING in PANC-1 cells. TLR3, MAVS, and STING were constitutively expressed and showed no difference after transfection with Poly(I:C)-HMW, Poly(I:C)-LMW, or Poly(dA:dT) ( Figure 4A). Then, these molecules were knocked down by siRNA, and the expression of these molecules decreased significantly at the mRNA level ( Figure 4B). Upon transfection of PANC-1 cells with cytosolic Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT), IFN-β secretion was markedly decreased after the knockdown of MAVS, but not of TRIF or STING (Figures 4C-E). Similar results were obtained in the human colorectal cancer cell line HCT-8 (Figures 4F-H).
Furthermore, we detected the activation of a downstream pathway involved in IFN-β secretion. We found that IRF-3 was markedly phosphorylated upon transfection of PANC-1 cells with Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT) in a time-dependent manner ( Figure 4I).
Taken together, these results indicate that human epithelial cancer cells respond to cytosolic nucleic acids via the RIG-I-MAVS-IRF3 signaling pathway.
as well as in PANC-1 cells stimulated with ISD and HSV60 ( Figure 5A). Interestingly, STING, TBK1, and IRF3 were not phosphorylated after transfection with 3 3 -cGAMP in these cancer cell lines (Figure 5A).
Upon stimulation, IRF3 is phosphorylated and translocated to the nucleus in immune cells. Thus, we transfected SiHa cells with ISD, HSV60, VACV70, Poly(dG:dC), and Poly(dA:dT) and confirmed whether IRF3 was translocated to the nucleus in these cells. We found that IRF3 was significantly translocated to the nucleus after transfection with ISD, HSV60, VACV70, and Poly(dG:dC), although the translocation was weak compared with that with Poly(dA:dT) ( Figure 5B). The same results were obtained in MDA-MB-231, HeLa, HCT-8, and PANC-1 cell lines ( Figure 5C).
Next, we transfected SiHa and PANC-1 cells with ISD, Poly(dG:dC), and Poly(dA:dT) and performed RNA sequencing. The results showed that the expression profiles in SiHa cells stimulated with ISD, Poly(dG:dC), and Poly(dA:dT) as well as in PANC-1 cells stimulated with Poly(dG:dC) and Poly(dA:dT) were the same as those in THP-1-derived macrophages stimulated with ISD and Poly(dG:dC) ( Figure 5D). Expression profiles in PANC-1 cells stimulated with ISD differed from those in other cells because these cells did not respond to ISD, as demonstrated by the lack of TBK1 and IRF3 phosphorylation ( Figure 5D). Interestingly, STING knockdown in SiHa cells suppressed the expression of IFN-λ1, ISG15, and CCL5 induced by ISD, HSV60, VACV70, and Poly(dG:dC); however, the expression of IFN-β was not decreased (Figures 5E,F). These data suggest that cGAS and STING intact human epithelial cancer cells sense cytosolic DNA through the cGAS-STING signaling pathway to produce cytokines, chemokines, and ISGs. Furthermore, novel pathways that control the secretion of IFNβ may exist.

DISCUSSION
We report that TLR3, TRIF, RIG-I, MDA5, LGP2, and MAVS were expressed in/on all 22 human epithelial cancer cell lines studied, but only cytosolic 5 -ppp-dsRNA, Poly(I:C)-HMW, Poly(I:C)-LMW, and/or Poly(dA:dT) could induce IFN-β secretion via the RIG-I-MAVS-IRF3 signaling pathway in most of the cell lines. Although both cGAS and STING were coexpressed in 10 of the 22 cell lines, none of these cell lines secreted IFN-β induced by cytosolic ISD, HSV60, VACV70, Poly(dG:dC), and 3 3 -cGAMP, irrespective of the expression pattern of cGAS and STING. Further experiments revealed that the cGAS-STING pathway was activated, as revealed by TBK1 and IRF3 phosphorylation and IFN-β and ISGs mRNA expression induced by cytosolic ISD, HSV60, VACV70, and Poly(dG:dC) in cGAS and STING intact cell lines. Therefore, most human epithelial cancer cell lines respond to cytosolic RNA through the RIG-I-MAVS-IRF3 signaling pathway, while cytosolic DNA was sensed by the cGAS-STING signaling pathway in cGAS and STING intact human epithelial cancer cell lines (Supplementary Figure 4).
RIG-I-like receptors play important roles in recognizing cytosolic RNA molecules. They have been found to be expressed Culture supernatants were harvested, and IFN-β secretion was measured by ELISA. All data are shown as the mean ± SD of at least three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). (F) HCT-8 cells were transfected using Lipofectamine RNAiMAX with siNC or siRNA specific for MAVS. Forty-eight hours later, RNA was extracted to detect the knockdown (KD) level by qPCR. (G-I) Silenced HCT-8 cells were transfected with Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT) for 18 h. Culture supernatants were harvested, and IFN-β secretion was measured by ELISA. All data are shown as mean ± SD of at least three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001). (J) PANC-1 cells were transfected with Poly(I:C)-HMW, Poly(I:C)-LMW, or Poly(dA:dT) for 3, 6, or 9 h. Next, they were lysed and subjected to western blotting to detect the expression of β-actin, RIG-I, and total and phosphorylated IRF3. Data are representative of at least three independent experiments.
in several human cancer cells and tissues (hepatocytes, intestinal epithelial cells, lung epithelial cells, primary human astrocytes, and glioblastoma), and these recognize cytosolic Poly(I:C) and viruses Hirata et al., 2007;Wang et al., 2009;Furr et al., 2010;Broquet et al., 2011;Glas et al., 2013;Sugimoto et al., 2014). We found that RIG-I, MDA5, LGP2, and MAVS were expressed in all human epithelial cancer cell lines analyzed in the current study. Results of functional analyses revealed that most of cancer cells responded to cytosolic Poly(I:C). This finding was consistent with that of previous studies. Additionally, cytosolic 5 -ppp-dsRNA and Poly(dA:dT) were found to activate most of the cell lines, as indicated by IRF3 phosphorylation and IFN-β secretion. Knockdown experiments showed that IFN-β secretion was inhibited by RIG-I and MAVS knockdown, demonstrating that most of cancer cells responded to cytosolic RNA through the RIG-I-MAVS-IRF3 signaling pathway. Moreover, we found that several cell lines (MDA-MB-231, MKN-45, LN-18, U87MG, and U118MG) sensed none of these cytosolic nucleic acids, indicating that hitherto unknown sensors might exist in these cell lines.
Several experimental studies have reported the expression of cGAS and/or STING in human melanoma, colorectal cancer, Merkel cell carcinoma, and ovarian cancer (Xia et al., 2016a,b;de Queiroz et al., 2019;Liu et al., 2020). However, their expression in other human cancer cells remains unknown. In the current study, we found that cGAS and STING were each expressed in approximately 59 and 73% of the cell lines examined, respectively, and cGAS and STING were co-expressed in only 45.5% of these cell lines. Although one study reported that cGAS and STING were highly expressed in various cancer tissues using bioinformatics analysis , their expression remains to be confirmed in more cell lines.
Previous studies and our present study reported that human epithelial cancer cell lines cannot respond to cytosolic DNA regardless of the expression pattern of cGAS and STING. Reportedly, lung adenocarcinoma-intrinsic glycogen branching enzyme (GBE1) antagonizes the expression and activation of STING . SOX2 promotes the degradation of STING protein in an autophagy-dependent manner in neck squamous cell carcinoma (Tan et al., 2018). Loss of MUS81 leads to the attenuation of STING-dependent type I interferon expression in prostate cancer cells (Ho et al., 2016). HER2 is strongly associated with STING and recruits AKT1 to directly phosphorylate TBK1 (Wu et al., 2019). Colon cancer cells hijack caspase-9 signaling to suppress the radiation-induced mitochondrial DNA-cGAS-STING sensing pathway and limit the secretion of type I IFNs (Han et al., 2020). However, in the present study, we found that cytosolic ISD, HSV60, VACV70, and Poly(dG:dC) induced the phosphorylation of TBK1 and IRF3, and the expression profiles in cGAS and STING intact human epithelial cancer cells induced by cytosolic DNA were the same as those in THP-1-derived macrophages. Further experiments revealed that the expression of IFN-λ1, ISG15, and CCL5 induced by cytosolic ISD, HSV60, VACV70, and Poly(dG:dC) was impaired by the knockdown of STING, while the expression of IFN-β was STING-independent. Thus, cGAS and STING intact human epithelial cancer cells can sense cytosolic DNA through the cGAS-STING signaling pathway, and there may be novel pathways and molecules to control the production and secretion of IFN-β .
In conclusion, we ascertained that most human cancer cells respond to cytosolic RNA through the RIG-I-MAVS-IRF3 signaling pathway, while the cGAS-STING pathway is activated despite the absence of IFN-β secretion in cGAS and STING intact human cancer cell lines. Our present findings are relevant in developing strategies for targeting nucleic acid receptors in cancer immunotherapy.

DATA AVAILABILITY STATEMENT
The raw data analyzed during the current study are available from the corresponding author upon reasonable request.