SUMMARY
STING is an ER-associated transmembrane protein that turns on and quickly turns off downstream signaling as it translocates from the ER to vesicles. How STING signaling is attenuated during trafficking remains poorly understood. Here, we show that trafficking-mediated STING degradation requires ER-exit, function of vacuolar ATPase complex, and late stage STING vesicles are sorted to Rab7-positive endolysosomes for degradation. Based on analysis of existing structures, we also identified the helix aa281-297 as a motif required for trafficking-mediated STING degradation. Immuno-EM reveals the size and clustering of STING vesicles and topology of STING on the vesicle. Importantly, blockade of trafficking-mediated STING degradation using bafilomycin A1 specifically enhanced cGAMP-mediated immune response and anti-tumor effect in mice. Together, our findings provide biochemical and imaging evidence for STING degradation by the lysosome, and pinpoint trafficking-mediated STING degradation as a previously unanticipated therapeutic target for enhancing STING signaling in cancer therapy.
In Brief
STING activation turns on and quickly turns off downstream signaling as it is trafficked through the secretory pathway. Gonugunta et al. found that trafficking-mediated STING degradation requires ER exit and sorting of STING vesicles to lysosomes for degradation. Blockade of STING degradation enhances STING signaling and anti-tumor response.
INTRODUCTION
Vertebrates express pattern-recognition receptors (PRRs) that detect microbes through pathogen-associated molecular patterns (PAMPs), which then activate interferon (IFN) and proinflammatory responses to eliminate the pathogen. As prolonged immune responses may be harmful to the host, innate immune signaling pathways are often tightly regulated to ensure robust and timely response against infection while minimizing risk associated with prolonged immune response. The cGAS-STING pathway responds to a wide variety of DNA pathogens by producing robust IFN response as soon as DNA is detected in the cytosol, but that response quickly dissipate through mechanisms that are poorly understood, but likely involves trafficking-mediated degradation of STING protein. A number of studies have implicated certain autophagy proteins (e.g. ULK1 and ATG9A) in negatively regulating STING signaling through interfering with STING-TBK1-IRF3 signaling complex assembly, but not degradation of STING protein (Konno et al., 2013; Saitoh et al., 2009). DNA stimulation-induced vesicles also do not have morphological characteristics of autophagosomes (Saitoh et al., 2009). STING is a transmembrane protein on the ER with the C-terminal cyclic GMP-AMP (cGAMP, produced by cGAS after DNA recognition) binding domain facing the cytosol. One important feature of STING signaling is that it is dynamically regulated during trafficking. We recently showed that STING ER exit is critical for turning on downstream immune signaling (Dobbs et al., 2015). It remains puzzling how STING signaling is turned off while trafficking from the ER to vesicles. Steady-state STING protein level is also tightly regulated by ubiquitination/deubiquitination through functions of iRhom2, and iRhom2-deficiency reduces STING protein level but does not affect trafficking-mediated STING degradation (Luo et al., 2016).
As STING is a critical mediator of IFN production, STING agonists such as cGAMP and other cyclic dinucleotides are being developed as vaccine adjuvants to elicit potent immune response (Fu et al., 2015; Li et al., 2013). STING agonists also elicit strong anti-tumor response by boosting host immune recognition of tumor antigens (Corrales et al., 2015; Woo et al., 2015). Despite tremendous interests in developing STING agonists as immune stimulating therapeutic agents, one inherent limitation of most, if not all, STING agonists is the transient nature of activated signaling. Thus, mechanistic understanding of how STING signaling is turned off is imperative with the hope that blockade of the ‘off’ pathway could potentially lead to enhanced or sustained STING signaling and more superior therapeutic benefits.
RESULTS
Trafficking-mediated STING degradation requires ER exit but not the downstream immune signaling cascade
We first transfected mouse embryonic fibroblast (MEF) with HT-DNA (herring testes DNA, a commonly used dsDNA ligand for cGAS) to activate the cGAS-STING pathway. As shown previously, endogenous STING protein is rapidly degraded after DNA stimulation. The timing of STING degradation follows TBK1 phosphorylation, a critical downstream signaling protein required for activation of Ifnb expression, as well as peak expression of Ifnb mRNA (Figure 1A–C). STING mRNA level was not affected by DNA stimulation (Figure 1B). We next established MEFs stably expressing mouse STING-GFP that allow convenient detection of STING-GFP degradation by fluorescence-activated cell sorting FACS (Dobbs et al., 2015). HT-DNA, cyclic dinucleotide such as cGAMP, c-di-GMP or DMXAA (a small molecule agonist of mouse STING) all triggered degradation of mouse STING-GFP or endogenous mouse STING in WT MEFs, suggesting that STING degradation requires activation by cyclic dinucleotide ligands, and upstream DNA and DNA sensor cGAS are dispensable (Figure 1D).
Figure 1. STING degradation is independent of downstream immune signaling.
(A) Immunoblots show kinetics of TBK1 phosphorylation and endogenous STING degradation in WT MEFs after HT-DNA stimulation. (B) Quantitative RT-PCR analysis of STING mRNA expression at 8 h post HT-DNA stimulation in WT MEFs. Y-axis shows fold increase compared to ‘Lipo’ (normalized to 1). (C) Quantitative RT-PCR analysis of Ifnb mRNA expression in a time course in wild type (WT) MEFs transfected with 1 μg HT-DNA. Y-axis shows fold increase compared to time zero. Ifnb mRNA values were normalized to Gapdh, and time zero value was set to 1 (same throughout). (D) FACS analysis of STING-GFP expression in Sting−/− MEFs reconstituted with mouse STING-GFP. Cells were transfected with lipofectamine alone (Mock, same throughout) or with indicated nucleic acid or STING agonists, and STING-GFP signal was measured by FACS 24 h after stimulation. (E) Immunoblots show endogenous STING degradation in WT, Tbk1−/−, Irf3−/− and cGas−/− MEFs after increasing amount of HT-DNA stimulation. (F–H) Signaling, trafficking and degradation analysis of WT and mutant human STING-HA rescue MEFs. Cells were mock treated or transfected with 1 μg HT-DNA. Ifnb mRNA expression (fold increase as in C) was measured by quantitative RT-PCR at 6 h (F). STING localization was visualized by fluorescent microscopy with cells fixed at 6 h (G). STING degradation at indicated times were measured by immunoblots (H). *p < 0.05, **p < 0.01 (same throughout). Data are representative of at least three independent experiments. Error bars, SEM. Unpaired t-test.
See also Figure S1.
After binding to cyclic dinucleotide, STING exits the ER, recruits TBK1, which phosphorylates STING at Serine 366 residue (Liu et al., 2015). TBK1 also phosphorylates itself and IRF3 leading to IFN expression. We transfected HT-DNA into Tbk1−/− or Irf3−/− MEFs and found that endogenous STING is still getting degraded (Figure 1E). A previous study also observed normal STING degradation after DNA stimulation in Tbk1−/− cells (Abe and Barber, 2014). Neither TBK1 nor IRF3 is degraded by DNA stimulation (Figure 1E). We next examined two key STING residuals, R232 (required for cGAMP binding and ER exit, (Zhang et al., 2013)) and S366 (phosphorylated by TBK1 and is required for downstream signaling, (Liu et al., 2015)), through stable expression of human STING in Sting−/− MEFs using a retroviral system (Dobbs et al., 2015). STING-WT (wild type) MEFs showed robust IFN response to HT-DNA stimulation, rapid translocation from the ER to vesicles and degradation by 6 h (Figure 1F, 1G). In contrast, STING-R232A, which cannot bind cGAMP (Zhang et al., 2013), did not activate IFN in respond to DNA stimulation, remained on the ER and did not degrade. Interestingly, STING-S366A also did not support DNA-mediated IFN activation as shown previously (Figure 1H, (Liu et al., 2015)). However, STING-S366A translocated to vesicles normally after DNA stimulation and is degraded normally (Figure 1G, 1H). We also confirmed that neither R232A or S366A mutants alter lysosome distributions (Figure S1A) or STING interaction with iRhom2 (Figure S1B), which was previously shown to regulate STING ER exit (Luo et al., 2016). Data from these experiments further suggest that as long as STING is activated by a ligand and begins trafficking, the degradation machinery will engage even in the absence of TBK1 or IRF3 or downstream signaling. R232A mutation blocks ligand binding and ER exit as well as subsequent signaling and degradation, whereas S366A mutation only blocks signaling but not trafficking or degradation.
Trafficking-mediated STING degradation requires V-ATPase function and acidified endolysosomes
We next examined whether trafficking-mediated STING degradation requires functions of the proteasome or the lysosome. We pretreated wild type MEFs with known inhibitors of each process, stimulated with HT-DNA, and measured degradation by immunoblot. We found that STING degradation is potently blocked when cells were treated with Bafilomycin A1 (BafA1), suggesting that lysosomes play an important role in trafficking-mediated STING degradation (Figure 2A). BafA1 did not block STING activation, since both phosphorylated (slower migrating band on immunoblots) and unphosphorylated STING accumulated in BafA1-treated cells (Figure 2A). BafA1 inhibits V-ATPase function and prevents acidification of endolysosomes. Chloroquine (CQ) partially blocked STING degradation, consistent with a role for lysosomes, although CQ also inhibits cGAS upstream of STING (An et al., 2015). In contrast, TBK1 inhibitor Compound II (CmpII) had no effect, and proteasome inhibitor MG132 had a small effect on STING degradation (Figure 2A). Another V-ATPase inhibitor Concanamycin A (ConA) also blocked STING degradation, while Brefeldin A (BFA, blocks ER exit) blocked STING activation (no slower migrating band) and degradation (Figure 2A, substantial STING degradation is not expected in the absent of trafficking). V-ATPase is a multi-subunits complex composed of a transmembrane base (V0) and an ATPase head (V1) facing the cytoplasm (Nishi and Forgac, 2002). We designed siRNAs to knockdown key components of the ATPase (V1b, V1c). siRNA knockdown of Atpase6v1b2 and Atpase6v1c1 also potently blocked STING-GFP degradation (Figure 2B). We also observed drastically increased accumulation of STING vesicles after DNA stimulation in cells treated with BafA1 compared to mock (Figure 2C, Figure S2A), further suggesting that BafA1 did not block STING ER exit, rather, it blocks STING vesicle degradation. We also stained the lysosomes with LysoTracker Red and show that, after DNA stimulation, both BafA1 and ConA treated cells show increased STING-GFP signal and decreased LysoTracker signal (Figure S2B), suggesting that neutralized lysosomes have impaired ability to degrade STING vesicles. Collectively, these data demonstrate that acidification of endolysosome is required for trafficking-mediated STING degradation and clearance of STING vesicles.
Figure 2. STING degradation requires acidified endolysosomes.
(A) Immunoblots show DNA-induced STING degradation in WT MEFs treated with various inhibitors. CmpII, Compound II (TBK1 inhibitor). MG132, proteasome inhibitor. CQ, chloroquine. BafA1, bafilomycin A1. BFA, brefedin A. CMA, concanamycin A. Cells were pre-treated with indicated inhibitor (on top) for 30 minutes before transfection with 1 μg HT-DNA. * indicates phosphorylated STING (Wang et al., 2016). (B) FACS analysis of DNA-induced STING-GFP degradation. STING-GFP MEFs were transfected with indicated siRNA (below); two days later cells were mock treated or transfected again with 1 μg HT-DNA. STING-GFP was measured by FACS 24 h after DNA transfection. Accumulative result is shown on the left. A representative set of FACS plots of si-CTL and si-Atp6v1c1 with and without HT-DNA stimulation are shown on the right. (C) Fluorescent micrographs show STING-GFP vesicles after HT-DNA stimulation in the presence or absence of BafA1. Green, STING-GFP. Red, endolysosome marker Rab7 staining. Data are representative of at least three independent experiments. Error bars, SEM. Unpaired t-test.
See also Figure S2.
Trafficking-mediated STING degradation does not require autophagy-related proteins
Previous studies showed that autophagy-related proteins such as ATG9A and ULK1 inhibit STING signaling through preventing TBK1 recruitment or IRF3 phosphorylation, respectively (Konno et al., 2013; Saitoh et al., 2009). Chaperone-mediated autophagy (CMA) may also play a role in STING degradation (Hu et al., 2016). We thus wanted to directly address whether trafficking-mediated STING degradation requires autophagy or CMA. We first used siRNA to knockdown genes encoding core proteins required for conventional autophagosome formation (Atg3, Atg5, Atg9a), a protein required for chaperone-mediated autophagy (Lamp2), and adaptor proteins required for selective autophagy (Calcoco2, Nbr1, Bnip3l, p62/Sqstm1, Tax1bp1). None of the knockdowns blocked endogenous STING degradation after ligand DMXAA stimulation despite good knockdown efficiency (Figure 3A, 3B). We next focused on CMA (Tasset and Cuervo, 2016). We treated cells with geldanamycin (GA) or 6-aminomicotinamide (6-AN) that are known to enhance CMA (Finn et al., 2005) or with BafA1 as a comparison. Of note, no inhibitor of CMA is known. We then stimulated cells with increasing dose of HT-DNA to capture broad range of STING degradation by Western blot (Figure 3C). BafA1 clearly blocked STING degradation. Both GA and 6-AN enhanced STING degradation compared to mock treated cells. Taken together, our data suggest that trafficking-mediated STING degradation does not require autophagy-related proteins. The exact cellular mechanism requires further study, and CMA is likely to play at least a partial role.
Figure 3. Autophagy-related proteins are not required for trafficking-mediated STING degradation.
(A) Immunoblot analysis of STING degradation after ligand stimulation. WT MEFs were transfected with control or specific siRNAs (top) and 48 hrs later stimulated with a cell-permeable STING ligand DMXAA for indicated amount of time (top). (B) Quantitative RT-PCR analysis of autophagy-related genes after siRNA treatment. Value for each gene knockdown efficiency was determined by the ratio of specific siRNA/si-control. Data from a representative set of experiment as A. (C) Immunoblot analysis of STING degradation after HT-DNA stimulation. WT MEFs were treated with indicated compound (left) for 30 minutes before transfection with different amounts of HT-DNA (500 ng to 2 ng in 1:1 dilutions). Bafilomycin A1 (BafA1), 20 nM. Geldanamycin (GA), 2 μM (Finn et al., 2005). 6-aminomicotinamide (6-AN), 50 mM (Finn et al., 2005). Data are representative of at least two independent experiments.
See also Figure S3.
We next analyzed whether ubiquitination of STING is required for degradation of STING vesicles. We mutated a single K150 to R (STING-K150R), or groups of K’s in the middle or at the C-terminus (KmidR, KendR) or all K’s (STING-K0). All four K mutants expressed well when retrovirally introduced into Sting−/− cells, localized properly to the ER, and translocated to vesicles after DNA stimulation (Figure S3A–C). Both KendR and K0 mutant failed to signal but degraded normally after DNA stimulation (Figure S3C, D). These data suggest that STING lysine ubiquitination is dispensable for ER to vesicle translocation and for trafficking-mediated degradation.
A STING motif required for trafficking-mediated degradation
We next analyzed published structures of STING C-terminus and identified two patches of negatively charged residuals, both on the exterior surface of the human STING dimer and towards the bottom facing the ER membrane, E282/D283 and E296/D297 (E281/D282 and E295/D296 in mouse STING, Figure 4A, 4B). These two patches are located at either end of the same helix aa281-297. We mutated these negatively changed residuals to alanine in STING-GFP and established stable expression cells in Sting−/− MEFs. We found that both E282A/D283A and E296A/D297A translocated from the ER to vesicles after stimulation, suggesting that these mutations do not block ER exit (Figure 4C). We then stimulated STING-GFP WT, E282A/D283A and E296A/D297A mutants with increasing amount of DNA and analyzed STING-GFP degradation by immunoblot. Both E282A/D283A and E296A/D297A mutants showed significant delay in DNA-stimulated degradation and enhanced TBK1 phosphorylation compared to WT, suggesting that both mutants activate enhanced downstream signaling (Figure 4D, Figure S4). We also stimulated these cells with cGAMP, and found that cGAMP-mediated STING degradation was also partially blocked by E282A/D283A and E296A/D297A mutants (Figure 4E). In addition, we analyzed gain-of-function STING mutant N154S that constitutively activates STING signaling, but still contains this motif (Figure S5). We stimulated Sting−/− MEFs reconstituted with WT or N154S with DNA and both degraded normally (Figure S5A). We also stimulated healthy control or N154S human fibroblasts with DNA and observed similar degradation of endogenous STING (Figure S5A). Together, these data indicate that the helix aa281-297 harbors a motif required for STING degradation.
Figure 4. A novel STING motif involved in trafficking-mediated degradation.
(A) Bottom view (left, surface charge) or side view (right, ribbon) of STING apo structure (4EF5). Arrow heads indicate E282/D283 and arrows indicate E296/D297. (B) Sequence alignment of human and mouse STING showing conservation of the helix aa281-297. (C) Microscopy images of WT or mutant STING-GFP in unstimulated cells or 6 h after DNA stimulation. WT or mutant STING-GFP was stably expressed in Sting−/− MEFs. These MEFs were unstimulated (top) or transfected with 1 μg HT-DNA and fixed 6 h later for microscopy. Green, STING-GFP. Blue, DAPI. (D) Immunoblots analysis of WT or mutant STING-GFP after stimulation with increasing amount of htDNA (500, 250, 125 ng) for 8 h. (E) FACS analysis of cGAMP-induced STING-GFP degradation. WT or mutant STING-GFP MEFs were transfected with indicated amount of cGAMP and GFP intensity was analyzed 24 h later by FACS. Data are representative of at least two independent experiments. Error bars, SEM. Unpaired t-test.
Late stage STING vesicles cluster and colocalize with endolysosome markers
After cyclic dinucleotide binding, STING translocates from the ER to cytoplasmic vesicles through ERGIC and Golgi apparatus. We previously showed that STING signaling occurs as soon as ER exit and translocation to the ERGIC, and that STING colocalizes extensively with ERGIC and Golgi apparatus during early stage of trafficking process (Dobbs et al., 2015). To better define late stage STING vesicle trafficking, we stimulated STING-GFP MEFs with HT-DNA and fixed the cells at 3 h and 7 h to enrich early and late stage vesicles, respectively. We then co-stained STING-GFP vesicles with early endosome marker Rab5, late endosome/lysosome marker Rab7, recycling endosome marker Rab11, as well as markers for ER, ERGIC and Golgi. As shown previously (Dobbs et al., 2015), unstimulated STING localizes on the ER, and early STING vesicles (‘DNA 3 h’) predominately colocalize with ERGIC and Golgi markers (Figure S6A). Interestingly, we observed strong colocalization of late stage STING vesicles (‘DNA 7 h’) with Rab7 (Figure 5A). Late stage STING vesicles did not colocalize with Rab5 or Rab11, suggesting that these vesicles are sorted to endolysosomes after they exited the Golgi (Figure S6B). We also stimulated STING-GFP cells with cGAMP, or Myc-mSTING cells with HT-DNA, and observed similar colocazation of STING vesicles with Rab7 (Figure 5B, Figure S6C). These data suggest that post-Golgi late stage STING vesicles are sorted to endolysosomes for degradation.
Figure 5. Late stage STING vesicles colocalize with endolysosome marker Rab7.
(A) Fluorescent micrographs show STING-GFP colocalization in MEFs. Cells were mock treated (Unstimulated) or transfected with 1 μg HT-DNA, fixed 7 h later and co-stained with markers for ER (Calnexin), ERGIC (P58), Golgi (GM130), Rab7 (late endosome/lysosome). Unstimulated and 3 h images are shown in Figure S6A. Quantitation of colocalization was calculated as Pearson’s correlation coefficient (r) shown on the right (n = 15). Dashed white box in each main image indicates enlarged area of interest shown below. (B) Fluorescent micrographs show STING-GFP colocalization with Rab7 after 7 h of cGAMP stimulation. (C) Late stage STING-GFP vesicles frequently encounter and fuse with lysosomes. STING-GFP MEFs were transfected with HT-DNA and time-laps live cell microscopy recording was started 6 h after DNA transfection (time 0′). Selected frames from Movie 2 are shown in C. Arrowheads indicate STING-GFP vesicles that are in contact with lysosomes. Note the gradual loss of GFP signal with time in the cell. (D, E) Topology of STING-GFP on vesicles by NanoGold electron microscopy. STING-GFP MEFs were grown on gridded glass bottom dishes, and stimulated with HT-DNA for 6 h. Cells were fixed and stained with anti-GFP antibody followed by secondary anti-mouse Nanogold/AF547 antibody (see Supplementary Method). Quantification of individual STING-GFP vesicle size and cluster size are shown in E. Data are representative of two independent experiments (at least four cells from each experiment were imaged in detail).
We next performed live cell time-laps microscopy using STING-GFP MEFs labeled with LysoTracker-Red to monitor early (1–3 h) and late (6–10 h) stage STING trafficking. At 30 minutes to 3 hours post DNA stimulation, STING-GFP moves from the ER to Golgi with enhanced GFP signal (likely due to local enrichment) showing very little overlap with lysosomes (Figure S7A, Movie 1). From 6 to 10 hours post DNA transfection, STING-GFP vesicles form large puncta that distribute evenly in the cytoplasm. These STING-GFP vesicles frequently encounter and fuse with lysosomes and gradually reduce in size while losing GFP signal (Figure 5C, Figure S7B, Movie 2–4).
We also performed NanoGold Immuno-EM with anti-GFP staining of DNA-stimulated STING-GFP MEFs to determine the size and ultrastructure of STING-GFP vesicles as well as topology of STING on the vesicle. The C-terminus of STING is expected to face the cytosol, but that has never been defined by electron microscopy. We found that a single STING-GFP vesicle is in average 110 nanometers in diameter, and that STING-GFP locates at the exterior surface of the single layer membrane vesicle, indeed facing the cytosol (Figure 5D, 5E). A previous study using conventional EM also found that DNA-induced vesicles to contain single membrane (Saitoh et al., 2009). STING-GFP vesicles often cluster into multi-vesicular structures that contain 2 to 12 vesicles per cluster, and are up to 820 nanometers in diameter, consistent with the large particle size observed by immunoflourscence microscopy.
Blockade of trafficking-mediated STING degradation by BafA1 enhances cGAMP-mediated immune signaling and anti-tumor response
Two major therapeutic benefits of STING signaling are innate immune activation and anti-tumor immune response. Repeated administration of STING agonists is often needed to achieve optimal response, due to the transient nature of STING-mediated IFN signaling (Corrales et al., 2015). We examined whether blockade of STING degradation would enhance signaling by retaining activated STING in the cytosol. We first transfected wild type MEFs with HT-DNA in the presence of increasing BafA1 concentration. BafA1 enhanced IFN and ISG expression induced by HT-DNA, but not by poly(I:C), at low concentration, and at high concentrations it became inhibitory likely due to nonspecific effects (Figure 6A). We also stimulated MEFs with cGAMP or DMXAA in the presence or absence of BafA1, and observed rapid degradation of endogenous STING induced by cGAMP and DMXAA, but not when BafA1 were also administered (Figure 6B). We then asked whether BafA1 could enhance cGAMP-mediated innate immune activation in mice and in primary human peripheral blood mononuclear cells (PBMCs). We treated wild type mice with lipofectamine (Lipo) alone, BafA1 alone, cGAMP or cGAMP+BafA1 by intraperitoneal (i.p.) injection, and measured immune gene expression in mouse splenocytes (Figure 6C). cGAMP induced IFN, ISGs and inflammatory genes in splenocytes 4 h after i.p. injection. BafA1 alone also induced expression of some genes (e.g Ifng, Cxcl10, Oasl2, Tnf). cGAMP+BafA1 induced broader and stronger immune gene expression compared to cGAMP or BafA1 alone, suggesting that BafA1 enhanced cGAMP-mediated STING activation in vivo (Figure 6C). We also isolated human PBMCs from healthy donors, and transfected cGAMP with or without BafA1. BafA1 alone induced fewer immune genes in human PBMCs. cGAMP+BafA1 again induced more robust immune activation than either treatment alone in PBMCs (Figure 6D). Collectively, these data suggest that blockade of trafficking-mediated STING degradation by BafA1 enhances cGAMP-mediated immune activation in vitro and in vivo.
Figure 6. BafA1 enhances cGAMP-mediated immune signaling.
(A) Quantitative RT-PCR analysis of Ifnb and Cxcl10 mRNA expression in WT MEFs treated with increasing concentration of BafA1 concurrent with HT-DNA or poly(I:C) transfection. (B) Immunoblots show cGAMP- or DMXAA-stimulated STING degradation and blockade by BafA1. WT MEFs were Treated with indicated reagents (top). Lipo, 1 μL. BafA1, 20 μM. cGAMP and DMXAA, 4 μg. (C) A heat map of quantitative RT-PCR array analysis of mouse immune genes. Each gene expression value was first normalized to Gapdh. Heat map was generated using GENE-E (the Broad Institute). (D) A heat map of quantitative RT-PCR array analysis of human immune genes. Human PBMCs were isolated from a healthy donor, treated with conditions shown on top and analyzed similarly as in C. Data are representative of at least two independent experiments. Error bars, SEM. Unpaired t-test.
We next evaluated blockade of STING degradation in cGAMP-mediated anti-tumor response using the B16 melanoma xenograft model. We implanted B16 cells subcutaneously (s.c.) in wild type mice. Approximate 5 days later, when tumor grows up to about 5 mm wide, we injected (i.t.) a single dose of carrier alone (Lipo), low (10 μg) or high (50 μg) dose cGAMP (complexed with lipofectamine). We also injected low dose cGAMP in combination with BafA1. In another condition, we injected (i.t.) the low dose cGAMP in three consecutive days (one injection per day). Both the high dose and multiple administration of the low dose cGAMP significantly inhibited tumor growth (Figure 7A). Single treatment of low dose cGAMP alone did not inhibit tumor growth. Remarkably, combined single administration of low dose cGAMP with BafA1 significantly inhibited tumor growth. Lipo+BafA1 did not inhibit tumor growth. In a separate experiment, we isolated tumors 6 hours after cGAMP and/or BafA1 injection and measured IFN and ISG expression. We found that ISGs such as Ifit1 and Cxcl10 were significantly increased in the cGAMP 10 μg+BafA1 condition compared to cGAMP 10 μg alone, suggesting that BafA1 enhanced cGAMP-mediated IFN signaling in the tumor (Figure 7B). To define the specificity of BafA1 for the STING pathway in anti-tumor response, we performed two additional experiments with the B16 melanoma model. We compared cGAMP versus cGAMP+BafA1 in Sting−/− mice, and found neither treatment inhibited tumor growth (Figure 7C). We also compared poly(I:C) versus poly(I:C)+BafA1. Poly(I:C) alone inhibited tumor growth at day 13, likely through the cytosolic RNA sensing pathways, but BafA1 did not enhance poly(I:C)-mediated anti-tumor response (Figure 7D). Together, these data demonstrated that BafA1 specifically enhances STING-mediated anti-tumor response in vivo. These studies also provide an important example that trafficking-mediated STING degradation can be targeted therapeutically to enhance STING agonist-mediated anti-tumor response.
Figure 7. BafA1 enhances cGAMP-mediated anti-tumor response.
(A, B) BafA1 enhances cGAMP-mediated anti-tumor response. 5×105 B16 cells were injected s.c. in wild type C57BL/6 mice. When tumor size reaches approximately 5 mm wide (or 5 days later), each treatment (as indicated) was injected i.t. Tumor size was measured twice per week (A, n=5). In a separate experiment, IFN and ISG expression was measured 6 h after cGAMP and/or BafA1 injection (B, n=5). BafA1, 15 ng per injection, same throughout. (C, D) BafA1-mediated anti-tumor effect is specific to the STING pathway. Similar to A, B16 cells were injected in Sting−/− mice and treated with cGAMP (C, n=5) or injected in C57BL/6 mice and treated with poly(I:C) (D, n=5). Data are representative of at least two independent experiments. Error bars, SEM. Unpaired t-test.
DISCUSSION
Here, we combined immunology, biochemistry and cell biology approaches to characterize trafficking-mediated STING degradation after activation and to demonstrate the therapeutic benefit of its blockade. Our results showed that post-Golgi STING vesicles are sorted to Rab7-positive endolysosomes. Degradation of STING vesicles requires lysosomes, but not autophagosomes. This is consistent with previous observation by regular EM that dsDNA-induced vesicles do not have morphological characteristics of autophagosomes such as double membranes (Saitoh et al., 2009). Our immuno-EM evidence also shows STING on the surface of single membrane vesicles, with the C-terminus exposed to the cytosol, which is important for recruiting downstream signaling components such as TBK1 and IRF3. Autophagy-related proteins such as ULK1 and ATG9A have been shown to negatively regulate STING signaling complex assembly (Konno et al., 2013; Saitoh et al., 2009). We found that STING degradation was not altered when we knocked down expression of core autophagy machinery or selective autophagy adaptor proteins. Thus, although certain autophagy proteins may be involved in STING signaling complex assembly, the process of autophagy and autophagosomes are not required for STING vesicle degradation.
How are STING vesicles directed to the lysosome? We observed dynamic interactions of STING vesicles with lysosomes via live cell imaging, where lysosomes either fuse or encapsulate clusters of STING vesicles and gradually eliminates all STING vesicles in a cell. This process could be stochastic or directed. A recent study suggested that CMA may be required for recruitment of STING vesicles to lysosomes (Hu et al., 2016). Although we did not find LAMP2 knockdown to block STING degradation, CMA-enhancing compounds did enhance STING degradation, suggesting the possibility that CMA is at least one of the cellular mechanisms directing STING vesicles to the lysosome. The next question is which motif in the STING protein is required for degradation? We identified a motif (helix aa281-297) containing two patches of negatively charged surface residuals (E281/D282 and E296/D297), when mutated, delays STING degradation. Interestingly, the same region was recently found to contain mutants, R281Q and R284G, that are associated with auto-inflammatory disease SAVI (Melki et al., 2017). These SAVI mutants in exon 7 are distinct from exon 5 mutations such as N154S and V155M, and may cause disease through a different mechanism. Further studies are needed to determine the role of these positively charged arginine residues in this region, and whether delayed STING degradation contributes to chronic activation of STING signaling in these patients.
Our experiments also revealed an intricate relationship between STING signaling and trafficking-mediated degradation. Both STING signaling and trafficking-mediated degradation require ER exit. STING signaling turns on immediately after ER exit and it remains active throughout the trafficking process until the vesicle stage, as TBK1 can be detected to co-localize with STING as early as ERGIC and as late as post-Golgi vesicles (Dobbs et al., 2015; Ishikawa et al., 2009). STING signaling turns off after engaging with acidified endolysosomes, degrading STING and possibility the associated pTBK1, although for pTBK1 we can not distinguish between degradation and de-phosphorylation. Trafficking-mediated STING degradation can be disengaged from signaling. Using various STING mutants and knockout cells, we found that as long as trafficking is initiated, STING will be rapidly degraded regardless of the presence of TBK1, IRF3 or downstream signaling, ubiquitination or S366 phosphorylation.
Importantly, our results pinpoint trafficking-mediated STING degradation as a previously unanticipated therapeutic target for interruption to boost STING signaling, and present an exciting opportunity for drug discovery. We envision that interruption of trafficking-mediated STING degradation would have broad therapeutic implications in vaccines and cancer. As a proof-of-principle, we showed that BafA1 and cGAMP combination therapy demonstrated enhanced immune activation and anti-tumor response in vivo, and that the therapeutic effect of BafA1 is specific to the STING pathway. Local administration of BafA1 functionally augmented cGAMP-mediated anti-tumor effect that would allow substantially reduced or less frequent cGAMP dosing while achieving similar therapeutic benefit. With increasing interests in developing STING agonists as vaccine adjuvants and anti-tumor therapy, we propose that blockade of STING degradation by compounds that act similar to BafA1 could be an exciting option as a combination therapy to greatly improve therapeutic benefits of STING agonists.
EXPERIMENTAL PROCEDURES
Cell Culture, mice and inhibitors
Wild type, Sting−/−, cGas−/−, Tbk1−/− and Irf3−/− MEFs were described previously (Dobbs et al., 2015). Mouse melanoma B16 cells were obtained from Noah Craft (UCLA). Bafilomycin A1 20 nM, Brefedin 40 μM, chloroquine 10 μM, Torin 10 nM, MG132 10 μM, Compound II 10 μg/mL, except noted otherwise. All experiments involving human and mouse materials were approved by the Institutional Review Board and Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center.
In vivo tumor study
5×105 B16 melanoma cells were injected subcutaneously (s.c.) into 6–8 weeks old wild type C57BL/6 or Sting−/− mice. When the tumor size reaches approximately 5 mm wide, one single dose of cGAMP or other treatments was injected intratumorly (i.t.). Tumor size was measured twice per week using calipers. Tumor volume was calculated as (length × width2)/2.
RNA isolation, quantitative RT-PCR analysis, cytokine-detection assay, Western blot and Immunoprecipitation
RNA isolation and qRT-PCR analysis were performed as described previously (1). Serum cytokines were measured by MILLIPLEX multiplex assay using Luminex (EMD Millipore) according to manufacture’s protocols. Western blots were performed as described previously (1). Co-immunoprecipitation experiments were performed as described previously (2).
Fluorescent and electron microscopy
Sting−/− MEFs were reconstituted with various WT or mutant STING-GFP (mouse or human, as indicated) as described before (Dobbs et al., 2015). For live cell time laps microscopy, STING-GFP MEFs grown on 35 mm glass bottom dish were transfected with 3 μg HT-DNA, treated with 50 nM LysoTracker Red (Invitrogen), and imaged in the Spinning Disk confocal microscope equipped with a heated stage and enclosed CO2 chamber for 3–4 hour durations. NanoGold immuno-EM was performed similar to procedures described in (Yamamoto and Masaki, 2010).
Statistical methods
Data are presented as the mean ± SEM. Graphpad Prism 6 was used for statistical analysis. Statistical tests performed were indicated in figure legend. *P < 0.05, **P < 0.01, and ***P < 0.001.
More detailed information please see SUPPLEMENTARY INFORMATION.
Supplementary Material
Early stage STING-GFP trafficking. Related to Figure 5. LysoTracker in Red. Recording begins at 0.5 h after DNA transfection of STING-GFP MEFs. Z stacks were acquired every 2 minutes. Movie shows 3D view in five frames (10 minutes of live cell movement) per second.
Late stage STING-GFP trafficking. Related to Figure 5. LysoTracker in Red. Recording begins at 6 h after DNA transfection of STING-GFP MEFs. Z stacks were acquired every 2 minutes. Movie shows 3D view in five frames (10 minutes of live cell movement) per second.
Late stage STING-GFP trafficking. Related to Figure 5. Similar to Movie 2, showing a different cell.
Late stage STING-GFP trafficking. Related to Figure 5. Similar to Movie 2, showing a different cell.
Highlights.
Trafficking-mediated STING degradation requires sorting of STING vesicles to lysosomes
Blockade of STING degradation enhances STING signaling and anti-tumor response.
Helix aa281-297 contains a motif required for STING degradation.
Trafficking-mediated STING degradation does not require downstream immune signaling.
Acknowledgments
We thank Glen Barber (Univ. Miami) for Sting−/− mice, Rolf Brekken (UTSW) for Tbk1−/− MEFs, Noah Craft (UCLA) for mouse melanoma B16 cells, Raphaela Goldbach-Mansky (NIAID) for SAVI patient fibroblasts. We also thank Abhijit Bugde at the UTSW live cell imaging core for assistance on time-laps microscopy, Anza Darehshouri at the UTSW EM core for assistance on Nanogold immuno-EM. We thank members of the Yan laboratory for helpful discussions. This work was supported by grants from the National Institute of Health (AR067135 to N.Y.) and the Burroughs Wellcome Fund (N.Y.). The authors have no conflict of interest.
Footnotes
AUTHOR CONTRIBUTIONS
V.K.G. and N.Y. designed the study. V.K.G. conducted the majority of experiments. T.S. performed mouse tumor experiment. V.P., K.Y., J.W., and N.D. helped with RNAi, immunoprecipitation, microscopy and gene expression analysis. N.Y. performed some of the microscopy experiments. V.K.G. and N.Y. wrote the manuscript with input from all authors.
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Associated Data
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Supplementary Materials
Early stage STING-GFP trafficking. Related to Figure 5. LysoTracker in Red. Recording begins at 0.5 h after DNA transfection of STING-GFP MEFs. Z stacks were acquired every 2 minutes. Movie shows 3D view in five frames (10 minutes of live cell movement) per second.
Late stage STING-GFP trafficking. Related to Figure 5. LysoTracker in Red. Recording begins at 6 h after DNA transfection of STING-GFP MEFs. Z stacks were acquired every 2 minutes. Movie shows 3D view in five frames (10 minutes of live cell movement) per second.
Late stage STING-GFP trafficking. Related to Figure 5. Similar to Movie 2, showing a different cell.
Late stage STING-GFP trafficking. Related to Figure 5. Similar to Movie 2, showing a different cell.