STING in Innate Immunity and Cancer: What’s the Buzz About?

STING (STimulator of INterferon Genes protein) acts as a sensor of cytosolic DNA. Bacteria/Virus or self-derived DNA in the cytosol activates the STING pathway and promotes the production of type I interferons (IFN-alpha and IFN-beta). STING also participates in cell death signaling through its association with MHC-II and the ERK pathway. STING has been suggested to interact with DDX58/RIG-I, MAVS, SSR2, RNF5, TRIM56, TBK1, IFIT1 and IFIT2. It generally localizes to the cytoplasm and membranes of the cell, ER, and mitochondria; however, in response to DNA stimulation, it translocates to the perinuclear region and interacts with TBK1 kinase. STING’s phosphorylation by TBK1 at Ser-358 results in STING activation. STING executes its role by sensing and binding cyclic di-GMP/c-di-GMP and cyclic GMP-AMP/cGAMP. This binding results in the activation of NF-kappa B and IRF3 transcriptional signaling pathways leading to the induction of Type I interferon response. Besides regulating immune responses to bacterial/viral pathogens, DNA sensing by innate immune cells has led to a better understanding of the mechanisms regulating autoimmunity and antitumor immune responses. STING regulated innate signaling has offered critical insights and new opportunities into the development of novel immunization regimes and therapeutics for the treatment of infection, autoimmune disorders, inflammation and cancer. [Barber, 2015].

STING Antibodies are essential to immunoassay based experiments. Novus Biologicals offers several antibodies for analysis of STING expression in human, mouse or rat species by Western blot (WB), ELISA, Immunohistochemistry (IHC), Flow cytometry (FLOW), and Immunocytochemistry/Immunofluorescence (ICC/IF) applications. Novus’ STING Antibodies are available in unconjugated and multiple conjugated formats.

STING Antibody [NBP2-24683] Immunohistochemistry analysis of STING protein in formalin-fixed paraffin-embedded mouse lung tissue section using STING antibody at 1:150 dilution with HRP-conjugated secondary. The signal was developed using DAB reagent and the nuclei were counterstained with hematoxylin. The antibody generated mainly a cytoplasmic staining in the bronchiolar and alveolar epithelial cells.		STING Antibody (723505) [MAB7169] Western blot analysis of STING protein in lysates from THP‑1 human acute monocytic leukemia and U937 human histiocytic lymphoma cell lines. STING antibody was used at 0.2 ug/ml concentration and the signal was detected with HRP-conjugated anti-mouse IgG secondary antibody (Catalog # HAF007). STING signal was detected at ~40 kDa position.
STING Antibody (723505) [MAB7169] ICC/IF analysis of U937 human histiocytic lymphoma cell line using STING antibody (clone 723505) at 3 ug/ml concentration (3 hours RT incubation) and NorthernLights (TM) 557-conjugated anti-Mouse IgG secondary antibody (red; catalog # NL007). Nuclei were counterstained with DAPI (blue) and STING was found to be localized to the cytoplasm.		STING Antibody (723505) [Alexa Fluor 488] [IC7169G] Flow Cytometry analysis of THP‑1 human acute monocytic leukemia cell line using AF488 conjugated STING antibody (clone 723505, filled histogram) or isotype control antibody (IC0041G, open histogram). This testing involved fixation using buffer catalog # FC004 and permeabilization with buffer catalog # FC005.

Novus Biologicals’ STING Antibodies have been cited in several publications from peer reviewed research journals.

In a recent study published in The Journal of Immunology, Sun et al 2015 used Novus Biological’ STING Antibody (NBP2-24683/IMG-6485A) for ICC/IF analysis of methanol fixed THP1 cells which were infected with Kaposi’s sarcoma–associated herpesvirus (KSHV) or human beta-herpesviruses (HCMV) and on murine bone marrow–derived dendritic cells (BMDCs) infected with alpha- herpesviruses/HSV-1 (MOI 3) and murine gammaherpesvirus 68 /MHV68 (MOI 20).  This study established that gamma-herpesviruses MHV68 and KSHV, in contrast to alpha- and beta-herpesviruses, only weakly induce innate immune responses through DNA-stimulated pathways. In another study, Berg and co-workers used a STING Antibody (NBP2-24548/ IMG-6422A) for ICC/IF staining of IL2/PHA-stimulated PBMCs which were further treated or not with Lipofectamine2000, and on the cells transfected with FITC-labeled DNA. They also used STING Antibody (MAB7196) for WB analysis in experiments involving IL2/PHA-stimulated PBMCs, and this study established that activated T cells detect intracellular DNA through IFI16 and recruit STING as well as TBK1. Hansen et al 2014 published a report in The EMBO Journal, wherein they employed STING Antibody (clone 723505) for Western blot analysis of PMA differentiated THP1 cells and human monocytederived macrophages (hMDM), and for ICC/IF analysis of PMA-differentiated THP1 macrophages which were transfected with FITC-labelled dsDNA or cyclic-di-AMP or were infected with Listeria monocytogenes (strain LO28, MOI25).

In a recent publication from Cell Reports, Barber’s team from University of Miami Miller School of Medicine (Florida USA) analyzed STING expression in tissue microarrays of normal and adenocarcinoma colon tissues with immunohistochemistry, and in several cell lines such as hTERT-BJ1 Telomerase Fibroblasts (hTERT), FHC, SW1116, SW480, SW1417, SW48, HT29, HT116, LoVo, LS123, LS174T and COLO205 using WB and ICC/IF applications. The authors established that STING mediated innate immune response is largely compromised in human colon cancers as well as many other types of human cancers mainly via silencing STING and/or synthase cGAS expression through epigenetic hypermethylation processes. They further suggested that the STING pathway may have a major function in suppressing colon tumorigenesis and that defects in STING signaling render cancer cells more susceptible to oncolytic viral infection. In another interesting study published in Cancer Immunology Research, Chandra et al. 2014 used a STING Antibody to analyze expression in primary breast tumors, metastases, and in 4T1 cell lines with bone marrow and HEK293 as positive control and negative control respectively.  This study used an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb) as the vaccine and c-di-GMP as the STING ligand in a metastatic breast cancer model 4T1, and established that c-di-GMP improves vaccine-based cancer immunotherapy through multiple signaling pathways.

More and more studies are being published on the STING pathway and it is emerging as an exciting target in innate immunity and cancer research. 

Novus Biologicals' offerings on STING target include:

• STING Antibodies
• STING Protein and Peptide
• STING Lysate

Compiled by: Subhash Gangar

References:

1. Barber GN. 2015. STING: infection, inflammation and cancer. Nat Rev Immunol. 15(12):760-70
2. Berg RK, Rahbek SH, Kofod-Olsen E et al. T cells detect intracellular DNA but fail to induce type I IFN responses: implications for restriction of HIV replication. PLoS One. 2014 Jan 3;9(1):e84513.
3. Sun C, Schattgen SA, Pisitkun P et al. 2015. Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection. J Immunol. 194(4):1819-31.
4. Hansen K, Prabakaran T, Laustsen A et al. 2014. Listeria monocytogenes induces IFNβ expression through an IFI16-, cGAS- and STING-dependent pathway. EMBO J.  33(15):1654-66.
5. Chandra D, Quispe-Tintaya W, Jahangir A et al. 2014. STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer. Cancer Immunol Res.  2(9):901-10.
6. Xia T, Konno H, Ahn J, Barber GN. 2016. Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis. Cell Rep. 14(2):282-97.