Abstract
Abstract Background: The DNA-sensing nucleotidyl transferase enzyme cyclic GMP-AMP synthase (cGAS), its second-messenger product cyclic GMP-AMP (cGAMP), and the cGAMP sensor Stimulator of interferon Genes (STING) form a major cytoplasmic DNA-sensing mechanism in human cells [Barber GN. Nat Rev Immunol, 2015]. cGAMP-activated STING oligomers bind TANK-binding kinase 1 (TBK1), translocate from the endoplasmic reticulum to perinuclear vesicles, and phosphorylate transcription factor interferon (IFN) regulatory factor 3 (IRF3), resulting in IRF3 nuclear translocation to induce expression of type I IFNs such as IFNβ. This pathway is critical for controlling innate antiviral immune responses, and is implicated in anti-tumor adaptive immunity, yet little is known regarding its function in human primary breast cancer (BC). Methods: Immunohistochemistry (IHC) staining for cGAS (ab224144), STING (CST13647), TBK1 (HPA045797), IRF3 (ab25950) and IFNβ (ab238675) was performed on tissue microarrays constructed from 18 normal human breast tissue samples and 197 primary BC samples to characterize in-situ protein expression. Positive controls were normal colon, MCF10A cells, testis tissue, MCF-7 cells and normal colon for cGAS, STING, TBK1, IRF3 and IFNβ, respectively. Substitution of primary antibodies with 0.5% normal goat serum served as negative controls. Scoring of extent of the IHC-stained area was 0 for no IHC signal, 1 for <10%, 2 for 10% to 50%, and 3 for >50% of tumor cells. IHC intensity was scored as 0 for no IHC signal, 1 for weak, 2 for moderate, and 3 for strong. A final IHC score was the product of extent score and intensity score, as described [Xia T, et al. Cell reports, 2016]. Categorical classification of high versus low final IHC scores was determined using the median as an unbiased cutoff. Associations between cGAS-STING pathway protein constituents was interrogated by Pearson’s test, and associations between STING pathway IHC expression and breast tumor clinicopathological features were by Chi-square. Results: Among 197 primary BCs, 51.5% were estrogen receptor (ER) positive/ human epidermal growth factor receptor 2 (HER2) negative, 28.4% were HER2 positive and 19.8% were triple negative BC (TNBC). Distinct cGAS-STING pathway expression patterns were observed among three BC subtypes (Table 1). Frequent high expression of cGAS-STING pathway constituents was observed in ER+/HER2- BC. By contrast, high cGAS expression was not associated with high STING expression or effector protein IFNβ in either HER2+ BC, or in TNBC. IRF3 and IFNβ expression was significantly positively associated with hormone receptor status (both P< .001); IFNβ expression was significantly inversely associated with higher grade (P=0.008) and higher mean Ki67 index (18 vs 10, P=0.005). High expression of IFNβ was positively correlated with higher levels of TBK1 and IRF3 (R=0.262 P<.001; R=0.347, P=<.001, respectively). Conclusion: To our knowledge, this is the first study to report cGAS-STING pathway in situ protein expression in primary BC, and demonstrates distinct expression patterns amongst different primary BC clinical subtypes. A limitation of this study is that IHC methods were insufficient for detecting post-translational modifications or translocation of STING pathway proteins. These results provide foundation for understanding the mechanisms of innate immune response in BC, as well as for developing new therapeutic strategies aimed at facilitating adaptive immune anti-tumor responses. Citation Format: Yu Zong, Xiaofei Liu, Glen N Barber, Mark D Pegram. cGAS-STING pathway protein expression in human primary breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-17.