STING agonist diABZI induces PANoptosis and DNA mediated acute respiratory distress syndrome (ARDS)

Stimulator of interferon genes (STING) contributes to immune responses against tumors and may control viral infection including SARS-CoV-2 infection. However, activation of the STING pathway by airway silica or smoke exposure leads to cell death, self-dsDNA release, and STING/type I IFN dependent acute lung inflammation/ARDS. The inflammatory response induced by a synthetic non-nucleotide-based diABZI STING agonist, in comparison to the natural cyclic dinucleotide cGAMP, is unknown. A low dose of diABZI (1 µg by endotracheal route for 3 consecutive days) triggered an acute neutrophilic inflammation, disruption of the respiratory barrier, DNA release with NET formation, PANoptosis cell death, and inflammatory cytokines with type I IFN dependent acute lung inflammation. Downstream upregulation of DNA sensors including cGAS, DDX41, IFI204, as well as NLRP3 and AIM2 inflammasomes, suggested a secondary inflammatory response to dsDNA as a danger signal. DNase I treatment, inhibition of NET formation together with an investigation in gene-deficient mice highlighted extracellular DNA and TLR9, but not cGAS, as central to diABZI-induced neutrophilic response. Therefore, activation of acute cell death with DNA release may lead to ARDS which may be modeled by diABZI. These results show that airway targeting by STING activator as a therapeutic strategy for infection may enhance lung inflammation with severe ARDS. Open in a separate windowSTING agonist diABZI induces neutrophilic lung inflammation and PANoptosis A, Airway STING priming induce a neutrophilic lung inflammation with epithelial barrier damage, double-stranded DNA release in the bronchoalvelolar space, cell death, NETosis and type I interferon release. B, 1. The diamidobenzimidazole (diABZI), a STING agonist is internalized into the cytoplasm through unknown receptor and induce the activation and dimerization of STING followed by TBK1/IRF3 phosporylation leading to type I IFN response. STING activation also leads to NF-kB activation and the production of pro-inflammatory cytokines TNFα and IL-6. 2. The activation of TNFR1 and IFNAR1 signaling pathway results in ZBP1 and RIPK3/ASC/CASP8 activation leading to MLKL phosphorylation and necroptosis induction. 3. This can also leads to Caspase-3 cleavage and apoptosis induction. 4. Self-dsDNA or mtDNA sensing by NLRP3 or AIM2 induces inflammsome formation leading to Gasdermin D cleavage enabling Gasdermin D pore formation and the release mature IL-1β and pyroptosis. NLRP3 inflammasome formation can be enhanced by the ZBP1/RIPK3/CASP8 complex. 5. A second signal of STING activation with diABZI induces cell death and the release of self-DNA which is sensed by cGAS and form 2′3′-cGAMP leading to STING hyper activation, the amplification of TBK1/IRF3 and NF-kB pathway and the subsequent production of IFN-I and inflammatory TNFα and IL-6. This also leads to IFI204 and DDX41 upregulation thus, amplifying the inflammatory loop. The upregulation of apoptosis, pyroptosis and necroptosis is indicative of STING-dependent PANoptosis. Subject terms: Cell death and immune response, Respiratory tract diseases     

CircNEIL3 mediates pyroptosis to influence lung adenocarcinoma radiotherapy by upregulating PIF1 through miR-1184 inhibition

Circular RNAs (circRNAs) belong to an abundant category of non-coding RNAs that are stable and specific, and thus have great potential in cancer treatment. However, little is known about the role of circRNAs during radiotherapy in lung adenocarcinoma (LUAD). Here, we established the expression profiles of 1,875 dysregulated circRNAs in non-irradiated and irradiated A549 cells and identified circNEIL3 as a significantly downregulated circRNA in A549 cells treated with 0, 2, or 4 Gy of radiation, respectively. Functional assays demonstrated that circNEIL3 knockdown promoted radiation-induced cell pyroptosis, whereas circNEIL3 overexpression had the opposite effects. Importantly, the effects of circNEIL3 overexpression on inhibiting pyroptosis were reversed by PIF1 knockdown. Mechanistically, circNEIL3-mediated pyroptosis was achieved through directly binding to miR-1184 as a sponge, thereby releasing the inhibition of miR-1184 on PIF1, which ultimately induces DNA damage and triggers AIM2 inflammasome activation. In vivo, circNEIL3 knockdown significantly enhanced the efficacy of radiotherapy as evidenced by decreases in tumor volume and weight. Collectively, the circNEIL3/miR-1184/PIF1 axis that mediate pyroptosis induction may be a novel, promising therapeutic strategy for the clinical treatment of lung cancer.Subject terms: Radiotherapy, Non-small-cell lung cancer     

Comprehensive molecular analysis of a four-pyroptosis-gene signature with prognosis and immune landscape in lung adenocarcinoma

Pyroptosis plays an important role in tumor immunity. However, the biological behavior and prognostic significance of pyroptosis remain unclear. We identified 41 pyroptosis regulators differently expressed in lung adenocarcinoma (LUAD). All cases of LUAD can be classified into two molecular subtypes using unsupervised clustering algorithm. Using multiple analyses, a four-pyroptosis-gene signature was constructed, and all LUAD patients were categorized as low-risk or high-risk with a longer overall survival (OS) time in the low-risk group(P < 0.001). This signature had power prognosis and stratification which was validated by six independent datasets and clinical subtypes. Besides, this signature showed distinct clinical outcomes, immune landscapes in different risk groups. Moreover, the low-risk group had a higher response against immunotherapy with a lower TIDE score. Importantly, this signature surpassed other biomarkers (TIDE, TMB, PD-L1) in predicting prognosis. Overall, the current study might help with precise prognostic prediction and crucial treatment strategies, eventually promoting tailored therapy for LUAD patients.     

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53^(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.     

The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures--specifically neuronal cells--from IR-induced damage, while the redox activity of APE1 does not appear to be involved.     

Small-molecule inhibition of APE1 induces apoptosis,pyroptosis, and necroptosis in non-small cell lung cancer

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.Subject terms: Non-small-cell lung cancer, Apoptosis     

Cell Type-Specific Subcellular Localization of Phospho-TBK1 in Response to Cytoplasmic Viral DNA

Cytoplasmic viral RNA and DNA are recognized by RIG-I-like receptors and DNA sensors that include DAI, IFI16, DDX41, and cGAS. The RNA and DNA sensors evoke innate immune responses through the IPS-1 and STING adaptors. IPS-1 and STING activate TBK1 kinase. TBK1 is phosphorylated in its activation loop, leading to IRF3/7 activation and Type I interferon (IFN) production. IPS-1 and STING localize to the mitochondria and endoplasmic reticulum, respectively, whereas it is unclear where phosphorylated TBK1 is localized in response to cytoplasmic viral DNA. Here, we investigated phospho-TBK1 (p-TBK1) subcellular localization using a p-TBK1-specific antibody. Stimulation with vertebrate DNA by transfection increased p-TBK1 levels. Interestingly, stimulation-induced p-TBK1 exhibited mitochondrial localization in HeLa and HepG2 cells and colocalized with mitochondrial IPS-1 and MFN-1. Hepatitis B virus DNA stimulation or herpes simplex virus type-1 infection also induced p-TBK1 mitochondrial localization in HeLa cells, indicating that cytoplasmic viral DNA induces p-TBK1 mitochondrial localization in HeLa cells. In contrast, p-TBK1 did not show mitochondrial localization in RAW264.7, L929, or T-23 cells, and most of p-TBK1 colocalized with STING in response to cytoplasmic DNA in those mammalian cells, indicating cell type-specific localization of p-TBK1 in response to cytoplasmic viral DNA. A previous knockout study showed that mouse IPS-1 was dispensable for Type I IFN production in response to cytoplasmic DNA. However, we found that knockdown of IPS-1 markedly reduced p-TBK1 levels in HeLa cells. Taken together, our data elucidated the cell type-specific subcellular localization of p-TBK1 and a cell type-specific role of IPS-1 in TBK1 activation in response to cytoplasmic viral DNA.     

Extracellular Vesicles-Encapsulated miR-153-3p Potentiate the Survival and Invasion of Lung Adenocarcinoma

Extracellular vesicles (EVs) play an essential role in the communication between cells and the tumor microenvironment. However, the effect of tumor-derived EVs on the growth and metastasis of lung adenocarcinoma (LUAD) remains to be explored. This study aimed to elucidate the role of miR-153-3p-EVs in the invasion and migration capabilities of LUAD cells and explore its mechanism through in vivo and in vitro experiments. We found that miR-153-3p was specifically and highly expressed in LUAD and its secreted EVs. Furthermore, the expression of BANCR was negatively regulated by miR-153-3p and identified as a target gene of miR-153-3p using luciferase reporter assays. Through further investigation, we found that the downregulation of BANCR activates the PI3K/AKT pathway and accelerates the process of epithelial-mesenchymal transition (EMT), which ultimately leads to the aggravation of LUAD. The orthotopic xenograft mouse model was established to illustrate the effect of miR-153-3p-EVs on LUAD. Animal studies showed that miR-153-3p-EVs accelerated tumor growth in mice. Besides, we found that miR-153-3p-EVs could damage the respiratory ability of mice and produce a mass of inflammatory cells around the lung tissue of mice. Nevertheless, antagomir-153-3p treatment could inhibit the deterioration of respiratory function and inhibit the growth of lung tumors in mice. In conclusion, our study reveals the potential molecular mechanism of miR-153-3p-EVs in the development of LUAD and provides a potential strategy for the treatment of LUAD.     

DNA疫苗抗原提呈的传感器分子研究新进展

DNA疫苗进入细胞后,除了转译成蛋白质抗原,通过MHC分子进行内源性或外源性抗原提呈外,近年来还发现可直接与相应的被称为核酸传感器分子,如TLR9、DAI、AIM2、STING、DDX41解旋酶和RNA聚合酶Ⅲ等结合,继而激活不同的免疫信号通路.基于DNA疫苗的传感器分子和信号通路研制免疫佐剂,可有效增强DNA疫苗的免疫原性.     

Regulation of cGAS-STING signalling in cancer: Approach for combination therapy

Innate immunity plays an important role not only during infection but also homeostatic role during stress conditions. Activation of the immune system including innate immune response plays a critical role in the initiation and progression of tumorigenesis. The innate immune sensor recognizes pathogen-associated molecular patterns (PAMPs) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (cGAS-STING) and induces type-1 immune response during viral and bacterial infection. cGAS-STING is regulated differently in conditions like cellular senescence and DNA damage in normal and tumor cells and is implicated in the progression of tumors from different origins. cGAS binds to cytoplasmic dsDNA and synthesize cyclic GMP-AMP (2’3’-cGAMP), which selectively activates STING and downstream IFN and NF-κB activation. We here reviewed the cGAS-STING signalling pathway and its cross-talk with other pathways to modulate tumorigenesis. Further, the review also focused on emerging studies that targeted the cGAS-STING pathway for developing targeted therapeutics and combinatorial regimens for cancer of different origins.     

Deubiquitinase USP35 restrains STING-mediated interferon signaling in ovarian cancer

Ovarian cancer is the most lethal malignant tumor of female reproductive system. It is well-known that induction of STING-mediated type I interferons can enhance the resultant antitumor activity. However, STING pathway is usually inactivated in cancer cells at multiple levels. Here, we identified deubiquitinase USP35 is upregulated in ovarian cancer tissues. High level of USP35 was correlated with diminished CD8⁺ T cell infiltration and poor prognosis in ovarian cancer patients. Mechanistically, we found that silencing USP35 reinforces the activation of STING-TBK1-IRF3 pathway and promotes the expression of type I interferons. Our data further showed that USP35 can directly deubiquitinate and inactivate STING. Interestingly, activation of STING promotes its binding to USP35 in a STING phosphorylation-dependent manner. Functionally, we found that knockdown of USP35 sensitizes ovarian cancer cells to the DNA-damage chemotherapeutic drug cisplatin. Overall, our study indicates that upregulation of USP35 may be a mechanism of the restricted STING activity in cancer cells, and highlights the significance of USP35 as a potential therapeutic target for ovarian cancer.Subject terms: Oncogenes, Cancer     

Linc00426 accelerates lung adenocarcinoma progression by regulating miR-455-5p as a molecular sponge

Increasing lines of evidence indicate the role of long non-coding RNAs (LncRNAs) in gene regulation and tumor development. Hence, it is important to elucidate the mechanisms of LncRNAs underlying the proliferation, metastasis, and invasion of lung adenocarcinoma (LUAD). We employed microarrays to screen LncRNAs in LUAD tissues with and without lymph node metastasis and revealed their effects on LUAD. Among them, Linc00426 was selected for further exploration in its expression, the biological significance, and the underlying molecular mechanisms. Linc00426 exhibits ectopic expression in LUAD tissues and cells. The ectopic expression has been clinically linked to tumor size, lymphatic metastasis, and tumor differentiation of patients with LUAD. The deregulation of Linc00426 contributes to a notable impairment in proliferation, invasion, metastasis, and epithelial–mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, the deregulation of Linc00426 could reduce cytoskeleton rearrangement and matrix metalloproteinase expression. Meanwhile, decreasing the level of Linc00426 or increasing miR-455-5p could down-regulate the level of UBE2V1. Thus, Linc00426 may act as a competing endogenous RNA (ceRNA) to abate miR-455-5p-dependent UBE2V1 reduction. We conclude that Linc00426 accelerates LUAD progression by acting as a molecular sponge to regulate miR-455-5p, and may be a potential novel tumor marker for LUAD.Subject terms: Prognostic markers, Tumour biomarkers     

INHA acts as a novel and potential biomarker in lung adenocarcinoma and shapes the immune-suppressive tumor microenvironment

BackgroundINHA expression has been correlated with the development, growth, and progression of multiple cancer types. However, the biological role of INHA has not been investigated in patients with lung adenocarcinoma (LUAD). Here, we performed a comprehensive bioinformatics analysis of the LUAD dataset to determine the mechanisms underlying the regulation of tumorigenesis by INHA.Materials and methodsINHA expression and clinical information of patients with LUAD were obtained from The Cancer Genome Atlas (TCGA) database. Protein levels in LUAD cell lines and human lung epithelial cells were examined by western blotting. Next, the prognostic value of INHA in LUAD was assessed using Cox regression analysis, while the potential biological functions and the impact on the immune microenvironment of INHA were investigated using gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA). Finally, the effect of INHA on LUAD cell proliferation and invasion was determined in vitro and in vivo.ResultsWe found significantly high mRNA and protein expression levels of INHA in LUAD tissues and cell lines. Additionally, a higher expression of INHA was linked to a shorter overall survival (OS) and a worse pathological stage, while INHA expression was associated with immune cell infiltration and immune-related markers in the LUAD tumor microenvironment. LUAD with high INHA expression tends to be a cold tumor. Furthermore, GO and KEGG enrichment analysis indicated that INHA-related genes were enriched in the cell adhesion and immune signaling pathways of LUAD. INHA promoted LUAD cell proliferation and invasion, in vitro and in vivo, by inducing the EGFR pathway.ConclusionOur findings revealed that INHA is overexpressed in LUAD and is linked to a poor prognosis. Our study demonstrates the potential of INHA as an immunotherapeutic and predictive biomarker in LUAD.