Nuclear Translocation Of p-STAT3 Research Articles

STAT3 Increases CVB3 Replication and Acute Pancreatitis and Myocarditis Pathology via Impeding Nuclear Translocation of STAT1 and Interferon-Stimulated Gene Expression.

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNβ as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Dimethyl itaconate inhibits antigen-specific Th17 cell responses and autoimmune inflammation via modulating NRF2/STAT3 signaling.

Pathogenic Th17 cells play a crucial role in autoimmune diseases like uveitis and its animal model, experimental autoimmune uveitis (EAU). Dimethyl itaconate (DMI) possesses potent anti-inflammatory effects. However, there is still a lack of knowledge about the role of DMI in regulating pathogenic Th17 cells and EAU. Here, we reported that intraperitoneal administration of DMI significantly inhibited the severity of EAU via selectively suppressing Th17 cell responses. Invitro antigen stimulation studies revealed that DMI dramatically decreased the frequencies and function of antigen-specific Th17, but not Th1, cells. Moreover, DMI hampered the differentiation of naive CD4+ T cells toward pathogenic Th17 cells. DMI-treated DCs produced less IL-1β, IL-6, and IL-23, and displayed an impaired ability to stimulate antigen-specific Th17 activation. Mechanistically, DMI activated the NRF2/HO-1 pathway and suppressed STAT3 signaling, which subsequently restrains p-STAT3 nuclear translocation, leading to decreased pathogenic Th17 cell responses. Thus, we have identified an important role for DMI in regulating pathogenic Th17 cells, supporting DMI as a promising therapy in Th17 cell-driven autoimmune diseases including uveitis.

α-Ketoglutaric acid ameliorates intervertebral disk degeneration by blocking the IL-6/JAK2/STAT3 pathway.

Intervertebral disk degeneration (IVDD) is the major cause of low back pain. Alpha-ketoglutaric acid (α-KG), an important intermediate in energy metabolism, has various functions, including epigenetic regulation, maintenance of redox homeostasis, and antiaging, but whether it can ameliorate IVDD has not been reported. Here, we examined the impacts of long-term administration of α-KG on aging-associated IVDD in adult rats. In vivo and in vitro experiments showed that α-KG supplementation effectively ameliorated IVDD in rats and the senescence of nucleus pulposus cells (NPCs). α-KG supplementation significantly attenuated senescence, apoptosis, and matrix metalloproteinase-13 (MMP-13) protein expression, and it increased the synthesis of aggrecan and collagen II in IL-1β-treated NPCs. In addition, α-KG supplementation reduced the levels of IL-6, phosphorylated JAK2 and STAT3, and the nuclear translocation of p-STAT3 in IL-1β-induced degenerating NPCs. The effects of α-KG were enhanced by AG490 in NPCs. The underlying mechanism may involve the inhibition of JAK2/STAT3 phosphorylation and the reduction of IL-6 expression. Our findings may help in the development of new therapeutic strategies for IVDD.NEW & NOTEWORTHY Alpha-ketoglutaric acid (α-KG) exerted its protective effect on nucleus pulposus cells' (NPCs) degeneration by inhibiting the senescence-associated secretory phenotype and extracellular matrix degradation. The possible mechanism may be associated with negatively regulating the JAK2/STAT3 phosphorylation and the decreased IL-6 expression, which could be explained by a blockage of the positive feedback control loop between IL-6 and JAK2/STAT3 pathway.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling

Ethnopharmacological relevanceCardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. Aim of this studyThe present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. MethodsThe structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. ResultsTwo new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. ConclusionTwo new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

Pretreatment with Eupatilin Attenuates Inflammation and Coagulation in Sepsis by Suppressing JAK2/STAT3 Signaling Pathway.

Sepsis is an aggressive and life-threatening organ dysfunction induced by infection. Excessive inflammation and coagulation contribute to the negative outcomes for sepsis, resulting in high morbidity and mortality. In this study, we explored whether Eupatilin could alleviate lung injury, reduce inflammation and coagulation during sepsis. We constructed an in vitro sepsis model by stimulating RAW264.7 cells with 1 μg/mL lipopolysaccharide (LPS) for 6 hours. The cells were divided into control group, LPS group, LPS+ Eupatilin (Eup) group, and Eup group to detect their cell activity and inflammatory cytokines and coagulation factor levels. Cells in LPS+Eup and Eup group were pretreated with Eupatilin (10μM) for 2 hours. In vivo, mice were divided into sham operation group, cecal ligation and puncture (CLP) group and Eup group. Mice in the CLP and Eup groups were pretreated with Eupatilin (10mg/kg) for 2 hours by gavage. Lung tissue and plasma were collected and inflammatory cytokines, coagulation factors and signaling were measured. In vitro, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and tissue factor (TF) expression in LPS-stimulated RAW264.7 cells was downregulated by Eupatilin (10μM). Furthermore, Eupatilin inhibited phosphorylation of the JAK2/STAT3 signaling pathway and suppressed p-STAT3 nuclear translocation. In vivo, Eupatilin increased the survival rate of the mice. In septic mice, plasma concentrations of TNF-α, IL-1β and IL-6, as well as TF, plasminogen activator inhibitor 1 (PAI-1), D-dimer, thrombin-antithrombin complex (TAT) and fibrinogen were improved by Eupatilin. Moreover, Eupatilin alleviated lung injury by improving the expression of inflammatory cytokines and TF, fibrin deposition and macrophage infiltration in lung tissue. Our results revealed that Eupatilin may modulate inflammation and coagulation indicators as well as improve lung injury in sepsis via the JAK2/STAT3 signaling pathway.

Bone Marrow Macrophages Induce Inflammation by Efferocytosis of Apoptotic Prostate Cancer Cells via HIF-1α Stabilization.

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present.

Effects of STAT3 Inhibitor BP-1-102 on The Proliferation, Invasiveness, Apoptosis and Neurosphere Formation of Glioma Cells in Vitro.

Malignant glioma, especially glioblastoma (GBM), has historically been associated with a low survival rate. The hyperactivation of STAT3 played a key role in GBM initiation and resistance to therapy; thus, there is an urgent requirement for novel STAT3 inhibitors. BP-1-102 was recently reported as a biochemical inhibitor of STAT3, but its roles and mechanism in biological behavior of glioma cells were still unclear. In this study, the effects of BP-1-102 on proliferation, apoptosis, invasion and neurosphere formation of glioma cell were investigated. Our results indicated that BP-1-102 inhibited the proliferation of U251 and A172 cells, and their IC50 values were 10.51 and 8.534 μM, respectively. Furthermore, BP-1-102 inhibited the invasion and migration abilities of U251 and A172 cells by decreasing the expression of matrix metallopeptidase 9, and induced glioma cell apoptosis by decreasing the expression of B-cell lymphoma-2. BP-1-102 also inhibited the formation of neurosphere. Mechanically, BP-1-102 reduced the phosphorylation of STAT3 and the p-STAT3's nuclear translocation in glioma cells. Thus, this study herein provided a potential drug for glioma therapy.

Higher efficacy of resveratrol against advanced breast cancer organoids: A comparison with that of clinically relevant drugs.

The lack of reliable drugs is a therapeutic challenge of advanced breast cancers (ABCs). Resveratrol (Res) exerts inhibitory effects on breast cancer cell lines and animal models, while its efficacy against individual breast cancer cases remains unknown. This study aims to use ABC-derived organoids (ABCOs) as the ex vivo therapeutic platform to clarify the effectiveness of resveratrol against different ABC subtypes. Immunohistochemical staining confirmed that the ABCOs maintained their original tumors' ER, PR, HER2, and Ki67 expression patterns. ABCO proliferation and viability tests showed >50% cell death rates in 79.2% (19/24) of Res-treated, 28.6% (2/7) fulvestrant-treated, 66.7% (4/6) paclitaxel-treated, and 66.7% (6/9) gemcitabine-treated ABCOs. pSTAT3 nuclear translocation was more frequent in Res-sensitive (17/19; 89.47%) than that (1/5; 20%) of Res-insensitive ABCOs, which were suppressed upon Res treatment. Statistical analysis revealed a close correlation of STAT3 activation with the efficacy of Res, but not related to tumor receptor expression patterns (ER, PR, HER2) and pathological classification. We demonstrate for the first time the higher efficacy and broader spectrum of Res against different subtypes of ABCOs in comparison with that of conventional antibreast cancer drugs, providing an alternative approach for better management of ABCs.

Vitamin E relieves chronic obstructive pulmonary disease by inhibiting COX2-mediated p-STAT3 nuclear translocation through the EGFR/MAPK signaling pathway

AbstractPatients with chronic obstructive pulmonary disease (COPD) are characterized by an imbalance between oxidant enzymes and antioxidant enzymes. In the present study, we explored the protective effect of vitamin E on COPD and the underlying mechanisms. Targets of vitamin E were predicted by bioinformatics analysis. After establishing cigarette smoke (CS)-induced COPD rats, the expression levels of epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX2), and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) were measured. Additionally, the effects of vitamin E on CS-induced COPD were explored by assessing inflammation, the reactive oxygen species (ROS), the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA), viability of human bronchial epithelioid (HBE) cells, and the expression of EGFR/MAPK pathway-related factors after loss- and gain- function assays. Vitamin E alleviated COPD. Vitamin E inhibited MAPK signaling pathway through decreasing EGFR expression. Additionally, vitamin E suppressed CS-induced HBE cell damage. Functionally, vitamin E attenuated CS-induced inflammation, apoptosis, and ROS by inhibiting the EGFR/MAPK axis, thereby inhibiting COX2-mediated p-STAT3 nuclear translocation. Moreover, overexpression of COX2 attenuated the protective effect of vitamin E on COPD rats. The present study shows that vitamin E inhibits the expression of COX2 by negatively regulating the EGFR/MAPK pathway, thereby inhibiting the translocation of phosphorylated STAT3 to the nucleus and relieving COPD.This paper provides new insights into the potential therapeutic effects of vitamin E on COPD. The authors show that vitamin E relieves chronic obstructive pulmonary disease (COPD) by negatively regulating the EGFR/MAPK pathway and inhibiting COX2-mediated translocation of phosphorylated STAT3 to the nucleus. Moreover, overexpression of COX2 reverses the protective effect of vitamin E in a rat model of COPD.

Mitigation Effects of Selenium Nanoparticles on Depression-Like Behavior Induced by Fluoride in Mice via the JAK2-STAT3 Pathway.

Depression is a mental health problem with typically high levels of distress and dysfunction, and 150 mg/L fluoride (F) can induce depression-like behavior. The development of depression is correlated with neuronal atrophy, insufficient secretion of monoamine neurotransmitters, extreme deviations from the normal microglial activation status, and immune-inflammatory response. Studies found that Se supplementation was related to the improvement of depression. In this study, we applied selenium nanoparticles (SeNPs) for F-induced depression disease mitigation by regulating the histopathology, metabolic index, genes, and protein expression related to the JAK2-STAT3 signaling pathway in vivo. Results showed that F and 2 mg Se/kg BW/day SeNPs lowered the dopamine (DA) content (P < 0.05), altered the microglial morphology, ramification index as well as solidity, and triggered the microglial neuroinflammatory response by increasing the p-STAT3 nuclear translocation (P < 0.01). Furthermore, F reduced the cortical Se content and the number of surviving neurons (P < 0.05), increasing the protein expressions of p-JAK2/JAK2 and p-STAT3/STAT3 of the cortex (P < 0.01), accompanied by the depression-like behavior. Importantly, 1 mg Se/kg BW/day SeNPs alleviated the microglial ramification index as well as solidity changes and decreased the interleukin-1β secretion induced by F by suppressing the p-STAT3 nuclear translocation (P < 0.01). Likewise, 1 mg Se/kg BW/day SeNPs restored the F-disturbed dopamine and noradrenaline secretion, increased the number of cortical surviving neurons, and reduced the vacuolation area, ultimately suppressing the occurrence of depression-like behavior through inhibiting the JAK2-STAT3 pathway activation. In conclusion, 1 mg Se/kg BW/day SeNPs have mitigation effects on the F-induced depression-like behavior. The mechanism of how SeNPs repair neural functions will benefit depression mitigation. This study also indicates that inhibiting the JAK/STAT pathway can be a promising novel treatment for depressive disorders.

Remodeling Chondroitin-6-Sulfate-Mediated Immune Exclusion Enhances Anti-PD-1 Response in Colorectal Cancer with Microsatellite Stability.

Metastatic microsatellite-stable (MSS) colorectal cancer rarely responds to immune checkpoint inhibitors (ICI). Metabolism heterogeneity in the tumor microenvironment (TME) presents obstacles to antitumor immune response. Combining transcriptome (The Cancer Genome Atlas MSS colorectal cancer, n = 383) and digital pathology (n = 96) analysis, we demonstrated a stroma metabolism-immune excluded subtype with poor prognosis in MSS colorectal cancer, which could be attributed to interaction between chondroitin-6-sulfate (C-6-S) metabolites and M2 macrophages, forming the "exclusion barrier" in the invasive margin. Furthermore, C-6-S derived from cancer-associated fibroblasts promoted co-nuclear translocation of pSTAT3 and GLI1, activating the JAK/STAT3 and Hedgehog pathways. In vivo experiments with C-6-S-targeted strategies decreased M2 macrophages and reprogrammed the immunosuppressive TME, leading to enhanced response to anti-PD-1 in MSS colorectal cancer. Therefore, C-6-S-induced immune exclusion represents an "immunometabolic checkpoint" that can be exploited for the application of combination strategies in MSS colorectal cancer ICI treatment.

Activation of CD44-Lipoprotein lipase axis in breast cancer stem cells promotes tumorigenesis

Breast cancer stem cells (CSCs) are distinct CD44+-subpopulations that are involved in metastasis and chemoresistance. However, the underlying molecular mechanism of CD44 in breast CSCs-mediated tumorigenesis remains elusive. We observed high CD44 expression in advanced-stage clinical breast tumor samples. CD44 activation in breast CSCs sorted from various triple negative breast cancer (TNBC) cell lines induced proliferation, migration, invasion, mammosphere formation that were reversed in presence of inhibitor, 4-methyl umbelliferone or CD44 silencing. CD44 activation in breast CSCs induced Src, Akt, and nuclear translocation of pSTAT3. PCR arrays revealed differential expression of a metabolic gene, Lipoprotein lipase (LPL), and transcription factor, SNAI3. Differential transcriptional regulation of LPL by pSTAT3 and SNAI3 was confirmed by promoter-reporter and chromatin immunoprecipitation analysis. Orthotopic xenograft murine breast tumor model revealed high tumorigenicity of CD24−/CD44+-breast CSCs as compared with CD24+-breast cancer cells. Furthermore, stable breast CSCs-CD44 shRNA and/or intratumoral administration of Tetrahydrolipstatin (LPL inhibitor) abrogated tumor progression and neoangiogenesis. Thus, LPL serves as a potential target for an efficacious therapeutics against aggressive breast cancer.

Da Cheng Qi Decoction Alleviates Cerulein-Stimulated AR42J Pancreatic Acinar Cell Injury via the JAK2/STAT3 Signaling Pathway.

Background Acute pancreatitis (AP) is a common acute abdomen inflammation, characterized by the dysregulation of digestive enzyme production and secretion. Many studies have shown that Da Cheng Qi Decoction (DCQD) is a secure, effective prescription on AP. In this study, cerulein-stimulated AR42J cells damage model was established to further explore the feasibility and underlying mechanism of DCQD as a potential inhibitor of JAK2/STAT3 pathway for the treatment of AP. Methods Cell viability of DCQD was measured using a cell counting Kit-8 assay. Pancreatic biochemical markers such as amylase, lipase, and C-reactive protein production were measured by assay kits, respectively. Cytokines (TNF-α, IL-6, IL-10, and IL-1β) were assayed by ELISA. Protein location and protein expression were detected by immunofluorescence staining and Western blotting, respectively. Gene expression was assessed by real-time PCR. For mechanistic analysis of the effect of DCQD on JAK2/STAT3 signaling pathway, selective JAK2 inhibitor (Fedratinib) and STAT3 inhibitor (Stattic) as well as STAT3 activator (Garcinone D) were used. Results DCQD protected cells by regulating cerulein-induced inflammation and reducing the secretion of pancreatic biochemical markers. Moreover, DCQD could not only inhibit the nuclear translocation of p-STAT3, but also decrease the mRNA expression of JAK2 and STAT3 as well as the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in protein level. Additionally, DCQD could regulate the mRNA and protein expression of JAK2/STAT3 downstream effectors, Bax and Bcl-XL. The activated effect of cerulein on JAK2/STAT3 pathway was also reversed by JAK2 inhibitor Fedratinib or STAT3 inhibitor Stattic. And the overexpression of JAK2/STAT3 pathway, via STAT3 activator Garcinone D, did exert damage on cells, which bore a resemblance to cerulein. Conclusion The activation of JAK2/STAT3 pathway may play a key role in the pathogenesis of cerulein-stimulated AR42J pancreatic acinar cell injury. DCQD could improve inflammatory cytokines and cell injury, which might be mediated by suppressing the activation of JAK2/STAT3 signaling pathway.

THAP9-AS1 Promotes Tumorigenesis and Reduces ROS Generation through the JAK2/STAT3 Signaling Pathway by Increasing SOCS3 Promoter Methylation in Osteosarcoma.

Increasing studies have demonstrated that dysfunction of long noncoding RNAs (lncRNAs) plays critical roles in the development of human cancers. THAP9-AS1 has been reported to be dysregulated and associated with tumor progression in some cancers. However, the function and mechanism of THAP9-AS1 in osteosarcoma (OS) remain unclear. In the present study, we found that the expression of THAP9-AS1 was significantly upregulated in OS tissues and associated with the advanced stage of tumors and poor prognosis of patients. Blast comparison results showed that the SOCS3 promoter region and THAP9-AS1 had base complementary pairing binding sites. The interactions between THAP9-AS1, DNA methyltransferases (DNMTs), and SOCS3 were assessed by RIP and ChIP assays. The results of methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) validated that THAP9-AS1 enhanced the methylation level of the SOCS3 promoter. The mRNA levels of SOCS3 in OS cells could be reversed by the demethylation agent 5-aza-2′-deoxycytidine. The mRNA expression of SOCS3 was downregulated in OS tissues and negatively correlated with THAP9-AS1 expression in tumors. Moreover, the western blot and immunofluorescence (IF) assay data showed that THAP9-AS1 activated the JAK2/STAT3 signaling pathway by upregulating p-JAK2 and p-STAT3 and the nuclear translocation of p-STAT3. Functionally, ectopic expression of THAP9-AS1 promoted cell proliferation, migration, and invasion and inhibited apoptosis, and this phenomenon could be reversed by SOCS3. Introduction of the JAK/STAT inhibitor AG490 partially abolished the stimulative effect of THAP9-AS1 on cellular processes. In addition, THAP9-AS1 decreased oxidative stress by reducing reactive oxygen species (ROS) and enhancing the mitochondrial membrane potential of OS cells via the SOCS3/JAK2/STAT3 pathway. Stable overexpression of THAP9-AS1 contributed to tumor growth and metastasis in vivo. In total, our findings suggested that upregulation of THAP9-AS1 might recruit DNMTs to epigenetically inhibit SOCS3, thereby activating the JAK2/STAT3 signaling pathway and oncogenesis of OS. These results provide novel insights for the understanding of OS progression.

STAT3 signaling statuses determine the fate of resveratrol-treated anaplastic thyroid cancer cells.

Anaplastic thyroid cancer/ATC is highly lethal malignancy without reliable chemotherapeutic drug. Resveratrol possesses anti-ATC activities but encounters resistance in some cases due to certain unknown reason(s). Because signal transducer and activator of transcription/STAT3 signaling is critical for ATC cell survival and the main molecular target of resveratrol, its roles in determining the fates of resveratrol-treated ATC cells were investigated here. Human THJ-11T, THJ-16 and THJ-21T ATC cell lines were treated by 100 μM resveratrol and their growth, statuses of STAT3 signaling and STAT3-related gene expression were examined. The relevance of STAT3 activation with resveratrol resistance was elucidated using STAT selective inhibitor AG490. Leukemia inhibitory factor/LIF expression and phosphorylated-STAT3/p-STAT3 nuclear translocation in ATC tissues were immunohistochemically analyzed. Resveratrol inhibited proliferation, p-STAT3 nuclear translocation as well as LIF and STAT3 expression of THJ-16T and THJ-21T but not THJ-21T cells which showed LIF upregulation and more frequent p-STAT3 nuclear translocation. AG490 significantly prevent p-STAT3 nuclear translocation, and reversed the resveratrol tolerance of THJ-11T cells. Immonohistochemical staining revealed 14.3% (4/28) of LIF and 3.6% (1/28) of p-STAT3 detection in noncancerous ATC-surrounding tissues, which increased to 89.5% (17/19) and 52.6% (10/19) respectively among ATC specimens. The correlative analysis indicated the relevance of LIF expression and STAT3 activation (r= 0.825; P< 0.01). The status of STAT3 activation and LIF expression are closely correlated with the therapeutic effect of resveratrol on ATCs. Frequent LIF upregulation and STAT3 activation are the unfavorable factors of ATCs and the potential targets of anti-ATC therapy.

TSLP regulates eotaxin-1 production by nasal epithelial cells from patients with eosinophilic CRSwNP.

Eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) is characterized by Th2-skewed inflammation with eosinophilic infiltration. Thymic stromal lymphopoietin (TSLP) promotes the development of allergic inflammation. Although increased TSLP is found in eCRSwNP, little is known about whether TSLP regulates eotaxin-1 production, a potent eosinophil chemoattractant that recruits and activates eosinophils. The aim of this study was to investigate the effects and mechanisms of TSLP in eotaxin-1 production in the eosinophilic inflammation of eCRSwNP. Human nasal epithelial cells (HNECs) from eCRSwNP patients were stimulated with recombinant human TSLP in the presence or absence of CYT387 (Janus kinase 1/2 inhibitor). Phosphorylated signal transducer activator of transcription 3 (p-STAT3) was measured by using immunocytochemistry. Eotaxin-1 expression was determined by using real-time PCR. Western blotting was used to detect the levels of p-STAT3 and eotaxin-1 protein. The treatment with TSLP induced STAT3 phosphorylation in HNECs, and promoted p-STAT3 nuclear translocation, leading to a time-dependent increase of eotaxin-1 expression. However, these effects were attenuated by CYT387 pretreatment. TSLP regulated eotaxin-1 production in HNECs via JAK1/2-STAT3 signaling, which might contribute to the eosinophilic inflammation of eCRSwNP.

Abstract 5436: Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells

Abstract BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in immune defense and plays an important role in biologic activities of cells including tumor cells. These effects are mediated by several signaling pathways, including the signal transducer and transcription activator 3 (STAT3), which plays a critical role in the development, growth, and metastasis of cancer. Inside cancer cells within the tumor microenvironment IL-6 activates (phosphorylates) STAT3. The P-STAT3 dimers translocate into the nucleus where it turns on many genes (e.g., BCL-XL, MCL-1, Myc, Cyclin D1/D2, VEGF, MMP-2, etc.) that are necessary for tumorigenesis. Since there are no commercially available IL-6 or STAT3 inhibitors, blocking IL-6/STAT3 signaling is a potential therapeutic strategy for cancer. SBT-100 is a novel sdAb which binds P-STAT3 to inhibit its translocation into the nuclei of cancer cells, thereby suppressing the cancers growth. METHODS: Human cancer cell lines obtained from ATCC. The STAT3 reporter assay (Promega, WI). IHC staining with IL-6 stimulation, the primary antibody (Ab) was STAT3 (124H6) mouse mAb (Cell Signaling), secondary Ab was anti-mouse IgG (H&amp;L), Alexa Fluor 488 (Cell Signaling), blocking Ab was SBT-100, and recombinant human IL-6 was from Peprotech. In vitro cancer cell suppression via MTT assay (3 day). Xenograft study, athymic nude mice were obtained from Envigo. RESULTS: The STAT3 reporter assay demonstrates that IL-6 activation of STAT3 is completely inhibited by 100ug/ml of SBT-100 (p&amp;lt;0.0012). SBT-100 shows significant (p&amp;lt;0.001) suppression of ten different human cancers in vitro which have constitutive expression of P-STAT3. Immunohistochemical (IHC) staining demonstrates that SBT-100 crosses the cell membrane and localizes within the cytoplasm of MDA-MB-231 cells. IHC staining of STAT3 shows IL-6 stimulation drives P-STAT3 into the nuclei of HEp-2 and PANC-1 cells and this translocation is inhibited by SBT-100. In vivo xenograft studies demonstrate that SBT-100 gives significant (p&amp;lt;0.001) tumor growth suppression of MDA-MB-231. CONCLUSION: SBT-100 inhibits IL-6 mediated function of P-STAT3 as demonstrated by two different assays. IHC staining demonstrates SBT-100 crosses the cell membrane to localize inside cancer cells. Based on its mechanism of action, SBT-100 can be a potential antibody targeted IL-6/STAT3 therapy for human cancer. Citation Format: Sunanda Singh, Genoveva Murillo, Avani Singh, Samara Singh, Meenakshi Parihar, Rajendra Mehta, Anjali Singh, Ashutosh Parihar. Novel single-domain antibody (sdAb), SBT-100, localizes in the cytoplasm to inhibit IL-6 mediated P-STAT3 nuclear translocation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5436.

Abstract 470: Epstein-Barr virus-encoded miR-BART5 modulates PIAS3-pSTAT3 and p21waf1 in gastric carcinoma cells

Abstract The ubiquitous Epstein-Barr virus (EBV) is associated with a subset of gastric carcinomas. MicroRNAs (miRs) are 22±2 nucleotide non-coding RNAs, and regulate various functions in cells. EBV-encoded miRs in gastric carcinoma cells have been identified, but the targets and roles of EBV-encoded miRs remain elusive. In the present study, we investigated cellular target of miR-BART5, and its possibility as an oncomiR. In naturally EBV-infected gastric carcinoma cells and EBV-positive gastric carcinoma tissues, the comprehensive EBV-miR profile revealed the expression of 22 EBV miRs (each having -3p and -5p) composed of miR-BART cluster 1 and cluster 2, without miR-BHRFs. Using bioinformatics analysis, we focused on miR-BART5 which shared seed sequence homology with hsa-miR-18a and miR-18b having oncomiR function, and protein inhibitor of activated STAT3 (PIAS3) mRNA. The western blot results confirmed that PIAS3 protein expression was reduced in miR-BART5-expressing gastric carcinoma cells (EBV-negative gastric carcinoma cells transfected with miR-BART5), compared with miR-control mock cells. Also PIAS3 expression was significantly lower in naturally EBV-infected gastric carcinoma cells than in EBV-negative gastric carcinoma or EBV-infected lymphoma cells. Moreover, there was a statistically significant inverse correlation between the expression of miR-BART5 and PIAS3. The luciferase reporter activity in cells diminished after co-transfection of pEZX-MT06 vector-PIAS3 3′UTR and miR-BART5, compared with co-transfection of empty vector and miR-control or co-transfection of empty vector and miR-BART5. When we tested twenty proteins as downstream candidates of miR-BART5, pSTAT3 increased and p21waf1 decreased in miR-BART5-expressing gastric carcinoma cells, compared with mock cells, and nuclear translocation of pSTAT3 was observed in miR-BART5-expressing gastric carcinoma cells. In a reverse context that naturally EBV-infected gastric carcinoma cells were transfected with anti-miR-BART5, protein levels were changed likewise; PIAS3 increased, pSTAT3 decreased and p21waf1 increased. However, there were no statistically significant differences in cellular biologic properties such as proliferation, apoptosis, invasion and migration between miR-BART5-expressing gastric carcinoma cells and mock cells, or between naturally EBV-infected gastric carcinoma cells transfected with anti-miR-BART5 and mock cells. Taken together, miR-BART5 not only targets directly PIAS3 and then modulates PIAS3-pSTAT3 axis, but also decreases p21waf1 protein, although it fails to alter cellular proliferation in gastric carcinoma cells. This suggests that a single viral microRNA is not sufficient to implement oncogenic function, despite directly targeting cellular protein involved in oncogenesis. Instead, a whole cluster like miR-BART cluster 1 or cluster 2 may be advocate to control cellular biologic property. Citation Format: Dong Ha Kim, Chan Jin Yoon, Jin-Seoub Kim, Sunyoung Park, Euno Choi, Jun Hee Woo, Mee Soo Chang. Epstein-Barr virus-encoded miR-BART5 modulates PIAS3-pSTAT3 and p21waf1 in gastric carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 470. doi:10.1158/1538-7445.AM2017-470

The Inhibitory Effects of HJC0416 on Liver Fibrogenesis in Activated Hepatic Stellate Cells

IntroductionThe STAT3 signaling pathway regulates activation of hepatic stellate cells (HSCs) and promotes its fibrogenic behavior. Using a fragment‐based drug design approach, our group has developed HJC0416, a novel orally bioavailable small‐molecule inhibitor of STAT3. HJC0416 was found to induce apoptosis and decreased tumor growth in breast cancer models. In the present study, we studied the antifibrogenic properties of HJC0416 in activated hepatic stellate cells (HSC), which are the major effector cells of liver fibrosis.MethodsHuman (LX‐2) and rat (HSC‐T6) hepatic stellate cell lines were used. Cell proliferation was measured by Alamar Blue assay. Cellular protein expression levels were determined by Western blotting and immunofluorescence. Cell cycle was assessed by flow cytometry. Yo‐pro‐1 staining was used to measure apoptosis.ResultsHJC0416 treatment significantly inhibited LX‐2 and HSC‐T6 cell proliferation in a dose‐dependent fashion, and induced apoptosis and S‐phase cell cycle arrest. HSC activation marker α‐smooth muscle actin was also attenuated. HJC0416 suppressed Tyr705‐phosphorylated STAT3 (pSTAT3) and impaired pSTAT3 nuclear translocation and transcriptional activity. STAT3 target genes cyclin D1 and c‐myc were down‐regulated by HJC0416. Static, a well‐known STAT3 inhibitor, confirmed that HSC proliferation was suppressed by STAT3 inhibition. Extracellular matrix (ECM) proteins collagen type I (Coll.I) and fibronectin (FN) are major components of hepatic scar, with TGFβ being the most potent stimulator for ECM production. Our data revealed that endogenous Coll.I and FN expression were suppressed by HJC0416 in a dose‐dependent manner; TGFβ‐stimulated Coll.I and FN production were prevented by pretreatment with HJC0416.ConclusionHJC0416 inhibits HSC activation via the STAT3 signaling pathway. HJC0416 represents a promising anti‐hepatic fibrogenic agent.Support or Funding InformationThis work was supported by grants P50 CA097007, P30 DA028821, R21 MH098344 (JZ) from the National Institutes of Health, Cancer Prevention Research Institute of Texas award, R. A. Welch Foundation Chemistry and Biology Collaborative Grant (JZ) from the Gulf Coast Consortia, and John Sealy Memorial Endowment Fund and the Center for Addiction Research (JZ) from the University of Texas Medical Branch.