STAT3 Activation In Response Research Articles

IL-6/gp130/STAT3 signaling contributed to the activation of the PERK arm of the unfolded protein response in response to chronic β-adrenergic stimulation

Prolonged activation of the PERK branch of the unfolded protein response (UPR) promotes cardiomyocytes apoptosis in response to chronic β-adrenergic stimulation. STAT3 plays a critical role in β-adrenergic functions in the heart. However, whether STAT3 contributed to β-adrenoceptor-mediated PERK activation and how β-adrenergic signaling activates STAT3 remains unclear. This study aimed to investigate whether STAT3-Y705 phosphorylation contributed to the PERK arm activation in cardiomyocytes and if IL-6/gp130 signaling was involved in the chronic β-AR-stimulation-induced STAT3 and PERK arm activation. We found that the PERK phosphorylation was positively associated with STAT3 activation. Wild-type STAT3 plasmids transfection activated the PERK/eIF2α/ATF4/CHOP pathway in cardiomyocytes while dominant negative Y705F STAT3 plasmids caused no obvious effect on PERK signaling. Stimulation with isoproterenol produced a significant increase in the level of IL-6 in the cardiomyocyte's supernatants, while IL-6 silence inhibited PERK phosphorylation but failed to attenuate STAT3 activation in response to isoproterenol stimulation. Gp130 silence attenuated isoproterenol-induced STAT3 activation and PERK phosphorylation. Inhibiting IL-6/gp130 pathway by bazedoxifene and inhibiting STAT3 by stattic both reversed isoproterenol-induced STAT3-Y705 phosphorylation, ROS production, PERK activation, IRE1α activation, and cardiomyocytes apoptosis in vitro. Bazedoxifene (5 mg/kg/day by oral gavage once a day) exhibited similar effect as carvedilol (10 mg/kg/day by oral gavage once a day) on attenuating chronic isoproterenol (30 mg/kg by abdominal injection once a day, 7 days) induced cardiac systolic dysfunction, cardiac hypertrophy and fibrosis in C57BL/6 mice. Meanwhile, bazedoxifene attenuates isoproterenol-induced STAT3-Y705 phosphorylation, PERK/eIF2α/ATF4/CHOP activation, IRE1α activation, and cardiomyocytes apoptosis to a similar extend as carvedilol in the cardiac tissue of mice. Our results showed that chronic β-adrenoceptor-mediated stimulation activated the STAT3 and PERK arm of the UPR at least partially via IL-6/gp130 pathway. Bazedoxifene has great potential to be used as an alternative to conventional β-blockers to attenuate β-adrenoceptor-mediated maladaptive UPR.

TRAF3 regulates IL-6 receptor signaling in T cells through the phosphatase PTPN22

Abstract IL-6 is an important signal three cytokine for T helper 17 (Th17) and T follicular helper (Tfh) differentiation. Previous work has suggested a possible role for TRAF3 in the function and/or differentiation of these two subsets, but no direct studies have yet been performed. In this study, we examine the role of TRAF3 in IL-6R signaling in T cells. TRAF3 regulates IL-2R and type I interferon receptor signaling in T cells by altering recruitment of the phosphatases PTPN2 and PTPN22, respectively. B cell TRAF3 curtails IL-6R signaling by recruiting PTPN22, thus we hypothesized that TRAF3 regulates T cell IL-6R signaling through a PTPN22-dependent mechanism. We investigated the role of T cell TRAF3 using a TRAF3-deficient human T cell lymphoma line and a T cells from a T cell-specific TRAF3 knockout mouse (T-Traf3−/−). We found reduced IL-6-induced STAT1 and STAT3 activation in human and mouse T cells. STAT1 and STAT3 activation are both required for full differentiation of mouse Tfh cells, whereas STAT3 is required for and STAT1 antagonizes Th17 differentiation. Interestingly, in vitrodifferentiation of naïve CD4 T cells to Th17 cells was not affected by TRAF3 deficiency. To determine the mechanism by which TRAF3 promotes STAT1 and STAT3 activation in response to IL-6R signaling, we treated WT and Traf3−/−T cells with IL-6 in the presence of PTPN22 inhibitor or vehicle control. We found that PTPN22 inhibition restored activation of STAT3, but did not restore activation of STAT1, suggesting different mechanisms of regulation. Together these data implicate TRAF3 in regulation of IL-6R signaling in a partly PTPN22-dependent manner, lending an additional level of understanding to TRAF3 regulation of Th17 and Tfh differentiation. Supported by NIH R01 AI123107, R01 AI162656, T32 AI007260

Matrix protein Tenascin-C promotes kidney fibrosis via STAT3 activation in response to tubular injury

Accumulating evidence indicates that the extracellular matrix (ECM) is not only a consequence of fibrosis, but also contributes to the progression of fibrosis, by creating a profibrotic microenvironment. Tenascin-C (TNC) is an ECM glycoprotein that contains multiple functional domains. We showed that following kidney injury, TNC was markedly induced in fibrotic areas in the kidney from both mouse models and humans with kidney diseases. Genetically deletion of TNC in mice significantly attenuated unilateral ureteral obstruction-induced kidney fibrosis. Further studies showed that TNC promoted the proliferation of kidney interstitial cells via STAT3 activation. TNC-expressing cells in fibrotic kidney were activated fibroblast 2 (Act.Fib2) subpopulation, according to a previously generated single nucleus RNA-seq dataset profiling kidney of mouse UUO model at day 14. To identify and characterize TNC-expressing cells, we generated a TNC-promoter-driven CreER2-IRES-eGFP knock-in mouse line and found that the TNC reporter eGFP was markedly induced in cells around injured tubules that had lost epithelial markers, suggesting TNC was induced in response to epithelium injury. Most of the eGFP-positive cells were both NG2 and PDGFRβ positive. These cells did not carry markers of progenitor cells or macrophages. In conclusion, this study provides strong evidence that matrix protein TNC contributes to kidney fibrosis. TNC pathway may serve as a potential therapeutic target for interstitial fibrosis and the progression of chronic kidney disease.

Helicobacter pylori Infection of Primary Human Monocytes Boosts Subsequent Immune Responses to LPS.

Infection with Helicobacter pylori (H. pylori) affects almost half of the world’s population and is a major cause of stomach cancer. Although immune cells react strongly to this gastric bacterium, H. pylori is still one of the rare pathogens that can evade elimination by the host and cause chronic inflammation. In the present study, we characterized the inflammatory response of primary human monocytes to repeated H. pylori infection and their responsiveness to an ensuing bacterial stimulus. We show that, although repeated stimulations with H. pylori do not result in an enhanced response, H. pylori-primed monocytes are hyper-responsive to an Escherichia coli-lipopolysaccharide (LPS) stimulation that takes place shortly after infection. This hyper-responsiveness to bacterial stimuli is observed upon infection with viable H. pylori only, while heat-killed H. pylori fails to boost both cytokine secretion and STAT activation in response to LPS. When the secondary challenge occurs several days after the primary infection with live bacteria, H. pylori-infected monocytes lose their hyper-responsiveness. The observation that H. pylori makes primary human monocytes more susceptible to subsequent/overlapping stimuli provides an important basis to better understand how H. pylori can maintain chronic inflammation and thus contribute to gastric cancer progression.

Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFR\u03b2 and affect PDGF-BB-induced STAT3 signalling

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor β (PDGFRβ) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRβ did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRβ lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

BackgroundH. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models.MethodsTo investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays.ResultsFollowing infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models.ConclusionOur findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

Bazedoxifene inhibits sustained STAT3 activation and increases survival in GBM

An important factor correlated with poor survival in glioblastoma (GBM) is the aberrant and persistent activation of STAT3, a critical transcription factor that regulates multiple genes with key roles in cell survival, proliferation, resistance to chemotherapy, and stem cell maintenance. The Interleukin-6 (IL6)-STAT3 signaling axis has been studied extensively in inflammation and cancer. However, it is not completely understood how high levels of activated STAT3 are sustained in tumors. Previously, we identified a novel mechanism of biphasic activation of STAT3 in response to gp130-linked cytokines, including IL6, in which activation of STAT3 is prolonged by circumventing the negative regulatory mechanisms induced by its initial activationTo target prolonged STAT3 activation, we used the small molecule inhibitor bazedoxifene (BZA), which blocks formation of the IL6 receptor-gp130 complex. Glioma stem-like cells (GSCs) are more tumorigenic and more resistant to therapy. STAT3 is a key driver of the expression of stem cell transcription factors, making it a therapeutically important target in GBM. We show that treating GSCs with BZA decreases their self-renewal capacity and the expression of GSC markers in vitro. Additionally, BZA crosses the blood-brain barrier and confers a survival advantage in an orthotopic syngeneic mouse model of GBM. Although IL6-STAT3 signaling is important for GSC survival, a therapeutic agent that inhibits this pathway without toxicity has yet to be identified. Our findings reveal a mechanism of sustained STAT3 signaling in GBM and reveal its role in GSC maintenance, and we identify BZA as a novel candidate for treating GBM.

Cutting Edge: IL-6-Driven Immune Dysregulation Is Strictly Dependent on IL-6R α-Chain Expression.

IL-6 binds to the IL-6R α-chain (IL-6Rα) and signals via the signal transducer gp130. Recently, IL-6 was found to also bind to the cell surface glycoprotein CD5, which would then engage gp130 in the absence of IL-6Rα. However, the biological relevance of this alternative pathway is under debate. In this study, we developed a mouse model, in which murine IL-6 is overexpressed in a CD11c-Cre-dependent manner. Transgenic mice developed a lethal immune dysregulation syndrome with increased numbers of Ly-6G+ neutrophils and Ly-6Chi monocytes/macrophages. IL-6 overexpression promoted activation of CD4+ T cells while suppressing CD5+ B-1a cell development. However, additional ablation of IL-6Rα protected IL-6-overexpressing mice from IL-6-triggered inflammation and fully phenocopied IL-6Rα-deficient mice without IL-6 overexpression. Mechanistically, IL-6Rα deficiency completely prevented downstream activation of STAT3 in response to IL-6. Altogether, our data clarify that IL-6Rα is the only biologically relevant receptor for IL-6 in mice.

MEK inhibition activates STAT signaling to increase breast cancer immunogenicity via MHC-I expression.

Aim: Immunotherapy and immune checkpoint inhibitors (ICI) have changed cancer care for many patients; however, breast cancers have exhibited minimal response to single agent ICI therapy. There is a significant need to identify novel targets capable of increasing cancer cell immunogenicity and response to ICIs in breast cancer. Mitogen activated protein kinase (MAPK) signaling is essential for many cellular processes but the relationship between MAPK signaling and cancer cell immunogenicity is less well understood. Recent reports suggest that MEK inhibition (MEKi) affects the tumor-immune microenvironment by altering the expression of interferon responsive PD-L1 and MHC-I through unknown mechanisms.Methods: Using western blotting and flow cytometry, we sought to determine whether MEKi affects JAK-STAT signaling upstream of PD-L1 and MHC-I expression in a panel of mouse mammary cancer and triple negative breast cancer cell lines.Results: The cell lines tested exhibited increased STAT activation in response to MEKi treatment. Furthermore, MEKi-induced MHC-I and PD-L1 expression are dependent upon STAT1 in MMTV-Neu cells. Interestingly, MEKi-induced STAT activation and interferon-responsive protein expression are abrogated with ErbB-family inhibitor co-treatment in MMTV-Neu cells, suggesting ErbB receptor signaling dependence, but not in basal-like cell lines. Importantly, analysis of basal-like breast cancer patient samples exhibited an inverse relationship between STAT1 and Ras/MAPK activation signatures.Conclusion: These findings suggest that MAPK signaling and STAT activation are inversely related in both mouse and human mammary tumors. This work also supports further study of MEKi to increase STAT signaling and potentially, immunotherapy responses through increased MHC-I and PD-L1 expression.

112-OR: Critical Role of Tyrosine Kinase 2 (TYK2) in Human Pancreatic Differentiation

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Genome wide association and fine mapping studies to identify causal variants predicted several loci in the human genome that are associated with T1D risk. One such candidate gene is a member of the JAK (Janus Kinase) family of tyrosine kinases, TYK2, which plays a critical role in intracellular signaling stimulated by cytokines through STATs. Loss-of-function variants of TYK2 are associated with protection against T1D. In line with this, silencing of TYK2 in beta cells attenuated inflammatory signaling pathways and apoptosis. To study the role of TYK2 in human beta-cell development, we now generated TYK2- knockout (KO) human iPSC lines using CRISPR-Cas9 genome editing and induced them to differentiate to pancreatic endocrine lineage. Our results suggest that absence of TYK2 does not compromise the pluripotency or the early pancreatic endoderm differentiation potential of iPSC. However, in the later stages of differentiation, the mRNA levels of key pancreatic TFs (PDX1(55±17%, p=0.03), NGN3(65±13%, p=0.008)), RFX6(58±14%, p=0.014), NKX6.1(39±19%, p=0.114) and NKX2.2(68±7%, p=0.0008)) were significantly reduced in the TYK2-KO cells. In the final stages of controlled induction to endocrine lineage TYK2-KO lines expressed lower levels of INS(53±9%, p=0.004), GCG(70±10.5%, p=0.002) and SST(62±8.6%, p=0.002) compared to control lines (mean ± SEM of 3 independent experiments). Furthermore immunostaining for INS, GCG and SST confirmed a lower number of endocrine hormone positive cells in TYK2-KO lines. In contrast to the wild type controls, the TYK2-KO iPSC-derived pancreatic cells displayed no activation of STAT1 and STAT2 and showed impaired STAT3 activation in response to IFNαor stimulation by polyinosinic-polycitidilic acid treatment, a viral infection mimetic. The present study suggests that TYK2 plays an important early role in pancreatic endocrine differentiation. Disclosure V. Chandra: None. H. Ibrahim: None. D. Balboa: None. T. Otonkoski: None.

IL-6 overexpression triggers inflammation through its only relevant receptor IL-6R

Abstract IL-6 is a pleiotropic cytokine that regulates development and function of variety immune cells. Here we used a novel mouse strain in which IL-6 can be overexpressed in a Cre-dependent manner and show that mice with CD11c-Cre mediated IL-6 overexpression succumb from systemic inflammation. High levels of IL-6 perturbed B and T cell development in primary lymphoid organs, bone marrow and thymus. Interestingly, IL-6-triggered inflammation promoted expansion of both Th17 and Treg cells, two cell types known for their reciprocal developmental requirements. However, the most dramatic increase was noted for myeloid cells, mostly neutrophils, which invaded spleen, thymus and blood of mice with IL-6 overexpression. Observed systemic inflammation ultimately led to mice mortality within 11 weeks of age. Mechanistically, IL-6 mediates its biological functions strictly through binding to IL-6R alpha chain followed by recruitment of the signal transducer gp130. Previously, an alternative pathway was suggested in which IL-6 can utilize CD5 to signal via gp130. However, when IL-6R was deleted in mice with IL-6 overexpression these mice were completely protected from IL-6- triggered pathology and fully phenocopied IL-6R deficient mice. In fact, IL-6R deficiency prevented downstream activation of STAT3 in response to IL-6. Together, our data suggest that IL-6R is the only biological relevant receptor for IL-6 in mice.

Ligand-induced IFNγ down regulation calibrates myeloid cell IFNγ responsiveness

Abstract Inflammation triggers production of type II IFN (IFNγ), which induces expression of gamma-activated genes (GAGs) that enhance antigen presentation and antimicrobial activity associated with M1-type macrophage activation. Negative regulation of these responses is important to prevent excessive macrophage activity and inflammation. We report here that stimulation with IFNγ transiently reduces myeloid cell IFNGR to raise the threshold for STAT1 activation in response to subsequent IFNγ. IFNγ production contributed to reduced monocyte surface IFNGR during the first day after systemic Listeria monocytogenes (Lm) infection. IFNγ treatment also reduced IFNGR staining on cultured bone marrow derived macrophages (BMDM) and dendritic cells (BMDCs), and human monocytes. These reductions in IFNGR were independent of type I IFNs, the primary trigger for IFNGR reductions later after Lm infection. Reductions in IFNGR correlated with reduced macrophage ifngr1 transcript abundance but this was independent of effects on the proximal Ifngr1 promoter. IFNGR down regulation following a brief exposure to IFNγ was further observed to transiently reduce myeloid cell sensitivity to subsequent IFNγ, measured as induction of phosphorylated (p)STAT1. Prolonged or elevated IFNγ could overcome the suppression of pSTAT1 activation. These data suggest transient IFNγ production reduces IFNGR to calibrate M1-type myeloid cell activation.

Exposure of Barrett's and esophageal adenocarcinoma cells to bile acids activates EGFR-STAT3 signaling axis via induction of APE1.

The development of Barret’s esophagus (BE) and its progression to esophageal adenocarcinoma (EAC) is highly linked to exposure to acidic bile salts due to chronic gastroesophageal reflux disease (GERD). In this study, we investigated the role of Apurinic/apyrimidinic endonuclease 1 /redox effector factor-1 (APE-1/REF-1) in STAT3 activation in response to EAC. Our results indicate that APE1 is constitutively overexpressed in EAC whereas its expression is transiently induced in response to acidic bile salts in non-neoplastic BE. Using overexpression or shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using an APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. EGFR phosphorylation (Y1068) was directly associated with APE1 levels and redox function. Co-immunoprecipitation and proximity ligation assays indicated that APE-1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following acidic bile salts treatment. ChIP assays indicated that acidic bile salts treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. The induction of APE-1 - STAT3 axis in acidic bile salts conditions provided a survival advantage and promoted cellular proliferation. In summary, our study provides multiple pieces of evidence supporting a critical role for APE1 induction in activating the EGFR-STAT3 signaling axis in response to acidic bile salts, the main risk factors for Barrett’s carcinogenesis.

Abstract LB-396: Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma cells

Abstract Background: Barrett's esophagus (BE) is a premalignant condition where columnar metaplasia replaces the stratified squamous epithelium of the distal esophagus and is regarded as the main risk factor for esophageal adenocarcinoma (EAC). BE develops as a consequence of chronic gastroesophageal reflux disease (GERD), where acid and bile salts abnormally refluxate from the stomach into the distal region of esophagus. APE1/REF-1 is a multifunctional protein that plays an essential role in DNA repair activity and in redox-dependent transcriptional regulation. STAT3 signaling has been implicated in regulating cell survival, angiogenesis, and metastasis. EGFR is well known to facilitate the tyrosine phosphorylation of STAT3 and initiates downstream signaling cascades that facilitate proliferation, tumorigenesis, metastasis, and chemo resistance. In this study, we investigated the role of APE1 in activation of STAT3 in response to acidic bile salts (ABS) in EAC. Methodology: Non-neoplastic Barrett's (CPA, BAR10T and CPB) and esophageal adenocarcinoma (OE33, FLO-1) cells were exposed to acidic bile salts (pH4.0 and 5.5, 100 and 200uM) for 20 minutes. The mRNA and protein expression levels of APE-1, STAT3 and target genes were evaluated by real time PCR and immunoblotting. STAT3 transcription activity was studied by luciferase reporter assay. Immunofluorescence and cytoplasmic-nuclear fractionation were used to examine localization of APE-1, EGFR and STAT3 after exposure to acidic bile salts. Results: We demonstrate that APE1 is constitutively overexpressed in EAC whereas its expression is induced in non-neoplastic BE in response to ABS. Using overexpression and shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. Phosphorylation of EGFR (Y1068) was directly associated with APE1 levels and redox function. By using co-immunoprecipitation and proximity ligation assays, we found that APE1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in the mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following ABS treatment. ChIP assays indicated that ABS-treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL, following ABS treatment. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. In summary, our results provide evidence that bile reflux induced APE1 is involved in the activation of EGFR-STAT3 signaling axis in BE and EAC. Conclusion: The redox activity of APE1 mediates EGFR- STAT3 activation as a survival mechanism in response to acidic bile salts and oxidative stress. Constitutive activation of APE1 in EAC may present a therapeutic opportunity to targeting redox vulnerabilities in cancer cells. Citation Format: Ajaz A. Bhat, Heng Lu, Mohammad Suotto, Chen Zheng, Dunfa Peng, Mohammad Haris, Abbes Belkhiri, Wael El-Rifai. Bile reflux-induced APE1 mediates activation of EGFR-STAT3 in barret's and esophageal adenocarcinoma 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 LB-396.

Tyr23 phosphorylation of Anxa2 enhances STAT3 activation and promotes proliferation and invasion of breast cancer cells.

Overexpression of Annexin A2 (Anxa2) is positively correlated with breast cancer progression, drug resistance, and poor prognosis of patients with breast cancer. Tyr23 Phosphorylation by Src-family tyrosine kinase is an important post-translational modification of Anxa2. This modification regulates the subcellular localization and functions of Anxa2 and has significant effects on cell proliferation, migration, and invasion. This study aims at revealing the association of Anxa2-Tyr23 phosphorylation in Anxa2-mediated acceleration of breast cancer progression and their elaborate molecular mechanisms. Cell biological function experiments were performed to determine the effects of Anxa2-Tyr23 Phosphorylation on breast cancer cell proliferation and invasion in vitro and metastasis in vivo. The interaction of Tyr23 phosphorylated Anxa2 and STAT3 was verified by co-immunoprecipitation assay. Related mRNA and protein expression levels of cyclin D1 and MMP2/9 and phosphorylation level of STAT3 were detected. Anxa2-Tyr23 phosphorylation is necessary for proliferation, invasion, and metastasis of breast cancer cells in vitro and in vivo. Tyr23 phosphorylated Anxa2 binds and enhances the sensitivity of STAT3 activation in response to IL-6, thereby increasing the protein and mRNA expression levels of cyclin D1 and MMP2/9 which are STAT3 key target genes and serve pivotal regulatory functions in cell proliferation and invasion, respectively. Our findings further confirmed the regulatory role of Anxa2 and revealed the direct relationship between Anxa2-Tyr23 phosphorylation and activation of STAT3. Moreover, this study provides novel insights into the function of Anxa2-Tyr23 phosphorylation in signal transduction for further understanding of the mechanism through which Anxa2 promotes the progression of breast cancer.

Signed, Sealed, Delivered: Microenvironmental Modulation of Extracellular Vesicle-Dependent Immunoregulation in the Lung.

Unconventional secretion and subsequent uptake of molecular cargo via extracellular vesicles (EVs) is an important mechanism by which cells can exert paracrine effects. While this phenomenon has been widely characterized in the context of their ability to promote inflammation, less is known about the ability of EVs to transfer immunosuppressive cargo. Maintenance of normal physiology in the lung requires suppression of potentially damaging inflammatory responses to the myriad of insults to which it is continually exposed. Recently, our laboratory has reported the ability of alveolar macrophages (AMs) to secrete suppressors of cytokine signaling (SOCS) proteins within microvesicles (MVs) and exosomes (Exos). Uptake of these EVs by alveolar epithelial cells (AECs) resulted in inhibition of pro-inflammatory STAT activation in response to cytokines. Moreover, AM packaging of SOCS within EVs could be rapidly tuned in response to exogenous or AEC-derived substances. In this article we will highlight gaps in knowledge regarding microenvironmental modulation of cargo packaging and utilization as well as EV secretion and uptake. Advances in these areas are critical for improving understanding of intercellular communication in the immune system and for therapeutic application of artificial vesicles aimed at treatment of diseases characterized by dysregulated inflammation.

STAT1 and NF-κB Inhibitors Diminish Basal Interferon-Stimulated Gene Expression and Improve the Productive Infection of Oncolytic HSV in MPNST Cells.

Interferon-stimulated genes (ISG) encode diverse proteins that mediate intrinsic antiviral resistance in infected cells. Here it was hypothesized that malignant peripheral nerve sheath tumor (MPNST) cells resist the productive infection of oncolytic herpes simplex virus (oHSV) through activation of the JAK/STAT1 pathway and resultant upregulation of ISGs. Multiple human and mouse MPNST cells were used to explore the relationship between STAT1 activation and the productive infection of Δγ134.5 oHSVs. STAT1 activation in response to oHSV infection was found to associate with diminished Δγ134.5 oHSVs replication and spread. Multiday pretreatment, but not cotreatment, with a JAK inhibitor significantly improved viral titer and spread. ISG expression was found to be elevated prior to infection and downregulated when treated with the inhibitor, suggesting that the JAK/STAT1 pathway is active prior to infection. Conversely, upregulation of ISG expression in normally permissive cells significantly decreased oHSV productivity. Finally, a possible link between NF-κB pathway activation and ISG expression was established through the expression of inhibitor of kB (IκB) which decreased basal STAT1 transcription and ISG expression. These results demonstrate that basal ISG expression prior to infection contributes to the resistance of Δγ134.5 oHSVs in MPNST cells. Although cancer-associated ISG expression has been previously reported to impart resistance to chemotherapy and radiotherapy, these data show that basal ISG expression also contributes to oncolytic HSV resistance. Mol Cancer Res; 14(5); 482-92. ©2016 AACR.

Defective STAT1 activation associated with impaired IFN-γ production in NK and T lymphocytes from metastatic melanoma patients treated with IL-2.

High dose (HD) IL-2 therapy has been used for almost two decades as an immunotherapy for metastatic melanoma. IL-2 promotes the proliferation and effector function of T and NK cells through the tyrosine phosphorylation and activation of signal transducer and activator of transcription factors (STAT), especially STAT5. However, whether any defects in STAT activation exist in T and NK lymphocytes from melanoma patients are under debate. Here, we measured the extent of HD IL-2-induced phosphorylation of STAT5 and STAT1 in lymphocyte subsets from metastatic melanoma patients and healthy controls at a single cell level using flow cytometry. We found no defects in IL-2-induced STAT5 phosphorylation and induction of proliferation in T and NK cell subsets in vitro. This was confirmed by measuring ex vivo STAT5 activation in whole blood collected from patients during their first bolus HD IL-2 infusion. IL-2 also induced STAT1 phosphorylation via IFN-γ receptors in T and NK cell subsets through the release of IFN-γ by CD56hi and CD56lo NK cells. Further analysis revealed that melanoma patients had a sub-optimal STAT1 activation response linked to lower IL-2-induced IFN-γ secretion in both CD56hi and CD56low NK cell subsets. STAT1 activation in response to IL-2 also showed an age-related decline in melanoma patients not linked to tumor burden indicating a premature loss of NK cell function. Taken together, these findings indicate that, although STAT5 activation is normal in metastatic melanoma patients in response to IL-2, indirect STAT1 activation is defective owing to deficiencies in the NK cell response to IL-2.

TRAF2 mediates JNK and STAT3 activation in response to IL-1β and IFNγ and facilitates apoptotic death of insulin-producing β-cells

Interleukin-1β (IL-1β) and interferon-γ (IFNγ) contribute to type 1 diabetes (T1D) by inducing β-cell death. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are adaptors that transduce signaling from a variety of membrane receptors including cytokine receptors. We show here that IL-1β and IFNγ upregulate the expression of TRAF2 in insulin-producing INS-1E cells and isolated rat pancreatic islets. siRNA-mediated knockdown (KD) of TRAF2 in INS-1E cells reduced IL-1β-induced phosphorylation of JNK1/2, but not of p38 or ERK1/2 mitogen-activated protein kinases. TRAF2 KD did not modulate NFκB activation by cytokines, but reduced cytokine-induced inducible nitric oxide synthase (iNOS) promotor activity and expression. We further observed that IFNγ-stimulated phosphorylation of STAT3 required TRAF2. KD of TRAF2 or STAT3 reduced cytokine-induced caspase 3/7 activation, but, intriguingly, potentiated cytokine-mediated loss of plasma membrane integrity and augmented the number of propidium iodide-positive cells. Finally, we found that TRAF2 KD increased cytokine-induced production of reactive oxygen species (ROS). In summary, our data suggest that TRAF2 is an important mediator of IL-1β and IFNγ signaling in pancreatic β-cells.

Retinal Caveolin-1 Modulates Neuroprotective Signaling.

Caveolin-1 (Cav-1), the scaffolding protein of caveolae, is expressed in several retinal cell types and is associated with ocular pathologies. Cav-1 modulates neuroinflammatory/neuroprotective responses to central nervous system injury. We have shown that loss of Cav-1 results in a blunted cytokine response in retinas challenged with inflammatory stimuli. As neuroinflammatory and neuroprotective signaling overlap in their cytokine production and downstream signaling pathways, we hypothesized that loss of Cav-1 may also suppress neuroprotective signaling in the retina. To test this, we subjected mice in which Cav-1 was deleted specifically in the retina to a neurodegenerative insult induced by sodium iodate (NaIO3) and measured STAT3 activation, a measure of neuroprotective signaling. Our results show that Cav-1 ablation blunts STAT3 activation induced by NaIO3. STAT3 activation in response to intravitreal administration of the IL-6 family cytokine, leukemia inhibitory factor (LIF), was not affected by Cav-1 deletion indicating a competent gp130 receptor response. Thus, Cav-1 modulates neuroprotective signaling by regulating the endogenous production of neuroprotective factors.