STAT3 Activator Research Articles

154 ER Stress Transcription Factor XBP1 Suppresses Intestinal Tumorigenesis

Background. The unfolded protein response (UPR) is a response mechanism to endoplasmic reticulum (ER) stress, which a cell is typically exposed upon protein misfolding. Intestinal epithelial cell (IEC) homeostasis critically depends upon an intact UPR and its perturbation due to deficiency of the key UPR transcription factor X-box binding protein-1 (XBP1) in IECs results in ER stress und spontaneous intestinal inflammation. Rare functional variants of XBP1 are associated with inflammatory bowel disease (IBD) and IBD patients typically exhibit unresolved ER stress in IECs. Long standing inflammation in IBD patients predisposes for colitis-associated cancer and cancer cells typically exhibit ER stress due to increased risk for protein misfolding as a consequence of the tumour microenvironment characterized by low oxygen, nutrient deprivation and pH changes. Aims. We hypothesised XBP1-deficiency and subsequent unresolved ER stress might affect intestinal regeneration, which may impact on tumourigenesis. Methods. NFκB and Stat3 activation was analysed by immunoblot and immunohistochemistry. Specific inhibitors of Stat3 (S3I-201) and NF κB (BAY11-7082), respectively, were administered to Xbp1flox/flox VillinCre mice. Epithelial proliferation was assessed by bromodeoxyuridine (BrdU) staining. Xbp1flox/flox VillinCre mice were analyzed at day 61 of azoxymethane (AOM) followed by three cycles of dextran sodium sulphate (DSS). Results. ER stress due to XBP1-deficiency results in a numerical increase of transit amplifying (TA) cells and consequently in an increased epithelial turn over. Xbp1-/-(IEC) mice activate Stat3, which localizes to TA cells, and its blockade via administration of the Stat3 inhibitor S3I-201 abrogates epithelial hyperregeneration. Xbp1-deletion in IECs leads to activation of NFκB and increased IL-6 secretion, a known activator of Stat3. Blockade of NFκB signaling via administration of the IKK2 inhibitor BAY11-7082 abrogated Stat3 activation in XBP1-deficient mice. NFκB and Stat3 promote colitis-associated cancer and XBP1deficient mice exhibit an increase in tumour burden in a model of colitis-associated cancer characterized by a 10-fold increase in tumour area as well as tumour number. Conclusions. Our data unravels XBP1 as an unexpected tumour suppressor in the intestinal epithelium, thereby opposing the current paradigm that cancer cells require an unimpaired UPR to survive in a stressful tumour microenvironment. Promotion of colitis-associated tumourigenesis is functionally linked to activation of NFκB and Stat3, which are potently engaged in XBP1deficient IECs. Our data show that unresolved ER stress in the intestinal epithelium increases the propensity to develop intestinal tumours.

STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells

Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The 'signal transducer and activator of transcription-3' (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.

Monocytes/macrophages support mammary tumor invasivity by co-secreting lineage-specific EGFR ligands and a STAT3 activator

BackgroundTumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors.MethodsFollowing exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients.ResultsPrimary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease.ConclusionsOur work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy.

Classical cadherins control survival through the gp130/Stat3 axis

Stat3 (Signal Transducer and Activator of Transcription-3) is activated by a number of receptor and nonreceptor tyrosine kinases. We recently demonstrated that engagement of E-cadherin, a calcium-dependent, cell to cell adhesion molecule which is often required for cells to remain tightly associated within the epithelium, also activates Stat3. We now examined the effect of two other classical cadherins, cadherin-11 and N-cadherin, whose expression often correlates with the epithelial to mesenchymal transition occurring in metastasis of carcinoma cells, upon Stat3 phosphorylation and activity. Our results indicate that engagement of these two cadherins also, can trigger a dramatic surge in Stat3 activity. This activation occurs through upregulation of members of the IL6 family of cytokines, and it is necessary for cell survival, proliferation and migration. Interestingly, our results also demonstrate for the first time that, in sharp contrast to Stat3, the activity of Erk (Extracellular Signal Regulated kinase) was unaffected by cadherin-11 engagement. Further examination indicated that, although IL6 was able to activate Erk in sparsely growing cells, IL6 could not induce an increase in Erk activity levels in densely growing cultures. Most importantly, cadherin-11 knock-down did allow Erk activation by IL6 at high densities, indicating that it is indeed cadherin engagement that prevents Erk activation by IL6. The fact that the three classical cadherins tested so far, E-cadherin, N-cadherin and cadherin11, which are present in essentially all tissues, actually activate Stat3 regardless of their role in metastasis, argues for Stat3 as a central survival, rather than invasion factor.

Identification of Steroid-Sensitive Gene-1/Ccdc80 as a JAK2-Binding Protein

The tyrosine kinase Janus kinase 2 (JAK2) is activated by many cytokine receptors, including receptors for GH, leptin, and erythropoietin. However, very few proteins have been identified as binding partners for JAK2. Using a yeast 2-hybrid screen, we identified steroid-sensitive gene-1 (SSG1)/coiled-coil domain-containing protein 80 (Ccdc80) as a JAK2-binding partner. We demonstrate that Ccdc80 preferentially binds activated, tyrosyl-phosphorylated JAK2 but not kinase-inactive JAK2 (K882E) in both yeast and mammalian systems. Ccdc80 is tyrosyl phosphorylated in the presence of JAK2. The binding of Ccdc80 to JAK2 occurs via 1 or more of the 3 DUDES/SRPX (DRO1-URB-DRS-Equarin-SRPUL/sushi repeat containing protein, x-linked) domain 5 domains of Ccdc80. Mutagenesis of the second DUDES domain suggests that the N-terminal third of the DUDES domain is sufficient for JAK2 binding. Ccdc80 does not alter the kinase activity of JAK2. However, Ccdc80 increases GH-dependent phosphorylation of Stat (signal transducer and activator of transcription) 5b on Tyr699 and substantially enhances both basal and GH-dependent phosphorylation/activation of Stat3 on Tyr705. Furthermore, Ccdc80 belongs to the group of proteins that function both in the intracellular compartment and are secreted. Secreted Ccdc80 associates with the extracellular matrix and is also found in the medium. A substantial portion of the Ccdc80 detected in the medium is cleaved. Finally, consistent with the DUDES domain serving as a JAK2-binding domain, we also demonstrate that another protein that contains a DUDES domain, SRPX2, binds preferentially to the activated tyrosyl-phosphorylated form of JAK2.

Application of Interleukin-22 Mediates Protection in Experimental Acetaminophen-Induced Acute Liver Injury

Acetaminophen (APAP, paracetamol)-induced hepatotoxicity, although treatable by timely application of N-acetylcysteine, can be fatal. Because it is among the common causes of acute liver failure in intensive care units and in light of its gradually increasing incidence, the need for novel therapeutic strategies aimed at severe intoxication is apparent. Recently, it has been shown that IL-22, a STAT3-activating cytokine, has the capability to mediate liver protection. Herein, the protective potential of IL-22 in murine APAP-induced hepatotoxicity was assessed. Intravenous administration of prophylactic IL-22 significantly reduced serum alanine aminotransferase levels and histopathologic damage in APAP-induced liver injury, a process that coincided with increased hepatocyte proliferation invivo. Concomitant gene expression analysis revealed hepatic induction of genes prototypically up-regulated by the IL-22/STAT3 axis, among others suppressor of cytokine signaling-3, lipocalin-2, and α1-antichymotrypsin. Notably, in a translational setting of therapeutic treatment 2 hours after APAP, IL-22 supported protection in the context of suboptimal N-acetylcysteine dosing. IL-22 likewise connected to augmented hepatocyte proliferation in this experimental setting. As detected by analysis of inflammatory cytokine production, systemically applied IL-22 did not display acute immunomodulation/stimulation in otherwise untreated or endotoxemic mice. Those latter observations clearly confirm acute tolerability of systemically applied IL-22. Observations presented altogether suggest that therapeutic IL-22 administration is a conceivable tissue-protective regimen aimed at hard-to-treat patients with severe APAP-induced hepatotoxicity.

Role of Stat3 Activation in Cell-Cell Interaction between B-Cell Lymphoma and Macrophages : The in vitro Study

Recently, tumor-associated macrophages (TAMs) have been of interest because of their protumoral functions. In patients with malignant lymphoma, an increased number of alternatively activated (M2) macrophages is closely associated with poor clinical prognosis. Signal transducer and activator of transcription 3 (Stat3) is an important molecule related to tumor development. Previously we demonstrated that Stat3 activation in primary central nervous system lymphoma cells was significantly associated with the number of M2 TAMs present. Here we report that direct contact with macrophages in culture was required for proliferation of B-cell lymphoma cell lines, and that M2 macrophages induced more proliferation than did M1 macrophages. Stat3 activation in lymphoma cells was involved in this cell-cell interaction. Cytokine array analysis demonstrated that complement 5a (C5a) was detected in supernatants of M2 macrophages, but not in those of M1 macrophages or lymphoma cells. Although we demonstrated M2 macrophage-derived C5a is one of growth factors and a Stat3 activator, C5a was not a predominant molecule associated to lymphoma cell activation induced by M2 macrophages. However, these findings provide novel insights into the molecular mechanism related to M2 macrophages and lymphoma cell interaction.

STAT3 inhibitors for cancer therapy

The signal transducer and activator of transcription STAT3 is a transcription factor which plays a key role in normal cell growth and is constitutively activated in about 70% of solid and hematological cancers. Activated STAT3 is phosphorylated on tyrosine and forms a dimer through phosphotyrosine/src homology 2 (SH2) domain interaction. The dimer enters the nucleus via interaction with importins and binds target genes. Inhibition of STAT3 results in the death of tumor cells, this indicates that it is a valuable target for anticancer strategies; a view that is corroborated by recent findings of activating mutations within the gene. Yet, there is still only a small number of STAT3 direct inhibitors; in addition, the high similarity of STAT3 with STAT1, another STAT family member mostly oriented toward apoptosis, cell death and defense against pathogens, requires that STAT3-inhibitors have no effect on STAT1. Specific STAT3 direct inhibitors consist of SH2 ligands, including G quartet oligodeoxynucleotides (ODN) and small molecules, they induce cell death in tumor cells in which STAT3 is activated. STAT3 can also be inhibited by decoy ODNs (dODN), which bind STAT3 and induce cell death. A specific STAT3 dODN which does not interfere with STAT1-mediated interferon-induced cell death has been designed pointing to the STAT3 DBD as a target for specific inhibition. Comprehensive analysis of this region is in progress in the laboratory to design DBD-targeting STAT3 inhibitors with STAT3/STAT1 discriminating ability.

BP-2-047, a Novel Small-Molecule Inhibitor of Stat3 Is Active in Myeloma Pre-Clinical Models: Rationale for Treatment of Stat3-Dependent Multiple Myeloma

AbstractAbstract 576Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character.We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures:No relevant conflicts of interest to declare.

NK Cell Deficiency in Job's Syndrome Patients

AbstractAbstract 3293 Background:Hyper-immunoglobulin E syndrome (Job's syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses from inappropriately mild immune responses to staphylococcal infection. Both sporadic and autosomal dominant Job's syndrome patients are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job's syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development and function in Job's syndrome patients has not been studied yet. Previous work performed in our lab employing STAT3 activating cytokines and STAT3 inhibitors showed that STAT3 activation plays a role in NK cell proliferation, survival, cytolytic activity, and regulation of expression of the activating receptor NKG2D. This finding prompted us to test whether aberrant STAT3 activation affects NK cell proliferation, survival and cytolytic activity, in NK cells of Job's syndrome patients. Objective:To assess proliferation, cytolytic activity and receptor expression on NK cells from Job's syndrome patients with dominant negative STAT3 mutations. Methods:NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cytolytic activity was evaluated by Calcein release assay. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by paired Student's t test using GraphPad Prism. Results:Flow-cytometric analysis of PBMCs showed significantly lower percentage of NK cells, identified as CD3− CD56+, in Job’s syndrome patients compared to normal donors. The expansion in response to membrane bound IL21 stimulation was found to be impaired in NK cells obtained from Job’s syndrome patients compared to those from normal donors. Expanded NK cells from Job’s syndrome patients had lower percentage of mature, CD56+CD16+NK cells compared to those from normal donors. We also found significantly reduced cytolytic activity in NK cells from Job’s syndrome patients. STAT3 activating cytokines IL10 and IL21 were also found to be impaired in their ability to induce NKG2D expression on NK cells obtained from Job’s syndrome patients. Conclusions:Our finding of deficient NK cell number and cytolytic activity in Job's syndrome patients is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job's syndrome patient's NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job's syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job's syndrome patients are also more prone to viral infections and lymphoma. Given the role of NK cells in the immune surveillance of virally infected and tumorigenic cells, low NK cell function may explain the proclivity of Job's syndrome patients to viral infections and lymphoma. Disclosures:No relevant conflicts of interest to declare.

The Liver as a Conductor of Systemic Iron Regulation: the Central Role of Hepcidin and BMP Signaling

AbstractAbstract SCI-22Iron is a necessary trace element found in nearly all living organisms and is critical to many cellular processes, including electron transport in the mitochondrial membrane, DNA synthesis, and hemoglobin synthesis. Iron deficiency leads to anemia, and excess iron can promote free radical formation and result in cellular injury (1). Thus, iron balance is tightly regulated to provide sufficient amounts of iron for critical cellular processes, while avoiding the detrimental effects of excess iron. Iron homeostasis is disrupted in many hematologic diseases, including hemochromatosis, the anemia of chronic disease, and β-thalassemia, affecting hundreds of millions of people worldwide. Hepcidin is a peptide hormone secreted by the liver that plays a central role in iron homeostasis. Hepcidin binding to ferroportin, the only known mammalian cellular iron exporter, leads to internalization and degradation of ferroportin in lysosomes, which decreases iron absorption from the diet and iron release from macrophages that recycle iron from senescent erythrocytes. As a central regulator of body iron metabolism, hepcidin is modulated by the same factors that alter iron homeostasis, including changes in body iron stores, inflammation, rate of erythropoiesis, anemia, and hypoxia. Hepcidin levels are increased in response to oral and parenteral iron loading and decreased under iron deficient conditions (2). Inflammation stimulates hepcidin expression by increasing IL-6 levels, which activates the JAK/STAT signaling cascade and binding of the transcriptional activator STAT3 to the hepcidin promoter (3, 4). In contrast, anemia and hypoxia are associated with decreased hepcidin levels (5). Studies of patients with autosomal recessive hemochromatosis, an iron overload disorder, have revealed that several hepatocyte proteins are required for appropriate hepcidin regulation, including those coded by HFE (6), TFR2 (7), and HJV (also called HFE2 and RGMc) (8). Mutations in either the hepcidin gene (HAMP) or HJV cause a more severe form of hemochromatosis that presents in the first or second decade of life. In juvenile hemochromatosis, patients who carry HJV mutations, urinary hepcidin is undetectable, suggesting that HJV is a key regulator of hepcidin (8). Disruption of Hjv in the mouse results in decreased hepcidin expression and increased iron deposition in the liver, pancreas, and heart but decreased iron levels in tissue macrophages (9, 10). HJV (RGMc) encodes hemojuvelin, a member of the repulsive guidance molecule family, which resides on the cell membrane as a glycosylphosphatidylinositol (GPI)-linked protein. It functions as a BMP6 coreceptor to activate hepcidin expression through a BMP/SMAD signaling pathway (11, 12, 13). Accordingly, disease-associated mutations in HJV result in decreased BMP/SMAD signaling and decreased hepcidin expression (11, 14). Hemojuvelin, acting through the BMP signaling pathway, thus plays an essential role in the regulation of hepcidin expression. BMP signaling also appears to be central in order for iron and inflammation to regulate hepcidin expression. Disclosures:Lin:Ferrumax Pharmaceuticals: Equity Ownership.

H. pyloriinduces the expression of Hath1 in gastric epithelial cells via interleukin-8/STAT3 phosphorylation while suppressing Hes1

Chronic gastritis associated with Helicobacter pylori is a leading cause of gastric intestinal metaplasia (IM), which arises from abnormal cell differentiation of the epithelium in the gastric mucosa. However, the mechanisms involved in H. pylori-mediated IM remain elusive. The aim of our study was to explore the effects and the underlying mechanisms of H. pylori on the abnormal expression of Hath1 and Sox2 and to reveal its relationship to the development of gastric IM. We found that Hath1 and Sox2 were overexpressed in gastric IM tissue. Hath1 expression was up-regulated, whereas Sox2 expression, which was independent of the CagA virulence factor, was down-regulated in gastric epithelial cells and coincided with increased IL-6 and IL-8 levels in the culture media. Stimulation with H. pylori-related cytokine IL-8, but not IL-6 or IL-1β, was induced by Hath1 expression in the gastric epithelial cells. Although IL-8 and IL-6 levels correlated with STAT3 (signal transducer and activator of transcription) phosphorylation before and after H. pylori eradication in the gastric mucosa, only the blocking of IL-8-induced STAT3 activation using AG490 or STAT3-targeting RNA interference altered Hath1 expression. Additionally, we found that H. pylori down-regulated Hes1, which is a direct downstream target gene of Notch signaling and a repressor of Hath1 expression. These findings suggest that H. pylori induced inflammation up-regulate Hath1 expression via interleukin-8/STAT3 (IL-8) phosphorylation while suppressing Hes1, which provides a novel molecular connection between a H. pylori infection and intestinal metaplasia.

The Transcription Factors Egr2 and Egr3 Are Essential for the Control of Inflammation and Antigen-Induced Proliferation of B and T Cells

SummaryLymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.

USP18 establishes the transcriptional and anti-proliferative interferon α/β differential

Type I IFNs (interferons) are pathogen-induced immunoregulatory cytokines that exert anti-viral and anti-proliferative activities through binding to a common cell-surface receptor. Among the 17 human IFN subtypes, IFNβ binds the IFNAR (IFNα receptor) 1/IFNAR2 receptor chains with particularly high affinity and is especially potent in select bioactivities (e.g. anti-proliferative and pro-apoptotic) when compared with IFNα2. However, no molecular basis has been ascribed to this differential action, since the two ligands are equipotent in immediate early signalling events. In the present study we report that IFNβ induces Stat (signal transducer and activator of transcription) phosphorylation and transcriptional activation of ISGs (interferon-stimulated genes), including two genes with pro-apoptotic functions, for a considerably longer time frame than does IFNα2. We show that the diversification of α2/β responses progressively builds up at the receptor level as a result of accumulating USP18 (ubiquitin specific protease 18), itself an ISG, which exerts its negative feedback action by taking advantage of the weakness of IFNα2 binding to the receptor. This represents a novel type of signalling regulation that diversifies the biological potential of IFNs α and β.

도두(刀豆)의 조혈증진 작용에 관한 연구

Objective : Canavalia gladiata DC semen (CGS) have been used to improve hematopoietic activity. In the current study, we investigated whether CGS regulate hemato-potentiating function using hematopoietic stem cells (HSCs) as a testing system. Methods : HSCs isolated from femur in mice with leukopenia and thrombocytopenia induced induced by CTX. Then, Real-time PCR was performed to measure the mRNA expression and hematopoietic related gene (EPO, IL-3, SCF, c-kit, GM-CSF), the phoaphorylation of GATA-1 and STAT-5a/b were observed by ELISA method, and the number of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E), semisolid clonogenic assay was performed. Result : When HSCs were treated with CGS, the expression of hematopoietic related genes (EPO, IL-3, SCF, c-kit, and GM-CSF) were significantly increased at the levels of mRNA as well as production in HSCs. Additionally, CGS enhanced phosphorylation of STAT-1 and signal transducer and activator of transcription-5a/b (STAT-5a/b) in HSCs. Furthermore, CGS significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Conclusion : These result suggest that CGS has hematopoietic enhancement via hematopoietic cytokine-mediated GATA-1/STAT-5a/b pathway.

Early-phase GVHD gene expression profile in target versus non-target tissues: kidney, a possible target?

GVHD is a major complication after allo-SCT. In GVHD, some tissues like liver, intestine and skin are infiltrated by donor T cells while others like muscle are not. The mechanism underlying targeted tropism of donor T cells is not fully understood. In the present study, we aim to explore differences in gene expression profile among target versus non-target tissues in a mouse model of GVHD based on chemotherapy conditioning. Expression levels of JAK-signal transducers and activators of transcription (STAT), CXCL1, ICAM1 and STAT3 were increased in the liver and remained unchanged (or decreased) in the muscle and kidney after conditioning. At the start of GVHD the expression levels of CXCL9, ITGb2, SAA3, MARCO, TLR and VCAM1 were significantly higher in the liver or kidney compared with the muscle of GVHD animals. Moreover, biological processes of inflammatory reactions, leukocyte migration, response to bacterium and chemotaxis followed the same pattern. Our data show that both chemotherapy and allogenicity exclusively induce expression of inflammatory genes in target tissues. Moreover, gene expression profile and histopathological findings in the kidney are similar to those observed in the liver of GVHD mice.

STAT3 Inhibition Overcomes Temozolomide Resistance in Glioblastoma by Downregulating MGMT Expression

Glioblastoma multiforme (GBM) is one of the most aggressive human tumors with a poor prognosis. Current standard treatment includes chemotherapy with the DNA-alkylating agent temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide-induced DNA damage due to elevated expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). Here, we show that upregulation of both MGMT and STAT3 was accompanied with acquisition of temozolomide resistance in the GBM cell line U87. Inactivation of STAT3 by inhibitor or short hairpin RNA (shRNA) downregulated MGMT expression in GBM cell lines. MGMT upregulation was not observed by the treatment of interleukin (IL)-6 which is a strong activator of STAT3. Contrarily, forced expressed MGMT could be downregulated by STAT3 inhibitor which was partially rescued by the proteasome inhibitor, MG132, suggesting the STAT3-mediated posttranscriptional regulation of the protein levels of MGMT. Immunohistochemical analysis of 44 malignant glioma specimens showed significant positive correlation between expression levels of MGMT and phosphorylated STAT3 (p-STAT3; P < 0.001, r = 0.58). Importantly, the levels of both MGMT and p-STAT3 were increased in the recurrence compared with the primary lesion in paired identical tumors of 12 cases. Finally, we showed that STAT3 inhibitor or STAT3 knockdown potentiated temozolomide efficacy in temozolomide-resistant GBM cell lines. Therefore, STAT3 inhibitor might be one of the candidate reagents for combination therapy with temozolomide for patients with temozolomide-resistant GBM.

Abstract B5: CpG-siRNA conjugates: Overcoming cancer immunoresistance

Abstract Small interfering RNA (siRNA) have been proved to be an effective tool for treating diseases caused by gene expression abnormalities. However, clinical relevance of siRNA technology is compromised by multiple obstacles, such as lack of cellular specificity, low efficiency of cellular uptake and/or poor cytoplasmic release. We have previously demonstrated that natural capability of certain immune cells to recognize non-methylated CpG motifs in DNA oligonucleotides can be harnessed for cell-specific siRNA delivery. The siRNA molecules conjugated to CpG oligonucleotides (CpG-siRNA) are efficiently internalized and bind to the intracellular Toll-like receptor 9 (TLR9) in human and mouse dendritic cells as well as in certain TLR9-positive tumor cells. We designed a CpG-siRNA targeting Stat3 (signal transducer and activator of transcription-3), a transcription factor, which is constitutively activated in majority of mouse and human cancers. Oncogenic Stat3 signaling regulates cancer cell proliferation, survival and promotes tumor immunoresistance. We have previously shown that CpG-Stat3 siRNA can eliminate Stat3 signaling in tumor-infiltrating myeloid cells, resulting in potent antitumor immunity. In the current study, we investigated the intracellular mechanism of action of CpG-siRNA conjugates. We have demonstrated that internalized CpG-siRNAs are recruited into endosomes by dynamin-dependent mechanism and bind to endosomal TLR9 receptor. Further events include uncoupling of siRNA from the CpG moiety by Dicer endonuclease, and rapid translocation of siRNA to endoplasmic reticulum for recruitment into functional RISC (RNA interference silencing complex). We confirmed the target gene silencing both in vitro and in vivo, and proved that it occurs through RNA interference mechanism. Our current results indicate that downstream effectors of TLR9 pathway may be involved in RISC assembly and/or activation. Overall, the CpG-siRNA strategy provides a novel opportunity for overcoming cancer immunoresistance with translational potential for therapy of various malignancies.

Genomic analysis of CD8 T cells during the early phases of the immune response (110.25)

Abstract The generation of long-lived memory CD8 T cells is critical for the development of effective vaccines and adoptive immunotherapy strategies. However, the molecular mechanisms by which a T cell transitions from naïve to memory remains poorly understood. In an effort to better understand the molecular pathways involved in the differentiation of CD8 T cells, we have analyzed ovalbumin-specific OTI CD8 T cells during the very early phases of the immune response at the genomic level. Following infection with Listeria monocytogenes expressing ovalbumin, OTI CD8 T cells from infected mice were sorted and subjected to microarray analysis. We show that while cell surface phenotype of OTI T cells remained largely unchanged shortly after activation, large scale changes occurred at the level of transcription. Expression levels of many families of genes involved in signaling networks in T cells, in addition to multiple immune response factors, were differentially expressed shortly after T cells were activated, but prior to the first division. Notably, expression of IL-21, a cytokine implicated in T cell memory, was transiently expressed with peak expression occurring at 12 hours post-stimulation. This was confirmed by intracellular staining for the IL-21 protein. Similarly, the peak of STAT3 expression coincided with IL-21, a known activator of STAT3. We are currently investigating autocrine IL-21 signaling in CD8 T cells and its role in memory development.

Abstract 44: Enhancement endometrial cancer cells malignant transformation by mutual interaction with mesenchymal stem cells through TGF-beta and SDF-1 signaling pathways

Abstract Designated to cyclic shedding, endometrial glandular stroma is rich for mesenchymal stem cells and may play crucial roles in cancer invasion. This study aimed to explore the interaction between endometrial cancer cells and endometrial mesenchymal stem cells (HEMSC). HEMSC were derived from human endometrium and were co-cultured with RL95-2 endometrial cancer cells. Transformation phenotypes including proliferation, migration, anchorage independent growth, colony formation and invasion were characterized. Cancer associated transforming growth factor (TGF)-beta1 and mesenchymal cell associated stromal cell-derived factor (SDF) and their down-stream signaling pathways were investigated. HEMSC had a similar surface marker profile as bone marrow mesenchymal stem cells, i.e. CD44(+), CD90(+) and CD34(-) and CD45(-) and readily be inducible to osteogenic and adipogenic differentiations. Upon coculture with HEMSC, RL95-2 cell secreted TGF-beta 1 which subsequently induced SDF1 secretion from HEMSC. The mutual interaction of these factors is associated with enhancement of cancer cell migration through the CXCR4 receptor and downstream transducer and activator of Stat3, with resulting over-expression of alpha-SMA and cytoskeleton re-organization. The study observed unprecedented interactions between cancer cells and mesenchymal stem cells with resulting enhancement of malignant transformation through mutual activation of cytokines and downstream signals. Mesenchymal stem cells either in adjacent to the lesion or home to the lesion can therefore be important activators of carcinogenesis and good therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 44. doi:1538-7445.AM2012-44