Signal Transducer And Activator Of Transcription 3 Ser727 Research Articles

Combined Catalpol and Tetramethylpyrazine Promote Axonal Plasticity in Alzheimer's Disease by Inducing Astrocytes to Secrete Exosomes Carrying CDK5 mRNA and Regulating STAT3 Phosphorylation.

Alzheimer's disease (AD) is a common progressive degenerative disease of the central nervous system in aging populations. This study aimed to investigate the effects of combined catalpol and tetramethylpyrazine (CT) in promoting axonal plasticity in AD and the potential underlying mechanism. Astrocytes were treated with different concentrations of compatible CT. Exosomes were collected and subjected to sequencing analysis, which was followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes. Amyloid precursor protein/presenilin 1 (APP/PS1) double-transfected male mice were used as the in vivo AD models. Astrocyte-derived exosomes that were transfected with cyclin-dependent kinase 5 (CDK5) or CT treatment were injected into the tail vein of mice. The levels of CDK5, synaptic plasticity marker protein neurofilament 200 (NF200), and growth-associated protein 43 (GAP-43) in the hippocampus of mice were compared in each group. Immunofluorescence staining was used to detect the localization of STAT3 and to visualize synaptic morphology via β-tubulin-III (TUBB3). Astrocyte-derived exosomes transfected with siCDK5 or treated with CT were co-cultured with HT-22 cells, which were untransfected or silenced for signal transducer and activator of transcription 3 (STAT3). Amyloid β-protein (Aβ)1-42 was induced in the in vitro AD models. The viability, apoptosis, and expression levels of NF200 and GAP-43 proteins in the hippocampal neurons of each group were compared. In total, 166 differentially expressed genes in CT-induced astrocyte-derived exosomes were included in the KEGG analysis, and they were found to be enriched in 12 pathways, mainly in axon guidance. CT treatment significantly increased the level of CDK5 mRNA in astrocyte-derived exosomes-these exosomes restored CDK5 mRNA and protein levels in the hippocampus of the in vivo AD model mice and the in vitro AD model; promoted p-STAT3 (Ser727), NF200 and GAP-43 proteins; and promoted the regeneration and extension of neuronal synapses. Silencing of CDK5 blocked both neuronal protection as well as induction of axonal plasticity in AD by CT-treated exosomes in vitro and in vivo. Moreover, silencing of STAT3 blocked both neuronal protection as well as induction of axonal plasticity in AD caused by CDK5 overexpression or CT-treated astrocyte-induced exosomes. CT promotes axonal plasticity in AD by inducing astrocytes to secrete exosomes carrying CDK5 mRNA and regulating STAT3 (Ser727) phosphorylation.

Discovery of the Highly Selective and Potent STAT3 Inhibitor for Pancreatic Cancer Treatment.

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Unfortunately, targeting STAT3 with small molecules has proven to be very challenging, and for full activation of STAT3, the cooperative phosphorylation of both tyrosine 705 (Tyr705) and serine 727 (Ser727) is needed. Further, a selective inhibitor of STAT3 dual phosphorylation has not been developed. Here, we identified a low nanomolar potency and highly selective small-molecule STAT3 inhibitor that simultaneously inhibits both STAT3 Tyr705 and Ser727 phosphorylation. YY002 potently inhibited STAT3-dependent tumor cell growth in vitro and achieved potent suppression of tumor growth and metastasis in vivo. More importantly, YY002 exhibited favorable pharmacokinetics, an acceptable safety profile, and superior antitumor efficacy compared to BBI608 (STAT3 inhibitor that has advanced into phase III trials). For the mechanism, YY002 is selectively bound to the STAT3 Src Homology 2 (SH2) domain over other STAT members, which strongly suppressed STAT3 nuclear and mitochondrial functions in STAT3-dependent cells. Collectively, this study suggests the potential of small-molecule STAT3 inhibitors as possible anticancer therapeutic agents.

Amelioration of TPA-induced skin inflammation by the leaf extract of Vernonia amygdalina involves ERK/STAT3 (Ser727) signaling inhibition

BackgroundUncontrolled inflammation causes health problems. Extracellular signal-regulated kinase (ERK) phosphorylates signal transducer and activator of transcription 3 (STAT3) at Ser727, resulting in inflammation. The leaf of Vernonia amygdalina (VA) is a medicinal herb for managing inflammation-associated diseases. Oral administration or topical application of VA leaf extract exerts anti-inflammatory effects in rat models. However, the anti-inflammatory mechanisms of the herb are not fully understood. PurposeIn this study, we aimed to investigate the involvement of ERK/STAT3 (Ser727) signaling in the anti-inflammatory effects of an ethanolic extract of VA leaves. Study design and methodsExtracts of VA leaves were prepared with different concentrations of ethanol. A LPS-stimulated RAW264.7 cell model was used for in vitro assays, and a TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model was employed for in vivo assays. The 95% ethanol extract of VA leaves (VAE) exerted the strongest inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages; thus it was selected for use in this study. Hematoxylin and eosin (H&E) staining was used to examine pathological conditions of mouse ear tissues. Griess reagent was employed to examine NO generation in cell cultures. Immunoblotting and ELISA were used to examine protein levels, and RT-qPCR was employed to examine mRNA levels. ResultsTopical application of VAE ameliorated mouse ear edema induced by TPA. VAE suppressed the phosphorylation of ERK (Thr202/Tyr204) and STAT3 (Ser727); and decreased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in the mouse ear tissues and in LPS-stimulated RAW 264.7 cells. VAE also inhibited NO production, and lowered mRNA levels of IL-6, IL-1β and TNF-α in the macrophages. ConclusionsVAE ameliorates TPA-induced mouse ear edema. Suppression of ERK/STAT3 (Ser727) signaling is involved in VAE's anti-inflammatory effects. These novel data provide further pharmacological justifications for the medicinal use of VA in treating inflammation-associated diseases, and lay the groundwork for developing VAE into a new anti-inflammatory agent.

Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

Brain injury-mediated induction of reactive astrocytes often leads to glial scar formation in damaged brain regions. Activation of signal transducer and activator of transcription 3 (STAT3), a member of the STAT family of transcription factors, plays a pivotal role in inducing reactive astrocytes and glial scar formation. Endothelin-1 (ET-1) is a vasoconstrictor peptide, and its levels increase in brain disorders and promote astrocytic proliferation through ETB receptors. To clarify the mechanisms underlying ET-1-mediated astrocytic proliferation, here we examined its effects on STAT3 in cultured rat astrocytes. ET-1 treatment stimulated Ser-727 phosphorylation of STAT3 in the astrocytes, but Tyr-705 phosphorylation was unaffected, and ET-induced STAT3 Ser-727 phosphorylation was reduced by the ETB antagonist BQ788. ET-1 stimulated STAT3 binding to its consensus DNA-binding motifs. Monitoring G1/S phase cell cycle transition through bromodeoxyuridine (BrdU) incorporation, we found that ET-1 increases BrdU incorporation into the astrocytic nucleus, indicating cell cycle progression. Of note, STAT3 chemical inhibition (with stattic or 5,15-diphenyl-porphine (5,15-DPP)) or siRNA-mediated STAT3 silencing reduced ET-induced BrdU incorporation. Moreover, ET-1 increased astrocytic expression levels of cyclin D1 and S-phase kinase-associated protein 2 (SKP2), which were reduced by stattic, 5,15-DPP, and STAT3 siRNA. ChIP-based PCR analysis revealed that ET-1 promotes the binding of SAT3 to the 5'-flanking regions of rat cyclin D1 and SKP2 genes. Our results suggest that STAT3-mediated regulation of cyclin D1 and SKP2 expression underlies ET-induced astrocytic proliferation.

Leptin attenuates cerebral ischemic injury in rats by modulating the mitochondrial electron transport chain via the mitochondrial STAT3 pathway.

BackgroundAccording to recent studies, leptin may exert a neuroprotective function by affecting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). During stress, STAT3 regulates mitochondrial oxidative stress and reduces apoptosis.ObjectiveIn the present study, we hypothesized that leptin increases STAT3 phosphorylation in the mitochondria and protects against mitochondrial oxidative stress in rats subjected to permanent middle cerebral artery occlusion (MCAO).ResultsLeptin reduced reactive oxygen species (ROS) production, and we confirmed that the mechanism underlying this change involved the enzymatic activities of mitochondrial respiratory chain complexes I and II. In addition, leptin increased the level of STAT3 Ser727 phosphorylation in the mitochondria.ConclusionsBased on these results, leptin may regulate mitochondrial respiratory chain enzymatic activities via mitochondria‐targeted STAT3 to reduce ROS production and protect brain tissues from mitochondrial oxidative stress during cerebral ischemia.

Roles of Pin1 as a Key Molecule for EMT Induction by Activation of STAT3 and NF-κB in Human Gallbladder Cancer.

Despite developments in multidisciplinary treatment, the prognosis for advanced gallbladder cancer (GBC) still is poor because of its rapid progression. Epithelial-mesenchymal transition (EMT) plays a central role in promoting tumor invasion and metastasis in malignancies thorough signal transducer and activator of transcription-3 (STAT3) and nuclear factor κB (NF-κB) activation. Whereas Pin1 mediates STAT3 and NF-κB activation, the involvement of Pin1 in GBC progression is unclear. Factors regulating Pin1-related STAT3 and NF-κB activation were evaluated using surgical specimens collected from 76 GBC patients, GBC cells, and orthotopic GBC xenograft mice. In the patients with GBC, high Pin1 expression in GBC was associated with aggressive tumor invasion and increased tumor metastasis, and was an independent factor for a poor prognosis. Pin1 expression was correlated with phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276), thereby activating STAT3 and NF-κB in GBC. Pin1-mediated STAT3 and NF-κB activation induced EMT in GBC. When Pin1 knockdown was performed in GBC cells, the phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276) was inhibited, and STAT3 and NF-κB activation was suppressed. Inactivation of STAT3 and NF-κB in Pin1-depleted cells decreased snail and zeb-2 expression, thereby reducing the rate of mesenchymal-like cells, suggesting that EMT was inhibited in GBC cells. PiB, a Pin1-specific inhibitor, inhibited EMT and reduced tumor cell invasion by inactivating STAT3 and NF-κB in vitro. Moreover, PiB treatment inhibited lymph node metastasis and intrahepatic metastasis in orthotopic GBC xenograft tumor in vivo. Pin1 accelerates GBC invasion and metastasis by activating STAT3 and NF-κB. Therefore, Pin1 inhibition by PiB is an excellent therapy for GBC by safely inhibiting its metastasis.

Abstract 925: Helicobacter pylori induces phosphorylation of STAT3 at Ser727 to promote mitophagy in gastric cancer cells

Abstract Chronic Helicobacter pylori (H. pylori) infection has been considered to be one of the high risk factors of gastric cancer, but the exact mechanism underlying H. pylori-mediated carcinogenesis has not been completely elucidated. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is implicated in inflammation-associated carcinogenesis. In a canonical STAT3 pathway, phosphorylation of STAT3 at Tyr705 is a major event of STAT3 activation. In the present study, we found that H. pylori induced phosphorylation of STAT3 at not only Tyr705 but also Ser727. Notably, STAT3 Ser727 phosphorylation occurred at late time independently of Tyr705 phosphorylation followed by migration to mitochondrion. It has been reported that mitophagy is a specialized form of autophagy that removes damaged mitochondria to maintain efficient cellular metabolism and reduces cellular stress. In our study, H. pylori infection induced mitophagy with upregulation of LC3 (microtubule-associated chain3), which is an autophagy marker, in mitochondria. H. pylori-induced Ser727 phosphorylation appears to regulate expression of LC3 based on the experiment utilizing the STAT3 mutant vector in which Ser727 is replaced by alanine, suggesting a potential link between mitophagy and STAT3 phosphorylation at Ser727. Furthermore, we found that STAT3 phosphorylated at Ser727 bound to Grim19 which is a component of complex I and heat shock protein 90 in mitochondria. How this binding contributes to mitopagy is under investigation. In conclusion, H. pylori induced STAT3 phosphorylation at Ser727 to promote mitophagy Citation Format: Juan-Yu Piao, Hee Geum Lee, Su-Jung Kim, Do-Hee Kim, Hye-Kyung Na, Young-Joon Surh. Helicobacter pylori induces phosphorylation of STAT3 at Ser727 to promote mitophagy in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 925. doi:10.1158/1538-7445.AM2015-925

Mitochondrial STAT3 contributes to transformation of Barrett's epithelial cells that express oncogenic Ras in a p53-independent fashion.

Metaplastic epithelial cells of Barrett's esophagus transformed by the combination of p53-knockdown and oncogenic Ras expression are known to activate signal transducer and activator of transcription 3 (STAT3). When phosphorylated at tyrosine 705 (Tyr705), STAT3 functions as a nuclear transcription factor that can contribute to oncogenesis. STAT3 phosphorylated at serine 727 (Ser727) localizes in mitochondria, but little is known about mitochondrial STAT3's contribution to carcinogenesis in Barrett's esophagus, which is the focus of this study. We introduced a constitutively active variant of human STAT3 (STAT3CA) into the following: 1) non-neoplastic Barrett's (BAR-T) cells; 2) BAR-T cells with p53 knockdown; and 3) BAR-T cells that express oncogenic H-Ras(G12V). STAT3CA transformed only the H-Ras(G12V)-expressing BAR-T cells (evidenced by loss of contact inhibition, formation of colonies in soft agar, and generation of tumors in immunodeficient mice), and did so in a p53-independent fashion. The transformed cells had elevated levels of both mitochondrial (Ser727) and nuclear (Tyr705) phospho-STAT3. Introduction of a STAT3CA construct with a mutated tyrosine phosphorylation site into H-Ras(G12V)-expressing Barrett's cells resulted in high levels of mitochondrial phospho-STAT3 (Ser727) with little or no nuclear phospho-STAT3 (Tyr705), and the cells still formed tumors in immunodeficient mice. Thus tyrosine phosphorylation of STAT3 is not required for tumor formation in Ras-expressing Barrett's cells. We conclude that mitochondrial STAT3 (Ser727) can contribute to oncogenesis in Barrett's cells that express oncogenic Ras. These findings suggest that agents targeting STAT3 might be useful for chemoprevention in patients with Barrett's esophagus.

Serine-727 phosphorylation activates hypothalamic STAT-3 independently from tyrosine-705 phosphorylation.

Transcriptional activity of signal transducer and activator of transcription-3 (STAT-3) is a key element in the central regulation of appetite and energy homeostasis. Activation of hypothalamic STAT-3 has been attributed to cytokine-promoted phosphorylation at tyrosine-705 (Tyr-705). In nonhypothalamic cells, STAT-3 is also phosphorylated at serine-727 (Ser-727), but the functional significance of Ser-727 in the regulation of hypothalamic STAT-3 is not known. We used 2 hypothalamic cell lines and analyzed the effects of various hormones on STAT-3-dependent reporter gene activity and observed that IFN-γ, epidermal growth factor (EGF), and bradykinin (BK) induce similar STAT-3 reporter activation. EGF and BK solely increased Ser-727 and IFN-γ increased Tyr-705 phosphorylation of STAT-3. Specific inhibition of ERK-1/2 activity blocked EGF- and BK-induced STAT-3 activation and Ser-727 phosphorylation. BK-induced ERK-1/2 activation occurred via EGF receptor transactivation. Consequently, the BK-mediated effects on STAT-3 were blocked by a specific EGF receptor antagonist. Next, we analyzed the effects of IFN-γ and EGF on the expression of the STAT-3-dependent genes thyroliberin-releasing hormone and suppressors of cytokine signaling-3. EGF but not IFN-γ enhanced thyroliberin-releasing hormone expression via STAT-3. With regard to suppressors of cytokine signaling-3, we observed prolonged expression induced by IFN-γ and a transient effect of EGF that required coactivation of the activator protein-1. Thus, EGF-promoted Ser-727 phosphorylation by ERK-1/2 is not only sufficient to fully activate hypothalamic STAT-3, but, in terms of targeted genes and required cofactors, entails distinct modes of STAT-3 actions compared with IFN-γ-induced Tyr-705 phosphorylation.

Thrombospondin peptide ABT-898 inhibits inflammation and angiogenesis in a colitis model.

To evaluate the efficacy of the improved thrombospondin mimetic peptide ABT-898 in a murine model of ulcerative colitis. The dextran sodium sulfate (DSS) was used for the induction of colitis in both TSP-1 deficient (TSP-1(-/-)) and wild type (WT) mice during 7 d. While mice were receiving the DSS dissolved in the drinking water, the ABT-898 peptide was dissolved in sterile 5% glucose solution and delivered using mini pumps subcutaneously implanted. Plasma samples were analyzed for interleukin (IL)-6 by ELISA assay and colonic tissues were harvested, fixed and processed for histological evaluation. Immunohistochemistry using antibodies for the detection of CD31 and MECA in endothelial cells was performed. Inflammation was graded in colonic sections and the number of microvessels in each lesion was assessed. Activation of signal transducer and activator of transcription 3 (STAT3) in colonic samples was quantified by immunohistochemistry and Western blotting using antibodies against total STAT3 and phosphorylated STAT3 (pSTAT3) (Ser727). Treatment with ABT-898 considerably diminished the inflammatory response in WT and TSP-1(-/-) mice (P < 0.0001 in both groups vs control). Identification of blood vessels highlighted by CD31/MECA immunohistochemistry, showed significantly reduced vessel counts in colitic lesions of WT and TSP-1(-/-) mice treated with ABT898 (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0002; WT controls/WT treated, P = 0.0005). Consistently, IL-6 was significantly diminished in plasma samples of TSP-1(-/-) and WT treated with the peptide when compared to the control mice (P = 0.0002 and P = 0.0148, respectively). pSTAT3 positive cells were quantified in WT and TSP-1(-/-) treated with ABT-898. A significant decrease in positive cells for pSTAT3 was observed in treated mice (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0089; WT/WT treated, P = 0.0110). These results were confirmed by Western blotting analyses showing lower levels of pSTAT3 in colitic lesions from mice treated with the peptide ABT-898. These findings indicate that the new peptide ABT-898 ameliorates inflammation and angiogenesis and might be a therapeutic alternative in IBD and inflammatory diseases.

Mitochondrial Localized Stat3 Promotes Breast Cancer Growth via Phosphorylation of Serine 727

Signal transducer and activator of transcription 3 (Stat3) is a key mediator in the development of many cancers. For 20 years, it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription factor activated by many cytokines. However, recent studies from this laboratory and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras-induced transformation of mouse embryo fibroblasts. The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed, we used 4T1 breast cancer cells, which form tumors that metastasize in immunocompetent mice. Substitution of Ser-727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, MLS-Stat3, has profound effects on tumor growth, complex I activity of the ETC, and accumulation of reactive oxygen species (ROS). Cells expressing MLS-Stat3(S727A) display slower tumor growth, decreased complex I activity of the ETC, and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor growth and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC.

Abstract 2592: Helicobacter pylori induces phosphorylation of STAT3 Tyr705 and Ser727 in human gastric epithelial cells.

Abstract Helicobacter pylroi (H. pylori) infection has been considered to be one of the high risk factors of gastritis and gastric cancer, but its pathogenic implication is still poorly understood. Recent studies indicate that constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated carcinogenesis. In our previous study, we found that the phosphorylation of STAT3 at Tyr705 was increased in H. pylori plus high salt diet-induced gastritis in a mouse model. The similar results were observed in human gastric AGS cells co-cultured with H. pylori. Interestingly, H. pylori induced phosphorylation of STAT3 at not only Tyr705 but also Ser727. However, the phosphorylation of two residues exhibited different profiles in human gastric epithelial cells upon H. pylori treatment. The phosphor-Tyr705 increased at early time of H. pylori infection and then decreased immediately, but phosphor-Ser727 increased thereafter which was first discovered in our present study. Phosphorylation of STAT3 is an essential event for inducing the expression of anti-apoptotic/survival proteins, such as Bcl-XL, BcL-2, survivin and c-Myc which contribute to gastric carcinogenesis. Levels of all these proteins were elevated in H. pylori-infected AGS cells. In conclusion, H. pylori induces phosphorylation of STAT3 at both Tyr705 and Ser727 residues. The role of phospohorylation of STAT3 at the Serine 727 residue in H. pylori-induced gastritis and gastric cancer is under investigation. Citation Format: Juan-Yu Piao, Hee-Geum Lee, Hyeon-Geum Ji, Hyeong-Joon Han, Hoang Kieu Chi Ngo, Do-Hee Kim, Su-Jung Kim, Sung-Young Lee, Young-Joon Surh. Helicobacter pylori induces phosphorylation of STAT3 Tyr705 and Ser727 in human gastric epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2592. doi:10.1158/1538-7445.AM2013-2592

Intranuclear Crosstalk between Extracellular Regulated Kinase1/2 and Signal Transducer and Activator of Transcription 3 Regulates JEG‐3 Choriocarcinoma Cell Invasion and Proliferation

Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation.

Abstract 2802: Selective inhibition of signal transducers and activators of transcription 3 overexpressing endometrial cancer cells, using HO-3867, a novel STAT3 inhibitor

Abstract OBJECTIVE: Signal transducer and activator of transcription 3 (STAT3) proteins have important roles in cancer cell survival and proliferation. Recent studies show constitutive activation of STAT3 is associated with tumor growth and oncogenic transformation in several human cancers; however, little is known about STAT3 expression in endometrial cancer. The objectives of this study were to examine the expression of the STAT family of proteins in endometrial cancer cell lines and to evaluate the efficacy of HO-3867, a novel STAT3 inhibitor. METHODS: Expression of STAT family proteins, cell cycle and apoptotic proteins were evaluated via western blot on Ishikawa, AN3-CA, RL95-2, HEC1B, HEC-1A, and SK-UT1B human endometrial cancer cell lines. Cell viability and proliferation of these cell lines were analyzed with MTT and nucleo counter assay after treatment with increasing concentrations of HO-3867. Cell cycle analysis and apoptosis were performed using flow cytometry and annexin V staining. An ubiquitination assay was used to examine the inhibition of CDK5 degradation. Finally, STAT3 over-expression or suppression experiments were performed using wild-type STAT3 cDNA and STAT3 siRNA. RESULTS: Expression of activated STAT1, 2, 5, and 6 was inconsistent among cell lines tested, while pSTAT3 Ser727 had consistently high expression. HO-3867 decreased cell viability through induced G2/M arrest and apoptosis within 24h of treatment. HO-3867 inhibited pSTAT3 Ser727 with no change in total STAT3. This was associated with an increase in expression of cell cycle related proteins p53 and p21, a decrease in anti-apoptotic proteins Bcl2 and Bcl-XL, as well as increased cleavage of caspases. After 24h of treatment with HO-3867, 55-88% of cells were apoptotic. The downregulation of Ser727 was, at least in part, due to an accelerated inhibition of ubiquitin-dependent CDK5 degradation, as a positive regulator of STAT3 Ser727. Suppression of STAT3 expression with siRNA significantly reduced cell survival. In addition, we confirmed that over-expression of STAT3 cDNA provides resistance to HO-3867. CONCLUSION: Endometrial cancer cells express high levels of pSTAT3 Ser727. HO-3867 induces cell cycle arrest and apoptosis by targeting pSTAT3 Ser727. Our findings suggest a specific role of serine-phosphorylated STAT3 that is independent of tyrosine phosphorylation. HO-3867 may be a potential adjunct to chemotherapy and radiation in the treatment of endometrial cancer. 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 2802. doi:1538-7445.AM2012-2802

Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation

Nutrient overload is associated with the development of obesity, insulin resistance, and type II diabetes. High plasma concentrations of amino acids have been found to correlate with insulin resistance. At the cellular level, excess amino acids impair insulin signaling, the mechanisms of which are not fully understood. Here, we report that STAT3 plays a key role in amino acid dampening of insulin signaling in hepatic cells. Excess amino acids inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis in mouse primary hepatocytes as well as in human hepatocarcinoma HepG2 cells. STAT3 knockdown protected insulin sensitivity from inhibition by amino acids. Amino acids stimulated the phosphorylation of STAT3 at Ser(727), but not Tyr(705). Replacement of the endogenous STAT3 with wild-type, but not S727A, recombinant STAT3 restored the ability of amino acids to inhibit insulin signaling, suggesting that Ser(727) phosphorylation was critical for STAT3-mediated amino acid effect. Furthermore, overexpression of STAT3-S727D was sufficient to inhibit insulin signaling in the absence of excess amino acids. Our results also indicated that mammalian target of rapamycin was likely responsible for the phosphorylation of STAT3 at Ser(727) in response to excess amino acids. Finally, we found that STAT3 activity and the expression of its target gene socs3, known to be involved in insulin resistance, were both stimulated by excess amino acids and inhibited by rapamycin. In conclusion, our study reveals STAT3 as a novel mediator of nutrient signals and identifies a Ser(727) phosphorylation-dependent and Tyr(705) phosphorylation-independent STAT3 activation mechanism in the modulation of insulin signaling.

Green Tea Polyphenol Epigallocatechin-3-Gallate Suppresses Collagen Production and Proliferation in Keloid Fibroblasts via Inhibition of the STAT3-Signaling Pathway

Keloids are benign skin tumors characterized by collagen accumulation and hyperproliferation of fibroblasts. To find an effective therapy for keloids, we explored the pharmacological potential of (-)-epigallocatechin-3-gallate (EGCG), a widely investigated tumor-preventive agent. When applied to normal and keloid fibroblasts (KFs) in vitro, proliferation and migration of KFs were more strongly suppressed by EGCG than normal fibroblast proliferation and migration (IC(50): 54.4 microM (keloid fibroblast (KF)) versus 63.0 microM (NF)). The level of Smad2/3, signal transducer and activator of transcription-3 (STAT3), and p38 phosphorylation is more enhanced in KFs, and EGCG inhibited phosphorylation of phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated protein kinase 1/2 (ERK1/2), and STAT3 (Tyr705 and Ser727). To evaluate the contribution of these pathways to keloid pathology, we treated KFs with specific inhibitors for PI3K, ERK1/2, or STAT3. Although a PI3K inhibitor significantly suppressed proliferation, PI3K and MEK/ERK inhibitors had a minor effect on migration and collagen production. However, a JAK2/STAT3 inhibitor and a STAT3 siRNA strongly suppressed proliferation, migration, and collagen production by KFs. We also found that treatment with EGCG suppressed growth and collagen production in the in vivo keloid model. This study demonstrates that EGCG suppresses the pathological characteristics of keloids through inhibition of the STAT3-signaling pathway. We propose that EGCG has potential in the treatment and prevention of keloids.

Cucurbitacin I (JSI-124) Has Potent Anti-Myeloma Effects Independent of Its Inhibition of JAK2-Induced STAT3 Activation.

Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. JSI-124 is a plant natural product identified previously as Cucurbitacin I, isolated from various plant families such as the Cucurbitaceae and Cruciferae and has been recently described as a specific inhibitor of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3). Based on the critical role of the IL-6/STAT3 pathway in MM we studied the effects of JSI-124 on different MM cells in vitro. Different human myeloma cell lines including MM1.S, IM9, OPM-2, RPMI-8226, ARH77 in addition to the murine 5TGM myeloma cell lines were incubated with increasing concentrations of JSI-124. The impact of JSI-124 on cell proliferation, cell cycle and induction of apoptosis was studied using [3H]-Thymidine incorporation, cell counting, flow cytometry and Annexin/PI staining and caspase 3, 8 and 9 activation. JSI-124 was able to inhibit proliferation and induce apoptosis in several MM cell lines, including MM1.S, IM9, OPM-2, RPMI-8226, ARH77, U266 and 5TGM in a dose- and time-dependent manner. JSI-124 lead to activation of caspase 3, 8 and 9 indicating involvement of both extrinsic and intrinsic apoptotic pathways. JSI-124-mediated induction of apoptosis was independent of JAK2/STAT3 inhibition as MM1.S and 5TGM cells, which lack constitutive STAT3 (Tyr705) activation were equally sensitive to JSI-124 compared to U266 cells which show a constitutive STAT3 Tyr705 phosphorylation. However, JSI-124 treatment was able to abrogate IL-6 and bone marrow stroma (BMSC)-induced STAT3 (Tyr705) activation in MM1.S cells. In addition we were able to observe JSI-124 dependent inhibition of constitutive STAT3 (Ser727) activation in MM cell lines. To further delineate the mechanism underlying its anti-myeloma effects we studied the impact of JSI-124 treatment on NF-κB, MAPK and PI3K pathways. Indeed, JSI-124 treatment resulted in inhibition of p-p65, p-MEK1,2 and p-Akt underscoring the effect of JSI-124 on STAT3-independent signaling. Our results indicate that JSI-124 is a powerful direct inhibitor of myeloma cells blocking constitutive and IL-6/BMSC-dependent STAT3 activation in addition to STAT3 independent signaling pathways. JSI-124 might therefore serve as a potent novel anti-myeloma agent targeting both myeloma cells and its bone marrow microenvironment. Further studies are warranted to evaluate the in vivo efficacy of JSI-124 and identify the STAT3 independent pathways contributing to myeloma cell growth and induction of apoptosis.

Activation of STAT3 by Gαs Distinctively Requires Protein Kinase A, JNK, and Phosphatidylinositol 3-Kinase

Signal transducer and activator of transcription 3 (STAT3) can be stimulated by several G(s)-coupled receptors, but the precise mechanism of action has not yet been elucidated. We therefore examined the ability of Galpha(s)Q226L (Galpha(s)QL), a constitutively active mutant of Galpha(s), to stimulate STAT3 Tyr705 and Ser727 phosphorylations in human embryonic kidney 293 cells. Apart from Galpha(s)QL, the stimulation of Galpha(s) by cholera toxin or beta2-adrenergic receptor and the activation of adenylyl cyclase by forskolin, (Sp)-cAMP, or dibutyryl-cAMP all promoted both STAT3 Tyr705 and Ser727 phosphorylations. Moreover, the removal of Galpha(s) by RNA interference significantly reduced the beta2-adrenergic receptor-mediated STAT3 phosphorylations, denoting its capacity to regulate STAT3 activation by a G protein-coupled receptor. The possible downstream signaling molecules involved were assessed by using specific inhibitors and dominant negative mutants. Induction of STAT3 Tyr705 and Ser727 phosphorylations by Galpha(s)QL was suppressed by inhibition of protein kinase A, Janus kinase 2/3, Rac1, c-Jun N-terminal kinase (JNK), or phosphatidylinositol 3-kinase, and a similar profile was observed in response to beta2-adrenergic receptor stimulation. In contrast to the Galpha16-mediated regulation of STAT3 in HEK 293 cells (Lo, R. K., Cheung, H., and Wong, Y. H. (2003) J. Biol. Chem. 278, 52154-52165), the Galpha(s)-mediated responses, including STAT3-driven luciferase activation, were resistant to inhibition of phospholipase Cbeta. Surprisingly, Galpha(s)-mediated phosphorylation at Tyr705, but not at Ser727, was resistant to inhibition of c-Src, Raf-1, and MEK1/2 as well as to the expression of dominant negative Ras. Therefore, as with other Galpha-mediated activations of STAT3, the stimulatory signal arising from Galpha(s) is transduced via multiple signaling pathways. However, unlike the mechanisms employed by Galpha(i) and Galpha(14/16), Galpha(s) distinctively requires protein kinase A, JNK, and phosphatidylinositol 3-kinase for STAT3 activation.

Heparin Synergistically Enhances Interleukin-11 Signaling through Up-regulation of the MAPK Pathway

Using an animal model of heparin-induced osteoporosis we previously demonstrated that heparin causes bone loss, in part, by increasing osteoclast number and activity. Furthermore, we found that, although heparin alone has no effect, it is able to synergistically enhance Interleukin-11 (IL-11)-induced signal transducer and activator of transcription 3 (STAT3) activation and thus increase osteoclast formation in vitro. In the present study, we examine the effect of various serine kinase inhibitors on the ability of heparin to act synergistically with IL-11. Inhibition of the c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or the phosphatidylinositol 3-kinase pathways had no effect on the ability of heparin to promote either IL-11-induced STAT3.DNA complex formation or osteoclast formation in vitro. In contrast, PD098059, a MAPK kinase inhibitor, completely abolished the synergy between heparin and IL-11. In an attempt to resolve the mechanism by which this was occurring, we examined the effect of heparin on STAT3 Ser-727 phosphorylation and extracellular signal-regulated kinases 1 and 2 (Erk1/2) activation, either in the presence or absence of IL-11. Heparin alone was found to have no effect on Ser-727 phosphorylation, nor did heparin alter the phosphorylation status of Ser-727 in the presence of IL-11. Heparin was, however, found to increase Erk1/2 activation in both a time- and dose-dependent manner. When taken together, these findings suggest that heparin enhances IL-11-induced STAT3 activation and thus osteoclast formation, by a mechanism that is independent of STAT3 Ser-727 phosphorylation but that involves up-regulation of the MAPK pathway.

BCR–ABL activates STAT3 via JAK and MEK pathways in human cells

Chronic myeloid leukaemia (CML) is characterised by a progression from a chronic towards an acute phase. We previously reported that signal transducer and activator of transcription 3 (STAT3), a major oncogenic signalling protein, is the target of p210-BCR-ABL in a murine embryonic stem (ES) cell model and in primary CD34+ CML cells. This activation was associated with inhibition of differentiation in ES cells. The present study found that BCR-ABL greatly phosphorylated STAT3 Ser727 residue and, to a lesser extent, Tyr705 residue in BCR-ABL-expressing cell lines (UT7-p210, MO7E-p210, and K562) and in primary CD34+ CML cells. Using BCR-ABL mutants, it was shown that BCR-ABL tyrosine kinase activity and its Tyr177 residue were necessary for STAT3 Ser727 phosphorylation. Constitutive STAT3 Tyr705 phosphorylation was associated with constitutive phosphorylation of Janus kinase (JAK)1 and JAK2, and was inhibited by the JAK inhibitor AG490, suggesting the involvement of JAK proteins in this process. Specific MEK [mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase] inhibitors PD98056 and UO126, as well as the use of a dominant-negative form of MEK1 abrogated STAT3 Ser727 phosphorylation, suggesting involvement of MAP-Kinase/Erk pathway. Inhibition of BCR-ABL with imatinib mesylate led to a dose-dependent downregulation of total STAT3 protein and mRNA, suggesting that BCR-ABL is involved in the transcriptional regulation of STAT3. Targeting JAK, MEK and STAT3 pathways could therefore be of therapeutic value, especially in advanced stage CML.