Tyrosine Phosphorylation Of STAT3 Research Articles

2-Ethoxystypandrone, a novel small-molecule STAT3 signaling inhibitor from Polygonum cuspidatum, inhibits cell growth and induces apoptosis of HCC cells and HCC Cancer stem cells

BackgroundSignal transducer and activator of transcription 3 (STAT3) is an oncogene constitutively activated in hepatocellular carcinoma (HCC) cells and HCC cancer stem cells (CSCs). Constitutively activated STAT3 plays a pivotal role in holding cancer stemness of HCC CSCs, which are essential for hepatoma initiation, relapse, metastasis and drug resistance. Therefore, STAT3 has been validated as a novel anti-cancer drug target and the strategies targeting HCC CSCs may bring new hopes to HCC therapy. This study aimed to isolate and identify small-molecule STAT3 signaling inhibitors targeting CSCs from the ethyl acetate (EtOAc) extract of the roots of Polygonum cuspidatum and to evaluate their in vitro anti-cancer activities.MethodsThe chemical components of the EtOAc extract and the subfractions of P. cuspidatum were isolated by using various column chromatographies on silical gel, Sephadex LH-20, and preparative HPLC. Their chemical structures were then determined on the basis of spectroscopic data including NMR, MS and IR analysis and their physicochemical properties. The inhibitory effects of the isolated compounds against STAT3 signaling were screened by a STAT3-dependent luciferase reporter gene assay. The tyrosine phosphorylation of STAT3 was examined by Western Blot analysis. In vitro anti-cancer effects of the STAT3 pathway inhibitor were further evaluated on cell growth of human HCC cells by a MTT assay, on self-renewal capacity of HCC CSCs by the tumorsphere formation assay, and on cell cycle and apoptosis by flow cytometry analysis, respectively.ResultsThe EtOAc extract of the roots of P. cuspidatum was investigated and a novel juglone analogue 2-ethoxystypandrone (1) along with seven known compounds (2–8) was isolated. Among the eight isolated compounds 1–8, 2-ethoxystypandrone was a novel and potent STAT3 signaling inhibitor (IC50 = 7.75 ± 0.18 μM), and inhibited the IL-6-induced and constitutive activation of phosphorylation of STAT3 in HCC cells. Moreover, 2-ethoxystypandrone inhibited cell survival of HCC cells (IC50 = 3.69 ± 0.51 μM ~ 20.36 ± 2.90 μM), blocked the tumorspheres formation (IC50 = 2.70 ± 0.28 μM), and induced apoptosis of HCC CSCs in a dose-dependent manner.ConclusionA novel juglone analogue 2-ethoxystypandrone was identified from the EtOAc extract of the roots of P. cuspidatum and was demonstrated to be a potent small-molecule STAT3 signaling inhibitor, which strongly blocked STAT3 activation, inhibited proliferation, and induced cell apoptosis of HCC cells and HCC CSCs. 2-Ethoxystypandrone as a STAT3 signaling inhibitor might be a promising lead compound for further development into an anti-CSCs drug.

2-Hydroxy-3-methylanthraquinone inhibits lung carcinoma cells through modulation of IL-6-induced JAK2/STAT3 pathway

Background2-hydroxy-3-methylanthraquinone (HMA), an anthraquinone monomer in traditional Chinese medicine Hedyotis diffusa, has been reported to inhibit the growth of several types of cancer, but its effect on lung cancer has not been adequately investigated. Hypothesis/PurposeThis study aimed to test the hypothesis that HMA inhibit the growth, migration, and invasion of lung cancer cells in part via downregulation of interleukin (IL)-6-induced JAK2/STAT3 pathway. MethodsGrowth and apoptosis of lung cancer cells were quantitated by CCK-8 assay and Annexin V-FITC/PI flow cytometric analysis, respectively. Migration and invasion of A549 cells were determined by wound-healing assay and transwell invasion assay, respectively. The effect of HMA on cytokines expression in A549 cells was evaluated by the cytokine antibody array assay. Gene expression and protein levels of related molecular markers were quantitated by real time-PCR and Western blot analysis, respectively. ResultsHMA significantly inhibited IL-6-stimulated growth and colony formation of A549 cells, increased the number of apoptotic cells, and inhibited invasion associated with downregulation of expression of IL-6-induced MMP-1, MMP-2, and MMP-9 genes. IL-6 increased the levels of tyrosine phosphorylation of JAK2 and STAT3 in A549 cells, which was reversed by HMA treatment. In addition, HMA reduced the expression of a series of inflammation-related cytokines in A549 cells supernatant, including IL-6, G-CSF, IL-6R, IL-8, MCP-1, RANTES, TNF-α. ConclusionThese results suggest that HMA may inhibit the growth and invasion of lung cancer cells in part via downregulation of IL-6-induced JAK2/STAT3 pathway.

Robustness and Information Transfer within IL-6-induced JAK/STAT Signalling

Cellular communication via intracellular signalling pathways is crucial. Expression and activation of signalling proteins is heterogenous between isogenic cells of the same cell-type. However, mechanisms evolved to enable sufficient communication and to ensure cellular functions. We use information theory to clarify mechanisms facilitating IL-6-induced JAK/STAT signalling despite cell-to-cell variability. We show that different mechanisms enabling robustness against variability complement each other. Early STAT3 activation is robust as long as cytokine concentrations are low. Robustness at high cytokine concentrations is ensured by high STAT3 expression or serine phosphorylation. Later the feedback-inhibitor SOCS3 increases robustness. Channel Capacity of JAK/STAT signalling is limited by cell-to-cell variability in STAT3 expression and is affected by the same mechanisms governing robustness. Increasing STAT3 amount increases Channel Capacity and robustness, whereas increasing STAT3 tyrosine phosphorylation reduces robustness but increases Channel Capacity. In summary, we elucidate mechanisms preventing dysregulated signalling by enabling reliable JAK/STAT signalling despite cell-to-cell heterogeneity.

Safety and Efficacy of HQP1351, a 3rd Generation Oral BCR-ABL Inhibitor in Patients with Tyrosine Kinase Inhibitor—Resistant Chronic Myelogenous Leukemia: Preliminary Results of Phase I Study

Background: HQP1351, a novel, potent 3rd generation TKI, was designed to treat patients with chronic myeloid leukemia (CML) resistant to current TKI-therapies including those with T315I mutation.Aims: A single-agent, open-label dose escalation and dose expansion Phase I study to assess the safety, preliminary efficacy, pharmacokinetic (PK) and pharmacodynamic (PD) properties of orally administered HQP1351 in the TKI-resistant patients with chronic phase (CP) or accelerated phase (AP) CML.Methods: HQP1351 was administered once every other day (QOD) in 28-days cycles at 11 dose cohorts ranging from 1mg to 60 mg. The eligible patients received treatments until disease progression or intolerable toxicities. The primary efficacy endpoint in the CML AP and CP patients, was complete hematological response (CHR) and major cytogenetic response (MCyR) respectively, MCyR includes partial cytogenetic response (PCyR) and complete cytogenetic response (CCyR).Blood samples were collected at various time points on Day 1-2 and Day 27-28 during cycle 1 for PK analyses. BCR-ABL inhibition was evaluated using tyrosine phosphorylation of CRKL and STAT5 in peripheral blood mononuclear cell (PBMCs) collected from the patients before and 4, 8, 24 and 48 hours post HQP1351 treatments on Day 1, 15 and 27 during cycle 1.Results:From November 2016 to July 2018, 70 patients (CP n=58 and AP n=12) enrolled received ≥1 cycle of HQP1351-treatment, only 1 patient withdrew from the study due to disease progression. Median age was 39 (range: 23-59) years. Median interval from CML diagnosis to starting HQP1351-treatment was 6.1 (1.1-14.7) years. Sixty-one (87%) patients received ≥2 prior lines of TKI-therapy. Fifty-three (76%) patients had BCR-ABL mutations and 45 (64%) had T315I mutation at baseline.With a median follow-up of 4.1 (1.0-21.2) cycles, HQP1351 was well-tolerated in all dose cohorts other than the 60mg cohort. In all patients, 67 (96%) patients experienced ≥1 treatment related adverse events (TRAEs) and 44 (63%) experienced TRAE(s) of grade 3-4 (Table 1). There was no patient withdrawal from the study because of TRAEs. Two out of 3 patients in the 60mg cohort experienced dose-limiting toxicity(DLT) and HQP1351 at 50mg QOD was considered as MTD.The anti-leukemic activities of HQP1351 were observed in this study. Sixty-five (93%) patients including 58 (100%) CP and 7 (58%) AP patients achieved CHR at the dose of 2mg to 60mg within 3 cycles. In 47 evaluable patients receiving HQP1351-treatment ≥3 cycles, 24 (51%) achieved MCyR including 21 (54%) CP and 3 (38%) AP patients at the dose of 12mg to 50mg, and 14 (30%) patients achieved CCyR including 12 (31%) CP and 2 (25%) AP patients. Total 6 (15%) CP patients achieved MMR. More than 65% of the patients achieved MCyR or MMR at the end of cycle 3. HQP1351 was highly active in patients with or without T315I mutation at baseline (Table 2).Following oral administration of HQP1351 at doses ranging from 1mg to 60 mg, the peak concentration was reached at 2-8 hrs. The elimination appeared to be linear with a mean terminal T1/2 of 15.3-36.5 hrs on Day 1, 18.8-42.5 hrs on Day 27, respectively. The ratios for AUC0-t and Cmax of HQP1351 on Day 27 versus Day 1 ranged from 1.03 to 2.12 and from 0.78 to 1.93, respectively. Thus, HQP1351 exhibited an approximately dose proportional increase in Cmax and AUC0-t following single or multiple oral administration dose ranging from 1 to 60 mg. PD study results demonstrated that reduction of CRKL phosphorylation was schedule and dose-dependent, ≥50% reduction was observed at doses ranging 12mg-60mg.Conclusions: The preliminary results of the phase 1 study showed that HQP1351, a novel 3rd-generation TKI, is safe and highly active in treatment of the TKI-resistant patients with CP-CML and AP-CML,with or without T315I mutation. DisclosuresChen:HealthQuest Pharma Inc.: Employment. Men:HealthQuest Pharma Inc.: Employment. Liu:HealthQuest Pharma Inc.: Employment. Sun:HealthQuest Pharma Inc.: Employment. JI:HealthQuest Pharma Inc.: Employment. Wang:HealthQuest Pharma Inc.: Employment. Hu:HealthQuest Pharma Inc.: Employment. Zou:HealthQuest Pharma Inc.: Employment. Yan:HealthQuest Pharma Inc.: Employment. Huang:Ascentage Pharma Group Inc.: Employment; HealthQuest Pharma Inc.: Employment. Yang:Ascentage Pharma Group Inc.: Employment. Zhai:Ascentage Pharma Group Inc.: Employment.

IL (Interleukin)-10-STAT3-Galectin-3 Axis Is Essential for Osteopontin-Producing Reparative Macrophage Polarization After Myocardial Infarction.

Both osteopontin (OPN) and galectin-3 have been implicated in phagocytic clearance of dead cells and reparative fibrosis during wound healing. CD206+ macrophages are involved in tissue repair through phagocytosis and fibrosis after myocardial infarction (MI). However, the relationship among OPN, galectin-3, and macrophage polarization in the context of MI remains unclear. The time course of Spp1 (encoding OPN) expression in the heart after MI showed a strong activation of Spp1 on day 3 after MI. To identify where in the body and in which cells the transcriptional activity of Spp1 increased after MI, we analyzed EGFP (enhanced green fluorescent protein)- Spp1 knockin reporter mice on day 3 after MI. The transcriptional activity of Spp1 increased only in CD206+ macrophages in the infarct myocardium, and most of CD206+ macrophages have strong transcriptional activation of Spp1 after MI. The temporal expression pattern of Lgal3 (encoding galectin-3) in cardiac macrophages after MI was similar to that of Spp1, and OPN is almost exclusively produced by galectin-3hiCD206+ macrophages. Although both interleukin (IL)-4 and IL-10 were reported to promote CD206+ macrophage-mediated cardiac repair after MI, IL-10- but not IL-4-stimulated CD11b+Ly6G- cells could differentiate into OPN-producing galectin-3hiCD206+ macrophages and showed enhanced phagocytic ability. Inhibition of STAT3 tyrosine phosphorylation suppressed IL-10-induced expression of intracellular galectin-3 and transcriptional activation of Spp1. Knockdown of galectin-3 suppressed their ability to differentiate into OPN-producing cells, but not STAT3 activation. The tyrosine phosphorylation of STAT3 and the appearance rate of galectin-3hiCD206+ cells on cardiac CD11b+Ly6G- cells in Spp1 knockout mice were the same as those in wild-type mice. Spp1 knockout mice showed vulnerability to developing post-MI left ventricular chamber dilatation and the terminal deoxynucleo-tidyltransferase 2'-Deoxyuridine-5'-triphosphate nick-end labeling (TUNEL)-positive cells in the infarcted myocardium after MI remained higher in number in Spp1 knockout mice than in wild-type mice. OPN is almost exclusively produced by galectin-3hiCD206+ macrophages, which specifically appear in the infarct myocardium after MI. The IL-10-STAT3-galectin-3 axis is essential for OPN-producing reparative macrophage polarization after myocardial infarction, and these macrophages contribute to tissue repair by promoting fibrosis and clearance of apoptotic cells. These results suggest that galectin-3 may contribute to reparative fibrosis in the infarct myocardium by controlling OPN levels.

RITA induces apoptosis in p53-null K562 leukemia cells by inhibiting STAT5, Akt, and NF-κB signaling pathways.

Targeting oncogenic signaling pathways by small molecules has emerged as a potential treatment strategy for cancer. reactivation of p53 and induction of tumor cell apoptosis (RITA) is a promising anticancer small molecule that reactivates p53 and induces exclusive apoptosis in tumor cells. Less well appreciated was the possible effect of small molecule RITA on p53-null leukemia cells. In this study, we demonstrated that RITA has potent antileukemic properties against p53-null chronic myeloid leukemia (CML)-derived K562 cells. RITA triggered apoptosis through caspase-9 and caspase-3 activation and poly (ADP-ribose) polymerase cleavage. RITA decreased STAT5 tyrosine phosphorylation, although it did not inhibit phosphorylation of the direct BCR-ABL substrate CrkL. Real-time PCR analysis showed that RITA downregulates antiapoptotic STAT5 target genes Bcl-xL and MCL-1. The downregulation of nuclear factor-κB (NF-κB), as evidenced by inhibition of IκB-α phosphorylation and its degradation, was associated with inhibition of Akt phosphorylation in RITA-treated cells. Furthermore, consistent with the decrease of mRNA levels, protein levels of the nuclear factor-κB-regulated antiapoptotic (cIAP1, XIAP, and Bcl-2) and proliferative (c-Myc) genes were downregulated by RITA in K562 cells. In conclusion, the ability of RITA to inhibit prosurvival signaling pathways in CML cells suggests a potential application of RITA in CML therapeutic protocols.

Aryl Hydrocarbon Receptor Promotes IL-10 Expression in Inflammatory Macrophages Through Src-STAT3 Signaling Pathway.

The aryl hydrocarbon receptor (AhR) is an important immune regulator with a role in inflammatory response. However, the role of AhR in IL-10 production by inflammatory macrophages is currently unknown. In this study, we investigated LPS-induced IL-10 expression in macrophages from AhR-KO mice and AhR-overexpressing RAW264.7 cells. AhR was highly expressed after LPS stimulation through NF-κB pathway. Loss of AhR resulted in reduced IL-10 expression in LPS-induced macrophages. Moreover, the IL-10 expression was elevated in LPS-induced AhR-overexpressing RAW264.7 cells. Maximal IL-10 expression was dependent on an AhR non-genomic pathway closely related to Src and STAT3. Furthermore, AhR-associated Src activity was responsible for tyrosine phosphorylation of STAT3 and IL-10 expression by inflammatory macrophages. Adoptive transfer of AhR-expressing macrophages protected mice against LPS-induced peritonitis associated with high IL-10 production. In conclusion, we identified the AhR-Src-STAT3-IL-10 signaling pathway as a critical pathway in the immune regulation of inflammatory macrophages, It suggests that AhR may be a potential therapeutic target in immune response.

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.

DBC1, Deleted in Breast Cancer 1, Is a Nuclear STAT5A-Interacting Protein in Adipocytes, but Its Primary Effects on GLUT4 Gene Expression and Lipolysis Are STAT5 Independent

STAT5A (signal transducer and activator of transcription 5A) is a transcription factor that plays a role in adipocyte development and function. Although the role of STAT5A in fat cell development is well characterized, its specific roles in mature fat cells are still largely unknown. Moreover, the potential role of STAT5A in metabolic diseases, in particular type 2 diabetes, remains unclear. To further understand the function of STAT5A in adipocytes, we employed a non-biased co-immunoprecipitation and mass-spectrometry-based approach to identify novel STAT5A-interacting proteins. One of the proteins we identified was DBC1 (deleted in breast cancer 1; also known as CCAR2). DBC1 is typically localized in the nucleus and has been primarily studied in tumor cells. However, the functions of DBC1 in adipocytes are relatively unknown. Using mouse fat cells, we confirmed that there is a physical association between endogenous STAT5A and DBC1 proteins under physiological conditions in the nucleus that is not dependent upon STAT5 tyrosine phosphorylation. Knockdown of DBC1 in 3T3-L1 adipocytes using siRNA did not affect the expression of several STAT5A target genes including socs3, cish, bcl-6, socs2, and igf-1. However, we did observe increased levels glycerol and free fatty acids released from adipocytes with reduced DBC1 following TNF (tumor necrosis factor) α stimulation. Although loss of DBC1 did not alter STAT5 transcriptional activity for the genes examined, it appears to modulate TNFα-mediated lipolysis in a STAT5-independent manner. In addition, DBC1 knockdown increased GLUT4 expression in murine fat cells but did not have a profound effect on TNFα-mediated changes in gene expression. On-going studies are being performed to elucidate the function of the DBC1/STAT5A interaction and to clarify the impact of DBC1 on lipolysis in adipocytes. Disclosure A. Able: None. A.J. Richard: None. J.M. Stephens: None.

Protein phosphatase 2A promotes IL-2R signaling in human regulatory T cells

Abstract Low-dose IL-2 therapy is a direct approach to boost Tregs and promote immune tolerance in autoimmune patients. The selective response of Tregs to low-dose IL-2 has been suggested to be due to high level of CD25 and activity of the Ser/Thr phosphatase, PP2A. Here we directly assessed the contribution of these two parameters in promoting IL-2 signaling in human Tregs. IL-2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells, directly demonstrating that high IL-2R levels promote the Treg response to low-dose IL-2. To evaluate the role of PP2A, expanded human Tregs were transduced with lentivirus to overexpress or knock down the PP2A catalytic subunit (PP2Ac). Overexpression of PP2Ac increased the level of all IL-2R subunits and promoted phosphorylation of Jak3 at Tyr980/981. Compared to control-transduced cells, PP2Ac-overexpressed Tregs were 3-fold more sensitive to IL-2 based on pSTAT5 activation. Increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5. Consistent with these findings, knockdown of PP2Ac in Tregs significantly reduced the pSTAT5 responses to IL-2. Overexpression of PP2Ac in Tregs also increased the expressions of proteins related to growth, survival, activation, and immunosuppressive function, and included several IL-2-regulated genes. Collectively, these findings indicate that PP2A promotes IL-2R signaling through multiple mechanisms, one related to control of IL-2R expression and another that may directly regulate IL-2R signaling.

IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from human skin mast cells by a STAT3-dependent mechanism.

Abstract Mast cells are tissue resident immune cells that cause allergies. The agents responsible for allergic reactions are pre-formed mediators like histamine and inflammatory lipids such as Prostaglandin D2 (PGD2) that are rapidly biosynthesized following crosslinking of FcɛRI with allergen. In humans, increased IL-6 levels are consistently correlated with allergic diseases like asthma and urticaria in which mast cells play a vital role. However, the exact role of IL-6 in allergic disease is not known. In this study, we show that human in situ-matured skin mast cells (huSMCs) express functional membrane-bound receptors for IL-6, and demonstrate our novel finding that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis. Treatment of huSMCs with IL-6 alone induced the phosphorylation of STAT3 at the activating tyrosine 705, and the expression of SOCS3 and VEGF mRNA. IL-6 alone did not induce PGD2 secretion, but significantly and dose-dependently enhanced PGD2 secretion from FcɛRI-activated huSMCs. Accordingly, IL-6 enhanced the FcɛRI-induced expression of COX-2, which is directly involved in PGD2 biosynthesis. In addition, phosphorylation of STAT3 at both tyrosine 705 and serine 727, which is required for maximal transcriptional activity, was increased in huSMCs treated with IL-6 + FcɛRI crosslinking. Moreover, the IL-6-mediated increase in FcɛRI-induced COX-2 expression was inhibited with C188-9, the small molecule inhibitor of STAT3 tyrosine phosphorylation. Together, these data demonstrate that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from huSMCs by a STAT3-dependent mechanism. Thus, we hypothesize that IL-6 contributes to allergic disease by enhancing the production of inflammatory PGD2 from mature mast cells.

Alantolactone suppresses APOC3 expression and alters lipid homeostasis in L02 liver cells

A high level of APOC3 expression is an independent risk factor for some lipid metabolism-related diseases, such as cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). This suggests that down-regulating APOC3 expression is a potential way of regulating lipid levels. In this study, we used luciferase reporter screening to identify a natural compound, alantolactone (ALA), that can inhibit the promoter activity of APOC3. ALA decreased APOC3 expression at both mRNA and protein levels. Then we pretreated L02 liver cells with oxLDL to investigate the function of ALA in lipid homeostasis. Intriguingly, ALA attenuated oxLDL-induced foam cell formation by reducing total cholesterol (TC) and triglyceride (TG) contents. Furthermore, these results could be reversed by overexpressing APOC3 protein. ALA inhibited tyrosine phosphorylation (Tyr705pho) of STAT3 to down-regulate APOC3 expression. Intriguingly, overexpression of a wild-type STAT3 or a constitutively active form of STAT3 (STAT3-C) up-regulated APOC3 expression and partly reversed the effect of ALA in oxLDL-induced L02 cells. Overexpression of wild-type STAT3 also increased TC but not TG contents in L02 cells. However, overexpression of STAT3-C significantly increased TC and TG contents, and the effect of ALA was partly attenuated by STAT3-C, although this was not statistically significant. These results suggest that ALA attenuates lipid accumulation through down-regulation of APOC3 expression, at least in part by inhibiting STAT3 signaling.

S-glutathionylation exerts opposing roles in the regulation of STAT1 and STAT3 signaling in reactive microglia.

STAT1 and STAT3 are two transcription factors involved in a lot of cellular functions such as immune response, proliferation, apoptosis, and cell survival. A number of literature evidences described a yin-yang relationship between activation of STAT1 and STAT3 in neurodegenerative disorders where STAT1 exerts a pro-apoptotic effect whereas STAT3 shows neuroprotective properties through the inhibition of apoptosis. Although the role of oxidative-stress in the pathogenesis of neurodegeneration is clearly described, its influence in the regulation of these pathways is poorly understood. Herein, we demonstrate that H2O2 rapidly induces phosphorylation of STAT1 whereas it is not able to influence phosphorylation of STAT3 in mouse microglia BV2 cells. The analysis of the molecular mechanism of STATs signaling reveals that H2O2 induces S-glutathionylation of both STAT1 and STAT3. The same post-translational event exerts an opposing role in the regulation of STAT1 and STAT3 signaling. These data not only confirm redox sensibility of STAT3 signaling but also reveal for the first time that STAT1 is susceptible to redox regulation. A deep study of the molecular mechanism of STAT1 redox regulation, identifies Cys324 and Cys492 as the main targets of S-glutathionylation and confirms that S-glutathionylation does not impair JAK2 mediated STAT1 tyrosine phosphorylation. These results demonstrate that both phosphorylation and glutathionylation contribute to activation of STAT1 during oxidative stress and underline that the same post-translation event exerts an opposing role in the regulation of STAT1 and STAT3 signaling in microglia cells.

Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation

Acute myeloid leukemia (AML) remains the most common form of acute leukemia among adults and accounts for a large number of leukemia-related deaths. Mutations in FMS-like tyrosine kinase 3 (FLT3) is one of the most prevalent findings in this heterogeneous disease. The major types of mutations in FLT3 can be categorized as internal tandem duplications (ITD) and point mutations. Recent studies suggest that ITDs not only occur in the juxtamembrane region as originally described, but also in the kinase domain. Although the juxtamembrane ITDs have been well characterized, the tyrosine kinase domain ITDs have not yet been thoroughly studied due to their recent discovery. For this reason, we compared ITD mutations in the juxtamembrane domain with those in the tyrosine kinase domain, as well as with the most common activating point mutation in the tyrosine kinase domain, D835Y. The purpose of this study was to understand whether it is the nature of the mutation or the location of the mutation that plays the main role in leukemogenesis. The various FLT3 mutants were expressed in the murine pro-B cell line Ba/F3 and examined for their capacity to form colonies in semisolid medium. The size and number of colonies formed by Ba/F3 cells expressing either the internal tandem duplication within juxtamembrane domain of the receptor (JMD-ITD) or the tyrosine kinase domain (TKD)-ITD were indistinguishable, while Ba/F3 cells expressing D835Y/FLT3 failed to form colonies. Cell proliferation and cell survival was also significantly higher in TKD-ITD expressing cells, compared to cells expressing D835Y/FLT3. Furthermore, TKD-ITD is capable of inducing phosphorylation of STAT5, while D835Y/FLT3 fails to induce tyrosine phosphorylation of STAT5. Other signal transduction pathways such as the RAS/ERK and the PI3K/AKT pathways were activated to the same level in TKD-ITD cells as compared to D835Y/FLT3 expressing cells. Taken together, our data suggest that TKD-ITD displays similar oncogenic potential to the JMD-ITD but a higher oncogenic potential than the D835Y point mutation.

Cancer/testis Antigen MAGEA3 Interacts with STAT1 and Remodels the Tumor Microenvironment.

Cancer-testis antigen MAGEA3, being restrictedly expressed in testis and various kinds of tumors, has long been considered as an ideal target for immunotherapy. In this study, we report that MAGEA3 interacts with STAT1 and regulates the expression of tyrosine phosphorylated STAT1 (pY-STAT1) in tumor cells. We show that pY-STAT1 is significantly up-regulated when MAGEA3 is silenced by MAGEA3-specific siRNA. RNA sequencing analysis identified 274 STAT1-related genes to be significantly altered in expression level in MAGEA3 knockdown cells. Further analysis of these differentially expressed genes with GO enrichment and KEGG pathway revealed that they are mainly enriched in plasma membrane, extracellular region and MHC class I protein complex, and involved in the interferon signaling pathways, immune response, antigen presentation and cell chemotaxis. The differentially expressed genes associated with chemokines, antigen presentation and vasculogenic mimicry formation were validated by biological experiments. Matrigel matrix-based tube formation assay showed that silencing MAGEA3 in tumor cells impairs tumor vasculogenic mimicry formation. These data indicate that MAGEA3 expression in tumor cells is associated with immune cells infiltration into tumor microenvironment and anti-tumor immune responses, implying that it may play an important role in tumor immune escape. Our findings reveal the potential impact of MAGEA3 on the immunosuppressive tumor microenvironment and will provide promising strategies for improving the efficacy of MAGEA3-targeted immunotherapy.

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cytokine-inducible SH2-containing protein (cish). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation.

Development of Erasin: a chromone-based STAT3 inhibitor which induces apoptosis in Erlotinib-resistant lung cancer cells

Inhibition of protein-protein interactions by small molecules offers tremendous opportunities for basic research and drug development. One of the fundamental challenges of this research field is the broad lack of available lead structures from nature. Here, we demonstrate that modifications of a chromone-based inhibitor of the Src homology 2 (SH2) domain of the transcription factor STAT5 confer inhibitory activity against STAT3. The binding mode of the most potent STAT3 inhibitor Erasin was analyzed by the investigation of structure-activity relationships, which was facilitated by chemical synthesis and biochemical activity analysis, in combination with molecular docking studies. Erasin inhibits tyrosine phosphorylation of STAT3 with selectivity over STAT5 and STAT1 in cell-based assays, and increases the apoptotic rate of cultured NSCLC cells in a STAT3-dependent manner. This ability of Erasin also extends to HCC-827 cells with acquired resistance against Erlotinib, a clinically used inhibitor of the EGF receptor. Our work validates chromone-based acylhydrazones as privileged structures for antagonizing STAT SH2 domains, and demonstrates that apoptosis can be induced in NSCLC cells with acquired Erlotinib resistance by direct inhibition of STAT3.

Drug-induced inhibition of phosphorylation of STAT5 overrides drug resistance in neoplastic mast cells.

Systemic mastocytosis (SM) is a mast cell (MC) neoplasm with complex pathology and a variable clinical course. In aggressive SM (ASM) and MC leukemia (MCL) responses to conventional drugs are poor and the prognosis is dismal. R763 is a multi-kinase inhibitor that blocks the activity of Aurora-kinase-A/B, ABL1, AKT and FLT3. We examined the effects of R763 on proliferation and survival of neoplastic MC. R763 produced dose-dependent inhibition of proliferation in the human MC lines HMC-1.1 (IC50 5-50 nM), HMC-1.2 (IC50 1-10 nM), ROSAKIT WT (IC50 1-10 nM), ROSAKIT D816V (IC50 50-500 nM) and MCPV-1.1 (IC50 100-1000 nM). Moreover, R763 induced growth inhibition in primary neoplastic MC in patients with ASM and MCL. Growth-inhibitory effects of R763 were accompanied by signs of apoptosis and a G2/M cell cycle arrest. R763 also inhibited phosphorylation of KIT, BTK, AKT and STAT5 in neoplastic MC. The most sensitive target appeared to be STAT5. In fact, tyrosine phosphorylation of STAT5 was inhibited by R763 at 10 nM. At this low concentration, R763 produced synergistic growth-inhibitory effects on neoplastic MC when combined with midostaurin or dasatinib. Together, R763 is a novel promising multi-kinase inhibitor that blocks STAT5 activation and thereby overrides drug-resistance in neoplastic MC.

Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

Constitutive JAK-STAT signaling drives the proliferation of most myeloproliferative neoplasms (MPN) and a subset of acute myeloid leukemia (AML), but persistence emerges with chronic exposure to JAK inhibitors. MPN and post-MPN AML are dependent on tyrosine phosphorylation of STATs, but the role of serine STAT1 phosphorylation remains unclear. We previously demonstrated that Mediator kinase inhibitor cortistatin A (CA) reduced proliferation of JAK2-mutant AML in vitro and in vivo and also suppressed CDK8-dependent phosphorylation of STAT1 at serine 727. Here we report that phosphorylation of STAT1 S727 promotes the proliferation of AML cells with JAK-STAT pathway activation. Inhibition of serine phosphorylation by CA promotes growth arrest and differentiation, inhibits colony formation in MPN patient samples and reduces allele burden in MPN mouse models. These results reveal that STAT1 pS727 regulates growth and differentiation in JAK-STAT activated neoplasms and suggest that Mediator kinase inhibition represents a therapeutic strategy to regulate JAK-STAT signaling.

Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation

BackgroundLung cancer is the leading cause of cancer death worldwide. However, the molecular mechanisms underlying lung cancer development have not been fully understood. The functions of histone deacetylases (HDACs), a class of total eighteen proteins (HDAC1–11 and SIRT1–7 in mammals) that deacetylate histones and non-histone proteins, in cancers are largely unknown.MethodsHdac7+/−/K-Ras mice and HDAC7-depleted human lung cancer cell lines were used as models for studying the function of Hdac7 gene in lung cancer. Kaplan-Meier survival analysis was performed to explore the relationship between HDAC7 expression and prognosis of human lung cancers. Recombinant lentivirus-mediated in vivo gene expression or knockdown, Western blotting, and pull-down assay were applied to investigate the underlying molecular mechanism by which Hdac7 promotes lung tumorigenesis.ResultsThe number and burden of lung tumor were dramatically reduced in Hdac7+/−/K-Ras mice compared to control K-Ras mice. Also, in Hdac7+/−/K-Ras mice, cell proliferation was significantly inhibited and apoptosis in lung tumors was greatly enhanced. Similarly, cell proliferation and anchorage-independent growth of human lung cancer cell lines expressing shHDAC7 were also significantly suppressed and apoptosis was dramatically elevated respectively. Mechanistic study revealed that Hdac7 mutation in mouse lung tumors or HDAC7 depletion in human tumor cell lines resulted in significantly enhanced acetylation and tyrosine-phosphorylation of Stat3 and HDAC7 protein directly interacted with and deacetylateed STAT3. The Hdac7 mutant-mediated inhibitory effects on lung tumorigenesis in mice and cell proliferation/soft agar colony formation of human lung cancer cell lines were respectively reversed by expressing dnStat3. Finally, the high HDAC7 mRNA level was found to be correlated with poor prognosis of human lung cancer patients.ConclusionOur study suggests that Hdac7 promotes lung tumorigenesis by inhibiting Stat3 activation via deacetylating Stat3 and may shed a light on the design of new therapeutic strategies for human lung cancer.