Blocked STAT3 Phosphorylation Research Articles

A newly-synthesized compound CP-07 alleviates microglia-mediated neuroinflammation and ischemic brain injury via inhibiting STAT3 phosphorylation.

Overactivated glial cells, especially microglia, are core components in the progression of pathologic neuroinflammation, and the application of anti-inflammatory reagents has been regarded as a potential therapy in the management of infarction/reperfusion (I/R) brain injury. This research aims to clarify the anti-inflammatory efect of a novel lipophilic compound N-(2-[4-tert-butylphenyl]-2-[pyrrolidine-1-yl]ethyl)-7-methyl-4-oxo-4H-chromene-2-carboxamide (named CP-07 in this study) in LPS-stimulated BV2 cell line and primary mouse microglia, and its therapeutic effect on I/R brain injury. Cell Counting Kit-8 assay was used to determine the maximal nontoxic dose of CP-07. The mRNA levels of representative proinflammatory cytokines were determined by quantitative real-time polymerase chain reaction both in vitro and in vivo. TTC staining was performed to calculate infarct volumes while behavioral tests were used to assess the neurological deficits at 24 h after middle cerebral artery occlusion (MCAO). Flow cytometry analysis and immunofluorescence staining were performed to calculate the percentage of pro-inflammatory microglia in vivo.A selective JAK2/STAT3 pathway inhibitor, AG490 was used to block STAT3 phosphorylation before the CP-07 anti-inflammation tests in vitro. CP-07 could effectively suppress the mRNA levels of IL-6, IL-1β, iNOS and TNF-α induced by lipopolysaccharide (LPS) in vitro, and markedly block the evaluation of the fluorescence intensity of Iba-1 in primary mouse microglia. In middle cerebral arteryocclusion models, intraperitoneal injection with 1 mg/kg CP-07 significantly reduced cerebral infarct volumes at 24 h after surgery compared with vehicle treatment group, and promoted the recovery of neurological functions in MCAO mice. Further studies validated that CP-07 administration reduced the percentage of CD86 positive microglia after I/R injury, and the expression level of p-STAT3 was also markedly reduced in both microglial cells and the penumbra tissues. Blocking STAT3 phosphorylation with AG490 could completely eliminate the anti-inflammatory effects of CP-07, at least in vitro. We showed that a newly synthesized compound, CP-07, could effectively reduce the inflammatory responses in LPS-stimulated BV2 cells and primary mouse microglia, and overproduction of cytokines in middle cerebral artery occlusion mouse models by inhibiting STAT3 phosphorylation, leading to a neuroprotective effect on I/R brain injury.

IL-6-Dependent STAT3 Activation and Induction of Proinflammatory Cytokines in Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6-dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4 + T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3-positive CD4 + T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3-positive cells were enriched in the peribiliary liver stroma and represented CD4 + T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.

BP‑1‑102 exerts antitumor effects on T‑cell acute lymphoblastic leukemia cells by suppressing the JAK2/STAT3/c‑Myc signaling pathway.

Drug resistance and relapse of T-cell acute lymphoblastic leukemia (T-ALL) remain significant concerns for physicians; hence, the development and screening of effective targeted drugs remain important. Considering that STAT3 is emerging as a potential therapeutic target for T-ALL, T-ALL cell lines (MOLT-4 and CUTLL1) were treated with BP-1-102, a small-molecule inhibitor that blocks STAT3 phosphorylation. Cell Counting Kit-8 assay and colony formation assay results showed that BP-1-102 inhibited T-ALL cell proliferation and colony formation. Flow cytometry and morphological results demonstrated that BP-1-102 dramatically induced apoptosis and caused cell cycle arrest at the G0/G1 phase in T-ALL cell lines. Western blotting results indicated that BP-1-102 suppressed the JAK2/STAT3/c-Myc pathway activity in T-ALL cell lines. In conclusion, BP-1-102 suppressed the JAK2/STAT3/c-Myc signaling pathway in T-ALL cells and exerted various antitumor effects, representing a promising targeted antitumor inhibitor.

The Stat3 inhibitor F0648-0027 is a potential therapeutic against rheumatoid arthritis

Rheumatoid arthritis (RA) is a disease characterized by chronic joint inflammation, pain and joint destruction, leading to alteration in activities of daily living, yet pathological mechanisms underlying the condition are not fully clarified. To date, various therapeutic agents have been developed as RA therapy including DMARDs and/or biological agents that target inflammatory cytokines or inhibit JAK. Here we asked whether inhibiting signal transducer and activator of transcription 3 (Stat3) activity would antagonize RA.Stat3 forms dimers when activated and undergoes nuclear translocalization; thus we screened approximately 4.9 million small compounds as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening. We identified 15 as strong candidates as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening from those compounds. Four of the 15 significantly inhibited expression of IL-6 and RANKL, both of which are direct targets of Stat3, induced by IL-6. Among four, one compound, F0648-0027, significantly inhibited arthritis development without apparent adverse effects in vivo in collagen-induced arthritis model mice. F0648-0027 also significantly blocked Stat3 phosphorylation and nuclear localization following IL-6 stimulation of fibroblasts. These data suggest that Stat3 is a target for collagen-induced arthritis in mice, and that F0648-0027 could serve as a therapeutic reagent against comparable conditions in humans.

Design, synthesis, and biological evaluation of benzo[b]thiophene 1,1-dioxide derivatives as potent STAT3 inhibitors.

As a member of the signal transducer and activator of transcription (STAT) family, STAT3 plays a critical role in several biological pathways such as cell proliferation, migration, survival, and differentiation. Due to abnormal continuous activation in tumors, inhibition of STAT3 has emerged as an attractive approach for the treatment of various cancer cells. Herein, we report a series of novel STAT3 inhibitors based on benzo[b]thiophene 1,1-dioxide scaffold and evaluated their anticancer potency. Among them, compound 8b exhibited the best activity against cancer cells. Compound 8b induced apoptosis and blocked the cell cycle. Meanwhile, 8b reduced intracellular ROS content and caused the loss of mitochondrial membrane potential. Further research revealed that 8b significantly blocked STAT3 phosphorylation and STAT3-dependent dual-luciferase reporter gene experiments showed that compound 8b has a marked inhibition of STAT3-mediated Firefly luciferase activity. Molecular modeling studies revealed compound 8b occupied the pocket well with the SH2 domain in a favorable conformation.

Paris saponin VII extracted from trillium tschonoskii suppresses proliferation and induces apoptosis of human colorectal cancer cells

Ethnopharmacological relevanceSaponins of many herbs could inhibit the growth of colorectal cancer cells. In the study, we investigated the effects of Paris saponin Ⅶ (PSⅦ), and elucidated its mechanism in colorectal carcinoma cells and a xenograft mouse model. Materials and methodsHT-29 and HCT-116 cells were treated with different concentrations of PSⅦ (0–100 μM). The effects of PSⅦ on HCT-116 cells were assessed using a microarray. Then, apoptotic cells were detected by flow cytometric analysis and apoptosis related protein expression was evaluated by Western blot. A xenograft model of nude mice was used to assess the effect of PSⅦ in vivo. ResultsMTT assay showed the IC50 values of PSⅦ for growth inhibition of HT-29 and HCT-116 cells were 1.02 ± 0.05 μM and 3.50 ± 0.79 μM respectively. Edu assay demonstrated that PSⅦ effectively suppressed the growth of HT-29 and HCT-116 cells. Treatment with 0–3 μM PSⅦ not only triggered apoptosis, but also activated caspase-3 and caspase-9 of HT-29 and HCT-116 cells in a concentration dependent manner. In parallel to the alterations, Bax and Cyto-c expression increased while Bcl-2 decreased. In nude mice, PSⅦ reduced the tumor size and induced the apoptosis of tumor cells. PSVII could suppress IL-6-induced phosphorylation of STAT3 in vitro and blocked STAT3 phosphorylation in vivo. ConclusionOur results suggest that PSVII suppressed the activation of IL-6/STAT3 pathway, consequently suppressed the growth and proliferation and triggered the apoptosis of CRC cells. These findings indicate that PSⅦ might be an effective tumouristatic agent for the treatment of colorectal cancer.

STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Heme oxygenase-1 directly binds STAT3 to control the generation of pathogenic Th17 cells during neutrophilic airway inflammation.

Specific JAK/STAT pathways play a critical role in the functional differentiation of distinct Th subsets. Previously, we showed that HO-1, a stress-inducible protein, inhibits Th17 cell differentiation and alleviates neutrophilic airway inflammation, but the responsible molecular basis remains unclear. We employed Th17-skewing differentiation and NEA mouse models to study the role of HO-1 in regulating IL-6-STAT3-RORγt/SOCS3 signaling pathway to control Th17 cell-mediated neutrophilic airway inflammation. The levels of cytokines and expressions of relative signaling molecules were measured by ELISA, western blot, and qPCR, respectively. Frequency of CD4+ IL-17A+ , CD4+ IL-6R+ , and CD4+ IL-23R+ cells was analyzed by FCM. The interaction between HO-1 and signaling pathway-related proteins was determined by co-immunoprecipitation and western blot. Here, we show that hemin-induced HO-1 overexpression is required to mediate this process. Specifically, HO-1 decreased STAT3 phosphorylation but not IL-6R/IL-23R expression or JAK1/JAK2 activation in CD4+ T cells. The effect was accompanied by co-inhibition of SOCS3, a negative feedback factor of STAT3 activation. HO-1 bound to three domains on STAT3 (DNA-binding, linker, and transactivation domains) to directly regulate STAT3 activation. Conversely, either forced expression of a constitutively active STAT3 mutant or application of small-interfering RNA (siRNA) for HO-1 reversed these effects. Our data suggest that HO-1 exerts its inhibitory effect on Th17 cell differentiation by directly associating and blocking STAT3 phosphorylation. We speculate that hemin may be a potential therapeutic candidate for the treatment of other types of immune and pulmonary inflammatory-related diseases.

Abstract 5813: Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway

Abstract RAB GTPases is involved in membrane trafficking vesicle formation and cell motility. Recently, exocytotic RABs received growing attention in cancer research. However, the molecular mechanism of exocytotic RABs in colorectal tumorigenicity remains unclear. In this study, we found RAB GTPase 3C (RAB3C) has the most significant association with higher pathological stage, tumor recurrence, frequent distant metastasis, and poor prognosis in 215 colorectal adenocarcinoma cohort using tissue microarray (TMA) analysis. In multivariate analyses, high RAB3C expression retained its independently prognostic significance for both overall survival (p=0.001) and disease-free survival (p < 0.001). Further experimental results showed increased migration and invasion ability in RAB3C overexpressing colon cancer cells compared with control group in vitro and in vivo. The cell culture medium collected from RAB3C overexpressing model could promote parental colon cancer cells further indicated that the metastasis-promoting role of RAB3C is exocytosis dependent. We then integrated the results from previously microarray and proteomics datasets and discovered that increased production of multiple cytokines in RAB3C overexpressing cell lines. Of these cytokines, IL-6 pathway is the top pathway corresponding to gene expression changes after RAB3C overexpression. Blocking of IL-6 function with IL-6 antibody or IL-6 knockdown significantly reduced the migration ability in RAB3C overexpressing colon cancer cells. Furthermore, secretion of IL-6 after RAB3C overexpression promoted the JAK2-STAT3 signaling with increased STAT3 phosphorylation levels to promote migration. In addition, Ruxolitinib, a JAK2 inhibitor, was found to significantly inhibited RAB3C induced colon cancer cell migration through blocking STAT3 phosphorylation. In conclusion, our study showed that RAB3C overexpression promoted tumor metastasis and is associated with poor prognosis of colon cancer patients through modulating the IL6 exocytosis of cancer cells to activate JAK2-STAT3 pathway. These findings suggest a new therapeutic strategy through the inhibition of STAT3 signaling pathway in RAB3C-IL-6 axis by Ruxolitinib to prevent metastasis of colon cancer. Note: This abstract was not presented at the meeting. Citation Format: Chi-Long Chen. Rab GTPase 3C promotes colon cancer metastasis via regulation of IL-6 secretion and STAT3 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5813. doi:10.1158/1538-7445.AM2017-5813

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide.

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2•-). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2•- blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2•- production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.

MPHOSPH1: a potential therapeutic target for hepatocellular carcinoma.

MPHOSPH1 is a critical kinesin protein that functions in cytokinesis. Here, we show that MPHOSPH1 is overexpressed in hepatocellular carcinoma (HCC) cells, where it is essential for proliferation. Attenuating MPHOSPH1 expression with a tumor-selective shRNA-expressing adenovirus (Ad-shMPP1) was sufficient to arrest HCC cell proliferation in a manner associated with an accumulation of multinucleated polyploid cells, induction of postmitotic apoptosis, and increased sensitivity to taxol cytotoxicity. Mechanistic investigations showed that attenuation of MPHOSPH1 stabilized p53, blocked STAT3 phosphorylation, and prolonged mitotic arrest. In a mouse subcutaneous xenograft model of HCC, tumoral injection of Ad-shMPP1 inhibited MPHOSPH1 expression and tumor growth in a manner correlated with induction of apoptosis. Combining Ad-shMPP1 injection with taxol administration enhanced antitumor efficacy relative to taxol alone. Furthermore, Ad-shMPP1 tail vein injection suppressed formation of orthotopic liver nodules and prevented hepatic dysfunction. Taken together, our results identify MPHOSPH1 as an oncogenic driver and candidate therapeutic target in HCC.

Abstract 3801: Small molecule inhibitor of Stat3 induces antitumor cell effects in vitro and antitumor effects in vivo against human glioma or breast cancer model

Abstract Aberrant activation of signal transducer and activator of transcription 3 (Stat3) is prevalent in many human cancers. Compelling evidence shows that constitutively-active Stat3 constitutes a point of convergence in oncogenic tyrosine kinase signaling, functioning as a master regulator of events crucial for tumorigenesis and malignant progression. We show herein that the small-molecule, SH5-07 abrogates constitutive activation of Stat3 and Stat3-dependent functions in human glioma and breast cancer cells. SH5-07 inhibits in vitro Stat3 DNA-binding activity with an IC50 value of 3.9 μM. Treatment of human glioma U251MG and breast cancer MDA-MB-231 cells that harbor aberrantly-active Stat3 with low micromolar SH5-07 blocks Stat3 phosphorylation, DNA-binding, and transcriptional activities in a time and dose-dependent manner. By contrast, SH5-07 shows little or no effect on the induction of pJAK2, pSrc, pErk1/2, and pShc in glioma and breast cancer cells. SH5-07 preferentially inhibited viability of human glioma and breast cancer cells that harbor aberrant Stat3 activity, with IC50 of 1.1-5.2 µM, compared to IC50 of 6.0-10.8 µM for cells that do not. Inhibition of aberrantly-active Stat3 by SH5-07 preferentially suppresses the anchorage-dependent and independent growth, induces apoptosis, and blocks migration and invasion in vitro of human glioma and breast cancer cells that harbor constitutively-active Stat3. Moreover, oral administration of SH5-07 inhibits the growth of human breast tumor xenografts in mice. Data together indicates SH5-07 is a promising small-molecule Stat3 inhibitor and a suitable drug candidate for clinical development. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Marcus Tius, James Turkson. Small molecule inhibitor of Stat3 induces antitumor cell effects in vitro and antitumor effects in vivo against human glioma or breast cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2014-3801

Metformin inhibits the IL-6-induced epithelial-mesenchymal transition and lung adenocarcinoma growth and metastasis.

ObjectiveEpithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. However, the underlying mechanisms of EMT in lung adenocarcinoma, and how this process might be inhibited, remain to be explored. This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.MethodsInvasion assay and MTT assay was performed to determine cell invasion and cell proliferation. Western blotting, immunofluorescence, real-time PCR, ELISA, and immunohistochemistry were performed to detect the expression of IL-6, E-cadherin, Vimentin, and p-STAT3.ResultsWe discovered that IL-6, via STAT3 phosphorylation, could promote lung adenocarcinoma cell invasion via EMT in vitro. This was supported by the inverse correlation between E-cadherin and IL-6 expression, positive correlation between IL-6 and vimentin mRNA expression and between STAT3 phosphorylation and IL-6 expression in tumor tissues. Importantly, metformin inhibited tumor growth and distant metastases in tumor-bearing nude mice and reversed IL-6-induced EMT both in vitro and in vivo. Furthermore, we found that blockade of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.ConclusionsCollectively, our present results show that enhanced IL-6 expression, via STAT3 phosphorylation, is a mechanism of EMT in lung adenocarcinoma. We found that metformin could inhibit IL-6-induced EMT possibly by blocking STAT3 phosphorylation.

A Specific STAT3-Binding Peptide Exerts Antiproliferative Effects and Antitumor Activity by Inhibiting STAT3 Phosphorylation and Signaling

STAT3 promotes the survival, proliferation, metastasis, immune escape, and drug resistance of cancer cells, making its targeting an appealing prospect. However, although multiple inhibitors of STAT3 and its regulatory or effector pathway elements have been developed, bioactive agents have been somewhat elusive. In this report, we report the identification of a specific STAT3-binding peptide (APTSTAT3) through phage display of a novel "aptide" library. APTSTAT3 bound STAT3 with high specificity and affinity (∼231 nmol/L). Addition of a cell-penetrating motif to the peptide to yield APTSTAT3-9R enabled uptake by murine B16F1 melanoma cells. Treatment of various types of cancer cells with APTSTAT3-9R blocked STAT3 phosphorylation and reduced expression of STAT targets, including cyclin D1, Bcl-xL, and survivin. As a result, APTSTAT3-9R suppressed the viability and proliferation of cancer cells. Furthermore, intratumoral injection of APTSTAT3-9R exerted potent antitumor activity in both xenograft and allograft tumor models. Our results offer a preclinical proof-of-concept for APTSTAT3 as a tractable agent for translation to target the broad array of cancers harboring constitutively activated STAT3.

JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells

Epithelial-mesenchymal transition (EMT), a key step in the early stages of cancer metastasis, is orchestrated by several signaling pathways, including IL-6/JAK/STAT3 and TGF-β/Smad signaling. However, an association between the two signaling pathways during the EMT process is largely unknown. Here, we focused on lung cancer and demonstrated that TGF-β1 induced the phosphorylation of Smad3 (p-Smad3), upregulation of Snail, a fibroblast-like morphology, and downregulation of E-cadherin as well as upregulation of vimentin in lung cancer cell lines. SIS3 (an inhibitor of Smad3) suppressed TGF-β1-induced activation of Smad3, upregulation of Snail and the EMT process. Importantly, the JAK2/STAT3-specific inhibitor AG490 blocked Stat3 phosphorylation, resulting in attenuated levels of TGF-β1-induced p-Smad3, Snail, MMP2, and Smad-mediated PAI-1 promoter reporter gene activity in A549 and H1650 cells. Subsequently, AG490 inhibited TGF-β-induced cell migration and invasion. Moreover, exogenous IL-6 treatment stimulated Stat3 activation, enhanced TGF-β-induced expression of p-Smad3 and Snail, aggravated the EMT process, and increased lung cancer cell migration and invasion induced by TGF-β1. Our findings show that the JAK/STAT3 pathway is required for TGF-β-induced EMT and cancer cell migration and invasion via upregulation of the expression of p-Smad3 and Snail, and the IL-6/JAK/STAT3 and TGF-β/Smad signaling synergistically enhance EMT in lung carcinomas. The present study suggests a novel rationale for inhibiting cancer metastasis using anti-IL-6/JAK/STAT3 and anti-TGF-β/Smad therapeutic strategies.

A perylene derivative regulates HIF-1α and Stat3 signaling pathways

It is becoming increasingly evident that improving the cure rate of many cancers will require treatment regimens hit more than one validated tumor targets. Developing an anti-cancer agent that targets two oncoproteins simultaneously is a promising strategy for accomplishing this goal. It would be expected to promote drug efficacy, reduce therapy-resistant without introducing additional toxic side effects. HIF-1α is a key regulator of the cellular response to hypoxia and is involved in tumor angiogenesis and cancer cell survival, glucose metabolism, and invasion. Stat3 has several oncogenic functions, including suppression of anti-tumor immune responses and promotion of inflammation. Recently, we have identified the perylene derivative, TEL03, as a dual inhibitor that targets both HIF-1α and Stat3. TEL03 blocks the expression of both HIF-1α and Stat3, regulated oncogenes (e.g., Bcl-2, VEGF, Glut1, and others) in cancer cells, and induces cancer cell apoptosis. The results demonstrated that: (i) TEL03 blocks Stat3 phosphorylation, and inhibits Stat3 transcriptional activity; and (ii) interferes the binding of HIF-1α to p300/CBP inducing its degradation by proteasomes under hypoxic conditions. Our in vivo tests showed that as a dual inhibitor, TEL03 dramatically inhibited tumor growth, and provided the evidence that targeting both HIF-1α and Stat3 simultaneously could be a promising strategy for breast and pancreatic cancer therapies.

Denileukin diftitox (ONTAK) induces a tolerogenic phenotype in dendritic cells and stimulates survival of resting Treg

AbstractDenileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen–specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator–dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinicaltrials.gov, #NCT00056134.

In Active Relapsing-Remitting Multiple Sclerosis, Effector T Cell Resistance to Adaptive T regs Involves IL-6–Mediated Signaling

Patients with multiple sclerosis (MS) manifest demyelination and neurodegeneration mediated in part by CD4(+) T cells that have escaped regulation. Resistance of pathogenic effector T cells (T(effs)) to suppression by regulatory T cells (T(regs)) has been demonstrated in several autoimmune diseases. Although impairment in T(reg) number and function has been observed in relapsing-remitting MS (RRMS), T(eff) resistance has not been well studied in this disease. To determine whether T(eff) resistance contributes to failed tolerance in RRMS, we performed T(reg) suppression assays with T(effs) from either RRMS patients not on immunomodulatory therapy or healthy individuals. T(eff) resistance was present in the T(effs) of RRMS patients with active disease but not from patients with inactive disease. Interleukin-6 (IL-6) and phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) promote T(eff) resistance to T(regs), and we found an increase in IL-6 receptor α (IL-6Rα) expression and elevated IL-6 signaling as measured by pSTAT3 in our RRMS subjects. Further, the impaired suppression in RRMS subjects correlated with an increase in IL-6Rα surface expression on CD4(+) T cells and an increase in pSTAT3 in response to IL-6. To address whether the enhanced pSTAT3 contributed to T(eff) resistance in active RRMS patients, we blocked STAT3 phosphorylation and found that impaired suppression was reversed. Therefore, enhanced IL-6R signaling through pSTAT3, in some cases through increased IL-6Rα expression, contributed to T(eff) resistance in active RRMS. These markers may aid in determining disease activity and responsiveness to immunomodulatory therapies in RRMS.

Abstract A123: TEL is a dual anticancer agent that targets Stat3 and HIF-1α.

Abstract We recently developed a dual anti-cancer agent, TEL (a perylence derivative) that independently targets both Stat3 and HIF-1α. Stat3 and HIF-1 have been demonstrated to be important targets for cancer therapy. Stat3 participates in oncogenesis through the up-regulation of genes encoding anti-apoptosis, cell-cycle regulators, and inducers of angiogenesis. Also, Stat3 suppresses anti-tumor immune responses and promotes inflammation-induced cancer, making it an attractive target. HIF-1 mediates the cellular response to hypoxia and activates the transcription of genes that are involved in angiogenesis, cell survival, glucose metabolism and invasion. HIF-1α was demonstrated to be overexpressed in many human cancers. Overexpression of HIF-1α, which results from intratumoral hypoxia and genetic alternations, has been associated with poor prognosis and treatment failure in a number of cancers. Our previous results demonstrated that targeting both Stat3 and HIF-1α together could improve tumor response, compared to either single agent alone, and reduce drug resistance and treatment failure. Recently, we have isolated or derived more than 20 molecules from Chinese medicinal herbs. Based on drug screening, we found that TEL selectively targets both HIF-1α and Stat3, significantly inhibiting activation of HIF-1α and p-Stat3, and blocking the expression of both HIF-1α and Stat3 regulated oncogenes (e.g. Bcl2, VEGF, Glut1, and others) in cancer cells, showing that it has great potential to be an anti-cancer agent. Our results demonstrated that (1) TEL inhibits both p-Stat3 and HIF-1α; (2) TEL strongly interacts with the residues of E612, S613 and E638 in the SH2 domain of Stat3 and disrupts the interaction between Stat3 and the phosphotyrosine-stimulating receptor, blocking Stat3 phosphorylation; (3) TEL directly binds to HIF-1 protein and inhibits the interaction between N803 of HIF-1α and p300/CBP under hypoxia condition, inducing the proteasomal degradation and blocking its transcription; and (4) TEL greatly induces cancer cell apoptosis by targeting Stat3 and HIF-1α, but does not cause toxicity in normal cells under the same condition. TEL is likely to have a favorable safety profile, as do many Chinese herbed medicines. Therefore, TEL not only has broad drug activity for cancer therapy, but may be well tolerated in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A123.

RISK and SAFE signaling pathway interactions in remote limb ischemic perconditioning in combination with local ischemic postconditioning

Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising methods to decrease ischemia-reperfusion (I/R) injury. We tested whether the use of the two procedures in combination led to an improvement in cardioprotection through a higher activation of survival signaling pathways. Rats exposed to myocardial I/R were allocated to one of the following four groups: Control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated 20min after coronary artery occlusion; IPost+RIPer, IPost and RIPer in combination. Infarct size was significantly reduced in both IPost and RIPer (34.25±3.36 and 24.69±6.02%, respectively) groups compared to Control (54.93±6.46%, both p<0.05). IPost+RIPer (infarct size=18.04±4.86%) was significantly more cardioprotective than IPost alone (p<0.05). RISK pathway (Akt, ERK1/2, and GSK-3β) activation was enhanced in IPost, RIPer, and IPost+RIPer groups compared to Control. IPost+RIPer did not enhance RISK pathway activation as compared to IPost alone, but instead increased phospho-STAT-3 levels, highlighting the crucial role of the SAFE pathway. In IPost+RIPer, a SAFE inhibitor (AG490) abolished cardioprotection and blocked both Akt and GSK-3β phosphorylations, whereas RISK inhibitors (wortmannin or U0126) abolished cardioprotection and blocked STAT-3 phosphorylation. In our experimental model, the combination of IPost and RIPer improved cardioprotection through the recruitment of the SAFE pathway. Our findings also indicate that cross talk exists between the RISK and SAFE pathways.