STAT3 DNA-binding Activity Research Articles

Potential role of NADPH oxidase-mediated activation of Jak2/Stat3 and mitogen-activated protein kinases and expression of TGF-β1 in the pathophysiology of acute pancreatitis

NADPH oxidase is potentially associated with acute pancreatitis by producing reactive oxygen species (ROS). We investigated whether NADPH oxidase mediates the activation of Janus kinase (Jak)2/signal transducers and activators of transcription (Stat)3 and mitogen-activated protein kinases (MAPKs) to induce the expression of transforming growth factor-β1 (TGF-β1) in cerulein-stimulated pancreatic acinar cells. AR42J cells were treated with an NADPH oxidase inhibitor diphenyleneiodonium (DPI) or a Jak2 inhibitor AG490. Other cells were transfected with antisense or sense oligonucleotides (AS or S ODNs) for NADPH oxidase subunit p22(phox) or p47(phox). TGF-β1 was determined by enzyme-linked immonosorbent assay. STAT3-DNA binding activity was measured by electrophoretic mobility shift assay. Levels of MAPKs as well as total and phospho-specific forms of Jak1/Stat3 were assessed by Western blot analysis. Cerulein induced increases in TGF-β1, Stat3-DNA binding activity and the activation of MAPKs in AR42J cells. AG490 suppressed these cerulein-induced changes, similar to inhibition by DPI. Cerulein-induced activation of Jak2/Stat3 and increases in MAPKs and TGF-β1 levels were inhibited in the cells transfected with AS ODN for p22(phox) and p47(phox) compared to S ODN controls. Inhibition of NADPH oxidase may be beneficial for prevention and treatment of pancreatitis by suppressing Jak2/Stat3 and MAPKs and expression of TGF-β1 in pancreatic acinar cells.

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

AbstractMicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF–induced granulocytic differentiation and enables G-CSF–dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, gene-specific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF–dependent differentiation. STAT3 protein expression, DNA-binding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b–mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells.

Abstract 1614: Genetic loss of PKC epsilon inhibits development and metastasis of prostate cancer in transgenic adenocarcinoma of mouse prostate model

Abstract Protein kinase C epsilon (PKC∈), a novel PKC isoform is over-expressed in prostate cancer (PCa) development in both human and mouse. PKC∈ expression correlates with the aggressiveness of human PCa. However, the role of PKC∈ plays in the course of development and metastasis of PCa in vivo is unknown. To obtain the first molecular genetic evidence that PKC∈ is linked to development and metastasis of PCa, we generated PKC∈-deleted mice by crossbreeding transgenic adenocarcinoma of mouse prostate (TRAMP) (TG//WT) mice with PKC∈ knock out (TG//KO) mice. Sixteen weeks old TG Wild-type (TG//WT), TG//PKC∈ Heterozygous (TG//Het) and TG//PKC∈ KO (TG//KO) were evaluated for PCa development and metastasis. A significant decrease in the PKC∈ protein level was observed in the prostate tissues of TG//Het and TG//KO mice compared to TG//WT mice. PKC∈ deletion in TRAMP mice did not affect on the expression of other PKC isoforms neither in prostate nor in brain tissues. We performed MicroPET/CT imaging, using a tumor selective radiopharmaceutical agent 124I-NM404, of 16 wks old mice from each of TG//WT, and TG//KO mice. Results illustrated a lack of focal uptake of 124I-NM404 in TG//KO mice compared to TG//WT mice. TG//WT mice showed metastasis in proximal lymph node as evident by uptake of 124I-NM404. Histopathology data revealed PCa metastasis into the lungs, livers and lymph nodes of TG//WT mice. However, no metastasis was observed in TG//Het and TG//KO mice suggesting the role of PKC∈ in development and metastasis. We have previously shown that PKC∈ associates with Stat3 and this association increases with the progression of the diseases in TRAMP mice and in human PCa. Deletion of PKC∈ in TRAMP mice inhibited 1) phosphorylated forms of both Stat3Ser727 and Stat3Tyr705, 2) DNA binding activity of Stat3, 3) expression levels of markers of proliferation (PI3K85 and PCNA), anti-apoptosis (BclxL), and metastasis (VEGF and COX-2) and 4) IL-6 receptor expression and serum IL-6 levels. To obtain clues about the genes regulated by PKC∈ and linked to the Stat3 signaling pathway, we performed focused PCR arrays of JAK/STAT signaling in excised PCa tissues from TG//WT and TG//KO mice. This array contained 84 genes related to JAK/STAT family members, the receptors that activate them, nuclear co-factor and co-activators associated with the Stat proteins, Stat inducible genes, and negative regulators. Data revealed a significant down-regulation in the m-RNA expression of CCAAT/enhancer binding protein (CEBP) β (2.5 fold), C-reactive protein (4.34 fold), epidermal growth factor receptor (EGFR) (4.54 fold), gp130 (2.5 fold), jun B (2.5 fold), and Stat3 (2.85 fold) in TG//KO mice compared to TG//WT mice. Taken together, these results provide first molecular genetic evidence of the role of PKC∈ in the development and metastasis of PCa in a transgenic mouse model. (Support: NIH Grant, CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2011-1614

Abstract 3544: Jak/Stat signaling: A potential target for pancreatic cancer management

Abstract NexrutineR (Nx) isolated from the bark of Phellodendron amurense is being sold as an anti-inflammatory herbal supplement over the counter in the US. Recent studies from our laboratory demonstrated its potential as an anti-prostatic agent by targeting multiple signaling pathways including Akt/CREB/NFkB/Cyclin D1/Cox-2 both in vitro and in vivo. Given the importance of some of these signaling pathways in multiple cancers, we explored its role in pancreatic cancer (PanCA) using variety of cell lines. Human pancreatic cancer cells that differ in the status of K-Ras (BxPC-3 with wild type K-Ras and Capan-2 with mutant K-Ras), non- tumorigenic HPNE-Ras, MIAPC and ASPC-1 were tested for growth inhibition, apoptosis, necrosis, Cox-2 expression and activity following treatment with Nx. Our data indicate that Nx can significantly inhibit proliferation of these cell lines differentially through induction of apoptosis and necrosis without any significant effect on cell cycle profile. Investigation of molecular mechanism of action identified a role for Jak1 and transcription factors NFkB and Stat3. Western blotting data show that Nx can inhibit the protein levels of pStat3 and Jak1 effectively without influencing the protein levels of total Stat3. Similarly Nx treatment decreased the DNA binding activities of both NFkB and Stat3. Chromatin immunoprecipitation assays revealed the binding of Stat3 to Cox-2 promoter. In addition combining Nx with Gemcitabine (standard of care treatment for PanCA) demonstrated strong synergistic growth inhibitory activity through modulation of Stat3. Overall, the present study unveils a novel interplay between Jak1/Stat3 and NFkB signaling in pancreatic cancer cells. These data further suggest that the targeted disruption of Jak1/Stat3 and NFkB may represent a valid therapeutic approach in this cancer. Supported by NCCAM (APK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3544. doi:10.1158/1538-7445.AM2011-3544

Abstract 1983: A cell-permeable Stat3 SH2 domain mimetic inhibitor of Stat3 activation inhibits growth of human breast tumor xenografts

Abstract Human tumors harbor multiple molecular aberrations that have become the driving force for uncontrolled cell proliferation and survival, and other tumor-associated processes. Constitutive activation of the Signal Transducer and Activator of Transcription (Stat) 3 protein is a frequent occurrence in many human cancers, including breast cancer. Given the role of aberrant Stat3 activity in human tumors, small-molecule Stat3 inhibitors would be useful novel therapeutics and tools for probing Stat3-mediated tumor processes. We herein report that a 28-mer peptide, SPI, composed of the amino acid residues 588-615 of the Stat3 SH2 domain, replicates Stat3 biochemical properties and functions as a Stat3 inhibitor. Studies show SPI and Stat3 (or Stat3 SH2 domain) both bind with similar affinities to known Stat3-binding phosphotyrosine (pY) peptide motifs, including the epidermal growth factor receptor (EGFR) peptides, pY1068EGFR and pY1086EGFR, and the high-affinity, interleukin-6 receptor (IL-6R)/gp130-derived peptide, GpYLPQTV-NH2. Consequently, SPI potently and selectively inhibits Stat3 SH2 domain:pY interactions and disrupts the binding of Stat3 to the IL-6R/gp130 peptide, GpYLPQTV-NH2. Fluorescence imaging studies show SPI is cell membrane-permeable. Treatment with SPI of malignant cells that harbor aberrant Stat3 activity resulted in the selective inhibition of constitutive Stat3 phosphorylation, and of Stat3 DNA-binding and transcriptional activities. By contrast, similar treatments have little or no effects on the induction of the Stat1, Stat5, and Erk1/2MAPK pathways in malignant cells or in EGF-stimulated fibroblasts. In contrast to normal cells, human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring constitutively-active Stat3 are highly sensitive to SPI and undergo extensive morphology changes, concomitant with the loss of viability and apoptosis. Significantly, treatment with SPI or its inverse sequence version strongly inhibited the growth of human breast tumor xenografts in mice, which is associated with the down-regulation of the expression of the known Stat3-regulated genes, Cyclin D1, Bcl-xL and Survivin. Our study identifies SPI as a novel molecular probe for interrogating Stat3 signaling and that functions as a selective Stat3 inhibitor, which induces antitumor response in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1983. doi:10.1158/1538-7445.AM2011-1983

Abstract 4512: A novel orally-bioavailable salicylic acid-based small molecular Stat3 inhibitor suppress growth of human breast tumor xenografts

Abstract With the critical role of aberrantly active Signal Transducer and Activator of Transcription (Stat) 3 protein in many human cancers, selective small molecular inhibitors targeting the dimerization event would be valuable as therapeutic agents and as chemical probes to clarify the complex biological functions of Stat3. By computational and structural analyses of the interaction between Stat3 and the lead dimerization disruptor, S3I-201.1066, we have designed a diverse set of analogs. We present evidence from in vitro studies that BP-1-102, a structural analog of S3I-201.1066, directly interacts with Stat3, with an affinity (KD) of 504 nM, inhibits in vitro Stat3 DNA-binding activity, with IC50 of 6.8 μM, and selectively inhibits Stat3 activity in human and mouse tumor cell lines that harbor constitutively-active Stat3. By contrast, BP-1-102 has little or no effect on pShc, pJak, pSrc, and pErk levels. Furthermore, BP-1-102 significantly and specifically suppressed the viability, proliferation, and survival of same malignant cell lines harboring aberrant Stat3 at low concentration, with little or no effect on normal NIH3T3 mouse fibroblasts or the mouse thymus epithelial stromal cells (TE-71) that do not harbor aberrant Stat3 activity. Furthermore, treatment of malignant cells harboring aberrant Stat3 activity with BP-1-102 down-regulated the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, and VEGF, which are known Stat3-regulated genes. These results together strongly suggest the anti-tumor cell effects of BP-1-102 is largely dependent on the inhibition of aberrant Stat3 activity. Significantly, BP-1-102 induced strong antitumor response in mouse xenografts of human breast cancer when administered via intravenous or oral gavage. Our studies identify a novel small-molecule that binds with a high affinity to Stat3 and that blocks Stat3 activation and function, and thereby induces antitumor response in human breast tumor xenografts harboring aberrant Stat3. Key Words: Stat3 inhibitor, antitumor, xenograft, BP-1-102, S3I-201.1066 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2011-4512

The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

BackgroundCurcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells.MethodsHuman and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting.ResultsOur data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway.ConclusionsThese data demonstrate that the novel curcumin analog FLLL32 has biologic activity against OSA cell lines through inhibition of STAT3 function and expression. Future work with FLLL32 will define the therapeutic potential of this compound in vivo.

A murine model of inducible, cardiac-specific deletion of STAT3: Its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning

Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~ 1 month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse ( MCM TG: STAT3 flox/flox ) by interbreeding STAT3 flox/flox mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG: STAT3 flox/flox mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG: STAT3 flox/flox mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-x L, c-FLIP L, c-FLIP S) proteins 24 h later; in contrast, in tamoxifen-treated MCM TG: STAT3 flox/flox mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35 days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIP L and c-FLIP S) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-x L).

(-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 бvia Stat3 in gastric cancer

To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer. Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditioned medium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay. There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490, VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited Stat3 nucleus translocation, and Stat3-DNA binding activity was also markedly decreased by EGCG. Furthermore, EGCG inhibited IL-6 induced vascular endothelial cell proliferation and tube formation in vitro and angiogenesis in vitro. EGCG inhibits IL-6-induced VEGF expression and angiogenesis via suppressing Stat3 activity in gastric cancer, which has provided a novel mechanistic insight into the anti-angiogenic activity of EGCG.

Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein.

To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation. Expression of CCN2/CTGF, p-STAT3, p-Smad3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reverse-transcriptase polymerase chain reaction. IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80, and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients' sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3) signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2/CTGF promoter. CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is down-regulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.

PML positively regulates interferon gamma signaling

PML, also known as TRIM19, belongs to the family encoding a characteristic RBCC/TRIM motif comprising several cysteine-rich zinc-binding domains (RING and B-boxes) and a coiled-coil domain. The RBCC domain and the covalent modification of PML by the small ubiquitin-like modifier (SUMO) are required for PML localization within the nuclear bodies (NBs). Analysis of PML−/− mice provided evidence for a physiological role of PML in apoptosis. Cells derived from these mice are defective in the induction of apoptosis by interferon (IFN). PML is expressed as a family of cytoplasmic and nuclear isoforms (PML I–VII) as a result of alternative splicing. Herein, we show that overexpression of all nuclear PML isoforms (I–VI) in human cells increased IFNγ-induced STAT1 phosphorylation, resulting in higher binding of STAT1 to DNA, higher activation of IFN-stimulated genes (ISGs), and an increase in the expression of their products. These effects, observed with IFNγ and not IFNα, required PML localization in the nucleus as they were not observed with the cytoplasmic isoform PMLVIIb or the cytoplasmic variants of PMLIV. They also necessitated PML SUMOylation and its RING finger domain. Conversely, downregulation of PML by RNA interference was accompanied by decrease in IFNγ-induced STAT1 phosphorylation, STAT1 DNA binding, transcription of ISGs and in the expression of their products. In addition, IFNγ-mediated STAT1 DNA-binding activity was decreased in PML−/− MEFs compared with wild-type MEFs. Taken together these results demonstrate that PML functions as a positive regulator of IFNγ signaling.

Identification of Purine-Scaffold Small-Molecule Inhibitors of Stat3 Activation by QSAR Studies.

To facilitate the discovery of clinically useful Stat3 inhibitors, computational analysis of the binding to Stat3 of the existing Stat3 dimerization disruptors and quantitative structure−activity relationships (QSAR) were pursued, by which a pharmacophore model was derived for predicting optimized Stat3 dimerization inhibitors. The 2,6,9-trisubstituted-purine scaffold was functionalized in order to access the three subpockets of the Stat3 SH2 domain surface and to derive potent Stat3-binding inhibitors. Select purine scaffolds showed good affinities (KD, 0.8−12 μM) for purified, nonphosphorylated Stat3 and inhibited Stat3 DNA-binding activity in vitro and intracellular phosphorylation at 20−60 μM. Furthermore, agents selectively suppressed viability of human prostate, breast and pancreatic cancer cells, and v-Src-transformed mouse fibroblasts that harbor aberrant Stat3 activity. Studies herein identified novel small-molecule trisubstituted purines as effective inhibitors of constitutively active Stat3 and of the viability of Stat3-dependent tumor cells, and are the first to validate the use of purine bases as templates for building novel Stat3 inhibitors.

STAT5 regulates glioma cell invasion by pathways dependent and independent of STAT5 DNA binding

STAT5 activation in primary glioma was analyzed using antibody directed against phosphorylated STAT5 (pSTAT5), in all samples robust pSTAT5 immunostaining was detected, predominantly in the nucleus. We then used immunofluorescence, transcription factor binding assays and western blotting to study EGF-modulated STAT5 activation in the human U87 glioma cell line. EGF was found to upregulate pSTAT5 levels and enhances STAT5 DNA-binding activity. To address the role of STAT5 in glioma cell invasion, resting and EGF-treated U87 cells were treated with siRNA directed against STAT5 and the extent of glioma cell migration into a Matrigel matrix was monitored. EGF treatment markedly enhanced matrix invasion, and knockdown of STAT5 expression with siRNA significantly downregulated matrix invasion by both EGF-stimulated and resting glioma cells. However, transfection with a decoy oligonucleotide abolished transcriptional activation of a STAT5 target gene but failed to inhibit matrix invasion in resting U87 cells. By contrast, the decoy nucleotide abolished EGF enhancement of matrix invasion. STAT5 therefore promotes glioma cell invasion via at least two different pathways: a pathway dependent of STAT5 DNA binding, and a second pathway independent of STAT5 DNA binding.

Excess Thyroid Hormone Inhibits Embryonic Neural Stem/Progenitor Cells Proliferation and Maintenance through STAT3 Signalling Pathway

Hyperthyroidism is prevalent during pregnancy, but little is known about the effects of excess thyroid hormone on the development of embryonic neural stem/progenitor cells (NSCs), and the mechanisms underlying these effects. Previous studies indicate that STAT3 plays a crucial role in determining NSC fate during neurodevelopment. In this study, we investigated the effects of a supraphysiological dose of 3,5,3'-L-triiodothyronine (T3) on the proliferation and maintenance of NSCs derived from embryonic day 13.5 mouse neocortex, and the involvement of STAT3 in this process. Our results suggest that excess T3 treatment inhibits NSC proliferation and maintenance. T3 decreased tyrosine phosphorylation of JAK1, JAK2 and STAT3, and subsequently inhibited STAT3-DNA binding activity. Furthermore, proliferation and maintenance of NSCs were decreased by inhibitors of JAKs and STAT3, indicating that the STAT3 signalling pathway is involved in the process of NSC proliferation and maintenance. Taken together, these results suggest that the STAT3 signalling pathway is involved in the process of T3-induced inhibition of embryonic NSC proliferation and maintenance. These findings provide data for understanding the effects of hyperthyroidism during pregnancy on fetal brain development, and the mechanisms underlying these effects.

The rGel/BLyS fusion toxin inhibits STAT3 signaling via down-regulation of interleukin-6 receptor in diffuse large B-cell lymphoma

Aberrant signal transducer and activator of transcription (STAT)3 signaling participates in the development and progress of human cancers. We previously generated a highly cytotoxic fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B-cells. In this study, we examined this fusion toxin for its ability to impact STAT3 signaling in diffuse large B-cell lymphoma (DLBCL). The activated B cell-like DLBCL lines were found to express higher levels of interleukin-6 receptor (IL-6R) and STAT3 than did the germinal center B cell-like DLBCL lines. Treatment of DLBCL cells with rGel/BLyS resulted in down-regulation of IL-6R and inhibited STAT3 phosphorylation, STAT3-DNA binding activity, and IL-6-inducible STAT3 reporter gene activity. In agreement with these results, we additionally found that rGel/BLyS down-regulated levels of several STAT3 targets (c-Myc, p21, Mcl-1, and Bcl-x L) and p-SYK, a positive regulator of STAT3. Inhibition of IL-6R-mediated STAT3 signaling by rGel/BLyS led to growth inhibition, triggered accumulation of cells in the sub-G 1 phase of the cell cycle, and induced apoptosis. Our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive DLBCL which is resistant to conventional chemotherapeutic regimens and STAT3 signaling pathway may be an attractive therapeutic target for non-Hodgkin's lymphoma.

281 Cancer: a less depressing outlook? Using antidepressants to induce autophagic programmed cell death in a resistant strain of Burkitt's lymphoma

STAT3, but not ERKs, mediates the IL-6–induced proliferation of renal cancer cells, ACHN and 769P.Although interleukin-6 (IL-6) has been suggested to function as an autocrine growth factor in renal cell carcinoma (RCC), the underlying mechanism responsible for the IL-6–induced proliferation of RCC has not been defined. The aim of this study was to characterize the signaling cascades mediating IL-6–induced proliferation and to investigate the use of effective novel interventions to block the IL-6–induced autocrine growth of renal cancer cells.IL-6–induced proliferation and intracellular signaling cascades were analyzed in four human renal cancer cell lines Caki-1, ACHN, 769P and A498. IL-6–induced activation of STAT3 (signal transducer and activator of transcription-1) and extracellular signal-regulated kinases (ERKs), and the effects of anti–IL-6 neutralizing antibody, Jak inhibitor AG 490, and MEK1 inhibitor PD 98059 were analyzed by Western blotting using phospho-specific antibodies. The DNA-binding activities of STATs were analyzed by EMSA. Apoptosis was determined by using nuclear staining and the TUNEL assay. Changes in the apoptosis-related proteins, bcl-2, bcl-xL, and bax were analyzed by Western blotting.IL-6 induced tyrosine phosphorylation and increased the DNA binding activity of STAT3 and, to a lesser extent, STAT1 in all cell lines except for Caki-1, which did not express the IL-6 receptor subunit gp130. ERKs were constitutively activated in all cell lines and the activation level was not up-regulated further by exogenously added IL-6 nor down-regulated by anti–IL-6 neutralizing antibody. IL-6–induced STAT3 tyrosine phosphorylation and DNA binding activity was inhibited by treatment with Jak specific inhibitor AG 490; however, it was not affected by the MEK1 inhibitor PD 98059. Moreover, treatment with AG 490 inhibited IL-6–induced proliferation of ACHN and 769P cells and induced apoptosis with the down-regulation of bcl-2 and the up-regulation of bax.This study identified STAT3, but not ERKs, to be a major mediator of IL-6–induced proliferation of renal cancer cells. Although ERKs were constitutively activated, ERKs were not found to be essential for the IL-6–induced proliferation and modulation of the STAT3 activity. Because the Jak specific inhibitor AG 490 effectively inhibited the IL-6–induced STAT3 activity and induced apoptosis, the blockade of the STAT3 signaling pathways is considered to be potentially useful as a novel therapeutic approach for RCC.

Functional Crosstalk between Type I and II Interferon through the Regulated Expression of STAT1

Author SummaryCells of the immune system release interferons (IFNs) in response to pathogens or tumor cells; these proteins signal to other immune cells to initiate the body's defense mechanisms. The two classes of IFNs—types I and II—have different receptors and distinct effects on the cells; however, there is “crosstalk” between them. In particular, small quantities of type I IFN can “prime” cells to produce a robust response to type II IFN. In this paper, we provide evidence to explain the molecular basis of this crosstalk. We show that continuous expression of the transcriptional activator c-Jun is responsible for producing basal, priming levels of a type I IFN; this signals to immune cells with the type I IFN receptor (IFNAR1) to maintain expression of STAT1 inside these cells. STAT1 is a key factor for immune cell responses to type II IFN. Thus, signaling by low levels of type I IFN primes the cells with sufficient STAT1 to respond robustly to a subsequent type II IFN signal. This work provides an alternative explanation of the priming phenomenon to a previous proposal that the ligand-bound type I receptor, IFNAR1, acts as a component of the type II IFN receptor.

Abstract 3795: STAT-3: A Novel Target of Diindolylmethane in Ovarian Cancer

Abstract Signal transducers and activation of transcription (STATs) are the important for growth and development. However defects in STAT's may lead to malignant transformation. STAT-3 is an oncogene which is over expressed in several cancers including ovarian cancer. We have previously demonstrated that Diindolylmethane (DIM), an active metabolite of Indole-3-Carbinol present in cruciferous vegetables, is effective in suppressing the proliferation of human ovarian cancer cells, however, the mechanism through which DIM reduces the growth of cancer cells was not clear. We hypothesized that DIM inhibits the growth of ovarian cancer cells by inhibiting STAT-3 activation. Our present studies demonstrate that DIM treatment inhibit the phosphorylation of STAT-3 at Tyr-705 and Ser-727 in a concentration-dependent manner in SKOV-3 and OVCAR-3 cells. Interestingly, DIM treatment also reduced the constitutive protein expression of STAT-3 in both the cell lines. In a time dependent study, we observed that STAT-3 inhibition was as early as 1h after DIM treatment. Next, we wanted to see whether DIM can block IL-6 mediated activation of STAT-3. Treatment of SKOV-3 or OVCAR-3 cells with 5ng/ml IL-6 for 15min increase the activation of STAT-3 by phosphorylation at Tyr705 by almost twenty fold. Our results reveal that DIM treatment almost completely blocked IL-6 induced phosphorylation of STAT-3 at Tyr705 in both SKOV-3 and OVCAR-3 cells and completely in TOV-21G cells. STAT-3 after activation translocate to nucleus, bind to DNA and is involved in the transcription of responsive genes such as Mcl-1, survivin and Bcl-2. Our results demonstrate that DIM inhibits the DNA binding activity of STAT-3 resulting in the reduced expression of Mcl-1, survivin and Bcl-2. Overexpression of STAT-3 by gene transfection abrogates the cell growth inhibitory effects of DIM. Taken together, our cellular results establish STAT-3 as a therapeutic target of DIM in ovarian cancer. Furthermore, our just completed in vivo studies indicate that oral administration of 2mg DIM per day for 6 weeks significantly suppressed the growth of SKOV-3 tumor xenografts in athymic nude mice. Whether SKOV-3 ovarian tumor growth suppression is through inhibition of STAT-3 in vivo is currently under investigation. [Supported in part by RO1 CA 106953 and CA 129038 awarded to S.K.S by NIH] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3795.

Abstract 616: R1507, the human monoclonal antibody targeting the insulin-like growth factor receptor (IGF-1R), in combination with cetuximab, can reverse acquired resistance to cetuximab in pancreatic and colon cancer models

Abstract Background: Cetuximab, the chimeric monoclonal antibody (mAb) directed against the human epidermal growth factor receptor (EGFR) is widely used for the treatment of colorectal cancer and is also effective in pancreatic cancer preclinical models. Acquired resistance to cetuximab is common and the IGF-1R signaling pathway is critically involved in this process. We hypothesized that the combined activation of EGFR and IGF1-R is responsible for the constitutive activation of the transcriptional factor STAT-3 which in turn results in acquired resistance to cetuximab in colorectal and pancreatic tumors. Targeting this activation of EGFR and IGF1-R using the combination of cetuximab and R1507 could potentially reverse this resistance. Methods: A model of pancreatic cancer (PC) cell line resistant in vivo to cetuximab was established by injecting orthotopically, luciferase- and GFP- expressing AsPC-1 human PC cells into nude mice. Once established these tumors in vivo, the mice were treated with cetuximab 500 µg ip bi-weekly until the 34th week, when tumor growth occurred despite cetuximab due to acquired resistance. The tumors were rapidly excised and the GFP- positive cells were selected to establish the cetuximab- resistant clone AsPC-1-CR. Two models of colon cancer (CRC) cell line with acquired resistance in vivo to cetuximab, SW480-CR and GEO-CR, were established as previously described (Bianco et al. Clin Cancer Res. 2008 14(16):5069-80). MTT assay was used to assess the antitumor activity of cetuximab, R1507, and the combination in vitro. Synergy was assessed according the combination-index method of Chou and Talalay. The expression of IGF1-R was studied by Western blot. STAT-3 activation was analyzed by EMSA. Inhibition of directional cell migration was studied in an in vitro wound-healing model. Results: The cetuximab- resistant PC and CRC cell lines, showed a significantly lower in vitro sensitivity to cetuximab when compared to the respective control cell lines. AsPC-1-CR and SW480-CR cells demonstrated a higher expression of IGF1-R. STAT-3 DNA binding activity was significantly increased in all of the cetuximab- resistant cells as compared with control cell lines. The addition of R1507 did not improve the antitumor activity of cetuximab in the control cell lines. However, the combination of cetuximab + R1507 reversed the resistance to cetuximab in the AsPC-1-CR and SW480-CR cell lines, thereby demonstrating synergistic antitumor activity in the setting of acquired resistance. R1507+cetuximab also significantly inhibited the migration of cetuximab- resistant CRC cells. Conclusion: IGF-1R inhibition using R1507 is a promising approach to restore the sensitivity to cetuximab in PC and CRC cells that have developed acquired resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 616.

Paracrine Factors from Human Placental Multipotent Mesenchymal Stromal Cells Protect Endothelium from Oxidative Injury via STAT3 and Manganese Superoxide Dismutase Activation1

Reactive oxygen species may cause oxidative damage in the placenta, yet some mechanisms must exist to reduce or prevent such damage. We investigated whether oxidative injury to placental endothelial cells is inhibited by activation of antioxidant enzymes by paracrine factors secreted by human placental multipotent mesenchymal stromal cells (hPMSC). hPMSC-conditioned medium and umbilical endothelial cells were assayed for cytokines and cytokine receptor expression by immunoassay and real-time PCR. Endothelial cell survival was evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay and caspase 3 activity assay. tert-Butyl hydroperoxide was used to induce oxidative injury in endothelial cells, with fluorescent microscopy and flow cytometry used to detect intracellular peroxides and cell apoptosis. Western blot, real-time PCR, STAT3 DNA-binding activity assay, and STAT3 siRNA were used to assess endothelial cell antioxidant enzymes. hPMSC-conditioned medium supported endothelial cell survival and reduced endothelial cell intracellular peroxides and apoptosis. hPMSCs expressed the transcripts of the interleukin (IL) 6 cytokine family, including IL6 and leukemia-inhibitory factor. hPMSC-conditioned medium activated STAT3 expression in endothelial cells, which was inhibited by neutralizing antibody to interleukin 6 signal transducer (IL6ST) but not to IL6 or leukemia-inhibitory factor. STAT3 siRNA or manganese superoxide dismutase (SOD2) siRNA transfected into endothelial cells inhibited the antiapoptotic effect of conditioned medium. SOD2 was significantly upregulated in endothelial cells by conditioned medium via STAT3 activation that, in turn, was inhibited by IL6ST-neutralizing antibody or STAT3 siRNA. Paracrine factors secreted by hPMSCs support endothelial cell survival. STAT3 activation and SOD2 production protect against oxidative stress-induced endothelial cell damage.