Regulation Of STAT3 Research Articles

Abstract 357: CYP3A4 Activates Stat3 in ER + breast cancer

Abstract CYP3A4 expression in breast cancer has been reported to correlate with nodal metastases and decreased overall survival, but its mechanisms in cancer progression are unknown. ERalpha has been reported to promote CYP3A4 expression. We have previously demonstrated that CYP3A4 is an arachidonic acid (AA) epoxygenase that activates breast cancer cell proliferation through biosynthesis of 14,15-epoxyeicosatrienoic acid (EET). Methods: Effects of 14,15-EET on Stat3 regulation were studied in MCF7 lines expressing CYP3A4 shRNA. Quantitative immunofluorescence (AQUA) interrogation of a tissue microarray was performed to evaluate CYP3A4 in cytoplasm, ERalpha and pY705Stat3 (pYStat3) in nuclei, and HER2 in cytoplasm/membrane. Values were transformed using the natural logarithm. These values were then averaged across the two spot sets. Pearson's correlation was calculated between the markers of interest. Results: Stat3 (Tyr 705) phosphorylation is inhibited by CYP3A4 silencing. CYP3A4 synthesizes 14,15-EET in breast cancer cells and silencing of Stat3 blocks breast cancer cell growth and abrogates 14,15-EET-induced proliferation, indicating that CYP3A4 requires 14,15-EET biosynthesis and Stat3 to induce cell growth. Silencing of CYP3A4 reduces nuclear pY705-Stat3, while 14,15-EET restores this signaling process. By preliminary immunofluorescence study of consecutive breast cancers (n=48) nuclear ERalpha was associated with cytoplasmic CYP3A4 with a correlation of r=0.7575 (p-value < 0.0001). Cytoplasmic CYP3A4 correlated with nuclear pYStat3, with a correlation of r=0.2708, approaching statistical significance (p = 0.0656). Cytoplasmic/membrane HER2, known to be induced by Stat3, was associated with nuclear pYStat3, exhibiting a correlation of r=0.4460 (p-value = 0.0015). Conclusions: ER+ breast cancer cell growth is therefore mediated, in part, by CYP3A4 biosynthesis of 14,15-EET that induces nuclear pY705-Stat3. These studies indicate that CYP3A4 is an AA epoxygenase that is expressed in ER+ breast cancer and promotes Stat3-mediated breast cancer cell growth, in part, through 14,15-EET biosynthesis. Activated Stat3 may mediate cytoplasmic/membrane HER2 expression. 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 357. doi:10.1158/1538-7445.AM2011-357

Abstract 2123: Cytoplasmic STAT1 expression level opposes disease-specific survival of sarcoma patients

Abstract Signal Transducer and Activator of Transcription 1 (STAT1) is a key mediator in the interferon gamma (IFN-γ) signaling pathway. It is well known that IFN-γ-activated STAT1 (pSTAT1) plays a critical role in the suppression of tumor development. However, STAT1 has also been shown to act as a tumor promoter in certain types of cancers. These findings indicate that STAT1 harbors dual functionality, functioning as either a tumor suppressor, or a promoter, a distinction that may ultimately depend on the tumor microenvironment. In this study, we hypothesized that the distribution of STAT1 (cytoplasmic vs nuclear) could affect disease-specific survival of sarcoma patients. STAT1 protein expression levels were examined in 123 soft tissue sarcoma samples from human patients by immunohistochemical staining. We observed a positive correlation between the nuclear STAT1 (pSTAT1) protein level and disease-specific survival. A statistically significant inverse correlation was also observed between the cytoplasmic STAT1 protein level and disease-specific survival in sarcoma patients. We have shown that STAT1 is negatively regulated by an interferon-stimulated gene, Interferon Regulatory Factor 8 (IRF8). Disruption of IRF8 function by ectopic expression of an IRF8 dominant negative mutant resulted in dramatic up-regulation of STAT1, down-regulation of Janus kinase 1 (JAK1) and pSTAT1, and increased metastatic potential of sarcoma cells in an experimental metastasis mouse model. In addition, using microRNA microarray analysis we have identified a possible mechanism for these changes through mi106b. The possibility of cytoplasmic STAT1 and nuclear STAT1 accumulation being responsible for the observed difference in disease-specific survival of sarcoma patients merits further study of the role STAT1's localization plays in cancer progression. Direct evidence of cytoplasmic STAT1 increasing tumor cell survival, as well as confirmation of our proposed mechanism for STAT1 regulation through IRF8 is still needed. Taken together, our research suggests that IRF8 is a negative regulator of cytoplasmic STAT1 and STAT1 is indeed a tumor promoter. 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 2123. doi:10.1158/1538-7445.AM2011-2123

Abstract 2047: Betulinic acid exerts anti-angiogenic activity via regulation of HIF-1 alpha and STAT3 in PC-3 prostate cancer cells under hypoxia

Abstract Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively overexpressed in several human cancer cells and related to various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined whether betulinic acid (BA), a triterpene from the bark of white birch, could inhibit hypoxia-mediated activation of STAT3 and HIF-1 alpha in androgen independent human prostate cancer PC-3 cells. Our results show that BA inhibited the transcriptional activities and nuclear accumulation of both HIF-1 alpha and STAT3 under hypoxia. In addition, BA significantly reduced cellular and secreted levels of hypoxia-induced vascular endothelial growth factor (VEGF), a critical angiogenic factor, in PC-3 cells. BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced PC-3 cells. Moreover, silencing HIF 1 alpha and STAT3 by siRNA transfection augmented the suppression of VEGF expression by BA under hypoxia. Mechanistically, BA inhibited the recruitment of HIF-1 alpha and STAT3 on the VEGF promoter. Taken together, these findings suggest that BA may exert angio-prevention via inactivation of hypoxia-induced STAT3 and HIF-1 alpha in PC-3 cells. The in vivo anti-angiogenic and anti-PCa efficacy are being evaluated. 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 2047. doi:10.1158/1538-7445.AM2011-2047

Abstract 1966: Regulation of RKIP function by Helicobacter pylori in gastric cancer cells

Abstract Raf kinase inhibitory protein (RKIP) is a negative regulator of the mitogen activated protein kinase (MAPK) cascade initiated by Raf-1 and nuclear factor kappa B (NF-κB) and is an activator of apoptosis in human cancers. STAT3 (signal transducer and activators of transcription) is often constitutively phosphorylated in gastric cancer. RKIP is a negative regulator of STAT3 and has been positively correlated with a higher survival rate in gastric cancer patients (Chatterjee, et al., Clin. Cancer Res., 14:2994-3001, 2008). Helicobacter pylori (H. pylori) infection is associated with high levels of activated STAT3 as well as activation of the transcription factor NF-κB in gastric epithelial cells, through mechanisms involving macrophage-derived IL-6. H. pylori infection led to a dose- and time-dependent increase in STAT3 activation and Snail, a protein that is a negative regulator of RKIP transcription. In addition, H. pylori and IL-6 treatment stimulated the phosphorylation of RKIP in a protein kinase C- (PKC) dependent manner. Interestingly, H. pylori stimulated RKIP transcription which was enhanced in the presence of the histone acetyl transferase, CBP, and inhibited in the presence of the PKC inhibitor. Surprisingly, RKIP stimulated STAT3 transcription in gastric cancer cells, an effect that was reversed in the presence of CBP. After H. pylori infection, RKIP localized to the nucleus and enhanced H. pylori-mediated apoptosis. Collectively, these results indicate that RKIP function is regulated by phosphorylation after H. pylori infection and acetylation by CBP. Moreover, our study demonstrates that RKIP post translational modifications regulate apoptosis induction (via phosphorylation) and inhibition of cell survival (via acetylation) in gastric cancer. These results also suggest the existence of a feedback loop between RKIP, H. pylori infection and STAT3 in gastric cancer. Supported, in part, by NIH COBRE Investigator Award Grant P20 RR17695 (DC and SM). 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 1966. doi:10.1158/1538-7445.AM2011-1966

Abstract 1323: Reciprocal regulation of Stat3 and caveolin-1 in normal fibroblasts and breast carcinoma lines

Abstract Membrane tyrosine kinases known to activate the signal transducer and activator of transcription-3 (Stat3) concentrate in caveolae, where they are sequestered in an inactive state through binding to the main caveolae protein, caveolin-1 (cav1). We previously demonstrated that cell-to-cell adhesion can cause a dramatic increase in Stat3 activity in cultured cells. Therefore, to examine the effect of cav1 upon Stat3, experiments were conducted at several confluences. Our results indicate that cav-1 downregulation through expression of anti-sense or shRNA constructs or treatment of cells with the pharmacological inhibitor, methyl-cyclo-dextran which destroys caveolae, activates Stat3 as well as Erk1/2, at all densities. Conversely, cav1 overexpression downregulates Stat3 and induces growth retardation or apoptosis in NIH3T3 fibroblasts and in breast cancer lines. In all cases, apoptosis was inhibited by co-expression of the constitutively active form of Stat3, Stat3C. Taken together, these findings point to cav1 as an inhibitor of Stat3 activity. In addition, it was previously demonstrated that cav1 upregulates p53, although the exact mechanism is unclear. Since Stat3 is known to inhibit p53 transcription through promotor binding, these data also point to the possibility that cav1 upregulation may, in fact, activate p53 through Stat3 inhibition. Our results further demonstrate for the first time that, in a feedback loop, Stat3 inhibition results in a dramatic increase in cav1 levels, indicating that Stat3 also downregulates cav1 expression. The above findings reveal the presence of a potent, negative regulatory loop between cav1, p53 and Stat3 that plays a crucial role in cellular survival. 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 1323. doi:10.1158/1538-7445.AM2011-1323

Epstein-Barr Virus LMP1 Activates EGFR, STAT3, and ERK through Effects on PKCδ

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the world's adult population and is linked to multiple malignancies, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). The EBV oncoprotein LMP1 induces transcription of the epidermal growth factor receptor (EGFR), which is expressed at high levels in NPC. EGFR transcription is induced by LMP1 through a p50 NFκB1-Bcl-3 complex, and Bcl-3 is induced by LMP1-mediated activation of STAT3. This study reveals that LMP1, through its carboxyl-terminal activation domain 1 (LMP1-CTAR1), activates both STAT3 and EGFR in a serum-independent manner with constitutive serine phosphorylation of STAT3. Upon treatment with EGF, the LMP1-CTAR1-induced EGFR was additionally phosphorylated and STAT3 became phosphorylated on tyrosine, concomitant with upregulation of a subset of STAT3 target genes. The kinase responsible for LMP1-CTAR1-mediated serine phosphorylation of STAT3 was identified to be PKCδ using specific RNAi, a dominant negative PKCδ, and the PKCδ inhibitor rottlerin. Interestingly, inhibition of PKCδ also inhibited constitutive phosphorylation of EGFR and LMP1-CTAR1-induced phosphorylation of ERK. Inhibition of PKCδ blocked LMP1-CTAR1-mediated transformation of Rat-1 cells, likely through the inhibition of ERK activation. These findings indicate that LMP1 activates multiple distinct signaling pathways and suggest that PKCδ functions as a master regulator of EGFR, STAT3, and ERK activation by LMP1-CTAR1.

Regulation of STAT signaling in mouse bone marrow derived dendritic cells by respiratory syncytial virus

Background/aimsDendritic cells (DCs) act as a portal for virus invasion as well as potent antigen-presenting cells (APCs) involved in the antiviral host response. Interferons (IFNs) are produced in response to bacterial and viral infection and activate innate immune responses to efficiently counteract and remove pathogenic invaders. Respiratory syncytial virus (RSV) could inhibit IFN-mediated signaling pathway in epithelial cells; however, the effects of RSV on IFN signaling in the dendritic cells (DCs) are still unknown. MethodsMouse bone marrow derived DCs (BMDCs) were mock or infected with RSV at different multiplicity of infection (MOI) for 24h, and then treated with different cytokines such as interferon-β (IFN-β), IFN-γ or interleukin-10 (IL-10). The mRNA expression of RSV nonstructural protein-1 (NS-1) and NS-2 was detected by RT-PCR. The expression of Janus family kinase-signal transducer and activator of transcription (JAK/STAT) signaling proteins was assessed by immunoblotting assays. The nuclear localization of specific signaling proteins was determined by immunofluorescence assay. ResultsIncreasing amounts of NS-1 or NS-2 mRNA expression in BMDCs were observed with infected RSV at increasing MOI, suggesting BMDCs were permissive for viral gene expression. Further examination of the IFN-β signaling cascade showed RSV infection increased the total cellular levels of STAT1 and STAT2 in BMDCs, but impaired the IFN-β-dependent phosphorylation and nuclear localization of STAT1 and STAT2. The inhibitory effects of RSV on STAT1 and STAT2 phosphorylation and translocation were abolished by UV inactivation. In contrast, RSV did not inhibit the IFN-γ-stimulated STAT1 phosphorylation and nuclear localization. IL-10-stimulated STAT3 phosphorylation was also unaffected by RSV. ConclusionsAs well as RSV inhibiting STAT protein levels through degradation mechanisms in epithelial cells, these findings demonstrate that RSV also can specifically inhibit the type I interferon response in BMDCs through regulation of STAT1 and STAT2 phosphorylation and nuclear translocation.

RA-inducible gene-I induction augments STAT1 activation to inhibit leukemia cell proliferation

RA-inducible gene I (RIG-I/DDX58) has been shown to activate IFN-β promoter stimulator 1 (IPS-1) on recognizing cytoplasmic viral RNAs. It is unclear how RIG-I functions within the IFN and/or RA signaling process in acute myeloid leukemia (AML) cells, however, where obvious RIG-I induction is observed. Here, we show that the RIG-I induction functionally contributes to IFN-α plus RA-triggered growth inhibition of AML cells. Interestingly, although RIG-I induction itself is under the regulation of STAT1, a major IFN intracellular signal mediator, under circumstances in which it does not stimulate IPS-1, it conversely augments STAT1 activation to induce IFN-stimulatory gene expression and inhibit leukemia cell proliferation. Thus, our results unveil a previously undescribed RIG-I activity in regulating the cellular proliferation of leukemia cells via STAT1, which is independent of its classic role of sensing viral invasion to trigger type I IFN transcription.

PS1-58 The role of STAT1 in regulation of mitochondrial gene expression

PS1-58 The role of STAT1 in regulation of mitochondrial gene expression

IL-6: Back to the future (ML01)

IL-6 was originally identified as a T cell-derived cytokine, which induces antibody production in B cells. A series of subsequent studies have revealed that IL-6 has a pleiotropic activity in various tissues and cells and its deregulated expression is responsible for several chronic inflammations and hemopoietic malignancies. Humanized antibody against 80kd IL-6R (Tocilizumab) has shown significant therapeutic effect in RA, JIA and Castleman's diseases. The antibody is effective even on anti-TNF unresponsive inflammatory diseases. Recently, TH17 is shown to be responsible for the pathogenesis of autoimmune diseases and IL-6 together with TGF-β are essential for the induction of TH17. We identified a new transcription factor required for Th-17 cell induction, which is induced by IL-6 and TGF-β. This molecule, aryl hydrocarbon receptor (Ahr) interacts with Stat1 and Stat5 and abrogate their negative activity in the induction of Th-17 cell differentiation. Experimental arthritis is completely abrogated in Ahr-KO mice as well as T cell-specific Ahr-deficient mice. In contrast, Ahr is shown to negatively requlate LPS-induced inflammatory cytokines production in macrophages by interacting with STAT-1. Thus, Ahr-KO mice become hyper-sensitive to LPS-induced septic shock. I will discuss here a novel pathways of the regulation of inflammation through the regulation of STAT1 with Ahr.

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.

The induction of STAT1 gene by activating transcription factor 3 contributes to pancreatic β-cell apoptosis and its dysfunction in streptozotocin-treated mice

It is well established that the IFN-γ/STAT1 pathway plays an important role in the pancreatic β-cell apoptosis that is observed in STZ-induced type 1 diabetes; however, the upstream regulatory proteins involved have not been understood. Here, we investigated whether activating transcription factor 3 (ATF3) affects STAT1-mediated β-cell dysfunction and apoptosis in streptozotocin-treated mice. To this, STZ (80 mg/kg, i.p.) was administered to wild-type and STAT1 −/− or IFN-γ −/− mice for 5 days and the mice were euthanized after 14 days. STZ-induced β-cell dysfunction and apoptosis were associated with increased STAT1/IRF-1 and ATF3 expression and were correlated with elevated IFN-γ levels. Genetic depletion using IFN-γ −/− or STAT1 −/− mice strongly inhibited the reduction of islet cell mass or insulin synthesis/secretion and the increase of β-cell apoptosis observed in STZ-treated wild-type mice. ATF3 overexpression, especially the C-terminal domain, strongly enhanced β-cell dysfunction and apoptosis by enhancing STAT1 activation and its accumulation, which were abolished with an ATF3-specific siRNA or C-terminal-deleted ATF3. The STZ induction of ATF3 was completely depleted in IFN-γ −/− mice, but not in STAT1 −/− mice. Furthermore, STAT1 did not affect ATF3 expression, but STAT1 depletion or its inactivation inhibited STZ-induced ATF3 nuclear translocation and β-cell apoptosis. Interestingly, ATF3 also increased STAT1 transcription by directly binding to a putative binding region (− 116 to − 96 bp) in the STAT1 promoter. Our results suggest that ATF3 functions as a potent upstream regulator of STAT1 and ATF3 may play a role in STZ-induced β-cell dysfunction by enhancing the steady state abundance of STAT1.

Abstract LB-236: Differential regulation of STAT3 in mammary gland involution as well as in mammary tumorigeneis

Abstract STAT3 (Signal Transducer and Activator of Transcription 3) is persistently activated in many human primary tumors as well as in cancer cell lines, including breast cancer. This activated STAT3 is associated with cell survival and proliferation, inhibition of apoptosis, activation of angiogenesis and suppression of immune surveillance. Thus, STAT3 is generally considered as a growth-promoting factor. However, STAT3 activation also plays an important role in initiation of apoptotic signaling, especially, in mammary epithelial cells during mammary gland involution. Conditional deletion of STAT3 is associated with delayed involution mainly due to inadequate induction of apoptosis in mammary epithelial cells. Additionally, STAT3 activation is associated with induction of growth arrest and apoptosis in mouse mammary epithelial cells, myeloid cells and granulocyte. Thus, STAT3 plays dual role as a growth promoter as well as a growth inhibitor. However, the molecular switch that is responsible for switching the STAT3- associated pro-oncogenic signal to pro-apoptotic signal is not known. In order to identify this molecular switch we screened for signaling molecules that regulate STAT3 as well as those that are regulated by STAT3 during mammary gland involution, in human primary breast tumor tissues, and in human breast cancer cell lines. Our results show that early in the onset of mammary gland involution there is a significant up regulation of STAT3 and SLC5A8, a Na+-coupled transporter for monocarboxylates, including butyrate an inhibitor of histone deacetylases (HDACs). Further, STAT3 expression during mammary gland involution was found to be predominantly acetylated and phosphorylated. However, in human breast cancer cell lines we found an increase in phosphorylated STAT3 with almost undetectable expression of SLC5A8 and significantly decreased expression of acetylated STAT3 expression. Heterologous expression of SLC5A8 in human mammary tumor cell lines resulted in a butyrate-dependent induction of apoptosis with an increase in acetylated forms of STAT3. These results suggest that SLC5A8 is the molecular switch that converts the pro-oncogenic STAT3 signal to a pro-apoptotic signal by mediating the butyrate entry into mammary epithelial cells and thus it activates death receptor signaling by inhibiting HDACs and activation of STAT3 acetylation during mammary gland involution. However, in cancer cells the expression of SLC5A8 is silenced and thus it no longer able to inhibits HDACs and activates Ac-Stat3 and death receptor signaling. Thus, our studies provide a molecular link between the STAT3-associated pro-apoptotic and pro-oncogenic signaling that observed in normal mammary epithelium and in human breast cancer, respectively. 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 LB-236.

Abstract 3791: Sulphoraphane inhibits STAT3 and its related genes in Du 145 cancer cells

Abstract Sulforaphane is a naturally occurring isothiocyanate compound mainly found in cruciferous vegetables. Sulforaphane has demonstrated cancer chemopreventive activities in a number of organ sites, including prostate. Mechanistic investigations have shown that SFN induces caspase-mediated apoptosis that is mediated through a rapid intracellular ROS generation. Since STAT3 signaling is crucial for cancer cell survival and ROS has been linked to inactivation of this transcriptional factor, we hypothesized that STAT3 may be an important target for SFN. Here, we investigated the effect of sulforaphane on the regulation of STAT3 in association of ROS in Du145 prostate cancer cells. We first confirmed that sulforaphane induced apoptosis by activation of caspase 8, 9, and 3, PARP cleavage and increase of TUNEL positive cells in Du145 cells, which were preceded for many hours by the increase of intracellular ROS, peaking at 30 minutes of exposure. We demonstrate for the first time that sulforaphane suppressed constitutive STAT3 activation and DNA-binding of STAT3 in a time- and dose-dependent manner in Du145 cells. These actions were also found in U266 multiple myeloma cells and U937 lymphoma cells. In addition, sulforaphane down-regulated the expression of STAT3-related genes such as bcl-2, bcl-xL, survivin, cyclin D1, and VEGF in Du145 cells. Furthermore, sulforaphane cross-linked JAK2, the upstream kinase of STAT3, in a time- and dose-dependent manner. Thiol-reducing agent dithiothreitol (DTT) reversed the inactivation of STAT3 and JAK2 induced by sulforaphane. Interestingly, N-acetyl cysteine (NAC), which leads to increased intracellular GSH synthesis, reversed the molecular patterns of PARP cleavage and inactivation of JAK2 and STAT3 induced by sulphoraphane. Taken together, these findings suggest that sulforaphane exposure leads to a rapid generation of ROS in Du145 cells and inhibition of JAK2/STAT3 pathway, followed by caspase-mediated apoptosis. Our data suggest that the JAK2/STAT3 pathway may be a novel target of SFN contributing to apoptosis. 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 3791.

Abstract 1657: HSP90 inhibition down regulates EGFR and its effector signaling proteins in pancreatic cancer cell lines

Abstract Rationale: Currently approved therapies, including EGFR inhibitors, have minimal effect on survival in pancreatic cancer (PC). HSP90 is a molecular chaperone that specifically enables activity of numerous protein kinases, including EGFR and other signaling proteins. We have reported that simultaneous inhibition of EGFR and HSP90 may be additive in inhibiting cell viability in PC cell lines. Here we report possible molecular mechanisms underlying HSP90 and EGFR inhibition. Methods: MiaPaCa-2 and PANC-1 were treated with EGFR inhibitor, compound 56 (CMP56) (5 − 35 μmol), and HSP90 inhibitor geldanamycin (250 nmol − 1μmol). Growth inhibition was evaluated using the MTT assay, and apoptosis was evaluated by caspase-3 and PARP cleavage. The effect on relevant cell signaling proteins was assessed by immunoblotting with specific antibodies directed against EGFR and effectors such as AKT, STAT-3 and JNK. Results: Growth inhibition was significantly (p < 0.05) higher in both MiaPaCa-2 and PANC1 cells treated with CMP56 and geldanamycin than in cells treated with either agent at various concentrations. The molecular effects are shown in table 1. There was a decrease in phosphorylation of EGFR in bothe cell lines after treatment with CMP56. Concomitant inhibition of EGFR and HSP90 was associated with decreased phosphorylation of ERK, JNK and AKT and with an increase in caspase-3, PARP cleavage in MiaPaCa-2 cells, but not in PANC-1 cells. Conclusions: These results suggest that HSP90 inhibition in MiaPaCa-2 cells induces down regulation of EGFR, STAT-3 and AKT, but not in PANC-1 cells. Higher concentrations of geldanamycin may be required in PANC-1cells. Combined inhibition of EGFR and HSP90 induced apoptosis additively. We are studying further to understand the other potential molecular pathways involved in this strategy.Table.Molecular effects of EGFR and HSP90 inhibition in PC Cell lines ↘ - Decrease, - No change, GEL - Geldanamycin (500 nmol), CMP-56 - Compound 56 (10 μmol)Protein of interest MiaPaCa-2 PANC-1 GELCMP56GEL + CMP56 GELCMP56GEL + CMP56EGFR↘-↘↘ --↘AKT↘-↘↘ --↘STAT-3↘-↘↘ --↘ 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 1657.

Abstract 4004: Reciprocal regulation of Stat3 and the caveolae protein, cav1

Abstract Caveolae are cholesterol-rich, flask-shaped invaginations of the plasma membrane with many roles in the cell, including the regulation of signal transduction. Caveolin 1 (Cav1) is the major protein responsible for the organization and maintenance of caveolae microdomains. Cav1 recruits many receptor and non-receptor tyrosine kinases and through binding to its scaffolding-domain, Cav1 sequesters the kinases in an inactive form, thereby preventing their involvement in signaling pathways. One important downstream target of many tyrosine kinases (Src, EGF, PDGF, IL6 and others) is the signal transducer and activator of transcription-3 (Stat3). Stat3 is a cytoplasmic signal transducer which is activated by tyrosine-705 phosphorylation by a number of kinases, then migrates to the nucleus to initiate transcription of genes involved in cell division and survival. Despite extensive evidence on the role of cav1 in signal transduction, its effect upon Stat3 is still obscure. We previously demonstrated that cell-to-cell adhesion, as occurs in confluent cultures, can cause a dramatic increase in Stat3 phosphorylation and activity in cultured cells (Oncogene 23:2600, MBC 16:3832). Therefore, to examine effects upon Stat3, cell density has to be taken into account. Our results now indicate that cav-1 downregulation through expression of an anti-sense construct, or treatment with the pharmacological inhibitor, methyl-cyclo-dextran which removes cholesterol from the membrane and destroys caveolae, caused a strong activation of Stat3 as well as Erk1/2, at all densities examined. Conversely, cav1 overexpression downregulated Stat3 and induced apoptosis in NIH3T3 fibroblasts both before and after transformation by the Simian Virus 40 Large Tumor antigen, as well as in HeLa cells. In all cases, apoptosis was prevented by co-expression of the constitutively active form of Stat3, Stat3C. Taken together, these findings point to cav1 as an inhibitor of Stat3 activity. It was previously demonstrated that cav1 upregulates p53 gene activity. Since Stat3 is known to inhibit p53 transcription by direct binding to the p53 promotor, these data also point to the possibility that cav1 may, in fact, activate p53 through Stat3 inhibition. Our results also demonstrate for the first time that, in a feedback loop, Stat3 inhibition following infection with an Adenovirus vector expressing a Stat3-specific, shRNA, results in a dramatic increase in cav1 levels, indicating that Stat3 also downregulates cav1 expression. Since p53 also upregulates cav1, and Stat3 blocks the p53 promotor, it is possible that Stat3 may block cav1 simply by downregulating p53, rather than downregulating the cav1 promotor directly. The above findings taken together reveal the presence of a potent, negative regulatory loop between cav1 and Stat3 activity that plays a crucial role in cellular survival. (supported by CIHR, CBCF-Ontario chapter, US Army breast cancer program NSERC and BCAK). 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 4004.

Estrogen regulates the expression and activity of serine proteases in mouse splenocytes (87.11)

Abstract In addition to digesting proteins, serine proteases also play critical roles in regulation of inflammatory responses, innate and adaptive immunity via proteolytic cleavage of protease-activated receptors (PARs) and/or signaling molecules such as NF-κB. Dysregulation of serine proteases activity may result in the development of variety of human diseases. We and others have shown that estrogen, an important immune modulator, promoted the production of proinflammatory molecules in different types of cell including murine splenocytes and peritoneal macrophages. Our recent report has demonstrated that serine proteases mediated proteolysis play a critical role in regulation of STAT1 and NF-κB activity in splenocytes from estrogen-treated mice. Inhibition of serine protease activity decreased the production of inflammatory molecules IFNγ and nitric oxide in splenocytes from estrogen-treated mice. In this study, we found that estrogen treatment increased the serine proteases activity in splenocytes. Further, we confirmed that neutrophils-derived elastase mRNA expression level is increased, and serine protease inhibitor SerpinB7 is decreased in estrogen-treated splenocytes when compared to placebo control. Together, our data suggest a novel mechanism underlying estrogen-mediated promotion of inflammatory responses. These studies may form the basis for developing new alternative therapies for estrogen-influenced autoimmune diseases by intervening the activity of serine proteases.

Microarray Analysis of Gene Expression in Laminar Tissue From Horses After Carbohydrate Overload

s Vol 30, No 2 (2010) 109 the disease process and that could serve as new targets for therapeutic intervention. Laminar Inflammatory Gene Expression in the Carbohydrate Overload Model of Equine Laminitis B.S. Leise, P.J. Johnson, R.R. Faleiros, S.J. Black, and J.K. Belknap, The Ohio State University College of Veterinary Medicine, Columbus, OH, University of Missouri College of Veterinary Medicine, Columbia, MO, Universidade Federal de Minas Gerais, Brazil, University of Massachusetts, Amherst, MA TAKE HOME MESSAGE Laminar inflammation is present but delayed in the CHO model of laminitis when compared to BWE. INTRODUCTION The purpose of this study was to determine if a similar pattern of laminar inflammation characterized by pro-inflammatory mediator expression occurs in the CHO model of laminitis as previously described in the BWE model. MATERIALS AND METHODS Real time-quantitative PCR for selected pro-inflammatory mediators was performed on laminar samples from sixteen horses administered carbohydrate (DEV group, n 1⁄4 8[onset of fever], OG1 group, n 1⁄4 8 [onest of lameness]), and eight horses administered water (CON) 24 hours after administration of deionized water. RESULTS Increased mRNA concentrations (P < 0.05) for IL-1b IL6, IL-12p35, COX-2, E-selectin, and ICAM-1 were present in laminae from horses with OG1 lameness, but not at the DEV time, when compared to the CON horses. No differences between the groups were found for IL-2, IL-10, TNFa, IFNg, or COX-1 at either the DEV or OG1 time points. DISCUSSION There is a marked difference in the temporal pattern of inflammatory events between the BWE and CHO, with many events peaking early in the developmental stages in the BWE model compared to the majority of events appearing to occur simultaneously at the onset of lameness in the CHO model. This suggests that, in addition to circulating inflammatory molecules, there may be a local phenomenon (i.e. alterations in blood flow or hypoxia) in the CHO model resulting in the simultaneous onset of multiple laminar events including endothelial activation, leukocyte emigration, and pro-inflammatory cytokine expression. CLINICAL RELEVANCE The delayed onset of laminar inflammatory events in the CHO laminitis model may indicate that the clinician has a substantial time period to address inflammatory signaling in septic horses at risk of laminitis prior to the onset of irreversible inflammatory injury. CONCLUSIONS Although the onset of cytokine expression is delayed in the CHO model compared to the BWE model a similar prominent laminar inflammatory response occurs at onset of lameness, further supporting the need to address inflammatory signaling in the acute clinical case of laminitis. Laminar Regulation of STAT1 and STAT3 in the Black Walnut Extract and Carbohydrate Induced Models of Laminitis B.S. Leise, M. Watts, E. Tanhoff, C. Yin, A. Pettigrew, P. Johnson, S. Black, and J. Belknap, The Ohio State University College of Veterinary Medicine, Columbus, OH, University of Missouri College of Veterinary Medicine, Columbia, MO, University of Massachusetts, Amherst, MA TAKE HOME MESSAGE Activation of STAT1 and STAT3 occurs in both the BWE and CHO model of laminitis and is a possible therapeutic target in laminitis. INTRODUCTION Similar to sepsis-related organ injury in humans, laminar injury in experimental models of laminitis is associated with marked increases in cytokine expression. JAK-STATs are well known cytokine activated signaling pathways that are reported to play an important role in regulating sepsis-related inflammation and damage in other species. The objective of this study was to determine the regulation of STAT1 and 3 signaling in the laminae in the BWE and CHO model of laminitis. MATERIALS AND METHODS Western hybridization analysis was performed to determine concentrations of tyrosineand serine-phosphorylated STAT1 and STAT3 from archived laminar tissue samples from previous BWE and CHO overload protocols. Real time-quantitative PCR was also performed to assess

Laminar Regulation of STAT1 and STAT3 in the Black Walnut Extract and Carbohydrate Induced Models of Laminitis

Laminar Regulation of STAT1 and STAT3 in the Black Walnut Extract and Carbohydrate Induced Models of Laminitis

Oct4 and LIF/Stat3 Additively Induce Krüppel Factors to Sustain Embryonic Stem Cell Self-Renewal

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.