Ca2+-independent phospholipase A2ß-derived PGE2 contributes to osteogenesis.

Bone modeling can be modulated by lipid signals such as arachidonic acid (AA) and its cyclooxygenase 2 (COX2) metabolite, prostaglandin E2 (PGE2), which are recognized mediators of optimal bone formation. Hydrolysis of AA from membrane glycerophospholipids is catalyzed by phospholipases A2 (PLA2s). We reported that mice deficient in the Ca2+- independent PLA2beta (iPLA2ß), encoded by Pla2g6, exhibit a low bone phenotype, but the cause for this remains to be identified. Here, we examined the mechanistic and molecular roles of iPLA2ß in bone formation using bone marrow stromal cells and calvarial osteoblasts from WT and iPLA2ß-deficient mice, and the MC3T3-E1 osteoblast precursor cell line. Our data reveal that transcription of osteogenic factors (Bmp2, Alpl, and Runx2) and osteogenesis are decreased with iPLA2ß-deficiency. These outcomes are corroborated and recapitulated in WT cells treated with a selective inhibitor of iPLA2 ß (10 µM S-BEL), and rescued in iPLA2ß-deficient cells by additions of 10 µM PGE2. Further, under osteogenic conditions we find that PGE2 production is through iPLA2ß activity and that this leads to induction of Runx2 and iPLA2ß transcription. These findings reveal a strong link between osteogenesis and iPLA2ß-derived lipids and raise the intriguing possibility that iPLA2ß-derived PGE2 participates in osteogenesis and in the regulation of Runx2 and also iPLA2ß.

Novel EGFR ectodomain mutations associated with ligand-independent activation and cetuximab resistance in head and neck cancer.

Epidermal growth factor receptor (EGFR) is a pro-tumorigenic receptor tyrosine kinase that facilitates growth for cancer cells that overexpress the receptor. Monoclonal anti-EGFR antibody Cetuximab (CTX) provides significant clinical benefit in patients with head and neck squamous cell carcinoma (HNSCC). Missense mutations in the ectodomain (ECD) of EGFR can be acquired under CTX treatment and mimic the effect of large deletions on spontaneous untethering and activation of the receptor. Little is known about the contribution of EGFR ECD mutations to EGFR activation and CTX resistance in HNSCC. We identified two concurrent non-synonymous missense mutations (G33S and N56K) mapping to domain I in or near the EGF binding pocket of the EGFR ECD in patient-derived HNSCC cells that were selected for CTX resistance through repeated exposure to the agent in an effort to mimic what may occur clinically. Structural modeling predicted that the G33S and N56K mutants would restrict adoption of a fully closed (tethered) and inactive EGFR conformation while not permitting association of EGFR with the EGF ligand or CTX. Binding studies confirmed that the mutant, untethered receptor displayed reduced affinity for both EGF and CTX but demonstrated sustained activation and presence at the cell surface with diminished internalization and sorting for endosomal degradation, leading to persistent downstream AKT signaling. Our results demonstrate that HNSCC cells can select for EGFR ECD mutations under CTX exposure that converge to trap the receptor in an open, ligand-independent, constitutively activated state. These mutants impede the receptor's competence to bind CTX possibly explaining certain cases of CTX treatment-induced or de novo resistance to CTX.

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms.

Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

Trastuzumab-Resistant HER2+ Breast Cancer Cells Retain Sensitivity to Poly (ADP-Ribose) Polymerase (PARP) Inhibition.

HER2-targeted therapies, such as trastuzumab, have increased the survival rates of HER2+ breast cancer patients. However, despite these therapies, many tumors eventually develop resistance to these therapies. Our lab previously reported an unexpected sensitivity of HER2+ breast cancer cells to poly (ADP-ribose) polymerase inhibitors (PARPi), agents that target homologous recombination (HR)-deficient tumors, independent of a DNA repair deficiency. In this study, we investigated whether HER2+ trastuzumab-resistant (TR) breast cancer cells were susceptible to PARPi and the mechanism behind PARPi induced cytotoxicity. We demonstrate that the PARPi ABT-888 (veliparib) decreased cell survival in vitro and tumor growth in vivo of HER2+ TR breast cancer cells. PARP-1 siRNA confirmed that cytotoxicity was due, in part, to PARP-1 inhibition. Furthermore, PARP-1 silencing had variable effects on the expression of several NF-κB-regulated genes. In particular, silencing PARP-1 inhibited NF-κB activity and reduced p65 binding at the IL8 promoter, which resulted in a decrease in IL8 mRNA and protein expression. Our results provide insight in the potential mechanism by which PARPi induces cytotoxicity in HER2+ breast cancer cells and support the testing of PARPi in patients with HER2+ breast cancer resistant to trastuzumab. Mol Cancer Ther; 17(5); 921-30. ©2018 AACR.

MicroRNA-31 is required for astrocyte specification.

Previously, we determined microRNA-31 (miR-31) is a noncoding tumor suppressive gene frequently deleted in glioblastoma (GBM); miR-31 suppresses tumor growth, in part, by limiting the activity of NF-κB. Herein, we expand our previous studies by characterizing the role of miR-31 during neural precursor cell (NPC) to astrocyte differentiation. We demonstrate that miR-31 expression and activity is suppressed in NPCs by stem cell factors such as Lin28, c-Myc, SOX2 and Oct4. However, during astrocytogenesis, miR-31 is induced by STAT3 and SMAD1/5/8, which mediate astrocyte differentiation. We determined miR-31 is required for terminal astrocyte differentiation, and that the loss of miR-31 impairs this process and/or prevents astrocyte maturation. We demonstrate that miR-31 promotes astrocyte development, in part, by reducing the levels of Lin28, a stem cell factor implicated in NPC renewal. These data suggest that miR-31 deletions may disrupt astrocyte development and/or homeostasis.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

Small G protein Rac GTPases regulate the maintenance of glioblastoma stem-like cells in vitro and in vivo.

Glioblastoma is the most common and aggressive malignant brain tumor in adults. The existence of glioblastoma stem cells (GSCs) or stem-like cells (stemloids) may account for its invasiveness and high recurrence. Rac proteins belong to the Rho small GTPase subfamily which regulates cell movement, proliferation, and survival. To investigate whether Rac proteins can serve as therapeutic targets for glioblastoma, especially for GSCs or stemloids, we examined the potential roles of Rac1, Rac2 and Rac3 on the properties of tumorspheres derived from glioblastoma cell lines. Tumorspheres are thought to be glioblastoma stem-like cells. We showed that Rac proteins promote the STAT3 and ERK activation and enhance cell proliferation and colony formation of glioblastoma stem-like cells. Knockdown of Rac proteins reduces the expression of GSC markers, such as CD133 and Sox2. The in vivo effects of Rac proteins in glioblastoma were further studied in zebrafish and in the mouse xenotransplantation model. Knocking-down Rac proteins abolished the angiogenesis effect induced by the injected tumorspheres in zebrafish model. In the CD133+-U373-tumorsphere xenotransplanted mouse model, suppression of Rac proteins decreased the incidence of tumor formation and inhibited the tumor growth. Moreover, knockdown of Rac proteins reduced the sphere forming efficiency of cells derived from these tumors. In conclusion, not only Rac1 but also Rac2 and 3 are important for glioblastoma tumorigenesis and can serve as the potential therapeutic targets against glioblastoma and its stem-like cells.

Protein kinase CK2 is important for the function of glioblastoma brain tumor initiating cells.

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α') and two regulatory (ß) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133- cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

Let-7 Status Is Crucial for PARP1 Expression in HER2-Overexpressing Breast Tumors.

HER2+ breast tumors have been shown to express elevated levels of PARP1 protein. Yet, the mechanism by which PARP1 is upregulated in HER2+ breast cancer is unknown. Here, knockdown of HER2 (ERBB2) in HER2+ breast cancer cells resulted in a reduction in PARP1 protein. Conversely, ectopic overexpression of HER2 in a non-HER2-overexpressing cell line resulted in increased PARP1 protein levels. Alterations in HER2 expression had no significant effect on PARP1 transcript levels. Instead, HER2 mRNA status was inversely correlated with let-7a miRNA levels in breast cancer cells. Ectopic expression of let-7a miRNA resulted in downregulation of PARP1 protein, whereas expression of the let-7a anti-miRNA increased PARP1 protein. Furthermore, luciferase assays demonstrate that let-7a regulates PARP1 via its 3'UTR. Importantly, let-7a was significantly lower in human HER2+ breast tumors compared with HER2- breast tumors and inversely correlated with PARP1 protein levels. Finally, HER2+ breast cancer cells exhibited similar cytotoxicity to ectopic let-7a expression as the PARP inhibitor veliparib (ABT-888). Collectively, these results reveal that increased PARP1 expression in HER2+ breast cancers is regulated by the let-7a miRNA, and that let-7a is a potential strategy to suppress PARP1 activity.Implications: This study reports the novel findings that HER2 increases PARP1 protein via suppression of the let-7a miRNA, which regulates the PARP1 3'-UTR. Moreover, HER2 status correlates with high PARP1 and low let-7a in breast cancer clinical specimens. Mol Cancer Res; 15(3); 340-7. ©2016 AACR.

STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE.

BACKGROUND: In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, mice genetically deficient in the transcription factor signal transducer and activator of transcription 4 (STAT4) are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ which act upstream and downstream of STAT4, respectively, does not ameliorate disease. These discordant findings imply that STAT4 may act in a non-canonical role during EAE. Recently, STAT4 has been shown to regulate GM-CSF production by CD4 T cells and this cytokine is necessary for the induction of EAE. However, it is not known if STAT4 controls GM-CSF production by both Th1 and Th17 effector CD4 T cells. METHODS: This study utilized the MOG(35-55) peptide immunization model of EAE. Intracellular cytokine staining and novel mixed bone marrow chimeric mice were used to study the CD4 T cell-intrinsic role of STAT4 during disease. STAT4 chromatin-immunoprecipitation (ChIP-PCR) experiments were performed to show STAT4 directly interacts with the Csf2 gene loci. RESULTS: Herein, we demonstrate that STAT4 controls CD4 T cell-intrinsic GM-CSF production by both Th1 and Th17 CD4 T cells during EAE as well as in vitro. Importantly, we show that STAT4 interacts with the Csf2 locus in MOG(35-55)-activated effector CD4 T cells demonstrating direct modulation of GM-CSF. CONCLUSIONS: Overall, these studies illustrate a previously unrecognized role of STAT4 to regulate GM-CSF production by not only Th1 cells, but also Th17 effector CD4 T cell subsets during EAE pathogenesis. Critically, these data highlight for the first time that STAT4 is able to modulate the effector profile of Th17 CD4 T cell subsets, which redefines our current understanding of STAT4 as a Th1-centric factor.

Loss of tumor suppressive microRNA-31 enhances TRADD/NF-κB signaling in glioblastoma.

Glioblastomas (GBMs) are deadly tumors of the central nervous system. Most GBM exhibit homozygous deletions of the CDKN2A and CDKN2B tumor suppressors at 9p21.3, although loss of CDKN2A/B alone is insufficient to drive gliomagenesis. MIR31HG, which encodes microRNA-31 (miR-31), is a novel non-coding tumor suppressor positioned adjacent to CDKN2A/B at 9p21.3. We have determined that miR-31 expression is compromised in >72% of all GBM, and for patients, this predicts significantly shortened survival times independent of CDKN2A/B status. We show that miR-31 inhibits NF-κB signaling by targeting TRADD, its upstream activator. Moreover, upon reintroduction, miR-31 significantly reduces tumor burden and lengthens survival times in animal models. As such, our work identifies loss of miR-31 as a novel non-coding tumor-driving event in GBM.

Snail2/Slug cooperates with Polycomb repressive complex 2 (PRC2) to regulate neural crest development.

Neural crest cells arise from the border of the neural plate and epidermal ectoderm, migrate extensively and differentiate into diverse cell types during vertebrate embryogenesis. Although much has been learnt about growth factor signals and gene regulatory networks that regulate neural crest development, limited information is available on how epigenetic mechanisms control this process. In this study, we show that Polycomb repressive complex 2 (PRC2) cooperates with the transcription factor Snail2/Slug to modulate neural crest development in Xenopus. The PRC2 core components Eed, Ezh2 and Suz12 are expressed in the neural crest cells and are required for neural crest marker expression. Knockdown of Ezh2, the catalytic subunit of PRC2 for histone H3K27 methylation, results in defects in neural crest specification, migration and craniofacial cartilage formation. EZH2 interacts directly with Snail2, and Snail2 fails to expand the neural crest domains in the absence of Ezh2. Chromatin immunoprecipitation analysis shows that Snail2 regulates EZH2 occupancy and histone H3K27 trimethylation levels at the promoter region of the Snail2 target E-cadherin. Our results indicate that Snail2 cooperates with EZH2 and PRC2 to control expression of the genes important for neural crest specification and migration during neural crest development.

NF-κB and STAT3 in glioblastoma: therapeutic targets coming of age.

Since we last addressed the roles of NF-κB and JAK/STAT3 signaling in glioblastoma (GBM) 5 years ago, tremendous strides have been made in the understanding of these two pathways in glioma biology. Contributing to prosurvival mechanisms, cancer stem cell maintenance and treatment resistance, both NF-κB and STAT3 have been characterized as major drivers of GBM. In this review, we address general improvements in the molecular understanding of GBM, the structure of NF-κB and STAT3 signaling, the ways in which these pathways contribute to GBM and advances in preclinical and clinical targeting of these two signaling cascades.

PERK-dependent activation of JAK1 and STAT3 contributes to endoplasmic reticulum stress-induced inflammation.

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.

RNAdigest: a web-based tool for the analysis and prediction of structure-specific RNAse digestion results.

Despite recent developments in analyzing RNA secondary structures, relatively few RNA structures have been determined. To date, many investigators have relied on the traditional method of using structure-specific RNAse enzymes to probe RNA secondary structures. However, if these data were combined with novel computational approaches, investigators would have an informative and valuable tool for RNA structural analysis. To this end, we created the web server "RNAdigest." RNAdigest uses mfold RNA structural models in order to predict the results of RNAse digestion experiments. Furthermore, RNAdigest also utilizes both RNA sequence and the experimental digestion patterns to formulate the constraints for predicting secondary structures of the RNA. Thus, RNAdigest allows for the structural interpretation of RNAse digestion experiments. Overall, RNAdigest simplifies RNAse digestion result analyses while allowing for the identification of unique fragments. These unique fragments can then be used for testing predicted mfold structures and for designing structural-specific DNA/RNA probes.

NF-κB-induced IL-6 ensures STAT3 activation and tumor aggressiveness in glioblastoma.

Glioblastoma (GBM) is the most aggressive, neurologically destructive and deadly tumor of the central nervous system (CNS). In GBM, the transcription factors NF-κB and STAT3 are aberrantly activated and associated with tumor cell proliferation, survival, invasion and chemoresistance. In addition, common activators of NF-κB and STAT3, including TNF-α and IL-6, respectively, are abundantly expressed in GBM tumors. Herein, we sought to elucidate the signaling crosstalk that occurs between the NF-κB and STAT3 pathways in GBM tumors. Using cultured GBM cell lines as well as primary human GBM xenografts, we elucidated the signaling crosstalk between the NF-κB and STAT3 pathways utilizing approaches that either a) reduce NF-κB p65 expression, b) inhibit NF-κB activation, c) interfere with IL-6 signaling, or d) inhibit STAT3 activation. Using the clinically relevant human GBM xenograft model, we assessed the efficacy of inhibiting NF-κB and/or STAT3 alone or in combination in mice bearing intracranial xenograft tumors in vivo. We demonstrate that TNF-α-induced activation of NF-κB is sufficient to induce IL-6 expression, activate STAT3, and elevate STAT3 target gene expression in GBM cell lines and human GBM xenografts in vitro. Moreover, the combined inhibition of NF-κB and STAT3 signaling significantly increases survival of mice bearing intracranial tumors. We propose that in GBM, the activation of NF-κB ensures subsequent STAT3 activation through the expression of IL-6. These data verify that pharmacological interventions to effectively inhibit the activity of both NF-κB and STAT3 transcription factors must be used in order to reduce glioma size and aggressiveness.

Activation of the NF-κB pathway by the STAT3 inhibitor JSI-124 in human glioblastoma cells.

Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-κB (NF-κB) pathway, via NF-κB p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-κB-driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-κB p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-κB and inducible knockdown of NF-κB p65, we found that JSI-124-induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-κB-dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects.

Protective role of STAT3 in NMDA and glutamate-induced neuronal death: negative regulatory effect of SOCS3.

The present study investigates the involvement of the IL-6 family of cytokines, activation of the transcription factor Signal Transducer and Activator of Transcription-3 (STAT3), and the role of Suppressor Of Cytokine Signaling-3 (SOCS3) in regulating excitotoxic neuronal death in vitro. Biochemical evidence demonstrates that in primary cortical neurons and SH-SY5Y neuroblastoma cells, IL-6 cytokine family members, OSM and IL-6 plus the soluble IL-6R (IL-6/R), prevent NMDA and glutamate-induced neuronal toxicity. As well, OSM and IL-6/R induce tyrosine and serine phosphorylation of STAT3 in primary cortical neurons and SH-SY5Y cells. Studies using Pyridine 6 (P6), a pan-JAK inhibitor, demonstrate that the protective effect of OSM and IL-6/R on neuronal death is mediated by the JAK/STAT3 signaling pathway. In parallel to STAT3 phosphorylation, OSM and IL-6/R induce SOCS3 expression at the mRNA and protein level. P6 treatment inhibits SOCS3 expression, indicating that STAT3 is required for OSM and IL-6/R-induced SOCS3 expression. Lentiviral delivery of SOCS3, an inhibitor of STAT3 signaling, into primary neurons and SH-SY5Y cells inhibits OSM and IL-6/R-induced phosphorylation of STAT3, and also reverses the protective effect of OSM and IL-6/R on NMDA and glutamate-induced neurotoxicity in primary cortical neurons. In addition, treatment with IL-6 cytokines increases expression of the anti-apoptotic protein Bcl-xL and induces activation of the Akt signaling pathway, which are also negatively regulated by SOCS3 expression. Thus, IL-6/R and OSM-induced SOCS3 expression may be an important factor limiting the neuroprotective effects of activated STAT3 against NMDA and glutamate-induced neurotoxicity.

Estrogen modulates NFκB signaling by enhancing IκBα levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-ß.

BACKGROUND: NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17ß-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2ß genes. ChIP analyses also demonstrated that ERß can be recruited to the promoters of MCP-1 and CINC-2ß during co-treatment with TNF-α and E2. CONCLUSIONS: These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.

Therapeutic potential of AZD1480 for the treatment of human glioblastoma.

Aberrant activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been implicated in glioblastoma (GBM) progression. To develop a therapeutic strategy to inhibit STAT-3 signaling, we have evaluated the effects of AZD1480, a pharmacologic inhibitor of JAK1 and JAK2. In this study, the in vitro efficacy of AZD1480 was tested in human and murine glioma cell lines. AZD1480 treatment effectively blocks constitutive and stimulus-induced JAK1, JAK2, and STAT-3 phosphorylation in both human and murine glioma cells, and leads to a decrease in cell proliferation and induction of apoptosis. Furthermore, we used human xenograft GBM samples as models for the study of JAK/STAT-3 signaling in vivo, because human GBM samples propagated as xenografts in nude mice retain both the hallmark genetic alterations and the invasive phenotype seen in vivo. In these xenograft tumors, JAK2 and STAT-3 are constitutively active, but levels vary among tumors, which is consistent with the heterogeneity of GBMs. AZD1480 inhibits constitutive and stimulus-induced phosphorylation of JAK2 and STAT-3 in these GBM xenograft tumors in vitro, downstream gene expression, and inhibits cell proliferation. Furthermore, AZD1480 suppresses STAT-3 activation in the glioma-initiating cell population in GBM tumors. In vivo, AZD1480 inhibits the growth of subcutaneous tumors and increases survival of mice bearing intracranial GBM tumors by inhibiting STAT-3 activity, indicating that pharmacologic inhibition of the JAK/STAT-3 pathway by AZD1480 should be considered for study in the treatment of patients with GBM tumors.