Pharmacological Inhibition Of ERK Research Articles

Soluble E-Cadherin Contributes to Airway Inflammation in Severe Asthma.

Impaired airway epithelial barrier and decreased expression of E-cadherin are key features of severe asthma. As a gatekeeper of the mucosa, E-cadherin can be cleaved from the cell surface and released into the apical lumen as a soluble form (sE-cadherin).This study was aimed to investigate the role of sE-cadherin in severe asthma.Induced sputum was obtained from healthy subjects and patients with asthma. Two murine models of severe asthma were established using either TDI (toluene diisocyanate) or OVA (ovalbumin)/CFA (complete Freund's adjuvants). The role of sE-cadherin in severe asthma was evaluated by intraperitoneal injection of DECMA-1, a neutralizing antibody against sE-cadherin. Mice or THP-1-derived macrophages were treated with recombinant sE-cadherin to explore the pro-inflammatory mechanism of sE-cadherin.Severe asthma patients had a significantly higher sputum sE-cadherin level than the health subjects with mild to moderate asthma, which were positively correlated with sputum HMGB1 level and glucocorticoid dosage required for daily control. Allergen exposure markedly increased sE-cadherin level in the bronchoalveolar lavage fluid in mice. Treatment of DECMA-1 significantly attenuated allergen-induced airway inflammation and hyperresponsivenes in both models of severe asthma. While exposure to recombinant sE-cadherin dramatically up-regulated VEGF expression in THP-1-derived macrophages, and increased neutophlil and eosinophil infiltration into the airway as well as the release of VEGF and IL-6 in mice, both of which can be suppressed by pharmacological inhibition of ERK signaling.Taken together, our data indicated that sE-cadherin contributed to the airway inflammation of severe asthma in an ERK-depedent pathway.

Citropten Inhibits Vascular Smooth Muscle Cell Proliferation and Migration via the TRPV1 Receptor.

Vascular smooth muscle cell (VSMC) proliferation and migration play critical roles in arterial remodeling. Citropten, a natural organic compound belonging to coumarin and its derivative classes, exhibits various biological activities. However, mechanisms by which citropten protects against vascular remodeling remain unknown. Therefore, in this study, we investigated the inhibitory effects of citropten on VSMC proliferation and migration under high-glucose (HG) stimulation. Citropten abolished the proliferation and migration of rat vascular smooth muscle cells (RVSMCs) in a concentration-dependent manner. Also, citropten inhibited the expression of proliferation-related proteins, including proliferating cell nuclear antigen (PCNA), cyclin E1, cyclin D1, and migration-related markers such as matrix metalloproteinase (MMP), MMP2 and MMP9, in a concentration-dependent manner. In addition, citropten inhibited the phosphorylation of ERK and AKT, as well as hypoxia-inducible factor-1α (HIF-1α) expression, mediated to the Krüppel-like factor 4 (KLF4) transcription factor. Using pharmacological inhibitors of ERK, AKT, and HIF-1α also strongly blocked the expression of MMP9, PCNA, and cyclin D1, as well as migration and the proliferation rate. Finally, molecular docking suggested that citropten docked onto the binding site of transient receptor potential vanilloid 1 (TRPV1), like epigallocatechin gallate (EGCG), a well-known agonist of TRPV1. These data suggest that citropten inhibits VSMC proliferation and migration by activating the TRPV1 channel.

Pharmacological ascorbate induces sustained mitochondrial dysfunction.

Pharmacological ascorbate (P-AscH-; high dose given intravenously) generates H2O2 that is selectively cytotoxic to cancer compared to normal cells. The RAS-RAF-ERK1/2 is a major signaling pathway in cancers carrying RAS mutations and is known to be activated by H2O2. Activated ERK1/2 also phosphorylates the GTPase dynamin-related protein (Drp1), which then stimulates mitochondrial fission. Although early generation of H2O2 leads to cytotoxicity of cancer cells, we hypothesized that sustained increases in H2O2 activate ERK-Drp1 signaling, leading to an adaptive response; inhibition of this pathway would enhance the toxicity of P-AscH-. Increases in phosphorylated ERK and Drp1 induced by P-AscH- were reversed with genetic and pharmacological inhibitors of ERK and Drp1, as well as in cells lacking functional mitochondria. P-AscH- increased Drp1 colocalization to mitochondria, decreased mitochondrial volume, increased disconnected components, and decreased mitochondrial length, suggesting an increase in mitochondrial fission 48h after treatment with P-AscH-. P-AscH- decreased clonogenic survival; this was enhanced by genetic and pharmacological inhibition of both ERK and Drp1. In murine tumor xenografts, the combination of P-AscH- and pharmacological inhibition of Drp1 increased overall survival. These results suggest that P-AscH- induces sustained changes in mitochondria, through activation of the ERK/Drp1 signaling pathway, an adaptive response. Inhibition of this pathway enhanced the toxicity P-AscH- to cancer cells.

Astrocytic ERK/STAT1 Signaling Contributes to Maintenance of Stress-Related Visceral Hypersensitivity in Rats

The rostral anterior cingulate cortex (rACC) has been found to be an important brain region in mediating visceral hypersensitivity. However, the underlying mechanisms remain unclear. This study aimed to explore the role of astrocytes in the maintenance of visceral hypersensitivity induced by chronic water avoidance stress (WAS) as well as the potential signaling pathway that activates astrocytes in the rACC. We found that ACC-reactive astrogliosis resulted in the overexpression of c-fos, TSP-1, and BDNF in stress-related visceral hypersensitivity rats. Visceral hypersensitivity was reversed by pharmacological inhibition of astrocytic activation after WAS, as were the overexpression of c-fos, TSP-1 and BDNF. Activation of the astrocytic Gi-pathway increased the visceral sensitivity and expression of c-fos, TSP-1, and BDNF. Visceral hypersensitivity was also ameliorated by the pharmacological inhibition of ERK and STAT1 phosphorylation after WAS. Furthermore, inhibition of the ERK-STAT1 cascade reduced astrocytic activation. These findings suggest that astrocytic ERK/STAT1 signaling in the rACC contributes to the maintenance of stress-related visceral hypersensitivity. PerspectiveVisceral hypersensitivity is a key factor in the pathophysiology of irritable bowel syndrome. This study highlights the important role of astrocytic ERK/STAT1 signaling in activating astrocytes in the rostral anterior cingulate cortex, which contributes to visceral hypersensitivity.

ERK inhibition in glioblastoma is associated with autophagy activation and tumorigenesis suppression.

Autophagy-dependent tumorigenic growth is one of the most commonly reported molecular mechanisms in glioblastoma (GBM) progression. However, the mechanistic correlation between autophagy and GBM is still largely unexplored, especially the roles of autophagy-related genes involved in GBM oncogenesis. In this study, we aimed to explore the genetic alterations that interact with both autophagic activity and GBM tumorigenesis, and to investigate the molecular mechanisms of autophagy involved in GBM cell death and survival. For this purpose, we systematically explored the alterations of autophagic molecules at the genome level in human GBM samples through deep RNA sequencing. The effect of genetic and pharmacologic inhibition of ERK on GBM growth in vitro and in vivo was researched. An image-based tracking analysis of LC3 using mCherry-eGFP-LC3 plasmid, and transmission electron microscopy were utilized to monitor autophagic flux. Immunoblot analysis was used to measure the related proteins. MAPK ERK expression was identified as one of the most probable autophagy-related transcriptional responses during GBM growth. The genetic and pharmacologic inhibition of ERK in vivo and in vitro led to cell death, demonstrating its critical role for GBM proliferation and survival. To our surprise, autophagic activities were excessively activated and resulted in cytodestructive effects on GBM cells upon ERK inhibitor treatment. Furthermore, based on the observation of downregulation of mTOR signaling, we speculated the ERK inhibitor-induced GBM cells death might depend on mTOR-mediated pathway, leading to autophagy dysregulation. Accordingly, the in vivo and in vitro experiments revealed that the mTOR inhibitor rapamycin further increased cell mortality and exhibited enhanced antitumor effect on GBM cells when co-treated with the ERK inhibitor. Our data creatively demonstrated that the autophagy-related regulator ERK maintains autophagic activity during GBM tumorigenesis via mTOR signaling pathway. The pharmacologic inhibition of both mTOR and ERK signaling exhibited synergistic therapeutic effect on GBM growth in vivo and in vitro, which has certain novelty and may provide a potential therapeutic approach for GBM treatment in the future.

IL11 is elevated in systemic sclerosis and IL11-dependent ERK signalling underlies TGFβ-mediated activation of dermal fibroblasts.

ObjectivesInterleukin 11 (IL11) is highly upregulated in skin and lung fibroblasts from patients with systemic sclerosis (SSc). Here we tested whether IL11 is mechanistically linked with activation of human dermal fibroblasts (HDFs) from patients with SSc or controls.MethodsWe measured serum IL11 levels in volunteers and patients with early diffuse SSc and manipulated IL11 signalling in HDFs using gain- and loss-of-function approaches that we combined with molecular and cellular phenotyping.ResultsIn patients with SSc, serum IL11 levels are elevated as compared with healthy controls. All transforming growth factor beta (TGFβ) isoforms induced IL11 secretion from HDFs, which highly express IL11 receptor α-subunit and the glycoprotein 130 (gp130) co-receptor, suggestive of an autocrine loop of IL11 activity in HDFs. IL11 stimulated ERK activation in HDFs and resulted in HDF-to-myofibroblast transformation and extracellular matrix secretion. The pro-fibrotic action of IL11 in HDFs appeared unrelated to STAT3 activity, independent of TGFβ upregulation and was not associated with phosphorylation of SMAD2/3. Inhibition of IL11 signalling using either a neutralizing antibody against IL11 or siRNA against IL11RA reduced TGFβ-induced HDF proliferation, matrix production and cell migration, which was phenocopied by pharmacological inhibition of ERK.ConclusionsThese data reveal that autocrine IL11-dependent ERK activity alone or downstream of TGFβ stimulation promotes fibrosis phenotypes in dermal fibroblasts and suggest IL11 as a potential therapeutic target in SSc.

Yolkin Isolated from Hen Egg Yolk as a Natural Immunoregulator, Activating Innate Immune Response in BMDM Macrophages.

One of the goals of biomedical sciences is to search and identify natural compounds that are safe, have no side effects, and possess immunostimulatory activity. It has been proven that medicines of natural origin can be effective agents, supporting the therapy of many diseases, not only in the weakened immune system of the body but also in the prevention of many diseases in healthy people. It has been shown that yolkin, a polypeptide complex isolated from hen egg yolk as a fraction accompanying immunoglobulin Y (IgY), possesses potential biological activity. However, the mechanism of its action has not been explained. The objective of this investigation was to examine the molecular mechanisms of innate immune response, activated in response to yolkin, in murine bone marrow-derived macrophages (BMDM). It was shown that yolkin induced phosphorylation of extracellular signal-kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) and upregulated expression and production of type I interferons, TNF-α (tumor necrosis factor α), and nitric oxide (NO), in BMDM cells. Using pharmacological inhibitors of ERK 1/2 and JNK kinases, we revealed that the JNK signaling cascade is required for yolkin-induced inducible NOS expression and upregulation of NO production in mouse macrophages. Using the TLR4-deficient BMDM cell line, we established that yolkin can activate macrophages in a TLR4-dependent manner. It was also shown that NO, TNF-α, and type I IFNs (α/β) produced by BMDM cells in response to yolkin triggered antiviral activity. These data indicate that yolkin affects the regulation of the immune system and antiviral response; therefore, it can be used as an effective immunostimulator of the innate immunity or as a supplement of the conventional therapy of immunodeficiency.

Synergistic antitumor activity of sorafenib and artesunate in hepatocellular carcinoma cells.

Sorafenib is currently the standard chemotherapy drug for treatment of advanced hepatocellular carcinoma (HCC). But its efficacy requires improvement, it is imperative to seek therapeutic strategies that combine sorafenib with other anticancer agents. In this study we investigated the synergistic anticancer effect of combining sorafenib and artesunate, an anti-malaria drug derivative, against HCC in vitro and in vivo. We first showed that artesunate (1-100 μM) alone dose-dependently inhibited the proliferation of five HCC cell lines tested with IC50 values of around 100 μM. Artesunate treatment dose-dependently increased the ROS level in both HuH7 and Hep3B cells; addition of NAC significantly ameliorated the antiproliferation effect of artesunate against HuH7 and Hep3B cells. Then we demonstrated that combination of sorafenib and artesunate exerted synergistic antiproliferation effect and induced synergistic apoptosis in HCC cell lines. In nude mice bearing Hep3B xenografts, combined administration of sorafenib and artesunate significantly enhanced the suppression on tumor growth. We further revealed that sorafenib dose-dependently decreased the levels of p-ERK and p-STAT3, whereas artesunate markedly increased the levels of p-ERK and p-STAT3 in HuH7 and Hep3B cells. When used in combination, sorafenib abolished artesunate-elevated levels of p-STAT3 and p-ERK. Moreover, pharmacological inhibition of ERK by inhibitor PD0325901 or STAT3 by inhibitor Stattic markedly enhanced the anticancer activity of artesunate, suggesting that suppression of ERK and STAT3 signaling by sorafenib contributes to the synergistic anticancer activity against HCC caused by combination of sorafenib and artesunate. Taken together, our results provide an evidence for possible use of sorafenib plus artesunate or artemisinin analogs for treatment of HCC in the future.

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.

Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling.

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3.

OR2 Ligation of HLA class II molecules promotes endothelial cell permeability through activated Src and ERK signals

Aim Microvascular inflammation, increased vascular permeability, and accumulation of intravascular mononuclear cells are characteristics of acute (AMR) and chronic (cAMR) antibody-mediated rejection. In solid organ transplantation, donor specific antibodies (DSA) are associated with AMR, and early or late graft loss. However, the mechanisms underlying how DSA against HLA class II (HLA II) activate the endothelium and mediate inflammation and vascular permeability are poorly understood. Methods We utilized recombinant Class II Transactivator (CIITA) sub-cloned with pAd/PL-DEST vector to achieve expression of HLA II on vascular endothelium, and then evaluated the effects of HLA II antibodies (anti-HLA II) on endothelial cell (EC) permeability. Immunoblotting was used to determine the activation of signaling pathways and confocal microscopy was used to determine vascular endothelial - Cadherin (VE-Cad) phosphorylation and internalization. Results Ligation of HLA II by anti-HLA II activated Src and ERK, induced stress fiber formation, and increased EC permeability. Anti-HLA II also stimulated VE-Cad phosphorylation at Tyr685 as well as its internalization, thereby disassembling intercellular junctions. Class II mediated stress fiber formation and EC permeability was abrogated by pharmacological inhibition of ERK (U0126), Src (PP2) or MLC2 (ML-7). Both pharmacological and siRNA inhibition of Src, but not ERK, inhibited class II-induced phosphorylation of VE-cCd at Tyr685 and consequent VE-Cad internalization. Conclusions In conclusion, our data show that HLA class II DSA promotes actin stress fiber formation via a Src/ERK/MLC2 dependent pathway. Further, class II crosslinking with antibodies induces rapid endocytosis of VE-Cad via Src-dependent tyrosine phosphorylation of VE-Cad, thereby disrupting the endothelial barrier function and contributing to vascular permeability.

Leptin induces matrix metalloproteinase 7 expression to promote ovarian cancer cell invasion by activating ERK and JNK pathways.

Leptin, an adipokine secreted by adipose tissue, induces cell invasion and metastasis. MMP7 is a member of the matrix metalloproteinase family that plays an important role in cell invasion. Here we evaluate the possible role and underlying mechanism of MMP7 in the leptin-mediated cell invasion in ovarian cancer cell lines. All experiments were carried out in cultured SKOV3, OVCAR3, and CaoV-3 ovarian cell lines. MMP7 expression was determined using the Western blot following treatment to various concentrations of leptin for defined time intervals. The activation of ERK, JNK, and P38 MAP kinases were determined using Western blotting. Wound healing and BD matrigel invasion assays were used to measure cell migration and invasion. The siRNA approach and pharmacological inhibitors of ERK and JNK pathway were used to confirm the receptor-dependent effect of leptin and a role for ERK and JNK pathway. Zymography assay was employed to determine MMP2 and MMP9 activation. Results show that leptin induces ERK1/2 and JNK1/2 activation and subsequently promotes MMP7 expression in SKOV3 (4.8 ± 0.14 fold of control, P < 0.01) and OVCAR3 (3.1 ± 0.19 fold of control, P < 0.01) ovarian cancer cell lines. These effects was reversed by knockdown of OB-Rb and/or pre-incubation with PD98059 (ERK1/2 inhibitor), SP600125 (JNK1/2 inhibitor). Gelatin zymography showed that MMP7 gene silencing attenuated leptin-induced MMP9 activation in SKOV3 cell line. Taken together, our results suggest new evidences for a modulatory effect of leptin in regulation of ovarian cancer cell invasion by stimulating MMP7 expression via ERK and JNK pathways.

Mild acidosis delays neutrophil apoptosis via multiple signaling pathways and acts in concert with inflammatory mediators.

Accumulating evidence indicates development of local extracellular acidosis in inflamed tissues in response to infection and tissue injury. Activation of infiltrating neutrophils contributes to a transient decrease in pH, which, in turn, triggers innate immunity. In this study, we investigated the impact of extracellular acidosis on neutrophil apoptosis, a critical determinant of the outcome of the inflammatory response and analyzed the underlying signaling pathways. Culture of human isolated neutrophils in mildly acidotic conditions (pH 6.5-7.0) resulted in activation of NF-κB; intracellular accumulation of cAMP; and phosphorylation of Akt, ERK, and p38 MAPK; and preservation of Mcl-1 expression. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor from the mitochondria to cytoplasm and nuclei, respectively and inhibited caspase-3 activity. Pharmacological inhibition of ERK, PI3K, NF-κB, or PKA partially reversed survival cues by extracellular acidosis and redirected neutrophils to apoptosis. Conversely, dibutyryl cAMP (100-500 μM) delayed apoptosis of neutrophils cultured at pH 7.4. Extracellular acidosis-generated survival cues were additive to the potent prosurvival signals from bacterial DNA, LPS, modified C-reactive protein, and serum amyloid A. Acidosis increased CpG DNA uptake by neutrophils and augmented phosphorylation of ERK and Akt, leading to preservation of Mcl-1 expression. Our results identified extracellular acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH can enhance neutrophil responses to inflammatory stimuli, thereby contributing to amplification or prolongation of the inflammatory response.

Phorbol esters dPPA/dPA promote furin expression involving transcription factor CEBPβ in neuronal cells.

Using high-throughput small molecule screening targeting furin gene, we identified that phorbol esters dPPA (12-Deoxyphorbol 13-phenylacetate 20-acetate) and dPA (12-Deoxyphorbol 13-acetate) significantly increased furin protein and mRNA expression in SH-SY5Y cells. This effect was prevented by PKC (protein kinase C) inhibitor calphostin C but not Ro318220, suggesting that the C1 domain, rather than the catalytic domain of PKC plays an important role. Luciferase assay revealed that nucleotides −7925 to −7426 were sufficient to mediate dPPA/dPA enhancement of furin P1 promoter activity. RNA interference of transcriptional factors CEBPβ (CCAAT/enhancer-binding protein β) and GATA1 revealed that knockdown of CEBPβ significantly attenuated the effect of dPPA on furin expression. Pharmacological inhibition of ERK and PI3K but not TGFβ receptor diminished the up-regulation of furin by dPPA. These results suggested that in neuronal cells, transcriptional activation of furin by dPPA/dPA may be initiated by C1 domain containing proteins including PKC; the intracellular signaling involves ERK and PI3K and transcription factor CEBPβ.

MicroRNA-194 Promotes Prostate Cancer Metastasis by Inhibiting SOCS2.

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial-mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194-driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194-stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194-driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021-34. ©2016 AACR.

Inhibition of IKK-NFκB pathway sensitizes lung cancer cell lines to radiation.

Objective: Cancer cell radioresistance is a stumbling block in radiation therapy. The activity in the nuclear factor kappa B (NFκB) pathway correlates with anti-apoptotic mechanisms and increased radioresistance. The IKK complex plays a major role in NFκB activation upon numerous signals. In this study, we examined the interaction between ionizing radiation (IR) and different members of the IKK-NFκB pathway, as well as upstream activators, RAF1, ERK, and AKT1.Methods: The effect of 4 Gy of IR on the expression of the RAF1-ERK-IKK-NFκB pathway was examined in A549 and H1299 lung cancer cell lines using Western blot analysis and confocal microscopy. We examined changes in radiation sensitivity using gene silencing or pharmacological inhibitors of ERK and IKKβ.Results: IKKα, IKKγ, and IκBα increased upon exposure to IR, thereby affecting nuclear levels of NFκB (phospho-p65). ERK inhibition or siRNA-mediated down-regulation of RAF1 suppressed the post-irradiation survival of the examined lung cancer cell lines. A similar effect was detected on survival upon silencing IKKα/IKKγ or inhibiting IKKβ.Conclusions: Exposure of lung cancer cells to IR results in NFκB activation via IKK. The genetic or pharmacological blockage of the RAF1-ERK-IKK-NFκB pathway sensitizes cells to therapeutic doses of radiation. Therefore, the IKK pathway is a promising target for therapeutic intervention in combination with radiotherapy.

Abstract IA08: Drugging undruggable kras: will myc do the trick?

Abstract KRAS oncogene mutations are found in ~95% of pancreatic ductal adenocarcinoma (PDAC) and are well validated drivers of PDAC initiation and maintenance. Consequently, a major goal for the field is the development of effective anti-KRAS therapies. However, despite more than three decades of intensive effort, no effective pharmacologic inhibitors of the KRAS have reached the clinic, prompting the widely held perception that KRAS is “undruggable”. However, with more advanced knowledge of KRAS function and new technological approaches, there is renewed hope that the time is ripe to revisit targeting KRAS. I will present our studies focused on the RAF-MEK-ERK mitogen-activated protein kinase cascade. This effector signaling network is a well-validated driver of KRAS-dependent cancer growth. Despite this key role, pharmacologic inhibitors of RAF and MEK have been ineffective in inhibiting growth of KRAS-mutated cancers, in large part due to dynamic signal reprogramming leading to ERK reactivation and bypass of inhibitor action. We therefore addressed whether direct ERK inhibition may be a more effective therapeutic approach. We identified a growth suppression mechanism due to pharmacologic inhibition of ERK that involves degradation of the MYC oncoprotein, activation of the p16INK4A-RB1 tumor suppressor pathway and induction of a senescence-like phenotype. We also applied unbiased genetic and chemical functional screens, as well as reverse phase protein array analyses, and identified the PI3K-AKT-mTOR signalling network as a key driver of pancreatic cancer resistance to ERK inhibition. Novel combination inhibitor approaches that synergistically enhanced ERK inhibitor anti-tumor activity were also identified. Finally, we determined that continued expression of KRAS was required for maintenance of MYC protein expression. We determined that previously identified mechanisms of RAS effector signaling alone did not account for MYC protein regulation in PDAC, prompting our search for novel protein kinases that regulate MYC protein degradation. Our efforts are centered defining pharmacologic strategies to accelerate MYC protein degradation as a novel approach for blocking mutant KRAS in PDAC. Citation Format: Channing J. Der.{Authors}. Drugging undruggable kras: will myc do the trick? [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr IA08.

Mild acidosis delays neutrophil apoptosis and enhances pro-survival signals from inflammatory mediators

Abstract Emerging evidence indicates that local acidosis associated with infection and tissue injury triggers innate and adaptive immunity. Activation of infiltrating neutrophils contributes to transient drop in pH. We investigated the impact of extracellular acidosis on neutrophil apoptosis, one of the check points in the outcome of the inflammatory response, and on the survival signals generated by bacterial DNA and serum amyloid A. Culture of human isolated neutrophils under mild acidosis (pH 6.5–7.0) resulted in concurrent activation of NF-κB, adenyl cyclase and the ERK and PI3K/Akt signaling pathways, leading to preservation of Mcl-1. Consequently, extracellular acidosis prevented disruption of mitochondrial transmembrane potential and translocation of cytochrome c, endonuclease G and apoptosis-inducing factor (AIF) from the mitochondria to cytoplasm and nuclei, respectively and attenuated activation of caspase-3. Pharmacological inhibition of ERK, PI3K, NF-κB and adenylcyclase partially reversed the anti-apoptotic action of acidosis. Furthermore, mild acidosis enhanced the pro-survival signal from bacterial DNA and serum amyloid A by enhancing ERK and PI3K-mediated inhibition of Mcl-1 degradation. Our results identify mild acidosis as a survival signal for neutrophils by suppressing the constitutive apoptotic machinery and suggest that transient decreases in local pH could contribute to amplification of inflammation. Funding Support: Supported by grants from CIHR (MOP-97742).

Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection.

MicroRNAs (miRNAs) have been well known to play diverse roles in viral infection at the level of posttranscriptional repression. However, much less is understood about the mechanism by which miRNAs are regulated during viral infection. It is likely that both host and virus contain factors to modulate miRNA expression. Here we report the up-regulation of microRNA-15b (miR-15b) in vitro upon infection with Japanese encephalitis virus (JEV). Analysis of miR-15b precursor, pri-miR-15b and pre-miR-15b, suggest that the regulation occurs transcriptionally. Further, we identified the transcriptional regulatory region of miR-15b that contains consensus binding motif for NF-κB subunit c-Rel and cAMP-response element binding protein (CREB), which are known as transcription factor to regulate gene expression. By promoter fusion and mutational analyses, we demonstrated that c-Rel and CREB bind directly to the promoter elements of miR-15b, which are responsible for miR-15b transcription in response to JEV infection. Finally, we showed that pharmacological inhibition of ERK and NF-κB signaling pathway blocked induction of miR-15b in JEV infection, suggesting important roles of ERK and NF-κB pathway in the regulation of miR-15b gene. Therefore, our observations indicate that induced expression of miR-15b is modulated by c-Rel and CREB in response to JEV infection.

Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways

The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.