Activation Of Elk-1 Research Articles

P38 MAPK pathway-dependent SUMOylation of Elk-1 and phosphorylation of PIAS2 correlate with the downregulation of Elk-1 activity in heat-stressed HeLa cells.

Stress-activated and mitogen-activated protein kinases (MAPKs) regulate gene expression by post-translational modifications of transcription factors. Elk-1, a transcription factor that regulates the expression of immediate early genes, is amenable to regulation by all the three mammalian MAPKs. In the present report, using inhibitors specific for different MAPK pathways, we show that during exposure of HeLa cells to heat stress, Elk-1 is SUMOylated with SUMO1 by p38 MAPK pathway-dependent mechanisms. Elk-1-phosphorylation levels were significantly reduced under similar conditions. We also show that transcriptional activity of Elk-1 as assessed by luciferase reporter expression and qPCR estimation of the expression of genes regulated by Elk-1 was downregulated upon exposure to heat stress; this downregulation was reversed when heat exposure was performed in the presence of either SB203580 (p38 MAPK inhibitor) or ginkgolic acid (inhibitor of SUMOylation). Elk-1 induced transcription is also regulated by PIAS2 which acts as a coactivator upon the activation of extracellular signal-regulated kinases (ERKs) and as a corepressor upon its phosphorylation by p38 MAPK. Since heat stress activates the p38 MAPK pathway, we determined if PIAS2 was phosphorylated in heat-stressed HeLa cells. Our studies indicate that in HeLa cells exposed to heat stress, PIAS2 is phosphorylated by p38 MAPK pathway-dependent mechanisms. Collectively, the results presented demonstrate that in heat-stressed HeLa cells, p38 MAPK pathway-dependent SUMOylation of Elk-1 and phosphorylation of PIAS2 correlate with the downregulation of transactivation by Elk-1.

Targeting SET to restore PP2A activity disrupts an oncogenic CIP2A-feedforward loop and impairs triple negative breast cancer progression.

BackgroundTriple-negative breast cancer (TNBC) remains difficult to be targeted. SET and cancerous inhibitor of protein phosphatase 2A (CIP2A) are intrinsic protein-interacting inhibitors of protein phosphatase 2A (PP2A) and frequently overexpressed in cancers, whereas reactivating PP2A activity has been postulated as an anti-cancer strategy. Here we explored this strategy in TNBC.MethodsData from The Cancer Genome Atlas (TCGA) database was analyzed. TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. The apoptotic cells were examined by flow cytometry and Western blot. A SET-PP2A protein-protein interaction antagonist TD19 was used to disrupt signal transduction. In vivo efficacy of TD19 was tested in MDA-MB-468-xenografted animal model.FindingsTCGA data revealed upregulation of SET and CIP2A and positive correlation of these two gene expressions in TNBC tumors. Ectopic SET or CIP2A increased cell viability, migration, and invasion of TNBC cells. Notably ERK inhibition increased PP2A activity. ERK activation is known crucial for Elk-1 activity, a transcriptional factor regulating CIP2A expression, we hypothesized an oncogenic feedforward loop consisting of pERK/pElk-1/CIP2A/PP2A. This loop was validated by knockdown of PP2A and ectopic expression of Elk-1, showing reciprocal changes in loop members. In addition, ectopic expression of SET increased pAkt, pERK, pElk-1 and CIP2A expressions, suggesting a positive linkage between SET and CIP2A signaling. Moreover, TD19 disrupted this CIP2A-feedforward loop by restoring PP2A activity, demonstrating in vitro and in vivo anti-cancer activity. Mechanistically, TD19 downregulated CIP2A mRNA via inhibiting pERK-mediated Elk-1 nuclear translocation thereby decreased Elk-1 binding to the CIP2A promoter.InterpretationThese findings suggested that a novel oncogenic CIP2A-feedforward loop contributes to TNBC progression and targeting SET to disrupt this oncogenic CIP2A loop showed therapeutic potential in TNBC.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. Small-molecule drug discovery and extensive biological characterization of a lead compound. We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.

Stimulation of TRPM3 channels increases the transcriptional activation potential of Elk-1 involving cytosolic Ca2+, extracellular signal-regulated protein kinase, and calcineurin

Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration.

Antidepressive effects of targeting ELK-1 signal transduction.

Depression, a devastating psychiatric disorder, is a leading cause of disability worldwide. Current antidepressants address specific symptoms of the disease, but there is vast room for improvement 1 . In this respect, new compounds that act beyond classical antidepressants to target signal transduction pathways governing synaptic plasticity and cellular resilience are highly warranted2-4. The extracellular signal-regulated kinase (ERK) pathway is implicated in mood regulation5-7, but its pleiotropic functions and lack of target specificity prohibit optimal drug development. Here, we identified the transcription factor ELK-1, an ERK downstream partner 8 , as a specific signaling module in the pathophysiology and treatment of depression that can be targeted independently of ERK. ELK1 mRNA was upregulated in postmortem hippocampal tissues from depressed suicides; in blood samples from depressed individuals, failure to reduce ELK1 expression was associated with resistance to treatment. In mice, hippocampal ELK-1 overexpression per se produced depressive behaviors; conversely, the selective inhibition of ELK-1 activation prevented depression-like molecular, plasticity and behavioral states induced by stress. Our work stresses the importance of target selectivity for a successful approach for signal-transduction-based antidepressants, singles out ELK-1 as a depression-relevant transducer downstream of ERK and brings proof-of-concept evidence for the druggability of ELK-1.

MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

17β-estradiol Induces MMP-9 and MMP-13 in TMJ Fibrochondrocytes via Estrogen Receptor α

Temporomandibular joint (TMJ) disorders, including degenerative TMJ disease, occur primarily in women of reproductive age. Previous studies showed elevated estrogen levels in subjects with TMJ disorders relative to controls and the presence of estrogen receptors α and β (ERα and ERβ) in TMJ fibrocartilage. Additionally, estrogen-induced overexpression of specific matrix metalloproteinases (MMPs), including MMP-9 and MMP-13, in TMJ fibrocartilage is accompanied by loss of extracellular matrices. However, the contribution of ERα and ERβ in estrogen-mediated induction of MMP-9 and MMP-13 and the signaling cascade leading to the upregulation of these MMPs have not been elucidated. Here, we show that specific siRNAs and selective ER antagonists effectively block ERα or ERβ expression in primary mouse TMJ fibrochondrocytes, but that only blockage of ERα suppresses MMP-9 and MMP-13 levels induced by 17β-estradiol (E2). Overexpression of ERα but not ERβ enhances E2-induced MMP-9. Using the same loss-of-function and gain-of-function approaches, we demonstrate that E2 stimulates ERK activation through ERα and that inhibition of ERK phosphorylation reduces E2-induced MMP-9. Furthermore, we reveal that E2 promotes NF-κB and ELK-1 activation through ERα/ERK signaling and that knockdown of either one decreases the respective activity of these signaling mediators and MMP-9 expression induced by E2, indicating that both contribute to E2/ERα/ERK-mediated MMP-9 upregulation. This is supported by findings in which mutated binding sites of either NF-κB or ELK-1 in the MMP-9 promoter lead to a significant reduction of E2-stimulated promoter activity. Our findings provide novel molecular mechanisms for the understanding of E2-mediated upregulation of MMPs, having implications to pathophysiologic TMJ cartilage matrix turnover that may yield therapeutic intervention targets for TMJ disorders.

Pharmacophore Identification of the Neuronal Cyclic AMP sensors PKA, Epac2 and NCS‐Rapgef2 Using Novel Adenine‐Based Compounds

In the neuroendocrine NS‐1 cell line, three separate cAMP signaling pathways mediate growth arrest (via Epac2 ‐‐ p38 MAPK), induction of neuronal genes and cell survival (via PKA ‐‐ CREB), and neurite outgrowth (via NCS‐Rapgef2 ‐‐ ERK) (see Emery et al., J. Biol. Chem. 289:10126–39, 2014). There is no identifiable cross‐talk between these three pathways, thus they are parcellated. These three cAMP signaling pathways are activated by a variety of Gs‐coupled receptors expressed in the CNS, which include the PACAP receptor PAC1 (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Jiang et al., eNeuro, ENEURO.0248–17.2017). Therefore, a signaling component unique to one pathway could represent a target for intracellular therapeutics to modulate aspects of neuronal behavior for CNS disorders related to addiction (e.g. D1‐dependent signaling to ERK) and anxiety (e.g. PACAP/PAC1 signaling). Using cell‐based assays to monitor the activation of each of the three pathways, we have sought to identify specific inhibitors and activators of NCS‐Rapgef2.Using a battery of biochemical and high‐content assays that distinguish signaling between the three cAMP sensors (PKA, Epac2 and NCS‐Rapgef2), we found that the adenylate cyclase inhibitor 9‐tetrahydrofuranyladenine (SQ,22536; IC50 for forskolin‐induced cAMP accumulation = 10 μM) also inhibits NCS‐Rapgef2 (IC50 = 170 μM, inhibiting 8‐Br‐cAMP‐induced Elk1 activation, reported in Emery et al., Mol Pharmacol 83, 95–105, 2013). To create compounds with selectivity and greater potency at NCS‐Rapgef2, we generated analogs of 9‐tetrahydrofuranyladenine at several positions of the adenine (6‐amino‐purine) moiety. In NS‐1 cells, we found that the compound N6‐phenyl‐9‐tetrahydrofuranyladenine (EL1101) has greatly reduced activity at adenylate cyclase (IC50 > 300 μM) and no detectable activity, either an agonist antagonist, for PKA‐ and Epac2‐dependent signaling. However, EL1101 inhibts 8‐Br‐cAMP‐induced ERK phosphorylation with a greater potently than 9‐tetrahydrofuranyladenine (IC50 = 10 μM for EL1101). At similar potencies (IC50 = 10 μM), EL1101 also blocked ERK phosphorylation by Gs‐coupled GPCR activation (PACAP/PAC1), adenylate cyclase activation by forskolin, and direct activation of NCS‐Rapgef2 with cell‐permeable cAMP analogs (8‐Br‐cAMP). We also employed a phenotypic high‐content assay (cAMP analog‐dependent neurite outgrowth) and found that EL1101 blocked the neuritogenic effect of cAMP analogs at a similar potency observed in biochemical assays (IC50 = 10 μM). We are currently exploring further derivatization of the 9‐tetrahydrofuranyladenine scaffold to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo to block aspects of D1 dopamine receptor‐dependent signaling caused by psychomotor stimulants that require ERK activation.Support or Funding InformationThis work was supported by National Institute of Mental Health Intramural Research Program Grants 1ZIAMH002386 (to L.E.E.) and 1ZIAMH002592 (to M.V.E.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.Support or Funding InformationAmerican Heart Association ‐ Grant‐in‐Aid 16GRNT31200034This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic Nicotine Exposure Reduces Antioxidant Function of Simvastatin in Renal Proximal Tubule Cells

We have previously reported that simvastatin exhibits antioxidant properties via extracellular signal-regulated kinase (ERK)/cAMP-response element binding (CREB) protein-dependent induction of heme oxygenase-1 (HO1) and chronic nicotine exposure inhibits ERK/CREB signaling in renal proximal tubule cells (through p66shc). Herein, whether nicotine dampens simvastatin-dependent HO1 induction was determined. Renal proximal tubule (NRK52E) cells were pre-treated with 200 μM nicotine for 24 h followed by 10 μM simvastatin. Promoter activity of HO1 and manganese superoxide dismutase (MnSOD) and activation of CREB and ERK (via ELK1) were determined in luciferase reporter assays. CREB and p66shc were modulated via genetic means. Nicotine suppressed simvastatin-dependent activation of HO1 and MnSOD promoters and activity of CREB and ELK1 via p66shc. Overexpression of CREB or knockdown of p66shc restored simvastatin-dependent induction of HO1 and MnSOD in the presence of nicotine. Antioxidant efficiency of simvastatin might be significantly lessened in smokers/E-cigarette users.

Identification of Secretory Leukoprotease Inhibitor As an Endogenous Negative Regulator in Allergic Effector Cells.

Mast cells, basophils, and eosinophils are central effectors in allergic inflammatory disorders. These cells secrete abundant serine proteases as well as chemical mediators and cytokines; however, the expression profiles and functions of their endogenous inhibitors remain elusive. We found that murine secretory leukoprotease inhibitor (SLPI) is expressed in basophils and eosinophils but in not in mast cells. SLPI-deficient (Slpi−/−) basophils produce more cytokines than wild-type mice after IgE stimulation. Although the deletion of SLPI in basophils did not affect the release of chemical mediators upon IgE stimulation, the enzymatic activity of the serine protease tryptase was increased in Slpi−/− basophils. Mice transferred with Slpi−/− basophils were highly sensitive to IgE-mediated chronic allergic inflammation. Eosinophils lacking SLPI showed greater interleukin-6 secretion and invasive activity upon lipopolysaccharide stimulation, and the expression of matrix metalloproteinase-9 by these eosinophils was increased without stimulation. The absence of SLPI increases JNK1 phosphorylation at the steady state, and augments the serine phosphorylation of JNK1-downstream ETS transcriptional factor Elk-1 in eosinophils upon stimulation. Of note, SLPI interacts with a scaffold protein, JNK-interacting protein 3 (JIP3), that constitutively binds to the cytoplasmic domain of toll-like receptor (TLR) 4, suggesting that SLPI controls Elk-1 activation via binding to JIP3 in eosinophils. Mice transferred with Slpi−/− eosinophils showed the exacerbation of chitin-induced allergic inflammation. These findings showed that SLPI is a negative regulator in allergic effector cells and suggested a novel inhibitory role of SLPI in the TLR4 signaling pathways.

MAP kinase p38 is a novel target of CacyBP/SIP phosphatase

Mitogen-activated protein (MAP) kinases are important players in cellular signaling pathways. Recently, it has been shown that CacyBP/SIP serves as a phosphatase for one of the MAP kinases, ERK1/2. Through dephosphorylation of this kinase CacyBP/SIP modulates the transcriptional activity of Elk-1 and the activity of the CREB-BDNF pathway. In this work, using NB2a cell lysate and recombinant proteins, we show that CacyBP/SIP binds and dephosphorylates another member of the MAP kinase family, p38. Analysis of recombinant full-length CacyBP/SIP and its three major domains, N-terminal, middle CS and C-terminal SGS, indicates that the middle CS domain is responsible for p38 dephosphorylation. Moreover, we show that CacyBP/SIP might be implicated in response to oxidative stress. Dephosphorylation of phospho-p38 by CacyBP/SIP in NB2a cells treated with hydrogen peroxide is much more effective than in control ones. In conclusion, involvement of CacyBP/SIP in the regulation of p38 kinase activity, in addition to that of ERK1/2, might point to the function of CacyBP/SIP in pro-survival and pro-apoptotic pathways.

The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Abstract 2876: The androgen receptor utilizes protein kinase docking sites to constitutively activate Elk1

Abstract The ETS domain transcription factor Elk1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of Elk1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and Elk1 with that for activation of Elk1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with Elk1 and to activate Elk1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with Elk1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of Elk1. We identified two sites on Elk1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on Elk1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with Elk1 and is exclusive of the interaction of Elk1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites. Citation Format: Rayna Rosati, Mugdha Patki, Selvakumar Dakshnamurthy, Venkatesh Chari, Thomas McFall, Manohar Ratnam. The androgen receptor utilizes protein kinase docking sites to constitutively activate Elk1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2876.

Abstract 781: Modeling the impact of somatic alterations across human cancers

Abstract Large-scale cancer genomics projects like The Cancer Genome Atlas have generated a comprehensive catalog of somatic mutations and copy number aberrations across many tumor types, but the role of most frequently altered genes remains obscure. To better model the impact of these alterations, we developed a novel computational strategy for exploiting parallel phosphoproteomics and mRNA sequencing data for large tumor sets by linking dysregulation of upstream signaling pathways with altered transcriptional response through the transcriptional circuitry. Our modeling allows us to interpret the impact of somatic alterations in terms of functional outcomes such as altered signaling and transcription factor (TF) activity. We used this novel machine learning strategy to train phosphoprotein-TF interaction models across 12 human cancers for which large reverse-phase protein array and RNA-seq data sets are available through TCGA. First, we used this approach to identify shared and cancer-specific roles of TF/signaling regulators across cancer types. Then we performed a statistical analysis to associate frequent somatic aberrations with alterations in inferred TF and signaling protein activities. From our analysis, we gained many novel insights into cancer biology. We identified both known (e.g FOXO1 for breast cancer) and novel TF regulators of cancer (e.g. ELK1 for head and neck cancer). Many of these identified TF regulators were significantly associated with survival outcome in bladder urothelial, renal cell clear and endometrial carcinoma. Next we performed a comprehensive cross-cancer analysis and identified relationships between somatic alterations and downstream transcriptional effects and signaling pathway activation. We observed that specific molecular aberrations have different functional consequences in different cancer types. For example, PIK3CA mutations are associated with altered activities of a diverse set of TFs across cancers that are involved in cell cycle, apoptosis, metabolism and MAPK/ERK signaling. Notably, in cell line models, we validated some of the altered TFs predicted by our model. We showed that PIK3CA mutation leads to ELK1 activation in breast and head and neck cancer models. We further found that different set of TFs are associated with a specific mutation in different cancers due to the different background of genomic aberrations in each cancer. For example, KRAS mutations are associated with a distinct set of TFs depending of cancer-specific co-mutation profiles (e.g. co-mutations of KRAS and TP53 in lung adenocarcinoma and co-mutations of KRAS and APC in colorectal cancer). Our analysis revealed both known and novel interactions of frequently altered genes with signaling pathways and transcriptional programs in a pan-cancer context. Patterns of co-alterations across cancers may provide new insights relevant to targeted therapy and may be crucial to optimizing combination therapies. Citation Format: Hatice U. Osmanbeyoglu, Eneda Toska, José Baselga, Christina Leslie. Modeling the impact of somatic alterations across human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 781.

A Novel Small Molecule Modulator of Amyloid Pathology.

Because traditional approaches to drug development for Alzheimer's disease are becoming increasingly expensive and in many cases disappointingly unsuccessful, alternative approaches are required to shift the paradigm. Following leads from investigations of dihydropyridine calcium channel blockers, we observed unique properties from a class of functionalized naphthyridines and sought to develop these as novel therapeutics that minimize amyloid pathology without the adverse effects associated with current therapeutics. Our data show methyl 2,4-dimethyl-5-oxo-5,6-dihydrobenzo[c][2,7]naphthyridine-1-carboxylate (BNC-1) significantly decreases amyloid burden in a well-established mouse model of amyloid pathology through a unique mechanism mediated by Elk-1, a transcriptional repressor of presenilin-1. Additionally, BNC-1 treatment leads to increased levels of synaptophysin and synapsin, markers of synaptic integrity, but does not adversely impact presenilin-2 or processing of Notch-1, thus avoiding negative off target effects associated with pan-gamma secretase inhibition. Overall, our data show BNC-1 significantly decreases amyloid burden and improves markers of synaptic integrity in a well-established mouse model of amyloid deposition by promoting phosphorylation and activation of Elk-1, a transcriptional repressor of presenilin-1 but not presenilin-2. These data suggest BNC-1 might be a novel, disease-modifying therapeutic that will alter the pathogenesis of Alzheimer's disease.

Silodosin inhibits prostate cancer cell growth via ELK1 inactivation and enhances the cytotoxic activity of gemcitabine

Biological significance of ELK1, a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation, in prostate cancer remains far from fully understood. In this study, we aim to investigate the role of ELK1 in tumor growth as well as the efficacy of a selective α1A-adrenergic blocker, silodosin, in ELK1 activity in prostate cancer cells. We first immunohistochemically determined the levels of phospho-ELK1 (p-ELK1) expression in radical prostatectomy specimens. We then assessed the effects of ELK1 knockdown via short hairpin RNA and silodosin on cell proliferation, migration, and invasion in prostate cancer lines. The levels of p-ELK1 expression were significantly higher in carcinoma than in benign (P < 0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (P = 0.002) as well as in HGPIN than in benign (P < 0.001). Kaplan-Meier and log-rank tests revealed that moderate-strong positivity of p-ELK1 in carcinomas tended to correlate with biochemical recurrence after radical prostatectomy (P = 0.098). In PC3 and DU145 expressing ELK1 (mRNA/protein) but no androgen receptor (AR), ELK1 silencing resulted in considerable decreases in the expression of c-fos as well as in cell migration/invasion and matrix metalloproteinase-2 expression, but not in cell viability. Silodosin treatment reduced the expression/activity of ELK1 in these cells as well as the viability of AR-positive LNCaP and C4-2 cells and the migration of both AR-positive and AR-negative cells, but not the viability of AR-negative or ELK1-negative cells. Interestingly, silodosin significantly increased sensitivity to gemcitabine, but not to cisplatin or docetaxel, even in AR-negative cells. ELK1 is likely to be activated in prostate cancer cells and promote tumor progression. Furthermore, silodosin that inactivates ELK1 in prostate cancer cells not only inhibits their growth but also enhances the cytotoxic activity of gemcitabine. Thus, ELK1 inhibition has the potential of being a therapeutic approach for prostate cancer.

Leucine-enkephalin promotes wound repair through the regulation of hemidesmosome dynamics and matrix metalloprotease

The skin responds to environmental stressors by coordinated actions of neuropeptides and their receptors. An endogenous peptide for δ-opioid receptor (DOPr), Leu-enkephalin (L-ENK), is expressed in the skin and its expression is altered in pathological conditions. Although the importance of DOPr is rapidly gaining recognition, the molecular mechanisms underlying its effects on wound healing are largely undefined. We show here that L-ENK induced activation of Erk, P90RSK, and Elk-1 and promoted the disruption of hemidesmosomes and the expression of matrix metalloprotease (MMP)-2 and MMP-9, important processes for wound healing. Treatment with Erk inhibitor blocked activation of P90RSK and Elk-1 and significantly blunted wound repair. Therefore, our results suggest that activation of Erk and its downstream effectors, P90RSK and Elk-1, are critical for DOPr-mediated skin homeostasis.

Grifolin directly targets ERK1/2 to epigenetically suppress cancer cell metastasis.

Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been reported by us and others to display potent antitumor effects. However, the molecular target of grifolin has not been identified and the underlying mechanism of action is not fully understood. Here, we report that the ERK1/2 protein kinases are direct molecular targets of grifolin. Molecular modeling, affinity chromatography and fluorescence quenching analyses showed that grifolin directly binds to ERK1/2. And in vitro and ex vivo kinase assay data further demonstrated that grifolin inhibited the kinase activities of ERK1/2. We found that grifolin suppressed adhesion, migration and invasion of high-metastatic cancer cells. The inhibitory effect of grifolin against tumor metastasis was further confirmed in a metastatic mouse model. We found that grifolin decreased phosphorylation of Elk1 at Ser383, and the protein as well as the mRNA level of DNMT1 was also down-regulated. By luciferase reporter and ChIP assay analyses, we confirmed that grifolin inhibited the transcription activity of Elk1 as well as its binding to the dnmt1 promoter region. Moreover, we report that significant increases in the mRNA levels of Timp2 and pten were induced by grifolin. Thus, our data suggest that grifolin exerts its anti-tumor activity by epigenetic reactivation of metastasis inhibitory-related genes through ERK1/2-Elk1-DNMT1 signaling. Grifolin may represent a promising therapeutic lead compound for intervention of cancer metastasis, and it may also be useful as an ERK1/2 kinase inhibitor as well as an epigenetic agent to further our understanding of DNMT1 function.