Inhibition Of ERK Phosphorylation Research Articles

2-Deoxyglucose Suppresses ERK Phosphorylation in LKB1 and Ras Wild-Type Non-Small Cell Lung Cancer Cells.

Tumor cells rely on aerobic glycolysis to generate ATP, namely the "Warburg" effect. 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, but its effect on cellular signaling pathways has not been fully elucidated. Herein, we sought to investigate the effect of 2-DG on ERK function in lung cancer cells. We found that 2-DG inhibits ERK phosphorylation in a time and dose-dependent manner in lung cancer cells. This inhibition requires functional LKB1. LKB1 knockdown in LKB1 wildtype cells correlated with an increase in the basal level of p-ERK. Restoration of LKB1 in LKB1-null cells significantly inhibits ERK activation. Blocking AMPK function with AMPK inhibitor, AMPK siRNA or DN-AMPK diminishes the inhibitory effect of 2-DG on ERK, suggesting that 2-DG—induced ERK inhibition is mediated by LKB1/AMPK signaling. Moreover, IGF1-induced ERK phosphorylation is significantly decreased by 2-DG. Conversely, a subset of oncogenic mutants of K-Ras, the main upstream regulator of ERK, blocks 2-DG—induced LKB1/AMPK signaling. These findings reveal the potential cross-talk between LKB1/AMPK and ERK signaling and help to better understand the mechanism of action of 2-DG.

Sprouty2 correlates with favorable prognosis of gastric adenocarcinoma via suppressing FGFR2-induced ERK phosphorylation and cancer progression.

Fibroblast growth factor receptor 2 (FGFR2) has been identified as a predictive biomarker for unfavorable prognosis of gastric adenocarcinoma. As a well-defined antagonist in FGFR2-induced RAS/ERK activation, ectopic expression of sprouty (SPRY) family was reported in several kinds of cancers except gastric cancer. To explore the clinical significance of SPRY family and its correlation with FGFR2, we detected the expression of FGFR2 and SPRY family in 104 cases of gastric adenocarcinoma and subsequently analyzed their correlations with clinicopathological factors and overall survival rates by univariate and multivariate analysis. As the result, we demonstrated that both FGFR2 high-expression and SPRY2 low-expression indicated poorer prognosis of gastric adenocarcinoma. SPRY2 low-expression was significantly associated with FGFR2 high-expression, positive lymphatic invasion and metastasis. We further proved that SPRY2 could suppress FGFR2-induced ERK phosphorylation, cell proliferation and invasion with experiments in vitro and in vivo. In conclusion, we demonstrated that SPRY2 low-expression is a biomarker for unfavorable prognosis in gastric adenocarcinoma. SPRY2 can antagonize FGFR2-induced proliferation and invasion via suppressing ERK phosphorylation in gastric cancer cells, indicating SPRY2 as a potential therapeutic target for gastric adenocarcinoma treatment.

Ibrutinib Abrogates TREM-1 Mediated Neutrophil Activation

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor on neutrophils (PMN) and important in the innate host defence against microbial pathogens. Here we examined the influence of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib on TREM-1 dependent activation of human PMNs. Firstly, ibrutinib specifically inhibited TREM-1 mediated PMN activation of the oxidative burst and CD62L shedding, whereas TLR mediated activation remained unaffected. Correspondingly, ibrutinib suppressed ERK phosphorylation after TREM-1, but not after TLR ligation. To clarify whether this TREM-1 specific effect of ibrutinib was also relevant in vivo, we treated mice with ibrutinib, and analyzed fungal clearance in vivo upon pulmonary challenge with A. fumigatus and PMN activation ex vivo upon TREM-1 or TLR ligation, again finding that TREM-1, but not TLR induced activation was suppressed. Equivalent results were obtained in PMNs of six lymphoma patients under treatment with ibrutinib. In summary, BTK plays an important role in TREM-1 mediated PMN activation, providing a potential mechanism for the frequently observed infectious complications in patients treated with ibrutinib, and fosters studies using this drug as a novel tool to modulate TREM-1 activity for therapeutic purposes, i. e. in sepsis or autoimmune diseases. DisclosuresHess:Pfizer: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding; Roche: Honoraria; Novartis: Honoraria.

Stanniocalcin 2 improved osteoblast differentiation via phosphorylation of ERK.

Mammalian stanniocalcin 2 (STC2) is a glycoprotein hormone with multiple functions. The present study determined the importance of STC2 in osteoblast differentiation. It was revealed that the expression of STC2 was increased during the differentiation of MC3T3-E1 cells to osteoblasts and that knockdown of STC2 reduced osteoblast differentiation and mineralization, whereas STC2 overexpression increased differentiation and mineralization. Knockdown of STC2 downregulated the expression of osteoblast-associated genes, including runt‑related transcription factor2, collagen typeIα1chain, osterix and osteocalcin. Overexpression of STC2 upregulated the expression of these osteoblastic genes. In addition, overexpression of STC2 enhanced the phosphorylation of extracellular signal‑regulated kinase1/2 (ERK1/2), whereas inhibition of ERK phosphorylation reduced osteoblast differentiation of MC3T3‑E1 cells overexpressing STC2. These findings indicated that STC2 may promote osteoblast differentiation and mineralization by regulating ERK activation.

Extrasynaptic NMDA receptor-induced tau overexpression mediates neuronal death through suppressing survival signaling ERK phosphorylation.

Intracellular accumulation of the hyperphosphorylated tau is a pathological hallmark in the brain of Alzheimer disease. Activation of extrasynaptic NMDA receptors (E-NMDARs) induces excitatory toxicity that is involved in Alzheimer's neurodegeneration. However, the intrinsic link between E-NMDARs and the tau-induced neuronal damage remains elusive. In the present study, we showed in cultured primary cortical neurons that activation of E-NMDA receptors but not synaptic NMDA receptors dramatically increased tau mRNA and protein levels, with a simultaneous neuronal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDARs, reversed E-NMDARs-induced tau overexpression. Activation of E-NMDARs in wild-type mouse brains resulted in neuron loss in hippocampus, whereas tau deletion in neuronal cultures and in the mouse brains rescued the E-NMDARs-induced neuronal death and degeneration. The E-NMDARs-induced tau overexpression was correlated with a reduced ERK phosphorylation, whereas the increased MEK activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation in vitro. Taking together, these results indicate that tau overexpression mediates the excitatory toxicity induced by E-NMDAR activation through inhibiting ERK phosphorylation.

Casearin D inhibits ERK phosphorylation and induces downregulation of cyclin D1 in HepG2 cells

Cancer is a public health problem which represents the second cause of death in the world. In this framework, it is necessary to identify novel compounds with antineoplastic potential. Plants are an important source for discovering novel compounds with pharmacological potential. In this study, we aimed to investigate the antiproliferative potential of isolated compounds from Casearia sylvestris on tumor cell lines. Crude extract effectively reduced cell viability of 4 tumor cell lines (HepG2, A549, U251-MG, and HT-144) after 48h treatment. HepG2 and HT-144 were the most responsive cells. Three fractions (aqueous ethanol, n-hexane and ethyl acetate) were tested against HepG2 and HT-144 cells and we observed that compounds with antiproliferative activity were concentrated in n-hexane and ethyl acetate fractions. The casearins A, G and J were isolated from n-hexane fraction, while casearin D was obtained from ethyl acetate fraction. We demonstrated that casearin D significantly inhibited the clonogenic capacity of HepG2 cells after 24h exposure indicating its antiproliferative activity. In addition, G1/S transition cell cycle arrest in HepG2 cells was also observed. These effects are related, at least in part, to ability of the casearin D in reducing ERK phosphorylation and cyclin D1 expression levels.

5α-Androstane-3α,17β-Diol Inhibits Neurotoxicity in SH-SY5Y Human Neuroblastoma Cells and Mouse Primary Cortical Neurons.

Low free T levels in men are associated with age-related cognitive decline and increased risk for neurotoxicity, resulting in disease. The mechanisms underlying these observations remain poorly defined. Although rapid, androgen receptor-dependent activation of ERK has been postulated as a neurotrophic and neuroprotective mechanism, actions of T metabolites such as 5α-androstane-3α,17β-diol (3α-diol) may also be involved. We investigated the influence of 3α-diol on the induction of ERK phosphorylation in SH-SY5Y human female neuroblastoma cells and primary cortical neurons from male and female mice. In SH-SY5Y cells, ERK phosphorylation was induced by 10 nM DHT, epidermal growth factor, hydrogen peroxide (H2O2), and acetylcholine. The addition of 10 nM 3α-diol, which did not itself activate ERK, significantly inhibited ERK phosphorylation induced by DHT, epidermal growth factor, or H2O2, but not acetylcholine. In both SH-SY5Y cells and primary cortical neurons, prolonged ERK phosphorylation and caspase-3 cleavage resulting from amyloid β-peptide 1-42 (Aβ42) exposure were inhibited by cotreatment with 3α-diol. 3α-diol also reduced the loss in cellular viability induced by Aβ42 or H2O2 in SH-SY5Y cells. These data suggest that T-mediated neuroprotection may occur via two distinct but complementary mechanisms: an initial rapid activation of ERK phosphorylation, followed by modulation via 3α-diol of the potentially adverse consequences of prolonged ERK activation.

Smooth muscle cell-specific Tgfbr1 deficiency promotes aortic aneurysm formation by stimulating multiple signaling events.

Transforming growth factor (TGF)-β signaling disorder has emerged as a common molecular signature for aortic aneurysm development. The timing of postnatal maturation plays a key role in dictating the biological outcome of TGF-β signaling disorders in the aortic wall. In this study, we investigated the impact of deficiency of TGFβ receptors on the structural homeostasis of mature aortas. We used an inducible Cre-loxP system driven by a Myh11 promoter to delete Tgfbr1, Tgfbr2, or both in smooth muscle cells (SMCs) of adult mice. TGFBR1 deficiency resulted in rapid and severe aneurysmal degeneration, with 100% penetrance of ascending thoracic aortas, whereas TGFBR2 deletion only caused mild aortic pathology with low (26%) lesion prevalence. Removal of TGFBR2 attenuated the aortic pathology caused by TGFBR1 deletion and correlated with a reduction of early ERK phosphorylation. In addition, the production of angiotensin (Ang)-converting enzyme was upregulated in TGFBR1 deficient aortas at the early stage of aneurysmal degeneration. Inhibition of ERK phosphorylation or blockade of AngII type I receptor AT1R prevented aneurysmal degeneration of TGFBR1 deficient aortas. In conclusion, loss of SMC-Tgfbr1 triggers multiple deleterious pathways, including abnormal TGFBR2, ERK, and AngII/AT1R signals that disrupt aortic wall homeostasis to cause aortic aneurysm formation.

A standardized bark extract of Pinus pinaster Aiton (Pycnogenol®) attenuated chronic obstructive pulmonary disease via Erk-sp1 signaling pathway

Ethnopharmacological relevanceA standardized bark extract of Pinus pinaster Aiton (Pycnogenol®; PYC) used as an herbal medicine to treat various diseases in Europe and North America. Aim of the studyThis study evaluates the ability of PYC to inhibit chronic obstructive pulmonary disease (COPD) in the cigarette smoke extract (CSE)-stimulated human airway epithelial cell line NCI-H292 and in a cigarette smoke (CS) and lipopolysaccharide (LPS)-induced mouse model. MethodsTo induce COPD, the mice intranasally received LPS on day 4 and were exposed to CS for 1h per day (total eight cigarettes per day) from days 1–7. The mice were administered PYC at a dose of 15mg/kg and 30mg/kg 1h before CS exposure. ResultsIn the CSE-stimulated NCI-H292 cells, PYC significantly inhibited Erk phosphorylation, sp1 expression, MUC5AC, and pro-inflammatory cytokines in a concentration-dependent manner, as evidenced by a reduction in their mRNA levels. Co-treatment with PYC and Erk inhibitors markedly reduced the levels inflammatory mediators compared to only PYC-treatment. In the COPD mice model, PYC decreased the inflammatory cell count and the levels of pro-inflammatory cytokines in the broncho-alveolar lavage fluid compared with COPD mice. PYC attenuated the recruitment of inflammatory cells in the airways and decreased the expression levels of Erk phosphorylation and sp1. PYC also inhibited the expression of myeloperoxidase and matrix metalloproteinases-9 in lung tissue. ConclusionOur results indicate that PYC inhibited the reduction in the inflammatory response in CSE-stimulated NCI-H292 cells and the COPD mouse model via the Erk-sp1 pathway. Therefore, we suggest that PYC has the potential to treat COPD.

The reduction of DSG1 in hereditary palmo-planter keratoderma causes the impaired differentiation and proliferation of sole epidermis

Striate palmo-planter keratoderma (sPPK) is a rare hereditary keratoderma characterized by linear or focal hyperkeratosis on physically stressed palm or sole skin, and caused by mutation of desmoglein1 (DSG1) or desmoplakin (DSP) gene. Here, we report a Japanese family with sPPK who presented linear or focal thickening of palm and sole skin, respectively. Genomic analyses of DSG1 of each affected kindred disclosed a single nucleotide deletion c.489delA in one allele, which leads to the truncation at the extracellular domain of DSG1. Histologically, the slightly widen intercellular spaces at spinous layer, the detachment at the tri-cellular junction at the granular layer and the characteristic marked hyperkeratosis of cornified layer were observed without any acantholytic phenotypes. In lesional skin, the expressions of keratins and related proteins vary alternately in accordant with sulci and cristae cutis in a stripe-like pattern. The K9, K2e and loricrin were particularly expressed in cristae cutis, whereas K14, K16, involucrin and DSG3 were more obvious in sulci cutis. The basal cell proliferation proved by ki67 expression is primarily restricted in cristae but not in sulci cutis. Recently, DSG1 is also suggested to govern the intracellular signals and to induce epidermal differentiation by inhibiting ERK phosphorylation through Erbin. Indeed, in our case, the expression of phosphorylated ERK (pERK) was decreased in cristae cutis in contrast to the increase in sulci cutis. These observations suggest the presence of downward signals through DGS1 to the activation of ERK in sulci cutis of basal layer under physiological stress. Consequently, sole skin may have the vertical stripe like differentiation in addition to the general horizontal differentiation.

A phase I dose-escalation study of TAK-733, an investigational oral MEK inhibitor, in patients with advanced solid tumors

SummaryPurpose TAK-733, an investigational, selective, allosteric MEK1/2 inhibitor, has demonstrated antitumor effects against multiple cancer cell lines and xenograft models. This first-in-human study investigated TAK-733 in patients with solid tumors. Methods Patients received oral TAK-733 once daily on days 1–21 in 28-day treatment cycles. Adverse events (AEs) were graded using the Common Terminology Criteria for AEs version 3.0. Response was assessed using RECIST v1.1. Blood samples for TAK-733 pharmacokinetics and pharmacodynamics (inhibition of ERK phosphorylation) were collected during cycle 1. Results Fifty-one patients received TAK-733 0.2–22 mg. Primary diagnoses included uveal melanoma (24 %), colon cancer (22 %), and cutaneous melanoma (10 %). Four patients had dose-limiting toxicities of dermatitis acneiform, plus fatigue and pustular rash in one patient, and stomatitis in one patient. The maximum tolerated dose was 16 mg. Common drug-related AEs included dermatitis acneiform (51 %), diarrhea (29 %), and increased blood creatine phosphokinase (20 %); grade ≥ 3 AEs were reported in 27 (53 %) patients. Median Tmax was 3 h; systemic exposure increased less than dose-proportionally over the dose range 0.2–22 mg. On day 21 maximum inhibition of ERK phosphorylation in peripheral blood mononuclear cells of 46–97 % was seen in patients receiving TAK-733 ≥ 8.4 mg. Among 41 response-evaluable patients, 2 (5 %) patients with cutaneous melanoma (one with BRAF L597R mutant melanoma) had partial responses. Conclusions TAK-733 had a generally manageable toxicity profile up to the maximum tolerated dose, and showed the anticipated pharmacodynamic effect of sustained inhibition of ERK phosphorylation. Limited antitumor activity was demonstrated. Further investigation is not currently planned.

Sulfur Dioxide Inhibits Extracellular Signal-regulated Kinase Signaling to Attenuate Vascular Smooth Muscle Cell Proliferation in Angiotensin II-induced Hypertensive Mice.

Background:Clarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2 has not been fully elucidated. This study was designed to investigate if SO2 inhibited VSMC proliferation in mice with hypertension induced by AngII.Methods:Thirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2 groups. Mice in AngII group and AngII + SO2 group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg−1·min−1. In addition, mice in AngII + SO2 received intraperitoneal injections of SO2 donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting.Results:In animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P-ERK induced by AngII (P < 0.05). In cell experiment, PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2 on VSMC proliferation (P < 0.05).Conclusions:ERK signaling is involved in the mechanisms by which SO2 inhibits VSMC proliferation in AngII-induced hypertensive mice via ERK signaling.

Endothelial progenitor cell number and ERK phosphorylation serve as predictive and prognostic biomarkers in advanced hepatocellular carcinoma patients treated with sorafenib

ABSTRACTSorafenib is an oral anti-angiogenic multi-kinase inhibitor used for systemic therapy in patients with advanced hepatocellular carcinoma (HCC) who are not suitable candidates for surgery or liver transplantation. An earlier study conducted with HCC tumor tissue suggested that ERK phosphorylation (pERK), a downstream target of sorafenib, may serve as a potential biomarker for therapeutic efficacy of sorafenib. However, no study thus far has utilized a minimal invasive procedure to predict HCC patient responsiveness to sorafenib. We evaluated the biomarker utility of circulating endothelial progenitor cells (EPCs) frequency and intracellular pERK levels in EPCs in peripheral blood obtained pre- and post-sorafenib therapy or after transarterial chemoembolistaion (TACE). A statistically significant reduction in the level of ERK phosphorylation and in the absolute number of EPCs was detected following in vivo sorafenib treatment (p < 0 .01 for both). In contrast, the decrease in the level of ERK phosphorylation and EPC number was either marginally significant or insignificant in patients treated with TACE (p = 0.05 and 0.06, respectively). In vitro sorafenib treatment of pre- and post-samples from the same patient cohort inhibited ERK phosphorylation levels in EPCs and decreased the number of EPCs at all doses tested (p = 0.01). Our findings support that the evaluation of both the circulating EPC frequency and the level of ERK phosphorylation in EPCs may serve as potential non-invasive biomarkers of sorafenib efficacy, both as predictor of treatment outcome and efficacy during drug treatment.

Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling.

Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation.

Role of WW Domain-containing Oxidoreductase WWOX in Driving T Cell Acute Lymphoblastic Leukemia Maturation

Whether tumor suppressor WWOX (WW domain-containing oxidoreductase) stimulates immune cell maturation is largely unknown. Here, we determined that Tyr-33-phosphorylated WWOX physically binds non-phosphorylated ERK and IκBα in immature acute lymphoblastic leukemia MOLT-4 T cells and in the naïve mouse spleen. The IκBα·ERK·WWOX complex was shown to localize, in part, in the mitochondria. WWOX prevents IκBα from proteasomal degradation. Upon stimulating MOLT-4 with ionophore A23187/phorbol myristate acetate, endogenous IκBα and ERK undergo rapid phosphorylation in <5 min, and subsequently WWOX is Tyr-33 and Tyr-287 de-phosphorylated and Ser-14 phosphorylated. Three hours later, IκBα starts to degrade, and ERK returns to basal or non-phosphorylation, and this lasts for the next 12 h. Finally, expression of CD3 and CD8 occurs in MOLT-4 along with reappearance of the IκBα·ERK·WWOX complex near 24 h. Inhibition of ERK phosphorylation by U0126 or IκBα degradation by MG132 prevents MOLT-4 maturation. By time-lapse FRET microscopy, IκBα·ERK·WWOX complex exhibits an increased binding strength by 1-2-fold after exposure to ionophore A23187/phorbol myristate acetate for 15-24 h. Meanwhile, a portion of ERK and WWOX relocates to the nucleus, suggesting their role in the induction of CD3 and CD8 expression in MOLT-4.

CD271 regulates the proliferation and motility of hypopharyngeal cancer cells.

CD271 (p75 neurotrophin receptor) plays both positive and negative roles in cancer development, depending on the cell type. We previously reported that CD271 is a marker for tumor initiation and is correlated with a poor prognosis in human hypopharyngeal cancer (HPC). To clarify the role of CD271 in HPC, we established HPC cell lines and knocked down the CD271 expression using siRNA. We found that CD271-knockdown completely suppressed the cells’ tumor-forming capability both in vivo and in vitro. CD271-knockdown also induced cell-cycle arrest in G0 and suppressed ERK phosphorylation. While treatment with an ERK inhibitor only partially inhibited cell growth, CDKN1C, which is required for maintenance of quiescence, was strongly upregulated in CD271-depleted HPC cells, and the double knockdown of CD271 and CDKN1C partially rescued the cells from G0 arrest. In addition, either CD271 depletion or the inhibition of CD271-RhoA signaling by TAT-Pep5 diminished the in vitro migration capability of the HPC cells. Collectively, CD271 initiates tumor formation by increasing the cell proliferation capacity through CDKN1C suppression and ERK-signaling activation, and by accelerating the migration signaling pathway in HPC.

A role of melatonin for occurrence of chronic obstructive pulmonary disease

A growth in prevalence of chronic obstructive pulmonary disease (COPD) is mainly related to continuing exposure of risk factors. COPD is a major and growing global health problem with great economic burden. Therefore, there is an urgent need to study molecular mechanisms of pathogenesis of COPD and to develop a new treatment strategy. Recent studies have revealed geroprotectors which could discover new therapeutic possibilities for COPD. Thus, use of melatonin could be an effective approach to treatment of COPD which could prevent the premature aging of the lungs. Melatonin is a neurohormone which regulates the human’s biological rhythm, metabolism, and immuno"neuroendocrine processes. Melatonin is produced mainly at night. At the same time, there is the peak activity of the immune system. One of the important functions of melatonin is its antioxidant activity. Oxidative stress occurs during acute exacerbations of asthma and COPD, whereas leukocyte number, and antioxidant enzymes and melatonin levels decrease. Exogenous melatonin could improve the oxidative stress and dyspnea and cause 1.6"fold reduction in 8"isoprostane concentration in COPD patients. Melatonin could also inhibit ERK phosphorylation and expression of Sp1 and MUC5AC due to inhibition of MAPK signaling pathway phosphorylation. Melatonin could improve sleep quality in COPD patients with concomitant sleep disorders. Furthermore, melatonin does not cause daytime sleepiness and respiratory disorders compared to conventional hypnotics which may worsen nighttime hypoxemia and respiratory failure in severe cases. Obviously, melatonin could be a potential agent for treatment of chronic airway diseases such as asthma and COPD. However, further long"term large"scale studies are needed to develop recommendations for safe use of melatonin in patients with COPD and sleep disorders.

Abstract 1247: ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant cancers

Abstract The clinical benefit of targeting the MAPK pathway is demonstrated by the success of RAF and MEK inhibitors in the treatment of melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms that result in reactivation of ERK signalling. Additionally, many BRAF- and KRAS-mutant cancers exhibit de novo resistance. As the direct targeting of ERK may overcome the limitations of RAF or MEK inhibitors, there are several ERK1/2 inhibitors in development. SCH772984 (SCH, Merck-Schering) is an example of an ERK1/2 inhibitor that in addition to inhibiting ERK catalytic activity also inhibits the phosphorylation of residues within the ERK activation loop. Several ERK1/2 inhibitors have been described that inhibit ERK catalytic activity without modulating ERK phosphorylation e.g GDC-0994 (GDC, Genentech). Here we aim to characterise the biological relevance of the different modes of ERK inhibition. We investigated the biochemical mechanism by which SCH inhibits ERK phosphorylation and demonstrated that it prevents the phosphorylation of ERK by MEK (T202/Y204), but does not affect MEK activity. We characterised the effects of the distinct modes of ERK inhibition on ERK substrates, cellular pERK levels, pathway feedback activation and sensitivities of cell lines with different genetic backgrounds. In BRAFV600E-mutant cells e.g A375 (melanoma) and Colo205 (colorectal), 16 hr treatment with SCH and GDC significantly inhibited the phosphorylation of RSK (ERK substrate). SCH significantly inhibited ERK phosphorylation, whereas GDC did not modulate pERK levels. Neither compound induced pathway feedback activation. In proliferation assays A375 and Colo205 cells had remarkably similar sensitivities to SCH (EC50 40 nM and 30 nM, respectively) and GDC (EC50 = 140 nM in both). In contrast to BRAF-mutant cells, SCH and GDC treatment conferred pMEK induction in the KRAS-mutant cell line HCT116 (while still significantly inhibiting RSK phosphorylation). This is likely to be attributable to the feedback-driven C-RAF activation commonly associated with MAPK pathway inhibition in KRAS-mutant cells. In addition to pMEK, GDC also conferred an induction of pERK. However, SCH overcame pathway feedback and inhibited the ERK phosphorylation. The inhibition of pathway feedback conferred by SCH was associated with increased sensitivity of HCT116 cells to SCH (EC50 = 30 nM) compared to GDC (EC50 = 1.1 μM). In a cell panel screen comprising 25 BRAF-mutant and 63 KRAS-mutant cell lines, SCH and GDC had similar activity in BRAF-mutant cell lines. However, although KRAS-mutant cell lines were sensitive to SCH, they were largely resistant to GDC. These data suggest that due to their ability to overcome MAPK inhibitor-induced pathway feedback, ERK inhibitors that also inhibit the phosphorylation of ERK would be more effective in KRAS-mutant cancer than those that inhibit ERK activity alone. Citation Format: Joanne M. Munck, Brent Graham, Juan Castro, Aurelie Courtin, Sandra Muench, Sharna Rich, Harpreet K. Saini, Jessica Brothwood, John Lyons, Neil T. Thompson, Nicola G. Wallis, Chris Murray. ERK1/2 inhibitors with distinct modes of inhibition confer different response profiles in KRAS mutant 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 1247.

Abstract 2324: FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells

Abstract BACKGROUND: Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. KRAS mutations are known to activate the RAF-MEK-ERK pathway. Fos-related antigen-1 (FRA1) is a MEK/ERK-dependent transcription factor and member of the AP-1 transcription factor superfamily. Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in immune-mediated tumor evasion. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation and FRA1 may be an important transcriptional mediator. METHODS: In order to assess the role of KRAS mutation independent of other somatic mutations and to begin to understand potential immune suppressive pathways operative in pulmonary premalignancy, premalignant human bronchial epithelial cell lines (HBEC) were used instead of human lung cancer cell lines. Four immortalized HBEC (2, 3, 7, and 11 cell lines) with KRAS v12-mutation (HBEC-KRAS) compared to control (HBEC-vector) were used to assess mRNA and protein expression levels of PD-L1 by RT-qPCR, flow cytometry, and western blot. Cell-lines were treated with MEK (ERK kinase) inhibitor (PD0325901) for 24 hours (h) up to 1μM. FRA1 was silenced by transfection with siRNA at 100nM. RESULTS: In comparing HBEC-KRAS to HBEC-vector (wild-type) cells, PD-L1 mRNA (1.3∼3.4 fold) and surface protein expressions (1.2∼2.3 fold by flow cytometry) were increased in all 4 cell lines and confirmed by western blot analyses. PD-L1 mRNA and protein levels were highest in HBEC3-KRAS. MEK inhibition resulted in decreased PD-L1 expression by 10 fold (HBEC3-vector) and 11 fold (HBEC3-KRAS) in mRNA levels and 3 fold (HBEC3-vector and HBEC3-KRAS) in surface protein levels. In comparing HBEC3-KRAS to HBEC3-vector, FRA1 increased mRNA (2.7 fold) and protein level expression (2.8 fold). In comparing HBEC3-KRAS to HBEC3-vector cells, FRA1 silencing (FRA1 siRNA) resulted in reduced PD-L1 (53% at 48 h; 73% at 72 h) and FRA1 (94% at 48 h; 99% at 72 h) by western blot. In comparing HBEC3-KRAS to HBEC3-vector cells, ERK phosphorylation increased 7.9 fold and FRA1 silencing led to inhibition of ERK phosphorylation by 2.8 fold at 72 h by western blot. CONCLUSIONS: Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates KRAS mutation driven up regulation of PD-L1 and at least in part through FRA1 dependence. Our data suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway through FRA1 and MEK/ERK-dependent transcriptional regulation. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential new combination immune therapies. Citation Format: Mi-Heon Lee, Jane Yanagawa, Tonya C Walser, Jonathan W. Goldman, Edward B. Garon, Gang Zeng, Sherven Sharma, Boning Gao, John Minna, Steven M. Dubinett, Jay M Lee. FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells. [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 2324.

Nifurtimox Is Effective Against Neural Tumor Cells and Is Synergistic with Buthionine Sulfoximine

Children with aggressive neural tumors have poor survival rates and novel therapies are needed. Previous studies have identified nifurtimox and buthionine sulfoximine (BSO) as effective agents in children with neuroblastoma and medulloblastoma. We hypothesized that nifurtimox would be effective against other neural tumor cells and would be synergistic with BSO. We determined neural tumor cell viability before and after treatment with nifurtimox using MTT assays. Assays for DNA ladder formation and poly-ADP ribose polymerase (PARP) cleavage were performed to measure the induction of apoptosis after nifurtimox treatment. Inhibition of intracellular signaling was measured by Western blot analysis of treated and untreated cells. Tumor cells were then treated with combinations of nifurtimox and BSO and evaluated for viability using MTT assays. All neural tumor cell lines were sensitive to nifurtimox, and IC50 values ranged from approximately 20 to 210 μM. Nifurtimox treatment inhibited ERK phosphorylation and induced apoptosis in tumor cells. Furthermore, the combination of nifurtimox and BSO demonstrated significant synergistic efficacy in all tested cell lines. Additional preclinical and clinical studies of the combination of nifurtimox and BSO in patients with neural tumors are warranted.