Mitogen-activated Protein/extracellular Signal-regulated Kinase Inhibitor Research Articles

Pharmacological postconditioning with Neuregulin-1 mimics the cardioprotective effects of ischaemic postconditioning via ErbB4-dependent activation of reperfusion injury salvage kinase pathway

BackgroundThe protective effect of Neuregulin-1 (NRG-1) on heart failure is well established. In this study, we assessed whether NRG-1 could protect the heart by mimicking the cardioprotective effects of ischaemic postconditioning (IP).MethodsWe used a myocardial reperfusion injury rat model in vivo to compare the cardioprotective effects of NRG-1(3 μg/kg, iv. at the onset of reperfusion) and IP. In Langendorff isolated heart perfusion experiments, we used the erythroblastic leukaemia viral oncogene homolog 4 (ErbB4) inhibitor AG1478, a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and a mitogen-activated protein/extracellular signal regulated kinase (MEK) inhibitor PD98059 to clarify whether the protective effects of NRG-1and IP depend on the NRG-1/ErbB4 signals and the reperfusion injury salvage kinase (RISK) pathway. Infarct size was detected by Evans blue and TTC. Apoptosis was detected by TUNEL assays. The expression of NRG-1/ErbB4 and downstream ERK1/2, AKT, AMPK and p70s6K were detected by western blotting. Hematoxylin/eosin (H&E) staining was used for histological analysis.ResultsWe found that NRG-1 and IP had similar effects on reducing myocardial infarct size and apoptosis in vivo. NRG-1 heart protein levels were upregulated in the IP group. Phosphorylation of AKT, ERK1/2 and ErbB4 were also increased in both the IP and NRG-1 groups. Furthermore, in Langendorff analyses, the ErbB4 inhibitor AG1478 suppressed the phosphorylation of ErbB4 and the RISK pathway and aggravated myocardial edema and fiber fracture, thereby inhibited the cardioprotective effects in both the IP and NRG-1 groups. For assessment of downstream signals, the PI3K inhibitor LY294002 and the MEK inhibitor PD98059 suppressed the phosphorylation of AKT and ERK1/2 respectively and abolished the cardioprotective effects induced by IP and NRG-1.ConclusionIn conclusion, both IP and NRG-1 could reduce infarct size and apoptosis through ErbB4-dependent activation of the RISK pathway in the same model; these results indicated the therapeutic potential of NRG-1 as a pharmacological postconditioning agent against myocardial reperfusion injury.

Brain Distribution of a Novel MEK Inhibitor E6201: Implications in the Treatment of Melanoma Brain Metastases.

Clinically meaningful efficacy in the treatment of brain tumors, including melanoma brain metastases (MBM), requires selection of a potent inhibitor against a suitable target, and adequate drug distribution to target sites in the brain. Deregulated constitutive signaling of mitogen-activated protein kinase (MAPK) pathway has been frequently observed in melanoma, and mitogen-activated protein/extracellular signal-regulated kinase (MEK) has been identified to be an important target. E6201 is a potent synthetic small-molecule MEK inhibitor. The purpose of this study was to evaluate brain distribution of E6201, and examine the impact of active efflux transport at the blood-brain barrier on the central nervous system (CNS) exposure of E6201. In vitro studies utilizing transfected Madin-Darby canine kidney II (MDCKII) cells indicate that E6201 is not a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp). In vivo studies also suggest a minimal involvement of P-gp and Bcrp in E6201's brain distribution. The total concentrations in brain were higher than in plasma, resulting in a brain-to-plasma AUC ratio (Kp) of 2.66 in wild-type mice. The brain distribution was modestly enhanced in Mdr1a/b-/-, Bcrp1-/-, and Mdr1a/b-/-Bcrp1-/- knockout mice. The nonspecific binding of E6201 was higher in brain compared with plasma. However, free-drug concentrations in brain following 40 mg/kg intravenous dose reach levels that exceed reported in vitro half-maximal inhibitory concentration (IC50) values, suggesting that E6201 may be efficacious in inhibiting MEK-driven brain tumors. The brain distribution characteristics of E6201 make it an attractive targeted agent for clinical testing in MBM, glioblastoma, and other CNS tumors that may be effectively targeted with inhibition of MEK signaling.

Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics.

Multiplexed isobaric tag based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of mitogen-activated protein/extracellular signal-regulated kinase (MEK) signaling in cancer and mitogen-activated protein kinase (MAPK)-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of tandem mass tag 10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ∼ 6000 proteins in each tissue, but significant protein-level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2 ) and quantified 10 562 phosphorylation events. Further analysis by phosphotyrosine peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of proline-x-serine-proline (PxSP) and serine-proline (SP) sites, consistent with extracellular-signal-regulated kinase (ERK) inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.

Abstract 744: Effective use of MDK/Midkine and MEK inhibitor to treat KRas mutated pulmonary adenocarcinoma cells

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide. Although a platinum-doublet regimen has become the standard of care, the benefit is modest and the 5-year survival rate has remained essentially unchanged over the past 3 decades. Recently, several molecular alterations have been defined as “driver mutations,“ such as mutations in epidermal growth factor receptor (EGFR), KRas, chimeric tyrosine kinases involving ALK, RET, ROS1. And molecular targeting therapies for lung adenocarcinoma, targeting mutant EGFR or ALK, RET and ROS1 fusions, limit non-tumor toxicity and extend survival time compared to the conventional chemotherapies. However, there is no effective molecularly targeted therapy for mutant KRas-driven lung adenocarcinoma, the most frequent type of lung adenocarcinoma in the Caucasian population. Therefore, specific therapies that target various lung tumor types are desperately needed. Midkine (MDK) is a heparin-binding growth factor that is highly expressed in many malignant tumors, including lung cancers. We have previously reported that a MDK inhibitor: iMDK suppresses non-small cell lung cancer expressing MDK in vitro and in vivo without harming normal cells. Importantly, iMDK inhibits the PI3 kinase / Akt pathway and induces apoptosis in MDK expressing non-small cell lung cancer cells. In the present study, we have investigated the combination effect of iMDK and a mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD0325901. The combination treatment of iMDK and PD0325901 more effectively suppressed cell viability and colony formation, increased caspase-3 activity and induced apoptosis than those of single use in H441 and H2009 pulmonary adenocarcinoma cells harboring a G12 KRas mutation. Here, we describe the efficacy of the use of iMDK and PD0325901 for patients with inoperable advanced lung cancer including pulmonary adenocarcinoma harboring KRas mutations. Citation Format: Takuya Fukazawa, Yutaka Maeda, Naomasa Ishida, Tomoki Yamatsuji, Munenori Takaoka, Minoru Haisa, Nagio Takigawa, Jeffery Whitsett, Yoshio Naomoto. Effective use of MDK/Midkine and MEK inhibitor to treat KRas mutated pulmonary adenocarcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 744. doi:10.1158/1538-7445.AM2014-744

Phosphoinositide 3-Kinase (PI3K) Pathway Alterations Are Associated with Histologic Subtypes and Are Predictive of Sensitivity to PI3K Inhibitors in Lung Cancer Preclinical Models

Class 1 phosphatidylinositol 3-kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here, we investigated biomarker strategies for PI3K pathway inhibitors in non-small-cell lung cancer (NSCLC). Molecular profiling for candidate PI3K predictive biomarkers was conducted on a collection of NSCLC tumor samples. Assays included comparative genomic hybridization, reverse-transcription polymerase chain reaction gene expression, mutation detection for PIK3CA and other oncogenes, PTEN immunohistochemistry, and FISH for PIK3CA copy number. In addition, a panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers. PIK3CA amplification was detected in 37% of squamous tumors and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and 0% of adenocarcinomas. Total loss of PTEN immunostaining was found in 21% of squamous tumors and 4% of adenocarcinomas. Cell lines harboring pathway alterations (receptor tyrosine kinase activation, PI3K mutation or amplification, and PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a mitogen-activated protein-extracellular signal-regulated kinase inhibitor had greater effects on cell viability than PI3K inhibition alone. Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with nonsquamous NSCLC patient populations.

Activation of MAP Kinase in Lumbar Spinothalamic Cells Is Required for Ejaculation

Ejaculation is a reflex controlled by a spinal ejaculation generator located in the lumbosacral spinal cord responsible for the coordination of genital sensory with autonomic and motor outputs that regulate ejaculation. In the male rat, a population of lumbar spinothalamic cells (LSt cells) comprises an essential component of the spinal ejaculation generator. LSt cells are activated with ejaculation, but the nature of the signal transduction pathways involved in this activation is unknown. Moreover, it is unknown if LSt cell activation is required for expression of ejaculation. The current study tested the hypothesis that ejaculatory reflexes are triggered via activation of the mitogen-activated protein (MAP) kinase signaling pathway in the LSt cells. Expression of phosphorylated extracellular signal-related kinases 1 and 2 (pERK) was investigated following mating behavior, or following ejaculation induced by electrical stimulation of the dorsal penile nerve (DPN) in anesthetized, spinalized male rats. Next, the effects of intrathecal or intraspinal delivery of Mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor U0126 on DPN stimulation-induced ejaculation was examined. Expression of pERK in LSt cells and associated areas was analyzed. Electromyographic recordings of the bulbocavernosus muscle were recorded in anesthetized, spinalized rats. Results indicate that the MAP kinase signaling pathway is activated in LSt cells following ejaculation in mating animals or induced by DPN stimulation in anesthetized, spinalized animals. Moreover, ERK activation in LSt cells is an essential trigger for ejaculation, as DPN stimulation-induced reflexes were absent following administration of MEK inhibitor in the L3-L4 spinal area. These data provide insight into the nature of the signal transduction pathways involved in the activation of ejaculation through LSt cells. The data demonstrate that ERK activation in LSt cells is essential for ejaculation and contribute to a more detailed understanding of the spinal generation of ejaculation.

Targeting Protein Translation in Human Non–Small Cell Lung Cancer via Combined MEK and Mammalian Target of Rapamycin Suppression

Lung cancer is a genetically heterogeneous disease characterized by the acquisition of somatic mutations in numerous protein kinases, including components of the rat sarcoma viral oncogene homolog (RAS) and AKT signaling cascades. These pathways intersect at various points, rendering this network highly redundant and suggesting that combined mitogen-activated protein/extracellular signal-regulated kinase (MEK) and mammalian target of rapamycin (mTOR) inhibition may be a promising drug combination that can overcome its intrinsic plasticity. The MEK inhibitors, CI-1040 or PD0325901, in combination with the mTOR inhibitor, rapamycin, or its analogue AP23573, exhibited dose-dependent synergism in human lung cancer cell lines that was associated with suppression of proliferation rather than enhancement of cell death. Concurrent suppression of MEK and mTOR inhibited ribosomal biogenesis by 40% within 24 h and was associated with a decreased polysome/monosome ratio that is indicative of reduced protein translation efficiency. Furthermore, the combination of PD0325901 and rapamycin was significantly superior to either drug alone or PD0325901 at the maximum tolerated dose in nude mice bearing human lung tumor xenografts or heterotransplants. Except for a PTEN mutant, all tumor models had sustained tumor regressions and minimal toxicity. These data (a) provide evidence that both pathways converge on factors that regulate translation initiation and (b) support therapeutic strategies in lung cancer that simultaneously suppress the RAS and AKT signaling network.

A citrus polymethoxyflavonoid, nobiletin, is a novel MEK inhibitor that exhibits antitumor metastasis in human fibrosarcoma HT-1080 cells

The activation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) is well known to be associated with tumor invasion and metastasis. We previously reported that a polymethoxyflavonoid, nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), derived from Citrus depressa (Hayata), inhibits the phosphorylation of MEK and thereby suppresses matrix metalloproteinase (MMP) expression in a tumor-metastasis stimulator, 12-O-tetradecanoyl phorbol 13-acetate (TPA)-stimulated human fibrosarcoma HT-1080 cells [Mol. Cancer Ther. 3 (2004) 839–847]. In the present study, we investigated whether or not nobiletin might directly influence MEK activity to exhibit the antitumor metastatic activity in vitro. MEK kinase assay using myelin basic protein (MBP) revealed that TPA-augmented MEK activity in HT-1080 cells and that the augmented MEK activity was diminished by nobiletin treatment. In addition, the decrease in MEK activity caused by nobiletin was found to inhibit the phosphorylation of extracellular regulated kinases (ERK), a downstream signaling factor for MEK. Furthermore, when an immunoprecipitated active MEK was incubated with nobiletin under cell-free conditions, nobiletin was found to inhibit the MEK-mediated MBP phosphorylation. In contrast, other citrus polymethoxyflavonoids such as 3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone (natsudaidain) and 3,5,6,7,8,3′,4′-heptamethoxyflavone, did not directly inhibit MEK activity. Moreover, natsudaidain and 3,5,6,7,8,3′,4′-heptamethoxyflavone exhibited no or less inhibitory effect than nobiletin on the proMMP-9/progelatinase B production in HT-1080 cells. Therefore, these results provide novel evidence that nobiletin directly inhibits MEK activity and decreases the sequential phosphorylation of ERK, exhibiting the antitumor metastatic activity by suppressing MMP expression in HT-1080 cells.