ERK Mitogen-activated Protein Kinase Cascade Research Articles

Targeting the ERK mitogen-activated protein kinase cascade for the treatment of KRAS-mutant pancreatic cancer.

Mutational activation of the KRAS oncogene is found in ~95% of pancreatic ductal adenocarcinoma (PDAC), the major form of pancreatic cancer. With substantial experimental evidence that continued aberrant KRAS function is essential for the maintenance of PDAC tumorigenic growth, the National Cancer Institute has identified the development of effective anti-KRAS therapies as one of four major initiatives for pancreatic cancer research. The recent clinical success in the development of an anti-KRAS therapy targeting one specific KRAS mutant (G12C) supports the significant potential impact of anti-KRAS therapies. However, KRASG12C mutations comprise only 2% of KRAS mutations in PDAC. Thus, there remains a dire need for additional therapeutic approaches for targeting the majority of KRAS-mutant PDAC. Among the different directions currently being pursued for anti-KRAS drug development, one of the most promising involves inhibitors of the key KRAS effector pathway, the three-tiered RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. We address the promises and challenges of targeting ERK MAPK signaling as an anti-KRAS therapy for PDAC. In particular, we also summarize the key role of the MYC transcription factor and oncoprotein in supporting ERK-dependent growth of KRAS-mutant PDAC.

The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction.

The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.

Inhibition of hTERT expression by MAP kinase inhibitor induces cell death in renal cell carcinoma

BackgroundHuman telomerase reverse transcriptase (hTERT) is one of the components of telomerase enzyme and its activity is associated with cell proliferation and differentiation. Extracellular signal regulated kinase (ERK)-mitogen activated protein kinase signaling pathway play an important role in hTERT expression. The present study was conducted to ascertain hTERT messenger RNA (mRNA) expression in renal cell carcinoma (RCC) and its association with clinicopathological characteristics. Further, we also explored hTERT targeting as possible tumor therapeutic. MethodsA total of 96 histopathologically confirmed RCC cases and corresponding normal tissues were subjected to hTERT gene expression using real-time PCR. Two RCC cell lines viz. ACHN and A498 were treated with MEK inhibitor (U0126) and hTERT mRNA expression, telomerase activity, cell viability, migration, and apoptosis were evaluated. ResultsThe hTERT mRNA levels were found to be significantly higher in RCC as compared with corresponding normal renal tissues (P = 0.040) as well as in high grades to that of low grades clear cell RCC (P = 0.008). Significantly diminished ERK phosphorylation and hTERT mRNA expression concomitantly reduced telomerase activity that was observed after U0126 treatment. Subsequently, cell viability and migration were significantly inhibited after treatment with U0126 in both the cell lines to that of control (P = 0.001). In addition, U0126-treated cells showed significantly increased apoptosis (P = 0.001) to that of controls. ConclusionHenceforth, this study infer that, hTERT expression can be used as a possible diagnostic marker for RCC, and inhibition of hTERT expression by hampering ERK-mitogen activated protein kinase cascade may be used as a promising anticancer target in RCC.

Chapter Four - Inhibitors of the ERK Mitogen-Activated Protein Kinase Cascade for Targeting RAS Mutant Cancers

Although recent success in identifying direct inhibitors of mutant Ras has begun to challenge the perception that Ras is "undruggable," the successful transition of these hits to the clinic remains uncertain. Therefore, current efforts to develop anti-Ras inhibitors are focused on indirect approaches, with inhibitors of the downstream effectors of Ras signaling having attracted the greatest current interest. Of the multitude of effectors, the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is arguably the most attractive target for these efforts. In this chapter, we review the evidence for a key driver role for the ERK MAPK cascade in RAS mutant cancers and the status of efforts to develop inhibitors of MEK1/2 and ERK1/2 to block this pathway.

Mechanism of lapatinib-mediated radiosensitization of breast cancer cells is primarily by inhibition of the Raf > MEK > ERK mitogen-activated protein kinase cascade and radiosensitization of lapatinib-resistant cells restored by direct inhibition of MEK

Background and purpose We recently showed that lapatinib, an EGFR/HER2 inhibitor, radiosensitized breast cancer cells of the basal and HER2+ subtypes. The purpose of this study was to identify the downstream signaling pathways responsible for lapatinib-mediated radiosensitization in breast cancer. Materials and methods Response of EGFR downstream signaling pathways was assessed by Western blot and clonogenic cell survival assays in breast tumor cells after irradiation (5 Gy), lapatinib, CI-1040, or combined treatment. Results In SUM102 cells, an EGFR+ basal breast cancer cell line, exposure to ionizing radiation elicited strong activation of ERK1/2 and JNK, which was blocked by lapatinib, and weak/no activation of p38, AKT or STAT3. Direct inhibition of MEK1 with CI-1040 resulted in 95% inhibition of surviving colonies when combined with radiation while inhibition of JNK with SP600125 had no effect. Lapatinib-mediated radiosensitization of SUM102 cells was completely abrogated with expression of constitutively active Raf. Treatment of lapatinib-resistant SUM185 cells with CI-1040 restored radiosensitization with 45% fewer surviving colonies when combined with radiation. Conclusions These data suggest that radiosensitization by lapatinib is mediated largely through inhibition of MEK/ERK and that direct inhibition of this pathway may provide an additional avenue of radiosensitization in EGFR+ or HER2+ breast cancers.

Mechanism of lapatinib-mediated radiosensitization of breast cancer cells is primarily by inhibition of the Raf>MEK>ERK mitogen-activated protein kinase cascade and radiosensitization of lapatinib-resistant cells restored by direct inhibition of MEK

Mechanism of lapatinib-mediated radiosensitization of breast cancer cells is primarily by inhibition of the Raf>MEK>ERK mitogen-activated protein kinase cascade and radiosensitization of lapatinib-resistant cells restored by direct inhibition of MEK

Advances in Targeting the Ras/Raf/MEK/Erk Mitogen-Activated Protein Kinase Cascade with MEK Inhibitors for Cancer Therapy

The identification of intracellular signaling cascades important for the growth and survival of cancer cells has led to the development of targeted cancer therapeutics aimed at blocking these signals. The mitogen-activated protein kinase (MAPK) pathway has a well-defined role in cancer biology and has been an important target in the development of targeted therapies. Recently, several small-molecule inhibitors of MAPK/extracellular signal-regulated kinase kinase (MEK), a key intermediary of MAPK signaling, have been developed and are currently being tested in clinical trials. Herein, we review the MAPK pathway, the development of small-molecule MEK inhibitors, and the results obtained to date with MEK inhibitors in human cancer trials.

The 5-HT- and FMRFa-activated signaling pathways interact at the level of the Erk MAPK cascade: Potential inhibitory constraints on memory formation

The sensorimotor synapse of Aplysia exhibits long-term facilitation (LTF) and long-term depression (LTD) elicited by the neuromodulator serotonin (5-HT) and the peptide Phe-Met-Arg-Phe-NH 2, respectively. 5-HT-induced LTF engages extracellular-regulated kinase (Erk) and CREB1, whereas FMRFa-induced LTD engages p38 MAPK (mitogen-activated protein kinase) and CREB2. The interaction of the 5-HT and FMRFa pathways was recently investigated in Aplysia at the level of gene expression. However, little is known about crosstalk of these pathways at the level of the second messenger cascades. We investigated the potential interaction of the 5-HT and FMRFa pathways at the level of the Erk cascade. We found that FMRFa inhibited basal Erk activity through p38 MAPK. FMRFa also inhibited 5-HT-induced phosphorylation of Erk and nuclear accumulation of phospho-ERK, suggesting that FMRFa may place inhibitory constraints on memory formation through regulation of the Erk MAPK cascade.

ABIN-2 forms a ternary complex with TPL-2 and NF-kappa B1 p105 and is essential for TPL-2 protein stability.

NF-kappa B1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-kappa B 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.

The Stability of the G Protein-coupled Receptor-β-Arrestin Interaction Determines the Mechanism and Functional Consequence of ERK Activation

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-beta-arrestin complexes, activated a beta-arrestin-bound pool of ERK2 more efficiently than alpha 1b and beta2 adrenergic receptors, which form transient receptor-beta-arrestin complexes. We next studied chimeric receptors in which the pattern of beta-arrestin binding was reversed by exchanging the C-terminal tails of the beta2 and V2 receptors. The ability of the V2 beta 2 and beta 2V2 chimeras to activate beta-arrestin-bound ERK2 corresponded to the pattern of beta-arrestin binding, suggesting that the stability of the receptor-beta-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2 beta 2 receptor, consistent with the cytosolic retention of beta-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2 beta 2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2 beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta-arrestin complex, controls the extent of beta-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.

Selective Inhibition of Heterotrimeric GsSignaling: TARGETING THE RECEPTOR-G PROTEIN INTERFACE USING A PEPTIDE MINIGENE ENCODING THE Gαs CARBOXYL TERMINUS

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.

β-Amyloid Activates the Mitogen-Activated Protein Kinase Cascade via Hippocampal α7 Nicotinic Acetylcholine Receptors:In VitroandIn VivoMechanisms Related to Alzheimer's Disease

Alzheimer's Disease (AD) is the most common of the senile dementias, the prevalence of which is increasing rapidly, with a projected 14 million affected worldwide by 2025. The signal transduction mechanisms that underlie the learning and memory derangements in AD are poorly understood. beta-Amyloid (Abeta) peptides are elevated in brain tissue of AD patients and are the principal component of amyloid plaques, a major criterion for postmortem diagnosis of the disease. Using acute and organotypic hippocampal slice preparations, we demonstrate that Abeta peptide 1-42 (Abeta42) couples to the mitogen-activated protein kinase (MAPK) cascade via alpha7 nicotinic acetylcholine receptors (nAChRs). In vivo elevation of Abeta, such as that exhibited in an animal model for AD, leads to the upregulation of alpha7 nAChR protein. alpha7 nAChR upregulation occurs concomitantly with the downregulation of the 42 kDa isoform of extracellular signal-regulated kinase (ERK2) MAPK in hippocampi of aged animals. The phosphorylation state of a transcriptional mediator of long-term potentiation and a downstream target of the ERK MAPK cascade, the cAMP-regulatory element binding (CREB) protein, were affected also. These findings support the model that derangement of hippocampus signal transduction cascades in AD arises as a consequence of increased Abeta burden and chronic activation of the ERK MAPK cascade in an alpha7 nAChR-dependent manner that eventually leads to the downregulation of ERK2 MAPK and decreased phosphorylation of CREB protein.

Temporal recruitment of the mSin3A-histone deacetylase corepressor complex to the ETS domain transcription factor Elk-1.

The transcriptional status of eukaryotic genes is determined by a balance between activation and repression mechanisms. The nuclear hormone receptors represent classical examples of transcription factors that can regulate this balance by recruiting corepressor and coactivator complexes in a ligand-dependent manner. Here, we demonstrate that the equilibrium between activation and repression via a single transcription factor, Elk-1, is altered following activation of the Erk mitogen-activated protein kinase cascade. In addition to its C-terminal transcriptional activation domain, Elk-1 contains an N-terminal transcriptional repression domain that can recruit the mSin3A-histone deacetylase 1 corepressor complex. Recruitment of this corepressor is enhanced in response to activation of the Erk pathway in vivo, and this recruitment correlates kinetically with the shutoff of one of its target promoters, c-fos. Elk-1 therefore undergoes temporal activator-repressor switching and contributes to both the activation and repression of target genes following growth factor stimulation.

Pasteurella multocida Toxin Stimulates Mitogen-activated Protein Kinase via Gq/11-dependent Transactivation of the Epidermal Growth Factor Receptor

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.

Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta

Mek is a dual-specificity kinase that activates the extracellular-signal-regulated (Erk) mitogen-activated protein (MAP) kinases upon agonist binding to receptors. The Erk MAP kinase cascade is involved in cell-fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single mek gene is present in Caenorhabditis elegans, Drosophila andXenopus, two mek homologs, Mek1 and Mek2, are present in the mammalian cascade. In the present study, we describe a mutant mouse line in which the mek1 gene has been disrupted by insertional mutagenesis. The null mutation was recessive lethal, as the homozygous mutant embryos died at 10.5 days of gestation. Histopathological analyses revealed a reduction in vascularization of the placenta that was due to a marked decrease of vascular endothelial cells in the labyrinthine region. The failure to establish a functional placenta probably explains the death of the mek1−/− embryos. Cell-migration assays indicated that mek1−/− fibroblasts could not be induced to migrate by fibronectin, although the levels of Mek2 protein and Erk activation were normal. Re-expression of Mek1 in the mutant mouse embryonic fibroblasts (MEFs) restored their ability to migrate. Our findings provide genetic evidence that establishes the unique role played by Mek1 in signal transduction. They also suggest that mek1 function is required for normal response to angiogenic signals that might promote vascularization of the labyrinthine region of the placenta.

JNK or IKK, AP-1 or NF-kappaB, which are the targets for MEK kinase 1 action?

MEKK1 (MEK kinase 1) is a mammalian serine/threonine kinase in the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) group (1). Being the first mammalian homolog of STE11, a MAPKKK that activates the pheromone responsive MAPK cascade of budding yeast, MEKK1 as its name indicates was thought to be an activator of the MAPK kinase (MAPKK) MEK1/2 and thus an activator of the ERK MAPK cascade. It therefore was rather surprising that titration experiments (2) or analysis of cells engineered to express MEKK1 from an inducible promoter (3) revealed that it is a far more potent activator of the JNK MAPK cascade. These observations made by using either the catalytic domain of MEKK1 (MEKK1Δ) or a 672-aa C-terminal fragment recently were confirmed by using full-length human MEKK1 (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work). Most importantly, when different mammalian MAPKKs were examined in vitro or in vivo for phosphorylation and activation by MEKK1, a MAPKK called JNKK1 (MKK4 or SEK1), whose function is JNK (and p38 MAPK) activation (4) was found to be the preferred MEKK1 substrate (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work). Based on specificity constants, MEKK1 phosphorylates JNKK1 45-fold more efficiently than it phosphorylates MEK1/2 (Y. Xia, Z. Wu, B. Su, B. Murray, and M.K., unpublished work), thus providing a clear biochemical explanation for the marked pro-JNK bias of MEKK1. Targets for JNK include transcription factors c-Jun and ATF2, which are components of the AP-1 dimer that are involved in induction of the c-jun protooncogene (5). JNK-mediated phosphorylation enhances the transcriptional activity of both c-Jun and ATF2 (6, 7). Correspondingly, MEKK1 expression plasmids are potent activators of a chimeric c-Jun-GAL4 transcription factor, in which the c-Jun activation domain is fused to the GAL4 DNA binding domain (8 …