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1.  Constitutive MEK1 Activation Rescues Anthrax Lethal Toxin-Induced Vascular Effects In Vivo▿  
Infection and Immunity  2010;78(12):5043-5053.
Anthrax lethal toxin (LT) increases vascular leakage in a number of mammalian models and in human anthrax disease. Using a zebrafish model, we determined that vascular delivery of LT increased permeability, which was phenocopied by treatment with a selective chemical inhibitor of MEK1 and MEK2 (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK). Here we investigate further the role of MEK1/phospho-ERK (pERK) in the action of LT. Overexpression of wild-type zebrafish MEK1 at high levels did not induce detrimental effects. However, a constitutively activated version, MEK1S219D,S223D (MEK1DD), induced early defects in embryonic development that correlated with increased ERK/MAPK phosphorylation. To bypass these early developmental defects and to provide a genetic tool for examining the action of lethal factor (LF), we generated inducible transgenic zebrafish lines expressing either wild-type or activated MEK1 under the control of a heat shock promoter. Remarkably, induction of MEK1DD transgene expression prior to LT delivery prevented vascular damage, while the wild-type MEK1 line did not. In the presence of both LT and MEK1DD transgene expression, cardiovascular development and function proceeded normally in most embryos. The resistance to microsphere leakage in transgenic animals demonstrated a protective role against LT-induced vascular permeability. A consistent increase in ERK phosphorylation among LT-resistant MEK1DD transgenic animals provided additional confirmation of transgene activation. These findings provide a novel genetic approach to examine mechanism of action of LT in vivo through one of its known targets. This approach may be generally applied to investigate additional pathogen-host interactions and to provide mechanistic insights into host signaling pathways affected by pathogen entry.
PMCID: PMC2981334  PMID: 20855511
2.  Spectrum of MEK1 and MEK2 gene mutations in cardio-facio-cutaneous syndrome and genotype–phenotype correlations 
Cardio-facio-cutaneous syndrome (CFCS) is a rare disease characterized by mental retardation, facial dysmorphisms, ectodermal abnormalities, heart defects and developmental delay. CFCS is genetically heterogeneous and mutations in the KRAS, BRAF, MAP2K1 (MEK1) and MAP2K2 (MEK2) genes, encoding for components of the RAS–mitogen activated protein kinase (MAPK) signaling pathway, have been identified in up to 90% of cases. Here we screened a cohort of 33 individuals with CFCS for MEK1 and MEK2 gene mutations to further explore their molecular spectrum in this disorder, and to analyze genotype–phenotype correlations. Three MEK1 and two MEK2 mutations were detected in six patients. Two missense MEK1 (L42F and Y130H) changes and one in-frame MEK2 (K63_E66del) deletion had not been reported earlier. All mutations were localized within exon 2 or 3. Together with the available records, the present data document that MEK1 mutations are relatively more frequent than those in MEK2, with exons 2 and 3 being mutational hot spots in both genes. Mutational analysis of the affected MEK1 and MEK2 exons did not reveal occurrence of mutations among 75 patients with Noonan syndrome, confirming the low prevalence of MEK gene defects in this disorder. Clinical review of known individuals with MEK1/MEK2 mutations suggests that these patients show dysmorphic features, ectodermal abnormalities and cognitive deficit similar to what was observed in BRAF-mutated patients and in the general CFCS population. Conversely, congenital heart defects, particularly mitral valve and septal defects, and ocular anomalies seem to be less frequent among MEK1/MEK2 mutation-positive patients.
PMCID: PMC2947095  PMID: 19156172
cardio-facio-cutaneous syndrome; MEK1; MEK2; BRAF
3.  Selective Role for Mek1 but not Mek2 in the Induction of Epidermal Neoplasia 
Cancer research  2009;69(9):3772-3778.
The Ras/Raf/Mek/Erk mitogen-activated protein kinase pathway regulates fundamental processes in normal and malignant cells, including proliferation, differentiation, and cell survival. Mutations in this pathway have been associated with carcinogenesis and developmental disorders, making Mek1 and Mek2 prime therapeutic targets. In this study, we examined the requirement for Mek1 and Mek2 in skin neoplasia using the two-step 7,12-dimethylbenz(a)anthraacene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) skin carcinogenesis model. Mice lacking epidermal Mek1 protein develop fewer papillomas than both wild-type and Mek2-null mice following DMBA/TPA treatment. Mek1 knockout mice had smaller papillomas, delayed tumor onset, and half the tumor burden of wild-type mice. Loss of one Mek1 allele, however, did not affect tumor development, indicating that one Mek1 allele is sufficient for normal papilloma formation. No difference in TPA-induced hyperproliferation, inflammation, or Erk activation was observed between wild-type, conditional Mek1 knockout, and Mek2-null mice, indicating that Mek1 findings were not due to a general failure of these processes. These data show that Mek1 is important for skin tumor development and that Mek2 cannot compensate for the loss of Mek1 function in this setting.
PMCID: PMC3576816  PMID: 19383924
4.  Mek2 Is Dispensable for Mouse Growth and Development 
Molecular and Cellular Biology  2003;23(14):4778-4787.
MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase 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 melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.
PMCID: PMC162209  PMID: 12832465
5.  MEK2 Is Sufficient but Not Necessary for Proliferation and Anchorage-Independent Growth of SK-MEL-28 Melanoma Cells 
PLoS ONE  2011;6(2):e17165.
Mitogen-activated protein kinase kinases (MKK or MEK) 1 and 2 are usually treated as redundant kinases. However, in assessing their relative contribution towards ERK-mediated biologic response investigators have relied on tests of necessity, not sufficiency. In response we developed a novel experimental model using lethal toxin (LeTx), an anthrax toxin-derived pan-MKK protease, and genetically engineered protease resistant MKK mutants (MKKcr) to test the sufficiency of MEK signaling in melanoma SK-MEL-28 cells. Surprisingly, ERK activity persisted in LeTx-treated cells expressing MEK2cr but not MEK1cr. Microarray analysis revealed non-overlapping downstream transcriptional targets of MEK1 and MEK2, and indicated a substantial rescue effect of MEK2cr on proliferation pathways. Furthermore, LeTx efficiently inhibited the cell proliferation and anchorage-independent growth of SK-MEL-28 cells expressing MKK1cr but not MEK2cr. These results indicate in SK-MEL-28 cells MEK1 and MEK2 signaling pathways are not redundant and interchangeable for cell proliferation. We conclude that in the absence of other MKK, MEK2 is sufficient for SK-MEL-28 cell proliferation. MEK1 conditionally compensates for loss of MEK2 only in the presence of other MKK.
PMCID: PMC3041822  PMID: 21365009
6.  Extracellular Signal-Regulated Kinases Modulate DNA Damage Response - A Contributing Factor to Using MEK Inhibitors in Cancer Therapy 
Current Medicinal Chemistry  2011;18(35):5476-5482.
The Raf-MEK-ERK pathway is commonly activated in human cancers, largely attributable to the extracellular signal-regulated kinases (ERKs) being a common downstream target of growth factor receptors, Ras, and Raf. Elevation of these up-stream signals occurs frequently in a variety of malignancies and ERK kinases play critical roles in promoting cell proliferation. Therefore, inhibition of MEK-mediated ERK activation is very appealing in cancer therapy. Consequently, numerous MEK inhibitors have been developed over the years. However, clinical trials have yet to produce overwhelming support for using MEK inhibitors in cancer therapy. Although complex reasons may have contributed to this outcome, an alternative possibility is that the MEK-ERK pathway may not solely provide proliferation signals to malignancies, the central scientific rationale in developing MEK inhibitors for cancer therapy. Recent developments may support this alternative possibility. Accumulating evidence now demonstrated that the MEK-ERK pathway contributes to the proper execution of cellular DNA damage response (DDR), a major pathway of tumor suppression. During DDR, the MEK-ERK pathway is commonly activated, which facilitates the proper activation of DDR checkpoints to prevent cell division. Inhibition of MEK-mediated ERK activation, therefore, compromises checkpoint activation. As a result, cells may continue to proliferate in the presence of DNA lesions, leading to the accumulation of mutations and thereby promoting tumorigenesis. Alternatively, reduction in checkpoint activation may prevent efficient repair of DNA damages, which may cause apoptosis or cell catastrophe, thereby enhancing chemotherapy’s efficacy. This review summarizes our current understanding of the participation of the ERK kinases in DDR.
PMCID: PMC3330700  PMID: 22087839
ERK1/2 kinases; DNA damage response (DDR); checkpoint activation; ATM; ATR.
7.  Activation of Erk and JNK MAPK pathways by acute swim stress in rat brain regions 
BMC Neuroscience  2004;5:36.
The mitogen-activated protein kinases (MAPKs) have been shown to participate in a wide array of cellular functions. A role for some MAPKs (e.g., extracellular signal-regulated kinase, Erk1/2) has been documented in response to certain physiological stimuli, such as ischemia, visceral pain and electroconvulsive shock. We recently demonstrated that restraint stress activates the Erk MAPK pathway, but not c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38MAPK, in several rat brain regions. In the present study, we investigated the effects of a different stressor, acute forced swim stress, on the phosphorylation (P) state of these MAPKs in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum. In addition, effects on the phosphorylation state of the upstream activators of the MAPKs, their respective MAPK kinases (MAPKKs; P-MEK1/2, P-MKK4 and P-MKK3/6), were determined. Finally, because the Erk pathway can activate c-AMP response element (CRE) binding (CREB) protein, and swim stress has recently been reported to enhance CREB phosphorylation, changes in P-CREB were also examined.
A single 15 min session of forced swimming increased P-Erk2 levels 2–3-fold in the neocortex, prefrontal cortex and striatum, but not in the hippocampus or amygdala. P-JNK levels (P-JNK1 and/or P-JNK2/3) were increased in all brain regions about 2–5-fold, whereas P-p38MAPK levels remained essentially unchanged. Surprisingly, levels of the phosphorylated MAPKKs, P-MEK1/2 and P-MKK4 (activators of the Erk and JNK pathways, respectively) were increased in all five brain regions, and much more dramatically (P-MEK1/2, 4.5 to > 100-fold; P-MKK4, 12 to ~300-fold). Consistent with the lack of forced swim on phosphorylation of p38MAPK, there appeared to be no change in levels of its activator, P-MKK3/6. P-CREB was increased in all but cortical (prefrontal, neocortex) areas.
Swim stress specifically and markedly enhanced the phosphorylation of the MAPKKs P-MEK1/2 and P-MKK4 in all brain regions tested without apparent alteration in the phosphorylation of P-MKK3/6. Curiously, phosphorylation of their cognate substrates (Erk and JNK) was increased to a much more modest extent, and in some brain regions was not altered. Similarly, there was a region-specific discrepancy between Erk and CREB phosphorylation. Possible explanations for these findings and comparison with the effects of restraint stress will be discussed.
PMCID: PMC526203  PMID: 15380027
8.  Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae 
Parasites & Vectors  2014;7:287.
Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.
We introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.
The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.
MEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.
PMCID: PMC4077580  PMID: 24957684
Anopheles; Mosquito; MAPK; Plasmodium; Malaria; Single nucleotide polymorphism; Immunity
9.  MEK targeting in N-RAS mutated metastatic melanoma 
Molecular Cancer  2014;13:45.
Gain of function mutations in B-RAF and N-RAS occur frequently in melanoma, leading to mitogen activating protein kinase (MAPK) pathway activation, and this pathway is the target of drugs in development. Our purpose was to study clinical characteristics of patients with mutations in this pathway and to determine activity of inhibitors of B-RAF and MEK in short term cultures grown from tumors of some of these patients.
Clinical and pathologic data were collected retrospectively on melanoma patients tested for B-RAF and N-RAS mutations at the Yale Cancer Center and associations with survival were determined. We studied in vitro activity of the pan-RAF inhibitor, RAF265, and the MEK inhibitor, MEK162, in 22 melanoma short term cultures. We further characterized the effect of MEK inhibition on apoptosis and growth of melanoma cultures.
In a cohort of 144 metastatic melanoma patients we found that patients with N-RAS mutant melanoma had a worse prognosis. These patients were more likely to have brain metastases at the time of presentation with metastatic disease than their N-RAS-wild-type counterparts. All N-RAS mutant melanoma cultures tested in our study (n = 7) were sensitive to MEK inhibition162. Exposure to MEK162 reduced ERK1/2 phosphorylation, and induced apoptosis. Clonogenic survival was significantly reduced in sensitive melanoma cell cultures.
The prognosis of patients with melanoma expressing constitutively active N-RAS is poor, consistent with studies performed at other institutions. N-RAS mutant melanoma cultures appear to be particularly sensitive to MEK162, supporting ongoing clinical trials with MEK162 in N-RAS mutated melanoma.
PMCID: PMC3945937  PMID: 24588908
Targeted therapy; Melanoma; N-RAS; MEK inhibitor; Apoptosis
10.  A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function. 
Molecular and Cellular Biology  1995;15(10):5214-5225.
Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.
PMCID: PMC230769  PMID: 7565670
11.  Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. 
Molecular and Cellular Biology  1993;13(11):6615-6620.
Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.
PMCID: PMC364724  PMID: 8413257
12.  PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C 
PLoS Genetics  2014;10(9):e1004582.
Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.
Author Summary
The specific response to environmental cues is crucial for cell differentiation and is often mediated by highly conserved eukaryotic MAP kinase (MAPK) pathways. How these pathways react specifically to huge numbers of different cues is still unclear, and current literature about adapter and scaffolding proteins remains scarce. However, gaining fundamental insight into molecular signaling determinants is pivotal for combating diseases with impaired signal transduction processes, such as Alzheimer's disease or cancer. Importantly, signal transduction can easily be studied in lower eukaryotes like filamentous fungi that are readily genetically tractable. The fungus Sordaria macrospora has a long history as an ideal model system for cell differentiation, and we show here that the proposed cell wall integrity (CWI) MAPK module of this fungus controls differentiation of sexual fruiting bodies, cell fusion, polar growth and cell wall stress response. We further discovered that developmental protein PRO40 binds the MAPK kinase kinase (MAPKKK), the MAPK kinase (MAPKK) and upstream activator protein kinase C (PKC1) of the CWI pathway and is required for MAK1 activity, thereby providing evidence that PRO40 is a scaffold protein. Collectively, our findings reveal a molecular role for a protein implicated in development, cell fusion, symbiosis, and pathogenicity in different fungi.
PMCID: PMC4154660  PMID: 25188365
13.  CInQ-03, a novel allosteric MEK inhibitor, suppresses cancer growth in vitro and in vivo  
Carcinogenesis  2013;34(5):1134-1143.
The mitogen-activated protein kinase kinase 1 and 2 signaling pathway is a major component of the RAS (Rat sarcoma)/RAF (Radpidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERKs (Extracellular signal-regulated kinases) signaling axis that regulates tumorigenesis and cancer cell growth. MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore, MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in mitogen-activated protein kinase signaling. Herein, we report the discovery of three novel MEK inhibitors, herein referred to as CInQ-01, CInQ-03 and CInQ-06. All three inhibitors were highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and anchorage-independent cell growth. The inhibitory activity was associated with markedly reduced phosphorylation of ERKs and ribosomal S6 kinases. Furthermore, administration of CInQ-03 inhibited colon cancer cell growth in an in vivo xenograft mouse model and showed no skin toxicity. Overall, these results suggest that these novel MEK inhibitors might be used for chemotherapy or prevention.
PMCID: PMC3643416  PMID: 23354306
14.  MEK-1 Activates C-Raf Through a Ras-Independent Mechanism 
Biochimica et biophysica acta  2013;1833(5):976-986.
C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2.
PMCID: PMC3608709  PMID: 23360980
Raf; Ras; MEK; ERK; MAPK; phosphorylation
15.  A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates* 
The Journal of biological chemistry  2003;278(35):32662-32672.
Specific docking interactions between MAPKs and their activating MAPK kinases (MKKs or MEKs) are crucial for efficient and accurate signal transmission. Here, we report the identification of a MAPK-docking site, or “D-site,” in the N terminus of human MKK4/JNKK1. This docking site conforms to the consensus sequence for known D-sites in other MKKs and contains the first of the two cleavage sites for anthrax lethal factor protease that have been found in the N terminus of MKK4. This docking site was both necessary and sufficient for the high affinity binding of the MAPKs JNK1, JNK2, JNK3, p38α, and p38β to MKK4. Mutations that altered conserved residues in this docking site reduced JNK/p38 binding. In addition, a peptide version of this docking site, as well as a peptide version of the JNK-binding site of the JIP-1 scaffold protein, inhibited both MKK4/JNK binding and MKK4-mediated phosphorylation of JNK1. These same peptides also inhibited JNK2-mediated phosphorylation of c-Jun and ATF2, suggesting that transcription factors, MKK4, and the JIP scaffold compete for docking to JNK. Finally, the selectivity of the MKK4, MEK1, and MEK2 D-sites for JNK versus ERK was quantified. The MEK1 and MEK2 D-sites displayed a strong selectivity for their cognate MAPK (ERK2) versus a non-cognate MAPK (JNK). In contrast, the MKK4 D-site exhibited only limited selectivity for JNK versus ERK.
PMCID: PMC3017503  PMID: 12788955
16.  Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics 
PLoS Medicine  2007;4(10):e316.
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.
Methods and Findings
We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.
Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation.
Editors' Summary
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC.
Why Was This Study Done?
Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR.
What Did the Researchers Do and Find?
The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth.
Additional Information.
Please access these Web sites via the online version of this summary at
A perspective by Ingo Mellinghoff discusses this article and two related research articles
Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
CancerQuest provides information on all aspects of cancer from Emory University (in several languages)
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Information for patients from Cancerbackup on erlotinib and gefitinib
PMCID: PMC2043013  PMID: 17973573
17.  A MEK Inhibitor Abrogates Myeloproliferative Disease in Kras Mutant Mice 
Science Translational Medicine  2011;3(76):76ra27.
Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are aggressive myeloproliferative neoplasms that are incurable with conventional chemotherapy. Mutations that deregulate Ras signaling play a central pathogenic role in both disorders, and Mx1-Cre, KrasLSL-G12D mice that express the Kras oncogene develop a fatal disease that closely mimics these two leukemias in humans. Activated Ras controls multiple downstream effectors, but the specific pathways that mediate the leukemogenic effects of hyperactive Ras are unknown. We used PD0325901, a highly selective pharmacological inhibitor of mitogen-activated protein kinase kinase (MEK), a downstream component of the Ras signaling network, to address how deregulated Raf/MEK/ERK signaling drives neoplasm formation in Mx1-Cre, KrasLSL-G12D mice. PD0325901 treatment induced a rapid and sustained reduction in leukocyte counts, enhanced erythropoiesis, prolonged mouse survival, and corrected the aberrant proliferation and differentiation of bone marrow progenitor cells. These responses were due to direct effects of PD0325901 on Kras mutant cells rather than to stimulation of normal hematopoietic cell proliferation. Consistent with the in vivo response, inhibition of MEK reversed the cytokine hypersensitivity characteristic of KrasG12D hematopoietic progenitor cells in vitro. Our data demonstrate that deregulated Raf/MEK/ERK signaling is integral to the growth of Kras-mediated myeloproliferative neoplasias, and further suggest that MEK inhibition could be a useful way to ameliorate functional hematologic abnormalities in patients with CMML and JMML.
PMCID: PMC3265440  PMID: 21451123
18.  Mitogenic growth signalling, DNA replication licensing, and survival are linked in prostate cancer 
British Journal of Cancer  2007;96(9):1384-1393.
Activation of mitogen/extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) growth signalling is coupled to increased cell proliferation in prostate cancer (PCa). Dysregulation of the DNA replication licensing pathway, a critical step in growth control downstream of transduction signalling pathways, is associated with development of PCa. In this study we have investigated linkages between the MEK5/ERK5 pathway and DNA replication licensing during prostate carcinogenesis. The effects of increased MEK5/ERK5 signalling on the expression of replication licensing factors Mcm2 and geminin and the proliferation marker Ki67 were studied in an ecdysone-inducible system expressing a constitutively activated mutant of MEK5 in EcR293 cells and in stable ERK5 over-expressing PC3 clones. In parallel, expression of these biomarkers in PCa biopsy specimens (n=58) was studied and compared to clinicopathological parameters. In both in vitro systems induction of MEK5 expression resulted in increased levels of phosphorylated ERK5 and Mcm2, geminin and Ki67 proteins. In PCa specimens average Mcm2 expression was greater than Ki67 and geminin expression (median labelling index (LI) 36.7, 18.1, and 3.4% respectively), consistent with their differential expression according to growth status (P<0.0001). Mcm2, geminin and Ki67 expression were significantly associated with Gleason grade (P=0.0002, P=0.0003, P=0.004); however there was no link with T or M stage. There was a significant relationship between increasing ERK5 expression and increasing Mcm2 (P=0.003) and Ki67 (P=0.009) expression, with non-significant trends seen with increasing MEK5 expression. There were significant associations between Gleason grade and the number of cells traversing G1 phase (Ki67LI-gemininLI; (P=0.001)), with high ERK5 levels associated with both an increase in replication licensed but non-cycling cells (Mcm2LI-Ki67LI; (P=0.01)) and accelerated cell cycle progression (gemininLI/Ki67LI; (P= 0.005)), all indicative of a shift towards increasing proliferative potential. While Mcm2 and Ki67 were both prognostic factors on univariate analysis, only Mcm2 remained an independent prognostic marker on multivariate analysis. Taken together, our data show that induction of MEK5/ERK5 signalling is linked to activation of the DNA replication licensing pathway in PCa, and that the strong prognostic value of MCM proteins may result from their function as relay stations coupling growth regulatory pathways to genome duplication.
PMCID: PMC2360172  PMID: 17406359
MEK5/ERK5; Mcm2; geminin; DNA replication licensing; prognosis; prostate cancer
19.  Mitogen-activated protein kinase (MAPK)-docking sites in MAPK kinases function as tethers that are crucial for MAPK regulation in vivo 
Cellular signalling  2005;18(1):123-134.
Docking sites on targets of mitogen-activated protein kinases (MAPKs) facilitate accurate and efficient substrate phosphorylation. MAPK/ERK kinases (MEKs, or MKKs), the upstream regulators of MAPKs, also contain N-terminal MAPK-docking sites, or ‘D-sites’; however, the in vivo functions of MEK D-sites are incompletely understood. Here we found that the ability of constitutively-active human MEK1 and MEK2 to stimulate ERK phosphorylation and to induce the neoplastic transformation of NIH 3T3 cells required the integrity of the D-site. In addition, D-site mutants of otherwise wild-type MEK1/2 were unable to anchor unphosphorylated ERK2 in the cytoplasm. ERK activation, cytoplasmic anchoring and release were completely retained in ‘swap’ mutants in which MEK2’s D-site was replaced with the D-site of MEK1 or yeast Ste7. Furthermore, these abilities were significantly retained when MEK2’s D-site was moved to its C-terminus, or replaced by an unrelated MAPK-binding domain taken from the Ets-1 transcription factor. We conclude that the D-sites in MEKs are crucial for the activation of their cognate MAPKs in vivo, and that their primary function is to tether their cognate MAPKs near the MEK’s kinase domain. This proximity effect is sufficient to explain the contribution that the D-site interaction makes to several biologically important signaling events.
PMCID: PMC3017502  PMID: 15979847
Mitogen-activated protein kinases; Signal transduction; Phosphorylation; Binding sites; Protein binding
20.  Sequential Activation of the MEK-Extracellular Signal-Regulated Kinase and MKK3/6-p38 Mitogen-Activated Protein Kinase Pathways Mediates Oncogenic ras-Induced Premature Senescence†  
Molecular and Cellular Biology  2002;22(10):3389-3403.
In primary mammalian cells, oncogenic ras induces premature senescence, depending on an active MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. It has been unclear how activation of the mitogenic MEK-ERK pathway by ras can confer growth inhibition. In this study, we have found that the stress-activated MAPK, p38, is also activated during the onset of ras-induced senescence in primary human fibroblasts. Constitutive activation of p38 by active MKK3 or MKK6 induces senescence. Oncogenic ras fails to provoke senescence when p38 activity is inhibited, suggesting that p38 activation is essential for ras-induced senescence. Furthermore, we have demonstrated that p38 activity is stimulated by ras as a result of an activated MEK-ERK pathway. Following activation of MEK and ERK, expression of oncogenic ras leads to the accumulation of active MKK3/6 and p38 activation in a MEK-dependent fashion and subsequently induces senescence. Active MEK1 induces the same set of changes and provokes senescence relying on active p38. Therefore, oncogenic ras provokes premature senescence by sequentially activating the MEK-ERK and MKK3/6-p38 pathways in normal, primary cells. These studies have defined the molecular events within the ras signaling cascade that lead to premature senescence and, thus, have provided new insights into how ras confers oncogenic transformation in primary cells.
PMCID: PMC133789  PMID: 11971971
21.  Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors 
BMC Cancer  2008;8:337.
The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.
Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.
We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.
MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.
PMCID: PMC2596176  PMID: 19014680
22.  The small molecule Mek1/2 inhibitor U0126 disrupts the chordamesoderm to notochord transition in zebrafish 
Key molecules involved in notochord differentiation and function have been identified through genetic analysis in zebrafish and mice, but MEK1 and 2 have so far not been implicated in this process due to early lethality (Mek1-/-) and functional redundancy (Mek2-/-) in the knockout animals.
Here, we reveal a potential role for Mek1/2 during notochord development by using the small molecule Mek1/2 inhibitor U0126 which blocks phosphorylation of the Mek1/2 target gene Erk1/2 in vivo. Applying the inhibitor from early gastrulation until the 18-somite stage produces a specific and consistent phenotype with lack of dark pigmentation, shorter tail and an abnormal, undulated notochord. Using morphological analysis, in situ hybridization, immunhistochemistry, TUNEL staining and electron microscopy, we demonstrate that in treated embryos the chordamesoderm to notochord transition is disrupted and identify disorganization in the medial layer of the perinotochordal basement mebrane as the probable cause of the undulations and bulges in the notochord. We also examined and excluded FGF as the upstream signal during this process.
Using the small chemical U0126, we have established a novel link between MAPK-signaling and notochord differentiation. Our phenotypic analysis suggests a potential connection between the MAPK-pathway, the COPI-mediated intracellular transport and/or the copper-dependent posttranslational regulatory processes during notochord differentiation.
PMCID: PMC2359734  PMID: 18419805
23.  AurkA inhibitors enhance the effects of B-RAF and MEK inhibitors in melanoma treatment 
Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment.
A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor). Cell proliferation was assessed using xCELLigence technology. Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis. Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination.
AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents. In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination. However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells.
These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations. Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma. However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence. Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.
PMCID: PMC4237855  PMID: 25074438
Melanoma; Aurora A kinase; Targeted therapy; Combined therapy; 3D-human skin reconstruction model
24.  Analysis of mRNA Profiles after MEK1/2 Inhibition in Human Pancreatic Cancer Cell Lines Reveals Pathways Involved in Drug Sensitivity 
Molecular cancer research : MCR  2012;10(12):1607-1619.
Mutationally activated KRAS, detected in approximately 90% of pancreatic ductal adenocarcinomas (PDA), has proven an intractable pharmacologic target to date. Consequently, efforts to treat KRAS-mutated cancers are focused on targeting RAS-regulated signaling pathways. In mouse models, expression of BRAFV600E combined with dominant-negative TP53 elicits PDA, and pharmacologic blockade of mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibits proliferation of human PDA-derived cell lines. To better understand the role of RAF→MEK→ERK signaling on PDA cell proliferation, we assessed the consequences of MEK inhibition on global patterns of mRNA expression and tumor cell proliferation in a panel of human PDA-derived cell lines. This analysis revealed that RAF→MEK→ERK signaling regulates mRNAs involved in cell-cycle control as well as regulators of the immune system. Linear regression analysis of relative drug sensitivity and mRNA expression revealed mRNAs and pathways correlating with relative drug sensitivity of the cell lines. Mice carrying orthotopically implanted pancreas tumors that were treated with MEK inhibitor displayed reduced tumor growth, concomitant with a reduction of cells in S phase. Furthermore, analysis of tumor mRNA expression revealed PDA cell lines to display similar baseline and MEK inhibitor mRNA expression profiles in vitro and in vivo. Among the proteins subject to downregulation following MEK inhibition, we identified c-MYC as a key driver of cell proliferation downstream of RAF→MEK→ERK signaling. Indeed, in some PDA cell lines, RNA interference–mediated silencing of c-MYC expression had antiproliferative effects similar to that of MEK inhibition, thereby highlighting the importance of c-MYC in key aspects of pancreatic cancer cell maintenance.
PMCID: PMC4261949  PMID: 22833572
25.  MEK inhibition induced downregulation of MRP1 and MRP3 expression in experimental hepatocellular carcinoma 
Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. Overexpression of ATP binding cassette (ABC) proteins correlates with activation of mitogen activated protein kinase (MAPK) pathway in HCC. Here, we systematically investigated the inhibition of MAPK pathway and its role in regulating HCC cell growth as well as ABC proteins MRP1 and MRP3 expression.
The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.
Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.
This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.
PMCID: PMC3558388  PMID: 23320839
Hepatocellular carcinoma; MEK; MRP1; MRP3; Multidrug resistance

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