MEK Partner Research Articles

Role of MEK partner-1 in cancer stemness through MEK/ERK pathway in cancerous neural stem cells, expressing EGFRviii

BackgroundGlioma stem cells (GSCs) are a major cause of the frequent relapse observed in glioma, due to their high drug resistance and their differentiation potential. Therefore, understanding the molecular mechanisms governing the ‘cancer stemness’ of GSCs will be particularly important for improving the prognosis of glioma patients.MethodsWe previously established cancerous neural stem cells (CNSCs) from immortalized human neural stem cells (F3 cells), using the H-Ras oncogene. In this study, we utilized the EGFRviii mutation, which frequently occurs in brain cancers, to establish another CNSC line (F3.EGFRviii), and characterized its stemness under spheroid culture.ResultsThe F3.EGFRviii cell line was highly tumorigenic in vitro and showed high ERK1/2 activity as well as expression of a variety of genes associated with cancer stemness, such as SOX2 and NANOG, under spheroid culture conditions. Through meta-analysis, PCR super-array, and subsequent biochemical assays, the induction of MEK partner-1 (MP1, encoded by the LAMTOR3 gene) was shown to play an important role in maintaining ERK1/2 activity during the acquisition of cancer stemness under spheroid culture conditions. High expression of this gene was also closely associated with poor prognosis in brain cancer.ConclusionThese data suggest that MP1 contributes to cancer stemness in EGFRviii-expressing glioma cells by driving ERK activity.

Abstract 4761: BCL2 induced by LAMTOR3-MAPK is a druggable target of chemoradioresistance in mesenchymal lung cancer

Abstract Metastasis and chemoresistance, which are main causes of lung cancer related death, have been major interest in cancer research. Recently, Epithelial-Mesenchymal Transition (EMT) a foremost process for acquiring metastatic properties has been demonstrated to be strongly link to chemoresistance. Due to limited understanding of mechanism for EMT mediated chemoresistance, druggable targets for chemoresistance remained unidentified yet. In this study, we established the mesenchymal like cancer cells (MLCCs) from A549 lung cancer cells with chronic exposure of TGFβ. With clinicogenomics database analysis and following validation with MLCC model, we determined that BCL2, of which expression was clearly induced and indicated a poor prognosis in the mesenchymal cancer patients, was responsible for high chemoresistance in MLCCs. Therefore combined treatment of chemotherapeutic drug and BH3 mimetic such as ABT-263 or ABT-737, remarkably sensitized MLCCs to chemo and radio therapy. Furthermore, BCL2 expression was governed by sustained ERK1 activity in MLCCs, which resulted from high level of MEK partner-1 (MP1) protein expression. Therefore, combined chemotherapy with small molecules (approved by FDA or clinically tested) targeting for MEK1 or BCL2 would be a clinically feasible approach to overcome EMT related chemoresistance. Citation Format: Ok-Seon Kwon, Soon-Ki Hong, Hyuk-Jin Cha. BCL2 induced by LAMTOR3-MAPK is a druggable target of chemoradioresistance in mesenchymal lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4761. doi:10.1158/1538-7445.AM2017-4761

BCL2 induced by LAMTOR3/MAPK is a druggable target of chemoradioresistance in mesenchymal lung cancer

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.

BPGAP1 spatially integrates JNK/ERK signaling crosstalk in oncogenesis.

Simultaneous hyperactivation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascades has been reported in carcinogenesis. However, how they are integrated to promote oncogenesis remains unknown. By analyzing breast invasive carcinoma database (The Cancer Genome Altas), we found that the mRNA expression levels of both JNK1 and ERK2 are positively correlated with the mRNA level of EEA1, an endosome associated protein, indicating the potential JNK/ERK crosstalk at endosome. Unbiased screen of different endosome-associated Rab GTPases reveals that late endosome serves as a unique platform to integrate JNK/ERK signaling. Furthermore, we identify that BPGAP1 (a BCH domain-containing, Cdc42GAP-like Rho GTPase-activating protein) promotes MEK partner 1 (MP1)-induced ERK activation on late endosome through scaffolding MP1/MEK1 complex. This regulatory function requires phosphorylation of BPGAP1 by JNK at its C terminal tail (Ser424) to unlock its autoinhibitory conformation. Consequently, phosphorylated BPGAP1 facilitates endosomal ERK signaling transduction to the nucleus, driving cell proliferation and transformation via the ERK-Myc-CyclinA axis. BPGAP1 therefore provides a crucial spatiotemporal checkpoint where JNK and MP1/MEK1 work in concert to regulate endosomal and nuclear ERK signaling in cell proliferation control.

The therapeutic effect and mechanism of niacin on acute lung injury in a rat model of hemorrhagic shock: Down-regulation of the reactive oxygen species-dependent nuclear factor κB pathway.

The purpose of the current study was to investigate the protective effect of niacin on acute lung injury by the down-regulation of the nuclear factor κB (NF-κB) pathway in hemorrhagic shock (HS) rats. HS was induced in male Sprague-Dawley rats by withdrawing blood to maintain a mean arterial pressure of 20 mm Hg to 25 mm Hg for 40 minutes. The rats were resuscitated by the reinfusion of the drawn blood, and a vehicle (HS), a low-dose of niacin (360 mg/kg, HS + LD-NA), or a high dose of niacin (1,080 mg/kg, HS + HD-NA) were administered orally. The survival of the subjects was observed for 72 hours, and a separate set of animals was killed at 6 hours after HS induction. We measured cytoplasmic phosphorylated inhibitor κB-α and inhibitor κB-α expressions, nuclear NF-κB p65 expression, NF-κB p65 DNA-binding activity, MEK partner 1 activity, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-8, nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide phosphate, reduced glutathione, glutathione disulfide, malondialdehyde levels, and histologic damage in the lung tissue. We also measured TNF-α, IL-6, and IL-8 levels in the serum. The survival rates of the sham, HS, HS + LD-NA, and HS + HD-NA groups were 6 of 6 (100%), 0 of 9 (0%), 1 of 9 (11.1%), and 3 of 9 (33.3%), respectively. A high dose of niacin increased lung NAD+, nicotinamide adenine dinucleotide phosphate levels, and glutathione-glutathione disulfide ratios; decreased lung malondialdehyde levels; down-regulated the NF-κB pathway; suppressed TNF-α, IL-6, and IL-8 levels in the lung tissue and serum; and attenuated histologic lung damage. A high dose of niacin attenuated lung inflammation, suppressed proinflammatory cytokine release, reduced histologic lung damage, and improved survival after HS in rats. Its therapeutic benefits were associated with the down-regulation of the reactive oxygen species-dependent NF-κB pathway.

Gastrin-induced proliferation involves MEK partner 1 (MP1)

The peptide hormone gastrin is an important factor for the maintenance and homeostasis of the gastric mucosa. We show that gastrin stimulates proliferation in a dose-dependent manner in the human gastric adenocarcinoma cell line AGS-GR. Furthermore, we demonstrate that the MAPK scaffold protein MEK partner 1 (MP1) is important for gastrin-induced phosphorylation of ERK1 and ERK2 and that MP1 promotes gastrin-induced proliferation of AGS-GR cells. Our results suggest a role of MP1 in gastrin-induced cellular responses involved in proliferation and homeostasis of the gastric mucosa.

The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells

MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1’s functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells. We find that MP1 is required for motility on fibronectin, but not for motility stimulated by serum or EGF. Surprisingly, MP1 appears not to function through its known binding partners MEK1 or PAK1, suggesting the existence of a novel pathway by which MP1 can regulate motility on fibronectin. MP1 may function by regulating the stability or expression of paxillin, a key regulator of motility.

Involvement of the MP1 scaffold protein in ERK signaling regulation during Drosophila wing development

Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved transduction pathways involved in many cellular processes. Kinase modules are associated with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Some of these scaffold proteins have been shown to be conserved, both in sequence and function. In mouse, the scaffold MP1 (MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously during embryonic and larval development. By in vitro and in vivo experiments, we show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing either dMP1 over-expression or dMP1 down-regulation by RNA interference highlight dMP1 function in the control of cell differentiation during development of the Drosophila wing.

35 ORAL Clinical attrition rates for kinase inhibitors in oncology

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.

Regulation of protein phosphorylation within the MKK1–ERK2 complex by MP1 and the MP1•P14 heterodimer

MEK partner 1 (MP1) and P14 are small proteins that modulate the Raf-MKK1/2-ERK1/2 pathway. To examine the biochemical basis for their function a fluorescent form of MP1 was prepared by labeling Cys-74 with fluorescein. Using this protein it was shown that MP1 binds unactivated ERK1, ERK2 and a constitutively active form of MKK1 (MKK1G7B) with dissociation constants of 9.7 ± 1.6, 3.3 ± 0.6 and 2.2 ± 0.5 μM, respectively. MP1 inhibits the ability of activated ERK2 to phosphorylate the transcription factor Ets-1. Both MP1 and the MP1•P14 complex inhibit the ability of activated ERK2 to phosphorylate MKK1G7B, thus impeding feedback inhibition. In contrast, MP1 and the P14•MP1 complex enhance the ability of MKK1G7B to phosphorylate ERK2, when ERK2 is present at a low concentration, but not when it is present at a high concentration. Thus, MP1 and the MP1•P14 complex have the potential to differentially modulate activating and inhibiting signals in the Raf-MKK1/2-ERK1/2 pathway.

The role of scaffold proteins in MEK/ERK signalling

Signal transduction networks allow cells to recognize and respond to changes in the extracellular environment. All eukaryotic cells have MAPK (mitogen-activated protein kinase) pathways that participate in diverse cellular functions, including differentiation, survival, transformation and movement. Five distinct groups of MAPKs have been characterized in mammals, the most extensively studied of which is the Ras/Raf/MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]/ERK cascade. Numerous stimuli, including growth factors and phorbol esters, activate MEK/ERK signalling. How disparate extracellular signals are translated by MEK/ERK into different cellular functions remains obscure. Originally identified in yeast, scaffold proteins are now recognized to contribute to the specificity of MEK/ERK pathways in mammalian cells. These scaffolds include KSR (kinase suppressor of Ras), beta-arrestin, MEK partner-1, Sef and IQGAP1. Scaffolds organize multiprotein signalling complexes. This targets MEK/ERK to specific substrates and facilitates communication with other pathways, thereby mediating diverse functions. The adaptor proteins regulate the kinetics, amplitude and localization of MEK/ERK signalling, providing an efficient mechanism that enables an individual extracellular stimulus to promote a specific biological response.

The MEK1 Scaffolding Protein MP1 Regulates Cell Spreading by Integrating PAK1 and Rho Signals

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response. Here we show that the putative scaffold protein MEK partner 1 (MP1) and its partner p14 regulate PAK1-dependent ERK activation during adhesion and cell spreading but are not required for ERK activation by platelet-derived growth factor. MP1 associates with active but not inactive PAK1 and controls PAK1 phosphorylation of MEK1. Our data further show that MP1, p14, and MEK1 serve to inhibit Rho/Rho kinase functions necessary for the turnover of adhesion structures and cell spreading and reveal a signal-channeling function for a MEK1/ERK scaffold in orchestrating cytoskeletal rearrangements important for cell motility.

MEK partner 1 (MP1): Regulation of oligomerization in MAP kinase signaling

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions. Gel filtration of cell lysates revealed two major MP1 peaks: a broad high molecular weight peak and a 28 kDa complex. An MP1 mutant that lost MEK1 binding no longer enhanced RasV12-stimulated ERK1 activity, and functioned as a dominant negative, consistent with the concept that MP1 function depends on facilitating these oligomerizations. Activation of the ERK pathway by serum or by RasV12 did not detectably affect MP1-MP1 dimerization or MP1-MEK1 interactions, but caused the dissociation of the MP1-ERK1 complex. Surprisingly, pharmacological inhibition of ERK activation did not restore the complex, suggesting that regulation of complex formation occurs independently of ERK phosphorylation. These results support the concept that MP1 functions as a regulator of MAP kinase signaling by binding to MEK1 and regulating its association with a larger signaling complex that may sequentially service multiple molecules of ERK.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade.

Signal transduction is controlled both by regulation of enzyme activation and by organization of enzymatic complexes with nonenzymatic adapters, scaffolds, and anchor proteins. The extracellular signal-regulated kinase (ERK) cascade is one of several evolutionarily conserved mitogen-activated protein (MAP) kinase cascades important in the regulation of growth, apoptosis, and differentiation. A two-hybrid screen was conducted to identify nonenzymatic components of this signaling cascade that might be important in regulating its activity. A protein called MP1 (MEK Partner 1) was identified that bound specifically to MEK1 and ERK1 and facilitated their activation. When overexpressed in cultured cells, MP1 enhanced activation of ERK1 and activation of a reporter driven by the transcription factor Elk-1. Expression of MP1 in cells increased binding of ERK1 to MEK1. MP1 apparently functions as an adapter to enhance the efficiency of the MAP kinase cascade.