Dual-specificity Protein Phosphatase Research Articles

Inhibition of miR-217 Protects Against Myocardial Ischemia–Reperfusion Injury Through Inactivating NF-κB and MAPK Pathways

Recent studies have demonstrated that miRNAs play a vital role in regulating myocardial ischemia/reperfusion injury (MIRI). MiR-217 has been proven to be implicated in cardiac diseases such as chronic heart failure and cardiac myxoma. However, the role of miR-217 in MIRI is not clear. A mouse MIRI model was established and the myocardial infarct size was evaluated by TTC staining. The expression level of miR-217 in I/R group was determined by real-time polymerase chain reaction. Subsequently, MIRI mice and H9C2 cells were administrated with miR-217 inhibitor in vivo and in vitro, respectively. The levels of TNF-α and IL-6 were measured by commercially available ELISA kits. Blood and cell samples were collected for the measurement of lactate dehydrogenase (LDH) level and caspase-3 activity. Cell viability was assessed with the CCK-8 assay. We then explored the detailed molecular mechanisms by TargetScan 7.1 database and further studies were performed to prove the prediction by dual-luciferase reporter assay. Larger stainless infarct areas were observed in the MIRI group, accompanied by inceased serum LDH activity, indicating the mouse MIRI model was successfully established. MiR-217 was up-regulated in MIRI mice and hypoxia/reoxygenation-treated H9C2 cells. MiR-217 knockdown alleviated the MIRI in MIRI mouse model, and also attenuated the myocardial hypoxia/reoxygenation injury in H9C2 cells. Moreover, dual specificity protein phosphatase 14 (DUSP14) was proved to be a target of miR-217. Besides, further study indicated that inhibition of miR-217 protected against MIRI through inactivating NF-κB and MAPK pathways via targeting DUSP14. MiR-217 inhibition protected against MIRI through inactivating NF-κB and MAPK pathways by targeting DUSP14. This study may provide valuable diagnostic and factors and therapeutic agents for MIRI.

Influence of dual-specificity protein phosphatase 5 on mechanical properties of rat cerebral and renal arterioles.

We recently reported that KO of Dual‐specificity protein phosphatase 5 (Dusp5) enhances myogenic reactivity and blood flow autoregulation in the cerebral and renal circulations in association with increased levels of pPKC and pERK1/2 in the cerebral and renal arteries and arterioles. In the kidney, hypertension‐related renal damage was significantly attenuated in Dusp5 KO rats. Elevations in pPKC and pERK1/2 promote calcium influx in VSMC and facilitate vasoconstriction. However, whether DUSP5 plays a role in altering the passive mechanical properties of cerebral and renal arterioles has never been investigated. In this study, we found that KO of Dusp5 did not alter body weights, kidney and brain weights, plasma glucose, and HbA1C levels. The expression of pERK is higher in the nucleus of primary VSMC isolated from Dusp5 KO rats. Dusp5 KO rats exhibited eutrophic vascular hypotrophy with smaller intracerebral parenchymal arterioles and renal interlobular arterioles without changing the wall‐to‐lumen ratios. These arterioles from Dusp5 KO rats displayed higher myogenic tones, better distensibility, greater compliance, and less stiffness compared with arterioles from WT control rats. VSMC of Dusp5 KO rats exhibited a stronger contractile capability. These results demonstrate, for the first time, that DUSP5 contributes to the regulation of the passive mechanical properties of cerebral and renal arterioles and provide new insights into the role of DUSP5 in vascular function, cancer, stroke, and other cardiovascular diseases.

Inhibition of miR-429 improves neurological recovery of traumatic brain injury mice and attenuates microglial neuroinflammation

BackgroundNeuroinflammation is a common therapeutic target for traumatic brain injury (TBI) due to its contribution to delayed secondary cell death and has the potential to occur for years after the initial insult. Previous studies demonstrate that miR-429 is up-regulated in the brain lesions of TBI mice, while its role in regulating neuroinflammation and brain injury remains largely unknown. MethodThe expression of miR-429 in LPS-activated microglia and microglia in TBI model was detected by RT-PCR. The effects of miR-429 inhibitors on LPS-activated microglia in vitro as well as neurological recovery and post-traumatic neuroinflammatory response in TBI model mice were detected in vivo. ResultsLPS and TBI significantly induce the up-expression of miR-429, inflammatory cytokines, MAPK-p38 and phosphorylated NF-κB in microglia, which were all inhibited by miR-429 inhibitors. Meanwhile, miR-429 inhibitors also attenuated the neurological impairment in TBI mice. Bioinformatics analysis showed that miR-429 could target and inhibit the expression of dual specificity protein phosphatase 1 (DUSP1), thus inhibiting the expression of MAPK-p38 and phosphorylated NF-κB. ConclusionmiR-429 plays a pro-inflammatory role in activated microglia by targeting DUSP1 signaling pathway. Inhibiting miR-429 can attenuate the inflammatory response of microglia and TBI-mediated brain damage.

Dual-specificity protein phosphatase DUSP4 regulates response to MEK inhibition in BRAF wild-type melanoma

BackgroundAiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.059) with docetaxel plus selumetinib. NRAS status did not associate with outcome. Here, the aim was to identify novel biomarkers of response to MEKi.MethodsA MEK 6 gene signature was quantified using NanoString and correlated with clinical outcomes. Two components of the gene signature were investigated by gene silencing in BRAF/NRAS wild-type melanoma cells.ResultsIn melanomas of patients on the selumetinib but not the placebo arm, two gene signature components, dual-specificity protein phosphatase 4 (DUSP4) and ETS translocation variant 4 (ETV4), were expressed more highly in responders than non-responders. In vitro, ETV4 depletion inhibited cell survival but did not influence sensitivity to MEKi selumetinib or trametinib. In contrast, DUSP4-depleted cells showed enhanced cell survival and increased resistance to both selumetinib and trametinib.ConclusionsETV4 and DUSP4 associated with clinical response to docetaxel plus selumetinib. DUSP4 depletion induced MEKi resistance, suggesting that DUSP4 is not only a biomarker but also a mediator of MEKi sensitivity.Clinical Trial RegistrationDOC-MEK (EudraCT no: 2009-018153-23).

Abstract C51: Targeted inhibition of DUSP1 and DUSP6 suppresses pancreatic adenocarcinoma cells’ growth and glucose metabolism via SAPK/JNK pathway activation

Abstract The highly malignant potential and aggressiveness of pancreatic adenocarcinoma cells is mainly promoted by oncogenic KRAS activation, which triggers numerous signaling pathways and controls key processes for tumor progression such as cell survival and metabolic reprograming. The major adjustments in metabolic reprogramming mediated by KRAS activity are controlled by MAP kinase activities, which are precisely regulated by protein phosphatases to coordinate complex signaling networks. The major class of phosphatases controlling MAPKs is represented by dual-specificity protein phosphatases (DUSPs). As DUSPs’ role in PDAC cells remains to be elucidated, here we investigated the impact of DUSPs on PDAC chemoresistance and metabolic reprogramming. PDAC cell lines (MIA PaCa-2 and PANC-1) stably expressing the Cas9 endonuclease were submitted to CRISPR using an Edit-R library (GE Dharmacon) of crRNAs targeting all DUSP genes with 3 crRNAs per gene. Phenotypic analysis was conducted to address PDAC cells’ glucose uptake and identify the most relevant targets. Glucose uptake and lactate production were performed by flow cytometry and colorimetric assay, respectively. DUSP1 and DUSP6 were inhibited through a pharmacologic and genetics approach. Gemcitabine response was performed in a 4-day MTT assay. In vitro assays such as cell viability, apoptosis, colony-forming assay, ROS production, glucose uptake rate, and protein expression were performed in three independent experiments. For gene expression, a PCR array with a set of 92 glucose metabolism-related genes has been performed. RNAseq data from pancreatic adenocarcinoma (data generated by the TCGA Research Network: http://cancergenome.nih.gov/) was analyzed by using the LinkedOmics platform (Vasaikar S, 2017). Among the whole DUSP family members, CRISPR screening revealed DUSP1 and DUSP6 as the major players in glucose uptake control in PDAC cell lines. BCI (inhibitor of DUSP1 and DUSP6) treatment of PDAC cells significantly modulated glucose uptake rates (p<0.002) and drastically reduced cell viability and survival (p<0.003), followed by a reduction in reactive oxygen species (ROS) production (p<0.05). DUSP1 and DUSP6 inhibition also provoked an increase on apoptosis (p<0.001) and sensitized cells to gemcitabine DUSPs inhibition by BCI promoted JNK activation but did not impact other MAPKs’ activity. Interestingly, we identified a differential gene expression signature of glucose metabolism and TCA cycle in both PCR array upon DUSP1 and DUSP6 modulation and from analysis of PDAC-TCGA data. Taken together, these data suggest that DUSP1 and DUSP6 may play an important role on PDAC cell survival through glycolytic and TCA cycle intermediates’ modulation. Additionally, these data point to BCI as a potential drug strategy to PDAC, which brings additional information to contribute to therapeutic target possibilities against this dismal disease. Citation Format: Pamela V. Andrade, Mariana T. Ruckert, Carlos Alberto O. Biagi Junior, Vanessa S. Silveira. Targeted inhibition of DUSP1 and DUSP6 suppresses pancreatic adenocarcinoma cells’ growth and glucose metabolism via SAPK/JNK pathway activation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C51.

CAMP Response Element Modulator α Induces Dual Specificity Protein Phosphatase 4 to Promote Effector T Cells in Juvenile-Onset Lupus

Effector CD4+ T cells with increased IL-17A and reduced IL-2 production contribute to tissue inflammation and organ damage in systemic lupus erythematosus (SLE). Increased expression of the transcription factor cAMP response element modulator (CREM) α promotes altered cytokine expression in SLE. The aim of this study was to investigate CREMα-mediated events favoring effector CD4+ T cells in health and disease. Using CRISPR/Cas9 genome editing and lentiviral transduction, we generated CREMα-deficient and CREMα-overexpressing Jurkat T cells. Gene expression and regulatory events were assessed using luciferase reporter assays and chromatin immunoprecipitation. Interaction between CREMα and p300 was investigated using proximity ligation assays, coimmunoprecipitation, and knockdown of p300. Gene expression profiles of modified cells were compared with CD4+ T cells from patients with juvenile-onset SLE. We show that CREMα induces dual specificity protein phosphatase (DUSP) 4 in effector CD4+ T cells through corecruitment of p300. The transcriptional coactivator p300 mediates histone acetylation at DUSP4, prompting increased gene expression. Using DUSP4 transfection models and genetically modified CREM-deficient and CREMα-overexpressing T cells, we demonstrate the molecular underpinnings by which DUSP4 induces IL-17A while limiting IL-2 expression. We demonstrate that CD4+ T cells from patients with juvenile-onset SLE share phenotypical features with CREMα-overexpressing CD4+ T cells, including increased DUSP4 expression and imbalanced IL-17A and IL-2 production. Taken together, we describe CREMα-mediated mechanisms that involve the transcriptional upregulation of DUSP4, leading to imbalanced cytokine production by effector T cells. Our findings identify the CREMα/DUSP4 axis as a promising candidate in the search for biomarkers and therapeutic targets in SLE.

Identification of an Apis cerana cerana MAP kinase phosphatase 3 gene (AccMKP3) in response to environmental stress.

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

MicroRNA-21 deficiency attenuated atherogenesis and decreased macrophage infiltration by targeting Dusp-8

Background and aimsAtherosclerosis is a chronic inflammatory disorder mediated by macrophage activation. MicroRNA-21 (miR-21) is a key regulator in the macrophage inflammatory response. However, the functional role of miR-21 in atherogenesis is far from clear. Methods and resultsHere, we report that miR-21 is significantly upregulated in mouse atherosclerotic plaques and peripheral monocytes from patients with coronary artery disease. Compared with miR-21+/+apoE−/− mice (apoE−/− mice), miR-21−/−apoE−/− (double knockout, DKO) mice showed less atherosclerotic lesions, reduced presence of macrophages, decreased smooth muscle cells(SMC) and collagen content in the aorta. We further explored the role of miR-21 in macrophage activation in vitro. Bone marrow-derived macrophages (BMDMs) from DKO mice not only exhibit impaired function of migration induced by chemokine (C–C motif) ligand 2 (CCL2) but also a weakened macrophage-endothelium interaction activated by tumor necrosis factor-α (TNF-α). However, atherogenic inflammatory cytokine secretion was not affected by miR-21 in vitro or in vivo. Additionally, miR-21 knockdown in BMDMs directly derepressed the expression of dual specificity protein phosphatase 8 (Dusp-8), a previously validated miR-21 target in cardiac fibroblasts, which negatively regulates mitogen-activated protein kinase (MAPK) signaling, particularly the p38-and c-Jun N-terminal kinase (JNK)-related signaling pathways. ConclusionsThese data demonstrate that inhibition of miR-21 may restrict the formation of atherosclerotic plaques partly by regulating macrophage migration and adhesion, while, reduced SMCs and collagen content in plaques may lead to a less stable phenotype with the progression of atherosclerosis. Thus, the absence of miR-21 reduces atherosclerotic lesions but may not represent all benefit in atherosclerosis development.

Dual-Specific Protein and Lipid Phosphatase PTEN and Its Biological Functions.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a 403-amino acid protein with an amino-terminal domain that shares sequence homology with the actin-binding protein tensin and the putative tyrosine-protein phosphatase auxilin. Crystal structure analysis of PTEN has revealed a C2 domain that binds to phospholipids in membranes and a phosphatase domain that displays dual-specific activity toward both tyrosine (Y), serine (S)/threonine (T), as well as lipid substrates in vitro. Characterized primarily as a lipid phosphatase, PTEN plays important roles in multiple cellular processes including cell growth/survival as well as metabolism.

Role of dual-specificity protein phosphatase DUSP10/MKP-5 in pulmonary fibrosis.

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP-5) is a member of the dual-specificity family of protein tyrosine phosphatases that negatively regulates p38 MAPK and the JNK. MKP-5-deficient mice exhibit improved muscle repair and reduced fibrosis in an animal model of muscular dystrophy. Here, we asked whether the effects of MKP-5 on muscle fibrosis extend to other tissues. Using a bleomycin-induced model of pulmonary fibrosis, we found that MKP-5-deficient mice were protected from the development of lung fibrosis, expressed reduced levels of hydroxyproline and fibrogenic genes, and displayed marked polarization towards an M1-macrophage phenotype. We showed that the profibrogenic effects of the transforming growth factor-β1 (TGF-β1) were inhibited in MKP-5-deficient lung fibroblasts. MKP-5-deficient fibroblasts exhibited enhanced p38 MAPK activity, impaired Smad3 phosphorylation, increased Smad7 levels, and decreased expression of fibrogenic genes. Myofibroblast differentiation was attenuated in MKP-5-deficient fibroblasts. Finally, we found that MKP-5 expression was increased in idiopathic pulmonary fibrosis (IPF)-derived lung fibroblasts but not in whole IPF lungs. These data suggest that MKP-5 plays an essential role in promoting lung fibrosis. Our results couple MKP-5 with the TGF-β1 signaling machinery and imply that MKP-5 inhibition may serve as a therapeutic target for human lung fibrosis.

Abstract P160: Dual-Specificity Protein Phosphatase 5 Regulates Renal Vessel Myogenic Response via Direct Modulating Extracellular Signal-Related Kinase and Protein Kinase C

Dual-specificity protein phosphatase 5 (DUSP5) is a serine-threonine phosphatase that regulates intracellular signal transduction by dephosphorylating extracellular signal-related kinase (ERK1/2) and protein kinase C (PKC). We previously reported that knockout (KO) of Dusp5 enhanced the myogenic response and autoregulation of cerebral and renal circulation in association with increased levels of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated protein kinase C (p-PKC) in the vessels. However, the influence of increased levels of p-ERK1/2 and p-PKC on vascular function is unknown. This study investigated whether Dusp5 regulates myogenic reactivity of freshly isolated middle cerebral arteries (MCA, ~140 um), cerebral penetrating arteries (PA, ~15 um), renal arcuate arterioles (AA, ~ 50 um), and interlobular arterioles (IA, ~25 um) of Dusp5 KO and WT rats via inhibition of ERK1/2 and PKC. The myogenic reactivity of both cerebral and renal vasculature was compared at different perfusion pressure. The inner diameters of IA and AA decreased to 87.24 ± 0.76 % and 92.81 ± 2.90 %, respectively, in Dusp5 KO rats (n=4), but only to 99.70 ± 2.76 % and 98.85 ± 2.79 %, respectively, in WT rats (n=4) when perfusion pressure was increased from 60 to 180 mmHg. Similar results were found in MCA and PA. Inhibition of ERK1/2 with FR 180204 (1 uM) or PKC with BIM III (300 nM) diminished myogenic reactivity to a greater extent in cerebral and renal vessels of Dusp5 KO versus WT rats. These results indicate that downregulation of DUSP5 expression increases the myogenic reactivity of both cerebral and renal arteries (both large and small vessels) by increasing p-ERK1/2 and p-PKC levels. Targeting Dusp5 may offer a novel approach for preventing hypertension-induced end-organ damage.

Knockout of Dual-Specificity Protein Phosphatase 5 Protects Against Hypertension-Induced Renal Injury.

Dual-specificity protein phosphatase 5 (DUSP5) is a member of the tyrosine-threonine phosphatase family with the ability to dephosphorylate and inactivate extracellular signal-related kinase (ERK). The present study investigates whether knockout (KO) of Dusp5 improves renal hemodynamics and protects against hypertension-induced renal injury. The renal expression of DUSP5 was reduced, and the levels of phosphorylated (p) ERK1/2 and p-protein kinase C (PKC) α were elevated in the KO rats. KO of Dusp5 enhanced the myogenic tone of the renal afferent arteriole and interlobular artery in vitro with or without induction of deoxycorticosterone acetate-salt hypertension. Inhibition of ERK1/2 and PKC diminished the myogenic response to a greater extent in Dusp5 KO rats. Autoregulation of renal blood flow was significantly impaired in hypertensive wild-type (WT) rats but remained intact in Dusp5 KO animals. Proteinuria was markedly decreased in hypertensive KO versus WT rats. The degree of glomerular injury was reduced, and the expression of nephrin in the glomerulus was higher in hypertensive Dusp5 KO rats. Renal fibrosis and medullary protein cast formation were attenuated in hypertensive Dusp5 KO rats in association with decreased expression of monocyte chemoattractant protein 1, transforming growth factor-β1, matrix metalloproteinase (MMP) 2, and MMP9. These results indicate that KO of Dusp5 protects against hypertension-induced renal injury, at least in part, by maintaining the myogenic tone of the renal vasculature and extending the range of renal blood flow autoregulation to higher pressures, which diminish glomerular injury, protein cast formation, macrophage infiltration, and epithelial-mesenchymal transformation in the kidney. SIGNIFICANCE STATEMENT: Dual-specificity protein phosphatase 5 (DUSP5) is a tyrosine-threonine phosphatase that inactivates extracellular signal-related kinase (ERK). We previously reported that knockout (KO) of Dusp5 enhanced the myogenic response and autoregulation in the cerebral circulation. The present study investigates whether KO of DUSP5 improves renal hemodynamics and protects against hypertension-induced renal injury. Downregulation of DUSP5 enhanced the myogenic tone of renal arteriole and artery and autoregulation of renal blood flow in association with reduced proteinuria, glomerular injury, and interstitial fibrosis after the induction of hypertension. Inhibition of ERK1/2 and protein kinase C diminished the myogenic response to a greater extent in Dusp5 KO rats. These results suggest that DUSP5 might be a viable drug target for the treatment of hypertension nephropathy.

Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells.

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to DUSP transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation. Potent DUSP gene inducers also include cannabinoids, which preserve DUSP activity in inflammatory conditions. Additionally, nucleotides activating P2X7 and P2Y13 nucleotide receptors behave as novel players in the regulation of DUSP function. They increase cell survival in stressful conditions, regulating DUSP protein turnover and inducing DUSP gene expression. In general terms, in the context of neural cells exposed to damaging conditions, the recovery of DUSP activity is neuroprotective and counteracts pro-apoptotic over-activation of p38 and JNK. In addition, remarkable changes in DUSP function take place during the onset of neuropathologies. The restoration of proper DUSP levels and recovery of MAPK homeostasis underlie the therapeutic effect, indicating that DUSPs can be relevant targets for brain diseases.

Functionalized nanoparticles containing MKP-1 agonists reduce periodontal bone loss.

Progress over of the past several years has elucidated a role for mitogen-activated protein kinase phosphatase to regulate periodontal inflammation yielding new possibilities for treatment of periodontal diseases. These studies aimed to determine if nanoparticles (NPs) loaded with a pharmacological agent that induces mitogen-activated protein kinase phosphatase have potential clinical utility for management of periodontal inflammation and alveolar bone. Polyethylene glycol (PEG)-polylactide (PLA) (PEG-PLA) NPs were loaded with auranofin (ARN), an antirheumatic drug, to induce mitogen-activated protein kinase phosphatase (MKP)-1 expression in vitro and in vivo. Release kinetics of ARN from NPs was performed by high performance liquid chromatography (HPLC). Fluorescent-labeled NPs were used to show uptake into macrophages by flow cytometry. Real-time quantitative polymerase chain reaction (qPCR) was used to determine dual specificity protein phosphatase (Dusp)-1 mRNA induction by Auranofin-loaded nanoparticles (ARN-NPs) and viability of ARN-NPs was determined by colorimetric in vitro assays. Functional in vitro assays were used to measure functional MKP-1 induction and preclinical models using Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced alveolar bone loss and microcomputed tomography was used to determine in vivo efficacy of functionalized ARN-NPs. Data indicated that ARN-NPs had reduced cytotoxicity compared with free ARN and Dusp1 mRNA and MKP-1 activity was significantly increased by ARN-NPs in vitro. Flow cytometry indicated rapid uptake into macrophages. Finally, significant bone loss reduction was observed with ARN-NPs compared with control NPs in vivo using an lipopolysaccharide-induced rat model of periodontitis. Results from these studies suggest that developing NPs functionalized with ARN have anti-inflammatory activities and may be a novel adjuvant therapeutic strategy to significantly improve periodontitis therapy and outcomes.

RAB14 activates MAPK signaling to promote bladder tumorigenesis.

Bladder cancer (BC) is a fatal invasive malignancy accounting for approximately 5% of all cancer deaths in humans; however, the underlying molecular mechanisms and potential targeted therapeutics for BC patients remain unclear. We report herein that RAB14 was overexpressed in BC tissues and cells with high metastatic potential and its abundance was significantly associated with lymph node metastasis (P = 0.001), a high-grade tumor stage (P = 0.009), poor differentiation (P < 0.001) and unfavorable prognoses of BC patients (P = 0.003, log-rank test). Interference by RAB14 mediated a reduction in the TWIST1 protein and inhibited cell migration and invasion (P < 0.05). Moreover, silencing RAB14 reduced cell proliferation and induced apoptosis in vitro and suppressed tumorigenesis in a mouse xenograft model. We demonstrated that RAB14-promoted BC cancer development and progression were associated with activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase signaling through upregulation of MAPK1/MAPK8 and downregulation of dual-specificity protein phosphatase 6/Src homology 2 domain containing transforming protein/Fos proto-oncogene, AP-1 transcription factor subunit (FOS). We provide evidence that RAB14 acts as a tumor promoter and modulates the invasion and metastatic potential of BC cells via activating the MAPK pathway.

Exploration of Proteins Involved in Acquisition of Resistance to Cetuximab

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (Mabs) show high efficacy in about 50% of colorectal cancer (CRC) patients with wild-type KRAS. However, &lt; 20% of patients with KRAS wild-type CRC have continued therapeutic effects with these agents, and acquired resistance to treatment has become a serious clinical problem. In this study, to clarify the factors related to acquisition of resistance to cetuximab (Cmab) and establish countermeasures against such acquired resistance, we conducted a comprehensive protein analysis via a proteomics approach using acquired resistance cell lines derived from Cmab-sensitive CRC cell lines and original cell lines. Cmab-acquired resistance cell lines were generated by continuous exposure of SW48 and C99 cell lines to Cmab. Expression of dCK and zinc finger and BTB domain-containing protein 41 (ZBTB41) increased more than 10-fold, and dual specificity protein phosphatase 3 (DUS3) expression decreased by less than 1/10 with acquisition of resistance to Cmab in both C99 and SW48 cell lines. Because overexpression of dCK is known as a positive indicator of efficacy of nucleoside analogs such as cytarabine or gemcitabine, it is considered that nucleoside analogs activated by dCK may be useful agents in treatment of cancers with acquired Cmab-resistance. In the future, we need to clarify the usefulness of these drugs for the treatment of Cmab resistant CRC and to assess the possibility of restoration of Cmab sensitivity by regulation of ZBTB41 and DUS3 expression.Keyword : cetuximab, colorectal cancer, acquired resistance, protein, dCK, ZBTB41

DUSP22 promotes senescence of HS-1 skin cancer cells through triggering MAPK signaling pathway.

Skin cancer severely threatens the public health. Dual-specificity protein phosphatase 22 (DUSP22) characterizes with multiple roles regulating cell growth, proliferation and gaining. This study aims to investigate the regulatory role of DUSP22 on aging of skin cancer cells and clarify molecular mechanisms, along with potential application value. HS-1 skin cancer cells were transfected with DUSP22 by liposome transfection approach. Western blot assay was used to evaluate the effects of DUSP22 on aging protein of HS-1 cells, and activation of mitogen-activated protein kinase cascade (MAPK) signal pathway. Real-time PCR (RT-PCR) and Western blot assay were used to examine the DUSP22 expression in HS-1 cancer cells. Meanwhile, the correlation between P53 protein and aging was also investigated. Transfection of DUSP22 plasmid significantly elevated DUSP22 expression in HS-1 skin cancer cells compared to un-transfected cells (p<0.05), and activated MAPK to induce cell aging. Transfection of small interfere RNA (siRNA) DUSP22 significantly suppressed DUSP22 expression in HS-1 cancer cells, inhibited MAPK signal pathway, and decreased aging proteins P53 and P21 compared to the untreated group (p<0.05). DUSP22 was downregulated in HS-1 skin cancer cells, with MAPK signal pathway inhibition, and lower aging protein P53 expression. DUSP22 expression was positively correlated with aging protein P53 (p<0.05). DUSP22 facilitated HS-1 skin cancer cell aging via activating MAPK signal pathway, possibly providing novel strategy against skin cancer.

Stressing out or stressing in: intracellular pathways for SAPK activation.

Stress-activated MAP kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPKs are not known. Our recent findings have begun to address two important and related questions. First, do various stresses activate a SAPK through common pathways initiated at the cell surface, or through alternative, intracellular inputs? Second, how does an activated SAPK mount a specific response appropriate to the particular stress experienced? Our work has uncovered the mechanisms by which two stresses, arsenite treatment and DNA damage, stimulate the yeast SAPKs Hog1 and Mpk1, respectively. We found that these stresses activate the SAPKs through intracellular inputs that modulate their basal phosphorylation, rather than by activation of the protein kinase cascades known to stimulate them. Both stresses act through targeting, in different ways, the tyrosine-specific or dual-specificity protein phosphatases that normally maintain the SAPKs in a low-activity state. Previous work has demonstrated that basal signal flux through SAPK pathways is important for the sensitivity and dynamic response to external signals. Our work reveals that basal activity of SAPKs is additionally important to allow SAPK activation by intracellular inputs that modulate that activity. Additionally, because different stressors may activate SAPKs by modulation of basal signal through inputs at distinct nodes along the canonical activation pathway, stress-specific SAPK outputs may be controlled, in part, by the specific intracellular mechanisms of their activation. Thus, understanding the intracellular pathways through which various stressors activate SAPKs is likely to provide insight into how they elicit physiologically coherent responses to the specific stress experienced.

The high NRF2 expression confers chemotherapy resistance partly through up-regulated DUSP1 in myelodysplastic syndromes.

Although cytarabine has been widely considered as one of the chemotherapy drugs for high-risk myelodysplastic syndromes (MDS), the overall response rate is only approximately 20-30%. Nuclear factor erythroid 2-related factor 2 (NRF2, also called NFE2L2) has been shown to play a pivotal role in preventing cancer cells from being affected by chemotherapy. However, it is not yet known whether NRF2 can be used as a prognostic biomarker in MDS, or whether elevated NRF2 levels are associated with cytarabine resistance. Here, we found that NRF2 expression levels in bone marrow from high-risk patients exceeded that of low-risk MDS patients. Importantly, high NRF2 levels are correlated with inferior outcomes in MDS patients (n=137). Downregulation of NRF2 by the inhibitor Luteolin, or lentiviral shRNA knockdown, enhanced the chemotherapeutic efficacy of cytarabine, while MDS cells treated by NRF2 agonist Sulforaphane showed increased resistance to cytarabine. More importantly, pharmacological inhibition of NRF2 could sensitize primary high-risk MDS cells to cytarabine treatment. Mechanistically, downregulation of dual specificity protein phosphatase 1, an NRF2 direct target gene, could abrogate cytarabine resistance in NRF2 elevated MDS cells. Silencing NRF2 or dual specificity protein phosphatase 1 also significantly sensitized cytarabine treatment and inhibited tumors in MDS cells transplanted mouse models in vivo. Our study suggests that targeting NRF2 in combination with conventional chemotherapy could pave the way for future therapy for high-risk MDS.

USP49 inhibits ischemia-reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1-JNK1/2 signaling.

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia-reperfusion (I/R) injury. However, the regulation of DUSP1-JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1-JNK1/2 signaling pathway with a protective role in cardiac I/R injury.