Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases.

An appropriate level of protein phosphorylation on tyrosine is essential for cells to react to extracellular stimuli and maintain cellular homeostasis. Faulty operation of signal pathways mediated by protein tyrosine phosphorylation causes numerous human diseases, which presents enormous opportunities for therapeutic intervention. While the importance of protein tyrosine kinases in orchestrating the tyrosine phosphorylation networks and in target-based drug discovery has long been recognized, the significance of protein tyrosine phosphatases (PTPs) in cellular signaling and disease biology has historically been underappreciated, due to a large extent to an erroneous assumption that they are largely constitutive and housekeeping enzymes. Here, we provide a comprehensive examination of a number of regulatory mechanisms, including redox modulation, allosteric regulation, and protein oligomerization, that control PTP activity. These regulatory mechanisms are integral to the myriad PTP-mediated biochemical events and reinforce the concept that PTPs are indispensable and specific modulators of cellular signaling. We also discuss how disruption of these PTP regulatory mechanisms can cause human diseases and how these diverse regulatory mechanisms can be exploited for novel therapeutic development.

Nuclear-Biased DUSP6 Expression is Associated with Cancer Spreading Including Brain Metastasis in Triple-Negative Breast Cancer.

DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.

Follicle-Stimulating Hormone (FSH)-dependent Regulation of Extracellular Regulated Kinase (ERK) Phosphorylation by the Mitogen-activated Protein (MAP) Kinase Phosphatase MKP3.

Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH promotes the differentiation and proliferation of GCs and is essential for fertility. We recently reported that ERK activation is necessary for FSH to induce key genes that define the preovulatory GC. This research focused on the phosphoregulation by FSH of ERK within GCs. FSH-stimulated ERK phosphorylation on Thr(202)/Tyr(204) was PKA-dependent, but MEK(Ser(217)/Ser(221)) phosphorylation was not regulated; rather, MEK was already active. However, treatment of GCs with the EGF receptor inhibitor AG1478, a dominant-negative RAS, an Src homology 2 domain-containing Tyr phosphatase inhibitor (NSC 87877), or the MEK inhibitor PD98059 blocked FSH-dependent ERK(Thr(202)/Tyr(204)) phosphorylation, demonstrating the requirement for upstream pathway components. We hypothesized that FSH via PKA enhances ERK phosphorylation by inhibiting the activity of a protein phosphatase that constitutively dephosphorylates ERK in the absence of FSH, allowing MEK-phosphorylated ERK to accumulate in the presence of FSH because of inactivation of the phosphatase. GCs treated with different phosphatase inhibitors permitted elimination of both Ser/Thr and Tyr phosphatases and implicated dual specificity phosphatases (DUSPs) in the dephosphorylation of ERK. Treatment with MAP kinase phosphatase (MKP3, DUSP6) inhibitors increased ERK(Thr(202)/Tyr(204)) phosphorylation in the absence of FSH to levels comparable with ERK phosphorylated in the presence of FSH. ERK co-immunoprecipitated with Myc-FLAG-tagged MKP3(DUSP6). GCs treated with MKP3(DUSP6) inhibitors blocked and PKA inhibitors enhanced dephosphorylation of recombinant ERK2-GST in an in vitro phosphatase assay. Together, these results suggest that FSH-stimulated ERK activation in GCs requires the PKA-dependent inactivation of MKP3(DUSP6).

Differential expression profiles and roles of inducible DUSPs and ERK1/2-specific constitutive DUSP6 and DUSP7 in microglia.

Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1/2 activity both in resting and LPS-stimulated microglia. In turn, DUSP6 suppressed the basal phosphorylation of ERK1/2, but exerted no suppressive effect on LPS-induced phosphorylation. Although DUSP6 is acknowledged as a negative regulator of the ERK1/2 pathway, such roles of DUSP6 need to be examined further in activated microglia.

Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice.

Insulin plays an important role in the control of hepatic glucose production. Insulin resistant states are commonly associated with excessive hepatic glucose production, which contributes to both fasting hyperglycaemia and exaggerated postprandial hyperglycaemia. In this regard, increased activity of phosphatases may contribute to the dysregulation of gluconeogenesis. Mitogen-activated protein kinase phosphatase-3 (MKP-3) is a key protein involved in the control of gluconeogenesis. MKP-3-mediated dephosphorylation activates FoxO1 (a member of the forkhead family of transcription factors) and subsequently promotes its nuclear translocation and binding to the promoters of gluconeogenic genes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). In this study, we investigated the effects of exercise training on the expression of MKP-3 and its interaction with FoxO1 in the livers of obese animals. We found that exercised obese mice had a lower expression of MKP-3 and FoxO1/MKP-3 association in the liver. Further, the exercise training decreased FoxO1 phosphorylation and protein levels of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and gluconeogenic enzymes (PEPCK and G6Pase). These molecular results were accompanied by physiological changes, including increased insulin sensitivity and reduced hyperglycaemia, which were not caused by reductions in total body mass. Similar results were also observed with oligonucleotide antisense (ASO) treatment. However, our results showed that only exercise training could reduce an obesity-induced increase in HNF-4α protein levels while ASO treatment alone had no effect. These findings could explain, at least in part, why additive effects of exercise training treatment and ASO treatment were not observed. Finally, the suppressive effects of exercise training on MKP-3 protein levels appear to be related, at least in part, to the reduced phosphorylation of Extracellular signal-regulated kinases (ERK) in the livers of obese mice.

In vivo structure-activity relationship studies support allosteric targeting of a dual specificity phosphatase.

Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of fibroblast growth factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. However, in vivo whole-organism screens of zebrafish identified compound 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogues to define the structure-activity relationship (SAR) of DUSP6 inhibition. In vivo high-content analysis in transgenic zebrafish, coupled with cell-based chemical complementation assays, identified structural features of the pharmacophore of 1 that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses.

Dual-specific phosphatase-6 (Dusp6) and ERK mediate AMPA receptor-induced oligodendrocyte death.

Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter.

Dual-specificity phosphatases: therapeutic targets in cancer therapy resistance.

PURPOSE: Therapy resistance is the principal obstacle to achieving cures in cancer patients and its successful tackling requires a deep understanding of the resistance mediators. Increasing evidence indicates that tumor phosphatases are novel and druggable targets in translational oncology and their modulation may hinder tumor growth and motility and potentiate therapeutic sensitivity in various neoplasms via regulation of various signal transduction pathways. Dual-specificity phosphatases (DUSPs) are key players of cell growth, survival and death and have essential roles in tumor initiation, malignant progression and therapy resistance through regulation of the MAPK signaling pathway. In this review, different aspects of DUSPs are discussed. METHODS: A comprehensive literature review was performed using various websites including PubMed. RESULTS: We provide mechanistic insights into the roles of well-known DUSPs in resistance to a wide range of cancer therapeutic approaches including chemotherapy, radiation and molecular targeted therapy in human malignancies. Moreover, we discuss the development of DUSP modulators, with a focus on DUSP1 and 6 inhibitors. Ultimately, the preclinical investigations of small molecule inhibitors of DUSP1 and 6 are outlined. CONCLUSION: Emerging evidence indicates that the DUSP family is aberrantly expressed in human malignancies and plays critical roles in determining sensitivity to a wide range of cancer therapeutic strategies through regulation of the MAPK signaling pathways. Consequently, targeting DUSPs and their downstream molecules can pave the way for more effective cancer therapies.

Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages.

The dual-specificity phosphatase 6 (Dusp6) functions as a feedback regulator of fibroblast growth factor (FGF) signaling to limit the activity of extracellular signal-regulated kinases (ERKs) 1 and 2. We have identified a small-molecule inhibitor of Dusp6-(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI)-using a transgenic zebrafish chemical screen. BCI treatment blocked Dusp6 activity and enhanced FGF target gene expression in zebrafish embryos. Docking simulations predicted an allosteric binding site for BCI within the phosphatase domain. In vitro studies supported a model in which BCI inhibits Dusp6 catalytic activation by ERK2 substrate binding. We used BCI treatment at varying developmental stages to uncover a temporal role for Dusp6 in restricting cardiac progenitors and controlling heart organ size. This study highlights the power of in vivo zebrafish chemical screens to identify new compounds targeting Dusp6, a component of the FGF signaling pathway that has eluded traditional high-throughput in vitro screens.

Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis.

FBXO31 is the substrate receptor of one of many CUL1-RING ubiquitin ligase (CRL1) complexes. Here, we show that low FBXO31 mRNA levels are associated with high pre-operative prostate-specific antigen (PSA) levels and Gleason grade in human prostate cancer. Mechanistically, the ubiquitin ligase CRL1FBXO31 promotes the ubiquitylation-mediated degradation of DUSP6, a dual specificity phosphatase that dephosphorylates and inactivates the extracellular-signal-regulated kinase-1 and -2 (ERK1/2). Depletion of FBXO31 stabilizes DUSP6, suppresses ERK signaling, and activates the PI3K-AKT signaling cascade. Moreover, deletion of FBXO31 promotes tumor development in a mouse orthotopic model of prostate cancer. Treatment with BCI, a small molecule inhibitor of DUSP6, suppresses AKT activation and prevents tumor formation, suggesting that the FBXO31 tumor suppressor activity is dependent on DUSP6. Taken together, our studies highlight the relevance of the FBXO31-DUSP6 axis in the regulation of ERK- and PI3K-AKT-mediated signaling pathways, as well as its therapeutic potential in prostate cancer.

Bioactivities of simplified adociaquinone B and naphthoquinone derivatives against Cdc25B, MKP-1, and MKP-3 phosphatases.

Some simplified adociaquinone B analogs and a series of 1,4-naphthoquinone derivatives were synthesized and tested against the three enzymes Cdc25B, MKP-1, and MKP-3. Cdc25B and MKP-1 in particular are enzymes overexpressed in human cancer cells, and they represent potential molecular targets for novel cancer chemotherapeutic treatments. A number of analogs exhibited significant inhibitory activity against these enzymes, and the bioassay data in addition to structure-activity relationships of these compounds will be discussed.

A cell-active inhibitor of mitogen-activated protein kinase phosphatases restores paclitaxel-induced apoptosis in dexamethasone-protected cancer cells.

Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression.

Targeted Inhibition of the Dual Specificity Phosphatases DUSP1 and DUSP6 Suppress MPNST Growth via JNK.

PURPOSE: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. EXPERIMENTAL DESIGN: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. RESULTS: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. CONCLUSIONS: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.

DUSP6 Inhibitor (E/Z)-BCI Hydrochloride Attenuates Lipopolysaccharide-Induced Inflammatory Responses in Murine Macrophage Cells via Activating the Nrf2 Signaling Axis and Inhibiting the NF-κB Pathway.

Macrophages play a fundamental role in human chronic diseases such as rheumatoid arthritis, atherosclerosis, and cancer. In the present study, we demonstrated that dual-specificity phosphatase 6 (DUSP6) was upregulated by lipopolysaccharide (LPS) treatment of macrophages. (E/Z)-BCI hydrochloride (BCI) functions as a small molecule inhibitor of DUSP6, and BCI treatment inhibited DUSP6 expression in LPS-activated macrophages. BCI treatment inhibited LPS-triggered inflammatory cytokine production, including IL-1ß and IL-6, but not TNF-α, and also affected macrophage polarization to an M1 phenotype. In addition, BCI treatment decreased reactive oxygen species (ROS) production and significantly elevated the levels of Nrf2. Interestingly, pharmacological inhibition of DUSP6 attenuated LPS-induced inflammatory responses was independent of extracellular signal-regulated kinase (ERK) signaling. Furthermore, BCI treatment inhibited phosphorylation of P65 and nuclear P65 expression in LPS-activated macrophages. These results demonstrated that pharmacological inhibition of DUSP6 attenuated LPS-induced inflammatory mediators and ROS production in macrophage cells via activating the Nrf2 signaling axis and inhibiting the NF-κB pathway. These anti-inflammatory effects indicated that BCI may be considered as a therapeutic agent for blocking inflammatory disorders.

Loss of MKP3 mediated by oxidative stress enhances tumorigenicity and chemoresistance of ovarian cancer cells.

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells.

Pharmacological inhibition of DUSP6 suppresses gastric cancer growth and metastasis and overcomes cisplatin resistance.

Gastric cancer (GC) is the second cause of cancer-related death. Cisplatin (CDDP) is widely used as the standard GC treatment, but relapse and metastasis are common because of intrinsic or acquired drug resistance. The mitogen-activated protein kinase phosphatases (MAPK)-extracellular signal regulated kinases (ERK) pathway contributes to GC progression and drug resistance, but targeting the MAPK-ERK pathway is challenging in GC therapy. Here, we demonstrated that dual-specificity phosphatases 6 (DUSP6) was overexpressed in GC and predicted poor overall survival and progression-free survival. Knockdown DUSP6 inhibited GC proliferation, migration, invasion and induced apoptosis. (E/Z)-BCI hydrochloride (BCI), a DUSP6 small molecule inhibitor, increased the activity of ERK but interestingly decreased the expression of ERK response genes in BGC823, SGC7901 and CDDP-resistant SGC7901/DDP cells. BCI also caused cell death through the DNA damage response (DDR) pathway. Moreover, BCI inhibited cell proliferation, migration and invasion in a receptor-independent manner and enhanced CDDP cytotoxicity at pharmacological concentrations in the GC cells. In vivo experiments further showed that BCI enhances the antitumor effects of CDDP in cell-based xenografts and PDX models. In summary, our findings indicated that disruption of DUSP6 by BCI enhanced CDDP-induced cell death and apoptosis in GC may partly through ERK and DDR pathways. Thus, this study suggests that DUSP6 is a potential prognostic biomarker and a promising target for GC therapy.

Hyperactivation of ERK by multiple mechanisms is toxic to RTK-RAS mutation-driven lung adenocarcinoma cells.

Synthetic lethality results when mutant KRAS and EGFR proteins are co-expressed in human lung adenocarcinoma (LUAD) cells, revealing the biological basis for mutual exclusivity of KRAS and EGFR mutations. We have now defined the biochemical events responsible for the toxic effects by combining pharmacological and genetic approaches and to show that signaling through extracellular signal-regulated kinases (ERK1/2) mediates the toxicity. These findings imply that tumors with mutant oncogenes in the RAS pathway must restrain the activity of ERK1/2 to avoid toxicities and enable tumor growth. A dual specificity phosphatase, DUSP6, that negatively regulates phosphorylation of (P)-ERK is up-regulated in EGFR- or KRAS-mutant LUAD, potentially protecting cells with mutations in the RAS signaling pathway, a proposal supported by experiments with DUSP6-specific siRNA and an inhibitory drug. Targeting DUSP6 or other negative regulators might offer a treatment strategy for certain cancers by inducing the toxic effects of RAS-mediated signaling.

Activation of ERK1/2 Causes Pazopanib Resistance via Downregulation of DUSP6 in Synovial Sarcoma Cells.

Synovial sarcoma (SS) is a rare high-grade malignant mesenchymal tumour with a relatively poor prognosis despite intensive multimodal therapy. Although pazopanib, a multi-kinase inhibitor, is often used for advanced SS, most cases eventually become resistant to pazopanib. In the present study, we investigated the mechanisms of acquired pazopanib resistance in SS. To examine acquired pazopanib resistance, two SS cell lines, SYO-1 and HS-SY-II, were isolated after multiple selection steps with increasing concentrations of pazopanib. SYO-1 was also used in vivo. Then, pazopanib-resistant clones were investigated to assess potential mechanisms of acquired pazopanib resistance. Stable pazopanib-resistant clones were established and exhibited enhanced cell cycle progression, cell growth with increased ERK1/2 phosphorylation, and higher sensitivity than parental cells to a MEK-inhibitor, trametinib, both in vitro and in vivo. Furthermore, addition of low-dose trametinib partially reversed the pazopanib resistance. In the pazopanib-resistant clones, dual specificity phosphatase 6 (DUSP6) was downregulated. Inhibition of DUSP6 expression in parental HS-SY-II cells partially recapitulated acquired pazopanib resistance. Acquired pazopanib resistance in SS was associated with activation of ERK1/2 through downregulation of DUSP6 expression. Simultaneous treatment with pazopanib and a MEK inhibitor could be a promising strategy to overcome pazopanib resistance in SS.

Disinhibition of the extracellular-signal-regulated kinase restores the amplification of circadian rhythms by lithium in cells from bipolar disorder patients.

UNLABELLED: Bipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs. CONTROLS: Pharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.