Extracellular Signal-regulated Kinase Target Research Articles

TRIM21 induces selective autophagic degradation of c-Myc and sensitizes regorafenib therapy in colorectal cancer

Kirsten rat sarcoma virus (KRAS) mutation is associated with malignant tumor transformation and drug resistance. However, the development of clinically effective targeted therapies for KRAS-mutant cancer has proven to be a formidable challenge. Here, we report that tripartite motif-containing protein 21 (TRIM21) functions as a target of extracellular signal-regulated kinase 2 (ERK2) in KRAS-mutant colorectal cancer (CRC), contributing to regorafenib therapy resistance. Mechanistically, TRIM21 directly interacts with and ubiquitinates v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) at lysine 148 (K148) via K63-linkage, enabling c-Myc to be targeted to the autophagy machinery for degradation, ultimately resulting in the downregulation of enolase 2 expression and inhibition of glycolysis. However, mutant KRAS (KRAS/MT)-driven mitogen-activated protein kinase (MAPK) signaling leads to the phosphorylation of TRIM21 (p-TRIM21) at Threonine 396 (T396) by ERK2, disrupting the interaction between TRIM21 and c-Myc and thereby preventing c-Myc from targeting autophagy for degradation. This enhances glycolysis and contributes to regorafenib resistance. Clinically, high p-TRIM21 (T396) is associated with an unfavorable prognosis. Targeting TRIM21 to disrupt KRAS/MT-driven phosphorylation using the antidepressant vilazodone shows potential for enhancing the efficacy of regorafenib in treating KRAS-mutant CRC in preclinical models. These findings are instrumental for KRAS-mutant CRC treatment aiming at activating TRIM21-mediated selective autophagic degradation of c-Myc.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH).Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression.Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

Novel TENM3-ALK fusion is an alternate mechanism for ALK activation in neuroblastoma.

The identification of molecular events underlying the pathogenesis of neuroblastoma can likely result in improved clinical outcomes for this disease. In this study, a translocation within chromosome 2p and 4q was found to bring about the formation of an in-frame fusion gene that was composed of portions of the teneurin transmembrane protein 3 (TENM3, also known as ODZ3) gene and the anaplastic lymphoma kinase (ALK) gene in tumor cells from patients with neuroblastoma. Expression of the full length TENM3-ALK cDNA in NIH-3T3 cells led to the formation of a fusion protein that: (1) possesses constitutive tyrosine kinase activity, (2) induces strong activation of the downstream targets of extracellular signal-regulated kinase (ERK), protein kinase B (a.k.a. AKT), and signal transducer and activator of transcription 3 (STAT3), (3) provokes oncogenic transformation in NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic mice, and (4) possesses sensitivity to ALK inhibitors in vitro and in vivo. Our findings demonstrated that patients with neuroblastoma may express a transforming fusion kinase, which is a promising candidate for a therapeutic target and a diagnostic molecular marker for neuroblastoma. The in-frame 5' partner gene that fuses with ALK has not been reported previously in neuroblastoma. Our data provide novel biological insights into the mechanism of ALK activation due to translocation, with implications for neuroblastoma tumorigenesis, and could be useful as a vital marker for the accurate diagnosis of this type of neuroblastoma.

Inhibition of Glutamate Release from Rat Cortical Nerve Terminals by Dehydrocorydaline, an Alkaloid from Corydalis yanhusuo.

Excessive release of glutamate induces excitotoxicity and causes neuronal damage in several neurodegenerative diseases. Natural products have emerged as potential neuroprotective agents for preventing and treating neurological disorders. Dehydrocorydaline (DHC), an active alkaloid compound isolated from Corydalis yanhusuo, possesses neuroprotective capacity. The present study investigated the effect of DHC on glutamate release using a rat brain cortical synaptosome model. Our results indicate that DHC inhibited 4-aminopyridine (4-AP)-evoked glutamate release and elevated intrasynaptosomal calcium levels. The inhibitory effect of DHC on 4-AP-evoked glutamate release was prevented in the presence of the vesicular transporter inhibitor bafilomycin A1 and the N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC but not the intracellular inhibitor of Ca2+ release dantrolene or the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157. Moreover, the inhibitory effect of DHC on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) inhibitor PD98059. Western blotting data in synaptosomes also showed that DHC significantly decreased the level of ERK1/2 phosphorylation and synaptic vesicle-associated protein synapsin I, the main presynaptic target of ERK. Together, these results suggest that DHC inhibits presynaptic glutamate release from cerebrocortical synaptosomes by suppressing presynaptic voltage-dependent Ca2+ entry and the MAPK/ERK/synapsin I signaling pathway.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

Alteration in cardiac PI3K/Akt/mTOR and ERK signaling pathways with the use of growth hormone and swimming, and the roles of miR21 and miR133.

Athletes misuse recombinant human growth hormone (r-hGH) to enhance their performance. Although r-hGH is known to increase cardiac hypertrophy, the underlying molecular mechanism remains unclear. The aim of the present study was to investigate the role of r-hGH in cardiac intracellular signaling pathways and of miR-21 and miR-133 expression in rat hearts during exercise. A total of 36 adult male Sprague-Dawley rats were divided into sedentary control (SC, n=9), swimming exercise (SE, n=8), r-hGH (GH, n=10) and swimming exercise plus r-hGH (SE-GH, n=9) groups. The exercise groups completed a 1-h swimming exercise 5 times a week for 8 weeks. Subcutaneous r-hGH was administered as 0.3 mg/kg/day. Phosphoinositide-3-kinase (PI3K), serine/threonine protein kinase 1 (AKT1), extracellular signal-regulated kinase (ERK), microRNA (miR)-21 and miR-133 expression was evaluated in ventricular muscle by real-time quantitative polymerase chain reaction. Protein expression of PI3K, AKT1, ERK and mechanistic target of rapamycin (mTOR) was also assessed by immunohistochemistry. Statistical differences were analyzed by two-way ANOVA. PI3K and AKT1 expression and the gene and protein levels was notably increased in the SE-GH group compared with in SC ventricular tissues (P<0.05). mTOR protein expression was higher in the GH, SE and SE-GH groups compared with in the SC group (P<0.05, <0.05 and <0.001, respectively). ERK gene/protein expression was similar across all groups. miR-21 and miR-133 levels in ventricular muscle were higher in the SE and GH groups than those in the SC group. In summary, growth hormone application coupled with swimming exercise appeared to affect the PI3K/AKT/mTOR signaling pathway in the left ventricular tissue of rats; however, ERK signaling pathway appeared inactive in physiological left ventricular hypertrophy caused by swimming and GH administration over 8 weeks. Furthermore, GH treatment resulted in increased miR-21 and miR-133 expression. Future study by our group will aim to assess the effects of higher dose GH treatment.

The Role of ERK Signaling in Experimental Autoimmune Encephalomyelitis.

Extracellular signal-regulated kinase (ERK) signaling plays a crucial role in regulating immune cell function and has been implicated in autoimmune disorders. To date, all commercially available inhibitors of ERK target upstream components, such as mitogen-activated protein (MAP) kinase/ERK kinase (MEKs), but not ERK itself. Here, we directly inhibit nuclear ERK translocation by a novel pharmacological approach (Glu-Pro-Glu (EPE) peptide), leading to an increase in cytosolic ERK phosphorylation during T helper (Th)17 cell differentiation. This was accompanied by diminished secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine influencing the encephalitogenicity of Th17 cells. Neither the production of the cytokine interleukin (IL)-17 nor the proliferation rate of T cells was affected by the EPE peptide. The in vivo effects of ERK inhibition were challenged in two independent variants of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Overall, ERK inhibition had only a very minor impact on the clinical disease course of EAE. This indicates that while ERK translocation might promote encephalitogenicity in T cells in vitro by facilitating GM-CSF production, this effect is overcome in more complex in vivo animal models of central nervous system (CNS) autoimmunity.

Ciproxifan, a histamine H3 receptor antagonist and inverse agonist, presynaptically inhibits glutamate release in rat hippocampus

Ciproxifan is an H3 receptor antagonist and inverse agonist with antipsychotic effects in several preclinical models; its effect on glutamate release has been investigated in the rat hippocampus. In a synaptosomal preparation, ciproxifan reduced 4-aminopyridine (4-AP)-evoked Ca2+-dependent glutamate release and cytosolic Ca2+ concentration elevation but did not affect the membrane potential. The inhibitory effect of ciproxifan on 4-AP-evoked glutamate release was prevented by the Gi/Go-protein inhibitor pertussis toxin and Cav2.2 (N-type) and Cav2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but was not affected by the intracellular Ca2+-release inhibitors dantrolene and CGP37157. Furthermore, the phospholipase A2 (PLA2) inhibitor OBAA, prostaglandin E2 (PGE2), PGE2 subtype 2 (EP2) receptor antagonist PF04418948, and extracellular signal-regulated kinase (ERK) inhibitor FR180204 eliminated the inhibitory effect of ciproxifan on glutamate release. Ciproxifan reduced the 4-AP-evoked phosphorylation of ERK and synapsin I, a presynaptic target of ERK. The ciproxifan-mediated inhibition of glutamate release was prevented in synaptosomes from synapsin I-deficient mice. Moreover, ciproxifan reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that ciproxifan, acting through the blockade of Gi/Go protein-coupled H3 receptors present on hippocampal nerve terminals, reduces voltage-dependent Ca2+ entry by diminishing PLA2/PGE2/EP2 receptor pathway, which subsequently suppresses the ERK/synapsin I cascade to decrease the evoked glutamate release.

Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis ofin vitrophosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites.In vitrophosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

Dynamics of Inductive ERK Signaling in the Drosophila Embryo

Transient activation of the highly conserved extracellular-signal-regulated kinase (ERK) establishes precise patterns of cell fates in developing tissues. Quantitative parameters of these transients are essentially unknown, but a growing number of studies suggest that changes in these parameters can lead to a broad spectrum of developmental abnormalities. We provide a detailed quantitative picture of an ERK-dependent inductive signaling event in the early Drosophila embryo, an experimental system that offers unique opportunities for high-throughput studies of developmental signaling. Our analysis reveals a spatiotemporal pulse of ERK activation that is consistent with a model in which transient production of a short-ranged ligand feeds into a simple signal interpretation system. The pulse of ERK signaling acts as a switch in controlling the expression of the ERK target gene. The quantitative approach that led to this model, based on the integration of data from fixed embryos and live imaging, can be extended to other developmental systems patterned by transient inductive signals.

Melanocortin 4 receptor activates ERK-cFos pathway to increase brain-derived neurotrophic factor expression in rat astrocytes and hypothalamus

Melanocortins are neuropeptides with well recognized anti-inflammatory and anti-apoptotic effects in the brain. Of the five melanocortin receptors (MCR), MC4R is abundantly expressed in the brain and is the only MCR present in astrocytes. We have previously shown that MC4R activation by the α-melanocyte stimulating hormone (α-MSH) analog, NDP-MSH, increased brain-derived neurotrophic factor (BDNF) expression through the classic cAMP–Protein kinase A–cAMP responsive element binding protein pathway in rat astrocytes. Now, we examined the participation of the mitogen activated protein kinases pathway in MC4R signaling. Rat cultured astrocytes treated with NDP-MSH 1 µM for 1 h showed increased BDNF expression. Inhibition of extracellular signal-regulated kinase (ERK) and ribosomal p90 S6 kinase (RSK), an ERK substrate, but not of p38 or JNK, prevented the increase in BDNF expression induced by NDP-MSH. Activation of MC4R increased cFos expression, a target of both ERK and RSK. ERK activation by MC4R involves cAMP, phosphoinositide-3 kinase (PI3K) and the non receptor tyrosine kinase, Src. Both PI3K and Src inhibition abolished NDP-MSH-induced BDNF expression. Moreover, we found that intraperitoneal injection of α-MSH induces BDNF and MC4R expression and activates ERK and cFos in male rat hypothalamus. Our results show for the first time that MC4R-induced BDNF expression in astrocytes involves ERK-RSK-cFos pathway which is dependent on PI3K and Src, and that melanocortins induce BDNF expression and ERK-cFos activation in rat hypothalamus.

Extracellular Regulated Kinase Phosphorylates Mitofusin 1 to Control Mitochondrial Morphology and Apoptosis

SummaryControlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1T562. Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.

Adhesion signaling promotes protease‑driven polyploidization of glioblastoma cells.

An increase in ploidy (polyploidization) causes genomic instability in cancer. However, the determinants for the increased DNA content of cancer cells have not yet been fully elucidated. In the present study, we investigated whether adhesion induces polyploidization in human U87MG glioblastoma cells. For this purpose, we employed expression vectors that reported transcriptional activation by signaling networks implicated in cancer. Signaling activation induced by intercellular integrin binding elicited both extracellular signal‑regulated kinase(ERK) and Notch target transcription. Upon the prolonged activation of both ERK and Notch target transcription induced by integrin binding to adhesion protein, cell cultures accumulated polyploid cells, as determined by cell DNA content distribution analysis and the quantification of polynucleated cells. This linked the transcriptional activation induced by integrin adhesion to the increased frequency of polyploidization. Accordingly, the inhibition of signaling decreased the extent of polyploidization mediated by protease‑driven intracellular invasion. Therefore, the findings of this study indicate that integrin adhesion induces polyploidization through the stimulation of glioblastoma cell invasiveness.

A Role for Mitogen- and Stress-Activated Kinase 1 in L-DOPA–Induced Dyskinesia and ∆FosB Expression

BackgroundAbnormal regulation of extracellular signal-regulated kinases 1 and 2 has been implicated in 3,4-dihydroxy-l-phenylalanine (L-DOPA)-induced dyskinesia (LID), a motor complication affecting Parkinson’s disease patients subjected to standard pharmacotherapy. We examined the involvement of mitogen- and stress-activated kinase 1 (MSK1), a downstream target of extracellular signal-regulated kinases 1 and 2, and an important regulator of transcription in LID. Methods6-Hydroxydopamine was used to produce a model of Parkinson’s disease in MSK1 knockout mice and in ∆FosB- or ∆cJun-overexpressing transgenic mice, which were assessed for LID following long-term L-DOPA administration. Biochemical processes were evaluated by Western blotting or immunofluorescence. Histone H3 phosphorylation was analyzed by chromatin immunoprecipitation followed by promotor-specific quantitative polymerase chain reaction. ResultsGenetic inactivation of MSK1 attenuated LID and reduced the phosphorylation of histone H3 at Ser10 in the striatum. Chromatin immunoprecipitation analysis showed that this reduction occurred at the level of the fosB gene promoter. In line with this observation, the accumulation of ∆FosB produced by chronic L-DOPA was reduced in MSK1 knockout. Moreover, inducible overexpression of ∆FosB in striatonigral medium spiny neurons exacerbated dyskinetic behavior, whereas overexpression of ∆cJun, which reduces ∆FosB-dependent transcriptional activation, counteracted LID. ConclusionsResults indicate that abnormal regulation of MSK1 contributes to the development of LID and to the concomitant increase in striatal ∆FosB, which may occur via increased histone H3 phosphorylation at the fosB promoter. Results also show that accumulation of ∆FosB in striatonigral neurons is causally related to the development of dyskinesia.

ERK/MAPK regulates ERRγ expression, transcriptional activity and receptor-mediated tamoxifen resistance in ER+ breast cancer.

Selective estrogen receptor modulators such as tamoxifen (TAM) significantly improve breast cancer-specific survival for women with estrogen receptor-positive (ER+) disease. However, resistance to TAM remains a major clinical problem. The resistant phenotype is usually not driven by loss or mutation of the estrogen receptor; instead, changes in multiple proliferative and/or survival pathways over-ride the inhibitory effects of TAM. Estrogen-related receptorγ (ERRγ) is an orphan member of the nuclear receptor superfamily that promotes TAM resistance in ER+ breast cancer cells. This study sought to clarify the mechanism(s) by which this orphan nuclear receptor is regulated, and hence affects TAM resistance. mRNA and protein expression/phosphorylation were monitored by RT-PCR and western blotting, respectively. Site-directed mutagenesis was used to disrupt consensus extracellular signal-regulated kinase (ERK) target sites. Cell proliferation and cell-cycle progression were measured by flow cytometric methods. ERRγ transcriptional activity was assessed by dual-luciferase promoter-reporter assays. We show that ERRγ protein levels are affected by the activation state of ERK/mitogen-activated protein kinase, and mutation of consensus ERK target sites impairs ERRγ-driven transcriptional activity and TAM resistance. These findings shed new light on the functional significance of ERRγ in ER+ breast cancer, and are the first to demonstrate a role for kinase regulation of this orphan nuclear receptor.

Quetiapine and aripiprazole signal differently to ERK, p90RSK and c-Fos in mouse frontal cortex and striatum: role of the EGF receptor

BackgroundSignaling pathways outside dopamine D2 receptor antagonism may govern the variable clinical profile of antipsychotic drugs (APD) in schizophrenia. One postulated mechanism causal to APD action may regulate synaptic plasticity and neuronal connectivity via the extracellular signal-regulated kinase (ERK) cascade that links G-protein coupled receptors (GPCR) and ErbB growth factor signaling, systems disturbed in schizophrenia. This was based upon our finding that the low D2 receptor affinity APD clozapine induced initial down-regulation and delayed epidermal growth factor receptor (EGFR or ErbB1) mediated activation of the cortical and striatal ERK response in vivo distinct from olanzapine or haloperidol. Here we map whether the second generation atypical APDs aripiprazole and quetiapine affect the EGFR-ERK pathway and its substrates p90RSK and c-Fos in mouse brain, given their divergent agonist and antagonist properties on dopaminergic transmission, respectively.ResultsIn prefrontal cortex, aripiprazole triggered triphasic ERK phosphorylation that was EGFR-independent but had no significant effect in striatum. Conversely quetiapine did not alter cortical ERK signaling but elevated striatal ERK levels in an EGFR-dependent manner. Induction of ERK by aripiprazole did not affect p90RSK signaling but quetiapine decreased RSK phosphorylation within 1-hour of administration. The transcription factor c-Fos by comparison was a direct target of ERK phosphorylation induced by aripiprazole in cortex and quetiapine in striatum with protein levels in temporal alignment with that of ERK.ConclusionsThese data indicate that aripiprazole and quetiapine signal to specific nuclear targets of ERK, which for quetiapine occurs via an EGFR-linked mechanism, possibly indicating involvement of this system in its action.

Abstract P6-04-17: The irreversible c-Jun N-terminal kinase (JNK) inhibitor, JNK-IN-8, sensitizes basal-like breast cancer cells to lapatinib

Abstract Basal-like breast cancers represent 15-20% of breast cancers diagnosed each year. These tumors appear earlier in life and have the worst prognosis due to high prevalence of metastasis. Endocrine and molecularly targeted therapies such as trastuzumab or lapatinib are ineffective against this tumor subtype. A high frequency of p53 mutations, low BRCA1 expression along with high epidermal growth factor receptor (EGFR) expression provide some insight into what types of targeted therapies may be useful for basal-like tumors in the future. We have recently shown that JNK2 mediates the expression of p53 and BRCA1 in response to EGFR activation to promote epithelial to mesenchymal transition and metastasis. Further, high JNK2 expression is associated with poorer survival in patients with basal-like breast cancer. These findings suggest that the JNK pathway may be a promising target for basal-type breast cancer treatment. Commonly used competitive ATP inhibitors of JNK have suffered from lack of specificity for JNK. However, the Gray laboratory recently developed highly specific covalent inhibitors of JNK (Zhang, et al. 2012). The agent showing the best properties is JNK-IN-8. Our data show that treatment of human and mouse basal-like, breast cancer cell lines with JNK-IN-8 sensitizes them to lapatinib. Single agents had minor affects on cell viability and cell cycle regulation, but combination treatment led to G2/M arrest and endoreduplication along with synergistic apoptotic responses. Further, the expression and electrophoretic mobility of mitotic arrest deficient 2 (MAD2) (a protein mediating chromosomal segregation) decreased after combination treatment which could explain the chromosomal duplication abnormalities observed. Combination treatment using lapatinib and JNK-IN-8 modulates extracellular signal-regulated kinase (ERK) phosphorylation unlike either drug alone suggesting that both JNK and ERK target G2/M check point proteins, and when perturbed, sensitize basal-type breast cancer cells to targeted therapies. We conclude that use of specific JNK inhibitors in tumors that are resistant to lapatinib, or perhaps trastuzumab, may sensitize them to treatments with these drugs, and may also prevent resistance to these drugs in tumors that were initially responsive. Considering that JNKs are nodes in receptor tyrosine kinase and many other signaling pathways, JNK-IN-8 treatment may improve the efficacy of other targeted therapies as well. Zhang, T. et al. “Discovery of potent and selective covalent inhibitors of JNK”. Chem Biol. 2012 Jan 27;19(1):140-54. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-17.

Increased activity of mitogen activated protein kinase pathway in flotillin-2 knockout mouse model

Flotillins are highly conserved and widely spread proteins that function in receptor tyrosine kinase signaling and membrane trafficking processes. Flotillin-1 and flotillin-2 have been shown to form both homo- and hetero-oligomers, and their cellular localization changes during signaling. Increased expression of flotillins has been detected in several types of cancer and shown to correlate with poor survival. Consistently, flotillin-2 knockout mice show a reduced formation of metastases in a breast cancer animal model. Our recent data have shown that flotillin-1 depletion results in diminished activation of the epidermal growth factor receptor and impairs its downstream signaling towards the mitogen activated protein kinases and the respective transcriptional response. Here we show that genetic ablation of flotillin-2 in a mouse model or its knockdown in cultured cells increases extracellular signal regulated kinase (ERK) activation. Furthermore, the downstream transcriptional targets of ERK and p53 are upregulated at both mRNA and protein levels. These data suggest that opposite effects are obtained upon ablation of one of the two flotillins, with flotillin-2 knockout/knockdown enhancing and flotillin-1 knockdown inhibiting ERK signaling. Due to their overexpression in cancers, flotillins may be considered as cancer therapy targets. However, our findings suggest that care needs to be taken when interfering with flotillin function, as undesired effects such as deregulation of growth-associated genes may emerge in certain cell types.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases expression of the gonadotropin-releasing hormone (GnRH) receptor in GnRH-producing GT1-7 cells overexpressing PACAP type I receptor

The present study demonstrates the action of pituitary adenylate cyclase-activating polypeptide (PACAP) on gonadotropin-releasing hormone (GnRH)-producing neuronal cells, GT1-7. Because we found the expression levels of PACAP type 1 receptor (PAC1R) to be low in these cells, we transfected them with PAC1R expression vector and observed the outcome. PACAP increased the activity of the serum response element (Sre) promoter, a target of extracellular signal-regulated kinase (ERK), as well as the cAMP response element (Cre) promoter in GT1-7 cells overexpressing PAC1R. We also observed ERK phosphorylation and cAMP accumulation upon PACAP stimulation. PACAP stimulated the promoter activity of GnRH receptor (GnRHR) with increasing levels of GnRHR proteins. Notably, the increase in GnRHR promoter activity from kisspeptin was potentiated in the presence of PACAP. A similar increasing effect of PACAP on the action of kisspeptin was observed for Cre promoter activity. On the other hand, the Sre promoter activated by kisspeptin was inhibited by co-treatment with kisspeptin and PACAP. Likewise, kisspeptin-increased GnRHR promoter activity and Cre promoter activity were both potentiated in the presence of cAMP, whereas the Sre promoter activated by kisspeptin was inhibited in the presence of cAMP. Our observations show that PACAP increases GnRHR expression and stimulates kisspeptin’s effect on GnRHR expression in association with the cAMP/PKA signaling pathway in GT1-7 cells overexpressing PAC1R. In addition, PACAP was shown to have an inhibitory effect on ERK-mediated kisspeptin action.

Enhancement role of host 12/15-lipoxygenase in melanoma progression

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.