Increased Phosphorylation Of ERK Research Articles

Paeonol Derivative, 6'-Methyl Paeonol, Attenuates Aβ-Induced Pathophysiology in Cortical Neurons and in an Alzheimer's Disease Mice Model.

Herbs themselves and various herbal medicines are great resources for discovering therapeutic drugs for various diseases, including Alzheimer's disease (AD), one of the common neurodegenerative diseases. Utilizing mouse primary cortical neurons and DiBAC4(3), a voltage-sensitive indicator, we have set up a drug screening system and identified an herbal extraction compound, paeonol, obtained from Paeonia lactiflora; this compound is able to ameliorate the abnormal depolarization induced by Aβ42 oligomers. Our aim was to further find effective paeonol derivatives since paeonol has been previously studied. 6'-Methyl paeonol, one of the six paeonol derivatives surveyed, is able to inhibit the abnormal depolarization induced by Aβ oligomers. Furthermore, 6'-methyl paeonol is able to alleviate the NMDA- and AMPA-induced depolarization. When a molecular mechanism was investigated, 6'-methyl paeonol was found to reverse the Aβ-induced increase in ERK phosphorylation. At the animal level, mice injected with 6'-methyl paeonol showed little change in their basic physical parameters compared to the control mice. 6'-Methyl paeonol was able to ameliorate the impairment of memory and learning behavior in J20 mice, an AD mouse model, as measured by the Morris water maze. Thus, paeonol derivatives could provide a structural foundation for developing and designing an effective compound with promising clinical benefits.

Med23 deficiency reprograms the tumor microenvironment to promote lung tumorigenesis

BackgroundLung cancer is the leading cause of cancer-related death worldwide. We previously found that Mediator complex subunit 23 (MED23) is important for the tumourigenicity of lung cancer cells with hyperactive Ras activity in vitro, although the in vivo function of MED23 in lung tumourigenesis remains to be explored.MethodsIn this study, we utilized well-characterized KrasG12D-driven non-small cell lung cancer mouse model to investigate the role of MED23 in lung cancer. The lung tumour progression was evaluated by H&E and IHC analysis. Western blotting and qRT-PCR assays were performed to detect changes in gene expression. Immune cells were analyzed by FACS technology. RNA-seq and reporter assays were conducted to explore the mechanism.ResultsWe observed that lung epithelial Med23 deletion by adeno-Cre resulted in a significant increase in KrasG12D tumour number and size, which was further verified with another mouse model with Med23 specifically deleted in alveolar type II cells. Mice with lung-specific Med23 deficiency also exhibited accelerated tumourigenesis, and a higher proliferation rate for tumour cells, along with increased ERK phosphorylation. Notably, the numbers of infiltrating CD4+ T cells and CD8+ T cells were significantly reduced in the lungs of Med23-deficient mice, while the numbers of myeloid-derived suppressor cells (MDSCs) and Treg cells were significantly increased, suggesting the enhanced immune escape capability of the Med23-deficient lung tumours. Transcriptomic analysis revealed that the downregulated genes in Med23-deficient lung tumour tissues were associated with the immune response. Specifically, Med23 deficiency may compromise the MHC-I complex formation, partially through down-regulating B2m expression.ConclusionsCollectively, these findings revealed that MED23 may negatively regulate Kras-induced lung tumourigenesis in vivo, which would improve the precise classification of KRAS-mutant lung cancer patients and provide new insights for clinical interventions.

Expression of the ether-a-gò-gò-related gene 1 channel during B and T lymphocyte development: role in BCR and TCR signaling.

The functional relevance of K+ and Ca2+ ion channels in the "Store Operated Calcium Entry" (SOCE) during B and T lymphocyte activation is well proven. However, their role in the process of T- and B- cell development and selection is still poorly defined. In this scenario, our aim was to characterize the expression of the ether à-go-go-related gene 1 (ERG1) and KV1.3 K+ channels during the early stages of mouse lymphopoiesis and analyze how they affect Ca2+signaling, or other signaling pathways, known to mediate selection and differentiation processes of lymphoid clones. We provide here evidence that the mouse (m)ERG1 is expressed in primary lymphoid organs, bone marrow (BM), and thymus of C57BL/6 and SV129 mice. This expression is particularly evident in the BM during the developmental stages of B cells, before the positive selection (large and small PreB). mERG1 is also expressed in all thymic subsets of both strains, when lymphocyte positive and negative selection occurs. Partially overlapping results were obtained for KV1.3 expression. mERG1 and KV1.3 were expressed at significantly higher levels in B-cell precursors of mice developing an experimental autoimmune encephalomyelitis (EAE). The pharmacological blockage of ERG1 channels with E4031 produced a significant reduction in intracellular Ca2+ after lymphocyte stimulation in the CD4+ and double-positive T-cell precursors' subsets. This suggests that ERG1 might contribute to maintaining the electrochemical gradient responsible for driving Ca2+ entry, during T-cell receptor signaling which sustains lymphocyte selection checkpoints. Such role mirrors that performed by the shaker-type KV1.3 potassium channel during the activation process of mature lymphocytes. No effects on Ca2+ signaling were observed either in B-cell precursors after blocking KV1.3 with PSORA-4. In the BM, the pharmacological blockage of ERG1 channels produced an increase in ERK phosphorylation, suggesting an effect of ERG1 in regulating B-lymphocyte precursor clones' proliferation and checkpoint escape. Overall, our results suggest a novel physiological function of ERG1 in the processes of differentiation and selection of lymphoid precursors, paving the way to further studies aimed at defining the expression and role of ERG1 channels in immune-based pathologies in addition to that during lymphocyte neoplastic transformation.

Host-derived growth factors drive ERK phosphorylation and MCL1 expression to promote osteosarcoma cell survival during metastatic lung colonization.

For patients with osteosarcoma, disease-related mortality most often results from lung metastasis-a phenomenon shared with many solid tumors. While established metastatic lesions behave aggressively, very few of the tumor cells that reach the lung will survive. By identifying mechanisms that facilitate survival of disseminated tumor cells, we can develop therapeutic strategies that prevent and treat metastasis. We analyzed single cell RNA-sequencing (scRNAseq) data from murine metastasis-bearing lungs to interrogate changes in both host and tumor cells during colonization. We used these data to elucidate pathways that become activated in cells that survive dissemination and identify candidate host-derived signals that drive activation. We validated these findings through live cell reporter systems, immunocytochemistry, and fluorescent immunohistochemistry. We then validated the functional relevance of key candidates using pharmacologic inhibition in models of metastatic osteosarcoma. Expression patterns suggest that the MAPK pathway is significantly elevated in early and established metastases. MAPK activity correlates with expression of anti-apoptotic genes, especially MCL1. Niche cells produce growth factors that increase ERK phosphorylation and MCL1 expression in tumor cells. Both early and established metastases are vulnerable to MCL1 inhibition, but not MEK inhibition in vivo. Combining MCL1 inhibition with chemotherapy both prevented colonization and eliminated established metastases in murine models of osteosarcoma. Niche-derived growth factors drive MAPK activity and MCL1 expression in osteosarcoma, promoting metastatic colonization. Although later metastases produce less MCL1, they remain dependent on it. MCL1 is a promising target for clinical trials in both human and canine patients.

Abstract P319: Eliminating Cardiotoxicity In Alpha1-adrenergic Receptor Antagonists

Introduction: Alpha-1 adrenergic receptor (α1-AR) antagonists (α1-blockers) are efficacious antihypertensives, but ALLHAT found that doxazosin (Dzn) doubled the risk of heart failure (HF). We previously demonstrated that α1-ARs, specifically the α1A-AR, protect the heart, explaining the worse outcomes with Dzn in ALLHAT. Interestingly, α1-ARs localize to and signal at the nucleus in cardiac myocytes (CM), whereas α1-ARs localize to the sarcolemma in smooth muscle (SM). Hypothesis: We hypothesize that a membrane impermeant α1-blocker would inhibit α1-ARs in SM, reducing blood pressure, but preserve protective α1-signaling in CMs, preventing HF. Methods: We developed an α1A-AR homology model to define the structural basis of α1-blocker binding and modified existing α1-blocker structure to reduce cardiotoxicity. Using this model, we designed 30 compounds, synthesized 9 in gram quantities, and tested 2 compounds. Results and Conclusions: Binding assays identified 2 novel high affinity α1-ligands; α1705 (IC50 = 5.2 [CI:3.5,7.6] nM, n=3) and α1707 (5.8 [CI:4.2,8.1] nM, n=3) (Dzn: 7.7 [CI:6.3,9.2] nM, n=6). Uptake assays in cultured CM indicated uptake of Prazosin (10 μM Prz: 230 nM in CMs detected by mass spectrometry), whereas α1705 and α1707 were not detected. In HEK cells expressing α1A-AR at the cell surface, Prz, α1705, and α1707 (all 2 μM) inhibited Ca2+ transients (90%, 76%, 88%) induced by the α1A agonist A61603 (20 μM). In CM, A61603 increased ERK phosphorylation (1.55±0.04 fold, n=7, P<0.0001), a survival signal, which was blocked by Prz (1.21±0.06 fold, n=7, P=NS), but not α1705 (1.72±0.13 fold, n=7, P<0.001). Further, A61603 (20 μM) inhibited H202-induced CM death (-17.4±7.6%, n=5), which was reversed by Prz (30.9±15.4%, n=5, P=0.035), but not α1705 (-0.94±11.6%, n=5, P=NS). In DOCA-salt hypertensive mice, infusion of Prz or α1705 (2 mg/kg/d, 3 d) lowered systolic pressure (Cont: +18.9 mmHg, n=1; Prz: 6.4±3.2 mmHg, n=3, α1705: -6.1±5.6 mmHg, n=3), and while Prz was detected in CMs following 4 d, α1705 was not. In conclusion, we have developed a novel membrane impermeant α1-blocker with reduced cardiotoxicity that could improve hypertension outcomes, especially in patients suffering from chronic kidney disease or benign prostatic hyperplasia.

Eucalyptol induces osteoblast differentiation through ERK phosphorylation in vitro and in vivo.

Eucalyptol (EU) is monoterpene oxide that is the main component of the essential oil extracted from aromatic plants such as Eucalyptus globules. EU has therapeutic effects such as antibacterial, anti-inflammatory and antioxidant in chronic diseases including inflammation disorder, respiratory disease, and diabetic disease. However, the effects of EU on osteoblast differentiation and bone diseases such as osteoporosis have not been studied. The present study investigated the effects of EU on osteoblast differentiation and bone formation. EU induces mRNA and protein expression of osteogenic genes in osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts. EU also promoted alkaline phosphatase (ALP) activity and mineralization. Here, the osteoblast differentiation effect of EU is completely reversed by ERK inhibitor. These results demonstrate that osteoblast differentiation effect of EU is mediated by ERK phosphorylation. The efficacy of EU on bone formation was investigated using surgical bone loss-induced animal models. EU dose-dependently promoted bone regeneration in zebrafish caudal fin rays. In the case of ovariectomized mice, EU increased ERK phosphorylation and ameliorated bone loss of femurs. These results indicate that EU ameliorates bone loss by promoting osteoblast differentiation through ERK phosphorylation. We suggest that EU, plant-derived monoterpenoid, may be useful for preventing bone loss. KEY MESSAGES: Eucalyptol (EU) increases osteoblast differentiation in pre-osteoblasts. EU up-regulates the osteogenic genes expression via ERK phosphorylation. EU promotes bone regeneration in partially amputated zebrafish fin rays. Oral administration of EU improves ovariectomy-induced bone loss and increases ERK phosphorylation.

FGF12 regulates cell cycle gene expression and promotes follicular granulosa cell proliferation through ERK phosphorylation in geese

The granulosa cells play an important role in the fate of follicular development or atresia in poultry. Fibroblast growth factor 12 (FGF12) is downregulated in atretic follicles and may be involved in regulating granulosa cell survival in previous studies, but its molecular mechanism remains unclear. In this study, FGF12 overexpression and knockdown models of goose granulosa cells were constructed to investigate its function. The downstream expression of the cell cycle pathway was analyzed by qPCR. Granulosa cell proliferative activity and apoptosis were detected by CCK8 and TUNEL. Protein phosphorylation levels of ERK and AKT were measured using Western blotting to analyze the key pathway of FGF12 regulation of granulosa cell proliferation. ERK protein phosphorylation inhibitor was added for further verification. After overexpression of FGF12, cell proliferation activity was increased, the expressions of cell cycle pathway genes CCND1, CCNA2, MAD2, and CHK1 were upregulated, the apoptosis of granulosa cell was decreased, and Caspase 3 gene and protein expression were downregulated. After the knockdown of FGF12, cell proliferation activity decreased, the expression of downstream genes in the cell cycle pathway was downregulated, the apoptosis of granulosa cells was increased, and the Bcl-2 gene and protein were downregulated. Overexpression of FGF12 promoted the synthesis of P4 and upregulates the expression of the STAR gene. Overexpression of FGF12 promoted ERK protein phosphorylation but did not affect AKT phosphorylation. The addition of ERK phosphorylation inhibitors resulted in the elimination of the increase in cell proliferative activity caused by FGF12 overexpression. In conclusion, FGF12 could promote proliferation and inhibit apoptosis of goose granulosa cells by increasing ERK phosphorylation.

Combination Therapy With MDM2 and MEK Inhibitors Is Effective in Patient-Derived Models of Lung Adenocarcinoma With Concurrent Oncogenic Drivers and MDM2 Amplification

IntroductionAlthough targeted therapies have revolutionized the therapeutic landscape of lung adenocarcinomas (LUADs), disease progression on single-agent targeted therapy against known oncogenic drivers is common, and therapeutic options after disease progression are limited. In patients with MDM2 amplification (MDM2amp) and a concurrent oncogenic driver alteration, we hypothesized that targeting of the tumor-suppressor pathway (by means of restoration of p53 using MDM2 inhibition) and simultaneous targeting of co-occurring MAPK oncogenic pathway might represent a more durably effective therapeutic strategy. MethodsWe evaluated genomic next-generation sequencing data using the Memorial Sloan Kettering Cancer Center-Integrated Mutation Profiling of Actionable Cancer Targets platform to nominate potential targets for combination therapy in LUAD. We investigated the small molecule MDM2 inhibitor milademetan in cell lines and patient-derived xenografts of LUAD with a known driver alteration and MDM2amp. ResultsOf 10,587 patient samples from 7121 patients with LUAD profiled by next-generation sequencing, 6% (410 of 7121) harbored MDM2amp. MDM2amp was significantly enriched among tumors with driver alterations in METex14 (36%, p < 0.001), EGFR (8%, p < 0.001), RET (12%, p < 0.01), and ALK (10%, p < 0.01). The combination of milademetan and the MEK inhibitor trametinib was synergistic in growth inhibition of ECLC5-GLx (TRIM33-RET/MDM2amp), LUAD12c (METex14/KRASG12S/MDM2amp), SW1573 (KRASG12C, TP53 wild type), and A549 (KRASG12S) cells and in increasing expression of proapoptotic proteins PUMA and BIM. Treatment of ECLC5-GLx and LUAD12c with single-agent milademetan increased ERK phosphorylation, consistent with previous data on ERK activation with MDM2 inhibition. This ERK activation was effectively suppressed by concomitant administration of trametinib. In contrast, ERK phosphorylation induced by milademetan was not suppressed by concurrent RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). In vivo, combination milademetan and trametinib was more effective than either agent alone in ECLC5-GLx, LX-285 (EGFRex19del/MDM2amp), L13BS1 (METex14/MDM2amp), and A549 (KRASG12S, TP53 wild type). ConclusionsCombined MDM2/MEK inhibition was found to have efficacy across multiple patient-derived LUAD models harboring MDM2amp and concurrent oncogenic drivers. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions activating the MAPK pathway, has evident clinical implications and will be investigated as part of a planned phase 1/2 clinical trial.

Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Generation and characterization of humanized affinity-matured EGFL6 antibodies for ovarian cancer therapy

ObjectivesEpidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer and promotes both endothelial cell proliferation/angiogenesis and cancer cell proliferation/metastasis. As such it has been implicated as a therapeutic target. As a secreted factor, EGFL6 is a candidate for antibody therapy. The objectives of this study were to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. MethodsA selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant humanized antibodies. These were screened and the lead candidate was affinity matured. Seven humanized affinity-matured EGFL6 antibodies were screened for their ability to block EGFL6 activity on cancer cells in vitro, two of which were selected and tested their therapeutic activity in vivo. ResultsHumanized affinity matured antibodies demonstrated high affinity for EGFL6 (150 pM to 2.67 nM). We found that several humanized affinity-matured EGFL6 antibodies specifically bound to recombinant, and native human EGFL6. Two lead antibodies were able to inhibit EGFL6-mediated (i) cancer cell migration, (ii) proliferation, and (iii) increase in ERK phosphorylation in cancer cells in vitro. Both lead antibodies restricted growth of an EGFL6 expressing ovarian cancer patient derived xenograft. Analysis of treated human tumor xenografts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis. ConclusionsOur studies confirm the ability of these humanized affinity-matured antibodies to neutralize EGFL6 and acting as a therapeutic to restrict cancer growth. This work supports the development of these antibody for first-in-human clinical trials.

Genetic removal of synaptic Zn2+ impairs cognition, alters neurotrophic signaling and induces neuronal hyperactivity.

Vesicular Zn2+ (zinc) is released at synapses and has been demonstrated to modulate neuronal responses. However, mechanisms through which dysregulation of zinc homeostasis may potentiate neuronal dysfunction and neurodegeneration are not well-understood. We previously reported that accumulation of soluble amyloid beta oligomers (AβO) at synapses correlates with synaptic loss and that AβO localization at synapses is regulated by synaptic activity and enhanced by the release of vesicular Zn2+ in the hippocampus, a brain region that deteriorates early in Alzheimer's disease (AD). Significantly, drugs regulating zinc homeostasis inhibit AβO accumulation and improve cognition in mouse models of AD. We used both sexes of a transgenic mouse model lacking synaptic Zn2+ (ZnT3KO) that develops AD-like cognitive impairment and neurodegeneration to study the effects of disruption of Zn2+ modulation of neurotransmission in cognition, protein expression and activation, and neuronal excitability. Here we report that the genetic removal of synaptic Zn2+ results in progressive impairment of hippocampal-dependent memory, reduces activity-dependent increase in Erk phosphorylation and BDNF mRNA, alters regulation of Erk activation by NMDAR subunits, increases neuronal spiking, and induces biochemical and morphological alterations consistent with increasing epileptiform activity and neurodegeneration as ZnT3KO mice age. Our study shows that disruption of synaptic Zn2+ triggers neurodegenerative processes and is a potential pathway through which AβO trigger altered expression of neurotrophic proteins, along with reduced hippocampal synaptic density and degenerating neurons, neuronal spiking activity, and cognitive impairment and supports efforts to develop therapeutics to preserve synaptic zinc homeostasis in the brain as potential treatments for AD.

Crosstalk between protein kinases AKT and ERK1/2 in human lung tumor-derived cell models.

There is no doubt that cell signaling manipulation is a key strategy for anticancer therapy. Furthermore, cell state determines drug response. Thus, establishing the relationship between cell state and therapeutic sensitivity is essential for the development of cancer therapies. In the era of personalized medicine, the use of patient-derived ex vivo cell models is a promising approach in the translation of key research findings into clinics. Here, we were focused on the non-oncogene dependencies of cell resistance to anticancer treatments. Signaling-related mechanisms of response to inhibitors of MEK/ERK and PI3K/AKT pathways (regulators of key cellular functions) were investigated using a panel of patients' lung tumor-derived cell lines with various stemness- and EMT-related markers, varying degrees of ERK1/2 and AKT phosphorylation, and response to anticancer treatment. The study of interactions between kinases was the goal of our research. Although MEK/ERK and PI3K/AKT interactions are thought to be cell line-specific, where oncogenic mutations have a decisive role, we demonstrated negative feedback loops between MEK/ERK and PI3K/AKT signaling pathways in all cell lines studied, regardless of genotype and phenotype differences. Our work showed that various and distinct inhibitors of ERK signaling - selumetinib, trametinib, and SCH772984 - increased AKT phosphorylation, and conversely, inhibitors of AKT - capivasertib, idelalisib, and AKT inhibitor VIII - increased ERK phosphorylation in both control and cisplatin-treated cells. Interaction between kinases, however, was dependent on cellular state. The feedback between ERK and AKT was attenuated by the focal adhesion kinase inhibitor PF573228, and in cells grown in suspension, showing the possible role of extracellular contacts in the regulation of crosstalk between kinases. Moreover, studies have shown that the interplay between MEK/ERK and PI3K/AKT signaling pathways may be dependent on the strength of the chemotherapeutic stimulus. The study highlights the importance of spatial location of the cells and the strength of the treatment during anticancer therapy.

Red algae-derived isofloridoside activates the sweet taste receptor T1R2/T1R3

Isofloridoside (IF) is a naturally occurring galactosylglycerol compound with a refreshing sweet taste. It is found in red algae, where it acts as a photosynthetic intermediate. However, the mechanism by which IF elicits sweetness has not been determined. In this study, we investigate the interaction between IF and the sweet taste receptor T1R2/T1R3. We show that IF causes an increase in intracellular Ca2+ concentration and an increase in Erk phosphorylation in T1R2/T1R3-expressing HEK293T cells, indicating that IF interacts with T1R2/T1R3. IF also activates endogenously expressed T1R2/T1R3 in mouse small intestinal endocrine L-cells, and promotes the secretion of the incretin glucagon-like peptide-1 (GLP-1). In silico docking simulations of IF and T1R2/T1R3 predict that IF forms hydrogen bonds with Tyr103 in the extracellular Venus flytrap domain of T1R2. This result is consistent with the binding mode of many other sweet-tasting molecules. We found that IF have the potential as a new alternative sweetener.

DAZAP1 facilitates the alternative splicing of KITLG to promote multiple myeloma cell proliferation via ERK signaling pathway.

Multiple myeloma (MM) is an incurable plasma cell malignancy, in which alternative pre-mRNA splicing (AS) acts as one of the key transcriptome modifier. The Deleted in Azoospermia-Associated Protein 1 (DAZAP1) is a splicing factor that has been identified as an oncogene in multiple cancers, yet its role in MM proliferation remains unclear. We first analyzed MM clinical databases and found that MM patients with elevated DAZAP1 had a poor survival. Furthermore, we overexpressed DAZAP1 by lentiviral transfection and utilized siRNA silencing the expression of DAZAP1 in MM cells. DAZAP1 promoted MM cell proliferation in vitro and accelerated MM xenograft tumor growth in vivo. KEGG pathway enrichment analysis showed that ERK signaling pathway was activated in DAZAP1-OE MM cells. The analyses of RIP-seq and RIP-qPCR revealed that DAZAP1 activated alternative splicing of KIT proto-oncogene ligand (KITLG) mRNA. Further study validated that DAZAP1 increased ERK phosphorylation via modulating alternative splicing of KITLG mRNA to promote MM cell proliferation. In conclusion, we establish DAZAP1 as a tumor-promoting gene with therapeutic potential and provide mechanistic insights into targeting DAZAP1 as a new strategy for the diagnosis and treatment of MM.

ERK activation modulates invasiveness and Reactive Oxygen Species (ROS) production in triple negative breast cancer cell lines

Triple negative breast cancer (TNBC) is the breast cancer subtype with the worst prognosis and still lacks a targeted therapy. In this study, we found increased ERK phosphorylation in TNBC cell lines and an important role for ERK in sustaining the migration of TNBC cells. Although ROS have been suggested to have an important role in sustaining MAPK signaling, antioxidant treatment increased ERK phosphorylation, probably suggesting increased invasive potential. Interestingly, treatment with PD0325901 (PD), a MEK inhibitor, decreased ROS levels in TNBC cells and decreased mitochondrial fragmentation in the MDAMB231 cell line. Our data supports an important role for MEK/ERK in TNBC, sustaining cellular migration, regulating mitochondrial dynamics and ROS production in this breast cancer subtype.

Lumican is elevated in the lung in human and experimental acute respiratory distress syndrome and promotes early fibrotic responses to lung injury

BackgroundFibroproliferative repair starts early in the inflammatory phase of acute respiratory distress syndrome (ARDS) and indicates a poor prognosis. Lumican, a small leucine-rich proteoglycan, is implicated in homeostasis and fibrogenesis, but its role in ARDS is unclear.MethodsBronchoalveolar lavage fluid (BALF) samples were obtained from ARDS patients (n = 55) enrolled within 24 h of diagnosis and mechanically ventilated (n = 20) and spontaneously breathing (n = 29) control subjects. Lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse models were intratracheally administered an adeno-associated virus (AAV) vector expressing lumican shRNA. Primary human lung fibroblasts (HLF) and small airway epithelial cells (SAECs) were cultured with tumour necrosis factor (TNF)-α or lumican. Luminex/ELISA, histochemistry/immunohistochemistry, immunofluorescence microscopy, quantitative real-time PCR, and western blotting were performed.ResultsLumican levels were significantly higher in the BALF of ARDS patients than in that of ventilated or spontaneously breathing controls (both p < 0.0001); they were correlated with the PaO2/FiO2 ratio and levels of proinflammatory cytokines (interleukin-6, interleukin-8, and TNF-α) and profibrotic factors (fibronectin, alpha-1 type I collagen [COL1A1], and alpha-1 type III collagen [COL3A1]). Lumican expression was enhanced in the alveolar walls and airway epithelium in the ALI mouse model. Murine lumican levels were also linked to proinflammatory and profibrotic cytokine levels in the BALF. In vitro, TNF-α induced the synthesis and secretion of lumican in HLF. In turn, lumican increased the expression of alpha-smooth muscle actin (α-SMA), COL1A1, and COL3A1 in HLF, upregulated α-SMA and COL3A1, downregulated E-cadherin, and caused spindle-shaped morphological changes in SAECs. Moreover, increased ERK phosphorylation and Slug were noted in both HLF and SAECs treated with lumican. In vivo, AAV-mediated knockdown of lumican inhibited the pulmonary production of fibronectin and COL3A1 and alleviated lung fibrotic lesions in LPS-challenged mice.ConclusionsPulmonary lumican levels were increased early in human and experimental ARDS and linked to disease severity and inflammatory fibrotic processes. Lumican triggers the transdifferentiation of lung fibroblasts into myofibroblasts and epithelial-mesenchymal transition in SAECs, possibly via the ERK/Slug pathway. Knockdown of pulmonary lumican attenuated extracellular matrix deposition in ALI mice. Overall, lumican promotes fibrotic responses in the early phase of ARDS, suggesting its potential as a therapeutic target.

Lack of Oestrogen Receptor Expression in Breast Cancer Cells Does Not Correlate with Kisspeptin Signalling and Migration.

Kisspeptin is an anti-metastatic mediator in many cancer types, acting through its receptor, KISS1R. However, controversy remains regarding its role in breast cancer since both pro- and anti-metastatic roles have been ascribed to it. In KISS1R overexpressing triple-negative breast cancer (TNBC) cells, stimulation has been associated with increased invasion and MMP-9 expression, leading to the suggestion that hormone receptor status determines the metastatic effects of kisspeptin. To assess the veracity of this claim, we compared endogenous KISS1R signalling and physiological output in the hormone receptor-negative MDA-MB-231 and BT-20 cell lines after KP-10 (shortest active kisspeptin peptide) stimulation. MDA-MB-231 cells are metastatic when implanted in mice while BT-20 are not and remain epithelial-like. We show that both cell lines express KISS1R mRNA and respond to KP-10 by elevating calcium mobilisation. However, KP-10 stimulation induced migration of MDA-MB-231, but not BT-20 cells, in a calcium-dependent manner. Moreover, only BT-20 cells responded to KP-10 by increasing ERK phosphorylation in a β-arrestin-dependent manner. Interestingly, both cell lines displayed different complements of β-arrestin 1 and 2 expression. Overall, our data shows that, in TNBC, it is not universally true that kisspeptin/KISS1R stimulate migration or pro-metastatic behaviour, as divergent responses were observed in the two TNBC lines tested. Whether this divergence is related to the observed differences in β-arrestin complements warrants further investigation and may enable further stratification of the ability of kisspeptin to influence breast tumour behaviour.

HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance. These variants may interact with each other to influence tumor pathophysiology, however, such interactions have not been fully elucidated. Additionally, the effect of target therapy for those variants also unclarified. In this study, we investigated the biological functions of a HER2 mutation (G776S mutation) of unknown pathological significance, which was detected together with APC mutation by cancer genome sequencing of samples from a colorectal cancer (CRC) patient. Transfection of the HER2 G776S mutation alone slightly increased the kinase activity and phosphorylation of HER2 protein, but did not activate HER2 downstream signaling or alter the cell phenotype. On the other hand, the HER2 G776S mutation was shown to have strong oncogenic potential when loss of APC function was accompanied. We revealed that loss of APC function increased Wnt pathway activity but also increased RAS–GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. In addition, afatinib, a pan-HER tyrosine kinase inhibitor, suppressed tumor growth in xenografts derived from HER2 G776S-transfected CRC cells. These findings suggest that this HER2 mutation in CRC may be a potential therapeutic target.

5HTR1E receptor interacts with Neurotrophic factor‐α1 and serotonin to activate two distinct signaling pathways

Neurotrophic factor‐α1/Carboxypeptidase E (NFα1/CPE) is a prohormone processing enzyme intracellularly and as a neurotrophic factor, extracellularly. Previously we showed that extracellular treatment of hippocampal neurons with NFα1/CPE protected them from oxidative stress‐induced cell death through ERK/BCL2 signaling. Based on these results, we hypothesized that NFα1/CPE activation of the ERK/BCL2 pathway to mediate neuroprotection should involve interaction with receptors at the cell membrane. We have identified 5‐hydroxytryptamine receptor 1E (5HTR1E), a Gi coupled serotonin receptor, as an extracellular binding partner of NFα1/CPE. This interaction was confirmed by co‐IP and pull‐down experiments followed by radioligand binding studies in HEK293‐5HTR1E overexpressing cells. Molecular modeling studies also predicted that NFα1/CPE interacted with HTR1E, not at the serotonin binding pocket, but via the extracellular loop 2 (ECL2) of 5HTR1E, forming 3 stable salt bridges with specific amino acid residues in NFα1/CPE. To identify the HTR1E downstream signaling pathways, we used HEK293 cells stably expressing 5HTR1E. First, we tested the dose dependent effect of both serotonin and NFα1/CPE on ERK, and cAMP signaling and found that both can increase ERK phosphorylation while cAMP pathway activation is serotonin dependent only. To further dissect the 5HTR1E mediated functions of both serotonin and NFα1/CPE we tested Gi, Gq, PKA, and PI3K inhibitors and observed that these proteins are not involved in NFα1/CPE mediated ERK phosphorylation, while serotonin‐stimulated ERK activation was Gi‐PKA dependent. We then explored the involvement of β‐arrestin, an alternative coupling mechanism with GPCRs for ERK activation. Studies in 5HTR1E expressing‐HEK293‐β‐arrestin KO cells revealed that NFα1/CPE‐5HTR1E complex was unable to induce ERK signaling in the absence of β‐arrestin proteins. Furthermore, molecular docking studies revealed a very strong coupling of b‐arrestin to intracellular loop (ICL) 2 and ICL3 of HTR1E. Using LDH cytotoxicity assay we demonstrated that NF‐α1/CPE was able to protect HEK‐293‐5HTR1E expressing cells against H2O2‐induced cytotoxicity, via increasing pro‐survival protein BCL2 expression; however, this protective effect of NFα1/CPE was diminished in the β‐arrestin KO cells. Our study identified two ligands for HTR1E: NFα1/CPE which interacts with HTR1E to activate β‐arrestin/ERK/BCL2 signaling pathway for protecting cells against oxidative stress; and serotonin which activated a Gi‐cAMP‐dependent pathway, the physiological role is under investigation.

Inhibition of Cdk5 increases osteoblast differentiation and bone mass and improves fracture healing

Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis, a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5 (Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by siRNA, genetic deletion using the Cre-loxP system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.