Src Protein Tyrosine Kinase Research Articles

Remote kidney and liver injury after transplantation of lung allografts in an allogeneic mouse model

Background: Remote organ dysfunction is common after lung transplantation and might negatively affect outcome. The local anesthetic ropivacaine was previously demonstrated to attenuate acute rejection after allogeneic lung transplantation in mice. We hypothesized that lung transplantation might result in detectable molecular signs of injury in kidneys and liver and that ropivacaine might attenuate this damage. Methods: Organs from C57BL/6 mice undergoing allogeneic orthotopic single-lung transplantation were procured at postoperative day five and analyzed using western blot and real-time quantitative polymerase chain reaction probing for Src protein tyrosine kinase, STAT3 and bax/bcl-2. During cold ischemia, the allograft had either been flushed with normal saline only or in combination with ropivacaine (1 µM). A non-transplanted group of animals served as baseline controls. Results: The allogeneic stimulus induced by transplantation lead to an increase in Src-phosphorylation and STAT3-expression in kidneys and livers of lung-transplanted mice compared to non-transplanted animals. Bax/bcl-2 as a marker of cellular apoptosis was not affected by the transplantation. In contrast to the findings in the transplanted lungs, the addition of ropivacaine did not have an effect on the examined markers of inflammation in the remote organs. Conclusions: The observed increase in the inflammatory signaling provides first insight into a possible mechanism, by which remote organ dysfunction after lung transplantation might occur.

Identifying p56lck SH2 Domain Inhibitors Using Molecular Docking and In Silico Scaffold Hopping

Bacterial infections are the second-leading cause of death, globally. The prevalence of antibacterial resistance has kept the demand strong for the development of new and potent drug candidates. It has been demonstrated that Src protein tyrosine kinases (TKs) play an important role in the regulation of inflammatory responses to tissue injury, which can trigger the onset of several severe diseases. We carried out a search for novel Src protein TK inhibitors, commencing from reported highly potent anti-bacterial compounds obtained using the Mannich reaction, using a combination of e-pharmacophore modeling, virtual screening, ensemble docking, and core hopping. The top-scoring compounds from ligand-based virtual screening were modified using protein structure-based design approaches, and their binding to the Src homology-2 domain of p56lck TK was predicted using ensemble molecular docking. We have prepared a database of 202 small molecules and have identified six novel top hits that can be subjected to further investigation. We have also performed in silico ADMET property prediction for the hit compounds. This combined computer-aided drug design approach can serve as a starting point for identifying novel TK inhibitors that could be further subjected to in vitro studies and validation of antimicrobial activity.

Integrated bioinformatics and network pharmacology to explore the therapeutic target and molecular mechanisms of Taxus chinensis against non-small cell lung cancer.

Taxus chinensis (TC) has tremendous therapeutic potential in alleviating non-small cell lung cancer (NSCLC), but the mechanism of action of TC remains unclear. Integrated bioinformatics and network pharmacology were employed in this study to explore the potential targets and molecular mechanism of TC against NSCLC. Data obtained from public databases were combined with appropriate bioinformatics tools to identify the common targets for TC and NSCLC. Common targets were uploaded to the Metascape database for gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway analyses. A protein-protein interaction network was established, and topological analysis was performed to obtain hub genes. The expression of the hub genes in NSCLC tissues and their consequent effects on the prognosis of patients with NSCLC were confirmed using the Human Protein Atlas database and appropriate bioinformatics tools. Molecular docking was used to verify the binding affinity between the active ingredients and hub targets. We found 401 common targets that were significantly enriched in the cancer, MAPK signaling, and PI3K/Akt signaling pathways. Proto-oncogene tyrosine-protein kinase Src (SRC), mitogen-activated protein kinase 1, phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1), AKT serine/threonine kinase 1 (AKT1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and lymphocyte-specific protein tyrosine kinase were identified as the hub genes. Immunohistochemical results confirmed that the expression of SRC, mitogen-activated protein kinase 1, PIK3R1, AKT1, and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha was upregulated in the NSCLC tissues, while survival analysis revealed the expression of SRC, AKT1, PIK3R1, and lymphocyte-specific protein tyrosine kinase was closely related to the prognosis of patients with NSCLC. Molecular docking results confirmed all bioactive ingredients present in TC strongly bound to hub targets. We concluded that TC exhibits an anti-NSCLC role through multi-target combination and multi-pathway cooperation.

Upregulation of the protein kinase Lyn is associated with renal injury in type 2 diabetes patients

Background The role of inflammation in the pathogenesis of type 2 diabetes mellitus (T2DM) is well established. Lyn, a member of the nonreceptor protein tyrosine kinase Src family, has been reported to modulate inflammatory signaling pathways. Methods Lyn expression was assessed in kidney biopsies of 11 patients with diabetic kidney disease (DKD) and in kidney tissues of streptozotocin (STZ)-induced DKD mice. 102 recruited T2DM patients were divided into three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. Twenty-one healthy volunteers were recruited as a control group. Clinical data, blood and urine samples of all individuals were collected for analysis. Results Lyn expression was augmented in the kidneys of DKD patients and STZ-induced diabetic mice. Compared with control and normoalbuminuria groups, both mRNA and protein expression of Lyn in peripheral blood mononuclear cells (PBMCs) in the macroalbuminuria group were significantly increased (p < .05). Elevated Lyn levels were independently related to urine albumin/urine creatinine ratio and were positively associated with key inflammatory factors, namely interleukin-1β, monocyte chemoattractant protein-1, and tumor necrosis factor-α. Additionally, Lyn exhibited a noteworthy connection with renal tubular injury indicators, specifically urinary neutrophil gelatinase-associated lipocalin and urinary retinol binding protein. ROC curve analysis showed that Lyn could predict albuminuria in diabetic patients with an area under the curve of 0.844 (95% CI: 0.764–0.924). Conclusion Lyn levels in PBMCs exhibited a positive correlation with the severity of albuminuria, renal tubular damage, and inflammatory responses. Hence, Lyn may be a compelling candidate for predicting albuminuria levels in diabetes.

Comparative proteomic and phosphoproteomic analysis reveals differential heat response mechanism in two congeneric oyster species

High-temperature stress caused by global climate change poses a significant threat to marine ectotherms. This study investigated the role of protein phosphorylation modifications in the molecular regulation network under heat stress in oysters, which are representative intertidal organisms that experience considerable temperature changes. Firstly, the study compared the extent of thermal damage between two congeneric oyster species, the relative heat-tolerant Crassostrea angulata (C. angulata) and heat-sensitive Crassostrea gigas (C. gigas), under sublethal temperature (37 °C) for 12 h, using various physiological and biochemical methods. Subsequently, the comparative proteomic and phosphoproteomic analyses revealed that high-temperature considerably regulated signal transduction, energy metabolism, protein synthesis, cell survival and apoptosis, and cytoskeleton remodeling through phosphorylation modifications of related receptors and kinases. Furthermore, the protein kinase A, mitogen-activated protein kinase 1, tyrosine-protein kinase Src, and serine/threonine kinase AKT, exhibiting differential phosphorylation modification patterns, were identified as hub regulators that may enhance glycolysis and TCA cycle to increase the energy supply, distribute protein synthesis, inhibit Caspase-dependent apoptosis activated by endogenous mitochondrial cytochrome release and maintain cytoskeletal stability, ultimately shaping the higher thermal resistance of C. angulata. This study represents the first investigation of protein phosphorylation dynamics in marine invertebrates under heat stress, reveals the molecular mechanisms underlying the differential thermal responses between two Crassostrea oysters at the phosphorylation level, and provides new insights into understanding phosphorylation-mediated molecular responses in marine organisms during environmental changes and predicting the adaptive potential in the context of global warming.

The ACE2 activator diminazene aceturate ameliorates colitis by repairing the gut-vascular barrier in mice.

Alleviating vascular barrier injury improves colitis. Angiotensin converting enzyme 2/angiotensin 1-7/Mas receptor (ACE2/Ang1-7/MasR) axis-related drugs have various biological properties, such as inhibition of inflammation and fibrosis, but their role in improving the gut-vascular barrier (GVB) has rarely been reported. This study aims to investigate the effects of diminazene aceturate (DIZE), an ACE2 activator, on vascular barrier damage in colitis. Mice were randomly divided into three groups: control, dextran sulfate sodium salt (DSS), and DIZE+DSS. Mice in the DSS group drank DSS for 8days starting on day 4. Mice in the DIZE+DSS group were pregavaged with DIZE for 3days and then drank DSS for 8days while continuing to be gavaged with DIZE for 4days. Mice were euthanized and samples were collected on the last day. Injury to colonic structure and colonic microvasculature was assessed by visual observation and appropriate staining. DSS-induced colonic and microvascular pathological damage in mice was substantially reversed by DIZE treatment. Molecular pathways were investigated by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme linked immunosorbent assay (ELISA). DSS treatment upregulated angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AT1R) protein, pro-inflammatory cytokines and inhibited tight junction-related protein expression. DIZE treatment activated ACE2/MasR protein expression and reversed epithelial barrier damage and inflammatory infiltration during DSS injury. In addition, DIZE treatment inhibited vascular endothelial growth factor A/vascular endothelial growth factor receptor 2/proto-oncogene tyrosine-protein kinase Src (VEGFA/VEGFR2/Src) pathway activation and restored vascular adhesion-linker protein vascular endothelial cadherin (VE-cadherin) expression during DSS injury. In conclusion, DIZE treatment ameliorated colitis, which was associated with balancing the two axes of the renin-angiotensin system (RAS) and repairing the GVB injury.

Targeting the Src N-terminal regulatory element in cancer.

The signaling pathways displayed by cancer cells are often composed by the same components than the physiological ones, yet the overall result is a pathological deregulation. The non-receptor protein tyrosine kinase Src is a good example. Src is the first described proto-oncogene and a demonstrated player in cancer progression, as it affects proliferation, invasion, survival, cancer stemness, and drug resistance. Src activation is linked to poor prognosis in many cancer types, yet mutations in this protein are rarely observed. In addition, being a demonstrated cancer target, unspecific inhibition of the kinase activity has proven inefficient in clinics since the inhibition of Src in non-cancerous cells results in unacceptable toxicity. Thus, there is a need for new target regions in Src that could inhibit Src activity only in certain cell types, e.g., cancer cells, while maintaining the normal physiological activity in healthy cells. The Src N-terminal regulatory element (SNRE) includes the poorly studied intrinsically disordered region with unique sequences for each of the members of the Src family. In this perspective, we discuss the non-canonical regulatory mechanisms involving the SNRE and their potential use as oncotargets.

A curcumin-nicotinoyl derivative and its transition metal complexes: synthesis, characterization, and in silico and in vitro biological behaviors.

Curcumin-nicotinoyl (Cur-Nic) was synthesized by the chemical modification of the curcumin structure, characterized, and used as a ligand for the synthesis of copper(II) and zinc(II) complexes. The biological activities of Cur-Nic and its metal complexes were predicted using the PASS and Swiss Target Prediction online software, respectively, and docking studies with tyrosine-protein kinase SRC were performed using the PyRx software to predict their anticancer activities. The toxicity effects of the complexes on a human breast cancer cell line (MCF-7) compared to a healthy breast cell line (MCF-10A) were investigated by the MTS assay. Although the metal complexes maintained the least toxicity against normal cells, the results indicated that compared to curcumin and Cur-Nic, the cytotoxicity toward cancer cells increased due to the complexation process. Moreover, the antibacterial evaluation of the compounds against a Gram-positive bacterium (MRSA) and a Gram-negative bacterium (E. coli) indicated that the Cu(II) complex and Cur-Nic were the best, respectively. Also, the Zn(II) complex was the most stable compound, and the Cu(II) complex was the best ROS scavenger based on the in vitro evaluation.

Curaxin CBL0137 inhibits endothelial inflammation and atherogenesis via suppression of the Src-YAP signalling axis.

Atherosclerotic vascular disease is the leading cause of mortality and morbidity worldwide. Our previous study uncovered that endothelium-specific knockdown of YAP suppresses atherogenesis, suggesting that YAP is a promising therapeutic target against atherosclerotic vascular disease. We established a drug screening platform, which aimed to identify new YAP inhibitors for anti-atherosclerotic treatment. Drug screening was performed by a luciferase reporter gene assay. RNA sequencing was performed to acquire the transcriptomic profile of CBL0137-treated endothelial cells. We assessed and validated the inhibitory effect of CBL0137 on YAP activity and inflammatory response in HUVECs and HAECs. We evaluated the vasoprotective effect of CBL0137 in vivo against plaque formation in ApoE-/- mice, using both disturbed flow-induced and chronic western diet-induced atherosclerotic models. We identified CBL0137 as a novel YAP inhibitor from an FDA drug library. CBL0137 inhibited YAP activity by restraining its phosphorylation at Y357. CBL0137 inhibited YAP activity to repress endothelial inflammation. Mechanistically, CBL0137 suppressed YAP phosphorylation at Y357 via the tyrosine-protein kinase Src. Furthermore, administration of CBL0137 ameliorated endothelial inflammation and the atherogenesis induced by disturbed flow and consumption of an atherogenic diet in ApoE-/- mice. To our knowledge, this is the first study to identify CBL0137 as a novel YAP inhibitor. We have demonstrated that pharmacologically targeting YAP by CBL0137 inhibits atherogenesis. The present results suggest that CBL0137 holds promise as a new drug for the treatment of atherosclerotic vascular disease.

Abstract 12286: A Novel Yap Inhibitor Cbl0137 Protects Against Endothelial Inflammation and Atherosclerosis

Background: Endothelial activation is a critical hallmark in the initiation of atherosclerosis. Our previous study found that YAP activation plays a significant role in induction of endothelial inflammation and formation of atherosclerotic plaques, suggesting that targeting YAP signaling pathway might be effective for the treatment of atherosclerotic cardiovascular disease. We have recently set up a reporter gene assay-based drug screening platform and identified CBL0137 as an inhibitor of YAP activity in human endothelial cells. Methods: We used 8*GTIIC luciferase reporter gene assay to screen a drug library and other small molecule compounds. We validated the inhibitory effect of CBL0137 on YAP activity by detecting the expression of YAP target genes and phosphorylation of YAP, which were measured by quantitative real-time polymerase chain reaction and western blotting, respectively. Subsequently, we treated human endothelial cells with CBL0137 (1 μM) for 4 hrs, and then isolated the total RNA and performed transcriptome sequencing. We explored the anti-inflammatory effects and underlying mechanisms of CBL0137 in primary endothelial cells. Moreover, we assessed whether CBL0137 could retard formation of atherosclerotic plaques in ApoE-/- mice using carotid artery partial ligation-induced and chronic western diet-induced atherosclerosis models. Results: We identified CBL0137 as a novel YAP inhibitor through screening the drug library. In human endothelial cells, CBL0137 inhibited the phosphorylation of YAP at Y357 to suppress YAP activity, and therefore repressed the expression of YAP target genes. In addition, CBL0137 restrained endothelial inflammatory response in a YAP-dependent manner. Furthermore, CBL0137 inhibited YAP phosphorylation at Y357 via the tyrosine-protein kinase Src. In animal experiments, administration of low doses of CBL0137 attenuated endothelial inflammation and atherosclerosis induced by disturbed flow or atherogenic diets in both male and female ApoE-/- mice. Conclusions: CBL0137 is a novel YAP inhibitor that protects against endothelial inflammation and atherogenesis. CBL0137 might be a promising anti-atherosclerosis drug, but further investigations including clinical trials are required.

Proteolysis Targeting Chimeras (PROTACs) Based on Promiscuous Kinase Inhibitor Synergistically Induce Cancer Cell Apoptosis Through Multiple Mechanisms

AbstractEleven new proteolysis targeting chimeras (PROTACs) based on promiscuous kinase inhibitor Sunitinib were designed, synthesized, and assessed for their anticancer activity against four human cancer cell lines A498, K562, HL‐60 and MCF‐7. Two known compounds were also prepared and used as control samples. The CCK‐8 assay results showed that a new PROTAC with a 1,4‐diaminobutane linker (PROTAC 10) exhibited significant antiproliferative activity against A498 (IC50=0.11 ± 0.01 μM) and K562 (IC50=0.59 ± 0.21 μM) cell lines. Western blot analysis showed that in addition to the previously reported G1 to S phase transition 1 (GSPT1), PROTAC 10 also reduced the protein level of fms‐like tyrosine kinase 3 (FLT3) and proto‐oncogene tyrosine protein kinase Src (SRC) in A498 cells. Moreover, PROTAC 10 regulated the protein levels of GSPT1 and SRC in a dose‐ and time‐dependent manner and induced degradation of GSPT1 in a ubiquitin‐proteasome‐dependent manner in K562 cells.

Prediction Targets of a Novel Sesquiterpene Lactone From Eupatorium lindleyanum DC. Against Viral Pneumonia Using Computational Pharmacology

Objective: This study employed computational pharmacology to explore the possible therapeutic targets and molecular mechanisms of F1012-2, a novel sesquiterpene lactone from Eupatorium lindleyanum DC. (EL), against viral pneumonia. Methods: With network pharmacology, we first looked into various databases for genes and proteins related to viral pneumonia, as well as the potential targets of F1012-2. By overlapping these 2 groups of genes, we acquired the candidate targets of F1012-2 against viral pneumonia. Afterward, enrichment analysis was performed to elucidate the interactive targets in the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. The protein–protein interaction (PPI) network was constructed through the STRING database, and the results were imported into Cytoscape software to search for the key genes by Network Analyzer. Finally, molecular docking analysis was used to further validate the candidate targets of F1012-2 against viral pneumonia. Results: A total of 110 target genes were found for the 3 compounds within F1012-2 (Eupalinolide G, [EG] Eupalinolide I, [EI] and Eupalinolide J [EJ]), while 4322 potential therapeutic targets were uncovered related to viral pneumonia, and the intersection of the 2 groups generated 78 target genes. Among the candidate genes, epidermal growth factor receptor (EGFR), interleukin-1beta (IL1B), tyrosine-protein kinase SRC, Caspase-3 (CASP3), and transcription factor and oncoprotein JUN have the highest degree, betweenness, and closeness, which could fit into binding with EG, EI, and EJ. The enrichment analysis indicated that the major pathways involved were sphingolipid, neurotrophin, tumor necrosis factor (TNF), and Toll-like receptor signaling pathways. Conclusion: These findings showed that by the combination of network pharmacology and molecular docking, we could speculate on the possible mechanism of F1012-2's effect on viral pneumonia, with EGFR, IL1B, SRC, and CASP3 as the promising targets.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide.

Changes of FLI-1 protein expression in mice with pulmonary endothelial barrier dysfunction following acute lung injury induced by lipopolysaccharide

Objective: To observe changes of Friend leukemia virus integration 1 (FLI-1) protein expression of pulmonary tissue in mice with pulmonary endothelial barrier dysfunction following acute lung injury (ALI) induced by lipopolysaccharide (LPS). Methods: The mouse model of ALI was established by injection of LPS (7.5 mg/kg, i.p. ). At 0 h, 12 h, 24 h and 48 h after LPS injection, pulmonary microvascular endothelial permeability and lung wet/dry weight ratio (W/D) were assessed. The contents of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were detected by ELISA method. The protein levels of FLI-1 and Src protein tyrosine kinase (SRC) were analyzed by Western blotting.Results: ①Pulmonary microvascular endothelial permeability at 12 h and 24 h were significantly higher than those of 0 h by 74.3% and 162.4%, respectively, while that of 48 h was lower than that of 24 h by 27.0% (P＜0.05). The W/D at 12 h and 24 h were significantly higher than those of 0 h by 50.1% and 122.9%, respectively, while that of 48 h was lower than that of 24 h by 10.7% (P＜0.05). ②The contents of IL-6 and TNF-α in BALF at 12 h and 24 h were significantly higher than those of 0 h, while those of 48 h were significantly lower than those of 24 h by 28.3% and 21.6% (P＜0.05), respectively. ③The protein level of FLI-1 in lung at 12 h and 24 h were down-regulated than those of 0 h by 20.4% and 56.9%, respectively, while that of 48 h was up-regulated than that of 24 h by 18.2% (P＜0.05). The protein level of SRC in lung at 12 h and 24 h were up-regulated than those of 0 h by 76.8% and 176.7%, respectively, while that of 48 h was down-regulated than that of 24 h by 33.4% (P＜0.05).④Same as the protein level of FLI-1 with the protein level of SRC in lung, pulmonary microvascular endothelial permeability was significantly negative correlated with the protein level of FLI-1 in lung, while it was significantly positive correlated with the protein level of SRC (P＜0.01). Conclusion: FLI-1 participates in the pathological proceeding of pulmonary endothelial barrier dysfunction following ALI induced by LPS.

Regulation of Fibroblast Cell Polarity by Src Tyrosine Kinase.

Src protein tyrosine kinases (SFKs) are a family of nonreceptor tyrosine kinases that are localized beneath the plasma membrane and are activated during cell adhesion, migration, and elongation. Due to their involvement in the activation of signal transduction cascades, SFKs have been suggested to play important roles in the determination of cell polarity during cell extension and elongation. However, the mechanism underlying Src-mediated polarity formation remains unclear. The present study was performed to investigate the mechanisms underlying Src-induced cell polarity formation and cell elongation using Src knockout fibroblasts (SYFs) together with an inhibitor of Src. Normal and Src knockout fibroblasts were also transfected with a wild-type c-Src, dominant negative c-Src, or constitutively active c-Src gene to analyze the changes in cell morphology. SYF cells cultured on a glass substrate elongated symmetrically into spindle-shaped cells, with the formation of focal adhesions at both ends of the cells. When normal fibroblasts were treated with Src Inhibitor No. 5, a selective inhibitor of Src tyrosine kinases, they elongated into symmetrical spindle-shaped cells, similar to SYF cells. These results suggest that cell polarity during extension and elongation may be regulated by SFKs and that the expression and regulation of Src are important for the formation of polarity during cell elongation.

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.FN7ZF7ULbDVsMsT6Sckwh1Video

PRRG4 promotes breast cancer metastasis through the recruitment of NEDD4 and downregulation of Robo1.

Metastasis is responsible for the death of most breast cancer patients. Robo1 has been implicated as a tumor suppressor for various cancers including breast cancer. However, it is not well understood how Robo1 expression is regulated during tumorigenesis. In this study, we uncovered that the transmembrane proline rich γ-carboxyglutamic acid protein 4 (PRRG4) promotes breast cancer metastasis by downregulating Robo1. Analysis of mRNA expression data in The Cancer Genome Atlas and immunohistochemistry assay on breast tumor samples showed that PRRG4 expression was higher in breast tumors than in normal breast tissues. Experiments with PRRG4 knockdown and overexpression revealed that PRRG4 promoted migration and invasion of breast cancer cells, and enhanced metastasis in an experimental metastasis model. Mechanistically, we found that PRRG4 via its LPSY and PPPY motifs recruited the E3 ubiquitin ligase NEDD4, which induced ubiquitination and degradation of Robo1, thus contributing to migration and invasion of breast cancer cells. In addition, PRRG4 interacted with and enhanced protein tyrosine kinase Src and FAK activation. Overall, our data support a model that PRRG4 via NEDD4 downregulates the Robo1, resulting in the activation of Src and FAK and promoting breast cancer metastasis. PRRG4 may be a novel target for treating metastatic breast cancer.

Rola midostauryny w leczeniu ostrej białaczki szpikowej z towarzyszącą mutacją FLT3

Acute myeloid leukemia (AML) is a highly heterogeneous disease. Mutation with internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is one of the two most common driver mu­tations in AML, the presence of which is associated with a poor outcome. Midostaurin (Mido) is a multi-targeted tyrosine kinase inhibitor that potently inhibits kinase FLT3, other kinases including platelet-derived growth factor receptors α (PDGFR- α ) and b (PDGFR- β ), tyrosine-protein kinase Src, KIT receptor tyrosine kinase, and vascular endothelial growth factor receptor (VEGFR). Mido is the first targeted therapy to demonstrate improved outcome, and as such it represents a game-changer. The RATIFY study demonstrated significant improvements in overall survival, and in event-free survival, when Mido was added to standard chemotherapy in patients with newly diagnosed FLT3-mutated AML. This paper reviews the current clinical evidence regarding Mido and its use in the induction and treatment of relapsed or refractory disease, and maintenance setting.

Glycogen Synthase Kinase 3β Regulates Antiviral Responses of TLR3 via TRAF2-Src Axis.

The protein tyrosine kinase Src regulates the synthesis of TLR3-mediated IFN-β via the TBK1-IFN regulatory factor 3 axis. However, the molecular mechanisms regulating Src activity in TLR3 signaling remain unclear. In this study, we report that GSK3β regulates Src phosphorylation via TNFR-associated factor 2 (TRAF2)-mediated Src ubiquitination. GSK3β deficiency in mouse embryonic fibroblasts significantly reduces polyinosinic:polycytidylic acid-induced IFN-β and IFN-stimulated gene expression, which is caused by diminished phosphorylation of Src at tyrosine 416. Src undergoes polyinosinic:polycytidylic acid-dependent lysine 63 chain ubiquitination, and TRAF2 is a direct E3 ligase for Src. Our study reveals novel mechanisms underlying TLR3-mediated antiviral responses mediated via the GSK3β-TRAF2-Src axis.

Spironolactone suppresses aldosterone-induced Kv1.5 expression by attenuating mineralocorticoid receptor-Nox1/2/4-mediated ROS generation in neonatal rat atrial myocytes

Our previous investigation indicated that angiotensin II (Ang II) enhances the expression of Kv1.5, a promising target for the treatment of atrial fibrillation (AF), by activating reactive oxygen species (ROS)-dependent phosphorylation of Smad 2/3 (forming P-Smad 2/3) and ERK 1/2 (forming P-ERK 1/2). A recent study indicated that aldosterone (Aldo) upregulates atrial Kv1.5 protein in a rat AF model, but the mechanism remains unknown. The present study aimed to clarify the mechanism underlying Aldo-induced Kv1.5 expression and to test whether spironolactone may modulate atrial Kv1.5. Our Western blot analysis indicated that the Aldo/mineralocorticoid receptor (MR) interacts with Ang II/AT1R in upregulating Kv1.5 expression in cultured neonatal atrial myocytes (NRAMs). Blockade of MR with spironolactone and of AT1R with losartan significantly suppressed Kv1.5 expression induction by combined Aldo and Ang II treatment. Aldo increased the protein expression of Nox1, Nox2 and Nox4, but this effect was abolished by spironolactone pretreatment. The Aldo-induced upregulation of Kv1.5 was also reversed by the Src protein tyrosine kinase family inhibitor PP2, the Nox2 inhibitor gp91ds-tat and the Nox1/Nox4 inhibitor GKT137831 but not by the Rac GTPase inhibitor NSC23766. Flow cytometry showed that the Aldo-induced ROS production was inhibited by spironolactone, PP2, gp91ds-tat and GKT137831. Spironolactone suppressed the Aldo-induced protein expression phosphorylated Src (P-Src), P-Smad 2/3 and P-ERK 1/2. In conclusion, we have demonstrated that spironolactone suppresses Aldo-induced Kv1.5 expression by attenuating MR-Nox1/2/4-mediated ROS generation in NRAMs.