Grb2-associated Binding Protein Research Articles

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.

Effect of exercise and diet intervention in NAFLD and NASH via GAB2 methylation

BackgroundNonalcoholic fatty liver disease (NAFLD) is a disorder that extends from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), which is effectively alleviated by lifestyle intervention. Nevertheless, DNA methylation mechanism underling the effect of environmental factors on NAFLD and NASH is still obscure. The aim of this study was to investigate the effect of exercise and diet intervention in NAFLD and NASH via DNA methylation of GAB2.MethodsMethylation of genomic DNA in human NAFLD was quantified using Infinium Methylation EPIC BeadChip assay after exercise (Ex), low carbohydrate diet (LCD) and exercise plus low carbohydrate diet (ELCD) intervention. The output Idat files were processed using ChAMP package. False discovery rate on genome-wide analysis of DNA methylation (q < 0.05), and cytosine-guanine dinucleotides (CpGs) which are located in promoters were used for subsequent analysis (|Δβ|≥ 0.1). K-means clustering was used to cluster differentially methylated genes according to 3D genome information from Human embryonic stem cell. To quantify DNA methylation and mRNA expression of GRB2 associated binding protein 2 (GAB2) in NASH mice after Ex, low fat diet (LFD) and exercise plus low fat diet (ELFD), MassARRAY EpiTYPER and quantitative reverse transcription polymerase chain reaction were used.ResultsBoth LCD and ELCD intervention on human NAFLD can induce same DNA methylation alterations at critical genes in blood, e.g., GAB2, which was also validated in liver and adipose of NASH mice after LFD and ELFD intervention. Moreover, methylation of CpG units (i.e., CpG_10.11.12) inversely correlated with mRNA expression GAB2 in adipose tissue of NASH mice after ELFD intervention.ConclusionsWe highlighted the susceptibility of DNA methylation in GAB2 to ELFD intervention, through which exercise and diet can protect against the progression of NAFLD and NASH on the genome level, and demonstrated that the DNA methylation variation in blood could mirror epigenetic signatures in target tissues of important biological function, i.e., liver and adipose tissue.Trialregistration International Standard Randomized Controlled Trial Number Register (ISRCTN42622771)

GAB1 alleviates septic lung injury by inhibiting the TLR4/ NF-κB pathway.

Sepsis, with its high morbidity and mortality, is a difficult problem in critical care medicine. The purpose of this study is to investigate the involvement of GRB2-associated binding protein 1 (GAB1) in septic lung injury. Lipopolysaccharide (LPS)-induced mouse model and A549 cell model were used to simulate septic lung injury. Haematoxylin and eosin (H&E) staining was used to observe the pathological changes. The terminal-deoxynucleotidyl transferase/(TdT)-mediated dUTP-biotin nick end labelling (TUNEL) staining and flow cytometry were used to detect apoptosis. The levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). In LPS-induced sepsis mice, GAB1 expression was markedly reduced, and GAB1 overexpression significantly attenuated cell apoptosis and decreased levels of macrophages, neutrophils, and inflammatory factors in the BALF. Our results also demonstrated that GAB1 overexpression significantly reduced LPS-induced apoptosis and inflammation of A549 cells. More importantly, GAB1 overexpression significantly inhibited the Toll-like receptor/ NFkappaB (TLR4/NF-κB) pathway, while silencing GAB1significantly activated the TLR4/NF-κB pathway and induced apoptosis and increased expression of inflammatory factors. However, the TLR4 inhibitor TAK-242 eliminated the effect of GAB1silencing on A549. In conclusion, GAB1 is a key regulator of sepsis by inhibiting TLR4/NF-κB mediated apoptosis and inflammation.

FoxO1-GAB1 axis regulates homing capacity and tonic AKT activity in chronic lymphocytic leukemia

AbstractRecirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2–associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the “tonic” AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Medulloblastoma: clinicopathological parameters, risk stratification, and survival analysis of immunohistochemically validated molecular subgroups

BackgroundMedulloblastoma (MB) is a heterogeneous disease, displaying distinct genetic profiles with specific molecular subgroups. This study aimed to validate MB molecular subgrouping using surrogate immunohistochemistry and associate molecular subgroups, histopathological types, and available clinicopathological parameters with overall survival (OS) and progression-free survival (PFS) of MB patients. This study included 40 MBs; immunohistochemical staining, using β-catenin and GRB2-Associated Binding Protein 1 (GAB1) antibodies, was used to classify MB cases into wingless signaling activated (WNT), sonic hedgehog (SHH), and non-WNT/SHH molecular subgroups. Nuclear morphometric analysis (for assessment of degree of anaplasia) and Kaplan-Meier survival curves were done.ResultsMB cases were classified into WNT (10%), SHH (30%), and non-WNT/SHH (60%) subgroups. Histopathological types differed significantly according to tumor location (p< 0.001), degree of anaplasia (p = 0.014), molecular subgroups (p < 0.001), and risk stratification (p = 0.008). Molecular subgroups differed significantly in age distribution (p = 0.031), tumor location (p< 0.001), histopathological variants (p < 0.001), and risk stratification (p < 0.001). OS was 77.5% and 50% after 1 and 2 years, while PFS was 65% and 27.5% after 1 and 2 years, respectively. OS and PFS were associated significantly with histopathological variants (p < 0.001 and 0.001), molecular subgroups (p = 0.012 and 0.005), and risk stratification (p < 0.001 and < 0.001), respectively.ConclusionsMedulloblastoma classification based on molecular subgroups, together with clinicopathological indicators, mainly histopathological types; accurately risk stratifies MB patients and predicts their survival.

Circ_0061012 contributes to IL-22-induced proliferation, migration and invasion in keratinocytes through miR-194-5p/GAB1 axis in psoriasis.

Psoriasis is a chronic inflammation-associated skin disorder featured by excessive proliferation and abnormal differentiation of keratinocytes. Here, we intended to investigate the role of circular RNA 0061012 (circ_0061012) in psoriasis progression. The expression of circ_0061012, SLMO2-ATP5E readthrough (SLMO2-ATP5E) messenger RNA (mRNA), microRNA-194-5p (miR-194-5p) and GRB2 associated binding protein 1 (GAB1) mRNA was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and metastasis were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell assays. Western blot assay was used to measure the protein levels of Ki67, matrix metallopeptidase 9 (MMP9) and GAB1. Dual-luciferase reporter assay and RNA immune co-precipitation (RIP) assay were used to verify the interaction between miR-194-5p and circ_0061012 or GAB1. Circ_0061012 abundance was significantly enhanced in lesional skin samples from psoriasis patients than that in normal skin specimens from healthy volunteers. Interleukin-22 (IL-22) treatment increased the expression of circ_0061012 in a dose-dependent manner. Circ_0061012 silencing alleviated IL-22-induced promoting effects in the proliferation, migration and invasion of HaCaT cells. Circ_0061012 interacted with miR-194-5p, and miR-194-5p knockdown counteracted circ_0061012 silencing-mediated influences in IL-22-induced HaCaT cells. GAB1 was a target of miR-194-5p in HaCaT cells, and miR-194-5p hampered proliferation and metastasis which were induced by IL-22 partly through targeting GAB1. Circ_0061012 elevated the expression of GAB1 through sponging miR-194-5p in HaCaT cells. Circ_0061012 accelerated IL-22-induced proliferation and metastasis in HaCaT cells through enhancing GAB1 expression via sponging miR-194-5p in psoriasis.

Loss of GRB2 associated binding protein 1 in arteriosclerosis obliterans promotes host autophagy.

Background:Arteriosclerosis obliterans (ASO) is a major cause of adult limb loss worldwide. Autophagy of vascular endothelial cell (VEC) contributes to the ASO progression. However, the molecular mechanism that controls VEC autophagy remains unclear. In this study, we aimed to explore the role of the GRB2 associated binding protein 1 (GAB1) in regulating VEC autophagy.Methods:In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression. Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima. Gain- and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1.Results:The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor (0.80 vs. 0.20, t = 6.43, P < 0.05). The expression level of GAB1 mRNA (1.00 vs. 0.24, t = 7.41, P < 0.05) and protein (0.72 vs. 0.21, t = 5.97, P < 0.05) was significantly decreased in ASO group as compared with the control group. Loss of GAB1 led to a remarkable decrease in LC3II (1.19 vs. 0.68, t = 5.99, P < 0.05), whereas overexpression of GAB1 significantly led to a decrease in LC3II level (0.41 vs. 0.93, t = 7.12, P < 0.05). Phosphorylation levels of JNK and p38 were significantly associated with gain- and loss-of-function of GAB1 protein.Conclusion:Loss of GAB1 promotes VEC autophagy which is associated with ASO. GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.

Depletion of circRNA circ_CDK14 inhibits osteosarcoma progression by regulating the miR-520a-3p/GAB1 axis.

Osteosarcoma (OS) is a lethal bone malignancy. Circular RNAs (circRNAs) have emerged as important regulators of OS development. CircRNA cyclin dependent kinase 14 (circ_CDK14) was reported to be a potential oncogene in OS. However, the mechanistic pathway by which circ_CDK14 functions in OS is largely unknown. The relative expression of circ_CDK14, microRNA (miR)-520a-3p, and GRB2 Associated Binding Protein 1 (GAB1) was evaluated by quantitative real-time PCR and western blot assays. Flow cytometry was employed to monitor cell cycle distribution and apoptosis. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were performed to assess cell viability and colony formation ability, respectively. Western blot assay was also used to detect the expression of apoptosis-related proteins. Transwell assay was carried out to monitor cell migration and invasion. Additionally, the target association between miR-520a-3p and circ_CDK14 or GAB1 was confirmed by a dual-luciferase reporter assay. Xenograft assay was applied to investigate the role of circ_CDK14 in vivo. Circ_CDK14 and GAB1 expression was upregulated, while miR-520a-3p was downregulated in OS tissues and cells. Circ_CDK14 depletion hindered OS cell proliferation, metastasis, and tumorigenesis while facilitated apoptosis, which were all ameliorated by miR-520a-3p inhibition. Circ_CDK14 could sponge miR-520a-3p. miR-520a-3p targeted GAB1 to repress OS cell proliferation and metastasis. Circ_CDK14 knockdown blocked OS tumor growth in vivo. Circ_CDK14 might positively affect OS development by modulating the miR-520a-3p/GAB1 axis.

Oncogenic circDHTKD1 promotes tumor growth and metastasis of oral squamous cell carcinoma in vitro and in vivo via upregulating miR-326-mediated GAB1.

Circular RNAs (circRNAs) have been extensively elucidated with regard to their significant implications in oral squamous cell carcinoma (OSCC). This study performed the functional investigation of circRNA dehydrogenase E1 and transketolase domain containing 1 (circDHTKD1) in OSCC. RNA expression levels of different molecules were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular behaviors were detected by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) for cell viability, colony formation assay for clonal capacity, flow cytometry for cell apoptosis, wound healing assay for migration, and transwell assay for migration/invasion. Western blot was used for analyzing protein expression. RNA pull-down and dual-luciferase reporter assays were applied to assess the binding between targets. A xenograft tumor model was established in nude mice for in vivo experiments. Our expression analysis revealed that circDHTKD1 was upregulated in OSCC tissues and cells. circDHTKD1 knockdown was shown to impede OSCC cell growth and metastasis but motivate apoptosis. Additionally, circDHTKD1 served as a microRNA-326 (miR-326) sponge and the function of circDHTKD1 was achieved by sponging miR-326 in OSCC cells. Also, miR-326 inhibited OSCC development via targeting GRB2-associated-binding protein 1 (GAB1). circDHTKD1 could sponge miR-326 to alter GAB1 expression. Furthermore, circDHTKD1 contributed to OSCC progression in vivo via the miR-326/GAB1 axis. These data disclosed a specific circDHTKD1/miR-326/GAB1 signal axis in governing the malignant progression of OSCC, showing the considerable possibility of circDHTKD1 as a predictive and therapeutic target for clinical diagnosis and treatment of OSCC.

The comparison of differentially expressed microRNAs in Bag-1 deficient and wild type MCF-7 breast cancer cells by small RNA sequencing.

The multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1 -mediated cell response, it has become essential to determine microRNA (miRNA) functions in breast cancer depending on Bag-1 expression level. This study aims to compare microRNA expression levels in wt and Bag-1 knockout (KO) MCF-7 breast cancer cells. hsa-miR-429 was selected as a potential miRNA in BAG-1KO MCF-7 cells because of the downregulation both in bioinformatics and validation qRT-PCR assay. According to predicted mRNA targets and functional enrichment analysis the ten hub proteins that are phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA), kinase insert domain receptor (KDR), GRB2 associated binding protein 1 (GAB1), Rac family small GTPase1 (RAC1), vascular endothelial growth factor A (VEGFA), Cbl proto-oncogene (CBL), syndecan 2 (SDC2), phospholipase C gamma 1 (PLCG1), E1A binding protein p300 (EP300), and CRK like proto-oncogene, adaptor protein (CRKL) were identified as targets of hsa-miR-429. The functional enrichment analysis showed that the most significant proteins were enriched in PI3K/Akt, focal adhesion, cytoskeleton regulation, proteoglycans in cancer, and Ras signaling pathways. It was determined that hsa-miR-429 targeted these pathways in Bag-1 deficient conditions and could be used as a potential therapeutic target in future studies.

Sustained Stimulation of β2AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway.

IntroductionThis study aimed to investigate the role of β2 adrenergic receptor (β2AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the β2AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression.MethodsH9C2 cells were treated with various protein inhibitors (CGP, β1AR inhibitor CGP20712; ICI, β2AR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the β2AR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot.ResultsGene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of β2AR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that β2AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex.ConclusionThe sustained stimulation of β2AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.

MicroRNA‑218 inhibits tumor angiogenesis of human renal cell carcinoma by targeting GAB2.

Renal cell carcinoma (RCC) is one of the most common malignant cancers in the adult urinary system worldwide. Tumor angiogenesis is a critical process during cancer progression, as it modulates carcinogenesis and metastasis. In recent years, microRNA-218 (miR-218) has been confirmed to play a crucial role in tumor suppression. However, the role of miR-218 in RCC angiogenesis remains unclear. In the present study, it was found that the expression of miR-218 was decreased in RCC tumor tissues and cell lines as detected by real-time PCR analysis. Tube formation assays and migration assays also confirmed that miR-218 inhibited the interaction between RCC cells and vascular endothelial cells by suppressing proangiogenic factor vascular endothelial growth factor A (VEGFA) in RCC cells. miR-218 also repressed the subcutaneous tumorigenesis of RCC cells in nude mice, and the corneal angiogenesis in rabbit eyes. The underlying molecular mechanism was elucidated; miR-218 targets GRB2-associated binding protein 2 (GAB2), thereby inhibiting the PI3K/AKT/mTOR/VEGFA pathway. These results provide new insights into the mechanism of RCC carcinogenesis and progression, suggesting that miRNA-218 may be a therapeutic target for the treatment of RCC.

GAB2 and GAB3 are expressed in a tumor stage-, grade- and histotype-dependent manner and are associated with shorter progression-free survival in ovarian cancer.

Ovarian cancer is the most lethal gynecological malignancy and molecular mechanisms of its progression and metastasis are not completely understood. Some members of GAB (GRB2-associated binding) protein family have been reported to be involved in tumor cell proliferation and metastasis in various cancer types. In the present study, we analyzed the expression of GAB proteins (GAB1, GAB2 and GAB3) in ovarian cancer compared to normal ovarian tissue, in terms of tumor stage, tumor grade and histological type. Differential expression analyses performed in R programming environment using multiple transcriptome datasets (n = 1449) showed that GAB1 expression is decreased in ovarian cancer independently of tumor stage, grade and histotype. Unlike GAB1, expression of GAB2 and GAB3 are increased from early stage to late stage and from low grade to high grade in epithelial ovarian cancer. GAB2 and GAB3 also showed histotype-dependent expression. GAB3 was computed as a top gene whose expression most significantly changed between tumor cells from primary tumor, metastases and ascites. High expression of GAB2 and GAB3 was shown to be associated with shorter progression-free survival in ovarian cancer. This study shows that GAB2 and GAB3 can be important regulators of tumor progression and metastasis in ovarian cancer.

Elevated expressions of SHP2 and GAB2 correlated with VEGF in eutopic and ectopic endometrium of women with ovarian endometriosis

Aims: Protein tyrosine phosphatase Src-homology-2-domain-containing phosphatase 2 (SHP2) and adaptor protein Grb2-associated binding protein 2 (GAB2) can bind to each other in various signal transduction. However, the expression of SHP2 and GAB2 have not been investigated in endometriosis. The aim of the study was to evaluate the expressions of SHP2 and GAB2, and explore the correlation with Ki67 and VEGF in ovarian endometriosis.Materials and methods: The protein expressions and localizations were assessed immunohistochemically in ectopic, eutopic endometrium and normal endometrium from patients with (n = 30) and without (n = 30) ovarian endometriosis.Results: SHP2 was mainly present in the endometrial glandular epithelium, with increased expression in eutopic endometrium and even higher expression in ectopic endometrium compared to control endometrium (p < .05). GAB2 was immunolocalized in endometrial epithelium and stroma, increasing its expression from control endometrium to eutopic and ectopic endometrium (p < .05). Positive correlation was found between SHP2 and GAB2 in endometrium (p < .01). SHP2 and GAB2 both positively correlated with VEGF (p < .05), but not Ki67 in endometrium.Conclusions: We provide the first evidence that the protein expressions of SHP2 and GAB2 were elevated in ectopic and eutopic endometrium, suggesting GAB2-SHP2 axis regulating VEGF might contribute to the pathomechanism of endometriosis.

G-CSF-dependent neutrophil differentiation requires downregulation of MAPK activities through the Gab2 signaling pathway.

Granulocyte colony-stimulating factor (G-CSF) stimulation of myeloid cells induced tyrosine-phosphorylation of cellular proteins. One of the tyrosine-phosphorylated proteins was found to be a scaffold protein, Grb2-associated binding protein 2 (Gab2). Another member of Gab family protein, Gab3, was exogenously overexpressed in neutrophil progenitor cells to make the Gab3 protein to compete with the endogenous Gab2 for the G-CSF-dependent signaling. In Gab3-overexpressed cells, the level of tyrosine phosphorylation of endogenous Gab2 by G-CSF stimulation was markedly downregulated, while the phosphorylation of Gab3 was significantly enhanced. The Gab3-overexpressed cells continuously proliferated in the medium containing G-CSF and lost the ability to differentiate to the mature neutrophil, characterized by the lobulated nucleus. The G-CSF stimulation-dependent tyrosine phosphorylation of Gab3, the association of SHP2 to Gab3 and the following mitogen-activated protein kinase (MAPK) activation were prolonged in the Gab3-overexpressed cells, compared to the parental cells, where the binding of SHP2 to Gab2 protein and thereby the activation of MAPK were not sustained after G-CSF stimulation. Inhibition of MAPK by pharmaceutical inhibitor restored the Gab3-overexpressed cells to the ability to differentiate to mature neutrophil. Therefore, G-CSF-dependent Gab2 phosphorylation and following its downregulation led the short-term MAPK activation. The downregulation of MAPK after transient Gab2 phosphorylation was necessary for the consequent neutrophil differentiation induced by G-CSF stimulation.

5-Bromo-3,4-dihydroxybenzaldehyde from Polysiphonia morrowii attenuate IgE/BSA-stimulated mast cell activation and passive cutaneous anaphylaxis in mice

The present study investigates the anti-allergic activity of the marine algal bromophenol, 3-bromo-4,5-dihydroxybenzaldehyde (BDB), isolated from Polysiphonia morrowii Harvey in immunoglobulin (Ig)E/bovine serum albumin (BSA)-stimulated mouse bone marrow-derived cultured mast cells (BMCMCs) and a passive cutaneous anaphylaxis (PCA) mice ear model. BDB effectively inhibited β-hexosaminidase release (IC50 = 80.12 µM), in IgE/BSA-stimulated BMCMCs without a cytotoxic response. Also, BDB down-regulated the expression or secretion of cytokines, interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α and the chemokine (thymus and activation-regulated chemokine (TARC). The above effects could be attributed to the dose-dependent decrease of FcεRI expression on the surface of BMCMCs and its stable IgE binding. Moreover, BDB suppressed the nuclear factor (NF)-κB and spleen tyrosine kinase (SYK)-linker for T-cell activation (LAT)-GRB2 associated binding protein 2 (Gab2) signaling axis activated by IgE/BSA stimulation. Furthermore, oral administration of BDB to IgE-sensitized mice effectively attenuated IgE-triggered PCA reaction. Collectively, the anti-allergic effects of BDB suggest its potential applicability as a candidate for in-depth test trials.

The miR-218/GAB2 axis regulates proliferation, invasion and EMT via the PI3K/AKT/GSK-3β pathway in prostate cancer

Altered expression of microRNA (miRNA) is associated with the occurrence and metastasis of various tumors. We previously found that miR-218 inhibits tumor angiogenesis through the RICTOR/VEGFA axis in prostate cancer (PCa). In this study, we determined that miR-218 also had a negative effect on cell growth, migration, and invasion ability in PCa. Our data showed that miR-218 bound to the Grb2-associated binding protein 2 (GAB2) 3'-UTR region and inhibited GAB2 expression. As a novel downstream target of miR-218, GAB2 has been reported to be involved in the occurrence and development of various human tumors, but its role in the progression and metastasis of PCa has not been addressed. We demonstrated for the first time that the expression of GAB2 in the PCa cell lines was increased, while knocking down GAB2 significantly inhibited cell growth, metastatic ability and EMT process in PCa. In addition, the recovery of GAB2 could reverse the changes in the biological function of PCa cells caused by the ectopic expression of miR-218. Mechanistically, miR-218-mediated GAB2 transcriptional suppression significantly inhibited the activity of the PI3K/AKT/GSK-3β pathway, whose abnormal activation was found to be related to the malignant progression of PCa. Taken together, our findings suggest that the miR-218/GAB2 axis may become a novel prognostic indicator and potential therapeutic target in PCa.

Mechanistic insights explain the transforming potential of the T507K substitution in the protein-tyrosine phosphatase SHP2

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Oncogenic Fusions May Be Frequently Present at Resistance of EGFR Tyrosine Kinase Inhibitors in Patients With NSCLC: A Brief Report

IntroductionDespite initial benefit, virtually all patients suffering from EGFR-mutant NSCLC experience acquired resistance to tyrosine kinase inhibitors (TKIs), driven by multiple mechanisms. Recent reports have identified oncogenic kinase fusions as off-target resistance mechanisms; however, these alterations have been rarely investigated at EGFR TKIs progression. MethodsPatients with EGFR-mutated metastatic NSCLC (N = 62) with tissue and plasma biopsies at EGFR TKI progression between January 2015 and June 2019, at a French hospital and optionally before progression, were identified from the prospective MATCH-R study (NCT02517892). Postprogression biopsy samples were analyzed for gene fusions using targeted gene panel sequencing, whole-exome sequencing, RNA sequencing, and comparative genomic hybridization array. ResultsSix gene fusions were detected in tumor progression biopsies under an EGFR TKI from 62 consecutive patients (9.7%) with EGFR-mutated advanced NSCLC. Among 31 patients progressing to first- or second-generation EGFR TKIs, one (3%) had an Eukaryotic translation initiation factor 4 gamma 2–GRB2 associated binding protein 1 (EIF4G2-GAB1) fusion. Among 31 patients progressing to the third-generation osimertinib, five (16%) presented oncogene fusions of fibroblast growth factor receptor 3–transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3) (n = 2), kinesin family member 5B–Ret proto-oncogene (KIF5B-RET) (n = 1), striatin–anaplastic lymphoma kinase (STRN-ALK) (n = 1), and zinc finger DHHC-Type palmitoyltransferase 20–Thr790Met (ZDHHC20-BRAF) (n = 1) transcripts. Out of two patients that received osimertinib at first-line, one acquired an FGFR3-TACC3 fusion at progression. In all patients, fusions co-occurred with the original activating EGFR mutation; however, among four patients with an acquired T790M mutation, three (75%) lost the T790M mutation. ConclusionsOncogenic fusions at the time of EGFR TKI resistance were identified at a relatively high frequency, mainly after the third-generation TKI osimertinib. Patients progressing to EGFR TKIs may have a new opportunity for targeted therapy when oncogenic fusions are identified.

LncRNA TUG1 alleviates sepsis-induced acute lung injury by targeting miR-34b-5p/GAB1

BackgroundSepsis-induced acute lung injury (ALI) is a clinical syndrome characterized by the injury of alveolar epithelium and pulmonary endothelial cells. This study aimed to investigate the regulation of long noncoding RNA (lncRNA) taurine up-regulated gene 1 (TUG1) in a murine ALI model and in primary murine pulmonary microvascular endothelial cells (PMVECs) stimulated with lipopolysaccharide (LPS).MethodsAdult C57BL/6 mice were intravenously injected with or without TUG1-expressiong adenoviral vector or control vector 1 week before the establishment of ALI model. PMVECs were transfected with TUG1-expressiong or control vectors followed by LPS stimulation. MiR-34b-5p was confirmed as a target of TUG1 using dual-luciferase reporter assay. GRB2 associated binding protein 1 (GAB1) was confirmed as a downstream target of miR-34b-5p using the same method. In the rescue experiment, PMVECs were co-transfected with TUG1-expressing vector and miR-34b-5p mimics (or control mimics) 24 h before LPS treatment.ResultsALI mice showed reduced levels of TUG1, pulmonary injury, and induced apoptosis and inflammation compared to the control group. The overexpression of TUG1 in ALI mice ameliorated sepsis-induced pulmonary injury, apoptosis and inflammation. TUG1 also showed protective effect in LPS-treated PMVECs. The expression of MiR-34b-5p was negatively correlated with the level of TUG1. TUG1-supressed apoptosis and inflammation in LPS-stimulated PMVECs were restored by miR-34b-5p overexpression. GAB1 was inversely regulated by miR-34b-5p but was positively correlated with TUG1 expression.ConclusionTUG1 alleviated sepsis-induced inflammation and apoptosis via targeting miR-34b-5p and GAB1. These findings suggested that TUG1 might be served as a therapeutic potential for the treatment of sepsis-induced ALI.