Grb2 Research Articles

The role of CD28 in adult T-cell leukaemia/lymphoma.

The role of CD28 in adult T-cell leukaemia/lymphoma.

Drug-Target Interaction Network Analysis of Gene-Phenotype Connectivity Maintained by Genistein.

Genistein is a type of isoflavone, which has been widely described as an antitumor agent in many cancers. The present study aimed to provide information on the mechanisms of genistein's activity and thus enable a wider range of targeted therapies in hepatitis B virus (HBV)-related liver cancer. We searched the DrugBank database for direct targets of genistein, which were then analyzed through the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database to predict their secondary protein targets. Thirteen primary protein targets of genistein and 209 secondary protein targets-associated genes were identified. The data were integrated into the network of protein targets-associated genes and visualized with the Cytoscape software. We further carried out GO (Gene Ontology) analysis and KEGG (Kyoto Encyclopedia of Gene and Genome) pathway analysis using DAVID (database for annotation, visualization, and integrated discovery) tool. The top 14 KEGG pathways were further assessed, and 19 overlapping genes derived from pathways of hepatitis B and cancer were discovered. The overlapping targets were further mapped in the online tool UALCAN to evaluate the survival rate of hepatocellular carcinoma (HCC) patients. We found that the overexpression of Grb2 (growth factor receptor-binding protein 2) (p < 0.0001) was linked to poor overall survival for liver HCC patients, followed by AKT1 (p = 0.0015) and PIK3CA (p = 0.0088). The present study analyzes the drug-target-disease network and may prove to be a useful tool in gene-phenotype connectivity for genistein in HBV-related liver cancer. Our data also pave the way for further research on Grb2 during the development of chronic HBV infection in liver cancer.

Protective effects of dioscin on vascular remodeling in pulmonary arterial hypertension via adjusting GRB2/ERK/PI3K-AKT signal

Pulmonary arterial hypertension (PAH) is a progressive and lethal cardiopulmonary. Pulmonary vascular remodeling (PVR) caused by excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) is the chief pathological feature of PAH. Dioscin is a natural product that possesses multiple pharmacological activities, but its effect on PAH remains unclear. In this study, effect of dioscin on vascular remodeling in PAH was assessed in hypoxia-induced PASMCs, hypoxia-induced and monocrotaline (MCT)-induced rats. Western blot, Real-time PCR and siRNA transfection tests were applied to evaluate the possible mechanisms of dioscin. In vitro experiments, results showed dioscin markedly inhibited the proliferation and migration, and promoted apoptosis of hypoxic PASMCs. In vivo, dioscin significantly decreased the right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI), and improved pulmonary vascular stenosis in rats induced by hypoxia or MCT. Molecular mechanism studies showed that dioscin significantly reduced the expression of growth factor receptor-bound protein 2 (GRB2). Subsequently, dioscin reduced the expressions of Ras, Cyclin D1, CDK4, c-Fos, PCNA and p-ERK to inhibit proliferation and migration of PASMCs, inhibited p-PI3K and p-AKT levels and increased Bax/Bcl2 ratio to promote cell apoptosis. GRB2 siRNA transfection in PASMCs further confirmed that the inhibitory action of dioscin in PAH was evoked by adjusting GRB2/ERK/PI3K-AKT signal. Taken together, our study indicated that dioscin attenuates PAH through adjusting GRB2/ERK/PI3K-AKT signal to inhibit PASMCs proliferation and migration, and promote apoptosis, and dioscin may be developed as a therapeutic strategy for treating PAH in the future.

Recombinant Chromosome 7 Driven by Maternal Chromosome 7 Pericentric Inversion in a Girl with Features of Silver-Russell Syndrome

Maternal uniparental disomy of chromosome 7 is present in 5–10% of patients with Silver-Russell syndrome (SRS), and duplication of 7p including GRB10 (Growth Factor Receptor-Bound Protein 10), an imprinted gene that affects pre-and postnatal growth retardation, has been associated with the SRS phenotype. Here, we report on a 17 year old girl referred to array-CGH analysis for short stature, psychomotor delay, and relative macrocephaly. Array-CGH analysis showed two copy number variants (CNVs): a ~12.7 Mb gain in 7p13-p11.2, involving GRB10 and an ~9 Mb loss in 7q11.21-q11.23. FISH experiments performed on the proband’s mother showed a chromosome 7 pericentric inversion that might have mediated the complex rearrangement harbored by the daughter. Indeed, we found that segmental duplications, of which chromosome 7 is highly enriched, mapped at the breakpoints of both the mother’s inversion and the daughter’s CNVs. We postulate that pairing of highly homologous sequences might have perturbed the correct meiotic chromosome segregation, leading to unbalanced outcomes and acting as the putative meiotic mechanism that was causative of the proband’s rearrangement. Comparison of the girl’s phenotype to those of patients with similar CNVs supports the presence of 7p in a locus associated with features of SRS syndrome.

Role of long noncoding RNA MEG3/miR-378/GRB2 axis in neuronal autophagy and neurological functional impairment in ischemic stroke

Autophagy has been shown to maintain neural system homeostasis during stroke. However, the molecular mechanisms underlying neuronal autophagy in ischemic stroke remain poorly understood. This study aims to investigate the regulatory mechanisms of the pathway consisting of MEG3 (maternally expressed gene 3), microRNA-378 (miR-378), and GRB2 (growth factor receptor-bound protein 2) in neuronal autophagy and neurological functional impairment in ischemic stroke. A mouse model of the middle cerebral artery occluded-induced ischemic stroke and an in vitro model of oxygen-glucose deprivation-induced neuronal injury were developed. To understand the role of the MEG3/miR-378/GRB2 axis in the neuronal regulation, the expression of proteins associated with autophagy in neurons was measured by Western blotting analysis, and neuron death was evaluated using a lactate dehydrogenase leakage rate test. First, it was found that the GRB2 gene, up-regulated in middle cerebral artery occluded-operated mice and oxygen-glucose deprivation-exposed neurons, was a target gene of miR-378. Next, miR-378 inhibited neuronal loss and neurological functional impairment in mice, as well as neuronal autophagy and neuronal death by silencing of GRB2. Confirmatory in vitro experiments showed that MEG3 could specifically bind to miR-378 and subsequently up-regulate the expression of GRB2, which in turn suppressed the activation of Akt/mTOR pathway. Taken together, these findings suggested that miR-378 might protect against neuronal autophagy and neurological functional impairment and proposed that a MEG3/miR-378/GRB2 regulatory axis contributed to better understanding of the pathophysiology of ischemic stroke.

Gene expression in urinary incontinence and pelvic organ prolapse: a review of literature.

To review current evidence on gene expression in women with urinary incontinence and pelvic organ prolapse (POP). Our literature review revealed numerous genes that are associated with urinary incontinence and POP. For overactive bladder and urge urinary incontinence, four genes were highlighted: adrenergic receptor β3, Rho guanine nucleotide exchange factor 10, Rho-associated coiled-coil containing protein kinase 2, and potassium two pore domain channel subfamily K member-1. For Stress Urinary incontinence (SUI), 13 genes were included: skin-derived antileukoproteinase, collagen type XVII alpha 1 chain, plakophilin 1, keratin 16, decorin, biglycan, protein bicaudal D homolog 2, growth factor receptor-bound protein 2, signal transducer and activator of transcription 3, apolipoprotein E, Golgi SNAP receptor complex member 1, fibromodulin, and glucocerebrosidase. For POP seven genes were identified: homeobox A13, matrix metallopeptidase 9, estrogen receptor 2, collagen type XIV alpha 1 chain, collagen type V alpha 1 chain, collagen type IV alpha 2 chain, and catenin beta 1. The current review highlights many genes which are potential biomarkers and targets for drug development.

Roles of Pyk2 in signal transduction after gonadotropin-releasing hormone receptor stimulation.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic neurons. It has been reported that GnRH treatment of cultured GnRH neurons (GT1-7 cells) activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in the activation of extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 (ERK1/2). In the present study, we first examined the possibility that GnRH treatment might activate epidermal growth factor receptor (EGFR). We found that activation of EGFR after GnRH treatment for 5 min was much less than after EGF or heparin-binding EGF treatment. Next, we examined whether or not Pyk2 bound to growth factor receptor-binding protein 2 (Grb2). We overexpressed FLAG-fused Pyk2 in GT1-7 cells, and immunoprecipitated Pyk2 using an anti-FLAG antibody. The binding of Pyk2 to Grb2 was detected only after GnRH treatment. In contrast, a site-directed mutant of Pyk2 wherein tyrosine 881 was mutated to phenylalanine did not bind to Grb2. Studies with small interfering RNA and inhibitors indicated that the activation of Grb2/Ras/Raf/MEK was a major pathway to ERK1/2 activation after the short-term treatment of GT1-7 cells with GnRH.

Epac1 Requires AMPK Phosphorylation to Regulate HMGB1 in the Retinal Vasculature.

PurposeTo investigate whether AMP-activated protein kinase (AMPK) is required for the reduction of high mobility group box 1 (HMGB1) by exchange proteins activated by cAMP 1 (Epac1) in the retinal vasculature.MethodsWe measured AMPK phosphorylation in normal and diabetic Epac1 floxed and cdh5/Epac1 Cre mice. We also treated primary human retinal endothelial cells (RECs) in normal (5-mM) or high (25-mM) glucose with an Epac1 agonist and AMPK or insulin-like growth factor receptor binding protein 3 siRNA. We measured protein levels of AMPK, sirtuin 1 (SIRT1), and HMGB1.ResultsAMPK phosphorylation was reduced in cdh5/Epac1 Cre mice, suggesting that Epac1 regulated AMPK actions. High-glucose culturing conditions reduced AMPK levels in RECs, but the levels were increased by the Epac1 agonist, supporting the idea that Epac1 regulates AMPK. The Epac1 agonist was not able to reduce HMGB1 levels or increase SIRT1 when AMPK was blocked by AMPK siRNA, thus demonstrating that Epac1 requires AMPK to regulate SIRT1 and HMGB1.ConclusionsEpac1 requires AMPK to increase SIRT1 and reduce HMGB1 in the diabetic retinal vasculature. This finding provides another pathway by which Epac1 may protect the retina during diabetes.

Aquaporin-1 facilitates proliferation and invasion of gastric cancer cells via GRB7-mediated ERK and Ras activation

ABSTRACT Gastric cancer, one of the most common malignant tumors of the digestive tract, is devoid of effective treatment owing to its highly invasive ability. Aquaporins (AQPs), transmembrane water channel proteins, has been shown to be involved in the malignancy of gastric cancer. This study aims to investigate the pathophysiological roles of AQP-1 in gastric cancer. We first demonstrated quantitative real-time polymerase chain reaction analysis and found up-regulation of AQP-1 in gastric cancer cell lines. Additionally, silence of AQP-1 inhibited cell proliferation via decrease of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2). Moreover, migration and invasion of gastric cancer cells were also suppressed by the interference of AQP-1. However, the tumorigenic mechanism of AQP-1 on gastric cancer is yet to be found. We demonstrated western blot analysis and found that knockdown of AQP-1 decreased protein expression of phospho (p)-GRB7 (growth factor receptor-bound protein 7) and led to a remarkable reduction of p-extracellular signal-regulated kinase (ERK) via inactivation of RAS. In general, our findings indicated that AQP-1 facilitates proliferation and invasion of gastric cancer cells via GRB7-mediated ERK and Ras activation, illuminating a novel AQP-1-RAS/ERK molecular axis as regulator in gastric cancer progression and suggesting potential implications in the treatment of gastric cancer.

The SLAMF3 rs509749 polymorphism correlates with malignant potential in multiple myeloma

The signaling lymphocytic activation molecule family 3 (SLAMF3) is highly expressed on plasma cells from patients with multiple myeloma (MM) and induces high malignant potential by ERK signaling mediated via the interaction with adaptor proteins SHP2 and GRB2. This study focused on the single-nucleotide polymorphism (SNP) of the SLAMF3 gene (rs509749, 1804A>G, M602V) in MM. The SNP G allele was a major type, and the frequencies of the GG, GA, and AA genotypes were 61.8%, 29.4%, and 8.8%, respectively, in patients with MM, which was almost the same as in healthy the control group in the Japanese population. Interestingly, patients with GG genotypes had significantly shorter overall survival times than patients with GA/AA genotypes. Consistent with those results, SLAMF3-overexpressing KMS-34 cells with the G allele (V602) had higher cell proliferation potential and were more resistant to anti-MM agents than those with the A allele (M602). When those cells were subcutaneously inoculated into NOG mice, tumor sizes in mice receiving V602 cells rapidly increased, and survival was significantly shorter than in mice injected with M602 cells. Furthermore, SLAMF3 V602 molecules bound more tightly to SHP2 and GRB2, with increased SHP2 and ERK phosphorylation compared with M602 cells. The mRNA expression of cell cycle-related genes (CCND1 and CCNE1) and anti-apoptotic genes (BCL2L and p21) was increased in V602 cells compared with M602 cells. The results thus suggested that the G allele of SLAMF3 SNP rs509749 may be associated with MM disease progression.

Early leucine programming on protein utilization and mTOR signaling by DNA methylation in zebrafish (Danio rerio)

BackgroundEarly nutritional programming affects a series of metabolism, growth and development in mammals. Fish also exhibit the developmental plasticity by early nutritional programming. However, little is known about the effect of early amino acid programming on growth and metabolism.MethodsIn the present study, zebrafish (Danio rerio) was used as the experimental animal to study whether early leucine stimulation can programmatically affect the mechanistic target of rapamycin (mTOR) signaling pathway, growth and metabolism in the later life, and to undercover the mechanism of epigenetic regulation. Zebrafish larvas at 3 days post hatching (dph) were raised with 1.0% leucine from 3 to 13 dph during the critical developmental stage, then back to normal water for 70 days (83 dph).ResultsThe growth performance and crude protein content of zebrafish in the early leucine programming group were increased, and consistent with the activation of the mTOR signaling pathway and the high expression of genes involved in the metabolism of amino acid and glycolipid. Furthermore, we compared the DNA methylation profiles between the control and leucine-stimulated zebrafish, and found that the methylation levels of CG-differentially methylated regions (DMGs) and CHH-DMGs of genes involved in mTOR signaling pathway were different between the two groups. With quantitative PCR analysis, the decreased methylation levels of CG type of Growth factor receptor-bound protein 10 (Grb10), eukaryotic translation initiation factor 4E (eIF4E) and mTOR genes of mTOR signaling pathway in the leucine programming group, might contribute to the enhanced gene expression.ConclusionsThe early leucine programming could improve the protein synthesis and growth, which might be attributed to the methylation of genes in mTOR pathway and the expression of genes involved in protein synthesis and glycolipid metabolism in zebrafish. These results could be beneficial for better understanding of the epigenetic regulatory mechanism of early nutritional programming.

NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation.

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 μM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.

Competing endogenous RNA network analysis for screening inflammation‑related long non‑coding RNAs for acute ischemic stroke.

Long non-coding RNAs (lncRNAs) represent potential biomarkers for the diagnosis and treatment of various diseases; however, the role of circulating acute ischemic stroke (AIS)-related lncRNAs remains relatively unknown. The present study aimed to screen crucial lncRNAs for AIS based on the competing endogenous RNA (ceRNA) hypothesis. The expression profile datasets for one mRNA, accession no. GSE16561, and four microRNAs (miRNAs), accession nos. GSE95204, GSE86291, GSE55937 and GSE110993, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified, and ClusterProfiler was used to interpret the function of the DEGs. Based on the protein-protein interaction (PPI) network and module analyses, hub DEGs were identified. A ceRNA network was established based on miRNA-mRNA or miRNA-lncRNA interaction pairs. In total, 2,041 DEGs and 5 DELs were identified between the AIS and controls samples in GSE16561, and 10 DEMs between at least two of the four miRNA expression profiles. A PPI network was constructed with 1,235 DEGs, among which 20 genes were suggested to be hub genes. The hub genes paxillin (PXN), FYN-proto-oncogene, Src family tyrosine kinase (FYN), ras homolog family member A (RHOA), STAT1, and growth factor receptor-bound protein 2 (GRB2), were amongst the most significantly enriched modules extracted from the PPI network. Functional analysis revealed that these hub genes were associated with inflammation-related signaling pathways. An AIS-related ceRNA network was constructed, in which 4 DELs were predicted to function as ceRNAs for 9 DEMs, to regulate the five identified hub genes; that is, minichromosome maintenance complex component 3 associated protein-antisense RNA 1 (MCM3AP-AS1)/long intergenic non-protein coding RNA 1089 (LINC01089)/hsa-miRNA (miR)-125a/FYN, inositol-tetrakisphosphate 1-kinase-antisense RNA 1 (ITPK1-AS1)/hsa-let-7i/RHOA/GRB2/STAT1, and human leukocyte antigen complex group 27 (HCG27)/hsa-miR-19a/PXN interaction axes. In conclusion, MCM3AP-AS1, LINC01089, ITPK1-AS1, and HCG27 may represent new biomarkers and underlying targets for the treatment of AIS.

The dynamics of free and phosphopeptide-bound Grb2-SH2 reveals two dynamically independent subdomains and an encounter complex with fuzzy interactions

The growth factor receptor-bound protein 2 (Grb2) is a key factor in the regulation of cell survival, proliferation, differentiation, and metabolism. In its structure, the central Src homology 2 (SH2) domain is flanked by two Src homology 3 (SH3). SH2 is the most important domain in the recognition of phosphotyrosines. Here, we present the first dynamical characterization of Grb2-SH2 domain in the free state and in the presence of phosphopeptide EpYINSQV at multiple timescales, which revealed valuable information to the understanding of phophotyrosine sensing mechanism. Grb2-SH2 presented two dynamically independent subdomains, subdomain I involved in pY recognition and subdomain II is the pY + 2 specificity pocket. Under semi-saturated concentrations of pY-pep we observed fuzzy interactions, which led to chemical exchange observed by NMR. This information was used to describe the encounter complex. The association with pY-pep is dynamic, involving fuzzy interactions and multiple conformations of pY-pep with negative and hydrophobic residues, creating an electrostatic-potential that drives the binding of pY-pep. The recognition face is wider than the binding site, with many residues beyond the central SH2 binding site participating in the association complex, which contribute to explain previously reported capability of Grb2 to recognize remote pY.

Y772 phosphorylation of EphA2 is responsible for EphA2-dependent NPC nasopharyngeal carcinoma growth by Shp2/Erk-1/2 signaling pathway

EphA2 is an important oncogenic protein and emerging drug target, but the oncogenic role and mechanism of ligand-independent phosphorylation of EphA2 at tyrosine 772 (pY772-EphA2) is unclear. In this study, we established nasopharyngeal carcinoma (NPC) cell lines with stable expression of exogenous EphA2 and EphA2-Y772A (phosphorylation inactivation) using endogenous EphA2-knockdown cells, and observed that pY772A EphA2 was responsible for EphA2-promoting NPC cell proliferation and anchorage-independent and in vivo growth in mice. Mechanistically, EphA2-Y772A mediated EphA2-activating Shp2/Erk-1/2 signaling pathway in the NPC cells, and Gab1 (Grb2-associated binder 1) and Grb2 (growth factor receptor-bound protein 2) were involved in pY772-EphA2 activating this signaling pathway. Our results further showed that Shp2/Erk-1/2 signaling mediated pY772-EphA2-promoting NPC cell proliferation and anchorage-independent growth. Moreover, we observed that EphA2 tyrosine kinase inhibitor ALW-II-41-27 inhibited pY772-EphA2 and EphA2-Y772A decreased the inhibitory effect of ALW-II-41-27 on NPC cell proliferation. Collectively, our results demonstrate that pY772-EphA2 is responsible for EphA2-dependent NPC cell growth in vitro and in vivo by activating Shp2/Erk-1/2 signaling pathway, and is a pharmacologic target of ALW-II-41-27, suggesting that pY772-EphA2 can serve as a therapeutic target in NPC and perhaps in other cancers.

Therapeutic efficacy of liposomal Grb2 antisense oligodeoxynucleotide (L-Grb2) in preclinical models of ovarian and uterine cancer.

Background: Adaptor proteins such as growth factor receptor-bound protein-2 (Grb2) play important roles in cancer cell signaling. In the present study, we examined the biological effects of liposomal antisense oligodeoxynucleotide that blocks Grb2 expression (L-Grb2) in gynecologic cancer models.Materials and Methods: Murine orthotopic models of ovarian (OVCAR5 and SKOV3ip1) and uterine (Hec1a) cancer were used to study the biological effects of L-Grb2 on tumor growth. In vitro experiments (cell viability assay, Western blot analysis, siRNA transfection, and reverse phase protein array) were carried out to elucidate the mechanisms and potential predictors of tumor response to L-Grb2.Findings: Treatment with L-Grb2 decreased tumor growth and metastasis in orthotopic models of ovarian cancer (OVCAR5, SKOV3ip1) by reducing angiogenesis and increasing apoptosis at a dose of 15 mg/kg with no effect on mouse body weight. Treatment with L-Grb2 and paclitaxel led to the greatest decrease in tumor weight (mean ± SEM, 0.17 g ± 0.10 g) compared with that in control mice (0.99 g ± 0.35 g). We also observed a reduction in tumor burden after treatment with L-Grb2 and the anti-VEGF antibody B-20 (86% decrease in tumor weight compared with that in controls). Ovarian cancer cells with ErbB2 amplification (OVCAR8 and SKOV3ip1) were the most sensitive to Grb2 downregulation. Reverse phase protein array analysis identified significant dysregulation of metabolites (LDHA, GAPDH, and TCA intermediates) in ovarian cancer cells after Grb2 downregulation.Interpretation: L-Grb2 has therapeutic efficacy in preclinical models of ovarian and uterine cancer. These findings support further clinical development of L-Grb2.

Low-magnitude vibration induces osteogenic differentiation of bone marrow mesenchymal stem cells via miR-378a-3p/Grb2 pathway to promote bone formation in a rat model of age-related bone loss.

The dysfunction of bone marrow mesenchymal stem cells (BMSCs) in osteogenic differentiation is one of the main causes of age-related bone loss. Our previous studies have shown that low-magnitude vibration (LMV) induces the osteogenic differentiation of BMSCs derived from ovariectomized osteoporotic rats. To investigate whether LMV promotes osteogenic differentiation of BMSCs and its underlying mechanisms in aged rats, 20-month-old female Sprague-Dawley rats (n=20) were randomly divided into LMV group (rats were vibrated at 0.3g and 90Hz for 30minutes, once daily, 5days a week until 12weeks for subsequent analysis, n=10), static group (rats were placed in the box on the vibration platform without vibration, n=10); 6-month-old female Sprague-Dawley rats were used as control (young group, n=10). The bone mineral density and bone strength of aged rats were significantly decreased compared with the young rats. Furthermore, the primary BMSCs isolated and cultured from the aged rats with the whole-bone marrow differential pasting method showed a decreased ability in osteogenic differentiation compared with that from the young rats. Then the differentially expressed miRNAs between the aged and young rat-derived BMSCs were screened by high-throughput sequencing and verified by qRT-PCR, and we found that miR-378a-3p was significantly downregulated in the aged rat-derived BMSCs compared with the young rat-derived BMSCs. By transfecting miRNA mimics and inhibitors, miR-378a-3p was confirmed to promote the expression levels of osteogenic genes (Runx2, ALP, Col I, and OCN) and ALP activity of the aged rat-derived BMSCs. Meanwhile, the expression levels of osteogenic genes and miR-378a-3p of aged rat-derived BMSCs were significantly upregulated by LMV (cells were vibrated at 0.3g and 90Hz for 30minutes a day, until 5days for subsequent analysis), while the LMV-induced osteogenic gene expression levels of aged rat-derived BMSCs were suppressed by miR-378a-3p inhibitors. Furthermore, the inhibition of growth factor receptor-bound protein 2 (Grb2) by miR-378a-3p and Grb2-siRNA promoted the LMV-induced osteogenic differentiation of aged rat-derived BMSCs. Additionally, LMV was found to promote bone mineral density and bone strength of aged rats in vivo, as well as upregulating the expression level of miR-378a-3p and downregulating the expression level of Grb2 of BMSCs from aged rats. These results suggest that LMV induces osteogenic differentiation of BMSCs through miR-378a-3p/Grb2 pathway to improve bone mineral density and mechanical properties in a rat model of age-related bone loss.

Lymecycline reverses acquired EGFR-TKI resistance in non–small-cell lung cancer by targeting GRB2

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were first-line treatments for NSCLC patients with EGFR-mutations. However, about 30 % of responders relapsed within six months because of acquired resistance. In this study, we used Connectivity Map (CMap) to discover a drug capable of reversing acquired EGFR-TKIs resistance. To investigate Lymecycline’s ability to reverse acquired EGFR-TKIs resistance, two Icotinib resistant cell lines were constructed. Lymecycline’s ability to suppress the proliferation of Icotinib resistant cells in vitro and in vivo was then evaluated. Molecular targets were predicted using network pharmacology and used to identify the molecular mechanism. Growth factor receptor-bound protein 2 (GRB2) is an EGFR-binding adaptor protein essential for EGFR phosphorylation and regulation of AKT/ERK/STAT3 signaling pathways. Lymecycline targeted GRB2 and inhibited the resistance of the cell cycle to EGFR-TKI, arresting disease progression and inducing apoptosis in cancer cells. Combined Lymecycline and Icotinib treatment produced a synergistic effect and induced apoptosis in HCC827R5 and PC9R10 cells. Cell proliferation in resistant cancer cells was significantly inhibited by the combined Lymecycline and Icotinib treatment in mouse models. Lymecycline inhibited the resistance of the cell cycle to EGFR-TKI and induced apoptosis in NSCLC by inhibiting EGFR phosphorylation and GRB2-mediated AKT/ERK/STAT3 signaling pathways. This provided strong support that Lymecycline when combined with EGFR targeting drugs, enhanced the efficacy of treatments for drug-resistant NSCLC.

2123-P: Targeted Deletion of Grb10 in Pancreatic ß-Cells Increases ß-Cell Mass via mTORC-Dependent Inhibition of Cell Dedifferentiation

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that negatively regulates insulin and mTORC1 signaling. Grb10 is highly expressed in mouse islets, but its role in β-cells function remains unclear. Here we show that Grb10 is highly expressed in human islets and the expression levels of Grb10 are markedly induced in the islets of diabetic mice as well as aged humans. β-cell-specific knockout of Grb10 in mice increased β-cell mass, reduced apoptosis, and improved glucose-stimulated insulin secretion. Mechanistically, knockout of Grb10 in β-cells enhanced mTORC1 signaling and inhibited β-cell dedifferentiation, which could be blocked by rapamycin administration in vivo. Our findings revealed a mechanism by which the Grb10/mTORC1 axis regulates β-cell dedifferentiation and function. Identification of key regulators of β-cell dedifferentiation will shed new light on the development of effective therapeutic strategies for treating both type 1 diabetes (T1D) and type 2 diabetes (T2D). Disclosure F. Liu: None. Funding National Natural Science Foundation of China (91749118, 81770775, 81730022); Planned Science and Technology Project of Hunan Province (2017RS3015)

Crystal structure of the SH3 domain of growth factor receptor-bound protein 2.

This study presents the crystal structure of the N-terminal SH3 (SH3N) domain of growth factor receptor-bound protein 2 (Grb2) at 2.5 Å resolution. Grb2 is a small (215-amino-acid) adaptor protein that is widely expressed and involved in signal transduction/cell communication. The crystal structure of full-length Grb2 has previously been reported (PDB entry 1gri). The structure of the isolated SH3N domain is consistent with the full-length structure. The structure of the isolated SH3N domain was solved at a higher resolution (2.5 Å compared with 3.1 Å for the previously deposited structure) and made it possible to resolve some of the loops that were missing in the full-length structure. In addition, interactions between the carboxy-terminal region of the SH3N domain and the Sos1-binding sites were observed in the structure of the isolated domain. Analysis of these interactions provided new information about the ligand-binding properties of the SH3N domain of Grb2.