Although the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non-small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation.

Four NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9.

As a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF.

Although the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a preclinical rationale for an ongoing phase 1 clinical trial of erlotinib plus INC-280 in EGFR-mutated NSCLC.

Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2-associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus-encoded protease 2A(pro), independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus-induced cleavage of GAB1 is beneficial to viral growth as the N-terminal proteolytic product of GAB1 (GAB1-N1-174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1-N1-174 fragment that enhances viral infectivity, at least in part, via activation of the ERK pathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication.

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

In recent decades, the stromal cell-derived factor-l (SDF-1) and Gab1 have been investigated to be involved in oncogenesis. However, it is scarcely reported that SDF-1-Gab1 pathway mediates proliferation and apoptosis in human chondrosarcoma (CS). In this study, we assessed the expression of Gab1 in 90 CS solid tumors by immunohistochemistry, immunoblotting, and qRT-PCR, and then, some in vitro assays were also applied to CS cells treated with SDF-1. We observed that the overexpression of Gab1 was positively correlated with lung metastasis and recurrence, and acts as an independent prognostic factor for CS patients. Gab1 expression was up-regulated in response to SDF-1 stimulation in CS cell line JJ012, SW1353, L3252. Overexpression of Gab1 increased Bcl-2/BAX ratio to promote cell growth via PI3K/AKT. On the other hand, silencing of Gab1 accelerated apoptosis and repressed the growth of CS cells, which further caused the inhibition of G1/S phase transition and decreased invasion capacity in CS cell lines. In vivo assay identified that the knockdown of Gab1 interfered with the tumor mass formation. In conclusion, our data identified overexpression of Gab1 in CS tissues, and Gab1 can be recommended as a novel biomarker for diagnosis and prognosis in patients with CS. Additionally, PI3K/AKT/Bcl-2/BAX axis was involved in Gab1-induced CS progression, indicating Gab1 might act as a new target for the treatment of CS patients.

Grb2-associated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.

During the cell transformation processes leading to erythroleukemia, erythroid progenitors often become erythropoietin (Epo)-independent for their proliferation. The biochemical events that could lead an erythroleukemic cell to growth factor-independence were investigated using spi-1 transgenic poerythroblasts. Spi-1/PU.1 is a myeloid and B-cell transcription factor of the ETS family and is activated by insertional mutagenesis during Friend erythroleukemia. Its overexpression in proerythroblasts induces their differentiation arrest without altering their erythropoietin requirement for proliferation (HS1 cells). At a later step, genetic alterations most probably occur allowing spi-1 transgenic poerythroblasts to proliferate in the absence of erythropoietin (HS2 cells). The signaling transduction pathways in HS1 and HS2 proerythroblasts were analyzed. The authors have previously shown that the Jak/STAT pathway was not activated in Epo-independent cells, but remained sensitive to Epo stimulation. In the present study, it is shown that the Epo-independent proliferation of HS2 cells requires active phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. In these cells, PI3K was constitutively associated with the molecular adapters Grb2 and Gab1, and with the phosphatases SHP-2 and SHIP. Moreover, PI3K activity was correlated with the constitutive phosphorylation of serine-threonine protein kinase (AKT) in HS2 cells. Lastly, a constitutive activation of the MAPKs extracellular signal-regulated kinases (ERK1/2) in HS2 cells was observed that occurs in a PI3K-independent manner, but depends strictly on the activity of the protein kinase C (PKC). These results suggest that constitutive activations of PI3K/AKT and PKC/MAPK pathways can act in synergy to lead a proerythroblast to proliferate without Epo.

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses leading to gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through interaction of the bacterial surface protein InlB with host Met receptor tyrosine kinase. Binding of InlB leads to phosphorylation of Met and the adapter Gab1 and to activation of host phosphoinositide (PI) 3-kinase. The mammalian ligand of Met, hepatocyte growth factor, promotes cell motility and morphogenesis in a manner dependent on phosphorylation of two docking site tyrosines at positions 1349 and 1356 in the receptor's cytoplasmic tail. Here we determined if these tyrosines were essential for Listeria entry. A derivative of the human cell line T47D stably expressing a truncated Met lacking most of its cytoplasmic domain was unable to support InlB-mediated signaling or entry. Surprisingly, cells expressing mutant Met containing phenylalanine substitutions in both tyrosines 1349 and 1356 (MetYF) allowed entry and InlB-induced Gab1 phosphorylation. However, in contrast to the situation in cells expressing wild-type Met, Gab1 phosphorylation in MetYF cells required PI 3-kinase activity. The Gab1 pleckstrin homology (PH) domain was constitutively associated with the plasma membrane of cells in a PI 3-kinase-dependent manner. Overexpression of the PH domain blocked entry of Listeria into cells expressing MetYF but not into cells expressing wild-type Met. Taken together, these results indicate that the docking site tyrosines are dispensable for internalization when membrane localization of Gab1 is constitutive. Distinct pathways of recruitment by phosphorylated tyrosines in Met and PH domain ligands in the membrane are redundant for bacterial entry.

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner.

Grb2-associated binder-1 (Gab1) is a docking protein closely related to insulin receptor substrates. We previously reported that tyrosine 1062 in RET receptor tyrosine kinase activated by glial cell line-derived neurotrophic factor (GDNF) represents a binding site for the Shc-Grb2-Gab1 complex, and that the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and SHP2 tyrosine phosphatase is associated with Gab1 in GDNF-treated cells. In the present study, we further analyzed the physiological roles of Gab1 downstream of RET, using Gab1 mutants that lack the binding sites for PI3K (Gab1 PI3K-m) or SHP-2 (Gab1 SHP2-m). Expression of Gab1 PI3K-m in SK-N-MC human primitive neuroectodermal tumor cells expressing wild-type RET markedly impaired Akt phosphorylation, Rac1 activation, and lamellipodia formation that were induced by GDNF whereas expression of Gab1 SHP2-m partially impaired Erk activation. Furthermore, expression of Gab1 PI3K-m, but not Gab1 SHP2-m, in TT human medullary thyroid carcinoma cells expressing RET with a multiple endocrine neoplasia 2A mutation enhanced cytochrome c release, and apoptosis induced by etoposide, suggesting that PI3K is involved in survival of TT cells via a mitochondrial pathway. These findings demonstrated that coupling of Gab1 to PI3K is important for biological responses in RET-expressing cells.

GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.

YopH is an exceptionally active tyrosine phosphatase that is essential for virulence of Yersinia pestis, the bacterium causing plague. YopH breaks down signal transduction mechanisms in immune cells and inhibits the immune response. Only a few substrates for YopH have been characterized so far, for instance p130Cas and Fyb, but in view of YopH potency and the great number of proteins involved in signalling pathways it is quite likely that more proteins are substrates of this phosphatase. In this respect, we show here YopH interaction with several proteins not shown before, such as Gab1, Gab2, p85, and Vav and analyse the domains of YopH involved in these interactions. Furthermore, we show that Gab1, Gab2 and Vav are not dephosphorylated by YopH, in contrast to Fyb, Lck, or p85, which are readily dephosphorylated by the phosphatase. These data suggests that YopH might exert its actions by interacting with adaptors involved in signal transduction pathways, what allows the phosphatase to reach and dephosphorylate its susbstrates.

Grb2-associated binder 1 (Gab1) and Gab2 play important roles in cancer cell signaling. In particular, it has been demonstrated that the upregulation of Gab2 may be correlated with the World Health Organization (WHO) grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time. However, the prognostic value of combined expression of Gab1 and Gab2 has not been explored. Gab1 and Gab2 expression in human gliomas and non-neoplastic brain tissues was measured by immunohistochemistry. Both the expression levels of Gab1 and Gab2 proteins in glioma tissues were significantly higher than those in non-neoplastic brain tissues (both P < 0.001). In addition, the overexpression of Gab1 and Gab2 proteins were both significantly associated with advanced WHO grades (both P < 0.001) and low KPS (both P = 0.01). Moreover, the overall survival of patients with high Gab1 protein expression or high Gab2 protein expression was obviously lower than those with low expressions (both P < 0.001). Notably, glioma patients with combined overexpression of Gab1 and Gab2 proteins (Gab1-high/Gab2-high) had shortest overall survival (P < 0.001). Furthermore, multivariate analysis showed that Gab1 expression (P = 0.01), Gab2 expression (P = 0.02), and combined expression of Gab1 and Gab2 (Gab1/Gab2, P = 0.006) were all independent prognostic factors for overall survival in glioma patients. Gab1 and Gab2 proteins are differentially expressed in glioma patients and closely correlated with the biological behavior of this malignancy. Combination of Gab1 and Gab2 expression may represent a promising biomarker for prognostication of human gliomas.

Contrary to the antimotogenic effect of NO in dedifferentiated vascular smooth muscle cells (VSMCs), we have reported that NO stimulates the motility of differentiated cultured VSMC isolated from adult rats. This process involves upregulation of protein tyrosine phosphatase SHP2, followed by downregulation of RhoA activity. In the present study, we tested the hypothesis that insulin alters the motogenic phenotype of cultured rat aortic smooth muscle cells exposed to NO from inhibition to stimulation of cell motility. We demonstrate for the first time that NO stimulates the motility of VSMCs cultured for several days in the presence but not the absence of insulin. Moreover, we show that NO blocks PDGF-induced cell motility in insulin-naive but not in insulin-treated cells. We also demonstrate that the scaffold adapter protein Gab1, considered a physiological activator of protein tyrosine phosphatase SHP2, increases cell motility in the presence but not the absence of insulin. In cells cultured in the presence of insulin, overexpression of Gab1 mimics, whereas a dominant-negative allele of Gab1 (Gab1YF) blocks, the motility-stimulatory effect of NO. Cotransfection experiments with dominant-negative Gab1 and wild-type SHP2 or wild-type Gab1 and dominant-negative SHP2 indicate that the two proteins work together as a functional unit to induce motility. Because chronic insulin can increase the levels of phosphatidylinositol 3 (PI3) kinase in several models of hyperinsulinemia, we also tested the potential involvement of this enzyme in mechanisms leading to increased cell motility. We found that the motogenic effect of NO, Gab1, and SHP2 was blocked by the selective PI3 kinase inhibitor LY294002, suggesting a requirement of PI3 kinase in mediating motogenesis. These observations may be relevant to molecular mechanisms related to the pathogenesis of vascular disease in hyperinsulinemic diabetes. The full text of this article is available online at http://www.circresaha.org.

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3 form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3 deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3 knockdown in bone marrow-derived macrophage cells (Gαi1/3 KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3 deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3 were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3 can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

The GRB2 associated binder 1 (GAB1) is an essential docking/adaptor protein for transmitting intracellular signals of the MET tyrosine kinase receptor activated by hepatocyte growth factor/scatter factor (HGF/SF). We found that in response to hours of HGF/SF treatment, the GAB1 protein level is degraded by a mechanism involving MET activity and the proteasomal machinery. We also showed that GAB1 is both multi- and poly-ubiquitinated in a CBL-dependent manner. A long term exposure to HGF/SF caused a more sustained down-regulation of GAB1 than of MET, associated with a loss of reactivation of the ERK MAP kinases to subsequent acute ligand treatment. These data demonstrate that GAB1 is ubiquitinated by CBL and degraded by the proteasome, and plays a role in negative-feedback regulation of HGF/SF-MET signaling.

OBJECTIVE

Glioma causes significant human mortalities annually. Molecularly-targeted therapy is a focus of glioma research.

METHODS

Grb2-associated binding 1 (Gab1) expression and microRNA-29a-3p ("miR-29a-3p") expression in human glioma cells and tissues were tested by Western blotting assay and qRT-PCR assay. shRNA/siRNA strategy was applied to silence Gab1 in human glioma cells. miR-29a or anti-sense miR-29a construct was transfected to human glioma cells. Cell proliferation was tested by BrdU ELISA assay and cell counting assay.

RESULTS

We show that expression of Gab1 was significantly elevated in human glioma tissues and cells, which correlated with downregulation of its putative microRNA: miR-29a-3p. In A172 glioma cells and primary human glioma cells, Gab1 shRNA/siRNA inhibited Akt-Erk activation and cell proliferation. Forced-expression of miR-29a-3p downregulated Gab1, inhibiting glioma cell proliferation, whereas miR-29a-3p was in-effective on cell proliferation in Gab1-silenced A172 cells. Furthermore, introduction of a 3'-untranslated region (3'-UTR) mutant Gab1 (UTR-G160A) blocked miR-29a-3p-induced inhibition on Akt signaling and A172 cell proliferation.

CONCLUSIONS

miR-29a-3p downregulation leads to Gab1 upregulation to promote glioma cell proliferation.

Aberrant hepatocyte growth factor (HGF)/MET signaling has been implicated in hepatocarcinogenesis, suggesting that MET may serve as an attractive therapeutic target in hepatocellular carcinoma. We sought to investigate the in vitro and in vivo antitumor activity of AMG 337, a potent and highly selective small molecule MET kinase inhibitor, in preclinical models of hepatocellular carcinoma. The antiproliferative activity of AMG 337 was evaluated across a panel of hepatocellular carcinoma cell lines in a viability assay. Daily oral administration was used to evaluate the in vivo antitumor activity of AMG 337 in two patient-derived xenograft (PDX) models of hepatocellular carcinoma (LI0612 and LI1078). AMG 337 exerted potent antiproliferative activity against 2 of 40 hepatocellular carcinoma cell lines, namely, MHCC97H (IC50, 0.015 μmol/L) and HCCLM3 (IC50, 0.025 μmol/L). Both sensitive cell lines showed MET amplification (MET/CEN-7 >2.0) assessed by FISH, and high MET expression (3+ IHC) assessed by IHC. AMG 337 potently inhibited p-MET in all cell lines with detectable levels of total MET. However, the dose-dependent inhibition of downstream effectors of HGF/MET signaling, including p-GAB1, p-AKT, and p-ERK, was limited to those cell lines sensitive to AMG 337 in a viability assay (MHCC97H and HCCLM3). AMG 337 significantly inhibited tumor growth at all doses tested in the MET-amplified and MET-high-expressing hepatocellular carcinoma PDX model LI0612 and had no effect on tumor growth in the non-MET-amplified and MET-low-expressing hepatocellular carcinoma PDX model LI1078. AMG 337 represents a promising and novel therapeutic strategy for targeting hepatocellular carcinomas with a dependence on HGF/MET signaling. Mol Cancer Ther; 15(6); 1227-37. ©2016 AACR.

Gestational diabetes mellitus (GDM) is a risk factor for abnormal heart development. Previous work showed that a decrease of myocardial cells and an increase of apoptotic cells leading to heart defects under hyperglycemia, and many genes and protein have been found to play important roles in cardiomyocyte apoptosis. However, there are still many blind nodes in HG-induced cardiac apoptosis. Our study showed that down-regulation of GAB1 occurred concurrently with HG-induced cardiomyocytes apoptosis and in the heart tissues of offspring of diabetic rats in vitro and in vivo. MTT and apoptosis assay showed GAB1 played a key role in mediating HG-induced apoptosis of cardiomyocytes. Down-regulation of XIAP and increased activities of Caspase3/7 was associated with GAB1-mediated cardiomyocyte apoptosis in response to HG treatment. Further study showed that the phosphorylation levels of AKT (Ser473) decreased after HG treatment. Over-expression of GAB1 resisted the reduction in AKT phosphorylation in response to HG. LY294002, which is an effective inhibitor of the PI3K/AKT signaling pathway, partly inhibited GAB1 to suppress apoptosis induced by HG in cardiomyocytes, and partly suppressed GAB1 to resist the decrease of XIAP in response to HG, indicating AKT signaling, XIAP, and Caspase3/7 participated in GAB1-mediated cardiomyocyte apoptosis in response to HG. Generally, we demonstrate a novel role of GAB1 and its down-stream signaling PI3K/AKT for modulating cardiomyocyte apoptosis in response to high glucose in vitro and vivo.

MicroRNA-200a (miR-200a) is frequently downregulated in most cancer types and plays an important role in carcinogenesis and cancer progression. In this study, we determined that miR-200a was downregulated in hepatocellular carcinoma (HCC) tissues and cell lines, consistent with the results of our previous study. Because a previous study suggested that downregulation of miR-200a is correlated with HCC metastasis, we aimed to elucidate the mechanism underlying the role of miR-200a in metastasis in HCC. Here we observed that overexpression of miR-200a resulted in suppression of HCC metastatic ability, including HCC cell migration, invasion, and metastasis, in vitro and in vivo. Furthermore, bioinformatics and luciferase reporter assays indicated that GAB1 is a direct target of miR-200a. Inhibition of GAB1 resulted in substantially decreased cell invasion and migration similar to that observed with overexpression of miR-200a in HCC cell lines, whereas restoration of GAB1 partially rescued the inhibitory effects of miR-200a. Taken together, these data provide novel information for comprehending the tumor-suppressive role of miR-200a in HCC pathogenesis through inhibition of GAB1 translation.

IL-4 induces Akt activation in macrophages, required for full M2 (alternative) polarization. We examined the roles of Gαi1 and Gαi3 in M2 polarization using multiple genetic methods. Methods and Results: In MEFs and primary murine BMDMs, Gαi1/3 shRNA, knockout or dominant negative mutations attenuated IL-4-induced IL4Rα endocytosis, Gab1 recruitment as well as Akt activation, leaving STAT6 signaling unaffected. Following IL-4 stimulation, Gαi1/3 proteins associated with the intracellular domain of IL-4Rα and the APPL1 adaptor, to mediate IL-4Rα endosomal traffic and Gab1-Akt activation in BMDMs. In contrast, gene silencing of Gαi1/3 with shRNA or knockout resulted in BMDMs that were refractory to IL-4-induced M2 polarization. Conversely, Gαi1/3-overexpressed BMDMs displayed preferred M2 response with IL-4 stimulation. In primary human macrophages IL-4-induced Akt activation and Th2 genes expression were inhibited with Gαi1/3 silencing, but augmented with Gαi1/3 overexpression. In Gαi1/3 double knockout (DKO) mice, M2 polarization, by injection of IL-4 complex or chitin, was potently inhibited. Moreover, in a murine model of asthma, ovalbumin-induced airway inflammation and hyperresponsiveness were largely impaired in Gαi1/3 DKO mice. Conclusion: These findings highlight novel and essential roles for Gαi1/3 in regulating IL-4-induced signaling, macrophage M2 polarization and allergic asthma response.

Keywords: Gαi1/3; IL-4; M2 polarization; allergic asthma response; signaling.

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.

OBJECTIVE

Some specific microRNAs (miRNAs) have been identified to regulate the tumorigenesis of non-small cell lung cancer (NSCLC). MiR-590-5p was found to involve in the carcinogenesis of human cancers. This study aims at exploring the role of miR-590-5p in the pathogenesis of NSCLC.

METHODS

The expressions of miR-590-5p and GAB1 were measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. The biological functions of miR-590-5p and GAB1 on cell viability and invasion were investigated through MTT and transwell assays. The binding site between miR-590-5p and GAB1 was verified by dual-luciferase reporter gene assay (DLR).

RESULTS

MiR-590-5p expression was downregulated in NSCLC. MiR-590-5p overexpression inhibited the proliferation and invasion of NSCLC cells. Furthermore, miR-590-5p was confirmed to directly target GAB1. GAB1 knockdown had the same effect as overexpression of miR-590-5p in NSCLC. Moreover, overexpression of GAB1 partially reversed the suppressive effect of miR-590-5p on NSCLC.

CONCLUSIONS

MiR-590-5p suppressed cell proliferation and invasion of NSCLC by inhibiting GAB1 expression, indicating that miR-590-5p was a suppressive miRNA in NSCLC.

A vital step in the development of heart failure is the transition from compensatory cardiac hypertrophy to decompensated dilated cardiomyopathy (DCM) during cardiac remodeling under mechanical or pathological stress. However, the molecular mechanisms underlying the development of DCM and heart failure remain incompletely understood. In the present study, we investigate whether Gab1, a scaffolding adaptor protein, protects against hemodynamic stress-induced DCM and heat failure. We first observed that the protein levels of Gab1 were markedly reduced in hearts from human patients with DCM and from mice with experimental viral myocarditis in which DCM developed. Next, we generated cardiac-specific Gab1 knockout mice (Gab1-cKO) and found that Gab-cKO mice developed DCM in hemodynamic stress-dependent and age-dependent manners. Under transverse aorta constriction (TAC), Gab1-cKO mice rapidly developed decompensated DCM and heart failure, whereas Gab1 wild-type littermates exhibited adaptive left ventricular hypertrophy without changes in cardiac function. Mechanistically, we showed that Gab1-cKO mouse hearts displayed severe mitochondrial damages and increased cardiomyocyte apoptosis. Loss of cardiac Gab1 in mice impaired Gab1 downstream MAPK signaling pathways in the heart under TAC. Gene profiles further revealed that ablation of Gab1 in heart disrupts the balance of anti- and pro-apoptotic genes in cardiomyocytes. These results demonstrate that cardiomyocyte Gab1 is a critical regulator of the compensatory cardiac response to aging and hemodynamic stress. These findings may provide new mechanistic insights and potential therapeutic target for DCM and heart failure.