Inhibition of insulin-like growth factor-I signaling by ethanol in neuronal cells

BACKGROUND: Ethanol inhibits insulin-like growth factor-I receptor (IGF-IR) activation. However, the potency of ethanol for inhibition of the IGF-IR and other receptor tyrosine kinases varies considerably among different cell types. We investigated the effect of ethanol on IGF-I signaling in several neuronal cell types. METHODS: IGF-I signaling was examined in SH-SY5Y neuroblastoma cells, primary cultured rat cerebellar granule neurons, and rat NG-108 neuroblastoma x glioma hybrids. The tyrosine phosphorylation of IGF-IR, IRS-2, Shc, and p42/p44 MAP kinase (MAPK), and the association of Grb-2 with Shc, were examined by immunoprecipitations and Western blotting. RESULTS: IGF-I-mediated tyrosine phosphorylation of MAPK was inhibited by ethanol in all cell lines. IGF-IR autophosphorylation was markedly inhibited by ethanol in SH-SY5Y cells, was only mildly inhibited in cerebellar granule neurons, and was unaffected in rat NG-108 cells. In vitro tyrosine autophosphorylation of immunopurified IGF-IR obtained from all cell lines was inhibited by ethanol. There was also differential ethanol sensitivity of IRS-2 and Shc phosphorylation, and the association of Shc with IRS-2, among the different cell types. CONCLUSIONS: The findings demonstrate that IGF-I-mediated MAPK activation is a sensitive target of ethanol in diverse neuronal cell types. The data are consistent with ethanol-induced inhibition of IGF-IR activity, although the extent of IGF-IR tyrosine autophosphorylation per se is a poor marker of the inhibitory action of ethanol on this receptor. Furthermore, despite uniform inhibition of MAPK in the different neuronal cell types, tyrosine phosphorylation of proximal mediators of the IGF-IR are differentially inhibited by ethanol.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.     

Ethanol inhibits insulin receptor tyrosine kinase

BACKGROUND: Ethanol inhibits insulin-like growth factor-I (IGF-I) and insulin signaling in various cell types. The tyrosine autophosphorylation of the IGF-I and insulin receptors appears to be a target for ethanol, as well as other receptor tyrosine kinases. METHODS AND RESULTS: The effect of ethanol on purified recombinant insulin receptor kinase activity was examined. A noncompetitive inhibition was observed at pharmacologically relevant concentrations of ethanol. Both peptide substrate tyrosine phosphorylation and kinase autophosphorylation are inhibited by ethanol. Near equivalent inhibition of kinase activity was noted for 300 mM methanol, 150 mM ethanol, 20 mM 1-propanol, and 10 mM 1-butanol. CONCLUSION: The findings identify a direct protein interaction site of ethanol, and provide insight into the mechanism by which ethanol inhibits receptor tyrosine kinase activity.     

SRC homology-2-containing protein tyrosine phosphatase-1 restrains cell proliferation in human medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is a rare tumor originating from thyroid parafollicular C cells, where, in the inherited form, constitutive activation of the RET protooncogene is responsible for unrestrained cell proliferation. We previously demonstrated that somatostatin (SRIF) reduces cell growth in the human MTC cell line TT, which expresses all SRIF receptor (SSTR) subtypes and responds differently to selective SSTR agonists. The antiproliferative mechanism of SRIF and its analogs in MTC is still unclear. Src homology-2-containing protein tyrosine phosphatase-1 (SHP-1), a cytoplasmic protein tyrosine phosphatase (PTP), is activated by somatotropin release-inhibiting factor and reduces mutated RET autophosphorylation in a heterologous system. In this study, we explore the role of PTP activation, in particular of SHP-1, in TT cells, where RET is constitutively activated. In TT cells, SRIF stimulated the PTP activity of SHP-1, which was associated with proliferation inhibition and with reduction in the MAPK pathway activation. Blockade of PTP activity with sodium orthovanadate induced cell proliferation and MAPK phosphorylation and blunted the inhibitory effects of SRIF. Moreover, SHP-1 associates with SSTR2 depending on its activation. By using a MAPK kinase inhibitor, we demonstrated that TT cell growth depends on MAPK pathway activation. Furthermore, in TT cells overexpressing SHP-1, cell proliferation and MAPK signaling were strongly down-regulated, whereas in TT cells transfected with a dominant negative form of SHP-1, cell proliferation and MAPK signaling were markedly induced. Our data demonstrate that SRIF inhibitory effects on TT cell proliferation are mediated, at least in part, by SHP-1, which acts through a MAPK-dependent mechanism.     

NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological responses

The signaling pathways that regulate smooth muscle cell migration and proliferation are incompletely understood. Smooth muscle cells express at least 3 families of receptor tyrosine kinases that mediate cell migration: platelet-derived growth factor (PDGF) receptors, the trk family of neurotrophin receptors, and insulin-like growth factor 1 receptor. The neurotrophin, nerve growth factor (NGF), and insulin-like growth factor 1 induce the migration but not the proliferation of smooth muscle cells, whereas PDGF-BB stimulates both responses. To determine whether distinct signaling pathways downstream of receptor tyrosine kinases specifically mediate smooth muscle cell migration or proliferation, the ligand-induced activation of different signaling pathways in smooth muscle cells was examined. NGF induces prolonged activation of the Shc/MAP kinase pathway and phospholipase Cgamma compared with PDGF-BB. The activation of phosphatidylinositol-3 kinase, however, was 10-fold greater in response to PDGF-BB compared with NGF. Insulin-like growth factor 1 activates only phosphatidylinositol-3 kinase. Pharmacological inhibitors of phosphatidylinositol-3 kinase, Wortmannin and LY294002, inhibit PDGF-BB and NGF-induced migration, whereas an inhibitor of MAP kinase kinase, PD98059, has no effect. Our results suggest that (1) different receptor tyrosine kinases use similar patterns of activation of signaling pathways to mediate distinct biological outcomes of cell migration and proliferation, (2) NGF activates signaling proteins in smooth muscle cells similar to those activated during NGF-induced neuronal differentiation, and (3) the combinatorial effects of different signaling pathways are important for the regulation of smooth muscle cell migration and proliferation. Further studies using mutant trk receptors will help to define the signal transduction pathways mediating NGF-induced smooth muscle cell migration.     

Caveolin-1 down-regulation inhibits insulin-like growth factor-I receptor signal transduction in H9C2 rat cardiomyoblasts

Caveolin (Cav)-1, the major caveolar protein, directly interacts with IGF-I receptor (IGF-IR) and its intracellular substrates. To determine the role of Cav-1 in IGF-IR signaling, we transfected H9C2 cells with small interfering RNA specific for Cav-1-siRNA. The selective down-regulation of Cav-1 (90%) was associated with a smaller reduction of Cav-2, whereas Cav-3 expression was unaffected. A significant reduction of IGF-IR tyrosine phosphorylation in Cav-1-siRNA H9C2 cells was found compared with H9C2 control cells (Ctr-siRNA). The reduced IGF-IR autophosphorylation resulted in a decrease of insulin receptor substrate-1, Shc, and Akt activation. In addition, in Cav-1-siRNA H9C2 cells, IGF-I did not prevent apoptosis, suggesting that Cav-1 is required to mediate the antiapoptotic effect of IGF-I in cardiomyoblasts. The down-regulation of Cav-1 decreased IGF-IR activation and affected the ability of IGF-I to prevent apoptosis after serum withdrawal also in human umbilical vein endothelial cells. These results demonstrate that: 1) Cav-1 down-regulation negatively affects IGF-IR tyrosine phosphorylation; 2) this effect causes a reduced activation of insulin receptor substrate-1, Shc, and Akt; and 3) Cav-1 is involved in IGF-IR antiapoptotic signaling after serum deprivation.     

Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor

STI 571 (formerly known as CGP 57148B) is a known inhibitor of the c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases. This compound is being evaluated in clinical trials for the treatment of chronic myelogenous leukemia. We sought to extend the activity profile of STI 571 by testing its ability to inhibit the tyrosine kinase activity of c-kit, a receptor structurally similar to PDGFR. We treated a c-kit expressing a human myeloid leukemia cell line, M-07e, with STI 571 before stimulation with Steel factor (SLF). STI 571 inhibited c-kit autophosphorylation, activation of mitogen-activated protein (MAP) kinase, and activation of Akt without altering total protein levels of c-kit, MAP kinase, or Akt. The concentration that produced 50% inhibition for these effects was approximately 100 nmol/L. STI 571 also significantly decreased SLF-dependent growth of M-07e cells in a dose-dependent manner and blocked the antiapoptotic activity of SLF. In contrast, the compound had no effect on MAP kinase activation or cellular proliferation in response to granulocyte-macrophage colony-stimulating factor. We also tested the activity of STI 571 in a human mast cell leukemia cell line (HMC-1), which has an activated mutant form of c-kit. STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than it did on ligand-dependent activation of the wild-type receptor. These findings show that STI 571 selectively inhibits c-kit tyrosine kinase activity and downstream activation of target proteins involved in cellular proliferation and survival. This compound may be useful in treating cancers associated with increased c-kit kinase activity.     

Ethanol-Induced Inhibition of Cell Proliferation Is Modulated by Insulin-Like Growth Factor-I Receptor Levels

Ethanol inhibits the tyrosine autophosphorylation of the insulin-like growth factor (IGF)-1 receptor, an action that correlates with the inhibition of IGF-I-stimulated cell proliferation [J. Biol. Chem . 268:21777–21782 (1993)l. In the current study, the IGF-I-dependent proliferation of mouse BALB/c3T3 cells was completely inhibited by ethanol, but the growth of BALB/c3T3 cells that overexpress the IGF-l receptor (p6 cells) was only partially inhibited by ethanol. BALB/ c3T3 cells that simultaneously overexpress both the IGF-I receptor and IGF-I were insensitive to growth inhibition by ethanol. In p6 cells, increasing concentrations of IGF-l overcame the inhibition of IGF-l receptor tyrosine autophosphorylation in the presence of ethanol. The importance of the IGF-I receptor as a specific target for ethanol was further investigated in C6 rat glioblastoma cells that respond mitogenically to both epidermal growth factor (EGF) and IGF-I. The mitogenic response of C6 cells to EGF was abrogated In cells expressing antisense mRNA to the IGF-l receptor. Thus, EGF action in these cells is dependent on activation of an IGF-I/lGF-I receptor au-tocrine pathway. Indeed, EGF stimulated an increase in IGF-l receptor levels by more than 100%. Ethanol completely inhibited the prollferation of C6 cells in response to either EGF or IGF-I. However, ethanol did not directly interfere with EGF receptor function, because EGF-induced cell proliferation was unaffected by ethanol when added exclusively during a 1-hr exposure to EGF. Ethanol did not interfere with the EGF-induced increase in IGF-I receptor expression. The addition of both EGF and IGF-I overcame the inhibitory action of ethanol. In conclusion, the potency of ethanol as an inhibitor of IGF-I-mediated cell proliferation correlates with the level of IGF-I receptors. In contrast to its effect on the IGF-I receptor, ethanol has no direct effect on EGF receptor activation.     

Ethanol Inhibition of Insulin Signaling in Hepatocellular Carcinoma Cells

Chronic ethanol toxicity impairs liver regeneration, inhibits DNA synthesis, and mutes cellular responses to growth factor stimulation. Previous studies demonstrated that the adverse effects of ethanol are mediated by inhibition of tyrosyi phosphorylation of the insulin receptor and the insulin receptor substrate-type 1 (IRS-1). However, overexpression of IRS-1 leads to increased DNA synthesis and cellular transformation due to constitutive activation of mitogen-activated protein (MAP) kinase. The present study examines the effects of ethanol on insulin signaling through IRS-1 in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, to determine whether such ceils were resistant to the inhibitory effects of ethanol. The results demonstrated that ethanol treatment (100 mM) caused 30 to 50% reductions in the levels of insulin-stimulated tyrosyi phosphorylation of the insulin receptor β-subunit, tyrosyi phosphorylation of IRS-1, phosphorylation of Erk2, association of phosphatidylinositol-3 kinase with tyrosyl-phosphorylated IRS-1, and MAP kinase and phosphatidylinositol-3 kinase activities. In contrast, ethanol treatment had no effect on epidermal growth factor-stimulated tyrosyi phosphorylation of She. Corresponding with the pronounced inhibition of MAP kinase, ethanol treatment resulted in 30 to 50% reductions in the expression levels of two important insulin-responsive genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and proliferating cell nuclear antigen (PCNA). The findings suggest that, in FOCUS hepatocellular carcinoma cells, which overexpress IRS-1, ethanol treatment substantially inhibits IRS-1 and MAP kinase signaling and growth-associated gene expression, but has no effect on She phosphorylation, which activates p21^ras through an IRS-1 independent pathway.     

Insulin-like growth factor-I-dependent signal transduction pathways leading to the induction of cell growth and differentiation of human neuroblastoma cell line SH-SY5Y: the roles of MAP kinase pathway and PI 3-kinase pathway

IGF-I regulates cell growth, differentiation, and survival in many cultured nerve cell lines. The present study was undertaken in the human neuroblastoma cell line, SH-SY5Y, to elucidate whether there are differences in the IGF-dependent signal transduction pathways that stimulate proliferation compared to those that induce differentiation. Quiescent SH-SY5Y cells were treated with IGF-I in the presence or absence of PD98059 (an inhibitor of MEK, a MAP kinase kinase) or LY294002 (an inhibitor of PI 3-kinase). Cell growth was assessed by measuring [3H]thymidine incorporation into DNA and cell number. Cell differentiation was assessed by measuring mRNA levels of NPY and neurite outgrowth. IGF-I both induced cell proliferation and differentiation. It stimulated tyrosine phosphorylation of the type I IGF receptor (IGF-IR) beta-subunit, IRS-I, IRS-2, and Shc, and these changes were associated with activation of Erk and Akt. PD98059 inhibited activation of Erk and LY294002 repressed activation of Akt in response to IGF-I, but did not affect tyrosine phosphorylation of the IGF-IR, IRS-1, IRS-2, or Shc. Each PD98059 and LY294002 inhibited IGF-I-dependent cell proliferation in a concentration-dependent manner. In contrast, each of these inhibitors only partially depressed NPY gene expression induced by IGF-I and slightly inhibited IGF-I-mediated neurite outgrowth; however, when both PD98059 and LY294002 were present, IGF-I-dependent NPY gene expression and neurite outgrowth were abolished completely. These results suggest that in these nerve cells, 1) the IGF-I signals through the MAP kinase pathway and PI-3 kinase pathway are independently essential to induce IGF-I-dependent growth, and 2) alternate activation of the MAP kinase pathway and PI 3-kinase pathway is sufficient for the cells to undergo IGF-I-dependent differentiation.     

Ethanol Promotes Cell Death by Inhibition of the Insulin-Like Growth Factor I Receptor

The mechanism by which chronic alcohol abuse induces widespread cell and tissue damage is unknown. Insulin-like growth factor I (IGF-I) is an important inhibitor of apoptosis in many cell types, in addition to its ability to stimulate proliferation. We have demonstrated previously (J, Biol. Chem . 268:21777–21782,1993; Lab. Invest . 71657–662, 1994) that ethanol in low concentrations inhibits the tyrosine auto-phosphorylation of the IGF-I receptor (IGF-IR) and IGF-I-mediated cell proliferation. We now demonstrate that ethanol reverses the antiapoptotic action of the IGF-IR in a tumor necrosis factor-a (TNF-α) model of apoptosis. In serum-depleted medium, IGF-I markedly protected BALB/c3T3 cells from TNF-α-induced apoptosis. Ethanol reversed the protective action of IGF-I, but did not enhance TNF-α killing in the absence of IGF-I. Hatf-maximal effective concentrations of ethanol were 5 to 10 mM. In the presence of 5 to 10% fetal bovine serum, TNF-α was cytotoxic for 3T3 cells only in the presence of ethanol. Mouse embryo fibroblasts with targeted knockout of the IGF-IR were completely insensitive to ethanol, in contrast with the ethanol-induced potentiation of apoptosis in wild-type cells. These results indicate that ethanol directly interacts with cellular factors that inhibit apoptosis and could provide a novel mechanism for ethanol-induced cytotoxicity in general.     

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF- induced PLC gamma tyrosine phosphorylation and inositol 1,4,5- trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism. (Gastroenterology 1997 Jun;112(6):2073-88)     

Regulation of human EGF receptor by lipids

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.     

Epidermal growth factor-induced activation of the insulin-like growth factor I receptor in rat hepatocytes

Insulin-like growth factor I (IGF-I) plays a critical role in the induction of cell cycle progression and survival in many cell types. However, there is minimal IGF-I binding to hepatocytes, and a role for IGF-I in hepatocyte signaling has not been elucidated. The dynamics of IGF-I receptor (IGF-IR) activation were examined in freshly isolated rat hepatocytes. IGF-I did not activate the IGF-IR. However, des(1-3)IGF-I, which weakly binds IGF binding protein-3 (IGFBP-3), induced IGF-IR phosphorylation. IGFBP-3 surface coating was identified by confocal immunofluorescence microscopy. In contrast with the inactivity of IGF-I, epidermal growth factor (EGF) induced the tyrosine phosphorylation of the IGF-IR in parallel with EGF receptor phosphorylation. Transactivation of the IGF-IR by EGF was inhibited by tyrphostin I-Ome-AG538, a tyrosine kinase inhibitor with high specificity for the IGF-IR. Src kinase inhibitors pyrazolopyrimidine PP-1 and PP-2 inhibited transactivation of the IGF-IR by EGF. EGF stimulated the tyrosine phosphorylation of Src, and induced its association with the IGF-IR. EGF-induced phosphorylations of insulin-related substrate (IRS)-1, IRS-2, Akt, and p42/44 mitogen-activated protein kinases (MAPKs) were inhibited variably by I-Ome-AG538. In conclusion, the data show an EGF- and Src-mediated transactivation pathway for IGF-IR activation in hepatocytes, and indicate a role for the IGF-IR in hepatocyte intracellular signaling. The findings also show a role for IGFBP-3 in the inhibition of IGF-I signaling in hepatocytes.     

Increased, not decreased activation of the insulin-like growth factor (IGF) receptor signalling pathway during ceramide-induced apoptosis.

The insulin-like growth factors (IGFs) are capable of blocking apoptosis in many cell lines in vitro. The IGF-I receptor (IGF-IR) is believed to mediate protective effects of the IGFs against apoptosis. To determine whether ceramide-mediated induction of apoptosis involved a decreased survival effect of the IGF-IR, apoptosis was induced in IGF-I receptor positive (R+) and negative (R-) murine fibroblasts by incubation with increasing doses of the sphingolipid analogue, C2 ceramide. Lower ceramide doses were required to induce death in receptor negative compared with receptor positive fibroblasts (P< 0.05 at ceramide doses of 2 microM or greater), not only corroborating evidence that the IGF-I receptor functions as a survival receptor, but also suggesting that ceramide is not inducing apoptosis by suppressing a survival effect of the IGF-IR. Ceramide has been reported to induce death through suppression of MAP kinase, and activation of JUN kinase signalling; since our initial data suggested that ceramide had not affected an anti-apoptotic signalling event of the IGF-IR, we monitored the activation of these enzymes. To our surprise, in the presence of ceramide, not only was JUN kinase activity increased, but so too was MAP kinase. Inhibition of MAP kinase, using the MEKK inhibitor, PD98059, significantly reduced ceramide-induced cell death (P< 0. 001). Ceramide also enhanced IGF-induced tyrosine phosphorylation of the IGF-I receptor and activated PI-3 kinase. The cumulative effects of these events resulted in increased progression to the G2 phase of the cell cycle, arrest without subsequent mitosis, and apoptosis. These results indicate that ceramide is capable of eliciting apparently contradictory events within a single cell type, and suggest that in the presence of an IGF-IR, survival is enhanced because ceramide can activate PI-3 kinase, believed to be an anti-apoptotic enzyme.     

A candidate targeting molecule of insulin-like growth factor-I receptor for gastrointestinal cancers

Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage.One new group of targets is tyrosine kinase receptors,which can be treated by several strategies,including small molecule tyrosine kinase inhibitors(TKIs) and monoclonal antibodies(mAbs).Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal(GI) carcinomas.The concept of targeting specif ic carcinogenic receptors has been validated by successful clinical application of many new drugs.Type I insulin-like growth factor(IGF) receptor(IGF-IR) signaling potently stimulates tumor progression and cellular differentiation,and is a promising new molecular target in human malignancies.In this review,we focus on this promising therapeutic target,IGF-IR.The IGF/IGF-IR axis is an important modifier of tumor cell proliferation,survival,growth,and treatment sensitivity in many malignant diseases,including human GI cancers.Preclinical studies demonstrated that downregulation of IGF-IR signals reversed the neoplastic phenotype and sensitized cells to anticancer treatments.These results were mainly obtained through our strategy of adenoviruses expressing dominant negative IGF-IR(IGF-IR/dn) against gastrointestinal cancers,including esophagus,stomach,colon,and pancreas.We also summarize a variety of strategies to interrupt the IGFs/IGF-IR axis and their preclinical experiences.Several mAbs and TKIs targeting IGF-IR have entered clinical trials,and early results have suggested that these agents have generally acceptable safety profiles as single agents.We summarize the advantages and disadvantages of each strategy and discuss the merits/demerits of dual targeting of IGF-IR and other growth factor receptors,including Her2 and the insulin receptor,as well as other alternatives and possible drug combinations.Thus,IGF-IR might be a candidate for a molecular therapeutic target in human GI carcinomas.     

A familial insulin-like growth factor-I receptor mutant leads to short stature: clinical and biochemical characterization

CONTEXT: IGF-I/IGF-I receptor (IGF-IR) signaling pathways play important roles in longitudinal growth. A novel Arg481Glu (R481Q) mutation in IGF-IR was detected in a family with intrauterine and postnatal growth retardation. OBJECTIVE: The objective of the study was to explore the mechanism whereby the R481Q mutation may be causative in growth retardation. PATIENTS: A 13-yr-old girl with short stature was studied for functional analysis of the R481Q mutation in the IGF-IR. RESULTS: Two members of a family who showed intrauterine and postnatal growth retardation, with increased serum IGF-I levels, demonstrated a substitution of arginine for glutamine at 481 (R481Q) in the IGF-IR. This mutation results in the formation of an altered fibronectin type III domain within the alpha-subunit. NIH-3T3 fibroblasts that overexpress the human wild-type or R481Q mutant IGF-IR demonstrated normal cell surface ligand binding by 125I-IGF-I binding assay. However, the fold increase of IGF-I stimulated tyrosine phosphorylation of the IGF-IR beta-subunit as well as downstream activation of ERK1/2 and Akt was reduced in cells overexpressing the mutant receptor. Additionally, basal and IGF-I-stimulated levels of cell proliferation were also reduced in cells overexpressing the mutant receptor. CONCLUSION: Our results demonstrate that NIH-3T3 cells overexpressing a mutant form of the Igf1r gene, in which arginine at 481 is substituted by glutamine, lead to reduced levels of the fold increase of IGF-IR beta-subunit phosphorylation as well as ERK1/2 and Akt phosphorylation and was accompanied by decreased cell proliferation. These results are postulated to be the cause of intrauterine and postnatal growth retardation in the described patients.     

Granulocyte-macrophage colony-stimulating factor activates microtubule-associated protein 2 kinase in neutrophils via a tyrosine kinase-dependent pathway.

Receptors of the hematopoietin superfamily, including the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, lack a tyrosine kinase domain as well as other sequences indicative of a known signaling mechanism. In this report, we identify the serine/threonine kinase, microtubule-associated protein 2 (MAP2) kinase, as an intermediate in the GM-CSF signal transduction pathway. Treatment of peripheral blood neutrophils or terminally differentiated HL-60 cells with GM-CSF induced a rapid and dose-dependent increase in MAP2 kinase activity. Maximal activity occurred within 5 minutes and the kinetics of the response varied depending on the target cell (prolonged in neutrophils and transient in neutrophilic HL-60 cells). MAP2 kinase activity in these cells correlates with the induction of a 42-Kd tyrosine phosphoprotein. Furthermore, tyrosine phosphorylation is necessary for MAP2 kinase activation since its activity is inhibited by treatment with the tyrosine kinase inhibitor, erbstatin analog. These data suggest that tyrosine phosphorylation is important in GM-CSF-mediated signal transduction and that MAP2 kinase activation may be a central biochemical event involved in its signaling.     

The role of insulin-like growth factor I and its receptor in cell growth, transformation, apoptosis, and chemoresistance in solid tumors

Insulin-like growth factor I (IGF-I) exerts pleiotropic effects on mammalian cells via stimulation of its receptor (IGF-IR), a receptor tyrosine kinase. In vivo, IGF-I acts both as a local tissue growth factor and as a circulating hormone. In oncological research, IGF-I has received increased attention as the activated IGF-I/IGF-IR system displays mitogeneic, transforming, and anti-apoptotic properties in various cell types by stimulating distinct intracellular signaling pathways. Recent data suggest that the anti-apoptotic effect of IGF-I may mediate decreased sensitivity to chemotherapeutic drugs in vitro and in vivo. Thus, targeting the IGF-I/IGF-IR system could serve as an approach to overcome clinical drug resistance in certain tumors. Received: 15 December 1998 / Accepted: 4 January 1999     

Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.