Insulin-like Growth factor-I Receptor Function Research Articles

IGF-IR internalizes with CAVEOLIN-1 and PTRF/CAVIN in HACAT cells

Background: Insulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified. Methodology/Principal Findings: We have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin coimmunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling. Conclusions/Significance: These data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

IGF-IR Internalizes with Caveolin-1 and PTRF/Cavin in Hacat Cells

BackgroundInsulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified.Methodology/Principal FindingsWe have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin co-immunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling.Conclusions/SignificanceThese data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

IGF-I and the aging mammalian brain

Insulin-like growth factors (IGFs) are important modulators of organismal life-span all along phylogeny. These growth factors are widely viewed as detrimental for long life by reducing tissue resistance to oxidative stress. However, IGF-I has been consistently shown to be a potent neuroprotective factor in mammals, and as such, a deterrent of brain aging. Conversely, recent data suggest that IGF-I may contribute to amyloid neurodegeneration underlying Alzheimer's disease. These opposing observations underline an incomplete understanding of the significance of this ancestral hormone pathway in relation to brain aging. It is possible that these opposite results are the consequence of using different experimental approaches. Thus, brain amyloid injury is reduced in mutant mice partially defective in IGF-I receptor function, whereas IGF-I is neuroprotective when administered to animal models of neurodegenerative disease or normal brain aging. This approach-dependent effect of IGF-I highlights a fundamental gap in our knowledge of the relationship between peripheral and brain IGF-I function and the actual biological impact of experimental modulation of brain IGF-I function. We suggest to directly address brain IGF-I function in the varying experimental approaches used to confirm that changes have taken place in the desired way.

Insulin‐like growth factor type I receptor gene expression and obesity in esophageal adenocarcinoma

The objective of this exploratory study was to evaluate the role of the insulin-like growth factor I receptor (IGF-IR) in esophageal adenocarcinoma (EADC). Using quantitative PCR, we studied IGF-IR mRNA expression in 52 well-characterized surgically resected EADC and matched histologically normal esophageal tissues, and examined IGF-IR expression levels in relation to clinicopathologic characteristics, body mass index (BMI), and the common IGF-IR polymorphism (G1013A), recently proposed to modify risk of obesity for EADC. Expression levels of IGF-IR mRNA were not significantly different between EADC and matched histologically normal esophageal epithelia. Although no significant associations were found between IGF-IR expression and BMI, tumor differentiation, stage or survival, when stratified by genotype, patients with the polymorphic A variant had significantly higher IGF-IR expression in EADC tissues compared with matched normal epithelia. These findings suggest that G1013A most likely modulates IGF-IR function, possibly by influencing gene transcription or mRNA stability, and represents a plausible mechanistic link underlying the association between obesity and esophageal malignancy.

PBK/TOPK promotes tumour cell proliferation through p38 MAPK activity and regulation of the DNA damage response

The contribution of the insulin-like growth-factor-I receptor (IGF-IR) to tumour progression is well documented. To identify new mediators of IGF-IR function in cancer, we recently isolated genes differentially expressed in cells overexpressing the IGF-IR. Among these was the serine/threonine kinase PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase), previously associated with highly proliferative cells and tissues. Here, we show that PBK is expressed at high levels in tumour cell lines compared with non-transformed cells. IGF-I could induce PBK expression only in transformed cells, whereas epidermal growth factor could induce PBK in non-transformed MCF-10A breast epithelial cells. Suppression of PBK expression using small interfering RNA did not prevent progression through the cell cycle, but caused decreased proliferation over time in culture, and reduced clonogenic growth in soft agarose. PBK knockdown impaired p38 activation after long-term stimulation with different growth factors and reduced DU145 cells motility. Suppressed PBK expression also resulted in an impaired response to DNA damage that was evident by the decreased generation of gamma-H2AX, increased DNA damage and decreased cell survival. Taken together, the data indicate that PBK is necessary for appropriate activation and function of the p38 pathway by growth factors. Thus, enhanced expression of PBK may facilitate tumour growth by mediating p38 activation and by helping cells to overcome DNA damage.

Crosstalk between PDGF and IGF-I receptors in rat liver myofibroblasts: implication for liver fibrogenesis

Insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF) have been identified as significant mitogens for liver myofibroblasts (LMFs), one of the cell populations playing a role in liver fibrogenesis. In the present work, we aimed to elucidate a possible interaction between PDGF receptor (PDGFR) and IGF-I receptor (IGF-IR) signaling in LMFs. Among different rat liver cells, PDGFR α- and β-subunits were mainly expressed in hepatic stellate cells and LMFs, and were upregulated during their in vitro cultivation. In LMFs, PDGF-BB (10?ng/ml) stimulated DNA synthesis approximately two-fold and this effect was similar to that of IGF-I. IGF-I and PDGF-BB differentially affected IGF-IR and PDGFR signaling. High concentrations of IGF-I decreased levels of IGF-IR and IRS-1 and inhibited the expression and activation of PDGFRα. PDGF-BB prevented IGF-I-induced downregulation of the IGF-IR, but did not affect expression of its cognate receptor subunits. Transphosphorylation of PDGFR and IGF-IR was not observed. PDGF effectively activated terminal MAP kinases, PI3 kinase and Akt kinase, whereas IGF-I demonstrated weaker effects. PLCγ1 was phosphorylated only in response to PDGF, but not to IGF-I. In rat LMFs, blockade of the IGF-IR via inhibition of the IGF-IR kinase completely abrogated IGF- and PDGF-induced mitogenesis and the ability of PDGF to phosphorylate PLCγ1. In conclusion, the presented data demonstrate that the PDGFR signaling requires a functional IGF-IR and that PDGF-BB stabilizes the IGF-IR function through preventing the IGF-I-induced downregulation of the IGF-IR. These interactions might be relevant in vivo for the fibroproliferative response during liver injury.

Mammalian target of rapamycin inhibitors activate the AKT kinase in multiple myeloma cells by up-regulating the insulin-like growth factor receptor/insulin receptor substrate-1/phosphatidylinositol 3-kinase cascade

Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and CCI-779, have shown preclinical potential as therapy for multiple myeloma. By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent serine phosphorylation of insulin receptor substrate-1 (IRS-1), they may enhance insulin-like growth factor-I (IGF-I) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/AKT activation. This may be a particular problem in multiple myeloma where IGF-I-induced activation of AKT is an important antiapoptotic cascade. We, therefore, studied AKT activation in multiple myeloma cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation, and PI3K activity in multiple myeloma cells and prolonged activation of AKT induced by exogenous IGF-I. CCI-779, used in a xenograft model, also resulted in multiple myeloma cell AKT activation in vivo. Blockade of IGF-I receptor function prevented rapamycin's activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1, enhanced IRS-1 association with IGF-I receptors, and prevented IRS-1 degradation. Although similarly blocking IRS-1 degradation, proteasome inhibitors did not activate AKT. Thus, mTOR inhibitors activate PI3-K/AKT in multiple myeloma cells; activation depends on basal IGF-R signaling; and enhanced IRS-1/IGF-I receptor interactions secondary to inhibited IRS-1 serine phosphorylation may play a role in activation of the cascade. In cotreatment experiments, rapamycin inhibited myeloma cell apoptosis induced by PS-341. These results provide a caveat for future use of mTOR inhibitors in myeloma patients if they are to be combined with apoptosis-inducing agents.

Gene expression profiles in cells transformed by overexpression of the IGF-I receptor

To identify genes associated with insulin-like growth factor-I receptor (IGF-IR)-mediated cellular transformation, we isolated genes that are differentially expressed in R- cells (derived from the IGF-IR knockout mouse) and R+ cells (R- cells that overexpress the IGF-IR). From these, 45 genes of known function were expressed at higher levels in R+ cells and 22 were expressed at higher levels in R- cells. Differential expression was confirmed by Northern blot analysis of R+ and R- cells. Genes expressed more abundantly in R+ cells are associated with (1) tumour growth and metastasis including, betaigH3, mts1, igfbp5 protease, and mystique; (2) cell division, including cyclin A1 and cdk1; (3) signal transduction, including pkcdeltabp and lmw-ptp; and (4) metabolism including ATPase H+ transporter and ferritin. In MCF-7 cells IGF-I induced expression of two genes, lasp-1 and mystique, which could contribute to metastasis. Lasp-1 expression required activity of the PI3-kinase signalling pathway. Mystique was highly expressed in metastatic but not in androgen-dependent prostate cancer cell lines and Mystique overexpression in MCF-7 cells promoted cell migration and invasion. We conclude that genes identified in this screen may mediate IGF-IR function in cancer progression.

Crosstalk Between PDGF and IGF-I Receptors in Rat Liver Myofibroblasts: Implication for Liver Fibrogenesis

Insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF) have been identified as significant mitogens for liver myofibroblasts (LMFs), one of the cell populations playing a role in liver fibrogenesis. In the present work we aimed to elucidate a possible interaction between PDGF receptor (PDGFR) and IGF-I receptor (IGF-IR) signaling in LMFs. Among different rat liver cells, PDGFR alpha- and beta-subunits were mainly expressed in hepatic stellate cells and LMFs, and were up-regulated during their in vitro cultivation. PDGF-BB (10 ng/mL) stimulated DNA synthesis approximately two-fold in LMFs and this effect was similar to that of IGF-I. IGF-I and PDGF-BB differentially affected IGF-IR and PDGFR signaling. High concentrations of IGF-I decreased IGF-IR and its principal docking protein IRS–1 as well as inhibited expression and activation of PDGFR-alpha. PDGF-BB prevented IGF-I-induced down-regulation of the IGF-IR, but did not affect expression of its cognate receptor subunits. Transphosphorylation of PDGFR and IGF-IR was not observed. PDGF effectively activated terminal MAP kinases (ERK1/2, p38 MAPK, JNK–1), in contrast to IGF-I, which demonstrated only weak effects. PLC-gamma–1 was activated only in response to PDGF, but not to IGF-I. Blockade of the IGF-IR via inhibition of the IGF-IR kinase completely abrogated IGF- and PDGF-induced mitogenesis of rat LMFs as well as the ability of PDGF to stimulate PLC-gamma–1 activity. In conclusion, the presented data demonstrate that the PDGFR signaling requires a functional IGF-IR and that PDGF-BB stabilizes the IGF-IR function through preventing the IGF-I-induced down-regulation of the IGF-IR. These interactions might be relevant in vivo for the fibroproliferative response during liver injury.

Ataxia-telangiectasia mutated gene controls insulin-like growth factor I receptor gene expression in a deoxyribonucleic acid damage response pathway via mechanisms involving zinc-finger transcription factors Sp1 and WT1.

The IGF-I receptor (IGF-IR) has a central role in cell cycle progression as well as in the establishment of the transformed phenotype. Increased expression of the IGF-IR gene, in addition, is correlated with acquisition of radioresistance for cell killing. The ataxia-telangiectasia mutated (ATM) gene product has a pivotal role in coordinating the cellular response to DNA damage. The present study was aimed at testing the hypothesis that the ability of ATM to coordinate the DNA damage response that will lead to cell survival or, alternatively, to apoptosis depends, to a significant extent, on its capacity to control IGF-IR gene expression. The potential involvement of ATM in regulation of IGF-IR expression and function was investigated in isogenic cells with and without ATM function [AT22IJE-T/pEBS7 (ATM -/-) and ATM-corrected AT22IJE-T/YZ5 (ATM +/+) cells and 293 human embryonic kidney cells transfected with small interfering RNAs targeted to ATM]. In addition, the effect of ATM on IGF-IR expression was assessed in nonisogenic cells with ATM function (HFF + human telomerase reverse transcriptase) and without ATM function (GM5823 + human telomerase reverse transcriptase). Results obtained showed that IGF-IR gene expression and IGF-IR promoter activity were largely reduced in ATM -/- cells. Addition of the radiomimetic agent neocarzinostatin for 4 h, however, induced a significant increase in IGF-IR levels in cells without ATM function. In addition, IGF-I-induced IGF-IR and insulin receptor substrate-1 phosphorylation were greatly impaired in ATM-deficient cells. Furthermore, we identified zinc-finger transcription factors Sp1 and WT1 as potential mediators of the effect of ATM on IGF-IR gene expression. The present data suggests that the IGF-IR gene is a novel downstream target in an ATM-mediated DNA damage response pathway. Deregulated expression of the IGF-IR gene after ionizing radiation may be linked to genomic instability and enhanced transforming capacity.

High expression level of insulin-like growth factor I receptor is associated with regional lymph node metastasis

4115 Background: Over-expression of the insulin-like growth factor I receptor (IGF-IR) has been found in many types of human cancers. In vivo, inhibition of the function of IGF-IR results in apoptosis and tumor growth attenuation, indicating that altered expression of IGF-IR may play an important role in human cancer. However, the role of IGF-IR in gastric cancer has not been explored. Methods: In this study, we determined the expression status of the IGF-IR in primary gastric cancer using immunohistochemistry. Results: In the 86 cases of resected gastric cancer examined, IGF-IR was over-expressed in 61.6% gastric tumor samples as compared to the adjacent cancer-free gastric mucosa (p=0.018). In patients with lymph node metastasis, high expression level of IGF-IR was detected in both the primary tumor site and metastatic tumor cells with lymph nodes (p=0.0102), implicating IGF-IR in the development of lymph node metastasis. It has been reported that the transcription factor Sp1 transactivates the IGF-IR p...

High expression level of insulin-like growth factor I receptor is associated with regional lymph node metastasis

4115 Background: Over-expression of the insulin-like growth factor I receptor (IGF-IR) has been found in many types of human cancers. In vivo, inhibition of the function of IGF-IR results in apoptosis and tumor growth attenuation, indicating that altered expression of IGF-IR may play an important role in human cancer. However, the role of IGF-IR in gastric cancer has not been explored. Methods: In this study, we determined the expression status of the IGF-IR in primary gastric cancer using immunohistochemistry. Results: In the 86 cases of resected gastric cancer examined, IGF-IR was over-expressed in 61.6% gastric tumor samples as compared to the adjacent cancer-free gastric mucosa (p=0.018). In patients with lymph node metastasis, high expression level of IGF-IR was detected in both the primary tumor site and metastatic tumor cells with lymph nodes (p=0.0102), implicating IGF-IR in the development of lymph node metastasis. It has been reported that the transcription factor Sp1 transactivates the IGF-IR promoter and regulates IGF-IR expression. We have previously showed that over-expression of Sp1 in gastric cancer associated with advanced stage and predicted poor survival. In the current study, the expression level of IGF-IR correlated with that of Sp1 (p=0.018). Further, the median survival for patients with low and high IGF-IR expression was 1242 days and 792 days, respectively. However, this survival difference was not statistically significant (p=0.16), indicating that a larger scale study is warranted to further determine the value of IGF-IR as a prognostic/survival marker. Conclusions: Taken together, our data suggest that alteration of IGF-IR expression is likely to play a significant role in the development and metastasis of gastric cancers. Although it is not an independent survival predictor, it is one of the essential down steam components of Sp1 that might affect patient outcome. No significant financial relationships to disclose.

Growth factor receptors as therapeutic targets: strategies to inhibit the insulin-like growth factor I receptor.

Neoplastic transformation is often related to abnormal activation of growth factor receptors and their signaling pathways. The concept of targeting specific tumorigenic receptors and/or signaling molecules has been validated by the development and successful clinical application of drugs acting against the epidermal growth factor receptor 2 (HER2/neu, Erb2), the epidermal growth factor receptor 1 (EGFR, HER1), the Brc-Abl kinase, the platelet-derived growth factor receptor, and c-kit. This review will focus on the next promising therapeutic target, the insulin-like growth factor I receptor (IGF-IR). IGF-IR has been implicated in a number of neoplastic diseases, including several common carcinomas. From a pharmaceutical standpoint, of particular importance is that IGF-IR appears to be required for many transforming agents (genetic, viral, chemical) to act, but is not obligatory for the function of normal adult cells. The tumorigenic potential of IGF-IR is mediated through its antiapoptotic and transforming signaling, and in some cases through induction of prometastatic pathways. Preclinical studies demonstrated that downregulation of IGF-IR reversed the neoplastic phenotype and sensitized cells to antitumor treatments. The strategies to block IGF-IR function employed anti-IGF-IR antibodies, small-molecule inhibitors of the IGF-IR tyrosine kinase, antisense oligodeoxynucleotides and antisense RNA, small inhibitory RNA, triple helix, dominant-negative mutants, and various compounds reducing ligand availability. The experience with these strategies combined with the knowledge gained with current anti-growth factor receptor drugs should streamline the development of anti-IGF-IR therapeutics.

Regulation of Hypoxia-Inducible Factor-1α, Vascular Endothelial Growth Factor, and Angiogenesis by an Insulin-Like Growth Factor-I Receptor Autocrine Loop in Human Pancreatic Cancer

Activation of the insulin-like growth factor-I receptor (IGF-IR) was recently shown to modulate angiogenesis by up-regulating the expression of vascular endothelial growth factor (VEGF). We hypothesized that inhibiting IGF-IR function would inhibit angiogenesis and growth of pancreatic cancer in vivo and sought to identify major signaling pathways regulated by IGF-IR in pancreatic cancer cells. Human pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative form of IGF-IR (IGF-IR DN) or an empty vector (pcDNA). In vitro, IGF-IR DN cells exhibited a decrease in both constitutive and inducible phosphorylation of IGF-IR and Erk1/2. Constitutive expression of nuclear hypoxia-inducible factor-1alpha and secreted VEGF (P < 0.01) protein levels also were significantly lower in IGF-IR DN cells than in pcDNA cells. In vivo, IGF-IR inhibition led to decreases in pancreatic tumor volume and weight, vessel density, and tumor cell proliferation (P < 0.01 for all) and increases in tumor cell apoptosis (P < 0.02). Our results suggest that autocrine activation of the IGF-IR system significantly affects VEGF expression and angiogenesis in human pancreatic cancer. Thus, IGF-IR may be a valid target in the treatment of pancreatic cancer.

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway.

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.

Alterations of the insulin-like growth factor system in patients with polycythemia vera

The molecular etiology of Polycythemia vera (PV) is still undetermined. Recently, enhanced tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) has been shown in PV bone marrow progenitors and peripheral blood mononuclear cells (PBMNC), and elevated levels of IGF binding protein-1 (IGFBP-1) in the serum of PV patients have been reported. To identify further alterations of circulating IGFBPs, the IGFBP profile in the serum of 12 PV patients was compared with age- and sex-matched controls by Western ligand blot (WLB), two-dimensional WLB, IGFBP-3 immunoblot and specific RIA for IGFBP-1, -2, -3 and IGFBP-4. To elucidate a role for the IGF-IR in the pathogenesis of PV, basal and IGF-I stimulated tyrosine phosphorylation of the IGF-IR β-subunit in PBMNC of PV patients or controls was determined by WLB. Furthermore, exons 2, 3 and 15-21 of the IGF-IR were screened for mutations by PCR-single strand conformation polymorphism analysis (PCR-SSCP). We found alterations of the IGFBP profile in the serum of eight out of 12 examined patients including elevated levels of IGFBP-1, -2 and -4, decreased levels of IGFBP-3 and an increase in IGFBP-3 fragment. However, no differences in tyrosine phosphorylation of the IGF-IR in PV patients, neither basal nor IGF-I induced, were detected. Furthermore, no mutations within the screened exons of the IGF-IR could be identified by PCR-SSCP. We conclude that there is no direct impairment of IGF-IR structure or function, but an altered IGFBP profile in a significant portion of PV patients which might contribute to the pathogenesis of PV in these patients.

Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth.

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (alphaIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 alphaIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92-98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the alphaIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble alphaIGF-IR scFvs, a prototype alphaIGF-IR scFv and its alternative type alphaIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alphaIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A--agarose chromatography. Levels of alphaIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that alphaIGF-IR scFv-Fc is a dimeric antibody. alphaIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10(8) M(-1), which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of alphaIGF-IR scFv-Fc (500 microg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alphaIGF-IR scFv-Fc is a first-generation recombinant alphaIGF-IR for the potential development of future alphaIGF-IR therapeutics.

Function of the IGF-I receptor in breast cancer.

The insulin-like growth factor-I receptor (IGF-IR) is a transmembrane tyrosine kinase regulating various biological processes such as proliferation, survival, transformation, differentiation, cell-cell and cell-substrate interactions. Different signaling pathways may underlie these pleiotropic effects. The specific pathways engaged depend on the number of activated IGF-IRs, availability of intracellular signal transducers, the action of negative regulators, and is influenced by extracellular modulators. Experimental and clinical data implicate the IGF-IR in breast cancer etiology. There is strong evidence linking hyperactivation of the IGF-IR with the early stages of breast cancer. In primary breast tumors, the IGF-IR is overexpressed and hyperphosphorylated, which correlates with radio-resistance and tumor recurrence. In vitro, the IGF-IR is often required for mitogenesis and transformation, and its overexpression or activation counteract effects of various pro-apoptotic treatments. In hormone-responsive breast cancer cells, IGF-IR function is strongly linked with estrogen receptor (ER) action. The IGF-IR and the ER are co-expressed in breast tumors. Moreover, estrogens stimulate the expression of the IGF-IR and its major signaling substrate IRS-1, while antiestrogens downregulate IGF-IR signaling, mainly by decreasing IRS-1 expression and function. On the other hand, overexpression of IRS-1 promotes estrogen-independence for growth and transformation. In ER-negative breast cancer cells, usually displaying a more aggressive phenotype, the levels of the IGF-IR and IRS-1 are often low and IGF is not mitogenic, yet the IGF-IR is still required for metastatic spread. Consequently, IGF-IR function in the late stages of breast cancer remains one of the most important questions to be addressed before rational anti-IGF-IR therapies are developed.

Membrane-associated Insulin-like Growth Factor-binding Protein-3 Inhibits Insulin-like Growth Factor-I-induced Insulin-like Growth Factor-I Receptor Signaling in Ishikawa Endometrial Cancer Cells

The function of cell surface-associated insulin-like growth factor-binding proteins (IGFBPs) is controversial. Both inhibition and facilitation of IGF action as well as IGF-independent effects have been reported. We examined the influence of endogenous cell surface-associated IGFBPs on IGF-I receptor (IGF-IR) function in Ishikawa endometrial cancer cells by comparing the effects of IGF-I and its truncated analog des-(1-3)-IGF-I on several components of the IGF-IR signal transduction pathway in the absence of significant amounts of soluble IGFBPs. IGF-I and des-(1-3)-IGF-I are known to have similar affinities for IGF-IR, although the affinity of des-(1-3)-IGF-I for IGFBPs is greatly reduced. Here we show that the two ligands were equipotent not only in IGF-IR binding but also in receptor activation in NIH 3T3 cells overexpressing IGF-IR and possessing a relatively small number of cell surface-associated IGFBPs. In contrast, des-(1-3)-IGF-I manifested a remarkably higher potency as compared with IGF-I in inducing short and middle term cellular responses in IGF-IR-transfected Ishikawa endometrial cancer cells possessing a high number of both the receptor and the cell membrane-bound IGFBP-3. Thus, this difference in the effects of IGF-I and des-(1-3)-IGF-I can be attributed to the attenuation of IGF-I-mediated IGF-IR signaling by membrane-bound IGFBP-3.

Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways Are Not Sufficient for Insulin-Like Growth Factor I-Induced Mitogenesis and Tumorigenesis

Insulin-like growth factor-I (IGF-I) and insulin are known to activate a signaling cascade involving ras --> kappa raf-1 --> mitogen-activated protein (MAP) kinase kinase (MEK) --> p42/p44 MAP kinase (Erk-1 and -2). Recent reports suggest that activation of this ras/MAP kinase pathway is involved in mitogenesis and c-fos transcription but is not required for insulin action on metabolic processes such as glycogen synthesis, lipogenesis, and GLUT-4-mediated glucose transport. Previously we and others have demonstrated that substitution of both tyrosines at positions 1250 and 1251 in the carboxy-terminal region of the human IGF-I receptor has relatively small effects on receptor and endogenous substrate phosphorylation but completely abrogated the ability of these cells to form tumors in nude mice or proliferate in response to IGF-I in culture. Replacement of the tyrosine at position 1316 also did not affect the kinase activity of the receptor with respect to autophosphorylation or phosphorylation of endogenous substrates but did reduce the ability of the receptor to mediate mitogenic or tumorigenic signals. To further characterize the role of these tyrosines in IGF-I receptor function, we have used three distinct approaches to examine the ras/MAP kinase pathway in IGF-I-induced mitogenesis and tumorigenesis in NIH-3T3 cells overexpressing wild-type and mutated IGF-I receptors: 1) tyrosine phosphorylation of the MAP kinases Erk-1 and -2; 2), mobility shifts indicative of MAP kinase phosphorylation; and 3) in vitro MAP kinase activation. We have also examined IGF-I-induced phosphatidylinositol (PI) 3-kinase activation in the same cell lines. By each method we show that the IGF-I-induced MAP kinase phosphorylation/activation and PI 3-kinase activation, are not different between cells overexpressing wild-type IGF-I receptors and cells carrying IGF-I receptors having tyrosine motifs replaced at positions 1250 and 1251. We conclude that mitogenic and tumorigenic signals involving tyrosine residues in the C-terminal domain of the IGF-I-receptor include pathways other than the MAP kinase and PI 3-kinase pathways.