Insulin-like Growth factor-I Receptor Kinase Research Articles

Molecular basis for differential recognition of an allosteric inhibitor by receptor tyrosine kinases.

Understanding kinase-inhibitor selectivity continues to be a major objective in kinase drug discovery. We probe the molecular basis of selectivity of an allosteric inhibitor (MSC1609119A-1) of the insulin-like growth factor-I receptor kinase (IGF1RK), which has been shown to be ineffective for the homologous insulin receptor kinase (IRK). Specifically, we investigated the structural and energetic basis of the allosteric binding of this inhibitor to each kinase by combining molecular modeling, molecular dynamics (MD) simulations, and thermodynamic calculations. We predict the inhibitor conformation in the binding pocket of IRK and highlight that the charged residues in the histidine-arginine-aspartic acid (HRD) and aspartic acid-phenylalanine-glycine (DFG) motifs and the nonpolar residues in the binding pocket govern inhibitor interactions in the allosteric pocket of each kinase. We suggest that the conformational changes in the IGF1RK residues M1054 and M1079, movement of the ⍺C-helix, and the conformational stabilization of the DFG motif favor the selectivity of the inhibitor toward IGF1RK. Our thermodynamic calculations reveal that the observed selectivity can be rationalized through differences observed in the electrostatic interaction energy of the inhibitor in each inhibitor/kinase complex and the hydrogen bonding interactions of the inhibitor with the residue V1063 in IGF1RK that are not attained with the corresponding residue V1060 in IRK. Overall, our study provides a rationale for the molecular basis of recognition of this allosteric inhibitor by IGF1RK and IRK, which is potentially useful in developing novel inhibitors with improved affinity and selectivity.

The IGF-Axis and Diabetic Retinopathy Before and After Gastric Bypass Surgery.

Laparoscopic gastric bypass (LGB) abruptly causes remission of type 2 diabetes (T2D). Such dramatic metabolic changes have previously been found to cause worsening of diabetic retinopathy (DR) and circulating insulin-like growth factor I (IGF-I) has been suggested as a causal mediator. We aimed to evaluate baseline imbalances in the circulating IGF-system and changes after LGB in patients with T2D. Prospective ocular examinations and measurement of the IGF-axis before and 3 and 12months after LGB. IGF-bioactivity was measured by cell-based IGF-I receptor (IGF-IR) kinase activation assay (bioactive IGF). Total IGF-I, IGF-II and IGF binding protein (IGFBP) 1 and 3 were determined by immunoassays. At baseline, 18 of 36 patients presented with DR. These patients had higher levels of bioactive IGF (p=0.03) than patients without DR and this association was strengthened in multivariate analysis (p=0.006). Three patients had worsening of DR, unrelated to other markers. In univariate analysis, bioactive IGF increased at 3months (p=0.05) but this change became insignificant in multivariate analysis (p=0.11). IGFBP-1 increased whereas IGFBP-3 and total IGF-II decreased at the two postoperative visits (p≤0.001). Total IGF-I showed no significant changes. HbA1c, glucose, HOMA-IR and lipids improved after surgery. Two patients did not complete the 12-month visit. In obese T2D patients, bioactive IGF is a potential biomarker for DR and levels tended to increase 3months after bariatric surgery. IGFBP-1 increased while IGFBP-3 and total IGF-II decreased postoperatively, but these changes were unassociated with the development of DR. Markers of the metabolic syndrome improved.

Effects of human insulin and insulin aspart preparations on levels of IGF-I, IGFBPs and IGF bioactivity in patients with type 1 diabetes.

BackgroundInsulin aspart (IAsp) and its biphasic preparations BIAsp50 and BIAsp70 (containing 50% and 70% IAsp, respectively) have distinct glucose-lowering properties as compared to human insulin (HI). We investigated whether this affected the circulating IGF-system which depends on the hepatic insulin exposure.MethodsIn a randomized, four-period crossover study, 19 patients with type 1 diabetes received identical doses (0.2 U/kg sc) of IAsp, BIAsp70, BIAsp50 and HI together with a standardized meal. Serum total IGF-I and IGFBP-1 to -3 were measured by immunoassays for nine hours post-prandially. Bioactive IGF was determined by an in-house, cell-based IGF-I receptor kinase activation (KIRA) assay.ResultsDespite marked differences in peripheral insulin concentrations and plasma glucose, the four insulin preparations resulted in parallel decreases in IGFBP-1 levels during the first 3 hours, and parallel increases during the last part of the study (3–9 hours). Thus, only minor significances were seen. Insulin aspart and human insulin resulted in a lower area under the curve (AUC) during the first 3 hours as compared to BIAsp70 (p = 0.009), and overall, human insulin resulted in a lower IGFBP-1 AUC than BIAsp70 (p = 0.025). Nevertheless, responses and AUCs of bioactive IGF were similar for all four insulin preparations. Changes in levels of bioactive IGF were inversely correlated to those of IGFBP-1, increasing during the first 3 hours, whereafter levels declined (-0.83 ≤ r ≤ -0.30; all p-values <0.05).Total IGF-I and IGFBP-3 remained stable during the 9 hours, whereas IGFBP-2 changed opposite of IGFBP-1, increasing after 3–4 hours whereafter levels gradually declined. The four insulin preparations resulted in similar profiles and AUCs of total IGF-I, IGFBP-2 and IGFBP-3.ConclusionsDespite distinct glucose-lowering properties, the tested insulin preparations had similar effects on IGF-I concentration and IGF bioactivity, IGFBP-2 and IGFBP-3 as compared to HI; only small differences in IGFBP-1 were seen and they did not affect bioactive IGF. Thus, insulin aspart containing preparation behaves as HI in regards to the circulating IGF-system. However, bioactive IGF appeared to be more sensitive to insulin exposure than total IGF-I. The physiological significance of this finding remains to be determined.Trial registrationNCT00888732

New strategies to overcome resistance to mammalian target of rapamycin inhibitors in breast cancer

To review the studies addressing mammalian target of rapamycin (mTOR) inhibitors in breast cancer and resistance to rapalogs. Preclinical and clinical studies have suggested mTOR inhibitors may help overcome the resistance to endocrine therapy and trastuzumab. Despite much interest, knowledge of the mechanism and molecular response to mTOR inhibitors is incomplete. Resistance to mTOR inhibitors has been explored in preclinical studies and can be defined as primary, associated with amplifications or mutations of different kinases, or secondary, in which rapalog activates the feedback loops involving the insulin-like growth factor I receptor (IGF-IR), platelet-derived growth factor receptor and mitogen-activated protein kinase (MAPK) pathway. Current clinical trials are testing the combinations of rapamycin with other kinase inhibitors including IGF-IR, phosphoinositide 3-kinase and MAPK-extracellular signal-regulated kinase inhibitors. Recent findings on the resistance to rapalogs have stimulated the assessment of combinations of inhibitors in clinical trials. This review summarizes the current knowledge of primary and secondary rapalog resistance, and the current efforts to overcome this resistance.

Resistin decreases insulin-like growth factor I–induced steroid production and insulin-like growth factor I receptor signaling in human granulosa cells

To identify resistin in human ovarian follicles and investigate the effect and the molecular mechanisms associated with resistin on steroidogenesis in human granulosa cells (GCs). The effects of recombinant human resistin on the secretion of progesterone (P) and estradiol (E2) by cultured human GCs were investigated. Academic institutions. Twenty infertile and healthy women undergoing IVF. Primary human GC cultures stimulated with recombinant human resistin (10 ng/mL). Determination of messenger RNA (mRNA) and protein expression of resistin in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry, respectively; measurement of P and E2 levels in the conditioned media by radioimmunoassay; determination of cell proliferation by tritiated thymidine incorporation; and analysis of signaling pathways activation by immunoblot analysis. Human GCs and theca cells express resistin. In primary human GCs, resistin decreases P and E2 secretion in response to insulin-like growth factor I (IGF-I). This was associated with a reduction in the P450 aromatase and P450scc (cholesterol side-chain cleavage cytochromes P450) (P450scc) protein levels but not those of 3β-hydroxysteroid dehydrogenase (3β-HSD) or steroidogenic acute regulatory protein (StAR) and with a decrease in IGF-I-induced IGF-I receptor and mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Resistin treatment does not affect IGF-I-induced cell proliferation and basal steroidogenesis (there is no IGF-I or follicle-stimulating hormone stimulation). In the basal state, resistin rapidly stimulates Akt and MAPK ERK1/2 and p38 phosphorylation in primary human GCs. Resistin is present in human GCs and theca cells. It decreases P and E2 secretion, P450scc and P450 aromatase protein levels, and IGF-IR signaling in response to IGF-I in primary human GCs.

Circulating IgGs May Modulate IGF-I Receptor Stimulating Activity in a Subset of Patients With Graves' Ophthalmopathy

There is a close association between levels of TSH binding inhibitory immunoglobulins (TBIIs) and Graves' ophthalmopathy (GO). In addition to the TSH receptor, the IGF-I receptor (IGF-IR) has been proposed to be a second autoantigen that plays a role in the pathogenesis of GO. The aim was to study relationships between TBII and serum IGF-IR stimulating activity in relationship to age in patients with GO. We performed a prospective study of 70 patients with GO (26 euthyroid, 39 subclinical hyperthyroid, 5 hyperthyroid; 8 males, 62 females; age, 47.9 ± 1.0 y). Patients were graded according to clinical activity score. IGF-IR stimulating activity was determined by IGF-IR kinase receptor activation assay; TBIIs were measured by immunoassay (Trak). Protein G magnetic beads were used to deplete serum of IgGs. TBII and clinical activity score were positively related (r = 0.30; P = .01). In subjects with TBII above mean +1 SD, IGF-IR stimulating activity was positively related to age (r = 0.43; P = .05), whereas such a relationship was absent for subjects with TBII below the mean +1 SD (r = -0.04; P = .81). Depletion of IgGs from sera of patients with both TBII above the mean +1 SD and IGF-IR stimulating activity above the mean -1 SD decreased IGF-IR stimulating activity, whereas depletion in patients with TBII above the mean +1 SD but IGF-IR stimulating activity below the mean -1 SD did not change IGF-IR stimulating activity. In subjects with TBII above the mean +1 SD, we observed an increase of IGF-IR stimulating activity with age. In a subgroup of these patients, depletion of IgGs significantly decreased IGF-IR stimulating activity, suggesting that, in a subset of patients with GO, IgGs may have IGF-IR stimulating activities.

Age‐related decline in osteoblastogenesis and 1α‐hydroxylase/CYP27B1 in human mesenchymal stem cells: stimulation by parathyroid hormone

With aging, there is a decline in bone mass and in osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro. Osteoblastogenesis can be stimulated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] and, in some hMSCs, by the precursor 25-hydroxyvitamin D(3) (25OHD(3) ). CYP27B1/1α-hydroxylase activates 25OHD(3) and, to a variable degree, hMSCs express CYP27B1. In this study, we tested the hypotheses (i) that age affects responsiveness to 25OHD(3) and expression/activity of CYP27B1 in hMSCs and (ii) that parathyroid hormone (PTH) upregulates CYP27B1 in hMSCs, as it does in renal cells. There were age-related declines in osteoblastogenesis (n=8, P=0.0286) and in CYP27B1 gene expression (n=27, r= -0.498; P=0.008) in hMSCs. Unlike hMSCs from young subjects (≤50 years), hMSCs from older subjects (≥55 years) were resistant to 25OHD(3) stimulation of osteoblastogenesis. PTH1-34 (100 nm) provided hMSCs with responsiveness to 25OHD(3) (P=0.0313, Wilcoxon matched pairs test) and with two episodes of increased 1,25(OH)(2) D(3) synthesis, of cAMP response element binding protein (CREB) activation, and of CYP27B1 upregulation. Both increases in CYP27B1 expression by PTH were obliterated by CREB-siRNA or KG-501 (which specifically inhibits the downstream binding of activated CREB). Only the second period of CREB signaling was diminished by AG1024, an inhibitor of insulin-like growth factor-I receptor kinase. Thus, PTH stimulated hMSCs from elders with responsiveness to 25OHD(3) by upregulating expression/activity of CYP27B1 and did so through CREB and IGF-I pathways.

IGF-I Stimulates Cooperative Interaction between the IGF-I Receptor and CSK Homologous Kinase that Regulates SHPS-1 Phosphorylation in Vascular Smooth Muscle Cells

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation.

The insulin-like growth factor-I receptor kinase inhibitor NVP-ADW742 sensitizes medulloblastoma to the effects of chemotherapy

Medulloblastoma is the most common malignant tumor of the central nervous system in children. The insulin-like growth factor-I receptor (IGF-IR) is an important growth factor for medulloblastoma. The novel IGF-I receptor (IGF-IR) kinase inhibitor NVP-ADW742 has in vitro activity against tumors. Daoy cells were treated with NVP-ADW742 combined with temozolomide, which is commonly used in the chemotherapy of medulloblastoma. The effects on proliferation were assayed by CCK-8 assay. Cell cycle status and apoptosis were assayed by FACS analysis. The IGF-IR signaling pathway was analyzed by RT2 Profiler™ PCR arrays and Western blotting. NVP-ADW742 inhibited IGF-IR-mediated proliferation with an IC50 of 11.12 µmol/l. The PCR array data suggested that 14 genes were down-regulated at the mRNA level after NVP-ADW742 treatment. Western blot analysis suggested that NVP-ADW742 induced early suppression of Akt, P38 and GSK-3β phosphorylation, as well as a decrease in the intracellular levels of PI3K, Akt, P38, GSK-3β and Bcl-2. Combined with NVP-ADW742 (2 µmol/l), IC50 of Daoy to temozolomide was decreased from 452.12 to 256.81 µmol/l. Cell apoptosis was enhanced from 16.18±2.47% to 23.20 ± 2.80%. G phase arrest was also found in both the temozolomide alone group and the temozolomide combined with NVP-ADW742 group. NVP-ADW742 inhibited the activation of PI3K, Akt, P38 and GSK-3β caused by temozolomide. NVP-ADW742 enhanced the chemosensitivity of Daoy to temozolomide in vitro, as a potent anti-tumor agent highly selective against IGF-IR.

GSK1838705A inhibits the insulin-like growth factor-1 receptor and anaplastic lymphoma kinase and shows antitumor activity in experimental models of human cancers

The insulin-like growth factor-I receptor (IGF-IR) signaling pathway is activated in various tumors, and inhibition of IGF-IR kinase provides a therapeutic opportunity in these patients. GSK1838705A is a small-molecule kinase inhibitor that inhibits IGF-IR and the insulin receptor with IC(50)s of 2.0 and 1.6 nmol/L, respectively. GSK1838705A blocks the in vitro proliferation of cell lines derived from solid and hematologic malignancies, including multiple myeloma and Ewing's sarcoma, and retards the growth of human tumor xenografts in vivo. Despite the inhibitory effect of GSK1838705A on insulin receptor, minimal effects on glucose homeostasis were observed at efficacious doses. GSK1838705A also inhibits the anaplastic lymphoma kinase (ALK), which drives the aberrant growth of anaplastic large-cell lymphomas, some neuroblastomas, and a subset of non-small cell lung cancers. GSK1838705A inhibits ALK, with an IC(50) of 0.5 nmol/L, and causes complete regression of ALK-dependent tumors in vivo at well-tolerated doses. GSK1838705A is therefore a promising antitumor agent for therapeutic use in human cancers.

A Novel Unidirectional Cross-Talk from the Insulin-Like Growth Factor-I Receptor to Leptin Receptor in Human Breast Cancer Cells

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells.

The insulin‐like growth factor‐I receptor inhibitor NVP‐AEW541 provokes cell cycle arrest and apoptosis in multiple myeloma cells

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of monoclonal plasma cells in the bone marrow (BM). Despite recent advances in the treatment, MM represents an incurable disease for which development of new therapies is required. We report the antimyeloma effect of NVP-AEW541, a small molecule that belongs to the pyrrolo[2,3-d]pyrimidine class, identified as a selective inhibitor of the insulin-like growth factor-I receptor (IGF-IR) in vitro kinase activity. NVP-AEW541 had a potent cytotoxic effect on fresh cells and in a murine MM model. NVP-AEW541 partially abrogated the proliferative advantage conferred by the coculture with BM stromal cells and the presence of growth factors produced by the BM microenvironment. In addition, NVP-AEW541 potentiated the action of drugs, such as bortezomib, lenalidomide, dexamethasone or melphalan. Moreover the triple combination of NVP-AEW541, dexamethasone and bortezomib resulted in a significant increase in growth inhibition. Mechanistic studies indicated that NVP-AEW541 provoked a marked cell cycle blockade accompanied by pRb downregulation. Interestingly, NVP-AEW541 increased the levels of p27 associated with a reduction in the CDK2 activity. Finally, NVP-AEW541 induced cell death through caspase-dependent and -independent mechanisms. All these data, suggest the potential effect of IGF-IR kinase inhibitors as therapeutic agents for MM patients.

Aldosterone Induces Elastin Production in Cardiac Fibroblasts through Activation of Insulin-Like Growth Factor-I Receptors in a Mineralocorticoid Receptor-Independent Manner

Aldosterone is known to regulate electrolyte homeostasis, but it may also contribute to other processes, including the maladaptive remodeling of postinfarct hearts. Because aldosterone has been implicated in the stimulation of collagen production in the heart, we investigated whether it would also affect elastin deposition in cultures of human cardiac fibroblasts. We first demonstrated that treatment with 1 to 50 nmol/L aldosterone leads to a significant increase in collagen type I mRNA levels and in subsequent collagen fiber deposition. Pretreatment of cells with the mineralocorticoid receptor antagonist spironolactone, but not with the glucocorticoid receptor antagonist RU 486, inhibited collagen synthesis in aldosterone-treated cultures. Most importantly, we demonstrated that aldosterone also increases elastin mRNA levels, tropoelastin synthesis, and elastic fiber deposition in a dose-dependent manner. Strikingly, neither spironolactone nor RU 486 eliminated aldosterone-induced increases in elastin production. We further discovered that the proelastogenic effect of aldosterone involves a rapid increase in tyrosine phosphorylation of the insulin-like growth factor-I receptor and that the insulin-like growth factor-I receptor kinase inhibitor AG1024 or an anti-insulin-like growth factor-I receptor-neutralizing antibody inhibits both insulin-like growth factor-I and aldosterone-induced elastogenesis. Thus, we have demonstrated for the first time that aldosterone, which stimulates collagen production through the mineralocorticoid receptor-dependent pathway, also increases elastogenesis via a parallel mineralocorticoid receptor-independent pathway involving I insulin-like growth factor-I receptor signaling.

A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor–dependent tumor growth in vivo

Insulin-like growth factor-I receptor (IGF-IR) and its ligands, IGF-I and IGF-II, are up-regulated in a variety of human cancers. In tumors, such as colorectal, non-small cell lung, ovarian, and pediatric cancers, which may drive their own growth and survival through autocrine IGF-II expression, the role of IGF-IR is especially critical. Here, we present a novel small-molecule IGF-IR kinase inhibitor, cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which displayed a cellular IC(50) of 19 nmol/L for inhibition of ligand-dependent autophosphorylation of human IGF-IR with 14-fold cellular selectivity relative to the human insulin receptor. PQIP showed minimal activity against a panel of 32 other protein kinases. It also abolished the ligand-induced activation of downstream phosphorylated AKT and phosphorylated extracellular signal-regulated kinase 1/2 in both IGF-IR transfectant cells and a GEO human colorectal cancer cell line. Analysis of GEO cells revealed a significant level of both phosphorylated IGF-IR and IGF-II expression. Furthermore, inactivation of IGF-II in conditioned GEO culture medium by a neutralizing antibody diminished IGF-IR activation, indicating the presence of a functional IGF-II/IGF-IR autocrine loop in GEO cells. Once daily oral dosing of PQIP induced robust antitumor efficacy in GEO xenografts. The antitumor efficacy correlated with the degree and duration of inhibition of tumor IGF-IR phosphorylation in vivo by this compound. Moreover, when mice were treated for 3 days with a dose of PQIP that maximally inhibited tumor growth, only minor changes in blood glucose were observed. Thus, PQIP represents a potent and selective IGF-IR kinase inhibitor that is especially efficacious in an IGF-II-driven human tumor model.

Autocrine insulin-like growth factor-I signaling promotes growth and survival of human acute myeloid leukemia cells via the phosphoinositide 3-kinase/Akt pathway

Insulin-like growth factor (IGF) signaling plays an important role in various human cancers. Therefore, the role of insulin-like growth factor I (IGF-I) signaling in growth and survival of acute myeloid leukemia (AML) cells was investigated. Expression of the IGF-I receptor (IGF-IR) and its ligand IGF-I were detected in a panel of human AML blasts and cell lines. IGF-I and insulin promoted the growth of human AML blasts in vitro and activated the phosphoinositide 3-kinase (PI3K)/Akt and the extracellular signal-regulated kinase (Erk) pathways. IGF-I-stimulated growth of AML blasts was blocked by an inhibitor of the PI3K/Akt pathway. Moreover, downregulation of the class Ia PI3K isoforms p110beta and p110delta by RNA interference impaired IGF-I-stimulated Akt activation, cell growth and survival in AML cells. Proliferation of a panel of AML cell lines and blasts isolated from patients with AML was inhibited by the IGF-IR kinase inhibitor NVP-AEW541 or by an IGF-IR neutralizing antibody. In addition to its antiproliferative effects, NVP-AEW541 sensitized primary AML blasts and cell lines to etoposide-induced apoptosis. Together, our data describe a novel role for autocrine IGF-I signaling in the growth and survival of primary AML cells. IGF-IR inhibitors in combination with chemotherapeutic agents may represent a novel approach to target human AML.

Insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 is active in breast cancer cells and enhances growth inhibition by herceptin through an increase in cell cycle arrest

21077 Background: Targeting ErbB2 with the monoclonal antibody trastuzumab (Herceptin) has shown to be active in breast cancer. However, a proportion of patients do not benefit from this treatment due to primary resistance. Mechanisms proposed for this resistance include activation of other receptors involved in proliferation as is the case of the insulin-like growth factor receptor (IGF-1R).Different strategies have been proposed for the inhibition of IGF-1R such as antibodies or small tyrosine-kinase inhibitors. In this study we analyzed the mechanism of action and the antiproliferative activity of an specific IGF-1R tyrosine-kinase inhibitor termed NVP-AEW541 alone and in combination with Herceptin. Methods: MCF7, SKBR3, BT474, and MDA-MB-231 breast cancer cells were used for this study. Proliferation and apoptosis were analyzed by MTT uptake assays or Annexin-V-FITC, respectively. The levels of different signalling proteins were analyzed by Western blotting. Microarray studies were performed using the HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA). Real time quantitative PCR was used to confirm the differentially regulated genes. Results: The IGF-1R was expressed in all the cell lines studied. Exogenous addition of IGF-1 increased the tyrosine phosphorylation of IGF-1R, and this was prevented by preincubation with NVP- AEW541. The latter also decreased MTT uptake, and provoked a slight increase in apoptosis of BT474 cells. Combination of NVP-AEW541 and Herceptin had a synergistic effect on the inhibition of BT474 proliferation. Cell cycle analyses indicated that the combined addition of NVP- AEW541 and Herceptin increased the number of cells in the G0/G1 phases. These data were complemented with an increase in p27 levels upon treatment with both compounds. Microarray analyses identified several genes whose expression was modified by each agent alone and by the combination. Conclusions: Our results indicate that the combined targeting of two receptor tyrosine kinases known to play important roles in tumor cell proliferation may be more efficient than individual targeting of these receptors. No significant financial relationships to disclose.

The insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 induces apoptosis in acute myeloid leukemia cells exhibiting autocrine insulin-like growth factor-I secretion

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34(+) cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.

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.

Signaling pathway of insulin-like growth factor-II as a target of molecular therapy for hepatoblastoma

To address the possibility that insulin-like growth factor (IGF)-II is a growth factor and its signaling pathway so as to develop a molecular therapy for hepatoblastoma. Huh-6 and HepG2, human hepatoblastoma cell lines, were used. IGF-II was added to the medium deprived of serum. Western blot analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Inhibitors of IGF-IR (piclopodophyllin, PPP), phosphatidyl-inositol (PI) 3-kinase (LY294002 and Wortmannin), or mitogen-activated protein (MAP) kinase (PD98059) were added to unveil the signaling pathway of IGF-II. Cells were analyzed morphologically with hematoxylin-eosin staining to reveal the mechanism of suppression of cell proliferation. IGF-II stimulated cells proliferated to 2.7 (269%+/-76%) (mean+/-SD) (Huh-6) and 2.1 (211%+/-85%) times (HepG2). IGF-IR was expressed in Huh-6 and HepG2. PPP suppressed the cell number to 44%+/-11% (Huh-6) and 39%+/-5% (HepG2). LY294002 and Wortmannin suppressed the cell number to 30%+/-5% (Huh-6), 44%+/-0.4% (HepG2), 49%+/-1.0% (Huh-6) and 46%+/-1.1% (HepG2), respectively. PD98059 suppressed the cell number to 33%+/-11% for HepG2 but not for Huh-6. When cell proliferation was prohibited, many Huh-6 and HepG2 cells were dead with pyknotic or fragmented nuclei, suggesting apoptosis. IGF-II was shown to be a growth factor of hepatoblastoma via IGF-I receptor and PI3 kinase which were good candidates for target of molecular therapy.

IGF-IR: Potential Role in Antitumor Agents

As insulin-like growth factor (IGF) signaling has been recognized to play an important role in human cancer, the IGF-I receptor (IGF-IR) is currently the focus of intensive research aimed at developing novel antitumor agents. The IGF system is frequently deregulated in cancer cells by the establishment of autocrine loops involving IGF-I or -II and/or IGF-IR over-expression. Moreover, epidemiological studies have suggested a link between elevated IGF levels and the development of major human malignancies, such as breast, colon, lung and prostate cancer. Experimental therapies aimed at inhibiting IGF signaling in human tumors involve various approaches, including neutralizing antibodies and pharmacological inhibitors of IGF-IR kinase activity. Although there are numerous reports describing the antitumor activity of such agents against human cancer cell lines propagated in vitro or in experimental animals, it remains unclear how soon the existing drugs will have a demonstrable effect in patients. In this review, we will discuss the evidence implicating the IGF signaling system in the pathology of human cancer and the various strategies that have so far been developed to target the IGF-IR.