Insulin-like growth factor-1 receptor and ErbB kinase inhibitor combinations block proliferation and induce apoptosis through cyclin D1 reduction and Bax activation

The insulin-like growth factor-1 receptor (IGF-1R) and ErbB family of receptors are receptor tyrosine kinases that play important roles in cancer. Lack of response and resistance to therapies targeting ErbB receptors occur and are often associated with activation of the IGF-1R pathway. Combinations of agents that inhibit IGF-1R and ErbB receptors have been shown to synergistically block cancer cell proliferation and xenograft tumor growth. To determine the mechanism by which targeting both IGF-1R and ErbB receptors causes synergistic effects on cell growth and survival, we investigated the effects of combinations of selective IGF-1R and ErbB kinase inhibitors on proliferative and apoptotic signaling. We identified A431 squamous cell carcinoma cells as most sensitive to combinations of ErbB and IGF-1R inhibitors. The inhibitor combinations resulted in not only blockade of A431 cell proliferation, but also induced apoptosis, which was not seen with either agent alone. Upon examining phosphorylation states and expression levels of proteins in the IGF-1R and ErbB signaling pathways, we found a correlation between the ability of combinations to inhibit proliferation and to decrease levels of phosphorylated Akt and cyclin D1. In addition, the massive cell death induced by combined IGF-1R/ErbB inhibition was associated with Mcl-1 reduction and Bax activation. Thus, targeting both IGF-1R and ErbB receptors simultaneously results in cell cycle arrest and apoptosis through combined effects on Akt, cyclin D1, and Bax activation.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.     

Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.     

Beta-arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression

Beta-arrestin1, which regulates many aspects of seven transmembrane receptor (7TMR) biology, has also been shown to serve as an adaptor, which brings Mdm2, an E3 ubiquitin ligase to the insulin-like growth factor-1 receptor (IGF-1R), leading to its proteasome-dependent destruction. Here we demonstrate that IGF-1R stimulation also leads to ubiquitination of beta-arrestin1, which regulates vesicular trafficking and activation of ERK1/2. This beta-arrestin1-dependent ERK activity can occur even when the classical tyrosine kinase signaling is impaired. siRNA-mediated suppression of beta-arrestin1 in human melanoma cells ablates IGF-1-stimulated ERK and prolongs the G1 phase of the cell cycle. These data suggest that beta-arrestin-dependent ERK signaling by the IGF-1R regulates cell cycle progression and may thus be an important regulator of the growth of normal and malignant cells.     

Age-related differences in insulin-like growth factor-1 receptor signaling regulates Akt/FOXO3a and ERK/Fos pathways in vascular smooth muscle cells

Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin-like growth factor-1 receptor (IGF-1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF-1R signaling changes with age in VSMC is not known. We previously found age-related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the upstream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age-related vascular pathology similar to humans, we compared IGF-1R expression in early passages of VSMC and found a constitutive activation of IGF-1R in VSMC from old compared to young rats, including IGF-1R expression and its tyrosine kinase activity. The link between IGF-1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF-1R with IGF-1 in young cells and attenuation of IGF-1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF-1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip-1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a-targets based on FOXO3a-siRNA treatment. Furthermore, IGF-1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF-1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis.     

The inhibition of angiogenesis and tumor growth by denbinobin is associated with the blocking of insulin-like growth factor-1 receptor signaling

Denbinobin, which is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla, has been demonstrated to display antitumor activity. Recent reports suggest that the enhanced activity of insulin-like growth factor-1 receptor (IGF-1R) is closely associated with tumor angiogenesis and growth. This study aims at investigating the roles of denbinobin in suppressing these effects and at further elucidating the underlying molecular mechanisms. In the present study, we used an in vivo xenograft model antitumor and the Matrigel implant assays to show that denbinobin suppresses lung adenocarcinoma A549 growth and microvessel formation. Additionally, crystal violet and capillary-like tube formation assays indicated that denbinobin selectively inhibits insulin-like growth factor-1 (IGF-1)-induced proliferation (GI50=1.3×10⁻⁸ M) and tube formation of human umbilical vascular endothelial cells (HUVECs) without influencing the effect of epidermal growth factor; vascular endothelial growth factor and basic fibroblast growth factor. Furthermore, denbinobin inhibited the IGF-1-induced migration of HUVECs in a concentration-dependent fashion. Western blotting and immunoprecipitation demonstrated that denbinobin causes more efficient inhibition of IGF-1-induced activation of IGF-1R and its downstream signaling targets, including , extracellular signal-regulated kinase, Akt, mTOR, p70S6K, 4EBP and cyclin D1. All of our results provide evidences that denbinobin suppresses the activation of IGF-1R and its downstream signaling pathway, which leads to the inhibition of angiogenesis. Our findings suggest that denbinobin may be a novel IGF-1R kinase inhibitor and has potential therapeutic abilities for angiogenesis-related diseases such as cancer.     

Gefitinib resistance in HCC mahlavu cells: upregulation of CD133 expression, activation of IGF-1R signaling pathway, and enhancement of IGF-1R nuclear translocation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer which accounts for more than half million deaths annually worldwide. While the incidence of HCC is still on the rise, options of treatment are limited and the overall survival rate is poor. The acquisition of cancer drug resistance remains one of the key hurdles to successful treatment. Clearly, a thorough understanding of the underlying mechanisms is needed for new strategies to design novel treatments and/or to improve the current therapies. In the present study, we examined the expression of cancer stem cell (CSC) marker CD133, the activation of insulin-like growth factor 1 receptor (IGF-1R) signaling, and the nuclear translocation of IGF-1R in HCC Mahlavu cells under the treatment of gefitinib, a cancer drug that inhibits epidermal growth factor receptor (EGFR) pathway. Our results demonstrated that Mahlavu cells exhibited strong gefitinib resistance and the CD133 expression level was dramatically increased (from 3.88% to 32%) after drug treatment. In addition, the gefitinib treated cells displayed increased levels of phosphorylation in IGF-1R and Akt, indicating the intensified activation of this cancer-associated signaling pathway. Moreover, we revealed that IGF-1R underwent nuclear translocation in gefitinib treated cells using confocal microscopy. The IGF-1R nuclear translocation was enhanced under gefitinib treatment and appeared in a dose-dependent manner. Our findings suggest that increased IGF-1R nuclear translocation after gefitinib treatment may contribute to the drug resistance and IGF1-R activation, which might also associate with the upregulation of CD133 expression.     

Multiple Signaling Pathways of the Insulin-Like Growth Factor 1 Receptor in Protection from Apoptosis

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.     

Transactivation of the EGF receptor mediates IGF-1-stimulated shc phosphorylation and ERK1/2 activation in COS-7 cells

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.     

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.     

A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor

Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase.     

RACK1 is an insulin-like growth factor 1 (IGF-1) receptor-interacting protein that can regulate IGF-1-mediated Akt activation and protection from cell death

The insulin receptor and insulin-like growth factor 1 receptor (IGF-1R), activated by their ligands, control metabolism, cell survival, and proliferation. Although the signaling pathways activated by these receptors are well characterized, regulation of their activity is poorly understood. To identify regulatory proteins we undertook a two-hybrid screen using the IGF-1R beta-chain as bait. This screen identified Receptor for Activated C Kinases (RACK1) as an IGF-1R-interacting protein. RACK1 also interacted with the IGF-1R in fibroblasts and MCF-7 cells and with endogenous insulin receptor in COS cells. Interaction with the IGF-1R did not require tyrosine kinase activity or receptor autophosphorylation but did require serine 1248 in the C terminus. Overexpression of RACK1 in either R+ fibroblasts or MCF-7 cells inhibited IGF-1-induced phosphorylation of Akt, whereas it enhanced phosphorylation of Erks and Jnks. Src, the p85 subunit of phosphatidylinositol 3-kinase, and SHP-2 were all associated with RACK1 in these cells. Interestingly, the proliferation of MCF-7 cells was enhanced by overexpression of RACK1, whereas IGF-1-mediated protection from etoposide killing was greatly reduced. Altogether the data indicate that RACK1 is an IGF-1R-interacting protein that can modulate receptor signaling and suggest that RACK1 has a particular role in regulating Akt activation and cell survival.     

Inhibitors of insulin-like growth factor-1 receptor tyrosine kinase are preferentially cytotoxic to nutrient-deprived pancreatic cancer cells

Chronic deprivation of nutrients is rare in normal tissues, however large areas of tumor are nutrient-starved and hypoxic due to a disorganized vascular system. Some cancers show an inherent ability to tolerate severe growth conditions. Therefore, we screened chemical compounds to identify cytotoxic agents that function preferentially in nutrient-deprived conditions. We found that AG1024, a specific inhibitor of insulin-like growth factor-1 receptor tyrosine kinase (IGF-1R), showed preferential cytotoxicity to human pancreatic cancer cells in nutrient-deprived conditions relative to cells in nutrient-sufficient conditions. The cytotoxicity of I-OMe-AG538 (another specific inhibitor of IGF-1R kinase) was also enhanced in nutrient-deprived cells. In addition, AG1024 and I-OMe-AG538 potently inhibited IGF-1R activation to nutrient-deprived cells. In contrast, conventional chemotherapeutic drugs, as well as inhibitors of PDGFR and EGFR kinases, elicited weak cytotoxicity. These data indicate that nutrient-deprived human pancreatic cancer cells have increased sensitivity to inhibition of IGF-1R activation. IGF-1R inhibitors offer a promising strategy for anticancer therapeutic approaches that are oriented toward tumor microenvironment.     

Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.     

Development of new insulin-like growth factor-1 receptor kinase inhibitors using catechol mimics

Because insulin-like growth factor-1 (IGF-1) and its receptor play a pivotal role in many cancers, it is an attractive target for the design of inhibitors. Here we present a new family of bioisostere inhibitors, based on the structure of AG 538. AG 538 is a substrate-competitive inhibitor of the IGF-1 receptor (IGF-1R), with an IC50 = 61 nM in a cell-free kinase assay (Blum, G., Gazit, A., and Levitzki, A. (2000) Biochemistry 39, 15705-15712). AG 538 is a low molecular weight compound containing two catechol rings, which are sensitive to oxidation in cells. We have therefore prepared and examined catechol bioisosteres of AG 538. These AG 538 bioisosteres possess similar biological properties to AG 538; they inhibit IGF-1R by a substrate-competitive mechanism and are non-competitive vis à vis ATP. They inhibit IGF-1R kinase activity in the sub-micromolar concentration range in cell-free assays. IGF-1 induced IGF-1R autophosphorylation; IRS-1 phosphorylation and protein kinase B activation are inhibited at a low micromolar concentration range when applied to intact cells. These inhibitors also block the formation of colonies in soft agar by prostate and breast cancer cells. The ability to replace catechol groups with a moiety that is more stable in cells may aid in developing non-catechol-containing substrate-competitive inhibitors targeted toward IGF-1R and possibly against other protein-tyrosine kinases.     

Cancer-associated fibroblasts promote epithelial-mesenchymal transition and EGFR-TKI resistance of non-small cell lung cancers via HGF/IGF-1/ANXA2 signaling

The involvement of the tumor stromal cells in acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism remains unclear. In the present study, we investigated the role and mechanism underlying Cancer-associated fibroblasts (CAFs) in TKI resistance of NSCLCs. In vitro and in vivo experiments showed that HCC827 and PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR-TKI gefitinib when cultured with CAFs isolated from NSCLC tissues. Moreover, we showed that CAFs could induce epithelial-mesenchymal transition (EMT) phenotype of HCC827 and PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition markers. Using proteomics-based method, we identified that CAFs significantly increased the expression of the Annexin A2 (ANXA2). More importantly, knockdown of ANXA2 completely reversed EMT phenotype and gefitinib resistance induced by CAFs. Furthermore, we found that CAFs increased the expression and phosphorylation of ANXA2 by secretion of growth factors HGF and IGF-1 and by activation of the corresponding receptors c-met and IGF-1R. Dual inhibition of HGF/c-met and IGF-1/IGF-1R pathways could significantly suppress ANXA2, and markedly reduced CAFs-induced EMT and gefitinib resistance. Taken together, these findings indicate that CAFs promote EGFR-TKIs resistance through HGF/IGF-1/ANXA2/EMT signaling and may be an ideal therapeutic target in NSCLCs with EGFR-activating mutations.