IGF-1R Tyrosine Kinase Research Articles

Selective Inhibition of Glycogen Synthase Kinase-3 by Facilitators of Insulin Response Promotes Proliferation and Survival in Multiple Myeloma Cells.

There is ample evidence for a central role of IGF-1R in multiple myeloma (MM) cell survival and resistance to apoptosis. Recently, we have demonstrated the use of inhibitors of the IGF-1R tyrosine kinase (RTK) activity as a promising therapeutic strategy in MM in vitro and in vivo (Stromberg et al Blood 2006,107:669–78, Menu et al Int J Cancer 2007,121:1857–61). In the present investigation, we have examined the role of glycogen synthase kinase-3 (GSK3), a target of the IGF-1R/PI 3-K/AKT pathway, in proliferation and apoptosis of MM. Unlike most kinases GSK3 is constitutively active, but may be inhibited by e.g. insulin, EGF and FGF. GSK3 is also a converging point of the Wnt and the IGF-1R/PI 3-K/AKT survival pathway. Reduction of GSK3 activity resulted from its phosphorylation at the regulatory residues Ser21 (α isoform) and Ser9 (β isoform) catalysed by AKT. GSK3 inhibitors LiCl, AR-A014418, the maleimides SB216763 and SB41528, and the highly selective aminopyrimidine derivatives CT98014 and CT99021 (Novartis, Emeryville, CA), were investigated for their effects on MM cells when growth and survival were challenged by cytotoxic stress and/or interference with different signaling proteins. Apoptosis induced by serum starvation was reduced by CT99021, an effect comparable to the survival-promoting effect of IGF-1. The selective GSK3 inhibitor also restored survival in MM cells induced by glucocorticoids and combinations increasing the susceptibility to apoptosis using PI 3-K inhibitors LY294002, the IGF-1R antagonist αIR3, the mTOR inhibitor rapamycin and the MEK inhibitor PD98059. In addition to the anti-apoptotic effect, CT99021 was also demonstrated to counteract G0/G1 accumulation induced by serum starvation, LY294002, αIR3 and rapamycin. However, accumulation of cells in the G2/M phase induced by doxorubicin and melphalan, were not counteracted by GSK3 inhibitors. Substrates of GSK3 most prominently include beta-catenin and the IκB-analog Bcl-3, both targeted for degradation upon GSK3 activation. Selective GSK inhibition restituted protein levels of beta-catenin, Bcl-3, p21 cip1/WAF1, p27kip1 and cyclin B1, D2 and E corroborating with the effects on survival and proliferation. These data demonstrate that targeting of the GSK3 using highly selective inhibitors is associated with potent anti-apoptotic effects and propagation through the cell cycle in MM.

HoxA9 Induces Insulin-Like Growth Factor-1 Receptor (IGF-1R) Expression in Acute Leukemia.

The homeobox (Hox) gene family encodes a group of transcription factors preferentially expressed during embryonic development and hematopoiesis. Overexpression of Hox genes is frequently associated with acute leukemia. HoxA9 is the most commonly overexpressed Hox gene in acute leukemia and forms part of a genetic signature that characterizes AML and ALL bearing MLL translocations. Despite frequent involvement in leukemia, pathways downstream of HoxA9 overexpression are not well characterized. In previous studies, we found that a B-lineage cell line bearing the MLL/AF4 oncogene, but lacking HoxA9 expression, retained B-lineage developmental capacity and normal apoptotic sensitivity to growth factor/stromal cell withdrawal. This led to the hypothesis that HoxA9 regulates pathways that promote leukemic cell proliferation and apoptotic resistance. This hypothesis was tested herein, by generating a conditional model of HoxA9 activity in the stromal cell dependent, HoxA9 negative pre-B ALL cell line, BLIN-2. HoxA9 was fused to the hormone binding domain of the estrogen receptor and cloned into a GFP/IRES retroviral construct. This construct was used to generate stably transduced BLIN-2/HoxA9:ER cells in which HoxA9 activity could be induced through the addition of the estrogen analog 4-hydroxytamoxifen (4HT) to the media. Cell fractionation experiments revealed that the presence of 4HT resulted in stabilization of HoxA9:ER protein and its nuclear localization in BLIN-2/HoxA9:ER cells. Electrophoretic mobility shifts performed with BLIN-2/HoxA9:ER cell nuclear extracts demonstrated that the HoxA9:ER fusion protein was capable of binding to the HoxA9 consensus DNA binding sequence. Conditional HoxA9 activation in BLIN-2/HoxA9:ER cells resulted in increased proliferation in the presence and absence of stromal cell support as compared with parental BLIN-2 cells, or BLIN-2 cells transduced with an empty vector (BLIN-2/MigR1). BLIN-2/HoxA9:ER cells survived in the absence of stromal support for an additional 3 to 5 days as compared to control cells. Stimulation of HoxA9 activity resulted in induction of IGF-1R protein expression on the surface of BLIN-2/HoxA9:ER cells. Induction of IGF-1R expression was further verified by western blot and RT-PCR analysis. HoxA9-mediated proliferative and survival effects were abrogated when BLIN-2/HoxA9:ER cells were treated with an IGF-1R tyrosine kinase inhibitor (AG1024) or with an IGF-1R monoclonal antibody (A12). IGF-1R expression correlated with endogenous HoxA9 expression in a small panel of MLL/AF4 positive ALL cell lines. Treatment of the MLL/AF4 positive cell line RS4;11 with the anti-IGF-1R antibody (A12) resulted in reduced proliferation. This effect was abrogated with supraphysiologic amounts of IGF-1. siRNA knock-down of endogenous HoxA9 expression in RS4;11 cells resulted in loss of IGF-1R expression. These data indicate that HoxA9 overexpression induces IGF-1R expression and subsequently promotes leukemic cell growth. These data provide novel mechanistic insight into how HoxA9 overexpression in acute leukemia promotes proliferation and apoptotic resistance through regulation of the IGF-1R pathway.

The insulin-like growth factor 1 receptor in cancer: Old focus, new future

The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and II clinical trials. Interactions of IGF-1R with insulin receptor, however, might complicate efficiency and tolerability of such drugs. This review describes mechanisms, recent developments and potential limitations of IGF-1R antibodies and tyrosine kinase inhibitors.

Identification of sensitivity markers for BMS-536924, an inhibitor for insulin-like growth factor-1 receptor

3506 Background: Insulin-like growth factor-1 receptor (IGF-1R) signaling is an important regulator of mitogenesis, transformation to the oncogenic phenotype and anti-apoptotic effects in malignant cells. Over-expression of IGF-1R, seen in many tumors, may confer a growth advantage or drug resistance. A potent small-molecule inhibitor (BMS-536924) of IGF-1R tyrosine kinase showed anti-tumor activity in sarcoma, prostate, colon and pancreatic tumor models. One of the integral goals in the development of BMS-536924 as a cancer therapeutic is to identify molecular biomarkers predictive of response to the drug that ultimately will aid in selecting the patients who are most likely to benefit. Methods: The sensitivity (IC50) to BMS-536924 was determined for a panel of 29 pediatric sarcoma and neuroblastoma cell lines. Both microarray and LC/MS based protein profiling were utilized to analyze the baseline gene or protein expression level. Drug treatment studies were performed using two rhabdomyosarcoma cell lines, Rh41 (sensitive to BMS-536924) and Rh36 (resistant to the drug) to identify markers that are modulated by BMS-536924. Results: (1). Sixteen out of the 29 cell lines were highly sensitive to BMS-536924; candidate markers that correlated with the sensitivity to BMS-536924 were identified by gene expression and protein profiling. (2). Histological correlation was also discovered, with specific subtypes of sarcoma having a low IC50 to BMS-536924. (3). Pathway analysis noted that some major candidate markers are common key steps in the EGF-R pathway and the IGF1-R pathway. This observation of cross-talk between the two pathways led to the hypothesis of synergy with combined inhibition of both pathways. Combination studies of BMS-536924 and EGFR inhibitors were performed and synergism was observed. (4). Markers modulated by BMS-536924 in a sensitive cell line were identified. Conclusions: This work has identified candidate markers correlating to BMS-536924 sensitivity in vitro. The possible mechanism of synergistic activity of IGF1-R and EGFR inhibitors will be presented. No significant financial relationships to disclose.

The PI3-K/AKT/mTOR pathway as a target for breast cancer therapy

3511 Background: The PI3K/Akt/mTOR pathway is found dysregulated in multiple tumours, including breast cancer (BC). RAD001 is a recently developed drug which blocks mTOR. To study this compound’s anti-tumour effect and interactions with the aforementioned pathway, we set up a translational research program which integrates basic and clinical research data. Methods: The preclinal studies performed in BC cell lines and xenografts included the use of proliferation assays, western blot (WB), small interfering RNA and immunohistochemistry (IHC) analysis. The clinical studies consisted in a phase I study with escalating doses and collection of tumour biospies at different time points Results: Our first achievement was validating the antibodies used in WB and IHC and directed towards different components of the PI3K/Akt/mTOR pathway. We first reported that sensitivity to RAD001 correlated to basal levels of p-Akt. We then observed that RAD001 inhibits mTOR downstream, pS6 and eIF4G, but increases the levels of p-Akt, both in vitro and in patients’tumour biopsies. We demonstrated that knock down of the pS6 kinase mimics the effect of RAD001. Indeed, by combining the PI3K inhibitors and the genomic approch of p85 dominant negative vector, we demonstrated that RAD001 induced p-Akt is through PI3K. Besides the p-Akt induction we also found a RAD001 induction of IRS-1, the pivot mediator of the Insulin like Growth factor-1 Receptor (IGF-1R). We reported that RAD001 resulted in a prolonged activation of IRS-1 and in a sustained association with the p85 subunit of PI3K. Thus RAD001 disrupts an inhibitory mechanism impinging on the PI3K signalling, resulting in sustained activation of the IGF-1R pathway. Accordingly we demonstrated that combination of RAD001 with coumpounds directed against the IGF-1R, i.e. both IGF-1R tyrosine kinase inhibitors and monoclonal antibodies, prevented RAD001 induced p-Akt and resulted in a supradditive growth inhibitory effects both in vitro and in MCF-7 human BC derived xenografts.We are now conducting a phase I study with a monoclonal antibody directed at the IGF-1R that blocks IGF-1R signalling. Conclusions: Dual inhibition of mTOR and IGF-1R completely blocks the PI3K/Akt/mTOR pathway and provide a mechanistic-based combined approach. Clinical studies in BC patients are being planned. No significant financial relationships to disclose.

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 potentiation of curcumin on insulin-like growth factor-1 action in MCF-7 human breast carcinoma cells

Curcumin has anticarcinogenic and chemopreventive properties in a variety of experimental cancer models. Our in vitro studies have shown that curcumin inhibits cell growth and induces apoptosis in MCF-7, a human breast carcinoma cell line. The insulin-like growth factor-1 (IGF-1) system, including IGFs (IGF-1 and IGF-2), IGF-1R (IGF-1 receptor) and IGFBPs (IGF binding proteins), has been implicated to play a critical role in the development of breast cancer. The aim of the present study was to investigate whether the growth inhibitory effects of curcumin were related to changes of the IGF-1 system in MCF-7 cells. IGF-1 at 50 μg/l in serum-free medium produced maximum proliferation and minimized apoptosis. However, curcumin exhibited a potent ability to blunt IGF-1-stimulated MCF-7 cell growth and reverse the IGF-1-induced apoptosis resistance. To determine whether curcumin intervenes in IGF-1 or IGFBP-3 secretion, MCF-7 cells were incubated in serum-free medium in the presence of various concentrations of curcumin for indicated time periods. Curcumin decreased the secretion of IGF-1 with a concomitant increase of IGFBP-3 in a dose-dependent manner. Receptor tyrosine kinase assays revealed that IGF-1-stimulated IGF-1R tyrosine kinase activation was also abrogated by curcumin in a dose-dependent manner. Real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction (RFQ-RT-PCR) further revealed that curcumin suppressed IGF-1R gene expression at transcriptional level. In conclusion, the inhibition of cell growth and induction of apoptosis by curcumin in MCF-7 cells might be mediated, at least partially, by its ability to down-regulate the IGF-1 axis.

In the absence of IGF-1 signaling, IFN-gamma induces T cell apoptosis (94.14)

Abstract IFNg exerts its biological activities by interacting with its cell surface receptor (IFNgR), which consists of two binding chains (R1) and two signal transducing chains (R2).The binding of IFNg to its receptor complex activates the JAK/STAT signal transduction pathway and induces the transcription of IFNg-inducible apoptotic genes. T cells that undergo Th1 polarization or malignant transformation become resistant to the antiproliferative effects of the IFNg/STAT1 pathway.This refractoriness is mainly due to R2 downregulation, due to ligand-independent internalization within clathrin-coated pits. We have previously demonstrated that, by inducing R2 internalization, IGF1 is a critical factors in limiting the IFNg/STAT1 pathway in T cells. Thus, the blockade of its signaling in T cells could hinder their intracellular R2 trafficking and reinstate their sensitivity to IFNg/STAT1 apoptotic signaling. To address this point, a retrovirus-based approach was exploited to stably express a dominant negative form of the IGF1R (IGF1R DN) in ST4 T cell line. Expression of IGF1R DN induced R2 cell surface accumulation and reinstated ST4 cell sensitivity to the IFNg/STAT1 apoptotic pathway in vitro and in vivo. Moreover, the combined exposure to an inhibitor of IGF1R tyrosine kinase activity (picropodophyllin, PPP) and IFNg induced STAT1-dependent apoptosis of three T cell lines (Jurkat ST4 and PF382).These results could be used in the elaboration of new therapeutic approaches based on IGF1 signaling blockade to overcome the resistance of malignant or autoreactive T cells to the apoptotic effect of IFNg.

Enhancement of Sensitivity of Human Lung Cancer Cell Line to TRAIL and Gefitinib by IGF-1R Blockade

Background: TRAIL is a cytokine that selectively induces apoptosis in various cancer cell lines. Gefitinib is new targeted drug applied in lung cancer that selectively inhibits EGFR tyrosine kinase. However, lung cancers have shown an initial or acquired resistance to these drugs. This study examined the effect of IGF-1R and its blockade on enhancing the sensitivity of lung cancer cell lines to TRAIL and gefitinib. Methods: Two lung cancer cell lines were used in this study. NCI H460 is very sensitive to TRAIL and gefitinib. On the other hand, A549 shows moderate resistance to TRAIL and gefitinib. The IGF-1R blockade was performed using adenoviruses expressing the dominant negative IGF-1R and shRNA to IGF-1R and AG1024 (IGF-1R tyrosine kinase inhibitor). Results: The adenovirus expressing dominant negative IGF-1R(950st) induced the increased expression of defective IGF-1R on the lung cancer cell surface, and the adenovirus-shIGF-1R effectively decreased the level of IGF-1R expression on cell surface. The genetic blockade of IGF-1R by the adenovirus-dnIGF-1R and AG1024 increased the sensitivity of A549 cells to TRAIL. The reduction of IGF-1R by transduction with ad-shIGF-1R also increased the sensitivity of the A549 cells to gefitinib. Conclusion: The blockade of IGF-1R through various mechanisms increased the sensitivity of the lung cancer cell line that was resistant to TRAIL and gefitinib. However, further studies using other cell lines showing acquired resistance as well as in vivo animal experiments will be needed.

BCR-ABL Activates IGF-1 Expression and Signaling in Chronic Myelogenous Leukemia Blast Crisis Cell Lines.

CML blast crisis is characterized by the continued presence of the Philadelphia chromosome, which expresses the P210 BCR-ABL fusion protein, and the acquisition of additional molecular and chromosomal alterations. Evolution from CML chronic phase to blast crisis is associated with loss of heterozygosity at chromosome region 1p36, which contains the putative tumor suppressor RIZ1. We found that RIZ1 expression decreases during progression from CML chronic phase to myeloid blast crisis and that forced RIZ1 expression in CML blast crisis (CML-BC) cell lines decreases proliferation, increases apoptosis, and enhances differentiation. Furthermore, we found that RIZ1 represses IGF-1 autocrine production and blocks the activation of the IGF-1 receptor, AKT, and ERK signaling pathways in CML-BC cell lines thereby implicating RIZ1 control of the IGF-1 pathway in the regulation of these phenotypes. As BCR-ABL induces a growth factor independent phenotype due to the activation of autocrine growth factor production, we analyzed IGF-1 expression in CML-BC cell lines following exposure to imatinib. We found that imatinib treatment reduced IGF-1 mRNA and extracellular IGF-1 suggesting that RIZ1 may suppress blast crisis by counteracting the ability of BCR-ABL activate RIZ1 regulated genes. We characterized the signaling pathways used by BCR-ABL to activate IGF-1 expression and found that the HCK inhibitor PP2 and STAT5b shRNA reduced IGF-1 expression whereas the JAK2 inhibitor AG490 increased IGF-1 expression. These results suggest that BCR-ABL activates HCK, which in turn activates STAT5b and induces IGF-1 expression. To confirm the importance of BCR-ABL induced IGF-1 signaling to CML-BC cell line viability, proliferation and apoptosis, we characterized these cellular phenotypes in the presence of IGF-1 receptor blocking antibody and the IGF-1 receptor tyrosine kinase inhibitor AG1024. Blockage of autocrine IGF-1 signaling in CML-BC cell lines using an anti-IGF-1 receptor blocking antibody reduced cell viability and decreased proliferation. AG1024 treatment of CML-BC cell lines decreased proliferation and induced apoptosis. Together these results demonstrate that RIZ1 counteracts the ability of BCR-ABL to induce IGF-1 signaling in CML-BC cell lines, which influences cell proliferation, apoptosis, and differentiation. Our findings highlight RIZ-1 and IGF-1 signaling pathways as potential therapeutic targets for treating CML blast crisis.

Ionizing radiation activates IGF-1R triggering a cytoprotective signaling by interfering with Ku-DNA binding and by modulating Ku86 expression via a p38 kinase-dependent mechanism

Ionizing radiation exposure results in the activation of several tyrosine kinase receptors that participate in radiation-induced DNA damage response and radioresistance. We previously showed that insulin-like growth factor 1 receptor (IGF-1R) inhibition enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) cells. In this paper, we demonstrate that in U1810 NSCLC cells gamma-radiation activates IGF-1R within 10 min, with a maximal activation effect 2 h post-irradiation. Impairment of IGF-1R tyrosine kinase activity enhances human lung cancer cells radiosensitivity by a mechanism that involves phosphatidylinositol 3-kinase (PI3-K) and p38 kinase. In an active form, IGF-1R binds and activates p38 kinase, promoting receptor signaling. Conversely, inhibition of IGF-1R phosphorylation results in IGF-1R/p38 complex disruption and p38 kinase inactivation. We have also demonstrated that in insulin-like growth factor-1-stimulated cells, Ku-DNA-binding activation is induced by ionizing radiation within 4 h, reaches a maximum level at 12 h and remains active up to 72 h. Blockade of IGF-1R activity or its downstream signaling through p38 kinase induces a decrease in radiation-mediated Ku-DNA-binding activation and downregulates the level of Ku86, without affecting Ku70 expression in the nucleus of U1810 cells. The IGF-1R signaling via PI3-K does not interfere with the p38 signaling, the Ku-DNA-binding activity or the level of Ku86. Our present study demonstrates for the first time that ionizing radiation activates IGF-1R. Inhibition of IGF-1R signaling via p38 kinase induces radiosensitivity by a novel mechanism involving nuclear Ku86.

Insulin-like growth factor-1 receptor and ligand targeting in head and neck squamous cell carcinoma

IGF-1 receptor (IGF-1R) signaling is associated with increased tumorigenesis of epithelial cancers. In light of recent epidemiological studies correlating high circulating levels of IGF-1 with increased risk of second primary tumors (SPTs) of the head and neck, we examined IGF system and epidermal growth factor receptor (EGFR) expression in human head and neck squamous cell carcinoma (HNSCC) matched pairs and a cross-section of HNSCC cell lines. Employing the latter, we demonstrated that IGF-1 stimulated S-phase transition in a PI 3-K/Akt and Erk-dependent manner in 5 of 8 cell lines, with Erk activation being dependent upon EGFR kinase activity. IGF-1 stimulated thymidine incorporation was inhibited by treatment with IGFBP-3, the IGF-1R tyrosine kinase inhibitor NVP-AEW541, or the EGFR tyrosine kinase inhibitor AG1478. Combining IGFBP-3 with NVP-AEW541 or AG1478 abrogated IGF-1 responses at 10-fold lower doses than either compound alone. These results demonstrate the potential for co-targeting the IGF system and EGFR in HNSCC.

IGF-1R tyrosine kinase expression and dependency in clones of IGF-1R knockout cells (R−)

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R−) represent a useful tool for molecular mapping of biological properties of the receptor. R− cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R− cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R− (R−s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R−s revealed expression of a 90 kDa protein being reactive to IGF-1R β-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R−s survival. Taken together, our study suggests that clones of R− express IGF-1R activity and dependency, which in turn may explain that R− can undergo spontaneous transformation.

Paradoxical effects of the phage display-derived peptide antagonist IGF-F1-1 on insulin-like growth factor-1 receptor signaling

The insulin-like growth factor binding proteins (IGFBPs) represent a unique class of IGF antagonists regulating the bioavailability of the IGFs extracellularly. Accordingly, they represent an important class of proteins for cancer therapeutics and chemoprevention. IGF-F1-1 is a cyclic hexadecapeptide identified by high throughput phage display that binds to the IGFBP-binding domain on IGF-1. It acts as an IGFBP-mimetic, capable of inhibiting IGF-1 binding to the IGFBPs. To further examine the utility of IGF-F1-1 as an IGF-1 antagonist we tested its ability to inhibit IGFBP-2 and IGFBP-3 binding to IGF-1, 125I-IGF-1 binding to IGF-1Rs and to block IGF-1 induced Akt activation, cell cycle changes and [ 3H]thymidine incorporation in MCF-7 cells. These biological activities were inhibited by treatment with IGFBP-2, wortmannin or the IGF-1R tyrosine kinase inhibitor, NVP-AEW541, but not by IGF-F1-1. Our findings confirm previous studies indicating that IGF-F1-1 is a weak antagonist of IGF-1 binding to the IGFBPs and the IGF-1R and suggest that it does not effectively inhibit downstream events stimulated by IGF-1. We further demonstrated that IGF-F1-1 treatment of MCF-7 cells results in the paradoxical activation of Akt, S-phase transition and [ 3H]thymidine incorporation. These results suggest that IGF-F1-1 is a weak agonist, exhibiting mitogenic actions. IGF-F1-1 may act in conjunction with IGF-1 at the IGF-1R or independently of IGF-1 at the IGF-1R or another receptor.

Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are mandatory. Most HCCs express insulin-like growth factors and their receptors (IGF-R). As IGF-1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the antineoplastic effects of inhibiting IGF-1R signaling in HCC cells by the novel IGF-1R tyrosine kinase inhibitor NVP-AEW541. Methods and results NVP-AEW541 induced a time- and dose-dependent growth inhibition in the human hepatoblastoma and hepatocellular carcinoma cell lines SK-Hep-1, Hep-3B, Hep-G2 and Huh-7. Measurement of LDH-release showed that the antineoplastic effect of NVP-AEW541 was not due to cytotoxicity. Instead NVP-AEW541 induced apoptosis as evidenced by both caspase-3 and -8 activation as well as by apoptosis-specific morphological and mitochondrial changes. In addition, nuclear degradation was monitored by DNA-laddering. NVP-AEW541-treatment suppressed the expression of the antiapoptotic proteins Bcl-2 and survivin, while the expression of the proapoptotic protein BAX was stimulated in a dose-dependent manner. Moreover, NVP-AEW541 arrested the cell cycle at the G1/S checkpoint. When NVP-AEW541 was combined with cytotoxic chemotherapy or with a specific epidermal growth factor receptor antibody additive antiproliferative effects were observed. Interpretation Inhibition of IGF-1R tyrosine kinase (IGF-1R-TK) by NVP-AEW541 induces growth inhibition, apoptosis and cell cycle arrest in human HCC cell lines without accompanying cytotoxicity. Thus, IGF-1R-TK inhibition may be a promising novel treatment approach in HCC.

The insulin-like growth factor receptor 1 is a promising target for novel treatment approaches in neuroendocrine gastrointestinal tumours

Gastrointestinal neuroendocrine tumours (NET) represent a heterogeneous tumour entity. The anti-neoplastic therapy of advanced NET disease is still unsatisfactory and innovative therapeutic approaches are needed. As NET frequently express insulin-like growth factors (IGFs) and their receptors (IGFR), known to promote survival, oncogenic transformation, tumour growth and spreading, the inhibition of the IGF/IGF-receptor system may offer possibilities for novel targeted treatment strategies of NET. Here, we studied the anti-neoplastic effects of an inhibition of the IGF-I receptor (IGF-1R) signalling in NET cells by the novel IGF-1R tyrosine kinase (TK) inhibitor NVP-AEW541, whose anti-neoplastic potency has not yet been tested in NET disease. Using two human NET cell lines with different growth characteristics, we demonstrated that NVP-AEW541 dose-dependently inhibited the proliferation of NET cells by inducing apoptosis and cell cycle arrest. Anti-neoplastic effects of NVP-AEW541 were also detected in primary cultures of human neuroendocrine gastrointestinal tumours. Apoptosis was characterized by activation of the apoptotic key enzyme, caspase-3, as well as by detection of changes in the expression of the pro- and anti-apoptotic proteins, BAX and Bcl-2, after NVP-AEW541 treatment. Cell cycle was arrested at the G1/S checkpoint. The anti-neoplastic effects of NVP-AEW541 involved the inactivation of ERK1/2. Induction of immediate cytotoxicity did not account for the anti-neoplastic effects of NVP-AEW541, as shown by measurement of lactate dehydrogenase release. Moreover, additive anti-neoplastic effects were observed when NVP-AEW541 was combined with cytostatics such as doxorubicin or the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin. This is the first report on the induction of apoptosis and cell cycle arrest by the IGF-1R-TK inhibitor, NVP-AEW541, in NET cells. The inhibition of the IGF/IGFR system appears to be a promising novel approach for future treatment strategies of NET disease.

IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells

AbstractEmerging evidence suggests the insulin-like growth factor-1 receptor (IGF-1R) to be an important mediator of tumor-cell survival and resistance to cytotoxic therapy in multiple myeloma (MM). Recently, members of the cyclolignan family have been shown to selectively inhibit the receptor tyrosine kinase (RTK) activity of the IGF-1R β-chain. The effects of the cyclolignan picropodophyllin (PPP) were studied in vitro using a panel of 13 MM cell lines and freshly purified tumor cells from 10 patients with MM. PPP clearly inhibited growth in all MM cell lines and primary MM samples cultured in the presence or absence of bone marrow stromal cells. PPP induced a profound accumulation of cells in the G2/M-phase and an increased apoptosis. Importantly, IGF-1, IGF-2, insulin, or IL-6 did not reduce the inhibitory effects of PPP. As demonstrated by in vitro kinase assays, PPP down-regulated the IGF-1 RTK activity without inhibiting the insulin RTK activity. This conferred decreased phosphorylation of Erk1/2 and reduced cyclin dependent kinase (CDK1) activity. In addition, the expression of mcl-1 and survivin was reduced. Taken together, we suggest that interfering with the IGF-1 RTK by using the cyclolignan PPP offers a novel and selective therapeutic strategy for MM.

Inhibiting the IGF-1 receptor tyrosine kinase with the cyclolignan PPP: an in vitro and in vivo study in the 5T33MM mouse model

AbstractInsulin-like growth factor 1 (IGF-1) plays a pleiotropic role in multiple myeloma (MM), that is, in survival, proliferation, chemotaxis, and angiogenesis. Strategies targeting the IGF-1 receptor (IGF-1R) may therefore be important to develop efficient anti-MM agents. In this work we investigated the effect of an IGF-1R tyrosine kinase (IGF-1RTK) inhibitor (picropodophyllin or PPP) in the 5T33MM mouse model. In vitro data showed that PPP reduced IGF-1R autophosphorylation and downstream ERK activation, leading to inhibition of IGF-1–stimulated proliferation and vascular endothelial growth factor (VEGF) secretion of MM cells. In an in vivo study, PPP reduced the bone marrow tumor burden and serum paraprotein in 5T33MM mice by 77% and 90%, respectively, compared to vehicle-treated animals. Angiogenesis was assessed by quantifying the microvessel density on CD31-stained paraffin sections and this was reduced by 60% in the PPP-treated group. In a separate survival experiment, Kaplan-Meier analysis demonstrated a significant increase in survival in PPP-treated 5T33MM animals compared to the vehicle controls (28 versus 18 days). These data suggest that the IGF-1RTK inhibitor PPP possesses a marked antitumor activity and strongly points to the possibility of using IGF-1R inhibitors in the treatment of MM.

Specific inhibition of the IGFR tyrosine kinase as a novel approach for targeted therapy in neuroendocrine gastrointestinal tumor disease

Background & Aims: Gastrointestinal neuroendocrine tumors (NET) represent a heterogeneous tumor entity. The antineoplastic treatment options of advanced neuroendocrine cancer disease are unsatisfactory. Hence, innovative therapeutic approaches are urgently needed. As NET cells frequently express insulin-like growth factors and their receptors (IGFR) which are known to promote survival, oncogenic transformation and tumor growth and spread, the IGF/IGFR system qualifies for novel targeted treatment approaches in NET disease. Here, we studied the antineoplastic effects of an inhibition of IGF–1 receptor (IGF–1R) signaling in NET cells by a novel IGF–1R tyrosine kinase (TK) inhibitor.

The insulin-like growth factor receptor is a promising target for innovative treatment approaches of hepatocellular cancer

Background & Aims: Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death worldwide. Due to very poor 5-year-survival new therapeutic approaches are uregently needed. Most HCCs express insulin-like growth factors and their receptors (IGFR). As IGF–1R-mediated signaling promotes survival, oncogenic transformation and tumor growth and spread, it represents a potential target for innovative treatment strategies of HCC. Here we studied the effects of a novel IGF–1R tyrosine kinase inhibitor in human HCC cells and focused on the antineoplastic efficacy of IGF-receptor inhibition alone as well as in combination with other growth factor receptor agents or cytostatics.