IGF Ligands Research Articles

Migratory capabilities of human umbilical cord blood-derived neural stem cells (HUCB-NSC) in vitro.

Many types of neural progenitors from various sources have been evaluated for therapy of CNS disorders. Prerequisite for success in cell therapy is the ability for transplanted cells to reach appropriate target such as stroke lesion. We have established neural stem cell line from human umbilical cord blood neural stem (HUCB-NSC). In the present study we evaluated migratory capabilities of cells (HUCB-NSC) and the presence of various migration-related receptors. Immunocytochemical analysis revealed abundant expression of CXCR4, PDGFR-alpha, PDGFR-beta, c-Met, VEGFR, IGF-1R and PSA-NCAM receptors in non-adherent population of HUCB-NSC cultured in serum free (SF) conditions (SF cells). Biological activity of selected receptors was confirmed by HUCB-NSC in vitro migration towards SDF-1 and IGF-1 ligands. Additionally, rat brain-derived homogenates have been assessed for their chemoattractive activity of HUCB-NSC. Our experiments unveiled that brain tissue was more attracted for HUCB-NSC than single ligands with higher potency of injured than intact brain. Moreover, adherent HUCB-NSC cultured in low serum (LS) conditions (LS cells) were employed to investigate an impact of different extracellular matrix (ECM) proteins on cell motility. It turned out that laminin provided most permissive microenvironment for cell migration, followed by fibronectin and gelatin. Unexpected nuclear localization of CXCR4 in SF cells prompted us to characterize intracellular pattern of this expression in relation to developmental stage of cells cultured in different conditions. Continuous culture of LS cells revealed cytoplasmatic pattern of CXCR4 expression while HUCB-NSC cultured in high serum conditions (HS cells) resulted in gradual translocation of CXCR4 from nucleus to cytoplasm and then to arising processes. Terminal differentiation of HUCB-NSC was followed by CXCR4 expression decline.

The Conserved Clusterin Gene Is Expressed in the Developing Choroid Plexus Under the Regulation of Notch But Not IGF Signaling in Zebrafish

Recent genome-wide association studies have implicated the clusterin gene in the etiology of Alzheimer's disease. The expression and function of clusterin in the developing brain, however, is poorly understood. In this study, we have characterized the zebrafish clusterin gene and determined its structural conservation, developmental expression, and physiological regulation. The structure of the zebrafish clusterin gene and protein is similar to its human orthologue. Biochemical assays show that zebrafish Clusterin is a secreted protein that cannot bind IGFs. In adult zebrafish, clusterin mRNA is detected in many tissues. In early development, clusterin mRNA becomes detectable at 12 h postfertilization, and its levels gradually increase thereafter. In situ hybridization analysis indicates that clusterin mRNA is specifically expressed in the developing diencephalic and myelencephalic choroid plexus. Among various stresses tested, heat shock, but not hypoxic or ionic stresses, increases the levels of clusterin mRNA. Inhibition of the IGF-I receptor-mediated signaling or overexpression of IGF ligands did not change clusterin mRNA levels. In comparison, inhibition or targeted knockdown of Notch signaling significantly increased clusterin mRNA expression in choroid plexus. These results suggest that clusterin is a marker of choroid plexus in zebrafish, and its expression in the developing choroid plexus is under the regulation of Notch but not IGF signaling.

Targeting the insulin-like growth factor pathway in rhabdomyosarcomas: rationale and future perspectives.

Rhabdomyosarcomas (RMS) are a heterogeneous group of tumors that share features of skeletal myogenesis and represent the most common pediatric soft tissue sarcoma. Even though significant advances have been achieved in RMS treatment, prognosis remains very poor for many patients. Several elements of the Insulin-like Growth Factor (IGF) pathway are involved in sarcomas, including RMS. The IGF2 ligand is highly expressed in most, if not all, RMS, and frequent overexpression of the receptor IGF1R is also found. This is confirmed here through mining expression profiling data of a large series of RMS samples. IGF signaling is implicated in the genesis, growth, proliferation, and metastasis of RMS. Blockade of this pathway is therefore a potential therapeutic strategy for the treatment of RMS. In this paper we examine the biological rationale for targeting the IGF pathway in RMS as well as the current associated preclinical and clinical experience.

High-Level IGF1R Expression Is Required for Leukemia Stem Cell Activity In T-ALL and Is Supported by Notch Signaling.

AbstractAbstract 3649T-cell acute lymphoblastic leukemia (T-ALL) is a lethal cancer of immature T cells that often shows aberrant activation of NOTCH1 and PI3K/Akt pathways. Although PI3K/Akt signaling is potentiated by PTEN mutations which occur in up to 20% of cases, upstream signals which initiate PI3K/Akt activation remain unclear. IGF1R is a receptor tyrosine kinase upstream of PI3K/Akt and Ras/Raf/MAPK signaling pathways with known roles in cancer cell growth and is expressed broadly in primary human T-ALL cells and established cell lines. We thus considered that IGF1R may represent a biologically relevant upstream activator of the PI3K/Akt pathway in T-ALL. Consistent with this idea, we demonstrate that treatment of human and mouse T-ALL cells with small molecule IGF1R kinase inhibitors and IGF1R blocking antibodies resulted in growth arrest of bulk cells. As well, genetic deletion of IGF1R by inducible Cre expression in mouse T-cell leukemias also leads to growth arrest and apoptosis of bulk cells.IGF1R and PI3K/Akt/mTOR signaling have also been described to promote self-renewal of normal and cancer stem cells, and thus we addressed whether IGF1R may play a role in leukemia stem cell (LSC) activity in T-ALL. Since outgrowth of bulk leukemia cells is required to detect LSC activity in vivo, we were not able to address IGF1R function in LSCs by genetic deletion; however, we elected to take advantage of a well-characterized hypomorphic allele of IGF1R carrying an integrated neo cassette within the second intron. IGF1Rneo/neo mice express full-length IGF1R protein, but at reduced levels (30-60% of wild-type). Despite this reduced expression level, we were able to generate lethal T-cell leukemias with 100% penetrance in primary recipients of IGF1Rneo/neo bone marrow transduced with activated NOTCH1 retrovirus, similar to what is observed with IGF1R+/+ bone marrow. Upon IV or intra-medullary injection into secondary congenic recipients, however, IGF1Rneo/neo leukemias exhibited a striking transplantation defect. Whereas IGF1R+/+ leukemias are consistently serially transplantable, 5 of 16 primary IGF1Rneo/neo leukemias failed to reconstitute disease in any secondary recipients, 5 produced disease in only a subset of secondary recipients, and 6 produced disease in all secondary recipients. Furthermore, treatment of IGF1R+/+ leukemia cells in vitro with IGF1R inhibitor prior to injection into secondary recipients prevented their transplantability, whereas treatment with PI3K and ERK inhibitors did not.Finally, given that inhibition of Notch signaling in human T-ALL cells compromises their ability to recapitulate disease in NOD/Scid recipients, we wondered whether this effect could be mediated by downregulation of IGF1R. In fact, we observed inhibition of Notch signaling, either by small molecule gamma-secretase inhibitor or transduction with dominant negative Mastermind, to result in a 2–3 fold decrease in IGF1R protein expression, corresponding to a 5 to 20-fold decrease in response to stimulation by IGF-1 ligand. Thus, these studies demonstrate high-level IGF1R signaling is required for LSC activity in T-ALL, and that Notch signaling may promote LSC function by supporting IGF1R expression. IGF1R inhibitors may therefore prove clinically useful in preventing disease relapse by compromising LSC self-renewal. Disclosures:Carboni:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment.

Testosterone-induced hypertrophy of L6 myoblasts is dependent upon Erk and mTOR

Testosterone increases the size and strength of skeletal muscle. This study further characterized the molecular mechanisms of the anabolic actions of testosterone on a rat myoblast cell line (L6 cells). Testosterone did not induce hypertrophy in L6 cells lacking the androgen receptor (AR). Hypertrophy was prevented by the AR antagonist bicalutamide and the mTOR inhibitor rapamycin. Testosterone induced Erk phosphorylation by 2 h, and mTOR autophosphorylation was elevated within 20 min; phosphorylation of p70S6 kinase was increased by 2 h. Inhibitors of Erk or PI3K blocked tesotosterone-induced hypertrophy. Erk phosphorylation returned to baseline when media containing testosterone was replaced at 16 h with fresh media lacking testosterone; when bicalutamide was added to testosterone-enriched media at 16 h, Erk phosphorylation remained elevated. Autophosphorylation of the IGF-1 receptor was minimally altered by testosterone at 20 min and unaffected at later time points; PI3K/PDK1-dependent phosphorylation of Akt was not altered by testosterone. These findings indicate that testosterone stimulates hypertrophy of L6 myoblasts through a mechanism that requires its binding to the AR and involves a signaling cascade dependent upon Erk and mTOR which is likely activated by substances released into the extracellular space which are not IGF-1 or other ligands for receptor tyrosine kinases.

Primary resistances to EGFR inhibitors (EGFRi) in HPV-positive and -negative oropharyngeal squamous cell carcinoma (OSCC).

10591 Background: HPV-positive and negative OSCC, despite a common EGFR expression, have a distinct biology and clinical behaviour, thus we hypothesize different mechanisms of primary resistance to EGFRi. Methods: PI3KCA, PTEN, HER2 and IGF-1R status was investigated in 17 HPV16-positive and 57 HPV16-negative fixed untreated OSCCs through FISH, sequencing, cDNA relative quantification by means of real-time PCR (method 2-ΔΔCt) and immunohistochemistry. Results: PI3KCA gene amplification involved only HPV16-negative (13%) cases, while a gain of PI3KCA gene copy number similarly occurred in HPV16-positive (29%) and negative (25%) OSCC. Activating mutations play a marginal role both in HPV16-positive (6%) and negative (2%) OSCC. An higher occurrence of PI3KCA transcript values, 31 folds > than the lower one, was found in HPV16-positive (60%) compared with negative (27%) OSCC (p=0.05). PTEN gene loss was restricted to HPV16-positive (12%) cases, while PTEN mutation similarly involved both HPV16-positive (9%) and negative (10%) OSSC. Overall, PI3KCA and PTEN alterations was more frequent in HPV16- positive (70%) than negative (44%) OSCC. Despite a statistically significant higher occurrence of an increased HER2 gene/chromosome 17 copy number in HPV16-negative (46%) than positive (18%) OSSC (p<0.004), no case showed HER2 expression. IGF-1R cytogenetic analysis revealed a statistically significant higher frequency of increased IGF- 1R/chromosome 15 copy number in HPV16-negative (41%) than positive (7%) OSCC (p=0.02). This is in keeping with the higher occurrence of IGF-1R transcript values, 30 folds > than the lower one, detected in HPV16-negative (73%) than positive (15%) cases (p<0.001). Most OSCC showed expression of the cognate ligands IGF1 and IGF2, however HPV16-negative cases expressed higher IGF1 levels than HPV16-positive OSCC. Conclusions: PI3KCA/PTEN alterations could be responsible to primary EGFRi resistance particularly in HPV16-positive tumors, while IGF-1R could be associated with resistance mainly in HPV16-negative OSCC. HER2 seems not to be involved. Based on our results, resistances to EGFRi should be tackled differently according to HPV tumor status. No significant financial relationships to disclose.

Abstract 2546: IGR-1R pathway role in oxaliplatin acquired resistance in colon cancer cell lines and its regulation by MMP-7 and IGFBP-2

Abstract Background: IGF-1R pathway and MMPs have been implicated in chemotherapy resistance in colorectal cancer. This receptor could be activated by IGF1 or IGF2 ligands, which bioavailability is regulated by IGFBPs, what could be degraded by MMPs. Objectives: To determine the basal levels and the modulation by chemotherapy (oxaliplatin), of the levels of IGF-1R, IGFBPs and MMPs in colon cancer cell lines (HT-29 (IC50: 6,46 uM) and LoVo (IC50: 0,25 uM) and their oxaliplatin resistant cell lines (HT-29OXAR3 (IC50: 29,86 uM) and LoVOXAR3 (2,45 uM)). To analyze the differences in apoptosis between parental and oxaliplatin resistant cell lines when are treated with oxaliplatin. Methods: MMP-7 levels were analyzed by RT-PCR, Western Blot and ELISA. Levels of IGF-1R, activated IGF-1R, IGFBP-2, and IGFBP-3 were determined by Western Blot and/or ELISA. Differences in cell viability were analyzed by MTT, and intrinsic (bcl-2, bax) and extrinsic (Fas, caspase 8) apoptotic pathways, by Western Blot, in parental and oxaliplatin resistant cell lines with dose-response experiments. Results: LoVo and LoVOXAR3 express low levels of MMP-7 and IGF-1R and high levels of IGFBP-2 compared with HT-29 and HT-29OXAR3. In addition, HT-29OXAR3 cells showed increased MMP-7, IGF-1R and pIGF-1R levels compared with HT-29 and lower levels of IGFBP-2. We also observed after oxaliplatin treatment in HT-29 cells a decrement of IGF-1R and IGFBP-2 and an increment in MMP-7 levels. In apoptotic experiments, FAS and caspase 8 (extrinsic pathway) were induced by oxaliplatin in LOVO (0.2 uM) and in LOVOXAR3 (20uM). Bcl-2 and bax (intrinsic pathway) were induced or decremented respectively in LOVO (2uM) and LOVOXAR3 (20uM). No changes in these extrinsic apoptotic proteins were observed in HT29 and HT29OXR3. In HT-29OXAR3 cell line higher doses of oxaliplatin (20 uM instead of 2uM) were needed to induce intrinsic apoptotic (bax) and antiapoptotic proteins (bcl-2) Conclusions: IGF-1R signalling is implicated in acquired oxaliplatin resistance and it is regulated by MMP-7 and IGFBP-2 in a different manner in our two models of cancer colon cell lines. The two apoptotic pathways were implicated in LoVo cell lines, but only intrinsic pathway regulates apoptosis in HT-29 cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2546.

Abstract 2427: Overexpression of insulin/IGF hybrid receptor as predictive biomarker of response to a humanized anti-insulin like growth factor-I receptor monoclonal antibody (CP751,871) in gastric and hepatocellular carcinoma cells

Abstract Insulin-like Growth factor I receptor (IGF-IR) is highly expressed in many human cancers and plays a major role in proliferation, survival, and metastasis. Accordingly, abundant IGF-1R targeting drugs, especially monoclonal antibody drugs, have been focused and developed due to their safety and efficacy. In order to define biomarkers correlating with the drug sensitivity, we screened 10 gastric and 10 hepatocellular cancer cell lines, for in vitro response to CP-751,871, anti-insulin like growth factor type 1 receptor antibody. Our results show that CP-751,871 inhibits IGF-1 ligand-dependent IGF-1R phosphorylation and downstream signaling of the two major IGF-1R pathways, mitogen-activated protein kinase and phosphatidylinositol 3′-kinase/AKt. Moreover, long-term exposure to CP-751,871 also caused to decrease phospho-stat 3 as well as phospho-Akt level and induced G1 phase cell cycle arrest in dose-dependent manner preferentially in the sensitive cells. Surprisingly, among these 20 cell lines, cells with IGF-IR and insulin receptor (IR) were significantly inhibited by CP-751,871 compared to cells with IGF-IR or IR overexpression only. Considering their highly homologous structure, we thereby determined if the effect of CP-751,871 had relevance to the level of IR/IGF1R hybrid receptors through co-immunoprecipitation with either CP-751,871 or IGF1R antibody. Its immunoblot analysis revealed that not only did sensitive cell lines, such as SNU719, SNU368, and HepG2, show relatively higher levels of IR/IGF1R hybrid, but also superior ability of CP-751,871 to recognize their IR/IGF1R heterodimer complexes. Furthermore, we could observe that the hybrid forms were recruited by IGF1 ligands but this IGF-I ligand dependent formation was blockaded by CP-751,871. Lastly, the treatment of these sensitive cell lines with CP-751,871 caused dimer dissociation. Therefore, our data suggest that the antiproliferative effect by CP-751,871 requires the co-expression of IGF-1R and IR and may be related with the expression level of hybrid Insulin/IGF-1 receptors in gastric and hepatocellular carcinoma cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2427.

Abstract 363: Therapeutic targeting of insulin receptor-A with a dual IGF-1R/IR inhibitor: Overcoming a potential escape mechanism from IGF-1R-specific signaling inhibition in insulin receptor-A transfected cancer cells

Abstract Type I Insulin-like Growth Factor Receptor (IGF-1R) is a tyrosine kinase receptor that is activated by binding to IGF-1 and IGF-II ligands; IGF-1R has been shown to play a role in cancer development and progression and therapies targeting IGF-1R have resulted in clinical benefit in cancer patients. Insulin Receptor (IR) which is closely related to IGF-1R, is expressed in normal tissues and tumors, and is expressed as two isoforms, IR-A and IR-B. IR-A exhibits mitogenic activity and is preferentially found in fetal tissue and cancer cells, whereas IR-B regulates glucose metabolism in response to insulin. Both IR isoforms bind insulin, and IR-A also binds IGF-II. Several cancer cell types that express IR-A also overexpress IGF-II, suggesting a possible autocrine loop that enhances tumor survival. Since IGF-1R antibodies bind specifically to IGF-1R and not to IR, IR signaling could represent a potential escape mechanism to antibody treatment through the IR-A pathway. BMS-754807, a small molecule inhibitor with dual activity against IGF-1R and IR, recently entered clinical development. It is anticipated that BMS-754807 can prevent or overcome therapeutic resistance resulting from increased IR-activated signaling. The goal of the presented studies was to demonstrate whether dual IGF-1R/IR inhibition by BMS-754807 may prevent IR-mediated resistance to IGF-1R inhibition. To test this hypothesis, a human rhabdomyosarcoma cell line, Rh41 expressing IGF-1R, but little IR, was engineered to express either IR-A (Rh41-IR-A) or IR-B (Rh41-IR-B). IR expression was confirmed by PCR analysis of specific IR isoforms. Rh41-IR-A cells showed phosphorylation of IR after stimulation with IGF-II and/or insulin. MAB391, a neutralizing monoclonal antibody to IGF-1R, inhibited the proliferation of parental Rh41 and Rh41-IR-B cells in vitro, but Rh41-IR-A cells exhibited no sensitivity to the IGF-1R mAb. Combination treatment with both IGF-1R and IR antibodies in Rh41-IR-A cells were strongly synergistic, suggesting dual receptor blocking is required for enhanced anti-tumor efficacy. Inhibition of IGF-1R and IR with BMS-754807 showed the same inhibitory activity in Rh41-IR-A cells as was observed in the parental Rh41 cells. Therefore, dual IGF-1R/IR inhibition by BMS-754807 treatment may overcome the ability of cancer cells to utilize the IR-A pathway and provide an advantage over IGF-1R specific antibodies in the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 363.

Abstract 2586: Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors

Abstract The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR) are transmembrane receptor tyrosine kinases that mediate proliferative and invasive cell signaling in cancer. Inhibition of either receptor reduces tumor growth in both mouse models and in human clinical studies. Blocking the EGFR pathway can induce compensatory activation of the IGFR pathway to drive tumor growth and IGFR inhibition can result in activation of EGFR signaling in preclinical models. Therefore, blocking both receptors simultaneously may achieve superior efficacy to blocking either pathway alone. We developed individual optimized Adnectins™ specific for blocking either EGFR or IGFR signaling and engineered them into a single protein that linked both Adnectins together to construct a bi-specific Adnectin targeting the EGFR and IGFR (EI-tandem). The bifunctional molecule blocked activation of EGFR and IGFR, inhibited both EGF and IGF-induced down-stream cell signaling (MAPK and AKT pathways) and was antiproliferative in human cancer cell lines. Potency of the EI-tandem was comparable to anti-EGFR and anti-IGFR antibodies. The EI-tandem demonstrated a synergistic inhibition of IGFR phosphorylation and down-stream cell signaling compared to Adnectins specific for only EGFR or IGFR alone. Although Adnectins bound to the EGFR at a site distinct from the clinically approved anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, they still blocked binding of EGF to the EGFR. PEGylated EI-tandem inhibited the growth of human tumor xenografts driven by both EGFR and IGFR signaling, degraded EGFR and IGFR, and reduced phosphorylation of EGFR in tumors. Treatment of mice with EI-tandem caused increases in levels of the circulating ligands TGFα and IGF1 resulting from blockade of their respective receptors and provided convenient soluble biomarkers of target suppression. These results show that a bifunctional Adnectin can confer improved biological activity compared to monospecific biologics in tumors where growth is driven by multiple growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2586.

Initial testing of a monoclonal antibody (IMC‐A12) against IGF‐1R by the pediatric preclinical testing program

Many childhood malignancies including sarcomas, neuroblastoma, and Wilms tumor show the presence of both, active, type-1-insulin-like growth factor receptor (IGF-1R), and the autocrine production of its ligands IGF-1/IGF-2. IMC-A12 is a fully human IgG1 antibody that prevents ligand binding to the IGF-1R. IMC-A12 was evaluated against the 23 cell lines of the Pediatric Preclinical Testing Program (PPTP) in vitro panel using 96 hr exposure at concentrations ranging from 0.01 nM to 0.1 microM. IMC-A12 was tested in vivo at a dose of 1 mg/mouse administered intraperitoneally twice weekly for 6 weeks. In vitro, IMC-A12-induced T/C values <50% in only three cell lines, a rhabdomyosarcoma cell line (Rh41) and two Ewing sarcoma cell lines (TC-71 and CHLA-9). In vivo, IMC-A12 induced significant differences in EFS distribution compared to control in 24 of 34 (71%) evaluable solid tumor xenografts. Using the PPTP "time to event" activity measure, IMC-A12 induced intermediate (n = 13) or high (n = 1) activity in 33 xenografts evaluable for this activity measure, including 6 of 6 rhabdomyosarcoma xenografts, 3 of 5 osteosarcoma xenografts, 2 of 5 neuroblastoma xenografts, and 1 of 5 Ewing sarcoma xenografts. The only objective response observed was observed in a rhabdomyosarcoma xenograft (Rh28) that achieved a maintained complete response. IMC-A12 demonstrated broad antitumor activity against the PPTP's in vivo solid tumor panels, with the activity primarily being tumor growth inhibition rather than tumor regression. IMC-A12 showed its greatest activity in vivo against the PPTP's rhabdomyosarcoma xenografts.

P46 Studies on the bivalent interaction of IGF-II with the insulin and type 1 IGF receptors

demonstrated the role of the IGF system in the regulation of trophoblast proliferation and survival and alterations in the IGF ligands or the receptors could have implications in the pathogenesis of trophoblastic diseases. The aim of this study was to investigate the role of the IGF system in trophoblast cell differentiation by evaluating the expression of genes of the IGF system during the process. We utilized the HTR8/Svneo extra villous trophoblast cell line and induced differentiation by stimulation with forskolin (FSK), an agonist of adenylyl cyclase, We found that cells treated with FSK 25mM for 72 hours secreted high levels of chorionic gonadotropin (hCG), a common differentiation marker of syncitiotrophoblast. Interestingly we found a hCG related increase in IGF-II and IGF-IIR mRNAs expression, suggesting an important role of this growth factor and its receptor during differentiation. To investigate the potential role of an autocrine IGF-II/IGF-IIR loop we obtained a stable IGF-IIR silenced HTR8/Svneo cell line by shRNA tranfection and induced differentiation aspreviously. After 72 hours of treatment with FSK we found a marked reduction in IGF-II mRNA levels, in comparison with the non-silenced cell line, together with a decrease in the expression in the IGF-I and Insulin receptors. In addition we observed a direct relation between IGF-IIR silencing with a delayed kinetic pattern of differentiation. In conclusion this study presents a first approximation to the autocrine role of IGFII/IGF-IIR system in trophoblastic cell differentiation.

Treatment of biliary tract cancer with NVP-AEW541: Mechanisms of action and resistance

To investigate in vitro treatment with NVP-AEW541, a small molecule inhibitor of insulin-like growth factor-1 receptor (IGF-1R), in biliary tract cancer (BTC), since this disease is associated with a poor prognosis due to wide resistance to chemotherapeutic agents and radiotherapy. Cell growth inhibition by NVP-AEW541 was studied in vitro in 7 human BTC cell lines by automated cell counting. In addition, the anti-tumoral mechanism of NVP-AEW541 was studied by Western blotting, cell cycle analysis and reverse transcription-polymerase chain reaction (RT-PCR). Anti-tumoral drug effect in combination with gemcitabine, 5-fluorouracil (5-FU) and Polo-like kinase 1 inhibitor BI2536 was also studied. In vitro treatment with NVP-AEW541 suppressed growth in all human BTC cell lines, however response was lower in gallbladder cancer. Treatment with NVP-AEW541 was associated with dephosphorylation of IGF-1R and AKT. In contrast, phosphorylation of p42/p44 and Stat3 and expression of Bcl-xL were inconsistently downregulated. In addition, treated cells showed cell cycle arrest at the G1/S-checkpoint and an increase in sub-G1 peak. Moreover, IGF-1R and its ligands IGF-1 and IGF-2 were co-expressed in RT-PCR, suggesting an autocrine loop of tumor cell activation. Combined with gemcitabine, NVP-AEW541 exerted synergistic effects, particularly at low concentrations, while effects of combination with 5-FU or BI 2536 were only additive. Our findings suggest that NVP-AEW541 is active against BTC in vitro and potentiates the efficacy of gemcitabine.

Abstract B253: The IGF1R inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-Stat3-HIF1 pathway in human glioblastoma cells

Abstract Glioblastomas (GBMs) typically develop hypoxic areas within the tumor mass, which for some cells results in necrosis, but for others leads to pleiotropic adaptive changes promoting tumor progression through activation of survival pathways, disruption of apoptosis, increase in invasive potential and neovascularization. The transcription factor HIF1 (hypoxia-inducible factor 1) is known to play a major role in directing this adaptive program, following stabilization of its inducible -subunit under hypoxic conditions; however, HIF1 activity can also be enhanced by oxygen-independent mechanisms upon activation of growth factor receptors and their downstream signal transduction pathways. The present study investigates a) the possible presence of a regulatory loop linking IGF1R and HIF1 in human glioblastoma cells; b) the role played by the signal transducer and activator of transcription 3 (Stat3) in this pathway; and c) the ability of the IGF1R inhibitor NVP-AEW541 (Novartis Pharma, Basel, CH) to disrupt this pathway. We show that activation of IGF1R by its ligand IGF1 causes Stat3 activation, increased levels of HIF1α and increased release of VEGF, the product of one of the major target genes for HIF1 transcriptional activity. Treatment with NVP-AEW541 reverses all these changes, and so does transfection of the cells with a dominant negative form of Stat3. Interestingly, we also observe that IGF-independent activation of HIF1, obtained in the presence of the hypoxia mimic CoCl2, results in IGF1R activation, thereby indicating that a two-way relationship exists between growth factor receptor activation and increase in HIF1 transcriptional activity; NVP-AEW541 treatment significantly reduces both HIF1α protein levels and IGF1R tyrosine-specific phosphorylation in CoCl2-treated cells. Because cells surviving under hypoxic condition often become resistant to radiotherapy and chemotherapy, modulation of the IGF1R-HIF1 pathway could be a viable approach to increase the effectiveness of conventional cytotoxic agents; as GBM typically presents hypoxic areas, this strategy might prove particularly valuable in improving the poor prognosis of patients affected by this tumor type. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B253.

Abstract A233: Generation of in vitro and in vivo tumor models driven by insulin-like growth factor receptor (IGF1R) and their use in the development of OSI-906, a selective IGF1R inhibitor

Abstract The emergence of insulin-like growth factor-1 receptor IGF1R as an important cancer target is evidenced by the growing number of clinical trials currently underway for both small molecule inhibitors and antibody therapeutics developed against it. In preclinical studies, perturbation in IGF1R signaling has been associated with tumor growth inhibition, blockade of metastasis, and enhanced sensitivity to therapeutic agents, including cytotoxic chemotherapeutics and molecular targeted agents. Currently agents directed against IGF1R are being evaluated in clinical trials for their therapeutic utility. Given the differential dependence on IGF1R exhibited by human tumors and xenograft lines, there is a need for additional preclinical models in which the relationship between genetic context and drug response in solid tumors can be better understood in an in vivo setting. To this end, we utilized our HER2 driven inducible breast cancer model to create a panel of genetically-engineered mouse tumors, each driven by the human IGF1R. Some of the models were also engineered to express a cognate ligand IGF2. In these models, tumors initially driven by human HER2 expression are re-programmed by the addition of the human IGF1R cDNA upon withdrawal of the original HER2 expression. Thus, the newly introduced IGF1R- IGF2 cDNA functionally maintains tumor survival by re-directing downstream signalling effectors in a process referred to as ‘Directed Complementation’ (DC). Using this approach, introduction of IGF1R strongly complemented tumor formation across the panel, whereas introduction of the ligands, either IGF1 or IGF2, alone or in combination did not. We recently showed that treatment of IGF1R/IGF2 DC tumors with OSI-906, a potent, selective orally active inhibitor of IGF1R, resulted in tumor regression within a week of treatment, with corresponding loss of phospho-IGF1R signaling (AACR 2009: Abstract # 2902, Lerner, et al). These results demonstrate the singular dependence of these tumors on IGF1R signaling, as well as the in vivo efficacy of the compound. Next, we sought to extend the utility of these validated tumor models to drug discovery by establishing IGF1R-dependent cell lines for high-throughput screening. Here we describe detailed molecular characterization of these cell lines derived from the IGF1R/IGF2 DC tumor models as well as their drug sensitivities. Importantly, we show that the IGF1R/IGF2 DC cell lines, like their parent tumors, retain activation of the pathway as well as sensitivity to OSI-906. Therefore, these tumor models and derived cell lines provide an effective preclinical tool for the development of targeted anti-IGF1R therapies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A233.

249 PROFILE OF MEMBERS OF IGF SYSTEM GENE EXPRESSION IN BOVINE IMMATURE OOCYTES: COMPARISON BETWEEN NELLORE (BOS INDICUS) AND HOLSTEIN (BOS TAURUS)

There is evidence that IGF system can be involved in the oocyte competence and, consequently, in the embryo development. To better understand possible differences between Bos taurus and Bos indicus in in vitro embryo development, the current work aimed to assess the expression of IGF ligands (IGF-1 and 2), types 1 and 2 IGF receptors (IGFR1 and 2), IGF-binding proteins 2 and 4 (IGFBP-2 and 4), and type A pregnancy-associated plasmatic protein (PAPP-A) mRNA in bovine immature oocytes fromBos Taurus and Bos indicus. Nellore and Holstein cows were submitted to ovum pickup, and the oocytes with grade 1, 2, and 3 were selected. To remove cumulus cells and pellucida zone, respectively, the oocytes were submitted to vortex (900 × g for 3 min) and protease treatment (Protease® from Streptomyces griseus, Sigma-Aldrich Corporation, St. Louis, MO, USA). Pools of 20 oocytes obtained from Nellore (n = 8) and Holstein (n = 4) cows were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA, USA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the RT and bovine-specific primers in the PCR. Expression of cyclophlin A (CYC-A) was used as internal control. The means of mRNA levels of target genes between the breeds were compared using t-test and Mann-Whitney test when the data had normal or not normal distribution, respectively. The means values of mRNA expression of IGF-1, IGF-2, IGF receptors (1 and 2), and IGFBP 2 and 4 were greater in Holstein (0.96 ± 0.21, 0.74 ± 0.27, 1.08 ± 0.04, 1.19 ± 0.5, 1.21 ± 0.23, 0.53 ± 0.15, respectively) compared with Nellore (0.48 ± 0.10, 0.07 ± 0.02, 0.04 ± 0.03, 0.06 ± 0.02, 0.05 ± 0.01, 0.03 ± 0.15, respectively; P &lt; 0.01). Never- theless, mRNA expression of PAPP-A was much greater in Nellore (28.10 ± 18.96) than in Holstein (1.32 ± 0.17; P &lt; 0.05). These results suggest that high expression of PAPP-A and low expression of IGFBP-2 and 4 could allow more efficiency on the degradation of IGFBP and increase the IGF bioavailability in the oocytes from Nellore as compared with Holstein cows. Satrapa, Simões, and Castilho received a fellowship and funding from FAPESP (São Paulo, Brazil).

Differential Roles of Insulin-like Growth Factor Receptor- and Insulin Receptor-mediated Signaling in the Phenotypes of Hepatocellular Carcinoma Cells

Intrahepatic and extrahepatic metastases are common findings in hepatocellular carcinoma (HCC). Insulin-like growth factor 2 (IGF2) expression is frequently induced in HCC, and serum IGF2 levels correlate with the presence of extrahepatic metastases. Yet, the role of IGF-induced signaling in the dissemination of HCC remains unclear. We have previously observed elevated IGF2 levels in tumors with metastatic potential in an HCC mouse model. Here, we demonstrate that inhibition of IGF2, or its receptor IGF1R, impairs the migration and invasion activities of murine HCC cells. Furthermore, inhibition of IGF1R also impairs the ability of HCC cells to colonize the lungs after introduction into the circulation through the tail vein but does not impair subcutaneous tumor growth. Collectively, these findings suggest that IGF1R-mediated signaling plays a causative role in tumor dissemination but is not required for tumor growth per se. Although previous studies indicate that IGF ligands can signal through IGF1R/insulin receptor (IR) heterodimers, and IR-A homodimers, we demonstrate that the IR is not required for invasion and metastasis by HCC cells. Finally, we identify matrix metalloproteinase 2 as a mediator of the invasive phenotype downstream of IGF1R-induced signaling. Thus, our studies demonstrate the importance of IGF2-induced signaling in the dissemination of HCC cells.

Targeted Gene Knockdown in Zebrafish Reveals Distinct Intraembryonic Functions for Insulin-Like Growth Factor II Signaling

IGF-II is the predominant IGF ligand regulating prenatal growth in all vertebrates, including humans, but its central role in placental development has confounded efforts to fully elucidate its functions within the embryo. Here we use a nonplacental model vertebrate (zebrafish) to interrogate the intraembryonic functions of IGF-II signaling. The zebrafish genome contains two coorthologs of mammalian IGF2 (igf2a, igf2b), which exhibit distinct patterns of expression during embryogenesis. Expression of igf2a mRNA is restricted to the notochord, primarily during segmentation/neurulation. By contrast, igf2b mRNA is expressed in midline tissues adjacent to the notochord, with additional sites of expression in the ventral forebrain, and the pronephros. To identify their intraembryonic functions, we suppressed the expression of each gene with morpholino oligonucleotides. Knockdown of igf2a led to defects in dorsal midline development, characterized by delayed segmentation, notochord undulations, and ventral curvature. Similarly, suppression of igf2b led to defects in dorsal midline development but also induced ectopic fusion of the nephron primordia, and defects in ventral forebrain development. Subsequent onset of severe body edema in igf2b, but not igf2a morphants, further suggested a distinct role for igf2b in development of the embryonic kidney. Simultaneous knockdown of both genes increased the severity of dorsal midline defects, confirming a conserved role for both genes in dorsal midline development. Collectively, these data provide evidence that the zebrafish orthologs of IGF2 function in dorsal midline development during segmentation/neurulation, whereas one paralog, igf2b, has evolved additional, distinct functions during subsequent organogenesis.

Insulin-Like Growth Factor Type 1 Receptor and Insulin Receptor Isoform Expression and Signaling in Mammary Epithelial Cells

The insulin receptor (IR) isoforms and the IGF type 1 receptor (IGF-1R) share a high degree of structural homology but differ in ligand binding kinetics and functions. We developed a highly specific quantitative PCR assay to quantify and compare IR-A, IR-B, and IGF-1R expression within an RNA population. We determined receptor expression in primary murine mammary epithelial cells (MECs) during postnatal development. Both IR isoform mRNAs were 3- to 16-fold higher than IGF-1R expression at all developmental times. IR protein was also 3- to 10-fold higher than IGF-1R protein; however, significantly less IGF-1R was found in hybrid receptors at early (49%) vs. late (79%) pregnancy, indicating that the amount of hybrid receptor is developmentally regulated. Despite high IR expression, IGF ligands were more effective than insulin in stimulating the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt pathway in acutely isolated MECs from virgin glands. Although approximately 40% of IR transcripts were the IGF-II-sensitive IR-A isoform, IGF-II failed to stimulate IR phosphorylation, and an IGF-1R-specific blocking antibody completely abrogated IGF-II-mediated Akt phosphorylation in the virgin MECs. Taken together, these data suggest that the IGF-1R is more active in signaling than the IR and is the predominant mediator of IGF actions in virgin MECs.

In Utero and Lactational Exposure to Nicotine Alters the Intra-Ovarian IGF System in Adult Female Rats

In humans there is evidence that in utero exposure to cigarette smoke results in decreased fertility in female offspring. We have demonstrated in rats that fetal and neonatal exposure to nicotine alone results in impaired fertility and increased follicular atresia in the adult female offspring. In mammals, the insulin-like growth factor-I and -II (IGF-I and -II) and their binding proteins (IGFBPs) are considered stimulators and inhibitors of follicular growth and maturation. Therefore we hypothesized that dysregulation of the intra-ovarian IGF system could be implicated in the impaired fertility observed in nicotine-exposed offspring. Nulliparous female Wistar rats were exposed to nicotine (1 mg/kg/d) for 2 weeks prior to mating until weaning. Ovaries were collected on the morning of estrus from sexually mature saline- and nicotine-exposed offspring. Protein expression of IGF ligands and receptors (IGFR-I and IGFR-II) were quantified by western blot and immunohistochemistry. The expression of IGF-I, IGF-II; IGFR-I, IGFR-II; and IGFBP1–6 mRNA in the ovary was deter- mined by semi-quantitative reverse transcriptase-PCR. Results showed that nicotine exposure significantly reduced IGF-I, IGF-II and IGFR-I protein expression (p < 0.01) relative to saline controls. Furthermore, nicotine-exposed offspring had significantly reduced IGFR-II mRNA expression (p < 0.01) in the ovary. Data from this study suggest that the decreased fertility and increased follicular atresia in nicotine-exposed animals may be due, in part, to disruption of IGF regulation in the ovary.