NT157 exhibits antineoplastic effects by targeting IRS and STAT3/5 signaling in multiple myeloma.

Multiple myeloma (MM) is a prevalent hematological malignancy with high recurrence and no definitive cure. The current study revisits the role of the IGF1/IGF1R axis in MM, introducing a novel inhibitor, NT157. The IGF1/IGF1R pathway is pivotal in MM, influencing cell survival, proliferation, and migration and impacting patient survival outcomes. NT157 targets intracellular proteins such as IRS and STAT proteins and demonstrates antineoplastic potential in hematological malignancies and solid tumors. In the present study, we assessed IGF1R signaling-related gene expression in MM patients and healthy donors, unveiling significant distinctions. MM cell lines displayed varying expression patterns of IGF1R-related proteins. A gene dependence analysis indicated the importance of targeting receptor and intracellular elements over autocrine IGF1. NT157 exhibited inhibitory effects on MM cell viability, clonal growth, cell cycle progression, and survival. Moreover, NT157 reduced IRS2 expression and STAT3, STAT5, and RPS6 activation and modulated oncogenes and tumor suppressors, fostering a tumor-suppressive molecular profile. In summary, our study demonstrates that the IGF1/IGF1R/IRS signaling axis is differentially activated in MM cells and the NT157's capacity to modulate crucial molecular targets, promoting antiproliferative effects and apoptosis in MM cells. NT157 may offer a multifaceted approach to enhance MM therapy.

Molecular Characterization of Patients with Cryptorchidism: Preliminary Search for an Expression Profile Related to That of Testicular Germ-Cell Tumors.

Cryptorchidism (CO) is a risk factor for the development of testicular germ-cell tumors (TGCT). This is supported by reports showing the persistence of gonocytes in CO patients. These cells are proposed to be related to the development of germ-cell neoplasia in situ (GCNIS), which is considered the precursor stage/lesion of TGCT. Therefore, it is proposed that some patients with CO could express some molecular markers related to TGCT. In this study, we analyzed testicular tissue samples from CO, TGCT, and controls. We determined the expression of POU5F1, PLAP, and KIT by immunohistochemistry and that of the hsa-miR-371-373 cluster, hsa-miR-367, and LATS2, PTEN, and IGFR1 genes by RT-qPCR. We then carried out a bioinformatic analysis to identify other possible candidate genes as tumor biomarkers. We found that 16.7% (2/12) of the CO patients presented increased expression of POU5F1, KIT, PLAP, hsa-miR-371-373, and hsa-miR-367 and decreased expression of LATS2 and IGF1R. Finally, the genes ARID4B, GALNT3, and KPNA6 were identified as other possible candidate tumor biomarkers. This is the first report describing the expression of the hsa-miR-371-373 cluster, hsa-miR-367, LATS2, and IGF1R in the testicular tissues of two CO patients with cells immune-positive to POU5F1, PLAP, and KIT, which is similar to what is observed in TGCT.

IGFBP7 Fuels the Glycolytic Metabolism in B-Cell Precursor Acute Lymphoblastic Leukemia by Sustaining Activation of the IGF1R-Akt-GLUT1 Axis.

Increased glycolytic metabolism plays an important role in B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL). We previously showed that IGFBP7 exerts mitogenic and prosuvival effects in ALL by promoting IGF1 receptor (IGF1R) permanence on the cell surface, thus prolonging Akt activation upon IGFs/insulin stimulation. Here, we show that sustained activation of the IGF1R-PI3K-Akt axis concurs with GLUT1 upregulation, which enhances energy metabolism and increases glycolytic metabolism in BCP-ALL. IGFBP7 neutralization with a monoclonal antibody or the pharmacological inhibition of the PI3K-Akt pathway was shown to abrogate this effect, restoring the physiological levels of GLUT1 on the cell surface. The metabolic effect described here may offer an additional mechanistic explanation for the strong negative impact seen in ALL cells in vitro and in vivo after the knockdown or antibody neutralization of IGFBP7, while reinforcing the notion that it is a valid target for future therapeutic interventions.

Ionizing Radiation and Estrogen Affecting Growth Factor Genes in an Experimental Breast Cancer Model.

Genes associated with growth factors were previously analyzed in a radiation- and estrogen-induced experimental breast cancer model. Such in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line, MCF-10F, to low doses of high linear energy transfer (LET) α particle radiation (150 keV/µm) and subsequent growth in the presence or absence of 17ß-estradiol. The MCF-10F cell line was analyzed in different stages of transformation after being irradiated with either a single 60 cGy dose or 60/60 cGy doses of alpha particles. In the present report, the profiling of differentially expressed genes associated with growth factors was analyzed in their relationship with clinical parameters. Thus, the results indicated that Fibroblast growth factor2 gene expression levels were higher in cells transformed by radiation or in the presence of ionizing radiation; whereas the fibroblast growth factor-binding protein 1gene expression was higher in the tumor cell line derived from this model. Such expressions were coincident with higher values in normal than malignant tissues and with estrogen receptor (ER) negative samples for both gene types. The results also showed that transforming growth factor alpha gene expression was higher in the tumor cell line than the tumorigenic A5 and the transformed A3 cell line, whereas the transforming growth factor beta receptor 3 gene expression was higher in A3 and A5 than in Tumor2 cell lines and the untreated controls and the E cell lines. Such gene expression was accompanied by results indicating negative and positive receptors for transforming growth factor alpha and the transforming growth factor beta receptor 3, respectively. Such expressions were low in malignant tissues when compared with benign ones. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expressions were found to be present in all BRCA patients that are BRCA-Basal, BRCA-LumA, and BRCA-LumB, except in BRCA-Her2 patients. The results also indicated that the insulin growth factor receptor gene expression was higher in the tumor cell line Tumor2 than in Alpha3 cells transformed by ionizing radiation only; then, the insulin growth factor receptor was higher in the A5 than E cell line. The insulin growth factor receptor gene expression was higher in breast cancer than in normal tissues in breast cancer patients. Furthermore, Fibroblast growth factor2, the fibroblast growth factor-binding protein 1, transforming growth factor alpha, the transforming growth factor beta receptor 3, and the insulin growth factor receptor gene expression levels were in stages 3 and 4 of breast cancer patients. It can be concluded that, by using gene technology and molecular information, it is possible to improve therapy and reduce the side effects of therapeutic radiation use. Knowing the different genes involved in breast cancer will make possible the improvement of clinical chemotherapy.

Type 1 Insulin-Like Growth Factor Receptor Nuclear Localization in High-Grade Glioma Cells Enhances Motility, Metabolism, and In Vivo Tumorigenesis.

Gliomas are the most frequent solid tumors in children. Among these, high-grade gliomas are less common in children than in adults, though they are similar in their aggressive clinical behavior. In adults, glioblastoma is the most lethal tumor of the central nervous system. Insulin-like growth factor 1 receptor (IGF1R) plays an important role in cancer biology, and its nuclear localization has been described as an adverse prognostic factor in different tumors. Previously, we have demonstrated that, in pediatric gliomas, IGF1R nuclear localization is significantly associated with high-grade tumors, worst clinical outcome, and increased risk of death. Herein we explore the role of IGF1R intracellular localization by comparing two glioblastoma cell lines that differ only in their IGF1R capacity to translocate to the nucleus. In vitro, IGF1R nuclear localization enhances glioblastoma cell motility and metabolism without affecting their proliferation. In vivo, IGF1R has the capacity to translocate to the nucleus and allows not only a higher proliferation rate and the earlier development of tumors but also renders the cells sensitive to OSI906 therapy. With this work, we provide evidence supporting the implications of the presence of IGF1R in the nucleus of glioma cells and a potential therapeutic opportunity for patients harboring gliomas with IGF1R nuclear localization.

Circ-LARP1B knockdown restrains the tumorigenicity and enhances radiosensitivity by regulating miR-578/IGF1R axis in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Circular RNA La Ribonucleoprotein 1B (circ-LARP1B) was reported to serve as an oncogene in many types of cancers. Radiotherapy (RT) is an important element of the multimodal treatment concept in malignancies. Here, this work aimed to investigate the role of circ-LARP1B in the tumorigenesis and radiosensitivity of hepatocellular carcinoma (HCC). PATIENTS OR MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of genes and proteins. In vitro experiments were conducted using cell counting Kit-8 (CCK-8), colony formation, EDU, transwell, and tube formation assays, respectively. Dual-luciferase reporter assay was employed to identify the target relationship between miR-578 and circ-LARP1B or IGF1R (insulin-like growth factor 1 receptor). In vivo assay was performed using murine xenograft model. RESULTS: Circ-LARP1B was highly expressed in HCC tissues and cells, and high expression of circ-LARP1B was closely associated with poor prognosis. Functional experiments demonstrated that circ-LARP1B silencing impaired cell proliferation, invasion, angiogenesis and reduced radioresistance in vitro. Mechanistically, circ-LARP1B could competitively bind with miR-578 to relieve the repression of miR-578 on the expression of its target gene IGF1R. Further rescue assay confirmed that miR-578 inhibition reversed the inhibitory effects of circ-LARP1B knockdown on HCC cell malignant phenotypes and radioresistance. Moreover, miR-578 overexpression restrained tumorigenicity and enhanced radiosensitivity in HCC cells, which were attenuated by IGF1R up-regulation. Besides that, circ-LARP1B knockdown impeded tumor growth and enhanced irradiation sensitivity in HCC in vivo. CONCLUSIONS: Circ-LARP1B knockdown restrained HCC tumorigenicity and enhanced radiosensitivity by regulating miR-578/IGF1R axis, providing a new target for the treatment of HCC.

Morphological and molecular effects of overexpressed GH on mice mammary gland.

Growth Hormone (GH) plays crucial roles in mammary gland development and growth, and its upregulation has been associated with breast cancer promotion and/or progression. To ascertain how high GH levels could promote mammary tissue oncogenic transformation, morphological characteristics and the expression of receptors involved in mammary growth, development and cancer, and of mitogenic mediators were analyzed in the mammary gland of virgin adult transgenic mice that overexpress GH. Whole mounting and histologic analysis evidenced that transgenic mice exhibit increased epithelial ductal elongation and enlarged ducts along with deficient branching and reduced number of alveolar structures compared to wild type mice. The number of differentiated alveolar structures was diminished in transgenic mice while the amount of terminal end buds (TEBs) did not differ between both groups of mice. GH, insulin-like growth factor 1 (IGF1) and GH receptor mRNA levels were augmented in GH-overexpressing mice breast tissue, as well as IGF1 receptor protein content. However, GH receptor protein levels were decreased in transgenic mice. Fundamental receptors for breast growth and development like progesterone receptor and epidermal growth factor receptor were also increased in mammary tissue from transgenic animals. In turn, the levels of the proliferation marker Ki67, cFOS and Cyclin D1 were increased in GH-overexpressing mice, while cJUN expression was decreased and cMYC did not vary. In conclusion, prolonged exposure to high GH levels induces morphological and molecular alterations in the mammary gland that affects its normal development. While these effects would not be tumorigenic per se, they might predispose to oncogenic transformation.

DDAB cationic lipid-mPEG, PCL copolymer hybrid nano-carrier synthesis and application for delivery of siRNA targeting IGF-1R into breast cancer cells.

BACKGROUND AND OBJECTIVE: To use siRNA molecule as a therapeutic agent in gene silencing, an efficient delivery system is necessary. Stability and clearance by reticuloendothelial of siRNA still remains the major challenges for clinical application. Herein, we could develop new lipid-polymer hybrid nanoparticles (LPHNP) as a siRNA carrier to silence insulin-like growth factor type I (IGF-1R) gene overexpression in MCF-7 human breast cancer cell line. METHODS: Dimethyldioctadecylammonium bromide-methoxy poly(ethylene glycol)-poly (ε-caprolactone) (DDAB-mPEG-PCL) LPHNPs were synthesized using a single step nanoprecipitation method and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM) microscope. Cytotoxicity of the nanoparticles was assessed in the MCF7 cell line using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Desired LPHNP-siRNA complex was determined using different Nitrogen:Phosphate ratio (N/P) ratios and gel retardation. To determine the encapsulation efficiency of siRNA (%) in LPHNP, its absorbance was measured. The effect of the siRNA-LPHNP complex on IGF-1R silencing was assessed by reverse transcription-polymerase chain reaction (RT-PCR) RESULTS: LPHNP was synthesized using a single-step sonication method with a size below 100 nM. The viability of cells treated with hybrid nanoparticles was significantly greater than the corresponding cationic lipid (P < 0.01). As demonstrated by gel retardation assay, efficient siRNA binding to LPHNP occurred at N/P equal to 40 and siRNA encapsulation efficiency was found to be 95% ± 4 at this ratio. LPHNP-IGF-1R siRNA complex could be able to down-regulate the target more efficiently when it compared with the corresponded controls (P < 0.001). CONCLUSION: In conclusion, our results suggest that DDAB cationic lipid and mPEG-PCL copolymer hybrid nanoparticle may be a good candidate for efficient siRNA delivery.

Case of 15q26-qter deletion associated with a Prader-Willi phenotype.

Prader-Willi syndrome (PWS) is one of the common neurogenetic disorders associated with intellectual disability. PWS involves a complex inheritance pattern and is caused by an absence of gene expression on the paternally inherited 15q11.2-q13 region, either due to deletion, maternal uniparental disomy or imprinting defect. The syndrome is characterized principally by severe neonatal hypotonia, a weak suck in infancy that is later followed by hyperphagia and obesity, developmental delay, intellectual disability and short stature. In the case of the chromosome 15q26-qter deletion syndrome or Drayer's syndrome, very few reports have been published. Its characteristics include intrauterine growth restriction, postnatal growth failure, varying degrees of intellectual disability, developmental delay, typical facial appearance and diaphragmatic hernia. The present paper describes a female patient in whom clinical findings were suggestive of PWS and deletion in the 15q26-qter region. Both karyotyping and methylation-specific polymerase chain reaction were shown to be normal. Nevertheless, fluorescence in situ hybridization showed a 15qter deletion that was later mapped by single nucleotide polymorphism (SNP)-array. The deleted genomic region involves the insulin-like growth factor-1 receptor (IGF1R) gene, which is related to short stature, developmental delay and intellectual disability. This case had various clinical characteristics in common with the cases of 15q26-qter deletionand characteristics compatible with PWS.

Oral Administration of Microencapsulated B. Longum BAA-999 and Lycopene Modulates IGF-1/IGF-1R/IGFBP3 Protein Expressions in a Colorectal Murine Model.

The Insulin-like growth factor-I/Insulin-like growth factor-I receptor (IGF-1/IGF-1R) system is a major determinant in colorectal cancer (CRC) pathogenesis. Probiotics (Bifidobacterium longum, BF) and lycopene (LYC) have been individually researched for their beneficial effects in the prevention of CRC. However, the effect of a combined treatment of microencapsulated BF and LYC on IGF-1/IGF-1R/IGFBPs (Insulin-like growth factor-binding proteins) expression in an azoxymethane (AOM)-dextran sulfate sodium (DSS)-induced CRC model have not been demonstrated. BF was microencapsulated by the spray drying technique, with high viability, and daily gavaged with LYC for 16 weeks to CD-1 mice in an AOM-DSS model. The results indicated that BF- and BF + LYC-treated groups had significantly lower inflammation grade, tumor incidence (13-38%) and adenocarcinoma (13-14%) incidence compared to the AOM + DSS group (80%), whereas LYC treatment only protected against inflammation grade and incidence. Caecal, colonic and fecal pH and ß-glucuronidase (ß-GA) values were significantly normalized by BF and LYC. Similarly, BF and BF + LYC treatments significantly reduced both the positive rate and expression grade of IGF-1 and IGF-1R proteins and normalized Insulin-like growth factor-binding protein-3 (IGFBP3) expression. Based on intestinal parameters related to the specific colon carcinogenesis in an AOM-DSS-induced model, LYC and microencapsulated BF supplementation resulted in a significant chemopreventive potential through the modulation of IGF-1/IGF-1R system.

Expression of Breast Cancer-Related Epitopes Targeting the IGF-1 Receptor in Chimeric Human Parvovirus B19 Virus-Like Particles.

Breast cancer is a worldwide health problem, and the complexity of the disease, as well as the lack of treatment specificity, generates an urgent need for developing prophylactic and therapeutic measures. Searching for novel epitope-based approaches able to induce tumour immunity, we designed virus-like particles (VLPs) derived from Human parvovirus B19 assembled of chimeric VP2 proteins displaying two epitopes from the insulin-like growth factor-1 receptor (IGF-1R). Here, we present the generation of two chimeric VP2s that retain the stability, solubility and conditions of purification and assembly of the native VP2. We generated versatile chimeric multiepitope anti-cancer vaccine candidates, which prevented and delayed tumour growth when used in a prophylactic scheme of 4 weekly immunizations prior to 4T1 cell inoculation in female BALB/c mice. The presence of specific antibodies against the displayed epitopes suggests their participation in the protective effect; in contrast, no significant proliferative T-cell responses were recorded following stimulation by specific epitopes. The results comprise an approach whereby fusing desired epitopes from cancer to the N-terminus of B19 VP2 protein can generate a library of chimeric VP2-desired epitopes for further assembly in a designed and personalized epitope delivery system.

Fibroblast deficiency of insulin-like growth factor 1 receptor type 1 (IGF1R) impairs initial steps of murine pheochromocytoma development.

Insulin-like growth factor 1 (IGF1) has a critical role in maintaining tumor phenotype and survival of already transformed murine pheochromocytoma (pheo) cells (MPC4/30) and it is required for the initial establishment of these tumors. However, the role of local IGF1/IGF1R system in tumor microenvironment has not been fully understood. In vivo, by subcutaneous injection of pheo cells in heterozygous IGF1R knockout mice (L/n), we found that the time of noticeable tumor appearance was delayed, and incidence was decreased in L/n group compared to control (L/L) mice. Once established, tumor proliferation, vascularization or growth rate did not differ between groups. In vitro, fibroblast from L/n and L/L mice were cultured to generate conditioned media (CM) and differential matrixes on which pheo cells were seeded. Proliferation rate was higher when pheo cells were cultured with CM, or in differential matrix generated by L/L murine fibroblasts. A diminished fibronectin (FN) expression and secretion from L/n fibroblast was associated with decreased expression of integrin subunits in tumor cells. Also, soluble factors as IGF1 and insulin-like growth factor binding protein 2 (IGFBP2) were reduced. Our data suggest that IGF1 signaling through IGF1R may contribute to tumor cells anchorage and survival by interaction with both matrix and soluble factors produced by tumor microenvironment fibroblasts.

IGF1R/IRS1 targeting has cytotoxic activity and inhibits PI3K/AKT/mTOR and MAPK signaling in acute lymphoblastic leukemia cells.

The IGF1R/IRS1 signaling is activated in acute lymphoblastic leukemia (ALL) and can be targeted by the pharmacological inhibitors NT157 (IGF1R-IRS1/2 inhibitor) and OSI-906 (IGF1R/IR inhibitor). Here we investigate the cellular and molecular effects of NT157 and OSI-906 in ALL cells. NT157 and OSI-906 treatment reduced viability, proliferation and cell cycle progression in ALL cell lines. Similarly, in primary samples of patients with ALL, both OSI-906 and NT157 reduced viability, but only NT157 induced apoptosis. NT157 and OSI-906 did not show cytotoxicity in primary samples from healthy donor. NT157 and OSI-906 significantly decreased Jurkat cell migration, but did not modulate Namalwa migration. Consistent with the more potent effect of NT157 on cells, NT157 significantly modulated expression of 25 genes related to the MAPK signaling pathway in Jurkat cells, including oncogenes and tumor suppressor genes. Both compounds inhibited mTOR and p70S6K activity, but only NT157 inhibited AKT and 4-EBP1 activation. In summary, in ALL cells, NT157 has cytotoxic activity, whereas OSI-906 is cytostatic. NT157 has a stronger effect on ALL cells, and thus the direct inhibition of IRS1 may be a potential therapeutic target in ALL.

Downregulation of lncRNA CCDC26 contributes to imatinib resistance in human gastrointestinal stromal tumors through IGF-1R upregulation

Imatinib is the first line of therapy for patients with metastatic or gastrointestinal stromal tumors (GIST). However, drug resistance limits the long-term effect of imatinib. Long non-coding RNAs (lncRNAs) are emerging as key players in regulating drug resistance in cancer. In this study, we investigated the association between lncRNA CCDC26 and IGF-1R in GIST and their involvement in drug resistance. Considering the key role of lncRNAs in drug resistance in cancer, we hypothesized that IGF-1R is regulated by lncRNAs. The expression of a series of reported drug resistance-related lncRNAs, including CCDC26, ARF, H19, NBR2, NEAT1, and HOTAIR, in GIST cells treated with imatinib H19 was examined at various time-points by qRT-PCR. Based on our results and published literature, CCDC26, a strongly down-regulated lncRNA following imatinib treatment, was chosen as our research target. GIST cells with high expression of CCDC26 were sensitive to imatinib treatment while knockdown of CCDC26 significantly increased the resistance to imatinib. Furthermore, we found that CCDC26 interacted with c-KIT by RNA pull down, and that CCDC26 knockdown up-regulated the expression of IGF-1R. Moreover, IGF-1R inhibition reversed CCDC26 knockdown-mediated imatinib resistance in GIST. These results indicated that treatments targeting CCDC26-IGF-1R axis would be useful in increasing sensitivity to imatinib in GIST.

Genetic Switches between Cancer and Emphysema Resolution of Cigarette-Smoke Induced Inflammation.

Cigarette smoke initiates an inflammatory response that has aftermath long after quitting. We segregated former smokers, according to their lung function and their co-founding diseases, in 3 groups: Cancer, Emphysema and COPD. Then we searched for outlier genes in intersections of Venn diagrams where we identified 6 subsets and 23 genes that may be responsible for disease outcome. Genes expressed in the cancer patients with or without emphysema (PPA subset) were BHLH, FPRL2, CD49D, DEADH, NRs4A3, MBLL, GNS, BE675435, ISGF-3, and FLJ23462. Patients with emphysema as co-founding disease, with or without cancer (APP), had only ANXA2 in common. Genes expressed only in non-cancer patients (AAP subset) of COPD group were IL-1A, SOX13, RPP38; TBXA2R, NPEPL1, CFLAR, TFEB, PRKCBP1, IGF1R, DDX11, and KCNAB1. HIV-1Rev was the gene expressed in cancer patients with emphysema (APA subset). Then, we also looked at out-layers genes significantly expressed in all patients (PPP subset with 5066 genes), the down-regulated in Emphysema were MMP9, PLUNC, CEACAM5, and NR4A1 while the up-regulated were F2R, COL15A1, PDE4C, and BGN. We chose genes and checked them at the protein level on immune cells, this showed that neutrophils from Cancer group had increased expression of CD49d, and their total number was also increased in bronchial-alveolar lavage (154%). Macrophages in the lung of patients with emphysema were associated with a significant increase of adhesion molecule CD58 and to significant CD95 decrease, indicating they do not die. Besides, macrophages downregulated MMP9 in the lung compared to blood macrophages. Overall, we find that cancer progression requires a stickier and greater number of neutrophils in the lung while emphysema requires stickier and longevous macrophages to lead matrix destruction, and together with higher expression of SOX13 and RPP38, may promote autoimmunity. We also identified two genes, ANXA2 and HIV1-rev, that may be a pivot between cancer and emphysema outcome of inflammation.

IGF-1R Regulates the Extracellular Level of Active MMP-2, Pathological Neovascularization, and Functionality in Retinas of OIR Mouse Model.

In ischemic proliferative diseases such as retinopathies, persistent hypoxia leads to the release of numerous neovascular factors that participate in the formation of abnormal vessels and eventually cause blindness. The upregulation and activation of metalloproteinases (MMP-2 and MMP-9) represent a final common pathway in this process. Although many regulators of the neovascular process have been identified, the complete role of the insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) appears to be significantly more complex. In this study, we used an oxygen-induced retinopathy (OIR) mouse model as well as an in vitro model of hypoxia to study the role of MMP-2 derived from Müller glial cells (MGCs) and its relation with the IGF-1/IGF-1R system. We demonstrated that MMP-2 protein expression increased in P17 OIR mice, which coincided with the active phase of the neovascular process. Also, glutamine synthetase (GS)-positive cells were also positive for MMP-2, whereas IGF-1R was expressed by GFAP-positive cells, indicating that both proteins were expressed in MGCs. In addition, in the OIR model a single intravitreal injection of the IGF-1R blocking antibody (αIR3) administered at P12 effectively prevented pathologic neovascularization, accelerated physiological revascularization, and improved retinal functionality at P17. Finally, in MGC supernatants, the blocking antibody abolished the IGF-1 effect on active MMP-2 under normoxic and hypoxic conditions without affecting the extracellular levels of pro-MMP-2. These results demonstrate, for the first time, that the IGF-1/IGF-1R system regulates active MMP-2 levels in MGCs, thus contributing to MEC remodeling during the retinal neovascular process.

Biomarkers of resistance to radiation therapy: a prospective study in cervical carcinoma.

BACKGROUND: Clinical parameters and proteins have recently been suggested as possible causes of radiotherapy (RT) resistance in cervical carcinoma (CC). The objective of the present study was to validate prognostic biomarkers of radiation resistance. METHODS: The present prospective study included patients undergoing RT with curative intent for histologically proven locally advanced squamous cell CC. Tissues and blood samples were systematically collected before RT initiation. Immuno-histochemistry was performed (IGF-IR α and ß, GAPDH, HIF-1 alpha, Survivin, GLUT1, CAIX, hTERT and HKII). Response to radiation was assessed through tumour response 3 months after RT completion, through overall survival (OS) and through progression-free survival (PFS). RESULTS: One hundred forty nine patients with a mean age of 46 years were included, with FIGO IIB (n = 53) and FIGO IIIB (n = 96) CCs. 61 patients were treated with exclusive RT + brachytherapy and 88 underwent chemo-radiotherapy + brachytherapy. Our findings suggest an association between hemoglobin level (Hb) (>11 g/dL) and 3 months complete response (p = 0.02). Hb level < 11 g/dL was associated with decreased PFS (p = 0.05) and OS (p = 0.08). Overexpression of IGF-1R ß was correlated with a decreased OS (p = 0.007). Overexpression of GLUT1 was marginally correlated with reduced OS (p = 0.05). PFS and OS were significantly improved in patients undergoing chemoradiation versus exclusive radiotherapy (PFS: p = 0.04; OS: p = 0.01). CONCLUSIONS: IGF-1R ß overexpression and Hb level (≤11 g/dl) were associated with poor prognosis, and thus appear to be possible interesting biomarkers of radiation resistance. Our results corroborate previous pre-clinical studies suggesting IGF-1R and hypoxia to be part of the biological pathways leading to radio-resistance.

Expanding Genetic and Functional Diagnoses of IGF1R Haploinsufficiencies.

BACKGROUND: The growth-promoting effects of IGF-I is mediated through the IGF-I receptor (IGF1R), a widely expressed cell-surface tyrosine kinase receptor. IGF1R copy number variants (CNV) can cause pre- and postnatal growth restriction or overgrowth. METHODS: Whole exome sequence (WES), chromosomal microarray, and targeted IGF1R gene analyses were performed on 3 unrelated children who share features of small for gestational age, short stature, and elevated serum IGF-I, but otherwise had clinical heterogeneity. Fluorescence-activated cell sorting (FACS) analysis of cell-surface IGF1R was performed on live primary cells derived from the patients. RESULTS: Two novel IGF1R CNV and a heterozygous IGF1R nonsense variant were identified in the 3 patients. One CNV (4.492 Mb) was successfully called from WES, utilizing eXome-Hidden Markov Model (XHMM) analysis. FACS analysis of cell-surface IGF1R on live primary cells derived from the patients demonstrated a ∼50% reduction in IGF1R availability associated with the haploinsufficiency state. CONCLUSION: In addition to conventional methods, IGF1R CNV can be identified from WES data. FACS analysis of live primary cells is a promising method for efficiently evaluating and screening for IGF1R haploinsufficiency. Further investigations are necessary to delineate how comparable IGF1R availability leads to the wide spectrum of clinical phenotypes and variable responsiveness to rhGH therapy.

IRS1/ß-Catenin Axis Is Activated and Induces MYC Expression in Acute Lymphoblastic Leukemia Cells.

Insulin-like growth factor 1 (IGF1) and its receptor IGF1R regulate normal cell growth and contribute to cell transformation through activation of downstream signaling pathways. In fibroblast cells, insulin receptor substrate 1 (IRS1), through IGF1 signaling, was found to be the key protein for nuclear translocation of ß-catenin and MYC transcription activation. We herein investigated the IRS1/ß-catenin axis in acute lymphoblastic leukemia (ALL) cells. Samples were obtained from 45 patients with ALL and 13 healthy donors. ALL cell lines were used. Gene expression was measured by quantitative PCR. Protein expression, associations, and cellular localization were evaluated by immunoprecipitation, subcellular fractionation, and confocal microscopy. Cells were submitted to IGF1 stimulation and/or IGF1R pharmacological inhibition (OSI-906). IRS1, ß-catenin, and MYC mRNA expression were significantly elevated in ALL patients, compared to normal controls. MYC mRNA expression positively correlated with ß-catenin and IRS1. Increased age and MYC expression negatively affected overall survival by univariate analysis. Total and phospho-IGF1R and IRS1, MYC and ß-catenin protein expression were higher in ALL cells, compared to normal peripheral blood mononuclear cells (PBMC). IRS1 and ß-catenin were found to be colocalized in the nuclei and the cytoplasm of ALL cell lines, whereas both proteins were only slightly detected in the cytoplasm of normal PBMC. In Jurkat cells, a constitutive IRS1 and ß-catenin protein interaction were observed; OSI-906 treatment decreased IGF1R tyrosine phosphorylation, IRS1 expression and phosphorylation, nuclear translocation of ß-catenin, IRS1 and ß-catenin association, and MYC protein expression. In conclusion, the IRS1/ß-catenin axis is activated in ALL cells. J. Cell. Biochem. 118: 1774-1781, 2017. © 2016 Wiley Periodicals, Inc.

IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis.

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1µM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.