Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.     

Metabotropic glutamate receptor 1 mediates melanocyte transformation via transactivation of insulin‐like growth factor 1 receptor

Our laboratory previously described the oncogenic properties of metabotropic glutamate receptor 1 (mGluR1) in melanocytes. mGluR1 transformed immortalized mouse melanocytes in vitro and induced vigorous tumor formation in vivo. Subsequently, we observed the activation of PI3K/AKT in mGluR1‐mediated melanocytic tumorigenesis in vivo. In particular, we identified AKT2 being the predominant isoform contributing to the activation of AKT. Suppression of Grm1 or AKT2 using an inducible Tet‐R siRNA system resulted in a 60 or 30% reduction, respectively, in in vivo tumorigenesis. We show that simultaneous downregulation of Grm1 plus AKT2 results in a reduction of approximately 80% in tumor volumes, suggesting that both mGluR1 and AKT2 contribute to the tumorigenic phenotype in vivo. The discrepancy between the mild in vitro transformation characteristics and the aggressive in vivo tumorigenic phenotypes of these stable mGluR1‐melanocytic clones led us to investigate the possible involvement of other growth factors. Here, we highlight a potential crosstalk network between mGluR1 and tyrosine kinase, insulin‐like growth factor 1 receptor (IGF‐1R).     

SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation

Increasing number of studies have shown nuclear localization of the insulin‐like growth factor 1 receptor (nIGF‐1R) in tumor cells and its links to adverse clinical outcome in various cancers. Any obvious cell physiological roles of nIGF‐1R have, however, still not been disclosed. Previously, we reported that IGF‐1R translocates to cell nucleus and modulates gene expression by binding to enhancers, provided that the receptor is SUMOylated. In this study, we constructed stable transfectants of wild type IGF1R (WT) and triple‐SUMO‐site‐mutated IGF1R (TSM) using igf1r knockout mouse fibroblasts (R‐). Cell clones (R‐WT and R‐TSM) expressing equal amounts of IGF‐1R were selected for experiments. Phosphorylation of IGF‐1R, Akt, and Erk upon IGF‐1 stimulation was equal in R‐WT and R‐TSM. WT was confirmed to enter nuclei. TSM did also undergo nuclear translocation, although to a lesser extent. This may be explained by that TSM heterodimerizes with insulin receptor, which is known to translocate to cell nuclei. R‐WT proliferated substantially faster than R‐TSM, which did not differ significantly from the empty vector control. Upon IGF‐1 stimulation G1‐S‐phase progression of R‐WT increased from 12 to 38%, compared to 13 to 20% of R‐TSM. The G1‐S progression of R‐WT correlated with increased expression of cyclin D1, A, and CDK2, as well as downregulation of p27. This suggests that SUMO‐IGF‐1R affects upstream mechanisms that control and coordinate expression of cell cycle regulators. Further studies to identify such SUMO‐IGF‐1R dependent mechanisms seem important.     

Low insulin‐like growth factor‐1 level predicts survival in humans with exceptional longevity

Attenuated growth hormone and insulin-like growth factor-1 (GH/IGF-1) signaling is associated with extended lifespan in several animal models. However, the effect of diminished GH/IGF-1 activity on survival in humans has not been confirmed. We tested the hypothesis that IGF-1 levels in nonagenarians (n = 184), measured at study enrollment, predict the duration of their incremental survival. In the Kaplan–Meier analysis, females with IGF-1 levels below the median (≤ 96 ng mL⁻¹) had significantly longer survival compared with females with levels above the median, P < 0.01. However, this survival advantage was not observed in males (P = 0.83). On the other hand, in both males and females with a history of cancer, lower IGF-1 levels predicted longer survival (P < 0.01). IGF-1 level remained a significant predictor of survival duration in linear regression models after multivariable adjustment in females (P = 0.01) and individuals with a history of cancer (P < 0.01). We show for the first time that low IGF-1 levels predict life expectancy in exceptionally long-lived individuals.     

Probing the disulfide folding pathway of insulin‐like growth factor‐I

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin‐like growth factor (IGF)‐I which when refolded in vitro produces the native three‐disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF‐I which contains a 13‐amino acid N‐terminal extension and a charge mutation at position 3 (Long‐ [Arg³]IGF‐I). Unlike IGF‐I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long‐[Arg³]GF‐I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long‐[Arg³]IGF‐I and IGF‐I, we acid‐trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non‐native intermediates, three native‐like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long‐[Arg³]IGF‐I; a single‐disulfide Cys¹⁸–Cys⁶¹ intermediate, an intermediate with Cys¹⁸–Cys⁶¹ and Cys⁶–Cys⁴⁸ disulfide bonds and another with Cys¹⁸–Cys⁶¹ and Cys⁴⁷–Cys⁵² disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys¹⁸‐Cys⁶¹, Cys⁶‐Cys⁴⁸ intermediate forms the native structure, not by the direct formation of the last (Cys⁴⁷‐Cys⁵²) disulfide bond, but by rearrangement via the Cys¹⁸–Cys⁶¹ intermediate and a productive Cys¹⁸–Cys⁶¹, Cys⁴⁷–Cys⁵² intermediate. In this pathway, the last disulfide bond to form involves Cys⁶ and Cys⁴⁸. Finally, we apply this pathway to IGF‐I and conclude that the divergence in the in vitro folding pathway of IGF‐I is caused by non‐native interactions involving Glu³ that stabilize the alternative structure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 693–703, 1999.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.     

Analysis of monosomy‐3 in immunomagnetically isolated circulating melanoma cells in uveal melanoma patients

Monosomy‐3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno‐FISH assay to detect chromosome‐3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non‐metastatic UM, of which 58% were positive for monosomy‐3. The monosomy‐3 status of CMC corresponded to the monosomy‐3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy‐3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow‐up period of 4 yr. This non‐invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.     

Transplant of insulin‐like growth factor‐1 expressing bone marrow stem cells improves functional regeneration of injured rat uterus by NF‐κB pathway

To investigate the potential beneficial effect of insulin‐like growth factor‐1 (IGF‐1) in BMSC transplantation therapy of uterus injury and the underlying molecular mechanisms, rat BMSCs were isolated and cultured. The relative expressions of IGF‐1 and IL‐10 were determined by RT‐PCR and immunoblotting. The secretory IL‐10 and released E2 were measured using ELISA kits. The relative vWF and α‐SMA expressions were determined by immunohistochemistry. The direct binding of NF‐κB subunit p50 with IL‐10 promoter was analysed by chromatin immunoprecipitation assay. The regulation of IL‐10 expression by p50 was interrogated by luciferase reporter assay. Our data demonstrated that IGF‐1 expression in BMSCs induced IL‐10 expression and secretion, which was further enhanced by E2‐PLGA. IGF‐1 overexpression improved BMSCs transplantation therapy in rat uterus injury. We further demonstrated that both inhibition and knockdown of p50 abolished IGF‐1‐induced expression and secretion of IL‐10 in BMSCs, which consequently compromised the IGF‐1 conferred therapeutic benefits against uterus injury. Furthermore, we elucidated that p50 regulated IL‐10 expression via direct association with its promoter. Our data suggested that transplantation of IGF‐1 overexpressing BMSCs improved functional regeneration of injured uterus by inducing IL‐10 expression and secretion via activation of NF‐κB signalling.     

Mediation of vertebrate life histories via insulin‐like growth factor‐1

Life‐history traits describe parameters associated with growth, size, survival, and reproduction. Life‐history variation is a hallmark of biological diversity, yet researchers commonly observe that one of the major axes of life‐history variation after controlling for body size involves trade‐offs among growth, reproduction, and longevity. This persistent pattern of covariation among these specific traits has engendered a search for shared mechanisms that could constrain or facilitate production of variation in life‐history strategies. Endocrine traits are one candidate mechanism that may underlie the integration of life history and other phenotypic traits. However, the vast majority of this research has been on the effects of steroid hormones such as glucocorticoids and androgens on life‐history trade‐offs. Here we propose an expansion of the focus on glucocorticoids and gonadal hormones and review the potential role of insulin‐like growth factor‐1 (IGF‐1) in shaping the adaptive integration of multiple life‐history traits. IGF‐1 is a polypeptide metabolic hormone largely produced by the liver. We summarize a vast array of research demonstrating that IGF‐1 levels are susceptible to environmental variation and that IGF‐1 can have potent stimulatory effects on somatic growth and reproduction but decrease lifespan. We review the few studies in natural populations that have measured plasma IGF‐1 concentrations and its associations with life‐history traits or other characteristics of the organism or its environment. We focus on two case studies that found support for the hypothesis that IGF‐1 mediates adaptive divergence in suites of life‐history traits in response to varying ecological conditions or artificial selection. We also examine what we view as potentially fruitful avenues of research on this topic, which until now has been rarely investigated by evolutionary ecologists. We discuss how IGF‐1 may facilitate adaptive plasticity in life‐history strategies in response to early environmental conditions and also how selection on loci controlling IGF‐1 signaling may mediate population divergence and eventual speciation. After consideration of the interactions among androgens, glucocorticoids, and IGF‐1 we suggest that IGF‐1 be considered a suitable candidate mechanism for mediating life‐history traits. Finally, we discuss what we can learn about IGF‐1 from studies in free‐ranging animals. The voluminous literature in laboratory and domesticated animals documenting relationships among IGF‐1, growth, reproduction, and lifespan demonstrates the potential for a number of new research questions to be asked in free‐ranging animals. Examining how IGF‐1 mediates life‐history traits in free‐ranging animals could lead to great insight into the mechanisms that influence life‐history variation.     

Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Insulin‐like growth factor 1 receptor signaling induced by supraphysiological doses of IGF‐1 in human peripheral blood lymphocytes

Insulin‐like growth factor‐1 (IGF‐1) mediates some of growth hormone anabolic functions through its receptor, IGF‐1R. Following ligand binding, intracellular signaling pathways are activated favouring proliferation, cell survival, tissue growth, development, and differentiation. IGF‐1 is included in the World Anti‐Doping Agency Prohibited List. While the evidence for IGF‐1 as performance‐enhancing substrate in healthy humans is still weak, clinical studies demonstrated that the endogenous growth hormone/IGF‐1 excess is associated with cardiovascular implications. Previously, we demonstrated that human peripheral blood lymphocytes represent a suitable system to identify a gene signature, related to dihydrotestosterone or IGF‐1 abuse, independent from the type of sport. In addition, in a proteomic study, we demonstrated that dihydrotestosterone hyperdosage affects cell motility and apoptosis. Here, we investigate the doping action of IGF‐1 by means of a differential proteomic approach and specific protein arrays, revealing an active cytoskeletal reorganization mediated by Stat‐1; moreover, IGF‐1 stimulation produces a sustained activation of different signaling pathways as well as an overproduction of cytokines positively related to immune response and inflammation. In conclusion, these data indicate that, following IGF‐1 hyperdosage, circulating peripheral blood lymphocytes could be more prone to transendothelial migration.     

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.