Rive Sarfstein

Metformin downregulates the insulin/IGF-I signaling pathway and inhibits different uterine serous carcinoma (USC) cells proliferation and migration in p53-dependent or -independent manners.

Accumulating epidemiological evidence shows that obesity is associated with an increased risk of several types of adult cancers, including endometrial cancer. Chronic hyperinsulinemia, a typical hallmark of diabetes, is one of the leading factors responsible for the obesity-cancer connection. Numerous cellular and circulating factors are involved in the biochemical chain of events leading from hyperinsulinemia and insulin resistance to increased cancer risk and, eventually, tumor development. Metformin is an oral anti-diabetic drug of the biguanide family used for treatment of type 2 diabetes. Recently, metformin was shown to exhibit anti-proliferative effects in ovarian and Type I endometrial cancer, although the mechanisms responsible for this non-classical metformin action remain unclear. The insulin-like growth factors (IGFs) play a prominent role in cancer biology and their mechanisms of action are tightly interconnected with the insulin signaling pathways. Given the cross-talk between the insulin and IGF signaling pathways, the aim of this study was to examine the hypothesis that the anti-proliferative actions of metformin in uterine serous carcinoma (USC) are potentially mediated via suppression of the IGF-I receptor (IGF-IR) pathway. Our results show that metformin interacts with the IGF pathway, and induces apoptosis and inhibition of proliferation and migration of USC cell lines with both wild type and mutant p53. Taken together, our results suggest that metformin therapy could be a novel and attractive therapeutic approach for human USC, a highly aggressive variant of endometrial cancer.

Assembly of the phagocyte NADPH oxidase complex : chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships

Phagocytes generate superoxide (O2.−) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH‐driven reduction of oxygen to O2.−, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1–212)‐Rac1 (1–192) is a potent activator in a cell‐free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox‐Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X‐ray scattering, and gel filtration. Based on this, a “propagated wave” model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.

Minireview: Nuclear Insulin and Insulin-like Growth Factor-1 Receptors: A Novel Paradigm in Signal Transduction

The specificity of the insulin receptor (InsR) and insulin-like growth factor-1 receptor (IGF1R) signaling pathways has been the focus of significant debate over the past few years. Recent evidence showing nuclear import and a direct transcriptional role for both InsR and IGF1R adds a new layer of complexity to this dialog. Hence, in addition to the classical roles associated with cell-surface receptors (eg, ligand binding, autophosphorylation of the tyrosine kinase domain, activation of insulin receptor substrate 1 (IRS-1) and additional substrates, protein-protein interactions with membrane and cytoplasm components), new data are consistent with nuclear (genomic) role(s) for both InsR and IGF1R. The present review provides a brief overview of the physical and functional similarities and differences between InsR and IGF1R and describes data from a number of laboratories providing evidence for a new layer of signaling regulation (ie, the ability of InsR and IGF1R to translocate to the cell nucleus and to elicit genomic activities usually associated with transcription factors). The ability of InsR and IGF1R to function as transcription factors, although poorly understood, constitutes a new paradigm in signal transduction. Although research on the role of nuclear InsR/IGF1R is still in its infancy, we believe that this rapidly developing area may have a major basic and translational impact on the fields of metabolism, diabetes, and cancer.

Insulin-like Growth Factor-I Receptor (IGF-IR) Translocates to Nucleus and Autoregulates IGF-IR Gene Expression in Breast Cancer Cells

The insulin-like growth factor (IGF) system plays an important role in mammary gland biology as well as in the etiology of breast cancer. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I and IGF-II, has emerged in recent years as a promising therapeutic target. The IGF and estrogen signaling pathways act in a synergistic manner in breast epithelial cells. The present study was aimed at investigating 1) the putative translocation of IGF-IR and the related insulin receptor (IR) to the nucleus in breast cancer cells, 2) the impact of IGF-IR and IR levels on IGF-IR biosynthesis in estrogen receptor (ER)-positive and ER-depleted breast cancer cells, and 3) the potential transcription factor role of IGF-IR in the specific context of IGF-IR gene regulation. We describe here a novel mechanism of autoregulation of IGF-IR gene expression by cellular IGF-IR, which is seemingly dependent on ER status. Regulation of the IGF-IR gene by IGF-IR protein is mediated at the level of transcription, as demonstrated by 1) binding assays (DNA affinity chromatography and ChIP) showing specific IGF-IR binding to IGF-IR promoter DNA and 2) transient transfection assays showing transactivation of the IGF-IR promoter by exogenous IGF-IR. The IR is also capable of translocating to the nucleus and binding the IGF-IR promoter in ER-depleted, but not in ER-positive, cells. However, transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation. Thus, whereas IGF-IR stimulated IGF-IR gene expression, IR inhibited IGF-IR promoter activity. In summary, we have identified a novel mechanism of IGF-IR gene autoregulation in breast cancer cells. The clinical implications of these findings and, in particular, the impact of IGF-IR/IR nuclear localization on targeted therapy require further investigation.

The intricate involvement of the Insulin-like growth factor receptor signaling in mild traumatic brain injury in mice

Insulin-like growth factor-1 (IGF-1) was suggested as a potential neuroprotective treatment for traumatic brain injury (TBI) induced damage (cognitive as well as cellular). The main goal of the present study was to evaluate the role of the IGF-1R activation in spatial memory outcome following mild traumatic brain injury. mTBI-induced phosphorylation of IGF-1R, AKT and ERK1/2, in mice hippocampus, which was inhibited when mice were pretreated with the selective IGF-1R inhibitor AG1024. IGF-1 administration prevented spatial memory deficits following mTBI. Surprisingly, blocking the IGF-1R signaling in mTBI mice did not augment the spatial memory deficit. In addition, this data imply an intriguing and complex role of the IGF-1 signaling axis in the cellular and behavioral events following mTBI.

Transcriptional regulation of the insulin-like growth factor-I receptor gene in breast cancer

The insulin-like growth factor-I receptor (IGF-IR) has an important role in normal mammary gland growth and morphogenesis. In addition, the IGF-IR has been implicated in the initiation and progression of breast cancer. Previous studies have indicated that acquisition of the malignant phenotype in breast cancer is initially IGF-IR dependent. Most breast cancer-derived cell lines and primary tumors express high levels of IGF-IR mRNA and protein, whereas metastatic stages are usually associated with a decrease in IGF-IR levels. Transcription of the IGF-IR gene is controlled by complex interactions involving DNA-binding and non DNA-binding transcription factors. This review highlights selected examples of tumor suppressors, including BRCA1, p53, and WT1, whose mechanism of action involves regulation of IGF-IR gene expression.

Long-acting insulin analogues elicit atypical signalling events mediated by the insulin receptor and insulin-like growth factor-I receptor

Aims/hypothesisInsulin analogues were developed to improve the pharmacological properties of injected insulin and to better mimic endogenous insulin output. However, certain insulin analogues have been suggested to display IGF-I-like biological activities. Furthermore, several recent epidemiological studies have suggested a potential increase in cancer risk for treatment of diabetes patients with long-acting analogue insulin glargine (A21Gly,B31Arg,B32Arg human insulin). Additional studies, however, reported no increased cancer risk. The purpose of the present study was to identify the receptor(s) and signal transduction pathways responsible for the biological actions of insulin glargine and insulin detemir (B29Lys[ε-tetradecanoyl],desB30 human insulin).MethodsThe colon cancer-derived cell line HCT116 was treated with increasing doses of insulin glargine, insulin detemir, regular insulin or IGF-I, and receptor activation was evaluated by immunoprecipitation assays. IGF-I receptor (IGF-IR) internalisation following insulin glargine treatment was assessed by confocal microscopy. Activation of the Akt and extracellular signal-regulated kinase pathways was evaluated by western blots. The anti-apoptotic effect of the analogues was measured by poly-(ADP ribose) polymerase antibody and annexin assays.ResultsWe found evidence for dual activation of the insulin receptor and IGF-IR by the analogues. Dose-dependency experiments showed that insulin glargine was able to phosphorylate the IGF-IR at fivefold lower doses than those required to activate the insulin receptor. We also showed that insulin glargine can lead to prolonged activation of the receptors and therefore promote abnormal signalling. Confocal imaging experiments showed that insulin glargine, but not regular insulin induced IGF-IR internalisation similarly to IGF-I. Finally, both analogues displayed IGF-I-like anti-apoptotic activities and stimulated cell cycle progression.Conclusions/interpretationOur data indicate that insulin glargine and insulin detemir display atypical signalling activities that differ from those elicited by regular insulin and involve activation of the anti-apoptotic IGF-IR.

The IGF Hormonal Network in Endometrial Cancer: Functions, Regulation, and Targeting Approaches

Epidemiological as well as clinical and experimental data identified the insulin-like growth factors (IGF1, IGF2) as important players in gynecological cancers in general, and endometrial tumors in particular. The IGF1 receptor (IGF1R), which mediates the proliferative and anti-apoptotic activities of both ligands, emerged in recent years as a promising therapeutic target in oncology. However, most clinical trials conducted so far led to mixed results, emphasizing the need to identify biomarkers that can predict responsiveness to anti-IGF1R-targeted therapies. This article will review recent data regarding the role and expression of IGF system components in endometrial cancer. In addition, we will review data on the interplay between the IGF signaling pathway and tumor suppressors p53 and breast cancer susceptibility gene-1 (BRCA1). Anti-oncogenes p53 and BRCA1 play a key role in the etiology of gynecological cancers and, therefore, their interaction with IGF1R is of high relevance in translational terms. A better understanding of the complex mechanisms underlying the regulation of the IGF system will improve our ability to develop effective treatment modalities for endometrial tumors.

The Mechanism of Action of the Histone Deacetylase Inhibitor Vorinostat Involves Interaction with the Insulin-Like Growth Factor Signaling Pathway

A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat.

p53 Regulates insulin-like growth factor-I receptor gene expression in uterine serous carcinoma and predicts responsiveness to an insulin-like growth factor-I receptor-directed targeted therapy.

The role of the insulin-like growth factors (IGF) in endometrial cancer has been well established. The IGF-I receptor (IGF-IR), which mediates the biological actions of IGF-I, is usually overexpressed in endometrial tumours. Uterine serous carcinoma (USC) constitutes a defined histological category among endometrial cancers. Mutation of the p53 gene appears early in the course of the disease and is considered a key event in the initiation of USC. The aim of the present study was to evaluate the potential interactions between p53 and the IGF-IR in USC. In addition, we investigated the role of p53 as a biomarker in IGF-IR targeted therapies. Immunohistochemical analysis in a collection of 35 USC specimens revealed that IGF-IR is highly expressed in primary and metastatic USC. Likewise, p53 was expressed in 85.7% of primary tumours and 100% of metastases. A significant negative correlation between p53 expression and survival was noticed. In addition, using USC-derived cell lines we provide evidence that p53 regulates IGF-IR gene expression via a mechanism that involves repression of the IGF-IR promoter. We show that the mechanism of action of p53 involves interaction with zinc finger protein Sp1, a potent transactivator of the IGF-IR gene. Finally, we demonstrate that USC tumours overexpressing p53 are more likely to benefit from anti-IGF-IR therapies. In summary, we provide evidence that p53 regulates IGF-IR gene expression in USC cells via a mechanism that involves repression of the IGF-IR promoter. The interplay between the p53 and IGF-I signalling pathways is of major basic and translational relevance.