Autocrine IGF-II-Associated Cancers: From a Rare Paraneoplastic Event to a Hallmark in Malignancy.

The paraneoplastic syndrome referred in the literature as non-islet-cell tumor hypoglycemia (NICTH) and extra-pancreatic tumor hypoglycemia (EPTH) was first reported almost a century ago, and the role of cancer-secreted IGF-II in causing this blood glucose-lowering condition has been widely established. The landscape emerging in the last few decades, based on molecular and cellular findings, supports a broader role for IGF-II in cancer biology beyond its involvement in the paraneoplastic syndrome. In particular, a few key findings are constantly observed during tumorigenesis, (a) a relative and absolute increase in fetal insulin receptor isoform (IRA) content, with (b) an increase in IGF-II high-molecular weight cancer-variants (big-IGF-II), and (c) a stage-progressive increase in the IGF-II autocrine signal in the cancer cell, mostly during the transition from benign to malignant growth. An increasing and still under-exploited combinatorial pattern of the IGF-II signal in cancer is shaping up in the literature with respect to its transducing receptorial system and effector intracellular network. Interestingly, while surgical and clinical reports have traditionally restricted IGF-II secretion to a small number of solid malignancies displaying paraneoplastic hypoglycemia, a retrospective literature analysis, along with publicly available expression data from patient-derived cancer cell lines conveyed in the present perspective, clearly suggests that IGF-II expression in cancer is a much more common event, especially in overt malignancy. These findings strengthen the view that (1) IGF-II expression/secretion in solid tumor-derived cancer cell lines and tissues is a broader and more common event compared to the reported IGF-II association to paraneoplastic hypoglycemia, and (2) IGF-II associates to the commonly observed autocrine loops in cancer cells while IGF-I cancer-promoting effects may be linked to its paracrine effects in the tumor microenvironment. Based on these evidence-centered considerations, making the autocrine IGF-II loop a hallmark for malignant cancer growth, we here propose the functional name of IGF-II secreting tumors (IGF-IIsT) to overcome the view that IGF-II secretion and pro-tumorigenic actions affect only a clinical sub-group of rare tumors with associated hypoglycemic symptoms. The proposed scenario provides an updated logical frame towards biologically sound therapeutic strategies and personalized therapeutic interventions for currently unaccounted IGF-II-producing cancers.

Reactivation of p53 mutants by prima-1 [corrected] in thyroid cancer cells.

Most undifferentiated thyroid carcinomas express p53 mutants and thereafter, are very resistant to chemotherapy. p53 reactivation and induction of massive apoptosis (Prima-1) is a compound restoring the tumor-suppressor activity of p53 mutants. We tested the effect of Prima-1 in thyroid cancer cells harboring p53 mutations. Increasing doses of Prima-1 reduced viability of thyroid cancer cells at a variable extent (range 20-80%). Prima-1 up-regulated p53 target genes (p21(WAF1) , BCL2-associated X protein (Bax), and murine double minute 2 (MDM2)), in BC-PAP and Hth-74 cells (expressing D259Y/K286E and K286E p53 mutants) but had no effect in SW1736 (p53 null) and TPC-1 (expressing wild-type p53) thyroid cancer cells. Prima-1 also increased the cytotoxic effects of either doxorubicin or cisplatin in thyroid cancer cells, including the chemo-resistant 8305C, Hth-74 and BC-PAP cells. Moreover, real-time PCR and Western blot indicated that Prima-1 increases the mRNA of thyroid-specific differentiation markers in thyroid cancer cells. Fluorescence-activated cell sorting analysis revealed that Prima-1 effect on thyroid cancer cells occurs via the enhancement of both cell cycle arrest and apoptosis. Small interfering RNA experiments indicated that Prima-1 effect is mediated by p53 mutants but not by the p53 paralog p73. Moreover, in C-643 thyroid cancer cells, forced to ectopically express wild-type p53, Prima-1 prevented the dominant negative effect of double K248Q/K286E p53 mutant. Finally, co-IP experiments indicated that in Hth-74 cells Prima-1 prevents the ability of p53 mutants to sequestrate the p53 paralog TAp73. These in vitro studies imply that p53 mutant reactivation by small compounds may become a novel anticancer therapy in undifferentiated thyroid carcinomas.

Thyroid Stem Cells But Not Differentiated Thyrocytes Are Sensitive to Slightly Increased Concentrations of Heavy Metals.

Thyroid cancer incidence is markedly increased in volcanic areas where residents are biocontaminated by chronic lifelong exposure to slightly increased metals in the environment. Metals can influence the biology of living cells by a variety of mechanisms, depending not only on the dose and length of exposure but also on the type and stage of differentiation of target cells. We explored the effect of five heavy metals (Cu, Hg, Pd, W and Zn) at nanomolar concentrations (the biocontamination level in residents of the volcanic area in Sicily where thyroid cancer is increased) on stimulating the proliferation of undifferentiated (thyrospheres) and differentiated human thyroid cells. Thyrosphere proliferation was significantly increased after exposure to each individual metal and a greater stimulating effect was observed when a mixture of the examined metals was used. No effect was seen in differentiated thyrocytes. For all metals, the dose-response curve followed a biphasic pattern that is typical of hormesis. Thyrosphere growth concerned the size rather than number, except with the metal mixture. An altered morphology was also observed in metal-treated thyrospheres. Metal-induced proliferation was due to activation of the ERK1/2 pathway, as confirmed by growth inhibition when ERK1/2 signaling was blocked. These studies show that stem/precursor thyroid cells are sensitive to small increases in environmental metal concentrations that are harmless for differentiated thyrocytes.

TAp73 alpha increases p53 tumor suppressor activity in thyroid cancer cells via the inhibition of Mdm2-mediated degradation.

p53 family proteins include p53 tumor suppressor, p63, and p73. Despite the high similarity in structure and function with p53, p63, and p73 function in tumor suppression is still controversial. Here, we show that TAp73alpha, a transcriptionally active p73 isoform, is able to synergize p53 tumor suppressor function in thyroid cancer cells. Indeed, depletion of p73 by small interfering RNA in thyroid cancer cells resulted in a reduced transcriptional activity of p53. Ectopic coexpression of both p53 and TAp73alpha in thyroid cancer cells resulted in increased transcription and tumor suppressor function compared with p53 or TAp73alpha alone, as well as in increased p53 protein levels. The enhancing effect of TAp73alpha on p53 activity is Mdm2 dependent because it is prevented by Mdm2 depletion by small interfering RNA. At least two mechanisms may explain the interference of TAp73alpha with p53 function. First, in thyroid cancer cells, TAp73alpha inhibits the effect of p53 on Mdm2 induction by antagonizing p53 at the Mdm2 promoter level. Second, a TAp73alpha mutant (G264W), which is devoid of DNA binding capability, is still able to increase p53 protein levels by competing with p53 for Mdm2 protein binding. Taken together, these results indicate that in thyroid cancer cells, TAp73alpha is able to increase p53 protein level and function by interfering with Mdm2-mediated p53 degradation. These results may be useful for designing gene therapies aimed at restoring a normal p53 function in thyroid cancer cells.

17beta-estradiol up-regulates the insulin-like growth factor receptor through a nongenotropic pathway in prostate cancer cells.

Prostate carcinomas frequently express estrogen receptors (ER), irrespective of androgen receptor (AR) expression; however, the role of ERs and estrogens in prostate cancer is controversial. We found that 17beta-estradiol (E(2)) is able to markedly up-regulate insulin-like growth factor (IGF)-I receptor (IGF-IR) mRNA and protein expression in both AR-positive (LNCaP cells) and AR-negative (PC-3 cells) prostate cancer cells. This effect occurs not only via ERalpha but also via ERbeta stimulation and is specific for IGF-IR because it does not involve the cognate insulin receptor. IGF-IR up-regulation is associated with increased IGF-IR phosphorylation and with increased mitogenic and motogenic activities in response to IGF-I. IGF-IR up-regulation by E(2) does not require ER binding to DNA and is poorly sensitive to antiestrogen blockade, whereas it is associated with the activation of cytosolic kinase cascades involving Src, extracellular signal-regulated kinase (ERK)-1/2, and, to a lesser extent, phosphatidylinositol 3-kinase and is sensitive to the inhibition of these kinases. In conclusion, our data indicate that estrogens may contribute to IGF system deregulation in prostate cancer through the activation of a nongenotropic pathway. Estrogens may have a role, therefore, in tumor progression to androgen independence. Inhibition of the IGF-IR or the Src-ERK pathway should be considered, therefore, as an adjuvant therapy in prostate cancer.

Identification of a novel EphB4 phosphodegron regulated by the autocrine IGFII/IRA axis in malignant mesothelioma.

Malignant mesothelioma is a deadly disease with limited therapeutic options. EphB4 is an oncogenic tyrosine kinase receptor expressed in malignant mesothelioma as well as in a variety of cancers. It is involved in tumor microenvironment mediating angiogenesis and invasive cellular effects via both EphrinB2 ligand-dependent and independent mechanisms. The molecular network underlying EphB4 oncogenic effects is still unclear. Here we show that EphB4 expression in malignant mesothelioma cells is markedly decreased upon neutralization of cancer-secreted IGF-II. In particular, we demonstrate that EphB4 protein expression in malignant mesothelioma cells depend upon a degradation rescue mechanism controlled by the autocrine IGF-II-insulin receptor-A specific signaling axis. We show that the regulation of EphB4 expression is linked to a competing post-translational modification of its carboxy-terminal tail via phosphorylation of its tyrosine 987 by the Insulin receptor isoform-A kinase-associated activity in response to the autocrine IGF-II stimuli. Neutralization of this autocrine-induced EphB4-phosphorylation by IGF-II associates with the increased ubiquitination of EphB4 carboxy-terminal tail and with its rapid degradation. We also describe a novel Ubiquitin binding motif in the targeted region as part of the identified EphB4 phosphodegron and provide 3D modeling data supporting a possible model for the acute EphB4 PTM-driven regulation by IGF-II. Altogether, these findings disclose a novel molecular mechanism for the maintenance of EphB4-expression in malignant mesothelioma cells and other IGF-II-secreting cancers (IGF2omas).

The Copper(II)-Assisted Connection between NGF and BDNF by Means of Nerve Growth Factor-Mimicking Short Peptides.

Nerve growth factor (NGF) is a protein necessary for development and maintenance of the sympathetic and sensory nervous systems. We have previously shown that the NGF N-terminus peptide NGF(1-14) is sufficient to activate TrkA signaling pathways essential for neuronal survival and to induce an increase in brain-derived neurotrophic factor (BDNF) expression. Cu2+ ions played a critical role in the modulation of the biological activity of NGF(1-14). Using computational, spectroscopic, and biochemical techniques, here we report on the ability of a newly synthesized peptide named d-NGF(1-15), which is the dimeric form of NGF(1-14), to interact with TrkA. We found that d-NGF(1-15) interacts with the TrkA-D5 domain and induces the activation of its signaling pathways. Copper binding to d-NGF(1-15) stabilizes the secondary structure of the peptides, suggesting a strengthening of the noncovalent interactions that allow for the molecular recognition of D5 domain of TrkA and the activation of the signaling pathways. Intriguingly, the signaling cascade induced by the NGF peptides ultimately involves cAMP response element-binding protein (CREB) activation and an increase in BDNF protein level, in keeping with our previous result showing an increase of BDNF mRNA. All these promising connections can pave the way for developing interesting novel drugs for neurodegenerative diseases.

Effect of low-dose tungsten on human thyroid stem/precursor cells and their progeny.

Thyroid cancer incidence is increased in volcanic areas where environment pollution biocontaminates residents. Tungsten (W) is the most increased heavy metal in drinking water of Mount Etna volcanic area where it exceeds the normal range in the urine of 27% inhabitants. The possible connection between increased tungsten and thyroid cancer has never been studied. We investigated in vitro the effect tungsten on both human thyrocytes in primary culture, thyrospheres (aggregates of stem/precursor thyroid cells) and thyrocytes differentiated from tungsten-exposed thyrospheres. Chronic exposure to low-dose (nanomolar range, as in the urines of volcanic area residents) soluble tungsten had major biological effects on thyroid stem/precursor cells, promoting growth with a biphasic (hormetic) dose-response and reducing apoptosis. No such effects were observed in mature thyrocytes. In addition, tungsten-exposed thyrospheres had abnormal expression of genes commonly altered also in thyroid cancer and increased activation of the DNA-repair proteins H2AX and 53BP1. Moreover, exposure to tungsten decreased thyrosphere differentiation, as indicated by the reduced expression of thyroid-specific genes in derived thyrocytes that also showed preneoplastic changes such as increased anchorage-independent growth, clonogenic growth and migration capacity. The mechanism of action of tungsten on thyroid stem/precursor cells is unclear but involves membrane G-proteins and activation of the ERK signaling pathway. These data indicate that chronic exposure to slightly increased tungsten, harmless for mature thyrocytes, importantly affects the biology of stem/precursor thyroid cells and of their progeny, inducing characteristics of preneoplastic transformation.

The role of insulin receptors and IGF-I receptors in cancer and other diseases.

There is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression of human cancer. In particular, the insulin-like growth factor receptor (IGF-IR), a tyrosine kinase receptor for IGF-I and IGF-II, has been well documented in cell culture, animal studies, and humans to play a role in malignant transformation, progression, protection from apoptosis, and metastasis. In addition, the hormone insulin (which is very closely related to the IGFs) and its tyrosine kinase receptor (the IR, which is very closely related to the IGR-IR) have been documented both in vitro and in vivo to play a key role in cancer biology. Indeed, several epidemiological studies have shown that insulin resistance status, characterized by hyperinsulinaemia, is associated with an increased risk for a number of malignancies, including carcinomas of the breast, prostate, colon and kidney. Recent data have elucidated some molecular mechanisms by which IR is involved in cancer. IR is over-expressed in several human malignancies. Interestingly, one of the two IR isoform (IR-A) is especially over-expressed in cancer. IR-A is the IR foetal isoform and has the peculiar characteristic to bind not only insulin but also IGF-II. In addition, the IR contributes to formation of hybrid receptors with the IGF-IR (HR). By binding to hybrid receptors, insulin may stimulate specific IGF-IR signalling pathways. Over-expression of IR-A is, therefore, a major mechanism of IGF system over-activation in cancer. In this respect, IR-A isoform and hybrid receptors should be regarded as potential molecular targets, in addition to IGF-IR, for novel anti-cancer therapy. These findings may have important implications for both the prevention and treatment of common human malignancies. They underline the concept that hyperinsulinaemia, associated with insulin resistance and obesity, should be treated by changes in life style and/or pharmacological approaches to avoid an increased risk for cancer. Moreover, native insulin and insulin analogue administration should be carefully evaluated in terms of the possible increase in cancer risk.

Amyloid Beta monomers regulate cyclic adenosine monophosphate response element binding protein functions by activating type-1 insulin-like growth factor receptors in neuronal cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with synaptic dysfunction, pathological accumulation of ß-amyloid (Aß), and neuronal loss. The self-association of Aß monomers into soluble oligomers seems to be crucial for the development of neurotoxicity (J. Neurochem., 00, 2007 and 1172). Aß oligomers have been suggested to compromise neuronal functions in AD by reducing the expression levels of the CREB target gene and brain-derived neurotrophic factor (BDNF) (J. Neurosci., 27, 2007 and 2628; Neurobiol. Aging, 36, 2015 and 20406 Mol. Neurodegener., 6, 2011 and 60). We previously reported a broad neuroprotective activity of physiological Aß monomers, involving the activation of type-1 insulin-like growth factor receptors (IGF-IRs) (J. Neurosci., 29, 2009 and 10582, Front Cell Neurosci., 9, 2015 and 297). We now provide evidence that Aß monomers, by activating the IGF-IR-stimulated PI3-K/AKT pathway, induce the activation of CREB in neurons and sustain BDNF transcription and release.

Functional responses and in vivo anti-tumour activity of h7C10: a humanised monoclonal antibody with neutralising activity against the insulin-like growth factor-1 (IGF-1) receptor and insulin/IGF-1 hybrid receptors.

A novel humanised monoclonal antibody (Mab, h7C10) was raised against the human insulin-like growth factor-1 receptor (IGF-1R); it exhibited potent inhibition of tumour growth in animal models. Further evaluation of its inhibitory activity at hybrid receptors (Hybrid-Rs) composed of the association between IGF-1R and insulin receptor (IR) was performed. Selective, potent and efficacious inhibition of [(125)I]IGF-1 binding as well as IGF-1- and IGF-2-mediated receptor phosphorylation was demonstrated at both IGF-1R and Hybrid-Rs, without activity at IR. Ligand-independent down-regulation of both IGF-1R and Hybrid-Rs was obtained upon long-term association with h7C10. In vivo evaluation was performed in a MDA-MB-231 xenograft mouse model, showing a 14-fold higher level of Hybrid-Rs as compared to IGF-1R. A more potent anti-tumoural response was obtained for h7C10 as compared to Mabs targeting solely IGF-1R or Hybrid-Rs. The herewith described neutralising properties of h7C10 as potent inhibitor of both IGF-1R and Hybrid-Rs are likely to participate in its anti-tumoural activities and maybe of interest for therapeutic applications.

Androgens up-regulate the insulin-like growth factor-I receptor in prostate cancer cells.

In this study, we show that androgens up-regulate insulin-like growth factor-I receptor (IGF-IR) expression and sensitize prostate cancer cells to the biological effects of IGF-I. Both dihydrotestosterone and the synthetic androgen R1881 induced an approximately 6-fold increase in IGF-IR expression in androgen receptor (AR)-positive prostate cancer cells LNCaP. In accordance with IGF-IR up-regulation, treatment with the nonmetabolizable androgen R1881 sensitized LNCaP cells to the mitogenic and motogenic effects of IGF-I, whereas an IGF-IR blocking antibody effectively inhibited these effects. By contrast, these androgens did not affect IGF-IR expression in AR-negative prostate cancer cells PC-3. Reintroduction of AR into PC-3 cells by stable transfection restored the androgen effect on IGF-IR up-regulation. R1881-induced IGF-IR up-regulation was partially inhibited by the AR antagonist Casodex (bicalutamide). Two other AR antagonists, cyproterone acetate and OH-flutamide, were much less effective. Androgen-induced IGF-IR up-regulation was not dependent on AR genomic activity, because two AR mutants, AR-C619Y and AR-C574R, devoid of DNA binding activity and transcriptional activity were still able to elicit IGF-IR up-regulation in HEK293 kidney cells in response to androgens. Moreover, androgen-induced IGF-IR up-regulation involves the activation of the Src-extracellular signal-regulated kinase pathway, because it was inhibited by both the Src inhibitor PP2 and the MEK-1 inhibitor PD98059. The present observations strongly suggest that AR activation may stimulate prostate cancer progression through the altered IGF-IR expression and IGF action. Anti-androgen therapy may be only partially effective, or almost ineffective, in blocking important biological effects of androgens, such as activation of the IGF system.

Chromosome 15 structural abnormalities: effect on IGF1R gene expression and function.

Insulin-like growth factor 1 receptor (IGF1R), mapping on the 15q26.3 chromosome, is required for normal embryonic and postnatal growth. The aim of the present study was to evaluate the IGF1R gene expression and function in three unrelated patients with chromosome 15 structural abnormalities. We report two male patients with the smallest 15q26.3 chromosome duplication described so far, and a female patient with ring chromosome 15 syndrome. Patient one, with a 568 kb pure duplication, had overgrowth, developmental delay, mental and psychomotor retardation, obesity, cryptorchidism, borderline low testis volume, severe oligoasthenoteratozoospermia and gynecomastia. We found a 1.8-fold increase in the IGF1R mRNA and a 1.3-fold increase in the IGF1R protein expression (P < 0.05). Patient two, with a 650 kb impure duplication, showed overgrowth, developmental delay, mild mental retardation, precocious puberty, low testicular volume and severe oligoasthenoteratozoospermia. The IGF1R mRNA and protein expression was similar to that of the control. Patient three, with a 46,XX r(15) (p10q26.2) karyotype, displayed intrauterine growth retardation, developmental delay, mental and psychomotor retardation. We found a <0.5-fold decrease in the IGF1R mRNA expression and an undetectable IGF1R activity. After reviewing the previously 96 published cases of chromosome 15q duplication, we found that neurological disorders, congenital cardiac defects, typical facial traits and gonadal abnormalities are the prominent features in patients with chromosome 15q duplication. Interestingly, patients with 15q deletion syndrome display similar features. We speculate that both the increased and decreased IGF1R gene expression may play a role in the etiology of neurological and gonadal disorders.

Peroxisomal proliferator-activated receptor-gamma agonists induce partial reversion of epithelial-mesenchymal transition in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is an extremely aggressive tumor characterized by marked epithelial mesenchymal transition, which leads, almost invariably, to death. Peroxisomal proliferator-activated receptor (PPAR)-gamma agonists have recently emerged as potential antineoplastic drugs. To establish whether ATC could be a target of PPAR gamma agonists, we first examined PPAR gamma protein expression in a panel of six ATC cell lines and then studied the biologic effects of two PPAR gamma agonists, ciglitazone and rosiglitazone, that belong to the class of thiazolidonediones. PPAR gamma protein was present and functional in all ATC cell lines. Both ciglitazone and rosiglitazone showed complex biological effects in ATC cells, including inhibition of anchorage-dependent and -independent growth and migration, and increased apoptosis rate. Rosiglitazone-induced growth inhibition was associated with cell cycle arrest and changes in cell cycle regulators, such as an increase of cyclin-dependent kinases inhibitors p21(cip1) and p27(kip1), a decrease of cyclin D1, and inactivation of Rb protein. Rosiglitazone-induced apoptosis was associated with a decrease of Bcl-X(L) expression and caspase-3 and -7 activation. Moreover, rosiglitazone antagonized IGF-I biological effects by up-regulating phosphatase and tensin homolog deleted from chromosome 10 with subsequent inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway. Finally, rosiglitazone increased the expression of thyroid-specific differentiation markers. In conclusions, these data suggest that PPAR gamma agonists induce a partial reversion of the epithelial mesenchymal transition in ATC cells by multiple mechanisms. PPAR gamma agonists may, therefore, have a role in the multimodal therapy currently used to slow down ATC growth and dissemination.

The Inorganic Side of NGF: Copper(II) and Zinc(II) Affect the NGF Mimicking Signaling of the N-Terminus Peptides Encompassing the Recognition Domain of TrkA Receptor.

The nerve growth factor (NGF) N-terminus peptide, NGF(1-14), and its acetylated form, Ac-NGF(1-14), were investigated to scrutinize the ability of this neurotrophin domain to mimic the whole protein. Theoretical calculations demonstrated that non-covalent forces assist the molecular recognition of TrkA receptor by both peptides. Combined parallel tempering/docking simulations discriminated the effect of the N-terminal acetylation on the recognition of NGF(1-14) by the domain 5 of TrkA (TrkA-D5). Experimental findings demonstrated that both NGF(1-14) and Ac-NGF(1-14) activate TrkA signaling pathways essential for neuronal survival. The NGF-induced TrkA internalization was slightly inhibited in the presence of Cu2+ and Zn2+ ions, whereas the metal ions elicited the NGF(1-14)-induced internalization of TrkA and no significant differences were found in the weak Ac-NGF(1-14)-induced receptor internalization. The crucial role of the metals was confirmed by experiments with the metal-chelator bathocuproine disulfonic acid, which showed different inhibitory effects in the signaling cascade, due to different metal affinity of NGF, NGF(1-14) and Ac-NGF(1-14). The NGF signaling cascade, activated by the two peptides, induced CREB phosphorylation, but the copper addition further stimulated the Akt, ERK and CREB phosphorylation in the presence of NGF and NGF(1-14) only. A dynamic and quick influx of both peptides into PC12 cells was tracked by live cell imaging with confocal microscopy. A significant role of copper ions was found in the modulation of peptide sub-cellular localization, especially at the nuclear level. Furthermore, a strong copper ionophoric ability of NGF(1-14) was measured. The Ac-NGF(1-14) peptide, which binds copper ions with a lower stability constant than NGF(1-14), exhibited a lower nuclear localization with respect to the total cellular uptake. These findings were correlated to the metal-induced increase of CREB and BDNF expression caused by NGF(1-14) stimulation. In summary, we here validated NGF(1-14) and Ac-NGF(1-14) as first examples of monomer and linear peptides able to activate the NGF-TrkA signaling cascade. Metal ions modulated the activity of both NGF protein and the NGF-mimicking peptides. Such findings demonstrated that NGF(1-14) sequence can reproduce the signal transduction of whole protein, therefore representing a very promising drug candidate for further pre-clinical studies.

In IGF-I receptor-deficient leiomyosarcoma cells autocrine IGF-II induces cell invasion and protection from apoptosis via the insulin receptor isoform A.

One of the two isoforms of the human insulin receptor (isoform A or IR-A) binds IGF-II with high affinity and is predominantly expressed in fetal tissues and malignant cells. We evaluated the biological relevance of IR-A in human myosarcoma cells. Six myosarcoma cell lines were studied. All produced high amounts of IGF-II and five of them predominantly expressed IR-A. SKUT-1 leiomyosarcoma cells, that do not express the IGF-IR, were identified as a suitable model to study the effects of IR-A in the absence of the interference of IGF-IR. In these cells, which express high levels of IR with an IR-A relative abundance of approximately 95%, IGF-II elicits biological effects exclusively via IR-A activation and IGF-I is almost ineffective. Blockade of autocrine IGF-II reduced unstimulated cell viability and migration. Although both insulin and IGF-II activate IR-A, these two ligands showed a different ability to activate different intracellular signaling pathways and to elicit different biological effects. Insulin was more potent than IGF-II in activating the PI3-K/Akt pathway and in protecting cells from apoptosis. In contrast, IGF-II was more potent than insulin in activating the Shc/ERK pathway and in stimulating cell migration. These data indicate that IGF-II sensitive IR-A is the predominant IR isoform in a variety of myosarcoma cells. In SKUT-1 leiomyoma cells this fetal IR isoform may vicariate the IGF-IR for cell response to both insulin and IGF-II. Acting on the same IR-A receptor IGF-II is more potent than insulin in stimulating cancer cell migration.

Monomeric ß-amyloid interacts with type-1 insulin-like growth factor receptors to provide energy supply to neurons.

ß-amyloid (Aß1-42) is produced by proteolytic cleavage of the transmembrane type-1 protein, amyloid precursor protein. Under pathological conditions, Aß1-42self-aggregates into oligomers, which cause synaptic dysfunction and neuronal loss, and are considered the culprit of Alzheimer's disease (AD). However, Aß1-42 is mainly monomeric at physiological concentrations, and the precise role of monomeric Aß1-42 in neuronal function is largely unknown. We report that the monomer of Aß1-42 activates type-1 insulin-like growth factor receptors and enhances glucose uptake in neurons and peripheral cells by promoting the translocation of the Glut3 glucose transporter from the cytosol to the plasma membrane. In neurons, activity-dependent glucose uptake was blunted after blocking endogenous Aß production, and re-established in the presence of cerebrospinal fluid Aß. APP-null neurons failed to enhance depolarization-stimulated glucose uptake unless exogenous monomeric Aß1-42 was added. These data suggest that Aß1-42 monomers were critical for maintaining neuronal glucose homeostasis. Accordingly, exogenous Aß1-42 monomers were able to rescue the low levels of glucose consumption observed in brain slices from AD mutant mice.

Interleukin-4 stimulates papillary thyroid cancer cell survival: implications in patients with thyroid cancer and concomitant Graves' disease.

IL-4, a pleiotropic cytokine mainly produced by activated helper T lymphocytes type 2 (Th2), is known to protect thyroid cells from autoimmune damage. Acting via its receptors (IL-4Ralpha), IL-4 has antiproliferative and apoptotic effects in many malignancies. Its effect in thyroid cancer is unknown. We found that surgical specimens of thyroid carcinomas express both IL-4Ralpha and IL-4 in the majority of cases. Thyroid glands affected by Graves' disease also express IL-4. We also studied a panel of eight thyroid cancer cell lines from different histotypes and found that thyroid cancer cells express high levels of IL-4Ralpha although they do not express IL-4. We then compared the biological effects of IL-4 in TPC-1, a thyroid cancer cell line, and in MCF-7 breast cancer cells. IL-4 very weakly stimulated thyroid cancer cell proliferation, but it was very effective in protecting thyroid cancer cells from apoptosis induced by staurosporin. The protective effect of IL-4 was similar in magnitude to that of IGF-I and was associated with up-regulation of the antiapoptotic molecule Bcl-2 and weak down-regulation of the proapoptotic molecule Bax. Moreover, IL-4 slightly potentiated the survival effect of IGF-I. In contrast, IL-4 reduced growth and induced apoptosis in MCF-7 cells. Taken together, these findings suggest that thyroid cancer cells receive significant protection from apoptosis by IL-4 produced in the thyroid gland by activated T lymphocytes when concomitant Graves' disease is present.

A novel autocrine loop involving IGF-II and the insulin receptor isoform-A stimulates growth of thyroid cancer.

The insulin receptor (IR) occurs in two isoforms (IR-A and IR-B) resulting from alternative splicing of exon 11 of the gene. The IR-A isoform is predominantly expressed in fetal tissues and malignant cells and binds IGF-II with high affinity. We previously observed that IRs are overexpressed in thyroid cancer cells; now we evaluated whether these cells preferentially express IR-A and produce IGF-II, which would activate a growth-promoting autocrine loop. The IR content ranged 6.0-52.6 ng/100 microg cell membrane protein in thyroid cancer primary cultures (n = 8) and permanent cell lines (n = 6) vs. 1.2-1.7 in normal thyroid cells (n = 11 primary cultures; P < 0.0001). IR-A isoform relative abundance ranged from 36-79% in cancer cells (with the highest values in undifferentiated cancers) vs. 27-39% in normal cells. Similar results were obtained in normal vs. cancer thyroid tissue specimens. IGF-II caused IR autophosphorylation with an ED(50) of 1.5-40.0 nM in cancer cells vs. more than 100 nM in normal cells; IGF-II affinity correlated with the relative abundance of IR-A (r = 0.628; P < 0.0001). IGF-II was expressed in all cancer cells, highly expressed in anaplastic cells, and less expressed in normal cells. In conclusion, malignant thyrocytes, especially when poorly differentiated, produce IGF-II and overexpress IR, predominantly as IGF-II-sensitive isoform A. A growth-promoting autocrine loop is activated, therefore, and may affect thyroid cancer biology.