Enhancement of insulin-like growth factor signaling in human breast cancer: estrogen regulation of insulin receptor substrate-1 expression in vitro and in vivo.

Cross-talk between insulin-like growth factor (IGF)- and estrogen receptor (ER)-signaling pathways results in synergistic growth. We show here that estrogen enhances IGF signaling by inducing expression of three key IGF-regulatory molecules, the type 1 IGF receptor (IGFR1) and its downstream signaling molecules, insulin receptor substrate (IRS)-1 and IRS-2. Estrogen induction of IGFR1 and IRS expression resulted in enhanced tyrosine phosphorylation of IRS-1 after IGF-I stimulation, followed by enhanced mitogen-activated protein kinase activation. To examine whether these pathways were similarly activated in vivo, we examined MCF-7 cells grown as xenografts in athymic mice. IRS-1 was expressed at high levels in estrogen-dependent growth of MCF-7 xenografts, but withdrawal of estrogen, which decreased tumor growth, resulted in a dramatic decrease in IRS-1 expression. Finally, we have shown that high IRS-1 expression is an indicator of early disease recurrence in ER-positive human primary breast tumors. Taken together, these data not only reinforce the concept of cross-talk between IGF- and ER-signaling pathways, but indicate that IGF molecules may be critical regulators of estrogen-mediated growth and breast cancer pathogenesis.     

Benzyl isothiocyanate inhibits basal and hepatocyte growth factor-stimulated migration of breast cancer cells

Benzyl isothiocyanate (BITC), which is found in cruciferous vegetables, has been shown to have anti-carcinogenic properties. Hepatocyte growth factor (HGF) has the ability to stimulate dissociation, migration, and invasion in various tumor cells, and abnormally increased expressions of HGF and its transmembrane tyrosine kinase receptor, c-Met, have previously been detected in human breast cancer, and are associated with high tumor grade and poor prognosis. In this study, in order to assess the mechanisms relevant to the BITC-induced regulation of breast cancer cell migration and invasion, MDA-MB-231 human breast cancer cells and 4T1 murine mammary carcinoma cells were cultured in the presence of 0-4?μmol/l BITC with or without 10?μg/l of HGF. BITC inhibited both the basal and HGF-induced migration of MDA-MB-231 and 4T1 cells in a dose-dependent manner. In MDA-MB-231 cells, BITC reduced both basal and HGF-induced secretion and activity of urokinase-type plasminogen activator (uPA). In addition, BITC increased the protein levels of plasminogen activator inhibitor-1. HGF stimulated c-Met and Akt phosphorylation, but did not affect the phosphorylation of extracellular signal-regulated kinase-1/2 or stress-activated protein/c-jun N-terminal kinase. BITC suppressed NF-κB activity and reduced the HGF-induced phosphorylation of c-Met and Akt in a dose-dependent manner. LY294002, a specific Akt inhibitor, reduced both basal and HGF-induced uPA secretion and migration of MDA-MB-231 cells. In this study, we demonstrated that BITC profoundly inhibits the migration and invasion of MDA-MB-231 cells, which is associated with reduced uPA activity, and also that these phenomena are accompanied by the suppression of Akt signaling.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation.

The insulin receptor substrates (IRSs)-1-4 play important roles in signal transduction emanating from the insulin and insulin-like growth factor (IGF)-I receptors. IRS-4 is the most recently characterized member, which has been found primarily in human cells and tissues. It interacts with SH2-containing proteins such as phosphatidylinositol 3'-kinase (PI3K), Grb2, Crk-II, and CrkL. In this study, we transfected IRS-4 in mouse NIH-3T3 cells that overexpress IGF-I receptors. Clones expressing IRS-4 showed enhanced cellular proliferation when cells were cultured in 1% fetal bovine serum without added IGF-I. Addition of IGF-I enhanced cellular proliferation in cells overexpressing the IGF-I receptor alone but had an even greater proliferative effect in cells overexpressing both the IGF-I receptors and IRS-4. When etoposide and methylmethane sulfonate (MMS), both DNA damaging agents, were added to the cells, they uniformly induced cell cycle arrest. Fluorescence-activated cell sorter analysis demonstrated that the arrest of the cell cycle occurred at the G(1) checkpoint, and furthermore no significant degree of apoptosis was demonstrated with the use of either agent. In cells, overexpressing IGF-I receptors alone, IGF-I addition enhanced cellular proliferation, even in the presence of etoposide and MMS. In cells overexpressing IGF-I receptors and IRS-4, the effect of IGF-I in overcoming the cell cycle arrest was even more pronounced. These results suggest that IRS-4 is implicated in the IGF-I receptor mitogenic signaling pathway.     

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.     

The N terminus of mannose 6-phosphate/insulin-like growth factor 2 receptor in regulation of fibrinolysis and cell migration

Leukocyte migration to sites of inflammation is a multistep process involving transient adhesion to the endothelium followed by cell surface-controlled proteolysis for transmigration through the vessel wall and chemotactic movement within tissues. One of the key players in this machinery appears to be the urokinase-type plasminogen activator (uPA)/uPA receptor system. The role of uPA and its receptor (CD87) in plasminogen (Plg) activation, cell adhesion, and chemotaxis is well established; however, less is known of how these activities are regulated. Here we provide evidence that the mannose 6-phosphate/insulin-like growth factor 2 receptor (CD222) controls CD87-mediated functions. Expression of human CD222 in CD222-/- mouse fibroblasts down-regulated Plg activation, cell adhesion, and chemotaxis induced by the uPA/CD87 system. In addition, we demonstrate that the N-terminal region of CD222, which is similar to the Plg-binding site of streptokinase, plays a crucial role in binding of CD87 and Plg. A peptide derived from this region in CD222 is able to disrupt the physical interaction of CD222 with CD87 and, furthermore, mimics the inhibitory effects of CD222 on CD87 functions. Taken together, our results indicate a novel role for CD222 in regulation of fibrinolysis, cell adhesion, and migration.     

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

Progesterone receptor regulation by retinoic acid in the human breast cancer cell line T-47D

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.     

Direct cross-talk of interleukin-6 and insulin signal transduction via insulin receptor substrate-1 in skeletal muscle cells

The exercise-induced interleukin (IL)-6 production and secretion within skeletal muscle fibers has raised the question of a putative tissue-specific function of IL-6 in the energy metabolism of the muscle during and after the exercise. In the present study, we followed the hypothesis that IL-6 signaling may directly interact with insulin receptor substrate (IRS)-1, a keystone in the insulin signaling cascade. We showed that IL-6 induces a rapid recruitment of IRS-1 to the IL-6 receptor complex in cultured skeletal muscle cells. Moreover, IL-6 induced a rapid and transient phosphorylation of Ser-318 of IRS-1 in muscle cells and in muscle tissue, but not in the liver of IL-6-treated mice, probably via the IL-6-induced co-recruitment of protein kinase C-delta. This Ser-318 phosphorylation improved insulin-stimulated Akt phosphorylation and glucose uptake in myotubes since transfection with an IRS-1/Glu-318 mutant simulating a permanent phospho-Ser-318 modification increased Akt phosphorylation and glucose uptake. Noteworthily, two inhibitory mechanisms of IL-6 on insulin action, phosphorylation of the inhibitory Ser-307 residue of IRS-1 and induction of SOCS-3 expression, were only found in liver but not in muscle of IL-6-treated mice. Thus, the data provided evidence for a possible molecular mechanism of the physiological metabolic effects of IL-6 in skeletal muscle, thereby exerting short term beneficial effects on insulin action.     

c-Raf-mediated inhibition of epidermal growth factor-stimulated cell migration.

Epidermal growth factor stimulates migration of a number of cell types, yet the signaling pathways that regulate epidermal growth factor-stimulated migration are poorly defined. In this report, we employ a transient transfection migration assay to assess the role of components of the Ras-mitogen-activated protein (MAP) kinase signaling pathway in epidermal growth factor-stimulated chemotaxis of rat embryo fibroblasts. Expression of dominant negative Ras blocks epidermal growth factor-mediated chemotaxis, while constitutively active Ras has no effect on chemokinesis or chemotaxis. PD98059 and U0126, inhibitors of MAP kinase kinase (MEK) activity, decreased epidermal growth factor-stimulated migration, while kinase-defective MEK1, an inhibitor of MAP kinase activation, enhanced migration. To understand the paradoxical effects of these molecules on epidermal growth factor-induced migration, we examined the role of c-Raf on migration. Expression of either wild type c-Raf or the catalytic domain of c-Raf effectively inhibited epidermal growth factor-stimulated cell migration. We suggest that, whereas Ras activity is necessary to promote epidermal growth factor-stimulated migration, sustained activation of c-Raf may be important in down-regulating migratory signaling pathways triggered by epidermal growth factor receptor activation. Further, activation of c-Raf upon inhibition of the MEK-MAP kinase pathway may contribute to the inhibition of cell migration observed with pharmacological MEK inhibitors.     

Secretion of Annexin II via activation of insulin receptor and insulin-like growth factor receptor

Annexin II is secreted into the extracellular environment, where, via interactions with specific proteases and extracellular matrix proteins, it participates in plasminogen activation, cell adhesion, and tumor metastasis and invasion. However, mechanisms regulating annexin II transport across the cellular membrane are unknown. In this study, we used coimmunoprecipitation to show that Annexin-II was bound to insulin and insulin-like growth factor-1 (IGF-1) receptors in PC12 cells and NIH-3T3 cells overexpressing insulin (NIH-3T3(IR)) or IGF-1 receptor (NIH-3T3(IGF-1R)). Stimulation of insulin and IGF-1 receptors by insulin caused a temporary dissociation of annexin II from these receptors, which was accompanied by an increased amount of extracellular annexin II detected in the media of PC12, NIH-3T3(IR), and NIH-3T3(IGF-1R) cells but not in that of untransfected NIH-3T3 cells. Activation of a different growth factor receptor, the platelet-derived growth factor receptor, did not produce such results. Tyrphostin AG1024, a tyrosine kinase inhibitor of insulin and IGF-1 receptor, was shown to inhibit annexin II secretion along with reduced receptor phosphorylation. Inhibitors of a few downstream signaling enzymes including phosphatidylinositol 3-kinase, pp60c-Src, and protein kinase C had no effect on insulin-induced annexin II secretion, suggesting a possible direct link between receptor activation and annexin II secretion. Immunocytochemistry revealed that insulin also induced transport of the membrane-bound form of annexin II to the outside layer of the cell membrane and appeared to promote cell aggregation. These results suggest that the insulin receptor and its signaling pathways may participate in molecular mechanisms mediating annexin II secretion.     

Role of insulin-like growth factors and the type I insulin-like growth factor receptor in the estrogen-stimulated proliferation of human breast cancer cells

Estrogen sensitizes the MCF-7 estrogen-responsive breast cancer cell line to the mitogenic effect of insulin and the insulin-like growth factors (IGFs). This sensitization is specific for estrogen and occurs at physiological concentrations of estradiol. Dose-response experiments with insulin, IGF-I, and IGF-II suggested that the sensitization is mediated through the type I IGF receptor. Binding experiments with 125I-IGF-I and hybridization of a type I IGF receptor probe to RNA showed that the levels of the type I IGF receptor and its mRNA are increased 7- and 6.5-fold, respectively, by estradiol. IGF-I and estradiol had similar synergistic effects on other estrogen-responsive breast cancer cell lines, but IGF-I alone increased the proliferation of the MDA MB-231 cell line which is not responsive to estrogens. These experiments suggest that an important mechanism by which estrogens stimulate the proliferation of hormone-dependent breast cancer cells involves sensitization to the proliferative effects of IGFs and that this may involve regulation of the type I IGF receptor.     

Progesterone receptor inhibits aromatase and inflammatory response pathways in breast cancer cells via ligand-dependent and ligand-independent mechanisms

Aromatase (product of CYP19 gene), the critical enzyme in estrogen biosynthesis, is up-regulated in 70% of all breast cancers and is highly correlated with cyclooxygenase 2 (COX-2), the rate-determining enzyme in prostanoid biosynthesis. Expression of COX-2 also is correlated with the oncogene HER-2/neu. The efficacy of current endocrine therapies for breast cancer is predicted only if the tumor contains significant amounts of estrogen receptor. Because the progesterone receptor (PR) is an estrogen-induced target gene, it has been suggested that its presence may serve as an indicator of estrogen receptor functional capacity and the differentiation state of the tumor. In the present study, we tested the hypothesis that PR serves a crucial protective role by antagonizing inflammatory response pathways in the breast. We observed that progesterone antagonized the stimulatory effects of cAMP and IL-1beta on aromatase, COX-2, and HER-2/neu expression in T47D breast cancer cells. These actions of progesterone were associated with increased expression of the nuclear factor-kappaB inhibitor, IkappaBalpha. In 28 breast cancer cell lines, IkappaBalpha expression was positively correlated with PR mRNA levels; overexpression of a phosphorylation-defective mutant of IkappaBalpha inhibited expression of aromatase, COX-2, and HER-2/neu. Moreover, in breast cancer cell lines cultured in the absence of progesterone, up-regulation of endogenous PR caused decreased expression of aromatase, COX-2, and HER-2/neu expression, whereas down-regulation of endogenous PR resulted in a marked induction of aromatase and HER-2/neu mRNA. Collectively, these findings suggest that PR plays an important antiinflammatory role in breast cancer cells via ligand-dependent and ligand-independent mechanisms.     

Induction of fascin spikes in breast cancer cells by activation of the insulin-like growth factor-I receptor

Insulin-like growth factor-I receptor (IGF-IR) signaling contributes to the formation of mammary carcinomas and has chiefly been studied with regard to the proliferative and anti-apoptotic effects of IGF-IR signaling. However, IGF-IR activation also affects the actin cytoskeleton and alterations in cell migratory behavior are of known importance for the malignant conversion and metastasis of epithelial cells. The actin-binding protein fascin is found in cell projections and spikes that are involved in the locomotion of mesenchymal cells. Fascin expression is typically low in normal epithelial cells, but is markedly upregulated in several types of carcinomas. Here, we also demonstrate increased fascin expression in breast carcinoma cell lines and adopt MCF-7 human mammary carcinoma cells that over-express wild-type or kinase-inactivated forms of the IGF-IR as a model system to test the hypothesis that IGF-IR activation induces fascin projections. We show that the time-dependent dissociation of cell colonies that occurs upon receptor activation by IGF-I involves the formation of dynamic, fascin-containing lateral cell projections that co-localize with ruffling membranes in association with protrusive activity and cell migratory phenotype. The molecular mechanism of these effects is completely dependent on IGF-IR tyrosine kinase activity and is mediated by a phosphatidylinositol (PI) 3-kinase-dependent process. In demonstrating transduction of fascin spike assembly by activation of a peptide growth factor receptor, these novel data reveal a wide role for fascin spikes in cell motility and provide new insight into the complex effects of IGF-IR signaling on actin cytoskeletal organization.     

A novel unidirectional cross-talk from the insulin-like growth factor-I receptor to leptin receptor in human breast cancer cells

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells.