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2.
Diabetes Obes Metab ; 12(5): 393-402, 2010 May.
Article in English | MEDLINE | ID: mdl-20415687

ABSTRACT

AIM: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. METHODS: The glycopeptide containing two additional non-proteinogenic amino acids was synthesized by standard solid-phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side-by-side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. RESULTS: The 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca) was designed to target a principal leptin/ObR-binding interface. E1/Aca induced leptin effects in ObR-positive cell lines at picomolar concentrations and readily crossed the blood-brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin-dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high-fat peanut diet in a dose-dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity-related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide-, but not leptin-treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. CONCLUSIONS: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin-deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.


Subject(s)
Blood-Brain Barrier/metabolism , Fertility/drug effects , Glycopeptides/agonists , Glycopeptides/pharmacology , Leptin/metabolism , Obesity/metabolism , Receptors, Leptin/agonists , Animals , Cell Proliferation/drug effects , Disease Models, Animal , Mice , Mice, Obese , Rats , Receptors, Leptin/metabolism , Weight Loss/drug effects
3.
Oncogene ; 27(4): 540-7, 2008 Jan 17.
Article in English | MEDLINE | ID: mdl-17653093

ABSTRACT

We reported previously that the obesity hormone leptin is overexpressed in breast cancer biopsies. Here, we investigated molecular mechanisms involved in this process, focusing on conditions that are associated with obesity, that is, hyperinsulinemia and induction of hypoxia. By using quantitative real-time PCR, immunofluorescent detection of proteins and enzyme-linked immunosorbent assays, we found that treatment of MCF-7 breast cancer cells with high doses of insulin or the hypoxia-mimetic agent CoCl2, or culturing the cells under hypoxic conditions significantly increased the expression of leptin mRNA and protein. Notably, the greatest leptin mRNA and protein expression were observed under combined hyperinsulinemia and hypoxia or hypoxia-mimetic treatments. Luciferase reporter assays suggested that increased leptin synthesis could be related to the activation of the leptin gene promoter. DNA affinity precipitation and chromatin immunoprecipitation experiments revealed that insulin, CoCl2 and/or hypoxia treatments augmented nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and increased its interaction with several upstream leptin regulatory sequences, especially with the proximal promoter containing four hypoxia-response elements and three GC-rich regions. By using reverse chromatin precipitation, we determined that loading of HIF-1alpha on the proximal leptin promoter concurred with the recruitment of p300, the major HIF coactivator, suggesting that the HIF/p300 complex is involved in leptin transcription. The importance of HIF-1alpha in insulin- and CoCl2-activated leptin mRNA and protein expression was confirmed using RNA interference.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Leptin/genetics , Active Transport, Cell Nucleus/drug effects , Binding Sites , Breast Neoplasms/metabolism , Cell Hypoxia/physiology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cobalt/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Insulin/pharmacology , Leptin/metabolism , Promoter Regions, Genetic/drug effects , Protein Binding , Transcriptional Activation/drug effects , Tumor Cells, Cultured , p300-CBP Transcription Factors/metabolism
4.
Ann Oncol ; 18 Suppl 6: vi116-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17591803

ABSTRACT

BACKGROUND: The obesity hormone, leptin, has been found to play a role in development and proliferation of normal and malignant tissues. Leptin activity is mediated through the leptin receptor (ObR) that is often expressed in different human cancer cells. Previously, we found that the expression of leptin and ObR can be stimulated by hypoxia-mimetic agents. The aim of this study was to analyze the abundance of and relationships among leptin, ObR and hypoxia-inducible factor-1alpha (HIF-1alpha, transcriptional regulator) in human colorectal cancer. MATERIALS AND METHODS: We investigated the expression of leptin, ObR and HIF-1alpha in colorectal cancer specimens from 135 patients who underwent curative resection. RESULTS: Immunoreactivity for leptin, ObR and HIF-1alpha protein was observed in 69 of 135 (51.1%), 129 of 135 (95.5%) and 88 of 135 (65.2%) of colorectal cancers, respectively. Statistically significant positive correlations were noted between leptin and HIF-1alpha (P = 0.005, r = 0.243), ObR and HIF-1alpha (P < 0.001, r = 0.325) as well as leptin and ObR (P < 0.001, r = 0.426) in the group of all patients as well as in various subgroups depending on clinicopathological features. CONCLUSIONS: The results indicate that the leptin system is overexpressed in human colorectal cancer and this overexpression appears to be associated with the abundance of HIF-1alpha.


Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leptin/biosynthesis , Leptin/genetics , Obesity/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/surgery , Disease Progression , Female , Gene Expression Regulation, Neoplastic/physiology , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Male , Middle Aged , Obesity/metabolism , Receptors, Leptin , Up-Regulation/genetics
5.
Ann Oncol ; 18 Suppl 6: vi81-5, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17591841

ABSTRACT

BACKGROUND: Insulin receptor substrate 1 (IRS-1) is a signaling molecule that exerts a key role in mediating cross talk between estrogen receptor alpha (ERalpha) and insulin-like growth factor 1 (IGF-1) in breast cancer cells. Previously, we demonstrated that a fraction of IRS-1 binds ERalpha, translocates to the nucleus, and modulates ERalpha-dependent transcription at estrogen response elements (ERE). Here, we studied structure-function relationships of the ERalpha:IRS-1 complex under IGF-1 and/or estradiol (E2) stimulation. MATERIALS AND METHODS: ERalpha and IRS-1 deletion mutants were used to analyze structural and functional ERalpha/IRS-1 interactions. IRS-1 binding to ERE and IRS-1 role in ERalpha-dependent ERE transcription was examined by chromatin immunoprecipitation and gene reporter analysis, respectively. The requirement for IRS-1 in ERalpha function was tested with RNAi technology. RESULTS: Nuclear translocation of IRS-1 was induced by E2, IGF-1, and a combination of both stimuli. ERalpha/IRS-1 binding was direct and involved the activation function-1 (AF-1)/DNA binding domain (DBD) region of ERalpha and two discrete regions of IRS-1 (the N-terminal pleckstrin homology domain and a region within the C-terminus). IRS-1 knock down abrogated IGF-1-dependent transcriptional activity of unliganded ERalpha, but induced the activity of liganded ERalpha. CONCLUSIONS: ERalpha/IRS-1 interactions are direct and involve the ERalpha AF-1/DBD domain and IRS-1 domains mapping within N- and C-terminus. IRS-1 may act as a repressor of liganded ERalpha and coactivator of unliganded ERalpha.


Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/physiology , Phosphoproteins/physiology , Active Transport, Cell Nucleus/genetics , Breast Neoplasms/genetics , Cell Line, Tumor , Estradiol/physiology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Humans , Insulin Receptor Substrate Proteins , Insulin-Like Growth Factor I/physiology , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/physiology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Receptor, Insulin/physiology , Receptors, Interferon/genetics , Receptors, Interferon/metabolism , Receptors, Interferon/physiology , Repressor Proteins/physiology , Structure-Activity Relationship
6.
Ann Oncol ; 18 Suppl 6: vi93-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17591843

ABSTRACT

BRCA1 and BRCA2 germline mutations contribute to a significant number of familial and hereditary breast and/or ovarian cancers. The proportion of high-risk families with breast and/or ovarian cancer cases due to mutations in these tumor suppressor genes varies widely among populations. In some population, a wide spectrum of different mutations in both genes are present, whereas in other groups specific mutations in BRCA1 and BRCA2 have been reported with high frequency. Most of these mutations are prevalent in restricted populations as consequence of a founder effect. The comparison of haplotypes between families with the same mutation can distinguish whether high-frequency alleles derive from an older or more recent single mutational event or whether they have arisen independently more than once. Here, we review some of the most well-known and significant examples of founder mutations in BRCA genes found in European and non-European populations. In conclusion, the identification of the ethnic group of families undergoing genetic counseling enables the geneticist and oncologist to make more specific choices, leading to simplify the clinical approach to genetic testing carried out on members of high-risk families. Futhermore, the high frequency of founder mutations, allowing to analyze a large number of cases, might provide accurate information regarding their penetrance.


Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Founder Effect , Mutation , Apoptosis Regulatory Proteins , Ethnicity/genetics , Genetic Testing , Humans
7.
Ann Oncol ; 18 Suppl 6: vi99-102, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17591844

ABSTRACT

BACKGROUND: The frequency and the type of BRCA1 mutations vary widely and might have different geographic and ethnic distribution. Most of these alterations are generally found in isolated populations as a consequence of the founder effect. The object of this study was to determine whether 4843delC, a deleterious mutation of the BRCA1 gene, might be due to a founder effect originating in the Sicilian region of Italy. This mutation was described by us for the first time and identified in two unrelated Sicilian families with hereditary breast/ovarian cancer. The two families were from the same geographical area (south-western area of Palermo, Sicily). The homogeneity of the ethnic group of the two families and the Single Nucleotide Polymorphism (SNPs) analysis of probands led us to perform a study of the allelotype of the various members. PATIENTS AND METHODS: The analysis of the haplotype of the probands and of several family members was conducted by means of a study of the highly polymorphic microsatellites within or flanking the BRCA1 gene. RESULTS: This analysis revealed the presence of a common allele associated with the mutation. CONCLUSIONS: We therefore conclude that 4843delC of the BRCA1 gene is a possible founder mutation in the Sicilian population.


Subject(s)
DNA Mutational Analysis , Founder Effect , Gene Deletion , Genes, BRCA1 , Breast Neoplasms/genetics , Female , Haplotypes , Humans , Male , Microsatellite Repeats , Ovarian Neoplasms/genetics , Pedigree , Sicily
8.
Ann Oncol ; 17 Suppl 7: vii27-9, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16760287

ABSTRACT

Estrogens exhibit important biological functions and influence several pathological processes of hormone-dependent diseases. The biological actions of estrogens require their interaction with two estrogen receptors (ER-alpha and ER-beta), which are ligand-dependent transcription factors. ER-alpha and ER-beta exhibit distinct tissue expression patterns as well as show different patterns of gene regulation. In addition, it has been suggested that ER-beta works as a counter partner of ER-alpha through inhibition of the transactivating functions of ER-alpha. For instance, ER-beta seems to play a different role in breast tumorigenesis than ER-alpha, as ER-beta decreased expression in breast cancer has been correlated with bad prognosis. Biological activities of ER-alpha and ER-beta could be controlled by a number of interacting proteins such as activators/inhibitors, ligand binding and kinases. We have previously reported that pRb2/p130, retinoblastoma related protein, could be involved in the silencing of ER-alpha gene during breast tumorigenesis. Here, we report that ER-beta and pRb2/p130 proteins co-immunoprecipitate in both nucleus and cytoplasm of MCF-7 breast cancer cells. Our hypothesis is that the interaction of pRb2/130 with ER-beta may have a functional significance in regulating ER-beta activity.


Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor beta/metabolism , Retinoblastoma-Like Protein p130/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , Humans , Immunoprecipitation , Molecular Sequence Data
9.
J Exp Clin Cancer Res ; 23(3): 385-94, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15595626

ABSTRACT

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is a multifunctional transmembrane tyrosine kinase that has been implicated in neoplastic transformation. The tumorigenic potential of IGF-IR relies on its strong anti-apoptotic and mitogenic activity. The growth and survival signals of IGF-IR are mediated through multiple intracellular pathways, many of which emanate from insulin receptor substrate 1 (IRS-1). In hormone-dependent breast cancer cells, IGF-IR and IRS-1 are often co-expressed with the estrogen receptor alpha (ERalpha), and IGF-I and ER systems are engaged in a powerful functional cross-talk. Most notably, activation of ERalpha upregulates the expression of IRS-1, IGF-IR, and IGF-1, which results in amplification of IGF-I responses. Reciprocally, stimulation of IGF-IR increases the phosphorylation and activity of ERalpha. In contrast, in ERalpha-negative breast cancer cells and tumors, the levels of IGF-IR and IRS-1 are often decreased and IGF-I is non-mitogenic. Our data suggest that defective IGF-IR signaling in ERalpha-negative cells is related, at least in part, to improper activation of the IRS-1/PI-3K/Akt/GSK-3 pathway and lack of Rb1 phosphorylation. These defects are partially reversed by re-expression of ERalpha. Interestingly, some non-mitogenic IGF-I responses, such as migration and invasion are retained in the absence of ERalpha, suggesting that IGF-IR function in breast cancer cells might depend on the ERalpha status. The understanding of how ERalpha may dictate IGF-I responses will help in devising rational anti-IGF-IR strategies for breast cancer treatment.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Estrogen Receptor alpha/physiology , Gene Expression Regulation, Neoplastic , Receptor, IGF Type 1/metabolism , Signal Transduction , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Humans , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Up-Regulation
10.
Horm Metab Res ; 36(5): 336-40, 2004 May.
Article in English | MEDLINE | ID: mdl-15156414

ABSTRACT

While obesity is a known risk factor for postmenopausal breast cancer, the molecular mechanisms involved are unclear. Systemic levels of leptin, the product of the ob (obesity) gene, are increased in obese individuals (body mass index, BMI, over 25) and are higher in women than men. Leptin has been found to stimulate the growth of breast cancer cells in vitro. Our goal was to determine whether leptin was 1) present in nipple aspirate fluid (NAF), and 2) whether NAF leptin levels were associated with a) levels in serum, b) obesity, and c) breast cancer. We collected and evaluated NAF specimens from 83 subjects and serum specimens from 49 subjects. NAF leptin was detectable in 16/41 (39 %) of premenopausal and 21/42 (50 %) postmenopausal subjects. NAF leptin was significantly lower (p = 0.042) in premenopausal than postmenopausal women with a BMI < 25, but not in those with a higher BMI. NAF leptin was significantly associated with BMI in premenopausal (p = 0.011) but not in postmenopausal women. Serum leptin was associated with BMI in both premenopausal and postmenopausal women (p = 0.0001 for both). NAF and serum leptin were associated in premenopausal (p = 0.02) but not postmenopausal women. Neither NAF nor serum leptin was associated with premenopausal or postmenopausal breast cancer. Our findings include that 1) leptin is present in the breast and detectable in a subset of NAF samples, 2) NAF leptin in premenopausal but not postmenopausal women parallels serum leptin levels, and 3) neither NAF nor serum levels of leptin were associated with premenopausal or postmenopausal breast cancer.


Subject(s)
Breast Neoplasms/epidemiology , Leptin/blood , Leptin/metabolism , Nipples/metabolism , Adult , Aged , Aged, 80 and over , Biopsy, Needle , Body Mass Index , Female , Humans , Middle Aged , Postmenopause , Premenopause
11.
Horm Metab Res ; 35(11-12): 794-801, 2003.
Article in English | MEDLINE | ID: mdl-14710360

ABSTRACT

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.


Subject(s)
Breast Neoplasms/pathology , Lymphatic Metastasis/pathology , Receptor, IGF Type 1/metabolism , Receptors, Estrogen/metabolism , Breast Neoplasms/physiopathology , Breast Neoplasms/surgery , Estrogen Receptor alpha , Estrogen Receptor beta , Female , Humans , Immunohistochemistry , Lymphatic Metastasis/physiopathology
12.
Biochem Biophys Res Commun ; 288(3): 685-9, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11676497

ABSTRACT

This study demonstrates how the potentiating effects of E2 on insulin signaling in ER-positive breast cancer cells are consequent to an enhanced IRS-1 expression [corrected]. It induces an increase of both PI-3K/AKT and ERK1/2 activities. A direct action of E2 in the regulating mouse IRS-1 gene is also investigated in both Chinese hamster ovary and MCF-7 cells that are transfected with mouse IRS-1 regulatory sequences. The authors have reported, for the first time, how E2 induction of IRS-1 mRNA was correlated with a direct positive regulatory role of E2 on the IRS-1 promoter. This effect seems to be not strictly related to the cell type.


Subject(s)
Estradiol/pharmacology , Gene Expression Regulation/drug effects , Insulin/physiology , Phosphoproteins/metabolism , Promoter Regions, Genetic/drug effects , Analysis of Variance , Breast Neoplasms , Humans , Insulin Receptor Substrate Proteins , Phosphoproteins/genetics , Phosphorylation , Promoter Regions, Genetic/genetics , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Tumor Cells, Cultured , Tyrosine/metabolism
13.
Cancer Res ; 61(18): 6747-54, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559546

ABSTRACT

The insulin-like growth factor I receptor (IGF-IR) is a ubiquitous and multifunctional tyrosine kinase that has been implicated in breast cancer development. In estrogen receptor (ER)-positive breast tumors, the levels of the IGF-IR and its substrate, insulin-receptor substrate 1 (IRS-1), are often elevated, and these characteristics have been linked with increased radioresistance and cancer recurrence. In vitro, activation of the IGF-IR/IRS-1 pathway in ER-positive cells improves growth and counteracts apoptosis induced by anticancer treatments. The function of the IGF-IR in hormone-independent breast cancer is not clear. ER-negative breast cancer cells often express low levels of the IGF-IR and fail to respond to IGF-I with mitogenesis. On the other hand, anti-IGF-IR strategies effectively reduced metastatic potential of different ER-negative cell lines, suggesting a role of this receptor in late stages of the disease. Here we examined IGF-IR signaling and function in ER-negative MDA-MB-231 breast cancer cells and their IGF-IR-overexpressing derivatives. We demonstrated that IGF-I acts as a chemoattractant for these cells. The extent of IGF-I-induced migration reflected IGF-IR levels and required the activation of phosphatidylinositol 3-kinase (PI-3K) and p38 kinases. The same pathways promoted IGF-I-dependent motility in ER-positive MCF-7 cells. In contrast with the positive effects on cell migration, IGF-I was unable to stimulate growth or improve survival in MDA-MB-231 cells, whereas it induced mitogenic and antiapoptotic effects in MCF-7 cells. Moreover, IGF-I partially restored growth in ER-positive cells treated with PI-3K and ERK1/ERK2 inhibitors, whereas it had no protective effects in ER-negative cells. The impaired IGF-I growth response of ER-negative cells was not caused by a low IGF-IR expression, defective IGF-IR tyrosine phosphorylation, or improper tyrosine phosphorylation of IRS-1. Also, the acute (15-min) IGF-I activation of PI-3 and Akt kinases was similar in ER-negative and ER-positive cells. However, a chronic (2-day) IGF-I exposure induced the PI-3K/Akt pathway only in MCF-7 cells. The reactivation of this pathway in ER-negative cells by overexpression of constitutively active Akt mutants was not sufficient to significantly improve proliferation or survival (with or without IGF-I), which indicated that other pathways are also required to support these functions. Our results suggest that in breast cancer cells, IGF-IR can control nonmitogenic processes regardless of the ER status, whereas IGF-IR growth-related functions may depend on ER expression.


Subject(s)
Breast Neoplasms/pathology , Protein Serine-Threonine Kinases , Receptor, IGF Type 1/physiology , Receptors, Estrogen/physiology , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Cell Division/drug effects , Cell Division/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cell Survival/drug effects , Cell Survival/physiology , Enzyme Activation , Humans , Insulin-Like Growth Factor I/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Receptor, IGF Type 1/biosynthesis , Receptor, IGF Type 1/genetics , Signal Transduction/drug effects , Signal Transduction/physiology , Transfection , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases
14.
J Biol Chem ; 276(43): 39892-7, 2001 Oct 26.
Article in English | MEDLINE | ID: mdl-11518717

ABSTRACT

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but the role of the IGF-IR in tumor metastasis is unclear. Here we studied the effects of the IGF-IR on intercellular connections mediated by the major epithelial adhesion protein, E-cadherin (E-cad). We found that IGF-IR overexpression markedly stimulated aggregation in E-cad-positive MCF-7 breast cancer cells, but not in E-cad-negative MDA-MB-231 cells. However, when the IGF-IR and E-cad were co-expressed in MDA-MB-231 cells, cell-cell adhesion was substantially increased. The IGF-IR-dependent cell-cell adhesion of MCF-7 cells was not related to altered expression of E-cad or alpha-, beta-, or gamma-catenins but coincided with the up-regulation of another element of the E-cad complex, zonula occludens-1 (ZO-1). ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. By co-immunoprecipitation, we found that ZO-1 associates with the E-cad complex and the IGF-IR. High levels of ZO-1 coincided with an increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas down-regulation of ZO-1 by the expression of an anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results suggested that one of the mechanisms by which the activated IGF-IR regulates E-cad-mediated cell-cell adhesion is overexpression of ZO-1 and the resulting stronger connections between the E-cad complex and the actin cytoskeleton. We hypothesize that in E-cad-positive cells, the IGF-IR may produce antimetastatic effects.


Subject(s)
Breast Neoplasms/metabolism , Cadherins/metabolism , Intercellular Junctions , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Receptor, IGF Type 1/metabolism , Cell Adhesion , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Models, Biological , Phosphoproteins/genetics , Protein Binding , Zonula Occludens-1 Protein
15.
J Mammary Gland Biol Neoplasia ; 5(1): 95-105, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10791772

ABSTRACT

The insulin-like growth factor-I receptor (IGF-IR) is a transmembrane tyrosine kinase regulating various biological processes such as proliferation, survival, transformation, differentiation, cell-cell and cell-substrate interactions. Different signaling pathways may underlie these pleiotropic effects. The specific pathways engaged depend on the number of activated IGF-IRs, availability of intracellular signal transducers, the action of negative regulators, and is influenced by extracellular modulators. Experimental and clinical data implicate the IGF-IR in breast cancer etiology. There is strong evidence linking hyperactivation of the IGF-IR with the early stages of breast cancer. In primary breast tumors, the IGF-IR is overexpressed and hyperphosphorylated, which correlates with radio-resistance and tumor recurrence. In vitro, the IGF-IR is often required for mitogenesis and transformation, and its overexpression or activation counteract effects of various pro-apoptotic treatments. In hormone-responsive breast cancer cells, IGF-IR function is strongly linked with estrogen receptor (ER) action. The IGF-IR and the ER are co-expressed in breast tumors. Moreover, estrogens stimulate the expression of the IGF-IR and its major signaling substrate IRS-1, while antiestrogens downregulate IGF-IR signaling, mainly by decreasing IRS-1 expression and function. On the other hand, overexpression of IRS-1 promotes estrogen-independence for growth and transformation. In ER-negative breast cancer cells, usually displaying a more aggressive phenotype, the levels of the IGF-IR and IRS-1 are often low and IGF is not mitogenic, yet the IGF-IR is still required for metastatic spread. Consequently, IGF-IR function in the late stages of breast cancer remains one of the most important questions to be addressed before rational anti-IGF-IR therapies are developed.


Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Receptor, IGF Type 1/physiology , Female , Humans , Insulin Receptor Substrate Proteins , Phosphoproteins/physiology , Receptor, IGF Type 1/chemistry , Receptor, IGF Type 1/genetics , Receptors, Estrogen/physiology , Signal Transduction
16.
Exp Cell Res ; 252(2): 439-48, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10527634

ABSTRACT

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with alpha5beta1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Movement , Proteins/metabolism , Receptors, Fibronectin/metabolism , Cell Adhesion , Female , Fibronectins , Humans , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Tumor Cells, Cultured , src Homology Domains
17.
Exp Cell Res ; 251(1): 244-55, 1999 Aug 25.
Article in English | MEDLINE | ID: mdl-10438590

ABSTRACT

Insulin-like growth factor I (IGF-I) promotes the motility of different cell types. We investigated the role of IGF-I receptor (IGF-IR) signaling in locomotion of MCF-7 breast cancer epithelial cells overexpressing the wild-type IGF-IR (MCF-7/IGF-IR). Stimulation of MCF-7/IGF-IR cells with 50 ng/ml IGF-I induced disruption of the polarized cell monolayer followed by morphological transition toward a mesenchymal phenotype. Immunofluorescence staining of the cells with rhodamine-phalloidin revealed rapid disassembly of actin fibers and development of a cortical actin meshwork. Activation of phosphatidylinositol (PI)3-kinase downstream of the IGF-IR was necessary for this process, as blocking PI 3-kinase activity with the specific inhibitor LY 294002 at 10 microM prevented disruption of the filamentous actin. In parallel, IGF-IR activation induced rapid and transient tyrosine dephosphorylation of focal adhesion proteins p125 focal adhesion kinase (FAK), p130 Crk-associated substrate (Cas), and paxillin. This process required phosphotyrosine phosphatase (PTP) activity, since pretreatment of the cells with 5 microM phenylarsine oxide (PAO), an inhibitor of PTPs, rescued FAK and its associated proteins Cas and paxillin from IGF-I-induced dephosphorylation. In addition, PAO-pretreated cells were refractory to IGF-I-induced morphological transition. Thus, our findings reveal a new function of the IGF-IR, the ability to depolarize epithelial cells. In MCF-7 cells, mechanisms of IGF-IR-mediated cell depolarization involve PI 3-kinase signaling and putative PTP activities.


Subject(s)
Actins/metabolism , Breast Neoplasms/pathology , Epithelial Cells/cytology , Membrane Potentials/physiology , Phosphotyrosine/metabolism , Proteins , Receptor, IGF Type 1/physiology , Breast Neoplasms/enzymology , Breast Neoplasms/metabolism , Breast Neoplasms/physiopathology , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cell Movement/drug effects , Cell Size/drug effects , Crk-Associated Substrate Protein , Cytoplasm/drug effects , Cytoplasm/enzymology , Cytoplasm/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/physiology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Insulin-Like Growth Factor I/pharmacology , Membrane Potentials/drug effects , Paxillin , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphoproteins/metabolism , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Retinoblastoma-Like Protein p130 , Signal Transduction/drug effects , Tumor Cells, Cultured
18.
Int J Cancer ; 81(2): 299-304, 1999 04 12.
Article in English | MEDLINE | ID: mdl-10188734

ABSTRACT

The pure antiestrogen ICI 182,780 inhibits insulin-like growth factor (IGF)-dependent proliferation in hormone-responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF-I receptor (IGF-IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF-IR signal transduction in MCF-7 breast cancer cells and in MCF-7-derived clones overexpressing either the IGF-IR or its 2 major substrates, insulin receptor substrate 1 (IRS-1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF-I-induced growth in all studied cells in a dose-dependent manner; however, the clones with the greatest IRS-1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS-1, we found that the antiestrogen reduced IRS-1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF-I. Moreover, in IRS-1-overexpressing cells, ICI 182,780 decreased IRS-1/p85 and IRS-1/GRB2 binding. The effects of ICI 182,780 on IGF-IR protein expression were not significant; however, the drug suppressed IGF-I-induced (but not basal) IGF-IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS-1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS-1 in breast tumors could contribute to the development of antiestrogen resistance.


Subject(s)
Breast Neoplasms/drug therapy , Estradiol/analogs & derivatives , Estrogen Antagonists/pharmacology , Insulin-Like Growth Factor Binding Proteins/drug effects , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Division/drug effects , Estradiol/pharmacology , Female , Fulvestrant , Humans , Insulin Receptor Substrate Proteins , Phosphoproteins/genetics , RNA, Messenger/biosynthesis , Receptor, Insulin/genetics , Signal Transduction/drug effects , Tumor Cells, Cultured
19.
Breast Cancer Res Treat ; 47(3): 255-67, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9516080

ABSTRACT

Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture. The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation. Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.


Subject(s)
Breast Neoplasms/ultrastructure , Receptor, IGF Type 1/physiology , Breast Neoplasms/pathology , Humans , Tumor Cells, Cultured
20.
Biochem Biophys Res Commun ; 253(2): 315-9, 1998 Dec 18.
Article in English | MEDLINE | ID: mdl-9878535

ABSTRACT

Cross-talk between steroid hormones and polypeptide growth factors regulates the growth of hormone-responsive breast cancer cells. For example, in the MCF-7 human breast cancer cell line, insulin up-regulates estrogen receptor (ER) content and binding capacity. Since the insulin receptor (IR) substrate 1 (IRS-1) is one of the core signaling elements transmitting mitogenic and metabolic effects of insulin, we investigated whether IRS-1 is also required for the insulin-induced function of the ER. The effects of insulin on the ER were compared in MCF-7 cells and MCF-7-derived cell lines with decreased levels (by approximately 80%) of IRS-1 due to the expression of IRS-1 antisense RNA. The severe IRS-1 deficiency in MCF-7 cells was associated with (1) reduced mitogenic response to 20 ng/ml insulin and 10% calf serum (CS), but not to 1 nM estradiol (E2); (2) loss of insulin-E2 synergism; (3) up-regulation of ER protein expression and binding capacity; and (4) loss of insulin-induced regulation of ER tyrosine phosphorylation. In conclusion, the data confirm the existence of the IR-ER cross-talk and suggest that IRS-1-dependent signaling may contribute to the negative regulation of the ER expression and function in MCF-7 cells.


Subject(s)
Breast Neoplasms/metabolism , Insulin/pharmacology , Phosphoproteins/physiology , Receptor, Insulin/metabolism , Receptors, Estrogen/metabolism , Signal Transduction/physiology , Culture Media, Conditioned/pharmacology , Down-Regulation/physiology , Estradiol/pharmacology , Growth Inhibitors/physiology , Humans , Insulin Receptor Substrate Proteins , Phosphorylation , Protein Binding/physiology , Receptor Cross-Talk/physiology , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/drug effects , Signal Transduction/drug effects , Tumor Cells, Cultured , Tyrosine/metabolism
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