ABSTRACT
Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr(1068)], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.
Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Epidermal Growth Factor/metabolism , Fibronectins/chemistry , Gene Expression Regulation, Neoplastic , Insulin-Like Growth Factor Binding Protein 3/metabolism , Biotinylation , Cell Line, Tumor , Gefitinib , Humans , Laminin/chemistry , MAP Kinase Signaling System , Phosphorylation , Quinazolines/pharmacology , Radioimmunoassay , rho-Associated Kinases/metabolismABSTRACT
The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.
Subject(s)
Proteins/metabolism , Receptor, IGF Type 2/metabolism , Amino Acid Sequence , Base Sequence , Chromatography, High Pressure Liquid , DNA Primers , Humans , Insulin-Like Growth Factor II , Molecular Sequence Data , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular , Polymerase Chain Reaction , Protein Binding , Protein Conformation , Proteins/chemistry , Proteins/genetics , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
The potent growth-promoting activity of insulin-like growth factor-II (IGF-II) is highly regulated during development but frequently up-regulated in tumors. Increased expression of the normally monoallelic (paternally expressed) mouse (Igf2) and human (IGF2) genes modify progression of intestinal adenoma in the Apc(Min/+) mouse and correlate with a high relative risk of human colorectal cancer susceptibility, respectively. We examined the functional consequence of Igf2 allelic dosage (null, monoallelic, and biallelic) on intestinal adenoma development in the Apc(Min/+) by breeding with mice with either disruption of Igf2 paternal allele or H19 maternal allele and used these models to evaluate an IGF-II-specific therapeutic intervention. Increased allelic Igf2 expression led to elongation of intestinal crypts, increased adenoma growth independent of systemic growth, and increased adenoma nuclear beta-catenin staining. By introducing a transgene expressing a soluble form of the full-length IGF-II/mannose 6-phosphate receptor (sIGF2R) in the intestine, which acts as a specific inhibitor of IGF-II ligand bioavailability (ligand trap), we show rescue of the Igf2-dependent intestinal and adenoma phenotype. This evidence shows the functional potency of allelic dosage of an epigenetically regulated gene in cancer and supports the application of an IGF-II ligand-specific therapeutic intervention in colorectal cancer.
Subject(s)
Adenomatous Polyposis Coli/genetics , Insulin-Like Growth Factor II/genetics , Receptor, IGF Type 2/metabolism , Adenomatous Polyposis Coli/metabolism , Adenomatous Polyposis Coli/pathology , Alleles , Animals , Cell Growth Processes/genetics , Crosses, Genetic , Disease Progression , Female , Gene Dosage , Genomic Imprinting , Insulin-Like Growth Factor II/antagonists & inhibitors , Insulin-Like Growth Factor II/biosynthesis , Insulin-Like Growth Factor II/metabolism , Ligands , Male , Mice , Mice, Inbred C57BL , Receptor, IGF Type 2/biosynthesis , Receptor, IGF Type 2/genetics , Transgenes , beta Catenin/metabolismABSTRACT
Ligands transported by the mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor (IGF2R) include IGF-II- and mannose 6-phosphate-modified proteins. Increased extracellular supply of IGF-II, either secondary to loss of the clearance function of IGF2R, loss of IGF binding protein function, or increased IGF2 gene expression, can lead to embryonic overgrowth and cancer promotion. Reduced supply of IGF-II is detrimental to tumor growth, and this suggests that gain of function of IGF-II is a molecular target for human cancer therapy. Domain 11 of IGF2R binds IGF-II with high specificity and affinity. Mutagenesis studies have shown that substitution of glutamic acid for lysine at residue 1554 results in a 6-fold higher affinity for IGF-II (20.5 nmol/L) than native domain 11 (119 nmol/L). Here, we generate a novel high-affinity IGF-II ligand trap by fusion of mutated human 11(E1554K) to a COOH-terminal human IgG1 Fc domain (11(E1554K)-Fc). The resulting homodimer has a significantly increased affinity for IGF-II (1.79 nmol/L) when measured by surface plasmon resonance. IGF-II signaling via the IGF-I receptor and the proliferative effect of IGF-II were specifically inhibited by 11(E1554K)-Fc in both HaCaT and Igf2(-/-) mouse embryonic fibroblast cells. These data confirm that a novel engineered and soluble IGF2R-11(E1554K)-Fc protein functions as an IGF-II-specific and high-affinity ligand trap in vitro and that this protein has potential application as an IGF-II antagonist for cancer therapy following in vivo experimental evaluation.
Subject(s)
Cell Proliferation , Fibroblasts/metabolism , Insulin-Like Growth Factor II/physiology , Keratinocytes/metabolism , Receptor, IGF Type 2/metabolism , Animals , Blotting, Western , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryo, Nonmammalian , Fibroblasts/cytology , Genetic Vectors , Humans , Insulin-Like Growth Factor II/genetics , Ligands , Mice , Mice, Knockout , Pichia/chemistry , Pichia/metabolism , Protein Binding , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptor, IGF Type 2/genetics , Receptors, Fc/genetics , Receptors, Fc/metabolism , Surface Plasmon Resonance , Thymidine/metabolismABSTRACT
In breast tumors IGF binding protein-2 (IGFBP-2) is elevated, and the presence of IGFBP-2 has been shown to correlate with malignancy. However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity. Exogenous IGFBP-2 acting via integrin receptors to reduce phosphatase and tensin homolog deleted from chromosome 10 (PTEN) levels protected these cells against death induced by various chemotherapeutic agents. This was dependent on a functional estrogen receptor (ER)-α because silencing ER-α blocked the ability of IGFBP-2 to confer cell survival. Loss of IGFBP-2 increased levels of PTEN and improved chemosensitivity of the cells, confirming its role as a survival factor. Silencing IGFBP-2 had no effect on the response to IGF-II, but responses to estrogen and tamoxifen were no longer observed due to loss of ER-α, which could be prevented by the inhibition of PTEN. Conversely, exogenous IGFBP-2 increased ER-α mRNA and protein in both normal and cancer cells via its interaction with integrin receptors. These actions of IGFBP-2 on ER-α involved the IGF-I receptor and activation of phosphatidylinositol 3-kinase in the cancer cells but were independent of this in normal breast cells. The production of IGFBP-2 by breast cancer cells enhances their proliferative potential, increases their survival, and protects them against chemotherapy-induced death. IGFBP-2 not only modulates IGFs and directly regulates PTEN but also has a role in maintaining ER-α expression.
Subject(s)
Estrogen Receptor alpha/genetics , Insulin-Like Growth Factor Binding Protein 2/physiology , Mammary Glands, Human/physiology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Drug Evaluation, Preclinical , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation/drug effects , Humans , Insulin-Like Growth Factor Binding Protein 2/antagonists & inhibitors , Insulin-Like Growth Factor Binding Protein 2/genetics , Insulin-Like Growth Factor Binding Protein 2/metabolism , Mammary Glands, Human/drug effects , Mammary Glands, Human/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , RNA, Small Interfering/pharmacologyABSTRACT
Recent evidence from epidemiology indicates that inter-individual variations in the growth hormone (GH)/IGF-I pathway affect the risk of individuals developing common epithelial cancers. This is supported by associations between normal common variants within genes from the pathway and these cancers, which excludes many potential confounding issues, such as reverse causality. This raises concern for the increasing numbers of patients treated with GH; although replacement therapy for GH-deficiency should aim to restore normality, which should then only incur a normal risk. The links with cancer also offers promising new opportunities. Clinical trials treating cancer patients with pharmaceuticals targeting the IGF-I receptor are well advanced with promising initial findings. In the future, there has to be much more emphasis within oncology on prevention and the GH/IGF-I pathway is one of few identified risk factors that are modifiable, not just by pharmaceutical, but also nutritional, interventions that may, in the long term, be more appropriate. Assessing the status of the GH/IGF-I pathway in individuals may also provide a means for targeting screening programs and preventative measures.
ABSTRACT
To gain a clearer insight into the mechanisms of skeletal muscle cell growth, differentiation and maintenance, we have developed a primary adult human skeletal muscle cell model. Cells were cultured from biopsies of rectus muscle from the anterior abdominal wall of patients undergoing elective surgery. Under differentiating conditions, all cultures formed myotubes, irrespective of initial myoblast number. Stimulation with both IGF-I and tumour necrosis factor alpha (TNFalpha) increased cellular proliferation but while IGF-I subsequently increased myoblast differentiation, via both hyperplasia and hypertrophy, TNFalpha inhibited the initiation of differentiation, but did not induce apoptosis. Addition of IGF-I stimulated both the MAP kinase and the phosphatidylinositide 3-kinase (PI 3-kinase) signalling pathways while treatment with TNFalpha preferentially led to MAP kinase activation although with a very different profile of activation compared to IGF-I. Data using the MEK inhibitor UO126 showed MAP kinase activity is not only needed for cellular proliferation but is also necessary for both the initiation and the progression of primary human myoblast differentiation. The PI 3-kinase pathway is also involved in differentiation, but activation of this pathway could not relieve inhibition of differentiation by TNFalpha or UO126. Our results show that the controlled temporal and amplitude of activation of multiple signalling pathways is needed for successful myoblast differentiation.
Subject(s)
Insulin-Like Growth Factor I/pharmacology , Muscle, Skeletal/cytology , Myoblasts, Skeletal/cytology , Protein Serine-Threonine Kinases , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacology , Adult , Aged , Apoptosis , Cell Culture Techniques , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Creatine Kinase/metabolism , Female , Glycogen Synthase Kinase 3/metabolism , Humans , Insulin-Like Growth Factor I/physiology , MAP Kinase Signaling System , Male , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Muscle Fibers, Skeletal/cytology , Muscle, Skeletal/drug effects , Myoblasts, Skeletal/drug effects , Myoblasts, Skeletal/enzymology , Neoplasms/pathology , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Tumor Necrosis Factor-alpha/physiologyABSTRACT
Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.