RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proteínas/metabolismo , Receptores de Fibronectina/metabolismo , Adesão Celular , Feminino , Fibronectinas , Humanos , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
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.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/patologia , Células Epiteliais/citologia , Potenciais da Membrana/fisiologia , Fosfotirosina/metabolismo , Proteínas , Receptor IGF Tipo 1/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Proteína Substrato Associada a Crk , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Paxilina , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteína p130 Retinoblastoma-Like , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Fulvestranto , Humanos , Proteínas Substratos do Receptor de Insulina , Fosfoproteínas/genética , RNA Mensageiro/biossíntese , Receptor de Insulina/genética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Neoplasias da Mama/ultraestrutura , Receptor IGF Tipo 1/fisiologia , Neoplasias da Mama/patologia , Humanos , Células Tumorais CultivadasRESUMO
Several polypeptide growth factors stimulate breast cancer growth and may be involved in tumor progression. However, the relative importance of diverse growth factor signaling pathways in the development and maintenance of the neoplastic phenotype is largely unknown. The activation of such growth factor receptors as the insulin-like growth factor I receptor (IGF-I R), erbB-type receptors (erbB Rs) and FGF receptors (FGF Rs) controls the phenotype of a model breast cancer cell line MCF-7. To evaluate the function of 2 post-receptor signaling molecules, insulin receptor substrate-1 (IRS-1) (a major substrate of the IGF-IR) and SHC (a common substrate of tyrosine kinase receptors), we developed several MCF-7-derived cell clones in which the synthesis of either IRS-1 or SHC was blocked by antisense RNA. In MCF-7 cells, down-regulation of IRS-1 by 80-85% strongly suppressed anchorage-dependent and -independent growth and induced apoptotic cell death under growth factor- and estrogen-reduced conditions. The reduction of SHC levels by approximately 50% resulted in the inhibition of monolayer and anchorage-independent growth but did not decrease cell survival. Importantly, cell aggregation and the ability of cells to survive on the extracellular matrix were inhibited in MCF-7/anti-SHC clones, but not in MCF-7/anti-IRS-1 clones. Cell motility toward IGF was not attenuated in any of the tested cell lines, but motility toward EGF was decreased in MCF-7/anti-SHC clones. Our results suggest that in MCF-7 cells: 1) both IRS-1 and SHC are implicated in the control of monolayer and anchorage-independent growth; 2) IRS-1 is critical to support cell survival; 3) SHC is involved in EGF-dependent motility; and 4) normal levels of SHC, but not IRS-1, are necessary for the formation and maintenance of cell-cell interactions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Neoplasias da Mama/patologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Apoptose , Western Blotting , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina , Fosfoproteínas/genética , Proteínas/genética , RNA Antissenso/farmacologia , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Somatomedinas/farmacologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais CultivadasRESUMO
The insulin-like growth factor I receptor (IGF-IR) is involved in the control of breast cancer cell growth. The cytostatic activity of tamoxifen (Tam), a nonsteroidal antiestrogen, is partially mediated through interference with IGF-I-R-dependent proliferation, yet the effects of Tam on IGF-IR intracellular signaling have never been elucidated. Consequently, we investigated how Tam modifies the IGF-IR signaling pathway in estrogen receptor-positive MCF-7 breast cancer cells and in MCF-7-derived clones overexpressing either the IGF-IR (MCF-7/IGF-IR cells) or its major substrate, IRS-1 (MCF-7/IRS-1 cells). MCF-7/IGF-IR and MCF-7/IRS-1 cells exhibit greatly reduced estrogen growth requirements but retain estrogen receptors and express sensitivity to antiestrogens comparable to that in the parental cells. In all tested cell lines, regardless of the amplification of IGF signaling, a 4-day treatment with 10 nM Tam produced a similar cytostatic effect. In MCF-7 and MCF-7/IGF-IR cells, growth inhibition by Tam was associated with the reduced tyrosine phosphorylation of the IGF-IR in the presence of IGF-I; however, the basal level of the IGF-IR remained unaffected. Moreover, Tam inhibited both basal and IGF-I-induced tyrosine phosphorylation of IRS-1, which was accompanied by down-regulation of IRS-1-associated phosphatidylinositol 3'-kinase activity and reduced IRS-1/growth factor receptor-bound protein 2 (GRB2) binding. In contrast, under the same treatment, tyrosine phosphorylation of Src-homology/collagen proteins (SHC; another substrate of the IGF-IR) and SHC/GRB2 binding were elevated. The protein levels of the IGF-IR and IRS-1 were not modified by Tam, whereas SHC protein expression was either not affected or moderately decreased by the treatment. In summary, this work provides the first evidence that in MCF-7 cells, cytostatic effects of Tam are associated with the modulation of IGF-IR signaling, specifically with: (a) down-regulation of IGF-I-induced tyrosine phosphorylation of the IGF-IR; (b) inhibition of IRS-1/phosphatidylinositol 3'-kinase signaling; and (c) up-regulation of SHC tyrosine phosphorylation and increased SHC/GRB2 binding. It is hypothesized that dephosphorylation of IRS-1 could be a major contributing factor in Tam cytostatic activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Receptor IGF Tipo 1/metabolismo , Tamoxifeno/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Células Tumorais CultivadasRESUMO
The insulin-like growth factor I receptor (IGF-IR) paracrine or autocrine loop plays an important role in the maintenance of breast cancer growth. Cancer cells contain several-fold higher levels of the IGF-IR than normal breast tissue; however, it is still not clear whether abnormally high activation of IGF-IR signaling may induce progression of the disease. To address this question, we have established several MCF-7-derived clones (MCF-7/IGF-IR cells) overexpressing the IGF-IR. We report here that overexpression of the IGF-IR did not modify sensitivity of cells to IGF-I; however, responsiveness to the ligand was moderately enhanced in most of the MCF-7/IGF-IR clones (measured by [3H]thymidine incorporation into DNA). All MCF-7/IGF-IR clones responded to the synergistic action of 1 nM estradiol (E2) and small amounts of IGF-I (up to 0.8 ng/ml). Exposure of cells to higher concentrations of IGF-I abolished estrogen requirements for stimulation of DNA synthesis in all MCF-7/IGF-IR clones, but not in the parental cells. The most important finding of this work was that the amplification of the IGF-IR induced cell-cell adhesion in MCF-7 cells. High levels of the IGF-IR promoted cell aggregation on Matrigel, allowed proliferation of cells within the aggregates, and protected clustered cells from death. In both MCF-7 and MCF-7/IGF-IR cells, IGF-I stimulated aggregation, whereas an anti-E cadherin antibody blocked cell-cell adhesion. Furthermore, immunofluorescence staining with specific antibodies revealed co-localization of the IGF-IR and E-cadherin at the points of cell-cell contacts. Moreover, the IGF-IR and its two substrates, insulin receptor substrate 1 and SHC, were contained within the E-cadherin complexes. Our results suggest that overexpressed IGF-IRs, by promoting the aggregation, growth, and survival of breast cancer cells, may accelerate the increase of tumor mass and may also prevent cell scattering.
Assuntos
Neoplasias da Mama/patologia , Caderinas/fisiologia , Adesão Celular , Estradiol/farmacologia , Receptor IGF Tipo 1/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/análise , Agregação Celular/efeitos dos fármacos , Divisão Celular , Sobrevivência Celular , Colágeno , Combinação de Medicamentos , Matriz Extracelular , Imunofluorescência , Humanos , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Laminina , Invasividade Neoplásica , Fosfoproteínas/análise , Fosforilação , Fosfotirosina/metabolismo , Proteínas/análise , Proteoglicanas , Receptor IGF Tipo 1/análise , Receptor IGF Tipo 1/genética , Transfecção , Células Tumorais CultivadasRESUMO
We studied the interactions of phosphorothioate oligodeoxynucleotides and heparin-binding growth factors. By means of a gel mobility shift assay, we demonstrated that phosphodiester and phosphorothioate homopolymers bound to basic fibroblast growth factor (bFGF). Binding of a probe phosphodiester oligodeoxynucleotide could also be shown for other proteins of the FGF family, including acidic fibroblast growth factor (aFGF), Kaposi's growth factor (FGF-4) as well as for the bFGF-related vascular endothelial growth factor, VEGF. No binding to epidermal growth factor (EGF) was observed. In addition, using a radioreceptor assay, we have shown that phosphorothioate homopolymers of cytidine and thymidine blocked binding of not only 125I-bFGF, but also of 125I-PDGF to NIH 3T3 cells, whereas phosphodiester oligodeoxynucleotides were ineffective. The extent of blockade of binding was dependent on the chain length of the phosphorothioate oligodeoxynucleotide. Furthermore, we have examined the effects of 18-mer phosphorothioate oligodeoxynucleotides of different sequences on 125I-bFGF binding to low and high affinity sites on both NIH 3T3 fibroblasts and DU-145 prostate cancer cells. Despite the fact that we have observed inhibition of bFGF binding by the 18-mer phosphorothioate oligodeoxynucleotides for both the high and low affinity classes of bFGF receptor, the inhibition was sequence-selective only for the high affinity receptors. We have also demonstrated that phosphorothioate homopolymers of cytidine and thymidine release bFGF bound to low affinity receptors in extracellular matrix (ECM). Finally, the most potent phosphorothioate oligodeoxynucleotides used in these experiments (e.g. SdC28) were inhibitors of bFGF-induced DNA synthesis in NIH 3T3 cells.
Assuntos
Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores de Superfície Celular/metabolismo , Tionucleotídeos/metabolismo , Células 3T3 , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Timidina/metabolismoRESUMO
Nine spontaneously transformed cell lines were isolated from embryo fibroblasts of mice and rats with different genotypes. In six cell lines highly tumorigenic cell variants were selected. At the start of culture all cell lines were of low or zero tumorigenicity. The same cells in a confluent monolayer in vitro had high contact inhibition of growth and proliferated in response to stimulation by growth factors. Tumour progression of the established lines was accompanied by significant changes of these properties. Clonal analysis of the six most malignant cell lines revealed their capacity to revert simultaneously to the non-tumorigenic state and to their initial growth characteristics. Frequencies of reversion to the non-tumorigenic phenotype were much higher than re-reversion to the tumorigenic phenotype. The reversions occurred in several sequential passages of transformed clones, with some variations in individual clones. These observations suppose that frequencies of tumour reversions are a constant genetic characteristic of every cell line.
Assuntos
Transformação Celular Neoplásica/genética , Células Tumorais Cultivadas/patologia , Animais , Linhagem Celular , Células Clonais , Testes Imunológicos de Citotoxicidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Substâncias de Crescimento/farmacologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos , Mitose/efeitos dos fármacos , Fenótipo , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Data are provided on the development of tumorigenicity in cells of 6 independently produced lines of mouse and rat embryonic fibroblasts during their prolonged passage in vitro. The data on the development of lysis of these cells by macrophages and activated lymphocytes during the same passage indicate that enhanced cell lysis of cells by natural effectors and, hence, cell recognition by them may be regarded as an obligatory manifestation of cell malignization.