RESUMO
Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Neoplasias da Mama/enzimologia , Carcinoma/enzimologia , Moduladores de Receptor Estrogênico/farmacologia , Fator 1 de Crescimento de Fibroblastos/fisiologia , Proteínas de Membrana/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Fosfotirosina/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxiciclina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genéticaRESUMO
ERalpha-negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key intermediate in the signal transduction pathways of these receptors. High levels of constitutive Raf kinase (Deltaraf) activity imparts ERalpha- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Deltaraf transfectants maintained in estrogen-depleted media showed greatly diminished responses to 17beta-estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays revealed a loss of ERalpha protein expression, and ribonuclease protection assays indicated that this correlated with loss of ERalpha message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfection assays with an estrogen-response element- reporter construct and Deltaraf or constitutively active MAPK kinase (DeltaMEK), no ligand- independent activation of ERalpha was observed. Transient expression of Deltaraf and double-label immunostaining showed ERalpha was lost in those cells that transiently expressed Deltaraf. Abrogation of Raf signaling via treatment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERalpha. Similar studies performed with MCF-7 cells overexpressing epidermal growth factor receptor or c-erbB-2 confirmed that hyperactivation of MAPK resulted in down-regulation of ERalpha that was reversible by MEK inhibition or transfection with dominant negative ERK1 and ERK2 constructs. These data suggest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for generation of an ERalpha-negative phenotype and, more importantly, that this process may be abrogated by inhibiting these pathways, thus restoring ERalpha expression.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Estrogênio/metabolismo , Northern Blotting , Western Blotting , Metilação de DNA , Ativação Enzimática , Receptores ErbB/genética , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Expressão Gênica , Humanos , Imuno-Histoquímica , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-raf/genética , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Ribonucleases , Transfecção , Células Tumorais CultivadasRESUMO
Angiopoietin-1 (Ang1) has been shown to act as an angiogenic promoter in embryonic angiogenesis by promoting vascular branching, pericyte recruitment and endothelial survival. We have investigated the role of Ang1 in tumour neovascularization under clinical conditions and in animal models. The expression of Ang1 in clinical breast cancer specimens was analysed by using laser-capture microdissection and reverse transcriptase-linked polymerase chain reaction (RT-PCR) on RNA isolated from the samples. Despite the expression of Ang1 in many human breast cancer cell lines, the gene was expressed in only three of 21 breast cancer clinical specimens, even though its receptor, Tie2, is abundant in the vasculature of all of these tumours. Ang1 was then overexpressed in a human breast cancer cell line (MCF-7) on its own and in conjunction with FGF1, an angiogenic factor shown to be able to increase the tumorigenicity of MCF-7 cells. High concentrations of Ang1 were produced in the conditioned media of the transfected cells (range 156-820 ng ml(-1)). However, in contrast to its physiological role as promoter of angiogenesis, overexpression of Ang1 did not enhance tumour growth, but instead caused up to a 3-fold retardation of tumour growth (P = 0.003).
Assuntos
Neoplasias da Mama/genética , Glicoproteínas de Membrana/genética , Angiopoietina-1 , Animais , Neoplasias da Mama/patologia , Células CHO , Divisão Celular/genética , Cricetinae , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , DNA Complementar/genética , Feminino , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Nus , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridine was consistently lower in lung tissues when compared with primary tumors, indicating that disseminated tumor cells were unable to maintain a high rate of proliferation. Increased angiogenesis resulting from FGF-1 production by the transfected cells with a concomitant increased rate of intravasation into developing blood vessels may be the underlying determinant of spontaneous micrometastasis produced by these cells when compared with parental MCF-7 cells.
Assuntos
Neoplasias da Mama/patologia , Fator 2 de Crescimento de Fibroblastos/genética , Neoplasias Pulmonares/secundário , Metástase Neoplásica/patologia , Animais , Neoplasias da Mama/genética , Feminino , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos Nus , Metástase Neoplásica/fisiopatologia , Ovariectomia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
We have previously shown that fibroblast growth factor (FGF)-1-, FGF-4-, or vascular endothelial growth factor (VEGF/VPF)-transfected MCF-7 breast carcinoma cells growing as tumors in nude mice are tamoxifen resistant and/or estrogen independent. These transfectants provide opportunity for study of in situ tumor-induced angiogenesis promoted by the individual angiogenic factors under growth-promoting versus growth-inhibiting hormonal conditions. In the present study, vessels in tumors harvested at varying times after tumor cell injection were immunohistochemically highlighted and vessel morphology and topography were scored on a scale of 0 to 4 by blinded observers. In tumors produced by all cell lines under all growth-promoting hormonal conditions, there was significantly increased abundance (P < 0.05) of edge-associated and intratumor microvessels, but not of stromally located microvessels, when compared with tumor nodules harvested under growth-inhibiting conditions, regardless of the identity of the angiogenic factor or the hormonal treatment. Image analysis of bromodeoxyuridine (BrdU)-labeled nuclei of tumors produced by all cell lines under all hormonal conditions harvested at early time points showed that mean labeling indices were highest for hormonal conditions that produced the most robust growth in that particular cell line, implying that a high BrdU labeling index is a predictor of future tumor growth in individual tumors. These results confirm previous studies that established the importance of neovascularization for tumor growth and provide validation for use of these cell lines to study the process of angiogenesis in vivo. Study of gene expression in endothelial cells in edge-associated or intratumor vessels using this model might reveal mechanisms important in tumor-induced angiogenesis in human breast cancer.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Carcinoma/irrigação sanguínea , Fatores de Crescimento Endotelial/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica , Linfocinas/fisiologia , Neovascularização Patológica/fisiopatologia , Animais , Neoplasias da Mama/genética , Bromodesoxiuridina , Carcinoma/genética , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Estradiol/farmacologia , Feminino , Fatores de Crescimento de Fibroblastos/genética , Humanos , Imuno-Histoquímica , Inflamação , Linfocinas/genética , Linfocinas/metabolismo , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Camundongos Nus , Microcirculação/anatomia & histologia , Microcirculação/efeitos dos fármacos , Transplante de Neoplasias , Neovascularização Patológica/diagnóstico , Ovariectomia , Prognóstico , Tamoxifeno/farmacologia , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Although the antiestrogen tamoxifen has been the mainstay of therapy for estrogen receptor (ER)-positive breast cancer, successful treatment of responsive tumors is often followed by the acquisition of tamoxifen resistance. Subsequently, only 30-40% of patients have a positive response to second hormonal therapies. This lack of response might be explained by mechanisms for tamoxifen resistance that sensitize ER pathways to small amounts of estrogenic activity present in tamoxifen or that bypass ER pathways completely. To elucidate one possible mechanism of tamoxifen resistance, we treated ovariectomized tumor-bearing mice injected with fibroblast growth factor (FGF)-transfected MCF-7 breast carcinoma cells with the steroidal antiestrogen ICI 182,780 or one of two aromatase inhibitors, 4-OHA or letrozole. These treatments did not slow estrogen-independent growth or prevent metastasis of tumors produced by FGF-transfected MCF-7 cells in ovariectomized nude mice. FGF-transfected cells had diminished responses to ICI 182,780 in vitro, suggesting that autocrine activity of the transfected FGF may be replacing estrogen as a mitogenic stimulus for tumor growth. ER levels in FGF transfectants were not down-regulated, and basal levels of transcripts for estrogen-induced genes or of ER-mediated transcription of estrogen response element (ERE) luciferase reporter constructs in the FGF expressing cells were not higher than parental cells, implying that altered hormonal responses are not due to down-regulation of ER or to FGF-mediated activation of ER. These studies indicate that estrogen independence may be achieved through FGF signaling pathways independent of ER pathways. If so, therapies directed at the operative mechanism might produce a therapeutic response or allow a response to a second course of antiestrogen treatment.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores da Aromatase , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Fatores de Crescimento de Fibroblastos/fisiologia , Tamoxifeno/uso terapêutico , Androstenodiona/análogos & derivados , Androstenodiona/uso terapêutico , Androstenodiona/toxicidade , Animais , Antineoplásicos/toxicidade , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cloranfenicol O-Acetiltransferase/biossíntese , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/toxicidade , Estradiol/uso terapêutico , Estradiol/toxicidade , Antagonistas de Estrogênios/uso terapêutico , Antagonistas de Estrogênios/toxicidade , Feminino , Fatores de Crescimento de Fibroblastos/biossíntese , Fulvestranto , Humanos , Letrozol , Luciferases/biossíntese , Camundongos , Camundongos Nus , Nitrilas/uso terapêutico , Nitrilas/toxicidade , Ovariectomia , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptores de Progesterona/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tamoxifeno/toxicidade , Transcrição Gênica , Transfecção , Transplante Heterólogo , Triazóis/uso terapêutico , Triazóis/toxicidade , Células Tumorais CultivadasRESUMO
To determine the extent to which autocrine effects of acidic fibroblast growth factor (FGF)-1 overexpression contribute to an increased malignant phenotype, FGF-1-transfected MCF-7 cells were retransfected with a FGF receptor (FGFR1) vector encoding a truncated dominant-negative receptor to inhibit autocrine FGF signal transduction. This transfection eliminated FGF signaling within the breast cancer cells without interfering with their ability to produce FGF-1, thereby allowing possible paracrine effects to still be observed in vivo. Truncated FGFR1 overexpression inhibited the acquired ability of FGF-1-overexpressing cells to form colonies in soft agar in estrogen-depleted or antiestrogen-containing medium. However, soft agar colony formation was still stimulated by estrogen treatment in cells expressing up to 6 x 10(5) truncated FGFR1 sites per cell. In vivo, truncated receptor expression severely inhibited the ability of the FGF-1-overexpressing cells to form tumors without estrogen in ovariectomized mice, indicating that the mitogenic effect of FGF-1 on the breast tumor cells was important in the estrogen-independent in vivo growth of these transfectants. However, rapid formation of large tumors was still observed in estrogen-supplemented mice injected with the truncated FGFR1-expressing cells, suggesting that the paracrine effects of FGF production could act in synergy with mitogenic effects mediated by estrogen. Truncated FGFR1-overexpressing cells also continued to form tumors in tamoxifen-treated mice, raising the possibility that the paracrine effects of FGF-1 expression may allow the partial agonist properties of this antiestrogen to be more readily observed. We conclude that autocrine effects of FGF-1 increase the ability of MCF-7 breast cancer cells to grow in vitro and in vivo under estrogen-depleted conditions but that paracrine effects of FGF-1 are also involved in the enhancement of tumor growth in estrogen-supplemented or tamoxifen-treated animals.
Assuntos
Comunicação Autócrina/fisiologia , Neoplasias da Mama/patologia , Antagonistas de Estrogênios/farmacologia , Estrogênios/fisiologia , Fator 1 de Crescimento de Fibroblastos/metabolismo , Comunicação Parácrina/fisiologia , Receptores Proteína Tirosina Quinases , Tamoxifeno/farmacologia , Animais , Northern Blotting , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , Primers do DNA/química , Resistencia a Medicamentos Antineoplásicos , Estrogênios/farmacologia , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
FGF-1 is expressed in a high proportion of breast tumors. While overexpression of FGF-4 in the MCF-7 breast carcinoma cell line confers the ability to form spontaneously metastasizing tumors in ovariectomized nude mice without estrogen supplementation and in mice that receive tamoxifen pellets, the response of a cell to individual FGFs can be controlled at multiple levels, and the significance of FGF-1 expression in human breast tumors is uncertain. To study the role of FGF-1, MCF-7 human breast cancer carcinoma cells, previously transfected with bacterial beta-galactosidase, were retransfected with FGF-1 expression vectors. FGF-1 transfectants formed large, vascularized tumors in ovariectomized nude mice without estrogen supplementation as well as in mice that received tamoxifen pellets. Lymphatic and pulmonary micrometastases were detected as deposits of X-gal-stained cells as early as 17 days after cell inoculation whereas no metastases were detected in estrogen-supplemented mice bearing similar-sized control tumors. When compared with controls, both clonal and polyclonal populations of FGF-1 overexpressing cells exhibited increased anchorage-independent growth and decreased population doubling times in estrogen-depleted or 4-hydroxytamoxifen containing medium. These results suggest that FGF signaling may be important in the transition of breast cancer cells from hormone-dependent to hormone-independent and from nonmetastatic to metastatic.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Carcinoma/irrigação sanguínea , Carcinoma/secundário , Antagonistas de Estrogênios/farmacologia , Fator 1 de Crescimento de Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Neovascularização Patológica/etiologia , Ovariectomia , Tamoxifeno/farmacologia , Animais , Permeabilidade Capilar , Adesão Celular , Divisão Celular/efeitos dos fármacos , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Vetores Genéticos , Humanos , Neoplasias Pulmonares/secundário , Metástase Linfática , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/genética , Fenótipo , RNA Mensageiro/metabolismo , Transfecção , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Overexpression of many growth factor receptors, as well as growth factors, has been shown to confer varying degrees of estrogen-independent growth on estrogen receptor (ER) positive breast cancer cells. The proto-oncogene Raf-1 is a key intermediate in the signal transduction pathway of many of these growth factor receptors, and when constitutively activated in fibroblasts is transforming. To examine the effects of Raf-1 kinase activity on the estrogen-dependent growth of human breast cancer cells, ER + MCF-7 breast cancer cells were stably transfected with an expression construct directing the expression of an amino-truncated protein having constitutive kinase activity. Expression of constitutively activated Raf in MCF-7 cells is incompatible with growth in the presence of estrogen; that is, cells down-regulate expression of the transfected Raf. Constitutive Raf activity does allow for growth of the cells in the absence of estrogen, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions. In addition, the high levels of Raf activity induce apoptosis in cells grown under either condition. This is a novel activity for Raf, and may occur because the levels of the constitutive Raf are extremely high in these cells.
Assuntos
Apoptose , Neoplasias da Mama/enzimologia , Estrogênios/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Meios de Cultura , Feminino , Humanos , Proteínas Serina-Treonina Quinases/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
To investigate the molecular mechanisms underlying the two- to three-fold induction of human transforming growth factor-alpha (hTGF-alpha) mRNA and two- to five-fold induction of hTGF-alpha protein observed following estrogen treatment of hormone-responsive human breast cancer cell lines, the hTGF-alpha promoter was assayed for ERE-like sequences able to mediate estrogen induction of a heterologous gene. Transient co-transfection of a chloramphenicol acetyl transferase (CAT) construct consisting of either 1100 bp or 330 bp of hTGF-alpha promoter sequence and an estrogen receptor expression vector into either COS-7 cells or hormonally responsive MCF-7 human breast cancer cells resulted in a two- to five-fold induction of CAT activity by estrogen. Although no consensus estrogen response element (ERE) exists in the hTGF-alpha promoter, a sequence consisting of two imperfect ERE palindromes separated by 20 bp is located at -200 to -252. This sequence was inserted into a mouse mammary tumor virus (MMTV) based CAT construct and assayed for its ability to confer estrogen regulation of CAT expression to a heterologous promoter. Transient co-transfection of this construct with an estrogen receptor expression vector into either COS-7 cells or MCF-7 cells resulted in an average 30-fold estrogen induction of CAT activity. Gel shift assays with human recombinant estrogen receptor (ER) and 32P-labelled fragments revealed that the ER could specifically bind to this sequence. These results indicate that this 53 bp sequence can function as an ERE, and is likely to be responsible for the observed induction of TGF-alpha message and protein in response to estrogen. These data also indicate that the level of estrogen inducibility mediated by this element may be positively or negatively modulated by interaction or competition with other transcription factors.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Fator de Crescimento Transformador alfa/genética , Animais , Sequência de Bases , Células COS , Carcinoma/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Sequência Consenso , DNA/metabolismo , Estradiol/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Vírus do Tumor Mamário do Camundongo , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Receptores de Estrogênio/fisiologia , Proteínas Recombinantes de Fusão , Sequências Repetitivas de Ácido Nucleico/genética , Transfecção , Células Tumorais CultivadasAssuntos
Indutores da Angiogênese/fisiologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Neovascularização Patológica/fisiopatologia , Progressão da Doença , Fatores de Crescimento Endotelial/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Humanos , Linfocinas/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Previously, we described FGF-1- or FGF-4-transfected MCF-7 breast carcinoma cells which are tumorigenic and metastatic in untreated or tamoxifen-treated ovariectomised nude mice. In this study, we have assessed the effects of AGM-1470, an antiangiogenic agent, and pentosan polysulphate (PPS), an agent that abrogates the effects of FGFs, on tumour growth and metastasis produced by these FGF-transfected MCF-7 cells. Untreated or tamoxifen-treated ovariectomised mice were injected with FGF-transfected cells, treated with AGM-1470 or PPS, and tumour growth and metastasis analysed. The sensitivity of FGF-transfected and parental MCF-7 cells to AGM-1470 or PPS was also determined in vitro. Both AGM-1470 and PPS inhibited tumour growth in otherwise untreated or tamoxifen-treated mice injected with either FGF- or FGF-4-transfected MCF-7 cells. This effect was more reliably seen in tamoxifen-treated animals. AGM-1470 was about 10(5) times less potent in inhibiting the anchorage-dependent growth of parental MCF-7 or FGF-transfected MCF-7 cells than in inhibiting the growth of human umbilical vein endothelial cells. PPS did not affect the in vitro growth of the transfectants or parental cells. Thus, the growth-inhibitory effect on tumours was in excess of the effect of either drug on the same cells in tissue culture, implying that stromal elements are important determinants of the effects of these drugs. There was a positive correlation between tumour size and the extent of proximal lymph node metastasis. However, neither drug had a significant effect on the extent of metastasis to proximal or distal lymph nodes or lungs. AGM-1470 or PPS may be helpful in cases of breast carcinoma in which angiogenesis is due to expression of FGFs by the tumour cells and may be more effective when combined with tamoxifen.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Fator 1 de Crescimento de Fibroblastos/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Poliéster Sulfúrico de Pentosana/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Sesquiterpenos/farmacologia , Animais , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/irrigação sanguínea , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cicloexanos , Implantes de Medicamento , Feminino , Fator 1 de Crescimento de Fibroblastos/biossíntese , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/biossíntese , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica/prevenção & controle , Neovascularização Patológica/prevenção & controle , O-(Cloroacetilcarbamoil)fumagilol , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Recombinantes/metabolismo , Tamoxifeno/administração & dosagem , Tamoxifeno/uso terapêutico , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
Progression of breast cancer from an estrogen-dependent, slowly growing tumor amenable to tamoxifen treatment to an aggressive, metastatic, estrogen-independent phenotype has been mimicked by the transfection of MCF-7 breast carcinoma cells with fibroblast growth factors 1 or 4. FGF-transfected cells are aggressively tumorigenic in ovariectomized or tamoxifen-treated nude mice, conditions under which the parental cells would not produce tumors. When detection of metastasis was enhanced by lacZ transfection, the FGF-transfected MCF-7 cells were reliably metastatic to lymph nodes and frequently metastatic to lungs, in further contrast to parental cells. An antiangiogenic drug, AGM-1470, given to mice bearing tumors produced by FGF-transfected MCF-7 cells, produced a decrease in tumor size. The decreased tumor size was not as marked as that produced by treatment with pentosan polysulfate, an agent which would abrogate all autocrine or paracrine effects of the transfected FGF. Thus, increased angiogenesis may be a component of the phenotypic change produced by the FGF transfection, but other autocrine or paracrine effects may also be important. Since a clonal FGF-4 and lacZ doubly-transfected cell line, MKL-4, progressively lost expression of the transfected lacZ gene in individual cells, we performed successive rounds of fluorescence-activated cell sorting to select high-expressing cells. High-expressing cell populations thus obtained rapidly lost expression of beta-gal activity in continued culture. High beta-gal expressing clonal cell lines of MKL-4 cells established by either one or two rounds of low-density cloning also lost lacZ expression with continued culture. Southern analysis of DNA from lacZ transfected cell lines showed the transfected sequences to be present and grossly intact in both high and low expressing populations. However, Northern analysis revealed that high-expressing populations of MKL-4 cells contained the most lacZ mRNA, implying that in the unstable MKL-4 cell line, individual cells are down-regulating mRNA levels of lacZ. Stable lacZ expression has been obtained in other FGF-transfected and parental MCF-7 cell lines using the same expression vector. Thus, the MKL-4 cell line is down-regulating mRNA encoding the transfected gene through a mechanism not dependent on the CMV promotor utilized in the expression vector. This evidence suggests that lacZ expression is not a benign modification in certain cells.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Carcinoma/irrigação sanguínea , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/fisiologia , Metástase Neoplásica/patologia , Neovascularização Patológica/patologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Divisão Celular , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Óperon Lac , Camundongos , Camundongos Nus , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
A c-erbB-2 expression vector was transfected into the estrogen receptor positive (ER+) MCF-7 human breast cancer cell line to determine if overexpression of this transmembrane tyrosine kinase could increase the malignant phenotype of this cell line. Loss of transfected c-erbB-2 expression was observed when cells were carried in medium containing estrogen. Homogeneous populations stably overexpressing levels of the 185 kDa c-erbB-2 observed in the SKBR-3 a breast cancer cell line which overexpresses c-erbB-2 as a result of gene amplification could be obtained by continually maintaining the transfected cell lines in estrogen-free conditions. Levels of constitutively activated c-erbB-2 varied among clonal isolates. Whereas some overexpressing lines did acquire the ability to form transient tumor nodules in ovariectomized nude mice without estrogen supplementation, as well as in mice that received the antiestrogen tamoxifen, one cell line that exhibited the highest levels of constitutively activated c-erbB-2 was able to form static tumors of a larger size under both conditions. This same cell line formed progressively growing tumors in estrogen-supplemented mice that were much larger than observed in mice injected with control cell lines, and also showed reduced sensitivity to antiestrogens in vitro, but it continued to have a low metastatic phenotype. These results suggest that signal transduction mediated by the c-erbB-2 tyrosine kinase can partially overcome the estrogen dependence of ER+breast cancer cells for growth and that c-erbB-2 overexpression confers a selective advantage to such cells in the absence of estrogen.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Estrogênios/fisiologia , Genes erbB-2 , Neoplasias Hormônio-Dependentes/genética , Tamoxifeno/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/genética , Estrogênios/deficiência , Estrogênios/metabolismo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hormônio-Dependentes/patologia , Fosforilação , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Sensibilidade e Especificidade , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Overexpression of epidermal growth factor receptor (EGFR) is correlated with loss of estrogen receptor and poor prognosis in breast cancer. To investigate this phenomenon, we transfected a cytomegalovirus expression vector directing the expression of EGFR into estrogen receptor-positive MCF-7 breast cancer cells and into a clone of MCF-7 cells previously transfected with transforming growth factor alpha. Cells arising from single clones or pooled polyclonal populations maintained in charcoal-stripped calf serum, a medium devoid of estrogen, overexpressed EGFR. Switching these cells to a medium containing fetal calf serum or charcoal-stripped calf serum plus 17 beta-estradiol resulted in the emergence of a population expressing low EGFR levels. Loss of expression was not a consequence of nonspecific repression of the cytomegalovirus promoter, because expression of the fibroblast growth factor (FGF)-4 complementary DNA in a similar vector was not lost in fetal calf serum. While loss of EGFR overexpression in fetal calf serum was seen at both the protein and mRNA levels, Southern blotting shows that this was not due to loss of the transfected gene. Subclones of a cell population with low EGFR expression were capable of increasing expression upon estrogen withdrawal, demonstrating that the changes in EGFR expression were reversible and suggesting a growth advantage conferred by EGFR overexpression under these restrictive growth conditions. Overexpression of EGFR did not result in loss of ER expression. These results suggest a role for overexpression of EGFR in the growth of estrogen receptor-positive breast cancer cells in the absence of estrogen.
Assuntos
Neoplasias da Mama/patologia , Receptores ErbB/metabolismo , Estradiol/farmacologia , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/metabolismo , Bovinos , Divisão Celular , Linhagem Celular , Meios de Cultura , Citomegalovirus , Receptores ErbB/biossíntese , Feminino , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/biossíntese , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transfecção , Células Tumorais CultivadasRESUMO
Fibroblast growth factors (FGFs) bind to cell membrane receptors and activate signal transduction pathways related to cell growth, angiogenesis, and tumorigenesis. FGFs have been shown to be abundantly expressed in some of the human tumors, which are known to be poorly responsive to radiation therapy. Using adrenal cortical carcinoma cells genetically engineered to express FGF-4, we have tested cellular survival following exposure to ionizing radiation. We report here that FGF-4 enhances cellular capacity to survive ionizing radiation. Furthermore, cell cycle analysis shows a pronounced increase in the duration of G2 arrest, suggesting perturbation of a cell cycle checkpoint. These findings implicate fibroblast growth factor-mediated signal transduction in cellular resistance of human tumors to radiation therapy.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Fase G2/efeitos dos fármacos , Proteínas Proto-Oncogênicas/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Fator 4 de Crescimento de Fibroblastos , Humanos , Células Tumorais CultivadasRESUMO
The MCF-7 human breast carcinoma cell line has been used as a recipient for eukaryotic plasmid expression vectors to determine the effects of growth factor and growth factor receptor overexpression on the estrogen-dependent, antiestrogen sensitive and poorly metastatic phenotypes exhibited by this line. Overexpression of some members of the erbB family of ligands and receptors were found to have some effects on these phenotypes. However, only when two members of the fibroblast growth factor family, FGF-1 and FGF-4, were overexpressed was progressive in vivo growth observed is either ovariectomized nude mice without estrogen supplementation or in mice that received tamoxifen treatment. FGF transfected cells also exhibited an increased ability to form micrometastases. The implications of these results with regard to the possible role of the paracrine and autocrine effects of angiogenic growth factor production in breast cancer progression are discussed.
Assuntos
Adenocarcinoma/patologia , Neoplasias da Mama/patologia , Estrogênios , Fatores de Crescimento de Fibroblastos/fisiologia , Neoplasias Hormônio-Dependentes/patologia , Transfecção , Células Tumorais Cultivadas , Adenocarcinoma/genética , Indutores da Angiogênese/fisiologia , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Progressão da Doença , Resistência a Medicamentos , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Feminino , Fatores de Crescimento de Fibroblastos/biossíntese , Fatores de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfocinas/biossíntese , Linfocinas/genética , Linfocinas/fisiologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias Hormônio-Dependentes/genética , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes de Fusão/biossíntese , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Células Tumorais Cultivadas/transplante , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Overexpression of some transmembrane tyrosine kinase growth factor receptors in breast and other tumors has been found to correlate with poor prognosis. Following the cloning of the first two members of the fibroblast growth factor family of receptors (FGFRs), amplification of these receptors in breast carcinomas was found. We have examined 23 breast carcinoma cell lines to determine the extent of expression of mRNA for fgfrs 1 through 4. All breast carcinoma cell lines examined expressed mRNA for at least one fgfr and several expressed high mRNA levels for a particular receptor. MDA-MB-134, an estrogen receptor-positive cell line, expressed very high levels of mRNA for fgfr-1 and elevated levels of mRNA for fgfr-4. This cell line was found to have an amplified fgfr-1 gene, but the gene for fgfr-4 was not amplified. MDA-MB-453 cells were found to express high levels of mRNA for fgfr-4 without amplification of the gene. MDA-MB-134 cells were examined for their response to FGF ligands. Tyrosine phosphorylation of a M(r) 150,000 protein resulted when MDA-MB-134 cells were treated with FGF-1 or FGF-2, implying the presence of a functional FGFR-1. MDA-MB-134 cells were growth-inhibited by picomolar concentrations of FGF-1 or FGF-2 in a dose-dependent manner under both anchorage-independent and anchorage-dependent conditions. These results may provide insight into the consequences of FGFR overexpression in breast tumors and the development of treatment modalities which use manipulation of growth factor responses.
Assuntos
Neoplasias da Mama/química , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptores de Fatores de Crescimento de Fibroblastos/análise , Anticorpos/análise , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Feminino , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Fosforilação , Fosfotirosina , Células Tumorais Cultivadas , Tirosina/análogos & derivados , Tirosina/imunologiaRESUMO
Successful antiestrogen treatment in patients with tamoxifen-responsive breast tumors is often followed by an outgrowth of tumors cells that are antiestrogen resistant, implying that estrogen-dependent tumors can become estrogen-independent. In an effect to mimic this progression, we have transfected fibroblast growth factor 4 into MCF-7 cells, a human breast carcinoma cell line that is estrogen-dependent for growth in nude mice. This transfection results in cell lines that form progressively growing, metastatic tumors when injected s.c. into untreated or tamoxifen-treated ovariectomized nude mice. In contrast to the parental cell line, growth of transfected cells in ovariectomized nude mice is stimulated by tamoxifen treatment and inhibited by estrogen treatment of the mice. Parental MCF-7 cells were transfected with an expression vector for beta-galactosidase, conferring the ability to convert the chromogenic substrate, 5-bromo-4-chloro-3-indoyl-beta-galactoside, to a blue color and allowing the detection of their presence within tumors developing after coinoculation with fibroblast growth factor 4-transfected cells. The fibroblast growth factor 4-transfected cells could support growth and metastasis of the beta-galactosidase-expressing parental cell line when both lines were coinjected into the same site in untreated or tamoxifen-treated, ovariectomized mice. These data suggest a possible role for fibroblast growth factors in the progression of breast tumors to an estrogen-independent, antiestrogen-resistant, metastatic phenotype. They also support a role for paracrine factors in mixed populations of tumor cells of differing states of malignant progression.
Assuntos
Neoplasias da Mama/fisiopatologia , Fatores de Crescimento de Fibroblastos/farmacologia , Neoplasias Experimentais/fisiopatologia , Proteínas Proto-Oncogênicas/farmacologia , Animais , Neoplasias da Mama/patologia , Divisão Celular , Estrogênios/farmacologia , Feminino , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Humanos , Técnicas In Vitro , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Ovariectomia , Proteínas Proto-Oncogênicas/genética , Tamoxifeno/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
We recently established transfectants of MCF-7 human breast cancer cells with fibroblast growth factor 4 (fgf-4) that showed rapid growth and spontaneous metastasis in ovariectomized and tamoxifen-treated nude mice. To establish a spontaneous metastatic model of human breast cancer cells in nude mice with a sensitive marker for detection of micrometastasis, the transfection of fgf-4 was combined with transfection of the bacterial lacZ gene encoding beta-galactosidase. MKL-4 cells, a lacZ transfectant of an fgf-4-transfected cell line, showed the same level of fgf-4 expression as parental cells and expressed a high level of beta-galactosidase activity. When MKL-4 cells were injected s.c. into female nude mice, rapidly growing tumors developed. Whole organ staining for beta-galactosidase activity was able to detect even small numbers of metastatic tumor cells. Micrometastases in lymph nodes, lung, and brain were detected 3 weeks after the tumor cell injections, the first time point tested. Within 12 weeks, metastases were observed in lymph nodes, lung, brain, kidney, perirenal fatty tissues, liver, spleen, retroperitoneum, heart, and gallbladder. The frequency of metastasis and number of foci were correlated with the volume of the primary tumors. The distribution of metastatic sites was similar to that in breast cancer patients. MKL-4 cells may be a useful model for studying the malignant progression of hormone-dependent breast cancer, antimetastatic drugs, or early events in metastasis.