RESUMO
We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, ß and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte-RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis.
Assuntos
Adipócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glândulas Mamárias Animais/embriologia , Morfogênese/fisiologia , Comunicação Parácrina/fisiologia , Receptores do Ácido Retinoico/metabolismo , Células 3T3-L1 , Animais , Proteínas de Transporte/metabolismo , Meios de Cultivo Condicionados/química , Citocinas/metabolismo , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imuno-Histoquímica , Glândulas Mamárias Animais/transplante , Camundongos , Camundongos Transgênicos , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
Downregulation of the cellular retinol-binding protein-I (CRBP-I) occurs in breast and other human cancers, but its significance is not well understood. Recently, we showed that restoration of CRBP-I expression in transformed MTSV1-7 breast epithelial cells increased retinoic receptor activity, inhibited anoikis, promoted acinar differentiation and inhibited tumorigenicity, suggesting that CRBP-I suppresses tumor progression. However, the mechanism underlying these effects of CRBP-I was not elucidated. Here we demonstrate, using genetic and pharmacological approaches, that CRBP-I inhibits, in a retinoic acid receptor-dependent manner, the PI3K/Akt survival pathway. Inhibition of PI3K/Akt was necessary and sufficient to explain the antitumor effects of CRBP-I and was mediated by decreased p85 regulatory and p110 catalytic subunit heterodimerization. We present evidence consistent with the idea that this effect is due to CRBP-I inhibition of p85 phosphorylation at Y688. To our knowledge, this is the first demonstration of PI3K regulation at the level of p85-p110 heterodimerization. These findings lead us to hypothesize that CRBP-I downregulation in cancer promotes tumor progression through inhibition of retinoic acid receptor activity and derepression of PI3K/Akt signaling via a novel mechanism.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Ácido Retinoico/fisiologia , Proteínas de Ligação ao Retinol/fisiologia , Transdução de Sinais/fisiologia , Mama , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular , Linhagem Celular Transformada , Dimerização , Progressão da Doença , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/metabolismo , Proteínas Celulares de Ligação ao Retinol , TransfecçãoRESUMO
Human breast cancer cell lines expressing the estrogen receptor alpha (ERalpha), all-trans-retinoic acid (ATRA) receptor alpha (RARalpha) and cellular retinoic acid binding protein II (CRABPII) genes are sensitive to ATRA-mediated growth inhibition. To study the relationship among ERalpha, RARalpha and CRABPII expression, the protein levels of each member were compared in five breast cancer cell lines (T47D, MCF-7, ZR-75-1, Hs587 T and MDA-MB-231 cells) and two immortalized nontumorigenic breast epithelial cell lines (MTSV1.7 and MCF-10A). ERalpha, RARalpha and CRABPII proteins were detected in T47D, MCF-7 and ZR-75-1 cells but not in other tested cell lines. RARalpha and CRABPII proteins were either reduced or undetectable in T47D/C4:2W and MCF-7/ADR cells with lost expression of ERalpha. Estradiol increased and anti-estrogens (tamoxifen and ICI 164,384) downregulated the expression of both RARalpha and CRABPII proteins in T47D and MCF-7 cells. RARalpha antagonist Ro-41-5253 inhibited CRABPII expression, but not RARalpha expression in estradiol-treated T47D and MCF-7 cells. Suppression of ERalpha by small interfering RNA (siRNA) reduced RARalpha and CRABPII gene expression and siRNA suppression of RARalpha reduced CRABPII expression while having no effect on ERalpha in T47D cells. Transient transfection of either RARalpha or ERalpha expression vectors increased CRABPII expression in MDA-MB-231 cells but only RARalpha, not ERalpha, activated hCRABPII promoter reporter. These results indicate that there is a gene activation pathway in which ERalpha drives RARalpha transcription and RARalpha drives CRABPII transcription in ERalpha-positive human breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores do Ácido Retinoico/metabolismo , Animais , Benzoatos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular , Chlorocebus aethiops , Cromanos/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Ativação Transcricional , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Retinoic acid suppresses cell growth and promotes cell differentiation, and pharmacological retinoic acid receptor (RAR) activation is anti-tumorigenic. This begs the question of whether chronic physiological RAR activation by endogenous retinoids is likewise anti-tumorigenic. RESULTS: To address this question, we generated transgenic mice in which expression of a ligand binding defective dominant negative RARalpha (RARalphaG303E) was under the control of the mouse mammary tumor virus (MMTV) promoter. The transgene was expressed in the lymphoid compartment and in the mammary epithelium. Observation of aging mice revealed that transgenic mice, unlike their wild type littermates, developed B cell lymphomas at high penetrance, with a median latency of 40 weeks. MMTV-RARalphaG303E lymphomas were high grade Pax-5+, surface H+L Ig negative, CD69+ and BCL6- and cytologically and phenotypically resembled human adult high grade (Burkitt's or lymphoblastic) lymphomas. We postulated that mammary tumors might arise after a long latency period as seen in other transgenic models of breast cancer. We tested this idea by transplanting transgenic epithelium into the cleared fat pads of wild type hosts, thus bypassing lymphomagenesis. At 17 months post-transplantation, a metastatic mammary adenocarcinoma developed in one of four transplanted glands whereas no tumors developed in sixteen of sixteen endogenous glands with wild type epithelium. CONCLUSION: These findings suggest that physiological RAR activity may normally suppress B lymphocyte and mammary epithelial cell growth and that global RAR inactivation is sufficient to initiate a stochastic process of tumor development requiring multiple transforming events. Our work makes available to the research community a new animal resource that should prove useful as an experimental model of aggressive sporadic lymphoma in immunologically uncompromised hosts. We anticipate that it may also prove useful as a model of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transformação Celular Neoplásica/genética , Genes Dominantes/genética , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/transplante , Glicina/genética , Glicina/metabolismo , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Receptor alfa de Ácido Retinoico , Taxa de SobrevidaRESUMO
BACKGROUND: The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer. RESULTS: Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event. CONCLUSION: CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.
Assuntos
Neoplasias da Mama/genética , Metilação de DNA , Proteínas de Ligação ao Retinol/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Evolução Molecular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Oncogenes/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao Retinol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
BACKGROUND: Retinoic acid receptor (RAR) activation induces cell differentiation and may antagonize cancer progression. Cellular retinol-binding protein I (CRBP-I) functions in retinol storage and its expression is lower in human cancers than in normal cells. We hypothesized that retinol storage might be linked to RAR activation and thus that lowered CRBP-I function might impair RAR activity and cell differentiation. METHODS: Sarcoma virus 40-immortalized human mammary epithelial cells (MTSV1-7) devoid of CRBP-I were transfected with wild-type CRBP-I or CRBP-I point mutants with low RA binding affinity. The subcellular localization of CRBP-I was investigated in these cells and in wild-type or CRBP-I null mouse mammary epithelial cells (MECs), using indirect immunofluorescence and sucrose gradient fractionation. RAR activity was assessed using reporter gene assays. Acinar differentiation and in vivo tumor growth were assessed in reconstituted basement membrane and athymic mice, respectively. RESULTS: In cells expressing wild-type CRBP-I but not the CRBP-I mutants, CRBP-I was found mainly in lipid droplets, the retinol storage organelle, and this localization was associated with promotion of retinol storage by wild-type CRBP-I only. RAR activity was higher and acinar differentiation was observed in cells expressing wild-type but not mutant CRBP-I. RAR antagonist treatment blocked and chronic RA treatment mimicked, the CRBP-I induction of cell differentiation. Finally, CRBP-I suppressed tumorigenicity in athymic mice. CONCLUSIONS: Physiologic RAR activation is dependent on CRBP-I-mediated retinol storage, and CRBP-I downregulation chronically compromises RAR activity, leading to loss of cell differentiation and tumor progression.
Assuntos
Neoplasias da Mama/metabolismo , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Animais , Diferenciação Celular , Progressão da Doença , Regulação para Baixo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Nus , Mutação Puntual , Receptores do Ácido Retinoico/genética , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Sacarose/metabolismo , TransfecçãoRESUMO
Retinoic acid (RA) receptor (RAR) beta2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RAR beta2 promoter and 5'-UTR is the underlying cause. Here we show that RAR alpha2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR alpha2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR alpha2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR alpha2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR alpha2 and RAR beta2. On the basis of a clonogenic assay, RAR alpha2 displayed ligand-dependent growth-suppressive activity similar to that of RARb eta2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR alpha2-transfected cells compared with 75 and 77%, respectively, in RAR beta2-transfected cells. We conclude that the silencing of the RAR alpha2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Receptores do Ácido Retinoico/genética , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Receptor alfa de Ácido Retinoico , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Plasminogen activation is a widespread cellular mechanism for localized extracellular proteolysis that is postulated to participate in many diverse physiological and pathological phenomena. The present review in intended as an introduction to the subject, and is by no means comprehensive, except for the section on breast cancer. Seral extensive reviews are available that should be consulted by intersted readers (1-6).