RESUMO
The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling axis is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of Nrf2 regulation has been focused at the protein level. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These RNA-sequencing data and subsequent mechanistic analyses led us to discover a novel role of HuR in regulating Nrf2 activity, and in the process, we further identified the related mRBP AUF1 as an additional novel Nrf2 regulator. Both mRBPs regulate Nrf2 activity by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export, whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using a Nrf2 activity-reporter zebrafish strain, we document that this post-transcriptional control of Nrf2 activity is conserved at the whole-vertebrate level.-Poganik, J. R., Long, M. J. C., Disare, M. T., Liu, X., Chang, S.-H., Hla, T., Aye, Y. Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Células Cultivadas , Proteína Semelhante a ELAV 1/genética , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neovascularização Patológica/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HCT116 , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Patológica/metabolismoRESUMO
Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types.
Assuntos
Proteínas E4 de Adenovirus/genética , Proteína Forkhead Box O1/genética , Transportador de Glucose Tipo 4/genética , Insulina/metabolismo , Proteínas E4 de Adenovirus/biossíntese , Adipócitos/metabolismo , Animais , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Humanos , Insulina/genética , Camundongos , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Transfecção , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Posttranscriptional gene regulation by miRNAs and RNA binding proteins (RBP) is important in development, physiology, and disease. To examine the interplay between miRNAs and the RBP ELAVL1 (HuR), we mapped miRNA binding sites at the transcriptome-wide scale in wild-type and Elavl1 knockout murine bone-marrow-derived macrophages. Proximity of ELAVL1 binding sites attenuated miRNA binding to transcripts and promoted gene expression. Transcripts that regulate angiogenesis and macrophage/endothelial crosstalk were preferentially targeted by miRNAs, suggesting that ELAVL1 promotes angiogenesis, at least in part by antagonism of miRNA function. We found that ELAVL1 antagonized binding of miR-27 to the 3' UTR of Zfp36 mRNA and alleviated miR-27-mediated suppression of the RBP ZFP36 (Tristetraprolin). Thus, the miR-27-regulated mechanism synchronizes the expression of ELAVL1 and ZFP36. This study provides a resource for systems-level interrogation of posttranscriptional gene regulation in macrophages, a key cell type in inflammation, angiogenesis, and tissue homeostasis.
Assuntos
Proteínas ELAV/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Células Cultivadas , Proteínas ELAV/genética , Proteína Semelhante a ELAV 1 , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMO
Posttranscriptional RNA regulation is important in determining the plasticity of cellular phenotypes. However, mechanisms of how RNA binding proteins (RBPs) influence cellular behavior are poorly understood. We show here that the RBP embryonic lethal abnormal vision like 1 (ELAVL1, also know as HuR) regulates the alternative splicing of eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), which encodes an eukaryotic translation initiation factor 4E transporter (4E-T) protein and suppresses the expression of capped mRNAs. In the absence of ELAVL1, skipping of exon 11 of Eif4enif1 forms the stable, short isoform, 4E-Ts. This alternative splicing event results in the formation of RNA processing bodies (PBs), enhanced turnover of angiogenic mRNAs, and suppressed sprouting behavior of vascular endothelial cells. Further, endothelial-specific Elavl1 knockout mice exhibited reduced revascularization after hind limb ischemia and tumor angiogenesis in oncogene-induced mammary cancer, resulting in attenuated blood flow and tumor growth, respectively. ELAVL1-regulated alternative splicing of Eif4enif1 leading to enhanced formation of PB and mRNA turnover constitutes a novel posttranscriptional mechanism critical for pathological angiogenesis.
Assuntos
Processamento Alternativo/fisiologia , Proteínas ELAV/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Proteína Semelhante a ELAV 1 , Éxons , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismoRESUMO
HuR is a ubiquitous nucleocytoplasmic RNA-binding protein that exerts pleiotropic effects on cell growth and tumorigenesis. In this study, we explored the impact of conditional, tissue-specific genetic deletion of HuR on intestinal growth and tumorigenesis in mice. Mice lacking intestinal expression of HuR (Hur (IKO) mice) displayed reduced levels of cell proliferation in the small intestine and increased sensitivity to doxorubicin-induced acute intestinal injury, as evidenced by decreased villus height and a compensatory shift in proliferating cells. In the context of Apc(min/+) mice, a transgenic model of intestinal tumorigenesis, intestinal deletion of the HuR gene caused a three-fold decrease in tumor burden characterized by reduced proliferation, increased apoptosis, and decreased expression of transcripts encoding antiapoptotic HuR target RNAs. Similarly, Hur(IKO) mice subjected to an inflammatory colon carcinogenesis protocol [azoxymethane and dextran sodium sulfate (AOM-DSS) administration] exhibited a two-fold decrease in tumor burden. Hur(IKO) mice showed no change in ileal Asbt expression, fecal bile acid excretion, or enterohepatic pool size that might explain the phenotype. Moreover, none of the HuR targets identified in Apc(min/+)Hur(IKO) were altered in AOM-DSS-treated Hur(IKO) mice, the latter of which exhibited increased apoptosis of colonic epithelial cells, where elevation of a unique set of HuR-targeted proapoptotic factors was documented. Taken together, our results promote the concept of epithelial HuR as a contextual modifier of proapoptotic gene expression in intestinal cancers, acting independently of bile acid metabolism to promote cancer. In the small intestine, epithelial HuR promotes expression of prosurvival transcripts that support Wnt-dependent tumorigenesis, whereas in the large intestine epithelial HuR indirectly downregulates certain proapoptotic RNAs to attenuate colitis-associated cancer. Cancer Res; 74(18); 5322-35. ©2014 AACR.
Assuntos
Neoplasias do Colo/patologia , Proteínas ELAV/fisiologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Animais , Apoptose/fisiologia , Processos de Crescimento Celular/fisiologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Camundongos , Camundongos KnockoutRESUMO
PURPOSE OF REVIEW: This review summarizes recent findings in the area of post-transcriptional regulation of gene expression during angiogenesis, also known as new blood vessel formation. Specifically, we focus on gene regulation by HuR, an RNA-binding protein (RBP), and microRNAs (miRNAs) and their interplay, which ultimately influences cellular phenotypes of cells involved in angiogenesis. RECENT FINDINGS: Recently, RBPs and miRNAs have emerged as key regulators of angiogenesis. We and others have demonstrated that the RBP HuR (a.k.a. Elavl1) stabilizes vascular endothelial growth factor-A mRNA, a potent angiogenic factor in the settings of tumor development and inflammation. However, several miRNAs were shown to modulate gene expression during developmental (miR-126), physiological (miR-126, miR-92a), and pathological angiogenesis (miR-200b, miR-132). Moreover, the interplay of HuR and miRNAs in the regulation of genes involved in angiogenesis was described. In addition, recent work suggests a new role of circulating miRNAs as paracrine mediators in angiogenesis. SUMMARY: The elucidation of novel posttranscriptional gene regulatory mechanisms has expanded our understanding of angiogenesis in physiological and pathological conditions. We anticipate that this knowledge will ultimately lead to new insights for discovering novel therapeutic strategies to control pathological angiogenesis.
Assuntos
Proteínas ELAV/fisiologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/fisiologia , Neovascularização Fisiológica/fisiologia , Humanos , Neovascularização Patológica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The cyclooxygenase/prostaglandin (COX/PG) signaling pathway is of central importance in inflammation and neoplasia. COX inhibitors are widely used for analgesia and also have demonstrated activity for cancer prophylaxis. However, cardiovascular toxicity associated with this drug class diminishes their clinical utility and motivates the development of safer approaches both for pain relief and cancer prevention. The terminal synthase microsomal PGE synthase-1 (mPGES-1) has attracted considerable attention as a potential target. Overexpression of mPGES-1 has been observed in both colorectal and breast cancers, and gene knockout and overexpression approaches have established a role for mPGES-1 in gastrointestinal carcinogenesis. Here we evaluate the contribution of mPGES-1 to mammary tumorigenesis using a gene knockout approach. Mice deficient in mPGES-1 were crossed with a strain in which breast cancer is driven by overexpression of human epidermal growth factor receptor 2 (HER2/neu). Loss of mPGES-1 was associated with a substantial reduction in intramammary PGE2 levels, aromatase activity, and angiogenesis in mammary glands from HER2/neu transgenic mice. Consistent with these findings, we observed a significant reduction in multiplicity of tumors ≥1mm in diameter, suggesting that mPGES-1 contributes to mammary tumor growth. Our data identify mPGES-1 as a potential anti-breast cancer target.
Assuntos
Deleção de Genes , Oxirredutases Intramoleculares/deficiência , Oxirredutases Intramoleculares/genética , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/patologia , Neovascularização Patológica/genética , Animais , Aromatase/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Transgênicos , Microvasos/metabolismo , Prostaglandina-E Sintases , Receptor ErbB-2/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
HuR, also known as Elavl1, is an RNA-binding protein that regulates embryonic development, progenitor cell survival, and cell stress responses. The role of HuR in angiogenesis is not known. Using a myeloid-specific HuR knock-out mouse model (Elavl1Mø KO), we show that HuR expression in bone marrow-derived macrophages (BMDMs) is needed to maintain the expression of genes enriched in AU-rich elements and U-rich elements in the 3'-UTR. In addition, BMDMs from Elavl1Mø KO mice also showed alterations in expression of several miRNAs. Interestingly, computational analysis suggested that miR-200b, which is up-regulated in Elavl1Mø KO BMDMs, interacts with myeloid mRNAs very close to the HuR binding sites, suggesting competitive regulation of gene expression. One such mRNA encodes vascular endothelial growth factor (VEGF)-A, a major regulator of angiogenesis. Immunoprecipitation of RNA-protein complexes and luciferase reporter assays indicate that HuR antagonizes the suppressive activity of miR-200b, down-regulates miR-200b expression, and promotes VEGF-A expression. Indeed, Vegf-a and other angiogenic regulatory transcripts were down-regulated in Elavl1Mø KO BMDMs. Interestingly, tumor growth, angiogenesis, vascular sprouting, branching, and permeability were significantly attenuated in Elavl1Mø KO mice, suggesting that HuR-regulated myeloid-derived factors modulate tumor angiogenesis in trans. Zebrafish embryos injected with an elavl1 morpholino oligomer or miR-200b mimic showed angiogenesis defects in the subintestinal vein plexus, and elavl1 mRNA rescued the repressive effect of miR-200b. In addition, miR-200b and HuR morpholino oligomer suppressed the activity of a zVEGF 3'-UTR luciferase reporter construct. Together, these studies reveal an evolutionarily conserved post-transcriptional mechanism involving competitive interactions between HuR and miR-200b that controls angiogenesis.
Assuntos
Proteínas ELAV/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Antígeno CD11b/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Deleção de Genes , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , Peixe-ZebraRESUMO
Once mRNAs are transcribed, spliced and transported to the cytoplasm, their fate is determined by the complex interplay of RNA binding proteins (RBPs) and microRNAs (miRNAs) that act on regulatory elements within the transcripts. The importance of post-transcriptional regulatory mechanisms in angiogenesis is underscored by the observation that perturbations in miRNAs and/or RBPs lead to profound phenotypic alterations in vascular development, homeostasis and disease, with current data suggesting that mRNAs for key angiogenic regulators (secreted factors and intracellular signaling intermediates) are subject to stringent post-transcriptional regulation by both RBPs and miRNAs. In addition, an intricate network of miRNAs and RBPs allow robust gene regulation in vascular cells. This review focuses on the miRNAs and RBPs which often cooperate to achieve precise spatial and temporal control of angiogenic regulatory genes.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Neovascularização Fisiológica/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement.
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/patologia , Inibidores da Angiogênese/uso terapêutico , Animais , Humanos , Fenótipo , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Caveolin-1, the signature protein of endothelial cell caveolae, has many important functions in vascular cells. Caveolae are thought to be the transcellular pathway by which plasma proteins cross normal capillary endothelium, but, unexpectedly, cav-1(-/-) mice, which lack caveolae, have increased permeability to plasma albumin. The acute increase in vascular permeability induced by agents such as vascular endothelial growth factor (VEGF)-A occurs through venules, not capillaries, and particularly through the vesiculo-vacuolar organelle (VVO), a unique structure composed of numerous interconnecting vesicles and vacuoles that together span the venular endothelium from lumen to ablumen. Furthermore, the hyperpermeable blood vessels found in pathological angiogenesis, mother vessels, are derived from venules. The present experiments made use of cav-1(-/-) mice to investigate the relationship between caveolae and VVOs and the roles of caveolin-1 in VVO structure in the acute vascular hyperpermeability induced by VEGF-A and in pathological angiogenesis and associated chronic vascular hyperpermeability. We found that VVOs expressed caveolin-1 variably but, in contrast to caveolae, were present in normal numbers and with apparently unaltered structure in cav-1(-/-) mice. Nonetheless, VEGF-A-induced hyperpermeability was strikingly reduced in cav-1(-/-) mice, as was pathological angiogenesis and associated chronic vascular hyperpermeability, whether induced by VEGF-A(164) or by a tumor. Thus, caveolin-1 is not necessary for VVO structure but may have important roles in regulating VVO function in acute vascular hyperpermeability and angiogenesis.
Assuntos
Permeabilidade Capilar/fisiologia , Caveolina 1/deficiência , Neovascularização Patológica/fisiopatologia , Adenoviridae , Animais , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Pele/irrigação sanguínea , Pele/patologia , Pele/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.
Assuntos
Catepsinas/genética , Inibidores de Cisteína Proteinase/farmacologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Vênulas/fisiologia , Animais , Catepsinas/antagonistas & inibidores , Cistatina A/genética , Cistatina A/farmacologia , Cistatina B/deficiência , Cistatina B/farmacologia , Camundongos , Camundongos Knockout , Camundongos Nus , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Reação em Cadeia da Polimerase , Vênulas/efeitos dos fármacosRESUMO
Cytochrome P450 aromatase (aromatase), a product of the CYP19 gene, catalyzes the synthesis of estrogens from androgens. Because aromatase-dependent estrogen biosynthesis has been linked to hormone-dependent breast carcinogenesis, it is important to elucidate the mechanisms that regulate CYP19 gene expression. The main objective of this study was to identify the receptors (EP) for prostaglandin E(2) (PGE(2)) that mediate the induction of CYP19 transcription in human adipocytes and breast cancer cells. Treatment with PGE(2) induced aromatase, an effect that was mimicked by either EP(2) or EP(4) agonists. Antagonists of EP(2) or EP(4) or small interference RNA-mediated down-regulation of these receptors suppressed PGE(2)-mediated induction of aromatase. PGE(2) via EP(2) and EP(4) stimulated the cAMP-->protein kinase A pathway resulting in enhanced interaction between P-CREB, p300, and the aromatase promoter I.3/II. Overexpressing a mutant form of p300 that lacks histone acetyltransferase activity suppressed PGE(2)-mediated induction of aromatase promoter activity. PGE(2) via EP(2) and EP(4) also caused a reduction in both the amounts of BRCA1 and the interaction between BRCA1 and the aromatase promoter I.3/II. Activation of the aromatase promoter by PGE(2) was suppressed by overexpressing wild-type BRCA1. Silencing of EP(2) or EP(4) also blocked PGE(2)-mediated induction of the progesterone receptor, a prototypic estrogen-response gene. In a mouse model, overexpressing COX-2 in the mammary gland, a known inducer of PGE(2) synthesis, led to increased aromatase mRNA and activity and reduced amounts of BRCA1; these effects were reversed by knocking out EP(2). Taken together, these results suggest that PGE(2) via EP(2) and EP(4) activates the cAMP-->PKA-->CREB pathway leading to enhanced CYP19 transcription and increased aromatase activity. Reciprocal changes in the interaction between BRCA1, p300, and the aromatase promoter I.3/II contributed to the inductive effects of PGE(2).
Assuntos
Adipócitos/enzimologia , Aromatase/biossíntese , Aromatase/genética , Proteína BRCA1/genética , Neoplasias da Mama/enzimologia , Proteína p300 Associada a E1A/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Receptores de Prostaglandina E/metabolismo , Adipócitos/metabolismo , Animais , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Humanos , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4RESUMO
The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in approximately 40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.
Assuntos
Ciclo-Oxigenase 2/deficiência , Neoplasias Mamárias Experimentais/genética , Receptor ErbB-2/genética , Animais , Ciclo-Oxigenase 2/genética , Feminino , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/enzimologia , Neovascularização Patológica/genéticaRESUMO
Expression of cyclooxygenase 2 (COX-2) in breast cancer correlates with poor prognosis, and COX-2 enzyme inhibitors reduce breast cancer incidence in humans. We recently showed that COX-2 overexpression in the mammary gland of transgenic mice induced mammary cancer. Because prostaglandin E2 (PGE2) is the major eicosanoid and because the EP2 subtype of the PGE2 receptor is highly expressed in the mammary tumors, we tested if this G protein-coupled receptor is required for tumorigenesis. We crossed the MMTV-COX-2 transgenic mice with Ep2-/- mice and studied tumor development in bigenic mice. Lack of EP2 receptor strongly suppressed COX-2-induced effects such as precocious development of the mammary gland in virgins and the development of mammary hyperplasia in multiparous female mice. Interestingly, the expression of amphiregulin, a potent mammary epithelial cell growth factor was down regulated in mammary glands of Ep2-/- mice. Total cyclic AMP (cAMP) levels were reduced in Ep2-/- mammary glands suggesting that PGE2 signaling via the EP2 receptor activates the Gs/cAMP/protein kinase A pathway. In mammary tumor cell lines, expression of the EP2 receptor followed by treatment with CAY10399, an EP2-specific agonist, strongly induced amphiregulin mRNA levels in a protein kinase A-dependent manner. These data suggest that PGE2 signaling via the EP2 receptor in mammary epithelial cells regulate mammary gland hyperplasia by the cAMP-dependent induction of amphiregulin. Inhibition of the EP2 pathway in the mammary gland may be a novel approach in the prevention and/or treatment of mammary cancer.
Assuntos
Glândulas Mamárias Animais/patologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Receptores de Prostaglandina E/fisiologia , Anfirregulina , Animais , Ciclo-Oxigenase 2 , Família de Proteínas EGF , Feminino , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/biossíntese , Hiperplasia , Endogamia , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Masculino , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , Receptores de Prostaglandina E/deficiência , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP2 , Transdução de SinaisRESUMO
Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Receptores de Prostaglandina E/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Sequência de Bases , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA , Feminino , Imunoprecipitação , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Receptores de Prostaglandina E Subtipo EP2 , Células Tumorais CultivadasRESUMO
HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene-induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.
Assuntos
Antígenos de Superfície/biossíntese , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas de Ligação a RNA/biossíntese , Animais , Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Análise Multivariada , PrognósticoRESUMO
Overexpression of human cyclooxygenase 2 (COX-2) in the mammary glands of transgenic mice induces tissue-specific tumorigenic transformation. However, the molecular mechanisms involved are not yet defined. Here we show that COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland. Microvessel density increased before visible tumor growth and exponentially during tumor progression. Inhibition of prostanoid synthesis with indomethacin strongly decreased microvessel density and inhibited tumor progression. Up-regulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue was also potently inhibited by indomethacin treatment, suggesting that prostanoids released from COX-2-expressing mammary epithelial cells induce angiogenesis. G protein-coupled receptors for the major product, prostaglandin E(2) (PGE(2)) EP(1-4), are expressed during mammary gland development, and EP(1,2,4) receptors were up-regulated in tumor tissue. PGE(2) stimulated the expression angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice. Such cells are tumorigenic in nude mice; however, treatment with Celecoxib, a COX-2-specific inhibitor, reduced tumor growth and microvessel density. These results define COX-2-derived PGE(2) as a potent inducer of angiogenic switch during mammary cancer progression.