RESUMO
Small nucleolar RNAs (snoRNAs) are noncoding RNAs that guide chemical modifications of structural RNAs. Whereas snoRNAs primarily localize in the nucleolus, where their canonical function is to target nascent ribosomal RNAs for 2'-O-methylation, recent studies provide evidence that snoRNAs traffic out of the nucleus. Furthermore, RNA-Seq data indicate that extracellular vesicles released from cells contain snoRNAs. However, it is not known whether snoRNA secretion is regulated or whether secreted snoRNAs are functional. Here, we show that inflammation stimulates secretion of Rpl13a snoRNAs U32a (SNORD32a), U33 (SNORD33), U34 (SNORD34), and U35a (SNORD35a) from cultured macrophages, in mice, and in human subjects. Secreted snoRNAs co-fractionate with extracellular vesicles and are taken up by recipient cells. In a murine parabiosis model, we demonstrate that snoRNAs travel through the circulation to function in distant tissues. These findings support a previously unappreciated link between inflammation and snoRNA secretion in mice and humans and uncover a potential role for secreted snoRNAs in cell-cell communication.
Assuntos
Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/química , RNA Nucleolar Pequeno/metabolismo , Proteínas Ribossômicas/fisiologia , Animais , Transporte Biológico , Nucléolo Celular/genética , Núcleo Celular/genética , Feminino , Humanos , Masculino , Metilação , Camundongos , Camundongos Knockout , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genéticaRESUMO
Endocrine therapy-resistant estrogen receptor-positive (ER(+)) breast cancer is the most common cause of breast cancer death. Miller and colleagues demonstrate that ligand-independent ER activity promotes the growth of breast cancer cells through CDK4/E2F. As an independent event, the phosphatidylinositol 3-kinase (PI3K) pathway is also upregulated in endocrine therapy-resistant cells. Promising preclinical evidence by several groups for the combination of an inhibitor of ligand-independent ER, fulvestrant, with PI3K inhibition, has led to the activation of trials evaluating this concept.
Assuntos
Neoplasias da Mama/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/deficiência , Animais , Feminino , HumanosRESUMO
INTRODUCTION: Inhibition of phosphatidylinositol-3-kinase (PI3K) induces apoptosis when combined with estrogen deprivation in estrogen receptor (ER)-positive breast cancer. The aims of the present study were to identify effective PI3K pathway inhibitor and endocrine therapy combinations, to evaluate the effect of PI3K pathway mutations and estrogen dependency on tumor response, and to determine the relevance of PIK3CA mutation in recurrent disease. METHODS: The PI3K catalytic subunit inhibitor BKM120, the mammalian target of rapamycin (mTOR) inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 were tested against ER-positive breast cancer cell lines before and after long-term estrogen deprivation (LTED). The impact of estradiol deprivation and the ER downregulator fulvestrant on PI3K pathway inhibitor-induced apoptosis was assessed. PIK3CA hotspot mutation analysis was performed in 51 recurrent or metastatic breast cancers and correlated with ER status and survival. RESULTS: Drug-induced apoptosis was most marked in short-term estrogen-deprived cells with PIK3CA mutation and phosphatase and tensin homolog loss. Apoptosis was most highly induced by BGT226, followed by BKM120, and then RAD001. Estradiol antagonized PI3K inhibitor-induced apoptosis following short-term estrogen deprivation, emphasizing a role for estrogen-deprivation therapy in promoting PI3K inhibitor activity in the first-line setting. ER-positive MCF7 LTED cells exhibited relative resistance to PI3K pathway inhibition that was reversed by fulvestrant. In contrast, T47D LTED cells exhibited ER loss and ER-independent PI3K agent sensitivity. PIK3CA mutation was prevalent in relapsed ER-positive disease (48%) and was associated with persistent ER positivity and a late relapse pattern. CONCLUSIONS: Estrogen deprivation increased the apoptotic effects of PI3K and dual PI3K/mTOR inhibitors in ER-positive disease, providing a rationale for PI3K/aromatase inhibitor combinations as first-line therapy. In LTED cells, differential effects on ER expression may be a relevant consideration. When ER was persistently expressed, fulvestrant strongly promoted PI3K drug activity. When ER was lost, PI3K inhibitor monotherapy was sufficient to induce high-level apoptosis. Although tumors with PIK3CA mutation had a late recurrence pattern, these mutations were common in metastatic disease and were most often associated with persistent ER expression. Targeting PIK3CA mutant tumors with a PI3K pathway inhibitor and fulvestrant is therefore a feasible strategy for aromatase-inhibitor-resistant ER-positive relapsed breast cancer.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Receptores de Estrogênio/metabolismo , Aminopiridinas/farmacologia , Apoptose/efeitos dos fármacos , Inibidores da Aromatase/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Estradiol/análogos & derivados , Estradiol/metabolismo , Estradiol/farmacologia , Estrogênios/metabolismo , Everolimo , Feminino , Fulvestranto , Humanos , Imidazóis/farmacologia , Morfolinas/farmacologia , Mutação , Recidiva Local de Neoplasia/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Endocrine therapy has been the most effective treatment modality for hormone receptor positive breast cancer. However, its efficacy has been limited by either de novo or acquired resistance. Recent data indicates that activation of the phosphatidylinositol 3-kinase (PI3K) signaling is associated with the poor outcome luminal B subtype of breast cancer and accompanied by the development of endocrine therapy resistance. Importantly, inhibition of PI3K pathway signaling in endocrine resistant breast cancer cell lines reduces cell survival and improves treatment response to endocrine agents. Interestingly, mutations in PIK3CA, the alpha catalytic subunit of the class IA PI3K, which renders cells dependent on PI3K pathway signaling, is the most common genetic abnormality identified in hormone receptor positive breast cancer. The synthetic lethality observed between estrogen deprivation and PI3K pathway inhibition in estrogen receptor positive (ER+) breast cancer cell lines provides further scientific rational to target both estrogen receptor and the PI3K pathway in order to improve the outcome of ER+ breast cancer.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Inibidores da Aromatase/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Mama/enzimologia , Estrogênios/metabolismo , Feminino , Humanos , Mutação , Neoplasias Hormônio-Dependentes/enzimologia , Fosfatidilinositol 3-Quinases/genética , Transdução de SinaisRESUMO
PURPOSE To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)-positive breast cancers in postmenopausal women. Materials and METHODS Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = -0.179; P = .05; and r = -0.389; P = .0005, respectively). CONCLUSION Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/sangue , Estrogênios/farmacologia , Perfilação da Expressão Gênica , Receptores de Estrogênio/análise , Idoso , Anastrozol , Neoplasias da Mama/sangue , Neoplasias da Mama/química , Feminino , Humanos , Antígeno Ki-67/análise , Pessoa de Meia-Idade , Nitrilas/uso terapêutico , Pós-Menopausa , Triazóis/uso terapêuticoRESUMO
Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)-positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110alpha and p110beta, in ER(+) breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110alpha RNAi inhibited growth and promoted apoptosis in all tested ER(+) breast cancer cells under estrogen deprived-conditions, whereas p110beta RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110alpha/p110beta inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER(+) breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110alpha and p110beta catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER(+) breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Estradiol/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Apoptose/genética , Neoplasias da Mama/genética , Processos de Crescimento Celular/genética , Classe I de Fosfatidilinositol 3-Quinases , Amplificação de Genes , Humanos , Imidazóis/farmacologia , Hibridização in Situ Fluorescente , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Quinolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Estrogênio/biossíntese , TransfecçãoRESUMO
Recent studies indicate that constitutive signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is a cause of treatment resistance in breast cancer patients. This implies that patients with tumors that exhibit aberrant PI3K signaling may benefit from targeted pathway inhibitors. The first agents to make it to the clinic are the rapamycin analogs. These compounds inhibit the downstream PI3K effector mTOR (mammalian target of rapamycin). A study presented in this issue of Breast Cancer Research suggests that recently developed inhibitors of phosphoinositide-dependent protein kinase 1, a more proximal target of the PI3K pathway, may provide an alternative route to effective PI3K pathway inhibition for breast cancer treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sirolimo/uso terapêuticoRESUMO
Activation of Ret, the receptor-tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), results in the recruitment and assembly of adaptor protein complexes that function to transduce signals downstream of the receptor. Here we identify Dok-6, a novel member of the Dok-4/5 subclass of the p62 Dok family of intracellular adaptor molecules, and characterize its interaction with Ret. Expression analysis reveals that Dok-6 is highly expressed in the developing central nervous system and is co-expressed with Ret in several locations, including sympathetic, sensory, and parasympathetic ganglia, as well as in the ureteric buds of the developing kidneys. Pull-down assays using the Dok-6 phosphotyrosine binding (PTB) domain and GDNF-activated Ret indicate that Dok-6 binds to the phosphorylated Ret Tyr(1062) residue. Moreover, ligand activation of Ret resulted in phosphorylation of tyrosine residue(s) located within the unique C terminus of Dok-6 predominantly through a Src-dependent mechanism, indicating that Dok-6 is a substrate of the Ret-Src signaling pathway. Interestingly, expression of Dok-6 potentiated GDNF-induced neurite outgrowth in GDNF family receptor alpha1 (GFRalpha1)-expressing Neuro2A cells that was dependent upon the C-terminal residues of Dok-6. Taken together, these data identify Dok-6 as a novel Dok-4/5-related adaptor molecule that may function in vivo to transduce signals that regulate Ret-mediated processes such as axonal projection.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Neuritos/ultraestrutura , Proteínas Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Embrião de Mamíferos , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas c-ret , Alinhamento de Sequência , Transdução de Sinais , Distribuição Tecidual , TransfecçãoRESUMO
Women with ErbB2-positive breast cancer have a poor prognosis, and frequently, chemotherapy treatment is ineffective. The ErbB2-targeted antibody trastuzumab improves survival when given with chemotherapy to patients with ErbB2-overexpressing metastatic disease, but treatment is not curative, and primary resistance is common. Postulated mechanisms of action for trastuzumab include immune-mediated cytotoxicity and receptor downmodulation. A study in this issue of Cancer Cell suggests that trastuzumab causes rapid activation of the PTEN lipid phosphatase, which in turn downregulates the phosphatidylinositol 3'-kinase (PI3K) pathway. Resistance to trastuzumab occurs when PTEN function is lost, suggesting that PTEN activation is a critical component of the therapeutic effect.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Monoéster Fosfórico Hidrolases/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Antineoplásicos/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Ativação Enzimática , Feminino , Genes Supressores de Tumor , Humanos , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , TrastuzumabRESUMO
The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.
Assuntos
Neurônios/citologia , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular , Humanos , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas/genética , Fosforilação , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/genética , Transfecção , Domínios de Homologia de srcRESUMO
Nerve growth factor (NGF) is required for the survival of developing sympathetic and sensory neurons. In the absence of NGF, these neurons undergo protein synthesis-dependent apoptosis. Ten years have gone by since the first reports of specific genes being upregulated during NGF deprivation-induced cell death. Over the last decade, a few additional genes (DP5, Bim, SM-20) have been added to a list that began with cyclin D1 and c-jun. In this chapter, we discuss the evidence that these genes act as regulators of neuronal cell death. We also suggest a hypothesis for how one gene, SM-20, may function to suppress a self-protection mechanism in NGF-deprived neurons.
Assuntos
Morte Celular/fisiologia , Proteínas de Ligação a DNA , Expressão Gênica/fisiologia , Fatores de Crescimento Neural/deficiência , Neurônios/citologia , Animais , Proteínas Reguladoras de Apoptose , Divisão Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Dioxigenases , História do Século XX , História do Século XXI , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Imuno-Histoquímica , Fatores de Crescimento Neural/história , Fatores de Crescimento Neural/fisiologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismoRESUMO
Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr(905) and Tyr(1062) was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr(1015) was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo.
Assuntos
Proteínas de Drosophila , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Fatores de Crescimento Neural/farmacologia , Neurturina , Fosforilação , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/química , Gânglio Cervical Superior/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Transmission electron microscopy reveals that the somatic testicular tissues and sperm ducts are elaborations of the epithelial lining of the tail coelom. The testes consist of closely packed spermatogonia embedded between specialized lateral field cells. These cells contain few organelles and appear to function mainly as a compartment boundary. Masses of spermatogenic cells are released into the tail coelom from the anterior end of the testes. The sperm ducts, lined by simple cuboidal ciliated epithelium, collect mature spermatozoa from the tail coelom and convey them to the blindly ending seminal vesicles. The sperm ducts also modify coelomic fluid entering them along with the spermatozoa. The seminal vesicles consist of a simple glandular lining epithelium embedded in the stratified epidermis. Secretions of the lining epithelium surround the enclosed sperm mass and correspond in position to a noncellular spermatophore coat visible by light microscopy around released sperm masses. Spermatophores leave the seminal vesicles through a temporary split that forms between microfilament-containing suture cells. Maturation of spermatozoa and filling of the seminal vesicles is cyclical, occurring late each day. © 1994 Wiley-Liss, Inc.