RESUMEN
Core binding factor (CBF) is a heterodimeric transcription factor containing one of three DNA-binding proteins of the Runt-related transcription factor family (RUNX1-3) and the non-DNA-binding protein, CBFß. RUNX1 and CBFß are the most common targets of chromosomal rearrangements in leukemia. CBF has been implicated in other cancer types; for example RUNX1 and RUNX2 are implicated in cancers of epithelial origin, including prostate, breast, and ovarian cancers. In these tumors, CBF is involved in maintaining the malignant phenotype and, when highly over-expressed, contributes to metastatic growth in bone. Herein, lentiviral delivery of CBFß-specific shRNAs was used to achieve a 95% reduction of CBFß in an ovarian cancer cell line. This drastic reduction in CBFß expression resulted in growth inhibition that was not associated with a cell cycle block or an increase in apoptosis. However, CBFß silencing resulted in increased autophagy and production of reactive oxygen species (ROS). Since sphingolipid and ceramide metabolism regulates non-apoptotic cell death, autophagy, and ROS production, fumonsin B1 (FB1), an inhibitor of ceramide synthase, was used to alter ceramide production in the CBFß-silenced cells. FB1 treatment inhibited the CBFß-dependent increase in autophagy and provided a modest increase in cell survival. To document alterations to sphingolipids in the CBFß-silenced cells, ceramide, and lactosylceramide levels were directly examined by mass spectrometry. Substantial increases in ceramide species and decreases in lactosylceramides were identified. Altogether, this report provides evidence that CBF transcriptional pathways control cellular survival, at least in part, through sphingolipid metabolism.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/deficiencia , Subunidad beta del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/genética , Esfingolípidos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis/genética , Autofagia/genética , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Lactosilceramidos/genética , Lactosilceramidos/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Esfingolípidos/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Ratones , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/patología , Proteína p14ARF Supresora de Tumor/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Proliferación Celular , Genes Supresores de Tumor , Humanos , Luciferasas/análisis , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/genética , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The Notch receptor pathway provides a paradigm for juxtacrine signaling pathways and controls stem cell function, developmental cell fate decisions, and cellular differentiation. The Notch pathway is constitutively activated in human cancers by chromosomal rearrangements, activating point mutations, or altered expression patterns. Therefore, the Notch pathway is the subject of chemotherapeutic intervention in a variety of human cancers. Notch receptor activation results in the gamma-secretase dependent proteolytic cleavage of the receptor to liberate the Notch intracellular domain that acts to mediate co-activator recruitment to the DNA binding transcription factor, CSL (CBF-1/RBP-Jκ, Su(H), Lag-1). Therapeutic targeting of the Notch pathway by gamma-secretase inhibitors prevents NICD production and regulates CSL-dependent transcriptional activity. To interrogate the loss of CSL activity in breast and prostate cancer cells, we used lentiviral-based shRNA knockdown of CSL. Knockdown of CSL expression was assessed by decreased DNA binding activity and resulted in decreased cell proliferation. In contrast, gamma-secretase inhibitor (GSI) treatment of these prostate and breast cancer cell lines resulted in minimal growth effects. PCR profiling of Notch pathway genes identified expression changes in few genes (Delta-like-1, Deltex-1, LMO2, and SH2D1A) after CSL knockdown. Consistent with differential effects of GSI on cell survival, GSI treatment failed to recapitulate the gene expression changes observed after CSL knockdown. Thus, CSL inhibition may provide a more effective mechanism to inhibit Notch-pathway dependent cancer cell proliferation as compared to GSI treatment.
Asunto(s)
Proliferación Celular , Regulación hacia Abajo/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Masculino , Ratones , Oligopéptidos/farmacología , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal/genética , Proteína Asociada a la Molécula de Señalización de la Activación LinfocitariaRESUMEN
Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.
Asunto(s)
Neoplasias de la Mama/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Subunidad beta del Factor de Unión al Sitio Principal/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Células 3T3 NIH , Trasplante de Neoplasias , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Interferencia de ARN , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
Type I phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) catalyzes the phosphorylation of phosphatidylinositol 4 phosphate [PI(4)P] at carbon 5, producing phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Phosphatidic acid (PA) activates PI4P5K in vitro and plays a central role in the activation of PIP5K pathways in vivo. This report demonstrates that actin fiber formation in murine fibroblasts involves PA activation of PIP5Ks and defines biochemical interactions between PA and the PIP5Ks. Inhibition of phospholipase D production of PA results in the loss of actin fibers. Overexpression of the beta isoform of the type I murine phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta) maintains actin fiber structure in the face of phospholipase D inhibition. PA activates mPIP5K-Ibeta by direct binding to mPIP5K-Ibeta through both electrostatic and hydrophobic interactions, with the fatty acid acyl chain length and degree of saturation acting as critical determinants of binding and activation. Furthermore, kinetic analysis suggests that phosphorylation of the PI(4)P substrate does not follow classical Michaelis-Menten kinetics. Instead, the kinetic data are consistent with a model in which mPIP5K-Ibeta initially binds to the lipid micelle and subsequently binds the PI(4)P substrate. In addition, the kinetics indicate substrate inhibition, suggesting that mPIP5K-Ibeta contains an inhibitory PI(4)P-binding site. These results suggest a model in which mPIP5K-Ibeta is surrounded by PI(4)P, but is unable to catalyze its conversion to PI(4,5)P2 unless PA is bound.
Asunto(s)
Ácidos Fosfatidicos/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Activación Enzimática , Cinética , Ratones , Células 3T3 NIH , Fosfolipasa D/metabolismo , Fosforilación , Unión Proteica , Transducción de SeñalRESUMEN
The RUNX transcription factors (RUNX1, RUNX2, and RUNX3) play essential roles in hematopoiesis and skeletal development. Consistent with these roles in differentiation and cell cycle, the activity of both RUNX1 and RUNX3 is perturbed in cancer. To determine a role for the RUNX factors in prostate biology, we investigated the expression of RUNX factors in prostate epithelial cell lines and normal prostate tissue. RUNX1, RUNX2, and RUNX3 were expressed in both normal prostate tissue and an immortalized, non-transformed cell line. We found that prostate cancer-derived cell lines expressed RUNX1 and RUNX2, but not RUNX3. Next, we sought to identify prostate-specific genes whose expression could be regulated by RUNX proteins. Four consensus RUNX sites are located within the prostate-specific antigen (PSA) regulatory region. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 is specifically bound to the PSA regulatory region in LNCaP cells. RUNX1 and RUNX2 activated the PSA regulatory region alone or cooperatively with prostate-derived ETS factor (PDEF) and RUNX1 physically associated with PDEF. Taken together, our results suggest that RUNX factors participate in prostate epithelial cell function and cooperate with an Ets transcription factor to regulate PSA gene expression.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Antígeno Prostático Específico/genética , Proteínas Proto-Oncogénicas c-ets/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética/fisiología , Secuencia de Bases , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Cartilla de ADN , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-ets/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gfi-1 and Gfi-1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C-terminal zinc-finger domain and may require an N-terminal SNAG domain (SNAIL/Gfi-1) to repress transcription, the mechanism by which Gfi-1 and Gfi-1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co-repressors N-CoR, SMRT, and mSin3A, and may act as a co-repressor for site-specific transcriptions factors. In this report we demonstrate that Gfi-1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. We observed that a portion of Gfi-1 and Gfi-1B associated with the nuclear matrix, as is the case with ETO. Moreover, Gfi-1 and ETO co-localize to punctate subnuclear structures. When co-expressed in mammalian cells, Gfi-1 associates with histone deacetylse-1 (HDAC-1), HDAC-2, and HDAC-3. These data identify ETO as a partner for Gfi-1 and Gfi-1B, and suggest that Gfi-1 proteins repress transcription through recruitment of histone deacetylase-containing complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/metabolismo , Matriz Nuclear/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Genes Reporteros/genética , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Filamentos Intermedios/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Proteínas Represoras/química , Transcripción Genética/fisiología , TransfecciónRESUMEN
Cloning and characterization of the 8;21 chromosomal breakpoint identified AML1 on chromosome 21 and ETO (MTG8) on chromosome 8, and the resultant chimeric gene product, AML-1/ETO. The ETO gene family now includes three human members encoding proteins composed of four evolutionarily conserved domains termed nervy homology regions (NHR) 1-4. ETO associates with N-CoR/Sin3a/HDAC complexes in vivo and acts as a corepressor for the promyelocytic zinc finger protein. Moreover, ETO is nuclear matrix attached at sites coincident with histone deacetylase enzymes and mSin3a. These data suggest that ETO proteins function as transcriptional corepressors. This review focuses on the ETO gene family in terms of expression and function. Specifically, the role of ETO as a co-repressor will be detailed. Additionally, the impact of this recent discovery on treatment of t(8;21)-containing leukemia will be discussed.
Asunto(s)
Proteínas de Unión al ADN/genética , Familia de Multigenes/genética , Proteínas Proto-Oncogénicas , Factores de Transcripción/genética , Animales , Sitios de Unión/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Regulación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/genética , Proteína 1 Compañera de Translocación de RUNX1 , Transcripción Genética/genéticaRESUMEN
The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.