RESUMEN
BACKGROUND: Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. METHODS: Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. RESULTS: A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). CONCLUSION: Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Animales , Ratones , Glioblastoma/patología , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Neoplasias Encefálicas/patologíaRESUMEN
Early stage localized prostate cancer (PCa) has an excellent prognosis; however, patient survival drops dramatically when PCa metastasizes. The molecular mechanisms underlying PCa metastasis are complex and remain unclear. Here, we examine the role of a new member of the fatty acid-binding protein (FABP) family, FABP12, in PCa progression. FABP12 is preferentially amplified and/or overexpressed in metastatic compared to primary tumors from both PCa patients and xenograft animal models. We show that FABP12 concurrently triggers metastatic phenotypes (induced epithelial-to-mesenchymal transition (EMT) leading to increased cell motility and invasion) and lipid bioenergetics (increased fatty acid uptake and accumulation, increased ATP production from fatty acid ß-oxidation) in PCa cells, supporting increased reliance on fatty acids for energy production. Mechanistically, we show that FABP12 is a driver of PPARγ activation which, in turn, regulates FABP12's role in lipid metabolism and PCa progression. Our results point to a novel role for a FABP-PPAR pathway in promoting PCa metastasis through induction of EMT and lipid bioenergetics.
Asunto(s)
Transformación Celular Neoplásica/patología , Metabolismo Energético , Transición Epitelial-Mesenquimal , Proteínas de Unión a Ácidos Grasos/metabolismo , Lípidos/química , PPAR gamma/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Dosificación de Gen , Humanos , Masculino , Invasividad Neoplásica , Metástasis de la Neoplasia , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2-4â¯cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2-4â¯cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2, Zar1, Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Desarrollo Embrionario/fisiología , Animales , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovario , ARN/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
AP-2 transcription factors play important roles in the regulation of gene expression during development. Four of the five members of the AP-2 family (AP-2α, AP-2ß, AP-2γ and AP-2δ) have previously been shown to be expressed in developing retina. Mouse knockouts have revealed roles for AP-2α, AP-2ß and AP-2δ in retinal cell specification and function. Here, we show that the fifth member of the AP-2 family, AP-2ε, is also expressed in amacrine cells in developing mammalian and chicken retina. Our data indicate that there are considerably fewer AP-2ε-positive cells in the developing mouse retina compared to AP-2α, AP-2ß and AP-2γ-positive cells, suggesting a specialized role for AP-2ε in a subset of amacrine cells. AP-2ε, which is restricted to the GABAergic amacrine lineage, is most commonly co-expressed with AP-2α and AP-2ß, especially at early stages of retinal development. Co-expression of AP-2ε and AP-2γ increases with differentiation. Analysis of previously published Drop-seq data from single retinal cells supports co-expression of multiple AP-2s in the same cell. Since AP-2s bind to their target sequences as either homodimers or heterodimers, our work suggests spatially- and temporally-coordinated roles for combinations of AP-2 transcription factors in amacrine cells during retinal development.
Asunto(s)
Células Amacrinas/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Retina/crecimiento & desarrollo , Factor de Transcripción AP-2/genética , Animales , Línea Celular , Línea Celular Tumoral , Pollos , Humanos , RatonesRESUMEN
BACKGROUND: AP-2δ is the most divergent member of the Activating Protein-2 (TFAP2) family of transcription factors. AP-2δ is restricted to specific regions of the CNS, including a subset of ganglion cells in the retina. Retinal ganglion cells (RGCs), the only output neurons of the retina, are responsible for transmitting the visual signal to the brain. RESULTS: AP-2δ knockout results in loss of Brn3c (Pou4f3) expression in AP-2δ -positive RGCs. While AP-2δ-/- mice have morphologically normal retinas at birth, there is a significant reduction in retinal ganglion cell numbers by P21, after eye opening. Chromatin immunoprecipitation indicates that Brn3c is a target of AP-2δ in the retina. Using fluorochrome-conjugated cholera toxin subunit B to trace ganglion cell axons from the eye to the major visual pathways in the brain, we found 87 % and 32 % decreases in ipsilateral and contralateral projections, respectively, to the superior colliculus in AP-2δ-/- mice. In agreement with anatomical data, visually evoked responses recorded from the brain confirmed that retinal outputs to the brain are compromised. CONCLUSIONS: AP-2δ is important for the maintenance of ganglion cell numbers in the retina. Loss of AP-2δ alters retinal axonal projections to visual centers of the brain, with ipsilaterial projections to the superior colliculus being the most dramatically affected. Our results have important implications for integration of the visual signal at the superior colliculus.
Asunto(s)
Axones/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Colículos Superiores/metabolismo , Factor de Transcripción AP-2/deficiencia , Animales , Apoptosis , Recuento de Células , Inmunoprecipitación de Cromatina , Visión de Colores , Adaptación a la Oscuridad , Potenciales Evocados Visuales/fisiología , Cuerpos Geniculados/citología , Cuerpos Geniculados/metabolismo , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Noqueados , Colículos Superiores/citología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/metabolismo , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción Brn-3C/metabolismoRESUMEN
Retinoic acid (RA), a metabolite of vitamin A, is required for the regulation of growth and development. Aberrant expression of molecules involved in RA signaling has been reported in various cancer types including glioblastoma multiforme (GBM). Cellular retinoic acid-binding protein 2 (CRABP2) has previously been shown to play a key role in the transport of RA to retinoic acid receptors (RARs) to activate their transcription regulatory activity. Here, we demonstrate that CRABP2 is predominantly located in the cytoplasm of GBM tumors. Cytoplasmic, but not nuclear, CRABP2 levels in GBM tumors are associated with poor patient survival. Treatment of malignant glioma cell lines with RA results in a dose-dependent increase in accumulation of CRABP2 in the cytoplasm. CRABP2 knockdown reduces proliferation rates of malignant glioma cells, and enhances RA-induced RAR activation. Levels of CRYAB, a small heat shock protein with anti-apoptotic activity, and GFAP, an astrocyte-specific intermediate filament protein, are greatly reduced in CRABP2-depleted cells. Restoration of CRYAB expression partially but significantly reversed the effect of CRABP2 depletion on RAR activation. Our combined in vivo and in vitro data indicate that: (i) CRABP2 is an important determinant of clinical outcome in GBM patients, and (ii) the mechanism of action of CRABP2 in GBM involves sequestration of RA in the cytoplasm and activation of an anti-apoptotic pathway, thereby enhancing proliferation and preventing RA-mediated cell death and differentiation. We propose that reducing CRABP2 levels may enhance the therapeutic index of RA in GBM patients.
Asunto(s)
Diferenciación Celular/fisiología , Citoplasma/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/metabolismo , Receptores de Ácido Retinoico/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Humanos , Pronóstico , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer. METHODS: CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis. RESULTS: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism. CONCLUSIONS: CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Receptores de Ácido Retinoico/genética , Biomarcadores de Tumor , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Espacio Intracelular/metabolismo , Modelos Biológicos , Clasificación del Tumor , Pronóstico , Transporte de Proteínas , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal , Tretinoina/farmacología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Malignant gliomas (MG), including grades III and IV astrocytomas, are the most common adult brain tumors. These tumors are highly aggressive with a median survival of less than 2 years. Nuclear factor I (NFI) is a family of transcription factors that regulates the expression of glial genes in the developing brain. We have previously shown that regulation of the brain fatty acid-binding protein (B-FABP; FABP7) and glial fibrillary acidic protein (GFAP) genes in MG cells is dependent on the phosphorylation state of NFI, with hypophosphorylation of NFI correlating with GFAP and B-FABP expression. Importantly, NFI phosphorylation is dependent on phosphatase activity that is enriched in GFAP/B-FABP+ve cells. Using chromatin immunoprecipitation, we show that NFI occupies the GFAP and B-FABP promoters in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. NFI occupancy, NFI-dependent transcriptional activity, and NFI phosphorylation are all modulated by the serine/threonine phosphatase calcineurin. Importantly, a cleaved form of calcineurin, associated with increased phosphatase activity, is specifically expressed in NFI-hypophosphorylated GFAP/B-FABP+ve MG cells. Calcineurin in GFAP/B-FABP+ve MG cells localizes to the nucleus. In contrast, calcineurin is primarily found in the cytoplasm of GFAP/B-FABP-ve cells, suggesting a dual mechanism for calcineurin activation in MG. Finally, our results demonstrate that calcineurin expression is up-regulated in areas of high infiltration/migration in grade IV astrocytoma tumor tissue. Our data suggest a critical role for calcineurin in NFI transcriptional regulation and in the determination of MG infiltrative properties.
Asunto(s)
Calcineurina/metabolismo , Glioma/enzimología , Factores de Transcripción NFI/metabolismo , Adulto , Astrocitoma/metabolismo , Astrocitoma/patología , Proteínas de Unión al Calcio/farmacología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Ciclosporina/farmacología , Glioma/patología , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ionomicina/farmacología , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Disabled-1 (Dab1) plays a key role in reelin-mediated neuronal migration during brain development. Tyrosine phosphorylation of Dab1 at two YQXI and two YXVP motifs recruits multiple SH2 domains, resulting in activation of a wide range of signaling cascades. However, the molecular mechanisms underlying the coordinated regulation of Dab1 downstream effectors remain poorly understood. Here, we show that alternative splicing results in inclusion of different combinations of YQXI and YXVP motifs in Dab1 isoforms during development. Dab1 variants with partial or complete loss of YQXI motifs are preferentially expressed at early developmental stages, whereas the commonly studied Dab1 is predominantly expressed at late developmental stages. Expression of Dab1 variants in 293T and Neuro2a cells reveals reduced levels or absence of tyrosine phosphorylation in variants that have lost one or both YQXI motifs. We further demonstrate that Dab1 variants differ in their abilities to activate Src and recruit distinct SH2 domains involved in specific downstream signaling pathways. We propose that coordinated expression of specific Dab1 isoforms in different populations of cells in the developing brain contributes to precise neuronal migration by modulating the activity of subsets of Dab1 downstream effectors.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Empalme Alternativo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular , Cartilla de ADN/genética , Exones , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes Neurológicos , Modelos Neurológicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Fosforilación , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Reelina , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transducción de Señal , Dominios Homologos srcRESUMEN
FABP7 has been implicated in tumour cell proliferation, cell migration, and poor prognosis in patients with high-grade astrocytoma and melanoma. In this study, we examine FABP7 expression in a cohort of 176 primary breast cancers by gene profiling and tissue microarray immunostaining. We show that FABP7 is significantly up-regulated in triple-negative breast cancer. Elevated FABP7 levels are associated with poor prognosis, absence of oestrogen and progesterone hormone receptors (ER, PR) and HER2, increased cell proliferation, and high tumour grade. Depletion of FABP7 in the ER/PR-negative cell line, MDA-MB-435S, significantly reduced cell growth rate and sensitized the cells to growth inhibition by omega-3 docosahexaenoic acid (DHA). A target of DHA-bound FABP7 in the nucleus is RXRß, a retinoid-activated nuclear receptor that functions as a transcription factor by either homodimerizing or heterodimerizing with other nuclear receptors such as PPARs. Based on our microarray data, RXRß, like FABP7, is an adverse prognostic factor for breast cancer. We propose that the DHA-FABP7-RXRß pathway promotes cell survival/proliferation in triple-negative breast cancer. Targeting this pathway may thus provide an alternate route for the treatment of triple-negative breast cancer.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/fisiología , Neoplasias de la Mama/patología , Proteínas Portadoras/fisiología , Proliferación Celular , Receptor beta X Retinoide/fisiología , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/fisiología , Adenocarcinoma/metabolismo , Adenocarcinoma/fisiopatología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/fisiopatología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Proteína de Unión a los Ácidos Grasos 7 , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Estimación de Kaplan-Meier , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites. PRINCIPAL FINDINGS: Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. CONCLUSIONS: These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment of Dab1-L-like and Dab1-E-like isoforms in retinoblastoma and neuroblastoma, respectively.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neoplasias del Sistema Nervioso/metabolismo , Retina/embriología , Retina/metabolismo , Animales , Secuencia de Bases , Sistema Nervioso Central/embriología , Embrión de Pollo , Exones , Humanos , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa/métodos , Isoformas de Proteínas , Proteína Reelina , Retinoblastoma/metabolismo , Homología de Secuencia de Ácido Nucleico , Familia-src Quinasas/metabolismoRESUMEN
Recent studies using animal models suggest that expression of FABP5 drives the stimulation of cell growth observed in estrogen receptor (ER)-negative breast cancer cells on exposure to retinoic acid (RA). The purpose of this study was to investigate the clinicopathological significance of FABP5 in breast cancer and to evaluate FABP5 as a prognostic marker and a possible novel therapeutic target in breast cancer. Gene expression microarray analysis revealed a significant correlation between elevated FABP5 RNA levels and ER/progesterone receptor (PR)-negative status, high tumor grade, and poor prognosis. Tissue microarray analysis demonstrated similar correlations with cytoplasmic FABP5 protein. Based on multivariate proportional regression analysis, cytoplasmic FABP5 is a significant and independent prognostic marker of overall survival and recurrence-free survival in breast cancer. The effects of FABP5 on tumor growth appear to be mediated primarily through cytoplasmic FABP, because no correlation was found between nuclear FABP5 and ER/PR-negative status, recurrence, and survival. FABP5 knockdown in breast cancer cell lines demonstrates a correlation between FABP5 levels and growth response to RA. We propose a model whereby growth-promoting FABP5 competes with growth-inhibiting CRABP2 for RA, with retention of RA in the cytoplasm by FABP5 preventing the inhibition of tumor growth.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Tretinoina/uso terapéutico , Anciano , Especificidad de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos/inmunología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Modelos Biológicos , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Ácido Retinoico/metabolismo , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Tretinoina/farmacologíaRESUMEN
Breast cancer is a heterogeneous disease characterized by diverse molecular signatures and a variable response to therapy. Clinical management of breast cancer is guided by the expression of estrogen and progesterone receptors and HER2 amplification. New prognostic and predictive markers, as well as additional targets for therapy, are needed for more effective management of this disease. Gene expression microarrays were probed with RNAs from 176 primary breast cancer samples and tissue microarrays immunostained with anti-DDX1 antibody, an antibody to DEAD box protein DDX1, a putative RNA-RNA and RNA-DNA unwinding protein normally found in the nucleus. Half of the patient cohort had experienced early relapse despite standard adjuvant therapy, but were otherwise matched for estrogen receptor and HER2 status, stage and duration of follow-up. Here, we identify DDX1 RNA overexpression as an independent prognostic marker for early recurrence in primary breast cancer, with a hazard ratio of 4.31 based on logrank analysis of Kaplan-Meier curves. Elevated levels of DDX1 protein in the cytoplasm also independently correlate with early recurrence with a hazard ratio of 1.90. In conclusion, our data indicate a strong and independent association between poor prognosis and deregulation of the DEAD box protein DDX1. We propose that elevated levels of DDX1 RNA or the presence of DDX1 in the cytoplasm could serve as an effective prognostic biomarker for early recurrence in primary breast cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Transporte de Proteínas , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Recurrencia , Análisis de SupervivenciaRESUMEN
The Reelin-Disabled-1 (Dab1) signaling pathway plays a key role in the positioning of neurons during brain development. Two alternatively spliced Dab1 isoforms have been identified in chick retina and brain: Dab1-E, expressed at early stages of development, and Dab1-L (commonly referred to as Dab1), expressed at later developmental stages. The well-studied Dab1-L serves as an adaptor protein linking Reelin signal to its downstream effectors; however, nothing is known regarding the role of Dab1-E. Here we show that Dab1-E is primarily expressed in proliferating retinal progenitor cells whereas Dab1-L is found exclusively in differentiated neuronal cells. In contrast to Dab1-L, which is tyrosine phosphorylated upon Reelin stimulation, Dab1-E is not tyrosine phosphorylated and may function independently of Reelin. Knockdown of Dab1-E in chick retina results in a significant reduction in the number of proliferating cells and promotes ganglion cell differentiation. Our results demonstrate a role for Dab1-E in the maintenance of the retinal progenitor pool and determination of cell fate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Pollos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/citología , Serina Endopeptidasas/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/genética , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Embrión de Pollo , Pollos/genética , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Mitosis , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Reelina , Retina/embriología , Retina/metabolismo , Retina/ultraestructura , Células Ganglionares de la Retina/citología , Serina Endopeptidasas/genética , Células Madre/citologíaRESUMEN
The underlying cause of human retinoblastoma is complete inactivation of both copies of the RB1 gene. Other chromosome abnormalities, with the most common being extra copies of chromosome arm 6p, are also observed in retinoblastoma. The RB protein has previously been shown to interact with TFAP2 transcription factors. Here, we show that TFAP2A and TFAP2B, which map to chromosome arm 6p, are expressed in the amacrine and horizontal cells of human retina. TFAP2A RNA can readily be detected in retinoblastoma cell lines and tumors; however, the great majority of retinoblastoma cell lines and tumors are completely devoid of TFAP2A protein and TFAP2B RNA/protein. Transfection of TFAP2A and TFAP2B expression constructs into retinoblastoma cells induces apoptosis and inhibits proliferation. Our results suggest that a consequence of loss of RB1 gene function in retinoblastoma cells is inactivation of TFAP2A and TFAP2B function. We propose that inability to differentiate along the amacrine/horizontal cell lineages may underlie retinoblastoma tumor formation.
Asunto(s)
Apoptosis , Feto/patología , Retina/patología , Neoplasias de la Retina/patología , Retinoblastoma/patología , Factor de Transcripción AP-2/metabolismo , Northern Blotting , Western Blotting , Diferenciación Celular , Feto/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , ARN Mensajero/genética , Retina/metabolismo , Neoplasias de la Retina/genética , Neoplasias de la Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-2/genética , Células Tumorales CultivadasRESUMEN
The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.
Asunto(s)
Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Otx/metabolismo , Retina/metabolismo , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Transactivadores/metabolismo , Arrestina/metabolismo , Línea Celular Tumoral , Preescolar , Feto , Glutamato Sintasa/metabolismo , Proteínas de Homeodominio/genética , Humanos , Antígeno Ki-67/metabolismo , Factores de Transcripción Otx/genética , Proteína Quinasa C-alfa/metabolismo , Retina/anatomía & histología , Neoplasias de la Retina/patología , Retinoblastoma/patología , Transactivadores/genéticaRESUMEN
Glial fibrillary acidic protein (GFAP), an intermediate filament protein normally found in astrocytes, and the radial glial marker brain fatty acid-binding protein (B-FABP; also known as FABP7) are co-expressed in malignant glioma cell lines and tumors. Nuclear factor I (NFI) recognition sites have been identified in the B-FABP and GFAP promoters, and transcription of both genes is believed to be regulated by NFI. Here, we study the role of the different members of the NFI family in regulating endogenous and ectopic B-FABP and GFAP gene transcription in human malignant glioma cells. We show by gel shifts that all four members of the NFI family (NFIA, NFIB, NFIC, and NFIX) bind to B-FABP and GFAP NFI consensus sites. Over-expression of NFIs, in conjunction with mutation analysis of NFI consensus sites using a reporter gene assay, supports a role for all four NFIs in the regulation of the GFAP and B-FABP genes. Knock-down of single or combined NFIs reveals promoter-dependent and promoter-context-dependent interaction patterns and suggests cross talk between the different members of the NFI family. Our data indicate that the NFI family of transcription factors plays a key role in the regulation of both the B-FABP and GFAP genes in malignant glioma cells.
Asunto(s)
Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/genética , Glioma/genética , Factores de Transcripción NFI/metabolismo , Proteínas Supresoras de Tumor/genética , Región de Flanqueo 3'/genética , Secuencia de Bases , Sitios de Unión/genética , Línea Celular Tumoral , Proteína de Unión a los Ácidos Grasos 7 , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Mutación , Factores de Transcripción NFI/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
AP-2 is a family of transcription factors that play important roles during embryonic development. Two AP-2 genes, AP-2alpha and AP-2beta, have previously been characterized in chick retina. Here, we demonstrate that a third member of the chicken AP-2 family, AP-2delta, is primarily expressed in the retina and brain, with highest levels at embryonic days 7 to 11. By in situ hybridization and immunohistochemical analysis, we show that AP-2delta RNA and protein are found in a subset of ganglion cells in embryonic chick retina. Co-immunostaining with anti-Brn3a and anti-AP-2delta antibodies indicates that approximately one-third of Brn3a-positive ganglion cells express AP-2delta. AP-2delta RNA but not AP-2delta protein is also found in cells located in the outer half of the inner nuclear layer. The spatial and temporal distribution of AP-2delta protein in the retina suggests a transient role in a subset of late-born ganglion cells likely involving axonal trafficking or pathfinding.
Asunto(s)
Proteínas del Ojo/biosíntesis , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/biosíntesis , ARN Mensajero/biosíntesis , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción AP-2/biosíntesis , Animales , Axones/metabolismo , Embrión de Pollo , Humanos , Células Ganglionares de la Retina/citologíaRESUMEN
Brain fatty acid-binding protein (B-FABP) is normally expressed in radial glial cells, where it plays a role in the establishment of the radial glial fiber network required for neuronal migration. B-FABP is also expressed in astrocytoma tumors and in some malignant glioma cell lines. To address the role of B-FABP in malignant glioma, we have studied the growth properties of clonal populations of malignant glioma cells modified for B-FABP expression. Here, we demonstrate that expression of B-FABP in B-FABP-negative malignant glioma cells is accompanied by the appearance of radial glial-like properties, such as increased migration and extended bipolar cell processes, as well as reduced transformation. Conversely, B-FABP depletion in B-FABP-positive malignant glioma cells results in decreased migration, reduction in cell processes, and a more transformed phenotype. Moreover, expression of B-FABP in astrocytomas is associated with regions of tumor infiltration and recurrence. Rather than being a direct manifestation of the tumorigenic process, we propose that the ability of high-grade astrocytoma cells to migrate long distances from the primary tumor reflects properties associated with their cell of origin. Thus, targeting B-FABP-expressing cells may make a significant impact on the treatment of these tumors.