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1.
ChemMedChem ; 19(18): e202400179, 2024 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-38861151

RESUMEN

The Eyes Absent (EYA) family of developmental proteins, often in partnership with the sine oculis (SIX) homeobox proteins, promote cancer metastasis and recurrence in numerous tumor types. In addition to being a transcriptional coactivator, EYA2 is a Tyr phosphatase that dephosphorylates H2AX which leads to repair instead of apoptosis upon DNA damage and ERß which inhibits the anti-tumor transcriptional activity of ERß. The SIX members of the EYA-SIX complex are difficult to target, therefore, we targeted the EYA2 to promote cell death and prevent cancer progression. We conducted structural optimization of a previously discovered allosteric inhibitor of EYA2, 9987, using the combination of in silico modeling, biochemical and cell-based assays. A new series of compounds was developed with significantly improved cellular activity and physiochemical properties desirable for brain targets. Specifically, compound 2 e showed >30-fold improvement in the medulloblastoma cell line D458, relative to 9987, while maintaining potent and selective inhibitory activity against EYA2 Tyr phosphatase activity and a good multiparameter optimization score for central nervous system drugs.


Asunto(s)
Antineoplásicos , Diseño de Fármacos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Proteínas Tirosina Fosfatasas , Humanos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Regulación Alostérica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Relación Estructura-Actividad , Línea Celular Tumoral , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a Droga , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química
2.
Neuro Oncol ; 25(12): 2287-2301, 2023 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-37486991

RESUMEN

BACKGROUND: Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. METHODS: Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. RESULTS: EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. CONCLUSIONS: Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.


Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Humanos , Niño , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Línea Celular Tumoral , Proteínas Tirosina Fosfatasas/genética , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Tirosina , Proteínas Nucleares/genética , Péptidos y Proteínas de Señalización Intracelular
3.
Neuro Oncol ; 19(6): 833-844, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-27932423

RESUMEN

Background: To elucidate molecular features associated with disproportionate survival of glioblastoma (GB) patients, we conducted deep genomic comparative analysis of a cohort of patients receiving standard therapy (surgery plus concurrent radiation and temozolomide); "GB outliers" were identified: long-term survivor of 33 months (LTS; n = 8) versus short-term survivor of 7 months (STS; n = 10). Methods: We implemented exome, RNA, whole genome sequencing, and DNA methylation for collection of deep genomic data from STS and LTS GB patients. Results: LTS GB showed frequent chromosomal gains in 4q12 (platelet derived growth factor receptor alpha and KIT) and 12q14.1 (cyclin-dependent kinase 4), and deletion in 19q13.33 (BAX, branched chain amino-acid transaminase 2, and cluster of differentiation 33). STS GB showed frequent deletion in 9p11.2 (forkhead box D4-like 2 and aquaporin 7 pseudogene 3) and 22q11.21 (Hypermethylated In Cancer 2). LTS GB showed 2-fold more frequent copy number deletions compared with STS GB. Gene expression differences showed the STS cohort with altered transcriptional regulators: activation of signal transducer and activator of transcription (STAT)5a/b, nuclear factor-kappaB (NF-κB), and interferon-gamma (IFNG), and inhibition of mitogen-activated protein kinase (MAPK1), extracellular signal-regulated kinase (ERK)1/2, and estrogen receptor (ESR)1. Expression-based biological concepts prominent in the STS cohort include metabolic processes, anaphase-promoting complex degradation, and immune processes associated with major histocompatibility complex class I antigen presentation; the LTS cohort features genes related to development, morphogenesis, and the mammalian target of rapamycin signaling pathway. Whole genome methylation analyses showed that a methylation signature of 89 probes distinctly separates LTS from STS GB tumors. Conclusion: We posit that genomic instability is associated with longer survival of GB (possibly with vulnerability to standard therapy); conversely, genomic and epigenetic signatures may identify patients where up-front entry into alternative, targeted regimens would be a preferred, more efficacious management.


Asunto(s)
Biomarcadores de Tumor/genética , Metilación de ADN , Genómica/métodos , Glioblastoma/genética , Glioblastoma/mortalidad , Sobrevivientes/estadística & datos numéricos , Transcriptoma , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia
4.
Hum Mutat ; 27(5): 427-35, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16619214

RESUMEN

Germline mutations in the human breast cancer susceptibility genes BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancer. In spite of the large number of sequence variants identified in BRCA1 and BRCA2 mutation analyses, many of these genetic alterations are still classified as variants of unknown significance (VUS). In this study, we evaluated 12 BRCA1/2 intronic variants in order to differentiate their pathogenic or polymorphic effects on the mRNA splicing process. We detected the existence of aberrant splicing in three BRCA1 variants (c.301-2delA/IVS6-2delA, c.441+1G>A/IVS7+1G>A, and c.4986+6T>G/IVS16+6T>G) and two BRCA2 variants (c.8487+1G>A/IVS19+1G>A and c.8632-2A>G/IVS20-2A>G). All but one of the aberrant transcripts arise from mutations affecting the conserved splice acceptor or donor sequences and all would be predicted to result in expression of truncated BRCA1 or BRCA2 proteins. However, we demonstrated that four of these splice-site mutations (i.e., c.301-2delA, c.441+1G>A, c.4986+6T>G, and c.8632-2A>G) with premature termination codons were highly unstable and were unlikely to encode for abundant expression of a mutant protein. Three variants of BRCA1 (c.212+3A>G/IVS5+3A>G, c.593+8A>G/IVS9+8A>G, and c.4986-20A>G/IVS16-20A>G) and four variants of BRCA2 (c.516-19C>T/IVS6-19C>T, c.7976-4_7976_3delTT/IVS17-4delTT, c.8487+19A>G/IVS19+19A>G, and c.9256- 18C>A/IVS24- 18C>A) in our studies show no effects on the normal splicing process, and they are considered to be benign polymorphic alterations. Our studies help to clarify the aberrant splicing in BRCA1 and BRCA2 as well as provide information that can be used clinically to help counsel breast/ovarian cancer prone families.


Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Intrones , Mutación , Empalme del ARN , ARN Mensajero/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Proteína BRCA1/química , Proteína BRCA2/química , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , Femenino , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético , Puromicina/farmacología , Sitios de Empalme de ARN , Análisis de Secuencia de Proteína
5.
PLoS One ; 8(8): e72134, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23967279

RESUMEN

Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the "Go or Grow" hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the "Go or Grow" hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a "growing-to-going" phenotype.


Asunto(s)
Glioma/genética , Glioma/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Ciclo Celular/genética , Movimiento Celular/genética , Proliferación Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioma/patología , Humanos , Inmunohistoquímica , Antígeno Ki-67/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo
6.
Mol Cancer Res ; 8(11): 1558-67, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20881009

RESUMEN

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Glioblastoma/metabolismo , Receptores del Factor de Necrosis Tumoral/biosíntesis , Proteína de Unión al GTP rac1/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Quinasa 2 de Adhesión Focal/deficiencia , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Inmunoprecipitación , Invasividad Neoplásica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transfección , Proteína de Unión al GTP rac1/deficiencia
7.
Neoplasia ; 11(4): 377-87, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19308292

RESUMEN

The invasive nature of malignant gliomas is a clinical problem rendering tumors incurable by conventional treatment modalities such as surgery, ionizing radiation, and temozolomide. Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) is a multifunctional adaptor protein, recruiting cytoplasmic signaling proteins and membrane receptors/transporters into functional complexes. This study revealed that NHERF-1 expression is increased in highly invasive cells that reside in the rim of glioblastoma multiforme (GBM) tumors and that NHERF-1 sustains glioma migration and invasion. Gene expression profiles were evaluated from laser capture-microdissected human GBM cells isolated from patient tumor cores and corresponding invaded white matter regions. The role of NHERF-1 in the migration and dispersion of GBM cell lines was examined by reducing its expression with small-interfering RNA followed by radial migration, three-dimensional collagen dispersion, immunofluorescence, and survival assays. The in situ expression of NHERF-1 protein was restricted to glioma cells and the vascular endothelium, with minimal to no detection in adjacent normal brain tissue. Depletion of NHERF-1 arrested migration and dispersion of glioma cell lines and caused an increase in cell-cell cohesiveness. Glioblastoma multiforme cells with depleted NHERF-1 evidenced a marked decrease in stress fibers, a larger cell size, and a more rounded shape with fewer cellular processes. When NHERF-1 expression was reduced, glioma cells became sensitized to temozolomide treatment resulting in increased apoptosis. Taken together, these results provide the first evidence for NHERF-1 as a participant in the highly invasive phenotype of malignant gliomas and implicate NHERF-1 as a possible therapeutic target for treatment of GBM.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Invasividad Neoplásica/genética , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Adhesión Celular/genética , Movimiento Celular/genética , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Glioblastoma/genética , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Rayos Láser , Microdisección , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiadores de Sodio-Hidrógeno/genética , Análisis de Matrices Tisulares , Transfección
8.
Summit Transl Bioinform ; 2008: 26-30, 2008 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21347122

RESUMEN

With the overwhelming volume of genomic and molecular information available on many databases nowadays, researchers need from bioinformaticians more than encouragement to refine their searches. We present here GeneRanker, an online system that allows researchers to obtain a ranked list of genes potentially related to a specific disease or biological process by combining gene-disease (or genebiological process) associations with protein-protein interactions extracted from the literature, using computational analysis of the protein network topology to more accurately rank the predicted associations. GeneRanker was evaluated in the context of brain cancer research, and is freely available online at http://www.generanker.org.

9.
Neoplasia ; 10(3): 235-43, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18320068

RESUMEN

FK506 binding protein 5 (FKBP5) belongs to a family of immunophilins named for their ability to bind immunosuppressive drugs, also known as peptidyl-prolyl cis-trans isomerases, and also with chaperones to help protein folding. Using glioma cDNA microarray analysis, we found that FKBP5 was overexpressed in glioma tumors. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. The roles of FKBP5 in glioma cells were then examined. We found that cell growth was suppressed after FKBP5 expression was inhibited by short interfering RNA transfection and enhanced by FKBP5 overexpression. Electrophoretic mobility shift assay showed that nuclear factor-kappa B (NF-kappaB) and DNA binding was enhanced by FKBP5 overexpression. The expression level of I-kappa B alpha and phosphorylated NF-kappaB was regulated by the expression of FKBP5. These data suggest that FKBP5 is involved in NF-kappaB pathway activation in glioma cells. In addition, FKBP5 overexpression in rapamycin-sensitive U87 cells blocked the cells' response to rapamycin treatment, whereas rapamycin-resistant glioma cells, both PTEN-positive and -negative, were synergistically sensitive to rapamycin after FKBP5 was knocked down, suggesting that the FKBP5 regulates glioma cell response to rapamycin treatment. In conclusion, our study demonstrates that FKBP5 plays an important role in glioma growth and chemoresistance through regulating signal transduction of the NF-kappaB pathway.


Asunto(s)
Antibióticos Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Resistencia a Antineoplásicos/genética , Glioma/patología , FN-kappa B/metabolismo , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus/fisiología , Apoptosis/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Glioma/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Proteínas de Unión a Tacrolimus/antagonistas & inhibidores , Proteínas de Unión a Tacrolimus/genética
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