RESUMO
The infiltration of glioma cells into adjacent tissue is one of the major obstacles in the therapeutic management of malignant brain tumours, in most cases precluding complete surgical resection. Consequently, malignant glioma patients almost invariably experience tumour recurrences. Within the brain, glioma cells migrate rapidly either amoeboidly or mesenchymally to invade surrounding structures, in dependence on the extracellular environment. In addition, radiotherapy, frequently applied as adjuvant therapeutic modality, may enhance tumour cell mobility. Here, we show that the receptor tyrosine kinase Mer (MerTK) is overexpressed in glioblastoma multiforme (GBM) and that this is accompanied with increased invasive potential. MerTK expression is maintained in primary GBM-derived tumour spheres under stem cell culture conditions but diminishes significantly in serum-containing cultures with concomitant downregulation of Nestin and Sox2. Depletion of MerTK disrupts the rounded morphology of glioma cells and decreases their invasive capacity. Furthermore, the expression and phosphorylation of myosin light chain 2 are strongly associated with MerTK activity, indicating that the effect of MerTK on glioma cell invasion is mediated by actomyosin contractility. Finally, DNA damage robustly triggers the upregulation and phosphorylation of MerTK, which protects cells from apoptosis. This effect is strongly impaired upon MerTK depletion or overexpression of an inactive MerTK mutant. Collectively, our data suggests that MerTK is a novel therapeutic target in the treatment of the malignant gliomas.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioblastoma/genética , Glioblastoma/patologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Linhagem Celular Tumoral , Forma Celular/genética , Sobrevivência Celular/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Regulação para Cima/genética , c-Mer Tirosina QuinaseRESUMO
Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation.
Assuntos
Astrócitos/metabolismo , Transformação Celular Neoplásica/patologia , Células-Tronco Neurais/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Animais , Astrócitos/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem da Célula , Transformação Celular Neoplásica/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Células-Tronco Neurais/patologia , Receptor Notch2/genéticaRESUMO
Altered expression of the eukaryotic translation initiation factor 3 (eIF3) subunit eIF3e/INT6 has been described in various types of human cancer, but the nature of its involvement in tumorigenesis is not yet clear. Using immunohistochemical analysis of 81 primary breast cancers, we found that high tumor grade correlated significantly with elevated cytoplasmic eIF3e level in epithelial tumor cells. Analysis of protein synthesis after siRNA-mediated knockdown in breast cancer cell lines indicated that eIF3e is not required for bulk translation. Microarray analysis of total and polysomal RNAs nonetheless identified distinct sets of mRNAs regulated either positively or negatively by eIF3e; functional classification of these revealed a marked enrichment of genes involved in cell proliferation, invasion and apoptosis. Validated mRNA targets regulated positively at the translational level by eIF3e included urokinase-type plasminogen activator and apoptotic regulator BCL-XL, whereas synthesis of proteins including the mitotic checkpoint component MAD2L1 was negatively regulated. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. Taken together, our study data suggest that eIF3e has a positive role in breast cancer progression. It regulates the translation, and in some cases abundance, of mRNAs involved in key aspects of cancer cell biology.
Assuntos
Neoplasias da Mama/genética , Fator de Iniciação 3 em Eucariotos/fisiologia , Proteínas de Choque Térmico/fisiologia , Oncogenes , Feminino , HumanosRESUMO
PURPOSE: The androgen sensitive prostate cancer cell line LNCaP is strongly positive for dihydrotestosterone (DHT) dependent telomerase activity, which is an important factor in cellular immortality and carcinogenesis. In this study we determined the potential of silibinin as an anticancer drug that down-regulates telomerase activity and prostate specific antigen (PSA) together with the co-activator of the androgen receptor prostate epithelium specific Ets transcription factor. MATERIALS AND METHODS: LNCaP cells were treated with various concentrations of silibinin in the presence or absence of 5alpha-DHT. We used real-time reverse transcriptase-polymerase chain reaction to quantify mRNA expression of PSA, prostate epithelium specific Ets transcription factor and the catalytic subunit of telomerase vs the housekeeping gene porphobilinogen deaminase with gene specific, dual labeled fluorescence probes. PSA secretion from LNCaP cells in conditioned medium was measured with an Elecsys System 2010 (Roche Diagnostics, Mannheim, Germany) and telomerase activity in extracts from LNCaP cells was measured with a TRAP (telomeric repeat amplification protocol) assay. RESULTS: Silibinin down-regulated PSA mRNA expression and PSA secretion in conditioned medium. Simultaneous stimulation with silibinin and 10(-8) M DHT also resulted in PSA down-regulation, whereas DHT alone increased PSA secretion. Telomerase catalytic subunit mRNA decreased significantly after silibinin stimulation. Telomerase activity was down-regulated by silibinin and stimulated by DHT. The 2 agents in combination resulted in telomerase down-regulation. CONCLUSIONS: The down-regulation of PSA by silibinin and its counteraction on DHT effects indicate that this compound can interact with the expression of genes that are regulated through the androgen receptor. Silibinin can also inhibit the telomerase activity that mediates cell immortality and carcinogenesis. The 2 effects underline the possible therapeutic use of silibinin as an antiproliferative agent in intervention for prostate cancer.
Assuntos
Antígeno Prostático Específico/antagonistas & inibidores , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/enzimologia , Silybum marianum , Silimarina/farmacologia , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Antígeno Prostático Específico/genética , Neoplasias da Próstata/metabolismo , RNA Mensageiro/biossíntese , Silibina , Telomerase/genética , Células Tumorais CultivadasRESUMO
RNA-binding proteins are involved in post-transcriptional processes like mRNA stabilization, post-transcriptional modification, and transport and have been suggested to play an important role in developmental gene regulation. We report here the cloning and characterization of Brunol4, a novel mouse cDNA closely related to the elav-type family of genes encoding for RNA-binding proteins and a subfamily recently named after the bruno gene of Drosophila. Murine Brunol4 is localized near the centromere of chromosome 18. The cDNA sequence of Brunol4 is separated by 12 introns and the size of Brunol4 may be around 250 kb due to the large size of several introns. Brunol4 expression is detectable in the developing embryo and, later on becomes mainly restricted to cerebral structures, in particular the cerebellum where it persists in the adult organism. We predict a role of Brunol4 and the respective human homologue in differentiation and maintenance of neuronal structures.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Animais , Sequência de Bases , Proteínas CELF , Mapeamento Cromossômico , DNA Complementar , Proteínas ELAV , Camundongos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.