RESUMEN
The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.
Asunto(s)
Fragmentos de Inmunoglobulinas/inmunología , Proteínas de Fusión Oncogénica/inmunología , Proteína Proto-Oncogénica c-fli-1/inmunología , Sarcoma de Ewing/genética , Sarcoma de Ewing/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Carcinoma Hepatocelular , Línea Celular Tumoral , Epítopos/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Neoplasias Hepáticas , Datos de Secuencia Molecular , Neuroblastoma , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Pliegue de Proteína , Proteína Proto-Oncogénica c-fli-1/química , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN , Activación Transcripcional , TransfecciónRESUMEN
Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Óseas/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Hipoxia/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Sarcoma de Ewing/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Técnicas para Inmunoenzimas , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/patología , Activación TranscripcionalRESUMEN
Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21(WAF1/CIP1) levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21(WAF1/CIP1) induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21(WAF1/CIP1). These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.
Asunto(s)
Genes p53 , Proteínas de Fusión Oncogénica/fisiología , Receptores Notch/fisiología , Sarcoma de Ewing/metabolismo , Factores de Transcripción/fisiología , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Cartilla de ADN , Técnica del Anticuerpo Fluorescente , Silenciador del Gen , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
The transmembrane 4 superfamily member KAI1/CD82, a metastasis suppressor, is correlated inversely with the progression and invasion of several tumors. It is capable of inhibiting metastasis without affecting tumorigenicity per se. KAI1/CD82 expression is down-regulated in the progression of common solid epithelial tumors of adulthood. Mutation of p53 is suggested to be involved in the modulation of KAI1. As little is known about its expression and possible prognostic impact in pediatric tumors, we investigated KAI1/CD82 expression in cell lines and primary tumor samples from pediatric tumors of neuroectodermal origin, neuroblastoma and Ewing's sarcoma family tumor. Twenty-four of 29 Ewing's sarcoma family tumor cell lines, independent of p53 status, showed KAI1 mRNA positivity by reverse transcription-PCR analysis in contrast to zero of eight neuroblastoma cell lines. Among 13 primary Ewing's sarcoma family tumor samples from patients with different disease extension, KAI1 mRNA expression was low as detected by reverse transcription-PCR. Twenty of 30 primary neuroblastoma specimens were KAI1-negative by immunofluorescence analysis whereas the remaining 10 gave weak to moderate staining patterns. There was no apparent correlation of KAI1 expression with any clinical or genetic features of the patients whose tumor samples were studied. Consequently, KAI1 may not be of prognostic relevance in this group of tumors although there may be some role for KAI1 modulation in the biology of these neuroectodermal tumors.