RESUMO
The purpose of our study was to investigate the prevalence of the PAX3/7-FKHR fusion genes and quantify the IGF2 gene expression in rhabdomyosarcoma (RMS) samples. Soft tissue sarcomas account 5% of childhood cancers and 50% of them are RMS. Morphological evaluation of pediatric RMS has defined two histological subtypes, embryonal (ERMS) and alveolar (ARMS). Chromosomal analyses have demonstrated two translocations associated with ARMS, resulting in the PAX3/7-FKHR rearrangements. Reverse transcriptase-polymerase chain reaction (RT-PCR) is extremely useful in the diagnosis of ARMS positive for these rearrangements. Additionally, several studies have shown a significant involvement of IGF pathway in the pathogenesis of RMS. The presence of PAX3/7-FKHR gene fusions was studied in 25 RMS samples from patients attending the IOP-GRAACC/UNIFESP and three RMS cell lines by RT-PCR. IGF2 gene expression was quantified by qPCR and related with clinic pathological parameters. Of the 25 samples, nine (36%) were ARMS and 16 (64%) were ERMS. PAX3/7-FKHR gene fusions expression was detected in 56% of ARMS tumor samples. IGF2 overexpression was observed in 80% of samples and could indicate an important role of this pathway in RMS biology.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Proteínas de Fusão Oncogênica , Fator de Transcrição PAX7/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/metabolismo , Adolescente , Linhagem Celular Tumoral , Criança , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Fator de Transcrição PAX3 , Fator de Transcrição PAX7/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Estudos Retrospectivos , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismoRESUMO
OBJECTIVE: Pax3 and Pax7 are closely related genes that are involved in commitment of cells to a myogenic lineage during skeletal muscle development and regeneration. Several Pax3 and Pax7 transcripts are expressed from the genes, generating different isoforms with potentially distinct DNA binding and transactivation properties. The aim of this study was to investigate the implication of Pax3 and Pax7 C-terminal isoforms during myogenic differentiation and tumorigenesis, since fusions involving these genes are commonly associated with alveolar rhabdomyosarcoma (ARMS). METHODS: Uncommitted (mouse mesenchymal stem cells, MSCs) and committed (C2C12) myogenic precursor cells were stably transfected with PAX3/FKHR and PAXC7/ FKHR fusion genes. We analysed gene and protein expression comparing the newly generated cells with the parental cells, to determine the functional importance of Pax3 and Pax7 C-terminal isoforms. RESULTS: We found that the transcript Pax3c was expressed at low levels in undifferentiated C2C12 and MSCs cells, but its expression levels increased considerably at later stages of differentiation. However, expression levels of Pax3d transcript increased only slightly after differentiation. Pax7 transcripts, present before differentiation in committed C2C12 cells, but absent in uncommitted MSCs, increased noticeably in MSCs after differentiation. We also found that the presence of PAX/FKHR fusions prevented both C2C12 and MSC cells from terminal myogenic differentiation and increased the expression of discrete endogenous Pax3/7 transcripts, in particular Pax3d and Pax7B. CONCLUSIONS: Our results suggest that both Pax3 and Pax7 transcripts are required for commitment of cells to the myogenic lineage, with each transcript having a distinct role. More specifically, the Pax3c isoform may be required for terminal myogenic differentiation whereas the Pax3d isoform may be involved in undifferentiated cell maintenance and/or proliferation.
Assuntos
Diferenciação Celular/fisiologia , Células Musculares/citologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/genética , Animais , Linhagem Celular , Linhagem da Célula/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Camundongos , Células Musculares/metabolismo , Fator de Transcrição PAX3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma Alveolar/metabolismo , TransfecçãoRESUMO
Rhabdomyosarcoma (RMS) is one of the most common extracranial solid tumours in children. Embryonal and alveolar subtypes of RMS present completely different genetic abnormalities. Embryonal RMS (eRMS) is characterised by loss of heterozygosity on the short arm of chromosome 11 (11p15.5), suggesting inactivation of a tumour-suppressor gene. In contrast, the majority (80-85%) of the alveolar RMS (aRMS) have the reciprocal chromosomal translocations 't(2;13)(q35;q14) or t(1;13)(p36;q14). t(2;13) appears in approximately 70% of patients with the alveolar subtype. The molecular counterpart of this translocation consists of the generation of a chimeric fusion gene involving the /PAX3/ gene located in chromosome 2 and a member of the fork-head family, /FOXO1/ (formerly /FKHR/), located in chromosome 13. A less frequent variant translocation t(1;13) involves another PAX family gene, /PAX7/, located in chromosome 1 and /FOXO1/ and is present in 10-15% of cases of the alveolar subtype in RMS. Recently, many studies focused on cancer have demonstrated the great potential of the genomic approach based on tumour expression profiles. These technologies permit the identification of new regulatory pathways. Molecular detection of minimal disease by a sensitive method could contribute to better treatment stratification in these patients. In RMS, the advances in the knowledge of the biological characteristics of the tumour are slowly translated into the clinical management of children with this tumour.
Assuntos
Rabdomiossarcoma/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Modelos Biológicos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma/diagnóstico , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/diagnóstico , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Transdução de Sinais , Translocação GenéticaRESUMO
The Snail family of genes comprise a group of transcription factors with characteristic zinc finger motifs. One of the members of this family is the Slug gene. Slug has been implicated in the development of neural crest in chick and Xenopus by antisense loss of function experiments. Here, we have generated functional derivatives of Xslug by constructing cDNAs that encode the Xslug protein fused with the transactivation domain of the virus-derived VP16 activator or with the repressor domain of the Drosophila Engrailed protein. Our results suggest that Xslug normally functions as a transcriptional repressor and that Xslug-VP16 behaves as a dominant negative of Xslug. In the present work, we confirm and extend previous results that suggest that Xslug has an important function in neural crest development, by controlling its own transcription. In addition we have uncovered a new function for Xslug. We show that Xslug is expressed in the dorsal mesendoderm at the beginning of gastrulation, where is it able to upregulate the expression of dorsal genes. On the other hand when Xslug is expressed outside of the organizer it represses the expression of ventral genes. Our results indicate that this effect on mesodermal patterning depends on BMP activity, showing that Xslug can directly control the transcription of BMP-4.