RESUMO
Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.
Assuntos
Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma Alveolar/genética , Translocação Genética/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Camundongos , Mioblastos/metabolismo , Mioblastos/patologia , RNA Mensageiro/biossíntese , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Alveolar/patologiaRESUMO
ETV7 is a human oncoprotein that cooperates with Eµ-MYC to promote pre-B-cell leukemia in mice. It is normally expressed in the bone marrow and fetal liver and is upregulated in primary leukemia, suggesting that it is involved in proper hematopoiesis and leukemogenesis. ETV7 has been deleted in most rodents, but is conserved in all other vertebrates, including the zebrafish, Danio rerio. In this report, we characterize the function of the zebrafish etv7 gene during erythropoiesis. Our results demonstrate that etv7 regulates the expression of the zebrafish lanosterol synthase (lss) gene, an essential gene in the cholesterol synthesis pathway. Furthermore, morpholino knockdown of etv7 leads to loss of hemoglobin-containing red blood cells, a phenotype that can be rescued by injection of exogenous cholesterol. We conclude that etv7 is essential for normal red blood cell development through regulation of the lss gene and the cholesterol synthesis pathway.
Assuntos
Vias Biossintéticas , Colesterol/biossíntese , Eritrócitos/metabolismo , Eritropoese , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Eritrócitos/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hemoglobinas/metabolismo , Humanos , Camundongos , Morfolinos/farmacologia , Proteínas Proto-Oncogênicas c-ets/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Globinas beta/genética , Globinas beta/metabolismoRESUMO
Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.
Assuntos
Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Rabdomiossarcoma Alveolar/genética , Animais , Compostos de Bifenilo/farmacologia , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Nitrofenóis/farmacologia , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Rabdomiossarcoma Alveolar/mortalidade , Rabdomiossarcoma Alveolar/patologia , Sulfonamidas/farmacologia , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children with an annual incidence of five new cases per million. Alveolar rhabdomyosarcoma (ARMS) is characterized by the t(2;13) or t(1;13) chromosomal translocations, which generate the PAX3-FOXO1 or PAX7-FOXO1 fusion genes, respectively. The oncogenic activity of PAX3-FOXO1 has been demonstrated in vitro and in vivo, yet expression of the fusion protein alone in primary myoblasts or a mouse model is insufficient for tumorigenic transformation. To identify genes cooperating with PAX3-FOXO1 in ARMS tumorigenesis, we generated a retroviral complementary DNA (cDNA) expression library from the Rh30 ARMS cell line. Arf-/- myoblasts expressing PAX3-FOXO1 and the retroviral cDNA library rapidly formed tumors after subcutaneous injection into NOD-SCID mice. Tumors formed by Arf-/-/PAX3-FOXO1/MarX-library myoblasts contained an unknown cDNA, encoding the C-terminus of the Homo sapiens hypothetical protein, FLJ10404, herein named IRIZIO. Expression of full length IRIZIO cDNA also cooperated with PAX3-FOXO1 in the transformation of Arf-/- myoblasts. Given that IRIZIO is expressed at increased levels in RMS, it might contribute to rhabdomyosarcomagenesis in humans.