RESUMO
Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Piridinas/farmacologia , Rabdomiossarcoma Alveolar/patologia , Animais , Linhagem Celular Tumoral , Linhagem da Célula , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) is a myogenic childhood sarcoma frequently associated with a translocation-mediated fusion gene, Pax3:Foxo1a. METHODS: We investigated the complementary role of Rb1 loss in aRMS tumor initiation and progression using conditional mouse models. RESULTS: Rb1 loss was not a necessary and sufficient mutational event for rhabdomyosarcomagenesis, nor a strong cooperative initiating mutation. Instead, Rb1 loss was a modifier of progression and increased anaplasia and pleomorphism. Whereas Pax3:Foxo1a expression was unaltered, biomarkers of aRMS versus embryonal rhabdomyosarcoma were both increased, questioning whether these diagnostic markers are reliable in the context of Rb1 loss. Genome-wide gene expression in Pax3:Foxo1a,Rb1 tumors more closely approximated aRMS than embryonal rhabdomyosarcoma. Intrinsic loss of pRb function in aRMS was evidenced by insensitivity to a Cdk4/6 inhibitor regardless of whether Rb1 was intact or null. This loss of function could be attributed to low baseline Rb1, pRb and phospho-pRb expression in aRMS tumors for which the Rb1 locus was intact. Pax3:Foxo1a RNA interference did not increase pRb or improve Cdk inhibitor sensitivity. Human aRMS shared the feature of low and/or heterogeneous tumor cell pRb expression. CONCLUSIONS: Rb1 loss from an already low pRb baseline is a significant disease modifier, raising the possibility that some cases of pleomorphic rhabdomyosarcoma may in fact be Pax3:Foxo1a-expressing aRMS with Rb1 or pRb loss of function.
RESUMO
PURPOSE: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. The alveolar subtype of rhabdomyosarcoma (ARMS) is a paradigm for refractory and incurable solid tumors because more than half of the children at diagnosis have either regional lymph node or distant metastases. These studies follow our previous observation that Interleukin-4 receptor α (IL-4Rα) is upregulated in both human and murine ARMS, and that the IL-4R signaling pathway may be a target for abrogating tumor progression. EXPERIMENTAL DESIGN: By in vitro biochemical and cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of IL-4 and IL-13 in IL-4R-mediated mitogenesis, myodifferentiation, and tumor progression. RESULTS: IL-4 and IL-13 ligands accelerated tumor cell growth and activated STAT6, Akt, or MAPK signaling pathways in the human RMS cell lines, RD and Rh30, as well as in mouse primary ARMS cell cultures. IL-4 and IL-13 treatment also decreased protein expression of myogenic differentiation factors MyoD and Myogenin, indicating a loss of muscle differentiation. Using a genetically engineered mouse model of ARMS, we have shown that inhibition of IL-4R signaling pathway with a neutralizing antibody has a profound effect on the frequency of lymph node and pulmonary metastases, resulting in significant survival extension in vivo. CONCLUSIONS: Our results indicate that an IL-4R-dependent signaling pathway regulates tumor cell progression in RMS, and inhibition of this pathway could be a promising adjuvant therapeutic approach.
Assuntos
Desdiferenciação Celular/genética , Transformação Celular Neoplásica/genética , Neoplasias Musculares/genética , Receptores de Interleucina-4/fisiologia , Rabdomiossarcoma/genética , Animais , Transformação Celular Neoplásica/induzido quimicamente , Células Cultivadas , Modelos Animais de Doenças , Genes p53 , Humanos , Camundongos , Camundongos Transgênicos , Mitógenos , Neoplasias Musculares/patologia , Fatores de Regulação Miogênica/genética , Metástase Neoplásica , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/genética , Receptores de Interleucina-4/genética , Rabdomiossarcoma/patologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Up-regulation of the expression of the gene C7orf24, encoding γ-glutamyl cyclotransferase, is a common event in cancers derived from various tissues, but its involvement in osteosarcomas (OS) has not yet been demonstrated. MATERIALS AND METHODS: The expression of C7orf24 was analyzed in human OS cell lines and primary tumor samples. The biological effects of C7orf24 on growth, motility, and invasion in the OS cell lines were investigated using siRNA for C7orf24. Genes related to the function of C7orf24 were sought by genome-wide gene expression profiling. RESULTS: The level of C7orf24 expression was much higher in the OS cell lines and OS primary tumors than in normal osteoblasts. Down-regulation of C7orf24 expression inhibited the growth of the cell lines in association with enhancement of cell-clustering. Treatment with C7orf24-siRNA inhibited cell motility and invasion. Gene ontology suggested the function of C7orf24 to be related to cell adhesion and protein transport. CONCLUSION: C7orf24 is also involved in the growth of OS, and is a potential biomarker for this type of tumor.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Proliferação de Células , Osteossarcoma/genética , gama-Glutamilciclotransferase/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias Ósseas/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , gama-Glutamilciclotransferase/antagonistas & inibidores , gama-Glutamilciclotransferase/metabolismoRESUMO
Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.
Assuntos
Ciclo Celular , Diferenciação Celular , Fibras Musculares Esqueléticas/metabolismo , Regeneração , Proteína do Retinoblastoma/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Cardiotoxinas/farmacologia , Camundongos , Camundongos Transgênicos , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteína do Retinoblastoma/genética , Células Satélites de Músculo Esquelético/citologia , Fatores de TempoRESUMO
Inhibition of the insulin-like growth factor 1 receptor (Igf1r) is an approach being taken in clinical trials to overcome the dismal outcome for metastatic alveolar rhabdomyosarcoma (ARMS), an aggressive muscle cancer of children and young adults. In our study, we address the potential mechanism(s) of Igf1r inhibitor resistance that might be anticipated for patients. Using a genetically engineered mouse model of ARMS, validated for active Igf1r signaling, we show that the prototypic Igf1r inhibitor NVP-AEW541 can inhibit cell growth and induce apoptosis in vitro in association with decreased Akt and Mapk phosphorylation. However, drug resistance in vivo is more common and is accompanied by Igf1r overexpression, Mapk reactivation, and Her2 overexpression. Her2 is found to form heterodimers with Igf1r in resistant primary tumor cell cultures, and stimulation with Igf2 leads to Her2 phosphorylation. The Her2 inhibitor lapatinib cooperates with NVP-AEW541 to reduce Igf1r phosphorylation and to inhibit cell growth even though lapatinib alone has little effect on growth. These results point to the potential therapeutic importance of simultaneous targeting of Igf1r and Her2 to abrogate resistance.
Assuntos
Pirimidinas/farmacologia , Pirróis/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Rabdomiossarcoma/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/patologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Lapatinib , Camundongos , Fosforilação/efeitos dos fármacos , Codorniz , Quinazolinas/farmacologia , Interferência de RNA , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Carga Tumoral/efeitos dos fármacos , Células Tumorais Cultivadas , Adulto JovemRESUMO
Embryonal rhabdomyosarcoma (eRMS) shows the most myodifferentiation among sarcomas, yet the precise cell of origin remains undefined. Using Ptch1, p53 and/or Rb1 conditional mouse models and controlling prenatal or postnatal myogenic cell of origin, we demonstrate that eRMS and undifferentiated pleomorphic sarcoma (UPS) lie in a continuum, with satellite cells predisposed to giving rise to UPS. Conversely, p53 loss in maturing myoblasts gives rise to eRMS, which have the highest myodifferentiation potential. Regardless of origin, Rb1 loss modifies tumor phenotype to mimic UPS. In human sarcomas that lack pathognomic chromosomal translocations, p53 loss of function is prevalent, whereas Shh or Rb1 alterations likely act primarily as modifiers. Thus, sarcoma phenotype is strongly influenced by cell of origin and mutational profile.
Assuntos
Rabdomiossarcoma Embrionário/patologia , Sarcoma/patologia , Animais , Diferenciação Celular , Linhagem da Célula , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes do Retinoblastoma , Genes p53 , Humanos , Camundongos , Mutação , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Rabdomiossarcoma Embrionário/genética , Sarcoma/genéticaRESUMO
Pituitary adenomas are classified into functioning and nonfunctioning (silent) tumors on the basis of hormone secretion. However, the mechanism of tumorigenesis and the cell of origin for pituitary adenoma subtypes remain to be elucidated. Employing a tamoxifen-inducible mouse model, we demonstrate that a novel postnatal Pax7(+) progenitor cell population in the pituitary gland gives rise to silent corticotroph macro-adenomas when the retinoblastoma tumor suppressor is conditionally deleted. While Pax transcriptional factors are critical for embryonic patterning as well as postnatal stem cell renewal for many organs, we have discovered that Pax7 marks a restricted cell population in the postnatal pituitary intermediate lobe. This Pax7(+) early progenitor cell population is overlapping but ontologically downstream of the Nestin(+) pituitary stem cell population, yet upstream of another newly discovered Myf6(+) late progenitor cell population. Interestingly, the Pax7(+) progenitor cell population is evolutionarily conserved in primates and humans, and Pax7 expression is maintained not only in murine tumors but also in human functioning and silent corticotropinomas. Taken together, our results strongly suggest that human silent corticotroph adenomas may in fact arise from a Pax7 lineage of the intermediate lobe, a region of the human pituitary bearing closer scientific interest as a reservoir of pituitary progenitor cells.
RESUMO
The highly aggressive muscle cancer alveolar rhabdomyosarcoma (ARMS) is one of the most common soft tissue sarcoma of childhood, yet the outcome for the unresectable and metastatic disease is dismal and unchanged for nearly three decades. To better understand the pathogenesis of this disease and to facilitate novel preclinical approaches, we previously developed a conditional mouse model of ARMS by faithfully recapitulating the genetic mutations observed in the human disease, i.e., activation of Pax3:Fkhr fusion gene with either p53 or Cdkn2a inactivation. In this report, we show that this model recapitulates the immunohistochemical profile and the rapid progression of the human disease. We show that Pax3:Fkhr expression increases during late preneoplasia but tumor cells undergoing metastasis are under apparent selection for Pax3:Fkhr expression. At a whole-genome level, a cross-species gene set enrichment analysis and metagene projection study showed that our mouse model is most similar to human ARMS when compared with other pediatric cancers. We have defined an expression profile conserved between mouse and human ARMS, as well as a Pax3:Fkhr signature, including the target gene, SKP2. We further identified 7 "druggable" kinases overexpressed across species. The data affirm the accuracy of this genetically engineered mouse model.
Assuntos
Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Alelos , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Progressão da Doença , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/biossíntese , Fatores de Transcrição Box Pareados/genética , Penetrância , Rabdomiossarcoma Alveolar/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/genéticaRESUMO
Bioluminescent reporter genes are sensitive in situ tools for following disease progression in preclinical models, albeit they are subject to scattering and absorption in deep tissues. We have generated a bicistronic Cre/LoxP reporter mouse line that pairs the expression of firefly luciferase with quantifiable expression of a human placental alkaline phosphatase that is secreted into the serum (SeAP). With the use of this dual-modality bioreporter with a novel, inducible Pax7-CreER line for tracking muscle satellite cells, we demonstrate the longitudinal kinetics of muscle stem cell turnover, accounting for a doubling of the signal from satellite cell and progeny every 3.93 wk in the transition from adolescence to early adulthood. We also show that this dual-modality bioreporter can be incorporated in preclinical cancer models, whereby SeAP activity is reflective of tumor burden. Thus, this dual bioreporter permits both spatial localization and accurate quantification of biological processes in vivo even when the tissue of interest is deep within the animal.
Assuntos
Células-Tronco Adultas/metabolismo , Genes Reporter , Sarcoma Experimental/genética , Sarcoma Experimental/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Fosfatase Alcalina/genética , Animais , Sequência de Bases , Primers do DNA/genética , Proteínas Ligadas por GPI , Humanos , Isoenzimas/genética , Luciferases de Vaga-Lume/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição PAX7/genéticaRESUMO
The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/enzimologia , Depsipeptídeos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Histona Desacetilases , Osteossarcoma/enzimologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias Ósseas/tratamento farmacológico , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Proteína Ligante Fas/efeitos dos fármacos , Proteína Ligante Fas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Osteossarcoma/tratamento farmacológicoRESUMO
We report the first case of a tumor of the Ewing sarcoma family of tumors arising from the urinary bladder 3 years after chemotherapy for acute lymphoblastic leukemia. A 16-year-old boy complained of macrohematuria and dysuria during the posttreatment follow up of his acute lymphoblastic leukemia. Ultrasonography and computed tomography revealed a 1-cm sized intravesical tumor. The tumor was transurethrally resected with no residual tumor at the margin. Histopathologic analyses revealed a small round blue cell tumor with positive staining for CD99 antibody. EWS-FLI1 fusion transcripts were detected in the tumor tissue by reverse transcriptase polymerase chain reaction. These findings support the diagnosis of Ewing sarcoma family of tumor. After adjuvant multidrug chemotherapy, the patient has shown no evidence of disease for more than 2 years.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Sarcoma de Ewing/cirurgia , Neoplasias da Bexiga Urinária/cirurgia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Terapia Combinada , Humanos , Masculino , Sarcoma de Ewing/tratamento farmacológico , Resultado do Tratamento , Neoplasias da Bexiga Urinária/tratamento farmacológicoRESUMO
Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.
Assuntos
Técnicas de Cultura de Células/métodos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Proteínas ras/metabolismo , Transformação Celular Neoplásica/genética , Células Cultivadas , Genes ras/genética , Humanos , Sarcoma/genética , Proteínas ras/genéticaRESUMO
We evaluated the ability of canine bone marrow stromal cells (cBMSCs) to regenerate bone in a cavity of the scapholunate created by curretage and freeze-thawing with liquid nitrogen (LN). Autologous BMSCs were harvested from the iliac crest and expanded in vitro. Their potential to differentiate into osteo-, chondro-, and adipogenic lineages was confirmed using a standard differentiation induction assay. LN-treated scapholunates showed no regeneration of bone tissue when the cavity was left alone, demonstrating severe collapse and deformity as observed in human Kienböck disease. A combination of beta-tri-calcium phosphate and a vascularized bone graft with autologous fibroblasts failed to regenerate bone in the LN-treated cavity. When the same procedure was performed using BMSCs, however, LN-treated scapholunates showed no collapse and deformity, and the cavity was completely filled with normal cancerous bone within 4 weeks. These results suggested the potential of using BMSCs to treat Kienböck disease.
Assuntos
Células da Medula Óssea/citologia , Transplante de Medula Óssea/métodos , Regeneração Óssea/fisiologia , Osso Semilunar/cirurgia , Osteonecrose/terapia , Células Estromais/citologia , Adipogenia/fisiologia , Animais , Células da Medula Óssea/fisiologia , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/uso terapêutico , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/efeitos dos fármacos , Condrogênese/fisiologia , Cães , Osso Semilunar/efeitos dos fármacos , Osso Semilunar/fisiologia , Imageamento por Ressonância Magnética/métodos , Nitrogênio/uso terapêutico , Osteocondrite/terapia , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Osteonecrose/diagnóstico por imagem , Osteonecrose/fisiopatologia , Radiografia , Células Estromais/transplante , Transplante AutólogoRESUMO
Synovial sarcoma is a malignant soft tissue tumor harboring a tumor-specific fusion gene, SYT-SSX, of which exon 10 of the SYT gene is fused to exon 6 of the SSX gene is the common form. Here we report a case of synovial sarcoma with a novel form of the SYT-SSX2 fusion transcript, in which 75 bases were inserted at the common fusion junction. Computer analyses revealed that 15 bases were from intron 10 of the SYT gene, and 10 from the end of intron 4, and 50 from exon 5 of the SSX2 gene. Precise analyses of genomic breakpoints in SYT and SSX2 loci revealed that the reciprocal translocation creating the fusion gene was associated with a large deletion in both loci. The structure of SYT-SSX2 suggests that the fusion transcript in this case was created using a cryptic splicing acceptor site 15 bases upstream of the genomic fusion point, incorporating intronic sequences in mature mRNA. Reexamination of two variant SYT-SSX2 genes reported previously revealed that unknown sequences inserted at the common junction points were derived from intron sequences, as in the present case.
Assuntos
Variação Genética , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Sarcoma Sinovial/química , Sarcoma Sinovial/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , DNA Complementar/genética , DNA de Neoplasias/análise , Éxons , Feminino , Humanos , Íntrons , Metástase Linfática , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Mensageiro/genética , Proteínas Recombinantes/análise , Sarcoma Sinovial/patologia , Sarcoma Sinovial/cirurgia , Análise de Sequência de DNA , Fatores de Tempo , Transcrição Gênica , Translocação GenéticaRESUMO
PURPOSE: Synovial sarcoma is a soft tissue sarcoma, the growth regulatory mechanisms of which are unknown. We investigated the involvement of fibroblast growth factor (FGF) signals in synovial sarcoma and evaluated the therapeutic effect of inhibiting the FGF signal. EXPERIMENTAL DESIGN: The expression of 22 FGF and 4 FGF receptor (FGFR) genes in 18 primary tumors and five cell lines of synovial sarcoma were analyzed by reverse transcription-PCR. Effects of recombinant FGF2, FGF8, and FGF18 for the activation of mitogen-activated protein kinase (MAPK) and the growth of synovial sarcoma cell lines were analyzed. Growth inhibitory effects of FGFR inhibitors on synovial sarcoma cell lines were investigated in vitro and in vivo. RESULTS: Synovial sarcoma cell lines expressed multiple FGF genes especially those expressed in neural tissues, among which FGF8 showed growth stimulatory effects in all synovial sarcoma cell lines. FGF signals in synovial sarcoma induced the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38MAPK but not c-Jun NH2-terminal kinase. Disruption of the FGF signaling pathway in synovial sarcoma by specific inhibitors of FGFR caused cell cycle arrest leading to significant growth inhibition both in vitro and in vivo. Growth inhibition by the FGFR inhibitor was associated with a down-regulation of phosphorylated ERK1/2 but not p38MAPK, and an ERK kinase inhibitor also showed growth inhibitory effects for synovial sarcoma, indicating that the growth stimulatory effect of FGF was transmitted through the ERK1/2. CONCLUSIONS: FGF signals have an important role in the growth of synovial sarcoma, and inhibitory molecules will be of potential use for molecular target therapy in synovial sarcoma.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Sarcoma Sinovial/patologia , Transdução de Sinais/fisiologia , Ureia/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Pirimidinas/farmacologia , Pirróis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma Sinovial/genética , Sarcoma Sinovial/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Ureia/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Osteosarcoma (OS) is the most prevalent malignant tumor among cases of Rothmund-Thomson syndrome (RTS) with germline mutations of the RECQL4 gene, a member of the RecQ helicase family. We investigated the involvement of the RECQL4 gene in the development of OS unrelated to RTS. RECQL4 mRNA was detected in 9 of 9 OS cell lines by Northern blotting and 26 of 26 OS tumors by RT-PCR. Direct sequencing of the entire coding region along with flanking splice junctions and 13 small (< 100 bp) introns in 71 OS tumors revealed 2 sites with a single-base change causing an amino acid change (G1814A for R355Q and C2474T for P441S) and one site with a 6 bp inframe deletion (4837-42delTGCACC for CT857-8del). Identical genotypes were found in corresponding normal tissues in all cases, and the frequency of each allele was not significantly different between OS and control populations. Our data indicate that the RECQL4 gene is not a frequent target for somatic mutations in sporadic OS unrelated to RTS.
Assuntos
Adenosina Trifosfatases/genética , Neoplasias Ósseas/genética , DNA Helicases/genética , Osteossarcoma/genética , Polimorfismo de Nucleotídeo Único/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Neoplasias Ósseas/enzimologia , Análise Mutacional de DNA , Primers do DNA , Humanos , Osteossarcoma/enzimologia , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , RecQ Helicases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Células Tumorais CultivadasRESUMO
Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.
Assuntos
Azacitidina/análogos & derivados , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Fatores de Transcrição/metabolismo , 5-Metilcitosina/química , Azacitidina/farmacologia , Sítios de Ligação , Western Blotting , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Linhagem Celular Tumoral , Condrócitos/metabolismo , Cromatina/metabolismo , Ilhas de CpG , Citosina/metabolismo , Decitabina , Genes Reporter , Humanos , Luciferases/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Sulfitos/farmacologia , Timina/química , Transcrição GênicaRESUMO
We investigated the expression of the Chondromodulin-I (ChM-I) gene, a putative tumor suppressor gene in cartilaginous tumors, by quantitative RT-PCR in 15 chondrosarcomas (CSs). Eight CSs expressed the ChM-I gene at the level higher than those in articular cartilage (positive cases), whereas the expression of the ChM-I gene in the remaining seven CSs was lower than those in articular cartilage (negative cases). All of five peripheral CS were positive, and the ChM-I positive tumors shared expression profiles of cartilage-related genes with articular cartilage cells. On the other hand, all of four central CSs without extramedullary lesions were negative, and the ChM-I negative tumors expressed the parathyroid hormone-related peptide gene at the lower level and the COL10A1 genes at the higher level than articular cartilage cells. Neither the histological grade nor the rate of recurrence showed clear association with the level of ChM-I gene expression. These results suggested that the expression of ChM-I gene in CS has no direct role in tumorigenesis but rather reflects the site of tumor development and therefore precursor of tumor cells.
Assuntos
Inibidores da Angiogênese/genética , Neoplasias Ósseas/genética , Condrossarcoma/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Agrecanas , Inibidores da Angiogênese/metabolismo , Cartilagem/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo IX/genética , Colágeno Tipo IX/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Proteoglicanas/genética , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2-7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.