Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 68
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Biol Rep ; 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31583563

RESUMO

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16Ink4a, which blocks cyclin-dependent kinase activity, and p19Arf, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFß-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31645346

RESUMO

Infantile myofibromatosis (IM) is an aggressive neoplasm composed of myofibroblast-like cells in children. Although typically localized, it can also present as multifocal disease, which represents a challenge for effective treatment. IM has previously been linked to activating somatic and germline point mutations in the PDGFRß tyrosine kinase encoded by the PDGFRB gene. Clinical panel-based targeted tumor sequencing of a tumor from a newborn with multifocal IM revealed a novel PDGFRB rearrangement, which was reported as being of unclear significance. Additional sequencing of cDNA from tumor and germline DNA confirmed a complex somatic/mosaic PDGFRB rearrangement with an apparent partial tandem duplication disrupting the juxtamembrane domain. Ectopic expression of cDNA encoding the mutant form of PDGFRB markedly enhanced cell proliferation of mouse embryo fibroblasts (MEFs) compared to wild-type PDGFRB and conferred tumor-forming capacity on nontumorigenic 10T1/2 fibroblasts. The mutated protein enhanced MAPK activation and retained sensitivity to the PDGFRß inhibitor imatinib. Our findings reveal a new mechanism by which PDGFRB can be activated in IM, suggest that therapy with tyrosine kinase inhibitors including imatinib may be beneficial, and raise the possibility that this receptor tyrosine kinase might be altered in a similar fashion in additional cases that would similarly present annotation challenges in clinical DNA sequencing analysis pipelines.

3.
Cancer Med ; 8(14): 6437-6448, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31456361

RESUMO

BACKGROUND: Previous studies of the prognostic importance of FOXO1 fusion status in patients with rhabdomyosarcoma (RMS) have had conflicting results. We re-examined risk stratification by adding FOXO1 status to traditional clinical prognostic factors in children with localized or metastatic RMS. METHODS: Data from six COG clinical trials (D9602, D9802, D9803, ARST0331, ARTS0431, ARST0531; two studies each for low-, intermediate- and high-risk patients) accruing previously untreated patients with RMS from 1997 to 2013 yielded 1727 evaluable patients. Survival tree regression for event-free survival (EFS) was conducted to recursively select prognostic factors for branching and split. Factors included were age, FOXO1, clinical group, histology, nodal status, number of metastatic sites, primary site, sex, tumor size, and presence of metastases in bone/bone marrow, soft tissue, effusions, lung, distant lymph nodes, and other sites. Definition and outcome of the proposed risk groups were compared to existing systems and cross-validated results. RESULTS: The 5-year EFS and overall survival (OS) for evaluable patients were 69% and 79%, respectively. Extent of disease (localized versus metastatic) was the first split (EFS 73% vs 30%; OS 84% vs. 42%). FOXO1 status (positive vs negative) was significant in the second split both for localized (EFS 52% vs 78%; OS 65% vs 88%) and metastatic disease (EFS 6% vs 46%; OS 19% vs 58%). CONCLUSIONS: After metastatic status, FOXO1 status is the most important prognostic factor in patients with RMS and improves risk stratification of patients with localized RMS. Our findings support incorporation of FOXO1 status in risk stratified clinical trials.

4.
Mol Cancer Res ; 17(9): 1854-1866, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31189690

RESUMO

Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFß selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFß signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFß induced ARF mRNA and protein, and ARF knockdown limited TGFß-mediated growth suppression. TGFß-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFß by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFß, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

5.
Genes Dev ; 33(11-12): 626-640, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30975722

RESUMO

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.


Assuntos
Carcinogênese , Desenvolvimento Muscular , Proteína MyoD/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Células Cultivadas , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Transição Epitelial-Mesenquimal , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Sequências Hélice-Alça-Hélice , Humanos , Proteína MyoD/química , Mioblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/química , Proteína 1 Relacionada a Twist/química
6.
Nat Rev Dis Primers ; 5(1): 1, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30617281

RESUMO

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. Decades of clinical and basic research have gradually improved our understanding of the pathophysiology of RMS and helped to optimize clinical care. The two major subtypes of RMS, originally characterized on the basis of light microscopic features, are driven by fundamentally different molecular mechanisms and pose distinct clinical challenges. Curative therapy depends on control of the primary tumour, which can arise at many distinct anatomical sites, as well as controlling disseminated disease that is known or assumed to be present in every case. Sophisticated risk stratification for children with RMS incorporates various clinical, pathological and molecular features, and that information is used to guide the application of multifaceted therapy. Such therapy has historically included cytotoxic chemotherapy as well as surgery, ionizing radiation or both. This Primer describes our current understanding of RMS epidemiology, disease susceptibility factors, disease mechanisms and elements of clinical care, including diagnostics, risk-based care of newly diagnosed and relapsed disease and the prevention and management of late effects in survivors. We also outline potential opportunities to further translate new biological insights into improved clinical outcomes.


Assuntos
Fatores Etários , Rabdomiossarcoma/diagnóstico , Rabdomiossarcoma/cirurgia , Humanos , Programas de Rastreamento/métodos , Qualidade de Vida/psicologia , Rabdomiossarcoma/fisiopatologia , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/fisiopatologia , Fatores de Risco
7.
J Clin Oncol ; 36(27): 2770-2777, 2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-30091945

RESUMO

Purpose Intermediate-risk rhabdomyosarcoma (RMS) includes patients with either nonmetastatic, unresected embryonal RMS (ERMS) with an unfavorable primary site or nonmetastatic alveolar RMS (ARMS). The primary aim of this study was to improve the outcome of patients with intermediate-risk RMS by substituting vincristine and irinotecan (VI) for half of vincristine, dactinomycin, and cyclophosphamide (VAC) courses. All patients received a lower dose of cyclophosphamide and earlier radiation therapy than in previous trials. Patients and Methods Patients were randomly assigned at study entry to either VAC (cumulative cyclophosphamide dose, 16.8 g/m2) or VAC/VI (cumulative cyclophosphamide dose, 8.4 g/m2) for 42 weeks of therapy. Radiation therapy started at week 4, with individualized local control plans permitted for patients younger than 24 months. The primary study end point was event-free survival (EFS). The study design had an 80% power (5% one-sided α-level) to detect an improved long-term EFS from 65% (with VAC) to 76% (with VAC/VI). Results A total of 448 eligible patients were enrolled in the study. At a median follow-up of 4.8 years, the 4-year EFS was 63% with VAC and 59% with VAC/VI ( P = .51), and 4-year overall survival was 73% for VAC and 72% for VAC/VI ( P = .80). Within the ARMS and ERMS subgroups, no difference in outcome by treatment arm was found. Severe hematologic toxicity was less common with VAC/VI therapy. Conclusion The addition of VI to VAC did not improve EFS or OS for patients with intermediate-risk RMS. VAC/VI had less hematologic toxicity and a lower cumulative cyclophosphamide dose, making VAC/VI an alternative standard therapy for intermediate-risk RMS.

8.
Cell Rep ; 24(1): 238-251, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-29972784

RESUMO

Identifying oncogenic drivers and tumor suppressors remains a challenge in many forms of cancer, including rhabdomyosarcoma. Anticipating gene expression alterations resulting from DNA copy-number variants to be particularly important, we developed a computational and experimental strategy incorporating a Bayesian algorithm and CRISPR/Cas9 "mini-pool" screen that enables both genome-scale assessment of disease genes and functional validation. The algorithm, called iExCN, identified 29 rhabdomyosarcoma drivers and suppressors enriched for cell-cycle and nucleic-acid-binding activities. Functional studies showed that many iExCN genes represent rhabdomyosarcoma line-specific or shared vulnerabilities. Complementary experiments addressed modes of action and demonstrated coordinated repression of multiple iExCN genes during skeletal muscle differentiation. Analysis of two separate cohorts revealed that the number of iExCN genes harboring copy-number alterations correlates with survival. Our findings highlight rhabdomyosarcoma as a cancer in which multiple drivers influence disease biology and demonstrate a generalizable capacity for iExCN to unmask disease genes in cancer.

9.
Elife ; 72018 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-29869612

RESUMO

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6-19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3, inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3/HES3, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.

10.
Clin Cancer Res ; 24(16): 3888-3897, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29691299

RESUMO

Purpose: A comprehensive analysis of the genomics of undifferentiated sarcomas (UDS) is lacking. We analyzed copy-number alterations and fusion status in patients with UDS prospectively treated on Children's Oncology Group protocol ARST0332.Experimental Design: Copy-number alterations were assessed by OncoScan FFPE Express on 32 UDS. Whole-exome and transcriptome libraries from eight tumors with sufficient archived material were sequenced on HiSeq (2 × 100 bp). Targeted RNA-sequencing using Archer chemistry was performed on two additional cases.Results: Five-year overall survival for patients with UDS was 83% (95% CI, 69%-97%) with risk-adapted therapy (surgery, chemotherapy, and radiotherapy). Both focal and arm-level copy-number alterations were common including gain of 1q (8/32, 25%) and loss of 1p (7/32, 22%), both of which occurred more often in clinically defined high-risk tumors. Tumors with both loss of 1p and gain of 1q carried an especially poor prognosis with a 5-year event-free survival of 20%. GISTIC analysis identified recurrent amplification of FGF1 on 5q31.3 (q = 0.03) and loss of CDKN2A and CDKN2B on 9p21.3 (q = 0.07). Known oncogenic fusions were identified in eight of 10 cases analyzed by next-generation sequencing.Conclusions: Pediatric UDS generally has a good outcome with risk-adapted therapy. A high-risk subset of patients whose tumors have copy-number loss of 1p and gain of 1q was identified with only 20% survival. Oncogenic fusions are common in UDS, and next-generation sequencing should be considered for children with UDS to refine the diagnosis and identify potentially targetable drivers. Clin Cancer Res; 24(16); 3888-97. ©2018 AACR.

11.
Cancer ; 124(9): 1973-1981, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29461635

RESUMO

BACKGROUND: Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior. METHODS: This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer. RESULTS: Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors. CONCLUSIONS: Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.

12.
Pediatr Blood Cancer ; 64(12)2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28521080

RESUMO

BACKGROUND: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) has historically been of prognostic and therapeutic importance. However, classification has been complicated by shifting histologic criteria required for an ARMS diagnosis. Children's Oncology Group (COG) studies after IRS-IV, which included the height of this diagnostic shift, showed both an increased number of ARMS and an increase in the proportion of fusion-negative ARMS. Following diagnostic standardization and histologic re-review of ARMS cases enrolled during this era, analysis of low-risk (D9602) and intermediate-risk (D9803) rhabdomyosarcoma (RMS) studies showed that fusion status rather than histology best predicts prognosis for patients with RMS. This analysis remains to be completed for patients with high-risk RMS. PROCEDURE: We re-reviewed cases on high-risk COG studies D9802 and ARST0431 with an enrollment diagnosis of ARMS. We compared the event-free survival (EFS) and overall survival by histology, PAX-FOXO1 fusion, and clinical risk factors (Oberlin score) for patients with metastatic RMS using the log-rank test. RESULTS: Histology re-review resulted in reclassification as ERMS for 12% of D9802 cases and 5% of ARST0431 cases. Fusion-negative RMS had a superior EFS to fusion-positive RMS; however, poorer outcome for metastatic RMS was most related to clinical risk factors including age, primary site, and number of metastatic sites. CONCLUSIONS: In contrast to low- or intermediate-risk RMS, in metastatic RMS, clinical risk factors have the most impact on patient outcome. PAX-FOXO1 fusion is more common in patients with a high Oberlin score, but fusion status is not an independent biomarker of prognosis.


Assuntos
Fusão Gênica , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Adolescente , Criança , Pré-Escolar , Intervalo Livre de Doença , Humanos , Lactente , Metástase Neoplásica , Rabdomiossarcoma Alveolar/mortalidade , Fatores de Risco , Adulto Jovem
13.
Sci Rep ; 6: 33429, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27642091

RESUMO

Mass spectrometry-based methods have been widely applied - often as the sole method - to detect mutations in human cancer specimens. We applied this approach to 52 childhood soft tissue sarcoma specimens in an attempt to identify potentially actionable mutations. This analysis revealed that 25% of the specimens harbored high-confidence calls for mutated alleles, including a mutation encoding FLT3(I836M) that was called in four cases. Given the surprisingly high frequency and unusual nature of some of the mutant alleles, we carried out ultra-deep next generation sequencing to confirm them. We confirmed only three mutations, which encoded NRAS(A18T), JAK3(V722I) and MET(R970C) in three specimens. Beyond highlighting those mutations, our findings demonstrate potential pitfalls of primarily utilizing a mass spectrometry-based approach to broadly screen for DNA sequence variants in archived, clinical-grade tumor specimens. Duplicate mass spectrometric analyses and confirmatory next generation sequencing can help diminish false positive calls, but this does not ameliorate potential false negatives due in part to evaluating a limited panel of sequence variants.


Assuntos
Espectrometria de Massas/métodos , Oncologia , Mutação/genética , Sarcoma/genética , Sequência de Bases , Linhagem Celular Tumoral , Criança , DNA de Neoplasias/metabolismo , GTP Fosfo-Hidrolases/genética , Frequência do Gene/genética , Humanos , Proteínas de Membrana/genética
14.
Pediatr Blood Cancer ; 63(9): 1557-62, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27198935

RESUMO

BACKGROUND: Rhabdomyosarcoma (RMS) is a rare, highly malignant tumor arising from primitive mesenchymal cells that differentiate into skeletal muscle. Relatively little is known about RMS susceptibility. Based on growing evidence regarding the role of early immunologic challenges on RMS development, we evaluated the role of infections and immunizations on this clinically significant pediatric malignancy. PROCEDURE: RMS cases (n = 322) were enrolled from the third trial coordinated by the Intergroup Rhabdomyosarcoma Study Group. Population-based controls (n = 322) were pair matched to cases on race, sex, and age. The following immunizations were assessed: diphtheria, pertussis, and tetanus (DPT); measles, mumps, and rubella; and oral polio vaccine. We also evaluated if immunizations were complete versus incomplete. We examined selected infections including chickenpox, mumps, pneumonia, scarlet fever, rubella, rubeola, pertussis, mononucleosis, and lung infections. Conditional logistic regression models were used to calculate an odds ratio (OR) and 95% confidence interval (CI) for each exposure, adjusted for maternal education and total annual income. RESULTS: Incomplete immunization schedules (OR = 5.30, 95% CI: 2.47-11.33) and incomplete DPT immunization (OR = 1.56, 95% CI: 1.06-2.29) were positively associated with childhood RMS. However, infections did not appear to be associated with childhood RMS. CONCLUSIONS: This is the largest study of RMS to date demonstrating a possible protective effect of immunizations against the development of childhood RMS. Further studies are needed to validate our findings. Our findings add to the growing body of literature, suggesting a protective role of routine vaccinations in childhood cancer and specifically in childhood RMS.


Assuntos
Infecção/complicações , Rabdomiossarcoma/prevenção & controle , Vacinação , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Rabdomiossarcoma/etiologia
15.
Cancer Genet ; 209(5): 182-94, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27132463

RESUMO

Sarcomas are a rare subgroup of pediatric cancers comprised of a variety of bone and soft-tissue tumors. While significant advances have been made in improving outcomes of patients with localized pediatric sarcomas since the addition of systemic chemotherapy to local control many decades ago, outcomes for patients with metastatic and relapsed sarcoma remain poor with few novel therapeutics identified to date. With the advent of new technologies to study cancer genomes, transcriptomes and epigenomes, our understanding of sarcoma biology has improved tremendously in a relatively short period of time. However, much remains to be accomplished in this arena especially with regard to translating all of this new knowledge to the bedside. To this end, a meeting was convened in Philadelphia, PA, on April 18, 2015 sponsored by the QuadW foundation, Children's Oncology Group and CureSearch for Children's Cancer that brought together sarcoma clinicians and scientists from North America to review the current state of pediatric sarcoma biology and ongoing/planned genomics based clinical trials in an effort to identify and bridge knowledge gaps that continue to exist at present. At the conclusion of the workshop, three key objectives that would significantly further our understanding of sarcoma were identified and a proposal was put forward to develop an all-encompassing pediatric sarcoma biology protocol that would address these specific needs. This review summarizes the proceedings of the workshop.


Assuntos
Sarcoma/genética , Pesquisa Médica Translacional , Animais , Protocolos Clínicos , Avaliação Pré-Clínica de Medicamentos , Epigenômica , Genômica , Mutação em Linhagem Germinativa , Humanos , Medicina de Precisão/tendências , Recidiva , Sarcoma/patologia , Sarcoma/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Sci Rep ; 6: 20376, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26847534

RESUMO

The transition from a committed progenitor cell to one that is actively differentiating represents a process that is fundamentally important in skeletal myogenesis. Although the expression and functional activation of myogenic regulatory transcription factors (MRFs) are well known to govern lineage commitment and differentiation, exactly how the first steps in differentiation are suppressed in a proliferating myoblast is much less clear. We used cultured mammalian myoblasts and an RNA interference library targeting 571 kinases to identify those that may repress muscle differentiation in proliferating myoblasts in the presence or absence of a sensitizing agent directed toward CDK4/6, a kinase previously established to impede muscle gene expression. We identified 55 kinases whose knockdown promoted myoblast differentiation, either independently or in conjunction with the sensitizer. A number of the hit kinases could be connected to known MRFs, directly or through one interaction node. Focusing on one hit, Mtor, we validated its role to impede differentiation in proliferating myoblasts and carried out mechanistic studies to show that it acts, in part, by a rapamycin-sensitive complex that involves Raptor. Our findings inform our understanding of kinases that can block the transition from lineage commitment to a differentiating state in myoblasts and offer a useful resource for others studying myogenic differentiation.


Assuntos
Desenvolvimento Muscular , Proteínas Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/metabolismo , Miogenina/metabolismo , Piperazinas/farmacologia , Análise Serial de Proteínas , Proteínas Quinases/química , Proteínas Quinases/genética , Piridinas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína Regulatória Associada a mTOR , Análise de Sequência de RNA , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética
17.
Pediatr Blood Cancer ; 63(4): 634-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26756883

RESUMO

BACKGROUND: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) is of prognostic and therapeutic importance. Criteria for classifying these entities evolved significantly from 1995 to 2013. ARMS is associated with inferior outcome; therefore, patients with alveolar histology have generally been excluded from low-risk therapy. However, patients with ARMS and low-risk stage and group (Stage 1, Group I/II/orbit III; or Stage 2/3, Group I/II) were eligible for the Children's Oncology Group (COG) low-risk rhabdomyosarcoma (RMS) study D9602 from 1997 to 1999. The characteristics and outcomes of these patients have not been previously reported, and the histology of these cases has not been reviewed using current criteria. PROCEDURE: We re-reviewed cases that were classified as ARMS on D9602 using current histologic criteria, determined PAX3/PAX7-FOXO1 fusion status, and compared these data with outcome for this unique group of patients. RESULTS: Thirty-eight patients with ARMS were enrolled onto D9602. Only one-third of cases with slides available for re-review (11/33) remained classified as ARMS by current histologic criteria. Most cases were reclassified as ERMS (17/33, 51.5%). Cases that remained classified as ARMS were typically fusion-positive (8/11, 73%), therefore current classification results in a similar rate of fusion-positive ARMS for all clinical risk groups. In conjunction with data from COG intermediate-risk treatment protocol D9803, our data demonstrate excellent outcomes for fusion-negative ARMS with otherwise low-risk clinical features. CONCLUSIONS: Patients with fusion-positive RMS with low-risk clinical features should be classified and treated as intermediate risk, while patients with fusion-negative ARMS could be appropriately treated with reduced intensity therapy.


Assuntos
Rabdomiossarcoma Alveolar/classificação , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Estimativa de Kaplan-Meier , Masculino , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma Embrionário/classificação , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/patologia
18.
Exp Eye Res ; 145: 224-229, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26778750

RESUMO

Arf encodes an important tumor suppressor, p19(Arf), which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19(Arf) expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfß, despite being isolated at a time when Tgfß has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrß as expression of Arf represses expression of Pdgfrß mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrß protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19(Arf) does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19(Arf) functions to inhibit PDGF-B driven proliferation ex vivo.


Assuntos
Proliferação de Células/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas Proto-Oncogênicas c-sis/fisiologia , Doenças Retinianas/fisiopatologia , Corpo Vítreo/citologia , Animais , Western Blotting , Ciclo Celular/fisiologia , Células Cultivadas , Camundongos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Supressora de Tumor p53 , Corpo Vítreo/efeitos dos fármacos
19.
Nat Commun ; 6: 8891, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26573325

RESUMO

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Sarcoma de Células Claras/genética , Estudos de Casos e Controles , Pré-Escolar , Feminino , Duplicação Gênica , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Lactente , Masculino , Mutação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Análise de Sequência de RNA , Sequências de Repetição em Tandem
20.
Clin Cancer Res ; 21(20): 4733-9, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26473193

RESUMO

PURPOSE: Pediatric rhabdomyosarcoma (RMS) has two common histologic subtypes: embryonal (ERMS) and alveolar (ARMS). PAX-FOXO1 fusion gene status is a more reliable prognostic marker than alveolar histology, whereas fusion gene-negative (FN) ARMS patients are clinically similar to ERMS patients. A five-gene expression signature (MG5) previously identified two diverse risk groups within the fusion gene-negative RMS (FN-RMS) patients, but this has not been independently validated. The goal of this study was to test whether expression of the MG5 metagene, measured using a technical platform that can be applied to routine pathology material, would correlate with outcome in a new cohort of patients with FN-RMS. EXPERIMENTAL DESIGN: Cases were taken from the Children's Oncology Group (COG) D9803 study of children with intermediate-risk RMS, and gene expression profiling for the MG5 genes was performed using the nCounter assay. The MG5 score was correlated with clinical and pathologic characteristics as well as overall and event-free survival. RESULTS: MG5 standardized score showed no significant association with any of the available clinicopathologic variables. The MG5 signature score showed a significant correlation with overall (N = 57; HR, 7.3; 95% CI, 1.9-27.0; P = 0.003) and failure-free survival (N = 57; HR, 6.1; 95% CI, 1.9-19.7; P = 0.002). CONCLUSIONS: This represents the first, validated molecular prognostic signature for children with FN-RMS who otherwise have intermediate-risk disease. The capacity to measure the expression of a small number of genes in routine pathology material and apply a simple mathematical formula to calculate the MG5 metagene score provides a clear path toward better risk stratification in future prospective clinical trials.


Assuntos
Proteínas de Fusão Oncogênica/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Embrionário/genética , Transcriptoma/genética , Adolescente , Adulto , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lactente , Masculino , Fatores de Transcrição Box Pareados/genética , Prognóstico , Fatores de Risco , Adulto Jovem
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA