RESUMEN
Rhabdomyosarcoma is the most common pediatric soft-tissue sarcoma, which includes two major subtypes, alveolar and embryonal rhabdomyosarcoma. The mechanism of its oncogenesis is largely unknown. However, the oncogenic process of rhabdomyosarcoma involves multi-stages of signaling protein dysregulation characterized by prolonged activation of tyrosine and serine/threonine kinases. To better understand this protein dysregulation, we evaluated the phosphorylation profiles of multiple tyrosine and serine/threonine kinases to identify whether these protein kinases are activated in rhabdomyosarcoma. We applied immunohistochemistry with phospho-specific antibodies to examine phosphorylation levels of selected receptor and non-receptor tyrosine kinases, mammalian target of rapamycin (mTOR), p70S6K, and protein kinase C (PKC) isozymes on alveolar and embryonal rhabdomyosarcoma tissue microarray slides. Our results showed that the phosphorylation levels of these kinases are elevated in some rhabdomyosarcoma tissues compared to normal tissues. Phosphorylation levels of receptor and non-receptor tyrosine kinases are elevated between 26 and 68% in alveolar rhabdomyosarcoma and between 24 and 71% in embryonal rhabdomyosarcoma, respectively, compared to normal tissues. In addition, phosphorylation levels of mTOR and its downstream targets, p70S6K, S6, and 4EBP1, are increased between 50 and 72% in both subtypes of rhabdomyosarcoma. Further, phosphorylation levels of PKCalpha, PKCdelta, PKCtheta, and PKCzeta/lambda are upregulated between 57 and 69% in alveolar rhabdomyosarcoma and between 43 and 72% in embryonal rhabdomyosarcoma. This is the first report to create a phosphorylation profile of tyrosine and serine/threonine kinases involved in the mTOR and PKC pathways of alveolar and embryonal rhabdomyosarcoma. These protein kinases may play roles in the development or tumor progression of rhabdomyosarcomas and thus may serve as novel targets for therapeutic intervention.
Asunto(s)
Proteínas Serina-Treonina Quinasas/análisis , Proteínas Tirosina Quinasas/análisis , Rabdomiosarcoma Alveolar/enzimología , Rabdomiosarcoma Embrionario/enzimología , Proteínas Adaptadoras Transductoras de Señales/análisis , Adolescente , Proteínas de Ciclo Celular , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Isoenzimas/análisis , Masculino , Estadificación de Neoplasias , Fosfoproteínas/análisis , Fosforilación , Proyectos Piloto , Proteína Quinasa C/análisis , Proteínas Quinasas/análisis , Proteínas Tirosina Quinasas Receptoras/análisis , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/patología , Proteína S6 Ribosómica/análisis , Proteínas Quinasas S6 Ribosómicas 70-kDa/análisis , Serina-Treonina Quinasas TOR , Análisis de Matrices TisularesRESUMEN
The CBL ubiquitin ligase targets a variety of activated tyrosine kinases (TKs) for degradation. Many TKs are mutationally or autocrine activated and/or often overexpressed at the mRNA and protein levels in acute leukemias. We hypothesized that CBL is mutated in patients with acute myeloid leukemia (AML). Four of 12 patients and the MOLM-13 cell line harbored c-CBL mutations, either RNA splicing mutations, missense mutations, or a nucleotide insertion. Additionally, 1 of the 12 patients harbored a missense mutation in the related CBL-b gene. Each c-CBL mutation involves the structurally important alpha-helix within the linker region, while the mutation in CBL-b was located in the Ub-E2 protein-binding RING finger. Short-interfering RNA knockdown of mutant c-CBL present in MOLM-13 cells was growth inhibitory. In summary, novel mutations in c-CBL and CBL-b have been identified in human AML and may represent potential targets for novel therapeutics.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Leucemia Mieloide Aguda/genética , Mutación Missense , Proteínas Proto-Oncogénicas c-cbl/genética , Empalme del ARN/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Masculino , Estructura Secundaria de Proteína/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll(PTD/WT) mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll(PTD/WT) mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.