RESUMO
The forkhead transcription factor FOXP1 is generally regarded as an oncogene in activated B cell-like diffuse large B-cell lymphoma. Previous studies have suggested that a small isoform of FOXP1 rather than full-length FOXP1, may possess this oncogenic activity. Corroborating those studies, we herein show that activated B cell-like diffuse large B-cell lymphoma cell lines and primary activated B cell-like diffuse large B-cell lymphoma cells predominantly express a small FOXP1 isoform, and that the 5'-end of the Foxp1 gene is a common insertion site in murine lymphomas in leukemia virus- and transposon-mediated insertional mutagenesis screens. By combined mass spectrometry, (quantative) reverse transcription polymerase chain reaction/sequencing, and small interfering ribonucleic acid-mediated gene silencing, we determined that the small FOXP1 isoform predominantly expressed in activated B cell-like diffuse large B-cell lymphoma lacks the N-terminal 100 amino acids of full-length FOXP1. Aberrant overexpression of this FOXP1 isoform (ΔN100) in primary human B cells revealed its oncogenic capacity; it repressed apoptosis and plasma cell differentiation. However, no difference in potency was found between this small FOXP1 isoform and full-length FOXP1. Furthermore, overexpression of full-length FOXP1 or this small FOXP1 isoform in primary B cells and diffuse large B-cell lymphoma cell lines resulted in similar gene regulation. Taken together, our data indicate that this small FOXP1 isoform and full-length FOXP1 have comparable oncogenic and transcriptional activity in human B cells, suggesting that aberrant expression or overexpression of FOXP1, irrespective of the specific isoform, contributes to lymphomagenesis. These novel insights further enhance the value of FOXP1 for the diagnostics, prognostics, and treatment of diffuse large B-cell lymphoma patients.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ativação Transcricional , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/química , Humanos , Memória Imunológica , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Mutagênese Insercional , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Isoformas de Proteínas , Proteínas Repressoras/químicaRESUMO
Chemokine-controlled migration plays a critical role in B-cell development, differentiation, and function, as well as in the pathogenesis of B-cell malignancies, including the plasma cell neoplasm multiple myeloma (MM). Here, we demonstrate that stimulation of B cells and MM cells with the chemokine stromal cell-derived factor-1 (SDF-1) induces strong migration and activation of the Ras-like GTPase Ral. Inhibition of Ral, by expression of the dominant negative RalN28 mutant or of RalBPDeltaGAP, a Ral effector mutant that sequesters active Ral, results in impaired SDF-1-induced migration of B cells and MM cells. Of the 2 Ral isoforms, RalA and RalB, RalB was found to mediate SDF-1-induced migration. We have recently shown that Btk, PLCgamma2, and Lyn/Syk mediate SDF-1-controlled B-cell migration; however, SDF-1-induced Ral activation is not affected in B cells deficient in these proteins. In addition, treatment with pharmacological inhibitors against PI3K and PLC or expression of dominant-negative Ras did not impair SDF-1-induced Ral activation. Taken together, these results reveal a novel function for Ral, that is, regulation of SDF-1-induced migration of B cells and MM cells, thereby providing new insights into the control of B-cell homeostasis, trafficking, and function, as well as into the pathogenesis of MM.
Assuntos
Linfócitos B/metabolismo , Movimento Celular , Quimiocina CXCL12/metabolismo , Leucemia Plasmocitária/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Quimiocina CXCL12/farmacologia , Galinhas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Genes Dominantes/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Plasmocitária/genética , Leucemia Plasmocitária/patologia , Mutação , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Proteínas ral de Ligação ao GTP/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis.