u-0126 and Lymphoma--Large-Cell--Anaplastic

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

Topics: Butadienes; Enzyme Inhibitors; Gene Knockdown Techniques; Hodgkin Disease; Humans; Jurkat Cells; K562 Cells; Ki-1 Antigen; Lymphoma, Large-Cell, Anaplastic; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Nitriles; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins c-jun

Abnormal expression of anaplastic lymphoma kinase (ALK) gene is an important pathogenic factor for anaplastic large cell lymphoma (ALCL). To study the function of ALK, an inducible short hairpin RNA (shRNA) system was stably introduced into cultured human ALCL cells. Inducing shRNA expression in the generated cells resulted in cellular ALK gene silencing and led to inactivation of multiple signaling pathways and growth arrest. Interestingly, a combination of ALK gene silencing with U0126, a kinase inhibitor specific for the extracellular signal-regulated kinases 1/2 pathway, resulted in an augmented reduction in cellular JunB expression. Functional studies indicated that combining ALK gene silencing with U0126 treatment provided a synergistic growth inhibition, which occurred faster and was more profound than with either treatment alone. This synergistic effect was also observed when measuring cell proliferation, apoptosis, and in vitro cell colony formation. Importantly, the combination of ALK gene silencing and U0126 had a prolonged inhibitory effect, preventing recovery of ALCL cell growth even after treatments were removed. Moreover, this synergistic inhibitory effect was confirmed in vivo using a mouse model with xenografted ALCL tumors. Our findings indicate that combining cellular ALK gene silencing with a low dose of U0126 may prove to be an effective and more specific therapeutic approach to treating ALCL.

Topics: Anaplastic Lymphoma Kinase; Animals; Apoptosis; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Combined Modality Therapy; Drug Synergism; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Gene Silencing; Humans; Lymphoma, Large-Cell, Anaplastic; Mice; Mice, Inbred BALB C; Mice, Nude; Nitriles; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; RNA, Small Interfering; Xenograft Model Antitumor Assays

Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL.. The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot.. Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin.. Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

Topics: Anaplastic Lymphoma Kinase; Apoptosis; Benzoquinones; Butadienes; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; G1 Phase; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lymphoma, Large-Cell, Anaplastic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Resting Phase, Cell Cycle; Time Factors