ABSTRACT
In neuronal cells, actin remodeling plays a well known role in neurite extension but is also deeply involved in the organization of intracellular structures, such as the Golgi apparatus. However, it is still not very clear which mechanisms may regulate actin dynamics at the different sites. In this report we show that high levels of the TTC3 protein, encoded by one of the genes of the Down Syndrome Critical Region (DCR), prevent neurite extension and disrupt Golgi compactness in differentiating primary neurons. These effects largely depend on the capability of TTC3 to promote actin polymerization through signaling pathways involving RhoA, ROCK, CIT-N and PIIa. However, the functional relationships between these molecules differ significantly if considering the TTC3 activity on neurite extension or on Golgi organization. Finally, our results reveal an unexpected stage-dependent requirement for F-actin in Golgi organization at different stages of neuronal differentiation.
Subject(s)
Actins/metabolism , Cell Differentiation/physiology , Golgi Apparatus/metabolism , Neurons/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases/metabolism , Actin Cytoskeleton/metabolism , Animals , Cells, Cultured , Hippocampus/metabolism , Neurites/metabolism , Rats , Ubiquitin-Protein Ligases/geneticsABSTRACT
Anaplastic lymphoma kinase (ALK) is constitutively activated in a number of human cancer types due to chromosomal translocations, point mutations, and gene amplification and has emerged as an excellent molecular target for cancer therapy. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity and cellular ALK tyrosine phosphorylation. It induced concentration-dependent growth inhibition/cytotoxicity of ALK-positive anaplastic large-cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cells, and displayed dose-dependent inhibition of ALK tyrosine phosphorylation in tumor xenografts in mice, with substantial target inhibition (>90%) for more than 12 hours following single oral dosing at 30 mg/kg. Dose-dependent antitumor activity was observed in ALK-positive ALCL, NSCLC, and neuroblastoma tumor xenografts in mice administered CEP-28122 orally, with complete/near complete tumor regressions observed following treatment at doses of 30 mg/kg twice daily or higher. Treatment of mice bearing Sup-M2 tumor xenografts for 4 weeks and primary human ALCL tumor grafts for 2 weeks at 55 or 100 mg/kg twice daily led to sustained tumor regression in all mice, with no tumor reemergence for more than 60 days postcessation of treatment. Conversely, CEP-28122 displayed marginal antitumor activity against ALK-negative human tumor xenografts under the same dosing regimens. Administration of CEP-28122 was well tolerated in mice and rats. In summary, CEP-28122 is a highly potent and selective orally active ALK inhibitor with a favorable pharmaceutical and pharmacokinetic profile and robust and selective pharmacologic efficacy against ALK-positive human cancer cells and tumor xenograft models in mice.
Subject(s)
Antineoplastic Agents/pharmacology , Benzocycloheptenes/pharmacology , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Xenograft Model Antitumor Assays , Administration, Oral , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Benzocycloheptenes/chemistry , Biological Availability , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Female , Humans , Immunoblotting , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, Nude , Mice, SCID , Molecular Structure , Neoplasms/metabolism , Neoplasms/pathology , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Neuroblastoma/pathology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/chemistry , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Acid sphingomyelinase (ASM) converts sphingomyelin (SM) into ceramide. Mutations in the ASM gene cause the mental retardation syndrome Niemann Pick type A (NPA), characterized as a lysosomal disorder because of the SM accumulation in these organelles. We here report that neurons from mice lacking ASM (ASMKO) present increased plasma membrane SM levels evident in detergent-resistant membranes. Paralleling this lipidic alteration, GPI-anchored proteins show an aberrant distribution in both axons and dendrites instead of the axonal enrichment observed in neurons from wild-type mice. Trafficking analysis suggests that this is due to defective internalization from dendrites. Increasing the SM content in wild-type neurons mimics these defects, whereas SM reduction in ASMKO neurons prevents their occurrence. Moreover, expression of active RhoA, which membrane attachment is affected by SM accumulation, rescues internalization rates in ASMKO neurons. These data unveil an unexpected role for ASM in neuronal plasma membrane organization and trafficking providing insight on the molecular mechanisms involved. They also suggest that deficiencies in such processes could be key pathological events in NPA disease.
