Abstract
Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy.
Highlights
Neuroblastoma (NB) is the most commonly diagnosed cancer in infants [1] and the most frequent solid extracranial neoplasm in children under five years of age [2,3]
In agreement with our previous observations in NB cell lines transiently transfected with synthetic siRNAs [9], these results indicate that high levels of DCL knockdown are required to reduce cell proliferation
In the present study we show that downregulation of the microtubule associated proteins (MAPs) DCL results in inhibition of neuroblastoma (NB) cell proliferation in vitro and in delayed NB tumor development in vivo
Summary
Neuroblastoma (NB) is the most commonly diagnosed cancer in infants [1] and the most frequent solid extracranial neoplasm in children under five years of age [2,3]. Great efforts have been done towards a more effective and less toxic therapy for NB. These efforts include the identification of novel molecular targets that play a crucial role in NB tumorigenic processes, such as proliferation, alternative energy metabolism, relative (acquired) resistance to apoptosis, angiogenesis and/or metastasis [6,7,8]. We have previously proposed the Doublecortin-like kinase (DCLK1) gene as an attractive molecular target for NB therapy [7,9]. DCLK-derived proteins belong to doublecortin (DCX) family and include the microtubule associated proteins (MAPs) DCLK-long and doublecortin-like (DCL). DCL and DCLK-long are highly expressed in neuroblasts and are vital for neuroblast proliferation, migration and differentiation [10,11]. Silencing of DCLK-derived MAPs results in cell-cycle arrest and apoptosis in NB cells [9,11]
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