Doublecortin undergo nucleocytoplasmic transport via the RanGTPase signaling to promote glioma progression

Abiola Abdulrahman Ayanlaja,Bo Cheng,Dianshuai Gao,Jie Wang,Guanquan Ji,Yue Gao,Lin Zhang,Ye Xiong,Kouminin Kanwore,Piniel Alphayo Kambey

doi:10.1186/s12964-019-0485-5

Abstract

BackgroundNuclear translocation of several oncogenic proteins have previously been reported, but neither the translocation of doublecortin (DCX) nor the mechanism involved has been studied. DCX is a neuronal microtubule-associated protein (MAP) that is crucial for adult neurogenesis and neuronal migration and has been associated with poor prognosis in gliomas.MethodsWe probed DCX expression in different grades of glioma tissues and conventional cells via western blotting. Then we analyzed the expression pattern in the Oncomine cancer profiling database. Confocal Immunofluorescence was used to detect DCX expression in the cellular compartments, while subcellular fractionation was probed via western blotting. Pulse shape height analysis was utilized to verify DCX localization in a larger population of cells. Co-immunoprecipitation was used in detecting DCX-import receptors interactions. To probe for DCX functions, stable cells expressing high DCX expression or knockdown were generated using CRISPR-Cas9 viral transfection, while plasmid site-directed mutant constructs were used to validate putative nuclear localization sequence (NLS) predicted via conventional algorithms and comparison with classical NLSs. in-silico modeling was performed to validate DCX interactions with import receptors via the selected putative NLS. Effects of DCX high expression, knockdown, mutation, and/or deletion of putative NLS sites were probed via Boyden’s invasion assay and wound healing migration assays, and viability was detected by CCK8 assays in-vitro, while xenograft tumor model was performed in nude mice.ResultsDCX undergoes nucleocytoplasmic movement via the RanGTPase signaling pathway with an NLS located on the N-terminus between serine47-tyrosine70. This translocation could be stimulated by MARK’s phosphorylation of the serine 47 residue flanking the NLS due to aberrant expression of glial cell line-derived neurotrophic factor (GDNF). High expression and nuclear accumulation of DCX improve invasive glioma abilities in-vitro and in-vivo. Moreover, knocking down or blocking DCX nuclear import attenuates invasiveness and proliferation of glioma cells.ConclusionCollectively, this study highlights a remarkable phenomenon in glioma, hence revealing potential glioma dependencies on DCX expression, which is amenable to targeted therapy.7MPie3i8fR4ak4tLMhQm1SVideo abstractGraphical

Highlights

Nuclear translocation of several oncogenic proteins have previously been reported, but neither the translocation of doublecortin (DCX) nor the mechanism involved has been studied
DCX expression was observed in grades II glioma tissues in the cohort of glioma samples probed with western blotting (WB) under similar condition, slightly higher than the non-neoplastic tissues, but significantly lower compared to high-grade gliomas (III and IV GBM tissues) (Fig. 1a)
Nuclear import of DCX contributes to glioma development Since DCX nuclear accumulation only occurs in glioma cells, we investigated the effects of DCX nucleocytoplasmic transport on glioma progression

Summary

Introduction

Nuclear translocation of several oncogenic proteins have previously been reported, but neither the translocation of doublecortin (DCX) nor the mechanism involved has been studied. There is increasing evidence that blocking the transport machinery of molecules into or out of the nuclear compartment may either attenuate or promote cancer progression [1]. It is becoming increasingly apparent since specific cancer-related proteins, such as the tumor suppressors p53 [4], retinoblastoma protein (Rb) [5], the paracrine/autocrine signaling molecule parathyroid hormone-related protein (PTHrP) [3], CD44 [6], and EGFR [7], have been reported to transport through the nuclear pore to either activate or deactivate relevant signaling cascades. Only a few prooncogenic molecules are known for nucleocytoplasmic movements

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