Functional Study Of The Receptor Tyrosine Phosphatase PTPRU And The Serine Kinase CDKL5 In Gliomas

PTPRU(receptor-type protein tyrosine phosphatase U)belongs to the R2B subfamily of protein phosphatase superfamily.Full-length PTPRU is composed of a MAM domain,an Ig-like domain,three FN? domains and two phosphatase domains.Full-length PTPRU functions as a tumor suppressor in certain cancer cell types.It promotes cell aggregation and inhibits cell spreading through hemophilic binding and suppresses cell proliferation and adhesion through tyrosine dephosphorylation of P-catenin and inhibition of subsequent downstream signaling.However,the expression and function of endogenous PTPRU in cancers,including malignant glioma,remains unknown.In the present study,we find that the expression of full-length PTPRU in glioma samples is relatively low when compared with abundant non-full-length PTPRU isoforms.One of these isoforms is particularly localized in the nucleus and ubiquitously expressed in several types of cancer cell lines and its expression is positively correlated with malignancy grade of glioma.Short hairpin RNA knockdown of endogeneous PTPRU in human and rat glioblastoma cell lines results in the suppression of proliferation,survival,cell cycle progression,invasion,migration,matrix-independent adhesion and vasculogenic mimicry in vitro,as well as intracranial tumor growth in vivo.Consistently,expression of proteins associated with cell cycle regulation,apoptosis resistance and cell motility is alltered following PTPRU knockdown.Both full-length and non-full-length PTPRU isoforms interact with ?-catenin in glioblastoma cells.Knockdown of PTPRU downregulates tyrosine phosphorylation of ?-catenin and inhibits ?-catenin/TCF-4 ineraction,transcriptional activity of ?-catenin/TCF4/LEF1 complex and expression of several target genes.Expression of a constitutive active LEF1/?-catenin fusion protein partly rescues ?-catenin transcriptional activity and cell migration in PTPRU-depleted cells.In addition,knockdown of PTPRU led to the increase of tyrosine phosphorylation of E3 ubiquitin ligase c-Cbl,c-Cbl/FAK interaction and FAK ubiquitination,which probably contributes to the destabilization of FAK and other focal adhesion proteins.Taken together,our findings demonstrate that endogeneous PTPRU is required for glioma growth and motility through regulating ?-catenin and focal adhesion signaling,which provides novel insight into mechanisms that can transform an anti-cancerous receptor phosphatase to an oncoprotein.Mutation in the CDKL5(cyclin-dependent kinase-like 5)gene is associated with severe neuronal developmental disorders,such as Rett syndrome accompanied by early onset of epilepsy or infantile spasm,and the function and underlying mechanism of CDKL5 protein in neuron have been broadly studied.CDKL5 is shown to inhibit proliferation and induce neuronal differentiation of neuroblastoma cells and its transcription is repressed by MYCN.In this study,we find that the expression of CDKL5 in neuronal and non-neuronal cells is different.The full-length CDKL5 is specifically expressed in neuron and neuroblastoma cells,whereas a nuclear localized CDKL5 isoform is exclusively expressed in glioblastoma cells and absent in neuron and glia.Knockdown of CDKL5 using specific short hairpin RNA inhibits glioblastoma cell proliferation,survival,migration and invasion in vitro and subcutaneous glioma growth in vivo.CDKL5 is highly expressed in glioblastoma stem-like cells enriched using serum-free neural stem cell medium.Glioblastoma cells lose stem-like properties and undergo multi-directional differentiation upon CDKL5 knockdown,as revealed by the tumor sphere formation assay and western blot analysis of glioma differentiation markers.Epigenetic marks involved in cell fate choice regulation are altered as well.CDKL5 depletion reduces the trimethylation of H3K9 and expression of Dnmtl and increases the dimethylation of H3K4,acetylation of H3K9 and expression of MeCP2.Furthermore,knockdown of CDKL5 enhances TRAIL-stimulated caspase-dependent apoptosis of U87 and A172 glioblastoma cells with upregulated DR5 expression but has no effect on U251 cells without DR5 upregulation and with significant Bcl-2 phosphorylation.In conclusion,these findings demonstrate that CDKL5 is important to maintain undifferentiated state and aggressiveness of glioblastoma cells.