The Effect And Mechanism Of Netrin-1 In Promoting Glioma Growth

Glioma is a most common and fatal malignant type of brain tumors.It is characterized by unregulated growth and aggressive infiltration,making it have no clear boundary between glioma and normal brain tissue,which is difficult to cure surgically.Based on the histological properties of gliomas,they can be classified into four grades,I,II,III,and IV,according to the World Health Organization for the classification of tumors of the central nervous system.Grade IV（gliomablastoma,GBM）is among the most malignant of glioma.The median prognosis for GBM is less than 15-20 months,with a 5-year survival of patients of no more than 5%.Recent studies have shown that,a variety of genetic abnormalities and aberrant cell signaling pathways have been implicated in glioma tumorigenesis,including dysfunction of p53,amplification of VEGF,loss of PTEN and so on.However,very little information regarding glioma tumorigenesis is available.Netrin-1 is a member of the laminin-related family of matrix-binding secreted proteins.It was first described as a guidance molecule for neuronal axon pathway finding.Initial studies showed that netrin-1 could interact with its receptors to mediate the attraction and repulsion of neuronal axons.Recently,netrin-1 has also been proposed to regulate cell survival and modulate tumorigenesis.Howerver,studies of the role of netrin-1 in glioma are limited,and most of them have focused exclusively on cell motility in vitro.Very recently,netrin-1 had been reported to mediate glioblastoma cell metastasis and angiogenesis.However,it remains unknown whether netrin-1 promotes glioma proliferation in human and,if so,which netrin-1 receptors regulate this cellular event.In our studies,we firstly measured netrin-1 protein and mRNA levels expression in 16paired fresh glioma samples.These data demonstrated that netrin-1 expression was significantly upregulated in glioma.Meanwhile,the analysis in Oncomine database further confirmed the conclusion.To better understand the correlation of netrin-1 with glioma progression,62formalin-fixed paraffin-embedded glioma specimens and 166 paired recurrence glioma speximens were analyzed for netrin-1 staining by IHC.The average netrin-1 staining signal showed a steady increasing trend from grade I to grade IV,and its expression has a positively correlation with glioma malignancy.We next investigated the association of netrin-1 expression with several known glioma parameters.Our analysis exhibited an obvious positive correlation between netrin-1 and Ki-67 index,but has on correlation with other parameters.The Spearman correlation coefficient confirmed that there was a moderate correlation between netrin-1expression and Ki-67 index.Receiver operating characteristics（ROC）curves demonstrated elevated netrin-1 expression were found to be independent factors for distinguishing low-grade gliomas and high-grade gliomas,suggesting that netrin-1 acting as a potential diagnostic marker for glioma.Secondly,suppressing netrin-1 expression reduced cell proliferation without affecting cell survival or apoptosis.Further studies have found that treatment with exogenous netrin-1enhanced glioma cell proliferation and activation of the NF-κB pathway,leading to the upregulation of c-Myc expression.To determine how netrin-1 initiated these cellular activities,we next examined the known netrin-1 receptors in glioma cells and clinical glioma specimens.We found that relatively high UNC5A expression was observed in the three glioma cell lines and glioma specimens,we hypothesized that UNC5A may be a key receptor involved in netrin-1-mediated tumorigenesis.Inhibition of UNC5A could not only abolish exogenous netrin-1-induced NF-κB p65^ser536 phosphorylation and c-Myc overexpression,but also abolish increase in netrin-1-induced cell proliferation.Lastly,we generated U251 cells that stably overexpressed the luciferase protein and stereotactically injected into the right striatum of recipient Balb/C nude mice.Through tracking the growth of tumor cells,we found the mice injected with shNetrin-1 cells exhibited relatively smaller tumor foci in the forehead.Then tumor sections were stained for Ki-67 and c-Myc expression by IHC,results showed that the number of Ki-67-positive cells and the expression of c-myc was significantly reduced in the shNetrin-1 tumors compared with the shCtrl tumors.Taken together,the results of our analyses reveal that netrin-1 is upregulated in glioma and plays an essential role in glioma proliferation.This effect is mediated by the UNC5A-NF-κB signaling pathway through activation of c-Myc expression to promote cell proliferation.