ARL4C Stabilized By AKT/mTOR Pathway Promotes The Invasion Of PTEN-deficient Primary Human Glioblastoma

Primary human glioblastoma（GBM）is the most common and malignant astrocytic glioma,accounting for about 90%of all brain tumors with poor prognosis in particular for those with mutation/deficiency in PTEN（phosphatase and tensin homolog deleted on chromosome 10）,a tumor suppressor,occurring in 40%human primary GBM.GBM bearing PTEN mutation/deletion is associated with increased invasiveness and drug resistance.The correlation between PTEN deficiency and poor prognosis of GBM patients suggests the importance to investigate the underlying mechanism.ARL4（ADP-ribosylation factor like-4）family proteins,including ARL4A,ARL4C and ARL4D have been implicated in essential cellular functions,such as spreading and migration and also exploited by cancer cells to disseminate and invade surrounding tissues.Recently,the expression of ARL4D is found to be post-transcriptionally regulated by AKT/mTOR pathway and linked to the status of PTEN in human glioma.Upon transfection of ARL4D into GBM cell line U87-MG,cell behavior remains unchanged,suggesting that increase in ARL4D abundance has no effect on tumor progression.Alterations in ARL4A are more frequent in papillomas and atypical papillomas of choroid plexus tumors.ARL4C is involved in the migration,invasion and proliferation of cancer cells and is considered as a therapeutic target in colorectal cancer,lung cancer and leiomyosarcoma.Moreover,ARL4C functions as a suppressor gene to inhibit the progression of ovarian cancer.The precise role of ARL4 family proteins in PTEN-deficient GBM remains unclear.In the present study,we screened and identified ARL4C was crucial in the progression of PTEN-deficient human primary GBM cells among ARL4 family.Mechanistically,loss of PTEN stabilized ARL4C protein through AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination detected by protein turnover assays and CO-IP.Functionally,ARL4C enhanced the progression of GBM cells in vitro and vivo.Moreover,microarray profiling and GST-pull down assay identified that ARL4C accelerated tumor mobility via RAC1-mediated filopodium formation.Importantly,overexpressing PTEN potently inhibited GBM tumor progression in vitro and vivo,while ARL4C overexpression reversed the tumor progression.Clinically,analyses with patients’specimens validated a negative correlation between PTEN and ARL4C expression.Elevated ARL4C expression and PTEN deficiency in tumor were associated with poorer disease-free survival and overall survival of GBM patients.Taken together,ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate.The main methods,results and conclusions of this study are as follows:1.High expression of ARL4C in PTEN-deficient primary GBM is associated with poor patient prognosis.1.1 ARL4C is highly expressed in primary PTEN-deficient primary GBM.Western blotting revealed that ARL4C is correlated with PTEN status in primary GBM cells and tissues,whereas no correlation is observed for ARL4A or ARL4D.Immunohistochemistry staining and analysis of both PTEN and ARL4C in the specimens from 128 primary GBM revealed a significant inverse correlation between the levels of ARL4C and PTEN（P=0.021）.These results indicated that ARL4C is closely related with PTEN status in primary GBM.1.2 The relationship between the expression level of ARL4C and clinical pathological parameters.The cutoff value of the IHC score was determined by relative risk analysis with the statistical software X-tile.The cutoff value was used to divide patients with ARL4C^low and ARL4C^high.Chi-square test revealed that ARL4C was positively correlated with Ki67 index（P=0.020）.It was no correlation with patient age（P=0.534）,gender（P=0.912）,tumor location（P=0.100）,KPS score（P=0.623）and EGFR status（P=0.232）.Furthermore,no significant correlatioship was observed in ARL4C expression and MGMT methlyation status on 64 GBM patients.1.3 ARL4C in combination with PTEN could serve as a valuable prognostic biomarker set for primary GBM patients.Kaplan-Meier survival analysis using Cohort-128 showed significantly shorter over survival rate（OS）（P=0.0030）and progression free survival rate（PFS）（P=0.0025）for the patients with ARL4Chigh.Moreover,survival analysis on 372 primary GBM patients from the TCGA-database consistently indicated that ARL4Chigh but PTEN deficiency was associated with poor OS（P=0.0288）and PFS（P=0.0069）.Univariate and multivariate analyses further indicated that ARL4Chigh but PTENlow was an independent prognostic indicator（P=0.004;P=0.0011）for OS of GBM patients.These data suggested that ARL4C promotes PETN-deficient GBM invasion through RAC1-mediated filopodium formation.2.AKT/mTOR pathway maintains ARL4C stability in PTEN-deficient primary GBM2.1 PTEN regulated ARL4C expression via AKT/mTOR pathway.PTEN loss results in persistent signaling through the phosphorylation of AKT.To examine the relationship between PTEN deficiency and ARL4C in primary GBM,we overexpressed PTEN in PTEN-deficient GBM cells（GBM-1,GBM-2 and U87-MG）and knocked down PTEN from PTEN-intact GBM cells（GBM-3 and LN229）We also overexpressed ARL4C in GBM-3 and LN229 GBM cells Overexpression of PTEN in PTEN-deficient GBM cells reduced ARL4C and p-AKT^S473 levels in these cells,whereas there was no significant difference in PTEN expression when ARL4C was overexpressed in PTEN-intact GBM cells（Supplementary Figure 3C）.In addition,ARL4C and p-AKT^S473levels were significantly increased when PTEN was silenced in PTEN-intact GBM cells.These results suggest that ARL4C may be regulated by PTEN through the phosphorylation of AKT.Inhibitors of AKT（MK-2206）or mTOR（CCI-779）significantly decreased the expression of ARL4C in PTEN-deficient GBM cells.Similar results were obtained with GBM-3 and LN229 cells in which PTEN was silenced by shRNA.2.2 AKT/mTOR pathway maintains ARL4C stability in GBM cells.No change in mRNA level of ARL4C in GBM treated with or without MK2206 or CCI-779,suggesting that the regulation of AKT/mTOR is dependent on the post-translational level.To examine the capacity of AKT/mTOR to stabilize ARL4C protein,we used cycloheximide（CHX）to measure the half-life of ARL4C in GBM cells upon inactivation of AKT or mTOR.Inhibition of AKT decreased the half-life of ARL4C after treatment with CHX.Similar results were observed when mTOR was inactivated by CCI-779 during treatment with CHX.Inactivation of AKT/mTOR pathway appears to destabilize ARL4C protein.The ubiquitination is a major reason in destabilization of proteins.To test the involvement of ubiquitination,we used a proteasome inhibitor MG-132 and found a full recovery of basal expression of ARL4C levels resulted from the inhibition by AKT inhibitor MK-2206 or mTOR inhibitor CCI-779.The ubiquitinated forms of ARL4C were increased in the presence of AKT inhibitor MK-2206 or mTOR inhibitor CCI-779 indicating AKT/mTOR signaling pathway is involved in the proteasomal degradation of ARL4C in GBM cells.Taken together,these data suggested AKT/mTOR pathway maintains ARL4C stability in PTEN-deficient GBM cells.3.ARL4C is crucial in the progression of PTEN deficient primary GBM.3.1 PTEN/ARL4C axis plays an important role in the progression of primary GBM.When overexpressed PTEN and/or ARL4C in PTEN-deficient GBM cells（GBM-1,GBM-2 and U87-MG）,we found that ARL4C increased GBM cell mobility and partially overcame the inhibitory effects of PTEN on tumor cell invasion and migration.Furthermore,bioluminescent analyses indicated that ARL4C overexpression partially compromised the inhibitory effects of PTEN overexpression on GBM growth in vivo.H&E staining and the analysis of mean invasive depth showed that ARL4C overexpression markedly increased the tumor invasiveness which was reduced by PTEN overexpression.Consequently,the survivals of mice bearing the xenografts overexpressing PTEN and ARL4C were markedly reduced as compared to those bearing the tumor overexpressing PTEN alone.These results indicate that PTEN loss promotes the progression of GBM through increasing ARL4C expression.3.2 Knock down or over express of ARL4C markedly changed the invasiveness and mobility of GBM cell in vitro.Knockdown of ARL4C in PTEN-deficient GBM cells（GBM-1,GBM-2 and U87-MG）reduced their mobility.Meanwhile,overexpression of ARL4C in PTEN-intact GBM cells（GBM-3 and LN229）significantly increased the cell mobility.3.3 ARL4C promotes tumor growth in 3D culture.In a 3D culture model,knock down ARL4C decreased proliferation of GBM cells in vitro,which is correlated with Ki67-index in PTEN-deficient GBM.Furthermore,xenografted tumors formed by ARL4C-knockdown GBM cells showed reduced proliferation as indicated by Ki67 staining.3.4 Disruption of ARL4C inhibits the tumorigenesis of PTEN-deficient GBM cells and prolongs the survival the mice bearing tumor.Bioluminescent imaging in those mice detected smaller tumors formed by shARL4C1and shARL4C2 GBM cells as compared to shCtrl ones.The survival of mice bearing tumors formed by sh ARL4C1 GBM cells and sh ARL4C2 ones was longer than that of those implanted with shCtrl ones.Histology revealed the decreased invasiveness of tumors formed by shARL4C2 cells.We then evaluated the depth of tumor invasion from the border of the tumor mass to the invaded cells and found that the invasiveness was remarkably decreased in the xenograft formed by shARL4C GBM cells as compared with that by shCtrl ones.These data suggested ARL4C is crucial in the progression of GBM4.ARL4C promotes PETN-deficient GBM invasion through RAC1-mediated filopodium formation4.1 ARL4C is correlated with invasive filopodium formation.We examined the filopodia of GBM-1 cells and found that filopodium number in ARL4C-overexpressing GBM-1 cells was markedly increased,while in sh ARL4C1 GBM-1cells the filopodium numbers were fewer.4.2 ARF6/RAC1 pathway is the main pathway of ARL4C-mediated filopodium formation.Gene chip assay revealed that ARF6,a protein recruits RAC GEFβ-PIX to activate RAC1,was the most differentially expressed in shctrl and shARL4C GBM cells in actin filaments gene set from GSEA and western blot confirme the result at protein level.We then evaluated the effect of ARL4C on RAC1 activity and found that in contrast to the consequence of overexpressing ARL4C knockdown of ARL4C inhibited RAC1 pathway.4.3 ARL4C/RAC1 axis was crucial in the filopodia formation.Treatment with 25μM NSC23766,a RAC1 inhibitor,markedly reduced RAC1 activity without changing total RAC1 level and cell growth.In addition,NSC32766 reversed the effect of ARL4C on GBM cell invasion and migration（Figure 5F）.Furthermore,the inhibition reduced the percentage of filopodium positive population in OE-ARL4C GBM-1cell.Thus,RAC1 appears to be a downstream molecule for ARL4C to regulate the formation of filopodia in PTEN-deficient GBM cells and their invasiveness.These data suggested that ARL4C promotes PETN-deficient GBM invasion through RAC1-mediated filopodium formation.Conclusion1.ARL4C was correlated with PTEN status in primary GBM among ARL4 family2.PTEN destabilized ARL4C expression via AKT/mTOR pathway.3.PTEN/ARL4C axis was crucial in the progression of GBM cell in vitro and vivo.4.ARL4C in combination with PTEN could serve as a valuable prognostic biomarker set for primary GBM patients.