KAP Regulates ROCK2 And Cdk2 In An RNA-Activated Glioblastoma Invasion Pathway

Research BackgroundGlioblastoma is the most common and most malignant intrinsic brain tumor in adults. At present, it is incurable. Despite maximal treatment, the median survival for glioblastoma patients remains 14 to 16 months. Although surgery is an important part of the therapeutic strategy for glioblastoma, these tumors cannot be cured surgically because of extensive invasion of the surrounding normal brain by tumor cells. Numerous investigations have examined the mechanisms underlying glioblastoma cell invasion. Receptor tyrosine kinase-activated signaling pathways, extracellular matrix molecules and other mechanisms have all been shown to play a role in this process.We recently reported the existence of a Cdc2-dependent invasion pathway in glioblastoma that is regulated by the cyclin-dependent kinase-associated phosphatase, KAP. KAP is a dual specificity phosphatase that dephosphorylates Cdk2,thereby inhibiting Cdk2 activity and cell cycle progression. In breast, prostate and liver cancers, KAP is overexpressed and reportedly increases tumorigenicity. This latter result is difficult to explain, given the inhibitory effect of KAP on cell cycle progression. One possible explanation comes from hepatocellular carcinoma, where KAP mRNA is overexpressed, but aberrantly spliced KAP mRNA transcripts encode KAP proteins that lack Cdk2-dephosphorylating activity. In glioblastoma, KAP mRNA is also overexpressed, but aberrant splicing of KAP leads to decreased KAP protein expression and the generation of a dominant negative form of KAP. The decrease in KAP activity leads to upregulation of Cdc2 expression and an increase in glioblastoma cell migration that can be antagonized by Cdc2 inhibition. The detailed mechanism underlying this phenomenon, however, has not been determined.In addition to splicing, posttranscriptional regulation of gene expression can be accomplished by RNA-mediated gene silencing. We and others have reported a prominent role for microRNAs in regulating growth and invasion in glioblastoma. MicroRNA 26a (mir-26a) has been reported to be amplified in glioblastoma and promotes glioblastoma growth by targeting PTEN, RB1 and MEKK2. In lung cancer, miR-26a promotes invasion by targeting PTEN, raising the possibility that a similar phenomenon may occur in glioblastoma. In nasopharyngeal carcinoma and hepatocellular carcinoma, however, miR-26a inhibits invasion and metastasis. Thus, the effect of miR-26a on invasion appears to be tissue specific, and the effect of miR-26a on glioblastoma invasion is unknown.We show here that both aberrant splicing and direct targeting of KAP mRNA by miR-26a decrease KAP expression in glioblastoma. We provide the first evidence that KAP binds and activates ROCK2, and that decreased KAP expression leads to decreased ROCK2 activity, increased Racl-dependent invasion and increased cyclin D1 expression. Decreased KAP expression also activates Cdk2, and we show that Cdk2 itself promotes glioblastoma invasion via an Rb/E2F/Cdc2-dependent pathway. This RNA-regulated KAP/ROCK2/Cdk2 pathway thus promotes invasion and cell cycle progression in PTEN-competent and PTEN-deficient glioblastoma cells.PurposeCyclin-dependent kinase-associated phosphatase, KAP, regulated glioblastoma invasion through Cdc2-dependent pathway, however, the mechanism of KAP regulating Cdc2 is not clear. We identify that KAP can bind the members of Rho kinase family, ROCK1,ROCK2, ROCKβ through Immunoprecipitation and mass spectrographic analysis, and that decreased KAP expression leads to decreased ROCK2 activity, activates Racl-dependent invasion pathway directly. Decreased KAP expression also activates Cdk2, and we show that Cdk2 itself promotes glioblastoma invasion via an Rb/E2F/Cdc2-dependent pathway. This RNA-regulated KAP/ROCK2/Cdk2 pathway thus promotes invasion and cell cycle progression in PTEN-competent and PTEN-deficient glioblastoma cells.Methods1. Glioblastoma stem-like cells (GSCs) were isolated from surgical glioblastoma specimens and maintained as tumorspheres in serum-free medium which were injected into brains of nude mic. We observe the migration distance of GSCs after treatment of Cdk2/Cdc2 inhibitor; Meanwhile, to assess the role of miR-26a on glioblastoma cell invasion, Cells overexpressing miR-26a invaded the brain much more extensively than control cells.2. Construct and clone pLenti-IRES-GFP vector with KAP, shKAP or miR-26a, Vectors were packaged in 293T cells. A control EGFP lentivirus was also prepared. LN229 and U251 GBM cells were transduced with KAP, shKAP, control or a miR-26a lentivirus, and stable cell lines were selected using blasticidin.3. We located KAP and ROCK1/2 in cells and identified the filopodia through immunocytochemistry.4. We tested the expression of KAP and miR-26a in different cell lines through real-timePCR.5. We tested the expression of KAP、 ROCKl/2、ROCKβ、Rac1、CDK2、CDK4、 CDK6、Rb、Cdc2、pCdc2、CyclinD1、Cadesman through western-blot.6. We determine the effects of miR-26a or KAP on GBM invision through transwell experiment.7. We test the ability of miR-26a binding to KAP 3’UTR through constructed luciferase reporter of KAP 3’UTR and mutant KAP 3’UTR.8. We determine the activated Racl by miR-26a and KAP through Racl activation kit and phosphalated sites through ROCK2 activation kit.Results1. miR-26a promotes glioblastoma invasion independent of PTEN.2. miR-26a promotes glioblastoma invasion by targeting KAP.3. KAP downregulation decreases ROCK2 activity and activates Racl.4. Cdk2 is activated by KAP downregulation and promotes invasion.5. KAP regulates phosphorylation of caldesmon and myosin light chain.6. Cdk2/Cdc2 inhibition decreases glioblastoma stem-like cell invasion.7. Cdk2/Cdc2 inhibition decreases glioblastoma stem-like cell invasion.Conclusions:1. miR-26a can target KAP mRNA 3’UTR directly;2. KAP knockdown can activated two different invision pathways, and promote GBM invision via Cdc2 dependent pathway.3. KAP and ROCK2 coexpressed in cytoplasm and co-activatived Racl;4. KAP knockdown can activate and phosphalate Cdk2,and promote GBM invision via Cdc2 dependent pathway5. In An RNA-Activated Glioblastoma Invasion Pathway. KAP regulates GBM invasion via ROCK2 and Cdk2, this is the novel invisive pathway independent PTEN.