Mechanism Of MLK3 And PI3Kβ Regulating Focal Adhesion Turnover And Cell Migration In Glioblastoma

BACKGROUND:The rapid migration and high invasiveness of glioblastoma(GBM)cells make it difficult to remove the operation altogether,and the tumor is prone to recurrence.Both MLK3 and PI3K p110β subunits are relevant regulators of tumor cell proliferation,migration,and invasion,but it is still unclear whether there is a synergistic regulatory effect between the two.In previous studies,it was found that targeting PI3Kβ and JNK can synergistically inhibit the proliferation and migration of glioblastoma cells in vitro and the growth of transplanted tumors by regulating the formation of lamellipodia and membrane folds,and the activities of Akt and FAK.Given that MLK3 can activate the JNK,p38,and ERK signaling pathways,we speculate that targeting MLK3 and PI3Kβ also have a synergistic inhibitory effect on the migration and invasion of glioblastoma cellsObjective:To explore the mechanism of MLK3 and PI3Kβ regulating glioblastoma cell migration.Methods:The specific inhibitors of PI3Kβ and MLK3,AZD6482 and URMC-099,were used to perform transwell cell invasion experiments on glioblastoma cells,and to evaluate the targeting of PI3Kβ and MLK3 to GBM cell migration and Impact of invasive function.Cellular immunofluorescence method was used to label focal adhesion protein,and the changes were observed under a laser scanning confocal microscope.Western blot was used to study the expression of focal adhesions and other related proteins on GBM after combined targeting of PI3Kβ and MLK3.Finally,tumor formation in nude mice were used to observe the tumor shrinkage of PI3Kβ and MLK3Results:The combination of AZD6482 and URMC-099 significantly inhibited the migration and invasion of GBM cells U-87 MG,and U-118 MG than the single drug;AZD6482 or URMC-099 alone could prevent lamellipodia and focal adhesion of GBM cells.The number of lamellipodia and focal adhesion spots on the cells was further reduced after the combination of the two drugs;The lamellipodia and focal adhesion of GBM cells could be blocked by reducing the phosphorylation levels of Akt,ERK,ROCK2 and Zyxin;Animal experiments also show that the combination of AZD6482 and URMC-099 also has a synergistic inhibitory effect on the growth of subcutaneous GBM xenografts in nude miceCONCLUSION:This study investigated the possibility of a combined drug strategy for GBM and found that the combined targeting of PI3Kβ and MLK3 has a synergistic inhibitory effect on GBM cell migration and invasion.It can reduce the phosphorylation levels of Akt,ERK,ROCK2,and Zyxin.Blocks the formation of lamellipodia and focal adhesion spots of GBM cells.Moreover,the combined targeting of PI3Kβ and MLK3 also has a synergistic inhibitory effect on the growth of subcutaneous GBM xenografts in nude mice.However,the dose of PI3Kβ and MLK3 inhibitors in animals still needs to be optimized,and another PI3Kβ that can penetrate the blood-brain barrier needs to be found inhibitors were used to evaluate the inhibitory effect of the combined targeting of PI3Kβ and MLK3 on the growth and invasion of orthotopic GBM xenografts.