Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities.

Simon Memmel,Lorenz Eing,Vladimir L Sukhorukov,Dmitri Sisario,Fábio L.R Ferreira,Caren Zöller,Cholpon S Djuzenova,Nicholas Becker,Heiko Zimmermann,Michael Flentje,Robin Heiden,Vanessa Fiedler,Markus Sauer,Astrid Katzer

doi:10.18632/oncotarget.16847

Abstract

High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

Highlights

Glioblastoma multiforme (GBM) is a high-grade astrocytoma associated with a median survival time of 15 months, even after surgical resection, chemotherapy and radiotherapy [1]
Using two different migration assays, Western blotting, conventional and super-resolution fluorescence microscopy we examine the effects of the dual PI3K/mTORinhibitor PI-103 alone and in combination with the heat shock protein 90 (Hsp90) inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two glioblastoma multiforme (GBM) cell lines (DK-MG and SNB19) markedly differing in their invasive capacity
Given the major roles of PI3K/mTOR and Hsp90 in regulating tumor cell motility, we evaluated in this study whether their inhibitors, PI-103 and NVP-AUY922 have potential as anti-migratory agents in GBM

Summary

Introduction

Glioblastoma multiforme (GBM) is a high-grade astrocytoma associated with a median survival time of 15 months, even after surgical resection, chemotherapy and radiotherapy [1]. More long-term survivors have been reported after combined chemo-radiotherapy, the high aggressiveness of GBM caused mainly by the diffuse infiltration of single tumor cells into the surrounding brain parenchyma makes complete tumor debulking virtually impossible [2, 3]. Better therapies for GBM patients are necessary, involving strategies to prevent cell migration and invasion, which would enhance the tumor response to local treatment. These challenges have led to extensive efforts in elucidating the regulatory mechanisms of GBM cell motility in vitro and in vivo. Frequent mutations in RTK, PTEN and p53 genes contribute to a radio- and chemoresistant phenotype and correlate with poor clinical outcome [6]

Methods

Results

Conclusion