gms | German Medical Science

Objective: The aim of the study was to evaluate the effect of the specific Axl-inhibitor BGB 324 on glioma cell proliferation, migration and tumor initiating capacity.

Method: Axl expression in glioblastoma cell lines was analyzed by microarrays, quantitative real time PCR, Western blot and flow cytometry. Axl-dependent signaling was analyzed by Western blot. Proliferation was measured by indirect luminometric analysis of ATP content. Migration was assessed in modified Boyden chamber assays. Attachment-independent growth as a measure of tumor initiating capacity was determined in soft agar assay. Data from inhibitor studies was validated by shRNA knockdown of Axl.

Results: We found Axl to be specifically overexpressed at the mRNA level in glioma cells with a differentiated phenotype as compared to cells with a stem-like phenotype. In these cells, Axl protein was strongly expressed at the cell surface and also released from the cells in its soluble form in a metalloproteinase-dependent manner. Axl's natural ligand, Gas 6, was also detected, indicating an autocrine stimulation loop in glioma cells. Notably, unprocessed Axl was also detected in the cells' conditioned media, suggesting an association of Axl with glioma-released exosomes. Gas 6-induced Erk 1/2 and Akt phosphorylation could be abrogated by interfering with Axl tyrosine kinase function, resulting in a dose-dependent decrease in proliferation and cell migration at inhibitor concentrations above 1 μM. Tumor initiating capacity in the presence of inhibitor was also significantly reduced by the inhibitor. Knockdown of Axl with specific shRNA confirmed the importance of Axl for these processes and the specificity of the inhibitor.

Conclusions: Targeting of the Axl receptor tyrosine kinase with specific compounds appears a promising strategy for the Axl-overexpressing, differentiated compartment of glioblastoma. However, the impact of Axl inhibition on the stem-like compartment of glioblastoma remains to be elucidated.