gms | German Medical Science

Objective: Despite multimodal therapy, GBM chemotherapy with Temozolomide (TMZ) remains inefficient due to chemoresistance. One possible mechanism of this observed resistance is due to proteolytic shedding of cell surface proteins. MMP and ADAM metalloproteases (MPs) are responsible for these cleavage reactions and are increased in GBM. Therefore they could potentially contribute to chemoresistance and the observed TMZ induced recurrence of glioblastoma.

Method: TMZ sensitivity was assessed in primary GBM cell lines in the absence or presence of hydroxamate MP inhibitors batimastat (BB-94) and marimastat (BB-2516). Induction of metalloproteases by TMZ was determined by quantitative real-time PCR (qPCR) and by activity assays. Effects of MPs on intracellular kinase (Akt/PI3K, ERK1/2) pathways were investigated by morphological analysis and Western blotting. TMZ sensitivity was analysed in cells with high and low MP expression levels. Invasion of TMZ and BB-94 treated cells was determined by Matrigel invasion assays. Potential substrates of MPs were determined by proteomic analysis of cell supernatants and confirmed by ELISA assays.

Results: Treatment of GBM cells with BB-94, but not BB-2516 increased sensitivity to TMZ induced cell death. Following TMZ treatment, MMP-1, 9, 14, and ADAM8 are increased at transcriptional and protein level. Mitochondrial effects of TMZ were independent of MP activities, but Akt and ERK1/2 phosphorylation in U87 cells was enhanced after TMZ treatment, and lowered in U87 cells bearing an ADAM8 knockdown. Application of UO126, a specific ERK1/2 inhibitor, abrogated TMZ-induced invasiveness by modulating the expression levels of MMP-1, MMP-9, and ADAM8. MP-dependent release of c-met and CD44 was determined in GBM cells treated with TMZ.

Conclusions: ADAM8 was identified as an important metalloprotease in conferring chemoresistance to GBM cells. ADAM8 expression modulates pAkt/PI3K and pERK1/2 and leads to increased survival and invasiveness, which could be mediated by cleavage of CD44 and/or c-Met. In recurrent GBM, ADAM8 levels are increased. The use of an ADAM8 inhibitor appears as a feasible treatment option to optimise TMZ chemotherapy and to prevent formation of recurrent glioblastoma in patients.