gms | German Medical Science

Objective: The objective of the current study was to identify central mediators of drug resistance in an Erlotinib-resistant, EGFR-amplified, EGFRvIII-positive in vitro- and in vivo model system.

Method: BS153 cells (EGFR-amplified, EGFRvIII-positive) were maintained in the presence of the EGFR TKI Erlotinib (Tarceva^®), giving rise to a resistant cell line BS153resE. The cell lines were compared for differentially activated signal transduction pathways by receptor tyrosine kinase arrays and Western blot analysis. Tumor initiation in vivo was compared by orthotopic injection of cells into nude mice. In vitro growth of both cell lines in response to pharmacological EGFR and PI3K inhibition and knockdown studies using small interfering RNA (siRNA) was assessed in proliferation assays.

Results: Erlotinib resistance in the cell model analyzed was associated with a strong upregulation of EGFRvIII at the protein level, while amplification of the egfr gene remained unchanged in the resistant BS153resE cells. In vivo, the Erlotinib-resistant phenotype led to delayed onset of tumor growth as well as to prolonged overall survival of mice injected with these cells, while the unusual morphology of tumors derived from BS153resE cells closely resembled that of parental BS153. On the molecular level we identified a significant decrease in phosphorylation of all major receptor tyrosine kinases (i.e. Met, PDGFRalpha, HER2) except for EGFR in BS153resE. When analyzing signaling molecules downstream of EGFR, we identified a strong up-regulation of phosphoinositide-3-kinase (PI3K), especially of its catalytical subunit p110delta. Targeting either PI3K with the inhibitor PX-866 or specifically targeting p110delta either pharmacologically with CAL-101 or siRNA reduced proliferation of the cells and resensitized BS153resE to Erlotinib in a dose-dependent manner.

Conclusions: Treatment failure of Erlotinib, frequently observed in glioma, is associated with up-regulation of the oncogenic EGFRvIII in the model of Erlotinib-resistant, EGFR-amplified cells presented in this study. Additionally, p110delta is identified as a central mediator of Erlotinib resistance downstream of EGFRvIII, offering a new target for combination therapy for EGFR-amplified glioblastoma.