Endocrine Abstracts

Introduction: Pancreatic neuroendocrine tumors (panNETs) are often inoperable at diagnosis. The mTORC1 inhibitor everolimus is approved for the treatment of advanced NETs. Unfortunately, the development of resistance against everolimus limits its clinical efficacy.

Aim: Our aim was to establish an everolimus-resistant panNET cell line to find common mechanisms of resistance.

Methods: Pancreatic neuroendocrine tumor cells (BON1) were treated for 24 weeks with 10 nM everolimus (Novartis). Medium supplemented with 10 nM everolimus was changed every 3 days. Two independent everolimus resistant BON1 cell lines were established (RR1 and RR2). Resistance was defined with an at least 2-fold higher IC₅₀, compared to the parental/control cell line.

Results: After 24 weeks of permanent exposure to everolimus both cell lines (RR1 and RR2) showed morphologic changes when compared to the control cell line. The control cell line showed sensitivity to everolimus with cellular survival declining to 54.70% (IC₅₀=34 nM) at 144 h treatment, whereas RR1 and RR2 showed resistance with cellular survival rates of 96.70% (IC₅₀=5200 nM) and 92.30% (IC₅₀=2500 nM), respectively. Western blot analysis showed different adaptive changes of the protein expression level in RR1 and RR2, but two prominent mutual features: The cell cycle component CDK1 (cdc2), which orchestrates the G1-S and G2-M progression was remarkably downregulated and the tyrosine kinase c-Met responsible for tumor cell motility, invasion and metastasis formation was upregulated. Consequently, flow cytometric analysis showed a significant cell cycle shift to G1 phase in RR1 and RR2 cells as well as a higher migration potential than control cells after everolimus treatment.

Conclusion: Everolimus resistance might be acquired by permanently altering the cell cycle and the migration potential. Combinatory treatment approaches to overcome resistance will be subject of further studies.