Abstract 2605: Metformin reduces growth of tumors generated from neuroblastoma stem cells in a xenograft mouse model; role of Cdc42 in mediating the effects
Authors: Ambrish Kumar, Donald J. DiPette, Ugra S. Singh
Journal: Cancer Research (2016)

Abstract Neuroblastoma, the most common malignant childhood cancer of the postganglionic sympathetic nervous system, is derived from the neural crest cells. Despite the standard therapy, the mortality rate remains high in children with neuroblastoma. Growing evidence confirm that cancer stem cells are responsible for drug resistance, and disease relapse. Hence targeting of cancer stem cells is an effective strategy to cure cancer. In the present study we tested if anti-diabetic drug metformin (N’, N’-dimethylbiguanide) has anti-survival effects against neuroblastoma stem cells. Using a panel of human neuroblastoma cell lines of different genetics- SH-SY5Y, SK-N-BE(2), IMR-32, NGP, and SK-N-F1 cells, we demonstrated that metformin dose-dependently reduced the protein expression of stem cell-specific transcription factors- sox2, oct4 and nanog. Neuroblastoma cells, in stem cell specific medium, formed compact and distinct spheroids which were enriched in stem cells. Addition of metformin (0.5 mM and onwards) significantly inhibited initiation of spheroids, and no spheroid formation was observed at 20 mM metformin. We further examined if metformin interfered with self-renewal and differentiation capacity of neuroblastoma stem cells. Our results demonstrated that metformin-treated primary spheroids lost their ability to form secondary spheroids in a metformin depleted medium (drug withdrawal experiment). However, addition of pharmacological inhibitor of Cdc42 (ML141) along with metformin increased the numbers of spheroids suggesting involvement of Cdc42 in spheroid formation. To further test the inhibitory effect of metformin on the tumorigenicity of neuroblastoma stem cells, we generated subcutaneous tumors by inoculating spheroids in athymic mice. Metformin (10, 30, and 100 mg/kg per mouse) was given daily by oral gavage, and tumor volume was measured. The size of tumors collected from mice fed with 30 and 100 mg/kg metformin was significantly reduced compared to tumors from metformin-untreated mice. In these tumors, metformin induced DNA fragmentation and apoptosis by activating caspase-3. The presence of cleaved caspase-3 signal in both sox2-expressing and sox2-nonexpressing cells indicated that metformin induced cell death in both normal tumor cells and tumor stem cells. These data validate the anti-survival activity of metformin against neuroblastoma stem cells. The fact that metformin is non-toxic and already approved by FDA to treat type 2 diabetes in children suggest metformin is a novel therapeutic drug to treat neuroblastoma. Citation Format: Ambrish Kumar, Donald J. DiPette, Ugra S. Singh. Metformin reduces growth of tumors generated from neuroblastoma stem cells in a xenograft mouse model; role of Cdc42 in mediating the effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2605.