Abstract B61: Targeting glycolysis and compensatory mitochondrial metabolism in neuroblastoma with FDA-approved ritonavir and metformin
Authors: Batra Surabhi , Rosen Steven T. , Shanmugam Mala 
Published: May 2015

One of the hallmarks of cancer cell development is increased dependence on glucose to fuel aerobic glycolysis for the increased production of cellular metabolites required for rapid cell division. [1] PET with fluorine-18 fluorodeoxyglucose (18F-FDG) exploits this increased aerobic glycolysis of malignant as compared with most normal cells. There is increasing literature to support the utility of PET scans in the diagnosis, staging and monitoring response of neuroblastoma (NBL) to treatment. Glucose is transported via a family of ATP-independent transporters (GLUTs). GLUTs 1, 3 and 4 are high affinity glucose transporters which have been shown to play roles in normal B cell glucose transport [2]. GLUT4 plays a critical role in facilitating glucose transport in hematological malignancies such as multiple myeloma [3]. This study focuses on identifying glucose transporters that may be aberrantly expressed in NBL cells. We have further used HIV protease inhibitor (ritonavir) that has an off-target inhibitory effect on GLUT4 to inhibit glucose transport in these cells. In addition we hypothesized that the cells, which are resistant to ritonavir, are sensitized upon treatment with a complex 1 inhibitor (metformin) that targets compensatory mitochondrial metabolism.

Methods: We tested the dependence of NBL cells on glucose by culturing six different cell lines under aerobic conditions in the presence and absence of glucose. These cell lines included both N-myc amplified and non-amplified cell lines. To determine the transporters involved in transport of glucose across the cell membrane we performed PCR and Western Blot for GLUT 1, 4 and 3, the major high affinity glucose transporters in various NBL cell lines. Based on the results of these tests and preliminary data we used lentivirus sh-RNA and knocked down GLUT 4 in the IMR 5 cell line. To further prove our hypothesis, we cultured cell lines in the presence and absence of metformin, ritonavir and both metformin and ritonavir both and determined viability using flow-cytometry analysis.

Results: The number of live cells in all the cell lines decreased considerably when deprived of glucose. Western Blot and PCR for various glucose transporters showed that GLUT 4 was highly expressed across all cell lines while the expression of GLUT 1 was variable. On knocking down GLUT 4 using lentivirus sh-RNA in the IMR 5 cell line we observed a large difference between the morphology of the 2 groups ( control sh-RNA and the ones with GLUT 4 knocked down). The cells where GLUT 4 had been knocked down were found to be more vacuolated and failed to form any neural processes which is a characteristic of this cell line at the end of 72 hours. We also observed that the cell line IMR5 (NMYC amplified) showed marked cell death in the presence of metformin and ritonavir compared to SHY5Y ( single copy of NMYC) where the cell death was not so remarkable.

Conclusions: NBL cells are highly dependent on glycolysis for their metabolism and to maintain viability. Inhibition of glycolysis through the inhibition of glucose flux into the cells via glucose transporters results in cell death. Further studies to validate this hypothesis and to test in in vivo are underway in our laboratory.

References:

1) Warburg O. On the origin of cancer cells. Science. 1956;123: 309-14.

2) Maratou, E., et al., Glucose transporter expression on the plasma membrane of resting and activated white blood cells. Eur J Clin Invest, 2007. 37(4): p. 282-90.

3) McBrayer, S.K., et al., Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy. Blood, 2012.

Citation Format: Surabhi Batra, Steven T. Rosen, Mala Shanmugam. Targeting glycolysis and compensatory mitochondrial metabolism in neuroblastoma with FDA-approved ritonavir and metformin. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B61.