Abstract 213: Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells
Authors: Senkowski Wojciech , Jarvius Malin , Kultima Kim , Rubin Jenny , Gustafsson Mats , Nygren Peter , Larsson Rolf , Fryknäs. Mårten 
Published: July 2016

Abstract
Inhibition of mitochondrial oxidative phosphorylation (OXPHOS) has recently emerged as a promising strategy for treatment of therapy-resistant cancer cells. These cells often reside within hypoxic tumor regions, where nutrient concentrations are low. Recently, OXPHOS inhibitors have been demonstrated to be toxic to quiescent, nutrient-deprived cells in multicellular tumor spheroids. Such spheroids, formed without medium exchange over the culture period, can serve as an appropriate model to mimic quiescent in vivo tumor regions. These spheroids exhibit low cell proliferation and comprise necrotic cores, contrary to commonly used spheroids cultured with frequent medium change.
We here aimed to characterize how quiescent cells respond to OXPHOS inhibition and thereby identify processes that could be co-targeted for enhanced toxicity. We treated HCT116 colon cancer cell line, grown as monolayer cultures and spheroids, with a range of OXPHOS inhibitors (n = 10, including FDA-approved drugs, e.g. nitazoxanide) and other compounds (n = 14) at escalating doses and in 4 biological replicates. Then, we obtained global gene expression profiles (n = 1149, including 144 vehicle controls) of all treatment conditions using L1000 Gene Expression Profiling method.
We found that upon exposure to OXPHOS inhibitors cells grown as nutrient-deprived spheroids significantly and in dose-dependent manner upregulate expression of genes involved in biosynthesis of cholesterol. This response was not observed for spheroids cultured with medium change or monolayer cell cultures. Thus, we were interested if simultaneous exposure to OXPHOS inhibitors and statins, inhibitors of mevalonate (cholesterol precursor) synthesis, would result in enhanced cytotoxic effects in quiescent, metabolically stressed cells.
We here demonstrate that combination of OXPHOS inhibitors and statins results in pronounced synergistic cytotoxicity in metabolically stressed spheroids. This effect was observed for various classes of OXPHOS inhibitors and different types of statins, indicating that the observed synergy was not a result of off-target effects. This notion was further strengthened by the finding that mevalonate largely abrogated the synergistic effects.
In conclusion, we here report that statins enhance the toxic effects of OXPHOS inhibitors in quiescent, metabolically stressed cells. Our results can serve as a foundation for further studies on targeting therapy-resistant and nutrient-deprived cancer cells by inhibition of OXPHOS. We also demonstrate, for the first time, that the L1000 Gene Expression Profiling can be used to study 3D cell cultures. Importantly, our findings underscore the importance of using a relevant cellular model for target discovery endeavors.
Citation Format: Wojciech Senkowski, Malin Jarvius, Kim Kultima, Jenny Rubin, Mats Gustafsson, Peter Nygren, Rolf Larsson, Mårten Fryknäs. Mitochondrial inhibitors and statins: a lethal combination for metabolically stressed cancer cells. [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 213.