Abstract 3781: A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning

Abstract

Abstract In the search for novel anticancer agents, the established principle is to harness differences between normal and tumor tissue. Thus, most of current chemotherapeutic agents target fast-proliferating cancerous cells. However, over the years such treatment strategies have proven less effective than initially expected. One of the main reasons for this is that conditions present in solid tumors, such as hypoxia, low glucose availability and high concentrations of metabolites, promote quiescent, highly resistant cell phenotypes. In our research, we aimed to target and exploit these tumor-specific conditions. To mimic the harsh conditions in a tumor, we used multicellular tumor spheroids (MCTS), which are known to simulate tumor microenvironment in vitro. We developed a novel method to easily form MCTS in 384-well format. There is only one spheroid per well formed and all are comparable in terms of size and shape. For the MCTS formation, we used colon carcinoma cell line, HCT116, with constitutive expression of green fluorescent protein (GFP). Then, we performed spheroid-based high-throughput drug screening using 1600 clinically active compounds. As a surrogate marker for cell viability, we measured mean spheroid GFP fluorescence intensity complemented with a standard resazurin-based assay. Active hits were tested in dose-response experiments in both spheroid and monolayer setup. We identified 12 compounds, which showed preferential activity against the MCTS model. We tested them in spheroid-based clonogenic assay and identified five compounds, which after 72 hrs treatment resulted in no clonogenicity at concentrations equal to monolayer-based IC50-values. Interestingly, all of these compounds have been previously reported to impair mitochondrial function. Three of them have been also reported as uncouplers of oxidative phosphorylation. We tested the influence of all five compounds on oxygen consumption rate. All, except one compound, caused irreversible shutdown of mitochondrial function. For further experiments, we decided to choose nitazoxanide - a clinically used anti-parasitic agent with excellent pharmacokinetics, bioavailability and safety profile. We show that treatment with nitazoxanide, at concentrations well below what is reached clinically, results in down regulation of c-myc, mTOR and Wnt signaling. Preliminary in vivo experiments show encouraging results. We conclude that MCTS-based screening identifies mitochondria as a potential target for cancer treatment and the anthelmintic drug nitazoxanide as a promising candidate. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2014-3781