Development of therapeutic strategies for quiescent tumor cell populations

Phenotypic screening is an effective approach to the discovery of small-molecule drugs. In this thesis, I have used cancer cells grown as 3D microtissues (multicellular spheroids) to study the effect of anticancer drugs and as drug discovery tools. Most clinically used cytotoxic drugs have only modest efficacy on spheroids, possibly explaining their limited efficacy on many solid tumors. We therefore used spheroids as targets for screening campaigns aimed to discover novel anti-cancer drugs. This work resulted in the identification of compounds that showed preferential cytotoxicity to spheroids compared to monolayer cultures. We also used multicellular spheroids to study regrowth of cells after cytotoxic therapy.

The compound VLX600 was identified in our spheroid screening work. This drug was found to induce autophagy and upregulation of glycolysis in tumor cells. Further work showed that VLX600 is an inhibitor of mitochondrial oxidative phosphorylation. The work has lead to the hypothesis that cells in the deep tumor parenchyme are sensitive to disturbances of energy metabolism due to lack of metabolic plasticity. Our studies demonstrated that VLX600 also decreased the levels of c-MYC. This phenomenon was also observed after treatment with other mitochondrial inhibitors. An additional screen was performed using glucose-deprived spheroids. This screen identified five molecules that showed selectivity to spheroids. All five drugs were found to inhibit mitochondrial respiration. The FDA-approved antiprotozoal drug nitazoxanide was chosen for further studies and may be a candidate for future clinical trials.

Increased glycolysis in tumor cells leads to the generation of metabolic acids and a subsequent acidification of the microenvironment of solid tumors. We found that tumor acidosis leads to induction of autophagy and present evidence that autophagy is a mechanism for cancer cells to adapt to an acidic environment.

We conclude from this work that multicellular spheroids can be used to screen for novel anticancer agents. Several drugs identified by this approach were found to be inhibitors of mitochondrial function. This finding suggests a therapeutic strategy to target quiescent tumor cells in metabolically compromised microenvironments.

List of scientific papers

I. The PI3K/mTOR inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumor cells after cytotoxic therapy. Emma Hernlund, Maria Hägg Olofsson, Walid Fayad, Mårten Fryknäs, Karolina Lesiak‐Mieczkowska, Xiaonan Zhang, Slavica Brnjic, Vivien Schmidt, Padraig D’Arcy, Tobias Sjöblom, Angelo De Milito, Rolf Larsson & Stig Linder. European Journal of Cancer 48, 396–406 (2012).
https://doi.org/10.1016/j.ejca.2011.11.013

II. Induction of mitochondrial dysfunction as a strategy for targeting tumour cells in metabolically compromised microenvironments. Xiaonan Zhang, Mårten Fryknäs, Emma Hernlund, Walid Fayad, Angelo De Milito, Maria Hägg Olofsson, Vladimir Gogvadze, Long Dang, Sven Påhlman, Leoni A. Kunz Schughart, Linda Rickardson, Padraig D’Arcy, Joachim Gullbo, Peter Nygren, Rolf Larsson & Stig Linder. Nature Communications, 5:3295 (2014).
https://doi.org/10.1038/ncomms4295

III. Autophagy is a protective mechanism for human melanoma cells under acidic stress. Maria Lucia Marino, Paola Pellegrini, Giuseppe Di Lernia, Mojgan Djavaheri-Mergny, Slavica Brnjic, Xiaonan Zhang, Maria Hägg, Stig Linder, Stefano Fais, Patrice Codogno and Angelo De‐Milito. Journal of Biological Chemistry 287, 30664–30676 (2012).
https://doi.org/10.1074/jbc.M112.339127

IV. Effects of mitochondrial inhibitors on c-Myc expression: consequences for drug discovery projects. Xiaonan Zhang, Padraig D'Arcy, Ganna Oliynykc, Marie Arsenian‐Henriksson and Stig Linder. [Manuscript]

V. Three-dimensional cell culture-based screening identifies the anthelminthic drug nitazoxanide as a candidate for treatment of solid tumors. Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, Ruben Isacson, Urban Höglund, Mats Gustafsson, Peter Nygren, Stig Linder, Rolf Larsson and Mårten Fryknäs. [Accepted]
https://doi.org/10.1158/1535-7163.MCT-14-0792