Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Mevalonate pathway provides ubiquinone to maintain pyrimidine 2 synthesis and survival in p53-deficient cancer cells exposed to metabolic 3 stress 4 5 Irem, Kaymak1, Carina, R., Maier1, Werner, Schmitz1, Andrew, D., Campbell2, 6 Beatrice, Dankworth1, Carsten, P., Ade1, Susanne, Walz3, Madelon, Paauwe2, 7 Charis, Kalogirou4, Hecham, Marouf1, Mathias, T., Rosenfeldt5,6, David, M., 8 Gay2,7, Grace, H., McGregor2,7, Owen, J., Sansom2 and Almut, Schulze1,6$# 9 10 1 Theodor-Boveri-Institute, Biocenter, Am Hubland, 97074 Würzburg, Germany 11 2 Cancer Research UK Beatson Institute, Garscube Estate Switchback Road 12 Bearsden Glasgow, G61 1BD 13 3 ComPrehensive Cancer Center Mainfranken, Core Unit Bioinformatics, 14 Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 15 4 DePartment of Urology, University Hospital Würzburg, Josef-Schneider-Str. 2, 16 97080 Würzburg 17 5 DePartment of Pathology, University HosPital Würzburg, Josef-Schneider-Str. 18 2, 97080 Würzburg 19 6 ComPrehensive Cancer Center Mainfranken, Josef-Schneider-Str.6, 97080 20 Würzburg, Germany 21 7Institute of Cancer Sciences, University of Glasgow, Garscube Estate, 22 Switchback Road, Bearsden, Glasgow, G61 1QH 23 24 #Corresponding author 25 email: [email protected] 26 $Current address: Division of Tumor Metabolism and Microenvironment, 27 German Cancer Research Center, Im Neuenheimer Feld 281, 69120 28 Heidelberg, Germany ([email protected]) 29 Phone: +49 6221 42 3423 30 31 Running Title: Mevalonate pathway supports ubiquinone synthesis in cancer 32 Conflict of interest: The authors declare no competing financial interests. 33 Keywords: cancer metabolism; colon cancer; p53; mevalonate pathway; 34 SREBP2; ubiquinone; pyrimidine synthesis 1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Abstract 2 Oncogene activation and loss of tumor suppressor function changes the 3 metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, 4 cancer cells adapt their metabolism to sustain growth and survival when access 5 to oxygen and nutrients is restricted, such as in poorly vascularized tumor 6 areas. We show here that p53-deficient colon cancer cells exposed to tumor- 7 like metabolic stress in spheroid culture activated the mevalonate pathway to 8 promote the synthesis of ubiquinone. This was essential to maintain 9 mitochondrial electron transport for respiration and pyrimidine synthesis in 10 metabolically compromised environments. Induction of mevalonate pathway 11 enzyme expression in the absence of p53 was mediated by accumulation and 12 stabilization of mature SREBP2. Mevalonate pathway inhibition by statins 13 blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and 14 apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, 15 ubiquinone produced by the mevalonate pathway was essential for the growth 16 of p53-deficient tumor organoids. In contrast, inhibition of intestinal 17 hyperproliferation by statins in an Apc/KrasG12D mutant mouse model was 18 independent of de novo pyrimidine synthesis. Our results highlight the 19 importance of the mevalonate pathway for maintaining mitochondrial electron 20 transfer and biosynthetic activity in cancer cells exposed to metabolic stress. 21 They also demonstrate that the metabolic output of this pathway depends on 22 both genetic and environmental context. 23 24 Significance: 25 p53-deficient cancer cells activate the mevalonate pathway via SREBP2 26 and promote the synthesis of ubiquinone that plays an essential role in reducing 27 oxidative stress and supports the synthesis of pyrimidine nucleotide 2 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Introduction 2 The metabolic activity of cancer cells is controlled by genetic alterations 3 and by the tumor microenvironment. Under metabolic stress, defined by 4 reduced access to nutrients and oxygen present in poorly vascularized solid 5 tumors, cancer cells need to adapt their metabolic activity to maintain cell 6 proliferation and survival. One important factor in the adaptation to metabolic 7 stress is the hypoxia inducible factor (HIF), which is stabilized and activated in 8 the absence of oxygen, and promotes the uptake of glucose and its 9 fermentation to lactate while reducing oxidative metabolism (1). However, poor 10 access to the vascular network not only reduces oxygen tension but also lowers 11 the availability of serum-derived nutrients. Therefore, cancer cells need to 12 undergo global rewiring of their metabolic activity to be able to adapt to these 13 conditions. 14 The p53 tumor suppressor is a master regulator of cellular metabolism 15 (2,3). It reduces glucose uptake (4) and alters glycolysis and modulates the flux 16 of metabolites into the pentose phosphate pathway (5-8). Conversely, p53 17 enhances mitochondrial metabolism by promoting the assembly of cytochrome 18 C oxidase (complex IV) and increasing respiration (9). It has been shown that 19 p53 allows cancer cells to adapt to nutrient deprivation, in particular the 20 absence of the amino acid serine and glutamine (10,11). Thus, loss of p53 21 function can increase the sensitivity of cancer cells towards metabolic stress, 22 resulting in a selective vulnerability that could be exploited therapeutically. 23 In this study, we have investigated the role of p53 in the regulation of 24 metabolic processes in colon cancer cells exposed to metabolic stress. In order 25 to recreate the simultaneous reduction in oxygen and nutrient availability found 26 in tumors, we cultured cancer cells as multicellular tumor spheroids. Under 27 these conditions, we find that p53-deficient cancer cells activate the expression 28 of enzymes of the mevalonate pathway via the sterol regulatory element 29 binding protein 2 (SREBP2). Moreover, inhibition of mevalonate pathway 30 activity with statins selectively induced apoptosis in p53-deficient cancer cells 31 exposed to metabolic stress. This effect was mediated by reduced generation 32 of ubiquinone (CoQ10), which p53-deficient cells require to maintain TCA cycle 33 activity, respiration and the synthesis of pyrimidine nucleotides. Our study thus 3 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 reveals a novel link between the regulation of isoprenoid synthesis and the 2 modulation of electron transfer mediated by ubiquinone in cancer cells. 3 Mevalonate pathway activity is essential for p53-deficient cancer cells to 4 proliferate and survive under the metabolic constraints of the tumor 5 microenvironment. 6 7 8 Materials and Methods 9 Tissue culture and reagents 10 HCT116 p53-isogenic cells were obtained from B. Vogelstein (Johns 11 Hopkins University, Baltimore) and HCT116 p21-isogenic cells from M. 12 Dobbelstein (Georg-August University, Göttingen). RKO p53-isogenic lines 13 were a gift from K.Vousden (Beatson Institute, Glasgow). All other cell lines 14 were from CRUK LRI Research Services, authenticated by STR profiling and 15 used at low passage. Unless stated otherwise, cells were cultured in DMEM 16 with 10% fetal bovine serum (FBS, Gibco), 4 mM L-glutamine and 1% penicillin- 17 streptomycin, at 37°C in a humidified incubator at 5% CO2 and regularly tested 18 for absence of mycoplasma. Etoposide, (R)-Mevalonic acid lithium salt, 19 SB216732, CHIR99021, simvastatin, zoledronic acid monohydrate, coenzyme 20 Q10, NAC, water-soluble cholesterol, uridine and 5-FU were all from Sigma. 21 MG132 and MK2206 were from Bertin Pharma, rapamycin from Cayman 22 Chemicals, mevastatin and YM-53601 from Biomol and nucleosides 23 (EmbryoMax 100x) from Merck-Milipore. 24 25 Spheroid formation, flow cytometry and histology 26 For spheroid formation, 10,000 cells/well were placed in 96-well ultralow 27 attachment plates (Corning® CORN7007) followed by centrifugation at 850g 28 for 10 min. Spheroids were cultured for 12-14 days, during which medium was 29 replaced every three days. 30 Monolayer and spheroid cells were incubated with 20 µM BrdU (Sigma) 31 for 24 hrs, trypsinized and fixed in 80% EtOH. Cells incubated in 2 M HCl with 32 0.5% Triton X-100 for 30 min at room temperature, neutralized with Na2B4O7. 33 and incubated with anti-BrdU-FITC antibodies (Biozol). Cells were washed, 4 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.