mevalonate pathway provides ubiquinone to maintain pyrimidine synthesis … · 8 promote the...

1

Mevalonate pathway provides ubiquinone to maintain pyrimidine 1

synthesis and survival in p53-deficient cancer cells exposed to metabolic 2

stress 3

4

Irem, Kaymak1, Carina, R., Maier1, Werner, Schmitz1, Andrew, D., Campbell2, 5

Beatrice, Dankworth1, Carsten, P., Ade1, Susanne, Walz3, Madelon, Paauwe2, 6

Charis, Kalogirou4, Hecham, Marouf1, Mathias, T., Rosenfeldt5,6, David, M., 7

Gay2,7, Grace, H., McGregor2,7, Owen, J., Sansom2 and Almut, Schulze1,6$# 8

9 1 Theodor-Boveri-Institute, Biocenter, Am Hubland, 97074 Würzburg, Germany 10 2 Cancer Research UK Beatson Institute, Garscube Estate Switchback Road 11

Bearsden Glasgow, G61 1BD 12 3 Comprehensive Cancer Center Mainfranken, Core Unit Bioinformatics, 13

Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 14 4 Department of Urology, University Hospital Würzburg, Josef-Schneider-Str. 2, 15

97080 Würzburg 16 5 Department of Pathology, University Hospital Würzburg, Josef-Schneider-Str. 17

2, 97080 Würzburg 18 6 Comprehensive Cancer Center Mainfranken, Josef-Schneider-Str.6, 97080 19

Würzburg, Germany 20 7Institute of Cancer Sciences, University of Glasgow, Garscube Estate, 21

Switchback Road, Bearsden, Glasgow, G61 1QH 22

23 #Corresponding author 24

email: [email protected] 25 $Current address: Division of Tumor Metabolism and Microenvironment, 26

German Cancer Research Center, Im Neuenheimer Feld 281, 69120 27

Heidelberg, Germany ([email protected]) 28

Phone: +49 6221 42 3423 29

30

Running Title: Mevalonate pathway supports ubiquinone synthesis in cancer 31

Conflict of interest: The authors declare no competing financial interests. 32

Keywords: cancer metabolism; colon cancer; p53; mevalonate pathway; 33

SREBP2; ubiquinone; pyrimidine synthesis 34

Research. on March 29, 2020. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 19, 2019; DOI: 10.1158/0008-5472.CAN-19-0650

http://cancerres.aacrjournals.org/

2

Abstract 1

Oncogene activation and loss of tumor suppressor function changes the 2

metabolic activity of cancer cells to drive unrestricted proliferation. Moreover, 3

cancer cells adapt their metabolism to sustain growth and survival when access 4

to oxygen and nutrients is restricted, such as in poorly vascularized tumor 5

areas. We show here that p53-deficient colon cancer cells exposed to tumor-6

like metabolic stress in spheroid culture activated the mevalonate pathway to 7

promote the synthesis of ubiquinone. This was essential to maintain 8

mitochondrial electron transport for respiration and pyrimidine synthesis in 9

metabolically compromised environments. Induction of mevalonate pathway 10

enzyme expression in the absence of p53 was mediated by accumulation and 11

stabilization of mature SREBP2. Mevalonate pathway inhibition by statins 12

blocked pyrimidine nucleotide biosynthesis and induced oxidative stress and 13

apoptosis in p53-deficient cancer cells in spheroid culture. Moreover, 14

ubiquinone produced by the mevalonate pathway was essential for the growth 15

of p53-deficient tumor organoids. In contrast, inhibition of intestinal 16

hyperproliferation by statins in an Apc/KrasG12D mutant mouse model was 17

independent of de novo pyrimidine synthesis. Our results highlight the 18

importance of the mevalonate pathway for maintaining mitochondrial electron 19

transfer and biosynthetic activity in cancer cells exposed to metabolic stress. 20

They also demonstrate that the metabolic output of this pathway depends on 21

both genetic and environmental context. 22

23

Significance: 24

p53-deficient cancer cells activate the mevalonate pathway via SREBP2 25

and promote the synthesis of ubiquinone that plays an essential role in reducing 26

oxidative stress and supports the synthesis of pyrimidine nucleotide 27




3

Introduction 1

The metabolic activity of cancer cells is controlled by genetic alterations 2

and by the tumor microenvironment. Under metabolic stress, defined by 3

reduced access to nutrients and oxygen present in poorly vascularized solid 4

tumors, cancer cells need to adapt their metabolic activity to maintain cell 5

proliferation and survival. One important factor in the adaptation to metabolic 6

stress is the hypoxia inducible factor (HIF), which is stabilized and activated in 7

the absence of oxygen, and promotes the uptake of glucose and its 8

fermentation to lactate while reducing oxidative metabolism (1). However, poor 9

access to the vascular network not only reduces oxygen tension but also lowers 10

the availability of serum-derived nutrients. Therefore, cancer cells need to 11

undergo global rewiring of their metabolic activity to be able to adapt to these 12

conditions. 13

The p53 tumor suppressor is a master regulator of cellular metabolism 14

(2,3). It reduces glucose uptake (4) and alters glycolysis and modulates the flux 15

of metabolites into the pentose phosphate pathway (5-8). Conversely, p53 16

enhances mitochondrial metabolism by promoting the assembly of cytochrome 17

C oxidase (complex IV) and increasing respiration (9). It has been shown that 18

p53 allows cancer cells to adapt to nutrient deprivation, in particular the 19

absence of the amino acid serine and glutamine (10,11). Thus, loss of p53 20

function can increase the sensitivity of cancer cells towards metabolic stress, 21

resulting in a selective vulnerability that could be exploited therapeutically. 22

In this study, we have investigated the role of p53 in the regulation of 23

metabolic processes in colon cancer cells exposed to metabolic stress. In order 24

to recreate the simultaneous reduction in oxygen and nutrient availability found 25

in tumors, we cultured cancer cells as multicellular tumor spheroids. Under 26

these conditions, we find that p53-deficient cancer cells activate the expression 27

of enzymes of the mevalonate pathway via the sterol regulatory element 28

binding protein 2 (SREBP2). Moreover, inhibition of mevalonate pathway 29

activity with statins selectively induced apoptosis in p53-deficient cancer cells 30

exposed to metabolic stress. This effect was mediated by reduced generation 31

of ubiquinone (CoQ10), which p53-deficient cells require to maintain TCA cycle 32

activity, respiration and the synthesis of pyrimidine nucleotides. Our study thus 33




4

reveals a novel link between the regulation of isoprenoid synthesis and the 1

modulation of electron transfer mediated by ubiquinone in cancer cells. 2

Mevalonate pathway activity is essential for p53-deficient cancer cells to 3

proliferate and survive under the metabolic constraints of the tumor 4

microenvironment. 5

6

7

Materials and Methods 8

Tissue culture and reagents 9

HCT116 p53-isogenic cells were obtained from B. Vogelstein (Johns 10

Hopkins University, Baltimore) and HCT116 p21-isogenic cells from M. 11

Dobbelstein (Georg-August University, Göttingen). RKO p53-isogenic lines 12

were a gift from K.Vousden (Beatson Institute, Glasgow). All other cell lines 13

were from CRUK LRI Research Services, authenticated by STR profiling and 14

used at low passage. Unless stated otherwise, cells were cultured in DMEM 15

with 10% fetal bovine serum (FBS, Gibco), 4 mM L-glutamine and 1% penicillin-16

streptomycin, at 37°C in a humidified incubator at 5% CO2 and regularly tested 17

for absence of mycoplasma. Etoposide, (R)-Mevalonic acid lithium salt, 18

SB216732, CHIR99021, simvastatin, zoledronic acid monohydrate, coenzyme 19

Q10, NAC, water-soluble cholesterol, uridine and 5-FU were all from Sigma. 20

MG132 and MK2206 were from Bertin Pharma, rapamycin from Cayman 21

Chemicals, mevastatin and YM-53601 from Biomol and nucleosides 22

(EmbryoMax 100x) from Merck-Milipore. 23

24

Spheroid formation, flow cytometry and histology 25

For spheroid formation, 10,000 cells/well were placed in 96-well ultralow 26

attachment plates (Corning® CORN7007) followed by centrifugation at 850g 27

for 10 min. Spheroids were cultured for 12-14 days, during which medium was 28

replaced every three days. 29

Monolayer and spheroid cells were incubated with 20 µM BrdU (Sigma) 30

for 24 hrs, trypsinized and fixed in 80% EtOH. Cells incubated in 2 M HCl with 31

0.5% Triton X-100 for 30 min at room temperature, neutralized with Na2B4O7. 32

and incubated with anti-BrdU-FITC antibodies (Biozol). Cells were washed, 33




5

treated with RNAse A (24 µg/ml) and propidium iodide (54 µM) for 30 min. 1

Analysis was performed on a BD FACSCanto II using FACSDIVATM software. 2

Spheroids were fixed with 3.7% paraformaldehyde, incubated in 70% 3

ethanol for 16 hrs, mixed with low-melting agarose and paraffin embedded. 4

4μm sections were deparaffinized and rehydrated. Antigen retrieval was 5

performed with citrate buffer (pH 6.0) in a microwave oven for 6 min. Sections 6

were stained with anti-Ki67 (SP6, Thermo Fischer) and anti-Cleaved Caspase 7

3 (Cell Signaling) in PBS/1% BSA at 4°C and biotinylated secondary antibody 8

(Vector Laboratories). Slides were developed with 3,3'-diaminobenzidine (Cell 9

Signaling) and counterstained with Gilmore 3 hematoxylin. For TUNEL staining, 10

sections were heated in citrate buffer (pH 6.0) for 2 min. TUNEL reactions were 11

developed for 1 hour (In Situ Cell Death Detection Kit, Sigma) and 12

counterstained with Hoechst (Sigma). Archival tumor tissue (8) was stained 13

with anti-Ki67 as above. 14

15

RNA sequencing 16

RNA was extracted using RNeasy columns (Qiagen) including DNase I 17

digestion. mRNA was isolated using NEBNext® Poly(A) mRNA Magnetic 18

Isolation Module and library preparation was performed with NEBNext® Ultra™ 19

RNA Library Prep Kit for Illumina following the manufacturer’s instructions. 20

Libraries were size-selected using Agencourt AMPure XP Beads (Beckman 21

Coulter) followed by amplification with 12 PCR cycles. Library quantification 22

and size determination was performed with an Experion system (Bio-Rad) and 23

libraries were sequenced with NextSeq500 (Illumina). 24

RNAseq data were analyzes as described in the supplementary 25

information and are available at GEO (GSE124189). 26

27

RNA extraction and RT-qPCR 28

Total RNA was isolated using PeqGOLD Trifast followed by reverse 29

transcription into cDNA using M-MLV Reverse Transcriptase (Promega) and 30

random hexamer primers. Real-time PCR was performed using Power-up 31

SYBR Green Master Mix (Thermo Fisher Scientific) using Quantitect primers 32

(Qiagen) or custom primers as followed: human ACTB forward 5’-33

GCCTCGCCTTTGCCGAT-3’ and reverse 5’-CGCGGCGATATCATCATCC-3’; 34




6

and human CDKN1A forward 5’-TCACTGTCTTGTACCCTTGTGC-3’ and 1

reverse 5’-CGTTTGGAGTGGTAGAAA-3’ (Sigma). All qPCR reactions were 2

performed in technical duplicate on three biologically independent replicate 3

samples. Relative mRNA amounts were calculated using the comparative CT 4

method after normalization to actin B (ACTB). 5

6

Western blotting 7

Cells were lysed in lysis buffer (1% Triton X100, 50 mM Tris pH 7.5, 300 8

mM NaCl, 1 mM EGTA, 1 mM DTT, 1 mM NaVO4 with protease inhibitors for 9

30 minutes and cleared by centrifugation and quantified using BCA assay 10

(Biovision). Nuclear extraction of SREBP2 was performed as previously 11

described (12). Proteins were separated on SDS-PAGE and blotted onto PVDF 12

membrane (Immobilon), blocked with blocking solution (LI-COR) and incubated 13

with primary and secondary antibodies. Signals were detected on an Odyssey 14

scanner and quantified using ImageJ. Antibodies used: p53 (DO-1), p21 (C-15

19), CCND1 (DSC-6) (from Santa Cruz), HMGCS-1 (#ab155787), histone-3 16

(#ab1791) (from Abcam), SREBP-2 (1D2), ABCA1 (from Novus), SREBP-2 17

(R&D Systems), GSK3 (4G-1E) (from Milipore), PDK1, ACSS2, p-GSK3a/b 18

(Ser21/9), S6 (5G10), p-S6 (Ser240/244), AKT, p-AKT (Ser473), PARP (from 19

Cell Signaling), beta-actin (AC-15), FDFT1, vinculin (from Sigma). Secondary 20

antibodies were from LI-COR Biosciences. 21

22

Stable isotope labelling and mass spectrometry 23

Monolayer cells or spheroids were washed with PBS and medium was 24

replaced with either complete medium or glucose-free medium with 25 mM 25

[U13C]-glucose (Cambridge Isotope Laboratories). Cells were incubated for the 26

indicated times, washed with cold 154 mM ammonium acetate and snap frozen. 27

For tissue extraction, 150 mg of frozen tissue was homogenized in 3 ml of H2O 28

using an UltraTurrax. 29

For water soluble metabolites, samples were extracted with ice-cold 30

MeOH/H2O (80/20, v/v) containing 0.1 µM lamivudine (Sigma) and separated 31

by centrifugation. Supernatants were transferred to a Strata® C18-E column 32

(Phenomenex) which has been conditioned with 1 ml of CH3CN and 1 ml of 33




7

MeOH/H2O (80/20, v/v). The eluate was dried and dissolved in 50 μl of a 1

mixture of CH3CN and 5 mM NH4OAc (25/75, v/v). 2

For cholesterol and ubiquinone, samples were extracted with ice-cold 3

MeOH/H2O (80/20, v/v) containing 1 µM CoQ9 (Sigma) and separated by 4

centrifugation. Supernatants were extracted twice with 0.4 ml of hexane, 5

collected and taken to dryness under nitrogen at 35°C. Samples were dissolved 6

in 150 µl of hexane and transferred to Strata® SI-1 columns (Phenomenex), 7

washed with 750 µl hexane and 500 ml hexane/acetic acid ethyl ether (18/1 8

v/v). Ubiquinone was eluted with 0.5 ml hexane/acetic acid ethylester (9/1, v/v). 9

Cholesterol was fully eluted with 0.5 ml hexane/acetic acid ethylester (9/1, v/v). 10

Eluates were dried under nitrogen at 35°C and dissolved in 50 µl iPrOH. 11

Metabolites were analyzed by LC-MS using setting provided in 12

supplementary methods. 13

14

Seahorse Assays 15

Spheroids were washed twice and transferred to XFe96 Spheroid 16

Microplates containing 160 µL of Seahorse XF Assay Medium supplemented 17

with 25 mM D-glucose and 10 mM sodium pyruvate at pH 7.4. Oxygen 18

consumption rates (OCR) were determined using an XF96e Extracellular Flux 19

Analyzer (Software Version 1.4) (Agilent) following manufacturer protocol. 20

During the experiment, 2 μM Oligomycin (Merck-Milipore), 0.5 µM FCCP 21

(Sigma) and 1 µM Rotenone/Antimycin A (Sigma) were injected. OCR of eight 22

biologically independent samples was normalized to spheroid area. 23

24

Organoid Culture 25

Mouse small intestines were isolated from wild-type, VillinCreERApcfl/fl or 26

VillinCreERApcfl/flKrasG12D/+ mice sacrificed three days post-induction with 27

tamoxifen, opened longitudinally and washed with PBS. Crypts were isolated 28

as described (13), mixed with 20 µl Matrigel (BD Bioscience) and plated in 24-29

well plates in Advanced DMEM/F12 (Thermo Fisher) supplemented with 1% 30

penicillin-streptomycin, 10 mM HEPES, 2 mM glutamine, N2 (Thermo Fisher), 31

B27 (Thermo Fisher), 100 ng/ml Noggin and 50 ng/ml EGF (both from 32

Peprotech). Growth factors were added every two days. Experiments were 33

performed on two biologically independent samples. Genotyping was 34




8

performed using the following primers: p53fl/fl 5’-1

GAAGACAGAAAAGGGGAGGG-3’ and 5’-AAGGGGTATGAGGGACAAGG-2

3’; KrasG12D 5’-GTCTTTCCCCAGCACAGTGC-3’, 5’-3

CTCTTGCCTACGCCACCAGCTC-3’ and 5’-4

AGCTAGCCACCATGGCTTGAGTAAGT CTGCA-3’. 5

6

Mice 7

All animal experiments were performed under UK Home Office 8

guidelines using project licences 70-8645 or 70-8646. Experimental protocols 9

were subject to the University of Glasgow animal welfare and ethical review 10

board approval. VillinCreER Apcfl/fl and VillinCreER Apcfl/fl KrasG12D/+ mice have 11

been described previously (14). For induction of intestinal hyperproliferation, 12

mice were given a single intraperitoneal injection of 80 mg/kg tamoxifen on one 13

occasion (VillinCreERApcfl/fl KrasG12D/+), or on two consecutive days 14

(VillinCreERApcfl/fl). Mice were treated with a daily dose of 50 mg/kg simvastatin 15

in 0.5% methylcellulose/5% DMSO or vehicle or a daily dose of 35 mg/kg 16

leflunomide in 100 µl 0.15% carboxymethylcellulose via oral gavage from one 17

day post initial tamoxifen injection. For 2H2O tracing, mice were exposed to 8% 18 2H2O in their drinking water for 4 days. Mice were given an intraperitoneal 19

injection of 250 µl of cell proliferation reagent (RPN201, GE 20

Healthcare/Amersham) 2 hrs prior to sacrifice and tissue sections were stained 21

for BrdU as described in supplementary methods. 22

23

Statistical analysis 24

Statistical details for each experiment are stated in the figure legends. 25

Graphs were generated using GraphPad Prism 6.0 (GraphPad software). 26

Unless otherwise indicated, statistical significance was calculated using the 27

unpaired two-tailed Student t-test. 28

29

30

Results 31

Spheroid culture induces tumor-like transcriptional signatures and leads 32

to complex metabolic reprogramming 33




9

To determine the influence of conditions of the tumor microenvironment 1

on colon cancer cells, we used isogenic HCT116 lines that are either wild type 2

(wt) for p53 or an isogenic derivative in which the TP53 gene had been deleted 3

by homologous recombination (15). These cells do not express detectable 4

levels of p53 and fail to induce p21 upon treatment with the DNA damaging 5

agent etoposide (Supplementary Fig. S1A). Both cell lines were cultured either 6

as subconfluent monolayers for 48 hours (MLC), or as large 3-dimensional 7

tumor spheroids (diameter >600 µm), thereby exposing cancer cells to 8

gradients of oxygen and nutrient depletion (16). Spheroid cultures (SPC) 9

showed an overall reduction in proliferation compared to MLC, which was 10

similar in both genotypes (Fig. 1A). However, staining for the proliferation 11

marker Ki67 revealed that p53 wt SPC show proliferation only in the outer 12

regions (Fig. 1B), while SPC of p53-deficient cells present Ki67 positivity 13

throughout their cross-sections (Fig. 1B). Similarly, subcutaneous xenograft 14

colon tumors formed by p53 wt HCT116 cells displayed more heterogenous 15

proliferation patterns compared to their p53-deficient counterparts (Fig. 1C). 16

This suggests that p53 is required for cell cycle arrest induced by the nutrient 17

and oxygen-depleted conditions found in SPC and tumors. 18

We next performed transcriptome analysis of p53 wt and deficient cells 19

cultured as SPC, MLC or xenograft tumors. Principal component analysis 20

(PCA) showed that global gene expression in SPC is more similar to those in 21

tumors rather than MLC (mainly in PC1 accounting for 80% of variance (Fig. 22

1D). Gene set enrichment analysis (GSEA) revealed reduced proliferation 23

(Hallmark_E2F_targets) and induction of interferon response and hypoxia 24

signatures as major transcriptional phenotypes in both SPC and tumors 25

compared to MLC (Fig. 1E and 1F). Moreover, analysis of the cell cycle 26

regulator cyclin D1 (CCND1) and the HIF target pyruvate dehydrogenase 27

kinase (PDK1) confirmed reduced proliferation and induction of hypoxia in SPC 28

compared to MLC (Fig. 1G). 29

We next performed metabolomic analysis to determine differences in 30

metabolism between genotypes and culture conditions (Supplementary Fig. 31

S1B). Stable isotope tracing using [U13C]-glucose showed that SPC increases 32

glucose-dependent lactate synthesis in both p53 wt and deficient cells (Fig. 33

1H). Time course experiments revealed that the labelling of TCA cycle 34




10

metabolites as well as alanine, glutamate and aspartate reached steady state 1

more rapidly in SPC compared to MLC, as maximal labelling was reached much 2

earlier (Supplementary Fig. S1C, D and E). Moreover, fractions of labelled 3

metabolites were reduced, indicating that the contribution of precursors other 4

that glucose, most likely glutamine, to the TCA cycle is higher in SPC compared 5

to MLC. We also found evidence for pyruvate-dependent anaplerosis, as M+3 6

isotopologues for succinate, fumarate and malate were formed more rapidly in 7

SPC compared to MLC (Supplementary Fig. S1D). This pyruvate-dependent 8

anaplerosis supported the production of aspartate, indicated by the high M+3 9

to M+2 ratio for aspartate in SPC (Fig. 1I). 10

While most metabolic differences between MLC and SPC were found in 11

both genotypes, the total levels of aspartate were higher in SPC from p53 wt 12

cells compared to p53-deficient SPC and also compared to MLC (Fig. 1J). 13

Aspartate is a precursor for pyrimidine nucleotide synthesis and thus essential 14

for proliferation (17). Consistently, glucose-derived labelling of uridine 15

monophosphate (UMP), a central metabolite in pyrimidine biosynthesis, while 16

overall reduced compared to MLC, was higher in p53-deficient SPC compared 17

to their wt counterparts (Supplementary Fig. S1F), potentially reflecting higher 18

demand of nucleotides for proliferation. 19

Together, transcriptomic and metabolic analyses demonstrated that 20

SPC induces hypoxic reprogramming of cellular metabolism in cancer cells. 21

However, oxidative reactions required to generate substrates for anabolic 22

reactions (i.e. aspartate) are still supported through anaplerosis. 23

24

Loss of p53 activates the mevalonate pathway via SREBP2 25

We next compared gene expression signatures between p53 wt and 26

deficient cells under the different culture conditions. The major signatures 27

associated with wt p53 status in all conditions were inflammation and interferon-28

a response (Supplementary Fig. S2A). Signatures associated with p53 29

deficiency in MLC mapped to TGF-b signaling, spermatogenesis and cell cycle 30

(Supplementary Fig. S2A, left part). In contrast, loss of p53 in SPC and 31

xenograft tumors resulted in the induction of genes associated with cholesterol 32

homeostasis (Supplementary Fig. S2A, middle and right part), many of which 33




11

are regulated by the SREBP transcription factors (18). Moreover, SREBP target 1

genes (Horton_SREBF_targets) (19) showed strong enrichment in p53-2

deficient cells in SPC and xenograft tumors but not in MLC (Fig. 2A), suggesting 3

that the combined effect of environment and loss of p53 leads to the 4

upregulation of these genes. As cholesterol homeostasis is preferentially 5

regulated by SREBP2, rather than the closely related SREBP1a or SREBP1c 6

isoforms (19), we next investigated expression of canonical SREBP2 target 7

genes. This showed increased expression of HMGCS1, MVD, HMGCR, 8

DHCR7 and FDFT1 mRNA in HCT116 SPC compared to ML, which was further 9

increased upon loss of p53 (Fig. 2B). Moreover, HMGCS1, FDFT1 and ACSS2 10

showed increased protein levels in p53-deficient SPC from a second isogenic 11

colon cancer cell line, RKO (Fig. 2C). 12

We also investigated whether expression of SREBP2 target genes is 13

associated with TP53 mutation in human colorectal adenocarcinoma (CRC). 14

Analysis of a TCGA dataset (20) revealed higher expression of canonical 15

SREBP2 targets in TP53 mutant tumors (Fig. 2D) and increased expression of 16

HMGCS1 in high grade CRC (Fig. 2E). Moreover, two colon cancer cell lines 17

expressing mutant TP53 (HT29 and DLD1), displayed stronger induction of 18

HMGCS1 expression upon SPC compared to p53 wt cell lines (LS174T and 19

LOVO) (Fig. 2F), corroborating that loss of normal p53 function either through 20

mutation or deletion increases the expression of mevalonate pathway genes. 21

Wild type p53 was shown to inhibit mevalonate pathway genes through 22

induction of the cholesterol transporter ABCA1 (21). In agreement with this 23

study, we found that ABCA1 mRNA expression was strongly reduced in p53-24

deficient cells both in MLC and SPC (Supplementary Fig. S2B). However, 25

levels of ABCA1 protein were higher in p53-deficient MLC and completely 26

absent in SPC (Supplementary Fig. S2C). Interestingly, ABCA1 is a target for 27

miRNA-33, which is encoded by an intron within the SREBF2 gene (22,23). It 28

is therefore possible that ABCA1 is repressed in SPC via a miRNA-dependent 29

mechanism. 30

To address the mechanism of mevalonate pathway regulation in our 31

system, we first confirmed that increased expression of HMGCS1 protein in 32

p53-deficient SPC is abolished upon shRNA-mediated silencing of SREBP2 33

(Supplementary Fig. S2D and E). We also established that p53-deficient SPC 34




12

contain high levels of the 55 kDa mature form of SREBP2 (Fig. 2G), which 1

represents the active transcription factor. Accumulation of mature SREBP2 and 2

enhanced target expression was also observed in MLC of p53-deficient 3

HCT116 cells cultured in lipid-reduced medium (Supplementary Fig. S2F and 4

G), a condition that induces SREBP processing (18). Nuclear accumulation of 5

mature SREBP2 is mediated by increased processing of the precursor or by 6

stabilization of the mature protein. As SREBP processing is induced by 7

mTORC1 (24,25), we first investigated the activity of this pathway. We found 8

that phosphorylation of the mTORC1 substrate p70S6K (indicated by the higher 9

relative abundance of the upper band) and its downstream target S6 ribosomal 10

protein (S6RB), is strongly increased in SPC compared to MLC (Fig. 2H and 11

Supplementary Fig. S2H). This was surprising as hypoxia, a major feature of 12

SPC, inhibits the mTORC1 pathway (26,27). Indeed, exposure of HCT116 MLC 13

to hypoxia decreased S6RB phosphorylation and slightly reduced HMGCS1 14

expression (Supplementary Fig. S2I). 15

As increased mTORC1 activity was observed in SPC from both 16

genotypes, we also addressed whether loss of p53 alters protein stability of 17

mature SREBP2. Treatment with the proteasome inhibitor MG132 only 18

increased mature SREBP2 in p53 wt SPC, confirming that mature SREBP2 is 19

more stable when p53 is absent (Fig. 2I). Mature SREBP2 is phosphorylated 20

by glycogen synthase kinase 3 (GSK3), leading to its ubiquitination and 21

degradation (28). We found an overall increase in GSK3 phosphorylation on 22

serine 21 (GSK3a) and serine 9 (GSK3b) in SPC compared to MLC, with a 23

further increase in p53-deficient cells (Fig. 2J), indicating reduced activity of the 24

kinase upon p53 loss. Consistently, treatment of p53 wt SPC with GSK3 25

inhibitors increased levels of mature SREBP2 and restored expression of 26

HMGCS1 mRNA to the same level found in p53-deficient cells (Fig. 2K and L). 27

Treatment with the mTORC1 inhibitor rapamycin reduced mature SREBP2 and 28

HMGCS1 mRNA in p53-deficient SPC (Fig. 2K and M). However, this was 29

independent of AKT, as treatment with MK2206 did not affect GSK3 or S6RB 30

phosphorylation (Supplementary Fig. S2J). 31




13

Together, this suggests that loss of p53 in SPC induces nuclear 1

accumulation of mature SREBP2 through activation of mTORC1 and inhibition 2

of GSK3. 3

4

Inhibition of mevalonate synthesis induces apoptosis in p53-deficient 5

spheroids 6

We next tested whether the mevalonate pathway contributes to cancer 7

cell survival in the metabolically compromised environment of SPC. We used 8

statins, a class of lipid-lowering drugs that inhibit the activity of HMGCR, the 9

rate limiting enzyme of the pathway (Fig. 3A). Statin treatment increased the 10

expression of SREBF target genes, due to inactivation of the negative feedback 11

loop (29), and resulted in global downregulation of cell cycle and epithelial to 12

mesenchymal transition (EMT) expression signatures regardless of genotype 13

(Supplementary Fig. S3A). Protein levels of the S-phase proteins cyclin A 14

(CCNA1) and aurora kinase A (AURKA) were also reduced (Supplementary 15

Fig. S3B), confirming that the mevalonate pathway contributes to proliferation 16

(30) and disruption of tissue architecture (31). 17

When investigating the effect of mevalonate pathway inhibitors on cell 18

viability, we found strong inhibition of proliferation in MLC, irrespective of 19

genotype (Supplementary Fig. S3C). In contrast, in SPC only p53-deficient cells 20

were sensitive to mevastatin treatment, indicated by TUNEL staining, while p53 21

wt cells were largely resistant to this treatment (Fig. 3B and C). Mevastatin-22

induced apoptosis in p53-deficient cells was blocked by addition of mevalonate, 23

the product of the HMGCR reaction (Fig. 3B and C), confirming the specificity 24

of the inhibitor. Apoptotic cells positive for TUNEL and cleaved caspase 3 were 25

mainly found in the core regions, where cells are experiencing the most severe 26

oxygen and nutrient depletion (Fig. 3B and Supplementary Fig. S3D), 27

suggesting that the mevalonate pathway supports cell viability under metabolic 28

stress. 29

As it has been shown that induction of p21 (CDKN1A) is required for the 30

p53-dependent remodeling of metabolism in response to serine deprivation in 31

colon cancer (10), we asked whether failure to induce p21 could be responsible 32

for the induction of apoptosis by statins in p53-deficient cells. However, while 33

short-term simvastatin treatment (24h) induced CDKN1A mRNA only in p53 wt 34




14

cells, both genotypes increased CDKN1A mRNA and protein expression after 1

longer statin exposure (72h) (Fig. 3D and E). This indicates that mevalonate 2

pathway inhibition induces p21 through a p53-independent mechanism. 3

Furthermore, statin treatment of SPC of p21-deficient HCT116 cells did not 4

induce apoptosis (Fig. 3F and G), confirming that p21 is dispensable for statin-5

resistance of p53 wt cells. 6

7

Mevalonate pathway inhibition blocks the production of ubiquinone 8

The mevalonate pathway facilitates the synthesis of isoprenoids, which 9

are substrates for cholesterol synthesis, protein prenylation as well as the 10

synthesis of dolichol, heme A and ubiquinone (Fig. 4A) (32). Ubiquinone 11

consists of a benzoquinone ring derived from tyrosine linked to a tail comprising 12

10 (human) or 9 (mouse) isoprenoid units and functions as electron transfer 13

molecule between the complexes of the respiratory chain (33). To determine 14

mevalonate pathway activity, we treated SPC of p53 wt and deficient cells with 15

[U13C]-glucose and determined label incorporation into different metabolites. 16

SPC of p53-deficient cells increase the incorporation of labelled carbons into 17

mevalonate, resulting in an overall increased abundance of this metabolite (Fig. 18

4B and C). In addition, p53-deficient SPC displayed overall higher levels of 19

acetyl-CoA, the substrate of the mevalonate pathway, without major difference 20

in the proportional labelling of the M+2 fraction, which is generated from citrate 21

(Supplementary Fig. S4A and B). However, despite the observed increase in 22

mevalonate synthesis, the amount of total and labelled cholesterol was much 23

lower in p53-deficient SPC compared to their wt counterparts (Fig. 4D and E). 24

In contrast, the amount of total and labelled ubiquinone was higher in p53-25

deficient cells, demonstrating a re-routing of metabolites into the ubiquinone 26

synthesis pathway (Fig. 4F and G). Treatment with simvastatin decreased the 27

amount of both metabolites and completely abolished their glucose-derived 28

labelling (Fig. 4D-F and Supplementary Fig. S4C-D). Importantly, isotopologue 29

peak distribution for cholesterol and ubiquinone were similar in both genotypes 30

(Fig. 4E and G), indicating comparable contribution of glucose to the acetyl-31

CoA pool (34). 32

To investigate whether a p53-dependent switch in the routing of 33

metabolites in the mevalonate pathway can also be observed in tumors, we 34




15

determined the abundance of cholesterol, 7-dihydroxy cholesterol (7-DHC) and 1

ubiquinone in xenograft colon tumors of p53 wt and deficient HCT116 cells. In 2

support of the results obtained in spheroid cultures, p53 wt colon tumors 3

displayed somewhat higher levels of cholesterol and 7-DHC compared to p53-4

deficient colon tumors. In contrast, levels of ubiquinone were overall higher in 5

p53-deficient colon tumors, although this difference failed to reach significance 6

(Fig. 4H). Together, these results suggest that loss of p53 alters mevalonate 7

pathway flux to support the production of ubiquinone. 8

9

Inhibition of ubiquinone synthesis impairs TCA cycle and respiration and 10

results in oxidative stress 11

Ubiquinone is an essential component of the mitochondrial electron 12

transport chain (ETC) where it shuttles electrons between NADH-CoQ 13

reductase (complex I) or succinate dehydrogenase (complex II) and CoQH2-14

cytochrome c reductase (complex III) (Fig. 5A). Using stable isotope tracing 15

with [U13C]-glucose, we found that simvastatin reduced labelled and unlabelled 16

fractions of aspartate and most TCA cycle metabolites in SPC from both 17

genotypes (Fig. 5B and Supplementary Fig. S5A). However, simvastatin 18

reduced basal and maximal oxygen consumption rates (OCR) in SPC of p53-19

deficient cells, which was rescued by mevalonate addition. In contrast, SPC 20

from p53 wt cells only displayed a small reduction in maximal respiration upon 21

statin treatment (Fig. 5C). 22

Reduced availability of oxygen as final electron acceptor of the electron 23

transport chain can lead to electron leakage and the formation of reactive 24

oxygen species (35). We therefore reasoned that inhibition of ubiquinone 25

synthesis could cause oxidative stress under the hypoxic conditions in SPC, 26

which could lead to the induction of apoptosis. Indeed, replenishing spheroid 27

cultures either with ubiquinone or the antioxidant N-acetyl-cysteine (NAC) was 28

as effective as mevalonate in preventing statin-induced apoptosis in SPC (Fig. 29

5D and E), while cell-permeable cholesterol had no effect (Supplementary Fig. 30

S5B). In addition, the viability of statin-treated MLC was not restored by the 31

addition of ubiquinone (Supplementary Fig. S5C), indicating that multiple 32

products of this pathway are needed to support the rapid proliferation of cancer 33

cells observed in MLC. 34




16

1

Production of ubiquinone by the mevalonate pathway supports 2

pyrimidine nucleotide biosynthesis 3

Ubiquinone also functions as an electron acceptor for dihydroorotate 4

dehydrogenase (DHODH), an essential enzyme for the generation of 5

pyrimidine nucleotides for DNA and RNA synthesis (Fig. 6A). Stable isotope 6

tracing showed higher incorporation of glucose-derived carbons into UMP in 7

SPC of p53-deficient cells (Fig. 6B). This was detected in the M+5 fraction, 8

representing labelling via ribose, but also in the M+7/M+8 fractions, 9

representing ribose plus either two or three labelled carbons derived from 10

aspartate (Fig. 6B). Treatment with statins significantly lowered labelling and 11

overall levels of UMP in SPC from both genotypes (Fig. 6B and C). This was 12

restored by supplementing statin-treated SPC with either mevalonate or 13

ubiquinone (Fig. 6C), confirming that ubiquinone is rate-limiting for pyrimidine 14

synthesis under these conditions. Moreover, addition of nucleosides or uridine, 15

which can readily be taken up by cells and used to replenish the nucleotide pool 16

via the salvage pathway, was sufficient to block the induction of apoptosis by 17

statins in p53-deficient SPC (Fig. 6D and Supplementary Fig. S6A). 18

The antimetabolite drug 5-fluoro-uracil (5-FU), which is standard-of-care 19

for advanced CRC, exerts its effect mostly through inhibition of thymidylate 20

synthase (TYMS) (36). TYMS converts dUMP to dTMP for DNA synthesis, and 21

5-FU treatment leads to DNA damage and cell death. We therefore investigated 22

whether statins alter 5-FU sensitivity of cancer cells under the metabolic 23

constraints of SPC. Interestingly, while p53 wt HCT116 cells showed 24

remarkable resistance towards 5-FU, most likely due to the low proliferation of 25

these cells in this condition, the drug sensitized the cells to simvastatin 26

treatment (Fig. 6E). In contrast, p53-deficient cells already showed induction of 27

apoptosis in response to statin alone, which was not further increased by 5-FU 28

(Fig. 6E). 29

Collectively, these results demonstrate that ubiquinone production by 30

the mevalonate pathway is essential for pyrimidine biosynthesis in cancer cells. 31

Inhibition of ubiquinone synthesis blocks the viability of p53-deficient cells 32

under the metabolic constraints of SPC. In contrast, p53 wt cells are initially 33




17

resistant to statin treatment but can be sensitized by the anti-metabolite 5-FU, 1

which blocks dTMP synthesis and causes DNA and RNA damage. 2

3

The metabolic output of the mevalonate pathway depends on 4

environmental context 5

To investigate the role of the mevalonate pathway under different 6

conditions resembling the tumor microenvironment, we used organoid cultures 7

of intestinal epithelial cells from mice carrying conditional alleles of Apc, Trp53 8

or KrasG12D together with VillinCreERT2. Efficient recombination of the Trp53 and 9

Kras locus were confirmed by PCR (Supplementary Fig. S7A). Placed in 10

organoid culture medium, these cells grow as large cysts without any signs of 11

differentiation (13). Interestingly, while simvastatin only had a minor effect on 12

the growth of Apc-deficient organoids, Apc/p53 double deficient cells showed a 13

severe reduction in organoid growth, which was fully restored by mevalonate 14

supplementation (Fig. 7A and B). Reduced organoid growth was accompanied 15

by induction PARP cleavage, a marker of apoptotic cell death (Supplementary 16

Fig. S7B). Similar results were also obtained for Apcfl/fl/KrasG12D and 17

Apcfl/fl/p53fl/fl/KrasG12 organoids (Fig. 7A and B), demonstrating that the deletion 18

of Trp53 sensitizes the organoids towards mevalonate pathway inhibition. 19

Moreover, inhibition of organoid growth in Apc/p53 deficient and Apcfl/fl/ 20

p53fl/fl/KrasG12D/+ cells was robustly restored by addition of either ubiquinone or 21

nucleosides (Fig. 7C and D), confirming that the provision of ubiquinone for 22

nucleotide biosynthesis is an essential function of the mevalonate pathway in 23

CRC tumor organoids. 24

We next assessed the ability of simvastatin to suppress intestinal 25

hyperproliferation induced by acute deletion of Apc and activation of Kras in 26

vivo. This was achieved by crossing mice carrying conditional alleles of Apc or 27

an activated allele of Kras (Apcfl/fl or Apcfl/fl;KrasG12D/+) to mice bearing the 28

VillinCreERT2 transgene (37). After induction of CRE-dependent recombination, 29

mice were treated for 4 days with simvastatin or vehicle and with D2O to assess 30

cholesterol and ubiquinone synthesis in vivo (38). Histological analysis of BrdU 31

positive cells demonstrated that simvastatin had no effect on proliferation in 32

Apc-deficient intestinal crypts, but blocked hyperproliferation induced by Kras 33

activation (Fig. 7E and F). Deuterium tracing revealed that the highly 34




18

proliferating intestinal crypts in VillinCREERT2Apcfl/fl;KrasG12D/+ mice exhibit high 1

levels of cholesterol synthesis, which was blocked by simvastatin (Fig. 7G). In 2

contrast, ubiquinone synthesis was already lower in intestines from double 3

mutant mice and not further reduced by simvastatin (Fig. 7G), indicating that 4

cholesterol rather than ubiquinone is the limiting metabolite produced by the 5

mevalonate pathway in this system. This was further corroborated by the 6

finding that hyperproliferation of intestinal crypts in 7

VillinCREERT2Apcfl/fl;KrasG12D/+ mice was insensitive to the DHODH inhibitor 8

leflunomide (Supplementary Fig. S7C and D), which has recently been shown 9

to block growth of breast cancer cells (39). Together, these results indicate that 10

de novo pyrimidine synthesis is dispensable for KRAS-induced intestinal 11

hyperproliferation and that the metabolic output of the mevalonate pathway 12

depends on genetic factors and microenvironmental context. 13

14

15

Discussion 16

Metabolic gradients in tumors are likely to simultaneously limit access to 17

oxygen and nutrients, making adaptation by metabolic compensation 18

challenging (40). One potential response of cancer cells to nutrient deprivation 19

is cell cycle arrest, which alleviates the metabolic demand of nucleotide 20

biosynthesis for DNA replication, allowing cancer cells to survive until nutrients 21

become available, for example after formation of new blood vessels or 22

engagement of metabolic symbiosis (41,42). Using spheroid cultures (SPC) as 23

model, we show here that p53 wt colon cancer cells respond to metabolic 24

deprivation by reducing proliferation. In contrast, p53-deficient CRC cells are 25

able to maintain proliferation in the spheroid center, where nutrient and oxygen 26

supply is restricted. Contrary to monolayer cultures, gene expression 27

signatures in SPC are characteristic of cell cycle arrest and induction of 28

hypoxia, similar to those found in tumor tissue. Moreover, stable isotope tracing 29

showed that SPC engage in hypoxic remodeling of their metabolism, with 30

reduced glucose oxidation, enhanced lactate production and increased TCA 31

cycle anaplerosis from pyruvate. Pyruvate anaplerosis promotes glutamine-32

independent growth of cancer cells (43) and supports aspartate synthesis in 33




19

succinate dehydrogenase (SDH) deficient cancer cells (44). We found that SPC 1

of p53-deficient colon cancer cells showed reduced aspartate levels, indicating 2

its enhanced usage for pyrimidine biosynthesis. 3

Importantly, p53-deficient CRC cells in SPC or grown as xenograft 4

tumors increase expression of mevalonate pathway enzymes and upregulation 5

of SREBP2 targets was observed in p53-mutant CRC patient samples and cell 6

lines. Previous studies have shown that mutant p53 can bind to SREBP2 and 7

promoting its transcriptional activity during disruption of mammary tissue 8

architecture (31), and that wt p53 represses the mevalonate pathway through 9

ABCA1-dependent inhibition of SREBP2 processing (21). We demonstrate 10

here that loss of p53 in SPC promotes expression of SREBP2 target genes by 11

activating mTORC1 signaling, which drives the processing of SREBP2 (24), 12

and by limiting the activity of GSK3, which controls the phosphorylation-13

dependent degradation of mature SREBP2 (28). The combination of mTORC1 14

activation and inhibition of GSK3 results in the accumulation of mature SREBP2 15

and increases the expression of its target genes. 16

Our study also shows that tumor-like metabolic stress alters the 17

sensitivity of cancer cells towards mevalonate pathway inhibition. In monolayer, 18

both genotypes were highly sensitive to mevalonate pathway inhibitors, most 19

likely because cells require cholesterol for rapid proliferation (32). However, 20

when exposed to metabolic stress, p53-proficient cells were largely resistant to 21

statin treatment, while p53-deficient cancer cells showed induction of 22

apoptosis. Cell death was restricted to the nutrient- and oxygen-deprived center 23

of the spheroids, indicating that the mevalonate pathway provides essential 24

metabolic functions under these conditions. Surprisingly, sensitivity towards 25

mevalonate pathway inhibition was independent of p21, suggesting that the 26

protective effect of wt p53 is independent of its role as transcriptional inducer 27

of this target. Indeed, it has been shown that an acetylation-deficient form of 28

p53 that is unable to induce p21 retains important tumor suppressive functions 29

(45). 30

We also demonstrate that p53-dependent metabolic rewiring of the 31

mevalonate pathway supports the synthesis of ubiquinone, an important 32

electron transport molecule of the ETC (46). Previous studies indicate that 33

nutrient deprivation increases dependency of cancer cells on ETC activity 34




20

(47,48), particularly for the generation of aspartate as precursor for pyrimidine 1

synthesis (49). DHODH, the enzyme converting dihydroorotate to orotate 2

during UMP synthesis, requires electron transfer via ubiquinone and has been 3

shown to support the growth of respiration-deficient tumors (39). This suggests 4

that ubiquinone synthesis by the mevalonate pathway supports pyrimidine 5

synthesis, particularly when efficient electron transport is hampered by low 6

oxygen availability. Ubiquinone deprivation also induces oxidative stress, 7

especially when demand for biosynthetic reactions that deliver electrons to the 8

ETC is high. We found that statin-induced cell death was prevented by 9

antioxidants or by the addition of nucleosides or uridine, which allow cells to 10

switch to the salvage pathway, suggesting that reducing de novo pyrimidine 11

synthesis prevents ROS formation and cell death. Furthermore, the anti-12

metabolite 5-FU, which blocks dTMP synthesis and induces DNA and RNA 13

damage, sensitized p53 wt SPC to statin treatment. 5-FU may impose 14

additional strain on pyrimidine biosynthesis and/or increase oxidative stress, 15

both of which would enhance the dependency of cancer cells on ubiquinone. 16

While clinical trials combining statins with 5-FU in CRC have produced some 17

promising results (50), our study suggests that p53 status could determine the 18

outcome of mevalonate pathway inhibition in CRC. 19

The dependence of p53-deficient cancer cells on mevalonate pathway 20

activity was also confirmed in apc-/- intestinal tumor organoids, where deletion 21

of p53, either alone or in combination with Kras activation, induced sensitivity 22

towards statin treatment. Addition of ubiquinone or nucleosides restored growth 23

of p53-deficient tumor organoids in the presence of statins, suggesting that 24

cells require mevalonate pathway-derived ubiquinone to counteract oxidative 25

stress and support biosynthetic reactions. Indeed, LGR5+ intestinal stem cells 26

are enriched for gene expression signatures linked to purine and pyrimidine 27

metabolism (51) and are highly dependent on mitochondrial metabolism (52). 28

We also found that statins block Kras-dependent hyperproliferation in 29

Apc-deficient intestinal crypts. However, in contrast to our findings in SPC and 30

organoids, this was associated with reduced cholesterol rather than ubiquinone 31

synthesis. Cholesterol is required for membrane synthesis (32) and could be 32

the major metabolic output of the mevalonate pathway in rapidly proliferating 33

tissues. Moreover, cells within the intestinal mucosa may not be exposed to 34




21

metabolic deprivation, as they have access to the nutrient-rich contents of the 1

intestinal lumen, including nucleosides generated by the degradation of diet-2

derived nucleic acids. 3

4

Together, our findings reveal a novel function of the mevalonate pathway 5

in supporting the synthesis of ubiquinone for electron transfer and pyrimidine 6

biosynthesis in p53-deficient cancer cells exposed to environmental stress. 7

However, our results also show that the dependence on mevalonate pathway-8

derived metabolites is determined by environmental context. Mevalonate 9

pathway inhibition may therefore be most effective under conditions of nutrient 10

and oxygen deprivation. Beneficial effects of mevalonate pathway inhibitors 11

have already been demonstrated in several cancer entities, including CRC 12

(53,54). The results of this study indicate that mevalonate pathway inhibitors 13

may need to be combined with treatments that induce metabolic stress, such 14

as anti-angiogenic therapy. 15

16

Acknowledgements 17

We thank B.Vogelstein (Johns Hopkins University, Baltimore), K.Vousden (The 18

Francis Crick Institute, London) and M.Dobbelstein (University Göttingen) for 19

providing p53 and p21 isogenic colon cancer cell lines, respectively. We thank 20

C.Schülein-Völk and U.Eilers for help with automated cell counting. We also 21

thank S.Janaki Raman and M.T.Snaebjörnsson for critically reading the 22

manuscript. This study was funded by grants from the German Research 23

Foundation FOR2314 and SCHU2670-1 (A.Schulze), the Graduate School of 24

Life Sciences Würzburg (I.Kaymak), GRK2243 (C.R.Maier), the Rosetrees 25

Trust (G.McGregor), the CRUK Grand Challenge (M.Paauwe and D.M.Gay) 26

and core funding to the Beatson Institute from Cancer Research UK (A17196) 27

(O.J.Sansom). 28

29

30

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Figure Legends: 1

Figure 1: Spheroid cultures replicate tumor-like transcriptional profiles 2

and show pyruvate-dependent anaplerosis 3

A) HCT116 p53+/+ and p53-/- cells were cultured as subconfluent monolayer 4

cultures (MLC) for 48 hrs or as multi-layered tumor spheroid cultures (SPC) for 5

14 days. Cells were incubated with BrdU for 24 hrs and analyzed by FACS. 6

B) HCT116 p53+/+ and p53-/- cells were cultured as SPC for 14 days, fixed and 7

embedded in paraffin. Histological sections were analyzed for expression of the 8

proliferation marker Ki67. Representative images of three spheroids analyzed 9

per condition are shown. 10

C) Analysis of proliferation in HCT116 p53+/+ and p53-/- xenograft tumor tissue 11

from (8) using Ki67. Representative images of tumors from six animals per 12

group are shown. 13

D) HCT116 p53+/+ and p53-/- cells were cultured as MLC for 48 hrs or as SPC 14

for 14 days. RNA was analyzed by RNA-SEQ and compared to RNA-SEQ data 15

from HCT116 p53+/+ and p53-/- cells grown as xenograft tumors (8). Principal 16

component analysis (PCA) shows overall higher similarity in gene expression 17

signatures between tumors (T) and SPC compared to MLC. 18

E) Gene set enrichment analysis (GSEA) comparing MLC and SPC cultures of 19

HCT116 p53+/+ and p53-/- cells. Enrichment plots for 20

HALLMARK_E2F_TARGETS, 21

BROWNE_INTERFERON_RESPONSE_GENES and 22

MANALO_HYPOXIA_UP are shown. 23

F) Enrichment plots for the same gene sets as in E comparing MLC and tumors 24

(T) of HCT116 p53+/+ and p53-/- cells. 25

G) Western blots (WB) showing levels of cyclin D1 (CCND1) and pyruvate 26

dehydrogenase kinase 1 (PDK1) in HCT116 p53+/+ or p53-/- cells grown as MLC 27

or SPC. Vinculin is shown as loading control. 28

H-J) HCT116 p53+/+ and p53-/- cells were cultured as MLC or SPC and labelled 29

for 16 hours with [U13C]-glucose. Cells were extracted and metabolites were 30

analyzed by LC-MS. Data show mean ±SEM of three independent biological 31

replicates. Results from time-resolved experiments are provided in Figure S1. 32

H) Relative peak intensities for lactate. 33




27

I) Ratios of M+3 and M+2 isotopologues for aspartate. 1

J) Relative peak intensities of aspartate. 2

3

Figure 2: Loss of p53 induces enzymes of the mevalonate pathway via 4

activation of SREBP2 5

A) Enrichment plots for HORTON_SREBF_TARGETS (19) for HCT116 p53+/+ 6

and p53-/- cells cultured as MLC, SPC or xenograft colon tumors. 7

B) Expression of canonical mevalonate pathway genes and SREBF2 in 8

HCT116 p53+/+ or p53-/- cells grown as MLC or SPC. Data show mean ±SEM 9

of three independent biological replicates. (*p<0.05; **p<0.01; ***p<0.001; 10

****p<0.0001, unpaired two-tailed Student’s t test). 11

C) Western blots showing levels of HMGCS1, FDFT1, ACSS2 and p53 in RKO 12

p53+/+ and RKO p53-/- cells grown as SPC. Vinculin is shown as loading control. 13

D) Combined z-score for the expression of canonical mevalonate pathway 14

genes (HMGCS1, HMGCR, MVD, DHCR7, ACSS2, FDFT1 and SREBF2) was 15

calculated for tumors from the TCGA colorectal adenocarcinoma dataset. 16

Mevalonate pathway signature values were compared between all p53 wt (n = 17

94) and p53 mutant (n = 88) tumors. p = 0.0051 was determined using an 18

unpaired two-tailed Student’s t test. 19

E) Expression of HMGCS1 in colorectal adenocarcinoma tumors from the 20

TCGA dataset according to stage (PT1-4). 21

F) Expression of HMGCS1 in TPP53 wt and mutant colon cancer cell lines 22

grown as MLC or SPC. 23

G) WB showing expression of HMGCS1 and mature SREBP2 in HCT116 p53+/+ 24

or p53-/- cells grown as MLC or SPC. Actin is shown as loading control. 25

H) WB showing levels of phosphorylated ribosomal protein S6 (P-S6RB) and 26

total ribosomal protein S6 expression (S6RB) in HCT116 p53+/+ or p53-/- cells 27

grown as MLC or SPC. Actin is shown as loading control. 28

I) SPC of HCT116 p53+/+ or p53-/- cells were treated with 20µM MG132 or 29

solvent for 1h. Levels of mature SREBP2 were detected by WB. Vinculin is 30

shown as loading control. 31

J) Western blots showing phosphorylation on GSK3a/b (serine 21/9) and total 32

GSK3a/b protein in HCT116 p53+/+ or p53-/- cells grown as MLC or SPC. 33




28

Numbers display P-GSK3/GSK3 signal ratio. Actin is shown as loading control. 1

Graph shows mean ±SEM ratios of biologically independent replicate SPC 2

samples (n=4, *p≤0.05 was determined using a paired two-tailed Student’s t 3

test). 4

K) HCT116 p53+/+ or p53-/- cells grown as SPC were treated with 20nM of 5

rapamycin (RAPA), 30 µM of SB216763 (SB) or 10 µM of CHIR99021 (CHIR) 6

for 24 hours and mature SREBP2 was analyzed by WB. Vinculin is shown as 7

loading control. Numbers show signal intensity for mSREBP normalized to 8

vinculin. 9

L) Expression of HMGCS1 mRNA in HCT116 p53+/+ or p53-/- cells grown as 10

spheroids and treated with 10 µM of CHIR99021 (CHIR) for 72 hours. Data 11

show mean ±SEM of three independent biological replicates. (*p<0.05, 12

unpaired two-tailed Student’s t test). 13

M) Effect of rapamycin on HMGCS1 expression in spheroid cultures of HCT116 14

p53+/+ or p53-/- cells. Data are presented as mean of duplicate samples. 15

16

Figure 3: The mevalonate pathway is essential for the survival of p53-17

deficient colon cancer cells 18

A) Diagram showing selected metabolites of the mevalonate pathway and 19

HMGCR, the molecular target of statins. 20

B) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with 10 µM 21

mevastatin (MST) or solvent (DMSO) either alone or in combination with 0.5 22

mM mevalonate (MVL) for 72 hours. Spheroids were fixed and histological 23

sections were analyzed for the presence of apoptotic cells by TUNEL staining. 24

Images show representative results of three spheroids analyzed per condition. 25

C) Quantitation of data shown in B. Data are presented as mean ±SEM of at 26

least 3 spheroids analyzed per condition. (**p<0.01, unpaired two-tailed 27

Student’s t test). 28

D) HCT116 p53+/+ and p53-/- cells grown as SPC were treated with 10µM 29

simvastatin (SIM) or solvent (DMSO) for 24 or 72 hours. Expression of p21 30

(CDKN1A) mRNA was determined by qPCR. Data show mean ±SEM of three 31

independent biological replicates. (*p<0.05; ***p<0.001, unpaired two-tailed 32





29

E) HCT116 p53+/+ and p53-/- cells were grown as SPC and treated with 10µM 1

simvastatin (SIM) or solvent (DMSO) either alone or in combination with 0.5 2

mM mevalonate for 72 hours. Expression of p21 protein was determined by 3

western blotting. Vinculin is shown as loading control. 4

F) HCT116 p21-/- cells were grown as SPC and treated with 10 µM simvastatin 5

(SIM) or solvent (DMSO) either alone or in combination with 0.5 mM 6

mevalonate (MVL) for 72 hours. Spheroids were fixed and histological sections 7

were analyzed for the presence of apoptotic cells by TUNEL staining. Images 8

show representative results of three spheroids analyzed per condition. 9

G) HCT116 p21+/+ or p21-/- cells were grown as SPC and treated with 10 µM 10

simvastatin (SIM) or solvent (DMSO) either alone or in combination with 0.5mM 11

mevalonate (MVL) for 72 hours. Expression of p21 protein was determined by 12

western blotting. Vinculin is shown as loading control. 13

14

Figure 4: Inhibition of mevalonate synthesis blocks the production of 15

ubiquinone in colon cancer cells 16

A) Schematic showing the branching of the mevalonate pathway into 17

cholesterol biosynthesis, the generation of isoprenoids for protein prenylation 18

and the synthesis of dolichol, heme A and ubiquinone (CoQ10). 19

B-C) HCT116 p53+/+ or p53-/- cells were grown as SPC and labelled with [U13C]-20

glucose for 16 hrs before extraction and analysis of mevalonate isotopologues. 21

Data show mean ±SEM of three independent biological replicates. 22

B) Relative peak intensities of labelled and unlabelled fractions for mevalonate. 23

C) Relative peak intensities of individual labelled fractions for mevalonate. 24

D-G) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with 10 µM 25

simvastatin (SIM) or solvent (DMSO) for 72 hrs. For the last 16 hours, cells 26

were labelled with [U13C]-glucose before cells were extracted and metabolites 27

were analyzed by LC-MS. Data show mean ±SEM of three independent 28

biological replicates. 29

D) Relative peak intensities of labelled and unlabelled fractions for cholesterol. 30

E) Relative peak intensities of individual isotopologues for cholesterol. 31

F) Relative peak intensities of labelled and unlabelled fractions for ubiquinone 32

(CoQ10). 33

G) Relative peak intensities of individual isotopologues for ubiquinone. 34




30

H) Xenograft tumors from HCT116 p53+/+ or p53-/- cells were extracted and 1

levels of cholesterol, 7-dihydroxycholesterol (7-DHC) and ubiquinone (CoQ10) 2

were determined by LC-MS. Data are shown as mean ±SEM of six p53+/+ and 3

five p53-/- colon tumors. (*p<0.05, unpaired two-tailed Student’s t test). 4

5

Figure 5: Inhibition of mevalonate synthesis blocks TCA cycle activity and 6

cellular respiration and induces oxidative stress 7

A) Diagram showing the role of ubiquinone (Q10) in electron transport within 8

the mitochondrial electron transport chain (ETC). 9


simvastatin (SIM) or solvent (DMSO) for 72 hrs. For the last 16 hours, cells 11

were labelled with [U13C]-glucose before cells were extracted and metabolite 12

levels were analyzed by LC-MS. Relative peak intensities of isotopologues for 13

aspartate are shown. Data show mean ±SEM of three independent biological 14

replicates. 15

C) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with 10 µM 16

simvastatin (SIM) or solvent (DMSO) either alone or in the presence of 0.5 mM 17

mevalonate for 72 hrs. Oxygen consumption rates (OCR) were determined 18

using the Seahorse Bioanalyzer. Oligomycin (oligo), FCCP and 19

rotenone/antimycin A (R/A) were added to determine ATP-dependent, maximal 20

and basal respiration. Data are presented as mean ± SEM of 12 spheroids 21

analyzed per condition. 22

D) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with 10 µM 23

simvastatin (SIM) or solvent (DMSO) either alone or in combination with 0.5 24

mM mevalonate (MVL), 10 µM ubiquinone (Q10) or 5 mM N-acetylcysteine 25

(NAC) for 72 hours. Spheroids were fixed and histological sections were 26

analyzed for the presence of apoptotic cells by TUNEL staining. Images show 27

representative results of three spheroids analyzed per condition. 28

E) Quantitation of data shown in D. Data are presented as mean ± SEM of at 29

least 3 spheroids analyzed per condition. (**p<0.01, unpaired two-tailed 30


32

Figure 6: Mevalonate pathway activity is essential for pyrimidine 33

nucleotide biosynthesis and survival in colon cancer cells 34




31

A) Diagram showing the role of ubiquinone (Q10) in the conversion of 1

dihydroorotate to orotate during pyrimidine biosynthesis. 2


simvastatin (SIM) or solvent (DMSO) for 72 hours. For the last 16 hours, cells 4

were labelled with [U13C]-glucose before metabolites were extracted and 5

analyzed by LC-MS. Relative peak intensities (left graph) and total labelled 6

fractions (right graph) for UMP and are shown. Data show mean ±SEM of three 7

independent biological replicates. (*p<0.05; **p<0.01, unpaired two-tailed 8

Student’s t test) 9

C) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with 10 µM 10

simvastatin (SIM) or solvent (DMSO) for 72 hours either alone or in combination 11

with 0.5 mM mevalonate (MVL) or 10 µM ubiquinone (Q10). For the last 16 12

hours, cells were labelled with [U13C]-glucose before metabolites were 13

extracted and analyzed by LC-MS. Relative peak intensities (left graph) and 14

total labelled fractions (right graph) for UMP and are shown. Data show mean 15

±SEM of three independent biological replicates. (*p<0.05; **p<0.01, unpaired 16

two-tailed Student’s t test). 17

D) HCT116 p53+/+ or p53-/- cells were grown as SPC and treated with solvent 18

(DMSO) or 10 µM simvastatin (SIM) either alone or in combination with 19

nucleosides (150 µM each of cytidine, guanosine, adenosine, uridine and 50µM 20

of thymidine = NCL) for 72 hours. Spheroids were fixed and histological 21

sections were analyzed for the presence of apoptotic cells by TUNEL staining. 22

Data are presented as mean ±SEM of at least 3 spheroids analyzed per 23

condition. (*p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test). 24

E) HCT116 p53+/+ or p53-/- cells were grown as SPCs and treated with solvent 25

(DMSO), 10 µM simvastatin (SIM), 10 µM 5-fluorouracil (5-FU) or a combination 26

of the two for 72 hours. Spheroids were analyzed by TUNEL staining. Data are 27

presented as mean ±SEM of at least 3 spheroids analyzed per condition. 28

(*p<0.05, ***p<0.001, unpaired two-tailed Student’s t test). 29

30

Figure 7: Simvastatin reduces growth of p53-deficient tumor organoids 31

and blocks proliferation in Apc/p53-deficient Kras-transformed intestinal 32

crypts 33




32

A) Primary mouse intestinal cells derived from VillinCREERT2Apcfl/fl, 1

VillinCREERT2Apcfl/fl;p53fl/fl, VillinCREERT2Apcfl/fl;KrasG12D or 2

VillinCREERT2Apcfl/fl;p53fl/fl;KrasG12D animals were used to generate organoid 3

cultures. Organoids were treated with 10 µM simvastatin (SIM) either alone or 4

in combinations with 0.5 mM mevalonate (MEV) for 48 hrs. Images show 5

representative microscopic fields from three independent replicate cultures. 6

B) Quantitation of data shown in (A). Data are presented as mean ±SEM of 7

microscopic fields from three independent cultures. (*p<0.05; **p<0.01, 8

****p<0.0001, unpaired two-tailed Student’s t test). 9

C) Apcfl/fl;p53fl/fl or Apcfl/fl;p53fl/fl;KrasG12D organoids were treated with 10 µM 10

simvastatin either alone or in combination with 10 µM ubiquinone (Q10) or 11

nucleosides (150 µM each of cytidine, guanosine, adenosine, uridine and 50µM 12

of thymidine) for 48 hrs. Images show representative microscopic fields from 13

three independent replicate cultures. 14

D) Quantitation of data shown in (C). Data are presented as mean ±SEM of 15

microscopic fields from three independent cultures. (****p<0.0001, unpaired 16

two-tailed Student’s t test). 17

E) VillinCREERT2;Apcfl/fl and VillinCREERT2;apcfl/fl;KrasG12D/+ mice were treated 18

with a single intraperitoneal injection of 80 mg/kg of tamoxifen on one occasion 19

(VillinCreERT2Apcfl/fl KrasG12D/+), or on two consecutive days 20

(VillinCreERT2Apcfl/fl). From one day post-induction, mice were treated with a 21

daily dose of 50 mg/kg simvastatin (in 0.5% methyl cellulose/ 5% DMSO). After 22

four days, mice were sacrificed and intestinal mucosa was fixed, paraffin 23

embedded and histological sections were stained for BrdU incorporation. 24

Representative images are shown. 25

F) Three intestinal crypts for each genotype and treatment were scored for 26

BrdU positive cells. 27

G) Fraction of cholesterol and ubiquinone (CoQ9) containing deuterated water 28

in intestinal mucosa from the different genotypes 29

F+G (*p<0.05 unpaired two-tailed Student’s t test). 30




Kaymak et al. Figure 1

I

p53+

/+p5

3-/-

p53+

/+p5

3-/-

0

200

400

600

800Lactate

M+0M+1M+2M+3

pea

k in

tens

ity/p

rot

SPCMLC

0.0

0.2

PC

2 (1

8%)

-0.2

PC1 (80%)

spheroids

tumors

monolayer

p53NULLp53WTp53NULLp53WTp53NULLp53WT

MLC

SPC

T

A

J

B

0.0

0.5

1.0

1.5

2.0

M+3

/M+2

Aspartate

p53+

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3-/-

p53+

/+p5

3-/-

SPCMLCp5

3+/+p5

3-/-

p53+

/+p5

3-/-

0

50

100

150

200

250

Aspartate

M+0M+1M+2M+3M+4

pea

k in

tens

ity/p

rot

SPCMLC

E F

p53+

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p53-/

-

p53+

/+p5

3-/-

0

50

100

%B

rdU

inco

rpor

atio

nD

MLC SPC

HALLMARK_E2F_TARGETS

MLC p53+/+

0.00.10.20.30.40.50.6

Enr

ichm

ent s

core

(ES

)

0.70.8

NES 3.61q≤0.05

MLC SPCp53+/+

0.0-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

BROWNE_INTERFERON_RESP_GENES

-0.6 NES -3.22q≤0.05

-0.7-0.8

MANALO_HYPOXIA_UP0.0

-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

MLC SPC

-0.6

p53+/+

-0.7

NES -3.12q≤0.05

MANALO_HYPOXIA_UP0.0

-0.1

-0.2

-0.3

-0.4

-0.5Enr

ichm

ent s

core

(ES

)

MLC Tp53+/+

NES -1.93 q≤0.05

Tp53+/+

0.0-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

-0.6

BROWNE_INTERFERON_RESP_GENES

NES -2.28q≤0.05

HALLMARK_E2F_TARGETS

MLC T

0.00.10.20.30.40.50.6

Enr

ichm

ent s

core

(ES

)

0.70.8

p53+/+

NES 5.67q≤0.05

Hp53+/+ p53-/-

MLC SPC

p53+/+ p53-/-

VCL

CCND1

PDK1

G

150μm

p53+/+ p53-/-Ki67 Ki67Ki67 Ki67

150μM150μM

spheroids

50μM 50μM

p53+/+ p53-/-Ki67 Ki67

xenograft tumorsC

SPCp53-/-

0.0-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

-0.6 NES -3.47q≤0.05-0.7

-0.8

0.0-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

SPC

-0.6

p53-/-

NES -3.32q≤0.05

SPC

0.00.10.20.30.40.50.6

Enr

ichm

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core

(ES

) 0.7

p53-/-

NES 3.0 q≤0.05

T

0.00.10.20.30.40.50.6

Enr

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core

(ES

)

0.70.8

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0.0

-0.1

-0.2

-0.3

-0.4

-0.5

Enr

ichm

ent s

core

(ES

)

MLC Tp53-/-

NES -1.80 q≤0.05

MLC Tp53-/-

0.0-0.1-0.2-0.3-0.4-0.5

Enr

ichm

ent s

core

(ES

)

-0.6

NES -2.24 q≤0.05

MLCSPC

MLC

MLCMLC

MLC

34 kDa

49 kDa

124 kDa





0.0-0.1-0.2-0.3-0.4-0.5-0.6

Enr

ichm

ent s

core

(ES

)

-0.7

HORTON_SREBF_TARGETS

NES -2.64q≤0.05

p53+/+ p53-/-

SpheroidsHORTON_SREBF_TARGETS

NES -1.8q=0.057

0.0-0.1-0.2-0.3-0.4-0.5-0.6

Enr

ichm

ent s

core

(ES

)

p53+/+ p53-/-

TumorsA

B

mSREBP2

p53+/+ p53-/-MLC SPC

p53+/+ p53-/-

ACTIN

HMGCS1

p53+/+p53-/-

GSK3α/β


p53+/+ p53-/-

ACTIN

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M

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Rel

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1.5

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A e

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4

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Enr

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0.2

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10

TP53 status

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M

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CON CHIRSBRAPA

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CON CHIRSBRAPA

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p53

p53+

/+p53-/-

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ACSS2

C

H

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8

10

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Rel

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1.0 0.93 3.05 4.35

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1.0 1.01.06 2.25 2.88 0.59 0.87 0.86

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VCL

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SPCI

1.0 2.32 1.0 0.84

CONRAPA

MLC SPC MLC SPC MLC SPC MLC SPC MLC SPC

NES 0.67q=0.991

124 kDa

53 kDa

79 kDa

48 kDa

57 kDa

57 kDa

42 kDa

60-78kDa 42 kDa

32 kDa

32 kDa 60-78kDa

124 kDa

60-78kDa

124 kDa

42 kDa

0.0

0.5

1.0

1.5

2.0

2.5 *

51 kDa47 kDa51 kDa47 kDa




DMSOSIM

24h

0

1

2

3

Rel

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RN

A e

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p53+/+ p53-/-0

2

4

6

Rel

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n

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*

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DMSOSIM

72h

D

DMSO SIM SIM + MVL

p21-

/-

110µm 110µm 110µm

F

CDKN1A CDKN1A


Acetyl-CoA

HMG-CoA

Mevalonate

Farnesyl-PP

Squalene

Cholesterol

HMGCRstatins

A

p53+/+ p53-/-DMSO SIM +

MVLSIM DMSO

p21

spheroid

SIM +MVL

SIM

p21+/+ p21-/-DMSO SIM +

MVLSIM DMSO

p21

spheroid

SIM +MVL

SIM

E

G

150µm

150µm

150µm

150µm

150µm

150µm

DMSO MST MST + MVL

purple=TUNELblue=DAPI

C

p53+

/+p5

3-/-

B

DMSOMST

MST + MVL

DMSOMST

MST + MVL

0

100

200

300

400

TUN

EL p

ositiv

e ce

lls/a

rea p53+/+

p53-/-**

VCL

VCL124 kDa

21 kDa

124 kDa

21 kDa





Acetyl-CoA

Mevalonate

Farnesyl-PP

Squalene

Cholesterol

Geranyl geranyl-PPDolicholHeme AUbiquinone (CoQ10)

A

0

50

100

150

Cholesterol

0

20

40

60

Ubiquinone (CoQ10)

unlabelledlabelled

M+0M+1M+2M+3M+4M+5M+6M+7M+8M+9M+1

0M+1

1M+1

2M+1

3M+1

4M+1

5M+1

6M+1

7M+1

8M+1

9M+2

0M+2

1M+2

2M+2

3M+2

4M+2

5M+2

6M+2

7M+2

8M+2

9M+3

0M+3

1M+3

2M+3

3M+3

4M+3

5M+3

6M+3

7M+3

8M+3

9M+4

0M+4

1M+4

2M+4

3M+4

4M+4

5M+4

6M+4

7M+4

8M+4

9M+5

00

1

2

3

1020304050

Ubiquinone (CoQ10)

p53+/+ DMSOp53-/- DMSO

p53+

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p53+

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p53-/

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p53-/

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p53+

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pea

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nsity

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t

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Glucose

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GF

ED

B

M+0 M+2 M+4 M+60.0

0.2

0.4

0.6p53+/+p53-/-

p53+/+ p53-/-0

100

200

300

400

7-DHC

p53+/+ p53-/-0

20

40

60

80

100

120

pea

k in

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ity (x

107 )/

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Cholesterol

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*H

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*

012345

50

100

M+0M+1M+2M+3M+4M+5M+6M+7M+8M+9M+1

0M+1

1M+1

2M+1

3M+1

4M+1

5M+1

6M+1

7M+1

8M+1

9M+2

0M+2

1M+2

2M+2

3M+2

4M+2

5M+2

6M+2

7

p53+/+ DMSOp53-/- DMSO

pea

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ity/p

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Cholesterol

pea

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labelled

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pea

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7-DHC





A B

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p53+

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p53-/

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p53-/

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0

100

200

300

400

Aspartate

M+0M+1M+2M+3M+4

C

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100

200

300

400

500

Time (minutes)

OC

R (p

mol

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0 50 100 150 2000

100

200

300

400

500

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OC

R (p

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are

a)

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150µm150µm150µm

150µm 150µm 150µm

D

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SIM +

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Q10

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100

200

300

400

500

TUN

EL p

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lls/a

rea **** ******p53+/+

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SIM + NAC

150µm

150µm

***

E

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SIM + MVL

150µm

150µm

pea

k in

tens

ity/p

rot

Q10

ATPSynthase

III IVI

II

cyt C

e- e-

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e-

H2O1/2 O2+H+

e-

matrix

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α-KG Malate

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intermembrane space




DMSO SIM

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DMSO SIM

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100

200

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rea

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SIM +

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0

100

200

300

400

TUN

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ll/are

a

**

*

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0

10

20

30

UMP

M+0M+1M+2M+3M+4M+5M+6M+7M+8M+9

0

2

4

6

8

10

UMP

**

*

p53+

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p53-/

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p53+

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p53-/

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p53+

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peak

inte

nsity

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t

pea

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tens

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rot

C

pea

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labelled

0

1

2

3

4

5

*

n.s.****

*

****

p53+

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p53+

/+ SIM

p53+

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+ MVL

p53+

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5

10

UMP UMP p

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t

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B

***

M+0M+1M+2M+3M+4M+5M+6M+7M+8M+9


Q10

ATPSynthase

DHODH

Dihydroorotatate Orotate

UMP

III IVI

II

cyt C

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matrix

carboamoylaspartate

A





A

B

DMSOSIM

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10

SIM+N

UCL0

500

1000

1500

Apcfl/fl p53fl/fl

****

****

****

DMSOSIM

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10

SIM+N

UCL0

500

1000

1500

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****

********

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200µm 200µm

200µm 200µm

200µm

200µm 200µm

200µm

200µm

200µm

200µm

200µm

200µm

200µm

200µm

200µm

200µm

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fl/fl p

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fl

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****

**

sphe

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200µm 200µm 200µm Apc

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0

20

40

60

80

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Apcfl/f

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Apcfl/f

l

Apcfl/f

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Apcfl/f

l KrasG12

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Cholesterol Ubiquinone (CoQ9)G

* * **

Apcfl/f

l

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D/+ + SIM




Published OnlineFirst November 19, 2019.Cancer Res Irem Kaymak, Carina Ramona Maier, Werner Schmitz, et al. exposed to metabolic stress

cellspyrimidine synthesis and survival in p53-deficient cancer Mevalonate pathway provides ubiquinone to maintain

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