Michael V. Berridge

Figure S4 from Mitochondrial Transfer Rescues Respiration to Support De Novo Pyrimidine Biosynthesis and Tumor Progression

Properties related to parental, rho0 and rho0 AOX cells

Figure S5 from Mitochondrial Transfer Rescues Respiration to Support De Novo Pyrimidine Biosynthesis and Tumor Progression

Gating strategy for flow cytometry

Figure S3 from Mitochondrial Transfer Rescues Respiration to Support De Novo Pyrimidine Biosynthesis and Tumor Progression

Gating strategy for flow cytometry

Cancer cells with severe defects in mitochondrial DNA (mtDNA) can import mitochondria via horizontal mitochondrial transfer to restore respiration. Mitochondrial respiration is necessary for the activity of dihydroorotate dehydrogenase (DHODH), an enzyme of the inner mitochondrial membrane that catalyzes the fourth step of de novo pyrimidine synthesis. In this study, we investigated the role of de novo synthesis of pyrimidines in driving tumor growth in mtDNA-deficient (ρ0) cells. Although ρ0 cells grafted in mice readily acquired mtDNA, this process was delayed in cells transfected with alternative oxidase (AOX), which combines the functions of mitochondrial respiratory complexes III and IV. The ρ0 AOX cells were glycolytic but maintained normal DHODH activity and pyrimidine production. Deletion of DHODH in a panel of tumor cells completely blocked or delayed tumor growth. The grafted ρ0 cells rapidly recruited tumor-promoting/stabilizing cells of the innate immune system, including protumor M2 macrophages, neutrophils, eosinophils, and mesenchymal stromal cells (MSC). The ρ0 cells recruited MSCs early after grafting, which were potential mitochondrial donors. Grafting MSCs together with ρ0 cancer cells into mice resulted in mitochondrial transfer from MSCs to cancer cells. Overall, these findings indicate that cancer cells with compromised mitochondrial function readily acquire mtDNA from other cells in the tumor microenvironment to restore DHODH-dependent respiration and de novo pyrimidine synthesis. The inhibition of tumor growth induced by blocking DHODH supports targeting pyrimidine synthesis as a potential widely applicable therapeutic approach.Significance:Mitochondrial complexes III and IV promote tumor progression by supporting de novo pyrimidine synthesis, requiring cancer cells devoid of mitochondrial DNA to recruit mitochondria from source cells to restore respiration in order to form tumors.

DHODH-dependent respiration delays acquisition of mtDNA in ρ0 cells. A, 4T1 ρ0 and 4T1 ρ0 AOX cells were subcutaneously injected into Balb/c mice at 106 per animal. On days 3, 5, 10, 15, 20, and 25, tumor tissue was retrieved from the mice, and sublines were established. Parental cells and sublines derived from 4T1 ρ0 and 4T1 ρ0 AOX cells were assessed for mtDNA using qPCR. B, Parental, ρ0 D15, and 4T1 ρ0 AOX D25 cells were assessed for the distribution of mtDNA polymorphism in tRNAArg, D-loop, and 16S rRNA in single cells of lines. C, Heatmap illustrates mRNA expression of mitochondrial and nuclear-encoded OXPHOS genes in the cell lines indicated. The patterns were generated by the median of clustering analysis of bulk RNA sequencing data. The vertical axis represents individual samples, whereas the horizontal axis shows individual transcripts. D, Protein expression of AOX was assessed in parental cells, and sublines were derived from ρ0 AOX. E and F, Routine and DHODH-dependent respiration of parental, ρ0, ρ0 AOX, and the derived sublines was evaluated using the Oxygraph. G and H, DHODH activity was assessed in parental and ρ0, ρ0 D3, ρ0 D15, and ρ0 AOX sublines and NAD+/NADH ratio for parental ρ0, ρ0 AOX. I, Parental and ρ0, ρ0 D3, ρ0 D5, ρ0 D15, ρ0 AOX, ρ0 AOX D15, ρ0 AOX D20, and ρ0 AOX D25 cells were evaluated by native blue gel electrophoresis for the assembly of respiratory complexes and supercomplexes using antibodies to relevant subunits as shown. J, Balb/c mice were grafted subcutaneously with parental, ρ0, and ρ0 AOX cells (106 per animal), and tumor volume was assessed on days as indicated using calipers (n = 3 for each group). Data are derived from at least three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA and the GraphPad Prism 8 software, considering differences with the P value of ≤0.05 as statistically significant. *, P value of 0.01 to 0.05; **, P value of 0.001 to 0.01; ***, P value of 0.0001 to 0.001; ****, P < 0.0001.

Absence of DHODH suppresses efficient tumor growth. A, Expression of DHODH in parental and DHODHKO cells (HCT116, MDA-MB-231, and PaTu 8902 cell lines) was assessed by Western blotting analysis. B–D, Routine and DHODH-dependent respiration was evaluated in parental and DHODHKO cells (B and C), as well as their proliferation (number of cells; D) at 72 hours in the presence and absence of uridine in the media. E, NSG mice were grafted subcutaneously with parental and DHODHKO cells (106 per animal), and tumor volume was assessed using calipers (n = 6 for each group). The experiments are representative of at least three independent repetitions (n ≥ 3). Statistical analysis was performed using an unpaired t test with GraphPad Prism 8 software, considering differences with a P value of ≤ 0.05 as statistically significant. Images are representative of three independent experiments of three separate biological replicates. *, P value of 0.01 to 0.05; **, P value of 0.001 to 0.01.