We therefore propose a scenario based
We therefore propose a scenario based on our experimental models and depicted schematically in Figure 7. In fully respiring cells, CI, CII, and DHODH transfer electrons to CoQ, which are then forwarded to CIII. This latter complex transfers electrons to CIV, which then produces water at the expense of molecular oxygen. CI, CIII, and CIV increase the proton-motive force in the form of ΔΨm,i that, among other functions, drives formation of ATP catalyzed by CV. In ρ0 cells, mtDNA is missing, resulting in the collapse of CI, CIII, and CIV, and in the assembly of CII and sub-CV. Under this scenario, DHODH cannot convert DHO to orotate, since CoQ cannot transfer electrons to CIII. ATP is generated by glycolysis and ΔΨm,i maintained by ATP cleavage by the ATPase activity of sub-CV; ρ0 Bioactive Compound Library form tumors only after acquisition of mtDNA from the host and restoration of respiration (Tan et al., 2015, Dong et al., 2017). DHODHKO cells lack tumor-forming capacity and cannot be “repaired” by mtDNA acquisition, because the absence DHODH precludes conversion of DHO to orotate. On the other hand, ρ0 AOX cells have normal DHODH-dependent respiration, since electrons generated by conversion of DHO to orotate are captured by CoQ and transferred to AOX, which substitutes for the combined activity of CIII and CIV. These cells efficiently form tumors. Finally, ATP5BKO cells that are highly glycolytic have normal DHODH-dependent respiration and form tumors faster than ρ0 cells. Together, our findings demonstrate an important role of DHODH and CoQ redox-cycling in tumor formation in cells with damaged mtDNA, resulting in mtDNA acquisition and restoration of respiration independent of OXPHOS-derived ATP. A typical consequence of OXPHOS dysfunction is the auxotrophy for pyruvate and uridine. Pyruvate as an exogenous electron acceptor is required to produce aspartate, a precursor of de novo pyrimidine biosynthesis, and uridine to complement defective DHODH-linked pyrimidine synthesis via salvage pathways (Loffler, 1980, King and Attardi, 1988, King and Attardi, 1989, Birsoy et al., 2015, Sullivan et al., 2015). We observed auxotrophy in D0–D15 cells, which was relieved by OXPHOS reconstitution following mtDNA transfer and prior to tumorigenesis, or in the case of uridine, by AOX expression. Given the efficacy of AOX-mediated restoration of tumorigenicity, this suggests that the DHODH dysfunction-induced defect in de novo pyrimidine synthesis could be a major obstacle for in vivo growth of respiration-compromised tumor cells, and that pyruvate might not be the limiting factor. Indeed, we consistently measured substantial pyruvate at the site of tumor growth throughout the course of the experiment, and levels of pyruvate in serum of mice and cancer patients were sufficient to support proliferation of ρ0 cells in vitro, although suboptimal. In addition, it has been reported that cancer cells deficient in the CII subunit SDHB are addicted to pyruvate (Cardaci et al., 2015, Lussey-Lepoutre et al., 2015), yet they readily form tumors in mice (Guzy et al., 2008, Bezawork-Geleta et al., 2018). Importantly, SDHB-deficient neoplasias are relatively common in humans and have unfavorable prognosis (King et al., 2011). These data suggest that pyruvate may not be limiting in vivo under all circumstances. To begin to place our results into context, we suggest that de novo pyrimidine biosynthesis, driven by functional OXPHOS, is crucial for tumor growth. This notion is supported by the failure of DHODHKO cells to form tumors, and by the recovery of tumorigenicity when AOX is expressed in ρ0 cells. Our results also suggest that DHODH represents a bottleneck for pyrimidine synthesis in non-respiring cells, although they do not rule out other limitations that might constrain in vivo growth in the absence of functional OXPHOS. While a deficiency in DHODH disrupts the pyrimidine biosynthesis pathway in a defined manner, expression of AOX restores DHODH activity as well as reactivating CoQ redox-cycling. AOX might also impact on additional metabolic pathways converging on the CoQ pool. It seems unlikely, however, that AOX indirectly supports the synthesis of aspartate via nascent CI. AOX expression did not affect the NADH/NAD+ ratio or content of aspartate in ρ0 cells, just as ρ0 AOX cells are auxotrophic for pyruvate. Previous studies have reported that aspartate can be limiting for tumor growth in vivo due to its inefficient import into cells and proposed that CI inhibition constrains tumor growth by limiting aspartate biosynthesis (Garcia-Bermudez et al., 2018, Sullivan et al., 2018). However, while growth retardation by CI inhibition was complete in vitro in the absence of pyruvate, it was incomplete when CI was targeted in vivo (Sullivan et al., 2018). The remaining proliferation in that case could perhaps be supported by extracellular pyruvate. To summarize, while this and other reports clearly show that pyrimidine biosynthesis is essential for tumor growth in multiple cancer models, the identity of the rate-limiting steps in various conditions deserves further investigation, as it will likely be affected by the environment and by the experimental model employed.