br Results br Discussion The importance of mitochondria for
Discussion The importance of mitochondria for the initiation and progression of tumorigenesis is now emerging. It is evident that, despite the well-known Warburg effect, tumors have active mitochondrial bioenergetic metabolism (Marin-Valencia et al., 2012, Hensley et al., 2016). Disruption of the ETC shows promise in cancer therapy (Rohlena et al., 2011, Zhang et al., 2014, Rohlenova et al., 2017). Cells with deleterious mtDNA mutations fail to form tumors (Park et al., 2009), and genetic ablation of OXPHOS restrains tumorigenesis (Weinberg et al., 2010). mtDNA-depleted (ρ0) tumor enzastaurin present a particularly instructive case. Having no functional OXPHOS, these cells cannot form tumors in mice unless they acquire host mtDNA (Tan et al., 2015) via horizontal transfer of whole mitochondria from the stroma (Dong et al., 2017). In this way, ρ0 cells give rise to palpable tumors only after a long initial lag period (Tan et al., 2015, Dong et al., 2017). While these studies suggest that functional OXPHOS is necessary for tumorigenesis, previously published data did not pinpoint exactly which aspect of OXPHOS function is essential. The key finding of the current study is that DHODH-driven pyrimidine biosynthesis, rather than OXPHOS-mediated ATP production, is essential for tumorigenesis. To further explore this issue, we developed a unique model that allowed us to characterize the link between OXPHOS function and tumor formation in unprecedented temporal detail. We followed the events associated with tumorigenesis of mtDNA-depleted ρ0 cells in mice during the initial lag period and throughout the various stages of tumor progression and demonstrate that the appearance of tumors coincides with OXPHOS reconstitution at days 15–25 post-grafting, after the end of a “dormant” period when mtDNA is replenished and OXPHOS machinery re-assembled. This detailed investigation was possible because cancer cells isolated at various time points in the 4T1ρ0 tumor model are remarkably stable in culture, and their properties, such as the level of mtDNA, respiration, or ETC assembly, did not change with time. This unexpected stability, observed by others in a different model (Picard et al., 2014), can be explained by the absence of selection pressure in the rich culture media containing uridine/pyruvate (see below). Surprisingly, we found that the best known OXPHOS function, production of ATP by ATP synthase, is not essential for tumorigenesis. While increased mitochondrial contribution to total ATP production was concurrent with OXPHOS reconstitution, ATP synthase assembly and appearance of tumors, the total ATP content in the cells and energy charge were not, in general, significantly decreased, but maintained by glycolysis with its much faster kinetics compared with OXPHOS (Koppenol et al., 2011). Most strikingly, cells deficient in ATP synthase were found to readily produce tumors. This suggests that mitochondrial ATP production is not limiting for tumor growth, at least at its earlier stages. Instead, we found that the important OXPHOS-related feature that promotes tumorigenesis is de novo pyrimidine synthesis, directly driven by respiration via DHODH, which converts DHO to orotate. This was clearly demonstrated by the failure of DHODH-deficient cells to form tumors, despite the fact that these cells show otherwise fully functional OXPHOS and normal ATP levels. Furthermore, the DHO/orotate ratio was increased in DHODHKO cells and non-respiring D0–D10 cells, and formation of UMP from glutamine was compromised. This indicates that the enzyme is inactive but is reactivated before tumors appear. The central role for DHODH in tumorigenesis is consistent with a recent report demonstrating upregulation of DHODH during the course of UV-induced skin tumorigenesis and its functional role therein (Hosseini et al., 2018). The link between OXPHOS and DHODH is maintained by CoQ redox-cycling (Gregoire et al., 1984, Ayer et al., 2015). Electrons, removed from DHO by DHODH, are transferred to CoQ yielding CoQH2, which is then re-oxidized at CIII. The CoQ redox cycle is broken in the absence of CIII/CIV activity when OXPHOS is non-functional due to mtDNA deficiency. This removes the only practical means of CoQ recovery in mammalian cells, while maintaining upstream sources of electrons for CoQ reduction such as CII or DHODH itself. These sources of electrons reduce CoQ to the maximal attainable level before they stall due to the lack of the electron acceptor. Indeed, in non-respiring D0–D10 cells CoQ was present predominantly in the reduced form, whereas the CoQH2/CoQ redox state decreased and approached parental cell values before the onset of tumor formation, coinciding with CIII/CIV reconstitution. AOX expression in ρ0 cells was sufficient to reactivate DHODH-driven respiration, normalize the CoQH2/CoQ and DHO/orotate ratios, reinitiate UMP synthesis both in vitro and in vivo, and restore tumorigenicity in the absence of mtDNA. Importantly, live labeling with 13C5,15N2-Gln confirmed the in vitro results in a mouse model derived from 4T1 ρ0 AOX cells 14 days after grafting the cells, i.e., at the stage when respiration is not recovered and tumor formation is largely dependent on AOX-propelled DHODH (Figures 6M–6P). It thus seems that lack of redox-cycling of CoQ, not OXPHOS deficiency per se, restricts tumorigenesis.