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  • As a functional ETC is

    2019-11-14

    As a functional ETC is required for DHODH catalysis (Loffler, Jockel, Schuster, & Becker, 1997; Rawls, Knecht, Diekert, Lill, & Loffler, 2000; Zameitat, Freymark, Dietz, Loffler, & Bolker, 2007), DHODH depends on the mitochondrial ETC to generate adequate concentrations of ubiquinone (Fig. 2). Cells lacking a fully functional mitochondrial ETC have an impaired ability to produce UMP (King & Attardi, 1989; Lane & Fan, 2015; Morais, Desjardins, Turmel, & Zinkewich-Peotti, 1988). Without a functional mitochondrial ETC, suramin must be supplemented with pyruvate and uridine to support proliferation (King & Attardi, 1989). This phenomenon is presumed to occur due to limited ubiquinone generation for DHODH catalysis. A cell line containing a dysfunctional mitochondrial ETC has been reported to be transformed with alternative ubiquinone oxidative enzymes. Once transformed, the cells were able to sustain growth in uridine- and pyruvate-free media (Perales-Clemente et al., 2008). This result suggests that cells containing a dysfunctional ETC may be indirectly inhibiting DHODH and depleting intracellular pyrimidine nucleotides necessary for growth. Previously, decreased DHODH activity has been observed with small molecule inhibitors of the ETC targeting cytochrome c oxidase (Beuneu et al., 2000). Additionally, inhibition of either DHODH or mitochondrial respiration complexes have resulted in similar cellular responses. For example, inhibition of either target induced p53 up-regulation (Khutornenko et al., 2010; Ladds et al., 2018). Collectively, these studies highlight that DHODH catalysis is dependent on a functional ETC or the presence of ubiquinone. This relationship has generated interest in the possible role DHODH may play in reactive oxygen species (ROS) homeostasis. The connection between DHODH and ROS in cancer is not well understood. Mitochondrial ROS are known to influence cellular proliferation and DHODH-catalyzed oxidation may affect mitochondrial ROS (Diebold & Chandel, 2016; Idelchik, Begley, Begley, & Melendez, 2017). A previous study showed that isolated mitochondria were capable of generating ROS through DHODH and that radical production was diminished by DHODH inhibitors (Hey-Mogensen, Goncalves, Orr, & Brand, 2014). This result implies that DHODH catalysis contributes to elevated ROS levels. However, knockdown of DHODH has been shown to increase the production of ROS and decrease the mitochondrial membrane potential (Fang et al., 2013). Furthermore, DHODH inhibitors were found to decrease the mitochondrial membrane potential (Koundinya et al., 2018). These conflicting results complicate the understanding of DHODH\'s role in ROS generation. It is possible that inhibitors of DHODH alter redox homeostasis in a context-dependent manner. It was found that cell lines most sensitive to DHODH inhibition consistently generated the lowest amount of ROS (Mohamad Fairus et al., 2017). This data implies that an antioxidant in combination with a DHODH inhibitor may be synergistic or at least additive. However, a DHODH inhibitor, teriflunomide, did not abrogate cell growth when the antioxidant pyrrolidine dithiocarbamate was co-administered (Hail Jr., Chen, Kepa, & Bushman, 2012). These studies highlight a correlation between ROS and DHODH catalysis, but the functional consequences appear to be context-dependent.
    Regulation of DHODH activity in cancer Regulation of DHODH activity occurs primarily through activation of de novo pyrimidine biosynthesis via the CAD complex. When cells are not preparing for growth, flux through the de novo pyrimidine pathway is slow and functions to generate RNA nucleotides primarily for protein synthesis (Coleman, Suttle, & Stark, 1977; Evans & Guy, 2004; Jones, 1980). In this state, flux is controlled through product feedback inhibition as high concentrations of uridine inhibit CAD. However, when cells prepare to divide, phosphorylation of CAD alters its affinity for uridine to overcome feedback inhibition (Carrey, Campbell, & Hardie, 1985; Sahay, Guy, Liu, & Evans, 1998). Selective phosphorylation of CAD at specific residues regulates flux through the de novo pyrimidine biosynthesis pathway (Huang & Graves, 2003). Flux through the pathway is increased when T456 of CAD is phosphorylated by mitogen-activated protein kinase (MAPK) or mechanistic target of rapamycin 1 complex (mTORC1) via S6 kinase (Ben-Sahra, Howell, Asara, & Manning, 2013; Robitaille et al., 2013; Sigoillot, Berkowski, Sigoillot, Kotsis, & Guy, 2003). After sufficient concentrations of nucleotides are reached, protein kinase A phosphorylates S1406 of CAD to down-regulate nucleotide biosynthesis (Kotsis et al., 2007). Overexpression of enzymes controlling CAD phosphorylation, such as MAPK or mTORC1, leads to increased flux through the pathway in cancer. For example, the breast cancer cell line MCF7 overexpressing MAPK kinase causes a 4-fold increase in the rate of de novo pyrimidine biosynthesis (Sigoillot, Sigoillot, & Guy, 2004). Modulation of flux through the pathway by mTORC1 has not been confirmed experimentally. In addition to phosphorylation, CAD localization also affects the rate of flux through the de novo pyrimidine pathway. Hindrance of CAD nuclear import has been found to decrease the rate of pyrimidine synthesis by 21% and decrease nucleotide concentrations by nearly 60% (Sigoillot et al., 2005). However, it is unclear how and why CAD localization affects de novo pyrimidine biosynthesis. Nonetheless, CAD\'s phosphorylation and localization play a significant role in regulation of DHODH activity.