We also performed a correlation analysis independent
We also performed a correlation analysis independent of annotated gene sets to identify potentially novel genes that are co-expressed with DHODH. Using the top 100 DHODH co-expressed genes in COAD, LGG, STAD, and PAAD diseases, we identified four common genes across all four diseases (Fig. 9). These genes are DNA polymerase delta 2 (POLD2), Tu translation elongation factor, mitochondrial (TUFM), peter pan homolog (PPAN), and ribosomal subunit RNA processing 9 (RRP9). POLD2 is an essential subunit of DNA polymerase delta, a complex that is involved in DNA replication and repair and is one of three major polymerases active in S-phase. DHODH may be connected to POLD2 primarily through providing a sufficient pool of pyrimidine nucleotides. Recent studies have connected DNA repair with de novo pyrimidine biosynthesis, although these have been through CAD (Brown, Spinelli, Asara, & Toker, 2017; Givechian, Garner, Garban, Rabizadeh, & Soon-Shiong, 2018). As its name suggests, TUFM participates in protein translation in the mitochondria. During the elongation phase, TUFM forms a complex with GTP and a charged tRNA. Upon correct pairing of the codon:anticodon, GTP is hydrolyzed and TUFM:GDP leaves the ribosome. In the context of cancer, the role of TUFM is not well understood. TUFM was identified as a target gene of Ronin, a transcriptional regulator important for embryonic stem cell self-renewal (Dejosez et al., 2010). Interestingly, reversible phosphorylation of the bacterial homolog of TUFM inhibited protein synthesis in quiescent cells, and this phosphorylation site is conserved in the mammalian homolog (Pereira, Gonzalez Jr., & Dworkin, 2015). TUFM has been shown to prevent the epithelial-to-mesenchymal transition and was observed to be successively down-regulated with increases in tumor stage in lung cancer (He et al., 2016). TUFM seems to play a role in differentiation status as does DHODH (see above). However, in colon cancer, TUFM appears to be considerably overexpressed and high expression correlates with poor prognosis (Shi et al., 2012). Additionally, TUFM may play a role in the transition of normal Estradiol Benzoate to benign and malignant tumors in colorectal tissues (Xi et al., 2017). In ovarian cancer, TUFM was upregulated in cell lines and tumor samples that were partially resistant to standard-of-care chemotherapies and could therefore act as a potential biomarker in predicting sensitivity to treatment (Cruz et al., 2017). PPAN resides in the nucleoli and participates in the maturation of 45S pre-RNA. PPAN expression can be stimulated by WNT signaling to trigger ribosomal biogenesis (Bugner, Tecza, Gessert, & Kuhl, 2011; Pfister, Keil, & Kuhl, 2015). Interestingly, high concentrations of leflunomide showed modulation of WNT/β-catenin signaling and was synergistic with an inhibitor of WNT secretion (Chen, Huang, et al., 2016). However, the concentration of leflunomide that was necessary to see these effects was much higher than what is required to see inhibition of DHODH, so it is likely that these observations are a result of an off-target effect of leflunomide (see below). It has been suggested that initiation of ribosomal biogenesis is a result of growth factor stimulation, as it occurs before S-phase entry (Sriskanthadevan-Pirahas, Lee, & Grewal, 2018). Overexpression of mutant RAS caused an increase in the expression of PPAN, which could be reversed by knockdown of MYC (Sriskanthadevan-Pirahas et al., 2018). However, in the presence of a MEK inhibitor and MYC overexpression, rRNA synthesis levels were not maintained, suggesting that RAS regulates ribosomal biogenesis through additional mechanisms - the mTOR pathway is one such possibility (Ghosh, Rideout, & Grewal, 2014; Sriskanthadevan-Pirahas et al., 2018). PPAN also localizes to the mitochondria where it acts as an anti-apoptotic factor independently of p53 (Pfister et al., 2015). Additionally, PPAN has been shown to be an important component of cellular differentiation for Xenopus laevis development (Bugner et al., 2011). Finally, the RRP9 gene codes for a vital U3 snoRP core protein known as U3-55 k, which is essential for ribosomal RNA synthesis (Chen, Blank, et al., 2016). Ribosomal biogenesis factors are predominantly controlled by S6 kinases, with RRP9 being one of them (Chauvin et al., 2014). S6 kinases are activated through phosphorylation by mTOR, itself a kinase that regulates cell growth and metabolism from a plethora of environmental stimuli (Laplante & Sabatini, 2012). Interestingly, mTOR has been shown to be associated with the histone deacetylase SIRT7, and SIRT7 was recently shown to deacetylate RRP9 (Chen, Blank, et al., 2016; Tsai, Greco, & Cristea, 2014). Both PPAN and RRP9 play a significant role in the regulation of ribosomal RNA synthesis and may be connected to DHODH through UMP synthesis as well as through upstream oncogenic signaling pathways. Although co-expression does not prove DHODH is directly interacting with these four targets, one might hypothesize a common functional relationship or transcriptional regulation that is worth investigating.