We previously showed that miR-23b* regulates proline oxidase (POX ) expression post-transcriptionally (Wei et al., Oncogene 2010). Interestingly, miR-23b, product of the same transcript as miR-23b*, inhibits the expression of glutaminase and the upregulation of glutaminase by c-Myc is mediated by its decrease of miR-23b. The well-recognized interconversions of glutamine and proline and the sharing of microRNAs from the same trascript was of considerable interest. To pursue these studies, we collaborated with Dr. Chi V. Dang of Johns Hopkins who has focused on the role of glutamine and its regulation by c-Myc (heretofore referred to as Myc) in tumor cells. Our current studies suggest that some of these cellular effects are due, in part, to Myc regulation of proline metabolism. Proline oxidase (POX), catalyzing the first step in proline catabolism is induced by p53 and PPARgamma. POX is a mitochondrial, metabolic tumor suppressor inhibiting proliferation and inducing apoptosis through the generation of reactive oxygen species. Recently we reported that the downregulation of POX expression in human kidney tumors is due to increased miR-23b* which inhibits POX translation. MiR-23b* is processed from the same transcript as miR-23b;the latter inhibits the translation of glutaminase, an enzyme mediating glutaminolysis. We now report that the oncogenic transcription factor Myc suppresses POX expression. Using human P493 B lymphoma cells which contain a tetracycline-repressible MYC construct and human PC3 prostate cancer cells with high levels of Myc, we showed that Myc suppressed POX not only at the transcriptional level, but more robustly at the translational level;the latter is mediated by upregulation of miR-23b*. The inhibition of growth in the absence of c-Myc was partially reversed by POX knockdown with siRNA, indicating the importance of suppression of proline catabolism in Myc-mediated proliferation and cell survival. Interestingly, Myc not only decreased proline catabolism and increased glutaminolysis, but also markedly increased the conversion of glutamine to proline as monitored by the production of 13C5,15N-proline from 13C5,15N2-glutamine. In contrast to its effects on POX, a proline-degradation enzyme, Myc markedly increased the enzymes of proline biosynthesis, including pyrroline-5-carboxylate synthase and pyrroline-5-carboxylate reductase 1. Although the conversion of glutamine to another nonessential amino acid, proline, may facilitate protein synthesis, preliminary results suggest that the biosynthesis of proline, like the degradation of proline, provides a metabolic switch modulating cell proliferation and metabolism. We pursued the possible metabolic link using both molecular and metabolic methodologies. Using P493 B lymphoma cells expressing Myc under the control of a tet-off promoter, we showed that Myc upregulates the expression not only of glutaminase, but also P5C synthase and P5C reductase 1. Additionally, not only does Myc downregulate POX, but also P5C dehydrogenase. Thus, Myc downregulates the enzymes of proline degradation and upregulates the enzymes of proline synthesis. Since we have previously described redox-mediated coupling between proline synthesis and glucose metabolism, we are examining the metabolic interlock catalyzed by the enzymes converting glutamine to proline. The interlock may may augment production of ribose for nucleotides and may augment glycolysis, i.e. the Warburg Effect while sparing pyruvate for biosynthesis. We also investigated the mechanism for the ROS signaling mediated by POX. Previously, we had considered that oxygen can be reduced by proline-derived electrons via FADH at the active site of POX. However, signaling ROS appears to be primarily from site III of the mitochondrial electron transport chain (ETC). Others using Sacchromyces have shown that POX electrons are transferred to ubiquinone. We showed that this is the case in human cancer cells. Furthermore, we showed that the presence of ubiquinone markedly increased POX catalytic rate as measured by the production of pyrroline-5-carboxylate from proline. Importantly, with the use of ETC inhibitors, we showed that ROS is generated from complex III. Since complex III can generate superoxide in mitochondrial matrix as well as in the intermembrane space, we co-transfected DLD-POX cells with SOD1 which can inhibit superoxide in the intermembrane space. Previous work showed that the mitochondrial apoptotic cascade can be blocked by co-expression of SOD2, so we tested other targets of POX using SOD1. First, we showed that ROS was inhibited, at least in part, by SOD1 expression. More importantly, we showed that the POX-mediated induction of Beclin, a protein activating the autophagic cascade, was inhibited by SOD1. Thus, the electrons from POX can generate ROS not only in the mitochondrial matrix, but also in the intermembrane space and the cytosol to contribute to redox-mediated signaling. These findings suggest that the regulated activity of proline oxidase (dehydrogenase) is an important source of ROS signaling.
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