Proline metabolism and cancer

Abstract

Proline plays a special role in cancer metabolism. Proline oxidase (POX), a.k.a. proline dehydrogenase (PRODH), is among a few genes induced rapidly and robustly by P53, the tumor suppressor. Ectopic expression of POX under control of tet-off promoter initiated mitochondrial apoptosis. The mechanism activated by POX is mediated by its production of ROS. In immunodeficient mice, POX overexpression markedly retarded growth of xenograft tumors. In human tumors of the digestive tract and kidney, POX was markedly decreased, suggesting that the suppressive effect of POX was downregulated. This was not due to POX gene mutations or hypermethylation. Instead, a microRNA, miR-23b*, expressed at high levels in tumors, was a potent inhibitor of POX expression. Furthermore, antagomirs of miR-23b* reversed the downregulated expression of POX and its tumor-suppressive effect, thereby providing a therapeutic strategy. POX not only responds to genotoxic stress, but also to inflammatory and metabolic stress. Depending on microenvironmental and temporal factors, POX can mediate oppositely-directed responses-programmed cell death, on the one hand, and survival, on the other.

Figure 1.

A schematic of the proline metabolic pathway and the “proline cycle.” Glutamic-gamma-semialdehyde (GSA) is in spontaneous equilibrium with its cyclized tautomer, delta¹-pyrroline-5-carboxylate (P5C); it is the obligate intermediate in the carbon interchange between the urea cycle and TCA cycle. The proline cycle transfers reducing equivalents to mitochondria; it is catalyzed by proline oxidase and P5C reductase. Proline oxidase is bound to mitochondrial inner membranes. Recent discoveries have identified 3 isozymes of P5C reductase (not shown) with distinct intracellular localization allowing for putative redox transfers within and between cellular compartments (9). The abbreviations shown are as follows: PRO, L-proline; P5C, delta¹-pyrroline-5-carboxylate; GLU, glutamate; GSA, glutamic-gamma-semialdehyde; ORN, ornithine; alpha-KG, alpha -ketoglutarate; TCA, tricarboxylic acid; NAD(P), nicotinamide adenine dinucleotide (phosphate). Enzymes are designated as follws: 1, P5C synthase; 2, P5C reductase (generic designation, there are 3 isozymes); 3, proline oxidase, a.k.a. proline dehydrogenase; 4, P5C dehydrogenase; 5, ornithine aminotransferase; 6, spontaneous.

Figure 2.

Schematic representation of the functions of proline oxidase (POX) in tumor suppression. It must be emphasized that the gene encoding POX is not a classical tumor suppressor gene. The markedly decreased or absent expression of POX in human tumors is not due to a somatic mutation or hypermethylation. Instead, POX is suppressed by a microRNA, miR-23b* which is markedly overexpressed in cancer. POX is induced by p53 and PPARgamma, reflecting genotoxic and inflammatory stress, respectively; it catalyzes the transfer of electrons from proline with intervening electron acceptors to reduce oxygen to form ROS (reactive oxygen species). ROS, by a number of mechanisms, increase proliferation, block the cell cycle and initiate apoptosis. Alternatively, alpha-KG, produced from proline by sequential dehydrogenations, destabilizes HIF-1alpha to block its proliferative signaling. These mechanisms suppressing tumors occur in the absence of metabolic stress (hypoxia and nutrient deprivation). Abbreviations are as follows: PPARgamma, peroxisome proliferator-activated receptor gamma; MAPK, mitogen-activated protein kinase; COX-2, cyclooxsygenase-2; GADD, growth arrest and DNA damage; PHD, prolyl hydroxylase; HIF, hypoxia inducible factor; VEGF, vascular endothelial growth factor; others are as shown in legend for figure 1.

Figure 3.

Role of POX signaling under conditions of metabolic stress. With glucose deprivation, AMPK is activated to increase POX expression whereas oxidized ligands from oxLDL bind to PPARgamma to induce POX. The ROS from POX activates autophagy for survival by upregulating beclin expression and to initiate autopahgy as demonstrated by the conversion of LC3 I to LC3 II in the formation of autophagosomes. Abbreviations are: oxLDL, oxidized low-density lipoprotein; LC3, microtubule associated protein 1 light chain 3; others are as shown in legend to figures 1 & 2.

Figure 4.

Timeline showing the role of POX in tumorigenesis. The timeline is simplified into 3 stages - stress, survival and proliferation. During the period of genotoxic or inflammatory stress, POX is induced to generate proline-dependent ROS which can initiate both apoptosis and autopahgy. During this period, nutrients (glucose and glutamine) are not perturbed. Malignant transformation occurs at the end of this period. The survival period is characterized by inadequate blood supply with resultant hypoxia and depletion of circulating substrates. Notably, cells are deprived of glucose and glutamine. During this period, POX is upregulated by AMPK, and proline (as well as hydroxyproline) is supplied by activation of matrix metalloproteinases to degrade extracellular matrix (ECM) in the microenvironment. ECM is predominantly collagen, rich in proline and hydroxyproline. POX can use proline as a source of ATP, metabolic intermediates, and ROS to signal autophagy. In the proliferation phase, vascularity has been restored and there is adequate oxygen and substrates. Tumors are “addicted” to glucose, generate ATP by glycolysis and use glucose and glutamine as precursors for cellular constituents necessary for growth. Under these conditions POX is downregulated through miR-23b* to minimize its apoptotic effects and to conserve proline for protein synthesis and the formation of extracellular matrix.