Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate.

Proline dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I-IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration.

Proline metabolism and cancer: emerging links to glutamine and collagen.

PURPOSE OF REVIEW: Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism. RECENT FINDINGS: The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production, storage and mobilization. SUMMARY: Mechanisms by which the proline regulatory axis modulates the cancer phenotype are being revealed. Proline can be synthesized from glutamine as well as derived from collagen degradation. The metabolism of proline serves as a source of energy during stress, provides signaling reactive oxygen species for epigenetic reprogramming and regulates redox homeostasis.

Mitogen activated protein (MAP) kinases: development of ATP and non-ATP dependent inhibitors.

Extracellular signals regulate most of the body's physiological functions through the MAP kinase signaling pathways. These MAP kinase signaling pathways are normally under tight regulation such that activation and inactivation occurs only when needed. However, aberrant regulation observed with naturally occurring mutations in specific signaling proteins often results in constitutive activation of the MAP kinases and is involved in several pathophysiological conditions, such as cancer, neurodegeneration, and inflammation. As such, much effort has been expended to develop inhibitory molecules of the MAP kinase signaling pathways. Several compounds have been identified that inhibit MAP kinase signaling by targeting receptors or other proteins upstream of the MAP kinases. The development of specific inhibitors of the MAP kinases themselves has been less successful and only a few compounds, which interfere with ATP binding, have been identified. A common problem with kinase inhibitors that compete with ATP binding is their lack of specificity. Thus, alternative approaches to inhibit MAP kinase function are being sought. The MAP kinase proteins contain docking domains that direct the interactions with a variety of substrate proteins. Using the 3-dimensional structure of MAP kinases and computer modeling, molecules that target specific docking domains and selectively disrupt substrate interactions are being developed. This non-ATP interfering approach may allow the selective inhibition of MAP kinase substrates involved in disease processes while preserving MAP kinase functions associated with normal cells.

Identification of novel extracellular signal-regulated kinase docking domain inhibitors.

The extracellular signal regulated kinase (ERK1 and ERK2) signal transduction pathways play a critical role in cell proliferation. Hyperactivation of the ERK proteins either through increased expression of membrane-bound growth factor receptors or genetic mutations of upstream proteins is thought to be involved in the pathogenesis of many human cancers. Thus, targeted inhibition of ERK signaling is viewed as a potential approach to prevent cancer cell proliferation. Currently, no specific inhibitors of the ERK proteins exist. Moreover, most kinase inhibitors lack specificity because they target the ATP binding region, which is well conserved among the protein kinase families. Taking advantage of recently identified ERK docking domains, which are reported to facilitate substrate protein interactions, we have used computer-aided drug design (CADD) to identify novel small molecular weight ERK inhibitors. Following a CADD screen of over 800 000 molecules, 80 potential compounds were selected and tested for activity in biological assays. Several compounds inhibited ERK-specific phosphorylation of ribosomal S6 kinase-1 (Rsk-1) or the ternary complex factor Elk-1 (TCF/Elk-1), both of which are involved in promoting cell proliferation. Active compounds showed a dose-dependent reduction in the proliferation of several cancer cell lines as measured by colony survival assays. Direct binding between the active compounds and ERK2 was indicated by fluorescence quenching. These active compounds may serve as lead candidates for development of novel specific inhibitors of ERK-substrate interactions involved in cell proliferation.

Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides.

The metabolism of the nonessential amino acid proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ(1)-pyrroline-5-carboxylate (P5C) or proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of proline was blocked by concomitant proline dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between proline and P5C rather than product proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy.

A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo.

In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.

Suppression of proliferation of two independent NF1 malignant peripheral nerve sheath tumor cell lines by the pan-ErbB inhibitor CI-1033.

Neurofibromatosis Type 1 (NF1) is characterized by the abnormal proliferation of neuroectodermal tissues and the development of certain tumors, particularly neurofibromas, which may progress into malignant peripheral nerve sheath tumors (MPNSTs). Effective pharmacological therapy for the treatment of NF1 tumors is currently unavailable and the prognosis for patients with MPNSTs is poor. Loss of neurofibromin correlates with increased expression of the epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases and these kinases have been shown to promote NF1 tumor-associated pathologies in vivo. We show here that while NF1 MPNST cells have higher EGFR expression levels and are more sensitive to EGF when compared to a non-NF1 MPNST cell line, the ability of the EGFR inhibitor gefitinib to selectively inhibit NF1 MPNST cell proliferation is marginal. We also show that NF1 MPNST proliferation correlates with activated ErbB2 and can be suppressed by nanomolar concentrations of the pan-ErbB inhibitor CI-1033 (canertinib). Consequently, targeting both EGFR and ErbB2 may prove an effective strategy for suppressing NF1 MPNST tumor growth in vivo.

Characterization of ATP-independent ERK inhibitors identified through in silico analysis of the active ERK2 structure.

The extracellular signal-regulated kinases (ERK1 and ERK2) are important mediators of cell proliferation. Constitutive activation of the ERK proteins plays a critical role in the proliferation of many human cancers. Taking advantage of recently identified substrate docking domains on ERK2, we have used computer-aided drug design (CADD) to identify novel low molecular weight compounds that interact with ERK2 in an ATP-independent manner and disrupt substrate-specific interactions. In the current study, a CADD screen of the 3D structure of active phosphorylated ERK2 protein was used to identify inhibitory compounds. We tested 13 compounds identified by the CADD screen in ERK-specific phosphorylation, cell proliferation, and binding assays. Of the 13 compounds tested, 4 compounds strongly inhibited ERK-mediated phosphorylation of ribosomal S6 kinase-1 (Rsk-1) and/or the transcription factor Elk-1 and inhibited the proliferation of HeLa cervical carcinoma cells with IC(50) values in the 2-10 microM range. These studies demonstrate that CADD can be used to identify lead compounds for development of novel non-ATP-dependent inhibitors selective for active ERK and its interactions with substrates involved in cancer cell proliferation.

Protein phosphatase 2A activity associated with Golgi membranes during the G2/M phase may regulate phosphorylation of ERK2.

The extracellular signal-regulated kinase (ERK) 1 and 2 proteins are mitogen-activated protein kinase (MAPK) members that regulate cell proliferation and differentiation. ERK proteins are activated exclusively by MAPK kinase 1 and 2 phosphorylation of threonine and tyrosine residues located within the conserved TXY MAPK activation motif. Although dual phosphorylation of Thr and Tyr residues confers full activation of ERK, in vitro studies suggest that a single phosphorylation on either Thr or Tyr may yield partial ERK activity. Previously, we have demonstrated that phosphorylation of the tyrosine residue (Tyr(P) ERK) may be involved in regulating the Golgi complex structure during the G2 and M phases of the cell cycle (Cha, H., and Shapiro, P. (2001) J. Cell Biol. 153, 1355-1368). In the present study, we examined mechanisms for generating Tyr(P) ERK by determining cell cycle-dependent changes in localized phosphatase activity. Using fractionated nuclei-free cell lysates, we find increased serine/threonine phosphatase activity associated with Golgi-enriched membranes in cells synchronized in the late G2/early M phase as compared with G1 phase cells. The addition of phosphatase inhibitors in combination with immunodepletion assays identified this activity to be related to protein phosphatase 2A (PP2A). The increased activity was accounted for by elevated PP2A association with mitotic Golgi membranes as well as increased catalytic activity after normalization of PP2A protein levels in the phosphatase assays. These data indicate that localized changes in PP2A activity may be involved in regulating proteins involved in Golgi disassembly as cells enter mitosis.