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Abstract Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.

Superoxide dismutases (SODs) play an important role in protecting plants against ROS toxicity induced by biotic and abiotic stress. Recent studies have shown that the SOD gene family is involved in plant growth and development; however, knowledge of the SOD gene family in tobacco is still limited. In the present study, the SOD gene family was systematically characterized in the tobacco genome. Based on the conserved motif and phylogenetic tree, 15 NtSOD genes were identified and classified into three subgroups, including 5 NtCSDs, 7 NtFSDs and 3 NtMSDs. The predicted results of the transport peptide or signal peptide were consistent with their subcellular localization. Most NtSOD genes showed relatively well-maintained exon-intron and motif structures in the same subgroup. An analysis of cis-acting elements in SOD gene promoters showed that NtSOD expression was regulated by plant hormones, defense and stress responses, and light. In addition, multiple transcription factors and miRNAs are predicted to be involved in the regulation of NtSOD gene expression. The qPCR results indicated specific spatial and temporal expression patterns of the NtSOD gene family in different tissues and developmental stages, and this gene family played an important role in protecting against heavy metal stress. The results of functional complementation tests in the yeast mutant suggested that NtCSD1a, NtFSD1e and NtMSD1b scavenge ROS produced by heavy metal stress. This study represents the first genome-wide analysis of the NtSOD gene family, which lays a foundation for a better understanding of the function of the NtSOD gene family and improving the tolerance of plants to heavy metal toxicity.

Ubiquitination and SUMOylation, which are posttranslational modifications, play prominent roles in regulating both protein expression and function in cells, as well as various cellular signal transduction pathways. Metabolic reprogramming often occurs in various diseases, especially cancer, which has become a new entry point for understanding cancer mechanisms and developing treatment methods. Ubiquitination or SUMOylation of protein substrates determines the fate of modified proteins. Through accurate and timely degradation and stabilization of the substrate, ubiquitination and SUMOylation widely control various crucial pathways and different proteins involved in cancer metabolic reprogramming. An understanding of the regulatory mechanisms of ubiquitination and SUMOylation of cell proteins may help us elucidate the molecular mechanism underlying cancer development and provide an important theory for new treatments. In this review, we summarize the processes of ubiquitination and SUMOylation and discuss how ubiquitination and SUMOylation affect cancer metabolism by regulating the key enzymes in the metabolic pathway, including glucose, lipid and amino acid metabolism, to finally reshape cancer metabolism.

[This corrects the article DOI: 10.1371/journal.pone.0106962.].

Artemisia annua produces artemisinin, an effective antimalarial drug. In recent decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and 13CO2 radioisotope study have shown that the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) genes from A. annua (AaDXS1, AaDXS2, and AaDXS3); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three DXS genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of AaDXS2 in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of AaDXS2, the promoter of AaDXS2 was cloned upstream of the β-glucuronidase gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that AaDXS2 was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that AaDXS2 might be the only member of the DXS family in A. annua that is involved in artemisinin biosynthesis.

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is a key enzyme in de novo nucleotide synthesis and nucleotide salvage synthesis pathways that are critical for purine and pyrimidine biosynthesis. Abnormally high expression of PRPS1 can cause many diseases, including hearing loss, hypotonia, and ataxia, in addition to being associated with neuroblastoma. However, the role of PRPS1 in neuroblastoma is still unclear. In this study, we found that PRPS1 was commonly expressed in neuroblastoma cells and was closely related to poor prognosis for cancer. Furthermore, down-regulation of PRPS1 inhibited neuroblastoma cell proliferation and tumor growth in vitro and in vivo via disturbing DNA synthesis. This study provides new insights into the treatment of neuroblastoma patients and new targets for drug development.

Histone methylation plays an important role in gene transcription and chromatin organization and is linked to the silencing of a number of critical tumor suppressor genes in tumorigenesis. G9a is a histone methyltransferase (HMTase) for histone H3 lysine 9. In this study, we investigated the role of G9a in neuroblastoma tumor growth together with the G9a inhibitor BIX01294. The exposure of neuroblastoma cells to BIX01294 resulted in the inhibition of cell growth and proliferation, and BIX01294 treatment resulted in the inhibition of the tumorigenicity of neuroblastoma cells in NOD/SCID mice. Therefore, G9a may be a potential therapeutic target in neuroblastoma. Moreover, we found several specific characteristics of autophagy after BIX01294 treatment, including the appearance of membranous vacuoles and microtubule-associated protein light chain 3 (LC3B). Similar results were observed in G9a-knockdown cells. In conclusion, our results demonstrated that G9a is a prognostic marker in neuroblastoma, and revealed a potential role of G9a in regulating the autophagy signaling pathway in neuroblastoma.

Leflunomide as an immunosuppressive drug is generally used in the treatment of rheumatoid arthritis. It inhibits DHODH (dihydroorotate dehydrogenase ), which is one of the essential enzymes in the de novo pyrimidine biosynthetic pathway. Here we showed that leflunomide significantly reduced cell proliferation and self-renewal activity. Annexin V-FITC/PI staining assay revealed that leflunomide induced S-phase cell cycle arrest, and promoted cell apoptosis. In vivo xenograft study in SCID mice showed that leflunomide inhibited tumor growth and development. We also observed that DHODH was commonly expressed in neuroblastoma. When treated with leflunomide, the neuroblastoma cell lines BE(2)-C, SK-N-DZ, and SK-N-F1 showed dramatic inhibition of DHODH at mRNA and protein levels. Considering the favorable toxicity profile and the successful clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in neuroblastoma.