Methionine restriction exposes a targetable redox vulnerability of triple-negative breast cancer cells by inducing thioredoxin reductase.

PURPOSE: Tumor cells are dependent on the glutathione and thioredoxin antioxidant pathways to survive oxidative stress. Since the essential amino acid methionine is converted to glutathione, we hypothesized that methionine restriction (MR) would deplete glutathione and render tumors dependent on the thioredoxin pathway and its rate-limiting enzyme thioredoxin reductase (TXNRD). METHODS: Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control or MR media and the effects on reactive oxygen species (ROS) and antioxidant signaling were examined. To determine the role of TXNRD in MR-induced cell death, TXNRD1 was inhibited by RNAi or the pan-TXNRD inhibitor auranofin, an antirheumatic agent. Metastatic and PDX TNBC mouse models were utilized to evaluate in vivo antitumor activity. RESULTS: MR rapidly and transiently increased ROS, depleted glutathione, and decreased the ratio of reduced glutathione/oxidized glutathione in TNBC cells. TXNRD1 mRNA and protein levels were induced by MR via a ROS-dependent mechanism mediated by the transcriptional regulators NRF2 and ATF4. MR dramatically sensitized TNBC cells to TXNRD1 silencing and the TXNRD inhibitor auranofin, as determined by crystal violet staining and caspase activity; these effects were suppressed by the antioxidant N-acetylcysteine. H-Ras-transformed MCF-10A cells, but not untransformed MCF-10A cells, were highly sensitive to the combination of auranofin and MR. Furthermore, dietary MR induced TXNRD1 expression in mammary tumors and enhanced the antitumor effects of auranofin in metastatic and PDX TNBC murine models. CONCLUSION: MR exposes a vulnerability of TNBC cells to the TXNRD inhibitor auranofin by increasing expression of its molecular target and creating a dependency on the thioredoxin pathway.

Genomics and Development of Lentinus tigrinus: A White-Rot Wood-Decaying Mushroom with Dimorphic Fruiting Bodies.

Lentinus tigrinus is a species of wood-decaying fungi (Polyporales) that has an agaricoid form (a gilled mushroom) and a secotioid form (puffball-like, with enclosed spore-bearing structures). Previous studies suggested that the secotioid form is conferred by a recessive allele of a single locus. We sequenced the genomes of one agaricoid (Aga) strain and one secotioid (Sec) strain (39.53-39.88 Mb, with 15,581-15,380 genes, respectively). We mated the Sec and Aga monokaryons, genotyped the progeny, and performed bulked segregant analysis (BSA). We also fruited three Sec/Sec and three Aga/Aga dikaryons, and sampled transcriptomes at four developmental stages. Using BSA, we identified 105 top candidate genes with nonsynonymous SNPs that cosegregate with fruiting body phenotype. Transcriptome analyses of Sec/Sec versus Aga/Aga dikaryons identified 907 differentially expressed genes (DEGs) along four developmental stages. On the basis of BSA and DEGs, the top 25 candidate genes related to fruiting body development span 1.5 Mb (4% of the genome), possibly on a single chromosome, although the precise locus that controls the secotioid phenotype is unresolved. The top candidates include genes encoding a cytochrome P450 and an ATP-dependent RNA helicase, which may play a role in development, based on studies in other fungi.

Nuclear populations of the multinucleate fungus of leafcutter ants can be dekaryotized and recombined to manipulate growth of nutritive hyphal nodules harvested by the ants.

We dekaryotized the multinucleate fungus Leucocoprinus gongylophorus, a symbiotic fungus cultivated vegetatively by leafcutter ants as their food. To track genetic changes resulting from dekaryotization (elimination of some nuclei from the multinuclear population), we developed two multiplex microsatellite fingerprinting panels (15 loci total), then characterized the allele profiles of 129 accessions generated by dekaryotization treatment. Genotype profiles of the 129 accessions confirmed allele loss expected by dekaryotization of the multinucleate fungus. We found no evidence for haploid and single-nucleus strains among the 129 accessions. Microscopy of fluorescently stained dekaryotized accessions revealed great variation in nuclei number between cells of the same vegetative mycelium, with cells containing typically between 3 and 15 nuclei/cell (average = 9.4 nuclei/cell; mode = 8). We distinguish four mycelial morphotypes among the dekaryotized accessions; some of these morphotypes had lost the full competence to produce gongylidia (nutritive hyphal-tip swellings consumed by leafcutter ants as food). In mycelial growth confrontations between different gongylidia-incompetent accessions, allele profiles suggest exchange of nuclei between dekaryotized accessions, restoring full gongylidia competence in some of these strains. The restoration of gongylidia competence after genetic exchange between dekaryotized strains suggests the hypothesis that complementary nuclei interact, or nuclear and cytoplasmic factors interact, to promote or enable gongylidia competence.

Species delimitation in Trametes: a comparison of ITS, RPB1, RPB2 and TEF1 gene phylogenies.

Trametes is a cosmopolitan genus of white rot polypores, including the "turkey tail" fungus, T. versicolor. Although Trametes is one of the most familiar genera of polypores, its species-level taxonomy is unsettled. The ITS region is the most commonly used molecular marker for species delimitation in fungi, but it has been shown to have a low molecular variation in Trametes resulting in poorly resolved phylogenies and unclear species boundaries, especially in the T. versicolor species complex (T. versicolor sensu stricto, T. ochracea, T. pubescens, T. ectypa). Here we evaluate the performance of three protein-coding genes (TEF1, RPB1, RPB2) for species delimitation and phylogenetic reconstruction in Trametes. We obtained 59 TEF1, 34 RPB1 and 55 RPB2 sequences from 69 individuals, focusing on the T. versicolor complex and performed phylogenetic analyses with maximum likelihood and parsimony methods. All three protein-coding genes outperformed ITS for separating species in the T. versicolor complex. The multigene phylogenetic analysis shows the highest amount of resolution and supported nodes separating T. ectypa, T. ochracea, T. pubescens and T. versicolor with strong support. In addition three slineages are resolved in the species complex of T. elegans. The T. elegans complex includes three species: T. elegans (based on material from Puerto Rico, Belize, the Philippines), T. aesculi (from North America) and T. repanda (from Papua New Guinea, the Philippines, Venezuela). The utility of gene markers varies, with TEF1 having the highest PCR and sequencing success rate and RPB1 offering the best backbone resolution for the genus.