Macrophage-derived IL-1β and TNF-α regulate arginine metabolism in neuroblastoma

© 2018 American Association for Cancer Research. Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1b and TNFa in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1b and TNFa established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1b and TNFa in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited

eIF4EP3L acts as a gatekeeper of TORC1 in activity-dependent muscle growth by regulating Mef2ca translational initiation

Muscle fiber size is activity-dependent and clinically important in ageing, bed-rest, and cachexia, where muscle weakening leads to disability, prolonged recovery times, and increased costs. Inactivity causes muscle wasting by triggering protein degradation and may simultaneously prevent protein synthesis. During development, muscle tissue grows by several mechanisms, including hypertrophy of existing fibers. As in other tissues, the TOR pathway plays a key role in promoting muscle protein synthesis by inhibition of eIF4EBPs (eukaryotic Initiation Factor 4E Binding Proteins), regulators of the translational initiation. Here, we tested the role of TOR-eIF4EBP in a novel zebrafish muscle inactivity model. Inactivity triggered up-regulation of eIF4EBP3L (a zebrafish homolog of eIF4EBP3) and diminished myosin and actin content, myofibrilogenesis, and fiber growth. The changes were accompanied by preferential reduction of the muscle transcription factor Mef2c, relative to Myod and Vinculin. Polysomal fractionation showed that Mef2c decrease was due to reduced translation of mef2ca mRNA. Loss of Mef2ca function reduced normal muscle growth and diminished the reduction in growth caused by inactivity. We identify eIF4EBP3L as a key regulator of Mef2c translation and protein level following inactivity; blocking eIF4EBP3L function increased Mef2ca translation. Such blockade also prevented the decline in mef2ca translation and level of Mef2c and slow myosin heavy chain proteins caused by inactivity. Conversely, overexpression of active eIF4EBP3L mimicked inactivity by decreasing the proportion of mef2ca mRNA in polysomes, the levels of Mef2c and slow myosin heavy chain, and myofibril content. Inhibiting the TOR pathway without the increase in eIF4EBP3L had a lesser effect on myofibrilogenesis and muscle size. These findings identify eIF4EBP3L as a key TOR-dependent regulator of muscle fiber size in response to activity. We suggest that by selectively inhibiting translational initiation of mef2ca and other mRNAs, eIF4EBP3L reprograms the translational profile of muscle, enabling it to adjust to new environmental conditions

Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response.

In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation

Muscle activity enhances zebrafish myofiber width and myosin level.

<p>Muscle activity was blocked either by adding MS222 to the fish water for 24<i>chrnd</i>^sb13/sb13 mutants lacking the acetylcholine receptor delta subunit, which were identified by their immotility. (A) Confocal stacks of slow MyHC immunostaining in 48 hpf embryos. Note the reduced myofibril content and poor bundling in inactive fish. White bars in the right-hand panels indicate minimal myofibrillar bundle width on each fiber. Fivefold more laser light was used to generate the lower right image. (B) Width of myofibrillar material in well-bundled regions of >5 myofibers in somite 17 were measured from >6 embryos in each condition. (C) Slow MyHC level relative to control in <i>n</i> = 15 embryos. (D and F) Western analysis of 48 hpf mutant or MS222-treated embryos, compared to respective controls. (E) Confocal stacks of embryos stained for general MyHC (A4.1025) in lateral (left image) and transverse (right image, somite indicated by white line) view. Graph shows relative MyHC level. <i>n</i> = 10 embryos. Bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice. Scale bar = 90 µm in (A, left), (E), and 23 µm in (A, right).</p

Active eIF4EBP3L regulates myofibrilogenesis following inactivity.

<p>Embryos were injected with the indicated MO, some exposed to MS222 for the last 17(A) or with plasmid DNA encoding a constitutively active eIF4EBP3L and GFP (5A3L-GFP) or empty vector (GFP) (B) were grown to 48–54 hpf and immunostained for slow MyHC. (A) BP3L MO1 prevents, and BP3L MO2 reduces, the decrease in slow MyHC caused by inactivity. Immunostaining (left panels) was quantified by confocal microscopy (right panel; <i>n</i> = 15 embryos/sample). (B) Overexpression of 5A3L reduces myofibril bundle width. Following heat shock, the size of slow myofibril bundles in GFP+ fibers was determined relative to their GFP− neighbors (<i>n</i> = 9 embryos/sample). Bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice. Scale bar = 23 µm.</p

Muscle inactivity down-regulates the TOR pathway and induces eIF4EBP3L mRNA.

<p>Wild-type (A–C, E) or <i>chrnd^sb13/+</i> incross zebrafish embryos were incubated with (grey bars) or without (black bars) MS222 for 17–24 h. At 48 hpf, embryos were analyzed by Western analysis (A), immunostaining (B), or qPCR relative to <i>actin</i> (20 embryos/sample; C). (A and B) Muscle inactivity reduces TORC1 activity. Phosphorylation levels of downstream TORC1 targets were reduced both in whole embryo (A) and in muscle tissue (B). Scale = 200 µm. Note that both eIF4EBP antisera likely detect eIF4EBP1, 2, and 3L as the epitope sequence is conserved between human and zebrafish (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679.s004" target="_blank">Figure S4C</a>). (C and E) Electrical inactivity specifically increased <i>eif4ebp3l</i> and <i>eif4ebp1</i> mRNA in whole embryos (C). Several zebrafish E3 ligase atrogenes showed similar increases in mRNA level (E). (D) Specific loss of muscle activity in acetylcholine receptor δ mutants (identified by their immotility) induces <i>eif4ebp3l</i>, but not <i>eif4ebp1</i> mRNA, compared to siblings. Actin served as a control (A, C–E) for normalization. Error bars represent SEM and samples were compared by <i>t</i> test. All experiments were repeated at least twice.</p

Muscle activity regulates translation of <i>mef2ca</i> mRNA.

<p>Zebrafish embryos were exposed to MS222, Rapamycin <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679-Drapeau1" target="_blank">[34]</a>, or vehicle control. (A and B) Muscle activity does not regulate Mef2 protein via the proteasomal or autophagic pathways. (A) Western blot of whole embryos (48 hpf; 50/sample) incubated with 100 nM MG132 or DMSO vehicle for 1 h prior to and then during 17 h MS222 treatment. (B) Quantification of Mef2c and full-length 50 kd Sqstm1 bands relative to Actin. (C) qPCR of the indicated mRNAs relative to <i>actin</i> following MS222 or vehicle (48 hpf 20 embryos/sample). (D) Schematic of separation of cytoplasmic extract on a sucrose gradient into light sub-polysome (SP) and heavy polysome (P) fractions to reveal fraction of mRNA in each. (E) Polysomal profiles of nucleic acid from the gradient of active control and inactive MS222 zebrafish. Peaks indicate 40 s, 60 s subunits, and 80 s monosomes. The amount of nucleic acid in the polysome (P) and subpolysome (SP) fractions were calculated from the areas between the dashed lines. (F) Differential regulation of muscle mRNAs by activity. Proportion of each specific mRNA in the P fraction, SP fraction, and unfractionated (total) was measured by qPCR. Activity-dependent change in translation rate at 48 hpf was determined as the ratio of polysomal/total in MS222 to polysome/total in control (70 embryos/sample). The level of each gene in the fraction was normalized to its level in the total to rule out change in transcription. To present the average from several experiments, the translation change was normalized to the relative change in <i>mef2ca</i>. Bars represent SEM (<i>n</i> = 4) and samples were compared by <i>t</i> test. All experiments were repeated at least twice.</p

Model of how muscle activity stimulates muscle growth through differential translational regulation.

<p>Previous studies indicated a role of TORC1 (a known cell size regulator) in activity-dependent muscle growth (1). TORC1 regulates growth by inhibiting the translation initiation inhibitor eIF4EBP (2) and by activation of S6K (3) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001679#pbio.1001679-Ishikawa1" target="_blank">[108]</a>, both leading to increased translation. In the absence of muscle activity, muscle fibers undergo atrophy, a reduction in size and strength. Both the FoxO/Atrogin-1 axis (4) and the calcineurin pathway (5) contribute to atrophy. We found that during muscle development lack of muscle activity increases the levels of eIF4EBP3L in muscle (6) and reduces the activity of TORC1 pathway (1), preventing eIF4EBP3L phosphorylation (2) and thereby activating it. High levels of active eIF4EBP3L prevent initiation of protein synthesis from specific mRNAs (7). Among mRNAs regulated by TORC1/eIF4EBP3L is that encoding the transcription factor Mef2ca, which is also regulated by calcineurin and is required for normal myofibrilogenesis and muscle growth (8). We hypothesize that other mRNAs are also differentially regulated by the activity/TORC1/eIF4EBP3L axis (9) and contribute to muscle homeostasis along with the FoxO and calcineurin pathways.</p