Glycolysis Inhibition of Autophagy Drives Malignancy in Ovarian Cancer: Exacerbation by IL-6 and Attenuation by Resveratrol.

peer reviewedCancer cells drive the glycolytic process towards the fermentation of pyruvate into lactate even in the presence of oxygen and functioning mitochondria, a phenomenon known as the "Warburg effect". Although not energetically efficient, glycolysis allows the cancer cell to synthesize the metabolites needed for cell duplication. Autophagy, a macromolecular degradation process, limits cell mass accumulation and opposes to cell proliferation as well as to cell migration. Cancer cells corrupt cancer-associated fibroblasts to release pro-inflammatory cytokines, which in turn promote glycolysis and support the metastatic dissemination of cancer cells. In mimicking in vitro this condition, we show that IL-6 promotes ovarian cancer cell migration only in the presence of glycolysis. The nutraceutical resveratrol (RV) counteracts glucose uptake and metabolism, reduces the production of reactive oxygen species consequent to excessive glycolysis, rescues the mitochondrial functional activity, and stimulates autophagy. Consistently, the lack of glucose as well as its metabolically inert analogue 2-deoxy-D-glucose (2-DG), which inhibits hexokinase 2 (HK2), trigger autophagy through mTOR inhibition, and prevents IL-6-induced cell migration. Of clinical relevance, bioinformatic analysis of The Cancer Genome Atlas dataset revealed that ovarian cancer patients bearing mutated TP53 with low expression of glycolytic markers and IL-6 receptor, together with markers of active autophagy, display a longer overall survival and are more responsive to platinum therapy. Taken together, our findings demonstrate that RV can counteract IL-6-promoted ovarian cancer progression by rescuing glycolysis-mediated inhibition of autophagy and support the view that targeting Warburg metabolism can be an effective strategy to limit the risk for cancer metastasis

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.

peer reviewedRegulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm(5)s(2) wobble uridine tRNA modification (U(34)-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U(34)-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U(34)-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis

Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

In the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway. METHODS: As an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting. RESULTS: In three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects. CONCLUSIONS: We demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses

Dopamine exacerbates mutant Huntingtin toxicity via oxidative-mediated inhibition of autophagy in SH-SY5Y neuroblastoma cells: Beneficial effects of anti-oxidant therapeutics

Neuronal cell death in Huntington's Disease (HD) is associated with the abnormal expansions of a polyglutamine (polyQ) tract in the huntingtin protein (Htt) at the N-terminus that causes the misfolding and aggregation of the mutated protein (mHtt). Autophagy-lysosomal degradation of Htt aggregates may protect the neurons in HD. HD patients eventually manifest parkinsonian-like symptoms, which underlie defects in the dopaminergic system. We hypothesized that dopamine (DA) exacerbates the toxicity in affected neurons by over-inducing an oxidative stress that negatively impinges on the autophagy clearance of mHtt and thus precipitating neuronal cell death. Here we show that the hyper-expression of mutant (>113/150) polyQ Htt is per se toxic to dopaminergic human neuroblastoma SH-SY5Y cells, and that DA exacerbates this toxicity leading to apoptosis and secondary necrosis. DA toxicity is mediated by ROS production (mainly anion superoxide) that elicits a block in the formation of autophagosomes. We found that the pre-incubation with N-Acetyl-l-Cysteine (a quinone reductase inducer) or Deferoxamine (an iron chelator) prevents the generation of ROS, restores the autophagy degradation of mHtt and preserves the cell viability in SH-SY5Y cells expressing the polyQ Htt and exposed to DA. The present findings suggest that DA-induced impairment of autophagy underlies the parkinsonism in HD patients. Our data provide a mechanistic explanation of the DA toxicity in dopaminergic neurons expressing the mHtt and support the use of anti-oxidative stress therapeutics to restore protective autophagy in order to slow down the neurodegeneration in HD patients

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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