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

Abstract

Background 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.

Identification of a long non‑coding RNA signature for predicting prognosis and biomarkers in lung adenocarcinoma

“…To explore the potential functions of AC124804.1 and MIR34AHG, the co-expressed protein-coding mRNAs of these lncRNAs were investigated using a Pearson correlation coefficient test followed by GO and KEGG functional enrichment analyses. These analyses demonstrated that the potential underlying molecular mechanisms of AC124804.1 function were pathways involving proteasome, peroxisome and SNARE interactions in vesicular transport, which have confirmed roles in tumorigenesis and progression in previous studies (37)(38)(39). MIR34AHG is the host gene of miR-34a and consequently may have biological functions associated with the miR-34 family, which may have suppressive functions in LUAD as previously demonstrated (40,41).…”

Promiscuous Roles of Autophagy and Proteasome in Neurodegenerative Proteinopathies

“…Remarkably, despite increasing autophagosome biogenesis, UPS inhibition does not affect autophagy flux [110]. This fits with recent evidence showing that UPS activity, rather than inhibition, promotes autophagy both by fostering the nuclear translocation of TFEB, and remarkably, by degrading mTOR itself, leading to its downregulation and detachment from the lysosomes [111]. In baseline conditions the turnover of the transmembrane autophagy protein Atg9 is also carried out by the UPS, while this is impeded upon autophagy-inducing stimuli such as starvation or rapamycin treatment [112].…”

“…In this functional setting, the merging between UPS and autophagy is defined as "proteaphagy". This is in line with studies indicating a reciprocal regulation between the two cell-clearing systems, with UPS behaving as a sentinel in sensing and regulating autophagy, and vice versa [68,[108][109][110][111][112][113]. In detail, UPS regulates the duration and amplitude of the autophagy response by controlling the stability of Unc-51 like autophagy activating kinase (ULK1)/Atg1 complex as well as mTOR and transcription factor EB (TFEB) kinases [108][109][110].…”

“…UPS may control autophagy dynamics also through regulating TFEB turnover, which upon de-phosphorylation and cytoplasm-to-nucleus shuttling, induces the expression of a variety of autophagy-related genes [110,111]. On the one hand, UPS inhibition promotes TFEB accumulation, de-phosphorylation and nuclear translocation, leading to an increased expression of downstream autophagy-related genes including LC3-II, cathepsin D, and LAMP1.…”

The role of ubiquitination and deubiquitination in cancer metabolism

“…Under nutrient rich conditions, activation of mTORC1 supports cancer cells growth. In periods of cellular stress, low levels of amino acids or absent ATP induces mTORC1 inhibition, which subsequently activates a compensatory mechanism named autophagy [25]. Autophagy is a highly regulated pathway essential for cell survival in nutrient-deprived conditions, complementing the classical pathways like glycolysis.…”