Proteasomal Degradation of the EWS-FLI1 Fusion Protein Is Regulated by a Single Lysine Residue

Abstract

Edited by George DeMartinoEHere, we identify the EWS-FLI1 protein as a substrate of the ubiquitin-proteasome system with a characteristic polyubiquitination pattern. Using a global protein stability approach, we determined the half-life of EWS-FLI1 to lie between 2 and 4 h, whereas full-length EWSR1 and FLI1 were more stable. By mass spectrometry, we identified two ubiquitin acceptor lysine residues of which only mutation of Lys-380 in the ETS domain of the FLI1 part abolished EWS-FLI1 ubiquitination and stabilized the protein posttranslationally. Expression of this highly stable mutant protein in Ewing cells while simultaneously depleting the endogenous wild type protein differentially modulates two subgroups of target genes to be either EWS-FLI1 protein-dependent or turnover-dependent. The majority of target genes are in an unaltered state and cannot be further activated. Our study provides novel insights into EWS-FLI1 turnover, a critical pathway in Ewing sarcoma pathogenesis, and lays new ground to develop novel therapeutic strategies in Ewing sarcoma. E-26 transformation-specific (ETS)2 family members are strong activators or repressors of transcription with a highly conserved ETS domain (1-3). ETS transcription factors (TFs) bind most commonly in complexes to a GGA core region to mediate gene expression (4, 5). Their main biological functions include regulation of differentiation, lineage determination of the hematopoietic system, and control of angiogenesis (6, 7). Most of the ETS family members have oncogenic potential because truncated or overexpressed ETS proteins have been linked to several cancer entities (8 -11). ERG and ETV1 are frequently fused to the TMPRSS2 promoter in prostate cancer, whereas ETV1 and ETV6 are implicated in leukemia (12, 13). Like other aberrant fusion proteins, they act as drivers of uncontrolled cell growth and survival (14, 15). However, most TFs do not harbor an enzymatic pocket and are therefore difficult to target directly. Novel strategies that uncover vulnerable sites in TFs are urgently needed to develop novel targeted therapies (16).Ewing sarcoma is a rare pediatric bone and soft tissue tumor with an aggressive behavior and prevalence to metastasize (17,18). Its main genetic abnormalities are EWS-ETS rearrangements, among them most commonly the EWS gene on chromosome 22 fused to FLI1 on chromosome 11, which results in expression of the chimeric transcription factor . Continuous expression of the fusion protein is crucial for tumor formation, progression, and maintenance (22, 23), and its down-regulation inhibits proliferation and reduces tumor cell growth (24 -26). EWS-FLI1 is thought to function mainly as a modulator to activate and repress a wide range of target genes but also as a regulator of splicing processes or as a component of large interaction networks (27-31). However, inhibition of a single downstream target gene has not been proven effective yet for Ewing sarcoma therapy.The turnover of most intracellular proteins is mediated via the ubiquitin-proteasome ...

Boosting mTOR-dependent autophagy via upstream TLR4-MyD88-MAPK signalling and downstream NF-κB pathway quenches intestinal inflammation and oxidative stress injury

“…AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins [ 38 ]. Moreover, 3-methyladenine (3-MA) inhibits class III PI3K and is widely used as an autophagy inhibitor in various studies [ 39 , 40 ]. Interestingly, our in vivo data showed that systemic administration of a mTOR inhibitor (AZD8055) or a mTOR-dependent autophagy stimulator (rapamycin) to LPS-treated mice significantly ameliorated gut inflammation and oxidant stress as shown by decreased body weight loss and histological damage, where the inhibition of autophagy by 3-MA in LPS-treated mice remarkably aggravated oxidative injury and histological damage.…”

Ubiquitin-Mediated Control of ETS Transcription Factors: Roles in Cancer and Development

“…Tumour cells characteristically express fusion proteins with an N-terminal region derived from EWS, a poorly characterised RNA-binding protein, and C-terminal region from FLI-1, or less frequently ERG, including the ETS domain. EWS-FLI-1 fusions are short-lived proteins that become ubiquitinated in the ETS domain and turned over by the UPS, although the E3 ubiquitin ligase responsible remains to be identified [ 60 ]. Of note, the same lysine (K334: = K388 in ETS-1) was also ubiquitinated on FLI-1.…”

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

“…The effects of EWSR1 on genes also targeted by the fusion protein are remarkably similar. Our result that hundreds of genes can be affected by both EWS-FLI1 and EWSR1 may be a lower estimate, because EWSR1 concentrations in the cell are quite high (~8 µM) and proteomics studies have indicated levels of endogenous EWS-FLI1 to be much lower (Elzi et al 2014;Hein et al 2015;Gierisch et al 2016). Consequently, siRNA knockdown may more effectively abolish the activity of EWS-FLI1, resulting in effects that were seen for more genes and changes to transcript levels that were generally greater than those found with the EWSR1 knockdown.…”