Ewing sarcoma is driven by fusion proteins containing a low-complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend leukemia virus integration 1). EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and self-assemble through its LC domain. The ability of RNA-binding proteins like EWSR1 to self-assemble or phase separate in cells has raised questions about the contribution of this process to EWS-FLI1 activity. We examined EWSR1 and EWS-FLI1 activity in Ewing sarcoma cells by siRNA-mediated knockdown and RNA-seq analysis. More transcripts were affected by the EWSR1 knockdown than expected and these included many EWS-FLI1 regulated genes. We reevaluated physical interactions between EWS-FLI1, EWSR1, and RNA Pol II, and used a cross-linking-based strategy to investigate protein assemblies associated with the proteins. The LC domain of EWS-FLI1 was required for the assemblies observed to form in cells. These results offer new insights into a protein assembly that may enable EWS-FLI1 to bind its wide network of protein partners and contribute to regulation of gene expression in Ewing sarcoma.
Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcription regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNAbinding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can selfassemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been vigorously studied and while many questions remain, the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, which suggested these proteins function closely together. We then studied the nature of interactions between EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for both the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions between EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell. 6 RNA SequencingA673 cells were transfected with 50 nM siRNA as described above. Cells were collected 72 hours posttransfection and total RNA was extracted using TRIzol reagent (ThermoFisher, Cat. #15596026). 1 µg of total RNA was prepared for sequencing using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490) to generate sequencing libraries according to manufacturer's instructions. Soft Agar Colony Formation AssayA673, SK-N-MC, and HEK293T/17 cells transfected with 50 nM siRNA were harvested 24 hours posttransfection. HEK293T/17 cells transfected with 50 nM siRNA and 2 µg of plasmid DNA were harvested 24 hours post-transfection. A673 and SK-N-MC cells were seeded at density of 1.0 x 10 5 cells. Cells were resuspended in 0.35% agarose in growth media and plated onto a bed of solidified 0.6% agarose in growth media. HEK293T/17 cells were seeded at a density of 20k to 30k cells. Cells were resuspended in 0.4% agarose in growth media onto a bed of solidified 0.6% agarose in growth media. A673 and SK-N-MC cells were grown at 37°C and 5% CO2 for 3-4 weeks, imaged, and then colonies were counted using ImageJ software. HEK293T/17 cells were grown at 37°C and 5% CO2 for 1-2 weeks, imaged, and then colonies were counted. Colonies with stained with 0.05% methylene blue. Cell Growth AssayA673 cells were reverse transfected at a density of 4.0 x 10 5 cells in 6-well dishes. Cells were collected by trypsinization and counted on a hemocytometer at 24, 48,...
Serous epithelial ovarian cancer metastasizes by direct seeding to form disseminated tumors within the peritoneal cavity and has an extremely poor clinical outcome with a 46.5% five-year survival rate. Resistance to anoikis (detachment-induced cell death) is a critical factor in the progression of this disease. We previously demonstrated that triple-negative breast cancer (TNBC) upregulates tryptophan-2,3-dioxygenase (TDO2), a rate limiting enzyme in tryptophan catabolism, under anchorage-independent conditions or by stimulation with an NFκB stimulating cocktail (IL1-β, TNFα). Indoleamine-pyrrole 2,3-dioxygenase (IDO1) is also a rate-limiting enzyme in the tryptophan catabolism pathway, & both TDO2 and ID01 can be expressed by tumors. Kynurenine, a metabolite of tryptophan catabolism acts in an autocrine fashion through the aryl hydrocarbon receptor to provide anti-apoptotic signals that enhance survival under anchorage independent conditions. Through paracrine action, kynurenine can suppress anti-tumor cytotoxic T-cell function. We hypothesize that tryptophan catabolism promotes ovarian cancer progression through these two mechanisms, primarily through TDO2. We mined the publically available Tothill Ovarian Cancer patient cohort (n=293) for correlations between TDO2 or IDO1 & ovarian cancer outcomes. The anoikis resistant serous ovarian cancer cell lines HEY, OV-1847, OVCA-420 & OVCA-433 were used to test levels of enzymes involved in tryptophan catabolism in vitro at baseline, in attached versus suspended conditions & following NFκB stimulation with IL1-β and TNFα. Three enzymes in the pathway; TDO2, IDO1 & Kynureninase (KYNU) were quantified by qRT-PCR & immunohistochemistry. Using a cytokine/chemokine array and follow-up ELISA, we also analyzed factors secreted by ovarian cancer cells surviving in suspension culture as compared to attached. In the ovarian cancer patient cohort, high TDO2 significantly correlated with higher stage disease (p=0.0033), increased recurrence rates (p<0.0001) & lower survival rates (p=0.0034), whereas IDO1 did not show significant correlations. The expression of tryptophan catabolizing enzymes was significantly increased in the suspended condition when compared to attached & following NFκB stimulation. Ovarian cancer cell lines in suspension have increased levels of IL-1α, G-CSF, MIF and IL-6 on arrays, & IL-6 was confirmed by ELISA. Data mining of patient specimens indicates that TDO2 may be the more relevant enzyme responsible for tryptophan metabolism in ovarian cancer since high TDO2 is correlated with higher clinical stage, disease progression & in recurrent disease, suggesting that TDO2 may facilitate disease progression. Tryptophan catabolizing enzymes are elevated in ovarian cancer cells under anchorage independent condition or inflammatory stimuli. Based on these findings, we believe that clinical trials targeting TDO2 in addition to IDO may be warranted for serous ovarian cancer. Citation Format: Lynelle P. Smith, Lucas M. Harrell, Jessica L. Christenson, Benjamin Bitler, Jill Slansky, Jennifer K. Richer. Tryptophan catabolism in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5137.
The protein FUS (FUSed in sarcoma) binds nascent RNA transcripts and all three nuclear RNA polymerases in metazoans. FUS undergoes phase separation to bind RNA polymerase II, which affects phosphorylation of the polymerase, recruitment of RNA processing factors, and transcription elongation and termination. This study investigates an activity of FUS targeting transcription but does not involve binding to the polymerase. An in vitro transcription assay revealed FUS prevented R-loops formed between the RNA transcript and DNA template, which could enhance transcription productivity for a non-eukaryote polymerase that FUS does not bind. The ability to bind RNA and phase separate was required for FUS activity. DRIP-seq analysis of human cells found endogenous activity matched shown by in vitro assays. FUS prevented R-loops at genomic locations and with sequence compositions consistent with previously characterized targets of FUS in cells. The model interpreted from these findings is that the abilities for RNA-binding and phase separation combine in FUS to prevent nucleic acid structures that disrupt chromosome stability and gene transcription.
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