Impact of EWS knockdown on LTBR mediated Gene Activation in human fibroblasts
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ABSTRACT:
PROVIDER: PRJNA432754 | ENA |
REPOSITORIES: ENA
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Project description:LTβR signaling is crucial for immune development, homeostasis, and inflammation. To identify protein determinants that control the quality of LTβR function, we use a novel proteomics approach to identify EWS as a novel signaling component of this pathway. LTβR signaling protein, TRAF3, controls the protein binding activity of EWS via formation of mutually exclusive protein-protein interactions. With transcriptomics and knockdown experiments we identify several pro-inflammatory genes with induction kinetics that are controlled by EWS during LTβR signaling. Given the link between gene induction responsiveness and mRNA decay, we show EWS controls the decay of these transcripts.
2019-03-01 | GSE110102 | GEO
Project description:Our objective is to clarify the function of EWS-POU5F1 chimera. Specifially, GBS6 cells were established from an undifferentiated bone sarcoma carrying translocation t(6;22)(p21;q12). The translocation resulted in a gene fusion between EWS and POU5F1. Gene expression analysis of t(6;22) undifferentiated sarcoma cell line GBS6 transfected with POU5F1 specific siRNA to investigate the function of EWS-POU5F1. Knockdown of EWS-POU5F1 using POU5F1 specific siRNAs. 3 control and 6 experimental replicates representing the same experiment repeated 3 times (1st, 2nd, 3rd).
2009-05-31 | E-GEOD-12320 | biostudies-arrayexpress
Project description:Ewing sarcoma (EWS) is a malignant pediatric bone cancer. Most Ewing sarcomas are driven by EWS-FLI1 oncogenic transcription factor that plays roles in transcriptional regulation, DNA damage response, cell cycle checkpoint control, and alternative splicing. USP1, a deubiquitylase which regulates DNA damage and replication stress responses, is overexpressed at both the mRNA and protein levels in EWS cell lines compared to human mesenchymal stem cells, the EWS cell of origin. The functional significance of high USP1 expression in Ewing sarcoma is not known. Here, we identify USP1 as a transcriptional target of EWS-FLI1 and a key regulator of EWS cell survival. We show that EWS-FLI1 knockdown decreases USP1 mRNA and protein levels. ChIP and ChIP-seq analyses show EWS-FLI1 occupancy on the USP1 promoter. Importantly, USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. We observe destabilization of Survivin (also known as BIRC5 or IAP4) and activation of caspases-3 and -7 following USP1 knockdown or inhibition in the absence of external DNA damage stimuli. Notably, EWS cells display hypersensitivity to combinatorial treatment of doxorubicin or etoposide, EWS standard of care drugs, and USP1 inhibitor compared to single agents alone. Together, our study demonstrates that USP1 is regulated by EWS-FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes EWS cells to standard of care chemotherapy.
2024-01-26 | PXD041033 | Pride
Project description:Lymphotoxin beta receptor (LTBR), a member of TNFRSF, was identified as a key regulator in the development, organization, and homeostasis of lymphoid tissues (Wolf, Seleznik, Zeller, & Heikenwalder, 2010). Recently, LTBR has been shown to play a role in tumor biology, both in solid tumors and in hematological cancers (Fernandes et al., 2016; Haybaeck et al., 2009; Lo et al., 2007). However, the role of LTBR in cancer biology is still controversial. From the perspective of tumor eradication, the combination of LIGHT and IFN-g can induce apoptotic cell death in HT29 cells and some other cell lines via the increase of intracellular reactive oxygen species (ROS) (Chang, Chao, Hsieh, & Lin, 2004; Zhang, Liu, Demchik, Zhai, & Yang, 2004). Artificial LTBR agonist was shown to induce tumor necrosis and decrease tumor burden in vivo in colon cancer or melanoma. (Hu et al., 2013), while recent studies revealed that LTBR mediated tumor progression, triggered by either LTα1b2 or LIGHT stimulation, leads to the recruitment of more infiltrating immune cells and the secretion of pro-survival inflammatory cyto-/chemokines (Ware, 2005). From this, the aim of the project is to compare the signalling changes between WT cells and HOIP absent Hep3B cells using a moTAP tagged LIGHT ligand. This ligand was pulled down by Flag IP, followed by Strep-tactin IP to explore the LTBR signalling complex with and without LUBAC. The result from this experiment would provide an unbiased picture of components of the LTBR-SC, also pointing out the differences between canonical NF-kB pathway and noncanonical Nf-kB pathway.
2025-04-01 | PXD034142 | Pride
Project description:The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis in many cancer types and has been associated with better treatment responses to immune-checkpoint blockade (ICB). However, the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses remain unclear. Lymphotoxin beta receptor (LTBR) signaling is a critical regulator of lymph node organogenesis and when combined with antiangiogenic and ICB treatment can augment tumor-associated HEV formation. Here we demonstrate that LTBR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTBR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTBR agonism on DC activation and maturation and associated DC- mediated T cell activation. Single agent LTBR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner; and the LTBR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies, respectively. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTBR agonism and lymphotoxin alpha (LT⍺) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTBR signaling in modulating the tumor microenvironment and inform future HEV/TLS- based strategies for cancer treatments.
2024-08-08 | GSE243170 | GEO
Project description:The pathognomonic EWS/ETS fusion transcription factors drive Ewing sarcoma (EWS) by orchestrating an oncogenic transcription program. Therapeutic targeting of EWS/ETS has not been successful; therefore identifying mediators of the EWS/ETS function could offer new therapeutic targets. Here we describe the dependency of chromatin reader BET bromodomain proteins in EWS/ETS driven transcription and investigate the potential of BET inhibitors in treating this lethal cancer. Similar to EWS/ETS fusions, knockdown of BET proteins BRD2/3/4 severely impaired the oncogenic phenotype of EWS cells. Notably, EWS/FLI1 and EWS/ERG was found to be in a transcriptional complex consisting of BRD4. RNA-Seq analysis upon BRD4 knockdown or its pharmacologic inhibition by the BET inhibitor JQ1 revealed an attenuated EWS/ETS transcriptional signature. In contrast to other reports, JQ1 reduced proliferation, and induced apoptosis through MYC-independent mechanism without affecting EWS/ETS protein levels, which was further confirmed by depleting BET proteins using PROTAC-BET degrader (BETd). Interestingly, polycomb repressive complex 2 (PRC2) associated factor PHF19 was downregulated by JQ1/BETd or BRD4 knockdown in multiple EWS cells. ChIP-seq analysis revealed occupancy of EWS/FLI1 at a distal regulatory element of PHF19 and its subsequent knockdown resulted in downregulation of PHF19 expression. Furthermore, deletion of PHF19 by CRISPR-Cas9 system lead to a decreased tumorigenic phenotype and increased sensitivity to JQ1. Importantly, PHF19 expression was associated with worse prognosis of Ewing sarcoma patients. In vivo, JQ1 demonstrated anti-tumor efficacy in multiple mouse xenograft models of EWS. Together, these results indicate that EWS/ETS require BET epigenetic reader proteins for its transcriptional program including PHF19 expression, which can be mitigated by BET inhibitors. Moreover, this study provides a clear rationale for the clinical utility of BET inhibitors in treating Ewing sarcoma.
2018-06-19 | GSE113604 | GEO
Project description:lncRNA Cfast knockdown in cardiac fibroblasts
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Project description:To study the effect of LTbR induction on spleen and duodenum transcriptome, we performed RNA sequencing of the spleen and intestine after Intravenous LTbR agonist AC H6 or isotype treatment. We analyzed differential gene expression.
2019-12-31 | GSE126115 | GEO
Project description:Our objective is to clarify the function of EWS-POU5F1 chimera. Specifially, GBS6 cells were established from an undifferentiated bone sarcoma carrying translocation t(6;22)(p21;q12). The translocation resulted in a gene fusion between EWS and POU5F1. Gene expression analysis of t(6;22) undifferentiated sarcoma cell line GBS6 transfected with POU5F1 specific siRNA to investigate the function of EWS-POU5F1.
2009-05-31 | GSE12320 | GEO
Project description:Although EWS/FLI-1 fusion protein is responsible for most EwingM-bM-^@M-^Ys sarcoma family tumors (ESFT), the function of native EWS remains largely unknown. Here, we first showed that EWS repressed protein expression in a tethering assay. mRNAs bound to EWS were determined by RNA-immunoprecipitation Chip assay, and one of them, proline-rich Akt substrate of 40 kDa (PRAS40) mRNA, directly interacted with EWS. The inhibitor of AKT, API-2, repressed ESFT cell proliferation. We demonstrate that EWS negatively regulated PRAS40 protein expression through binding to PRAS40 3M-bM-^@M-^YUTR. Furthermore, PRAS40 knockdown inhibited the proliferation and metastatic potential of ESFT cells. Cytoplasmic lysates or whole cell lysates were prepared from HeLa S3 cells transfected with pFLAG-EWS , and incubated with anti-FLAG M2 Affinity Gel (Sigma) at 4M-BM-0C for 2 h. RNAs from lysates and immunoprecipitates were analysed using GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix).
2012-01-16 | E-GEOD-29313 | biostudies-arrayexpress