scholarly journals Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma

NAR Cancer ◽  
2022 ◽  
Vol 4 (1) ◽  
Author(s):  
Garrett T Graham ◽  
Saravana P Selvanathan ◽  
Stefan K Zöllner ◽  
Emily Stahl ◽  
Adam Shlien ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ewing Sarcoma ◽  
Effective Means ◽  
Gc Content ◽  
Human Mesenchymal Stem Cells ◽  
Bone Cancer ◽  
Mrna Splicing ◽  
Cell Of Origin ◽  
Pediatric Tumor ◽  
Round Blue Cell Tumor

ABSTRACT Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines.

scholarly journals The Transcription Factor FEZF1, a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5668
Author(s):  
Laura García-García ◽  
Enrique Fernández-Tabanera ◽  
Saint T. Cervera ◽  
Raquel M. Melero-Fernández de Mera ◽  
Santiago Josa ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Sarcoma Cell ◽  
Critical Pathways ◽  
Pediatric Tumor ◽  
Sarcoma Cells

Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.

scholarly journals Hippo pathway effectors YAP1/TAZ induce a EWS-FLI1-opposing gene signature and associate with disease progression in Ewing Sarcoma

2019 ◽  
Author(s):  
Pablo Rodríguez-Núñez ◽  
Laura Romero-Pérez ◽  
Ana T. Amaral ◽  
Pilar Puerto-Camacho ◽  
Carmen Jordán ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Fusion Protein ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hippo Pathway ◽  
Protein A ◽  
Gene Signature ◽  
Cell Of Origin

AbstractYAP1 and TAZ (WWTR1) oncoproteins are the final transducers of Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing Sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involvingEWSR1gene, andFLI1as the most common partner. The fusion protein is a potent driver of oncogenesis but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome.We did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation ofRASSF1locus -a regulator of Hippo pathway- was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation ofRASSF1correlated with the transcriptional silencing of the tumor suppressor isoformRASFF1A, and transcriptional activation of the protumorigenic isoformRASSF1Cpromoting YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells, and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be partly explained by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS.

scholarly journals HOTAIRprimes the Ewing sarcoma family of tumors for tumorigenesis via epigenetic dysregulation involving LSD1

2018 ◽  
Author(s):  
Hasan Siddiqui ◽  
Julia Selich-Anderson ◽  
Joshua Felgenhauer ◽  
James Fitch ◽  
Vijay Nadella ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Colony Formation ◽  
Fusion Gene ◽  
Tumor Xenograft ◽  
Cell Physiology ◽  
Cell Of Origin

AbstractThe EWS-FLI1 fusion protein drives oncogenesis in the Ewing sarcoma family of tumors (ESFT) in humans, but its toxicity in normal cells requires additional cellular events for oncogenesis. We show that the lncRNAHOTAIRmaintains cell viability in the presence of EWS-FLI1 and redirects epigenetic regulation in ESFT.HOTAIRis consistently overexpressed in ESFTs and is not driven by EWS-FLI1. Repression ofHOTAIRin ESFT cell lines significantly reduces anchorage-independent colony formation in vitro and impairs tumor xenograft growth in vivo. Overexpression ofHOTAIRin human mesenchymal stem cells (hMSCs), a putative cell of origin of ESFT, and IMR90 cells induces colony formation. Critically, HOTAIR-expressing hMSCs and IMR90 cells remain viable with subsequentEWS-FLI1expression.HOTAIRinduces histone modifications and gene repression through interaction with the epigenetic modifier LSD1 in ESFT cell lines and hTERT-hMSCs. Our findings suggest thatHOTAIRmaintains ESFT viability through epigenetic dysregulation.SignificanceWhile theEWS-FLI1fusion gene was determined to be the oncogenic driver in the overwhelming majority of ESFT, it is toxic to cell physiology and requires one or more additional molecular events to maintain cell viability. As these tumors have surprisingly few genetic mutations at diagnosis, epigenetic changes have been considered to be such an event, but the mechanism by which these changes are driven remains unclear. Our work shows thatHOTAIRis consistently expressed among ESFT and induces epigenetic and gene expression changes that cooperate in tumorigenesis. Furthermore, expression ofHOTAIRallows for cell viability in the setting of subsequentEWS-FLI1expression. Our findings elucidate new steps of malignant transformation in this cancer and identify novel therapeutic targets.

scholarly journals Epigenetic Regulation of the Tumour Suppressor RASSF1A in Bone Cancer Cells: DNA Methylation Study

2021 ◽  
Vol 8 (8) ◽  
pp. 453-459
Author(s):  
Amal Majed Alenad
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Promoter Methylation ◽  
Human Cancer ◽  
Gene Promoter ◽  
Bone Cancer ◽  
Rassf1a Gene ◽  
Frequent Event

Objective: Osteosarcoma is a bone cancer that affects children and adolescents. The RASSF1A is a tumor suppressor capable of mediating the regulation of cell cycle arrest, migration, including apoptosis. It is the most continually silenced gene that contributes to human cancer. Furthermore, RASSF1A functions as a scaffold protein that can regulate microtubules network and bind apoptotic kinases MST1 and MST2 via the Sav-RASSF-Hippo domain. Epigenetic inactivation of genes by DNA methylation is a key factor regulating gene expression and genomic stability. Our aim was to study the RASSF1A gene promoter methylation in three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines. Materials and Methods: Three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines were used to study RASSF1A gene promoter methylation, using bisulphite conversion of DNA, followed by methylation-specific polymerase chain reaction (PCR) Results: The RASSFIA’s gene promoter methylation was established as a frequent event. Hypermethylation of RASSF1A promoter, was detected in five out of six studied cell lines. Conclusions: These results demonstrated that altering the Sav-RASSF1-Hippo may be accomplished through hypermethylation of RASSF1A and may play an essential role in Ewing’s sarcoma and Osteosarcoma. The methylation pattern of Sav-RASSF1-Hippo tumor suppressor pathway in human bone cancer along with RASSF1A expression with its effector proteins merits further investigation.  This may reveal how the RASSFIA has a physiological signal transduction, including how the process of its deregulation can contribute to transformation of the cell, eventually leading to the incorporation of novel therapeutic options with improved prognosis for bone cancer.

scholarly journals Metastasis of Ewing Sarcoma to the Pancreas: Case Report and Literature Review

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Hyma Polimera ◽  
Prashanth Moku ◽  
Shady Piedra Abusharar ◽  
Monali Vasekar ◽  
Jayakrishna Chintanaboina
Keyword(s):  
Ewing Sarcoma ◽  
Rare Case ◽  
Bone Cancer ◽  
Positive Outcome ◽  
Tumor Burden ◽  
Response To Treatment ◽  
Effective Management ◽  
Review Of The Literature ◽  
Appropriate Treatment ◽  
Prompt Diagnosis

Ewing sarcoma (ES) is a highly aggressive malignant bone cancer. ES is part of the Ewing sarcoma family of tumors (ESFT), which express characteristic t(11;22) translocation as well as higher levels of CD99. Given that metastasis and tumor burden are significant prognostic factors in patient’s response to treatment, prompt diagnosis is needed to effectively treat ESFT patients. However, the challenges in classifying and characterizing ESFT complicate effective management and treatment of ES. In this report, we present a rare case of ES metastasis to the pancreas. Upon review of the literature, we found 39 cases of ESFT involving the pancreas, but only 3 were metastatic to the pancreas while the remaining cases of ESFT primarily originated from the pancreas. Given the rarity of such metastasis, the positive outcome in our patient’s case may explain the importance of prompt diagnosis in order to initiate appropriate treatment.

scholarly journals Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines.

1991 ◽  
Vol 11 (4) ◽  
pp. 1854-1860 ◽  
Author(s):  
N P Shah ◽  
O N Witte ◽  
C T Denny
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Philadelphia Chromosome ◽  
Gc Content ◽  
Cellular Transformation ◽  
Nucleotide Sequence Analysis ◽  
Coding Sequences ◽  
Gene Assay

The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.

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