scholarly journals Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 328 ◽  
Author(s):  
Ramona Palombo ◽  
Veronica Verdile ◽  
Maria Paola Paronetto
Keyword(s):  
Alternative Splicing ◽  
Ewing Sarcoma ◽  
Splicing Factors ◽  
Mrna Variants ◽  
Aggressive Tumor ◽  
Chemotherapeutic Treatment ◽  
Sarcoma Cells ◽  
Combinatorial Mechanism ◽  
Worse Prognosis

Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors

Science ◽  
1994 ◽  
Vol 265 (5179) ◽  
pp. 1706-1709 ◽  
Author(s):  
J. Caceres ◽  
S Stamm ◽  
D. Helfman ◽  
A. Krainer
Keyword(s):  
Alternative Splicing ◽  
Splicing Factors

scholarly journals CAPER-α alternative splicing regulates the expression of vascular endothelial growth factor165 in Ewing sarcoma cells

Cancer ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2106-2116 ◽  
Author(s):  
Gangxiong Huang ◽  
Zhichao Zhou ◽  
Hua Wang ◽  
Eugenie S. Kleinerman
Keyword(s):  
Alternative Splicing ◽  
Ewing Sarcoma ◽  
Vascular Endothelial ◽  
Sarcoma Cells

scholarly journals Therapeutic Potential of EWSR1–FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3783
Author(s):  
Saint T. Cervera ◽  
Carlos Rodríguez-Martín ◽  
Enrique Fernández-Tabanera ◽  
Raquel M. Melero-Fernández de Mera ◽  
Matias Morin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ewing Sarcoma ◽  
Therapeutic Potential ◽  
Bone Cancer ◽  
Genetic Inactivation ◽  
Complete Inactivation ◽  
Oncogenic Driver ◽  
Sarcoma Cells ◽  
Block Cell Proliferation

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1–FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1–FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1–FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1–FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1–FLI1 at the cell level should be considered a therapeutic approach to develop in the future.

scholarly journals EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

2020 ◽  
Vol 48 (20) ◽  
pp. 11434-11451
Author(s):  
Xianping Shi ◽  
Yueyuan Zheng ◽  
Liling Jiang ◽  
Bo Zhou ◽  
Wei Yang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Ewing Sarcoma ◽  
Gene Mutations ◽  
Mapk Signaling ◽  
Genome Wide ◽  
Regulatory Loop ◽  
Sarcoma Cells

Abstract Core regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and adolescents carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC ‘trio’ constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other's super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, CRC-downstream genes and signaling pathways may contain potential therapeutic targets for this malignancy.

scholarly journals Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 5902-5910 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Jesse Quintero ◽  
Eric S. Reid ◽  
Christian Gocke ◽  
Carl C. Baker
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Splicing Factors ◽  
Bovine Papillomavirus ◽  
Splicing Pattern ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

scholarly journals EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

2020 ◽  
Author(s):  
Xianping Shi ◽  
Yueyuan Zheng ◽  
Liling Jiang ◽  
Bo Zhou ◽  
Wei Yang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Ewing Sarcoma ◽  
Gene Mutations ◽  
Mapk Signaling ◽  
Genome Wide ◽  
Children And Young Adults ◽  
Regulatory Loop ◽  
Sarcoma Cells

AbstractCore regulatory circuitry (CRC)-dependent transcriptional network is critical for developmental tumors in children and young adults carrying few gene mutations. However, whether and how CRC contributes to transcription regulation in Ewing sarcoma is unknown. Here, we identify and functionally validate a CRC “trio” constituted by three transcription factors (TFs): KLF15, TCF4 and NKX2-2, in Ewing sarcoma cells. Epigenomic analyses demonstrate that EWS-FLI1, the primary fusion driver for this cancer, directly establishes super-enhancers of each of these three TFs to activate their transcription. In turn, KLF15, TCF4 and NKX2-2 co-bind to their own and each other’s super-enhancers and promoters, forming an inter-connected auto-regulatory loop. Functionally, CRC factors contribute significantly to cell proliferation of Ewing sarcoma both in vitro and in vivo, and are all overexpressed in this cancer. Mechanistically, CRC factors exhibit prominent capacity of co-regulating the epigenome in cooperation with EWS-FLI1, occupying 77.2% of promoters and 55.6% of enhancers genome-wide. Downstream, CRC TFs coordinately regulate gene expression networks in Ewing sarcoma, directly controlling important signaling pathways for cancer, such as lipid metabolism pathway, PI3K/AKT and MAPK signaling pathways. Together, molecular characterization of the oncogenic CRC model advances our understanding of the biology of Ewing sarcoma. Moreover, this study identifies CRC-downstream genes and signaling pathways, which may contain potential targets for therapeutic intervention for this malignancy.

scholarly journals Automated compound testing in zebrafish xenografts identifies combined MCL-1 and BCL-XL inhibition to be effective against Ewing sarcoma

2021 ◽  
Author(s):  
Sarah Grissenberger ◽  
Caterina Sturtzel ◽  
Andrea Wenninger-Weinzierl ◽  
Eva Scheuringer ◽  
Lisa Bierbaumer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ewing Sarcoma ◽  
Transcriptional Analysis ◽  
Size Analysis ◽  
Sarcoma Cell ◽  
Current Standard ◽  
Sarcoma Cells ◽  
Effective Drugs ◽  
Sarcoma Treatment

Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.

scholarly journals hnRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells

2017 ◽  
Vol 45 (21) ◽  
pp. 12270-12284 ◽  
Author(s):  
Ilaria Passacantilli ◽  
Paola Frisone ◽  
Elisa De Paola ◽  
Marco Fidaleo ◽  
Maria Paola Paronetto
Keyword(s):  
Alternative Splicing ◽  
Ewing Sarcoma ◽  
Mtor Pathway ◽  
Sarcoma Cells

scholarly journals Secondary motifs enable concentration-dependent regulation by Rbfox family proteins

2019 ◽  
Author(s):  
Bridget E. Begg ◽  
Marvin Jens ◽  
Peter Y. Wang ◽  
Christopher B. Burge
Keyword(s):  
Alternative Splicing ◽  
Rna Splicing ◽  
Neuronal Development ◽  
Splicing Factors ◽  
Reporter Assay ◽  
Animal Development ◽  
High Affinity ◽  
Second Wave ◽  
Relevant Range

AbstractThe Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart, and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity, but just half of Rbfox CLIP peaks contain a GCAUG motif. We incubated recombinant RBFOX2 with over 60,000 transcriptomic sequences to reveal significant binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX concentrations. We find that many of these “secondary motifs” bind Rbfox robustly in vivo and that several together can exert regulation comparable to a GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.

scholarly journals Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

1996 ◽  
Vol 16 (5) ◽  
pp. 2325-2331 ◽  
Author(s):  
R R Gontarek ◽  
D Derse
Keyword(s):  
Alternative Splicing ◽  
Exon Skipping ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Exonic Splicing Enhancer ◽  
Exon Inclusion ◽  
Alternative Mrna Splicing ◽  
Splicing Enhancer

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Sign in / Sign up

Export Citation Format

Share Document