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Frezza V, Chellini L, Riccioni V, Bonvissuto D, Palombo R, Paronetto M. DHX9 helicase impacts on splicing decisions by modulating U2 snRNP recruitment in Ewing sarcoma cells. Nucleic Acids Res 2025; 53:gkaf068. [PMID: 39970297 PMCID: PMC11826090 DOI: 10.1093/nar/gkaf068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Revised: 12/17/2024] [Accepted: 01/30/2025] [Indexed: 02/17/2025] Open
Abstract
Ewing sarcomas (ESs) are biologically aggressive tumours of bone and soft tissues caused by chromosomal translocations yielding in-frame fusion proteins driving the neoplastic transformation. The DNA/RNA helicase DHX9 is an important regulator of cellular processes often deregulated in cancer. Using transcriptome profiling, our study reveals cancer-relevant genes whose splicing is modulated by DHX9. Immunodepletion experiments demonstrate that DHX9 impacts on the recruitment of U2 small nuclear RNP (snRNP) onto the pre-mRNA. Analysis of structure and sequence features of DHX9 target exons reveal that DHX9-sensitive exons display shorter flanking introns and contain HNRNPC and TIA1 consensus motifs. A prominent target of DHX9 is exon 11 in the Cortactin (CTTN) gene, which is alternatively spliced to generate isoforms with different activities in cell migration and tumour invasion. Alternative inclusion of the exon 11 in CTTN gene is one of the most recurrent isoform switches in multiple cancer types, thus highlighting the pivotal role of DHX9 in defining the tumour phenotype. Biochemical analyses reveal that DHX9 binding promotes the recruitment of U2snRNP, SF3B1, and SF3A2 to the splice sites flanking exon 11. These findings uncover a new role of DHX9 in the control of co-transcriptional splicing in ES, which may represent a new druggable target to counteract ES malignancy.
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Affiliation(s)
- Valentina Frezza
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia IRCCS, Via del Fosso di Fiorano, 64, 00143 Rome, Italy
| | - Lidia Chellini
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia IRCCS, Via del Fosso di Fiorano, 64, 00143 Rome, Italy
| | - Veronica Riccioni
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia IRCCS, Via del Fosso di Fiorano, 64, 00143 Rome, Italy
| | - Davide Bonvissuto
- Section of Human Anatomy, Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
| | - Ramona Palombo
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia IRCCS, Via del Fosso di Fiorano, 64, 00143 Rome, Italy
| | - Maria Paola Paronetto
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia IRCCS, Via del Fosso di Fiorano, 64, 00143 Rome, Italy
- Department of Movement, Human and Health Sciences, University of Rome “Foro Italico”, Piazza Lauro de Bosis 6, 00135, Rome, Italy
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Naro C, Ruta V, Sette C. Splicing dysregulation: hallmark and therapeutic opportunity in pancreatic cancer. Trends Mol Med 2024:S1471-4914(24)00308-3. [PMID: 39648052 DOI: 10.1016/j.molmed.2024.11.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2024] [Revised: 11/12/2024] [Accepted: 11/13/2024] [Indexed: 12/10/2024]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by dismal prognosis. Late diagnosis, resistance to chemotherapy, and lack of efficacious targeted therapies render PDAC almost untreatable. Dysregulation of splicing, the process that excises the introns from nascent transcripts, is emerging as a hallmark of PDAC and a possible vulnerability of this devastating cancer. Splicing factors are deregulated in PDAC and contribute to all steps of tumorigenesis, from inflammation-related early events to metastasis and acquisition of chemoresistance. At the same time, splicing dysregulation offers a therapeutic opportunity to target cancer-specific vulnerabilities. We discuss mounting evidence that splicing plays a key role in PDAC and the opportunities that this essential process offers for developing new targeted therapies.
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Affiliation(s)
- Chiara Naro
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168 Rome, Italy; Gemelli Science and Technology Park (GSTeP) Organoids Research Core Facility, Fondazione Policlinico A. Gemelli, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 00168 Rome, Italy
| | - Veronica Ruta
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168 Rome, Italy
| | - Claudio Sette
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168 Rome, Italy; Gemelli Science and Technology Park (GSTeP) Organoids Research Core Facility, Fondazione Policlinico A. Gemelli, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 00168 Rome, Italy.
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Uchida Y, Kurimoto R, Chiba T, Matsushima T, Oda G, Onishi I, Takeuchi Y, Gotoh N, Asahara H. RNA binding protein ZCCHC24 promotes tumorigenicity in triple-negative breast cancer. EMBO Rep 2024; 25:5352-5382. [PMID: 39420119 PMCID: PMC11624195 DOI: 10.1038/s44319-024-00282-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2024] [Revised: 08/28/2024] [Accepted: 09/26/2024] [Indexed: 10/19/2024] Open
Abstract
Triple-negative breast cancer (TNBC) lacks the expression of hormone and HER2 receptors and is highly malignant with no effective therapeutic targets. In TNBC, the cancer stem-like cell (CSC) population is considered to be the main cause of resistance to treatment. Thus, the therapeutic targeting of this population could substantially improve patient survival. Here, we identify the RNA-binding protein ZCCHC24 as enriched in the mesenchymal-like TNBC population. ZCCHC24 promotes the expression of a set of genes related to tumorigenicity and treatment resistance by directly binding to the cis-element "UGUWHWWA" in their mRNAs, thereby stabilizing them. One of the ZCCHC24 targets, ZEB1, is a transcription factor that promotes the expression of cancer stemness genes and reciprocally induces ZCCHC24 expression. ZCCHC24 knockdown by siRNAs shows a therapeutic effect and reduces the mesenchymal-like cell population in TNBC patient-derived xenografts. ZCCHC24 knockdown also has additive effects with the BET inhibitor JQ1 in suppressing tumor growth in TNBC patient-derived xenografts.
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Affiliation(s)
- Yutaro Uchida
- Department of Systems Biomedicine, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Ryota Kurimoto
- Department of Systems Biomedicine, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Tomoki Chiba
- Department of Systems Biomedicine, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Takahide Matsushima
- Department of Systems Biomedicine, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Goshi Oda
- Department of Surgery, Breast Surgery, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Iichiroh Onishi
- Department of Comprehensive Pathology, Institute of Science Tokyo, Tokyo, 113-8510, Japan
| | - Yasuto Takeuchi
- Division of Cancer Cell Biology, Kanazawa University, Kanazawa, 920-1192, Japan
- Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, 920-1192, Japan
| | - Noriko Gotoh
- Division of Cancer Cell Biology, Kanazawa University, Kanazawa, 920-1192, Japan
- Institute for Frontier Science Initiative, Kanazawa University, Kanazawa, 920-1192, Japan
| | - Hiroshi Asahara
- Department of Systems Biomedicine, Institute of Science Tokyo, Tokyo, 113-8510, Japan.
- Department of Molecular and Cellular Biology, Scripps Research, La Jolla, CA, 92037, USA.
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4
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Gutman T, Tuller T. Computational Analysis of MDR1 Variants Predicts Effect on Cancer Cells via their Effect on mRNA Folding. PLoS Comput Biol 2024; 20:e1012685. [PMID: 39724131 DOI: 10.1371/journal.pcbi.1012685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Accepted: 11/29/2024] [Indexed: 12/28/2024] Open
Abstract
The P-glycoprotein efflux pump, encoded by the MDR1 gene, is an ATP-driven transporter capable of expelling a diverse array of compounds from cells. Overexpression of this protein is implicated in the multi-drug resistant phenotype observed in various cancers. Numerous studies have attempted to decipher the impact of genetic variants within MDR1 on P-glycoprotein expression, functional activity, and clinical outcomes in cancer patients. Among these, three specific single nucleotide polymorphisms-T1236C, T2677G, and T3435C - have been the focus of extensive research efforts, primarily through in vitro cell line models and clinical cohort analyses. However, the findings from these studies have been remarkably contradictory. In this study, we employ a computational, data-driven approach to systematically evaluate the effects of these three variants on principal stages of the gene expression process. Leveraging current knowledge of gene regulatory mechanisms, we elucidate potential mechanisms by which these variants could modulate P-glycoprotein levels and function. Our findings suggest that all three variants significantly change the mRNA folding in their vicinity. This change in mRNA structure is predicted to increase local translation elongation rates, but not to change the protein expression. Nonetheless, the increased translation rate near T3435C is predicted to affect the protein's co-translational folding trajectory in the region of the second ATP binding domain. This potentially impacts P-glycoprotein conformation and function. Our study demonstrates the value of computational approaches in elucidating the functional consequences of genetic variants. This framework provides new insights into the molecular mechanisms of MDR1 variants and their potential impact on cancer prognosis and treatment resistance. Furthermore, we introduce an approach which can be systematically applied to identify mutations potentially affecting mRNA folding in pathology. We demonstrate the utility of this approach on both ClinVar and TCGA and identify hundreds of disease related variants that modify mRNA folding at essential positions.
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MESH Headings
- Humans
- ATP Binding Cassette Transporter, Subfamily B/genetics
- ATP Binding Cassette Transporter, Subfamily B/metabolism
- ATP Binding Cassette Transporter, Subfamily B/chemistry
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Polymorphism, Single Nucleotide/genetics
- Neoplasms/genetics
- Neoplasms/metabolism
- Computational Biology
- RNA Folding/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Gene Expression Regulation, Neoplastic
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Affiliation(s)
- Tal Gutman
- Department of Biomedical Engineering, the Engineering Faculty, Tel Aviv University, Tel-Aviv, Israel
| | - Tamir Tuller
- Department of Biomedical Engineering, the Engineering Faculty, Tel Aviv University, Tel-Aviv, Israel
- The Sagol School of Neuroscience, Tel-Aviv University, Tel Aviv, Israel
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Anczukow O, Allain FHT, Angarola BL, Black DL, Brooks AN, Cheng C, Conesa A, Crosse EI, Eyras E, Guccione E, Lu SX, Neugebauer KM, Sehgal P, Song X, Tothova Z, Valcárcel J, Weeks KM, Yeo GW, Thomas-Tikhonenko A. Steering research on mRNA splicing in cancer towards clinical translation. Nat Rev Cancer 2024; 24:887-905. [PMID: 39384951 DOI: 10.1038/s41568-024-00750-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 08/27/2024] [Indexed: 10/11/2024]
Abstract
Splicing factors are affected by recurrent somatic mutations and copy number variations in several types of haematologic and solid malignancies, which is often seen as prima facie evidence that splicing aberrations can drive cancer initiation and progression. However, numerous spliceosome components also 'moonlight' in DNA repair and other cellular processes, making their precise role in cancer difficult to pinpoint. Still, few would deny that dysregulated mRNA splicing is a pervasive feature of most cancers. Correctly interpreting these molecular fingerprints can reveal novel tumour vulnerabilities and untapped therapeutic opportunities. Yet multiple technological challenges, lingering misconceptions, and outstanding questions hinder clinical translation. To start with, the general landscape of splicing aberrations in cancer is not well defined, due to limitations of short-read RNA sequencing not adept at resolving complete mRNA isoforms, as well as the shallow read depth inherent in long-read RNA-sequencing, especially at single-cell level. Although individual cancer-associated isoforms are known to contribute to cancer progression, widespread splicing alterations could be an equally important and, perhaps, more readily actionable feature of human cancers. This is to say that in addition to 'repairing' mis-spliced transcripts, possible therapeutic avenues include exacerbating splicing aberration with small-molecule spliceosome inhibitors, targeting recurrent splicing aberrations with synthetic lethal approaches, and training the immune system to recognize splicing-derived neoantigens.
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Affiliation(s)
- Olga Anczukow
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
| | - Frédéric H-T Allain
- Department of Biology, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland
| | | | - Douglas L Black
- Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA
| | - Angela N Brooks
- Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA, USA
| | - Chonghui Cheng
- Department of Molecular and Human Genetics, Lester & Sue Breast Center, Baylor College of Medicine, Houston, TX, USA
| | - Ana Conesa
- Institute for Integrative Systems Biology, Spanish National Research Council, Paterna, Spain
| | - Edie I Crosse
- Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA
| | - Eduardo Eyras
- Shine-Dalgarno Centre for RNA Innovation, Australian National University, Canberra, Australian Capital Territory, Australia
| | - Ernesto Guccione
- Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY, USA
| | - Sydney X Lu
- Department of Medicine, Stanford Medical School, Palo Alto, CA, USA
| | - Karla M Neugebauer
- Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA
| | - Priyanka Sehgal
- Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | - Xiao Song
- Department of Neurology, Northwestern University, Chicago, IL, USA
| | - Zuzana Tothova
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | - Juan Valcárcel
- Centre for Genomic Regulation, Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
| | - Kevin M Weeks
- Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA
| | - Gene W Yeo
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA
| | - Andrei Thomas-Tikhonenko
- Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
- Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
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Shen J, Shentu J, Zhong C, Huang Q, Duan S. RNA splicing factor RBFOX2 is a key factor in the progression of cancer and cardiomyopathy. Clin Transl Med 2024; 14:e1788. [PMID: 39243148 PMCID: PMC11380049 DOI: 10.1002/ctm2.1788] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2024] [Revised: 07/15/2024] [Accepted: 07/19/2024] [Indexed: 09/09/2024] Open
Abstract
BACKGROUND Alternative splicing of pre-mRNA is a fundamental regulatory process in multicellular eukaryotes, significantly contributing to the diversification of the human proteome. RNA-binding fox-1 homologue 2 (RBFOX2), a member of the evolutionarily conserved RBFOX family, has emerged as a critical splicing regulator, playing a pivotal role in the alternative splicing of pre-mRNA. This review provides a comprehensive analysis of RBFOX2, elucidating its splicing activity through direct and indirect binding mechanisms. RBFOX2 exerts substantial influence over the alternative splicing of numerous transcripts, thereby shaping essential cellular processes such as differentiation and development. MAIN BODY OF THE ABSTRACT Dysregulation of RBFOX2-mediated alternative splicing has been closely linked to a spectrum of cardiovascular diseases and malignant tumours, underscoring its potential as a therapeutic target. Despite significant progress, current research faces notable challenges. The complete structural characterisation of RBFOX2 remains elusive, limiting in-depth exploration beyond its RNA-recognition motif. Furthermore, the scarcity of studies focusing on RBFOX2-targeting drugs poses a hindrance to translating research findings into clinical applications. CONCLUSION This review critically assesses the existing body of knowledge on RBFOX2, highlighting research gaps and limitations. By delineating these areas, this analysis not only serves as a foundational reference for future studies but also provides strategic insights for bridging these gaps. Addressing these challenges will be instrumental in unlocking the full therapeutic potential of RBFOX2, paving the way for innovative and effective treatments in various diseases.
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Affiliation(s)
- Jinze Shen
- Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang ProvinceSchool of MedicineHangzhou City UniversityHangzhouChina
| | - Jianqiao Shentu
- Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang ProvinceSchool of MedicineHangzhou City UniversityHangzhouChina
| | - Chenming Zhong
- Medical Genetics Center, School of MedicineNingbo UniversityNingboChina
| | - Qiankai Huang
- Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang ProvinceSchool of MedicineHangzhou City UniversityHangzhouChina
| | - Shiwei Duan
- Key Laboratory of Novel Targets and Drug Study for Neural Repair of Zhejiang ProvinceSchool of MedicineHangzhou City UniversityHangzhouChina
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7
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Cordaro A, Barreca MM, Zichittella C, Loria M, Anello D, Arena G, Sciaraffa N, Coronnello C, Pizzolanti G, Alessandro R, Conigliaro A. Regulatory role of lncH19 in RAC1 alternative splicing: implication for RAC1B expression in colorectal cancer. J Exp Clin Cancer Res 2024; 43:217. [PMID: 39098911 PMCID: PMC11299361 DOI: 10.1186/s13046-024-03139-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2024] [Accepted: 07/26/2024] [Indexed: 08/06/2024] Open
Abstract
Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.
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Affiliation(s)
- Aurora Cordaro
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
| | - Maria Magdalena Barreca
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
| | - Chiara Zichittella
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
| | - Marco Loria
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
| | - Denise Anello
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
| | - Goffredo Arena
- McGill University Health Centre, Montréal, Canada
- Fondazione Istituto G. Giglio di Cefalù, Cefalù, Italy
| | | | | | - Giuseppe Pizzolanti
- Dipartimento di Promozione della Salute, Materno-Infantile, di Medicina Interna e Specialistica di Eccellenza "G. D'Alessandro", PROMISE, University of Palermo, Palermo, 90127, Italy
- AteN Center-Advanced Technologies Network Center, University of Palermo, Palermo, 90128, Italy
| | - Riccardo Alessandro
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy
- Institute for Biomedical Research and Innovation (IRIB), National Research Council (CNR), Palermo, Italy
| | - Alice Conigliaro
- Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy.
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8
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Rosemann J, Pyko J, Jacob R, Macho J, Kappler M, Eckert AW, Haemmerle M, Gutschner T. NANOS1 restricts oral cancer cell motility and TGF-ß signaling. Eur J Cell Biol 2024; 103:151400. [PMID: 38401491 DOI: 10.1016/j.ejcb.2024.151400] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2023] [Revised: 02/04/2024] [Accepted: 02/20/2024] [Indexed: 02/26/2024] Open
Abstract
Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells.
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Affiliation(s)
- Julia Rosemann
- Institute of Molecular Medicine, Section for RNA biology and pathogenesis, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Jonas Pyko
- Institute of Molecular Medicine, Section for RNA biology and pathogenesis, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Roland Jacob
- Institute of Molecular Medicine, Section for RNA biology and pathogenesis, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Jana Macho
- Institute of Molecular Medicine, Section for RNA biology and pathogenesis, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Matthias Kappler
- Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Alexander W Eckert
- Department of Cranio Maxillofacial Surgery, Paracelsus Medical University, Nuremberg 90471, Germany
| | - Monika Haemmerle
- Institute of Pathology, Section for Experimental Pathology, Martin Luther University Halle-Wittenberg, Halle 06120, Germany
| | - Tony Gutschner
- Institute of Molecular Medicine, Section for RNA biology and pathogenesis, Martin Luther University Halle-Wittenberg, Halle 06120, Germany.
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9
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Zhang YE, Stuelten CH. Alternative splicing in EMT and TGF-β signaling during cancer progression. Semin Cancer Biol 2024; 101:1-11. [PMID: 38614376 PMCID: PMC11180579 DOI: 10.1016/j.semcancer.2024.04.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Revised: 11/20/2023] [Accepted: 04/04/2024] [Indexed: 04/15/2024]
Abstract
Epithelial to mesenchymal transition (EMT) is a physiological process during development where epithelial cells transform to acquire mesenchymal characteristics, which allows them to migrate and colonize secondary tissues. Many cellular signaling pathways and master transcriptional factors exert a myriad of controls to fine tune this vital process to meet various developmental and physiological needs. Adding to the complexity of this network are post-transcriptional and post-translational regulations. Among them, alternative splicing has been shown to play important roles to drive EMT-associated phenotypic changes, including actin cytoskeleton remodeling, cell-cell junction changes, cell motility and invasiveness. In advanced cancers, transforming growth factor-β (TGF-β) is a major inducer of EMT and is associated with tumor cell metastasis, cancer stem cell self-renewal, and drug resistance. This review aims to provide an overview of recent discoveries regarding alternative splicing events and the involvement of splicing factors in the EMT and TGF-β signaling. It will emphasize the importance of various splicing factors involved in EMT and explore their regulatory mechanisms.
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Affiliation(s)
- Ying E Zhang
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
| | - Christina H Stuelten
- Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA
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10
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Huang F, Jin L, Zhang X, Wang M, Zhou C. Integrated pan-cancer analysis reveals the immunological and prognostic potential of RBFOX2 in human tumors. Front Pharmacol 2024; 15:1302134. [PMID: 38881877 PMCID: PMC11176534 DOI: 10.3389/fphar.2024.1302134] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2023] [Accepted: 05/08/2024] [Indexed: 06/18/2024] Open
Abstract
Background The role of RNA-binding fox one homolog 2 (RBFOX2) in the progression of multiple tumors is increasingly supported by evidence. However, the unclearness pertaining to the expression of RBFOX2, its prognostic potential, and its correlation with the tumor microenvironment (TME) in pan-cancer persists. This study aims to comprehensively investigate the immunological prognostic value of RBFOX2. Methods The Cancer Genome Atlas Gene Expression Omnibus Genotype-Tissue Expression (GTEx), TIMER2.0, Kaplan-Meier (K-M) Plotter, University of Alabama at Birmingham Cancer data analysis Portal (UALCAN), cbioportal, and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) were utilized for a systematic analysis of RBFOX2. This analysis included studying its expression, prognostic value, DNA methylation, enrichment analysis, immune infiltration cells, and immune-related genes. Additionally, qRT-PCR, CCK-8, colony formation, transwell assays, and immunohistochemistry were employed to analyze the expression and biological function of RBFOX2 in liver cancer. Results Variations in RBFOX2 expression have been observed across diverse tumors and have been identified as indicators of unfavorable prognosis. It is closely linked to immune infiltration cells, immune checkpoints, chemokines, and chemokine receptors in the TME. Higher levels of RBFOX2 have been significantly associated with low response and poor prognosis in patients with non-small cell lung cancer (NSCLC) and melanoma who receive immunotherapy. Furthermore, the DNA methylation of RBFOX2 varies across different types of cancer and has shown better prognosis in patients with BLCA, BRCA, CESC, COAD, DLBC, HNSC, LAML, LGG, LUAD, PAAD, SKCM and THYM. Interestingly, RBFOX2 expression was found to be lower in hepatocellular carcinoma (HCC) patients' tumor tissues compared to their paired adjacent tissues. In vitro studies have shown that knockdown of RBFOX2 significantly promotes the growth and metastasis of liver cancer cells. Conclusion This study investigates the correlation between DNA methylation, prognostic value, and immune cell infiltration with the expression of RBFOX2 in pan-cancer and indicates its potential role to inhibit metastasis of liver cancer.
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Affiliation(s)
- Fengxian Huang
- Department of Radiation Oncology, Shaanxi Provincial People's Hospital, Xi'an, China
| | - Long Jin
- Department of Radiation Oncology, Shaanxi Provincial People's Hospital, Xi'an, China
| | - Xinyue Zhang
- Department of Radiation Oncology, Shaanxi Provincial People's Hospital, Xi'an, China
| | - Min Wang
- Department of Science and Education, Xi'an Children's Hospital Affiliated of Xi'an Jiaotong University, Xi'an, China
| | - Congya Zhou
- Department of Radiation Oncology, Shaanxi Provincial People's Hospital, Xi'an, China
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11
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Dai C, Cui X, Wang J, Dong B, Gao H, Cheng M, Jiang F. CX‑5461 potentiates imatinib‑induced apoptosis in K562 cells by stimulating KIF1B expression. Exp Ther Med 2024; 27:107. [PMID: 38356673 PMCID: PMC10865453 DOI: 10.3892/etm.2024.12395] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2023] [Accepted: 11/29/2023] [Indexed: 02/16/2024] Open
Abstract
The selective RNA polymerase I inhibitor CX-5461 has been shown to be effective in treating some types of leukemic disorders. Emerging evidence suggests that combined treatments with CX-5461 and other chemotherapeutic agents may achieve enhanced effectiveness as compared with monotherapies. Currently, pharmacodynamic properties of the combination of CX-5461 with tyrosine kinase inhibitors remain to be explored. The present study tested whether CX-5461 could potentiate the effect of imatinib in the human chronic myeloid leukemia cell line K562, which is p53-deficient. It was demonstrated that CX-5461 at 100 nM, which was non-cytotoxic in K562 cells, potentiated the pro-apoptotic effect of imatinib. Mechanistically, the present study identified that the upregulated expression of kinesin family member 1B (KIF1B) gene might be involved in mediating the pro-apoptotic effect of imatinib/CX-5461 combination. Under the present experimental settings, however, neither CX-5461 nor imatinib alone exhibited a significant effect on KIF1B expression. Moreover, using other leukemic cell lines, it was demonstrated that regulation of KIF1B expression by imatinib/CX-5461 was not a ubiquitous phenomenon in leukemic cells and should be studied in a cell type-specific manner. In conclusion, the results suggested that the synergistic interaction between CX-5461 and imatinib may be of potential clinical value for the treatment of tyrosine kinase inhibitor-resistant chronic myeloid leukemia.
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Affiliation(s)
- Chaochao Dai
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
| | - Xiaopei Cui
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
| | - Jie Wang
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
| | - Bo Dong
- Department of Cardiology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, P.R. China
| | - Haiqing Gao
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
| | - Mei Cheng
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
| | - Fan Jiang
- Shandong Key Laboratory of Cardiovascular Proteomics and Department of Geriatric Medicine, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China
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12
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den Hollander P, Maddela JJ, Mani SA. Spatial and Temporal Relationship between Epithelial-Mesenchymal Transition (EMT) and Stem Cells in Cancer. Clin Chem 2024; 70:190-205. [PMID: 38175600 PMCID: PMC11246550 DOI: 10.1093/clinchem/hvad197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2023] [Accepted: 11/02/2023] [Indexed: 01/05/2024]
Abstract
BACKGROUND Epithelial-mesenchymal transition (EMT) is often linked with carcinogenesis. However, EMT is also important for embryo development and only reactivates in cancer. Connecting how EMT occurs during embryonic development and in cancer could help us further understand the root mechanisms of cancer diseases. CONTENT There are key regulatory elements that contribute to EMT and the induction and maintenance of stem cell properties during embryogenesis, tissue regeneration, and carcinogenesis. Here, we explore the implications of EMT in the different stages of embryogenesis and tissue development. We especially highlight the necessity of EMT in the mesodermal formation and in neural crest cells. Through EMT, these cells gain epithelial-mesenchymal plasticity (EMP). With this transition, crucial morphological changes occur to progress through the metastatic cascade as well as tissue regeneration after an injury. Stem-like cells, including cancer stem cells, are generated from EMT and during this process upregulate factors necessary for stem cell maintenance. Hence, it is important to understand the key regulators allowing stem cell awakening in cancer, which increases plasticity and promotes treatment resistance, to develop strategies targeting this cell population and improve patient outcomes. SUMMARY EMT involves multifaceted regulation to allow the fluidity needed to facilitate adaptation. This regulatory mechanism, plasticity, involves many cooperating transcription factors. Additionally, posttranslational modifications, such as splicing, activate the correct isoforms for either epithelial or mesenchymal specificity. Moreover, epigenetic regulation also occurs, such as acetylation and methylation. Downstream signaling ultimately results in the EMT which promotes tissue generation/regeneration and cancer progression.
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Affiliation(s)
- Petra den Hollander
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI, United States
- Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, United States
| | - Joanna Joyce Maddela
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI, United States
- Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, United States
| | - Sendurai A Mani
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI, United States
- Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI, United States
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13
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Maurin M, Ranjouri M, Megino-Luque C, Newberg JY, Du D, Martin K, Miner RE, Prater MS, Wee DKB, Centeno B, Pruett-Miller SM, Stewart P, Fleming JB, Yu X, Bravo-Cordero JJ, Guccione E, Black MA, Mann KM. RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing. Nat Commun 2023; 14:8444. [PMID: 38114498 PMCID: PMC10730836 DOI: 10.1038/s41467-023-44126-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Accepted: 11/30/2023] [Indexed: 12/21/2023] Open
Abstract
RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.
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Affiliation(s)
- Michelle Maurin
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | | | - Cristina Megino-Luque
- Division of Hematology and Oncology, Department of Medicine, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Justin Y Newberg
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Dongliang Du
- Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Katelyn Martin
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Robert E Miner
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Mollie S Prater
- Department of Cell and Molecular Biology and Center for Advanced Genome Engineering (CAGE), St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Dave Keng Boon Wee
- Institute for Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Republic of Singapore
| | - Barbara Centeno
- Department of Anatomic Pathology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Shondra M Pruett-Miller
- Department of Cell and Molecular Biology and Center for Advanced Genome Engineering (CAGE), St. Jude Children's Research Hospital, Memphis, TN, 38105, USA
| | - Paul Stewart
- Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Jason B Fleming
- Department of Gastrointestinal Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Xiaoqing Yu
- Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, 33612, USA
| | - Jose Javier Bravo-Cordero
- Division of Hematology and Oncology, Department of Medicine, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Ernesto Guccione
- Center for OncoGenomics and Innovative Therapeutics (COGIT), Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
- Center for Therapeutics Discovery, Department of Oncological Sciences and Pharmacological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Michael A Black
- Department of Biochemistry, University of Otago, Dunedin, 9054, New Zealand
| | - Karen M Mann
- Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA.
- Department of Gastrointestinal Oncology, Moffitt Cancer Center, Tampa, FL, 33612, USA.
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14
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Kwon MJ. Role of epithelial splicing regulatory protein 1 in cancer progression. Cancer Cell Int 2023; 23:331. [PMID: 38110955 PMCID: PMC10729575 DOI: 10.1186/s12935-023-03180-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2023] [Accepted: 12/12/2023] [Indexed: 12/20/2023] Open
Abstract
As aberrant alternative splicing by either dysregulation or mutations of splicing factors contributes to cancer initiation and progression, splicing factors are emerging as potential therapeutic targets for cancer therapy. Therefore, pharmacological modulators targeting splicing factors have been under development. Epithelial splicing regulatory protein 1 (ESRP1) is an epithelial cell-specific splicing factor, whose downregulation is associated with epithelial-mesenchymal transition (EMT) by regulating alternative splicing of multiple genes, such as CD44, CTNND1, ENAH, and FGFR2. Consistent with the downregulation of ESRP1 during EMT, it has been initially revealed that high ESRP1 expression is associated with favorable prognosis and ESRP1 plays a tumor-suppressive role in cancer progression. However, ESRP1 has been found to promote cancer progression in some cancers, such as breast and ovarian cancers, indicating that it plays a dual role in cancer progression depending on the type of cancer. Furthermore, recent studies have reported that ESRP1 affects tumor growth by regulating the metabolism of tumor cells or immune cell infiltration in the tumor microenvironment, suggesting the novel roles of ESRP1 in addition to EMT. ESRP1 expression was also associated with response to anticancer drugs. This review describes current understanding of the roles and mechanisms of ESRP1 in cancer progression, and further discusses the emerging novel roles of ESRP1 in cancer and recent attempts to target splicing factors for cancer therapy.
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Affiliation(s)
- Mi Jeong Kwon
- Vessel-Organ Interaction Research Center (MRC), College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea.
- BK21 FOUR KNU Community-Based Intelligent Novel Drug Discovery Education Unit, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu, 41566, Republic of Korea.
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15
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Wang Y, Yang Y, Yang Y, Dang Y, Guo Z, Zhuang Q, Zheng X, Wang F, Cheng N, Liu X, Guo W, Zhao B. Hypoxia induces hepatocellular carcinoma metastasis via the HIF-1α/METTL16/lnc-CSMD1-7/RBFOX2 axis. iScience 2023; 26:108495. [PMID: 38089592 PMCID: PMC10711500 DOI: 10.1016/j.isci.2023.108495] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 10/06/2023] [Accepted: 11/16/2023] [Indexed: 01/23/2025] Open
Abstract
Hypoxic microenvironment is clinically associated with metastasis and poor prognosis of numerous cancers. The mechanisms by which intratumoral hypoxia regulates metastasis are not fully understood. Our study identifies a downregulation of Lnc-CSMD1-7 in hepatocellular carcinoma (HCC) and correlated with poor prognosis of HCC patients. Lnc-CSMD1-7 negatively regulated HCC cell migration and invasion in vitro and suppressed lung metastasis in vivo. Mechanistically, Lnc-CSMD1-7 directly binds to RBFOX2, thereby affecting RBFOX2-regulated alternative splicing in epithelial and mesenchymal-specific events. More importantly, hypoxic microenvironment and m6A methylation mediate the downregulation of Lnc-CSMD1-7 expression. Specifically, hypoxia transcriptionally upregulates the expression of the m6A methyltransferase METTL16 via HIF-1α, and METTL16 directly binds to Lnc-CSMD1-7 and downregulates the RNA stability of Lnc-CSMD1-7 via m6A methylation, ultimately promoting HCC metastasis. Our findings highlight the regulatory function of the METTL16/Lnc-CSMD1-7/RBFOX2 axis in modulating hypoxia-induced HCC progression, which may provide potential prognostic and therapeutic targets for HCC treatment.
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Affiliation(s)
- Yingchao Wang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
| | - Yong Yang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
- Fujian Medical University Cancer Hospital, Fujian Cancer Hospital, Fuzhou, China
| | - Ye Yang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
| | - Yuan Dang
- Fujian Medical University Cancer Hospital, Fujian Cancer Hospital, Fuzhou, China
| | - Zhiting Guo
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
| | - Qiuyu Zhuang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
| | - Xiaoyuan Zheng
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
| | - Fei Wang
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
| | - Niangmei Cheng
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
| | - Xiaolong Liu
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
| | - Wuhua Guo
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
- Department of Interventional Radiology, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou, Fujian, China
| | - Bixing Zhao
- The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P.R. China
- Mengchao Med-X Center, Fuzhou University, Fuzhou 350116, P.R. China
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16
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Maltseva D, Tonevitsky A. RNA-binding proteins regulating the CD44 alternative splicing. Front Mol Biosci 2023; 10:1326148. [PMID: 38106992 PMCID: PMC10722200 DOI: 10.3389/fmolb.2023.1326148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2023] [Accepted: 11/15/2023] [Indexed: 12/19/2023] Open
Abstract
Alternative splicing is often deregulated in cancer, and cancer-specific isoform switches are part of the oncogenic transformation of cells. Accumulating evidence indicates that isoforms of the multifunctional cell-surface glycoprotein CD44 play different roles in cancer cells as compared to normal cells. In particular, the shift of CD44 isoforms is required for epithelial to mesenchymal transition (EMT) and is crucial for the maintenance of pluripotency in normal human cells and the acquisition of cancer stem cells phenotype for malignant cells. The growing and seemingly promising use of splicing inhibitors for treating cancer and other pathologies gives hope for the prospect of using such an approach to regulate CD44 alternative splicing. This review integrates current knowledge about regulating CD44 alternative splicing by RNA-binding proteins.
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Affiliation(s)
- Diana Maltseva
- Faculty of Biology and Biotechnology, HSE University, Moscow, Russia
| | - Alexander Tonevitsky
- Faculty of Biology and Biotechnology, HSE University, Moscow, Russia
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
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17
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Moss ND, Wells KL, Theis A, Kim YK, Spigelman AF, Liu X, MacDonald PE, Sussel L. Modulation of insulin secretion by RBFOX2-mediated alternative splicing. Nat Commun 2023; 14:7732. [PMID: 38007492 PMCID: PMC10676425 DOI: 10.1038/s41467-023-43605-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 11/15/2023] [Indexed: 11/27/2023] Open
Abstract
Insulin secretion is a tightly regulated process that is vital for maintaining blood glucose homeostasis. Although the molecular components of insulin granule trafficking and secretion are well established, how they are regulated to rapidly fine-tune secretion in response to changing environmental conditions is not well characterized. Recent studies have determined that dysregulation of RNA-binding proteins (RBPs) and aberrant mRNA splicing occurs at the onset of diabetes. We demonstrate that the RBP, RBFOX2, is a critical regulator of insulin secretion through the alternative splicing of genes required for insulin granule docking and exocytosis. Conditional mutation of Rbfox2 in the mouse pancreas results in decreased insulin secretion and impaired blood glucose homeostasis. Consistent with defects in secretion, we observe reduced insulin granule docking and corresponding splicing defects in the SNARE complex components. These findings identify an additional mechanism for modulating insulin secretion in both healthy and dysfunctional pancreatic β cells.
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Affiliation(s)
- Nicole D Moss
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Kristen L Wells
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Alexandra Theis
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Yong-Kyung Kim
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Aliya F Spigelman
- Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
| | - Xiong Liu
- Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
| | - Patrick E MacDonald
- Department of Pharmacology and Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
| | - Lori Sussel
- Barbara Davis Center for Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
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18
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Li S, Chen Y, Xie Y, Zhan H, Zeng Y, Zeng K, Wang L, Zhan Z, Li C, Zhao L, Chen X, Tan Y, Wang Z, Bu J, Song Y, Deng F, Zhou A. FBXO7 Confers Mesenchymal Properties and Chemoresistance in Glioblastoma by Controlling Rbfox2-Mediated Alternative Splicing. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2303561. [PMID: 37822160 PMCID: PMC10667838 DOI: 10.1002/advs.202303561] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 09/06/2023] [Indexed: 10/13/2023]
Abstract
Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.
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Affiliation(s)
- Shangbiao Li
- Department of Radiation OncologyZhujiang HospitalSouthern Medical UniversityGuangzhou510280China
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Yanwen Chen
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Yuxin Xie
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Hongchao Zhan
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Yu Zeng
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Kunlin Zeng
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Li Wang
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Ziling Zhan
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Cuiying Li
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Liqian Zhao
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Xiaoxia Chen
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Yujing Tan
- Department of Radiation OncologyZhujiang HospitalSouthern Medical UniversityGuangzhou510280China
| | - Zhongyong Wang
- Department of NeurosurgeryThe Second Affiliated Hospital of Soochow UniversitySuzhou215004China
| | - Junguo Bu
- Department of Radiation OncologyZhujiang HospitalSouthern Medical UniversityGuangzhou510280China
| | - Ye Song
- Department of NeurosurgeryNanfang HospitalSouthern Medical UniversityGuangzhou510515China
| | - Fan Deng
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
| | - Aidong Zhou
- Department of Radiation OncologyZhujiang HospitalSouthern Medical UniversityGuangzhou510280China
- Department of Cell BiologySchool of Basic Medical ScienceSouthern Medical UniversityGuangzhou510515China
- Guangdong Province Key Laboratory of Molecular Tumor PathologySouthern Medical UniversityGuangzhou510515China
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19
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Li L, Zheng J, Oltean S. Regulation of Epithelial-Mesenchymal Transitions by Alternative Splicing: Potential New Area for Cancer Therapeutics. Genes (Basel) 2023; 14:2001. [PMID: 38002944 PMCID: PMC10671305 DOI: 10.3390/genes14112001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2023] [Revised: 10/23/2023] [Accepted: 10/25/2023] [Indexed: 11/26/2023] Open
Abstract
The epithelial-mesenchymal transition (EMT) is a complicated biological process in which cells with epithelial phenotype are transformed into mesenchymal cells with loss of cell polarity and cell-cell adhesion and gain of the ability to migrate. EMT and the reverse mesenchymal-epithelial transitions (METs) are present during cancer progression and metastasis. Using the dynamic switch between EMT and MET, tumour cells can migrate to neighbouring organs or metastasize in the distance and develop resistance to traditional chemotherapy and targeted drug treatments. Growing evidence shows that reversing or inhibiting EMT may be an advantageous approach for suppressing the migration of tumour cells or distant metastasis. Among different levels of modulation of EMT, alternative splicing (AS) plays an important role. An in-depth understanding of the role of AS and EMT in cancer is not only helpful to better understand the occurrence and regulation of EMT in cancer progression, but also may provide new therapeutic strategies. This review will present and discuss various splice variants and splicing factors that have been shown to play a crucial role in EMT.
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Affiliation(s)
| | | | - Sebastian Oltean
- Department of Clinical and Biomedical Sciences, Faculty of Health and Life Sciences, University of Exeter Medical School, Exeter EX1 2LU, UK; (L.L.)
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20
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Datta S, Cao W, Skillman M, Wu M. Hypoplastic Left Heart Syndrome: Signaling & Molecular Perspectives, and the Road Ahead. Int J Mol Sci 2023; 24:15249. [PMID: 37894928 PMCID: PMC10607600 DOI: 10.3390/ijms242015249] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 10/07/2023] [Accepted: 10/12/2023] [Indexed: 10/29/2023] Open
Abstract
Hypoplastic left heart syndrome (HLHS) is a lethal congenital heart disease (CHD) affecting 8-25 per 100,000 neonates globally. Clinical interventions, primarily surgical, have improved the life expectancy of the affected subjects substantially over the years. However, the etiological basis of HLHS remains fundamentally unclear to this day. Based upon the existing paradigm of studies, HLHS exhibits a multifactorial mode of etiology mediated by a complicated course of genetic and signaling cascade. This review presents a detailed outline of the HLHS phenotype, the prenatal and postnatal risks, and the signaling and molecular mechanisms driving HLHS pathogenesis. The review discusses the potential limitations and future perspectives of studies that can be undertaken to address the existing scientific gap. Mechanistic studies to explain HLHS etiology will potentially elucidate novel druggable targets and empower the development of therapeutic regimens against HLHS in the future.
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Affiliation(s)
| | | | | | - Mingfu Wu
- Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, USA; (S.D.); (W.C.); (M.S.)
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21
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Liu B, Song A, Gui P, Wang J, Pan Y, Li C, Li S, Zhang Y, Jiang T, Xu Y, Pei D, Song J. Long noncoding RNA LINC01594 inhibits the CELF6-mediated splicing of oncogenic CD44 variants to promote colorectal cancer metastasis. Cell Death Dis 2023; 14:427. [PMID: 37452042 PMCID: PMC10349055 DOI: 10.1038/s41419-023-05924-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 06/16/2023] [Accepted: 06/23/2023] [Indexed: 07/18/2023]
Abstract
Long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis and tumor metastasis. However, the underlying mechanisms of lncRNAs in colorectal cancer (CRC) need further exploration. By using data from The Cancer Genome Atlas (TCGA) and GEO databases, we identified a novel CRC-related lncRNA, LINC01594, that is significantly upregulated in CRC and associated with poor prognosis. In vitro and in vivo, gain- and loss-of-function experiments demonstrated that LINC01594 promotes metastasis in CRC. LINC01594 functions as a DNMT1 scaffold, increasing the level of CELF6 promoter methylation. LINC01594 also competitively binds the transcription factor p53, decreasing CELF6 expression. This inhibited the exon skipping of CD44 V4-V7 induced by CELF6. In summary, this study highlights a novel CRC biomarker and therapeutic target, LINC01594, and the findings suggest that the LINC01594-CELF6-CD44 axis might serve as a biomarker and therapeutic target in CRC.
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Affiliation(s)
- Bowen Liu
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Angxi Song
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Pengkun Gui
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Jin Wang
- Department of Pathology, Xuzhou Medical University. No. 209, Tongshan Road, Yunlong District, Xuzhou, 221004, China
| | - Yaojie Pan
- Department of Medical Oncology, Zhejiang Provincial People's Hospital. No. 158, Shangtang Road, Xiacheng District, Zhejiang, 310000, China
| | - Chao Li
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Shuai Li
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Yi Zhang
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Tao Jiang
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Yixin Xu
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China
| | - Dongsheng Pei
- Department of Pathology, Xuzhou Medical University. No. 209, Tongshan Road, Yunlong District, Xuzhou, 221004, China.
| | - Jun Song
- Department of General Surgery, The Affiliated Hospital of Xuzhou Medical University. No. 99, Huaihai West Road, Quanshan District, Xuzhou, 221006, China.
- Institute of Digestive Diseases, Xuzhou Medical University. No. 84, Huaihai West Road, Quanshan District, Xuzhou, 221002, China.
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22
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Derham JM, Kalsotra A. The discovery, function, and regulation of epithelial splicing regulatory proteins (ESRP) 1 and 2. Biochem Soc Trans 2023; 51:1097-1109. [PMID: 37314029 PMCID: PMC11298080 DOI: 10.1042/bst20221124] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Revised: 06/01/2023] [Accepted: 06/05/2023] [Indexed: 06/15/2023]
Abstract
Alternative splicing is a broad and evolutionarily conserved mechanism to diversify gene expression and functionality. The process relies on RNA binding proteins (RBPs) to recognize and bind target sequences in pre-mRNAs, which allows for the inclusion or skipping of various alternative exons. One recently discovered family of RBPs is the epithelial splicing regulatory proteins (ESRP) 1 and 2. Here, we discuss the structure and physiological function of the ESRPs in a variety of contexts. We emphasize the current understanding of their splicing activities, using the classic example of fibroblast growth factor receptor 2 mutually exclusive splicing. We also describe the mechanistic roles of ESRPs in coordinating the splicing and functional output of key signaling pathways that support the maintenance of, or shift between, epithelial and mesenchymal cell states. In particular, we highlight their functions in the development of mammalian limbs, the inner ear, and craniofacial structure while discussing the genetic and biochemical evidence that showcases their conserved roles in tissue regeneration, disease, and cancer pathogenesis.
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Affiliation(s)
- Jessica M. Derham
- Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Auinash Kalsotra
- Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL, USA
- Cancer Center @ Illinois, University of Illinois Urbana-Champaign, Urbana, IL, USA
- Carl R. Woese Institute of Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA
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23
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Biswal SR, Singh M, Dwibedy SLL, Kumari S, Muthuswamy S, Kumar A, Kumar S. Deciphering the RNA-binding protein interaction with the mRNAs encoded from human chromosome 15q11.2 BP1-BP2 microdeletion region. Funct Integr Genomics 2023; 23:174. [PMID: 37219715 DOI: 10.1007/s10142-023-01105-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Revised: 05/12/2023] [Accepted: 05/15/2023] [Indexed: 05/24/2023]
Abstract
Microdeletion of the 15q11.2 BP1-BP2 region, also known as Burnside-Butler susceptibility region, is associated with phenotypes like delayed developmental language abilities along with motor skill disabilities, combined with behavioral and emotional problems. The 15q11.2 microdeletion region harbors four evolutionarily conserved and non-imprinted protein-coding genes: NIPA1, NIPA2, CYFIP1, and TUBGCP5. This microdeletion is a rare copy number variation frequently associated with several pathogenic conditions in humans. The aim of this study is to investigate the RNA-binding proteins binding with the four genes present in 15q11.2 BP1-BP2 microdeletion region. The results of this study will help to better understand the molecular intricacies of the Burnside-Butler Syndrome and also the possible involvement of these interactions in the disease aetiology. Our results of enhanced crosslinking and immunoprecipitation data analysis indicate that most of the RBPs interacting with the 15q11.2 region are involved in the post-transcriptional regulation of the concerned genes. The RBPs binding to this region are found from the in silico analysis, and the interaction of RBPs like FASTKD2 and EFTUD2 with exon-intron junction sequence of CYFIP1 and TUBGCP5 has also been validated by combined EMSA and western blotting experiment. The exon-intron junction binding nature of these proteins suggests their potential involvement in splicing process. This study may help to understand the intricate relationship of RBPs with mRNAs within this region, along with their functional significance in normal development, and lack thereof, in neurodevelopmental disorders. This understanding will help in the formulation of better therapeutic approaches.
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Affiliation(s)
- Smruti Rekha Biswal
- Department of Life Science, National Institute of Technology (NIT), Rourkela, Odisha, 769008, India
| | - Mandakini Singh
- Department of Life Science, National Institute of Technology (NIT), Rourkela, Odisha, 769008, India
| | | | - Subhadra Kumari
- Department of Life Science, National Institute of Technology (NIT), Rourkela, Odisha, 769008, India
| | - Srinivasan Muthuswamy
- Department of Life Science, National Institute of Technology (NIT), Rourkela, Odisha, 769008, India
| | - Ajay Kumar
- Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India
| | - Santosh Kumar
- Department of Life Science, National Institute of Technology (NIT), Rourkela, Odisha, 769008, India.
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24
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Jbara A, Lin KT, Stossel C, Siegfried Z, Shqerat H, Amar-Schwartz A, Elyada E, Mogilevsky M, Raitses-Gurevich M, Johnson JL, Yaron TM, Ovadia O, Jang GH, Danan-Gotthold M, Cantley LC, Levanon EY, Gallinger S, Krainer AR, Golan T, Karni R. RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer. Nature 2023; 617:147-153. [PMID: 36949200 PMCID: PMC10156590 DOI: 10.1038/s41586-023-05820-3] [Citation(s) in RCA: 42] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Accepted: 02/10/2023] [Indexed: 03/24/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1-3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.
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Affiliation(s)
- Amina Jbara
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Kuan-Ting Lin
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
| | - Chani Stossel
- Division of Oncology, Sheba Medical Center Tel Hashomer, Ramat-Gan, Israel
| | - Zahava Siegfried
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Haya Shqerat
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Adi Amar-Schwartz
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Ela Elyada
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Maxim Mogilevsky
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | | | - Jared L Johnson
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Tomer M Yaron
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Department of Medicine, Weill Cornell Medicine, New York, NY, USA
- Englander Institute for Precision Medicine, Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA
- Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA
| | - Ofek Ovadia
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel
| | - Gun Ho Jang
- Department of Surgery, University of Toronto, Toronto, Ontario, Canada
| | - Miri Danan-Gotthold
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
| | - Lewis C Cantley
- Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA
- Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Erez Y Levanon
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
| | - Steven Gallinger
- Department of Surgery, University of Toronto, Toronto, Ontario, Canada
| | | | - Talia Golan
- Division of Oncology, Sheba Medical Center Tel Hashomer, Ramat-Gan, Israel
| | - Rotem Karni
- Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
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25
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Lu B, Li X, Miao W, Liu Q, Li R, Cui C, Gao Q, Lian R. LncRNA ZFAS1 promotes laryngeal cancer progression through RBFOX2-mediated MENA alternative splicing. ENVIRONMENTAL TOXICOLOGY 2023; 38:522-533. [PMID: 36336961 DOI: 10.1002/tox.23695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 09/22/2022] [Accepted: 10/13/2022] [Indexed: 06/16/2023]
Abstract
Laryngeal cancer (LC) is the most common aggressive malignancy of the head and neck. LncRNA ZNFX1 antisense RNA 1 (ZFAS1) displays oncogenic properties in head and neck squamous cell carcinoma, but its regulatory role in laryngeal cancer progression remains obscure. Here, we found that ZFAS1 expression in laryngeal cancer tissues and cells was higher than that in adjacent normal tissues and normal nasopharyngeal epithelial cells. Highly expressed ZFAS1 was associated with advanced lymph node metastasis stages and clinical stages. ZFAS1 overexpression promoted LC cell proliferation, invasion, and N-cadherin and Vimentin expression, and suppressed E-cadherin expression. While ZFAS1 knockdown played an opposite role. Mechanistically, ZFAS1 stabilized RNA binding fox-1 homolog 2 (RBFOX2) protein expression by binding to RBFOX2, and RBFOX2 overexpression reversed the effect of ZFAS1 silence on cell functions. Moreover, highly expressed RBFOX2 led to skipping of MENA exon 11a and generating a pro-invasive isoform (MENAINV ). MENAINV overexpression effectively abolished the inhibitory effect of RBFOX2 knockdown on cell malignant progression. Furthermore, Hep2 cells infected with lentivirus-mediated ZFAS1 shRNA or negative control shRNA were subcutaneously injected into mice to assess the role of ZFAS1 in tumor growth. And the data showed that silencing ZFAS1 in vivo hindered xenograft tumor growth. In conclusion, silencing ZFAS1 alleviated malignant progression of laryngeal cancer cells and mouse xenograft tumor growth by regulating RBFOX2-mediated alternative splicing of MENA.
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Affiliation(s)
- Baocai Lu
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Xiao Li
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Wenjie Miao
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Qi Liu
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Ruixue Li
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Can Cui
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Qingzu Gao
- Department of Pathology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
| | - Rong Lian
- Department of Otolaryngology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan, China
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26
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Akcan TS, Vilov S, Heinig M. Predictive model of transcriptional elongation control identifies trans regulatory factors from chromatin signatures. Nucleic Acids Res 2023; 51:1608-1624. [PMID: 36727445 PMCID: PMC9976927 DOI: 10.1093/nar/gkac1272] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Revised: 12/09/2022] [Accepted: 01/12/2023] [Indexed: 02/03/2023] Open
Abstract
Promoter-proximal Polymerase II (Pol II) pausing is a key rate-limiting step for gene expression. DNA and RNA-binding trans-acting factors regulating the extent of pausing have been identified. However, we lack a quantitative model of how interactions of these factors determine pausing, therefore the relative importance of implicated factors is unknown. Moreover, previously unknown regulators might exist. Here we address this gap with a machine learning model that accurately predicts the extent of promoter-proximal Pol II pausing from large-scale genome and transcriptome binding maps and gene annotation and sequence composition features. We demonstrate high accuracy and generalizability of the model by validation on an independent cell line which reveals the model's cell line agnostic character. Model interpretation in light of prior knowledge about molecular functions of regulatory factors confirms the interconnection of pausing with other RNA processing steps. Harnessing underlying feature contributions, we assess the relative importance of each factor, quantify their predictive effects and systematically identify previously unknown regulators of pausing. We additionally identify 16 previously unknown 7SK ncRNA interacting RNA-binding proteins predictive of pausing. Our work provides a framework to further our understanding of the regulation of the critical early steps in transcriptional elongation.
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Affiliation(s)
- Toray S Akcan
- Institute of Computational Biology, Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.,Department of Computer Science, TUM School of Computation, Information and Technology, Technical University Munich, Munich, Germany
| | - Sergey Vilov
- Institute of Computational Biology, Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany
| | - Matthias Heinig
- Institute of Computational Biology, Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.,Department of Computer Science, TUM School of Computation, Information and Technology, Technical University Munich, Munich, Germany.,DZHK (German Centre for Cardiovascular Research), Munich Heart Association, Partner Site Munich, 10785 Berlin, Germany
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27
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Bareche Y, Kelly D, Abbas-Aghababazadeh F, Nakano M, Esfahani PN, Tkachuk D, Mohammad H, Samstein R, Lee CH, Morris LGT, Bedard PL, Haibe-Kains B, Stagg J. Leveraging big data of immune checkpoint blockade response identifies novel potential targets. Ann Oncol 2022; 33:1304-1317. [PMID: 36055464 DOI: 10.1016/j.annonc.2022.08.084] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2022] [Revised: 08/03/2022] [Accepted: 08/22/2022] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND The development of immune checkpoint blockade (ICB) has changed the way we treat various cancers. While ICB produces durable survival benefits in a number of malignancies, a large proportion of treated patients do not derive clinical benefit. Recent clinical profiling studies have shed light on molecular features and mechanisms that modulate response to ICB. Nevertheless, none of these identified molecular features were investigated in large enough cohorts to be of clinical value. MATERIALS AND METHODS Literature review was carried out to identify relevant studies including clinical dataset of patients treated with ICB [anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) or the combination] and available sequencing data. Tumor mutational burden (TMB) and 37 previously reported gene expression (GE) signatures were computed with respect to the original publication. Biomarker association with ICB response (IR) and survival (progression-free survival/overall survival) was investigated separately within each study and combined together for meta-analysis. RESULTS We carried out a comparative meta-analysis of genomic and transcriptomic biomarkers of IRs in over 3600 patients across 12 tumor types and implemented an open-source web application (predictIO.ca) for exploration. TMB and 21/37 gene signatures were predictive of IRs across tumor types. We next developed a de novo GE signature (PredictIO) from our pan-cancer analysis and demonstrated its superior predictive value over other biomarkers. To identify novel targets, we computed the T-cell dysfunction score for each gene within PredictIO and their ability to predict dual PD-1/CTLA-4 blockade in mice. Two genes, F2RL1 (encoding protease-activated receptor-2) and RBFOX2 (encoding RNA-binding motif protein 9), were concurrently associated with worse ICB clinical outcomes, T-cell dysfunction in ICB-naive patients and resistance to dual PD-1/CTLA-4 blockade in preclinical models. CONCLUSION Our study highlights the potential of large-scale meta-analyses in identifying novel biomarkers and potential therapeutic targets for cancer immunotherapy.
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Affiliation(s)
- Y Bareche
- Faculty of Pharmacy, Université de Montréal, Montreal, Canada; Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Institut du Cancer de Montréal, Montreal, Canada
| | - D Kelly
- Princess Margaret Cancer Centre, University Health Network, Division of Medical Oncology and Hematology, Toronto, Canada
| | - F Abbas-Aghababazadeh
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada
| | - M Nakano
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada
| | - P N Esfahani
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada
| | - D Tkachuk
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada
| | - H Mohammad
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada
| | - R Samstein
- Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, USA
| | - C-H Lee
- Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, USA
| | - L G T Morris
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA
| | - P L Bedard
- Princess Margaret Cancer Centre, University Health Network, Division of Medical Oncology and Hematology, Toronto, Canada
| | - B Haibe-Kains
- Princess Margaret Bioinformatics and Computational Genomics Laboratory, University Health Network, Toronto, Canada; Princess Margaret Cancer Centre, University Health Network, Toronto, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Canada; Department ofComputer Science, University of Toronto, Toronto, Canada; Department ofOntario Institute for Cancer Research, Toronto, Canada; Department ofVector Institute for Artificial Intelligence, Toronto, Canada; Department ofBiostatistics Division, Dalla Lana School of Public Health, Toronto, Canada.
| | - J Stagg
- Faculty of Pharmacy, Université de Montréal, Montreal, Canada; Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Institut du Cancer de Montréal, Montreal, Canada.
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28
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Characterization of alternative mRNA splicing in cultured cell populations representing progressive stages of human fetal kidney development. Sci Rep 2022; 12:19548. [PMID: 36380228 PMCID: PMC9666651 DOI: 10.1038/s41598-022-24147-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Accepted: 11/10/2022] [Indexed: 11/16/2022] Open
Abstract
Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme-a transient kidney-specific progenitor state-undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms' tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms' tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms' tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.
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29
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Zhang J, Chen S, Wei S, Cheng S, Shi R, Zhao R, Zhang W, Zhang Q, Hua T, Feng D, Yu Z, Wang H. CircRAPGEF5 interacts with RBFOX2 to confer ferroptosis resistance by modulating alternative splicing of TFRC in endometrial cancer. Redox Biol 2022; 57:102493. [PMID: 36182807 PMCID: PMC9526237 DOI: 10.1016/j.redox.2022.102493] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2022] [Revised: 09/21/2022] [Accepted: 09/23/2022] [Indexed: 11/21/2022] Open
Abstract
Endometrial cancer (EC) is one of the most common gynecological cancers. Ferroptosis is a newly identified form of cell death characterized by iron-dependent lipid peroxide accumulation. Circular RNAs (circRNAs) have emerged as critical regulators for cancer development. However, circRNA-mediated modulation of ferroptosis in EC is yet to be clarified. In this study, we found that circRAPGEF5 expression was elevated in EC tissues compared to the normal endometrial tissues. In vitro and in vivo functional analysis demonstrated that circRAPGEF5 facilitates rapid proliferation of EC cells. RNA binding protein fox-1 homolog 2 (RBFOX2), a splicing regulator, was identified as the protein interacts with circRAPGEF5. Further studies revealed that circRAPGEF5 can bind to the Fox-1 C-terminal domain of RBFOX2 and induces specific exon exclusion of TFRC through obstructing the binding of RBFOX2 to pre-mRNA. As a result, elevated levels of circRAPGEF5 lead to ferroptosis resistance via the decreased labile iron pool and attenuated lipid peroxide production in EC cells. Additionally, a series of gain- and loss-of-function experiments demonstrated that knocking down or overexpressing RBFOX2 reversed the effects of knocking down or overexpressing circRAPGEF5 in EC cells. Finally, it is revealed that circRAPGEF5 promote the formation of TFRC with exon-4 skipping and confer ferroptosis resistance in EC cells through the interaction with RBFOX2. Collectively, these findings provide new insight into the molecular mechanism in which circRNAs mediate mediates ferroptosis via modulating alternative splicing, and circRAPGEF5/RBFOX2 splicing axis could be a promising therapeutic target for treating EC.
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Affiliation(s)
- Jun Zhang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Shuaijun Chen
- Department of Pathology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430014, China
| | - Sitian Wei
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Shuangshuang Cheng
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Rui Shi
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Rong Zhao
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Wei Zhang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Qi Zhang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Teng Hua
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Dilu Feng
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China
| | - Zhicheng Yu
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China.
| | - Hongbo Wang
- Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, China; Clinical Research Center of Cancer Immunotherapy, Wuhan, Hubei, 430022, China.
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Trelford CB, Dagnino L, Di Guglielmo GM. Transforming growth factor-β in tumour development. Front Mol Biosci 2022; 9:991612. [PMID: 36267157 PMCID: PMC9577372 DOI: 10.3389/fmolb.2022.991612] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Accepted: 09/15/2022] [Indexed: 11/14/2022] Open
Abstract
Transforming growth factor-β (TGFβ) is a ubiquitous cytokine essential for embryonic development and postnatal tissue homeostasis. TGFβ signalling regulates several biological processes including cell growth, proliferation, apoptosis, immune function, and tissue repair following injury. Aberrant TGFβ signalling has been implicated in tumour progression and metastasis. Tumour cells, in conjunction with their microenvironment, may augment tumourigenesis using TGFβ to induce epithelial-mesenchymal transition, angiogenesis, lymphangiogenesis, immune suppression, and autophagy. Therapies that target TGFβ synthesis, TGFβ-TGFβ receptor complexes or TGFβ receptor kinase activity have proven successful in tissue culture and in animal models, yet, due to limited understanding of TGFβ biology, the outcomes of clinical trials are poor. Here, we review TGFβ signalling pathways, the biology of TGFβ during tumourigenesis, and how protein quality control pathways contribute to the tumour-promoting outcomes of TGFβ signalling.
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Affiliation(s)
- Charles B. Trelford
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
| | - Lina Dagnino
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
- Department of Oncology, Children’s Health Research Institute and Lawson Health Research Institute, London, ON, Canada
| | - Gianni M. Di Guglielmo
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada
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Chetta M, Tarsitano M, Oro M, Rivieccio M, Bukvic N. An in silico pipeline approach uncovers a potentially intricate network involving spike SARS-CoV-2 RNA, RNA vaccines, host RNA-binding proteins (RBPs), and host miRNAs at the cellular level. J Genet Eng Biotechnol 2022; 20:129. [PMID: 36066672 PMCID: PMC9446605 DOI: 10.1186/s43141-022-00413-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 08/25/2022] [Indexed: 11/10/2022]
Abstract
BACKGROUND In the last 2 years, we have been fighting against SARS-CoV-2 viral infection, which continues to claim victims all over the world. The entire scientific community has been mobilized in an attempt to stop and eradicate the infection. A well-known feature of RNA viruses is their high mutational rate, particularly in specific gene regions. The SARS-CoV-2 S protein is also affected by these changes, allowing viruses to adapt and spread more easily. The vaccines developed using mRNA coding protein S undoubtedly contributed to the "fight" against the COVID-19 pandemic even though the presence of new variants in the spike protein could result in protein conformational changes, which could affect vaccine immunogenicity and thus vaccine effectiveness. RESULTS The study presents the findings of an in silico analysis using various bioinformatics tools finding conserved sequences inside SARS-CoV-2 S protein (encoding mRNA) same as in the vaccine RNA sequences that could be targeted by specific host RNA-binding proteins (RBPs). According to the results an interesting scenario emerges involving host RBPs competition and subtraction. The presence of viral RNA in cytoplasm could be a new tool in the virus's armory, allowing it to improve its chances of survival by altering cell gene expression and thus interfering with host cell processes. In silico analysis was used also to evaluate the presence of similar human miRNA sequences within RBPs motifs that can modulate human RNA expression. Increased cytoplasmic availability of exogenous RNA fragments derived from RNA physiological degradation could potentially mimic the effect of host human miRNAs within the cell, causing modulation of the host cell network. CONCLUSIONS Our in silico analysis could aid in shedding light on the potential effects of exogenous RNA (i.e. viruses and vaccines), thereby improving our understanding of the cellular interactions between virus and host biomolecules. Finally, using the computational approach, it is possible to obtain a safety assessment of RNA-based vaccines as well as indications for use in specific clinical conditions.
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Affiliation(s)
- Massimiliano Chetta
- AORN A. Cardarelli-Dipartimento delle Tecnologie Avanzate Diagnostico-Terapeutiche e dei Servizi sanitari-U.O.C. Genetica Medica e di Laboratorio, Via A. Cardarelli 9, 80131, Napoli, Italy.
| | - Marina Tarsitano
- AORN A. Cardarelli-Dipartimento delle Tecnologie Avanzate Diagnostico-Terapeutiche e dei Servizi sanitari-U.O.C. Genetica Medica e di Laboratorio, Via A. Cardarelli 9, 80131, Napoli, Italy
| | - Maria Oro
- AORN A. Cardarelli-Dipartimento delle Tecnologie Avanzate Diagnostico-Terapeutiche e dei Servizi sanitari-U.O.C. Genetica Medica e di Laboratorio, Via A. Cardarelli 9, 80131, Napoli, Italy
| | - Maria Rivieccio
- AORN A. Cardarelli-Dipartimento delle Tecnologie Avanzate Diagnostico-Terapeutiche e dei Servizi sanitari-U.O.C. Genetica Medica e di Laboratorio, Via A. Cardarelli 9, 80131, Napoli, Italy
| | - Nenad Bukvic
- AOUC "Policlinico di Bari"-UOC Lab. di Genetica Medica, Piazza Giulio Cesare 11, 70124, Bari, Italy
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32
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Homan RA, Jadhav AM, Conway LP, Parker CG. A Chemical Proteomic Map of Heme-Protein Interactions. J Am Chem Soc 2022; 144:15013-15019. [PMID: 35960875 PMCID: PMC9811995 DOI: 10.1021/jacs.2c06104] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Heme is an essential cofactor for many human proteins as well as the primary transporter of oxygen in blood. Recent studies have also established heme as a signaling molecule, imparting its effects through binding with protein partners rather than through reactivity of its metal center. However, the comprehensive annotation of such heme-binding proteins in the human proteome remains incomplete. Here, we describe a strategy which utilizes a heme-based photoaffinity probe integrated with quantitative proteomics to map heme-protein interactions across the proteome. In these studies, we identified 350+ unique heme-protein interactions, the vast majority of which were heretofore unknown and consist of targets from diverse functional classes, including transporters, receptors, enzymes, transcription factors, and chaperones. Among these proteins is the immune-related interleukin receptor-associated kinase 1 (IRAK1), where we provide preliminary evidence that heme agonizes its catalytic activity. Our findings should improve the current understanding of heme's regulation as well as its signaling functions and facilitate new insights of its roles in human disease.
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Affiliation(s)
- Rick A. Homan
- Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
| | - Appaso M. Jadhav
- Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
| | - Louis P. Conway
- Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
| | - Christopher G. Parker
- Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
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Pan YJ, Liu BW, Pei DS. The Role of Alternative Splicing in Cancer: Regulatory Mechanism, Therapeutic Strategy, and Bioinformatics Application. DNA Cell Biol 2022; 41:790-809. [PMID: 35947859 DOI: 10.1089/dna.2022.0322] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
[Formula: see text] Alternative splicing (AS) can generate distinct transcripts and subsequent isoforms that play differential functions from the same pre-mRNA. Recently, increasing numbers of studies have emerged, unmasking the association between AS and cancer. In this review, we arranged AS events that are closely related to cancer progression and presented promising treatments based on AS for cancer therapy. Obtaining proliferative capacity, acquiring invasive properties, gaining angiogenic features, shifting metabolic ability, and getting immune escape inclination are all splicing events involved in biological processes. Spliceosome-targeted and antisense oligonucleotide technologies are two novel strategies that are hopeful in tumor therapy. In addition, bioinformatics applications based on AS were summarized for better prediction and elucidation of regulatory routines mingled in. Together, we aimed to provide a better understanding of complicated AS events associated with cancer biology and reveal AS a promising target of cancer treatment in the future.
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Affiliation(s)
- Yao-Jie Pan
- Department of Pathology, Laboratory of Clinical and Experimental Pathology, Xuzhou Medical University, Xuzhou, China
| | - Bo-Wen Liu
- Department of General Surgery, Xuzhou Medical University, Xuzhou, China
| | - Dong-Sheng Pei
- Department of Pathology, Laboratory of Clinical and Experimental Pathology, Xuzhou Medical University, Xuzhou, China
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van den Bosch QCC, Nguyen JQN, Brands T, van den Bosch TPP, Verdijk RM, Paridaens D, Naus NC, de Klein A, Kiliç E, Brosens E. FOXD1 Is a Transcription Factor Important for Uveal Melanocyte Development and Associated with High-Risk Uveal Melanoma. Cancers (Basel) 2022; 14:cancers14153668. [PMID: 35954332 PMCID: PMC9367502 DOI: 10.3390/cancers14153668] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Revised: 07/12/2022] [Accepted: 07/22/2022] [Indexed: 11/27/2022] Open
Abstract
Simple Summary Despite successful treatment of primary uveal melanoma (UM), metastases still occur in approximately 50% of the patients. Unfortunately, little is known about the mechanism behind metastasized UM. By reanalyzing publicly available single-cell RNA sequencing data of embryonic zebrafish larvae and validating the results with UM data, we have identified five transcription regulators of interest: ELL2, KDM5B, REXO4, RBFOX2 and FOXD1. The most significant finding is FOXD1, which is nearly exclusively expressed in high-risk UM and is associated with poor survival. FOXD1 is a novel gene which could be involved in the metastatic capability of UM. Elucidating its function and role in metastatic UM could help to understand and develop treatment for UM. Abstract Uveal melanoma (UM) is a deadly ocular malignancy, originating from uveal melanocytes. Although much is known regarding prognostication in UM, the exact mechanism of metastasis is mostly unknown. Metastatic tumor cells are known to express a more stem-like RNA profile which is seen often in cell-specific embryonic development to induce tumor progression. Here, we identified novel transcription regulators by reanalyzing publicly available single cell RNA sequencing experiments. We identified five transcription regulators of interest: ELL2, KDM5B, REXO4, RBFOX2 and FOXD1. Our most significant finding is FOXD1, as this gene is nearly exclusively expressed in high-risk UM and its expression is associated with a poor prognosis. Even within the BAP1-mutated UM, the expression of FOXD1 is correlated with poor survival. FOXD1 is a novel factor which could potentially be involved in the metastatic capacity of high-risk UM. Elucidating the function of FOXD1 in UM could provide insight into the malignant transformation of uveal melanocytes, especially in high-risk UM.
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Affiliation(s)
- Quincy C. C. van den Bosch
- Department of Ophthalmology, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (Q.C.C.v.d.B.); (J.Q.N.N.); (T.B.); (N.C.N.)
- Department of Clinical Genetics, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands;
| | - Josephine Q. N. Nguyen
- Department of Ophthalmology, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (Q.C.C.v.d.B.); (J.Q.N.N.); (T.B.); (N.C.N.)
- Department of Clinical Genetics, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands;
| | - Tom Brands
- Department of Ophthalmology, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (Q.C.C.v.d.B.); (J.Q.N.N.); (T.B.); (N.C.N.)
- Department of Clinical Genetics, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands;
| | - Thierry P. P. van den Bosch
- Department of Pathology, Section Ophthalmic Pathology, Erasmus MC Cancer Institute, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (T.P.P.v.d.B.); (R.M.V.)
| | - Robert M. Verdijk
- Department of Pathology, Section Ophthalmic Pathology, Erasmus MC Cancer Institute, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (T.P.P.v.d.B.); (R.M.V.)
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Dion Paridaens
- The Rotterdam Eye Hospital, 3011 BH Rotterdam, The Netherlands;
| | - Nicole C. Naus
- Department of Ophthalmology, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (Q.C.C.v.d.B.); (J.Q.N.N.); (T.B.); (N.C.N.)
| | - Annelies de Klein
- Department of Clinical Genetics, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands;
| | - Emine Kiliç
- Department of Ophthalmology, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; (Q.C.C.v.d.B.); (J.Q.N.N.); (T.B.); (N.C.N.)
- Correspondence: (E.K.); (E.B.); Tel.: +31-107030683 (E.B.)
| | - Erwin Brosens
- Department of Clinical Genetics, Erasmus MC Cancer Center, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands;
- Correspondence: (E.K.); (E.B.); Tel.: +31-107030683 (E.B.)
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LncRNA MIAT Promotes Spinal Cord Injury Recovery in Rats by Regulating RBFOX2-Mediated Alternative Splicing of MCL-1. Mol Neurobiol 2022; 59:4854-4868. [PMID: 35641779 DOI: 10.1007/s12035-022-02896-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 05/21/2022] [Indexed: 10/18/2022]
Abstract
LncRNA myocardial infarction-associated transcript (MIAT) alleviates acute spinal cord injury (ASCI)-induced neuronal cell apoptosis, but the specific mechanism of it involved in regulating SCI progression needs further exploration. Here, a SCI rat model was established, followed by administration with adenovirus-mediated MIAT overexpression vector (Ad-MIAT) alone or together with Ad-RBFOX2 (RNA binding fox-1 homolog 2). The data indicated that MIAT overexpression promoted motor function recovery, improved morphology of injured tissues, and restrained neuron loss and cell apoptosis in SCI rats. Then, PC-12 cells were treated with H2O2 to induce cell injury. And highly expressed MIAT suppressed H2O2-caused decrease in cell viability and increase in cell apoptosis. MIAT stabilized RBFOX2 protein expression by binding to RBFOX2, thereby promoting RBFOX2-induced upregulation of anti-apoptotic MCL-1L (myeloid cell leukemia sequence 1) and reduction of pro-apoptotic MCL-1S. And silencing RBFOX2 in vitro blocked the inhibitory effect of MIAT on cell apoptosis. Moreover, MCL-1-specific steric-blocking oligonucleotides (SBOs) were used to transfer the MCL-1 pre-mRNA splicing pattern from MCL-1L to MCL-1S. SBOs reversed the protection effect of RBFOX2 overexpression on H2O2-induced cell injury. Furthermore, overexpression of MCL-1L instead of MCL-1S facilitated autophagy activation in H2O2-stimulated cells. Interestingly, co-overexpression of MIAT and RBFOX2 had a better promoting effect on SCI recovery. In conclusion, MIAT mitigated SCI by promoting RBFOX2-mediated alternative splicing of MCL-1. Our findings might provide a promising therapeutic target for SCI.
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Alternative Splicing of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Is Regulated by RBFOX2 in Lymphoid Malignancies. Mol Cell Biol 2022; 42:e0050321. [PMID: 35404107 PMCID: PMC9119065 DOI: 10.1128/mcb.00503-21] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans-regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT-specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.
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37
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Forastieri C, Italia M, Toffolo E, Romito E, Bonasoni MP, Ranzani V, Bodega B, Rusconi F, Battaglioli E. Evolution Increases Primates Brain Complexity Extending RbFOX1 Splicing Activity to LSD1 Modulation. J Neurosci 2022; 42:3689-3703. [PMID: 35351830 PMCID: PMC9087731 DOI: 10.1523/jneurosci.1782-21.2022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 02/28/2022] [Accepted: 03/03/2022] [Indexed: 11/21/2022] Open
Abstract
Recent branching (100 MYA) of the mammalian evolutionary tree has enhanced brain complexity and functions at the putative cost of increased emotional circuitry vulnerability. Thus, to better understand psychopathology, a burden for the modern society, novel approaches should exploit evolutionary aspects of psychiatric-relevant molecular pathways. A handful of genes is nowadays tightly associated to psychiatric disorders. Among them, neuronal-enriched RbFOX1 modifies the activity of synaptic regulators in response to neuronal activity, keeping excitability within healthy domains. We here dissect a higher primates-restricted interaction between RbFOX1 and the transcriptional corepressor Lysine Specific Demethylase 1 (LSD1/KDM1A). A single nucleotide variation (AA to AG) in LSD1 gene appeared in higher primates and humans, endowing RbFOX1 with the ability to promote the alternative usage of a novel 3' AG splice site, which extends LSD1 exon E9 in the upstream intron (E9-long). Exon E9-long regulates LSD1 levels by Nonsense-Mediated mRNA Decay. As reintroduction of the archaic LSD1 variant (AA) abolishes E9-long splicing, the novel 3' AG splice site is necessary for RbFOX1 to control LSD1 levels. LSD1 is a homeostatic immediate early genes (IEGs) regulator playing a relevant part in environmental stress-response. In primates and humans, inclusion of LSD1 as RbFOX1 target provides RbFOX1 with the additional ability to regulate the IEGs. These data, together with extensive RbFOX1 involvement in psychiatric disorders and its stress-dependent regulation in male mice, suggest the RbFOX1-LSD1-IEGs axis as an evolutionary recent psychiatric-relevant pathway. Notably, outside the nervous system, RbFOX2-dependent LSD1 modulation could be a candidate deregulated mechanism in cancer.SIGNIFICANCE STATEMENT To be better understood, anxiety and depression need large human genetics studies aimed at further resolving the often ambiguous, aberrant neuronal pathomechanisms that impact corticolimbic circuitry physiology. Several genetic associations of the alternative splicing regulator RbFOX1 with psychiatric conditions suggest homeostatic unbalance as a neuronal signature of psychopathology. Here we move a step forward, characterizing a disease-relevant higher primates-specific pathway by which RbFOX1 acquires the ability to regulate neuronal levels of Lysine Specific Demethylase 1, an epigenetic modulator of environmental stress response. Thus, two brain-enriched enzymes, independently shown to homeostatically protect neurons with a clear readout in terms of emotional behavior in lower mammals, establish in higher primates and humans a new functional cooperation enhancing the complexity of environmental adaptation and stress vulnerability.
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Affiliation(s)
- Chiara Forastieri
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
| | - Maria Italia
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
| | - Emanuela Toffolo
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
| | - Elena Romito
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
| | | | - Valeria Ranzani
- Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi," Milano, 20122, Italy
| | - Beatrice Bodega
- Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi," Milano, 20122, Italy
- Department of Biosciences, Università degli Studi di Milano, Milano, 20133, Italy
| | - Francesco Rusconi
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
| | - Elena Battaglioli
- Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Segrate, 20090, Italy
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Transcriptional and post-transcriptional control of epithelial-mesenchymal plasticity: why so many regulators? Cell Mol Life Sci 2022; 79:182. [PMID: 35278142 PMCID: PMC8918127 DOI: 10.1007/s00018-022-04199-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 01/18/2022] [Accepted: 02/07/2022] [Indexed: 12/12/2022]
Abstract
The dynamic transition between epithelial-like and mesenchymal-like cell states has been a focus for extensive investigation for decades, reflective of the importance of Epithelial-Mesenchymal Transition (EMT) through development, in the adult, and the contributing role EMT has to pathologies including metastasis and fibrosis. Not surprisingly, regulation of the complex genetic networks that underlie EMT have been attributed to multiple transcription factors and microRNAs. What is surprising, however, are the sheer number of different regulators (hundreds of transcription factors and microRNAs) for which critical roles have been described. This review seeks not to collate these studies, but to provide a perspective on the fundamental question of whether it is really feasible that so many regulators play important roles and if so, what does this tell us about EMT and more generally, the genetic machinery that controls complex biological processes.
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39
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Verma SK, Deshmukh V, Thatcher K, Belanger KK, Rhyner A, Meng S, Holcomb R, Bressan M, Martin J, Cooke J, Wythe J, Widen S, Lincoln J, Kuyumcu-Martinez M. RBFOX2 is required for establishing RNA regulatory networks essential for heart development. Nucleic Acids Res 2022; 50:2270-2286. [PMID: 35137168 PMCID: PMC8881802 DOI: 10.1093/nar/gkac055] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 01/14/2022] [Accepted: 01/25/2022] [Indexed: 12/14/2022] Open
Abstract
Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.
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Affiliation(s)
- Sunil K Verma
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Vaibhav Deshmukh
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Kaitlyn Thatcher
- Department of Pediatrics, Medical College of Wisconsin, Division of Pediatric Cardiology, The Herma Heart Institute, Children's WI, Milwaukee, WI 53226, USA
| | - KarryAnne K Belanger
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Alexander M Rhyner
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
- Center for Organ Repair and Renewal, Baylor College of Medicine, Houston, TX 77030, USA
| | - Shu Meng
- Houston Methodist Research Institute, Department of Cardiovascular Sciences, Houston, TX 77030, USA
| | - Richard Joshua Holcomb
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Michael Bressan
- Department of Cell Biology and Physiology, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC27599, USA
| | - James F Martin
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
- Center for Organ Repair and Renewal, Baylor College of Medicine, Houston, TX 77030, USA
- Cardiomyocyte Renewal Lab;Texas Heart Institute, Houston, TX77030, USA
| | - John P Cooke
- Houston Methodist Research Institute, Department of Cardiovascular Sciences, Houston, TX 77030, USA
| | - Joshua D Wythe
- Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
- Center for Organ Repair and Renewal, Baylor College of Medicine, Houston, TX 77030, USA
- Cardiomyocyte Renewal Lab;Texas Heart Institute, Houston, TX77030, USA
| | - Steven G Widen
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Joy Lincoln
- Department of Pediatrics, Medical College of Wisconsin, Division of Pediatric Cardiology, The Herma Heart Institute, Children's WI, Milwaukee, WI 53226, USA
| | - Muge N Kuyumcu-Martinez
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA
- Department of Neuroscience, Cell Biology and Anatomy, Institute for Translational Sciences, University of Texas Medical Branch, 301 University Blvd. Galveston, TX 77555, USA
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40
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Choi S, Cho N, Kim KK. Non-canonical splice junction processing increases the diversity of RBFOX2 splicing isoforms. Int J Biochem Cell Biol 2022; 144:106172. [PMID: 35124219 DOI: 10.1016/j.biocel.2022.106172] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Revised: 01/23/2022] [Accepted: 02/01/2022] [Indexed: 12/13/2022]
Abstract
The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.
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Affiliation(s)
- Sunkyung Choi
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea
| | - Namjoon Cho
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea
| | - Kee K Kim
- Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, 34134, Republic of Korea.
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41
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Ou X, Zhou X, Li J, Ye J, Liu H, Fang D, Cai Q, Cai S, He Y, Xu J. p53-Induced LINC00893 Regulates RBFOX2 Stability to Suppress Gastric Cancer Progression. Front Cell Dev Biol 2022; 9:796451. [PMID: 35127712 PMCID: PMC8807521 DOI: 10.3389/fcell.2021.796451] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2021] [Accepted: 12/29/2021] [Indexed: 01/07/2023] Open
Abstract
Long noncoding RNAs (lncRNAs) have been reported to regulate diverse tumorigenic processes. However, little is known about long intergenic non-protein coding RNA 00893 (LINC00893) and its role in gastric cancer (GC). Herein we investigated its biological functions and molecular mechanism in GC. LINC00893 was decreased in GC tissues but significantly elevated in AGS cells after treatment with Nutlin-3. In GC patients, it was found that low expression of LINC00893 was correlated with tumor growth, metastasis and poor survival. Functionally, overexpression of LINC00893 suppressed the proliferation, migration and invasion of GC cells. Mechanistically, LINC00893 regulated the expression of epithelial-mesenchymal transition (EMT)-related proteins by binding to RNA binding fox-1 homolog 2 (RBFOX2) and promoting its ubiquitin-mediated degradation, thus suppressing the EMT and related functions of GC. In addition, the transcription factor p53 can regulate the expression of LINC00893 in an indirect way. Taken together, these results suggested that LINC00893 regulated by p53 repressed GC proliferation, migration and invasion by functioning as a binding site for RBFOX2 to regulate its stability and the expression of EMT-related proteins. LINC00893 acts as a tumor-inhibiting lncRNA that is induced by p53 in GC and regulates EMT by binding to RBFOX2, thus providing a novel experimental basis for the clinical treatment of GC.
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Affiliation(s)
- Xinde Ou
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Xingyu Zhou
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Jin Li
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Digestive Disease Center, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China
| | - Jinning Ye
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Haohan Liu
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Deliang Fang
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Qinbo Cai
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Shirong Cai
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Yulong He
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Digestive Disease Center, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, China
- *Correspondence: Yulong He, ; Jianbo Xu,
| | - Jianbo Xu
- Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- *Correspondence: Yulong He, ; Jianbo Xu,
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42
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Siena ÁDD, Barros IID, Storti CB, de Biagi Júnior CAO, da Costa Carvalho LA, Maria-Engler SS, Sousa JDF, Silva WA. Upregulation of the novel lncRNA U731166 is associated with migration, invasion and vemurafenib resistance in melanoma. J Cell Mol Med 2022; 26:671-683. [PMID: 35040264 PMCID: PMC8817119 DOI: 10.1111/jcmm.16987] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 08/30/2021] [Accepted: 09/08/2021] [Indexed: 12/18/2022] Open
Abstract
Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis‐related processes. In this sense, we found the intergenic lncRNA U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA U73166 for cancer processes. Additionally, using naïve and vemurafenib‐resistant cell lines and data from a patient before and after resistance, we found that vemurafenib‐resistant samples had a higher expression of lncRNA U73166. Also, we retrieved data from the literature that indicates lncRNA U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA U73166 in metastasis development. We also pointed herein the lncRNA U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.
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Affiliation(s)
- Ádamo Davi Diógenes Siena
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Cell Based Therapy, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil
| | - Isabela Ichihara de Barros
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Cell Based Therapy, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil
| | - Camila Baldin Storti
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Cell Based Therapy, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil
| | - Carlos Alberto Oliveira de Biagi Júnior
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Cell Based Therapy, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil
| | | | - Silvya Stuchi Maria-Engler
- Department of Clinical and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo (USP), São Paulo, Brazil
| | | | - Wilson Araújo Silva
- Department of Genetics, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Cell Based Therapy, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Center for Integrative Systems Biology-CISBi, NAP/USP, Ribeirão Preto Medical School, University of São Paulo (USP), Ribeirão Preto, Brazil.,Institute for Cancer Research, Cidade dos Lagos, Guarapuava, Brazil
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Öther-Gee Pohl S, Myant KB. Alternative RNA splicing in tumour heterogeneity, plasticity and therapy. Dis Model Mech 2022; 15:dmm049233. [PMID: 35014671 PMCID: PMC8764416 DOI: 10.1242/dmm.049233] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Alternative splicing is a process by which a single gene is able to encode multiple different protein isoforms. It is regulated by the inclusion or exclusion of introns and exons that are joined in different patterns prior to protein translation, thus enabling transcriptomic and proteomic diversity. It is now widely accepted that alternative splicing is dysregulated across nearly all cancer types. This widespread dysregulation means that nearly all cellular processes are affected - these include processes synonymous with the hallmarks of cancer - evasion of apoptosis, tissue invasion and metastasis, altered cellular metabolism, genome instability and drug resistance. Emerging evidence indicates that the dysregulation of alternative splicing also promotes a permissive environment for increased tumour heterogeneity and cellular plasticity. These are fundamental regulators of a patient's response to therapy. In this Review, we introduce the mechanisms of alternative splicing and the role of aberrant splicing in cancer, with particular focus on newfound evidence of alternative splicing promoting tumour heterogeneity, cellular plasticity and altered metabolism. We discuss recent in vivo models generated to study alternative splicing and the importance of these for understanding complex tumourigenic processes. Finally, we review the effects of alternative splicing on immune evasion, cell death and genome instability, and how targeting these might enhance therapeutic efficacy.
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Affiliation(s)
| | - Kevin B. Myant
- Cancer Research UK Edinburgh Centre, Institute of Genetics of Cancer, The University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
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44
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Lyu J, Cheng C. Regulation of Alternative Splicing during Epithelial-Mesenchymal Transition. Cells Tissues Organs 2022; 211:238-251. [PMID: 34348273 PMCID: PMC8741878 DOI: 10.1159/000518249] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Accepted: 06/28/2021] [Indexed: 01/03/2023] Open
Abstract
Alternative splicing is an essential mechanism of gene regulation, giving rise to remarkable protein diversity in higher eukaryotes. Epithelial-mesenchymal transition (EMT) is a developmental process that plays an essential role in metazoan embryogenesis. Recent studies have revealed that alternative splicing serves as a fundamental layer of regulation that governs cells to undergo EMT. In this review, we summarize recent findings on the functional impact of alternative splicing in EMT and EMT-associated activities. We then discuss the regulatory mechanisms that control alternative splicing changes during EMT.
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Affiliation(s)
- Jingyi Lyu
- Lester and Sue Smith Breast Center, Department of Molecular
& Human Genetics, Department of Molecular & Cellular Biology, Baylor College
of Medicine, Houston, TX 77030, USA,Integrative Molecular and Biomedical Sciences Graduate
Program, Baylor College of Medicine, Houston, TX 77030, USA
| | - Chonghui Cheng
- Lester and Sue Smith Breast Center, Department of Molecular
& Human Genetics, Department of Molecular & Cellular Biology, Baylor College
of Medicine, Houston, TX 77030, USA,Integrative Molecular and Biomedical Sciences Graduate
Program, Baylor College of Medicine, Houston, TX 77030, USA.,To whom correspondence should be addressed:
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45
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Naro C, De Musso M, Delle Monache F, Panzeri V, de la Grange P, Sette C. The oncogenic kinase NEK2 regulates an RBFOX2-dependent pro-mesenchymal splicing program in triple-negative breast cancer cells. J Exp Clin Cancer Res 2021; 40:397. [PMID: 34930366 PMCID: PMC8686545 DOI: 10.1186/s13046-021-02210-3] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 12/06/2021] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND Triple-negative breast cancer (TNBC) is the most heterogeneous and malignant subtype of breast cancer (BC). TNBC is defined by the absence of expression of estrogen, progesterone and HER2 receptors and lacks efficacious targeted therapies. NEK2 is an oncogenic kinase that is significantly upregulated in TNBC, thereby representing a promising therapeutic target. NEK2 localizes in the nucleus and promotes oncogenic splice variants in different cancer cells. Notably, alternative splicing (AS) dysregulation has recently emerged as a featuring trait of TNBC that contributes to its aggressive phenotype. METHODS To investigate whether NEK2 modulates TNBC transcriptome we performed RNA-sequencing analyses in a representative TNBC cell line (MDA-MB-231) and results were validated in multiple TNBC cell lines. Bioinformatics and functional analyses were carried out to elucidate the mechanism of splicing regulation by NEK2. Data from The Cancer Genome Atlas were mined to evaluate the potential of NEK2-sensitive exons as markers to identify the TNBC subtype and to assess their prognostic value. RESULTS Transcriptome analysis revealed a widespread impact of NEK2 on the transcriptome of TNBC cells, with 1830 AS events that are susceptible to its expression. NEK2 regulates the inclusion of cassette exons in splice variants that discriminate TNBC from other BC and that correlate with poor prognosis, suggesting that this kinase contributes to the TNBC-specific splicing program. NEK2 elicits its effects by modulating the expression of the splicing factor RBFOX2, a well-known regulator of epithelial to mesenchymal transition (EMT). Accordingly, NEK2 splicing-regulated genes are enriched in functional terms related to cell adhesion and contractile cytoskeleton and NEK2 depletion in mesenchymal TNBC cells induces phenotypic and molecular traits typical of epithelial cells. Remarkably, depletion of select NEK2-sensitive splice-variants that are prognostic in TNBC patients is sufficient to interfere with TNBC cell morphology and motility, suggesting that NEK2 orchestrates a pro-mesenchymal splicing program that modulates migratory and invasive properties of TNBC cells. CONCLUSIONS Our study uncovers an extensive splicing program modulated by NEK2 involving splice variants that confer an invasive phenotype to TNBCs and that might represent, together with NEK2 itself, valuable therapeutic targets for this disease.
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Affiliation(s)
- Chiara Naro
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168, Rome, Italy.
- Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy.
| | - Monica De Musso
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168, Rome, Italy
- Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy
| | - Francesca Delle Monache
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168, Rome, Italy
- Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy
| | - Valentina Panzeri
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168, Rome, Italy
- Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy
| | | | - Claudio Sette
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, 00168, Rome, Italy.
- Fondazione Santa Lucia, IRCCS, Rome, Italy.
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Marima R, Francies FZ, Hull R, Molefi T, Oyomno M, Khanyile R, Mbatha S, Mabongo M, Owen Bates D, Dlamini Z. MicroRNA and Alternative mRNA Splicing Events in Cancer Drug Response/Resistance: Potent Therapeutic Targets. Biomedicines 2021; 9:1818. [PMID: 34944633 PMCID: PMC8698559 DOI: 10.3390/biomedicines9121818] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 11/24/2021] [Accepted: 11/29/2021] [Indexed: 12/24/2022] Open
Abstract
Cancer is a multifaceted disease that involves several molecular mechanisms including changes in gene expression. Two important processes altered in cancer that lead to changes in gene expression include altered microRNA (miRNA) expression and aberrant splicing events. MiRNAs are short non-coding RNAs that play a central role in regulating RNA silencing and gene expression. Alternative splicing increases the diversity of the proteome by producing several different spliced mRNAs from a single gene for translation. MiRNA expression and alternative splicing events are rigorously regulated processes. Dysregulation of miRNA and splicing events promote carcinogenesis and drug resistance in cancers including breast, cervical, prostate, colorectal, ovarian and leukemia. Alternative splicing may change the target mRNA 3'UTR binding site. This alteration can affect the produced protein and may ultimately affect the drug affinity of target proteins, eventually leading to drug resistance. Drug resistance can be caused by intrinsic and extrinsic factors. The interplay between miRNA and alternative splicing is largely due to splicing resulting in altered 3'UTR targeted binding of miRNAs. This can result in the altered targeting of these isoforms and altered drug targets and drug resistance. Furthermore, the increasing prevalence of cancer drug resistance poses a substantial challenge in the management of the disease. Henceforth, molecular alterations have become highly attractive drug targets to reverse the aberrant effects of miRNAs and splicing events that promote malignancy and drug resistance. While the miRNA-mRNA splicing interplay in cancer drug resistance remains largely to be elucidated, this review focuses on miRNA and alternative mRNA splicing (AS) events in breast, cervical, prostate, colorectal and ovarian cancer, as well as leukemia, and the role these events play in drug resistance. MiRNA induced cancer drug resistance; alternative mRNA splicing (AS) in cancer drug resistance; the interplay between AS and miRNA in chemoresistance will be discussed. Despite this great potential, the interplay between aberrant splicing events and miRNA is understudied but holds great potential in deciphering miRNA-mediated drug resistance.
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Affiliation(s)
- Rahaba Marima
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
| | - Flavia Zita Francies
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
| | - Rodney Hull
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
| | - Thulo Molefi
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Department of Medical Oncology, Steve Biko Academic Hospital, University of Pretoria, Hatfield, Pretoria 0028, South Africa
| | - Meryl Oyomno
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Department of Surgery, Steve Biko Academic Hospital, University of Pretoria, Hatfield, Pretoria 0028, South Africa
| | - Richard Khanyile
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Department of Medical Oncology, Steve Biko Academic Hospital, University of Pretoria, Hatfield, Pretoria 0028, South Africa
| | - Sikhumbuzo Mbatha
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Department of Surgery, Steve Biko Academic Hospital, University of Pretoria, Hatfield, Pretoria 0028, South Africa
| | - Mzubanzi Mabongo
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Department of Maxillofacial and Oral Surgery, School of Dentistry, University of Pretoria, Hatfield, Pretoria 0028, South Africa
| | - David Owen Bates
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
- Centre for Cancer Sciences, Division of Cancer and Stem Cells, Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK
| | - Zodwa Dlamini
- SAMRC Precision Oncology Research Unit (PORU), Pan African Cancer Research Institute (PACRI), University of Pretoria, Hatfiel, Pretoria 0028, South Africa; (R.M.); (F.Z.F.); (R.H.); (T.M.); (M.O.); (R.K.); (S.M.); (M.M.); (D.O.B.)
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47
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Ebrahimie E, Rahimirad S, Tahsili M, Mohammadi-Dehcheshmeh M. Alternative RNA splicing in stem cells and cancer stem cells: Importance of transcript-based expression analysis. World J Stem Cells 2021; 13:1394-1416. [PMID: 34786151 PMCID: PMC8567453 DOI: 10.4252/wjsc.v13.i10.1394] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 06/21/2021] [Accepted: 09/14/2021] [Indexed: 02/06/2023] Open
Abstract
Alternative ribonucleic acid (RNA) splicing can lead to the assembly of different protein isoforms with distinctive functions. The outcome of alternative splicing (AS) can result in a complete loss of function or the acquisition of new functions. There is a gap in knowledge of abnormal RNA splice variants promoting cancer stem cells (CSCs), and their prospective contribution in cancer progression. AS directly regulates the self-renewal features of stem cells (SCs) and stem-like cancer cells. Notably, octamer-binding transcription factor 4A spliced variant of octamer-binding transcription factor 4 contributes to maintaining stemness properties in both SCs and CSCs. The epithelial to mesenchymal transition pathway regulates the AS events in CSCs to maintain stemness. The alternative spliced variants of CSCs markers, including cluster of differentiation 44, aldehyde dehydrogenase, and doublecortin-like kinase, α6β1 integrin, have pivotal roles in increasing self-renewal properties and maintaining the pluripotency of CSCs. Various splicing analysis tools are considered in this study. LeafCutter software can be considered as the best tool for differential splicing analysis and identification of the type of splicing events. Additionally, LeafCutter can be used for efficient mapping splicing quantitative trait loci. Altogether, the accumulating evidence re-enforces the fact that gene and protein expression need to be investigated in parallel with alternative splice variants.
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Affiliation(s)
- Esmaeil Ebrahimie
- School of Animal and Veterinary Sciences, The University of Adelaide, Adelaide 5005, South Australia, Australia
- La Trobe Genomics Research Platform, School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Melbourne 3086, Australia
- School of Biosciences, The University of Melbourne, Melbourne 3010, Australia,
| | - Samira Rahimirad
- Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran 1497716316, Iran
- Division of Urology, Department of Surgery, McGill University and the Research Institute of the McGill University Health Centre, Montreal H4A 3J1, Quebec, Canada
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Kim JH, Jeong K, Li J, Murphy JM, Vukadin L, Stone JK, Richard A, Tran J, Gillespie GY, Flemington EK, Sobol RW, Lim STS, Ahn EYE. SON drives oncogenic RNA splicing in glioblastoma by regulating PTBP1/PTBP2 switching and RBFOX2 activity. Nat Commun 2021; 12:5551. [PMID: 34548489 PMCID: PMC8455679 DOI: 10.1038/s41467-021-25892-x] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Accepted: 09/01/2021] [Indexed: 12/15/2022] Open
Abstract
While dysregulation of RNA splicing has been recognized as an emerging target for cancer therapy, the functional significance of RNA splicing and individual splicing factors in brain tumors is poorly understood. Here, we identify SON as a master regulator that activates PTBP1-mediated oncogenic splicing while suppressing RBFOX2-mediated non-oncogenic neuronal splicing in glioblastoma multiforme (GBM). SON is overexpressed in GBM patients and SON knockdown causes failure in intron removal from the PTBP1 transcript, resulting in PTBP1 downregulation and inhibition of its downstream oncogenic splicing. Furthermore, SON forms a complex with hnRNP A2B1 and antagonizes RBFOX2, which leads to skipping of RBFOX2-targeted cassette exons, including the PTBP2 neuronal exon. SON knockdown inhibits proliferation and clonogenicity of GBM cells in vitro and significantly suppresses tumor growth in orthotopic xenografts in vivo. Collectively, our study reveals that SON-mediated RNA splicing is a GBM vulnerability, implicating SON as a potential therapeutic target in brain tumors.
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Affiliation(s)
- Jung-Hyun Kim
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
- Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA
| | - Kyuho Jeong
- Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA
| | - Jianfeng Li
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
- Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA
| | - James M Murphy
- Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA
| | - Lana Vukadin
- Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Joshua K Stone
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
| | - Alexander Richard
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
| | - Johnny Tran
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
| | - G Yancey Gillespie
- Department of Neurosurgery, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Erik K Flemington
- Department of Pathology, Tulane University School of Medicine, Tulane Cancer Center, New Orleans, LA, USA
| | - Robert W Sobol
- Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA.
- Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL, USA.
| | - Ssang-Teak Steve Lim
- Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL, USA.
| | - Eun-Young Erin Ahn
- Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
- O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.
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49
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Freitag N, Xie Y, Adam LM, Borowski S, Blois SM, Barrientos G. Expression of the alternative splicing regulator Rbfox2 during placental development is differentially regulated in preeclampsia mouse models. Am J Reprod Immunol 2021; 86:e13491. [PMID: 34363260 DOI: 10.1111/aji.13491] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 07/21/2021] [Accepted: 08/03/2021] [Indexed: 12/14/2022] Open
Abstract
PROBLEM Proper placental development is pivotal to ensure healthy pregnancy outcomes. Among the multiple cellular mechanisms involved in the orchestration of this process, little is known on the role of alternative splicing events in the modulation of trophoblast cell biology. Here, we evaluated the expression of the alternative splicing regulator Rbfox2 in the pre- and post-placentation period in mouse pregnancies in both healthy and pathological settings. METHOD OF STUDY Immunofluorescence analysis of Rbfox2 expression in mouse implantation sites collected during the pre-placentation period (E5-E7) and post-placentation (E13). RESULTS We identified a progressive increase of Rbfox2 levels throughout the peri-implantation period with a shift from a cytoplasmatic expression on E5-E6 to a predominantly nuclear expression on E7, together with a prominent expression of this factor in both subcellular compartments of the primitive placenta. Our results further showed that in contrast to healthy gestations, Rbfox2 expression decreased in preeclamptic models during the post-placentation period. Finally, we further demonstrated enhanced expression of Rbfox2 proteins in allogeneic pregnancy compared to syngeneic models. CONCLUSIONS Our findings uncover a novel role for Rbfox2-controlled splicing events in the modulation of trophoblast function, with potential implications for the pathogenesis of preeclampsia and other pregnancy complications originated from defective placentation.
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Affiliation(s)
- Nancy Freitag
- Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Experimental and Clinical Research Center, a Cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, AG GlycoImmunology, and the Charité - Universitätsmedizin Berlin, Berlin, Germany.,Department for Psychosomatic Medicine, Charité - Universitätsmedizin Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Yiran Xie
- Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Reproductive Medicine Center, Taihe Hospital, Hubei Medical University, Shiyan, China
| | - Lisa-Marie Adam
- Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Sophia Borowski
- Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Experimental and Clinical Research Center, a Cooperation between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, AG GlycoImmunology, and the Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Sandra M Blois
- Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Gabriela Barrientos
- Laboratorio de Medicina Experimental, Hospital Alemán, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
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50
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Mochizuki Y, Funayama R, Shirota M, Kikukawa Y, Ohira M, Karasawa H, Kobayashi M, Ohnuma S, Unno M, Nakayama K. Alternative microexon splicing by RBFOX2 and PTBP1 is associated with metastasis in colorectal cancer. Int J Cancer 2021; 149:1787-1800. [PMID: 34346508 DOI: 10.1002/ijc.33758] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2021] [Revised: 07/28/2021] [Accepted: 07/30/2021] [Indexed: 11/09/2022]
Abstract
The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.
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Affiliation(s)
- Yasushi Mochizuki
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan.,Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Ryo Funayama
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Matsuyuki Shirota
- Division of Interdisciplinary Medical Science, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Yuna Kikukawa
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Masahiro Ohira
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Hideaki Karasawa
- Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Minoru Kobayashi
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan.,Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Shinobu Ohnuma
- Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Michiaki Unno
- Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan
| | - Keiko Nakayama
- Department of Cell Proliferation, ART, Graduate School of Medicine, Tohoku University, Sendai, Japan
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