5d9666fd8e5fff977765902508ad0924 pmc_34638328.pdf 8495f05c26ae0749c6fe5c551a713db8bbf18ae6 pmc_34638328.pdf c3b82c13c942963203fc6e63b1db50e1d231f21063c6257c59ba16cf260eec07 pmc_34638328.pdf Title: Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro Subject: Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determinant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited to genes involved in the maintenance of the identity of neural progenitors. We combined analysis of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression are associated to shorter overall survival of neuroblastoma patients, even when those patients with the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma. Keywords: ZRF1; neuronal differentiation; epigenetics; pediatric cancer; neuroblastoma Author: Carlos Jiménez, Roberta Antonelli, Marc Masanas, Aroa Soriano, Laura Devis-Jauregui, Jessica Camacho, Ainara Magdaleno, Gabriela Guillén, Raquel Hladun, Luz Jubierre, Josep Roma, David Llobet-Navas, Josep Sánchez de Toledo, Lucas Moreno, Soledad Gallego and Miguel F. Segura Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Oct 9 07:23:16 2021 CEST ModDate: Sat Oct 9 07:26:38 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4004860 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CIQLLF+VnURWPalladioL Type 1 Custom yes yes no 10 0 WFSDPI+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 EJDAIH+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 IGPJEC+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 ANHOCF+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 GIFXFT+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 41 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 63 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 67 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 71 0 VORBJF+CMSY10 Type 1 Builtin yes yes no 86 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 90 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 97 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 110 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 113 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 116 0 CJLMOK+TimesNewRoman CID TrueType Identity-H yes yes yes 127 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 133 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 136 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 139 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 150 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 153 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 156 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 169 0 CJLHKD+PalatinoLinotype CID TrueType Identity-H yes yes yes 172 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 178 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 181 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 192 0 CJLHKD+PalatinoLinotype CID TrueType Identity-H yes yes yes 195 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 201 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 204 0 CJLHKC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 215 0 CJLHKE+PalatinoLinotype TrueType WinAnsi yes yes no 218 0 CJLHKG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 221 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 234 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:39:09 CEST RepresentationInformation: pmc_34638328.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:05:50 CEST Size: 4004860 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 408 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Analysis of Neuroblastoma Gene Expression Datasets Destination: subsection.2.1 Item: Title: Cell Lines Destination: subsection.2.2 Item: Title: Western Blot Analysis Destination: subsection.2.3 Item: Title: Proliferation and Colony Formation Assays Destination: subsection.2.4 Item: Title: Migration and Invasion Assays Destination: subsection.2.5 Item: Title: Differentiation Assays Destination: subsection.2.6 Item: Title: Vectors and Lentiviral Infection Destination: subsection.2.7 Item: Title: ZRF1 Overexpression Experiments Destination: subsection.2.8 Item: Title: ZRF1 Knockdown Experiments Destination: subsection.2.9 Item: Title: Results Destination: section.3 Children: Item: Title: ZRF1 Is an Independent Prognostic Factor in Neuroblastoma Destination: subsection.3.1 Item: Title: ZRF1 Is Not Sufficient to Enhance Neuroblastoma Aggressiveness Destination: subsection.3.2 Item: Title: ZRF1 Knockdown Does Not Impair Neuroblastoma Proliferation and Reveals Inconsistencies between Different Gene Silencing Methodologies Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Neuronal Differentiation-Related Epigenetic Regulator ZRF1 Has Independent Prognostic Value in Neuroblastoma but Is Functionally Dispensable In Vitro Author: Carlos Jiménez, Roberta Antonelli, Marc Masanas, Aroa Soriano, Laura Devis-Jauregui, Jessica Camacho, Ainara Magdaleno, Gabriela Guillén, Raquel Hladun, Luz Jubierre, Josep Roma, David Llobet-Navas, Josep Sánchez de Toledo, Lucas Moreno, Soledad Gallego and Miguel F. Segura Subject: Neuroblastoma is a pediatric tumor of the peripheral nervous system that accounts for up to ~15% of all cancer-related deaths in children. Recently, it has become evident that epigenetic deregulation is a relevant event in pediatric tumors such as high-risk neuroblastomas, and a determinant for processes, such as cell differentiation blockade and sustained proliferation, which promote tumor progression and resistance to current therapies. Thus, a better understanding of epigenetic factors implicated in the aggressive behavior of neuroblastoma cells is crucial for the development of better treatments. In this study, we characterized the role of ZRF1, an epigenetic activator recruited to genes involved in the maintenance of the identity of neural progenitors. We combined analysis of patient sample expression datasets with loss- and gain-of-function studies on neuroblastoma cell lines. Functional analyses revealed that ZRF1 is functionally dispensable for those cellular functions related to cell differentiation, proliferation, migration, and invasion, and does not affect the cellular response to chemotherapeutic agents. However, we found that high levels of ZRF1 mRNA expression are associated to shorter overall survival of neuroblastoma patients, even when those patients with the most common molecular alterations used as prognostic factors are removed from the analyses, thereby suggesting that ZRF1 expression could be used as an independent prognostic factor in neuroblastoma. Keywords: ZRF1; neuronal differentiation; epigenetics; pediatric cancer; neuroblastoma Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Oct 09 07:23:16 CEST 2021 ModDate: Sat Oct 09 07:26:38 CEST 2021 ID: 0x72f7058d4fe80aa585b17d6e43d67bb4, 0x72f7058d4fe80aa585b17d6e43d67bb4 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: GIFXFT+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 178 FontDescriptor: FontName: GIFXFT+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CRJHCU+PazoMath-Italic FontSubset: true FirstChar: 98 LastChar: 98 FontDescriptor: FontName: CRJHCU+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: EJDAIH+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: EJDAIH+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true 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