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1.
Neurooncol Adv ; 3(1): vdab056, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34056606

RESUMEN

BACKGROUND: MYCN has been an attractive therapeutic target in neuroblastoma considering the widespread amplification of the MYCN locus in neuroblastoma, and its established role in neuroblastoma development and progression. Thus, understanding neuroblastoma-specific control of MYCN expression at the transcriptional and post-transcriptional level would lead to identification of novel MYCN-dependent oncogenic pathways and potential therapeutic strategies. METHODS: By performing loss- and gain-of-function experiments of the neuroblastoma hotspot locus 6p22.3 derived lncRNAs CASC15-003 and NBAT1, together with coimmunoprecipitation and immunoblotting of MYCN, we have shown that both lncRNAs post-translationally control the expression of MYCN through regulating a deubiquitinase enzyme USP36. USP36 oncogenic properties were investigated using cancer cell lines and in vivo models. RNA-seq analysis of loss-of-function experiments of CASC15-003/NBAT1/MYCN/USP36 and JQ1-treated neuroblastoma cells uncovered MYCN-dependent oncogenic pathways. RESULTS: We show that NBAT1/CASC15-003 control the stability of MYCN protein through their common interacting protein partner USP36. USP36 harbors oncogenic properties and its higher expression in neuroblastoma patients correlates with poor prognosis, and its downregulation significantly reduces tumor growth in neuroblastoma cell lines and xenograft models. Unbiased integration of RNA-seq data from CASC15-003, NBAT1, USP36, and MYCN knockdowns and neuroblastoma cells treated with MYCN inhibitor JQ1, identified genes that are jointly regulated by the NBAT1/CASC15-003/USP36/MYCN pathway. Functional experiments on one of the target genes, COL18A1, revealed its role in the NBAT1/CASC15-003-dependent cell adhesion feature in neuroblastoma cells. CONCLUSION: Our data show post-translational regulation of MYCN by NBAT1/CASC15-003/USP36, which represents a new regulatory layer in the complex multilayered gene regulatory network that controls MYCN expression.

2.
NAR Cancer ; 3(1): zcab002, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-34316698

RESUMEN

Despite the rapid improvements in unveiling the importance of lncRNAs in all aspects of cancer biology, there is still a void in mechanistic understanding of their role in the DNA damage response. Here we explored the potential role of the oncogenic lncRNA SCAT7 (ELF3-AS1) in the maintenance of genome integrity. We show that SCAT7 is upregulated in response to DNA-damaging drugs like cisplatin and camptothecin, where SCAT7 expression is required to promote cell survival. SCAT7 silencing leads to decreased proliferation of cisplatin-resistant cells in vitro and in vivo through interfering with cell cycle checkpoints and DNA repair molecular pathways. SCAT7 regulates ATR signaling, promoting homologous recombination. Importantly, SCAT7 also takes part in proteasome-mediated topoisomerase I (TOP1) degradation, and its depletion causes an accumulation of TOP1-cc structures responsible for the high levels of intrinsic DNA damage. Thus, our data demonstrate that SCAT7 is an important constituent of the DNA damage response pathway and serves as a potential therapeutic target for hard-to-treat drug resistant cancers.

3.
Oncogene ; 40(13): 2463-2478, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33674747

RESUMEN

Recent advances in genomics unraveled several actionable mutational drivers in lung cancer, leading to promising therapies such as tyrosine kinase inhibitors and immune checkpoint inhibitors. However, the tumors' acquired resistance to the newly-developed as well as existing therapies restricts life quality improvements. Therefore, we investigated the noncoding portion of the human transcriptome in search of alternative actionable targets. We identified an antisense transcript, LY6K-AS, with elevated expression in lung adenocarcinoma (LUAD) patients, and its higher expression in LUAD patients predicts poor survival outcomes. LY6K-AS abrogation interfered with the mitotic progression of lung cancer cells resulting in unfaithful chromosomal segregation. LY6K-AS interacts with and stabilizes 14-3-3 proteins to regulate the transcription of kinetochore and mitotic checkpoint proteins. We also show that LY6K-AS regulates the levels of histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of kinetochore members. Cisplatin treatment and LY6K-AS silencing affect many common pathways enriched in cell cycle-related functions. LY6K-AS silencing affects the growth of xenografts derived from wildtype and cisplatin-resistant lung cancer cells. Collectively, these data indicate that LY6K-AS silencing is a promising therapeutic option for LUAD that inhibits oncogenic mitotic progression.


Asunto(s)
Proteínas 14-3-3/genética , Adenocarcinoma del Pulmón/genética , Antígenos Ly/genética , ARN Largo no Codificante/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/efectos de los fármacos , Proliferación Celular/genética , Cisplatino/farmacología , Femenino , Proteínas Ligadas a GPI/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Histonas/genética , Humanos , Masculino , Ratones , Mitosis/genética , Pronóstico , Transcriptoma/genética
4.
Cancer Res ; 81(6): 1457-1471, 2021 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-33372039

RESUMEN

Neuroblastoma has a low mutation rate for the p53 gene. Alternative ways of p53 inactivation have been proposed in neuroblastoma, such as abnormal cytoplasmic accumulation of wild-type p53. However, mechanisms leading to p53 inactivation via cytoplasmic accumulation are not well investigated. Here we show that the neuroblastoma risk-associated locus 6p22.3-derived tumor suppressor NBAT1 is a p53-responsive lncRNA that regulates p53 subcellular levels. Low expression of NBAT1 provided resistance to genotoxic drugs by promoting p53 accumulation in cytoplasm and loss from mitochondrial and nuclear compartments. Depletion of NBAT1 altered CRM1 function and contributed to the loss of p53-dependent nuclear gene expression during genotoxic drug treatment. CRM1 inhibition rescued p53-dependent nuclear functions and sensitized NBAT1-depleted cells to genotoxic drugs. Combined inhibition of CRM1 and MDM2 was even more effective in sensitizing aggressive neuroblastoma cells with p53 cytoplasmic accumulation. Thus, our mechanistic studies uncover an NBAT1-dependent CRM1/MDM2-based potential combination therapy for patients with high-risk neuroblastoma. SIGNIFICANCE: This study shows how a p53-responsive lncRNA mediates chemotherapeutic response by modulating nuclear p53 pathways and identifies a potential treatment strategy for patients with high-risk neuroblastoma.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/genética , Neuroblastoma/tratamiento farmacológico , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Fraccionamiento Celular , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Masculino , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/cirugía , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Largo no Codificante/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Exportina 1
5.
Epigenetics ; 14(11): 1125-1140, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31216925

RESUMEN

EZH2 is overexpressed in poor-prognostic chronic lymphocytic leukaemia (CLL) cases, acting as an oncogene; however, thus far, the EZH2 target genes in CLL have not been disclosed. In this study, using ChIP-sequencing, we identified EZH2 and H3K27me3 target genes in two prognostic subgroups of CLL with distinct prognosis and outcome, i.e., cases with unmutated (U-CLL, n = 6) or mutated IGHV genes (M-CLL, n = 6). While the majority of oncogenic pathways were equally enriched for EZH2 target genes in both prognostic subgroups, PI3K pathway genes were differentially bound by EZH2 in U-CLL versus M-CLL. The occupancy of EZH2 for selected PI3K pathway target genes was validated in additional CLL samples (n = 16) and CLL cell lines using siRNA-mediated EZH2 downregulation and ChIP assays. Intriguingly, we found that EZH2 directly binds to the IGF1R promoter along with MYC and upregulates IGF1R expression in U-CLL, leading to downstream PI3K activation. By investigating an independent CLL cohort (n = 96), a positive correlation was observed between EZH2 and IGF1R expression with higher levels in U-CLL compared to M-CLL. Accordingly, siRNA-mediated downregulation of either EZH2, MYC or IGF1R and treatment with EZH2 and MYC pharmacological inhibitors in the HG3 CLL cell line induced a significant reduction in PI3K pathway activation. In conclusion, we characterize for the first time EZH2 target genes in CLL revealing a hitherto unknown implication of EZH2 in modulating the PI3K pathway in a non-canonical, PRC2-independent way, with potential therapeutic implications considering that PI3K inhibitors are effective therapeutic agents for CLL.


Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Linfoide/genética , Transducción de Señal , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Humanos , Leucemia Linfoide/metabolismo , Leucemia Linfoide/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Regulación hacia Arriba
6.
Nat Commun ; 9(1): 883, 2018 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-29491376

RESUMEN

Despite improvement in our understanding of long noncoding RNAs (lncRNAs) role in cancer, efforts to find clinically relevant cancer-associated lncRNAs are still lacking. Here, using nascent RNA capture sequencing, we identify 1145 temporally expressed S-phase-enriched lncRNAs. Among these, 570 lncRNAs show significant differential expression in at least one tumor type across TCGA data sets. Systematic clinical investigation of 14 Pan-Cancer data sets identified 633 independent prognostic markers. Silencing of the top differentially expressed and clinically relevant S-phase-enriched lncRNAs in several cancer models affects crucial cancer cell hallmarks. Mechanistic investigations on SCAT7 in multiple cancer types reveal that it interacts with hnRNPK/YBX1 complex and affects cancer cell hallmarks through the regulation of FGF/FGFR and its downstream PI3K/AKT and MAPK pathways. We also implement a LNA-antisense oligo-based strategy to treat cancer cell line and patient-derived tumor (PDX) xenografts. Thus, this study provides a comprehensive list of lncRNA-based oncogenic drivers with potential prognostic value.


Asunto(s)
Neoplasias/genética , ARN Largo no Codificante/genética , Fase S , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/fisiopatología , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ARN , Proteína 1 de Unión a la Caja Y/genética , Proteína 1 de Unión a la Caja Y/metabolismo
7.
Cancer Cell ; 33(3): 417-434.e7, 2018 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-29533783

RESUMEN

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , ARN Largo no Codificante/genética , Factor de Transcripción SOX9/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , Neuroblastoma/genética , Neuroblastoma/patología , Ubiquitina Tiolesterasa/genética
8.
Clin Epigenetics ; 8: 106, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27777635

RESUMEN

BACKGROUND: Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq) is a robust and powerful method for analyzing methylated CpG-rich regions with complete genome-wide coverage. In chronic lymphocytic leukemia (CLL), the role of CpG methylated regions associated with transcribed long noncoding RNAs (lncRNA) and repetitive genomic elements are poorly understood. Based on MBD-Seq, we characterized the global methylation profile of high CpG-rich regions in different CLL prognostic subgroups based on IGHV mutational status. RESULTS: Our study identified 5800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal controls. From cllDMGs, 40 % of hypermethylated and 60 % of hypomethylated genes were mapped to noncoding RNAs. In addition, we found that the major repetitive elements such as short interspersed elements (SINE) and long interspersed elements (LINE) have a high percentage of cllDMRs (differentially methylated regions) in IGHV subgroups compared to normal controls. Finally, two novel lncRNAs (hypermethylated CRNDE and hypomethylated AC012065.7) were validated in an independent CLL sample cohort (48 samples) compared with 6 normal sorted B cell samples using quantitative pyrosequencing analysis. The methylation levels showed an inverse correlation to gene expression levels analyzed by real-time quantitative PCR. Notably, survival analysis revealed that hypermethylation of CRNDE and hypomethylation of AC012065.7 correlated with an inferior outcome. CONCLUSIONS: Thus, our comprehensive methylation analysis by MBD-Seq provided novel hyper and hypomethylated long noncoding RNAs, repetitive elements, along with protein coding genes as potential epigenetic-based CLL-signature genes involved in disease pathogenesis and prognosis.


Asunto(s)
Metilación de ADN , Epigenómica/métodos , Leucemia Linfocítica Crónica de Células B/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Islas de CpG , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Elementos de Nucleótido Esparcido Largo , Pronóstico , ARN Mensajero/genética , Análisis de Secuencia de ADN/métodos , Elementos de Nucleótido Esparcido Corto , Análisis de Supervivencia
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