RESUMEN
The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.
Asunto(s)
Empalme Alternativo , Transición Epitelial-Mesenquimal , MicroARNs , Transición Epitelial-Mesenquimal/genética , Humanos , MicroARNs/metabolismo , MicroARNs/genética , Línea Celular Tumoral , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/metabolismo , Factores de Empalme Serina-Arginina/genética , Regulación Neoplásica de la Expresión Génica , Proliferación Celular/genética , Apoptosis/genética , Factor de Crecimiento Transformador beta/metabolismo , AnimalesRESUMEN
BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.
Asunto(s)
Neuroblastoma , Animales , Humanos , Lactante , Ratones , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes myc , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Recurrencia Local de Neoplasia/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/metabolismoRESUMEN
The IGF2 mRNA-binding protein 1 (IGF2BP1) is a non-catalytic post-transcriptional enhancer of tumor growth upregulated and associated with adverse prognosis in solid cancers. However, conserved effector pathway(s) and the feasibility of targeting IGF2BP1 in cancer remained elusive. We reveal that IGF2BP1 is a post-transcriptional enhancer of the E2F-driven hallmark in solid cancers. IGF2BP1 promotes G1/S cell cycle transition by stabilizing mRNAs encoding positive regulators of this checkpoint like E2F1. This IGF2BP1-driven shortening of the G1 cell cycle phase relies on 3'UTR-, miRNA- and m6A-dependent regulation and suggests enhancement of cell cycle progression by m6A-modifications across cancers. In addition to E2F transcription factors, IGF2BP1 also stabilizes E2F-driven transcripts directly indicating post-transcriptional 'super'-enhancer role of the protein in E2F-driven gene expression in cancer. The small molecule BTYNB disrupts this enhancer function by impairing IGF2BP1-RNA association. Consistently, BTYNB interferes with E2F-driven gene expression and tumor growth in experimental mouse tumor models.
Asunto(s)
Factores de Transcripción E2F/genética , Neoplasias/genética , Proteínas de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Neoplasias/patología , Proteínas de Unión al ARN/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias Ováricas/etiología , Neoplasias Ováricas/metabolismo , Proteínas de Unión al ARN/genética , Transducción de Señal , Familia-src Quinasas/metabolismo , Uniones Adherentes/genética , Uniones Adherentes/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Dominios Homologos src , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
The oncofetal mRNA-binding protein IGF2BP1 and the transcriptional regulator SRF modulate gene expression in cancer. In cancer cells, we demonstrate that IGF2BP1 promotes the expression of SRF in a conserved and N6-methyladenosine (m6A)-dependent manner by impairing the miRNA-directed decay of the SRF mRNA. This results in enhanced SRF-dependent transcriptional activity and promotes tumor cell growth and invasion. At the post-transcriptional level, IGF2BP1 sustains the expression of various SRF-target genes. The majority of these SRF/IGF2BP1-enhanced genes, including PDLIM7 and FOXK1, show conserved upregulation with SRF and IGF2BP1 synthesis in cancer. PDLIM7 and FOXK1 promote tumor cell growth and were reported to enhance cell invasion. Consistently, 35 SRF/IGF2BP1-dependent genes showing conserved association with SRF and IGF2BP1 expression indicate a poor overall survival probability in ovarian, liver and lung cancer. In conclusion, these findings identify the SRF/IGF2BP1-, miRNome- and m6A-dependent control of gene expression as a conserved oncogenic driver network in cancer.
Asunto(s)
MicroARNs/genética , Proteínas de Unión al ARN/genética , Factor de Respuesta Sérica/genética , Adenosina/análogos & derivados , Adenosina/genética , Animales , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The oncofetal IGF2 mRNA binding proteins (IGF2BPs) are upregulated in most cancers but their paralogue-specific roles in tumor cells remain poorly understood. In a panel of five cancer-derived cell lines, IGF2BP1 shows highly conserved oncogenic potential. Consistently, the deletion of IGF2BP1 impairs the growth and metastasis of ovarian cancer-derived cells in nude mice. Gene expression analyses in ovarian cancer-derived cells reveal that the knockdown of IGF2BPs is associated with the downregulation of mRNAs that are prone to miRNA regulation. All three IGF2BPs preferentially associate upstream of miRNA binding sites (MBSs) in the 3'UTR of mRNAs. The downregulation of mRNAs co-regulated by miRNAs and IGF2BP1 is abrogated at low miRNA abundance or when miRNAs are depleted. IGF2BP1 associates with these target mRNAs in RISC-free complexes and its deletion enhances their association with AGO2. The knockdown of most miRNA-regulated target mRNAs of IGF2BP1 impairs tumor cell properties. In four primary cancers, elevated synthesis of these target mRNAs is largely associated with upregulated IGF2BP1 mRNA levels. In ovarian cancer, the enhanced expression of IGF2BP1 and most of its miRNA-controlled target mRNAs is associated with poor prognosis. In conclusion, these findings indicate that IGF2BP1 enhances an aggressive tumor cell phenotype by antagonizing miRNA-impaired gene expression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/fisiología , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Eliminación de Gen , Humanos , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , Neoplasias/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fenotipo , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
In primary neurons, the oncofetal RNA-binding protein IGF2BP1 (IGF2 mRNA-binding protein 1) controls spatially restricted ß-actin (ACTB) mRNA translation and modulates growth cone guidance. In cultured tumor-derived cells, IGF2BP1 was shown to regulate the formation of lamellipodia and invadopodia. However, how and via which target mRNAs IGF2BP1 controls the motility of tumor-derived cells has remained elusive. In this study, we reveal that IGF2BP1 promotes the velocity and directionality of tumor-derived cell migration by determining the cytoplasmic fate of two novel target mRNAs: MAPK4 and PTEN. Inhibition of MAPK4 mRNA translation by IGF2BP1 antagonizes MK5 activation and prevents phosphorylation of HSP27, which sequesters actin monomers available for F-actin polymerization. Consequently, HSP27-ACTB association is reduced, mobilizing cellular G-actin for polymerization in order to promote the velocity of cell migration. At the same time, stabilization of the PTEN mRNA by IGF2BP1 enhances PTEN expression and antagonizes PIP(3)-directed signaling. This enforces the directionality of cell migration in a RAC1-dependent manner by preventing additional lamellipodia from forming and sustaining cell polarization intrinsically. IGF2BP1 thus promotes the velocity and persistence of tumor cell migration by controlling the expression of signaling proteins. This fine-tunes and connects intracellular signaling networks in order to enhance actin dynamics and cell polarization.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Actinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular/fisiología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Fosforilación , ARN Mensajero/metabolismoRESUMEN
The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/genética , Línea Celular Tumoral , Movimiento Celular , Femenino , Células HEK293 , Proteína HMGA2/antagonistas & inhibidores , Proteína HMGA2/metabolismo , Humanos , MicroARNs/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/fisiopatología , Isoformas de ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Estrés Fisiológico/genética , Proteínas Portadoras/metabolismo , Línea Celular , ADN Helicasas , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de SeñalRESUMEN
The post-transcriptional control of gene expression mediated by RNA-binding proteins (RBPs), long non-coding RNAs (lncRNAs) as well as miRNAs is essential to determine tumor cell fate and thus is a major determinant in cancerogenesis. The IGF2 mRNA binding protein family (IGF2BPs) comprises three RBPs. Two members of the family, IGF2BP1 and IGF2BP3, are bona fide oncofetal proteins, which are de novo synthesized in various human cancers. In vitro studies revealed that IGF2BPs serve as post-transcriptional fine-tuners modulating the expression of genes implicated in the control of tumor cell proliferation, survival, chemo-resistance and metastasis. Consistently, the expression of both IGF2BP family members was reported to correlate with an overall poor prognosis and metastasis in various human cancers. Due to the fact that most reports used a pan-IGF2BP antibody for studying IGF2BP expression in cancer, paralogue-specific functions can barely be evaluated at present. Nonetheless, the accordance of IGF2BPs' role in promoting an aggressive phenotype of tumor-derived cells in vitro and their upregulated expression in aggressive malignancies provides strong evidence that IGF2BPs are powerful post-transcriptional oncogenes enhancing tumor growth, drug-resistance and metastasis. This suggests IGF2BPs as powerful biomarkers and candidate targets for cancer therapy.
Asunto(s)
Antígenos de Neoplasias/genética , Transformación Celular Neoplásica/genética , Neoplasias/patología , Proteínas de Unión al ARN/genética , Proliferación Celular , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Neoplasias/genética , Proteínas de Unión al ARN/biosíntesisRESUMEN
Loss of fragile X mental retardation protein (FMRP) causes synaptic dysfunction and intellectual disability. FMRP is an RNA-binding protein that controls the translation or turnover of a subset of mRNAs. Identifying these target transcripts is an important step toward understanding the pathology of the disease. Here, we show that FMRP regulates actin organization and neurite outgrowth via the armadillo protein p0071. In mouse embryonic fibroblasts (MEFs) lacking FMRP (Fmr1-), the actin cytoskeleton was markedly reorganized with reduced stress fibers and F-actin/G-actin ratios compared to fibroblasts re-expressing the protein. FMRP interfered with the translation of the p0071 mRNA in a 3'-UTR-dependent manner. Accordingly, FMRP-depleted cells revealed elevated levels of p0071 protein. The knockdown of p0071 in Fmr1- fibroblasts restored stress fibers and an elongated cell shape, thus rescuing the Fmr1- phenotype, whereas overexpression of p0071 in Fmr1+ cells mimicked the Fmr1- phenotype. Moreover, p0071 and FMRP regulated neurite outgrowth and branching in a diametrically opposed way in agreement with the negative regulation of p0071 by FMRP. These results identify p0071 as an important and novel FMRP target and strongly suggest that impaired actin cytoskeletal functions mediated by an excess of p0071 are key aspects underlying the fragile X syndrome.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuritas/metabolismo , Placofilinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Placofilinas/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) controls the migration and invasiveness of primary as well as tumor-derived cells in vitro. Whether the protein also modulates epithelial-mesenchymal-transition (EMT), a hallmark of tumor progression involved in tumor cell dissemination, remained elusive. In this study, we reveal that IGF2BP1 enhances mesenchymal-like cell properties in tumor-derived cells by promoting the expression of the transcriptional regulators LEF1 and SLUG (SNAI2). IGF2BP1 associates with LEF1 transcripts and prevents their degradation in a 3'-UTR-dependent manner resulting in an upregulation of LEF1 expression. LEF1 promotes transcription of the mesenchymal marker fibronectin by associating with the fibronectin 1 promoter. Moreover, LEF1 enforces the synthesis of the 'EMT-driving' transcriptional regulator SNAI2. Accordingly, IGF2BP1 knockdown causes MET-like (mesenchymal-epithelial-transition) morphological changes, enhances the formation of cell-cell contacts and reduces cell migration in various mesenchymal-like tumor-derived cells. However, in epithelial-like tumor-derived cells characterized by a lack or low abundance of IGF2BP1, the protein fails to induce EMT. These findings identify IGF2BP1 as a pro-mesenchymal post-transcriptional determinant, which sustains the synthesis of 'EMT-driving' transcriptional regulators, mesenchymal markers and enhances tumor cell motility. This supports previous reports, suggesting a role of IGF2BP1 in tumor cell dissemination.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Mesodermo/citología , Neoplasias/fisiopatología , Proteínas de Unión al ARN/fisiología , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Fibronectinas/biosíntesis , Fibronectinas/genética , Células HEK293 , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Neoplasias/patología , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The insulin-like growth factor-2 mRNA-binding proteins 1, 2, and 3 (IGF2BP1, IGF2BP2, IGF2BP3) belong to a conserved family of RNA-binding, oncofetal proteins. Several studies have shown that these proteins act in various important aspects of cell function, such as cell polarization, migration, morphology, metabolism, proliferation and differentiation. In this review, we discuss the IGF2BP family's role in cancer biology and how this correlates with their proposed functions during embryogenesis. IGF2BPs are mainly expressed in the embryo, in contrast with comparatively lower or negotiable levels in adult tissues. IGF2BP1 and IGF2BP3 have been found to be re-expressed in several aggressive cancer types. Control of IGF2BPs' expression is not well understood; however, let-7 microRNAs, ß-catenin (CTNNB1) and MYC have been proposed to be involved in their regulation. In contrast to many other RNA-binding proteins, IGF2BPs are almost exclusively observed in the cytoplasm where they associate with target mRNAs in cytoplasmic ribonucleoprotein complexes (mRNPs). During development, IGF2BPs are required for proper nerve cell migration and morphological development, presumably involving the control of cytoskeletal remodeling and dynamics, respectively. Likewise, IGF2BPs modulate cell polarization, adhesion and migration in tumor-derived cells. Moreover, they are highly associated with cancer metastasis and the expression of oncogenic factors (KRAS, MYC and MDR1). However, a pro-metastatic role of IGF2BPs remains controversial due to the lack of 'classical' in vivo studies. Nonetheless, IGF2BPs could provide valuable targets in cancer treatment with many of their in vivo roles to be fully elucidated.
Asunto(s)
Desarrollo Embrionario/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Modelos Biológicos , Neoplasias/fisiopatología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Gránulos Citoplasmáticos/metabolismo , Humanos , Familia de Multigenes/genética , Neoplasias/metabolismo , Neuronas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , beta Catenina/metabolismoRESUMEN
Cytokinesis requires the spatio-temporal coordination of cell-cycle control and cytoskeletal reorganization. Members of the Rho-family of GTPases are crucial regulators of this process and assembly of the contractile ring depends on local activation of Rho signalling. Here, we show that the armadillo protein p0071, unlike its relative p120(ctn), is localized at the midbody during cytokinesis and is essential for cell division. Both knockdown and overexpression of p0071 interfered with normal cell growth and survival due to cytokinesis defects with formation of multinucleated cells and induction of apoptosis. This failure of cytokinesis seemingly correlated with the deregulation of Rho activity in response to altered p0071 expression. The function of p0071 in regulating Rho activity occurred through an association of p0071 with RhoA, as well as the physical and functional interaction of p0071 with Ect2, the one Rho guanine-nucleotide exchange factor (GEF) essential for cytokinesis. These findings support an essential role for p0071 in spatially regulating restricted Rho signalling during cytokinesis.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Citocinesis , Placofilinas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoA/metabolismo , Animales , Centrosoma/metabolismo , Regulación hacia Abajo , Humanos , Ratones , Células 3T3 NIH , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Huso Acromático/metabolismoRESUMEN
We show the effects of the three purine derivatives, caffeine, theophylline, and istradefylline, on cAMP production by adenylyl cyclase 5 (ADCY5)-overexpressing cell lines. A comparison of cAMP levels was performed for ADCY5 wild-type and R418W mutant cells. ADCY5-catalyzed cAMP production was reduced with all three purine derivatives, while the most pronounced effects on cAMP reduction were observed for ADCY5 R418W mutant cells. The gain-of-function ADCY5 R418W mutant is characterized by an increased catalytic activity resulting in elevated cAMP levels that cause kinetic disorders or dyskinesia in patients. Based on our findings in ADCY5 cells, a slow-release formulation of theophylline was administered to a preschool-aged patient with ADCY5-related dyskinesia. A striking improvement of symptoms was observed, outperforming the effects of caffeine that had previously been administered to the same patient. We suggest considering theophylline as an alternative therapeutic option to treat ADCY5-related dyskinesia in patients.
Asunto(s)
Discinesias , Teofilina , Humanos , Preescolar , Cafeína , Inhibidores de Fosfodiesterasa , Broncodilatadores , Diuréticos , Vasodilatadores , Agitación PsicomotoraRESUMEN
The analysis of protein-RNA association in vitro commonly involves radiolabeled in vitro transcribed RNAs. Nucleotides labeled with near-infrared (NIR) dyes provide promising alternatives for studying protein-RNA binding in vitro. However, it remained elusive whether random labeling of RNA probes by NIR dyes interferes with protein binding. Here, we demonstrate that infrared scanning allows the detection of randomly NIR-labeled RNA probes in the low femtomole range. The analyses of eight distinct protein-RNA complexes by electrophoretic mobility shift assay, filter binding, or UV crosslinking revealed that protein binding specificity remains unaffected by random NIR labeling. Accordingly, NIR probes allowed the rapid identification of the short noncoding Y3-RNA as a novel RNA target of ZBP1 (zipcode binding protein). Whereas binding of ZBP1 to the ACTB-zipcode and Y3 was exclusive, the protein formed a trimeric complex with the La protein and Y3. This was dissociated in the presence of Y5 RNA, resulting in the formation of ZBP1/Y3 and La/Y5 complexes. Hence, ZBP1 apparently resides in at least two distinct cellular RNPs: mRNA-containing mRNPs or Y3-containing yRNPs. In conclusion, our findings indicate that randomly labeled NIR probes provide a powerful tool for the rapid and sensitive analysis of protein-RNA binding in vitro. In contrast to radiolabeled RNAs, NIR probes remain stable for months, do not pose any safety considerations, and enable the significantly expedited analysis of experimental data due to fast read technologies available. The most prominent advantage of probes labeled by NIR dyes is the option to color-code distinct transcripts, allowing the unbiased identification of distinct protein-RNA complexes in one sample.
Asunto(s)
Proteínas Aviares/metabolismo , ARN Citoplasmático Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas Aviares/aislamiento & purificación , Línea Celular , Pollos , Ensayo de Cambio de Movilidad Electroforética , Humanos , Inmunoprecipitación , Unión Proteica/genética , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
The RNA-binding protein IGF2BP1 (IGF-II mRNA binding protein 1) stabilizes the c-myc RNA by associating with the Coding Region instability Determinant (CRD). If and how other proteins cooperate with IGF2BP1 in promoting stabilization of the c-myc mRNA via the CRD remained elusive. Here, we identify various RNA-binding proteins that associate with IGF2BP1 in an RNA-dependent fashion. Four of these proteins (HNRNPU, SYNCRIP, YBX1, and DHX9) were essential to ensure stabilization of the c-myc mRNA via the CRD. These factors associate with IGF2BP1 in a CRD-dependent manner, co-distribute with IGF2BP1 in non-polysomal fractions comprising c-myc mRNA, and colocalize with IGF2BP1 in the cytoplasm. A selective shift of relative c-myc mRNA levels to the polysomal fraction is observed upon IGF2BP1 knockdown. These findings suggest that IGF2BP1 in complex with at least four proteins promotes CRD-mediated mRNA stabilization. Complex formation at the CRD presumably limits the transfer of c-myc mRNA to the polysomal fraction and subsequent translation-coupled decay.
Asunto(s)
Citoplasma/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Sitios de Unión , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transfección , Proteína 1 de Unión a la Caja YRESUMEN
An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein-mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.
Asunto(s)
Proteínas Aviares/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Animales , Pollos , Gránulos Citoplasmáticos/metabolismo , Humanos , Unión Proteica , Transporte de Proteínas , Eliminación de Secuencia/genéticaRESUMEN
Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3'-untranslated region of the beta-actin mRNA known as the 'zipcode'. ZBP1 promotes translocation of the beta-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. It is not known how the ZBP1-RNA complex achieves asymmetric protein sorting by localizing beta-actin mRNA. Here we show that chicken ZBP1 modulates the translation of beta-actin mRNA. ZBP1 associates with the beta-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Translation only occurs when the ZBP1-RNA complex reaches its destination at the periphery of the cell. At the endpoint of mRNA transport, the protein kinase Src promotes translation by phosphorylating a key tyrosine residue in ZBP1 that is required for binding to RNA. These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth.
Asunto(s)
Actinas/análisis , Actinas/biosíntesis , Proteínas Aviares/metabolismo , Polaridad Celular , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas de Unión al ARN/metabolismo , Actinas/genética , Animales , Proteínas Aviares/genética , Línea Celular , Pollos , Proteínas de Unión al ADN , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
MEX3A belongs to the MEX3 (Muscle EXcess) protein family consisting of four members (MEX3A-D) in humans. Characteristic for MEX3 proteins is their domain structure with 2 HNRNPK homology (KH) domains mediating RNA binding and a C-terminal really interesting new gene (RING) domain that harbors E3 ligase function. In agreement with their domain composition, MEX3 proteins were reported to modulate both RNA fate and protein ubiquitination. MEX3 paralogs exhibit an oncofetal expression pattern, they are severely downregulated postnatally, and re-expression is observed in various malignancies. Enforced expression of MEX3 proteins in various cancers correlates with poor prognosis, emphasizing their oncogenic potential. The latter is supported by MEX3A's impact on proliferation, self-renewal as well as migration of tumor cells in vitro and tumor growth in xenograft studies.