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1.
bioRxiv ; 2023 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-37292633

RESUMEN

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

2.
Sci Adv ; 8(43): eabo1304, 2022 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-36306353

RESUMEN

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

3.
PLoS Pathog ; 18(4): e1010401, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35363834

RESUMEN

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.


Asunto(s)
Virus BK , Infecciones por Polyomavirus , Poliomavirus , Virus BK/genética , Humanos , Poliomavirus/genética , Infecciones por Polyomavirus/genética , Empalme del ARN , Virus 40 de los Simios/genética
4.
Mol Cell ; 81(19): 4041-4058.e15, 2021 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-34624217

RESUMEN

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proliferación Celular , Proteínas Inmediatas-Precoces/metabolismo , Mitosis , Células Neoplásicas Circulantes/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Regiones no Traducidas 3' , Animales , Antineoplásicos/farmacología , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina , Femenino , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Células HEK293 , Humanos , Proteínas Inmediatas-Precoces/genética , Indoles/farmacología , Células MCF-7 , Ratones Endogámicos NOD , Ratones SCID , Mitosis/efectos de los fármacos , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/patología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Fenilacetatos/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Estructuras R-Loop , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transducción de Señal , Elongación de la Transcripción Genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32503981

RESUMEN

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Roturas del ADN de Doble Cadena , Recombinación Homóloga , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/metabolismo , Animales , Línea Celular Tumoral , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Resistencia a Antineoplásicos/genética , Técnicas de Silenciamiento del Gen , Humanos , Metilación , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Science ; 367(6485): 1468-1473, 2020 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-32029688

RESUMEN

Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.


Asunto(s)
Neoplasias de la Mama/patología , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Proteínas Ribosómicas/genética , Animales , Neoplasias de la Mama/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Análisis de Secuencia de ARN
7.
Genome Biol ; 21(1): 33, 2020 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-32039742

RESUMEN

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.


Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Resistencia a Antineoplásicos , Procesamiento Postranscripcional del ARN , Tristetraprolina/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Células THP-1 , Transcriptoma , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
8.
Nat Commun ; 11(1): 164, 2020 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-31919360

RESUMEN

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.


Asunto(s)
Predisposición Genética a la Enfermedad/genética , Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/patogenicidad , Gripe Humana/genética , Gripe Humana/patología , Células A549 , Proteínas Adaptadoras Transductoras de Señales/genética , Antivirales/farmacología , Sistemas CRISPR-Cas , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dibenzotiepinas , Estudio de Asociación del Genoma Completo , Humanos , Proteínas de la Membrana/genética , Metiltransferasas/metabolismo , Morfolinas , Proteínas del Tejido Nervioso/genética , Oxazinas/farmacología , Piridinas/farmacología , Piridonas , Tiepinas/farmacología , Triazinas/farmacología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Internalización del Virus
9.
Nucleic Acid Ther ; 28(5): 273-284, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30133337

RESUMEN

The Ebola virus is a zoonotic pathogen that can cause severe hemorrhagic fever in humans, with up to 90% lethality. The deadly 2014 Ebola outbreak quickly made an unprecedented impact on human lives. While several vaccines and therapeutics are under development, current approaches contain several limitations, such as virus mutational escape, need for formulation or refrigeration, poor scalability, long lead-time, and high cost. To address these challenges, we developed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to target critical Ebola viral proteins and the human intracellular host protein Niemann-Pick C1 (NPC1), required for viral entry into infected cells. We generated noninfectious viral luciferase reporter assays to identify LNA ASOs that inhibit translation of Ebola viral proteins in vitro and in human cells. We demonstrated specific inhibition of key Ebola genes VP24 and nucleoprotein, which inhibit a proper immune response and promote Ebola virus replication, respectively. We also identified LNA ASOs targeting human host factor NPC1 and demonstrated reduced infection by chimeric vesicular stomatitis virus harboring the Ebola glycoprotein, which directly binds to NPC1 for viral infection. These results support further in vivo testing of LNA ASOs in infectious Ebola virus disease animal models as potential therapeutic modalities for treatment of Ebola.


Asunto(s)
Fiebre Hemorrágica Ebola/genética , Proteína Niemann-Pick C1/genética , Oligonucleótidos Antisentido/genética , Proteínas Virales/genética , Animales , Modelos Animales de Enfermedad , Ebolavirus/genética , Ebolavirus/patogenicidad , Fiebre Hemorrágica Ebola/terapia , Fiebre Hemorrágica Ebola/virología , Humanos , Inmunidad Innata/genética , Ratones , Proteína Niemann-Pick C1/antagonistas & inhibidores , Nucleoproteínas/antagonistas & inhibidores , Nucleoproteínas/genética , Oligonucleótidos/genética , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Primates/virología , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/genética
10.
Methods Mol Biol ; 1686: 251-264, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29030826

RESUMEN

Quiescence (G0) is defined as an assortment of cell cycle arrested states that exhibit distinct properties. Leukemias harbor a subpopulation of G0 cells that can be enriched by growth factor deprivation or serum starvation. Target site reporters with shortened poly(A) tails show translation activation by microRNAs, via a noncanonical mechanism, when introduced into the nucleus of G0 cells. This is because recruitment by the activation causing FXR1a-microRNA-protein complex (FXR1a-microRNP) is nuclear and requires shortened poly(A) tails to avoid repressive factors and canonical translation. When introduced into the cytoplasm, target mRNAs and microRNAs are directed toward repression rather than translation activation. Leukemic cell lines are difficult to transfect but can be routinely nucleofected-where in vitro transcribed mRNA reporters and microRNAs are introduced into the nucleus of G0 leukemic cells. Nucleofection of a microRNA target reporter and either cognate, targeting microRNA, or control microRNA, into the nucleus of G0 cells, enables analysis of translation activation by microRNAs in G0. We discuss a modified protocol that we developed for transfection of mRNAs along with microRNAs to test translation regulation by microRNAs in G0 leukemic cells.


Asunto(s)
Leucemia Monocítica Aguda/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Fase de Descanso del Ciclo Celular , Humanos , Leucemia Monocítica Aguda/genética , ARN Mensajero/genética
11.
Proc Natl Acad Sci U S A ; 114(25): E4961-E4970, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28584122

RESUMEN

The reversible state of proliferative arrest known as "cellular quiescence" plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA-processing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1-dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest.


Asunto(s)
Vías Biosintéticas/genética , Proliferación Celular/genética , Carioferinas/genética , MicroARNs/genética , Receptores Citoplasmáticos y Nucleares/genética , Vías Biosintéticas/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citoplasma/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Guanosina/análogos & derivados , Guanosina/farmacología , Células HEK293 , Células HeLa , Humanos , Proteína Exportina 1
12.
RNA Biol ; 14(2): 137-145, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27911187

RESUMEN

Eukaryotic protein synthesis is a multifaceted process that requires coordination of a set of translation factors in a particular cellular state. During normal growth and proliferation, cells generally make their proteome via conventional translation that utilizes canonical translation factors. When faced with environmental stress such as growth factor deprivation, or in response to biological cues such as developmental signals, cells can reduce canonical translation. In this situation, cells adapt alternative modes of translation to make specific proteins necessary for required biological functions under these distinct conditions. To date, a number of alternative translation mechanisms have been reported, which include non-canonical, cap dependent translation and cap independent translation such as IRES mediated translation. Here, we discuss one of the alternative modes of translation mediated by a specialized microRNA complex, FXR1a-microRNP that promotes non-canonical, cap dependent translation in quiescent conditions, where canonical translation is reduced due to low mTOR activity.


Asunto(s)
Proteínas Argonautas/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Factor 4G Eucariótico de Iniciación/metabolismo , Exorribonucleasas/metabolismo , Regulación de la Expresión Génica , Humanos , Poli A/genética , Unión Proteica , Biosíntesis de Proteínas , Proteínas de Unión a Caperuzas de ARN/metabolismo , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TOR/metabolismo
13.
Mol Cell ; 61(5): 760-773, 2016 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-26942679

RESUMEN

MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNA-protein complex (microRNP) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling, and therefore reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation, and thereby for the oocyte immature state. P97 interacts with 3' UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5' caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.


Asunto(s)
Senescencia Celular , MicroARNs/metabolismo , Oocitos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sitios de Unión , Línea Celular , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , MicroARNs/genética , Proteómica/métodos , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleoproteínas/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Xenopus laevis
14.
Mol Cancer Ther ; 15(1): 142-53, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26637368

RESUMEN

Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication.


Asunto(s)
Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Metabolómica , Ratones , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
15.
Proc Natl Acad Sci U S A ; 111(41): E4315-22, 2014 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-25261552

RESUMEN

Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met(i)) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.


Asunto(s)
Puntos de Control del Ciclo Celular , Factores Eucarióticos de Iniciación/genética , Regulación hacia Arriba , Animales , Línea Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Humanos , Ratones , Oocitos/citología , Oocitos/metabolismo , Fosforilación , Biosíntesis de Proteínas , ARN de Transferencia de Metionina , Xenopus laevis
16.
Genes Dev ; 27(23): 2543-8, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24298054

RESUMEN

Insulin-like growth factor 2 (IGF2), a developmentally regulated and maternally imprinted gene, is frequently overexpressed in pediatric cancers. Although loss of imprinting (LOI) at fetal promoters contributes to increased IGF2 in tumors, the magnitude of IGF2 expression suggests the involvement of additional regulatory mechanisms. A microRNA (miRNA) screen of primary Wilms' tumors identified specific overexpression of miR-483-5p, which is embedded within the IGF2 gene. Unexpectedly, the IGF2 mRNA itself is transcriptionally up-regulated by miR-483-5p. A nuclear pool of miR-483-5p binds directly to the 5' untranslated region (UTR) of fetal IGF2 mRNA, enhancing the association of the RNA helicase DHX9 to the IGF2 transcript and promoting IGF2 transcription. Ectopic expression of miR-483-5p in IGF2-dependent sarcoma cells is correlated with increased tumorigenesis in vivo. Together, these observations suggest a functional positive feedback loop of an intronic miRNA on transcription of its host gene.


Asunto(s)
Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , Intrones , MicroARNs/metabolismo , Regiones Promotoras Genéticas/genética , Regiones no Traducidas 5'/genética , Línea Celular , Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , Feto/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Unión Proteica , ARN Mensajero/metabolismo
17.
Adv Exp Med Biol ; 768: 97-126, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23224967

RESUMEN

MicroRNAs are small noncoding RNA regulatory molecules that control gene expression by guiding associated effector complexes to other RNAs via sequence-specific recognition of target sites. Misregulation of microRNAs leads to a wide range of diseases including cancers, inflammatory and developmental disorders. MicroRNAs were found to mediate deadenylation-dependent decay and translational repression of messages through partially complementary microRNA target sites in the 3'-UTR (untranslated region). A growing series of studies has demonstrated that microRNAs and their associated complexes (microRNPs) elicit alternate functions that enable stimulation of gene expression in addition to their assigned repressive roles. These reports, discussed in this chapter, indicate that microRNA-mediated effects via natural 3' and 5'-UTRs can be selective and controlled, dictated by the RNA sequence context, associated complex, and cellular conditions. Similar to the effects of repression, upregulated gene expression by microRNAs varies from small refinements to significant amplifications in expression. An emerging theme from this literature is that microRNAs have a versatile range of abilities to manipulate post-transcriptional control mechanisms leading to controlled gene expression. These studies reveal new potentials for microRNPs in gene expression control that develop as responses to specific cellular conditions.


Asunto(s)
Expresión Génica , MicroARNs/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Ribonucleoproteínas/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Bacterias/genética , Bacterias/metabolismo , Humanos , MicroARNs/metabolismo , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Activación Transcripcional
18.
Genes Dev ; 26(13): 1459-72, 2012 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-22751500

RESUMEN

Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFßR2 and the TGFß signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFß pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.


Asunto(s)
Línea Celular , Glioblastoma/metabolismo , MicroARNs/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Animales , Glioblastoma/genética , Humanos , Ratones , MicroARNs/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
19.
RNA Biol ; 9(6): 871-80, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22699554

RESUMEN

MicroRNAs are small non-coding RNA regulators of gene expression that play important roles in critical biological processes, including cell division, self-renewal and cell state maintenance. Their deregulation leads to extensive clinical consequences in tumorigenesis. Cancers demonstrate heterogeneity in their cell states implicated in their resistance and resurgence. Apart from proliferating cells, cancers harbor a small proportion of assorted quiescent cells that resist conventional therapeutics and contribute to cancer recurrence. MicroRNA expression, targets, microRNPs (microRNA-protein complexes) and their functions have been demonstrated to be regulated in distinct tumor cell states and as an adaptive response to stress signals in tumor-unfavorable environments. In turn, altered microRNPs and their modified post-transcriptional mechanisms of gene expression may contribute to tumor resistance and influence tumor progression. An understanding of distinct microRNA mechanisms in cancer cells would provide extensive insights into the versatile roles of microRNAs in the perpetuation of tumors and indicate potential therapeutic avenues.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias/patología , Interferencia de ARN , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Proliferación Celular , Humanos , MicroARNs/fisiología , Neoplasias/genética , Neoplasias/metabolismo , Estabilidad del ARN
20.
Wiley Interdiscip Rev RNA ; 3(3): 311-30, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22072587

RESUMEN

MicroRNAs are small non-coding RNA guide molecules that regulate gene expression via association with effector complexes and sequence-specific recognition of target sites on other RNAs; misregulated microRNA expression and functions are linked to a variety of tumors, developmental disorders, and immune disease. MicroRNAs have primarily been demonstrated to mediate posttranscriptional downregulation of expression; translational repression, and deadenylation-dependent decay of messages through partially complementary microRNA target sites in mRNA untranslated regions (UTRs). However, an emerging assortment of studies, discussed in this review, reveal that microRNAs and their associated protein complexes (microribonucleoproteins or microRNPs) can additionally function to posttranscriptionally stimulate gene expression by direct and indirect mechanisms. These reports indicate that microRNA-mediated effects can be selective, regulated by the RNA sequence context, and associated with RNP factors and cellular conditions. Like repression, translation upregulation by microRNAs has been observed to range from fine-tuning effects to significant alterations in expression. These studies uncover remarkable, new abilities of microRNAs and associated microRNPs in gene expression control and underscore the importance of regulation, in cis and trans, in directing appropriate microRNP responses.


Asunto(s)
MicroARNs/metabolismo , Regulación hacia Arriba , Animales , Regulación de la Expresión Génica , Humanos , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Ribonucleoproteínas/metabolismo , Activación Transcripcional
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