RESUMEN
Several kinds of stress promote the formation of three-stranded RNA:DNA hybrids called R-loops. Insufficient clearance of these structures promotes genomic instability and DNA damage, which ultimately contribute to the establishment of cancer phenotypes. Paraspeckle assemblies participate in R-loop resolution and preserve genome stability, however, the main determinants of this mechanism are still unknown. This study finds that in Multiple Myeloma (MM), AATF/Che-1 (Che-1), an RNA-binding protein fundamental to transcription regulation, interacts with paraspeckles via the lncRNA NEAT1_2 (NEAT1) and directly localizes on R-loops. We systematically show that depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids. We provide evidence that such failure to resolve R-loops causes sustained activation of a systemic inflammatory response characterized by an interferon (IFN) gene expression signature. Furthermore, elevated levels of R-loops and of mRNA for paraspeckle genes in patient cells are linearly correlated with Multiple Myeloma progression. Moreover, increased interferon gene expression signature in patients is associated with markedly poor prognosis. Taken together, our study indicates that Che-1/NEAT1 cooperation prevents excessive inflammatory signaling in Multiple Myeloma by facilitating the clearance of R-loops. Further studies on different cancer types are needed to test if this mechanism is ubiquitously conserved and fundamental for cell homeostasis.
Asunto(s)
Mieloma Múltiple , ARN Largo no Codificante , Humanos , Estructuras R-Loop , Mieloma Múltiple/genética , Paraspeckles , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Interferones/genética , Proteínas Represoras/metabolismo , Proteínas Reguladoras de la Apoptosis/genéticaRESUMEN
Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , ADN Ribosómico/genética , Genes de ARNr/genética , ARN Polimerasa I/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/patología , Daño del ADN , ADN Ribosómico/metabolismo , Homeostasis , Humanos , Fosforilación , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Ribosomas/metabolismoRESUMEN
Despite progress in treating B-cell precursor acute lymphoblastic leukemia (BCP-ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high-risk relapsed patients. Che-1/AATF (Che-1) is an RNA polymerase II-binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che-1 is overexpressed in pediatric BCP-ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP-ALL cells. Furthermore, we report that c-Myc regulates Che-1 expression by direct binding to its promoter and describe a strict correlation between Che-1 expression and c-Myc expression. RNA-seq analyses upon Che-1 or c-Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP-seq experiments suggest that Che-1 acts as a downstream effector of c-Myc. These results identify the pivotal role of Che-1 in the control of BCP-ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP-ALL.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/genética , Línea Celular Tumoral , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Regiones Promotoras Genéticas/genéticaRESUMEN
Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Mieloma Múltiple/patología , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Femenino , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones Desnudos , Mieloma Múltiple/metabolismo , Complejos Multiproteicos/metabolismo , Fosforilación , Proteínas Represoras/genética , Estrés Fisiológico , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
To combat threats posed by DNA damage, cells have evolved mechanisms, collectively termed DNA damage response (DDR). These mechanisms detect DNA lesions, signal their presence, and promote their repair. Centrosomes integrate G2/M checkpoint control and repair signals in response to genotoxic stress, acting as an efficient control mechanism when G2/M checkpoint function fails and mitosis begins in the presence of damaged DNA. Che-1 is an RNA polymerase II-binding protein involved in the regulation of gene transcription, induction of cell proliferation, and DDR. Here we provide evidence that in addition to its nuclear localization, Che-1 localizes at interphase centrosomes, where it accumulates following DNA damage or spindle poisons. We show that Che-1 depletion generates supernumerary centrosomes, multinucleated cells, and multipolar spindle formation. Notably, Che-1 depletion abolishes the ability of Chk1 to bind pericentrin and to localize at centrosomes, which, in its turn, deregulates the activation of centrosomal cyclin B-Cdk1 and advances entry into mitosis. Our results reinforce the notion that Che-1 plays an important role in DDR and that its contribution seems to be relevant for the spindle assembly checkpoint.
Asunto(s)
Antígenos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Centrosoma/metabolismo , Cromosomas Humanos/metabolismo , Daño del ADN , Mitosis/fisiología , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Antígenos/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromosomas Humanos/genética , Ciclina B/genética , Ciclina B/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Humanos , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas Quinasas/genética , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. METHODS: Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser316, Ser320 and Ser321) in 308-325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. RESULTS: Here, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. CONCLUSIONS: Che-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Quinasa de la Caseína II/metabolismo , Proteínas Represoras/metabolismo , Proliferación Celular/fisiología , Humanos , Fosforilación , TransfecciónRESUMEN
Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of "active chromatin" by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.
Asunto(s)
Mieloma Múltiple , Proteínas Nucleares , Proliferación Celular , Cromatina , Humanos , Mieloma Múltiple/genética , Proteínas Nucleares/genética , Factores de Transcripción/genéticaRESUMEN
Deptor is an important protein that belongs to the mTORC1 and mTORC2 complexes, able to interact with mTOR and to inhibit its kinase activity. As a natural mTOR inhibitor, Deptor is involved in several molecular pathways, such as cell growth, apoptosis, autophagy and ER stress response. For this reason, Deptor seems to play an important role in controlling cellular homeostasis. Despite several recent insights characterizing Deptor functions and regulation, its complete role within cells has not yet been completely clarified. Indeed, quite recently, Deptor has been associated with chromatin, and it has been demonstrated having a role in transcriptional regulation, controlling in such way endoplasmatic reticulum activity.From all these observations it is not surprising that Deptor can behave either as an oncogene or oncosuppressor, depending on the cell- or tissue-contexts. This review highlights recent progresses made in our understanding of the many activities of Deptor, describing its transcriptional and post-transcriptional regulation in different cancer cell types. Moreover, here we discuss the possibility of using compounds able to inhibit Deptor or to disrupt its interaction with mTOR as novel approaches for cancer therapy.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Autofagia , Proliferación Celular , Estrés del Retículo Endoplásmico , Humanos , Especificidad de Órganos , Transducción de Señal , Activación TranscripcionalRESUMEN
BACKGROUND: Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. METHODS: The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. RESULTS: Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. CONCLUSIONS: We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Neoplasias Colorrectales/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Represoras/genética , Hipoxia de la Célula , Proliferación Celular , Neoplasias Colorrectales/química , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Células HCT116 , Células HT29 , Humanos , Estabilidad Proteica , Análisis de Secuencia de ARNRESUMEN
Multiple myeloma (MM) is a malignant disorder of plasma cells characterized by active production and secretion of monoclonal immunoglobulins (IgG), thus rendering cells prone to endoplasmic reticulum (ER) stress. For this reason, MM cell survival requires to maintain ER homeostasis at basal levels. Deptor is an mTOR binding protein, belonging to the mTORC1 and mTORC2 complexes. It was reported that Deptor is overexpressed in MM cells where it inhibits mTOR kinase activity and promotes cell survival by activating Akt signaling. Here we identify Deptor as a nuclear protein, able to bind DNA and regulate transcription in MM cells. In particular, we found that Deptor plays an important role in the maintenance of the ER network, sustaining the expression of several genes involved in this pathway. In agreement with this, Deptor depletion induces ER stress and synergizes the effect of the proteasome inhibitor bortezomib (Bz) in MM cells. These findings provide important new insights in the ER stress control in MM cells.