RESUMEN
Cancer stem cells (CSCs) are a subpopulation of cancer cells endowed with high tumorigenic, chemoresistant and metastatic potential. Nongenetic mechanisms of acquired resistance are increasingly being discovered, but molecular insights into the evolutionary process of CSCs are limited. Here, we show that type I interferons (IFNs-I) function as molecular hubs of resistance during immunogenic chemotherapy, triggering the epigenetic regulator demethylase 1B (KDM1B) to promote an adaptive, yet reversible, transcriptional rewiring of cancer cells towards stemness and immune escape. Accordingly, KDM1B inhibition prevents the appearance of IFN-I-induced CSCs, both in vitro and in vivo. Notably, IFN-I-induced CSCs are heterogeneous in terms of multidrug resistance, plasticity, invasiveness and immunogenicity. Moreover, in breast cancer (BC) patients receiving anthracycline-based chemotherapy, KDM1B positively correlated with CSC signatures. Our study identifies an IFN-I â KDM1B axis as a potent engine of cancer cell reprogramming, supporting KDM1B targeting as an attractive adjunctive to immunogenic drugs to prevent CSC expansion and increase the long-term benefit of therapy.
Asunto(s)
Neoplasias de la Mama , Epigénesis Genética , Histona Demetilasas , Interferón Tipo I , Antraciclinas/metabolismo , Antraciclinas/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Histona Demetilasas/metabolismo , Humanos , Interferón Tipo I/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
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
DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
Asunto(s)
Metilación de ADN , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Timina ADN Glicosilasa/metabolismo , 5-Metilcitosina/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Citidina Desaminasa/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Timina ADN Glicosilasa/genética , Transcripción 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
Che-1 is a RNA polymerase II-binding protein involved in the transcription of E2F target genes and induction of cell proliferation. Here we show that Che-1 contributes to DNA damage response and that its depletion sensitizes cells to anticancer agents. The checkpoint kinases ATM/ATR and Chk2 interact with Che-1 and promote its phosphorylation and accumulation in response to DNA damage. These Che-1 modifications induce a specific recruitment of Che-1 on the TP53 and p21 promoters. Interestingly, it has a profound effect on the basal expression of p53, which is preserved following DNA damage. Notably, Che-1 contributes to the maintenance of the G2/M checkpoint induced by DNA damage. These findings identify a mechanism by which checkpoint kinases regulate responses to DNA damage.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Genes p53 , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/fisiología , Animales , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , División Celular , Quinasa de Punto de Control 2 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Fase G2 , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Arteriovenous malformations (AVMs) are abnormal connections between arteries and veins that bypass the capillary system. Among AVMs, uterine ones are very rare, and it is not possible to have clear data on their incidence, as a good part of the patients remain clinically asymptomatic. Uterine AVMs consist of abnormal communications between branches of the uterine artery and the myometrial venous plexus. They can lead to significant bleeding, resulting in severe anemia and the need for transfusions. Both medical and surgical therapeutic approaches are described in the literature; as regards surgical treatments, the hysteroscopic excision of the endometrial mass represents a conservative and minimally invasive approach. However, there are no reported cases in the literature of AVMs treated using a hysteroscopic approach under local anesthesia and in an office setting. In this article, we propose the case of a young woman diagnosed with postpartum uterine AVM, treated using a 16 Fr miniresectoscope (GUBBINI system; Tontarra Medizintechnik®, Tuttlingen, Germany) in an office setting with a pain control protocol (pericervical infiltration and nitrous oxide via bucconasal mask). No complications occurred, and the woman was discharged immediately after the procedure. Finally, the patient was asked how tolerable and acceptable the procedure was compared to expectations; the woman defined the procedure as very bearable and well tolerated. The outpatient treatment, with an adequate pain control protocol, proved to be less invasive for the woman, did not require narcosis and hospital admission, but was equally therapeutic and effective compared to the treatment performed in the operating room.
RESUMEN
DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Histona Desacetilasas/metabolismo , Proteínas Represoras , Proteína de Retinoblastoma/fisiología , Células 3T3 , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , División Celular , Línea Celular , Secuencia Conservada , Factores de Transcripción E2F , Glutatión/metabolismo , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Humanos , Ratones , Modelos Biológicos , Mutación , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de la radiación , Proteínas Portadoras/genética , Proteínas Portadoras/efectos de la radiación , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Activación Enzimática/efectos de la radiación , Genes Supresores de Tumor , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oligonucleótidos Antisentido , Fosforilación , Proteína de la Leucemia Promielocítica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Serina , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Rayos UltravioletaRESUMEN
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
Che-1 is a nuclear protein involved in the regulation of gene transcription and cell proliferation. It has also been shown to localize to the cytoplasm of postmitotic neuronal cells, where it is able to interact with the microtubule-associated protein tau. Cyclin-dependent kinase 5 (Cdk5) is a postmitotic proline-directed serine/threonine kinase that hyperphosphorylates tau under pathological conditions. We observed that Che-1 overexpression induces Cdk5 expression both at the mRNA and protein levels. Furthermore, we show that Che-1 directly interacts with Cdk5 protein in vivo. Cdk5/Che-1 complex formation does not compete with Cdk5/p35 interaction, thus Che-1 is able to bind the active kinase complex. Finally, we demonstrated that Che-1 is itself a Cdk5 substrate.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica , Expresión Génica/fisiología , Neuronas/fisiología , Factores de Transcripción/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Cerebelo/citología , Quinasa 5 Dependiente de la Ciclina/genética , Regulación de la Expresión Génica/genética , Humanos , Inmunoprecipitación/métodos , Ratones , Proteínas Nucleares , Ratas , Ratas Wistar , Factores de Transcripción/genética , Transfección/métodosRESUMEN
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
The p53 protein is a key player in a wide range of protein networks that allow the state of "good health" of the cell. Not surprisingly, mutations of the TP53 gene are one of the most common alterations associated to cancer cells. Mutated forms of p53 (mtp53) not only lose the ability to protect the integrity of the genetic heritage of the cell but also acquire pro-oncogenic functions, behaving like dangerous accelerators of transformation and tumor progression. In recent years, many studies focused on investigating possible strategies aiming to counteract this mutant p53 "gain of function" but the results have not always been satisfactory. Che-1/AATF is a nuclear protein that binds to RNA polymerase II and plays a role in multiple fundamental processes, including control of transcription, cell cycle regulation, DNA damage response, and apoptosis. Several studies showed Che-1/AATF as an important endogenous regulator of p53 expression and activity in a variety of biological processes. Notably, this same regulation was more recently observed also on mtp53. The depletion of Che-1/AATF strongly reduces the expression of mutant p53 in several tumors in vitro and in vivo, making the cells an easier target for chemotherapy treatments. In this mini review, we report an overview of Che-1/AATF functions and discuss a possible role of Che-1/AATF in cancer therapy, with particular regard to its action on p53/mtp53.
RESUMEN
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.
Asunto(s)
Retículo Endoplásmico , Homeostasis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Animales , Antineoplásicos/farmacología , Bortezomib/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Transgénicos , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Interferencia de ARNRESUMEN
RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II). It is selectively down-regulated upon treatment with doxorubicin (dox). Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation. To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening. It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified. This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit. Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4. Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction. We show that RPB3 expression is regulated during muscle differentiation. Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects. These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription.
Asunto(s)
Diferenciación Celular/fisiología , Miogenina/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Sitios de Unión , Western Blotting , Diferenciación Celular/genética , Línea Celular , Humanos , Ratones , Miocardio/citología , Miocardio/metabolismo , Miogenina/genética , Unión Proteica , ARN Polimerasa II/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
PURPOSE: To investigate the possible existence of an antiapoptotic cross-talk between HER-2 and antiapoptotic Bcl-2 family members. EXPERIMENTAL DESIGN: Bcl-2 and Bcl-XL expression and apoptosis induction were analyzed in HER-2 gene-amplified (BT474) and nonamplified (ZR 75-1) breast cancer cell lines exposed to trastuzumab, alone or in combination with either Bcl-2/Bcl-XL bispecific antisense oligonucleotides (AS-4625) or the small-molecule Bcl-2 antagonist HA14-1. RESULTS: In addition to HER-2 and epidermal growth factor receptor, trastuzumab down-regulated Bcl-2, but not Bcl-XL, protein, and mRNA expression in BT474 cells. Interestingly, trastuzumab-induced down-regulation of HER-2 and Bcl-2 was also observed in three of five and two of three breast cancer patients undergoing trastuzumab treatment, respectively. Despite Bcl-2 down-regulation, however, trastuzumab only marginally increased the rate of apoptosis (7.3 +/- 3.5%). We therefore investigated whether a combination of AS-4625 and trastuzumab might increase proapoptotic efficiency. AS-4625 treatment of BT474 cells decreased both Bcl-2 and Bcl-XL expression, resulting in a 21 +/- 7% net apoptosis induction; the combination of AS-4625 followed by trastuzumab resulted in a significantly stronger induction of apoptosis (37 +/- 6%, P <0.01) that was not observed with the reverse treatment sequence (trastuzumab followed by AS-4625). Similar results were obtained with the Bcl-2 antagonist HA14-1; indeed, exposure of BT474 cells to HA14-1 followed by trastuzumab resulted in a striking proapoptotic synergism (combination index=0.58 +/- 0.18), as assessed by isobologram analysis. CONCLUSIONS: Altogether our findings suggest that combined targeting of HER-2 and Bcl-2 may represent a novel, rational approach to more effective breast cancer therapy.