196,017 research outputs found
Functional role of the zinc finger factor ZNF281 in DNA damage response
No full textLa sopravvivenza degli organismi dipende dall'accurata trasmissione dell'informazione genetica durante i processi che portano alla divisione cellulare. Questa fedele trasmissione richiede non soloun'elevata precisione nella replicazione del DNA e nella perfetta segregazione cromosomica, ma anche la capacità di sopravvivere ai danni al DNA spontanei o indotti minimizzando il numero di mutazioni ereditabili dalle cellule figlie. Le cellule sono constantemente soggette agli effetti citotossici e mutageni degli agenti in grado di causare danno al DNA. Per rispondere e difendersi da questa minaccia, le cellule eucariotiche hanno evoluto dei processi che sono collettivamente catalogati come risposta al danno al DNA (DDR). La DDR è una complessa via di trasduzione del segnale che ha la capacità di riconoscere la presenza di danni al DNA e trasdurre questa informazione nella cellula al fine di attivare i processi di riparo. Infatti, le cellule possiedono diversi enzimi capaci di rimodellare e riparare il DNA, comunque la loro attività deve essere spazialmente e temporalmente regolata per ottimizzare il riparo del DNA e prevenire potenziali e deleterie alterazioni nella struttura del DNA. In questi ultimi anni considerevoli progressi sono stati fatti per delucidare le componenti e i processi che caratterizzano la DDR eucariotica. Nonostante questo, un punto centrale in questo campo di studio rimane l'identificazione dei geni e delle proteine che controllano l'espressione delle proteine coinvolte nella DDR. Di interesse è la recente scoperta di un numero crescente di fattori di trascrizione che in grado di regolare direttamente la riparazione del DNA e che fanno parte come componenti integrali del macchinario di riparo stesso con meccanismi indipendenti dalla loro abilità di promuovere e/o reprimere la trascrizione. Infatti, gli agenti di danno al DNA (radiazioni, chemioterapia, agenti genotossici) sono in grado di promuovere la traslocazione di alcuni fattori di trascrizione direttamente sulle lesioni al DNA, dove partecipano all'attivazione del riparo al DNA. ZNF281 è un fattore di trascrizione appartenente alla tipologia degli "zinc finger" coinvolto nel mantenimento della staminalità cellulare e nei processi di transizione epiteliale-mesenchimale (EMT). In questo studio vengono analizzati i ruoli di ZNF281 durante la DDR. Infatti, riportiamo che l'espressione di ZNF281 aumenta sia a livello di mRNA, che di proteina dopo stress genotossico indotto da agenti chimici che causano danno al DNA (etoposide, doxorubicina, camptotecina) in diversi sistemi quali le cellule tumorali, cellule di cheratinociti primari e nell’epidermide del Mus musculus. Il comet assay dimostra che il riparo al DNA è ritardato nelle cellule silenziate per l'espressione di ZNF281 e trattate con etoposide. Inoltre, l'analisi con RT profiler array ha dimostrato che l'espressione di dieci geni coinvolti nella risposta al danno al DNA è diminuita nelle cellule trattate con etoposide e silenziate per l'espressione di ZNF281. In linea con questa scoperta, XRCC2 e XRCC2, due geni che prendono parte rispettivamente ai processi di ricombinazione omologa (HR) e Non Homologous End Joining (NHEJ), sono trascrizionalmente attivati da ZNF281 attraverso un meccanismo dipendente dal legame al DNA come dimostrato da esperimenti di saggio della luciferasi e di immunoprecipitazione della cromatina (ChIP). Inoltre, ZNF281 è un co-fattore della proteina c-Myc per l'attivazione dell'espressione della nucleolina e della ciclina B1; mentre c-Myc, anche se in grado di legare promotori di XRCC2 e XRCC4, non è capace di promuovere la trascrizione o di modificare l'attività di ZNF281 sui promotori di questi due geni. L'analisi bioinformatica di una coorte di 1971 pazienti affetti da cancro alla mammella rivela una correlazione statisticamente significativa tra l'espressione di ZNF281 e di XRCC2. Inoltre l'analisi proteomica e il proximity ligation assay (PLA) dimostrano che ZNF281 interagisce con DNA-PK, un'importante proteina del processo di DDR, suggerendo per ZNF281, un ruolo indipendente dalla trascrizione. In sintesi, i risultati discussi in questo lavoro, evidenziano per la prima volta il coinvolgimento di ZNF281 nella risposta cellulare a stress genotossici attraverso un controllo esercitato sull'espressione di geni che agiscono in differenti meccanismi di riparo al danno e attraverso la sua interazione con DNA-PK.The survival of organisms depends on the accurate transmission of genetic information from one cell to its daughters. Such faithful transmission requires not only extreme accuracy in replication of DNA and precision in chromosome distribution, but also the ability to survive spontaneous and induced DNA damage while minimizing the number of heritable mutations. Therefore, cells are constantly under threat from the cytotoxic and mutagenic effects of DNA damaging agents. To respond to these threats, eukaryotes have evolved the DNA damage response (DDR). The DDR is a complex array of different mechanisms that have the ability to sense DNA damage and transduce this information to the cell in order to modulate cellular responses to DNA damage. Cells possess several enzymatic tools capable of remodeling and repairing DNA; however, their activities must be tightly regulated in a temporal, spatial, and DNA lesion-appropriate fashion to optimize repair and prevent unnecessary and potentially deleterious alterations in the structure of DNA during normal cellular processes. During the past several years, considerable progress has been made in elucidating the components and the processes of the eukaryotic DDR. A central issue in this field, which remains to be understood in greater detail, is the identification of the controllers of the expression of DDR proteins. Interestingly, in recent years an increasing number of studies have revealed that several TFs regulate DNA repair directly and can function as integral components of the repair machinery itself in a transcription independent fashion. In fact, DNA damage-inducing insults (irradiation, chemotherapy drugs) promote translocation of some TFs directly to DNA lesions, where they actively facilitate DNA repair. ZNF281 is a zinc finger transcription factor involved in the control of cellular stemness and Epithelial Mesenchymal Transition (EMT). In this study we analyze the roles of ZNF281 during DDR. We report that ZNF281 expression increased after genotoxic stress caused by DNA damaging drugs (Etoposide, Doxorubicin, Camptothecin) in cancer cell lines, normal keratinocytes and in mouse skin in vivo. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with Etoposide. Furthermore, RT profiler array analysis demonstrated that the expression of ten DDR genes was down-regulated in cells treated with Etoposide and silenced for ZNF281. In line with these findings, XRCC2 and XRCC4, two genes that take part in Homologous Recombination (HR) and Non Homologous End Joining (NHEJ) respectively, were transcriptionally activated by ZNF281 through a DNA binding-dependent mechanism as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation (ChIP) experiments. In addition, ZNF281 works as a c-Myc co-factor to stimulate the expression of nucleolin and cyclin B1; instead c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and XRCC2. Moreover proteomic analysis and Proximity Ligation Assay (PLA) demonstrated that ZNF281 interacts with DNA-PK, an important protein of DDR, suggesting a transcription-independent role of ZNF281 in DDR. Our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms and through interaction with with corecomponents of DNA repair pathways
The DNA/RNA helicase DHX9 contributes to the transcriptional program of the androgen receptor in prostate cancer
No full textBackground: Prostate cancer (PC) is the most commonly diagnosed male malignancy and an important cause of mortality. Androgen deprivation therapy is the first line treatment but, unfortunately, a large part of patients evolves to a castration-resistant stage, for which no effective cure is currently available. The DNA/RNA helicase DHX9 is emerging as an important regulator of cellular processes that are often deregulated in cancer. Methods: To investigate whether DHX9 modulates PC cell transcriptome we performed RNA-sequencing analyses upon DHX9 silencing in the androgen-responsive cell line LNCaP. Bioinformatics and functional analyses were carried out to elucidate the mechanism of gene expression regulation by DHX9. Data from The Cancer Genome Atlas were mined to evaluate the potential role of DHX9 in PC. Results: We found that up-regulation of DHX9 correlates with advanced stage and is associated with poor prognosis of PC patients. High-throughput RNA-sequencing analysis revealed that depletion of DHX9 in androgen-sensitive LNCaP cells affects expression of hundreds of genes, which significantly overlap with known targets of the Androgen Receptor (AR). Notably, AR binds to the DHX9 promoter and induces its expression, while Enzalutamide-mediated inhibition of AR activity represses DHX9 expression. Moreover, DHX9 interacts with AR in LNCaP cells and its depletion significantly reduced the recruitment of AR to the promoter region of target genes and the ability of AR to promote their expression in response to 5α-dihydrotestosterone. Consistently, silencing of DXH9 negatively affected androgen-induced PC cell proliferation and migration. Conclusions: Collectively, our data uncover a new role of DHX9 in the control of the AR transcriptional program and establish the existence of an oncogenic DHX9/AR axis, which may represent a new druggable target to counteract PC progression
Activation of miR200 by c-Myb depends on ZEB1 expression and miR200 promoter methylation
No full textTumor progression to metastasis is a complex, sequential process that requires proliferation, resistance to apoptosis, motility and invasion to colonize at distant sites. The acquisition of these features implies a phenotypic plasticity by tumor cells that must adapt to different conditions by modulating several signaling pathways1 during the journey to the final site of metastasis. Several transcription factors and microRNA play a role in tumor progression, but less is known about the control of their expression during this process. Here, we demonstrate by ectopic expression and gene silencing that the proto-oncogene c-Myb activates the expression of the 5 members of miR200 family (miR200b, miR200a, miR429, miR200c and miR141) that are involved in the control of epithelial-mesenchymal transition (EMT) and metastasis in many types of cancers. Transcriptional activation of miR200 by c-Myb occurs through binding to myb binding sites located in the promoter regions of miR200 genes on human chromosomes 1 and 12. Furthermore, when c-Myb and the transcriptional repressor ZEB1 are co-expressed, as at the onset EMT, the repression by ZEB1 prevails over the activation by c-Myb, and the expression of miR200 is inhibited. We also demonstrate that during EMT induced by TGF-β, the promoters of miR200 genes are methylated, and their transcription is repressed regardless of the presence of repressors such as ZEB1 and activators such as c-Myb. Finally, we find a correlation between the expression of c-Myb and that of four out of 5 miR200 in a data set of 207 breast cancer patients. © 2013 Landes Bioscience
ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining
No full textEfficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies
Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors
No full textBrain tumors are a heterogeneous group of neoplasms ranging from almost benign to highly aggressive phenotypes. The malignancy of these tumors mostly relies on gene expression reprogramming, which is frequently accompanied by the aberrant regulation of RNA processing mechanisms. In brain tumors, defects in alternative splicing result either from the dysregulation of expression and activity of splicing factors, or from mutations in the genes encoding splicing machinery components. Aberrant splicing regulation can generate dysfunctional proteins that lead to modification of fundamental physiological cellular processes, thus contributing to the development or progression of brain tumors. Herein, we summarize the current knowledge on splicing abnormalities in brain tumors and how these alterations contribute to the disease by sustaining proliferative signaling, escaping growth suppressors, or establishing a tumor microenvironment that fosters angiogenesis and intercellular communications. Lastly, we review recent efforts aimed at developing novel splicing-targeted cancer therapies, which employ oligonucleotide-based approaches or chemical modulators of alternative splicing that elicit an impact on brain tumor biology
Neuroblastoma: oncogenic mechanisms and therapeutic exploitation of necroptosis
No full textNeuroblastoma (NB) is the most common extracranial childhood tumor classified in five stages (1, 2, 3, 4 and 4S), two of which (3 and 4) identify chemotherapy-resistant, highly aggressive disease. High-risk NB frequently displays MYCN amplification, mutations in ALK and ATRX, and genomic rearrangements in TERT genes. These NB subtypes are also characterized by reduced susceptibility to programmed cell death induced by chemotherapeutic drugs. The latter feature is a major cause of failure in the treatment of advanced NB patients. Thus, proper reactivation of apoptosis or of other types of programmed cell death pathways in response to treatment is relevant for the clinical management of aggressive forms of NB. In this short review, we will discuss the most relevant genomic rearrangements that define high-risk NB and the role that destabilization of p53 and p73 can have in NB aggressiveness. In addition, we will propose a strategy to stabilize p53 and p73 by using specific inhibitors of their ubiquitin-dependent degradation. Finally, we will introduce necroptosis as an alternative strategy to kill NB cells and increase tumor immunogenicity
ZNF281 contributes to the DNA damage response by controlling the expression of XRCC2 and XRCC4
No full textZNF281 is a zinc-finger factor involved in the control of cellular stemness and epithelial-mesenchymal transition (EMT). Here, we report that ZNF281 expression increased after genotoxic stress caused by DNA-damaging drugs. Comet assays demonstrated that DNA repair was delayed in cells silenced for the expression of ZNF281 and treated with etoposide. Furthermore, the expression of 10 DNA damage response genes was downregulated in cells treated with etoposide and silenced for ZNF281. In line with this finding, XRCC2 and XRCC4, two genes that take part in homologous recombination and non-homologous end joining, respectively, were transcriptionally activated by ZNF281 through a DNA-binding-dependent mechanism, as demonstrated by luciferase assays and Chromatin crosslinking ImmunoPrecipitation experiments. c-Myc, which also binds to the promoters of XRCC2 and XRCC4, was unable to promote their transcription or to modify ZNF281 activity. Of interest, bioinformatic analysis of 1971 breast cancer patients disclosed a significant correlation between the expression of ZNF281 and that of XRCC2. In summary, our data highlight, for the first time, the involvement of ZNF281 in the cellular response to genotoxic stress through the control exercised on the expression of genes that act in different repair mechanisms
Activation of miR200 by c-Myb depends on ZEB1 expression and miR200 promoter methylation
No full textTumor progression to metastasis is a complex, sequential process that requires proliferation, resistance to apoptosis, motility and invasion to colonize at distant sites. The acquisition of these features implies a phenotypic plasticity by tumor cells that must adapt to different conditions by modulating several signaling pathways (1) during the journey to the final site of metastasis. Several transcription factors and microRNA play a role in tumor progression, but less is known about the control of their expression during this process. Here, we demonstrate by ectopic expression and gene silencing that the proto-oncogene c-Myb activates the expression of the 5 members of miR200 family (miR200b, miR200a, miR429, miR200c and miR141) that are involved in the control of epithelial-mesenchymal transition (EMT) and metastasis in many types of cancers. Transcriptional activation of miR200 by c-Myb occurs through binding to myb binding sites located in the promoter regions of miR200 genes on human chromosomes 1 and 12. Furthermore, when c-Myb and the transcriptional repressor ZEB1 are co-expressed, as at the onset EMT, the repression by ZEB1 prevails over the activation by c-Myb, and the expression of miR200 is inhibited. We also demonstrate that during EMT induced by TGF-β, the promoters of miR200 genes are methylated, and their transcription is repressed regardless of the presence of repressors such as ZEB1 and activators such as c-Myb. Finally, we find a correlation between the expression of c-Myb and that of four out of 5 miR200 in a data set of 207 breast cancer patients
c-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer
No full textThe splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis
Impairment of FOXM1 expression in mesenchymal cells from patients with myeloid neoplasms, de novo and therapy-related, may compromise their ability to support hematopoiesis
No full textBone marrow mesenchymal stem cells (BM-MSCs) exhibit multiple abnormalities in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including reduced proliferative and clonogenic capacity, altered morphology, impaired immunoregulatory properties and capacity to support hematopoiesis. Here, we investigated expression of the FOXM1 gene, a transcription factor driving G2/M gene expression, in BM-MSCs isolated from patients with MDS and AML, de novo and therapy-related, compared to BM-MSCs isolated from healthy donors (HD). We observed a statistically significant downregulation of FOXM1 expression in BM-MSCs isolated from MDS and AML patients, as compared to controls. In parallel, expression of FOXM1 mitotic targets (CCNB1, CDC20, PLK1 and NDC80) was suppressed in patients' BM-MSCs, as compared to HD. No differences in the expression of FOXM1 and its mitotic targets were observed in BM-mononuclear cells from the different sources. From a functional standpoint, silencing of FOXM1 mRNA in healthy MSC induced a significant decrease in the expression of its targets. In this line, healthy MSC silenced for FOXM1 showed an impaired ability to support hematopoiesis in vitro. These findings suggest that deregulation of FOXM1 may be involved in the senescent phenotype observed in MSC derived from myeloid neoplasms
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