RESUMEN
Genetic studies in mice and human cancers established BCL11B as a haploinsufficient tumor suppressor gene. Paradoxically, BCL11B is overexpressed in some human cancers where its knockdown is synthetic lethal. We identified the BCL11B protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro DNA repair assays demonstrated that both BCL11B and a small recombinant BCL11B213-560 protein lacking transcription regulation potential can stimulate the enzymatic activities of two base excision repair (BER) enzymes: NTHL1 and Pol ß. In cells, BCL11B is rapidly recruited to sites of DNA damage caused by laser microirradiation. BCL11B knockdown delays, whereas ectopic expression of BCL11B213-560 accelerates, the repair of oxidative DNA damage. Inactivation of one BCL11B allele in TK6 lymphoblastoid cells causes an increase in spontaneous and radiation-induced mutation rates. In turn, ectopic expression of BCL11B213-560 cooperates with the RAS oncogene in cell transformation by reducing DNA damage and cellular senescence. These findings indicate that BCL11B functions as a BER accessory factor, safeguarding normal cells from acquiring mutations. Paradoxically, it also enables the survival of cancer cells that would otherwise undergo senescence or apoptosis due to oxidative DNA damage resulting from the elevated production of reactive oxygen species.
Asunto(s)
Reparación por Escisión , Proteínas Represoras , Animales , Humanos , Ratones , Daño del ADN , Reparación del ADN/genética , Genes Supresores de Tumor , Oncogenes , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Base excision repair is initiated by DNA glycosylases that recognize specific altered bases. DNA glycosylases for oxidized bases carry both a glycosylase activity that removes the faulty base and an apyrimidinic/apurinic lyase activity that introduces a single-strand DNA incision. In particular, the CUT domains within the CUX1 and CUX2 proteins were recently shown to interact with the 8-oxoguanine (8-oxoG) DNA glycosylase and stimulate its enzymatic activities. SATB1, which contains two CUT domains, was originally characterized as a T cell-specific genome organizer whose aberrant overexpression in breast cancer can promote tumor progression. Here we investigated the involvement of SATB1 in DNA repair. SATB1 knockdown caused a delay in DNA repair following exposure to H2O2, an increase in OGG1-sensitive oxidized bases within genomic DNA, and a decrease in 8-oxoG cleavage activity in cell extracts. In parallel, we observed an increase in phospho-CHK1 and γ-H2AX levels and a decrease in DNA synthesis. Conversely, ectopic expression of SATB1 accelerated DNA repair and reduced the levels of oxidized bases in genomic DNA. Moreover, an enhanced GFP-SATB1 fusion protein was rapidly recruited to laser microirradiation-induced DNA damage. Using purified proteins, we showed that SATB1 interacts directly with OGG1, increases its binding to 8-oxoG-containing DNA, promotes Schiff base formation, and stimulates its glycosylase and apyrimidinic/apurinic lyase enzymatic activities. Structure/function analysis demonstrated that CUT domains, but not the homeodomain, are responsible for the stimulation of OGG1. Together, these results identify another CUT domain protein that functions both as a transcription factor and an accessory factor in base excision repair.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , ADN Glicosilasas/química , ADN Glicosilasas/genética , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Células Jurkat , Proteínas de Unión a la Región de Fijación a la Matriz/química , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Factores de TranscripciónRESUMEN
The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1âº/â» MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.
Asunto(s)
Daño del ADN , Reparación del ADN/fisiología , Genes ras/fisiología , Proteínas de Homeodominio/fisiología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Células Cultivadas , Senescencia Celular/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Mamarias Experimentales/genética , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de TranscripciónRESUMEN
CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.
Asunto(s)
Corteza Cerebral/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Homeodominio/metabolismo , Neuronas/metabolismo , Animales , Línea Celular , Corteza Cerebral/citología , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Ratones , Neuronas/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Ratas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The p110 Cut homeobox 1 (CUX1) transcription factor regulates genes involved in DNA replication and chromosome segregation. Using a genome-wide-approach, we now demonstrate that CUX1 also modulates the constitutive expression of DNA damage response genes, including ones encoding ATM and ATR, as well as proteins involved in DNA damage-induced activation of, and signaling through, these kinases. Consistently, RNAi knockdown or genetic inactivation of CUX1 reduced ATM/ATR expression and negatively impacted hallmark protective responses mediated by ATM and ATR following exposure to ionizing radiation (IR) and UV, respectively. Specifically, abrogation of CUX1 strongly reduced ATM autophosphorylation after IR, in turn causing substantial decreases in (i) levels of phospho-Chk2 and p53, (ii) γ-H2AX and Rad51 DNA damage foci and (iii) the efficiency of DNA strand break repair. Similarly remarkable reductions in ATR-dependent responses, including phosphorylation of Chk1 and H2AX, were observed post-UV. Finally, multiple cell cycle checkpoints and clonogenic survival were compromised in CUX1 knockdown cells. Our results indicate that CUX1 regulates a transcriptional program that is necessary to mount an efficient response to mutagenic insult. Thus, CUX1 ensures not only the proper duplication and segregation of the genetic material, but also the preservation of its integrity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/fisiología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Puntos de Control del Ciclo Celular , Supervivencia Celular , Células Cultivadas , Roturas del ADN , Reparación del ADN/genética , Regulación de la Expresión Génica , Inestabilidad Genómica , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Interferencia de ARN , Recombinasa Rad51/análisis , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2(+) subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.
Asunto(s)
Ciclo Celular , Inestabilidad Cromosómica/fisiología , Proteínas de Homeodominio/fisiología , Mitosis/fisiología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Línea Celular , Replicación del ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Factores de TranscripciónRESUMEN
BACKGROUND: Overexpression of the Cut homeobox 1 gene, CUX1, inversely correlates with patient survival in breast cancers. Cell-based assays and molecular studies have revealed that transcriptional regulation by CUX1 involves mostly the proteolytically processed p110 isoform. As there is no antibody specific to p110 CUX1 only, an alternate strategy must be employed to identify its targets. RESULTS: We expressed physiological levels of a tagged-p110 CUX1 protein and performed chromatin affinity purification followed by hybridization on ENCODE and promoter arrays. Targets were validated by chromatin immunoprecipitation and transcriptional regulation by CUX1 was analyzed in expression profiling and RT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression. Approximately 47% and 14% of CUX1 binding sites were respectively mapped less than 4 Kbp, or more than 40 Kbp, away from a transcription start site. More genes exhibited changes in expression following CUX1 knockdown than p110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4% of putative targets identified on the ENCODE and promoter arrays respectively. This proportion increased to 11.2% for targets with 2 binding sites or more. Transcriptional repression was observed in a slightly higher proportion of target genes. The CUX1 consensus binding motif, ATCRAT, was found at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1 consensus motifs present on the array were bound in vivo. The presence of a consensus binding motif did not have an impact on whether a target gene was repressed or activated. Interestingly, the distance between a binding site and a transcription start site did not significantly reduced the ability of CUX1 to regulate a target gene. Moreover, CUX1 not only was able to regulate the next adjacent gene, but also regulated the gene located beyond this one as well as the gene located further away in the opposite direction. CONCLUSION: Our results demonstrate that p110 CUX1 can activate or repress transcription when bound at a distance and can regulate more than one gene on certain genomic loci.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Proteínas Represoras/genética , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Isoformas de Proteínas/metabolismo , Análisis de Matrices Tisulares , Factores de Transcripción , Sitio de Iniciación de la Transcripción , Activación Transcripcional/fisiologíaRESUMEN
Cancer cells, in which the RAS and PI3K pathways are activated, produce high levels of reactive oxygen species (ROS), which cause oxidative DNA damage and ultimately cellular senescence. This process has been documented in tissue culture, mouse models, and human pre-cancerous lesions. In this context, cellular senescence functions as a tumour suppressor mechanism. Some rare cancer cells, however, manage to adapt to avoid senescence and continue to proliferate. One well-documented mode of adaptation involves increased production of antioxidants often associated with inactivation of the KEAP1 tumour suppressor gene and the resulting upregulation of the NRF2 transcription factor. In this review, we detail an alternative mode of adaptation to oxidative DNA damage induced by ROS: the increased activity of the base excision repair (BER) pathway, achieved through the enhanced expression of BER enzymes and DNA repair accessory factors. These proteins, exemplified here by the CUT domain proteins CUX1, CUX2, and SATB1, stimulate the activity of BER enzymes. The ensued accelerated repair of oxidative DNA damage enables cancer cells to avoid senescence despite high ROS levels. As a by-product of this adaptation, these cancer cells exhibit increased resistance to genotoxic treatments including ionizing radiation, temozolomide, and cisplatin. Moreover, considering the intrinsic error rate associated with DNA repair and translesion synthesis, the elevated number of oxidative DNA lesions caused by high ROS leads to the accumulation of mutations in the cancer cell population, thereby contributing to tumour heterogeneity and eventually to the acquisition of resistance, a major obstacle to clinical treatment.
RESUMEN
We identified the BCL11A protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro, DNA repair assays demonstrate that both BCL11A and a small recombinant BCL11A160-520 protein that is devoid of DNA binding and transcription regulatory domains can stimulate the enzymatic activities of two base excision repair enzymes: NTHL1 and DNA Pol ß. Increased DNA repair efficiency, in particular of the base excision repair pathway, is essential for many cancer cells to proliferate in the presence of elevated reactive oxygen species (ROS) produced by cancer-associated metabolic changes. BCL11A is highly expressed in triple-negative breast cancers (TNBC) where its knockdown was reported to reduce clonogenicity and cause tumour regression. We show that BCL11A knockdown in TNBC cells delays repair of oxidative DNA damage, increases the number of oxidized bases and abasic sites in genomic DNA, slows down proliferation and induces cellular senescence. These phenotypes are rescued by ectopic expression of the short BCL11A160-520 protein. We further show that the BCL11A160-520 protein accelerates the repair of oxidative DNA damage and cooperates with RAS in cell transformation assays, thereby enabling cells to avoid senescence and continue to proliferate in the presence of high ROS levels.
RESUMEN
The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Fase G1/fisiología , Proteínas de Homeodominio/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteína Quinasa CDC2/genética , Línea Celular , Núcleo Celular/genética , Ciclina B/genética , ADN/genética , ADN/metabolismo , Proteínas de Homeodominio/genética , Humanos , Ratones , Mutación Missense , Proteínas Nucleares/genética , Fosforilación/fisiología , Unión Proteica/fisiología , Proteínas Represoras/genéticaRESUMEN
Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). We have identified an interaction between stefin B and nucleosomes, specifically with histones H2A.Z, H2B, and H3. In synchronized T98G cells, stefin B co-immunoprecipitated with histone H3, predominantly in the G(1) phase of the cell cycle. Stefin B-deficient mouse embryonic fibroblasts entered S phase earlier than wild type mouse embryonic fibroblasts. In contrast, increased expression of stefin B in the nucleus delayed cell cycle progression in T98G cells. The delay in cell cycle progression was associated with the inhibition of cathepsin L in the nucleus, as judged from the decreased cleavage of the CUX1 transcription factor. In vitro, inhibition of cathepsin L by stefin B was potentiated in the presence of histones, whereas histones alone did not affect the cathepsin L activity. Interaction of stefin B with the Met-75 truncated form of cathepsin L in the nucleus was confirmed by fluorescence resonance energy transfer experiments in the living cells. Stefin B could thus play an important role in regulating the proteolytic activity of cathepsin L in the nucleus, protecting substrates such as transcription factors from its proteolytic processing.
Asunto(s)
Catepsina L/metabolismo , Núcleo Celular/metabolismo , Cistatina B/metabolismo , Regulación de la Expresión Génica , Histonas/química , Animales , Ciclo Celular , Línea Celular Tumoral , Citosol/metabolismo , Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Histonas/metabolismo , Humanos , Ratones , Modelos BiológicosRESUMEN
Recent studies revealed that CUT domains function as accessory factors that accelerate DNA repair by stimulating the enzymatic activities of the base excision repair enzymes OGG1, APE1, and DNA pol ß. Strikingly, the role of CUT domain proteins in DNA repair is exploited by cancer cells to facilitate their survival. Cancer cells in which the RAS pathway is activated produce an excess of reactive oxygen species (ROS) which, if not counterbalanced by increased production of antioxidants, causes sustained oxidative DNA damage and, ultimately, cell senescence. These cancer cells can adapt by increasing their capacity to repair oxidative DNA damage in part through elevated expression of CUT domain proteins such as CUX1, CUX2, or SATB1. In particular, CUX1 overexpression was shown to cooperate with RAS in the formation of mammary and lung tumors in mice. Conversely, knockdown of CUX1, CUX2, or SATB1 was found to be synthetic lethal in cancer cells exhibiting high ROS levels as a consequence of activating mutations in KRAS, HRAS, BRAF, or EGFR. Importantly, as a byproduct of their adaptation, cancer cells that overexpress CUT domain proteins exhibit increased resistance to genotoxic treatments such as ionizing radiation, temozolomide, and cisplatin.
RESUMEN
The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Reparación del ADN , Dominios y Motivos de Interacción de Proteínas , Sitios de Unión , Línea Celular , Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Activación Enzimática , Técnicas de Inactivación de Genes , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Unión Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.
Asunto(s)
Cadherinas/genética , Movimiento Celular , Regulación hacia Abajo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transducción de Señal , Animales , Bovinos , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Inmunoprecipitación de Cromatina , Perros , Regulación de la Expresión Génica , Genes Reporteros , Estudio de Asociación del Genoma Completo , Proteínas de Homeodominio/genética , Humanos , Uniones Intercelulares/metabolismo , Ratones , Proteínas Nucleares/genética , Ocludina , Regiones Promotoras Genéticas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Caracoles/genética , Factores de Transcripción , Transcripción Genética , Activación TranscripcionalRESUMEN
Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).
Asunto(s)
Replicación del ADN/genética , Proteínas de Homeodominio/fisiología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Activación Transcripcional , Animales , Línea Celular , Cromatina/aislamiento & purificación , Cromatografía de Afinidad , Genes cdc , Genómica , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Fase S/genética , Análisis de Secuencia de ADN , Factores de TranscripciónRESUMEN
BACKGROUND: Lats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation. RESULTS: Genome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1. CONCLUSION: While inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.
Asunto(s)
Genes Supresores de Tumor , Proteínas de Homeodominio/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Femenino , Proteínas de Homeodominio/biosíntesis , Humanos , Ratones , Ratones Transgénicos , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Represoras/biosíntesis , Transcripción Genética , Activación TranscripcionalRESUMEN
Proteolytic processing by cathepsin L generates p110 Cut homeobox 1 (CUX1) at the end of the G(1) phase, whereas an alternative transcript encodes p75 CUX1. These short CUX1 isoforms were reported to be overexpressed in cancer cells, and transgenic mice overexpressing the p75 isoform were found to develop myeloproliferative disease-like myeloid leukemias. In the present study, we report that the neutrophil elastase can also generate a short CUX1 isoform in the MV4;11 acute myeloid leukemia cell line. Proteolytic processing was so efficient that the full-length CUX1 protein was detected only when cells were maintained in the presence of the specific elastase inhibitor III. In agreement with these findings, higher levels of the processed cyclin E isoforms were also detected in MV4;11 cells. Reappearance of full-length cyclin E and CUX1 could be induced upon the treatment of MV4;11 cells with the differentiation inducer phorbol 12-myristate 13-acetate or, unexpectedly, following overexpression of a short recombinant CUX1 protein. In both cases, the mechanism involved transcriptional repression of the neutrophil elastase gene. This result revealed a negative feedback loop whereby CUX1 shuts down the expression of the protease that cleaves it. Overall, the findings in MV4;11 and other cancer cells suggest that various mechanisms are used in cancer to favor the expression of short CUX1 isoforms.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucemia Mieloide/enzimología , Elastasa de Leucocito/metabolismo , Procesamiento Proteico-Postraduccional , Extractos Celulares , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclina E/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/química , Humanos , Leucemia Mieloide/patología , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Fase S/fisiología , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Pruebas de Carcinogenicidad , Proliferación Celular , Transformación Celular Neoplásica , Células Cultivadas , Centrifugación , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A2 , Ciclinas/genética , Ciclinas/metabolismo , Femenino , Fibroblastos/citología , Genes ras , Proteínas de Homeodominio/genética , Ratones , Ratones Mutantes , Ratones Desnudos , Proteínas Nucleares/genética , Isoformas de Proteínas , Proteínas Represoras/genética , Timidina/metabolismo , Factores de Transcripción/genéticaRESUMEN
Somatic mutations of the CUT-like homeobox 1 (CUX1) gene (CUX1 MT) can be found in myeloid neoplasms (MNs), in particular, in myelodysplastic syndromes (MDSs). The CUX1 locus is also deleted in 3 of 4 MN cases with -7/del(7q). A cohort of 1480 MN patients was used to characterize clinical features and clonal hierarchy associated with CUX1 MT and CUX1 deletions (CUX1 DEL) and to analyze their functional consequences in vitro. CUX1 MT were present in 4% of chronic MNs. CUX1 DEL were preferentially found in advanced cases (6%). Most MDS and acute myeloid leukemia (AML) patients with -7/del(7q) and up to 15% of MDS patients and 5% of AML patients diploid for the CUX1 locus exhibited downmodulated CUX1 expression. In 75% of mutant cases, CUX1 MT were heterozygous, whereas microdeletions and homozygous and compound-heterozygous mutations were less common. CUX MT/DEL were associated with worse survival compared with CUX1 WT Within the clonal hierarchy, 1 of 3 CUX1 MT served as founder events often followed by secondary BCOR and ASXL1 subclonal hits, whereas TET2 was the most common ancestral lesion, followed by subclonal CUX1 MT Comet assay of patients' bone marrow progenitor cells and leukemic cell lines performed in various experimental conditions revealed that frameshift mutations, hemizygous deletions, or experimental CUX1 knockdown decrease the repair of oxidized bases. These functional findings may explain why samples with either CUX1 MT or low CUX1 expression coincided with significantly higher numbers of somatic hits by whole-exome sequencing. Our findings implicate the DNA repair dysfunction resulting from CUX1 lesions in the pathogenesis of MNs, in which they lead to a mutator phenotype.
Asunto(s)
Susceptibilidad a Enfermedades , Proteínas de Homeodominio/genética , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/etiología , Proteínas Represoras/genética , Factores de Transcripción/genética , Biomarcadores , Aberraciones Cromosómicas , Evolución Clonal/genética , Femenino , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/metabolismo , Humanos , Estimación de Kaplan-Meier , Leucocitos Mononucleares , Pérdida de Heterocigocidad , Masculino , Mutación , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Fenotipo , Polimorfismo de Nucleótido Simple , Pronóstico , Proteínas Represoras/metabolismo , Eliminación de Secuencia , Factores de Transcripción/metabolismoRESUMEN
Cux (Cut homeobox) genes are present in all metazoans. Early reports described many phenotypes caused by cut mutations in Drosophila melanogaster. In vertebrates, CUX1 was originally characterized as the CCAAT-displacement protein (CDP). Another line of investigation revealed the presence of CUX1 within a multi-protein complex called the histone nuclear factor D (HiNF-D). Recent studies led to the identification of several CUX1 isoforms with distinct DNA binding and transcriptional properties. While the CCAAT-displacement activity was implicated in the transcriptional repression of several genes, some CUX1 isoforms were found to participate in the transcriptional activation of some genes. The expression and activity of CUX1 was shown to be regulated through the cell cycle and to be a target of TGF-beta signaling. Mechanisms of regulation include alternative transcription initiation, proteolytic processing, phosphorylation and acetylation. Cell-based assays have established a role for CUX1 in the control of cell cycle progression, cell motility and invasion. In the mouse, gene inactivation as well as over-expression in transgenic mice has revealed phenotypes in multiple organs and cell types. While some phenotypes could be explained by the presumed functions of CUX1 in the affected cells, other phenotypes invoked non-cell-autonomous effects that suggest regulatory functions with an impact on cell-cell interactions. The implication of CUX1 in cancer was suggested first from its over-expression in primary tumors and cancer cell lines and was later confirmed in mouse models.