RESUMEN
Recent studies have described a DNA damage tolerance pathway choice that involves a competition between PrimPol-mediated repriming and fork reversal. Screening different translesion DNA synthesis (TLS) polymerases by the use of tools for their depletion, we identified a unique role of Pol ι in regulating such a pathway choice. Pol ι deficiency unleashes PrimPol-dependent repriming, which accelerates DNA replication in a pathway that is epistatic with ZRANB3 knockdown. In Pol ι-depleted cells, the excess participation of PrimPol in nascent DNA elongation reduces replication stress signals, but thereby also checkpoint activation in S phase, triggering chromosome instability in M phase. This TLS-independent function of Pol ι requires its PCNA-interacting but not its polymerase domain. Our findings unravel an unanticipated role of Pol ι in protecting the genome stability of cells from detrimental changes in DNA replication dynamics caused by PrimPol.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN , Humanos , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , ADN/metabolismo , Reparación del ADN , Daño del ADN , Inestabilidad Cromosómica , ADN Primasa/genética , ADN Primasa/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismoRESUMEN
p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Replicación del ADN/genética , Antígeno Nuclear de Célula en Proliferación/genética , Quinasas Ciclina-Dependientes/genética , Humanos , Inhibidores de Proteínas Quinasas/metabolismo , Fase S/genéticaRESUMEN
Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.
Asunto(s)
Replicación del ADN/genética , Recombinación Homóloga/genética , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ubiquitinación/genética , Muerte Celular/genética , Línea Celular , Línea Celular Tumoral , ADN/genética , Daño del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Células HCT116 , Células HEK293 , Humanos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Understanding the mechanism of metastatic dissemination is crucial for the rational design of novel therapeutics. The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with human breast cancer aggressiveness although the underlying mechanisms are still unclear. Here, shRNA-mediated SPARC knockdown greatly reduced primary tumor growth and completely abolished lung colonization of murine 4T1 and LM3 breast malignant cells implanted in syngeneic BALB/c mice. A comprehensive study including global transcriptomic analysis followed by biological validations confirmed that SPARC induces primary tumor growth by enhancing cell cycle and by promoting a COX-2-mediated expansion of myeloid-derived suppressor cells (MDSC). The role of SPARC in metastasis involved a COX-2-independent enhancement of cell disengagement from the primary tumor and adherence to the lungs that fostered metastasis implantation. Interestingly, SPARC-driven gene expression signatures obtained from these murine models predicted the clinical outcome of patients with HER2-enriched breast cancer subtypes. In total, the results reveal that SPARC and its downstream effectors are attractive targets for antimetastatic therapies in breast cancer.Implications: These findings shed light on the prometastatic role of SPARC, a key protein expressed by breast cancer cells and surrounding stroma, with important consequences for disease outcome. Mol Cancer Res; 15(3); 304-16. ©2016 AACR.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Osteonectina/metabolismo , Receptor ErbB-2/metabolismo , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Osteonectina/genética , Pronóstico , Receptor ErbB-2/genética , Resultado del TratamientoRESUMEN
The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.
Asunto(s)
División Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Replicación del ADN , ADN/biosíntesis , Inestabilidad Genómica , Células Cultivadas , HumanosRESUMEN
DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.
Asunto(s)
Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Células Cultivadas , ADN/química , ADN/genética , ADN/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Recombinación Homóloga , Humanos , Células K562 , Conformación de Ácido Nucleico , Interferencia de ARN , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , ADN Polimerasa iotaRESUMEN
After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Primasa/fisiología , Proteínas de Unión al ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , ADN/efectos de la radiación , Enzimas Multifuncionales/fisiología , Recombinasa Rad51/fisiología , Rayos Ultravioleta , Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Progresión de la Enfermedad , Relación Dosis-Respuesta en la Radiación , Células HeLa , Humanos , Proteína Homóloga de MRE11 , ARN Interferente Pequeño/metabolismoRESUMEN
Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Replicación del ADN/efectos de la radiación , Inestabilidad Genómica , Rayos Ultravioleta , Línea Celular , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S/genética , Estrés FisiológicoRESUMEN
Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.
Asunto(s)
Apoptosis/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Daño del ADN , Lactonas/farmacología , Sesquiterpenos/farmacología , Proliferación Celular/efectos de los fármacos , Ciclina B1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Marcadores Genéticos , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The checkpoint kinases Chk1 and ATR are broadly known for their role in the response to the accumulation of damaged DNA. Because Chk1 activation requires its phosphorylation by ATR, it is expected that ATR or Chk1 down-regulation should cause similar alterations in the signals triggered by DNA lesions. Intriguingly, we found that Chk1, but not ATR, promotes the progression of replication forks after UV irradiation. Strikingly, this role of Chk1 is independent of its kinase-domain and of its partnership with Claspin. Instead, we demonstrate that the ability of Chk1 to promote replication fork progression on damaged DNA templates relies on its recently identified proliferating cell nuclear antigen-interacting motif, which is required for its release from chromatin after DNA damage. Also supporting the importance of Chk1 release, a histone H2B-Chk1 chimera, which is permanently immobilized in chromatin, is unable to promote the replication of damaged DNA. Moreover, inefficient chromatin dissociation of Chk1 impairs the efficient recruitment of the specialized DNA polymerase η (pol η) to replication-associated foci after UV. Given the critical role of pol η during translesion DNA synthesis (TLS), these findings unveil an unforeseen facet of the regulation by Chk1 of DNA replication. This kinase-independent role of Chk1 is exclusively associated to the maintenance of active replication forks after UV irradiation in a manner in which Chk1 release prompts TLS to avoid replication stalling.