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The final sentence of the Acknowledgements should be as follows: This work was supported by grants from Instituto de Salud Carlos III (BA15/00092), Spanish Ministry of Economy and Competitiveness/EU-ERDF (SAF2016-80626-R, SAF2013-49149-R, BFU2014-51672-REDC), Fundación CajaCanarias (AP2015/008) to RF, and the Australian National Health and Medical Research (NHMRC program grant to SRL and KKK (APP1017028).
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This corrects the article DOI: 10.1038/onc.2017.21.
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Correct control of DNA replication is crucial to maintain genomic stability in dividing cells. Inappropriate re-licensing of replicated origins is associated with chromosomal instability (CIN), a hallmark of cancer progression that at the same time provides potential opportunities for therapeutic intervention. Geminin is a critical inhibitor of the DNA replication licensing factor Cdt1. To properly achieve its functions, Geminin levels are tightly regulated through the cell cycle by ubiquitin-dependent proteasomal degradation, but the de-ubiquitinating enzymes (DUBs) involved had not been identified. Here we report that DUB3 and USP7 control human Geminin. Overexpression of either DUB3 or USP7 increases Geminin levels through reduced ubiquitination. Conversely, depletion of DUB3 or USP7 reduces Geminin levels, and DUB3 knockdown increases re-replication events, analogous to the effect of Geminin depletion. In exploring potential clinical implications, we found that USP7 and Geminin are strongly correlated in a cohort of invasive breast cancers (P<1.01E-08). As expected, Geminin expression is highly prognostic. Interestingly, we found a non-monotonic relationship between USP7 and breast cancer-specific survival, with both very low or high levels of USP7 associated with poor outcome, independent of estrogen receptor status. Altogether, our data identify DUB3 and USP7 as factors that regulate DNA replication by controlling Geminin protein stability, and suggest that USP7 may be involved in Geminin dysregulation during breast cancer progression.
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Neoplasias da Mama/enzimologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Endopeptidases/metabolismo , Geminina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Ciclo Celular , Linhagem Celular Tumoral , Instabilidade Cromossômica , Replicação do DNA/fisiologia , Progressão da Doença , Endopeptidases/genética , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Invasividade Neoplásica , Prognóstico , Estabilidade Proteica , RNA Interferente Pequeno/genética , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina , UbiquitinaçãoRESUMO
Stresses such as hypoxia, nutrient deprivation and acidification disturb protein folding in the endoplasmic reticulum (ER) and activate the Unfolded Protein Response (UPR) to trigger adaptive responses through the effectors, PERK, IRE1 and ATF6. Most of these responses relate to ER homoeostasis; however, here we show that the PERK branch of the UPR also controls DNA replication. Treatment of cells with the non-genotoxic UPR agonist thapsigargin led to a rapid inhibition of DNA synthesis that was attributable to a combination of DNA replication fork slowing and reduced replication origin firing. DNA synthesis inhibition was dependent on the UPR effector PERK and was associated with phosphorylation of the checkpoint adaptor protein Claspin and activation of the Chk1 effector kinase, both of which occurred in the absence of detectable DNA damage. Remarkably, thapsigargin did not inhibit bulk DNA synthesis or activate Chk1 in cells depleted of Claspin, or when Chk1 was depleted or subject to chemical inhibition. In each case thapsigargin-resistant DNA synthesis was due to an increase in replication origin firing that compensated for reduced fork progression. Taken together, our results unveil a new aspect of PERK function and previously unknown roles for Claspin and Chk1 as negative regulators of DNA replication in the absence of genotoxic stress. Because tumour cells proliferate in suboptimal environments, and frequently show evidence of UPR activation, this pathway could modulate the response to DNA replication-targeted chemotherapies.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Replicação do DNA/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Humanos , Transfecção , eIF-2 Quinase/genéticaRESUMO
The DNA damage checkpoint is essential for the maintenance of genome integrity after genotoxic stress, and also for cell survival in eukaryotes. Claspin has a key role in the ATR (ATM and Rad3-related)-Chk1 branch of the DNA damage checkpoint and is also required for correct DNA replication. To achieve properly these functions, Claspin is tightly regulated by ubiquitinin-dependent proteasomal degradation, which controls Claspin levels in a DNA-damage- and cell-cycle-dependent manner. Here, we identified a new regulator of Claspin, the ubiquitin-specific peptidase 29, USP29. Downregulation of USP29 destabilizes Claspin, whereas its overexpression promotes an increase in Claspin levels. USP29 interacts with Claspin and is able to deubiquitinate it both in vivo and in vitro. Most importantly, USP29 knockdown results in an impaired phosphorylation of Chk1 after DNA damage and USP29-depleted cells show a major defect in the S-phase progression. With these results, we identified USP29 as a new player in the ATR-Chk1 pathway and the control of DNA replication.
