Map of synthetic rescue interactions for the Fanconi anemia DNA repair pathway identifies USP48

Genome Instability and Cance

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs

The roles of p27Kip1 and DNA damage signalling in the chemotherapy-induced delayed cell cycle checkpoint

DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR-dependent checkpoint response, followed by a later DDR-independent, but p27Kip1-dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA-PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two-step response was DDR-dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA-PKcs resulted in cell-cycle re-entry despite the increased levels of p27Kip1 at all time points analyzed. We further investigated the regulation of p27Kip1 protein levels in the particular setting. Our results showed that the protein status of p27Kip1 is mainly determined by p38-MAPK, whereas the role of SKP2 is less significant in the doxoroubicin-treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy-induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied. © 2010 The Authors. Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Loss of p14ARF confers resistance to heat shock- and oxidative stress-mediated cell death by upregulating β-catenin

The p14ARF is a key tumor suppressor induced mainly by oncogenic stimuli. Although p14ARF does not seem to respond to DNA damage, there are very few data regarding its role in other forms of stress, such as heat shock (HS) and oxidative stress (OS). Here, we report that suppression of p14ARF increased resistance to cell death when cells were treated with H2O2 or subjected to HS. In this setting, protection from cell death was mediated by elevated levels and activity of β-catenin, as downregulation of β-catenin alleviated the protective role of p14 ARF silencing. Moreover, Hsp70 was shown to regulate β-catenin protein levels by interacting with p14ARF, suggesting that Hsp70, p14ARF and β-catenin form a regulatory network. This novel pathway triggers cell death signals when cells are exposed to HS and OS. © 2010 UICC

Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or " oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies. Copyright © American Society for Investigative Pathology

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16 INK4A, a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload. © 2013 Macmillan Publishers Limited. All rights reserved

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic ‘hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload

Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours. © 2013 Macmillan Publishers Limited. All rights reserved

Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.</p