DNA REPLICATION STRESS IN CANCER AND STEM CELLS
Our research focuses on the molecular characterization of DNA replication stress and its contribution to genome instability. We aim to understand the mechanistic basis of genome rearrangements arising during perturbed DNA replication in stem- and somatic cells, affecting various aspects of human disease and most specifically cancer. These studies take advantage of an established technological platform, ranging from standard molecular and cell biology methods to specialized single-molecule in vivo analysis of replication intermediates.
Structural insights into DNA replication stress in cancer onset
DNA damage response is a critical anti-tumour barrier that prevents the proliferation of cells with potentially hazardous genetic alterations. It acts early in tumorigenesis and its activation was observed already in pre-cancerous lesions of various organs. The activation of the DNA damage checkpoint in these lesions was ascribed to oncogene-induced deregulation of DNA synthesis, or “replication stress”. Although the indirect consequences of replication stress, i.e. cell cycle arrest and senescence, have been elucidated to some extent, our understanding of the underlying molecular events is extremely vague. This is mainly due to the lack of information on the in vivo DNA structures generated under such conditions.
The replication stress phenotype can be reproduced in cell culture by overexpression of various oncogenes influencing DNA replication, or by inactivation of crucial cell cycle controls, leading to over-replication of the genome. We have exploited these systems to identify oncogene-associated defects in DNA replication. Overexpression of different oncogenes (e.g. CyclinE and CDC25A) has a substantial effect on bulk DNA synthesis (Figure 1A-B) and leads to a marked slow-down of individual replication forks, measured by FACS analysis and DNA fiber labelling, respectively. Furthermore, electron microscopic analysis1 reveals the accumulation of aberrant replication intermediates. When oncogene activation is coupled to inactivation of cell cycle checkpoints, unscheduled processing of these unusual intermediates leads to massive DNA breakage and full DDR activation, associated with premature mitotic entry2. Using a similar set of approaches we have also recently characterized the molecular consequences of "re-replication" (Figure 1C), a deregulation of replication initiation program that is frequently associated with tumorigenesis3. We are currently extending our studies to a broad spectrum of oncogenes, to possibly extract common molecular mechanisms underlying tumorigenesis from its earliest stages. Furthermore, we have recently started investigating replication-transcription interference, which is postulated to result from the accumulation of toxic DNA-RNA hybrids (R-loops) and was recently shown to underlie the tumorigenic potential of several oncogenes.
Figure 1. Flow cytometric/single molecule analysis of DNA replication stress by oncogene activation or deregulated initiation. (A) FACS-based distinction of γH2AX patterns after Cdc25A induction. Red and green signals indicate cells with pan-nuclear γH2AX and γH2AX foci respectively, indicative of active DNA damage response. (B) FACS analysis after Cdc25A induction shows accumulation of cells with γH2AX foci and pan-nuclear staining. Pan-nuclear γH2AX is associated with replicative arrest. (C) Representative DNA tract labelled with CldU (red) for 2 h and IdU (green) for 30 min and spread by DNA fiber assay to identify re-replication events. This molecule shows two "re-replication" events (yellow tracts) in close proximity.
1) K. J. Neelsen, A. Ray Chaudhuri, C. Follonier, R. Herrador andM. Lopes(2014). Visualization and interpretation of eukaryotic DNA replication intermediates by electron microscopy in vivo. In "Functional Analysis of DNA and Chromatin", Humana Press, ed. J. C. Stockert. Methods in Molecular Biology, 1094:177-208
2) K. J. Neelsen, I. M. Y. Zanini, R. Herrador and M. Lopes (2013). Oncogenes induce genotoxic stress by mitotic processing of unusual replication intermediates. Journal of Cell Biology, 6: 699-708
3) K.J. Neelsen, I.M.Y. Zanini, S. Mijic, R. Herrador, R. Zellweger, A. Ray Chaudhuri, K.D. Creavin, J.J. Blow and M. Lopes(2013). Deregulated origin licensing leads to chromosomal breaks by re-replication of a gapped DNA template. Genes and Development, 27:2537-42
Uncovering the structural determinants of DNA replication stress induced by cancer chemotherapeutics
DNA replication interference is one of the most common strategies employed in the cancer chemotherapy. Although many of these drugs have been used in the clinics for decades, their molecular mechanism of action is often poorly understood, preventing the informed selection of appropriate chemotherapeutic regimens for different tumors and the development of potent combinatorial treatments. We have successfully used our established experimental platform for DNA replication studies of several chemotherapeutic drugs, uncovering surprising alterations of replication fork architecture and refining established models of their action. For example, although replication-induced DSB have long been postulated to mediate the cytotoxicity of topoisomerase inhibitors, we showed that an active control of replication fork progression and architecture – by replication fork slowing and reversal (Figure 2A) - can protect normal and cancer cells from these treatments when these drugs are used at clinically relevant doses4. More recently, we showed that replication fork remodeling is a conserved, global response to a full range of genotoxic drugs (Figure 2B), covering practically all different strategies of replication interference typically used in cancer chemotherapy5. In fact, we have provided solid evidence that these unusual intermediates (reversed forks) accumulate also in the face of endogenous obstacles to replication6, and that they are transient and genome-protective7. We are currently attempting to elucidate the cellular factors playing a role in the formation and resolution of these structures, as they represent potential targets for cancer chemotherapy. Among these factors, we are actively investigating homologous recombination and Fanconi anemia factors, as they were all recently shown to promote genome integrity via replication fork protection, by yet-elusive mechanisms. Furthermore, we are specifically investigating the peculiar transactions that allow replication forks to bypass and repair inter-strand crosslinks. These are particularly toxic adducts – induced by byproducts of cell metabolism, as well as several chemotherapeutic drugs – which should represent roadblocks to the replication process, but are in fact mysteriously bypassed at high efficiency during genome replication of human cells.
Figure 2. Mild genotoxic treatments, including cancer chemotherapeutics, result in frequent replication fork reversal. (A) Electron micrograph of a representative reversed replication fork from U2OS cells treated for 1h with 20nM ETP. P indicates the parental duplex, D indicates daughter duplexes and R indicates the regressed arm. (B) Frequency of reversed replication forks in U2OS cells either untreated (NT) and upon the indicated treatments. In brackets, the total number of analyzed molecules. Above each column, the percentage of reversed forks is indicated.
4) A. Ray Chaudhuri, Y. Hashimoto, R. Herrador, K.J. Neelsen,D. Fachinetti, R. Bermejo, A. Cocito, V. Costanzo and M. Lopes(2012). Topoisomerase I poisoning results in PARP-mediated replication fork reversal. Nature Structural and Molecular Biology19: 417–423
5) R. Zellweger, D. Dalcher, K. Mutreja, J. A. Schmid, R. Herrador, M. Berti, A. Vindigniand M. Lopes (2015). Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells. Journal of Cell Biology 208:563-79
6) A. Ray Chaudhuri, A. K. Ahuja, R. HerradorandM. Lopes(2015). PARG prevents the accumulation of unusual replication structures during unperturbed S phase. Mol Cell Biol. 35:856-65
7) K. J. Neelsen andM. Lopes(2015). Replication fork reversal in eukaryotes: from dead end to dynamic response. Nature Reviews Mol Cell Biol, 16:207-20
DNA replication stress in stem cells
Embryonic stem cells (ESCs) have the unique ability to self-renew and are capable of differentiating into multiple cell types. In contrast, adult stem cells – such as hematopoietic stem cells (HSCs) - repopulate specific tissues and their exhaustion has been linked to ageing, but the underlying molecular mechanisms are largely unknown. Several knockout-mouse models have uncovered a role for numerous DNA repair factors in ageing and cancer. Besides well-known repair activities, conditional deletion of the ATR gene - which is a central factor activated in response to DNA replication stress - causes depletion of the stem cell niche, suggesting that stem cells need to protect their genomes during active proliferation. We are investigating the intriguing connection between replication stress and aging, applying some of our most revealing approaches to different populations of stem cells. We have recently shown in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on ATR and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51 (Figure 3A). Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress – which surprisingly do not impair the mitotic process - are rapidly lost at the onset of differentiation and result from the rapid transition through the G1 phase, which is strictly required to maintain pluripotency. In this context, when cell cycle checkpoints are mostly inactive and numerous DNA lesions are channeled into replication, fork slowing and reversal are strictly required to avoid chromosomal breakage and represent an effective alternative strategy of genome maintenance, compatible with the high proliferation of these cells (Figure 3B)8. We are now expanding these studies, by actively investigating whether similar surprising phenomena can be observed in early embryogenesis of other organisms. Furthermore - in light of the recently proposed links between adult stem cell proliferation, DNA damage and cancer - we are studying whether activation of quiescent stem cells leads to detectable replication stress phenotypes.
Figure 3. Replication stress markers in embryonic stem cells and their alternative, replication-coupled strategy for genome maintenance. (A)Immunofluorescence staining for the DNA damage-marker γH2AX, and for chromatin binding of the ssDNA-binding proteins RPA32 and RAD51 in E3.5 blastocysts. The results indicate activation of DNA damage response and accumulation of ssDNA in unperturbed mouse embryonic stem cells (ESCs) within their natural environment. (B) A model depicting differential control of genome stability in ESCs and proliferating somatic cells. Under-replicated regions and residual DNA damage are unavoidably present at the end of each S phase in both ESCs and somatic cells. Differentiated cells have prolonged gap phases, assemble 53BP1 nuclear bodies and repair most of these lesions prior to S phase entry. However, owing to the brief gap phases, ESCs channel a high number of these lesions into the following S phase and protect genome integrity by extensive fork reversal and replication-coupled repair.
8) A. K. Ahuja, K. Jodkowska, F. Teloni, A. H. Bizard, R. Zellweger, R. Herrador, S. Ortega, I. D. Hickson, M. Altmeyer, J. Mendez and M. Lopes (2016). A short G1 phase imposes constitutive replication stress and fork remodeling in mouse embryonic stem cells. Nature Communications, doi:10.1038/ncomms10660
A powerful technological platform to support structural DNA investigations worldwide
Thanks to the rare combination of specialized approaches, our laboratory has also been very actively involved in collaborative efforts worldwide that require structural insights into DNA metabolism. Several projects have reached publication stage, while several other collaborative efforts are ongoing.