Review DNA Replication and Oncogene-Induced Replicative Stress

DNA Replication and Oncogene-Induced Review Replicative Stress

Stephanie A. Hills and John F.X. Diffley* caused by replication of a damaged DNA template, for example caused by reactive oxygen species [13,14], but oncogene-induced replicative stress in cultured cells is independent of oxidative stress [3]. Instead recent data suggest Summary that activated oncogenes might have a more direct effect upon the regulation of DNA replication, and this will be the DNA replication must be tightly regulated to ensure that focus of our review. We begin by summarising our current the genome is accurately duplicated during each cell understanding of replication initiation and its regulation, cycle. When these regulatory mechanisms fail, replicative focussing on metazoans, before describing potential mech- stress and DNA damage ensue. Activated oncogenes pro- anisms for oncogene-induced replicative stress and how mote replicative stress, inducing a DNA damage response they trigger DNA damage. Finally we discuss how replicative (DDR) early in tumorigenesis. Senescence or apoptosis stress might promote the types of genomic instability result, forming a barrier against tumour progression. This commonly seen in cancers and how the causes of replicative may provide a selective pressure for acquisition of muta- stress might change as cancer evolves. tions in the DDR pathway during tumorigenesis. Despite its potential importance in early cancer development, the DNA Replication Initiation and Its Regulation precise nature of oncogene-induced replicative stress The eukaryotic cell cycle is driven by periodic oscillations in remains poorly understood. Here, we review our current the activity of cyclin-dependent kinase (CDK), due to periodic, understanding of replication initiation and its regulation, antiphase oscillations of cyclin synthesis and the anaphase- describe mechanisms by which activated oncogenes promoting complex/cyclosome (APC/C), which targets might interfere with these processes and discuss how cyclins for degradation. APC/C activity is high and CDK activ- replicative stress might contribute to the genomic insta- ity low from late mitosis through G1 phase. G1 cyclins (cyclin bility seen in cancers. D in metazoans), which accumulate in response to mitogenic signalling, trigger the phosphorylation and inactivation of the Introduction retinoblastoma protein (RB), a cell cycle inhibitor. This re- Genomic instability occurs in almost all human cancers, but leases the transcriptional activator E2F (Figure 1A; reviewed whether it is an active force during cancer evolution or in [15]), which promotes expression of the G1/S and S phase simply a consequence of tumour progression has been a cyclins (cyclins E and A, which bind to CDK2 in G1/S, in meta- subject of debate [1,2]. Evidence now suggests that DNA zoans) along with various other proteins required for S phase damage occurs early in tumorigenesis in a wide array of progression, including the replication factors Cdc6 and Cdt1, cancers [3,4]. This is before signs of telomere attrition or discussed below. High CDK activity drives entry into S phase hypoxia, which have long been known to cause DNA dam- and inhibits APC/C activity. High CDK activity is maintained age [4–8]. Instead it has been proposed that activated onco- through G2 phase and mitosis. Ultimately, mitotic cyclin– genes, selected for their ability to promote proliferation, CDK (cyclin B/Cdk1 in metazoans) promotes its own inactiva- cause replicative stress. This replicative stress, character- tion by reactivating the APC/C, which triggers mitotic exit and ised by increased numbers of stalled and collapsed re- the start of the low CDK period of the next cell cycle. plication forks, accounts for early DNA damage [3,4].In Replication must be strictly coordinated with the cell cycle precancerous lesions, this DNA damage triggers a strong to ensure faithful duplication of the genome. The first step in DNA damage response (DDR), which induces senescence replication initiation is the assembly of prereplicative com- or apoptosis, forming a barrier against tumour progression plexes (pre-RCs) at replication origins, a process known as [9,10]. Only when further genetic or epigenetic changes licensing. During licensing, the core replicative helicase down-regulate the DDR, usually via the tumour suppressor component, the hexameric minichromosome maintenance p53 pathway, is tumorigenesis able to proceed. Patterns of 2-7 (MCM) complex, is loaded around double-stranded DNA damage and oncogenic mutations in mouse cancer DNA as an inactive double hexamer (Figure 1B) [16–18]. models and human tumours support the oncogene-induced Loading MCM requires several other pre-RC factors: the replicative stress model over other models of oncogene- six subunit origin recognition complex (ORC; subunits induced senescence, such as that mediated by the tumour Orc1-6), Cdc6 and Cdt1. At the G1/S transition, two kinases, suppressor ARF (reviewed in [11,12]). Therefore in the early CDK and Dbf4-dependent kinase (DDK), activate the MCM stages of tumorigenesis, replicative stress appears to be helicase, which involves the recruitment of Cdc45 and the responsible for generating genomic instability and a DDR heterotetrameric GINS complex to form the CMG complex to limit cancer development, and this in turn provides the [19–21]. The conversion of pre-RCs into bidirectional repli- selective pressure for acquisition of further mutations that somes requires a host of other factors, including Sld2 promote tumour progression. (RecQL4 in metazoans), Sld3 (Treslin/TICRR in metazoans), Given its apparent key role in the early stages of cancer Sld7 (currently no clear orthologue identified, but MTBP development, surprisingly little is known about the nature may perform a similar role in metazoans [22]), Mcm10 and of oncogene-induced replicative stress. It is possible it is Dpb11 (TopBP1 in metazoans) (Figure 1B).

Cancer Research UK London Research Institute, Clare Hall Preventing Re-Replication Laboratories, South Mimms, Herts, EN6 3LD, UK. To ensure replication happens once and only once during *E-mail: John.Difﬂ[email protected] the cell cycle, the two steps of replication initiation, origin Current Biology Vol 24 No 10 R436

Figure 1. The G1/S transition and replication AB initiation. ORC Cdt1 (A) During G1, mitogenic stimulation triggers Ras-dependent signalling, which activates Mitogenic signalling cyclin D transcription and cyclin D/CDK4 Cdc6 assembly. Cyclin D/CDK4 phosphorylates and inactivates RB, which releases E2F from Ras inhibition. E2F then promotes expression of Origin MCM2-7 various genes required for S phase entry. Cyclin D/CDK4 p16/INK4A licensing Cyclin E is an E2F target, which completes phosphorylation and inactivation of RB, thereby providing positive feedback to drive Rb HPV E7 Myc cells into S phase. The RB–E2F pathway becomes deregulated by various oncogenes (in green) and tumour suppressor genes (in E2F red), to promote S phase entry and cell proliferation. (B) Replication initiation occurs in two temporally separated steps, origin licensing Cdc25A Cyclin E/CDK2 S phase factors Sld2 and origin firing. Origin licensing occurs from Sld3 CDK late mitosis to the end of G1, when CDK Sld7 DDK activity is low. ORC, Cdc6 and Cdt1 cooperate S phase entry Mcm10 to load the MCM2-7 complex onto chromatin Dpb11 Origin Pol as an inactive head-to-head double hexamer firing P around double-stranded DNA. At the G1/S P transition, origin firing begins, driven by the P action of two kinases, CDK and DDK. The con- GINS version of an inactive double hexamer into two P Cdc45 functional replisomes involves several firing P factors, including Sld2, Sld3, Sld7, Mcm10, P Dpb11 and DNA polymerase ε, which together aid the recruitment of Cdc45 and GINS to Current Biology form the CMG complex, which stimulates the helicase activity of the MCM2-7 complex. Ori- gins do not fire synchronously during S phase and the time of origin firing is associated with the time of association of firing factors. licensing and firing, are temporally separated. This separa- to replication-coupled PCNA-mediated ubiquitination and tion is coordinated by changes in CDK activity. High CDK proteolysis by the CRL4Cdt2 pathway [47–49]. Because activity triggers origin firing and marks the start of S phase. APC/C activity and the onset of DNA replication are both CDKs also inhibit assembly of pre-RCs so once an origin regulated by CDK, these two mechanisms are indirectly has fired it cannot be relicensed until the cell has exited the CDK-dependent. subsequent mitosis, ensuring origins fire just once in each cell cycle. CDK phosphorylates and inhibits various pre-RC The Licensing Checkpoint components: in the budding yeast Saccharomyces cerevi- In yeast, if pre-RC assembly is partially inhibited during G1 siae, CDK phosphorylation causes ubiquitin-mediated pro- phase, cells enter S phase with reduced numbers of origins teolysis of Cdc6 by CRL1Cdc4 [23,24], nuclear export of the and this causes genomic instability, as discussed below MCM complex [25,26] and inactivation of Orc2 and Orc6 [50–53]. However, if origin licensing during G1 phase is [27–30]. In mammalian cells, CDK phosphorylation of pre- inhibited in human cells by expression of non-degradable RC components can also play a direct role in blocking geminin [54] or depletion of pre-RC factors [55–58], cells licensing (Figure 2). Phosphorylated Cdt1 and Orc1 are arrest in G1 phase. How pre-RC assembly is monitored by targeted for ubiquitin-mediated degradation by CRL1Skp2 this ‘licensing checkpoint’ and how this signal is transduced [31–33] and phosphorylation and ubiquitylation of Orc1 can to downstream targets is still unclear. However, it is clear cause its nuclear export during S and G2 phase [34]. Orc2 that licensing inhibition results in a reduction in cyclin chromatin binding is inhibited upon CDK phosphorylation E/CDK2 activity and concomitant Rb hypophosphorylation [32,35]. Cdc6 is also phosphorylated by CDK during S phase [56–58], suggesting that the checkpoint works by maintain- but how this inhibits pre-RC assembly is still unclear. Ectopic ing repression of E2F to prevent S phase entry. This has overexpressed Cdc6 is exported from the nucleus during S been linked to an increase in levels of the CDK inhibitors phase [36,37], and consistent with this a pool of endogenous p27 and p21 [57], an independent reduction in cyclin D Cdc6 has been shown to be exported [38]. However, the expression [56] and a loss of CDK2-activating phosphoryla- chromatin-bound fraction of endogenous Cdc6 remains tion and nuclear accumulation [58]. Maintenance of the unaltered throughout S phase [38–40]. licensing checkpoint also requires p53, since p53 depletion There are two further mechanisms to inhibit origin from licensing-deficient cells overcomes G1 arrest and licensing outside of G1 phase in metazoans, and both act allows progression into S phase [58]. on the pre-RC factor Cdt1 (Figure 2; reviewed in [41]). First, geminin, an APC/C target that is present from S through Temporal Program of Replication to M phase, binds Cdt1 and inhibits its chromatin bind- Origins do not all fire simultaneously at the onset of S phase, ing [42–46]. Second, chromatin-bound Cdt1 is subjected but follow a predetermined temporal pattern [59,60].In Special Issue R437

budding yeast, limiting levels of ﬁring factors, including Cdc45, PCNA Sld3, Sld2, Sld7, Dpb11 and Dbf4 [61,62], are recruited prefer- Geminin

Cul4-DDB1

entially to early-firing origins during G1 and only to late-firing Degradation Sequesters origins during S phase [63,64], resulting in temporally ordered Cyclin A/CDK2 origin firing (discussed in [65]). In human cells, at least Cdc45 Orc1 Cdt2 also appears to be limiting for replication [66]. P P Skp2 Replicative Stress Following Inappropriate Origin SCF -mediated degradation Nuclear export P Licensing or Firing Orc2 Skp2 Cdt1 SCF -mediated degradation Together, the regulatory pathways described ensure that the Chromatin binding inhibition Cdc6 P genome is accurately duplicated in each cell cycle. If any of Nuclear export? these pathways are disrupted, origin usage during S phase Current Biology will be altered. In this section we summarise evidence indicating that replicative stress and genome instability can be Figure 2. Prevention of re-replication in metazoans. caused by reduced origin usage, increased origin usage or CDKs inhibit origin licensing by phosphorylating several members of re-use of origins in a single cell cycle. We also discuss the pre-RC. Phosphorylation of Cdc6 may cause its nuclear export; each of these with respect to oncogenesis. phosphorylation of Orc2 inhibits its chromatin binding; phosphorylation of Orc1 and Cdt1 targets them for ubiquitin-mediated degradation Skp2 Origin Under-Usage by CRL1 , and phosphorylation and ubiquitylation of Orc1 also causes nuclear export. Additionally, Cdt1 is degraded by the Reducing the levels of pre-RC factors or increasing the levels CRL4Cdt2 pathway via replication-coupled PCNA-mediated ubiquitina- of licensing inhibitors can result in fewer MCM complexes tion and is sequestered by geminin, preventing its chromatin binding. being loaded onto DNA and fewer origins firing in S phase. In budding yeast, which lacks a licensing checkpoint, increasing G1 CDK activity by overexpressing a G1 cyclin or deleting a CDK inhibitor inhibits the assembly of pre- yeast and human cells. The mechanism by which reduced RCs and causes cells to enter S phase with reduced numbers licensing generates damage is unknown. One possibility is of active origins [52,53]. The licensing checkpoint prevents S that each fork has to cover a greater distance, which might phase entry in normal mammalian cells with reduced origin increase the chance of replication fork stalling. Once a fork licensing. In cancer cells, however, the licensing checkpoint stalls, there will also be fewer dormant origins available for is often compromised, presumably because the p53 and Rb/ rescue, and therefore the stalled fork may persist, increasing E2F pathways that mediate the checkpoint are frequently the likelihood of the fork collapsing and DSBs developing. deregulated, either by mutation or through oncogenic signal- The reduction in origin usage may also increase the prob- ling (reviewed in [15]). Thus, when licensing is inhibited, ability that cells will enter mitosis with incompletely cancer cells enter S phase with reduced numbers of func- replicated DNA. The presence of replication intermediates tional replication origins [54,55,58]. will prevent efficient chromosome segregation and cause In yeast, inhibition of origin licensing causes genomic chromosome breakage during mitosis [79]. Mitotic abnor- instability; entering S phase with too few active origins malities, including lagging chromosomes, anaphase bridges causes double-strand breaks (DSBs), increased recombina- and acentric chromosomes as well as micronuclei have been tion and gross chromosomal rearrangements [50–53]. In hu- shown to develop in response to licensing inhibition, through man cancer cells lacking a functional licensing checkpoint, CDK misregulation or MCM depletion [52,76,80]. licensing inhibition causes DNA damage, an abortive S Mouse models of licensing deficiency have been devel- phase and apoptosis [54,55,58]. oped where the numbers of licensed origins are reduced Despite the fact that MCMs are loaded onto DNA in 3–10- but the animals are still viable. Both the Mcm4 chaos mutant fold excess over those used for normal origin firing [67–69], and the Mcm2IRES-CreERT2 mouse are cancer-prone, devel- reducing licensing still affects genome stability. This is oping a variety of tumours dependent on genetic back- because these excess or ‘dormant’ origins [70] are required ground [77,81,82]. Tumorigenesis is accelerated in a to act as back-ups in times of replication fork stalling and p53-deficient background suggesting that the DDR acts as collapse during S phase, since origins cannot be licensed an anti-cancer barrier in response to replicative stress [77]. at this stage (reviewed in [71,72]). There is evidence that This suggests, at least in mouse models, that licensing inhi- extra origins can be selected for activation in G1 in response bition could be a driving force behind tumorigenesis. to replicative stress in the previous S phase, which may be In most human cancers, the pathways that control CDK2 important in cells experiencing chronic replicative stress activation during G1 become deregulated (Figure 1A; [73]. Nonetheless, it is possible to deplete MCMs to such a reviewed in [83,84]). In addition to driving uncontrolled prolif- level that normal replication is not perturbed, and a licensing eration, this might also limit the low CDK ‘window’ during G1 checkpoint is not activated in normal mammalian cells, but phase, resulting in reduced levels of origin licensing, similar the number of dormant origins is reduced. These cells are to the situation in yeast [52,53]. Cyclin E, a G1/S cyclin and hypersensitive to exogenous forms of replicative stress, positive regulator of CDK2, is frequently deregulated in which increase the frequency of fork stalling, resulting in malignancies, associated with gene amplification or more DNA lesions and checkpoint activation [70,74–76]. They commonly with disruption of the pathways that control its also tend to show higher levels of spontaneous DNA damage periodicity [85,86]. Overexpression of cyclin E in human presumably because there is more fork stalling even in an cells causes genomic instability and specifically inhibits unchallenged S phase [75–78]. MCM chromatin binding during G1, causing a reduction in Thus, entering S phase with a reduced number of potential origin firing during S phase [87,88]. Longer replication origins promotes DNA damage and genome instability in tracks and increased fork stalling are seen upon cyclin E Current Biology Vol 24 No 10 R438

overexpression, both characteristic of licensing inhibition oncogenes also causes a hyperproliferative phenotype by [3,89]. Although these experiments were performed on accelerating the G1/S transition (Figure 1A), suggestive of cancer cells, which presumably lack a functional licensing deregulated CDK activation. In fact, in Xenopus egg extracts, checkpoint, one might predict that overexpression of cyclin simply increasing CDK activity causes late origins to fire E would override the checkpoint and promote S phase entry early [101], so this seems a likely explanation for increased with insufficiently licensed origins even in cells with an other- origin firing in response to oncogenic activity. wise functional licensing checkpoint. Consistent with this, The oncogene Myc may influence origin activation more overexpression of various oncogenes seems to compromise directly, since it binds DNA close to replication origins, the licensing checkpoint; HPV E7 sensitises cells to MCM interacts with members of the pre-RC and colocalises with depletion [56], a KRAS mutant sensitises cells to Cdc6 replication foci in early S phase [99]. Myc is known to antag- depletion [90] and Myc overexpression sensitises cells to onise the CDK inhibitor p27 [102] and therefore has been Orc1 depletion [91]. It will be interesting to investigate suggested to lower the threshold of CDK required locally at whether these and other oncogenes also indirectly inhibit replication origins [93].InXenopus egg extracts, adding licensing, by deregulating CDK activity in order to promote p27 to levels that would normally inhibit replication rescues cell cycle entry. over-initiation following excess Myc addition, suggesting that p27 counteracts the effects of Myc [93]. Origin Over-Usage Alternatively, activation of supernumerary replication Increasing the number of active origins in S phase or disturb- origins by oncogenes may be a secondary consequence of ing the temporal program of origin activation can also pro- some other form of replication stress. As discussed above, mote genomic instability. In budding yeast, increasing dormant origins can be activated by factors which slow or levels of the normally limiting firing factors causes increased stall replication forks, thus reducing inter-origin distance firing of replication origins in early S phase, and this is asso- [74,75]. This might go some way to explaining why cyclin E ciated with DNA damage and reduced viability [61,62,92]. overexpression appears to have dual effects on replication Similarly, increasing the level of Cdc45 in Xenopus causes initiation, both reducing licensing and increasing origin firing, increased early origin firing and a DDR [93]. While transient if these effects occur sequentially. Alternatively these con- overexpression of Cdc45 in human cells causes increased flicting results might be explained by the way in which cyclin origin firing, constitutive overexpression results in reduced E is upregulated in different systems. Cyclin E expression is proliferative capacity, suggesting that increased early origin normally restricted to S phase but if overexpression causes it firing is detrimental for cells [66]. to become deregulated relative to the cell cycle, this would Experiments aimed to directly manipulate the replication limit the low CDK period in G1 and inhibit licensing. In timing program in a variety of organisms suggest that origin contrast, if cyclin E overexpression causes higher levels over-usage can promote genomic instability. Increased specifically in S phase, increased origin activity would ensue. origin activity may exhaust substrates required for replica- Oncogene-induced replicative stress shows many of tion, such as deoxynucleoside triphosphates (dNTPs; the characteristics of stress induced by increased origin discussed in [94]) and RPA [95]. This would presumably firing. For example, replicative stress induced by some result in reduced fork speed and an increased chance of oncogenes can be rescued by addition of exogenous nucle- fork stalling. Asymmetric forks, which are thought to be an osides, suggesting nucleotide levels may have become indicator of fork stalling, are seen when origin activity is depleted, perhaps as a consequence of origin over-usage directly increased in yeast and DNA damage can be rescued [10,89,99,100]. In addition cyclin E overexpression has by increasing dNTP levels in vivo [61]. RPA is not only limiting been shown to specifically increase the extent of replica- for replication but also has a protective role in preventing tion–transcription interference, and cyclin E-induced replica- DNA damage; RPA coats both newly generated single- tive stress can be limited using transcription inhibitors [100]. stranded DNA (ssDNA) at ongoing replication forks and stretches of ssDNA persisting at stalled forks, shielding Origin Re-Usage it from nucleases. Excessive origin firing can deplete RPA If inappropriate re-licensing of replication origins occurs and therefore increase DSB formation at unprotected from S to mitosis, when CDK levels are high, origins may ssDNA [95]. fire more than once within a single cell cycle and this can Increased numbers of replicons may clash with other pro- result in re-replication of DNA. cesses that occur on DNA, such as transcription. Transcrip- In eukaryotes, there are several overlapping mechanisms tion occurs during S phase but, like replication, is temporally acting to prevent re-licensing, as already discussed. The and spatially regulated in such a manner that it is generally redundancy in the system suggests that disrupting one separated from replication territories [96,97]. Even so, there such pathway should not result in re-replication. This is are situations, even in an unperturbed S phase, where repli- certainly true in budding yeast; only when all three CDK cation and transcription machineries encounter one another regulatory pathways are inhibited, by expression of pre-RC and this causes replication forks to stall until the block is mutants refractory to CDK regulation, is significant re-repli- bypassed (reviewed in [98]). An increase in the number of cation observed [27]. Similarly, in mammalian cells, overex- forks due to origin over-usage or disruption of the temporal pression of the CDK targets Cdc6 or ORC does not cause regulation of replication may increase the chance of replica- substantial re-replication [34,36,103,104]. The third target tion–transcription collisions and thus increase the chance of of regulation in metazoans, Cdt1, is more tightly regulated, fork collapse and DNA damage. being inhibited by CDK, geminin and CRL4-dependent Overexpression of several oncogenes, including cyclin E, degradation, suggesting its deregulation might be more HPV E6 and E7, Myc and Ras, reduces inter-origin distance harmful to the cell [41]. Consistent with this, in Xenopus in human cells, indicating that, at least in localised regions, egg extracts, Drosophila and mammalian cells, excess origin firing is increased [10,89,99,100]. Each of these Cdt1 alone can induce re-replication, indicating that the Special Issue R439

Cdc6 and ORC regulatory pathways are not sufficient and, since the Rb/E2F pathway is frequently deregulated to block re-replication when Cdt1 is overexpressed through oncogenic signalling, this might explain why overex- [43,46,104–112]. Interestingly, this response to Cdt1 overexpression of licensing proteins is so common. In fact, Cdc6 pression is seen in cancer cells but not in normal primary or has been shown to be directly upregulated in response to immortalised human cells [103,104,107]. In fact in normal Ras, cyclin E and Mos overexpression [3,10,129]. Moreover, mammalian cells, like yeast, it seems that extensive re-repli- cyclin E and Ras can cause re-replication in human cells cation only occurs when all three inhibitory targets of the pre- when over-expressed [3,10]. RC are overexpressed [113]. The propensity of cancer cells to re-replicate upon deregulation of Cdt1 suggests that Replicative Stress Promotes Genomic Instability some of the normal, redundant regulation of licensing has Several mechanisms have been proposed to explain how been compromised. This may be due, in part, to the fact replicative stress leads to genome instability. Common that many pre-RC factors are already overexpressed in fragile sites (CFSs) are segments of the genome prone to cancer (reviewed in [114] and discussed below). breakage when cells are exposed to replicative stress, and Re-replication can cause cell death and genome insta- are already expressed (i.e. broken) in precancerous lesions bility [28,46,115]. In yeast, Xenopus egg extracts and when the DDR is still active, suggesting DNA damage at human cells, re-replication causes checkpoint activation these sites is one of the earliest steps in tumorigenesis associated with the appearance of ssDNA and DSBs [3,4]. These sites tend to occur in late-replicating, AT-rich [43,46,104,106,107,116–120]. In addition, there is evidence regions and are often associated with large genes. to suggest that normal but not cancer cells possess a sepa- Accordingly, some CFSs are particularly susceptible to repli- rate checkpoint that limits re-replication in its very early cation–transcription interference, since replication and stages [104,107]. This ATR-mediated checkpoint may detect transcription often occur simultaneously on long open ssDNA that accumulates early on during re-replication due to reading frames [130]. Activated oncogenes that increase extensive DNA unwinding by the replicative helicase at the frequency of origin firing or alter the replication timing re-licensed origins, uncoupled from DNA synthesis, perhaps program could thereby increase the chance of collisions due to an imbalance in levels of polymerases and replication and therefore the chance of fork stalling and collapse. origins [107]. In contrast, for many CFSs the instability of the site does Precisely how re-replication causes DNA damage and not correlate with its expression level [131]. Instead, it activates checkpoints is still unclear, but several mecha- appears these sites are intrinsically difficult to replicate. nisms have been proposed. If re-initiation of replication is Many CFSs are prone to breakage under reduced licensing infrequent, the resulting replication forks will be relatively conditions, for example after MCM depletion [132]. This is isolated and therefore will have to travel some distance because CFSs appear to be either origin-deficient [133] or before meeting another fork, which increases the chance of activate all dormant origins under unchallenged conditions fork stalling (discussed in [121]). Since the fork is unlikely due to AT-rich sequences that promote fork stalling [132]. to be rescued by a neighbouring fork, the stalled fork will Recently other genomic regions have been found to be persist, increasing the likelihood of fork collapse and DNA particularly sensitive to replicative stress; these have been breakage. If re-initiation from any origin is a frequent event, termed early-replicating fragile sites (ERFSs) and are located there is the chance that replication forks will end up ‘chasing’ in highly transcribed, repetitive and CpG-rich regions. Here, one another along DNA, potentially causing head-to-tail replication–transcription collisions certainly appear to play a collisions and irreversible fork stalling, as has been seen in role in instability. Similarly to CFSs, these sites have also Xenopus egg extracts supplemented with excess Cdt1 been shown to be sensitive to oncogene activation [134]. [117]. Deregulated origin firing can also rapidly generate As well as preferentially targeting CFSs and ERFSs, all ssDNA gaps due to fork collapse, which act as obstacles types of replicative stress discussed in this review can for re-replicating forks and induce further fork stalling, induce genome-wide fork stalling. This can result in collapse and DSBs [122]. immediate fork collapse and breakage during S phase but Origin re-firing may increase the number of replication many replication intermediates, particularly at CFSs, persist forks during S phase, in a similar manner to origin over- into mitosis [135,136], presumably because they replicate usage. In turn it might also cause replication elongation late and remain shielded by RPA [95]. Frequently the sister factors to become depleted or clashes with transcription. chromatids remain interlinked due to under-replication or Alternatively, origin re-firing and the associated replicative unresolved replication structures [135] and these are stress may inhibit origin firing via activation of DNA damage resolved in mitosis by structure-specific nucleases that checkpoints [72], which might lead to an overall reduction in cleave the DNA to prevent formation of anaphase bridges, origin firing, and the potential problems associated with this generating DSBs [137,138]. In addition, condensation of (see above). chromosomes harbouring unresolved intermediates may It has been proposed that re-replication has some role to also be particularly prone to breakage [136]. play during cancer progression. Licensing factors are The DSBs generated as a result of replicative stress are frequently upregulated in cancer cells [100,103,119,123– precursors for genomic instability; in fission yeast, expres- 125]. This is not simply due to increased cell proliferation sion of an inducible replication fork barrier resulted in fork since it does not correlate with other proliferation markers, stalling and site-specific gross chromosomal rearrange- such as Ki-67, suggesting the increased levels may cause ments [139]. In sporadic cancers, chromosomal instability misregulation of the replication licensing system. The pre- (CIN) is the most common form of instability, characterised RC factors Cdt1 and Cdc6 can both act as oncogenes; by changes in chromosomal structure and number (reviewed transgenic Cdt1 mice and mice injected with Cdt1- or in [12]). These structural changes, including translocations, Cdc6-overexpressing cells develop tumours [119,126,127]. amplifications and deletions, can be explained by error- Many pre-RC factor encoding genes are E2F targets [128] prone repair of DSBs. Non-homologous end joining and Current Biology Vol 24 No 10 R440

Figure 3. Copy number increase of replication AB 35 Breast invasive carcinoma factors in cancer. Serous ovarian cancer Colon and rectum adenocarcinoma (A) Frequency (as percent) of ampliﬁcation 30 Glioblastoma of various genes involved in initiating Lung squamous cell carcinoma Prostate adenocarcinoma DNA replication. Data were taken from the 25 Sarcoma Memorial Sloan-Kettering Cancer Center’s cBioPortal web site (http://www.cbioportal.

20 org/public-portal/), which collates data from original sources [144,151–155]. (B) Diagram of human chromosome 8 showing the posi- 15 tions of MTBP, MYC and RECQL4.

5 compaction in mitosis, they can incur MTBP (8q24.12) 0 multiple DSBs and yet still have the MYC (8q24.21)

Cdt1 Dbf4 potential to be incorporated into Orc1 Orc2 Orc3 Orc4 Orc5 Orc6 MYC Cdc6 Cdc7 Mcm2 Mcm3 Mcm4 Mcm5 Mcm6 Mcm7 MTBP Cdc45 TICRR Mcm10 CCND1 TopBP1 GAPDH RecQL4 RECQL4 (8q24.3) the genome, resulting in clustering of Dbf4B (Drf1) GINS1 (Psf1) GINS2 (Psf2) GINS3 (Psf3) GINS4 (Sld5) Chromosome 8 breakpoints [147]. The structural rearrangements Current Biology caused by replicative stress can go onto cause problems in mitosis, when structurally abnormal chromosomes microhomology-mediated end joining generate structural (usually acentric or dicentric) become misegregated [79].In rearrangements and loss of material, as well as commonly this manner structural chromosomal instability can promote observed microhomology at breakpoints. Replicative repair numerical chromosomal instability. In addition, further pathways, known to occur in yeast, have been proposed to complex chromosomal rearrangements can be generated explain the complex structural rearrangements and genomic by subsequent breakage-fusion-bridge cycles [148]. duplications prevalent in cancer [140,141]. This involves a Although oncogene-induced replicative stress is clearly stalled replication fork continuing replication on a new tem- important for generating genomic instability early on in plate mediated by microhomology. The low processivity of tumorigenesis, other factors such as telomere attrition and the polymerase causes frequent fork stalling and potentially hypoxia are also likely to play significant roles, particularly many rounds of template switching. Recently, break- at later stages. induced replication has been shown to occur in human cells and to be responsible for a significant proportion of genomic Replicative Stress during Cancer Evolution amplifications in cyclin E-overexpressing cell lines [142]. Even single oncogenes can induce replicative stress by Although the choice of error-prone pathways might seem different mechanisms depending on context. For example, surprising, it is possible the homologous recombination cyclin E overexpression has been shown to inhibit licensing, machinery becomes overwhelmed by large numbers of increase origin activation and induce re-replication in DSBs, as is likely the case under conditions of replicative different experiments [3,88,89,100] and Ras promotes both stress (reviewed in [143]). origin over-usage and re-usage [10]. Thus, it is unlikely that Independent of its cause, since all types of replicative a single type of replicative stress defines any cancer; in stress generate stalled forks and DSBs, the types of CIN fact, evidence suggests that the causes of replicative stress generated will be very similar. Origin deficiency is likely to might be quite dynamic during tumorigenesis. increase the occurrence of under-replicated DNA and Different mechanisms of replicative stress may also be therefore may promote genomic loss. Re-replication specif- more or less important at different stages of tumour evolu- ically promotes amplification, because when two forks tion. A vigorous DNA damage response, presumably acti- collapse within a re-replication bubble, the structure gener- vated by replicative stress, can already be seen at one of ated promotes non-allelic homologous recombination the earliest detectable stages of cancer, hyperplasia [3,4]. [121]. It seems that certain regions of the genome show a Levels of pre-RC factors Cdc6 and Cdt1 at this stage, higher frequency of amplification in cancer [144] and there however, are relatively low [119], suggesting that this replica- is evidence that chromatin architecture might affect the like- tive stress is unlikely to be due to re-replication. It may be lihood of re-replication and copy number gain [145]. that, in these earliest stages, replicative stress arises from High throughput sequencing has recently revealed the reduced origin licensing. Indeed, acute expression of cyclin extent of CIN, highlighting extensive intrachromosomal E has been shown to reduce licensing and subsequently rearrangements and small copy number changes restricted reduce DNA synthesis within several hours [88], while ex- to subchromosomal regions, termed chromothripsis [146]. periments which have shown increased origin firing and re- Replicative stress, through its preferential targeting of replication have generally examined cells after several days CFSs, might start to explain this clustering [143]. Break- of cyclin E overexpression [3,80,89,100]. Re-replication, induced replicative repair can also help explain the complex and perhaps increased origin firing, probably arises slightly structural rearrangements that are seen. In addition it is later during tumour development. This hyperactivity of known that micronuclei, initially generated by an aberrant origins requires increased transcription of pre-RC factors mitosis, are particularly prone to replicative stress because through oncogene-induced upregulation of the Rb/E2F they have poor nuclear import of replication and repair fac- pathway and, in human tumour samples, the pre-RC factors tors. When these under-replicated chromosomes undergo Cdc6 and Cdt1 only become upregulated from the stage of Special Issue R441

Figure 4. Oncogene activation causes repli- Oncogene activation cative stress and genomic instability. Oncogene activation can interfere with replication initiation in various ways (orange Increased/deregulated Deregulated p53 and boxes) causing origin over-usage, under- CDK2 activity Rb/E2F pathways usage or re-usage; the circles on DNA repre- sent replication origins with green circles being origins that normally ﬁre during S phase and red circles denoting dormant origins. This Reduced licensing results in replicative stress (red boxes), Increased expression causing fork stalling or collapse which can of licensing factors lead to DNA damage in S phase or when cells Licensing checkpoint enter mitosis with under-replicated DNA. Dashed arrows indicate the ways in which origin re-usage can cause similar effects to origin over-usage if re-ﬁring is occurring frequently. A couple of measures used to Origin over-usage Origin under-usage Origin re-usage restrict replicative stress are also highlighted (green boxes). See text for full details.

Replication factor Replication– Long replication Head-to-tail fork exhaustion transcription collisions tracks collisions dysplasia, well after the DDR is first detected. Moreover, levels of both Cdc6 and Cdt1 increased further on Dormant origin activation Enter mitosis with under- progression from dysplasia to carci- Fork stalling/collapse replicated DNA noma, which did not correlate with a simple increase in proliferation rate [119]. In addition, the transformation capability of Cdc6 and Cdt1 overexpression is greater if non-primary cells DNA DSBs are used, which are more reflective of the dysplastic stage [119]. Genomic instability Although replicative stress is a hall- Current Biology mark of early tumour progression, and this can be mimicked by manipulating levels of replication factors, mutations in replication factors are rare in cancer (Figure 3A). This is provide the substrates for selection of novel mutations not surprising when one considers that replicative stress contributing to rapid evolution and genetic heterogeneity is actually detrimental to the cell, initially inducing apoptosis in tumours. or senescence. Also, although replicative stress specifically drives genomic instability, which is important for tumour Concluding Remarks and Future Perspectives progression, there is evidence to suggest that primary cells Evidence suggests that oncogenes deregulate replication already have high enough mutation rates to account for the initiation and in turn drive genomic instability via various number of mutations required to transform cells [2]. There- mechanisms (Figure 4). Although it now seems likely that fore, it seems unlikely that mutations that drive replicative oncogene-induced replicative stress has a role to play in stress would ever be selected alone. Instead oncogenes, early progression of many tumours, further work is required selected for their ability to promote proliferation, generate to understand exactly which types of replicative stress replicative stress as a by-product, due to upregulation of contribute towards DNA damage seen in precancerous le- the Rb/E2F pathway and increased CDK activity, and this sions. The presence of replicative stress in cancer cells has in turn promotes genomic instability. However, replication interesting implications for cancer therapies. It may explain factors occasionally become amplified, and this is generally why certain tumour types stop responding to traditional as part of an oncogene cluster, for example Cdc6 is genotoxic therapeutics, since replicative stress selects for frequently amplified with ERBB2 [119], and RecQL4 and mutations that overcome the DDR. In addition, it may open MTBP are frequently co-amplified with Myc (Figure 3B). It up new possibilities for therapeutic targets; for example, in is interesting to consider the possibility that amplification advanced tumours where the DDR has been bypassed, the of some limiting replication factors along with known remaining active components of the DDR pathway may aid oncogenes may increase the fitness of these transformed the tumour in coping with continuing replicative stress by cells and thus contribute to oncogenesis. promoting fork stability and repair. Here, targeting other As mutations in p53 and other DDR factors are selected genes that constitute the DDR pathway may actually sup- in response to oncogene-induced replicative stress, exten- press tumorigenesis [149,150]. sive genomic instability develops. The acquisition of further mutations in oncogenes and tumour suppressor genes References may reduce the tumour’s dependency on replicative stress 1. Lengauer, C., Kinzler, K.W., and Vogelstein, B. (1998). Genetic instabilities in human cancers. Nature 396, 643–649. as a driving factor for tumour progression. Even so, repli- 2. Tomlinson, I., and Bodmer, W. (1999). Selection, the mutation rate and cative stress will continue to generate DNA damage and cancer: ensuring that the tail does not wag the dog. Nat. Med. 5, 11–12. Current Biology Vol 24 No 10 R442

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