Review
The complex basis underlying common
fragile site instability in cancer
Efrat Ozeri-Galai*, Assaf C. Bester* and Batsheva Kerem
Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University,
Jerusalem 91904, Israel
Common fragile sites (CFSs) were characterized almost
30 years ago as sites undergoing genomic instability in
cancer. Recently, in vitro studies have found that onco-
gene-induced replication stress leads to CFS instability.
In vivo, CFSs were found to be preferentially unstable
during early stages of cancer development and to leave a
unique signature of instability. It is now increasingly
clear that, along the spectrum of replication features
characterizing CFSs, failure of origin activation is a com-
mon feature. This and other features of CFSs, together
with the replication stress characterizing early stages of
cancer development, lead to incomplete replication that
results in genomic instability preferentially at CFSs.
Here, we review the shared and unique characteristics
of CFSs, their underlying causes and their implications,
particularly with respect to the development of cancer.
The involvement of CFSs in cancer
CFSs were first described in 1984 as gaps and constrictions
in metaphase chromosomes of cells grown under mild
replication stress conditions [1]. The expression level of
a CFS is measured by the frequency of the gaps and
constrictions at that site on metaphase chromosomes.
CFSs are present in all individuals and are considered
to be part of the normal chromosomal structure. For some
CFSs in humans, an ortholog has been found in mice [2–5]
and primates [6]. Several studies suggested that CFSs are
conserved even in the yeast Saccharomyces cerevisiae.
These studies found regions in the yeast genome that
are slow replicating [7] or are potential impediments to
replication forks [8,9]. At present, 87 CFSs are listed in the
NCBI gene database (http://www.ncbi.nlm.nih.gov/gene),
but the exact number depends on the inducer, cell type and
method of analysis. One study analyzed 25 000 meta-
phases in lymphocytes and reported 230 CFSs; however,
most of these sites were infrequently expressed [10].
In vitro, most CFSs are induced using low concentrations
of aphidicolin, an inhibitor of polymerase a, d and e [11,12].
Under these conditions, CFSs are hot spots for sister chro-
matid exchange and show high frequency of translocations
and deletions in somatic cell hybrid systems [13]. Thus, DNA
breakage at CFSs leads to the genomic instability of these
sites. In vivo, CFSs correlate with chromosomal breakpoints
in tumors [14,15], and were found to be involved in deletions
Corresponding author: Kerem, B. (batshevak@mail.savion.huji.ac.il)
Keywords: fragile sites; replication; cancer; genomic instability; DNA damage;
dormant origins.
*
These authors contributed equally to this manuscript.
0168-9525/$ – see front matter ß 2012 Published by Elsevier Ltd. doi:10.1016/j.tig.2012.02.006 Trends in Genetics, June 2012, Vol. 28, No. 6
of tumor suppressor genes and genomic amplification of
oncogenes [16–18]. In addition, CFSs are preferential hot-
spots for integrations of viral DNA, which can lead to cancer
development [19–22]. Recently, several studies have dem-
onstrated that oncogene expression leads to replication
stress [23–26]. Indeed, owing to the sensitivity of CFSs to
replication stress, the genomic instability at CFSs precedes
the instability in other genomic regions [26]. In recent years,
studies have revealed a role for checkpoint and DNA dam-
age response (DDR) proteins in the mechanisms controlling
CFS stability (Table 1).
In this review, we focus on recent results that shed new
light on the sequence and replication features of CFSs and
the mechanisms leading to their instability under replica-
tion stress, such as during cancer development. Specifical-
ly, we discuss new findings from studies using the high-
resolution DNA combing approach to analyze the replica-
tion dynamics along CFS regions; we also examine the role
of CFSs in genomic instability in cancer.
Sequence characteristics of CFSs
For many years, no specific DNA sequences characterizing
CFSs were found. However, examination of the DNA se-
quence and structural characteristics showed that they are
enriched in sequences with high DNA helix flexibility in
the twist angle [27,28]. These flexible sequences were
found to comprise interrupted AT-dinucleotide repeats
(AT-rich) with a potential to form stable DNA secondary
structures that might lead to inhibition of DNA replication
[29] (Box 1). Recent studies described below in more detail
demonstrate that these AT-rich flexibility sequences can
lead to replication arrest and DNA breakage.
An in vitro study of the replication pattern along DNA
fragments from the CFS FRA16D showed that the DNA
polymerase is arrested near AT-rich sequences [30]. Inter-
estingly, the addition of the Werner helicase (Box 2), which
is implicated in the resolution of DNA secondary structures,
alleviated the polymerase stalling at these sequences [30].
This result supports the role of secondary structures formed
at AT-rich sequences in DNA replication perturbation. A
second study analyzed the fragility of sequences from
FRA16D in a yeast system. In this study, a YAC-containing
the sequence of the FRA16D region was found to break at
AT-rich sequences, even in the absence of replication stress.
Moreover, using a two-dimensional gel, it was demonstrated
that AT repeats can lead to replication fork arrest [31].
These two studies demonstrate that AT-rich sequences
are difficult to replicate, probably as a result of secondary
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Review Trends in Genetics June 2012, Vol. 28, No. 6

Table 1. Proteins involved in CFS stability under replication stress conditions

Protein General known Suggested function in CFS stability Experimental method Effect on CFS expression Refs
function under replication stress conditions (increase vs decrease)
ATR DNA damage Replication stress response and Downregulation, knockout, Increase [79,80]
transducer maintenance of stalled fork stability kinase-dead ATR, Seckel
syndrome cells
CHK1 DNA damage Replication stress response Downregulation Increase [81]
checkpoint
ATM DNA damage DSB repair Gene mutation Increase only with [82]
transducer ATR downregulation
BRCA1 DNA damage G2–M checkpoint Gene mutation, knockout, Increase [83]
mediator downregulation
FANCD2 DNA repair DNA repair Gene mutation, downregulation Increase [84]
XLF DNA repair Non-homologous end-joining (NHEJ) Gene mutation, downregulation Increase [85]
RAD51 DNA repair Homologous recombination (HR) Downregulation Increase [86]
DNA-PK DNA repair NHEJ Downregulation Increase [86]
Ligase IV DNA repair NHEJ Downregulation Increase [86]
SMC1 Cohesin subunit Postreplicative DSB repair Downregulation Increase [87]
Claspin S-phase checkpoint Maintaining stalled fork stability Downregulation Increase [88]
HUS1 Cell cycle checkpoint Intra S-phase checkpoint and Knockout Increase [89]
DNA repair
WRN Helicase Resolving DNA secondary structures Gene mutation, downregulation Increase [90]
BLM Helicase Resolving DNA secondary structures Gene mutation Increase [91]
Topo I Topoisomerase Regulating the coupling between the Inhibition by betulinic acid Decrease [92]
helicase and polymerase and/or camptothecin
Topo I Topoisomerase Preventing interference between Downregulation Increase [93]
replication and transcription

structure formation that subsequently leads to DNA break-
age. Furthermore, an indication for the role of AT-rich
sequences in DNA instability in vivo was found when
sequences at recurrent cancer breakpoints occurring in
CFSs were shown to harbor DNA flexibility (AT-rich)
sequences [32–34]. In addition, proteins that are important
for the resolution of DNA secondary structures, such as
helicases and topoisomerase I, were shown to modulate
CFS stability (Box 2 and Table 1). Recently, direct evidence
for replication fork arrest at AT-rich sequences was provided
by analyzing the endogenous FRA16C region in human cells
[35], discussed in more detail below.
Box 1. Sequence characteristics of CFSs
CFSs are mapped based on their appearance on metaphase
chromosomes. Most CFSs have only been mapped cytogenetically,
and only 35 of them were mapped more accurately by molecular
cloning using fluorescence in situ hybridization (FISH) analysis [71].
A large number of cloned CFSs map to large genomic regions of
several hundred kb. Several studies have suggested that, although
CFSs map to large regions, there are ‘hot spots’ of instability that
can be defined as the core of the fragile region [32,57,67,94]. For
most CFSs, the core of fragility has not yet been identified, which
makes the determination of DNA sequences specific to CFSs a
difficult task. Although CFSs do not share specific sequences,
analysis of the flexibility of the twist angle between each two base
pairs along the DNA sequence revealed high flexibility sequences in
the CFSs FRA3B, FRA7H and FRA7G [28]. The high flexibility
sequences that were analyzed for sequence composition are rich
with interrupted runs of AT-dinucleotide repeats [29]. In subsequent
years, more cloned CFSs were analyzed and also found to be
enriched for AT-rich flexible sequences [27]. In silico and in vitro
analyses revealed that AT-rich sequences longer than 200 bp have
the potential to form stable secondary structures [29,95]. The
structural characteristics of AT-rich flexibility sequences can lead
to replication perturbation and thus CFS instability.
296
Replication features of CFSs
Replication timing studies
Numerous mechanisms underlying CFS sensitivity to rep-
lication stress have been proposed years ago, including late
timing of replication, widely separated origins and DNA
sequences that are unusually difficult to replicate [36].
Analyzing the unique replication pattern of CFSs is im-
portant for understanding the replication features leading
to their sensitivity to replication stress, which results in
their instability. The replication pattern of several CFSs
was initially analyzed using replication timing methods
and showed a variety of replication patterns for different
CFSs. FRA3B [36,37] and FRA16D [38] start replication
very late during S-phase in unperturbed cells. A mild
replication stress generated by aphidicolin treatment
leads, in some cases, to incomplete replication even in
G2 cells [36,38,39]. Exploring the replication time on
metaphase chromosomes showed that the replication of
FRA3B is asynchronous, and that only the late-replicating
allele showed gaps and constrictions [37].
In other fragile sites (FRA7H [40], FRA7G [16], FRA1H
and FRA2G [41]), replication starts during early to mid
S-phase. In these sites, a significant delay in replication
timing between adjacent regions was found. These results
indicate that there is an intrinsic delay in the replication
progression in these fragile sites. For FRA1H and FRA2G,
it was found that by late S-phase, approximately half of the
fragile site regions were still unreplicated [41]. The repli-
cation delay at FRA7H and FRA7G was shown to further
increase under replication stress by the addition of aphi-
dicolin treatment [16,40]. The delay in replication at these
fragile sites could result from slow progression of the
replication fork throughout the region or from stalling of
the replication fork at specific sites.
Review
Box 2. Proteins involved in resolution of DNA secondary
structures required for CFS stability
Several proteins that are important for the resolution of DNA
secondary structures or for the stability of stalled forks have been
shown to play a role in CFS stability. Downregulation of the Werner
helicase, which is implicated in the resolution of secondary
structures leading to replication fork arrest, leads to gaps and
constrictions on metaphase chromosomes even in unstressed cells,
and it is important to maintain fragile site stability under normal
conditions [90]. Another helicase implicated in CFS expression is
BLM, the helicase deficient in Bloom’s syndrome. Spontaneously
occurring chromosome aberrations in Bloom’s syndrome cells were
found to correlate with bands harboring fragile sites, oncogenes and
breakpoints involved in cancer rearrangements [91]. Another key
enzyme that is important for the integrity of DNA replication is
topoisomerase I (Topo I). In vitro studies showed that aphidicolin
treatment leads to uncoupling between the polymerase and the
helicase, resulting in long stretches of single-stranded DNA (ssDNA)
that can form secondary structures, such as hairpins or cruciforms,
at AT-rich sequences. Partial inhibition of Topo I can alleviate the
polymerase–helicase uncoupling and so reduce the probability of
the formation of secondary structures. Indeed, inhibition of Topo I
results in a reduction of gaps and breaks at CFSs induced by
aphidicolin [92]. Another study investigated the function of Topo I in
preventing the formation of RNA–DNA hybrids during transcription,
which probably prevents the interference between replication and
transcription [93]. Interestingly, the results show that Topo I
depletion by short hairpin RNA induced an increase in the frequency
of the CFS expression level. The different effects of the various
functions of Topo I on CFS expression still need to be investigated.
Downregulation of claspin, a mediator protein involved in Chk1
activation, leads to CFS instability, even under normal growth
conditions [88]. The yeast homolog of claspin, Mrc1, localizes to
replication forks during normal S-phase [96]. Mrc1 may form a
pausing complex with Tof1 that binds to stalled forks and maintains
their stability by preventing progression of the replication fork
complex upon arrest of DNA synthesis [96]. In addition, it was
demonstrated that, in an Mrc1 mutant, repeat sequences that form
DNA secondary structures, show higher levels of instability [97].
These results suggest that the role of claspin in maintaining the
stability of forks stalled at DNA secondary structures is important for
CFS stability. Other proteins involved in CFS stability are listed in
Table 1 (main text).
These replication timing analyses suggest that late or
delayed replication of the fragile region is part of the
mechanism leading to the gaps and constrictions at meta-
phase chromosomes of cells grown under replication stress
conditions. Because the cell cycle checkpoints are per-
turbed in many cancers, the unreplicated regions within
CFSs cannot lead to cell cycle arrest; thus, the cell proceeds
to mitosis with incomplete DNA replication at CFSs,
resulting in chromosomal instability. However, many
regions in the genome replicate very late during S-phase,
and replication of at least 1% of the genome extends into G2
[42], indicating that late replication itself is insufficient to
generate fragility.
DNA combing and nascent strand studies
Only in recent years, with the development of powerful
techniques for the analysis of DNA replication, has a more
detailed description of CFS replication features emerged.
To date, the replication dynamics along three CFSs,
FRA3B, FRA16C and FRA6E, have been studied using
two of these novel methods: DNA combing [43] and nascent
strand DNA analysis [44].
Trends in Genetics June 2012, Vol. 28, No. 6
FRA3B
FRA3B is mapped over a large genomic region exceeding 3
Mb. Within this region lies the large tumor suppressor
gene, fragile histidine triad (FHIT), extending over 1.5 Mb.
DNA combing analysis of 1.6 Mb overlapping FHIT dem-
onstrated that the replication rate and fork stalling fre-
quency along this site are similar to that along the whole
genome [45]. A correlation between the origin pattern and
the expression level of FRA3B was found. Analysis in
lymphocyte cells, where FRA3B is highly expressed,
showed that a large region (700 kb) within the fragile site
had no origins of replication, even following replication
stress. By contrast, in fibroblasts, low FRA3B expression
levels correlate with multiple initiation and termination
events that are evenly distributed all along the fragile
region. Thus, the paucity in origins along FRA3B plays
a role in its instability.
These results are also consistent with findings from a
genome-wide analysis of replication timing, in both lym-
phocytes and fibroblasts, using the Repli-Seq technique
[46]. The Repli-seq method does not allow mapping of exact
origin locations, but it can distinguish between origin-poor
regions and regions with evenly distributed active origins.
Analysis of CFS expression showed that the repertoire of
CFSs expressed in fibroblasts is different from that of
lymphocytes [47]. Although most of the highly expressed
CFSs in fibroblasts were previously described in lympho-
cytes, their expression levels were much lower [10,47]. The
initiation profiles from the Repli-seq analysis for another
major fragile site in lymphocytes, FRA16D, and the two
strongest CFSs in fibroblasts showed patterns similar to
FRA3B. The expression level of FRA16D was also lower in
fibroblasts compared with lymphocytes [45]. Nevertheless,
FRA3B and FRA16D were still among the 12 most
expressed CFSs in fibroblasts, representing 2.4% and
5.5% of the breaks, respectively [47]. The expression of
FRA3B and FRA16D in cells in which the origin density in
the fragile region is similar to the whole genome indicates
that features of these fragile sites other than paucity of
origins must contribute to their instability.
The origins of replication at FRA3B were also assessed
using nascent strand analysis followed by microarray
quantification [48]. This study found four inefficient origins
in a region of 50 kb within the 700 kb origin-poor region in
lymphocytes. Thus, both studies suggest that, in lympho-
cytes, there is a large genomic region within FRA3B that
has very low efficiency of origin activation. The combina-
tion of late replication with low efficiency of origins could
explain the incomplete replication of the FRA3B region in
G2–M, leading to its instability.
FRA16C
A recent DNA combing study analyzing the replication
pattern along 600 kb within the CFS FRA16C identified
four AT-rich flexibility sequences over a length of 400 bp
[35]. The analysis revealed a slower replication rate and
shorter origin distance at the fragile site region compared
with the whole genome under normal growth conditions,
indicating that additional origins are activated. Because
dormant origins are normally activated following replica-
tion stress, the replication pattern of FRA16C indicates
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that replication of this region is inherently perturbed.
However, following aphidicolin treatment, no additional
origin activation was detected at the FRA16C region. In
fact, the interorigin distance in FRA16C under normal
growth conditions was shorter than the interorigin dis-
tance in the whole genome under replication stress. These
results imply that the FRA16C region activates all avail-
able dormant origins under normal growth conditions.
Under normal growth conditions as well as under repli-
cation stress, the FRA16C region shows higher levels of
replication fork arrest compared with the whole genome.
These arrest sites were not randomly distributed but were
preferentially close to the AT-rich sequences. These results
support a model in which the replication at FRA16C is
intrinsically perturbed, even under normal growth condi-
tions, owing to AT-rich sequences. The stability of the
fragile site under normal conditions is maintained by
dormant origin activation, which allows the completion
of DNA replication. Under replication stress conditions,
the replication is further perturbed and more replication
forks arrest at AT-rich sequences. However, no additional
origins are available, leading to replication delay and
fragile site instability. The replication dynamics in this
CFS demonstrate the role of the AT-rich sequences in the
replication perturbation along CFSs. This replication per-
turbation, together with the inability to activate dormant
origins under replication stress, probably leads to incom-
plete replication, destabilizing the CFS.
FRA6E
FRA6E harbors the large parkinson protein 2 (PARK2)
gene, which extends over 1.3 Mb. The replication pattern of
approximately 1 Mb of FRA6E containing part of PARK2
was analyzed using DNA combing [49]. The analyzed
region of this fragile site contains relatively long AT-rich
sequences (400–1383 bp), along which the replication rate
of the fragile site was slower, the origin distance was
shorter and a higher frequency of fork arrests was found
compared with the whole genome. A comparison between
the replication dynamics along FRA6E and a specific
early-replicating non-fragile region showed no significant
difference in these parameters. These results suggest that
features other than replication dynamics, such as late
replication, are important components of CFS identity.
In addition, an approximately 500-kb region of FRA6E
contained few activated origins compared with the flank-
ing regions. These results suggest that both replication
arrests and paucity of origin activation together lead to the
replication stress sensitivity of FRA6E. Therefore, FRA6E
represents a combination of the replication patterns of the
two CFSs described above. Further high-resolution analy-
sis of FRA6E as well as other CFSs is required to evaluate
the entire spectrum of replication patterns leading to the
replication stress sensitivity of CFSs.
Mechanisms leading to CFS instability
Several lines of evidence suggest that no single mechanism
can account for the instability of all CFSs. Different poten-
tial characteristics of CFSs have been proposed over the
years, but none are common to all of the cloned CFSs
(Figure 1).
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Transcripti R
TRENDS in Genetics
Figure 1. The molecular characteristics of common fragile sites (CFSs) and the
interactions among them. The main characteristics of CFSs are presented around
the circle. Lines in the interior of the circle connect these different characteristics.
The color code indicates characteristics that have been shown to contribute
together to the expression of CFSs. Abbreviations: DDR, DNA damage response;
FADO, failure in activation of dormant origins.
One characteristic of CFSs contributing to instability is
colocalization with very large genes. Some of the large CFS
genes [such as FHIT and WW domain containing oxidore-
ductase (WWOX)] play important roles in cancer develop-
ment. It has been proposed that interference between the
replication and transcription processes of these large genes
could explain CFS instability. Evidence supporting this
hypothesis was found in a recent study that showed a
correlation between the instability of CFSs and the expres-
sion level of the underlying large genes. Moreover, the
levels of RNA:DNA hybrids formed at CFSs harboring
large genes were shown to modulate CFS instability
[50]. However, given that only approximately half of the
CFS regions are associated with very large genes, this
mechanism cannot explain the fragility of all CFSs [51].
Several recent studies focused on the chromatin struc-
ture of CFSs indicated that they are characterized by a
specific nucleosome binding pattern and a compact chro-
matin structure, both of which can influence the replication
pattern of CFSs (Box 3). However, these are not common
features of all CFSs. Thus, condensed chromatin may
explain the instability of a subtype of CFSs or contribute
to CFS instability together with an additional feature, such
as fork stalling or origin paucity.
The DNA replication studies described above demon-
strate that, in all studied CFSs, the replication pattern
differs from that of the whole genome. However, there are
clearly distinct replication features for different fragile
sites, all resulting in high sensitivity to replication stress.
We propose that the key characteristics of the CFS region
underlying their replication traits can best be viewed as a
range. At one end are fragile sites harboring a large region
with inefficient origins. At the other end are fragile sites
containing sequences that form secondary structures im-
peding the replication fork progression. These sequences
lead to intrinsic replication perturbation and dormant
origin activation, even under normal growth conditions.
It is possible that there are also some CFSs between these
Review
Box 3. Chromatin organization at CFSs
Chromatin structure plays a role in replication, transcription and
repair processes [98], and so may impact CFS stability. The
chromatin structure is influenced by nucleosome position, histones
and other DNA-binding proteins as well as by histone modifications,
such as specific methylations and acetylations.
In humans, most nucleosomes are not found at fixed sites but are
randomly distributed. However, analysis of the nucleosome posi-
tions at the breakpoint regions of FRA3B showed that nucleosomes
at these sites are located in fixed positions (termed ‘phased’
nucleosomes) [99]. DNA sequences associated in vivo with the
phased nucleosomes do not lead to phased nucleosomes in in vitro
reconstitution experiments. These results suggest that other factors
contribute to the arrangement of nucleosomes at FRA3B [99]. This
pattern of nucleosomes dictates which sequences will always be
either wrapped around the histones or in the linker regions. For
example, if the origins of replication are always wrapped around the
histones, this could interfere with the binding of origin complexes
and delay replication initiation [98].
In another study, CFSs were found to be characterized by
hypoacetylation of histones. Analysis of six of the molecularly cloned
CFSs showed that five had lower levels of acetylated histones
compared with the average in the genome implying that the
chromatin structure was relatively condensed. Moreover, relaxing
the chromatin by treatment with the histone deacetylase (HDAC)
inhibitor, TSA, or with 5-Aza, an inhibitor of DNA methylation, led to a
decrease in CFS expression [100]. This result supports the role of
condensed chromatin structure in CFS instability. High levels of
acetylation have been associated with access to factors needed to
promote the binding of pre-replication complexes to replication
origins [101]. In addition, relaxed chromatin is important for enabling
efficient repair of DNA damage [102]. Thus, the compact nature of the
chromatin at CFSs might impair origin assembly and/or DNA damage
repair, leading to CFS expression. The results of these two studies can
help define the characteristics leading to the unique replication
pattern of CFSs and their sensitivity to replication stress.
two extremes. These fragile sites might contain both diffi-
cult to replicate sequences and inefficient origins. Howev-
er, in all cases, the CFS region fails to activate additional
origins under replication stress conditions, leading to rep-
lication delay and, in some cases, incomplete replication in
G2 cells. From the CFSs analyzed so far, it seems that,
despite the different replication features, late and/or
delayed replication and limited origin activation are
shared by all CFSs and result in their genomic instability.
Genome instability of CFSs during cancer development
Molecular mapping of fragile sites reveals that genomic
instability in CFSs alters the expression of important
cancer genes in different cancer types. FRA3B, the most
studied CFS, maps to intron 5 of the tumor suppressor gene
FHIT [52]. This CFS is altered in a variety of human
cancers, including lung, breast, head and neck, stomach
and pancreatic carcinomas [53]. Interestingly, integrations
of human papilloma virus (HPV) in cervical cancer cells
were also found in FRA3B [54]. FRA16D also harbors the
very large tumor-suppressor gene, WWOX [32,55]. Dele-
tions leading to loss of heterozygosity (LOH) in FRA16D
were found in different cancer types [56]. Deletions and
LOH in other CFSs are also associated with cancer, includ-
ing FRA6E [57] and FRA9E [58]. A more recent analysis of
LOH in 20 patients with premalignant Barrett’s esophagus
revealed copy number losses associated with many CFSs
(FRA3B, FRA9A/9C, FRA4D, FRA5E, FRA1K, FRAXC,
FRA16D and FRA12B) [59].
Trends in Genetics June 2012, Vol. 28, No. 6
CFSs are involved in amplifications of genomic regions
along the chromosome, leading to multiple gene copies and,
hence, elevated expression of those genes [60]. The ampli-
fication of MET, which involves breakpoints in FRA7G, as
well as oncogene amplifications in FRA11F, FRA2H,
FRA2S, FRA2G, FRA2C and FRA7I [61–64], were shown
to be involved in cancer development. CFSs are also in-
volved in the formation of translocations. One example
is the translocation between 3p14.2 and 8q24.1,
t(3;8)(pl4.2;q24.13), identified in a family with high inci-
dence of clear renal cell carcinoma (RCC) [65]. The trans-
location breakpoint on chromosome 3 is located in the
tumor-suppressor FHIT, close to the core of FRA3B
[66,67]. Recently, the breakpoints leading to a RET–PTC
translocation, which is frequently found in papillary thy-
roid carcinoma, were located in the CFSs FRA10C and
FRA10G [68]. Furthermore, treatment of a thyroid epithe-
lial cell line with a CFS inducer leads to spontaneous RET–
PTC rearrangements in vitro [68]. Altogether, a large
number of studies, mainly analyzing chromosomal insta-
bility in tumor cells, indicate that CFSs are unstable
during cancer development. However, until recently it
remained unclear whether genomic instability of CFSs
in cancer results simply from their inherent instability
or if it is a result of positive selection driven by altered
cancer genes mapped within CFSs, leading to their over-
representation.
One way to answer this question is to look at analyses of
oncogene amplification in a variety of cancers. The break-
age–fusion–bridge (BFB) model predicts that amplification
is achieved by cycles of chromosomal breaks and fusions,
leading to amplification of genomic regions close to the
chromosomal breakpoints [60]. According to this model,
double-strand breaks (DSBs) could occur anywhere be-
tween the centromere and the amplified region. In vitro
studies showed that the chromosomal breakpoints, located
at the boundaries of BFB amplifications, are set by breaks
within CFSs [69]. Evidence for a role of CFSs in the BFB
mechanism leading to oncogene amplification in vivo was
found in a cell line derived from human gastric carcinoma
[70]. Analysis of the breakpoints that led to MET amplifi-
cation revealed a ‘ladder-like’ structure and an inverted
repeat organization of the amplicon, with FRA7G at its
boundaries, as predicted by the BFB model [16]. MET
amplification induced by FRA7G instability was later
found in primary esophageal adenocarcinoma tumor cells
[18]. Similarly, additional fragile sites (FRA11F, FRA2H,
FRA2S, FRA2C and FRA7I) are involved in BFB-type
oncogene amplifications in different cancer types [61–64].
The finding of fragile sites at the boundary of the
amplicons shows that the DSBs involved in these BFB
amplifications occur preferentially in the fragile regions.
Furthermore, these results indicate that oncogene ampli-
fications are indeed the outcome of an intrinsic instability
conferred by the fragile site sequences, because, in the case
of intrachromosomal amplification of large genomic
regions, the breaks are distant from the targeted genes
and so are not affected by selection [16].
Additional corroboration for the supposition that the
unstable nature of CFSs drives chromosomal instability in
cancer emerged recently from an elegant study [71] in
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which a large-scale analysis of 746 cancer cell lines
revealed an instability signature in CFSs that differed
significantly from the signature normally found in cancer
genes [71]. The deletions in recessive cancer genes were
enriched with homozygous deletions that confer selective
growth advantage, whereas CFS deletions were character-
ized by small hemizygous deletions, indicating an in-
creased local rate of DNA breakage. Importantly, the
hemizygous signature of the fragile sites was also found
in fragile sites harboring tumor suppressor genes, such as
FRA3B and FRA16D, that did not result in homozygous
inactivation of the underlying genes. However, by chance,
some regions were converted to homozygous deletions by a
small or large deletion on the other parental chromosome
[71]. This study suggests that the instability in fragile sites
is the result of their intrinsic features regardless of their
gene content.
Another type of cancer-related instability involves the
integration of foreign DNA, especially viral DNA, into the
genome. Such integrations can act as a causative agent or a
cofactor in tumorigenicity. HPV, the most important can-
cer-related virus, is integrated in the genome of most
cervical cancer cells [72]. The first reported HPV integra-
tion into a CFS was in FRA3B, near the RCC translocation
breakpoint [54]. Further analyses revealed preferential
HPV integrations into CFSs throughout the genome in
primary cervical tumors [19–22]. Because HPV integrates
passively into the genome and is not directed by viral
genes, it was important to determine the molecular basis
for the preferential integration of HPV into CFSs. Recent-
ly, the expression of the HPV-16 E6 and E7 oncogenes was
found to confer replication stress leading to DNA DSBs and
genomic instability preferentially at CFSs [23]. This sug-
gests that, following HPV infection, the host cells suffer
from replication stress, leading preferentially to DSBs in
CFSs. The integration of the viral DNA is a result of
incorrect ligation of the two double-stranded ends of the
broken host chromosome and the viral DNA.
Another type of viral integration that increases the risk
of cancer development is the integration of retroviral vec-
tors used in gene therapy trials [73]. A large-scale analysis
of a retroviral vector based on the murine leukemia virus
revealed preferential integrations of the retroviral DNA
into CFSs [74], indicating perturbed replication following
retroviral infection. Altogether, findings in recent years
indicate that CFSs are preferentially unstable during early
stages of cancer development. Because instability in CFSs
alters the expression of important cancer genes, it is
important to understand the molecular mechanisms lead-
ing to instability of CFSs during cancer development.
CFSs are preferentially unstable during early stages of
cancer development
To shed light on the events leading to genomic instability in
cancer, several studies focused on the early stages of cancer
development. Large-scale LOH and comparative genome
hybridization analyses during early stages of different
cancers revealed that the instability in CFSs precedes
the instability in other genomic regions [25,59]. Similar
results were obtained in genome-wide analyses in precan-
cerous experimental models [75]. These findings suggest a
300
Trends in Genetics June 2012, Vol. 28, No. 6
new model in which replication stress is an important
mechanism leading to genomic instability during early
stages of cancer development. This proposes new targets
for cancer therapy based on modulation of the replication
stress response [76].
For many years, the mechanism leading to the instabil-
ity of CFSs in cancer remained largely unknown. However,
recent work has shown that activation of oncogenes leads
to perturbed DNA replication, resulting in hyper-replica-
tion, slow replication fork progression and activation of
dormant origins of replication [23,24,77,78]. This onco-
gene-induced replication stress might explain the prefer-
ential genomic instability in CFSs. Importantly, oncogene-
induced replication stress was found to result from insuffi-
cient nucleotide synthesis required to support normal DNA
replication, which is consistent with in vitro DNA breaks in
CFSs induced by perturbing the nucleotide balance and
biosynthesis through application of hydroxyurea or BrdU,
as well as through folate deprivation [23]. Understanding
the molecular basis characterizing the different fragile
sites will shed light on chromosomal instability in cancer.
Concluding remarks
Recent advances in technologies enabling whole-genome
instability and DNA replication analyses have led to a
breakthrough in understanding the mechanisms leading
to the instability of CFSs during the early stages of cancer
development. These analyses revealed that replication
stress that occurs during cancer development leads to
preferential instability along the replication-sensitive
CFS regions. Instability in CFSs in cancer may lead to
deletions, translocations, amplifications and integrations
of viral DNA. Furthermore, it is now increasingly clear that
CFSs are a collection of heterogeneous sites along the
genome that share features leading to their sensitivity
to replication stress. However, various mechanisms might
underlie the basis of this sensitivity in different subgroups
of CFSs (Figure 1). It will be important to investigate the
interplay between the different mechanisms and their
synergistic effects on CFS stability in vitro and in vivo.
Further studies are needed to unfold the complex basis of
CFS stability and its relevance to cancer development and
other human diseases.
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