Download DNA replication-associated lesions: importance in early

1352
Biochemical Society Transactions (2007) Volume 35, part 5
DNA replication-associated lesions: importance
in early tumorigenesis and cancer therapy
E. Petermann* and T. Helleday*†1
*Radiation Oncology and Biology, University of Oxford, Oxford OX3 7LJ, U.K., and †Department of Genetics, Microbiology and Toxicology, Stockholm
University, S-106 91 Stockholm, Sweden
Abstract
DNA lesions resulting from impaired progression of replication forks are implicated in genetic instability and
tumorigenesis. Because the cellular response to these lesions poses an important tumorigenesis barrier,
the responsible signalling and repair pathways are often mutated or inactive in tumours. Here, we discuss
how such deficiencies can in turn be exploited for cancer therapy.
Replication-associated lesions
During every round of DNA replication, moving replication
forks encounter countless obstacles and lesions on the
DNA template, such as DNA-bound proteins, difficult to
replicate secondary structures or unrepaired DNA damage
[1]. This can result in prolonged stalling of the fork and,
for example after collision with unrepaired SSBs (singlestrand breaks), fork collapse and the generation of lethal
DNA DSBs (double-strand breaks) [2]. Such replicationassociated lesions are implicated in genomic instability and
tumorigenesis, but can also be exploited for cancer therapy,
especially because cancer cells are highly proliferating and
often defective in the DNA damage signalling or DNA
repair pathways that deal with these lesions.
Replication-associated lesions in early
tumorigenesis
Recent studies suggest the existence of tumorigenesis
barriers that slow or inhibit the progression of pre-neoplastic
lesions to neoplasia (tumours). One such barrier involves
oncogene-induced DNA replication stress. This replication
stress leads to activation of DNA damage response pathways
involving the ATM (ataxia telangiectasia mutated)–Chk2
(checkpoint kinase 2) and the ATR (ataxia telangiectasia
mutated- and Rad3-related)–Chk1 signalling cascades, arrest
in S- and G2 -phases of the cell cycle and apoptosis. The
activation of the DNA damage response precedes genomic
instability in tumour development [3,4]. It was observed that
overexpression of the oncogene cyclin E, which is involved
in origin licensing, leads to altered replication dynamics,
formation of single-stranded DNA, phosphorylation of
Key words: cancer therapy, DNA replication, homologous recombination, poly(ADP-ribose)
polymerase (PARP), repair, tumorigenesis.
Abbreviations used: ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutatedand Rad3-related; BRCA2, breast-cancer susceptibility gene 2; Chk2, checkpoint kinase 2; DNAPK, DNA-dependent protein kinase; DSB, double-strand break; HR, homologous recombination;
NHEJ, non-homologous end joining; PARP, poly(ADP-ribose) polymerase; RNAi, RNA interference;
SSB, single-strand break.
1
To whom correspondence should be addressed (email [email protected]).
C The
C 2007 Biochemical Society
Authors Journal compilation the checkpoint kinase Chk1 and replication-associated
DSBs [3,5]. Early pre-cancerous lesions were found to
display allelic imbalance at specific chromosomal loci termed
‘common fragile sites’ [4], indicating DNA breakage at these
loci, which is normally observed after partial inhibition of
replication fork progression [6]. These results suggest that,
in pre-cancerous lesions, deregulated origin firing can lead to
impaired replication fork progression, which is in turn sensed
as replication stress. The exact relationship between origin
firing and replication stress requires further investigation.
A second tumorigenesis barrier is mediated by oncogeneinduced senescence [7–9]. Recently, a link between these
two barriers was shown by demonstrating that senescence
induced by overexpression of several oncogenes, including
cyclin E, is dependent on the replication stress-induced
DNA damage response described above [5].
This suggests the existence of selection for cells with
mutated or inactivated DNA damage response pathways
for tumour progression. Accordingly, loss of the checkpoint
factors 53BP1 and Chk2, in addition to mutant p53, was
found in more advanced cancer stages [4]. Loss of checkpoint
factors, again, may also lead to perturbed replication
dynamics and replication-associated DSBs, which might
further increase genomic instability [6]. In addition, cancer
cells are very often defective in DNA repair mechanisms that
deal with replication-associated DNA damage, such as HR
(homologous recombination) repair [10,11]. In the following,
we discuss how defects in DNA repair mechanisms can
be exploited to amplify endogenous replication-associated
lesions for cancer therapy.
Pathways of repairing
replication-associated lesions
A major pathway for the repair and restart of collapsed
replication forks is HR, a complex pathway that repairs
one- or two-ended DSBs, and possibly other damaged fork
structures, by utilizing homologous sequences on the sister
chromatid [2,12–14]. The HR factor BRCA2 (breast-cancer
susceptibility gene 2) has also been proposed to be involved
Cancer
in overcoming replication blocks [15]. HR is regulated
by several signalling pathways. For example, the DNA
damage response kinase Chk1 interacts with Rad51 and
is required for Rad51 foci formation and HR repair of
collapsed replication forks [16]. The ATM checkpoint kinase
has also been implicated in HR repair of DNA breaks that
arise from the collision of replication forks with DNA SSBs
[17].
An alternative pathway for the repair of DSBs is NHEJ
(non-homologous end joining), which directly rejoins
DSB ends by ligation, with little requirement for sequence
homology. Although NHEJ is involved in repairing DSBs
resulting from fork collapse during S-phase, it plays a less
important role than HR [12].
Exploiting replication-associated lesions
for therapy
A new concept for cancer therapy is to amplify endogenous
tumour-specific DNA lesions, to specifically kill tumour
cells. This can be achieved by inhibition of DNA repair.
For example, inhibitors of PARP-1 [poly(ADP-ribose)
polymerase-1] are widely used to decrease the efficiency of
SSB repair. An increased amount of SSBs is likely to lead to
more collapsed replication forks that require recombination
repair to restart. Indeed, HR has a very important role in
PARP-1-defective cells, as inhibition or loss of PARP-1
is associated with a hyper-recombinogenic phenotype as
indicated by a high level of sister chromatid exchanges and
Rad51 foci [18,19]. Equally, cells defective in recombination
may therefore be more sensitive to inhibition of PARP-1.
This idea was put into practice for cells that are
mutated in the breast-cancer susceptibility genes BRCA1 or
BRCA2, which encode proteins involved in HR repair [10].
Heterozygous carriers of a mutation in either gene have a
considerably increased risk of breast or ovarian cancers that
arise from cells that have lost the wild-type copy. The loss
of HR leads to alterations in DSB repair and thus accelerates
genetic instability, which is likely to drive cancer development
[10]. Cell lines homozygous for either the BRCA1 or
BRCA2 mutation are highly sensitive to PARP inhibitors
[20,21]. These cells are 100–1000-fold more sensitive to PARP
inhibitors than the heterozygote or the wild-type cell lines,
and PARP inhibitors can even induce regression of tumours
derived from the homozygous mutated cells. siRNA (small
interfering RNA)-mediated depletion of BRCA2 in MCF7
(wild-type p53) and MDA-MB-231 (mutated p53) breast
cancer cell lines also results in sensitivity to PARP inhibitormediated cytotoxicity [21]. This shows that BRCA2 defective
breast cancers can be specifically targeted using inhibitors of
PARP-1 alone, a treatment that is likely to be highly tumourspecific since only the tumours (which are BRCA2−/− ) in the
BRCA2+/− patients are completely defective in HR repair.
The use of an inhibitor of a DNA repair enzyme alone, in the
absence of an exogenous DNA-damaging agent, to selectively
kill a tumour represents a new concept in cancer treatment.
Clinical trials using PARP inhibitors alone are under way [22].
If tumours are not inherently deficient in HR, downregulation of HR activity in combination with PARP
inhibitors could be a suitable therapeutic strategy to increase
replication-associated lesions. A variety of proteins involved
in HR were recently investigated for their impact on the cytotoxicity of the PARP inhibitors KU0058684 and KU0058948
as proof of principle. RNAi (RNA interference)-mediated
silencing of or deficiency in the HR factors Rad51, Rad54,
Dss1 and RPA1, the DNA damage signalling proteins ATM,
ATR, Chk1, Chk2 and Nbs1 and components of the Fanconi’s
anaemia signalling pathway led to increased sensitivity to
PARP inhibition [23]. For therapy, small molecule inhibitors
are more desirable than RNAi. Rad51 expression can also
be down-regulated by inhibiting c-Abl kinase with imatinib
mesylate (Gleevec), resulting in cellular sensitization at least
to DNA-damaging agents [24]. Inhibitors of Chk1 (e.g.
UCN-01) are widely under investigation for cancer therapy.
Chk1 activity is not only required for HR repair of collapsed
replication forks, but also for maintaining high rates of replication fork progression, via a currently unknown mechanism
that is HR-independent but might involve maintenance of the
replisome [6,25]. Chk1 inhibition leads to rapid accumulation
of replication-associated DNA DSBs [26], which could result
from a combination of accumulating replication-associated
lesions and inability to repair them by HR. These results
highlight the importance of Chk1 in preventing DNA damage
during replication, which makes it highly interesting as a
potential target for cancer therapy. ATM inhibitors presents
another possibility of indirectly inhibiting HR. PARP-1−/−
cells are sensitive to the ATM inhibitor KU55933, and ATMdeficient cells are conversely sensitive to the PARP inhibitor
4-amino-1,8-napthalamide. Inhibition of PARP leads to
ATM activation, and PARP inhibitor-induced HR repair is
abolished in ATM-inhibited cells [17].
NHEJ is also targeted for cancer therapy, by using
inhibitors of DNA-PK (DNA-dependent protein kinase),
a central NHEJ factor. However, it is not clear whether
inhibition of NHEJ, which is less important during S-phase
than HR, presents a possibility to increase the cytotoxicity
of replication-associated lesions, and the combination of
DNA-PK and PARP inhibitors did not reduce survival in
mouse embryonic fibroblasts [27].
In conclusion, DNA replication-associated lesions
are commonly formed during tumorigenesis and can be
efficiently exploited for targeted therapy. A future challenge
will be to gain further insight into the origins and nature of
lesions involved in tumorigenesis and to identify components
of DNA damage signalling or DNA repair pathways
that are suitable for targeting to specifically kill cancer
cells.
We thank the Swedish Cancer Society, the Swedish Children’s
Cancer Foundation, the Swedish Research Council, the Swedish
Pain Relief Foundation, the Medical Research Council and Cancer
Research UK for supporting this work financially.
C The
C 2007 Biochemical Society
Authors Journal compilation 1353
1354
Biochemical Society Transactions (2007) Volume 35, part 5
References
1 Mirkin, E.V. and Mirkin, S.M. (2007) Microbiol. Mol. Biol. Rev. 71, 13–35
2 Helleday, T., Lo, J., van Gent, D.C. and Engelward, B.P. (2007) DNA Repair
6, 923–935
3 Bartkova, J., Horejsi, Z., Koed, K., Kramer, A., Tort, F., Zieger, K., Guldberg,
P., Sehested, M., Nesland, J.M., Lukas, C. et al. (2005) Nature 434,
864–870
4 Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P., Zacharatos, P., Kotsinas, A.,
Liloglou, T., Venere, M., Ditullio, Jr, R.A., Kastrinakis, N.G., Levy, B. et al.
(2005) Nature 434, 907–913
5 Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D., Issaeva,
N., Vassiliou, L.V., Kolettas, E., Niforou, K., Zoumpourlis, V.C. et al. (2006)
Nature 444, 633–637
6 Petermann, E. and Caldecott, K.W. (2006) Cell Cycle 5, 2203–2209
7 Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M.,
Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W. et al. (2005) Nature
436, 725–730
8 Collado, M., Gil, J., Efeyan, A., Guerra, C., Schuhmacher, A.J., Barradas, M.,
Benguria, A., Zaballos, A., Flores, J.M., Barbacid, M. et al. (2005) Nature
436, 642
9 Michaloglou, C., Vredeveld, L.C., Soengas, M.S., Denoyelle, C., Kuilman,
T., van der Horst, C.M., Majoor, D.M., Shay, J.W., Mooi, W.J. and Peeper,
D.S. (2005) Nature 436, 720–724
10 Venkitaraman, A.R. (2002) Cell 108, 171–182
11 Kennedy, R.D. and D’Andrea, A.D. (2006) J. Clin. Oncol. 24,
3799–3808
12 Lundin, C., Erixon, K., Arnaudeau, C., Schultz, N., Jenssen, D., Meuth, M.
and Helleday, T. (2002) Mol. Cell. Biol. 22, 5869–5878
13 Arnaudeau, C., Lundin, C. and Helleday, T. (2001) J. Mol. Biol. 307,
1235–1245
14 Saleh-Gohari, N., Bryant, H.E., Schultz, N., Parker, K.M., Cassel, T.N. and
Helleday, T. (2005) Mol. Cell. Biol. 25, 7158–7169
C The
C 2007 Biochemical Society
Authors Journal compilation 15 Shivji, M.K. and Venkitaraman, A.R. (2004) DNA Repair 3, 835–843
16 Sorensen, C.S., Hansen, L.T., Dziegielewski, J., Syljuasen, R.G.,
Lundin, C., Bartek, J. and Helleday, T. (2005) Nat. Cell Biol. 7,
195–201
17 Bryant, H.E. and Helleday, T. (2006) Nucleic Acids Res. 34,
1685–1691
18 Oikawa, A., Tohda, H., Kanai, M., Miwa, M. and Sugimura, T. (1980)
Biochem. Biophys. Res. Commun. 97, 1311–1316
19 Schultz, N., Lopez, E., Saleh-Gohari, N. and Helleday, T. (2003)
Nucleic Acids Res. 31, 4959–4964
20 Farmer, H., McCabe, N., Lord, C.J., Tutt, A.N., Johnson, D.A., Richardson,
T.B., Santarosa, M., Dillon, K.J., Hickson, I., Knights, C. et al. (2005) Nature
434, 917–921
21 Bryant, H.E., Schultz, N., Thomas, H.D., Parker, K.M., Flower, D., Lopez, E.,
Kyle, S., Meuth, M., Curtin, N.J. and Helleday, T. (2005) Nature 434,
913–917
22 Ratnam, K. and Low, J.A. (2007) Clin. Cancer Res. 13, 1383–1388
23 McCabe, N., Turner, N.C., Lord, C.J., Kluzek, K., Bialkowska, A., Swift, S.,
Giavara, S., O’Connor, M.J., Tutt, A.N., Zdzienicka, M.Z. et al. (2006)
Cancer Res. 66, 8109–8115
24 Bristow, R.G., Ozcelik, H., Jalali, F., Chan, N. and Vesprini, D. (2007)
Radiother. Oncol. 83, 220–230
25 Petermann, E., Maya-Mendoza, A., Zachos, G., Gillespie, D.A., Jackson,
D.A. and Caldecott, K.W. (2006) Mol. Cell. Biol. 26, 3319–3326
26 Syljuasen, R.G., Sorensen, C.S., Hansen, L.T., Fugger, K., Lundin, C.,
Johansson, F., Helleday, T., Sehested, M., Lukas, J. and Bartek, J. (2005)
Mol. Cell. Biol. 25, 3553–3562
27 Veuger, S.J., Curtin, N.J., Richardson, C.J., Smith, G.C. and Durkacz, B.W.
(2003) Cancer Res. 63, 6008–6015
Received 11 June 2007
doi:10.1042/BST0351352