114CANCER CELL : FEBRUARY 2005
nesis (Lozano and Zambetti, 2005).
These data are offset by the knowledge
that p21 mutations in human cancers are
rare. Besides the possibility of redundant
function of other p53 targets, another
explanation for the contradictory data is
that decreases in p21 stability through
deletion or mutation of WISp39 are more
common than p21 alterations in tumor
development. The regulation of Wisp39
as a function of the cell cycle is also likely.
Counterintuitive to the role of p21 in
cell cycle arrest, p21 has also been
implicated as a positive regulator of cell
survival. For example, loss of p21 sensi-
tizes cells to undergo uncoordinated
DNA replication and death induced by
anticancer drugs (Waldman et al., 1996).
Because these cells tolerate expression
of p21 in the first place, the p21 in these
cells may not be functional in its cell
cycle-inhibiting capacity.The phosphory-
lation status of p21 differs in different cell
lines and may regulate these activities
(Li et al., 2002). Nonetheless, since p21
is overexpressed in some advanced
human tumors, downregulation of p21
protein may facilitate more efficient
chemotherapy (Seoane et al., 2002).The
newfound role of Hsp90 and WISp39 in
the stabilization of p21 certainly adds
more validity to the belief that targeting
Hsp90 could be used to treat cancer,
since inhibition of Hsp90 may reduce
p21 levels.WISp39 confers specificity on
the ability of Hsp90 to promote stability
of p21, so we could imagine that an
agent that specifically blocks WISp39
interaction with Hsp90 will serve only to
target p21 degradation without affecting
other Hsp90 targets. This scenario may
be useful in some treatment schemes
where tumor cells are dependent on p21
Many questions remain to be
addressed. How does this complex pro-
tect p21 from degradation? How does
the trimeric complex allow p21 interac-
tion with the cyclin-cdk machinery? Does
it affect p21 phosphorylation and interac-
tions with other proteins? This study rais-
es a number of intriguing questions that
will need to be addressed in the future.
Geng Liu and
The University of Texas M.D. Anderson
Cancer Center, Department of
Molecular Genetics, Section of Cancer
Genetics, 1515 Holcombe Boulevard,
Houston, Texas 77030
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P R E V I E W S
Pioneering and painstaking work over
the last 15 years has established that
mutations in up to 11 different genes
could lead to FA (Joenje et al., 1997;
Strathdee et al., 1992). The identity of
nine of these genes is now known, and
recent studies have confirmed the genet-
ic view that they all code for components
of a single tumor suppressor pathway.
Although the primary sequences of the
FA proteins reveal very little about how
they function, we know that most of them
(FANCA, B, C, E, F, G, and L) interact to
form a nuclear complex (FA nuclear com-
plex) (reviewed in Joenje and Patel,
2001; D’Andrea and Grompe, 2003).
However, not until the publication of a
landmark paper in 2001 did we develop a
comprehensive outline of the FA path-
way (Garcia-Higuera et al., 2001). This
seminal study showed that the FA
nuclear complex is essential for the acti-
vation of a newly identified key FA pro-
tein, FANCD2.The activation step
resulted in the conjugation of one ubiqui-
tin polypeptide to a single specific lysine
residue (K561) on the FANCD2 protein.
The consequences of this modification
are to direct FANCD2 to DNA replication
or damage-induced nuclear foci.It is very
likely that at these sites, FANCD2 directs
DNA repair.Despite the evident progress
in the FA field over the last few years,
there are still many questions that need
to be resolved if we wish to gain a com-
plete molecular understanding of the FA
pathway. We will need to know more
about the precise DNA repair activity in
which the FA proteins participate.We will
need to understand the functional rele-
vance of the interactions between the
“core” FA pathway and other tumor sup-
“Dub”bing a tumor suppressor pathway
The autosomal recessive disease Fanconi anemia (FA) causes bone marrow failure and a hugely increased propensity to
develop cancer. Cells from FA patients are prone to chromosome breakage, indicating that FA gene products are required
to ensure genomic integrity. Most of the identified FA proteins are components of a nuclear complex whose principal func-
tion is to activate FANCD2 by monoubiquitination. Monoubiquitinated FANCD2 accumulates at sites of genome damage,
where it probably functions to facilitate DNA repair. A recent paper in Molecular Cell (Nijman et al., 2005) reports the identi-
fication of an enzyme that is responsible for regulating the FA pathway by deactivating FANCD2.
CANCER CELL : FEBRUARY 2005115
pressor gene products such as BRCA1,
BRCA2 (readers should be aware that
biallelic germline mutations in BRCA2 do
lead to a phenotype that is akin to FA;
Howlett et al., 2002), ATM, NBS1, and
the Bloom’s syndrome helicase. Finally,
we will need to establish how the FA
pathway is regulated in cells, what is the
precise mechanism for switching it on,
and how is it switched off.It is this impor-
tant latter question that is addressed in a
paper published in a recent issue of
Molecular Cell (Nijman et al., 2005).
A collaborative study between
Bernard’s and D’Andrea’s
describes a novel approach to identify a
modulator of the FA pathway. As men-
tioned above, the activation of FANCD2
by monoubiquitination is a key step;
loss of this modification disables the
whole pathway. Like phosphorylation,
monoubiqutination is a dynamic and
reversible process whereby an enzyme
cascade conjugates ubiquitin to a target
protein, and a family of enzymes, the
deubiquitinating enzymes, or DUBs for
short, are potent at removing this modi-
fication. A gene family-specific siRNA
library was used to screen for siRNA
that results in an accumulation of acti-
vated FANCD2. These screens lead to
the identification of siRNA that resulted
in the persistence of monoubiquitinated
FANCD2. The siRNA corresponded to
the USP1 gene, and further studies with
additional siRNA confirmed that they
specifically led to the knockdown of this
DUB enzyme. The USP1 gene and pro-
tein is under cell cycle regulation, accu-
mulating throughout the S phase and
diminishing near the point at which
FANCD2 is deubiquitinated. Following
DNA damage, both USP1 and FANCD2
are bound to chromatin, where they
interact. Some functional work indicates
that USP1 knockdown reduces chromo-
some breakage induced by mitomycin C
treatment, perhaps because of an
attenuated inactivation of FA pathway.
Collectively, the data supports a key
role for USP1 in shutting down the FA
pathway, acting directly on ubiquitinated
FANCD2 on chromatin (Figure 1).
However, this study raises a raft of
new questions. Why isn’t all of the
FANCD2 that is associated with USP1
deubiquitinated—is there another level of
regulation that impacts on the activity of
the enzyme? Is FANCD2 the only sub-
strate for this enzyme, or are other
monoubiquitinated proteins such as
PCNA also its substrates? Recent stud-
ies have indicated that the FA proteins
facilitate some forms of homologous
recombination and error-prone repair
(Niedzwiedz et al., 2004; Yamamoto et
al., 2005);does inactivation of USP1 lead
to an enhancement of these repair
processes? Although FA is a rare condi-
tion, somatic inactivation of the FA path-
way occurs in
(Taniguchi et al., 2003;Tischkowitz et al.,
2003). Could induction of USP1 reduce
the efficacy of the FA DNA damage
response in some instances? Finally,
modulating the activity of the FA pathway
has obvious therapeutic implications. Its
inactivation leads to marked sensitivity to
certain chemotherapeutic agents such as
cisplatin. Getting a better molecular
understanding of how the pathway is
switched on or off would provide potential
therapeutic targets.The current work now
sets the stage nicely for studies to unrav-
el the regulation of this conserved and
fundamental tumor suppressor pathway.
Ketan J. Patel1,2,*
1MRC Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, United
2Department of Medicine, University of
Cambridge, Addenbrooke’s Hospital,
Hills Road, Cambridge CB2 2SP, United
D’Andrea, A.D., and Grompe, M. (2003). Nat.
Rev.Cancer 3, 23–34.
Garcia-Higuera, I., Taniguchi, T., Ganesan, S.,
Meyn, M.S., Timmers, C., Hejna, J., Grompe, M.,
and D’Andrea, A.D.(2001).Mol.Cell 7, 249–262.
Howlett, N.G., Taniguchi, T., Olson, S., Cox, B.,
Waisfisz, Q., De Die-Smulders, C., Persky, N.,
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Joenje, H., Oostra, A.B., Wijker, M., di Summa,
F.M., van Berkel, C.G., Rooimans, M.A., Ebell,
W., van Weel, M., Pronk, J.C., Buchwald, M., and
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Nijman, S.M.B., Huang, T.T., Dirac, A.M.G.,
Brummelkamp, T.R., Kerkhoven, R.M., D’Andrea,
A.D., and Bernards, R. (2005). Mol. Cell 17,
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Taniguchi, T., Tischkowitz, M., Ameziane, N.,
Hodgson, S.V., Mathew, C.G., Joenje, H., Mok,
S.C., and D’Andrea, A.D. (2003). Nat. Med. 9,
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Winter, J.P., Harris, R., Taniguchi, T., D’Andrea,
A., Hodgson, S.V., Mathew, C.G., and Joenje, H.
Yamamoto, K., Hirano, S., Ishiai, M., Morishima,
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et al.(2005).Mol.Cell.Biol.25, 34–43.
P R E V I E W S
Figure 1. Current outline of the FA pathway
(1) DNA damage results in the activation of
the FA pathway; this step is not well under-
stood. (2) The FA nuclear complex consisting
of most of the known FA proteins activates
FANCD2 by the conjugation of a single ubiq-
uitin residue. Modified FANCD2 binds to
chromatin surrounding damaged DNA,
where it may function to direct DNA repair.
(3) At this site, USP1 deubiquitinates FANCD2,
resulting in its inactivation and release from