doi: 10.1242/10.1242/jcs.00276
Journal of Cell Science 116, 773-783 (2003)
Copyright © 2003 The Company of Biologists Limited
doi: 10.1242/jcs.00276
Prolyl isomerase Pin1: a catalyst for oncogenesis and a potential therapeutic target in cancer
Akihide Ryo,
Yih-Cherng Liou,
Kun Ping Lu* and
Gerburg Wulf*
Cancer Biology Program, Division of Hematology/Oncology, Department of
Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, MA 02215, USA
*
Authors for correspondence (e-mail:
klu{at}caregroup.harvard.edu;
gwulf{at}caregroup.harvard.edu)
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Summary
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Phosphorylation of proteins on serine or threonine residues preceding
proline (Ser/Thr-Pro) is a major intracellular signaling mechanism. The
phosphorylated Ser/Thr-Pro motifs in a certain subset of phosphoproteins are
isomerized specifically by the peptidyl-prolyl cis-trans isomerase Pin1. This
post-phosphorylation isomerization can lead to conformational changes in the
substrate proteins and modulate their functions. Pin1 interacts with a number
of mitotic phosphoproteins, and plays a critical role in mitotic regulation.
Recent work indicates that Pin1 is overexpressed in many human cancers and
plays an important role in oncogenesis. Pin1 regulates the expression of
cyclin D1 by cooperating with Ras signaling and inhibiting the interaction of
ß-catenin with the tumor suppressor APC and also directly stabilizing
cyclin D1 protein. Furthermore, PIN1 is an E2F target gene essential
for the Neu/Ras-induced transformation of mammary epithelial cells. Pin1 is
also a critical regulator of the tumor suppressor p53 during DNA damage
response. Given its role in cell growth control and oncogenesis, Pin1 could
represent a new anti-cancer target.
Key words: Phosphorylation, Prolyl-isomerase, Pin1, Post-phosphorylation regulation, Signal transduction, Oncogenesis
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Introduction
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The phosphorylation of proteins on serine or threonine residues that
immediately precede proline residues (Ser/Thr-Pro) is an important signaling
mechanism controlling many cellular processes, such as cell cycle regulation,
transcription, cell differentiation and proliferation
(Blume-Jensen and Hunter,
2001
; Hunter,
1995; Hunter,
1998; Lu, K. P. et al.,
2002b). The deregulation of this elaborate signaling mechanism can
result in cell transformation and oncogenesis. Therefore, precise pre- and
post-phosphorylation regulatory mechanisms have evolved that maintain
steady-state intracellular signaling.
Ser/Thr-Pro motifs are the major phosphorylation sites for a large
superfamily of `proline-directed' kinases, including cyclin-dependent kinases
(CDKs), mitogen-activated protein kinases (MAPKs) and glycogen synthase kinase
3ß (GSK-3ß), and conversely they are dephosphorylated by Ser/Thr
phosphatases, including PP2A, FCP1 and calcineurin
(Lu, K. P. et al., 2002b).
Furthermore, MAPK ERK2 and CDK2, as well as the Ser/Thr phosphatase PP2A, are
conformation specific, preferentially phosphorylating/dephosphorylating the
trans isomer (Brown et al.,
1999; Weiwad et al.,
2000; Zhou et al.,
2000). Ser/Thr phosphorylation has for a long time been believed
to regulate the function of proteins by altering their conformations; however,
little is known about the actual conformational changes and their importance.
The identification and characterization of a peptidyl-prolyl cis/trans
isomerase (PPIase), Pin1, has led to the discovery of a novel
post-phosphorylation regulatory mechanism
(Lu et al., 1996;
Ranganathan et al., 1997;
Yaffe et al., 1997). Pin1
binds to and isomerizes the peptidyl-prolyl bond in specific phosphorylated
Ser/Thr-Pro motifs and thereby induces conformational changes in its target
proteins (Albert et al., 1999;
Arevalo-Rodriguez et al., 2000;
Hsu et al., 2001;
Kops et al., 2002;
Liou et al., 2002;
Lu et al., 1999a;
Lu et al., 1999b;
Ryo et al., 2002;
Ryo et al., 2001;
Shen et al., 1998;
Stukenberg and Kirschner,
2001; Wu et al.,
2000; Wulf et al.,
2002; Wulf et al.,
2001; Yaffe et al.,
1997; Zacchi et al.,
2002; Zheng et al.,
2002; Zhou et al.,
2000). These conformational changes can have profound effects on
the function of Pin1 substrates, modulating their activity, phosphorylation
status, protein-protein interactions, subcellular localization and stability
(Fig. 1). For example, Pin1 can
bind to and induce conformational changes in the mitotic phosphatase Cdc25C
and the microtubule-binding protein tau, after they have been phosphorylated
on specific Ser/Thr-Pro motifs. Such conformational changes can directly
inhibit the ability of phosphorylated Cdc25C to dephosphorylate and activate
Cdc2 (Shen et al., 1998;
Stukenberg and Kirschner,
2001; Zhou et al.,
2000), or restore the ability of phosphorylated tau to promote
microtubule assembly (Lu et al.,
1999a). Furthermore, such conformational changes can also regulate
the dephosphorylation of Cdc25C and tau because phosphatases such as PP2A
dephosphorylate only the trans isoform of phosphorylated Ser/Thr-Pro motifs
(Zhou et al., 2000). Thus,
phosphorylation-dependent prolyl isomerization is a new post-phosphorylation
signaling mechanism.
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Fig. 1. A novel post-phosphorylation regulatory mechanism in phosphorylation
signaling. Phosphorylation of proteins by proline-directed kinases (e.g. CDKs,
MAPKs, GSK-3ß) creates binding sites for the prolyl-isomerase Pin1 (1st
step). Subsequent prolyl-isomerization by Pin1 induces conformational changes
and thereby regulates the function of target proteins (2nd step).
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Pin1 was originally identified in a yeast two-hybrid screen as a human
protein that interacts physically and functionally with a mitotic kinase and
was the first peptidyl-prolyl cis/trans isomerase (PPIase) shown to be
essential for cell division in yeast and human cells
(Lu et al., 1996). Pin1
homologues are highly conserved in eukaryotes
(Huang et al., 2001;
Landrieu et al., 2000;
Metzner et al., 2001;
Yao et al., 2001;
Zhou et al., 1999), and the
budding yeast homologue, Ess1p/Ptrf1p, was isolated a long time ago but did
not have any previously known activity
(Hanes et al., 1989;
Hani et al., 1995). With the
exception of the plant enzymes, which appear to contain only PPIase domains,
most other Pin1-type PPIases also contain an N-terminal WW domain. The
function of the WW domain is to target the enzyme to its substrates, where the
PPIase domain is both sufficient and necessary to catalyze the conformational
change and to carry out the essential function of this enzyme. Depletion of
Pin1 causes mitotic arrest and apoptosis in budding yeast and tumor cell lines
(Lu et al., 1996), and Pin1 is
required for the DNA replication checkpoint and G2/M transition in
Xenopus extracts (Winkler et
al., 2000). Pin1 has been shown to be involved in the regulation
of many other cellular events, such as cell cycle progression, transcriptional
regulation and cell proliferation and differentiation
(Albert et al., 1999;
Arevalo-Rodriguez et al., 2000;
Crenshaw et al., 1998;
Gerez et al., 2000;
Hani et al., 1999;
Hsu et al., 2001;
Kamimoto et al., 2001;
Lavoie et al., 2001;
Liou et al., 2002;
Liu et al., 2001;
Messenger et al., 2002;
Morris et al., 1999;
Pathan et al., 2001;
Patra et al., 1999;
Rippmann et al., 2000;
Ryo et al., 2002;
Ryo et al., 2001;
Shen et al., 1998;
Wu et al., 2000;
Wulf et al., 2001).
Furthermore, it is involved in the DNA damage response, regulating p53
function (Wulf et al., 2002;
Zacchi et al., 2002;
Zheng et al., 2002).
Moreover, it is also involved in Alzheimer's disease
(Lu et al., 1999a;
Zhou et al., 2000) and cancer
(Liou et al., 2002;
Ryo et al., 2002;
Ryo et al., 2001;
Wulf et al., 2001).
Here we review recent studies demonstrating the role of Pin1 in cell growth
control and oncogenesis and discuss the feasibility of Pin1 as a potential
therapeutic target for anti-cancer treatment. Comprehensive recent reviews on
function and regulation of Pin1 (Lu, K. P.
et al., 2002b; Zhou et al.,
1999), as well as its specific role in transcription
(Shaw, 2002) and in
Alzheimer's disease (Lu, K. P. et al.,
2002a), are available elsewhere.
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Regulation of cyclin D1 expression
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At least 20 proteins have been shown to be Pin1 targets
(Table 1). Of these known Pin1
substrates, cyclin D1 is the most extensively studied both in vitro and in
vivo (Liou et al., 2002;
Ryo et al., 2002;
Ryo et al., 2001;
Wulf et al., 2001). The
connection between Pin1 and cyclin D1 was originally identified in screens for
Pin1 expression in human breast cancer tissues
(Wulf et al., 2001). Indeed,
Pin1 is overexpressed in several human cancers, including breast, lung and
prostate cancers and its expression levels positively correlate with the tumor
grade in breast cancer (Ryo et al.,
2001; Wulf et al.,
2001). In addition, Pin1 is overexpressed in all breast cancer
cell lines examined when compared with non-transformed or primary mammary
epithelial cells (Wulf et al.,
2001), and Pin1 is one of the genes suppressed by overexpression
of wild-type BRCA1 (MacLachlan et al.,
2000).
Elevated Pin1 levels in breast cancer significantly correlate with cyclin
D1 overexpression (Ryo et al.,
2002; Ryo et al.,
2001; Wulf et al.,
2001). In fact, we have found that Pin1 levels in cyclin D1
overexpressing tumors are on average about twice as high as those in
cyclin-D1-negative tumors. This is significant given that cyclin D1 is an
essential downstream target for mammary tumorigenesis. Cyclin D1 is
overexpressed in about half of breast cancer patients
(Bartkova et al., 1994;
Gillett et al., 1994).
Overexpression of cyclin D1 contributes to cell transformation
(Alt et al., 2000;
Hinds et al., 1994),
inhibition of cyclin D1 expression by antisense expression causes growth
arrest in tumor (Arber et al.,
1997; Driscoll et al.,
1997; Kornmann et al.,
1998; Schrump et al.,
1996) and disruption of the cyclin D1 gene in mice completely
suppresses the ability of Ha-Ras or Her2/Neu to induce tumor development in
mammary glands (Yu et al.,
2001). In breast cancer tissues, Her2/Neu overexpression
correlates with Pin1 overexpression, although this correlation did not reach
statistical significance, probably because of the small number of
Her2/Neu-positive patients in the study
(Wulf et al., 2001). It is of
interest, though, that Pin1 levels were 1.7-2 times higher in patients who are
either Her2/Neu positive, or negative for estrogen receptor expression
(Wulf et al., 2001). Further
studies in larger cohorts may clarify the relationship between Pin1 expression
and these unfavorable biochemical markers, and establish whether Pin1
expression would be a useful additional marker for breast cancer
prognosis.
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Cooperation with the Ras/AP-1 signaling pathway
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The AP-1 complex, which includes transcription factors such as Jun, Fos and
FosB, regulates a wide range of cellular processes, including cell
proliferation, cell death, survival and differentiation, and has a binding
site in the cyclin D1 promoter (Shaulian
and Karin, 2002). The significance of Jun activation for cell
proliferation and cyclin D1 induction has been studied in mice lacking AP-1
components. Fos/FosB double knockout and Jun knockout mouse
embryonic fibroblasts (MEFs) have reduced cyclin D1 levels and display severe
proliferative defects (Behrens et al.,
1999; Brown et al.,
1998; Shaulian and Karin,
2001; Shaulian and Karin,
2002). Ras signaling activates the proline-directed MAP kinases
p38 and JNK/SAPK, which phosphorylate Jun on two critical N-terminal Ser-Pro
motifs at Ser63 and Ser73, thereby enhancing its transcriptional activity
(Albanese et al., 1999;
Albanese et al., 1995;
Bakiri et al., 2000;
Binetruy et al., 1991;
Derijard et al., 1994;
Shaulian and Karin, 2001;
Whitmarsh and Davis, 1996).
How the activity of Jun is further regulated after phosphorylation has not
been known until recently.
We have found that Pin1 not only binds phosphorylated Jun but also
increases its ability to activate the cyclin D1 promoter in cooperation either
with activated JNK or oncogenic Ha-Ras. In contrast, inhibition of endogenous
Pin1 reduces the transcriptional activity of phosphorylated Jun, indicating
that endogenous Pin1 is also required for optimal activation. Thus, Pin1 is a
potent modulator of phosphorylated Jun in inducing cyclin D1 expression,
presumably by regulating the conformation of the phosphorylated Ser-Pro motifs
in Jun (Wulf et al., 2001).
Jun is basically a positive regulator of cell proliferation
(Behrens et al., 1999;
Brown et al., 1998;
Shaulian and Karin, 2001;
Shaulian and Karin, 2002),
and the Pin1-induced conformational changes in Jun potentially affect its
ability to form homo- or hetero-dimers and/or its DNA binding activity.
However, further studies are necessary to define the molecular mechanisms by
which Pin1 affects Jun function.
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Activation of the Wnt/ß-catenin pathway
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Wnt/ß-catenin signaling is involved in control of gene expression,
cell adhesion and cell polarity (Kinzler
and Vogelstein, 1996; Moon et
al., 2002; Morin,
1999; Polakis,
2000). Activation of the Wnt/ß-catenin signaling pathway is a
major feature of human cancers (Kinzler
and Vogelstein, 1996; Polakis,
2000). Deregulation of this signaling pathway has been found in a
subset of human malignancies that carry mutations in proteins participating in
this pathway, such as APC (adenomatous polyposis coli), axin and
ß-catenin itself (Kinzler and
Vogelstein, 1996; Polakis,
2000; Satoh et al.,
2000). The end result of this aberrant activation is always the
cytosolic stabilization of ß-catenin, which enhances the transcription of
a number of target genes, including the cyclin D1 gene and Myc, which
can lead to oncogenesis (Behrens et al.,
1996; He et al.,
1998; Mann et al.,
1999; Molenaar et al.,
1996; Tetsu and McCormick,
1999). Mutations in APC or ß-catenin are often found in
certain tumor types, such as colon cancer
(Kinzler and Vogelstein, 1996;
Morin, 1999;
Polakis, 2000), but they are
rarely observed in others, such as breast cancer
(Jonsson et al., 2000;
Lin et al., 2000;
Schlosshauer et al., 2000).
However, there is compelling evidence for a crucial role for ß-catenin
signaling in the tumorigenesis of breast cancer
(Jonsson et al., 2000;
Lin et al., 2000;
Roose et al., 1999;
Schlosshauer et al., 2000).
Furthermore, ß-catenin levels are significantly upregulated and are a
strong and independent prognostic factor in human breast cancer patients
(Lin et al., 2000).
A differential display screen for genes regulated by Pin1 that compared
inducible Pin1-overexpressing breast cancer MCF-7 cells and control cells
identified 17 known genes (Ryo et al.,
2001). Interestingly, four of the 12 genes whose expression is
upregulated are targets of ß-catenin and its transcriptional partner TCF:
those encoding cyclin D1, Myc, PPAR-delta and fibronectin
(Gradl et al., 1999;
He et al., 1999;
He et al., 1998;
Tetsu and McCormick, 1999).
Overexpression or depletion of Pin1 in cell lines has been shown to regulate
the stability and subcellular localization of ß-catenin
(Ryo et al., 2001). In
addition, Pin1 can activate the cyclin D1 promoter not only through AP-1 sites
but also through TCF-binding sites that are present
(Ryo et al., 2001).
Upregulation of Pin1 in breast cancer strongly correlates with increased
ß-catenin levels in the tumors examined, whereas ß-catenin levels
are decreased in Pin1-knockout mouse tissues
(Ryo et al., 2001).
How does Pin1 regulate ß-catenin levels? As shown above,
conformational changes caused by Pin1-catalyzed prolyl-isomerization can
affect protein stability, phosphorylation status and protein-protein
interactions (Hsu et al.,
2001; Kops et al.,
2002; Liou et al.,
2002; Lu et al.,
1999a; Lu et al.,
1999b; Ryo et al.,
2002; Ryo et al.,
2001; Shen et al.,
1998; Stukenberg and
Kirschner, 2001; Wulf et al.,
2002; Wulf et al.,
2001; Yaffe et al.,
1997; Zacchi et al.,
2002; Zheng et al.,
2002; Zhou et al.,
2000). Pin1 binds exclusively to phosphorylated ß-catenin,
and its binding site has been mapped to the pSer246-Pro motif
(Ryo et al., 2001). This
motif is located at an exposed loop region between the two helixes at the
third armadillo repeat interface, and it is next to the surface that interacts
with APC in the three-dimensional structure of ß-catenin
(Graham et al., 2000;
Huber et al., 1997;
von Kries et al., 2000). APC
is the shuttling protein that exports nuclear ß-catenin to the cytoplasm
for degradation (Bienz, 2002;
Henderson, 2000;
Neufeld et al., 2000;
Rosin-Arbesfeld et al.,
2000). Mutations of Phe253 and Phe293 in
ß-catenin abolish its ability to bind to APC
(Graham et al., 2000;
Huber et al., 1997;
von Kries et al., 2000).
Similarly, Pin1 binding and isomerization specifically inhibits the
interaction between ß-catenin and APC, resulting in the nuclear
accumulation and stabilization of ß-catenin. Pin1-dependent
prolyl-isomerization thus appears to be a novel mechanism for the regulation
of ß-catenin—APC interaction. Given the overexpression of Pin1 in
many cancers, this mechanism might up-regulate ß-catenin activity in
tumors such as breast cancer, in which APC and/or ß-catenin mutations are
not common (Ryo et al.,
2001).
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Regulation of cyclin D1 protein levels
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Pin1-knockout mice were originally reported to develop normally
and to have no phenotype (Fujimori et al.,
1999). More recent analyses indicate that these mice display a
range of cell proliferative abnormalities, including decreased body size,
testicular atrophy and retinal degeneration, a phenotype strongly reminiscent
of the cyclin-D1-knockout mice (Liou et
al., 2002). Most strikingly, the breast epithelial compartment in
Pin1-null mice cannot undergo the massive proliferative changes
associated with pregnancy. Importantly, cyclin D1 protein levels are
significantly decreased in every tissue that displays a severe phenotype
(Liou et al., 2002). These
results strongly suggest that Pin1 is essential for the regulation of cyclin
D1 in vivo. Interestingly, mouse models in which AP-1 or ß-catenin/APC
function is perturbed do not display defects associated with lack of cyclin D1
(Behrens et al., 1999;
Brown et al., 1998;
Haegel et al., 1995;
Shaulian and Karin, 2001),
indicating that other Pin1-dependent mechanisms for regulating cyclin D1
exist. Indeed, cyclin D1 mRNA levels in Pin1-knockout MEFs are
significantly reduced probably because of defective Jun/AP-1 and
ß-catenin/TCF pathways. However, the reduction of cyclin D1 protein
levels is disproportionally greater (Liou
et al., 2002). Phosphorylation of cyclin D1 by GSK-3ß on
Thr286-Pro site regulates turnover and localization of cyclin D1 by enhancing
its binding to CRM1, a nuclear exporter of cyclin D1, which leads to
degradation of cyclin D1 in the cytoplasm
(Alt et al., 2000;
Diehl et al., 1998;
Diehl et al., 1997;
Fukuda et al., 1997). Pin1 can
bind to and presumably isomerize the phosphorylated Thr286-Pro motif in cyclin
D1, which may inhibit its interaction with CRM1. This would stabilize cyclin
D1 by preventing its nuclear export and proteolysis in the cytoplasm
(Liou et al., 2002). Pin1 thus
seems to regulate cyclin D1 function at both the transcriptional and
post-translational levels, and this may explain why the Pin1-null
phenotype resembles the cyclin-D1-null phenotype. It is also consistent with
the finding that Pin1-deficient MEFs cannot effectively restart proliferation
in response to serum stimulation after G0 arrest
(Fujimori et al., 1999). These
results indicate that Pin1 is required for progression through G0 to S in
addition to mitosis.
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Regulation of pin1 transcription and function by oncogenic
pathways
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How is Pin1 activated in cancer cells? The PIN1 promoter sequence
has neither a TATA nor CAAT box but has two putative GC boxes and three
putative E2F-binding sites (Ryo et al.,
2002). Indeed, E2F family proteins activate the PIN1
promoter through these E2F-binding sites. E2F proteins also bind the
PIN1 promoter in vitro and in vivo, and increased Pin1 levels in
breast cancer cell lines correlate with an increase in binding of E2F to the
PIN1 promoter. Moreover, overexpression of E2F enhances PIN1
promoter activity and mRNA levels in breast cancer cells. In common with many
other E2F-target genes (Fry et al.,
1997; Nevins,
2001; Ohtani et al.,
1995), PIN1 transcription and its protein levels
fluctuate during cell cycle progression in non-neoplastic cells
(Ryo et al., 2002) but not in
transformed cells (Shen et al.,
1998). Interestingly, aberrantly high E2F1 levels have been
described in breast cancer (Zhang et al.,
2000), and therefore it is possible that deregulation of E2F plays
a key role in the upregulation of Pin1 in breast cancer. Since deregulation of
the Rb/E2F pathway is also found in many other cancer types and contributes to
the oncogenesis of a number of human cancers
(Johnson and Schneider-Broussard,
1998; Nevins,
2001; Weinberg,
1995), deregulation of the Rb/E2F pathway may cause Pin1
overexpression in other cancer cells.
In addition to being transcriptionally regulated, Pin1 is also regulated by
post-translational controls. One such regulatory mechanism is phosphorylation.
Phosphorylation of the Pin1 WW domain inhibits its ability to bind target
proteins and regulates the subcellular localization of Pin1
(Lu, P. J. et al., 2002).
Dephosphorylated Pin1 accumulates during the G2/M transition in HeLa cells,
whereas in G1 and S phase phosphorylated Pin1 is predominant
(Lu, P. J. et al., 2002). In
human breast tumors, the dephosphorylated, and presumably active, form of Pin1
accumulates (Wulf et al.,
2001). It will be important to identify the specific kinase
responsible because, theoretically, this kinase would be able to inhibit Pin1
function, thereby suppressing its ability to activate oncogenic pathways as
described above. Phosphospecific antibodies will help us to assess more
accurately the ratio of phosphorylated to dephosphorylated Pin1 and may become
an important tool for identifying the kinase/phosphatase activities regulating
the phosphorylation status of Pin1. Finally, Pin1 levels have been shown to be
decreased upon prolonged exposure to the microtubule-targeting drug Taxol,
which can apparently be prevented by some proteasome inhibitors; this suggests
that Pin1 is also subjected to proteolytic regulation
(Basu et al., 2002). However,
direct evidence for such a regulatory mechanism has not yet been provided.
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Pin1 overexpression and cell transformation
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Recent data support the notion that Pin1 can at least partially transform
mammary epithelial cells (Ryo et al.,
2002). Pin1 confers anchorage-independent cell growth on the
non-transformed mammary epithelial cell line MCF-10A
(Ryo et al., 2002).
Furthermore, its overexpression interferes with normal cell differentiation
and acinal formation in a three-dimensional matrigel assay
(Muthuswamy et al., 2001;
Petersen et al., 1992;
Ryo et al., 2002). However,
Pin1 overexpression does not affect cell growth or cell morphology under
normal culture conditions. Overexpression of Pin1 might thus trigger some
early events during cell transformation
(Ryo et al., 2002), although
it remains to be determined whether Pin1 itself is sufficient for
transformation in vivo. It is possible that elevated Pin1 levels become
oncogenic only after a `first hit', i.e. activation of an oncogenic pathway
that leads to substrate phosphorylation that allows Pin1 to exert this
function. The observation that Pin1 greatly enhances and facilitates
transformation by oncogenic Neu and Ras in mammary epithelial cells is
consistent with this hypothesis (Ryo et
al., 2002).
Neu or Ras signaling is frequently deregulated in breast cancers, although
mutations and amplifications of these genes are rarely observed
(Andrechek and Muller, 2000;
Harari and Yarden, 2000).
Transgenic overexpression of MMTV-Ha-Ras or MMTV-Neu potently induces mammary
tumors by stimulating cyclin D1. However, transgenic overexpression of
MMTV-cyclin D1 is much less tumorigenic
(Muller et al., 1988;
Sinn et al., 1987;
Wang et al., 1994). In
addition, constitutive overexpression of cyclin D1 alone cannot transform
MCF-10A cells, nor is it sufficient to prevent G1 arrest induced by EGF
deprivation (Chou et al.,
1999). These discrepancies could be explained by the findings that
cyclin D1 is regulated not only by transcriptional activation but also by the
post-translational stabilization described above. In contrast to wild-type
cyclin D1, the mutant cyclin D1T286A is stable and functions as a
constitutively active mutant that can potently transform fibroblasts
(Alt et al., 2000). Both
transcriptional activation and post-translational stabilization of cyclin D1
thus seem to be critical for tumor development induced by Neu/Ras
signaling.
Similarly to cyclin D1, Pin1 is highly overexpressed in the mammary glands
of transgenic mice that overexpress MMTV-Neu or MMTV-Ha-Ras
(Muller et al., 1988;
Ryo et al., 2002;
Sinn et al., 1987).
Inhibition of Pin1 by a dominant negative mutant or an antisense construct
dramatically reduces both cell proliferation and the transformation induced by
the Neu and Ras oncogenes. This reduction can be reversed by expression of the
constitutively active cyclin D1 T286A mutant that is resistant to Pin1
inhibition (Ryo et al.,
2002). These results suggest that cyclin D1 is a specific
downstream target of Pin1 for oncogenesis. Cyclin D1 is overexpressed in 50%
of all breast cancers, but genetic amplification accounts for only 10% of this
overexpression (Sutherland and Musgrove,
2002). Pin1 therefore probably plays an important role in
maintaining cyclin D1 levels sufficient for transformation of mammary
epithelial cells.
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Regulation of p53 in the DNA damage response
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The tumor suppressor protein p53 regulates multiple cellular functions,
including cell cycle checkpoints, genomic stability and apoptosis
(Colman et al., 2000;
Lakin and Jackson, 1999;
Meek, 1999;
Ryan et al., 2001;
Taylor and Stark, 2001;
Wahl and Carr, 2001). DNA
damage leads to the stabilization and accumulation of p53, which plays a
pivotal role in transcriptional activation of the cell cycle inhibitor p21 and
cell cycle arrest. This prolonged half-life of p53 is at least partially due
to its dissociation from the ubiquitin ligase MDM2. The increase in p53
stability and/or transcriptional activity depends critically on its
phosphorylation on multiple serine/threonine residues, including those
preceding prolines (Abraham et al.,
2000; Blaydes et al.,
2001; Bulavin et al.,
1999; Buschmann et al.,
2001; Milner et al.,
1990; Sakaguchi et al.,
1998; Sanchez-Prieto et al.,
2000; Turenne et al.,
2001). Recent results indicate a new role of Pin1 in the DNA
damage response. Pin1 is an indispensable positive regulator of p53 in
response to DNA damage induced by genotoxic drug treatment or UV radiation
(Zacchi et al., 2002;
Zheng et al., 2002) or
ionizing radiation (Wulf et al.,
2002; Zacchi,
2002). DNA damage enhances the specific interaction between Pin1
and p53, which depends on the WW domain in Pin1 and specific phosphorylated
Ser-Pro motifs in p53. Pin1 binds to p53 at phosphorylated Ser33 and Ser46
following exposure to ionizing radiation
(Wulf et al., 2002) and at
phosphorylated Ser33, Thr81 and Ser315 following exposure to UV radiation or
genotoxic drug treatment (Zacchi et al.,
2002; Zheng et al.,
2002). More importantly, Pin1 is required for the stabilization of
the p53 protein after DNA damage, preventing p53 from binding to its ubiquitin
ligase, MDM2 (Wulf et al.,
2002; Zacchi et al.,
2002). In addition, Pin1 increases the transcriptional activity of
p53 towards the p21 and MDM2 promoters
(Wulf et al., 2002;
Zacchi et al., 2002;
Zheng et al., 2002). Given
that phosphorylation of p53 by various kinases, such as Chk1, ATM and MAP
kinases, is believed to play a central role in induction of p53 by DNA damage,
these results indicate that Pin1-dependent phosphorylation-dependent
isomerization is a new and critical mechanism to control p53 stability and
function after it has been phosphorylated.
The physiological consequences of Pin1 loss in normal cells are still
controversial. Zheng et al. (Zheng et
al., 2002) and Wulf et al.
(Wulf et al., 2002) show that
Pin1 is required for maintaining the DNA-damage cell cycle checkpoint by
inducing the cell cycle inhibitor p21, which will protect cells from DNA
damage-induced cell death (Wulf et al.,
2002; Zheng et al.,
2002). In contrast, Zacchi et al. claim that Pin1 accelerates
apoptosis by enhancing pro-apoptotic genes downstream of p53, such as Bax and
DR2/Killer (Zacchi et al.,
2002). This discrepancy may reflect the fact that p53 induces sets
of genes required for cell cycle arrest and apoptosis, depending on the
cellular context and intensity and timing of the respective DNA damage
(Colman et al., 2000;
Lakin and Jackson, 1999;
Meek, 1999;
Ryan et al., 2001;
Taylor and Stark, 2001;
Wahl and Carr, 2001). Careful
in vivo analysis of the effects of different types of genotoxic insult may
clarify whether Pin1-mediated prolyl isomerization of p53 directs the cells in
a given cell towards apoptosis or towards cell cycle arrest.
These results are initially counterintuitive: why would a protein that
amplifies oncogenic signals also activate a tumor suppressor gene?
Proline-directed phosphorylation plays an important role in both the promotion
and suppression of oncogenesis, and therefore Pin1 is likely to be involved in
both processes. It has been well established that many proteins are involved
in both processes. For example, transcription factors such as Myc and E2F
family members participate in a complex signaling network that regulates cell
growth, differentiation, cell survival and apoptosis in non-malignant cells.
Only in a permissive environment will overexpressed or mutated forms of these
proteins contribute to carcinogenesis
(Oster et al., 2002;
Trimarchi and Lees, 2002;
Zhou and Hurlin, 2001).
Therefore, it may be important to distinguish the physiological function of
Pin1 in normal cells from its pathological role in cancers where Pin1 is
deregulated. It is likely that, under physiological conditions in normal
cells, Pin1-mediated p53 regulation is important for cell cycle checkpoint
regulation and the maintenance of genomic stability. In cancer cells, however,
this mechanism may be defective because oncogenic signalling pathways induced
by Pin1 overexpression may override the DNA damage repair mechanisms and/or
because p53 is absent or mutated in many cancer cell types. Interestingly,
Pin1 can also stabilize p53 mutants with the same efficacy as the wild-type
protein (G.W. and K.P.L., unpublished results). Since a cellular environment
in which Myc and/or Ras expression is deregulated can favor the selection of
p53 mutations (Chikatsu et al.,
2002) and since some p53 mutants function as dominant negative
mutants (de Vries et al.,
2002; Monti et al.,
2002), Pin1 overexpression in the context of a mutated
p53 gene might even contribute to genomic instability in cancer
cells. However, further studies are needed to define the physiological and
pathological roles of Pin1-mediated p53 regulation.
|
Is Pin1 an oncogene or a catalyst for oncogenic activation?
|
|---|
A defining feature of oncogenes is that their mutation or amplification is
associated with oncogenesis. To date, genetic alterations of the PIN1
gene in cancer cells have not yet been described. However, Pin1 levels are
upregulated in cancer cells, probably as a result of the deregulation of the
E2F/Rb pathway, which occurs in >80% of human malignancies
(Nevins, 2001). The activity
of Pin1 depends critically on the phosphorylation status of its substrate
proteins (Liou et al., 2002;
Lu et al., 1999b;
Ryo et al., 2001;
Shen et al., 1998;
Stukenberg and Kirschner,
2001; Wulf et al.,
2001; Yaffe et al.,
1997; Zhou et al.,
2000). Without prior phosphorylation of these targets on
Ser/Thr-Pro motifs, Pin1 cannot bind and catalyze prolyl-isomerization. In
cancer cells, a wave of serine and threonine phosphorylation occurs as a
result of oncogenic signaling
(Blume-Jensen and Hunter,
2001; Hunter,
1995). We propose that Pin1 overexpression cooperates with these
activated kinases to promote cell proliferation and transformation. In this
model, Pin1 would respond to and translate oncogenic signaling into the actual
events of cancer cell growth. Whereas Pin1 itself may not be sufficient for
complete cell transformation, it would be an indispensable translator and
amplifier of oncogenic signal transduction. The fact that Pin1 depends on the
presence of oncogenes such as Ras or Neu to transform cells fully in vitro is
consistent with this idea. This means that the action of Pin1 might depend
entirely on the cellular context and may vary significantly with cell type,
proliferative status and age. For example, Pin1-knockout mice develop
normally and do not show any significant phenotype at a young age. However,
after several months, the mice exhibit age-dependent proliferative disorders
in specific tissues (Liou et al.,
2002). Studies on the incidence and distribution of tumor
development in Pin1-transgenic or Pin1-deficient mice in the
presence or absence of other oncogenes will be important to address the role
of Pin1 in oncogenesis in vivo.
Pin1 thus functions at multiple steps in oncogenic signaling pathways as an
`oncogenic catalyst' (Fig. 2).
It collaborates with Ras/JNK signaling to increase the transcriptional
activity of Jun towards cyclin D1 (Wulf
et al., 2001). It also activates ß-catenin, which can induce
the transcription of the cyclin D1 gene, Jun and Myc
(Behrens et al., 1996;
He et al., 1998;
Mann et al., 1999;
Molenaar et al., 1996;
Ryo et al., 2002;
Tetsu and McCormick, 1999).
In addition, Myc can enhance cyclin D1 function by inducing Cdk4 expression
(Hermeking et al., 2000) and
also directly induce E2F family genes
(Leone et al., 2000;
Sears et al., 1997). These
molecules act synergistically to regulate cyclin D1 and E2F function. Finally,
Pin1 itself is further upregulated by E2F activation in a positive
feedback loop (Ryo et al.,
2002) (Fig. 2). The
amplification of this positive feedback pathway may play a role in aberrant
cell proliferation and oncogenesis.
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|
Fig. 2. Pin1 functions as a critical catalyst for integrating multiple oncogenic
signaling pathways. Pin1 gene expression is induced by growth factor signaling
through Neu and Ras signaling. Ras signaling induces JNK/SAPK activity to
phosphorylate Jun. Subsequently, Pin1 binds to and isomerizes phosphorylated
Jun to enhance its transcriptional activity. In parallel, Pin1 activates the
ß-catenin pathway by preventing ß-catenin binding to APC, which can
induce Jun gene expression. These signaling cascades eventually lead to an
increase in cyclin D1 transcription. Furthermore, Pin1 also directly binds to
and stabilizes cyclin D1 protein. In addition, Pin1 can induce the c-Myc gene
through the activation of the ß-catenin pathway, which can then enhance
cyclin D1 function by inducing Cdk4 gene expression and/or directly activate
E2F family genes. Finally, E2F can induce Pin1 expression in a positive
feedback loop involving the cyclin D1/E2F pathway.
|
|
|
Therapeutic implications
|
|---|
Several lines of evidence suggest that inhibition of Pin1 can suppress
oncogenesis, offering an attractive option for anti-cancer therapy. First,
Pin1 has an extraordinarily high substrate specificity and well-defined active
site (Lu et al., 1999b;
Ranganathan et al., 1997;
Shen et al., 1998;
Verdecia et al., 2000;
Yaffe et al., 1997).
Historically, it has been much easier to develop inhibitors specific for an
enzyme, such as Pin1, than for a non-enzymatic protein, such as cyclin D1.
Second, Pin1 is overexpressed in cancer cells and can potentiate the function
of some oncogenes (Ryo et al.,
2002; Ryo et al.,
2001; Wulf et al.,
2001). Third, overexpression of Pin1 can confer transforming
properties on mammary epithelial cells and also enhance transformed phenotypes
of mammary epithelial cells induced by Neu and Ras
(Ryo et al., 2002). Fourth,
depletion of Pin1 using antisense PIN1 or dominant negative Pin1 causes cancer
cells to enter mitotic block and apoptosis in transient transfection
(Lu et al., 1996;
Lu, P. J. et al., 2002;
Rippmann et al., 2000).
Recent reports that Pin1 associates with the anti-apoptotic protein Bcl-2 in
mitosis (Basu et al., 2002;
Pathan et al., 2001) suggest
that apoptosis induced by Pin1 inhibition may occur via modulation of Bcl-2
function, although the biological significance of this interaction remains to
be elucidated. Fifth, inhibition of Pin1 by stable expression of dominant
negative Pin1 suppresses the transformed phenotypes induced by Ras/Neu, which
can be reversed by the constitutively active cyclin D1 mutant that is
resistant to Pin1 inhibition (Ryo et al.,
2002). Finally, since Pin1-knockout mice do reach
adulthood despite some cell proliferative abnormalities, especially in old age
(Fujimori et al., 1999;
Liou et al., 2002), an
anti-Pin1 therapy might not have general toxic effects.
The feasibility of therapeutic inhibition of Pin1 has not yet been
explored. In contrast to cyclophilins and FK506-binding proteins, where highly
specific inhibitors are well characterized and widely used clinically
(Fischer, 1994;
Hunter, 1998;
Schreiber, 1991), the only
known Pin1 inhibitor is Juglone (Hennig et
al., 1998). Juglone covalently inactivates a unique cysteine
residue in the active site of Pin1-type and parvulin-type isomerases. Juglone
has some anti-cancer activity and has been used as a Pin1 inhibitor in several
studies in cells (Chao et al.,
2001, He et al.,
2001; Rippmann et al.,
2000). However, given that Juglone potently inhibits many other
proteins and enzymes (Chao et al.,
2001; Duhaiman,
1996; Munday and Munday,
2000; Muto et al.,
1987), it is unlikely to be Pin1 specific in the cell. Therefore,
there is a need for the development of Pin1-specific inhibitors. In addition
to providing powerful tools for dissecting Pin1 function in vivo, such
Pin1-specific inhibitors may open a new avenue for anticancer treatment. They
may themselves be highly effective anticancer drugs or become valuable
adjuncts to established chemotherapeutic regimen.
|
Conclusions and perspectives
|
|---|
It has become evident that phosphorylation-dependent prolyl isomerization
is a previously uncharacterized post-phosphorylation signaling mechanism in
cell proliferation and transformation. Following phosphorylation induced by
oncogenic signaling pathways, Pin1 catalyzed prolyl-isomerization is
able to induce conformational changes and thereby to regulate the function of
phosphorylated proteins. Interestingly, the level and activity of Pin1 itself
are also upregulated by oncogenic pathways. Therefore, Pin1 may function as a
critical catalyst that amplifies and translates multiple oncogenic signaling
mechanisms during oncogenesis. Inhibition of Pin1 may thus provide a unique
way of disrupting oncogenic pathways and therefore become an appealing target
for novel anticancer therapies. However, further experiments, including in
vivo studies using Pin1-knockout and Pin1-transgenic mouse models in the
presence or absence of other oncogenes are necessary to elucidate the function
and regulation of Pin1 in cell growth regulation and oncogenesis.
|
Acknowledgments
|
|---|
We are grateful to B. Neel, L. Cantley, T. Hunter, J. Nevins, C. Sherr, P.
Sicinski, J. Brugge, M. Yamamoto and M. Yoshida for constructive discussions
and/or suggestions and to K. Perrem and X. Zhou for their important
contributions. A.R. is a Leukemia and Lymphoma Society Special Fellow, Y.-C.L.
is a Canadian Institute of Health Research Fellow and G.W. is a recipient of a
Mentored Clinical Investigator Award from NIH (CA093655). K.P.L. is a Pew
Scholar and a Leukemia and Lymphoma Society Scholar. The studies performed in
the authors' laboratory were supported by NIH grants GM56230, GM58556 and
AG17870 to K.P.L.
|
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Summary
Introduction
