CDK inhibitors: cell cycle regulators and beyond.
ABSTRACT First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways, the cyclin-CDK inhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 have emerged as multifaceted proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering their subcellular localization, protein-protein interactions, and stability. These functions are essential for the maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to tumor suppression.
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ABSTRACT: Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G1 phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G1 phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.Molecules and Cells 11/2014; · 2.24 Impact Factor
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ABSTRACT: PCTAIRE1 is a cyclin-dependent kinase family protein that has been implicated in spermatogenesis. Although we recently revealed the function of PCTAIRE1 in tumorigenesis of epithelial carcinoma cells, its tumorigenic function in melanoma remains unclear. Interrogation of the Oncomine database revealed that malignant melanoma showed up-regulation of PCTAIRE1 mRNA compared to normal skin and benign melanocytic nevus tissues. In the melanoma cell lines A2058 and SK-MEL-28, PCTAIRE1 gene knockdown using siRNA or shRNA diminished melanoma cell proliferation as assessed by cellular ATP levels, cell counting and clonogenic assays. Moreover, FACS analyses of annexin V-PI staining and DNA content showed that PCTAIRE1 knockdown caused apoptosis in A2058 cells. In contrast, PCTAIRE1 does not appear to be involved in the proliferation of immortalized human keratinocyte HaCaT cells. Depletion of PCTAIRE1 by siRNA/shRNA led to p27 accumulation in melanoma cells but not HaCaT cells. In tumor xenografts of melanoma A2058 cells, conditional knockdown of PCTAIRE1 restored p27 protein expression and suppressed tumor growth. Our findings reveal a crucial role for PCTAIRE1 in regulating p27 protein levels and tumor growth in melanoma cells, suggesting that PCTAIRE1 could provide a target for melanoma treatment.Oncoscience. 01/2014; 1(10):624-33.
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ABSTRACT: TOX is a nuclear factor essential for the development of CD4(+) T cells in the thymus. It is normally expressed in low amounts in mature CD4(+) T cells of the skin and the peripheral blood. We have recently discovered that the transcript levels of TOX were significantly increased in mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma (CTCL) as compared with normal skin or benign inflammatory skin. However, its involvement in advanced CTCL and its biological effects on CTCL pathogenesis have not been explored. In this study, we demonstrate that the TOX expression is also enhanced significantly in primary CD4(+)CD7(-) cells from patients with Sézary syndrome, a leukemic variant of CTCL, and that high TOX transcript levels correlate with increased disease-specific mortality. Stable knockdown of TOX in CTCL cells promoted apoptosis and reduced cell cycle progression, leading to less cell viability and colony-forming ability in vitro, and reduced tumor growth in vivo. Furthermore, TOX knockdown significantly increased two cyclin-dependent kinase inhibitors, CDKN1B and CDKN1C. Finally, blocking CDKN1B and CDKN1C reversed growth inhibition of TOX knockdown. Collectively, these findings provide strong evidence that aberrant TOX activation is a critical oncogenic event for CTCL. Copyright © 2014 American Society of Hematology.Blood 12/2014; · 9.78 Impact Factor
Cell Cycle Regulators and Beyond
Arnaud Besson,1Steven F. Dowdy,2and James M. Roberts3,*
1Universite ´ de Toulouse – LBCMCP and CNRS - UMR5088, Toulouse, France
2Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, University of California, San Diego,
School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA
3Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
First identified as cell cycle inhibitors mediating the growth inhibitory cues of upstream signaling pathways,
thecyclin-CDKinhibitorsoftheCip/Kipfamilyp21Cip1,p27Kip1,and p57Kip2haveemergedas multifaceted
proteins with functions beyond cell cycle regulation. In addition to regulating the cell cycle, Cip/Kip proteins
play important roles in apoptosis, transcriptional regulation, cell fate determination, cell migration and
cytoskeletal dynamics. A complex phosphorylation network modulates Cip/Kip protein functions by altering
their subcellular localization, protein-protein interactions, and stability. These functions are essential for the
maintenance of normal cell and tissue homeostasis, in processes ranging from embryonic development to
General Features of Cip/Kip Proteins
Progression through the cell-division cycle is regulated by the
coordinated activities of cyclin/cyclin-dependent kinases (CDK)
complexes. One level of regulation of these cyclin-CDK com-
plexes is provided by their binding to CDK inhibitors (CKIs). In
metazoans, two CKI gene families have been defined based
on their evolutionary origins, structure, and CDK specificities.
The INK4 gene family encodes p16INK4a, p15INK4b, p18INK4c,
and p19INK4d, all of which bind to CDK4 and CDK6 and inhibit
their kinase activities by interfering with their association with
D-type cyclins (Sherr and Roberts, 1999). In contrast, CKIs of
the Cip/Kip family bind to both cyclin and CDK subunits and
can modulate the activities of cyclin D-, E-, A-, and B-CDK com-
plexes (Sherr and Roberts, 1999). The Cip/Kip family members
p21Cip1/Waf1/Sdi1(p21, encoded by cdkn1a) (el-Deiry et al.,
1993; Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993),
p27Kip1(p27, cdkn1b) (Polyak et al., 1994a; Polyak et al.,
1994b; Toyoshima and Hunter, 1994), and p57Kip2(p57, cdkn1c)
(Lee et al., 1995; Matsuoka et al., 1995) share a conserved
N-terminal domain that mediates binding to cyclins and CDKs
but diverge in the remainder of their sequence, suggesting
that each of these proteins could have distinct functions and
A vast body of literature has described the importance of p21,
p27, and p57 in restraining proliferation during development, dif-
ferentiation, and response to cellular stresses (Sherr and Rob-
erts, 1999), although each has specific biological functions that
distinguish it from the other family members. Thus, different anti-
proliferative signals tend to cause elevated expression of only
a subset of the Cip/Kip proteins. For example, p21 is an impor-
tant transcriptional target of p53 and mediates DNA-damage-
and Tyner, 1999). In contrast to p21, p27 expression is usually
elevated in mitogen-starved cells and other quiescent states,
and the protein is rapidly downregulated as cells enter the cell
cycle (Besson et al., 2006; Coats et al., 1996). Several lines of
evidence point toward an important role for p57 in the regulation
of the cell cycle during embryonic development. Unlike its ubiq-
uitous siblings, p57 has a tissue-restricted expression pattern
et al., 1995). The transcriptional regulation of p57 is mediated by
factors that play critical roles during embryogenesis such as
Notch/Hes1, MyoD, BMP-2 and -6, and p73 (Blint et al., 2002;
Georgia et al., 2006; Gosselet et al., 2007; Vaccarello et al.,
2006). The cdkn1c gene is also an imprinted gene with preferred
expression of the maternal allele (Matsuoka et al., 1996), which
is recognized as a general mechanism to regulate embryonic
growth (Andrews et al., 2007). Importantly, p57 is the only CKI
to be required for embryonic development, as most mice lacking
the cdkn1c gene have multiple developmental abnormalities and
die at birth (Yan et al., 1997; Zhang et al., 1997).
The importance of the Cip/Kip proteins in cell-cycle regulation
is underscored by the phenotypes of the knockout mice for each
size and multiple organ hyperplasia, revealing the importance of
p27 in limiting growth (Fero et al., 1996). Although mice lacking
p21 do not display an overt hyperproliferative disorder, p21?/?
cells fail to undergo DNA-damage-induced cell-cycle arrest
and can reach higher saturation density (Deng et al., 1995).
Embryos lacking p57 exhibit hyperplasia in several organs and
delayed differentiation, probably due to failure to exit the cell
cycle in a timely fashion (Zhang et al., 1997).
Although p21, p27, and p57 were initially considered as tumor
suppressors based on their ability to block cell proliferation, it
rapidly became clear that the situation was not so simple. p21,
p27, and p57 are also involved in the regulation of cellular pro-
cesses beyond cell-cycle regulation, including transcription,
circumstances. Moreover, it appears that the loss or subversion
of the regulatory mechanisms governing Cip/Kip proteins may
lead to the specific loss of the tumor suppressor function of
the CKI while maintaining the oncogenic ones. Herein, we will
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
examine these various functions, how they are regulated,
and their significance in vivo, especially in the context of tumor-
Cip/Kip Proteins in Cell-Cycle Regulation
Cip/Kip proteins were initially characterized as strict inhibitors of
all cyclin-CDK complexes, albeit displaying lower affinity toward
cyclin B-CDK1 (Sherr and Roberts, 1999). The crystal structure
of the N-terminal cyclin and CDK-binding domains of p27 (aa
22–106) bound to cyclin A-CDK2 revealed that the CKI occludes
a substrate interaction domain on the cyclin subunit and inserts
itself in the catalytic cleft of the CDK, thereby preventing ATP
binding and catalytic activity (Russo et al., 1996). However, sub-
sequent studies reported that p21, p27, and p57 participated in
the assembly of catalytically active cyclin D-CDK4/6 complexes
(LaBaer et al., 1997). Therefore, not only could Cip/Kip proteins
promote cyclin D-dependent events, but the sequestration of
CKIs into cyclin D-CDK4/6 complexes could allow the down-
stream activation of cyclin E-CDK2 (Cheng et al., 1998; Perez-
Roger et al., 1999; Polyak et al., 1994a; Reynisdottir et al.,
1995). The latter is confirmed in cyclin D1 and D2 knockout
mice, which display reduced CDK2-associated kinase activity,
likely due to the increased availability of Cip/Kip proteins to
bind to CDK2 (Geng et al., 2001; Perez-Roger et al., 1999). Nev-
ertheless, there are other reports demonstrating inhibition of
cyclin D-CDK4/6 complexes by Cip/Kip proteins, and therefore
it was anticipated that the effect of these CKIs on CDK activity
would be modulated by other factors.
The Cip/Kip proteins are intrinsically unstructured, adopting
specific tertiary conformations only after binding to other
proteins (Adkins and Lumb, 2002; Esteve et al., 2003; Lacy
et al., 2004). This conformational flexibility suggests that phos-
phorylation events and protein-protein interactions may modify
the folding of the CKIs, thereby modulating their ability to inhibit
cyclin-CDK complexes. Likewise, it may explain why CKIs are
various cellular functions.
Indeed, it appears that the binding specificity of Cip/Kip pro-
teins is modulated by their phosphorylation on distinct residues,
and their potency to inhibit cyclin-CDK complexes can be mod-
ified by binding to other proteins. For example, phosphorylation
of p21 on Thr-57 (by CDK2 or glycogen synthase kinase 3b
[GSK3b]) increases the ability of p21 to bind to cyclin B1-CDK1
complexes at the G2/M transition, without inhibiting the com-
plexes, thus promoting cell-cycle progression (Dash and
El-Deiry, 2005). Likewise, the phosphorylation of p27 on Tyr-
74, -88 and/or -89bySrc, Lyn, orAbl, greatly decreased the abil-
ity of p27 to inhibit CDK2 containing complexes, asTyr-88 ispart
of the 310-helix that normally inserts into the ATP-binding site of
the CDK (Chu et al., 2007; Grimmler et al., 2007). In addition, p27
phosphorylation on Tyr-88 and -89 was reported to decrease its
affinity for CDK2 while increasing that for CDK4 complexes
(Kardinal et al., 2006). A recent report suggested that Tyr-88
phosphorylation was cell-cycle regulated and modulated the
ability of p27 to inhibit cyclin D-CDK4 complexes (James et al.,
2008). p27 was a potent cyclin D-CDK4 inhibitor in quiescent
cells, but not in cycling cells, in which it was tyrosine phosphor-
ylated. Moreover, the weak inhibitory form of p27, phosphory-
lated on Tyr-88, could be converted to a potent inhibitor bytreat-
ment with protein tyrosine phosphatase (PTP) (James et al.,
2008). However, the physiological significance of this regulatory
pathway of p27 in vivo remains to be investigated. Several other
phospho-sites on p21 and p27 also indirectly affect the ability of
these proteins to bind to and inhibit cyclin-CDK complexes by
controlling their subcellular localization (see below) (Borriello
et al., 2007; Child and Mann, 2006).
A number of proteins can either enhance or diminish the inhib-
itory effect of CKIs on cyclin-CDK complexes by forming quater-
nary complexes and potentially altering the conformation of the
CKI bound to these complexes. Human papillomavirus-16 E7
protein can bind to p21 and p27 and abrogate their inhibitory ac-
tivity toward CDK2-containing complexes (Funk et al., 1997;
Jones et al., 1997). The nuclear protein Set/TAF1 (Template-
activating factor-1) was found to associate with the C-terminal
part of p21, which reversed the inhibition of cyclin E-CDK2 and
enhanced the inhibition of cyclin B-CDK1 complexes (Canela
et al., 2003; Estanyol et al., 1999). Two other proteins, TOK1a
(p21 and CDK-associated protein-1) (Ono et al., 2000) and the
multifunctional domain protein TSG101 (Tumor susceptibility
gene-101) (Oh et al., 2002), can also associate with p21 to en-
hance cyclin-CDK inhibition. Further studies are warranted to
elucidate how the binding of these proteins may alter the confor-
mation of the CKI bound to cyclin-CDK complexes and to deter-
minehowsignificant theseinteractions areinthecontrolof p21’s
function. The general conclusion is that Cip/Kip proteins are
potent inhibitors of cyclin-CDK complexes, although most of
the findings in recent years have shown that their inhibitory
potential is dependent on cellular context and regulated via
phosphorylations and protein-protein interactions.
Cip/Kip proteins also modulate cell-cycle progression inde-
pendently of cyclins and CDKs via the inhibition of components
of the replication machinery. p21 was first reported to bind to
proliferating cell nuclear antigen (PCNA), a DNA polymerase
d processivity factor, via its C terminus (aa 143–160), thereby
blocking processive DNA synthesis (Luo et al., 1995). This func-
tion of p21 is modulated by phosphorylations on Ser-145 (by
PKB/Akt [protein kinase B] and possibly PKA), Ser-146 (by
PKC z), or Ser-160 (by PKC), which prevent p21 from binding
to PCNA (Child and Mann, 2006). p57 was subsequently found
to interact with PCNA (via aa 271–275), preventing its activity
and blocking DNA replication (Watanabe et al., 1998). Although
no interaction between p27 and PCNA has been reported, p27
may also inhibit DNA synthesis via the interaction and inhibition
of minichromosome maintenance-7 (MCM7), a subunit of the
MCM2-7 replication fork helicase, an activity that lies within the
C-terminal part of p27 (aa 144–198) (Nallamshetty et al., 2005).
Regulation of p21 and p27 Localization
Phosphorylation on various amino acids controls many aspects
of Cip/Kip protein biology, not only by altering the Cip/Kip pro-
teins’ affinity for specific cyclin-CDK complexes and other pro-
teins, but also their stability (reviewed in Borriello et al., 2007;
Child and Mann, 2006), and their subcellular localization. Phos-
phorylation of p21 on two sites, Thr-145 and Ser-153, by PKB/
Akt and PKC, respectively, promote the cytoplasmic retention
of p21 (Child and Mann, 2006; Rodriguez-Vilarrupla et al.,
2005; Zhou etal., 2001).The first site isproximal to p21’snuclear
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
localization sequence (NLS) and prevents the interaction with
importin when phosphorylated, and the second one occludes
nuclear import (Child and Mann, 2006; Rodriguez-Vilarrupla
et al., 2005; Zhou et al., 2001). Three phosphorylation sites on
p27 were found to cause cytoplasmic localization. Ser-10 phos-
phorylation, which stabilizes the protein in quiescent cells,
causes its export from the nucleus in G1 phase by providing
a binding site for CRM1/exportin1 (Besson et al., 2006; Connor
et al., 2003; Rodier et al., 2001). In quiescent cells, Mirk/Dirk ki-
nase has been proposed to phosphorylate this site, whereas in
with stathmin (KIS), PKB/Akt, and extracellular signal-regulated
kinase-2 (ERK2) as the physiologically relevant in vivo kinases
(Besson et al., 2004a). On the other hand, phosphorylation of
Thr-157 (a site not conserved in the mouse protein) by PKB/
Akt or Thr-198 by PKB/Akt or p90 ribosomal S6-kinase
(p90RSK) causes retention of p27 in the cytoplasm by promoting
the association of p27 with 14-3-3, which prevents p27 from
interacting with importin a and transport in the nucleus (Fujita
et al., 2003; Liang et al., 2002; Sekimoto et al., 2004). The extent
to which altered subcellular localization modulates the many
biological effects of Kip/Cip proteins is an area of considerable
interest and active investigation.
Cip/Kip Proteins, Tumor Suppressors,.and
There is a great deal of evidence, both from clinical studies and
p27, and p57. However, the full extent to which p27, and proba-
timated by the study of mouse models that treated these CKIs
solely as CDK inhibitors. Only very recently has the development
of a new mouse model allowed us to ask whether CDK-indepen-
dent functions of p27 need to be considered in order to under-
stand the biological effects of p27. Those studies revealed that
the CDK-independent functions of p27 play significant roles,
not only in normal development, but also during tumorigenesis.
The p27?/?mice, which spontaneously develop adenomas of
theintermediate lobeof the pituitary gland andare moresuscep-
tible to tumorigenesis induced by chemical carcinogens or irra-
diation, gave firm evidence for a tumor suppressor role of p27
(Fero et al., 1996, 1998; Kiyokawa et al., 1996; Nakayama
et al., 1996). In fact, the loss of one allele of cdkn1b is sufficient
over, the tumor-prone phenotypes caused by the loss of other
tumor suppressor genes, such as Rb (Retinoblastoma), PTEN
(phosphatase and tensin homolog deleted on chromosome
ten), p16INK4a, p18INK4c, or APC (adenomatous poliposis coli),
are enhanced by the simultaneous loss of p27 (Di Cristofano
et al., 2001; Franklin et al., 2000; Malumbres et al., 2000;
Martin-Caballero et al., 2004; Park et al., 1999). In rats, an in-
herited syndrome of multiple endocrine neoplasia (MEN) was
linked to a germline nonsense mutation in the cdkn1b gene,
and germline mutations in the human cdkn1b gene are also
associated with MEN (Georgitsi et al., 2007; Pellegata et al.,
2006). p27 expression is a prognostic marker for clinical out-
come in human cancers. Low amounts of nuclear p27 protein
are frequently observed in a broad array of human malignancies,
including carcinomas of the breast, colon, prostate, ovary, lung,
tumor aggressiveness (Besson et al., 2004a; Blain et al., 2003;
Slingerland and Pagano, 2000). However, unlike the classic
tumor suppressor genes Rb or p53, inactivating mutations of
the cdkn1b gene in tumors are extremely rare (Blain et al.,
2003; Fero et al., 1998; Ponce-Castaneda et al., 1995; Slinger-
land and Pagano, 2000). Instead, p27 is downregulated by other
mechanisms, including proteolytic degradation, decreased
transcription, or by cytoplasmic mislocalization. In addition, the
inhibition of p27 expression by miRNAs in glioblastomas and
prostate carcinoma cell lines was recently described, and this
might constitute yet another way to decrease p27 levels in
tumors (Galardi et al., 2007; le Sage et al., 2007).
Increasing evidence points to the importance of subcellular
localization in the control of p27’s function and raises the possi-
bility that cytoplasmic p27 may actively contribute to tumorigen-
esis (Besson et al., 2004a). This is supported by the fact that
elevated cytoplasmic localization of p27 is a negative prognostic
factor in subsets of certain tumor types, including carcinomas of
the breast, cervix, esophagus, ovary, uterus, some leukemias
and lymphomas, and in melanomas (Besson et al., 2004a; Blain
et al., 2003; Denicourt et al., 2007; Qi et al., 2006; Rosen et al.,
2005; Slingerland and Pagano, 2000). In contrast, mice express-
ing p27S10A, which is mostly nuclear, were partially resistant to
urethane-induced tumorigenesis despite reduced p27 protein
levels (Besson et al., 2006).
tate cancer models, p27+/?mice were more prone to tumorigen-
esis than p27?/?animals, suggesting an active contribution of
the remaining p27 allele during tumor development (Gao et al.,
2004; Muraoka et al., 2002). Moreover, a knockin mouse model
in which point mutations in p27 prevent its interaction with
cyclins and CDKs (p27CK?) revealed that in contrast to p27?/?
mice, which spontaneously develop only pituitary tumors,
p27CK?mice developed hyperplastic lesions and tumors in mul-
tiple organs, including the lung, retina, pituitary, ovary, adrenals,
spleen, and lymphomas (Besson et al., 2007). Although the
of p27, they did not address whether that effect was exerted in
the nucleus or cytoplasm, which should be the focus of future
studies. Overall, there are clear indications, both from clinical
studies and animal models, that p27 has a tumor suppressor
function which is exerted in the nucleus via its inhibitory inter-
actions with cyclin-CDK enzymes. Collectively, the data also
suggest that p27 can promote oncogenesis and this occurs
independently of its interaction with cyclins and CDKs, most
likely in the cell cytoplasm.
In the lung and retina of p27CK?mice, the development of tu-
mors was associated with the expansion of stem/progenitor cell
populations (Besson et al., 2007). Thus, this mouse model indi-
cates that p27, independently of its role as a CDKinhibitor, could
function as an oncogene in vivo, possibly by deregulating the
proliferation and/or differentiation of stem/progenitor cells.
Therefore, a reasonable hypothesis to account for the lack of
mutations in the cdkn1b gene in human cancers is that there is
a selection both for the loss of the CDK-inhibitory, tumor-sup-
pressive function of p27 in the nucleus and for the maintenance
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
of thecyclin-CDK-independent,oncogenicfunctions of p27.The
misregulation of stem/progenitor cells by p27 could constitute
an important pathway by which it promotes tumorigenesis. The
CDK-independent functions ofp27arejustbeginningto bestud-
ied, and the mechanism by which they can promote tumorigen-
esis and stem-cell amplification remains to be evaluated. In
particular, whether the participation of p27 in tumor initiation
and progression relies on single or multiple distinct functions of
the protein remain unknown.
asstrong,andthemanyreportsoffer acontrasting viewonp21’s
role during tumorigenesis. Although p21?/?mice were initially
described to remain tumor-free until 7 months of age, a subse-
quent study found that they were susceptible to spontaneous
tumorigenesis at an average age of 16 months and developed
mostly histiocytic sarcomas, hemangiomas, and lymphomas
(Dengetal.,1995;Martin-Caballero etal.,2001).Also supporting
a tumor suppressor role for p21, the tumor-prone phenotype
caused by the loss of p18INK4cor APC was enhanced by con-
comitant loss of p21 (Malumbres et al., 2000). On the other
hand, loss of p21 actually delayed tumor development in
ATM?/?, p53?/?, or irradiated wild-type mice; this was attributed
to increased apoptosis of tumor cells in the absence of p21
(De la Cueva et al., 2006; Martin-Caballero et al., 2001; Wang
et al., 1997). Likewise, PDGF-induced gliomagenesis was dra-
matically reduced in mice lacking p21 (Liu et al., 2007). The
tumor-promoting effect in this model required the cyclin-binding
domain of p21 to increase the amount of nuclear cyclin D1 (Liu
et al., 2007), providing a clear example of tumorigenic role for
p21 dependent on its cyclin-CDK regulatory function. Indeed,
binding of p21 to cyclin D1 prevents its export from the nucleus
and its subsequent degradation in the cytoplasm (Alt et al.,
2002). Therefore, as with p27, the study of the p21-null mouse
may have led to a misunderstanding of the complex role that
tion of p21 separation-of-function mutations is likely to offer
significant new insights into the roles of p21 in tumors.
Indeed, clinical studies analyzing human tumors have also
in the cdkn1a gene are exceedingly rare (Roninson, 2002). While
loss of p21 is a negative prognostic marker in some cancer
types, it appears that overexpression or cytoplasmic localization
of p21 is a marker of poor prognosis and aggressive tumors in
carcinomas of the pancreas, breast, prostate, ovary, cervix,
and in glioblastomas (Biankin et al., 2001; Roninson, 2002).
Thus, it appears likely that similar to p27, exclusion of p21
from the nucleus results in the loss of its tumor-suppressor
function while selectively maintaining other tumor-promoting
There is ample evidence indicating a tumor-suppressor role
for p57, and, unlike its siblings, no oncogenic activity has been
through several mechanisms, including maternal-specific loss of
heterozygosity (LOH); loss of imprinting; promoter methylation in
carcinomas of the lung, gastrointestinal tract, liver, pancreas,
breast, head, and neck; acute myeloid leukemia; and others
(Higashimoto et al., 2006; Kikuchi et al., 2002; Kobatake et al.,
2004; Kondo et al., 1996; Lai et al., 2000). The perinatal lethality
of mice lacking p57 has limited further investigations to deter-
mine the significance of p57 as a tumor suppressor in the adult
in vivo, and conditional p57 mutant animals have not yet been
constructed. However, in one study in which approximately
10% of p57-null animals survived to adulthood, no spontaneous
tumors were observed at the age of 5 months (Yan et al., 1997).
It is not known whether the tumor-suppressor function of p57
domains unique to p57 (PAPA repeats and QT domain in human
p57), whose functions remain unclear, also participate. Cdkn1c
(encoding p57), a maternally expressed gene, resides within an
imprinted gene cluster on chromosome 11p15 that has been
implicated in the development of Beckwith-Wiedemann syn-
drome (BWS). BWS is a heterogeneous overgrowth syndrome
associated with various developmental defects and increased
risk of embryonal tumor development (Weksberg et al., 2005).
Cdkn1c mutations are found in 5%–10% of sporadic BWS cases
2005). In addition, maternal-specific methylation of a chromatin
insulator, KvDMR1, is lost in 30%–50% of BWS cases, causing
mutations rather than complete deletion or silencing of the p57
gene, and the majority of these mutations are located outside
of the cyclin-CDK regulatory region in the C-terminal part of the
protein (Bhuiyan et al., 1999; Hatada et al., 1997; Lee et al.,
1997; O’Keefe et al., 1997). These support the hypothesis that
the role of p57 in tumorigenesis may extend beyond cyclin-
Cip/Kip Proteins and Apoptosis
CKIs can modulate apoptosis in various ways, depending on the
cellular context and the pathway they target (Figure 1). Numer-
ous reports have suggested proapoptotic roles for p21 or p27
by overexpression in cancer cell lines or in response to antican-
cer agents; however, in these studies, p21 was overexpressed
using adenoviral vectors, and their relevance to physiological
conditions is uncertain. One way by which Cip/Kip proteins pro-
tect against apoptosis is via their CDK inhibitory activity. This
was first shown in endothelial cells, where caspase-mediated
cleavage of p21 and p27 upregulates CDK2 activity, thereby
enhancing the apoptotic program (Levkau et al., 1998). Indeed,
both dominant-negative CDK2 and a caspase-resistant mutant
of p21 suppressed apoptosis in this model. However, most of
the studies investigating a role for Cip/Kip proteins in apoptosis
didnottestwhetherthiswaslinked toCDKregulation, andfuture
experiments should aim at exploring how exactly the CKIs are
working in this process. There are also numerous in vivo models
of apoptosis being increased in CKI-deficient mice, but again
whether these phenotypes were CDK mediated was not deter-
mined. For example, several of the phenotypes of the p57-null
mice were shown to be a consequence of increased apoptosis
attributed to a failure to exit the cell cycle and differentiate
(Yan et al., 1997; Zhang et al., 1997). Likewise, p27 has been
reported to play a protective role in safeguarding normal tissues
from excessive apoptosis during inflammatory injury (Opha-
scharoensuk et al., 1998). This ability of CKIs to limit or prevent
apoptosis is also particularly relevant in cancer therapies, and
induction of p21 (by p53) and p27 have been associated with
resistance to apoptosis induced by cytotoxic drugs or irradiation
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
(De la Cueva et al., 2006; Eymin and Brambilla, 2004; Martin-
Caballero et al., 2001; St Croix et al., 1996; Wang et al., 1997).
Theimpact ofCKIsonapoptosismay notbeexplained entirely
by CDK regulation. For instance, p57 could either promote or in-
hibit apoptosis. In both cases, this was independent of its role in
cyclin-CDK regulation: p57 and a mutant lacking the ability to
interact with cyclins and CDKs were equally able to promote
staurosporine-induced apoptosis, which involved the transloca-
tion of p57 to mitochondria (Vlachos et al., 2007). On the other
hand, p57 was found to bind via its QT domain (aa 238–316) to
the stress-activated kinase JNK1/SAPK, inhibiting its kinase
activity, and expression of the QT domain was sufficient to block
UV- or MEKK1-induced apoptosis, mediated by JNK1 (Chang
et al., 2003). Substantial evidence points to a role for p21 in pro-
regulation, and protein-protein interactions; this constitutes the
best-characterized oncogenic function of the protein (Roninson,
2002). p21 could block Fas-mediated cell death by directly bind-
ing via its N terminus to pro-caspase-3, which prevented its con-
version to the active caspase form (Suzuki et al., 1998). p21 can
prevent stress-induced apoptosis mediated by the JNK and p38
signaling pathways by acting at two distinct levels. First, p21
binds to and inhibits the activity of the MAPKKK ASK1/MEKK5
(Huang et al., 2003). Second, like p57, p21 can bind to JNK
kinases through its cyclin-CDK binding domain, which both
inhibits JNK activity and prevents JNK activation by upstream
kinases (Shim et al., 1996). So far, there have not been any
reports to suggest that p27 can directly interfere with pro- or
Cip/Kip Proteins in Transcriptional Regulation
Similar to the regulation of apoptosis, Cip/Kip proteins can re-
press transcription indirectly by inhibiting cyclin-CDK com-
plexes, in turn preventing the phosphorylation of Rb-family pro-
teins (p107, p110, and p130). In their hypophosphorylated state,
Rb-related proteins sequester E2F family members, thereby re-
pressing their transcriptional targets (Sherr and Roberts, 1999).
In addition, Cip/Kip proteins can also modulate the activity of
transcription factors through direct binding to transcription fac-
tors (Figure 2). The N-terminal cyclin-CDK binding region of
p57 can interact with MyoD, protecting MyoD from degradation
and thus promoting transactivation of muscle-specific genes
(Reynaud et al., 2000). Interestingly, a similar mechanism is ob-
served between p27 and Neurogenin-2, and p27-mediated sta-
bilization of Neurogenin-2 promotes the differentiation of neuro-
nal progenitors in the cortex (Nguyen et al., 2006). This
interaction is evolutionarily conserved, since in Xenopus primary
neurogenesis also relies on the stabilization of X-NGNR1, a Neu-
rogenin-2 homolog, by p27Xic(Vernon et al., 2003).
p21 is also a potent regulator of several transcription factors.
Through direct binding, p21 inhibits the activities of E2F1,
c-Myc, and STAT3 (Coqueret and Gascan, 2000; Delavaine and
La Thangue, 1999; Kitaura et al., 2000). On the other hand, p21
can stimulate p300/Creb-binding protein (CBP) histone acetyl
transferase complex-mediated transcriptional activation (Snow-
den et al.,2000). Through direct binding to p300, p21 can disrupt
the activity of the CRD1-transcriptional-repression domain of
p300 and derepress transcription of target genes (Snowden
et al., 2000). The biological significance of the role of p21 in tran-
scriptional regulation is still poorly understood. For example,
Figure 2. Transcriptional Regulation by Cip/Kip Proteins
The CKIs p21, p27, and p57 can repress E2F-mediated transcription indirectly
via theinhibition of cyclin-CDK complexes,thereby maintaining Rb-family pro-
teins (Rb/p110, p107, and p130) in a hypophosphorylated state in which they
sequester E2F transcription factors. Cip/Kip proteins also regulate transcrip-
tions factors directly; for instance, p57 and p27 can interact via their N termini
with MyoD and Neurogenin-2 (Ngn-2), respectively, stabilizing them and pro-
moting transcription of their target genes. On the other hand, p21 binds to
E2F1, c-Myc, and STAT3 to inhibit their activities. p21 may also derepress
p300/CBP targets by inhibiting the transcriptional repression domain of p300.
Figure 1. Cip/Kip Proteins and Apoptosis
Cip/Kip proteins can inhibit apoptosis indirectly via the inhibition of
cyclin-CDK complexes, and p21 and p27 are targeted for cleavage
by caspases to promote cyclin-CDK activation during the apoptotic
process. Upregulation of the CKIs by cytotoxic agents can partici-
pate in the resistance of tumor cells to anticancer treatments. p21
and p57 may also directly prevent the induction of apoptosis by in-
terfering with activation of the stress-signaling pathways; for in-
stance, both bind to and inhibit the activity of JNK1/SAPK, and
p21 can also inhibit ASK1/MEKK5. p21 may also block the process-
ing of pro-caspase-3 into its active form.
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
p21, by associating with E2F1, mediates the transcriptional re-
pression of Wnt genes downstream of Notch to regulates kerati-
nocyte fate and growth (Devgan et al., 2005). One could envision
that in tumors in which p21 is highly expressed, this function
could play a significant role in regulating tumor progression.
Some indications suggest that p21 participates in the transcrip-
tional changes taking place in cells that undergo DNA-damage-
induced senescence following treatment with chemotherapeutic
agents, and these cells secrete a number of factors that promote
growth and survival of surrounding tumor cells (Roninson, 2003).
Cip/Kip Proteins in the Regulation of Rho Signaling
and Cytoskeletal Dynamics
Studies in yeast first suggested a direct functional link between
CDK inhibitors and regulators of cytoskeletal organization. Dur-
ing vegetative growth, the CDK inhibitor Far1 binds to and se-
questers Cdc24, a guanine-nucleotide exchange factor (GEF)
for Cdc42, in the nucleus. Activation of Cln/Cdc28 in the G1
releasing Cdc24, which is then exported to the bud site on the
plasma membrane where it regulates cytoskeletal organization
and cell polarity (Shimada et al.,2000). On the other hand, during
plex, and Far1 is required for the recruitment of Cdc24 to sites of
Gbgactivation at the cell cortex for polarized growth in response
to pheromones (Butty et al., 1998; Shimada et al., 2000).
In mammals, the Rho family of GTPases, composed of at least
20 members, regulates multiple signaling pathways with pleio-
tropic cellular responses. Rho and its effector Rho-kinase
(ROCK) are best known for their role in the regulation of actin-
stress-fiber formation, focal-adhesion assembly, as well as ac-
tin-myosin contractility (Etienne-Manneville and Hall, 2002).
Stress-fiber assembly is mediated by the activation of LIM-ki-
nase (LIMK) by ROCK, which in turn phosphorylates and inacti-
vates the actin-depolymerization factor cofilin to promote
stress-fiber formation. On the other hand, Rac and its effector
p21-activated kinase (PAK) induce actin rearrangements that
control the formation of lamellipodia and new focal contacts at
the cell edges (Ridley et al., 2003). Cell migration requires a tight
balance of Rho and Rac activities, and the dynamic activation
dinated. Indeed, insufficient levels of Rho-GTP will inhibit migra-
tion by preventing cells from achieving the level of adhesiveness
and traction needed for movement (Nobes and Hall, 1999; Wor-
adhesion and prevents focal adhesion turnover, resulting in the
inhibition of cell migration (Ren et al., 2000; Sahai et al., 2001;
Vial et al., 2003).
In a remarkable example of convergent evolution, CKI-medi-
ated regulation of cytoskeletal dynamics arose independently
in metazoans. Thus, all three Cip/Kip proteins inhibit the Rho/
ROCK/LIMK/Cofilin signaling pathway, albeit acting at distinct
levels (Figure 3) (Besson et al., 2004a). Interestingly, Rho can
also negatively regulate the levels of the p27 and p21 proteins
(Hu et al., 1999; Olson et al., 1998; Vidal et al., 2002). Thus,
Rho and p21 and p27 could participate in a negative-feedback
loop. The network of interactions between CDK inhibitors and
the Rho signaling pathway could very well represent a basic
mechanism to coordinate cytoskeletal functions with cell
p57 was found to interact with LIMK1 without inhibiting its ac-
tivity, and overexpression of p57 resulted in nuclear localization
of LIMK1 (Yokoo et al., 2003). This was accompanied by loss of
LIMK1-associated stress fibers, suggesting that the p57-LIMK1
other report, knockdown of p57 delayed the migration of neu-
rons in the cortical plate during mouse development; however,
whether this resulted from the sequestration of LIMK1 was not
investigated (Itoh et al., 2007).
Cytoplasmic p21 has been shown to bind and inhibit the Rho-
kinase ROCK1 (Lee and Helfman, 2003; Tanaka et al., 2002).
p21-mediated ROCK1 inhibition promoted neurite extension of
neuroblastoma cells and hyppocampal neurons in vitro (Tanaka
et al., 2002). Moreover, in rat, the transduction of a TAT-p21 fu-
sion protein at the site of spinal-cord injury promoted axonal re-
(Tanaka et al., 2004).
p21 inhibition of ROCK may also be selected for during tumor
progression to enhance cell motility. Ras-transformed cells are
highly motile and invasive, despite having high levels of Rho ac-
tivity. In fact, these cells have found ways to uncouple the effec-
tor pathways downstream of Rho. For instance, Ras-induced
ERK signaling uncouples Rho from inducing actin stress fibers
etal., 2001). Alternatively, Ras activation can induce p21 expres-
Once in the cytoplasm, p21 inhibits ROCK activity, thereby un-
coupling Rho from stress-fiber formation (Lee and Helfman,
2003). Further studies of tumor cells with high levels of cytoplas-
the acquisition of a motile and invasive phenotype.
The involvement of p27 in the regulation of migration was first
reportedin hepatocellular carcinoma cells,in whichtransduction
of a TAT-p27 protein induced migration (Nagahara et al., 1998).
Moreover, a recent report has shown that expression of even
subphysiological levelsofcytoplasmic p27inmelanoma cellsre-
sults in a profound increase in metastatic potential in vivo (Deni-
court etal., 2007). In these cells, both the export of p27 to the cy-
toplasm through its phosphorylation on Ser-10 and a region in
the C-terminal half of p27 were required for hepatocyte-
growth-factor-induced migration (McAllister etal., 2003). Further
analysis showed that the motility of p27-null mouse fibroblasts
was impaired due to an increase in RhoA activity and that normal
migration could be restored by inhibiting ROCK (Besson et al.,
2004b). Indeed, p27?/?cells had increased numbers of stress fi-
bers and focal adhesions and elevated levels of Rho-GTP (Bes-
son et al., 2004b). Overexpression experiments revealed that
p27 interacted with RhoA, thereby preventing RhoA activation
by interfering with RhoA binding to its GEFs (Besson et al.,
2004b). The regulation of RhoA by p27 was critical for proper mi-
gration of neuronal progenitor cells in the subventricular cortex
of developing mice (Kawauchi et al., 2006; Nguyen et al.,
2006). Thus, CKIs may be crucial for proper neurogenesis, given
that neuronal migration in the cerebral cortex is regulated by p27
in the subventricular zone and by p57 in the cortical plate.
These results indicate that p27 regulates cell migration by pre-
venting Rho activation. Different migratory responses, either
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
inhibition or promotion, have been observed in various cell types
by manipulating p27 levels (Besson et al., 2004a). It is therefore
possible that the effect of RhoA inhibition by p27 could vary, de-
pending on the cellular context, and reflect distinct requirements
for Rho and Rac for migration. For instance, cells that migrate
with an elongated morphology require Rac activity, whereas
cells migrating with a rounded morphology depend on Rho/
ROCK signals (Sahai and Marshall, 2003). In breast cancer and
glioblastoma cell lines, cytoplasmic p27-mediated inhibition of
RhoA activity promoted motility and invasion, and knockdown
of p27 impaired tumorigenicity and invasiveness of tumor xeno-
grafts in mice (Wu et al., 2006). Recently, cyclin D1 was found to
promote cell motility in a p27-dependent manner (Li et al., 2006).
Cyclin D1is overexpressed is several types of cancers in human,
and this correlates with tumor metastasis in some tumors. Thus,
itispossiblethat sequestration of p27in cyclin D1-CDK4/6 com-
plexes could both inactivate the cell-cycle inhibitory function of
p27 while promoting its role in cell migration.
While moststudies havefocusedonthe effect ofp27inhibition
of RhoA on migration, a new study suggests that p27 could be
more widely involved in the modulation of the pleiotropic effects
of RhoA signals. Indeed, RhoA was recently found to positively
regulate PTEN, the lipid phosphatase best known to counteract
phosphatidyl inositol 3-kinase (PI 3-K) activation. It turns out that
PI 3-K can negatively regulate PTEN in part via a pathway involv-
ing PKB/Akt-induced cytoplasmic localization of p27, which in-
hibits RhoA (Papakonstanti et al., 2007). Thus, cytoplasmic
p27 could participate in the inactivation of the tumor suppressor
PTEN. p27 inhibits RhoA activation by preventing the RhoA/GEF
interaction. However, p27 did not interfere equally with the activ-
ity of all GEFs tested, suggesting that p27 may only inhibit RhoA
activation by select upstream pathways (Besson et al., 2004b;
A.B. and J.M.R., unpublished data).
Although the factthat Cip/Kip proteins canaffect both the cell-
division cycle or cytoskeletal structure may represent functions
that are executed independently of one another, a more interest-
ing hypothesis is that this organization underlies a pathway that
serves to coordinately regulate both processes. Indeed, it is
common to observe an alternation between episodes of cell
movement and periods of cell proliferation. Some examples in-
clude neural-crest cell migration, which occurs without concom-
itant cell division (Perris, 1997); woundhealing, during whichker-
atinocytes first migrate into the wound bed before beginning to
proliferate (Martin, 1997); and gastrulation, during which time
cell division is suppressed by a prolongation of the cell cycle
(Nance and Priess, 2002). Cell migration and cell division are of-
ten activated by the same upstream signaling pathways (Besson
et al., 2004a; Collins et al., 1999; Frey et al., 2004), but the mech-
anisms that determine whether a cell chooses to migrate or di-
vide are not well understood. That CKIs directly modulate both
processes and their regulation by mitogenic stimuli suggests
that they may also regulate the choice between cell movement
and cell proliferation. For instance, high levels of p27 would
inhibit cell proliferation and promote cell migration and,
Figure 3. Regulation of Rho Signaling and Cytoskeletal Dynamics by Cip/Kip Proteins
In the nucleus, Cip/Kip proteins primarily function to restrict the activities of cyclin-CDK complexes. Phosphorylation of p27 on Ser-10 promotes its binding tothe
exportin CRM1 and nuclear export. On the other hand, phosphorylations on T157 (by Akt) or T198 (by Akt or p90Rsk) promote binding to 14-3-3 proteins and
prevent the reentry of p27 in the nucleus. In the cytosol, p27 can bind to RhoA, preventing its activation by its GEFs (guanine-nucleotide exchange factors), lead-
ing to decreased actin stress fiber and focal-adhesion formation and resulting in several cell types in increased migration, invasion, and metastasis. PI3K-AKT
induction of cytoplasmic localization of p27 is also involved in the inhibition of PTEN activation via p27-mediated inhibition of the RhoA-ROCK pathway. p21 cy-
toplasmic localization is induced by phosphorylation on T145 and S153 by Akt and PKC, respectively; however, how p21 is exported from the nucleus is unclear.
Cytoplasmicp21canbindtoROCK,inhibitingitskinase activity, resulting indecreasedactinstressfibers formation. Themechanism ofnucleo-cytoplasmic shut-
tling of p57 is unknown at this point. Cytoplasmic p57 can bind to LIMK and induce its translocation into the nucleus, resulting in loss of actin stress fibers.
Developmental Cell 14, February 2008 ª2008 Elsevier Inc.
conversely, low levels of p27 would stimulate proliferation and
inhibit cell movement. Misregulation of p27 in tumors may result
in the simultaneous promotion of both cell proliferation and cell
migration and therefore contributes to the invasive phenotype
of aggressive cancers.
Over the years, it has become evident that CKIs of the Cip/Kip
family are not merely involved in limiting cell division but are mul-
tifunctional proteins playing key roles in the regulation of the cell
cycle, cell survival, transcription, differentiation, cytoskeletal dy-
namics, cell migration, and probably other, as yet undiscovered,
functions. The importance of the cyclin-CDK-independent func-
tions of these CKIs is just beginning to be grasped, and much
work remains to be done before we have a clear understanding
of the regulation and interplay between the various roles of these
proteins. For example, why some terminally differentiated cells,
like neurons, express cyclins, CDKs, and their inhibitors remains
a mystery. An attractive hypothesis is that CKIs may act as mo-
lecular switches to coordinate cell proliferation with the other
cellular processes they govern.
From the perspective of cancer biology, CKIs play a dual role
during tumorigenesis, acting as both tumor suppressors and
oncogenes. Indeed, in tumors the dysregulation of various sig-
naling pathways, such as PKB/Akt, inactivates the tumor-sup-
pressor functions of these proteins but maintains or even exac-
erbates the oncogenic ones. Thus, for therapeutic purposes,
simply increasing the expression of these CKIs may not be
beneficial and could have consequences opposite to those
intended. A major challenge is now to gain the knowledge that
will permit the specific targeting the oncogenic functions of
CKIs while maintaining or restoring the tumor suppressive func-
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pour la Recherche sur le Cancer. S.F.D. is an Investigator of the Howard
Hughes Medical Institute. J.M.R. is supported by NIH grant 1R01CA118043.
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