Less Understood Issues: P21cip1 in Mitosis and Its Therapeutic Potential

REVIEW Less understood issues: p21Cip1 in mitosis and its therapeutic potential

N-N Kreis, F Louwen and J Yuan

p21Cip1 is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/ cyclin B1, and is important for a ﬁne-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will brieﬂy summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.

Oncogene (2015) 34, 1758–1767; doi:10.1038/onc.2014.133; published online 26 May 2014

INTRODUCTION arguing about the role of p21 as a classical tumor suppressor. The progression through the cell cycle is accurately regulated. This notion has been revised by a later study reporting that Loss of cell cycle control promotes tumorigenesis and is one of the p21-knockout mice were, at an average age of 16 months, hallmarks of cancer cells.1 Key regulators of the cell cycle are a susceptible to the development of spontaneous tumors like family of serine/threonine kinases: cyclin-dependent kinases (Cdk). histiocytic sarcomas, hemangiomas, B-cell lymphomas and lung The Cdks act at different stages of the cell cycle and are carcinomas as well as to severe glomerulonephritis at an average 13 responsible for the transition from one cell cycle phase to the of 9.6 months. In addition, the combined knockout of p21 Kip1 next.2,3 They are thus strictly controlled, among others, by positive and p27 in mice further increased the occurrence of 14 regulators like cyclins, which can be counteracted by negative spontaneous tumors, strongly suggestive of p21 as a tumor regulators the Cdk inhibitors (CKI).2,3 CKIs are divided into two suppressor in vivo. fi fi individual families on the basis of their structure and specificities. p21, encoded by CDKN1A, was the rst identi ed member of The first family, containing p15INK4b, p16INK4a, p18INK4c and the CKIs. It was independently isolated by several groups and p19INK4d (Inhibitors of Cdk4), binds and inhibits only Cdk4/Cdk6 named as a result of its distinct functions as a subunit of the cyclin D1 immunocomplex (p21),15,16 a wild-type p53-activated fragment complexes. The second family is comprised of Cip/Kip proteins 17 18 (Cdk-interacting protein/kinase-inhibitory protein) and character- (WAF1), a Cdk-interacting protein (Cip1), a senescent cell- derived inhibitor-1 (sdi1),19 a melanoma differentiation-associated ized by its ability to bind to a much broader range of Cdks as well 20 as cyclin subunits. This class includes p21Cip1 (hereafter referred to gene 6 (mda6) and also as Cdk2-associated protein-20 in mouse fi 21 22 fl as p21), p27Kip1 and p57Kip2, who are evolutionally highly broblasts (CAP20). The numerous names of p21 re ect its conserved and are mostly able to substitute each other.4–6 p21 complexity and manifold capabilities, correlate with an increasing and p57Kip2 are uniquely expressed in mammals, whereas p27Kip1 appreciation of its importance in a wide variety of cellular is conserved throughout different species. It is found in budding processes. yeast (Sic1), fission yeast (Rum1), Drosophila (Dacapo) and Xenopus (Xic1).7 CKIs have been thoroughly investigated in vivo: mice lacking STRUCTURE OF P21 functional CKIs reveal a broad spectrum of phenotypes.8 Interest- In free solution, p21 is an intrinsically unstructured protein with ingly, p57Kip2 is the only member required for embryonic 15–20% of secondary structure and a lack of tertiary structure.23–25 development. About 90% of the p57Kip2-knockout mice were Conformational analysis suggests that posttranslational modifica- embryonic or neonatal lethal.9 Of the INK4 inhibitors, only tions of p21 like phosphorylation may alter its folding, subcellular p16INK4a-knockout mice showed an increased incidence of localization and mediate different binding partners, referred to as spontaneous and carcinogen-induced tumor development.10,11 a ‘folding-on-binding mechanism’.24 Through its ‘binding promis- However, p21-knockout mice remained tumor-free upon 7 months cuity’ and diversity,23,26 p21 is able to bind to different Cdks that with a defective G1 checkpoint in mouse embryonic fibroblasts,12 have a sequence homology of 57–83% as well as to varying cyclins

Department of Gynecology and Obstetrics, J.W. Goethe-University, Frankfurt, Germany. Correspondence: Dr N-N Kreis or Dr J Yuan, Department of Gynecology and Obstetrics, J.W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. E-mail: [email protected] or [email protected] Received 13 March 2014; revised 16 April 2014; accepted 16 April 2014; published online 26 May 2014 p21 in mitosis N-N Kreis et al 1759 with a homology of 25–42%.24 With its Cdk binding domain (K proliferating cell nuclear antigen (PCNA), leading to an inhibition 27 29,32 site, amino acids 53–58) and the 310 helix, p21 binds to the Cdks, of the DNA synthesis. A second weaker cyclin binding domain whereby the residue Tyr-77 (Y77) of p21 blocks the ATP binding exists at the C-terminus (Cy2),27 overlapping with the PCNA pocket of the Cdks.28,29 The structural adaptability of p21 is binding region (Figure 1). There are also two nuclear export mediated through a stretchable linker helix domain at the signals,33 a nuclear localization signal34 and different degradation N-terminus between the conserved cyclin binding domain (Cy1) motifs like D-box and F-box in p21. In addition, the binding and the K site. The dynamic linker helix domain is required to fit of different proteins like calmodulin causes its nuclear the spectrum of distances between the distinct Cdk/cyclin translocation.35,36 Furthermore, p21 is acetylated at its N-terminus complexes.30,31 The N-terminus of p21 is important for the and is hence not a substrate for N-ubiquitylation.37 The association with the Cdks as well as the cyclin subunits. By modification of p21 impacts its structure and further influences contrast, the C-terminus of p21 mediates the binding to its function.

Figure 1. Illustration of the p21 structure. The LH-domain (linker helix) and the 310 helix with amino acid Y77 (Tyr-77) are indicated. For references see main text. Cy1 and Cy2, cyclin binding domains; F-box and D-box, destruction motifs; K, Cdk binding domain; NES, nuclear export signal; NLS, nuclear localization signal; PCNA, proliferating cell nuclear antigen; S2-ACE, acetylation of Ser-2.

Table 1. Phosphorylation sites of p21

Amino acid Kinase Function Ref

Thr-57 Cdk2/cyclin A Critical residue for the interaction with cyclin B1, promoting the Cdk1/cyclin B1 activity in G2/M 39 ERK2 In vitro phosphorylation site, cytoplasmic translocation of p21, simultaneous phosphorylation of Thr-57 and 44 Ser-130 leads to degradation of p21 GSK3β In vitro phosphorylation site, destabilization of p21 in endothelial cells 80 JNK1/p38α In vitro phosphorylation site, increased p21 stability mediated through Mst-1/2 after disruption of actin 75,76 cytoskeleton Ser-98 ASK1 Binding to and inhibition of ASK1 51 JNK1 In vitro phosphorylation site, abolished interaction of ASK1 and p21 after JNK1 inhibitor treatment 51 Ser-114 GSK3β UV-induced ATR-activation and p21 degradation (CRL4Cdt2), PCNA-dependent 62,81 Ser-130 Cdk2/cyclin E Decreased p21 stability via ubiquitin-dependent degradation (SCFSkp2), phosphorylation of Ser-130 stimulates 60 the ligation of p21 to ubiquitin Cdk6/cyclin K Essential for the cyclin K-mediated release of a p21-imposed G1 arrest, phosphorylatable form unable to 40 associate with Cdk2 in vivo ERK2 In vitro phosphorylation site, cytoplasmic translocation of p21, simultaneous phosphorylation of Thr-57 and Ser- 44 130 leads to degradation of p21 JNK1/p38α Increase in p21 stability in response to TGF-β 75 Thr-145 Akt1/PKB Cytoplasmic translocation of p21 in HER2/neu expressing cells, prevents binding to PCNA and decreases binding 41,45 of Cdk2/4 in endothelial cells DAPK3/ZIP Increased p21 stability, identified by phage-peptide display 77 Pim-1 Increased p21 stability, nuclear localization of p21 in H1299 lung cancer cells, inhibition of PCNA binding 46,49 Pim-2 Increased p21 stability in the nucleus, inhibition of cell proliferation in HCT116 cells at G1/S phase 42 PKA In vitro phosphorylation, inhibition of PCNA binding in Sf9 cells 50 Ser-146 Akt1/PKB Increased p21 stability, protection of glioblastoma cell lines from taxol-induced apoptosis, promotion of cell 79 survival Lats2 Cytoplasmic degradation of p21 in response to UV irradiation and promotion of apoptosis 82 NDR1/2 Knockdown of NDR1/2 results in G1 arrest and increased p21 stability 43 Pim-1 Indirect phosphorylation through Pim-1 overexpression, cytoplasmic translocation of p21 in H1299 lung cancer 46 cells, maintaining PCNA association PKC Inhibition of PCNA binding, in vivo detection with phospho-specific sera in Sf9 cells 50 PKCδ Increased p21 stability 78 PKCζ Decreased p21 stability in response to insulin 83 Ser-153 Mirk/Dyrk1 Cytoplasmic translocation of p21 blocks apoptosis during differentiation of C2C12 myoblasts 47 PKC Cytoplasmic translocation of p21 and disrupted stress fibers, binding of calmodulin induces nuclear 48 accumulation and inhibition of PKC-dependent phosphorylation Ser-160 PKC In vitro phosphorylation site, inhibition of PCNA binding in Sf9 cells 50 Abbreviations: ASK1, apoptosis signal-regulating kinase 1; Cdk1/2/4/6, cyclin-dependent kinase 1/2/4/6; CRL4Cdt2, Cullin 4-RING ubiquitin ligase/Cdc10- dependent transcript 2; DAPK3/ZIP, death-associated protein kinase 3; Dyrk1, dual-specificity tyrosine phosphorylation-regulated kinase 1; ERK2, extracellular signal-regulated kinase 2; GSK3β, glycogen synthase kinase 3β; HER2/neu, human epidermal growth factor receptor 2; JNK1, Jun N-terminal protein kinase 1; Lats2, large tumor suppressor 2; NDR1/2, nuclear-Dbf2-related kinase 1/2; Mirk, minibrain-related kinase; Mst-1/2, mammalian sterile 20-like-1/2 kinase; PCNA, proliferating cell nuclear antigen; PKA/B/C, protein kinase A/B/C; SCFSkp2, Skp1-Cullin-F-box/S phase kinase-associated protein 2; Sf9, Spodoptera frugiperda; TGF-β, transforming growth factor-β. Reversible phosphorylation serves as a mechanism for posttranslational regulation of p21. The function and responsible kinase are indicated.

© 2015 Macmillan Publishers Limited Oncogene (2015) 1758 – 1767 p21 in mitosis N-N Kreis et al 1760 FUNCTIONAL REGULATION OF P21 DURING THE CELL CYCLE cytoplasm.68 It targets cytoplasmic p21 and is crucial for the The functional regulation of p21 is more sophisticated than motility control of cells. It remains to be defined whether 68 initially assumed and relies basically on two posttranslational phosphorylation is required for this event. modifications: phosphorylation and ubiquitylation. Posttranscrip- For ubiquitin-independent proteolysis, free p21 is able to bind directly to various proteasome complexes like 20S (C8α),69 26S70 tional regulations of p21 by microRNA or RNA binding proteins are 71,72 summarized by Jung et al..38 and PA28γ/REGγ. Besides, knockdown of PA28γ/REGγ with siRNA causes a G2/M arrest.72 MDM2 and MDMX (murine double minute 2/X), independent of their E3 ligase activity, have been Phosphorylation shown to be able to promote the degradation of p21 in the G1 The process of reversible phosphorylation serves as a tool for the and early S phase.73,74 In addition, two less well-known E3 ligases posttranslational regulation of p21 and controls p21’s activity, of p21, p53RFP (p53-inducible RING-finger protein) and MKRN1 localization, stability and degradation. Phosphorylation of p21 at (Makorin RING finger protein 1), promote apoptosis upon DNA different sites promotes p21 on one hand to form several damage.53 complexes and on the other hand prevents it from binding to Moreover, phosphorylation of p21 by distinct kinases has been 29 distinct partners. p21, composed of 164 amino acids, contains known to have either a positive impact on the protein stability 13 serines (Ser), 8 threonines (Thr) and 2 tyrosines (Tyr). This including Thr-57,75,76 Ser-130,75 Thr-14542,46,49,77 and Ser- results in 14% possible phosphorylatable amino acid residues and 14643,78,79 or a negative impact like Thr-57,44,80 Ser-114,62,81 Ser- potential changes in the control of p21. Known phosphorylation 13044,60 and Ser-146.82,83 Taken together, p21’s protein stability sites of p21 are illustrated in Table 1. Important phosphorylatable and turnover are tightly controlled in an orchestrated and residues for cell cycle regulation are Thr-57,39 Ser-130,40 coordinated manner by diverse pathways upon different Thr-14541,42 and Ser-146.43 Importantly, phosphorylations that situations to allow its multifaceted biological roles. cause the cytoplasmic translocation of p21 like Thr-57,44 Ser-130,44 Thr-145,45 Ser-14646 and Ser-15347,48 have been reported to affect the therapeutic response, as discussed below. Further, other GENERAL PHYSIOLOGICAL FUNCTIONS OF P21 phosphorylations of p21 result in either the disruption of its p21 is a multifunctional, broad-acting protein with key roles in cell PCNA binding41,46,49,50 or modulate its interaction with apoptosis cycle regulation, apoptosis, differentiation, cell migration, cytos- signal-regulating kinase 1 (ASK1).51 keletal dynamics, transcription, DNA repair, onset of senescence and aging.38,84 p21 is considered to be the master regulator of the 6 Degradation cellular stress response system. Recent studies have evinced an additional important role of p21 in reprogramming of induced The p21 expression level is critical for the precise regulation of the pluripotent stem cells.85,86 The multiple functions of p21 are activity of its diverse binding partners. As a short-lived protein,52 excellently summarized and discussed in several review p21 offers the opportunity to rapidly respond to intrinsic – articles.6,38,84,87 89 p21 has a pathophysiological role in different and extrinsic alterations. Its turnover is controlled by ubiquitin- 90 87 91 92 diseases, like cancer, atherosclerosis and HIV infection. dependent and -independent pathways and the different p21 is regulated through p53-dependent17 and -independent subcellular pools of p21 are individually regulated, reviewed 93,94 53 pathways. p53 is able to bind to the p21 promoter and directly excellently by Starostina and Kipreos. On the other hand, p21 activate its expression.95 Independent of p53, a plenty of can be stabilized and protected against proteasomal degradation β γ 54 transcriptional activators, such as TGF- , EGF, IL6, IFN- , NGF, through binding to nucleophosmin/B23, WISp39/Hsp90 (heat 93,94,96 55 56 and oncogenes, such as Ras or c-myc, regulate p21. p21 was shock protein) or cyclin D1 mediated by Ras. originally described as a strict inhibitor of the Cdk family16 and a For ubiquitin-dependent proteolysis, which is the principle classical tumor suppressor supported by the fact that p21- degradation pathway, p21 is targeted by different E3 ligases. knockout mice obtained spontaneous tumors.13,14 First contra- Crucial factors for the recognition of p21 by an E3 ligase are its dictions arose in 1997, when it was discovered that low fi subcellular localization, binding partner, site-speci c phosphoryla- concentrations of p21 led to an increase in the assembly of 57,58 fi tion and the cell cycle phase. The E3 speci city is provided by Cdk4/6 and cyclin D complexes.97,98 Hence, the word inhibitor is in different tertiary structures of p21 adopted through its binding to some cases a ‘confusing misnomer’.99 As highlighted in Figure 2, 53 fi varied proteins. Three E3 ligases have been identi ed to be recent investigations postulate that p21 acts also as an oncogene important for p21 degradation in the nucleus: SCFSkp2 (Skp1- 100 ‘ – through its cytoplasmic translocation indicating its antagonistic Cullin-F-box/S phase kinase-associated protein 2),59 61 CRL4Cdt2 ’ – duality contributing, among others, to an inhibition of (Cullin 4-RING ubiquitin ligase/Cdc10-dependent transcript 2)62 64 96 ‘ ’ Cdc20 apoptosis. Given its function as a linchpin of cell fate, p21 is and APC/C (anaphase-promoting complex/cyclosome, cell responsible for the fine-tuned balance between cell cycle arrest/ 65 division cycle 20). During the G1/S transition, the E3 ligase repair/survival, apoptosis and senescence/quiescence.82,101,102 The Skp2 59–61 SCF targets phosphorylated p21 bound to Cdk2/cyclin E. dual ‘character’ of p21, mainly depending on its subcellular The phosphorylation at Ser-130 by it is not strictly required, but localization, is also reflected in the difficulties of utilizing it as a Skp2 60 increases the degradation efficiency of p21 by SCF . therapeutic target, as detailed below. Consequently, reduced expression of SCFSkp2, induced for example by deficiency of Forkhead Box M1 (FoxM1), impairs the degradation of p21 and enhances its nuclear level.66 UV P21 AND THE CELL CYCLE CONTROL irradiation-induced degradation of p21 physically bound to PCNA This versatile protein is able to control the cell cycle through and phosphorylated at Ser-114 in the S phase is mediated by different mechanisms: directly through binding of different – CRL4Cdt2.62 64 In prometaphase, p21 is recognized at its D-box proteins, like Cdks18 and PCNA32 or indirectly at the transcriptional motif by APC/CCdc20 when bound to Cdk1/cyclin B,65 which is level.103,104 The complexity of the regulatory role of p21 is counteracted by the overexpression of Cdk2, at least in highlighted by the work of Ferrándiz et al.,104 which carried out cardiomyocytes.67 Interestingly, Skp2 silencing induces a slight gene expression profiling with a zinc-inducible p21 expression accumulation of p21 probably representing another degradation system. After conditional induction of p21 in the human leukemia pathway of p21 during mitosis.65 Recently, it has been shown that cell line K562, the expression of 350 genes was altered and the a fourth E3 ligase CRL2LRR1 (Cullin 2-RING ubiquitin ligase/leucine- vast majority of the genes were downregulated.104 The gene rich repeat protein) is acting only and specialized in the distribution displays that 19% of the regulated genes are

Figure 2. Functions of p21 associated with its cellular context. The dual behavior of p21 is highlighted depending on its localization. Nuclear p21 acts mainly as a tumor suppressor, whereas cytoplasmic p21 behaves like an oncogene. The potential functions of p21, which can have either a tumor-suppressive or an oncogenic impact, are denoted with an asterisk (*). important for cell cycle progression. p21 acts as a transcriptional Cep170, a maternal centriole marker.119 Knockdown of p21 in co-repressor of various genes such as Cdk2 and cyclin E2 in the S murine myeloblasts triggers centrosome-mediated chromosomal phase as well as Cdk1, cyclin B1, Cdc25C (cell division cycle 25C), instability and contributes to cytokinesis failures and/or poly- Wee1, Polo-like kinase 1 (Plk1), Aurora B and MCAK (mitotic ploidization, which could be rescued by using two different centromere-associated kinesin) in mitosis, to name a few.104 These Cdk1 inhibitors,119 confirming the observation that p21-deficient observations are in line with a previous study that p21 expression tumor cells display an accumulation of abnormal centrosomes selectively inhibits a set of genes involved in mitosis, DNA followed by failed cytokinesis after ionizing radiation.107 Depletion replication, segregation and repair.103 Therefore, p21 indirectly of p21 in human hematopoietic cells also induces abnormal regulates mitotic genes and consequently the cell cycle arrest. centrosome numbers, deformed nuclear architecture and polyploidy.120 In line with these observations, it has been demonstrated that p21 is indeed required for retaining chromo- FUNCTION OF P21 DURING G2/M somal stability in mice.121–123 The data suggest that p21 is a p21 is not only a central regulator of the interphase and a guardian for a faithful G2/M transition by maintaining the G2 transducer of stress stimuli in the DNA damage response pathway checkpoint and centrosome integrity, which are a prerequisite for but also an important teammate at the G2/M transition and in an errorless start into mitosis. mitotic progression. Function of p21 during mitosis Function of p21 at the G2/M transition p21 was reported to be an inefficient inhibitor of Cdk1/cyclin B1 p21 shows a bimodal periodicity peaking in G1 and G2 at in vitro124 and to have a low binding affinity to Cdk1 in mRNA105 as well as at protein level.106 Most of the functional work asynchronous fibroblasts,125 leading to the anticipation that p21 of p21 during G2 is related to the induction of p21 in response to may be not an effective inhibitor for Cdk1/cyclin B1 in vivo. genotoxic stress and to the maintenance of the G2 checkpoint in However, there are several indications pointing to a direct role of – human fibroblasts as well as in tumor cells,107 111 often associated p21 involved in the regulation of the Cdk1/cyclin B1 activity in with reduced Cdk1/cyclin B1 kinase activity and/or enhanced mitosis in the absence of DNA damage. Functionally, Medema – binding ability toward Cdk1/cyclin B1.112 117 p21 is required to et al.117 showed that an inducible overexpression of p21 in tumor maintain the G2 arrest after DNA damage by blocking the cells inhibits the mitotic entry. Nontransformed fibroblasts interaction of Cdk1 with Cdk-activating kinase, thus inhibiting the expressing low amounts of p21 display more mitotic cells leading activating phosphorylation of Cdk1 at Thr-161.118 p21 causes the to the hypothesis for a shorter G2 phase or a prolonged mitotic nuclear retention of inactive Cdk1/cyclin B1 complexes in human duration.106 Catalytically, phosphorylated p21 is known to be fibroblasts in response to genotoxic stress.115 The binding of involved in the regulation of the activity of Cdk1/cyclin B1.39 endogenous p21, not ectopic overexpressed p21, blocks the A significant reduction in Cdk1/cyclin B1 kinase activity has activation of Cdk1/cyclin B1 and its recruitment to the been observed in tumor cells upon enforced expression of centrosomes.115 An alternative way of mediating the G2 arrest is p21.113,117,118,126 Spatially, p21 accumulates in the nucleus at the reducing Emi1 (early mitotic inhibitor 1), an inhibitor of the mitotic onset in human fibroblasts and associates with Cdk/cyclin anaphase-promoting complex, in response to DNA damage.110 A and Cdk/cyclin B1 complexes.106 Phenotypically, overexpression In addition, p21 deficiency stimulates centriole overduplication of p21 induces polyploidy and a partial inhibition of cyclin leading to an aberrant centrosome number evidenced by staining B1-associated kinase activity.111,113 On the other hand, knockout

© 2015 Macmillan Publishers Limited Oncogene (2015) 1758 – 1767 p21 in mitosis N-N Kreis et al 1762 of p21 also generates polyploidy, especially after treatment with that p21 is important for a fine-tuned regulation of mitosis DNA-damaging agents or irradiation127 or when the mitotic through the surveillance of the Cdk1 activity.130 spindle is disrupted with nocodazole in tumor cells and in The Cdk1 activity is mainly controlled throughout mitosis by fibroblasts.107,109,128 degradation of its regulatory subunit cyclin B1.131 The ubiquitin- Given the elevated level of p21 during G2/M,105,106 the dependent degradation of cyclin B1 is required for an inactivation pronounced phosphorylated band of p21 in early mitosis39,106 of Cdk1, which enables the mitotic exit132–134 and a stepwise and the in vivo inhibition of Cdk1 by p21,126,129 we have removal of its activity.135 Loss of p21 leads to a relatively systematically studied the functional importance of p21 during hyperactive Cdk1 causing mitotic defects.130 Consistent with our this cell cycle phase. We demonstrate that p21 is indeed observation, Pomerening et al.136 show that cells expressing abundantly available during mitosis by showing a slow-migrat- constitutively active Cdk1 exit a mitosis-like state without finishing ing/phosphorylated band in tumor cells regardless of the p53 cytokinesis. Low levels of nondegradable cyclin B1 arrest cells in status.130 It has been reported that the phosphorylation of p21 on telophase without undergoing cytokinesis, suggesting the impor- Thr-57 by Cdk2/cyclin A at the G2/M transition increases its tance of different thresholds of the Cdk1 activity at different binding affinity to Cdk1/cyclin B1, whereas cyclin B1 is phos- stages of mitosis, whereby the onset of cytokinesis is not blocked phorylated at Ser-126, leading further to an increased kinase but defective, demonstrated by connecting cells via a cytoplasmic activity of Cdk1.39 In our experimental settings, p21 properly binds bridge.137,138 The treatment with a Cdk1 inhibitor initiates to Cdk1/cyclin B1 and is able to inhibit its kinase activity.130 From cytokinesis with nonsegregating chromatin.137,138 Moreover, a synchronization experiments and tracking of living cells with time- metaphase arrest with high Cdk1 activity has been observed in lapse microscopy, we have observed that the presence of p21 Xenopus or HeLa cells treated with a proteasome inhibitor or facilitates a faithful progression throughout mitosis. Its deficiency expressing nondegradable cyclin B.139 Upon addition of flavopir- results in a prolonged mitotic duration by about 20%. Further idol, a Cdk1 inhibitor, these cells exit mitosis and undergo analysis of time-lapse microscopy data indicates that metaphase, cytokinesis even in the presence of remaining high levels of cyclin anaphase and cytokinesis are extended in HCT116 cells without B.139 Interestingly, high Cdk1 activity before anaphase blocks the p21.130 In addition, the loss of p21 induces mitotic defects such as accumulation of Aurora B and MKLP1 at the cleavage furrow and chromosome segregation errors and cytokinesis failures in various on the spindle midzone resulting in failed cytokinesis.140,141 The p21-depleted cell lines. Interestingly, the central spindle localiza- results are in agreement with our observations that cancer cells tion of Aurora B, the kinase of the chromosomal passenger without p21 show mitotic defects like lagging chromosomes, complex, is deregulated in cells in the absence of p21. Moreover, failed localization of Aurora B as well as MKLP1 and cytokinesis the mitotic kinesin-like protein 1 (MKLP1), a member of the failures. This phenotype could be partially rescued by inhibiting centralspindlin complex, is also delocalized at the central spindle. Cdk1 with the selective inhibitor RO-3306 or by adding back In line with previous observations, our data underscore the notion p21.130 These data clearly indicate that a relatively high level of

Figure 3. p21 is required for a smooth and errorless mitotic progression. (a) The Cdk1/cyclin B1 activity is precisely controlled and mitosis progresses properly in the presence of p21, in collaboration with the degradation of cyclin B1. (b) The absence of p21 causes a hyperactive Cdk1/cyclin B1, leading to further defective mitosis. Scale bar, 5 μm. (c) p21 is increased in stressed mitotic tumor cells, which promotes the survival of tumor cells and manages their exit from mitosis with cytokinesis failures. Increased cytoplasmic p21 inhibits apoptosis, possibly also facilitates induction of senescence, metastasis and therapy resistance.

Oncogene (2015) 1758 – 1767 © 2015 Macmillan Publishers Limited p21 in mitosis N-N Kreis et al 1763 Cdk1, resulting from nondegradable cyclin B1 or deficiency of p21, Given the dual behavior of p21, different strategies have been is the reason for defective mitosis and cytokinesis. Defects considered to target it: induction versus downregulation.153 The in chromosome segregation or failed cytokinesis could lead idea of an unspecific induction of p21, to reconstitute its tumor further to chromosomal instability and consequently promoting suppressor activities, through histone deacetylase or proteasome tumorigenesis. inhibitors is based on its anti-proliferative and pro-apoptotic Functional interaction of p21 with Aurora B has been recently functions.87,146,153 The induction of p21 is also used as a drug reported.142 Aurora B deficiency leads to an enhanced expression response marker in various clinical trials.153 It is noteworthy to of p21 and an increased pool of Cdk1 bound to it, resulting in mention that generally increasing p21 as an anticancer therapy delayed DNA replication and premature mitotic exit from bears some risks, as increased p21 is not only localized in the prometaphase. This phenomenon is restored upon depletion of nucleus but also in the cytoplasm, which further contributes to p21 in Aurora B-deficient cells. Aurora B represses indirectly the tumor cell survival by inhibiting stress-induced as well as 96 154 expression of p21 at the transcriptional level probably through Fas-mediated apoptosis and by promoting cell migration. phosphorylation and suppression of p53.142 These data highlight The enhanced migration in response to cytoplasmic p21 is that the amount of p21 is crucial and determines the fate of a cell. associated with metastasis via the inhibition of the RhoA-pathway 154,155 High levels of p21 achieved after DNA damage induce a G2 and a changed phosphorylation level of cofilin. arrest,114,115 whereas intermediate levels enable a mitotic entry Another critical aspect to be considered is the role of p21 in but result in a premature mitotic exit in the absence of Aurora maintaining stem cells and promoting senescence (Figure 2). p21 142 130 142 maintains the quiescence of hematopoietic stem cells and its B. In support of our observation, Trakala et al. suggest that 156 p21 deficiency may also prolong the duration of mitosis in normal proliferation is increased in p21-knockout mice. This capability of p21 makes itself as an oncogene by preserving the self-renewal cells and p21 may have important functions during mitosis by 157 6 regulating the activity of Cdk1. of leukemia stem cells, summarized by Warfel and El-Deiry. Taken together, the proper regulation of p21 is crucial in mitosis Cancer stem cells have been suggested to promote tumorigenesis 158 fl (Figure 3a). It directly influences the Cdk1 activity and indirectly and to be the seeds for metastasis. Indeed, p21 in uences the activity and localization of Aurora B, the two key mitotic cancer stem cells showing that loss of p21 may promote breast cancer epithelial mesenchymal transition and stem cell features kinases. Loss of p21 leads to the deregulation of their downstream 159 substrates and induces defective mitosis (Figure 3b). Moreover, in vivo. Moreover, senescence, initiated through the induction of p21,89 was anticipated to have a tumor-suppressive function, as p21 determines the fate of stressed mitotic cells, for example, 160 tumor cells with deregulated kinases, or tumor cells treated with it causes a permanent growth arrest. However, recent studies mitotic agents (Figure 3c). Especially, tumor cells face enormous reveal that senescent cells secrete many factors, like interleukins mitotic stress when they are exposed to mitotic agents like or matrix metalloproteinases, which in turn affect neighboring cells, promote tissue repair and tumor progression.161 In addition, paclitaxel or mitotic kinase inhibitors, such as Plk1 or Aurora 162 – cellular senescence can contribute to therapy resistance. p21 kinase inhibitors, which are already in clinical trials.143 145 Most of could function as a double-edged sword in maintaining stem cell these drugs cause an increase of mitotic p21 in surviving tumor renewal and in promoting senescence. cells.130 This enhanced p21 promotes the survival of treated tumor The subcellular localization of p21 is responsible for its cells and manages eventually the exit of mitosis with cytokinesis contradictory effects (Figure 2) and the variable reports about failures. Subsequently, increased p21 localizes in the cytoplasm, the clinical outcomes. All studies, in vitro and in vivo, provide which blocks apoptosis, facilitates possibly the induction of evidence that especially cytoplasmic p21 has an oncogenic senescence and metastasis, resulting in therapy resistance. More potential. Cytoplasmic p21 is frequently found in tumors and is work is needed to delineate the function, the regulation of linked to aggressiveness and a poor prognosis of tumor localization and turnover, and interaction partners of this patients.87,89,99 It also affects the response to chemo- and increased pool of p21 in stressed mitotic normal as well as cancer radiotherapy146 leading to drug resistance.99,163 Cytoplasmic cells. A better molecular understanding of p21 in mitosis, localization of p21 is mainly ascribed to phosphorylation of especially under stressed conditions, will shed new light in the multiple kinases at distinct phosphorylation sites within p21, like fi selective and ef cient application of mitotic anticancer drugs. Thr-57,44 Ser-130,44 Thr-145,45 Ser-14646 and Ser-15347,48 (Table 1). The first shown and best-studied phosphorylation with impact on ’ 41,45,79 TRANSLATING IN TUMOR THERAPY p21 s localization is Thr-145 by Akt/PKB (protein kinase B). It has been established that cytoplasmic p21 conducts an inhibition p21 has been regarded as tumor suppressor by regulating the 89 of several caspases and apoptotic effectors, like pro-caspase-3, cell cycle and maintaining genomic stability. Mutations in the caspase-8, caspase-10, ASK1 and stress-activated protein kinase,87 p21 gene CDKN1A are extremely rare,89 yet some epigenetic 79,164–167 146 heading further toward the resistance to tumor therapy alterations have been reported. p21 is often deregulated in and consequently worse prognosis of tumor patients.168–171 The human cancer by its reduced level, partially through loss of oncogenic potential of p21 raises the concept of its silencing,99,153 17,147 functional tumor suppressors such as p53, hyperactive specifically targeting increased cytoplasmic-localized p21.172,173 148 oncogenes like c-myc or a loss-of-function via cytoplasmic Several small-molecule inhibitors against p21 have been 149 translocation. Human fibroblasts as well as colorectal cancer identified and investigated:163 butyrolactone,174 LLW10,175 cells without p21 have been shown to be defective in maintaining sorafenib176 and UC2288.90,163 They showed some effects, yet 106,107,150 G1 and G2 checkpoints. Loss of p21 promoting tumor unspecific. It is highly desired to identify specific inhibitors to 13,14 development was demonstrated by knockout mice studies selectively target the cytoplasmic pool of p21, by blocking its with accelerated tumor formation and metastasis upon gamma- nuclear-cytoplasmic shuttling or promoting its degradation in the irradiation.151 Moreover, reduced expression of p21 was correlated cytoplasm. Notably, upon mitotic drugs, p21 is stabilized and with the malignancy of melanoma and metastasis.152 On the other increased in the cytoplasm (our unpublished data), where it hand, cytoplasmic-localized p21 acts as an oncogene with possibly acts as an anti-apoptotic, pro-survival and pro-migration ‘anti-apoptotic gain-of-function’ pointing to a role in factor, which offers a platform for p21 inhibition. It will thus be tumorigenesis.100,149 Low levels of p21 bind to Cdk4/6 stimulating crucial to selectively interfere with the capability of p21, namely, cell cycle progression.97,98 p21 is deregulated in human cancer, to preserve its ability of cell cycle control and genome stability, and its divergent function as a tumor suppressor or an oncogene and to avoid its ability in promoting migration, senescence, cancer depends on the cellular context and the circumstances.87 stem cell renewal, anti-apoptosis and pro-survival. In this context,

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