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One of the main engines that drives cellular transformation is the loss of proper control of the mammalian cell cycle. The cyclin-dependent kinase inhibitor p21 (also known as p21WAF1/Cip1) promotes cell cycle arrest in response to many stimuli. It is well positioned to function as both a sensor and an effector of multiple anti-proliferative signals. This Review focuses on recent advances in our understanding of the regulation of p21 and its biological functions with emphasis on its p53-independent tumour suppressor activities and paradoxical tumour-promoting activities, and their implications in cancer.
Higher eukaryotes have evolved multiple checkpoint mechanisms to monitor and respond to cellular perturbations, halting cellular progression until errors are fixed or the environment becomes permissible to the faithful transmission of genetic material1. Perturbations in checkpoint mechanisms are detrimental to the integrity of the genome, promote cancer development2 and significantly affect the efficacy of anticancer treatment3. The tumour suppressor protein p53 mediates the DNA damage-induced checkpoint through the transactivation of various growth inhibitory or apoptotic genes. Among these, the small 165 amino acid protein p21 (also known as p21WAF1/Cip1) mediates p53-dependent G1 growth arrest4,5. Earlier studies supported the view that p21 suppresses tumours by promoting cell cycle arrest in response to various stimuli. Additionally, substantial evidence from biochemical and genetic studies indicates that p21 acts as a master effector of multiple tumour suppressor pathways for promoting anti-proliferative activities that are independent of the classical p53 tumour suppressor pathway (FIG. 1). Despite its profound role in halting cellular proliferation and its ability to promote differentiation and cellular senescence, recent studies suggest that, under certain conditions, p21 can promote cellular proliferation and oncogenicity6. Consequently, p21 is often misregulated in human cancers, but its expression, depending on the cellular context and circumstances, suggests that it can act as a tumour suppressor or as an oncogene (TABLE 1).
p21 mediates its various biological activities primarily by binding to and inhibiting the kinase activity of the cyclin-dependent kinases (CDKs) CDK2 and CDK1 (also known as CDC2) leading to growth arrest at specific stages in the cell cycle (FIG. 2). In addition, by binding to proliferating cell nuclear antigen (PCNA), p21 interferes with PCNA-dependent DNA polymerase activity, thereby inhibiting DNA replication and modulating various PCNA-dependent DNA repair processes. In this Review we discuss recent advances concerning the complex role of p21 in the development of cancer. We describe the various effector functions of p21 that allow it to exert its biological activities. We further describe our current understanding of the various mechanisms that control p21 expression, both transcriptionally and post-transcriptionally, and how deregulation of these mechanisms may contribute to tumorigenesis.
p21-mediated growth inhibition has been attributed to two main activities that depend on two non-overlapping structural domains: the carboxy-terminal PCNA-binding domain and the amino-terminal CDK–cyclin inhibitory domain7,8. Through binding to PCNA, p21 competes for PCNA binding with DNA polymerase-δ and several other proteins involved in DNA synthesis, thus directly inhibiting DNA synthesis9.
p21 belongs to the Cip and Kip family of CDK inhibitors that includes p21, p27 and p57. These inhibit the kinase activity of broad but not identical classes of CDK–cyclin complexes through their N-terminal homologous sequences. p21 also inhibits CDK activity indirectly by interfering with the activating phosphorylation of CDK1 and CDK2 in the activation segment by an unidentified mechanism10–12. p21 binds the cyclin subunit through a conserved Cy1 motif in the N-terminal half and through a weaker and redundant Cy2 motif in the C-terminal half13. It also interacts with the CDK subunit through a separate CDK-binding site in the N-terminal half13. Through its Cy motifs, p21 disrupts the interaction between CDK and substrates that bind to CDK–cyclin through similar Cy motifs, such as RBL1 (also known as p107) and RBL2 (also known as p130), retinoblastoma (Rb) family proteins and CDC25C14–16. CDC25C, a tyrosine phosphatase that dephosphorylates the cyclin B-bound CDK1 that is required for entry into mitosis, can in turn alleviate CDK inhibition by competing with p21 for cyclin binding through the Cy motif16.
p21 inhibits cell cycle progression primarily through the inhibition of CDK2 activity, which is required not only for the phosphorylation of RB with the consequent release and activation of E2f-dependent gene expression, but also for the firing of replication origins and for the activity of proteins directly involved in DNA synthesis17. Although this activity is shared by other CDK inhibitors such as p27 and p57, biochemical and genetic evidence suggest that they have distinct roles in tumorigenesis18. Nevertheless, p21 is uniquely positioned to function as a central inhibitor of CDK2 that is activated in response to a variety of cellular and environmental signals to promote tumour suppressor activities (FIG. 1). Experimental evidence however, suggests that the proliferation of some human cancer cells does not require active CDK2 (REF. 19). Moreover, targeted deletion of Cdk2 indicates that CDK2 is dispensable for cell cycle inhibition by p21 (REF. 20). CDK1, at least in some tissues, may be the crucial target of p21 in tumorigenesis27 because p21 effectively inhibits the kinase activity of CDK1 both in unstressed cells and after genotoxic stresses, leading to growth arrest in the G2 phase of the cell cycle21–26 (FIG. 2).
Microarray-based studies suggest that p21 expression positively correlates with the suppression of genes that are important for cell cycle progression and the induction of genes associated with senescence28. Although p21-induced changes in gene expression can be explained by the inhibition of CDK2 activity by p21, several studies support additional roles for p21 that are independent of CDK2 or RB. For example, p21 associates directly with E2F1 and suppresses its transcriptional activity29 (FIG. 2). In response to notch 1 activation, p21 suppresses E2F1-dependent Wnt4 expression, thereby controlling cellular growth30. p21 also binds to and represses the transcription factor signal transducer and activator of transcription 3 (STAT3)31, thereby inhibiting cytokine-stimulated and STAT3-dependent gene expression. Similarly, p21 represses MYC-dependent transcription by associating with the N-terminus of MYC and interfering with MYC–MAX dimerization32. In turn, MYC disrupts the PCNA–p21 interaction, thus alleviating p21-dependent inhibition of PCNA and DNA synthesis32.
The ability of p21 to promote cell cycle inhibition may also depend on its ability to mediate p53-dependent gene repression, as p21 is both necessary and sufficient for p53-dependent repression of genes regulating cell cycle progression, including CDC25C, CDC2, CHEK1, CCNB1 (which encodes cyclin B1), TERT (which encodes telomerase reverse transcriptase) and the anti-apoptotic gene BIRC5 (survivin)33,34. CDC2, CHEK1 and TERT are repressed by p21 through the inhibition of CDK2-mediated phosphorylation of RB- and E2f-dependent transcription34–36. Additionally, by inhibiting CDK2, p21 inhibits the induction of CDC2 and CCNB1 indirectly, as the expression of these genes at the G1/S transition is mediated by the NF-Y transcription factor following its phosphorylation by CDK2 (REFS 37,38).
p21 also activates gene transcription by de-repressing p300–CREBBP (CREB-binding protein)39. Because p300–CREBBP cooperates with multiple factors to promote the transcriptional induction of CDKN1A (the gene encoding p21) in response to a variety of stimuli (see below), de-repression of p300–CREBBP by p21 seems to be part of a positive feedback loop that amplifies p21 expression. The p21-dependent activation of p300–CREBBP-driven gene transcription has a significant role in regulating oestrogen receptor-α (ERα)-dependent gene expression, thereby inducing the differentiation of ERα-positive cells40. This is important as p21 upregulation is sufficient to prevent the growth of ERα-positive breast cancer cells41 and may affect the efficacies of anti-oestrogen treatments.
Although best known for its growth-inhibitory functions, p21 also inhibits apoptosis, which might account for its paradoxical oncogenic activities6 (discussed below). Through its ability to promote cell cycle inhibition, especially in the face of genotoxic insults or microtubule-destabilizing agents, p21 protects cells from apoptosis because an active cell cycle is required to sense these agents and trigger apoptosis. The cytostatic effect of p21 with the consequent inhibition of apoptosis, however, is counteracted by several mechanisms. For example, the cellular response can be switched from cell cycle arrest to apoptosis by the selective transcriptional repression of CDKN1A, the selective activation of pro-apoptotic genes or defects in p21 expression downstream of p53 (REFS 42–44). Furthermore, and as discussed below, post-translational modifications of p21 such as its phosphorylation (which affects protein stability45–47 or cytoplasmic localization45,48 of p21) and its cleavage by caspase 3 (REF. 49) also account for the differential effects on cell cycle arrest versus apoptosis.
p21 can protect against apoptosis in response to other stimuli such as those induced by growth factor deprivation, p53 overexpression or during the differentiation of monocytes6. Under these conditions, apoptosis does not depend on cell cycle progression, so the anti-apoptotic activity of p21 cannot be attributed to its cytostatic effects. Instead, it may rely on the ability of p21 to regulate gene transcription through its multiple protein–protein interactions or through its roles in DNA repair (described below). For example, cytoplasmically localized p21 binds to and inhibits the activity of proteins directly involved in the induction of apoptosis, including procaspase 3, caspase 8, caspase 10, stress-activated protein kinases (SAPKs) and apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5)6,50 (FIG. 2). Furthermore, p21 can mediate the upregulation of genes encoding secreted factors with anti-apoptotic activities6,50. p21 also suppresses the induction of pro-apoptotic genes by MYC and E2F1 through direct binding and inhibition of their transactivation functions50. The potential requirement for CDK activity for the induction of pro-apoptotic genes by MYC or E2F1, however, cannot be ruled out. Knock-in mice expressing p21 mutants that cannot suppress the transcription of genes or that fail to bind to or inhibit the transactivation functions of MYC or E2F1 will help to elucidate the contribution of these different effector functions of p21 to blocking apoptosis.
Paradoxically, p21 might also promote apoptosis through both p53-dependent and p53-independent mechanisms under certain cellular stresses. Exactly how p21 promotes apoptosis is not clear, but might depend on both p53-dependent and p53-independent upregulation of the pro-apoptotic protein BAX, activation of members of the tumour necrosis factor family of death receptors or effects on DNA repair51. In several of the studies that indicated a pro-apoptotic role for p21, it was shown only that apoptosis concurred with induction of p21 without determining whether p21 is required for the induction of apoptosis. Thus, a careful analysis is needed to investigate the exact role of p21 under these conditions.
p21 has a significant role in modulating DNA repair processes. First, by inhibiting cell cycle progression, p21 allows DNA repair to proceed while inhibiting apoptosis. Secondly, p21 can compete for PCNA binding with several PCNA-reliant proteins that are directly involved in DNA repair processes9 (FIG. 2). For example, p21 interferes with PCNA–DNMT1, which is required not only for DNA synthesis but also for DNA repair52,53. Additionally, a p21 or p21-derived PCNA-interacting peptide inhibits mismatch repair54 and PCNA-dependent base excision repair55 indicating that the p21–PCNA interaction is sufficient for p21 to inhibit these DNA repair processes. Moreover, p21 modulates translesion DNA synthesis, which is important for bypassing stalled replication forks, by inhibiting PCNA monoubiquitylation56,57.
Recent evidence suggests that p21 may also regulate nucleotide excision repair (NER) although its exact role has been controversial58. Defects in NER genes account for the rare genetic disorder xeroderma pigmentosum, which is characterized by an increased frequency of skin cancer59. The xeroderma pigmentosum group E gene product DDB2, a significant player in recognizing DNA damage in NER and a component of the CRL4 (cullin–RING ligase 4) E3 ubiquitin ligase complex, promotes p53 degradation in ultraviolet-irradiated cells with the consequent downregulation of p21 (REF. 60). Significantly, downregulation or deletion of Cdkn1a in NER-deficient Ddb2−/− mouse embryonic fibroblasts restores NER activity, suggesting that p21 represses NER activity60. Additionally, in ultraviolet-irradiated cells61,62, as well as in several neoplastic cell lines irradiated with ionizing radiation63, p21 is proteolytically degraded through the action of another member of the CRL4 E3 ubiquitin ligase family, CRLCDT2 (also known as DTL), by a mechanism that requires the physical interaction of p21 with PCNA. Thus, the CRL4 E3 ubiquitin ligases seem to promote NER by downregulating p21, both transcriptionally (through the degradation of p53 through DDB2) and post-transcriptionally (through PCNA-dependent degradation of p21 through CRLCDT2). Given the significant role of the various DNA repair processes in protecting against cancer, future work using DNA repair animal models will be useful in elucidating the extent to which p21 modulates DNA repair processes and whether this activity contributes to its tumour-suppressing or tumour-promoting activities.
The transcriptional regulation of p21 has been extensively studied64. In this Review we focus on recent advances in our understanding of the transcriptional activation and repression of CDKN1A (FIG. 3). In diploid, non-immortalized, non-transformed cells oncogenic Ras activates CDKN1A transcription through both p53-dependent and p53-independent mechanisms. The p53-independent transactivation of CDKN1A by activated Ras requires the transcription factor E2F1 (REF. 65). E2F1 and E2F3 strongly activate CDKN1A transcription by binding to cis-acting elements between −119 to +16 of CDKN1A66,67. Raf, a downstream effector of Ras, also transactivates CDKN1A independently of p53 (REF. 68). Oncogenic Ras and Raf, however, induce p21-dependent senescence69,70 and other genetic mutations are necessary for bypassing oncogene-induced senescence, which, like apoptosis, is a significant barrier to tumorigenesis71. The significant role of p21 in promoting HRAS-induced senescence is underscored by the finding that Cdkn1a deletion cooperates with activated HRAS to promote tumours in mice72–75. If p21 inhibits CDKs, how does HRAS or Raf transform cells or promote tumours when it induces p21 and cellular senescence? The answer to this question came from the discovery that RHOD, a small GTPase and a downstream effector that is required for the transforming activity of HRAS76,77, suppresses CDKN1A trans-activation in response to HRAS stimulation78. In fact, RHOD is dispensable for HRAS-induced DNA synthesis in serum-starved Cdkn1a−/− fibroblasts, indicating that the primary role of RHOD is to suppress p21 induction by HRAS78. Recent work suggest that the HRAS–ARF–p53–p21 senescence circuitry can be disrupted by the expression of ID1 (REF. 75), a helix–loop–helix transcription regulator that is overexpressed in a number of solid tumours79, 80 and whose expression positively correlates with advanced disease and poor prognosis in prostate81,82, ovarian83 and breast cancer79. ID1 appears to render cells refractory to growth inhibition by p21 (REF. 75). How ID1 prevents growth inhibition despite high levels of p21 remains unclear. However, given that p21 expression is frequently increased in human cancer (TABLE 1), understanding the mechanisms by which growth inhibition is prevented despite high levels of p21 will provide significant insight into the development and progression of various human cancers.
Besides mitogen-dependent transactivation through the HRAS–Raf–Mapk pathway, CDKN1A transcription is also activated by several nuclear receptors including retinoid receptors, vitamin D receptors and androgen receptors. These operate independently of p53 through binding to their cognate responsive elements in the CDKN1A promoter64. The transcription factors SP1, SP3, AP2, CCAAT/enhancer binding protein-α (C/EBPα), C/EBPβ, BETA2 (also known as NEUROD1), GAX (also known as MOX2), homeobox A10 (HOXA10), STATs and myoblast determination protein 1 (MYOD1) also control CDKN1A transcription and upregulate p21 in response to a plethora of stimuli and anticancer agents (FIG. 3). Several of the transcriptional inducers of p21, such as nerve growth factor (NGF), progesterone, Ca2+ or the transcription factors BETA2 and MYOD1, cooperate with the transcriptional co-activator p300–CREBBP to activate the CDKN1A promoter64.
Several members of the Krüppel-like transcription factor (Klf) family, which are key transcriptional regulators of proliferation and differentiation84, also regulate the transcription of CDKN1A by p53-independent mechanisms. These transcription factors bind to GC boxes and upregulate or downregulate target gene transcription. Of particular interest is KLF6, a tumour suppressor that is frequently inactivated or downregulated in human tumours including prostate85,86, lung87, hepatic88 and colon89. KLF6 binds two GC boxes located about 120 bp upstream from the transcription start site of CDKN1A and cooperates with p300–CREBBP to activate CDKN1A transcription85,90. Interestingly, KLF6 also activates transcription of the transforming growth factor-β (TGFβ) receptors91, indicating that KLF6, TGFβ and p21 are in the same tumour suppressor pathway (FIG. 3).
KLF4, which is expressed in epithelial tissues, is frequently downregulated in gastrointestinal, colorectal and bladder cancers, and its tumour suppressor activities partially depend on its ability to induce p21 expression92. In colorectal cancer, KLF4 downregulation or inactivation is associated with a similar reduction in p21 expression93. In response to DNA damage, KLF4 is induced by p53 and synergizes with p53 to activate the CDKN1A promoter94. In fact, through induction of p21, KLF4 mediates a DNA damage-induced and p53-dependent G1/S checkpoint95. Intriguingly, KLF4 directly suppresses the TP53 promoter and exhibits paradoxical oncogenic activities through its ability to suppress senescence in response to oncogenic HRAS activation96. Thus, a complex pattern of p21 and p53 regulation by KLF4 may determine the role of KLF4 in oncogenesis92.
The transcription of CDKN1A is also regulated by CDX2, a member of the caudal-related homeobox gene family that is involved (along with CDX1) in intestinal development, proliferation and differentiation97. CDX2 is a tumour suppressor that is downregulated during colorectal carcinogenesis98,99. Ectopic CDX2 expression inhibits the proliferation of colorectal cancer cells, induces the differentiation of undifferentiated intestinal epithelial cells99,100 and induces p21 in human colon cancer cells through transactivation of the CDKN1A promoter101. p21 is also downregulated during colorectal tumorigenesis (TABLE 1), which is probably a direct result of CDX2 downregulation or inactivation, as stronger expression of CDX2 and p21 is observed mostly in tumour patches with higher levels of differentiation98,99,102,103. Strikingly, CDX2 activates KLF4 transcription104 and the CDX2 gene itself is regulated by another tumour suppressor gene, APC (adenomatous polyposis coli)105. Consistently, colon cancer cells with mutations in APC or CTNNB1 (which endodes β-catenin) exhibit lower expression levels both of CDX2 and KLF4 (REFS 104,105). Thus, the APC–CDX2–KLF4–p21 axis is a multilayered tumour suppressor pathway that regulates p21 expression (FIG. 3).
Whereas the deregulated expression of p21 in cancer often correlates with the loss of function of transcriptional activators of p21 (including p53), upregulation or gain of function mutations in genes that repress CDKN1A transcription may also contribute to cancer development. For example, it is likely that the transcriptional repression of CDKN1A by MYC (FIG. 3) plays a part in the development of tumours in which MYC is overexpressed. This may be important in ERα-positive breast tumours in which oestrogen-dependent upregulation of MYC and the subsequent downregulation of p21 promote cell proliferation, and disruption of the MYC–p21 circuit contributes to the resistance to anti-oestrogen therapies106.
Interestingly, MYC induces the transcription of AP4, a transcription factor that is frequently increased in colonic progenitor cells and in colorectal cancer and is capable of repressing CDKN1A transcription107. Significantly, AP4 overexpression inhibits p53-mediated cell cycle arrest, sensitizes cells to DNA damage-induced apoptosis and can suppress TGFβ-dependent CDKN1A transactivation107. Abrogating the growth-inhibitory functions of TGFβ is a hallmark of many cancers108, so it is tempting to speculate that AP4, and other factors that inhibit p21 post-transcriptionally and abrogate TGFβ-induced growth arrest, such as the newly identified microRNA cluster miR-106b-25 (REF. 109), may contribute to the development of these cancers.
Although much of the control of p21 is at the transcriptional level, recent work suggests that post-transcriptional control of p21 is equally important. In actively dividing cells, p21 is an unstable protein with a half-life of about 20 to 60 minutes. Newly synthesized p21 protein is protected from proteasomal degradation by the activity of FKBPL (also known as WISP39), an adaptor that recruits HSP90 to p21 (REF. 110). Importantly, cells depleted of WISP39 fail to upregulate p21 in response to DNA damage, indicating that the transcriptional control of CDKN1A is insufficient to upregulate p21 after DNA damage in the absence of p21 stabilization110.
Three E3 ubiquitin ligase complexes, SCFSKP2 (SKP1–CUL1–SKP2), CRL4CDT2 (CUL4A or CUL4B–DDB1–CDT2 (DDB1 is DNA damage-binding protein 1)) and APC/CCDC20 (anaphase-promoting complex (APC)–cell division cycle 20), promote the proteolysis of p21 through the proteasome at specific stages in an unperturbed cell cycle (FIG. 4). SCFSKP2, CRL4CDT2 and APC/CCDC20 promote the ubiquitylation and degradation of p21 only when it is bound by complexes of CDK2 with cyclin E or cyclin A, PCNA, or complexes of CDK1 with cyclin A or cyclin B, respectively. p21 that is not bound to CDK or PCNA, however, is degraded independently of ubiquitin by interaction of its C terminus with the C8α subunit of the 20S proteasome111,112, but this method of p21 degradation does not occur in all cell types113. Ubiquitin-independent proteolysis of p21 does not require the ubiquitin-binding 19S proteasomal lid and instead is dependent on the REGγ subunit of the proteasome113,114. Various factors and signalling molecules affect the stability of p21 to affect cell cycle progression. For example, TGFβ and bone morphogenetic protein 2 (BMP2) suppress the growth of human colon cancer cells partly owing to increased p21 protein stability, although the mechanism is poorly understood115,116. In response to oxidative stress, the activation of JNK1 (JUN amino-terminal kinase 1) promotes growth arrest by inhibiting p21 ubiquitylation117–119. Additionally, a number of tumour viruses regulate p21 stability and affect cell cycle progression and apoptosis (BOX 1).
Many viral proteins affect the stability or post-transcriptional regulation of p21, thereby affecting cellular proliferation. For example, the human papilloma virus (HPV) E6 protein can downregulate p21 independently of p53 (REFS 173–176). Although E6 is essential for the oncogenic activity of HPV and has anti-apoptotic activities, under some conditions, such as DNA damage175, E6 downregulates p21 to promote apoptosis177. The adeno-associated virus type 2, a helper-dependent human parvovirus, preferentially downregulates p21 protein in HPV-infected cells with a concomitant increase in cyclin-dependent kinase 2 (CDK2)–cyclin E activity but prevents further progression through S phase, thus favouring the replication of the adeno-associated virus type 2 (REF. 178). The hepatitis C virus core protein inhibits p21 post-transcriptionally, alleviates CDK2 inhibition and contributes to hepatitis C virus-mediated tumorigenesis179. Finally, the K cyclin encoded by the human herpesvirus 8 promotes p21 phosphorylation at Ser130 by CDK6 without affecting its stability or nuclear –cytoplasmic localization180. Interestingly, although the phosphorylation of p21 at Ser130 by CDK2 targets it for ubiquitylation by the SCFSKP2 (SKP1–CUL1–SKP2) E3 ubiquitin ligase complex and degradation127, p21 phosphorylation by CDK6–cyclin K prevents p21 association with CDK2, thus alleviating a p21-imposed G1 arrest180. Although the mechanism by which these viral proteins affect p21 stability or activity is largely unknown, these findings demonstrate that targeting p21 is a common mechanism by which these viruses regulate cell cycle progression and apoptosis.
Several proteins involved in the ubiquitin-dependent proteolysis of p21 are upregulated in a variety of human tumours, suggesting that p21 downregulation may account for some of the oncogenic properties of these proteins. For example, SKP2, an F box protein that is the substrate recognition factor of the SCFSKP2 E3 ubiquitin ligase complex, which is necessary for the degradation of p21 at the G1/S transition and during S phase of the cell cycle, is oncogenic and frequently upregulated in human cancers120. Similarly, CDT2, a substrate recognition factor for p21 degradation61,121 by the CRL4CDT2 ubiquitin ligase complex, is overexpressed in breast cancer122 and in primary hepatocellular carcinomas, especially at advanced stages123. Finally, CUL4A (which encodes the CUL4A E3 subunit of the CRL4CDT2 ubiquitin ligase complex) is overexpressed in breast cancers and hepatocellular carcinomas124,125. It will be of interest to test whether the upregulation of these oncogenes causes p21 downregulation and whether p21 downregulation contributes to their oncogenic activity.
Whereas the growth-inhibitory functions of p21 are associated with its nuclear localization, the anti-apoptotic or oncogenic activities of p21 (described below) are frequently associated with its cytoplasmic accumulation. In fact, cytoplasmic expression of p21 is common in human malignancies and correlates positively with aggressive tumours and poor prognosis (TABLE 1). Multiple protein kinases catalyse the phosphorylation of p21 to regulate its stability and localization in the cell126. Phosphorylation of p21 at Ser130 by CDK2–cyclin E, for example, promotes its binding to SKP2, leading to its ubiquitylation and subsequent proteolysis, and thus promotes cellular progression at the G1/S transition and during S phase of the cell cycle127.
Phosphorylation of p21 at Thr145 in the PCNA-binding site by AKT1 (also known as PKB) disrupts its binding with PCNA45,128, induces its cytoplasmic accumulation and is required for ERBB2-mediated proliferation of breast cancer cells and breast carcinogenesis48,129,130. Similarly, the overexpression of the IKKβ (inhibitor of nuclear factor-κB kinase-β), which is seen in some human breast cancers, is associated with AKT1 phosphorylation and the cytoplasmic accumulation of p21 (REF. 131) (FIG. 2). The cytoplasmic accumulation of p21 promotes cell survival through the inhibition of cytoplasmically localized apoptosis-related proteins, and promotes cellular proliferation through both the alleviation of CDK2 and PCNA inhibition and the assembly of the D-type cyclins (D1, D2 and D3) with CDK4 and CDK6 (FIG. 2). Because AKT1 phosphorylates and inhibits glycogen synthase kinase-3β (GSK3β), which phosphorylates cyclin D1 at Thr286 and promotes its degradation132, AKT1-mediated assembly of complexes of cyclin D1 with CDK4 or CDK6 is facilitated by the stabilization of cyclin D1.
In endothelial cells, however, AKT1-mediated phosphorylation of p21 at Thr145 does not affect p21 localization, although it disrupts its interaction with PCNA, decreases CDK2 inhibition and promotes endothelial cell proliferation128. On the other hand, in serum- stimulated endothelial cells, GSK3β phosphorylates p21 at Thr57 and promotes its degradation133 by an unidentified mechanism. The contradictory effects of AKT1- and GSK3β-mediated phosphorylation of p21 at Thr145 and Thr57, respectively, on the fate of p21 may be explained by cell type differences or additional cell type-specific modifications on p21. Furthermore, the regulation of p21 by AKT1 and GSK3β in endothelial cells may have a role in promoting neovascularization and metastasis.
In addition to Thr145, AKT1 phosphorylates p21 at Ser146, also leading to the stabilization of p21 and cell survival45. p21 can also be phosphorylated at Ser146 by protein kinase C (PKC). However, it is unclear whether this phosphorylation is catalysed by PKCδ to stabilize p21 (REF. 47) or PKCζ to destabilize p21 (REF. 134). The explanation for this contradiction may lie in the cellular context in which these PKC isoforms are activated and on other proteins that affect p21 phosphorylation.
Much of our understanding about the role of p21 in cancer has come from knockout mouse studies combined with biochemical and functional analysis of cells in culture. Groundbreaking work came from the initial discovery of p21 as a potential mediator of the tumour suppressor activity of p53 (REF. 135). Subsequent work showed that, although deletion of Cdkn1a in mice abrogated DNA damage-induced and p53-dependent growth arrest, it had no effect on p53-dependent apoptosis4,5. p21 could not, therefore, account for all the tumour suppressor activities of p53. Nevertheless, p21 is a major determinant of tumour protection by p53 (REF. 136), as Cdkn1a deletion drastically accelerated tumour formation in mice expressing a mutant form of p53 (Trp53R172P+/+) that is incapable of inducing apoptosis but retains partial growth arrest activity137.
The first genetic evidence supporting a tumour suppressor activity for p21 came from the discovery that Cdkn1a−/− mice developed spontaneous tumours138. The late onset of these tumours (average age of 16 months) compared with those arising in mice deficient in other tumour suppressor genes such as Trp53 (REFS 139,140), p16 (REF. 141) or Arf (REF. 142) suggests that the loss of Cdkn1a by itself is insufficient to promote malignancy. Although many human cancers such as colorectal, cervical, head and neck, and small-cell lung cancers are associated with reduced p21 expression (TABLE 1), the extreme rarity of loss-of-function mutations in CDKN1A in human cancer143–145 argues that p21 may not be a classical tumour suppressor. Instead, p21 synergizes with tumour suppressors and antagonizes oncogenes to protect against cancer (TABLE 2). Furthermore, Cdkn1a deficiency accelerates the development of chemically induced tumours in mice146–149. Additional in vivo evidence for tumour suppressor activity for p21 comes from studies using the transplantation of Cdkn1a−/− cells in mice with defined genetic alterations. For example, although the leukaemogenic fusion protein AML1–ETO (AML1 is also known as RUNX1) does not promote leukaemia without secondary mutations, fetal liver haematopoietic cells isolated from Cdkn1a−/− mice and transduced with AML1–ETO promoted leukaemogensis when transplanted into mice150. Cdkn1a deficiency also cooperates with the co-expression of HRAS and MYC151, the expression of BCR–ABL1 (BCR is breakpoint cluster region) (REF. 152) or with Ink4 deletion153 to promote transformation and proliferation of cells in culture. Together, these data are consistent with the multi-step tumorigenesis theory and a role for p21 in this process.
A significant insight into the role of p21 in tumour suppression came from a study by Shen et al.154 demonstrating a prominent tumour suppressor role for p21 in a genomically unstable background. Cdkn1a deficiency cooperated with the loss of the DNA damage checkpoint protein ATM (ataxia–telangiectasia mutated) in promoting aneuploidy that preceded tumour development154. Furthermore, although malignancies developing in the aforementioned Trp53R172P+/+ mice retain stable genomes, lymphomas and sarcomas arising in Trp53R172P+/+;Cdkn1a−/− mice had an earlier onset and exhibited chromosomal aberrations and marked aneuploidy137. The finding that p21 downregulation inversely correlates with microsatellite instability in colorectal cancer, irrespective of the p53 status155,156, adds support to the conclusion that the loss of protection against genomic instability by p21 contributes to human malignancy.
p21 also promotes genomic stability in stem cells, both maintaining the self-renewal capacity of stem cells (BOX 2), and possibly contributing to its oncogenic potential (discussed below). For example, although haematopoietic stem cells (HSCs) derived from mice that are engineered to express PML–RAR (retinoic acid receptor) — the initiating oncogene of human acute promyelocytic leukaemia (APL)157 — exhibit relatively moderate DNA damage foci, those derived from PML–RAR;Cdkn1a−/− mice exhibit a significantly higher rate of DNA damage foci, with more than 95% of cells exhibiting multiple foci per cell158. Thus, at least in the context of overexpression of this oncogene, p21 seems to limit DNA damage and protect against genomic instability in HSCs. Although there is currently no evidence to suggest that the increase in genomic instability in the absence of p21 in HSCs results in increased tumorigenesis, it is conceivable that the acquisition of additional genetic alterations, under these circumstances, may uncover a protective role for p21.
Recent evidence suggests that p21 is crucial for maintaining stem cell potential by restricting stem cell self-renewal in various tissues146,181–183. This is best understood in the haematopoietic system where, under homeostatic conditions,Cdkn1a−/− mice exhibit increased absolute numbers and proliferation of haematopoietic stem cells181.Cdkn1a−/− haematopoietic stem cells, however, rapidly lose their stem cell potential following serial bone marrow repopulation. Premature death, owing to haematopoietic cell depletion, ensues when these animals are exposed to acute genotoxic stress. Thus, restricted proliferation is a prerequisite for long-term stem cell potential and p21, through its ability to suppress the cell cycle, is a crucial determinant of stem cell pool persistence in vivo181. However, in response to cytokines, Cdkn1a−/− bone marrow progenitor cells exhibit decreased proliferation184,185. Consequently, it was hypothesized that p21 has distinct roles in subcompartments of the haematopoietic lineages, inhibiting the proliferation of stem cells but stimulating the proliferation of progenitor cells181. This dichotomy may reflect the differential role of p21 in inhibiting cyclin-dependent kinase (CDK) complexes in stem cells but promoting the assembly of complexes of D-type cyclins with CDK4 and CDK6 (REF. 163) in their progeny.
Although some studies suggest that the lack of p21, and the consequent increase in stem cell populations (for example, in keratinocyte stem cells) is strongly associated with increased susceptibility to carcinogenesis146,149,186, a recent study suggests that it does not contribute to carcinogenesis187. Nevertheless, p21 was recently shown to be crucial for maintaining the self-renewal capacity of leukaemia stem cells that were derived from mice expressing the leukaemia-associated oncogene PML RAR (retinoic acid receptor) by protecting them from exhaustion in stressful conditions158. The results demonstrate that p21 is important for the maintenance, rather than the initiation, of at least a subset of malignancies. They also suggest that this activity of p21 may vary depending on the specific genetic alterations.
The simple view that p21 acts as a tumour suppressor has been complicated by the finding that p21 can exhibit oncogenic activities6,159. p21 is overexpressed in a variety of human cancers including prostate, cervical, breast and squamous cell carcinomas and, in many cases, p21 upregulation correlates positively with tumour grade, invasiveness and aggressiveness and is a poor prognostic indicator (TABLE 1). As mentioned above, in some of these cases p21 is cytoplasmic so its oncogenic function might be dependent on non-traditional cytoplasmic targets of the protein. Although there is little or no direct evidence to suggest that p21 upregulation contributes to the development of these cancers, it may affect the responsiveness to chemotherapy and radiotherapy160.
The theory that p21 may function as an oncogene under certain circumstances is supported by a limited number of mouse genetic studies that showed that Cdkn1a deletion suppressed the development of spontaneous lymphomas arising in Trp53−/− (REF. 161) and Atm−/− (REF. 162) mice and radiation-induced lymphomas arising in wild-type138 and Trp53−/− (REF. 161) mice. Interestingly, lymphomas arising in Cdkn1a−/− mice exhibit a high rate of apoptosis, suggesting that the anti-apoptotic activity of p21 is pro-tumorigenic6. Why such an oncogenic activity is only manifested in lymphomas is unclear, but lymphocytes may be particularly sensitive to the anti-apoptotic activity of p21. Because p21 is crucial for cellular differentiation, it is possible that reduced tumorigenesis in the absence of p21 is due to a block in cell differentiation at a stage in which the cells cannot proliferate.
As discussed, p21 can also promote oncogenesis independently of its anti-apoptotic activity by promoting the assembly of complexes of cyclin D with CDK4 or CDK6 without inhibiting their kinase activity163. For example, p21 promotes oligodendrogliomas only when it can form complexes with cyclin D1 (REF. 164). p21-mediated nuclear retention of cyclin D1 protects cyclin D1 from cytoplasmic degradation165 and promotes its association with and activation of CDK4 and CDK6. In fact, constitutively nuclear cyclin D1 (cyclin D1T286A) restored the development of oligodendrogliomas in Cdkn1a−/− mice only if the cyclin D1 could complex with CDK4 (REF. 4). The sequestration of p21 by CDK4–cyclin D and CDK6–cyclin D may also promote oncogenesis by freeing CDK2 from inhibitory p21. This is demonstrated by the ability of T cell leukaemia virus type 1 (HTLV-1) to bypass the G1/S arrest through binding of p21 to CDK4–cyclin D2 and the consequent activation of CDK2 (REF. 166). The ability of p21 (at low stoichiometric concentrations) to promote the activity of CDK4–cyclin D and CDK6–cyclin D may explain why tumour suppression by p21 varies with its expression level or the genetic background — the loss of a single Cdkn1a allele (but not homozygous deletion), for example, accelerated tumour growth in mice carrying the Wnt1 transgene167.
Several anticancer agents such as histone deacetylase (HDAC) inhibitors function, at least partly, through their ability to promote the induction of p21 (REF. 168). Other agents such as statins, which are routinely used to lower cholesterol levels, exhibit profound anti-proliferative capacity by inducing p21 (REF. 169) and are being investigated for their anti-tumorigenic activities170. The complex network regulating p21 activity and biological functions, however, warrants caution with regard to its application for cancer therapy. The various effects of p21 on gene regulation and its role in genomic stability, apoptosis, senescence and DNA repair may not only contribute to cancer development but also profoundly affect the efficacy of DNA-damaging agents or other anticancer drugs that induce p21. The challenge lies in selectively inhibiting only the oncogenic activities of p21 and not its tumour suppressor functions. Therefore, the development of agents that interfere with the ability of p21 to assemble CDK4–cyclin D and CDK6–cyclin D complexes but retain its ability to suppress CDK2 or CDK1 may be an attractive line of investigation. Alternatively, it may be beneficial, instead of targeting p21 per se, to selectively target factors upstream or downstream of p21 that affect these particular aspects of p21 function. Drugs that can specifically inhibit the anti-apoptotic functions of p21 may be especially effective when combined with other drugs that are capable of inducing p21, such as DNA-damaging agents.
Significant recent advances have been made in elucidating the various players that are involved in p21 degradation and the various post-translational modifications that affect the stability and cellular localization of p21. Biochemical and structural studies of the various ubiquitin ligase complexes directly involved in p21 proteolysis under different conditions will undoubtedly help the development of selective inhibitors for these ligases and provide a platform for the development of a new generation of anticancer agents. Furthermore, DNA-damaging agents that may selectively inhibit AKT1 activity may not only deprive tumours of the pro-survival functions of AKT1, they are also likely to destabilize p21 leading to augmentation of their apoptotic effects. This possibility is supported by a study in which the DNA-damaging agent aminoflavone induced apoptosis of MCF7 breast cancer cells only at concentrations at which it reduced AKT1 activity and destabilized p21 (REF. 46).
An alternative therapeutic approach may take advantage of the ability of p21 to induce senescence in tumours. Recent work suggests that tumour regression can be achieved through the reactivation of senescence, for example by restoring p53 function171 or through the inactivation of MYC in tumours with functional p53 (REF. 172). Although MYC inactivation upregulated p21 only in a subset of tumours, the results demonstrate that activation of senescence is not only feasible but also a promising approach to tumour regression in vivo. Even in tumours that retain high levels of p21, it may still be possible to induce tumour regression through the reactivation of senescence. However, this possibility will require a greater understanding of the various players (such as that described for the transcription factor ID1) that can abrogate p21-induced senescence despite high levels of p21. p21 expression in these tumours can potentially be exploited for therapy by targeting ID1 or similar molecules, leading to the reactivation of senescence downstream of p21. Finally, advances in our understanding of the precise role of p21 in modulating DNA repair processes under various conditions are urgently needed and may shed more light on the role of p21 in the development and treatment of cancer.
Owing to the extensive literature concerning the regulation and activity of p21, it was impossible to account for many interesting findings in a single Review. We therefore apologize to colleagues whose work was not cited. This work was supported by grants from the National Institutes of Health (Cancer Training Grant T32CA009109 for T.A. and R01CA89406 for A.D.).
APC | BIRC5 | CCNA2 | CCNB1 | CDC2 | CDKN1A | CHEK1 | KLF6 | Wnt4
UniProtKB: http://www.uniprot.orgAKT1 | AP4 | ARF | ATM | BAX | BMP2 | caspase 8 | caspase 10 | CBP | CDC20 | CDC25 | CDK1 | CDK2 | CDK4 | CDK6 | CDX1 | CDX2 | DDB2 | DTL | E2F1 | E2F3 | ERBB2 | ETO | IKKβ| INK1 | KLF4 | MAP3K5 | MAX | MYC | NEUROD1 | NGF | notch 1 | p21 | p27 | p53 | p57 | PCNA | procaspase 3 | RB | RBL1 | RBL2 | RHOD | SKP2 | STAT3
Anindya Dutta’s homepage: http://mexico.bioch.virginia.edu/