Regulating the p53 system through ubiquitination

Abstract

The tumor suppressor p53 is tightly controlled at low levels in cells by constant ubiquitination and proteasomal degradation. In response to stresses, ubiquitination of p53 is inhibited through diverse pathways, depending on the nature of the stimulus and cell type. This leads to the accumulation and activation of p53, which induces cell cycle arrest and/or apoptosis to prevent cells from transformation. Many studies have indicated that defects of the p53 system are present in most, if not all, human tumor cells. Meanwhile, significant progress has been made in understanding the molecular mechanisms of p53 ubiquitination and the regulation of the p53 system. Therefore, it is possible now to consider targeting ubiquitination as a means to regulate and reactivate p53 in tumors. Emerging evidence suggests that inhibiting the E3 activity of Mdm2, blocking the interaction of p53 and Mdm2, and restoring the function of mutated p53 are potential effective strategies to kill certain tumor cells selectively. It is conceivable that new chemotherapeutic agents based on these studies will be generated in the not-so-distant future.

Introduction

P53 tumor suppressor system

Since its discovery in 1979 as an SV40 large T antigen-binding protein, p53 has been the subject of intense study, especially after 1989, when it was demonstrated that mutation of p53 is associated with human cancers (Baker et al., 1989; Nigro et al., 1989; Takahashi et al., 1989). It has become clear now that p53 is a major ‘guardian of the genome’ (Lane, 1992). In response to stresses such as DNA damage and abnormal proliferation, p53 is accumulated and activated in cells, leading to cell cycle arrest or apoptosis. In doing so, it facilitates the repair of damaged DNA or eliminates irreversibly damaged or abnormally growing cells to prevent potential transformation. Under certain conditions cell cycle block protects cells from apoptosis, while under other circumstances, cells undergo growth arrest and apoptosis. Recent studies in fly and nematode indicated that induction of cell death following genotoxic challenges appears to be the ancestral function of p53, whereas the growth arrest may be a more recently acquired activity by p53 in higher organisms (Derry et al., 2001; Sogame et al., 2003). The importance of p53 in safeguarding genomes has been clearly demonstrated by genetic and clinical studies. P53-deficient mice are highly tumor susceptible, although it is not clear yet why the majority of tumors in these mice are lymphoma and skin cancer. Germline mutations in p53 result in Li-Fraumeni syndrome, a hereditary syndrome that predisposes individuals to various tumors. Moreover, about 50% of human cancers carry mutated p53, and human tumor cells retaining wild-type p53 often have defects in activating or responding to p53. It is likely that all cancer cells have a dysfunctional p53 system, which includes p53 and associated proteins as well as its upstream regulators and downstream effectors.

P53 as a specific transcription factor

As a sequence-specific transcription factor, p53 has a central sequence-specific DNA-binding domain. This domain has also been shown to interact with the ASPP (Ankyrin repeat, SH3 domain, and proline-rich domain containing protein) family proteins (ASPP1 and ASPP2), and that the interaction allows p53 to activate preferably transcription from proapoptotic genes such as Bax (Samuels-Lev et al., 2001). The N-terminal domain of p53 is responsible for interacting with basic transcription factors (such as hTAFII-31 and hTAFII-70) and transcription coactivators p300/CBP and PCAF, which also have histone acetyl transferases activity. Its C-terminus participates in its oligomerization and regulates the transactivation activity of the N-terminal domain, although the mechanisms of regulation remain the subject of intense investigation (Vousden, 2002). Association with other proteins and post-transcriptional modifications modulate the transcriptional activity of p53. While p300 and PCAF are acetyl transferases that acetylate p53 C-terminal lysine residues such as K382, another p53-binding protein SIRT1 (human Sir2) is a deacetylase that can specifically deacetylate p53 K382 (Luo et al., 2001; Vaziri et al., 2001; Langley et al., 2002). In cells subjected to DNA-damaging agents, p53 is acetylated at K382 and 320 (Sakaguchi et al., 1998; Abraham et al., 2000). It has also been shown that acetylation by p300 and PCAF enhance the transactivation activity of p53 in cells, whereas deacetylation of p53 suppresses such activity. However, p300/CBP and PCAF also promote histone acetylation and facilitate the access of promoters by transcription factors (Liu et al., 2003). Therefore, it is possible that the enhancement of transactivation is largely due to the recruitment of p300 and PCAF to p53-bound promoter regions. Phosphorylation of p53 at certain sites such as Ser15 enhances its interaction with p300/CBP and PCAF (Lambert et al., 1998), suggesting acetylation may have an important role in activating p53-responsive genes by DNA-damaging agents, which induce phosphorylation of p53 at multiple sites. Expression of oncogenic Ras in primary human cells causes phosphorylation of p53 at Ser33 and Ser46 by p38 MAPK (Bulavin et al., 2002). Interestingly, the activity of p38 MAPK is negatively regulated by phosphatase PPM1D, whose expression is upregulated by p53. This forms a negative feedback loop to modulate p53 activity in normal cells. It has been shown that the inactivation of p38 MAPK or overexpression of PPM1D significantly reduced p53-dependent transactivation. Consistently, approximately 11% of primary breast tumors have amplified PPM1D gene and retain wild-type p53. Phosphorylation of p53 at Ser 33, Thr 81, and Ser 315 leads to the generation of binding sites for Pin1, a peptidylprolyl isomerase that recognizes phosphorylated Ser/Thr-Pro motif (Zacchi et al., 2002; Zheng et al., 2002). Overexpression of the Pin1 enhances p53-dependent transactivation, gene expression, and apoptosis in an enzyme activity-dependent manner. In a Pin1-inducible cell line, the expression of Pin1 did not change the level of p53, but nevertheless increased the expression of p53 target genes (Zheng et al., 2002), suggesting that Pin1 directly affects the transactivation ability of p53 through inducing conformation change.

P53-regulated genes and cellular responses

Through the consensus binding sequence at their regulatory regions, p53 directly regulates the expression of many genes to modulate various cellular processes. For example, induction of cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 leads to G1 cell cycle arrest. The increased expression of GADD45 and 14-3-3σ participate in p53-driven G2 arrest (Colman et al., 2000). A number of p53-induced genes, such as Apaf1, p53AIP1, and proapoptotic members of the Bcl-2 family (Bax, Bid, Noxa, and PUMA), are directly involved in the mitochondria-dependent cell death pathway (Vousden and Lu, 2002). Deficiency or downregulation of each of these molecules results in decreased sensitivity to p53-dependent apoptosis under certain circumstances, indicating p53 utilizes a number of different molecules to activate the intrinsic apoptotic pathway. Elevated p53 also induces the expression of several death receptors and related molecules such as Fas, DR5, PIDD, p53DRL1 (Tanikawa et al., 2003), and TRAF4 (Sax and El-Deiry, 2003), whose overexpression induces or enhances apoptotic cell death. In addition, p53 has been shown to repress the expression of a number of genes and at least some of them, such as cyclin B1 and surviving, are negative regulators of apoptosis (Krause et al., 2000; Hoffman et al., 2002). Therefore, p53 may kill cells through the coordinated action of many genes and the contribution of each molecule depends on the cell type and stimulus. To fully understand the mechanism of p53-mediated apoptosis in particular cells, it is necessary to examine the global transcriptional changes induced by the activation of p53 in the cells. This has become possible with the completion of human genome project, production of high-quality microarrays, and improvement of techniques such as chromatin immunoprecipitation (ChIP) analysis. Intriguingly, one such study using ovarian cancer cells infected with p53-expressing adenovirus indicated that about 80% of the putative p53-responsive genes are in fact repressed by p53 (Mirza et al., 2003). It will be interesting to determine whether and how these identified genes are involved in p53-induced cellular responses in these tumor cells.

P53 siblings: cooperation and antagonism

The two p53 family members p73 and p63 share considerable homology with p53 and have similar domain structures (Jost et al., 1997; Kaghad et al., 1997; Yang et al., 1998). It is therefore not surprising that p73 and p63 can increase the expression of some p53 target genes, such as p21WAF−1 and Bax, and induce growth arrest and apoptosis under certain circumstances. It has also been shown that stress conditions such as radiation that upregulate p53 can also modulate p73 and p63 levels (Gong et al., 1999; Liefer et al., 2000). However, p63 and p73 have their own unique roles in development. In contrast to the essentially normal development in p53-deficient mice, p63−/− mice have defects in limb and epithelial development, whereas p73−/− mice have defects in neurological and immunological systems. Furthermore, the p73 and p63 genes can give rise to multiple isoforms through alternative splicing and utilization of different promoters. Notably, utilization of these second promoters leads to the production of p73 and p63 that lack their N-terminal transactivation domains, which act as dominant-negative inhibitors of p53 through heterodimerization and competition of DNA-binding sites. Moreover, p53 activates the transcription from the second promoter of p73 and therefore increases the production of truncated p73. This represents yet another negative feedback loop that modulates p53 activity (Yang et al., 2002). It has also been shown that downregulation of the N-terminally truncated p63 promotes UV-induced apoptosis in epidermis (Liefer et al., 2000), suggesting that the dominant-negative p63 modulates the cellular response to p53. Interestingly, the dominant-negative p73 is highly expressed in neuroblastoma cells (Douc-Rasy et al., 2002), and its increased expression is correlated with decreased apoptosis in tumor tissues and poor prognosis (Casciano et al., 2002). It has also been found that the level of N-terminally truncated p63 is elevated in squamous cell carcinoma due to p63 gene amplification (Hibi et al., 2000). Thus, p73 and p63 truncations likely act as dominant-negative inhibitors of p53 to promote malignant transformation. Intriguingly, combined loss of p73 and p63 prevents p53-dependent apoptosis, apparently due to the inability of p53 to upregulate apoptosis-related genes such as Bax (Flores et al., 2002). Although the mechanisms of such cooperation are not yet known, this work indicated that the p53 siblings also function as transcription cofactors to positively regulate p53 activity.

Regulation of p53 through ubiquitination

Ubiquitination and proteasomal degradation controls intracellular p53 level

Ubiquitin is a 76 amino-acid protein that can be covalently attached to Lys on target proteins through the sequential actions of enzymes E1, E2, and E3 (Pickart, 2001; Weissman, 2001). E3s, also known as ubiquitin protein ligases, recognize substrates and therefore are the primary specificity determining factors in ubiquitination. Once modified with chains of four or more ubiquitins linked through Lys 48 of ubiquitin (poly- or multiubiquitin) proteins can be efficiently targeted to the 26S proteasome, an ATP-dependent proteolytic complex, where they are degraded. Ubiquitination also directly affects the function and location of many target proteins either through monoubiquitination (a single ubiquitin added to one or more Lys on a protein) or through the generation of chains of ubiquitin linked through Lys other than K48 (such as K63) (Hicke, 2001). Thus, the ubiquitination and proteasomal degradation system plays an important role in controlling the levels and activities of myriad cellular proteins and in this way regulates a wide range of basic cellular processes such as cell cycle progression and signal transduction. As p53 is a potent cell cycle inhibitor and apoptosis inducer, it is not unexpected that its level and activity are tightly controlled. Early studies demonstrated that the half-life of p53 is only about 30 min (Maltzman and Czyzyk, 1984). DNA-damaging agents increased the half-life to several hours, leading to its accumulation.

The first direct evidence of a role for ubiquitination in downregulating p53 came from studies on the mechanism of cell transformation by human papillomavirus (HPV). Work by Howley and co-workers led to the determination that p53 degradation was increased in cells expressing E6 from oncogenic strains of HPV. This led to the discovery of E6-associated protein (E6-AP) as an ubiquitin ligase and to a model in which E6 usurped this normal cellular protein and redirected it towards p53 (Scheffner et al., 1993). Although E6/E6-AP appears to only regulate p53 in cells infected by oncogenic forms of HPV, these findings represented one of the first examples of in vivo ubiquitination leading to depletion of a normal cellular protein, and provided the first molecular signature for a family of ubiquitin ligases, the HECT (homologous to E6-AP carboxyl terminus) E3s. Moreover, HPV is not the only virus that has evolved strategies to cope with the p53 system in host cells. E1B55K and E4orf6 of human adenovirus target p53 for degradation through cooperation with a cullin-containing complex that consists of Cul5, elongins B and C, and the RING finger protein Rbx1 (Querido et al., 2001). Evidence of a role for ubiquitination in p53 turnover in cells not expressing HPV E6 was facilitated by the development of proteasome inhibitors in the mid-1990s. Treatment of cells with the peptide aldehyde proteasome inhibitor MG132 resulted in an increase in p53 levels together with a marked increase in ubiquitinated p53 (Maki et al., 1996). These results indicated that p53 is rapidly degraded by proteasomes and raised the possibility that modulating the ubiquitination-dependent degradation is a major mechanism for cells to control p53 level.

Mdm2 is a major negative regulator of p53

Mdm2 was first identified as the product of a gene amplified in a spontaneously transformed murine cell line. The 90-kDa protein is able to inhibit the transcriptional activity of p53 by binding to its transactivation domain and concealing it from basic transcriptional machinery and coactivators (Momand et al., 1992). The inhibition has also been attributed to the intrinsic transcriptional repressive activity of N-terminal region of Mdm2 and more recently to the recruitment of a transcriptional corepressor named CtBP2 by Mdm2 (Mirnezami et al., 2003). It was also found that Mdm2 promotes the ubiquitination and degradation of p53 and that the experimental overexpression of Mdm2 results in decreases in the level and activity of p53 (Haupt et al., 1997; Kubbutat et al., 1997). Further studies demonstrated that Mdm2 is a ubiquitin ligase (Honda et al., 1997) and the activity of Mdm2 towards p53 both in vitro (Fang et al., 2000; Honda and Yasuda, 2000) and in cells (Fang et al., 2000) was mediated by its C-terminal RING finger. Thus, overexpression of Mdm2 provides an alternative to p53 mutation as a means of inactivating p53 and allows the development of tumors that retain wild-type p53.

The RING finger motif is characterized by a group of conserved Cys and His residues that form a crossbrace structure conformed by the binding of two zinc ions. This compact structure interacts with E2 enzymes and facilitates ubiquitination of bound substrates. In the case of Mdm2, its intrinsic ligase activity also serves to target its own ubiquitination and degradation (Fang et al., 2000). The specificity of Mdm2 E3 activity has been illustrated by the observation that it also binds to p63 and p73, but does not promote their ubiquitination and degradation (Zeng et al., 1999; Balint and Vousden, 2001). Part of this specificity may be due to a cluster of six Lys residues found near the C terminus of p53 but are absent from the other family members. These residues are believed to represent the ubiquitination sites on p53 (Lohrum et al., 2001). Interestingly, while a substitution of a heterologous RING finger for that of Mdm2 still allows for Mdm2 self-ubiquitination, such a substitution does not result in targeting of p53, suggesting that the RING finger may also contribute to determining the specificity of Mdm2 and is important for targeting of p53 (Fang et al., 2000).

One unresolved issue is whether Mdm2 by itself is sufficient to mediate the formation of multiubiquitin chains on p53. Some in vitro studies have suggested that Mdm2 can only mediate the addition of single ubiquitin to multiple Lys residues, which would be insufficient to target the protein for proteasomal degradation. One possibility that has been suggested is that the role of Mdm2 monoubiquitination is to provide a signal leading to the export of p53 to the cytosol where polyubiquitination and proteasomal degradation would occur. However, nuclear export is not required for proteasomal degradation of p53 (Xirodimas et al., 2001). Another possibility is that Mdm2 needs additional factors to promote polyubiquitination of p53 in the nucleus. Such a factor that facilitates polyubiquitination has been referred as an E4 (Koegl et al., 1999). A recent study reported that p300 has ubiquitin ligase activity for p53 and promotes the polyubiquitination of p53 together with Mdm2 (Grossman et al., 2003), suggesting that p300 could be an E4 for p53. Since p300 is a transcription coactivator, it is conceivable that Mdm2 promotes the polyubiquitination and degradation of p53 that is engaging transcriptional machinery, while it only leads to monoubiquitination of the p53 molecules that are not actively involved in transcription. As of this writing, it remains to be determined with certainty to what extent the in vitro observations of monoubiquitination by Mdm2 truly reflect the in vivo setting.

Pirh2 has recently been identified as another RING finger protein that binds to p53 and mediates its ubiquitination independent of Mdm2 (Leng et al., 2003). Downregulation of endogenous Pirh2 increases p53, whereas expression of Pirh2 leads to a decrease in p53, resulting in the repression of p53-induced transactivation and reverse of growth inhibition. The existence of an additional E3 for p53 provides a potential explanation for the observation that the kinase JNK can target p53 ubiquitination and degradation in an Mdm2-independent manner (Fuchs et al., 1998). Given the important roles of JNK in cellular signal transduction, it is very interesting to examine whether Pirh2 is regulated by JNK and whether they are involved in p53 regulation under physiological or pathological conditions. Like Mdm2, Pirh2 is also upregulated by p53 at transcriptional level. Pirh2 binds to a region (residues 82–292) distinct from that to which Mdm2 binds (residues 1–51). Thus, it is conceivable that Mdm2 and Pirh2 can bind to a single p53 molecule and function in a cooperative manner to mediate its ubiquitination.

MdmX as a partner and regulator of Mdm2

MdmX (also known as Mdm4) is a p53-binding protein that shares extensive homology with Mdm2, including a RING finger at its C-terminus (Shvarts et al., 1996) and an N-terminal region that binds p53. Unlike Mdm2, MdmX is not increased following genotoxic stimulation, and it does not effectively promote p53 ubiquitination (Stad et al., 2001). Domain swap experiments suggest that both the RING finger and a central region within Mdm2 contribute to the difference in activity between the two proteins (Meulmeester et al., 2003). Nevertheless, as is the case for Mdm2, MdmX deficiency leads to embryonic death that is rescued by the loss of p53 (Jones et al., 1995; Montes de Oca Luna et al., 1995; Parant et al., 2001; Finch et al., 2002; Migliorini et al., 2002), indicating that MdmX is also a critical negative regulator of p53 in vivo. Accordingly, downregulation of MdmX causes a decrease of Mdm2 and increase of p53, which increases the sensitivity of cells to UV-induced apoptosis (Finch et al., 2002; Gu et al., 2002). Furthermore, amplification and overexpression of the MdmX gene were found in certain human malignant gliomas that have wild-type p53 (Riemenschneider et al., 1999). The molecular basis of these observations has been investigated using p53−/−MdmX−/− MEFs. The expression of MdmX in these cells increased the half-life of Mdm2 and enhanced the degradation of transfected p53 (Gu et al., 2002). A potential explanation is provided by the observation that formation of the heterodimer selectively inhibits Mdm2 autoubiquitination, but not ubiquitination of p53 by Mdm2. Since the RING finger regions of Mdm2 and MdmX are also required for their dimerization (Tanimura et al., 1999), resolving the three-dimensional structure of the heterodimer should enhance our understanding of these results. In addition, other mechanisms may contribute to the effect of MdmX in cells. For example, transfection of MdmX to certain cultured cells led to the retention of ubiquitinated p53 in the nucleus (Stad et al., 2001), suggesting that MdmX functions through preventing nuclear export of p53. Enforced expression of MdmX can inhibit Mdm2-mediated p53 ubiquitination and degradation, presumably through competing with Mdm2 in binding with p53 (Stad et al., 2001). Thus, while the formation of Mdm2–Mdmx dimers may favor p53 ubiquitination, higher levels of this protein may serve a dominant-negative function in preventing access of Mdm2 to p53. To understand the effects of changing the relative levels of Mdm2 and MdmX, it is important to consider that their effects may be occurring in the context of tetramerization of p53. Each p53 tetramer could theoretically bind multiple Mdm2/MdmX. Furthermore, it has recently been reported that MdmX has low in vitro E3 activity (about 0.1–1% of that observed with Mdm2), which may participate in its autoubiquitination as well as the ubiquitination of Mdm2 and p53 (Badciong and Haas, 2002). The in vivo significance of this, given the generally negative data for MdmX as an E3, remains to be determined.

Alternative reading frame (ARF) generates an ubiquitination inhibitor

ARF is a small basic protein (pI>12) encoded by the INK4a locus that also encodes the cyclin-dependent kinase inhibitor p16INK4a (Lowe and Sherr, 2003). Shortly after its identification, both human and mouse ARF (p14ARF and p19 ARF, respectively) were found to interact with Mdm2 and to block Mdm2-mediated p53 degradation (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998). This was attributed to the direct inhibition of Mdm2 E3 activity by ARF (Honda and Yasuda, 1999; Midgley et al., 2000). Intriguingly, it was shown recently that the expression of ARF promotes Mdm2-mediated ubiquitination of MdmX (Pan and Chen, 2003). Since MdmX is important for the stability of Mdm2, it is conceivable that this may contribute to the inhibition of Mdm2 E3 activity toward p53 following stresses that induce ARF in cells. It was also found that experimentally overexpressed ARF resides in the nucleolus and could induce the nucleolar localization of Mdm2, presumably by revealing an otherwise cryptic nucleolar localization signal in the Mdm2 RING finger. This could result in the inhibition of p53 degradation, due to separation of Mdm2 from p53 or possibly by preventing nuclear export of the Mdm2/p53 complex (Tao and Levine, 1999; Weber et al., 1999; Zhang and Xiong, 1999). However, the essential role of nucleolar retention is challenged by the finding that some mutated ARFs do not induce growth arrest despite their ability to retain Mdm2 in the nucleolus (Korgaonkar et al., 2002). Moreover, various truncated p14ARFs that contain the 1–29 region did not necessarily accumulate in the nucleolus, but nevertheless stabilized Mdm2 and p53 (Llanos et al., 2001). In addition, in certain cell lines, induction of ARF leads to stabilization of p53 and cell cycle arrest without the relocation of Mdm2 to the nucleolus (Lin and Lowe, 2001; Llanos et al., 2001). Therefore, relocation to the nucleolus is not essential for the inhibition of Mdm2 by ARF in all cells, although it may contribute to the suppression of Mdm2 under certain circumstances.

Axis of regulation: diverse signals from multiple pathways

The ARF–Mdm2–p53 axis is regulated at multiple levels through many different mechanisms (Figure 1). Genotoxic stimuli are able to activate several kinases (including Chk1, Chk2, ATM, and ATR) that phosphorylate the N-terminus of p53 at multiple sites (such as residues 15, 20, 33, 37, and 46) (Appella and Anderson, 2001). This not only enhances transactivation activity of p53 noted above but also prevents its interaction with Mdm2, leading to the inhibition of ubiquitination. It has been shown that the oncogene promyelocytic leukemia gene (PML) protects p53 from Mdm2-mediated ubiquitination through prolonging the phosphorylation at serine 20 (Louria-Hayon et al., 2003). There are additional mechanisms to inhibit p53 ubiquitination. A potential tumor suppressor ING1 appears to stabilize p53 by competing with Mdm2 for binding to p53 (Leung et al., 2002). A number of Lys residues at the C-terminus of p53 are the targets of both acetylation and ubiquitination. Their acetylation inhibits Mdm2-mediated p53 ubiquitination (Li et al., 2002b). While p53-associated p300/CBP and PCAF are likely involved in the acetylation, Mdm2-recruited histone deacetylase (HDAC)1 can deacetylate p53, allowing for its ubiquitination and degradation (Ito et al., 2002). Deubiquitination enzymes could also regulate the ubiquitination status of p53. One such enzyme, HAUSP, has been shown to deubiquitinate specifically and stabilize p53 by removing its ubiquitin modifications (Li et al., 2002a). It is interesting to examine how PML, ING1, and HAUSP are regulated following genotoxic stimuli since the physiological significance of these molecules in regulating p53 remains to be determined.

Figure 1
figure1

Ubiquitination of p53 and its regulation

The level and activity of Mdm2 is also regulated by a variety of signals. It has been shown that the p53 inducer nitric oxide downregulates Mdm2 at a post-transcriptional level (Wang et al., 2002). In response to DNA damage, ATM phosphorylates Mdm2 on Ser395, which impedes Mdm2-mediated nuclear export and degradation of p53 (Maya et al., 2001). ATM can also activate c-Abl, which phosphorylates Mdm2 at Tyr394 and prevents its interaction with p53 (Goldberg et al., 2002). However, phosphorylation may also enhance the activity of Mdm2 under certain circumstances. For example, the growth factor-activated Akt phosphorylates Mdm2 at Ser166 and Ser186, which promotes nuclear translocation of Mdm2, leading to increased p53 ubiquitination and degradation (Mayo and Donner, 2001; Zhou et al., 2001). This may contribute to the antiapoptotic action of growth factors such as IGF-1 and EGF. Interacting with other proteins provides another way to modulate the activity of Mdm2. In addition to ARF, the E3 activity of Mdm2 is inhibited by ribosomal protein L11, resulting in the stabilization and activation of p53 (Lohrum et al., 2003). Since the interaction between Mdm2 and L11 is enhanced after treating cells with low doses of actinomycin D, such interaction may represent a mechanism for cells to cope with perturbations in ribosome protein synthesis. Interestingly, tumor susceptibility gene 101 product (TSG101), an E2-like ubiquitin-binding protein that has an important role in endosomal trafficking and downregulation of membrane receptors (Lu et al., 2003), also binds and regulates Mdm2 (Li et al., 2001). The overexpression of TSG101 resulted in an increase in Mdm2 and a reciprocal decrease of p53 in cells. This regulation appears to be important as TSG101 deficiency leads to early embryonic death and accumulation of p53 in embryo cells (Ruland et al., 2001). It is not clear yet how the regulation of Mdm2 contributes to the proposed tumor suppressor function of TSG 101.

Although it does not directly respond to genotoxic stresses, ARF is induced by hyperproliferative signals, such as oncogene activation, to induce the accumulation of p53 and suppress cell transformation. One of the molecules that connect ARF with cell proliferation is E2F, whose activity is regulated by retinoblastoma (Rb) protein, a target of the Cdks. Hypophosphorylated Rb binds with E2F and the resultant E2F/Rb complex inhibits ARF expression (Rowland et al., 2002). During proliferation, activated Cdks phosphorylate Rb leading to the release of E2F, which promotes ARF upregulation. The importance of ARF in tumor development was clearly illustrated by the studies with transgenic mice expressing Myc under the control of Ig μ heavy-chain promoter (Eμ). The Eμ-Myc transgenic mice usually develop lymphoma by 1 year of age. However, when the transgene was expressed in Arf−/− mice, aggressive disseminated lymphomas developed rapidly and mice died within a few weeks after birth (Eischen et al., 1999; Schmitt et al., 1999). In addition, the expression of ARF is negatively regulated by a transcription repressor Bmi-1 (Twist) (Jacobs et al., 1999; Maestro et al., 1999). Interestingly, recent studies demonstrated that there is a selective loss of self-renewing adult hematopoietic stem cells in Bmi-1-deficient mice (Park et al., 2003). The expression of ARF is increased in bone marrow cells from these mice and infection with viruses expressing ARF killed hematopoietic stem cells from wild-type mice, but not those from p53 deficient mice, suggesting that increased levels of ARF and p53 are responsible for the loss of hematopoietic stem cells in Bmi-1-deficient mice.

It was noted that ARF could suppress the proliferation of MEFs that lack both p53 and Mdm2 (Weber et al., 2000). One possible reason for the suppression is that ARF can induce the expression of certain antiproliferative genes, which may reduce the rate of DNA synthesis (Yarbrough et al., 2002; Kuo et al., 2003). In addition, inhibition of ribosomal RNA processing by ARF may contribute to its antiproliferation activity (Sugimoto et al., 2003). It is also conceivable that ARF could inhibit ubiquitination processes by E3s other than Mdm2 or affect localization of other transcription factors. The importance of these nonoverlapping activities of ARF, Mdm2, and p53 is illustrated by the observation that, compared to mice lacking ARF or p53 alone, mice deficient in all three genes develop a much broader spectrum of tumors and the tumors can appear simultaneously at independent sites (Weber et al., 2000). Thus, ARF, Mdm2, and p53 represent components of a complicated system that prevents the formation of tumors through multiple mechanisms.

Targeting ubiquitination: therapeutic exploration of the p53 system

Blocking the Mdm2 and p53 interaction

It has become clear over the last decade that apoptosis is a critical fail-safe mechanism in mammalian cells (Green and Evan, 2002). To prevent the generation of cancer cells, apoptotic pathways are activated in response to oncogenic conditions such as improper activation of oncogenes and DNA damage. This leads to cell death or renders them more susceptible to apoptotic stimuli. During the course of transformation, cells acquire defects in the apoptosis machinery. These alterations allow them to survive oncogenic changes and provide them survival advantages, such as protection against hypoxia, decreased dependence on growth factors, and increased tolerance to genetic alterations. Since the p53 system is responsible for sensing oncogenic changes and determining the fate of affected cells, it is likely to be dysfunctional in all tumors. In fact, tumor cells retaining wild-type p53 usually have defects in activating or responding to p53. Notably, overexpression of Mdm2 occurs in about 10% of human tumors and loss of ARF is found in even more tumor cells (Lowe and Sherr, 2003). In these tumors, inhibition of Mdm2 function should result in p53 accumulation, leading to growth arrest or apoptosis. One attractive strategy to inhibit ubiquitination of p53 by Mdm2 is to block their interaction. An Mdm2-binding sequence in p53 has been identified (including residues 18–23) that likely forms a helical conformation upon binding to Mdm2 (Picksley et al., 1994). Moreover, these studies have shown that p53-derived peptides (such as residues 15–29) bind to Mdm2 (with Kd ranging from 60 to 700 nM), and thus provided much needed basic knowledge for designing inhibitors. Using phage display, a 12-residue peptide was found to be almost 30-fold more potent than the original p53-derived peptide in binding Mdm2 (Bottger et al., 1997). When expressed as a fusion protein with thioredoxin in cells, the peptide increased the level of p53, activated p53-responsive genes, and led to cell cycle arrest. These results indicated that disrupting Mdm2 and p53 interaction could be an effective way to reactivate p53. A number of other peptides were also designed to achieve more stable structures and/or higher affinity for Mdm2. Intriguingly, substituting residues corresponding to p53 Tyr22 and Trp23 with phosphonomethylphenylalanine and 6-chloro-tryptophan generated a peptide that is about 1780-fold more potent than the original p53-derived peptide in binding with Mdm2 (Garcia-Echeverria et al., 2000; Chene et al., 2002). The peptide activated the p53 system and induced apoptosis when added directly to cultured tumor cell lines. However, it has been reported that p53 C-terminus-derived Mdm2-binding peptides have p53-independent cytotoxicity to transformed cells (Kanovsky et al., 2001), suggesting that under certain circumstances these peptides may act by means other than just blocking p53 degradation. There have also been efforts to identify natural products and synthetic small molecules that could impede binding of p53 and Mdm2. For example, a fungal polypeptide named chlorofusin is an effective inhibitor of p53 and Mdm2 interaction in vitro (Duncan et al., 2001). A synthetic polycyclic compound that mimics Phe19 and Trp23 of p53 cause the accumulation of p53 and induces apoptosis in cell lines that harbor wild-type p53 (Zhao et al., 2002). While these compounds could be good leads for further investigation, questions have been raised concerning whether small molecules could effectively block the interaction of the two macromolecules. Hopefully, high-throughput screening of chemical libraries could identify new compounds that are more potent and effective as potential therapeutic agents.

Inhibiting E3s that ubiquitinate p53

Since the E3 activity of Mdm2 is essential for the ubiquitination and degradation of p53, inhibition of the ligase activity would also increase the intracellular level of p53 and potentially kill tumor cells. This may be a more feasible approach because many enzyme inhibitors have been successfully developed in the past and ARF, an endogenous inhibitor of Mdm2 E3 activity, already exists. While the therapeutic potential of ARF remains to be further explored, a number of laboratories have begun to look for small molecules that act on Mdm2 and inhibit its E3 activity. It has been reported that screening chemical libraries identified three structurally unrelated compounds that specifically inhibit Mdm2-mediated p53 ubiquitination in vitro (Lai et al., 2002). They appear to bind to a common site on Mdm2 and do not impede the Mdm2 interaction with p53 or E2 (Ubc4). Although the actual data were not shown in the article, the authors indicated that these compounds do not inhibit the autoubiquitination of Mdm2. It will be interesting to know whether these compounds could cause the accumulation of active p53 in cells and selectively kill tumor cells.

Cervical cancer is one of the most common malignancies in woman. Many studies have demonstrated that infection with oncogenic forms of HPV, such as type 16 and 18, is the major risk factor for the cancer. Since E6 protein from these types of HPV can associate with E6-AP to promote the ubiquitination and degradation of p53, the accumulation and activation of p53 following the expression of oncogenes, such as HPV-encoded E7, are inhibited (Scheffner et al., 1993). This explains why the majority of cervical cancer cells retain wild-type p53 and suggests that an increase of p53 in these cells may be able to prevent cell transformation and kill cancer cells. The latter predication has been demonstrated in cervical carcinoma cell lines carrying integrated HPV. Low doses of the RNA synthesis inhibitor actinomycin D and nuclear export inhibitor leptomycin B can induce the accumulation of p53 in these cells, leading to transcription of p53-responsive genes and p53-dependent apoptosis (Hietanen et al., 2000). Therefore, it is conceivable that developing small molecules that interfere with the interaction of p53 and E6-E6AP or that inhibit the E3 activity of E6-E6AP might be effective strategies to prevent and treat cervical cancer.

Rescuing mutated p53: is ubiquitination involved?

A large number of studies have revealed two unique features of p53 mutation in human tumor cells. First, most of the mutations are single amino-acid substitutions that do not cause the loss of protein expression. As a result, cancer cells usually have high levels of mutated p53. There is evidence indicating that at least certain p53 mutations provide transformed cells with survival advantages or directly promote transformation. Second, the great majority of identified p53 mutations are located in the core (DNA-binding) domain, where they are often directly involved in DNA–protein interactions or are critical for the structure of p53. Since tumor cells harboring mutated p53 often retain the ability to respond to wild-type p53, restoring the activity of p53 mutants should suppress their growth or even induce apoptosis. The reversibility of mutated p53 was first illustrated by the finding that p53 harboring V143A mutation is inactive at 37°C, but can be activated at lower temperature (32°C). Through genetic screening and direct mutagenesis, it was documented that introducing a second mutation could restore the activity of certain p53 mutants. Moreover, C-terminal truncations of p53 and short synthetic peptides that bind to the p53 core domain are also effective in inducing p53 activities (Selivanova et al., 1999; Friedler et al., 2002). These studies suggest that restoring the wild-type function to mutated p53 could be an effective strategy for cancer therapy. Since peptides are generally not ideal drugs, efforts have been made to look for small molecules that can help the folding of mutated p53 and therefore restore its function (Foster et al., 1999; Bykov et al., 2002). For example, ellipticine and derived compounds can restore the transactivation activity of p53 (Peng et al., 2003). However, it has not been determined whether they act directly upon mutated p53. From high-throughput screening, a compound named CP-31398 was found to stabilize a p53 epitope in vitro, increase transcriptional active p53 in cells harboring mutant p53, and slow tumor growth in mice (Bykov et al., 2002), suggesting it could be a prototype of new anticancer drugs. As the effects of CP-31398 and the core domain-interacting peptides on mutated p53 depend on new protein synthesis, it is likely that they all bind to newly synthesized p53 and facilitate its folding. Unexpectedly, CP-31398 also stabilizes wild-type p53 and increases its intracellular level, leading to cell cycle arrest or apoptosis (Luu et al., 2002; Wang et al., 2003). Further studies demonstrated that the compound inhibits p53 ubiquitination in an Mdm2 and p53 phosphorylation-independent manner. These results raise a number of interesting issues. Among them, is the effect of CP-31398 on mutated p53 related to inhibition of ubiquitination? What are the molecular mechanisms of inhibiting p53 ubiquitination by the compound? Can this compound be used for the treatment of tumors with wild-type p53?

Other successful and potential strategies

Targeting other components of the p53 system can also potentially result in the accumulation and activation of p53. For example, inhibiting the phosphatase PPM1D or blocking the cooperation between p300 and Mdm2 would likely increase the intracellular level of p53. Since certain p53 mutants promote cell transformation, accelerating their degradation could kill tumor cells or make them more susceptible to chemotherapeutic agents. It has been shown that a HDAC inhibitor selectively downregulates mutated, but not wild-type p53, apparently through inhibiting its interaction with the chaperone protein Hsp90 (Yu et al., 2002). It is worth noting that there are a large number of genes encoding deubiquitinating enzymes in the genomes, and at least some of them participate in the regulation of ubiquitination through removing ubiquitin from specific targeting proteins (Wing, 2003). The finding that HAUSP specifically interacts with p53 and regulates its intracellular level in an isopeptidase activity-dependent manner suggests that it could be a target for intervention (Li et al., 2002a). Interestingly, a proteasome inhibitor PS-341 has demonstrated antitumor activity in various animal models and shown promise as an effective therapeutic for certain human neoplasms such as multiple myeloma. Although its mechanisms of inducing tumor cells apoptosis are not yet well understood, PS-341 causes accumulation of p53 and increases p53 phosphorylation in tumor cells (Hideshima et al., 2003). In certain cell lines, a dominant-negative form of p53 blocks proteasome inhibitor-induced apoptosis (including PS-341) (Lopes et al., 1997). Therefore, it is likely that increased p53 contributes to PS-341-induced tumor cell apoptosis, although the drug can still kill tumor cells in the absence of functional p53 through other mechanisms, such as activating JNK and disrupting cell cycle machinery.

Conclusions

The p53 system plays a critical role in safeguarding the genome and preventing cell transformation. While p53 is subjected to many post-translational modifications, ubiquitination is a central theme in its regulation. Signals transduced through diverse intracellular pathways act on the ARF–Mdm2–p53 axis to modulate the level and activity of p53 through ubiquitination. Given the involvement of the p53 system in most, if not all, human tumors, targeting the ubiquitination process should be an effective strategy to prevent and treat cancers. Intriguingly, while a number of proteasome inhibitors have been developed, little progress has been made in finding specific inhibitors for E1, E2, and E3. Inhibitors of E3s, such as Mdm2 and E6/E6-AP, are particularly desirable for experimental and therapeutic purposes. With the significant advance in our understanding of the molecular mechanisms of ubiquitination and promising results from PS-341, it is conceivable that we will witness the generation of more chemopreventive and chemotherapeutic agents targeting the ubiquitination of the p53 system in the coming years.

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Acknowledgements

We would like to thank Drs Mary Perry and Kevin Lorick for critical reading and editing of the manuscript. We apologize to colleagues whose important contributions have been cited only indirectly due to space limitation. This publication has been partially funded with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400.

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Yang, Y., Li, CC. & Weissman, A. Regulating the p53 system through ubiquitination. Oncogene 23, 2096–2106 (2004). https://doi.org/10.1038/sj.onc.1207411

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Keywords

  • p53
  • Mdm2
  • ubiquitination
  • molecular targeting
  • apoptosis

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