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Convergence of p53 and TGF-beta signaling networks
Cancer Letters 213 (2004) 129–138 www.elsevier.com/locate/canlet
Mini review
Convergence of p53 and TGF-beta signaling networks Sirio Dupont, Luca Zacchigna, Maddalena Adorno, Sandra Soligo, Dino Volpin, Stefano Piccolo*, Michelangelo Cordenonsi* Department of Histology Microbiology and Medical iotechnologies, Section of Histology and Embryology, University of Padua, viale Colombo 3, 35121 Padua, Italy Received 25 May 2004; accepted 3 June 2004
Abstract p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: p53; TGF-beta; Growth control; Cancer; Embryonic development
1. Introduction Members of the TGF-beta growth factor family are fundamental signals regulating cell behaviour in a variety of cellular contexts [1–4]. In adult tissues, TGF-beta conveys crucial cytostatic signals to epithelial, immune and other cells types; the loss of this response is the hallmark of many cancers [5]. * Corresponding authors. Address: DHMMB, Histology and Embryology, University of Padua, Viale Colombo 3, 35100 Padua, Italy. Tel.: C39-049-8276098; fax: C39-049-8276079. E-mail address: [email protected] (S. Piccolo).
Research over more than a decade has brought us a fairly satisfactory view of how the TGF-beta signal is transduced from the cell surface to the nucleus. TGF-beta ligands bind to cognate serine/threonine kinase receptors leading, intracellularly, to phosphorylation and activation of the Receptor-Smads signal transducers (R-Smad), such as Smad2 and Smad3. Once activated, the R-Smads translocate into the nucleus to control gene expression in concert with Smad4. The Smads bind to DNA with low affinity and, unless their consensus is reiterated, they require transcription factors as partners for specific interaction with target promoters [6]. The archetypal
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.06.008
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Smad partner is FAST-1 (forkhead activin signal transducer), which lacks intrinsic transcriptional activity, but is essential for targeting the Smads to some developmentally relevant promoters, such as Mix.2 [7–10]. One may expect that such remarkably simple transduction machinery may leave little room for flexibility and lead to a rather stardardized gene expression profile. In contrast, TGF-beta can control hundreds of target genes in different cells [11–13]. Although the mechanisms by which such transcriptional plasticity is attained are unknown, they must entail the interaction of the Smads with other ‘informational nodes’ of the cell. These nodes should be able to integrate multiple inputs from inside and outside the cell setting the ‘context’ in which the TGF-beta signal must act.
2. TGF-beta and growth control In epithelial cells, TGF-beta controls cell growth by the up-regulation of the cyclin-dependent-kinase inhibitors p15INK4b and p21WAF1 [14,15] and the concomitant repression of the growth promoting transcription factors c-MYC, Id1, Id2 and Id3 [16–18]. In normal cells, the anti-mitogenic activities of TGF-beta prevail over inputs favoring proliferation whereas this program fails to be activated in cancer cells. The resistance to TGF-beta induced cytostasis can be acquired in some cancers through inactivation of the genes encoding for TGF-beta receptors and Smads [19]. However, the vast majority of cancers lose selectively the growth-inhibitory response to TGF-beta in the presence of intact TGF-beta receptors and Smads, leaving other responses fully operative. Indeed, advanced carcinomas typically express high levels of TGF-beta to foster their own invasive/prometastatic abilities [20,21]. Therefore, as loss of TGFbeta responsiveness is rare in cancer, it is of great interest to determine how cancer cells acquire selective resistance to TGF-beta in growth-inhibition. Conceivably, loss of TGF-beta tumor suppressing effects should occur by genetic modifications in unknown Smad effectors or downstream molecules. The identification of these modulators is therefore essential for understanding tumor incidence and progression.
3. Identification of p53 as Smad partner p53 was isolated independently in this laboratory and by Atsushi Suzuki’s group in unbiased screens for biologically active molecules expressed during embryonic development [22,23]. The two groups searched for genes endowed with biological activity within small pools of cDNAs (n!100) from embryonic libraries using Xenopus assays. Curiously, the same result was obtained by quite distinct screening strategies. One group set to identify mouse genes whose expression promoted the activation of mesoderm and endoderm markers in embryonic cells [22], which is a typical TGF-beta-related effect in frog embryos. The second group screened for molecules able to transform the most anterior neural tissue into posterior brain or spinal cord [23]. During Xenopus development, this ‘posteriorizing’ activity emanates from the mesoderm cells underlying the developing CNS (such as those of the notocord and somites) [24]. In both screens, sib-selection revealed that p53 was, unexpectedly, the biologically active cDNA [22,23]. Injection of titrated doses of p53 mRNA in Xenopus cells causes them to adopt mesodermal and endodermal fates, in a dose-dependent fashion. Molecularly, these inductions are direct (that is, cycloheximide insensitive) and require p53-dependent transcription [22,23]. Importantly, p53 requires Smad activity to engage its TGF-beta-like effects [22,23]. This suggests that overexpression of p53 can make cells responsive to otherwise sub-threshold amounts of endogenous TGF-beta ligands. Along the same line, it will be interesting to check whether TGF-beta and unbalanced levels of p53 may cooperate during mammalian development; indeed, mice mutant for the p53-inhibitor mdm-2 [25,26] die very early during embryonic development (around E6.5), leaving unaddressed the possibility that the expression of early mesoderm/endoderm markers (whose induction is controlled by Nodal, a TGF-beta-related ligand) may be perturbed in this genetic background as an effect of increased p53 levels. Of note, a closer comparison of the immediate gene responses triggered by Smad2 and different doses of p53 mRNA in Xenopus cells reveals a large, but not complete overlap of activity, being p53 unable to activate some TGF-beta targets (such as goosecoid,
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Wnt8 and Xnr-1) [22,23]. This finding reveals that p53 acts selectively on a subset of TGF-beta target promoters, rather than as general enhancer of TGF-beta responses. Interestingly, biochemical experiments unveiled that the mechanism of this selectivity is dual: p53 associates with Smad2 and Smad3 in vivo in a TGF-beta-dependent manner but, at the same time, p53 must contact its own cognate DNA site on a promoter to facilitate a robust TGF-beta induced transcription [22]. This model is supported by two observations. First, the Mix.2 and PAI-1 promoters, two paradigms of TGF-betainduced transcription, contain a p53-binding element in proximity of the TGF-beta enhancer. Mutation of these p53-elements—or depletion of endogenous p53 by siRNA—quantitatively inhibits the inducibility of these reporters by TGF-beta. Conversely, overexpression of p53 leads to a synergistic increase in TGF-beta-induced transcription of the same reporters. Second, point-mutations disrupting p53 DNA binding activity do block its biological activity in Xenopus assays [22]. In other words, p53 is unable to enter into a Smad-activated transcriptional complex unless it can bind to neighboring cognate DNA sequences. Perhaps, the affinity of the interaction between p53 and Smad is low and can occur efficiently, at physiological concentrations, only when the two proteins are brought close on promoter DNA. Alternatively, only ‘activated’/DNA-bound p53 may be competent to interact with the Smads. Whatever the mechanisms, it is the concomitant presence of a Smad-regulated enhancer and of a p53 binding site that ultimately defines a gene under joined-control of p53 and TGF-beta.
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leading to the assembly of a more stable and specific multifactorial complex [29] (Fig. 1). Although the domains of p53 required for interacting with the Smads are unknown, an interesting conclusion emerges from the published studies: the C-terminus of p53 plays an inhibitory function in TGF-beta-gene responses. Indeed, in mesoderm
4. A new twist for the p53 C-terminus? p53 binds the N-terminal MH1 domain of Smad2, that includes the Smads’ DNA-binding site and also contains the binding domain for other transcription factors, including JunD, LEF-1 and FoxO [15, 22,27,28]. This raises the intriguing possibility that these other proteins may modulate the interaction between p53 and Smads. In the p53/Smad2 complex, the C-terminal MH2 domain of Smad2 is free to interact with FAST-1 and Smad4. This suggests that Smad2 may bridge the DNA-bound p53 and FAST-1
Fig. 1. A model depicting the convergence of p53 and Smads in TGF-beta-mediated gene transcription. Upon TGF-beta stimulation, Smad2 moves to the nucleus where it associates with Smad4 and specific DNA binding co-factors, such as FAST1, and the resulting complex binds to a specific promoter sequence. p53 and the activated Smad complex bind to each other and to their cognate sites on DNA, leading to a synergistic transcriptional activation. Other components of the TGF-beta signaling cascade, transcriptional cofactors and the basal transcriptional machinery were omitted for simplicity.
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induction assays, injection of mRNA encoding either for a deletion of the last 30 aminoacids of Xp53 or for mouse p53AS (a naturally occurring alternatively spliced RNA generating a C-terminal variant) display a substantially higher efficiency than injection of comparable amounts of wild-type/regularly spliced p53 (p53R) [22,23]. This finding is intriguing as this represents the first clear indication on the physiological function of the p53 C-terminus in vivo [30]. Early reports showed that deletion of C-terminal residues had a dramatic increase in p53 ability to bind to naked DNA, in gel-shift assays [31]. In contrast, recent results found that these C-terminally deleted variants of p53 are actually much weaker than intact p53 in binding to chromatinized promoters in vitro and in vivo [32]. Moreover, in vivo experiments showed that p53AS and C-terminally deleted p53 have growthinhibiting ability comparable to regular-p53 [33] and display weaker effects on apoptosis [34,35]. Finding a role for the p53 C-terminus in the Xenopus model system paves the way for addressing the regulatory function of many post-translational modifications impinging on p53 in the context of TGF-beta gene responses.
5. A developmental role for p53 p53 is not only instrumental for fostering TGF-beta gene responses but it is also required for correct TGF-beta responsiveness. Indeed, Xenopus cells depleted of p53 become refractive to activin, and p53 knock-down in frog embryos inhibits mesoderm formation and patterning [22]. These studies suggest that the endogenous TGF-beta signals are by themselves insufficient, but require the concomitant presence of p53, for turning-on effectively several of their targets. Finding a developmental role for p53 is obviously surprising, given that p53 knock-out mice develop normally [36]. One possible explanation for this discrepancy is offered by the finding that different p53 family members are expressed—and remarkably abundant—in early mouse embryos and may compensate for loss of p53 [22]. This is not the case in lower vertebrates where p53 is the only member of its family expressed during early embryogenesis [37–40]. Of note, p63 and p73 are critical for embryonic development [41]. Recent work demonstrated that
p53-related proteins may converge to regulate common biological processes and, in so doing, one may receive assistance from its siblings [42,43]. One shared biological property may be the ability to bind and to cooperate with Smads. Indeed, p73 can also bind the Smads and different isoforms of p63/p73 can synergize with Smads in promoting a very robust increase in TGF-beta induced transcription of cloned reporter constructs [22]. In addition to redundancy, it has been proposed that the different developmental phenotypes caused by lack of p53 in frog and mammals may also reflect distinct developmental strategies among vertebrate embryos [29]. Indeed, amphibian embryos develop rapidly, whereas viviparity imposes that gastrulation is a late event in mammals, taking place days after fertilization. Thus, mammals might have evolved systems that bypass the requirement of p53 function. However, this would contrast not only with the high level of expression of all p53 family members at early stages, but also with the phylogenetic conservation of the p53 binding elements in frog, human and mouse Mix.2-related genes at the 3 0 of their Smad enhancer (Cordenonsi and Piccolo, unpublished). A particularly intriguing issue is whether a cooperation between p53 family members and TGF-beta signals may underlie some of the developmental roles of p63 and p73. p63 is essential for the maintenance of stem cells in several epithelial tissues, such as skin, prostate and breast, and for the stratification of the skin [44,45] whereas p73 is essential for neurogenesis, pheromone signaling and inflammation [46]. Clearly, genetic interaction studies and biochemical experiments will be required to understand precisely the developmental links between p63 and p73 with TGF-beta/Smad signaling.
6. Interplay of p53 and TGF-beta in the control of cell growth Irrespective of its conservation, one attractive hypothesis is that the link between p53 and TGF-beta in frog development may have served as a prelude for its tumor suppressing roles in longer-living vertebrates [29]. Indeed, it has been shown that p53 can be crucial for TGF-beta-induced growth arrest in mammalian cells. Lack of p53, either transient
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(upon siRNA transfection) or genetic (using p53K/K MEFs), involves a defective cytostatic response to TGF-beta and the concomitant incapacity to turn-on efficiently the expression of the CDK inhibitor p21WAF1 in some cell types [22]. Interestingly, this may have consequences for some slowly-proliferating adult stem cells, as shown for mouse hematopoietic progenitors, whose expansion in vitro is antagonized by TGF-beta in a p53-dependent manner [22]. The finding of an involvement of p53 in TGF-beta induced growth arrest is intriguing but it is apparently at odd with the observation that HaCaT keratinocytes, carrying inactivated p53 alleles, do respond to TGFbeta-promoted cytostasis [47]; yet, siRNA against p63 in HaCaT keratinocytes leads to an attenuated induction of p21 by TGF-beta [22]. This finding suggests that p53 may be the prominent regulator of TGF-beta induced growth inhibition only in some cells, such as those expressing mainly p53, or comparable amounts of the three family members. In contrast, it has been calculated that p53 is a fraction of p63/p73 in several epithelial cells, suggesting that p63/p73 isoforms may easily take-over in case of p53 loss [48]. In this context, it would be important to systematically correlate TGF-beta responsiveness with the expression levels and genetic status of p53 family members in primary cancers and cell lines. Finally, it must be recalled that the cell’s genetic make-up has a pivotal role in regulating TGF-betagene responses [6] and therefore mechanisms other than redundancy might be taken into account to compensate for p53 deficiencies.
7. How many jointly-controlled genes? The link between p53 and TGF-beta in development and somatic cell-growth is intriguing but opens many new issues that need to be addressed. A crucial one is: what are the genes under joint control of p53 and TGF-beta? As shown in Fig. 1, the cooperating effects of p53 toward TGF-beta responses are delivered through a p53-binding element in the vicinity of a Smad enhancer on a target promoter. Unfortunately, a TGF-beta responsive element has been characterized only in a very restricted number of promoters. p53 contributes to
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TGF-beta-mediated activation of the PAI-1 and Mix.2 genes, that bear a functional p53-binding element. In contrast, we could not identify a p53-binding element in the known regulatory regions of p53-independent TGF-beta targets, such as goosecoid and TIEG [22]. Despite the lack of systematic characterization of Smad enhancers, gene expression profiles of immediate TGF-beta target genes have been carried out for various cells by several laboratories [11–13]. Interestingly, in a bioinformatic cross-comparison between the regulatory sequences of these 800 TGF-beta targets and a genomic database of p53-targets [49], we identified about 200 genes putatively co-regulated. Strikingly, clustering these p53-positive genes suggests that only two cellular responses might be predicted to be under a global control of p53 family members and TGF-beta, namely, growth inhibition and, surprisingly, extracellular matrix remodelling and attachment (Cordenonsi and Piccolo, unpublished). It is temptive to speculate that these genes may be part of the pro-invasive phenotypes triggered by TGF-beta in advanced carcinomas, a context in which TGF-beta fosters migration, epithelial– mesenchymal transdifferentiation and induction of tissue specific metastasis [2,19,50,51]. Interestingly, p53 family members have been shown to be instrumental for cell movements and migration through a 3D collagen gel [52]. Although p53 itself is unlikely to be involved in these processes, several reports have indicated DeltaNp63 as a marker of malignant esophageal, breast and lung carcinomas [53–56]. Although, DeltaNp63 is an isoform of p63 that lacks the canonical N-terminal transactivation domain, it can display some transcriptional activation abilities [57,58]. DeltaNp63 can be oncogenic, as it antagonizes p53 activity, but it is still able to cooperate with the Smads [22,59]. Thus, it will be interesting to test whether overexpression of DeltaNp63 may constitute a step toward the gain of a proinvasive TGF-beta response in some tumors. In contrast to cytostasis and control of extracellular environment, processes such as TGF-beta-controlled apoptosis, metabolism and cytoskeletal remodeling are unlikely to be under global joined-control of p53 family members and TGF-beta (Cordenonsi and Piccolo, unpublished).
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8. p53, TGF-beta and cancer The p53/TGF-beta connection suggests that, during tumorigenesis, genetic or epigenetic modifications in p53 may contribute to the gain of resistance to TGF-beta induced growth inhibition. The role of p53 and TGF-beta has been studied only independently in the mouse skin model of chemical carcinogenesis, the best understood system for epithelial tumor progression [60]. In this system, rather than influencing the incidence of benign tumors, p53 acts to prevent transition from papilloma to carcinoma, such that in p53-null mice the frequency of malignant conversion is dramatically augmented [61]. A similar phenotype is observed in mice overexpressing a dominant negative form of type II TGF-beta receptor in the basal/follicular cells of the skin [62]. These results raise the tantalizing possibility that the selective loss of the cytostatic response to TGF-beta may coincide with loss of p53. Indeed, p53 mutations in chemically induced mouse skin tumors appear to occur at the benign-malignant transition [63,64]. An aspect related to cancer that merits further discussion is the possible role of mutant-p53. p53 mutations possess transforming activity and are found with very high frequency in common human malignancies [65]. Given the role of wild-type p53 in TGFbeta gene responses, one might predict that blockade of endogenous p53 functions with a mutant p53 allele would interfere with TGF-beta sensitivity. Indeed, this hypothesis is supported by experiments performed in the early 1990s, where introduction of a p53G132F allele in nonneoplastic BALB/MK keratinocytes triggered an attenuated response to TGF-beta [66]. However, the biological functions and the operating principles of mutant p53 remain poorly understood. Indeed, during cancer progression, there is a strong selective pressure for maintaining a mutant-p53 expression at high levels and losing the second p53 wild-type allele [67]. This suggests that mutant-p53 is endowed with dominant gain-of function properties going above and beyond its antagonist activity over the wild-type protein. For instance, it will be very important to establish whether mutant p53 does inhibit Smad activity directly or, oppositely, by acting downstream/in parallel to TGFbeta induced cytostasis.
As originally shown by Vize and co-workers, the developing frog embryo may provide useful insights into the study of mutant p53, as localized expression of p53R175H mRNA leads to an impaired terminal differentiation and the growth of ‘tumor-like’ masses that fail to be incorporated in normal tissues [68]. Once again, this result parallels with the dramatic effects of p53-deficiency on cancer cell differentiation [61,69]. Why the role of p53 in differentiation becomes apparent only in the context of developing tumors—as well as the involvement of TGF-beta in these processes—remains a mystery.
9. Regulation of p53 activity and TGF-beta gene responses: hints and challenges from embryonic development Distinct intracellular and extracellular stimuli, such as several types of stress, mitogens, and activation of oncogenes, converge on p53 to promote its transcriptional activity [65]. p53 is therefore an important element of the cellular context, able to rapidly integrate distinct inputs in TGF-beta gene responses. Precedents for this type of combinatorial control have been described in Drosophila embryos. During specification of mesoderm precursors of the fly, transcription of even-skipped requires local activators to support the effect of dpp/TGF-beta/ MAD signaling, being the latter insufficient by itself [70]. Therefore, in light of our results, integration of context-dependent activators and TGF-beta signals may be a general and conserved strategy to restrict the expression of key downstream effectors of TGF-beta only to specific-competent cells. There is no direct study on the spatial/temporal distribution and activity of p53 during embryogenesis. One possibility is that p53 may be equally active in all the cells of the early embryo. In this case, p53 may contribute to the TGF-beta response by broadening the effectiveness of the endogenous Nodal signals. p53 might be envisioned also as a competence factor, because its absence would render the Smad complex insufficient to reach the minimal threshold for promoter activation in vivo. Alternatively, p53 protein/mRNA stability may be patterned during embryogenesis; this is attractive because it may provide a mechanism to lock the activation of certain
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Fig. 2. Simplified model illustrating the regulatory interactions between molecules involved in the G1/S-phase transition.
genes only to restricted zones of competence. Consistent with this notion is the intriguing finding that p53-dependent transcription from a cloned p53-reporter is higher in the more vegetal cells, i.e. those devoted to form endoderm or mesoderm in response to TGF-beta, than in animal cells, the prospective ectoderm [23]. A prominent modulator of p53 is the E3-ubiquitin ligase mdm2, which promotes p53 degradation and cytoplasmic retention [71]. In the frog, mdm2 is expressed early during embryogenesis and its RNA decreases at the time of mesoderm specification, suggesting that it may play a role in regulating the timing of p53 activity in the embryo [72]. An additional way to regulate p53 may occur via E2F proteins, in keeping with the notion that loss of Rb or overexpression of E2F1 result in increased levels of p53 [73–75] (Fig. 2). It is unclear at present whether this pathway exists in early embryos to regulate p53 activity, as p19/14ARF was found only in mammals. However, a recent report has challenged this notion, suggesting that ARF may have been present in the common ancestors of mammals and birds [76]. Interestingly, E2F has been isolated in an expression cloning screen for genes able to activate posterior development, an approach very similar to that one used to isolate p53, and p53
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and E2F do share the ability to activate posterior homeobox genes [23,77]. Perhaps the most attractive scenario is that p53 activity itself may be regulated during embryogenesis. In recent years, a great emphasis has been placed on the potential role of post-translational modifications as ‘activators’ of p53 from a latent to a more active status. Exciting biochemical studies have shown that activation of p53 is not a single event, but rather entails a series of coordinated/sequential events whose nature is governed by the type and intensity of the activating input [78]. These studies provide a mechanistic basis for the capability of p53 to act as sensor of multiple pathways [65]. It is very unlikely that DNA damage, radiations and oxidative stress can play a major physiological role in regulating p53 activity in early embryos. More appealing is the idea that more developmentally relevant signaling cascades may regulate p53 activation in vivo. Notably, while the relevance of p53’s covalent modifications remain scattered in the case of the response to DNA damage, it is plausible that post-translational modifications may be indeed critical for other p53 responses, also in light of the evolutionary conservation of the p53 activation machinery. In conclusion, the merging of p53 and TGF-beta signaling networks unveils a new way to link TGF-beta with other pathways within the cell. Integrating information on growth, stress and cellular signaling within TGF-beta gene responses may provide useful insights for cancer therapy. Loss of p53 may have a causal relationship with the selective loss of TGF-beta tumor suppressive effects in some cancers; conversely, p53 family members may be attractive targets for therapeutic intervention to selectively restore or invalidate some of the pleiotropic roles of TGF-beta in malignancies.
Acknowledgements We apologize to those researchers whose work has not been cited because of space constraints. We thank G. Bressan for comments. Our work is supported by AIRC, Telethon, ISS and MIUR. SD is recipient of a post-doctoral fellowship from University of Padua.
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Mini review
Convergence of p53 and TGF-beta signaling networks Sirio Dupont, Luca Zacchigna, Maddalena Adorno, Sandra Soligo, Dino Volpin, Stefano Piccolo*, Michelangelo Cordenonsi* Department of Histology Microbiology and Medical iotechnologies, Section of Histology and Embryology, University of Padua, viale Colombo 3, 35121 Padua, Italy Received 25 May 2004; accepted 3 June 2004
Abstract p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: p53; TGF-beta; Growth control; Cancer; Embryonic development
1. Introduction Members of the TGF-beta growth factor family are fundamental signals regulating cell behaviour in a variety of cellular contexts [1–4]. In adult tissues, TGF-beta conveys crucial cytostatic signals to epithelial, immune and other cells types; the loss of this response is the hallmark of many cancers [5]. * Corresponding authors. Address: DHMMB, Histology and Embryology, University of Padua, Viale Colombo 3, 35100 Padua, Italy. Tel.: C39-049-8276098; fax: C39-049-8276079. E-mail address: [email protected] (S. Piccolo).
Research over more than a decade has brought us a fairly satisfactory view of how the TGF-beta signal is transduced from the cell surface to the nucleus. TGF-beta ligands bind to cognate serine/threonine kinase receptors leading, intracellularly, to phosphorylation and activation of the Receptor-Smads signal transducers (R-Smad), such as Smad2 and Smad3. Once activated, the R-Smads translocate into the nucleus to control gene expression in concert with Smad4. The Smads bind to DNA with low affinity and, unless their consensus is reiterated, they require transcription factors as partners for specific interaction with target promoters [6]. The archetypal
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.06.008
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Smad partner is FAST-1 (forkhead activin signal transducer), which lacks intrinsic transcriptional activity, but is essential for targeting the Smads to some developmentally relevant promoters, such as Mix.2 [7–10]. One may expect that such remarkably simple transduction machinery may leave little room for flexibility and lead to a rather stardardized gene expression profile. In contrast, TGF-beta can control hundreds of target genes in different cells [11–13]. Although the mechanisms by which such transcriptional plasticity is attained are unknown, they must entail the interaction of the Smads with other ‘informational nodes’ of the cell. These nodes should be able to integrate multiple inputs from inside and outside the cell setting the ‘context’ in which the TGF-beta signal must act.
2. TGF-beta and growth control In epithelial cells, TGF-beta controls cell growth by the up-regulation of the cyclin-dependent-kinase inhibitors p15INK4b and p21WAF1 [14,15] and the concomitant repression of the growth promoting transcription factors c-MYC, Id1, Id2 and Id3 [16–18]. In normal cells, the anti-mitogenic activities of TGF-beta prevail over inputs favoring proliferation whereas this program fails to be activated in cancer cells. The resistance to TGF-beta induced cytostasis can be acquired in some cancers through inactivation of the genes encoding for TGF-beta receptors and Smads [19]. However, the vast majority of cancers lose selectively the growth-inhibitory response to TGF-beta in the presence of intact TGF-beta receptors and Smads, leaving other responses fully operative. Indeed, advanced carcinomas typically express high levels of TGF-beta to foster their own invasive/prometastatic abilities [20,21]. Therefore, as loss of TGFbeta responsiveness is rare in cancer, it is of great interest to determine how cancer cells acquire selective resistance to TGF-beta in growth-inhibition. Conceivably, loss of TGF-beta tumor suppressing effects should occur by genetic modifications in unknown Smad effectors or downstream molecules. The identification of these modulators is therefore essential for understanding tumor incidence and progression.
3. Identification of p53 as Smad partner p53 was isolated independently in this laboratory and by Atsushi Suzuki’s group in unbiased screens for biologically active molecules expressed during embryonic development [22,23]. The two groups searched for genes endowed with biological activity within small pools of cDNAs (n!100) from embryonic libraries using Xenopus assays. Curiously, the same result was obtained by quite distinct screening strategies. One group set to identify mouse genes whose expression promoted the activation of mesoderm and endoderm markers in embryonic cells [22], which is a typical TGF-beta-related effect in frog embryos. The second group screened for molecules able to transform the most anterior neural tissue into posterior brain or spinal cord [23]. During Xenopus development, this ‘posteriorizing’ activity emanates from the mesoderm cells underlying the developing CNS (such as those of the notocord and somites) [24]. In both screens, sib-selection revealed that p53 was, unexpectedly, the biologically active cDNA [22,23]. Injection of titrated doses of p53 mRNA in Xenopus cells causes them to adopt mesodermal and endodermal fates, in a dose-dependent fashion. Molecularly, these inductions are direct (that is, cycloheximide insensitive) and require p53-dependent transcription [22,23]. Importantly, p53 requires Smad activity to engage its TGF-beta-like effects [22,23]. This suggests that overexpression of p53 can make cells responsive to otherwise sub-threshold amounts of endogenous TGF-beta ligands. Along the same line, it will be interesting to check whether TGF-beta and unbalanced levels of p53 may cooperate during mammalian development; indeed, mice mutant for the p53-inhibitor mdm-2 [25,26] die very early during embryonic development (around E6.5), leaving unaddressed the possibility that the expression of early mesoderm/endoderm markers (whose induction is controlled by Nodal, a TGF-beta-related ligand) may be perturbed in this genetic background as an effect of increased p53 levels. Of note, a closer comparison of the immediate gene responses triggered by Smad2 and different doses of p53 mRNA in Xenopus cells reveals a large, but not complete overlap of activity, being p53 unable to activate some TGF-beta targets (such as goosecoid,
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Wnt8 and Xnr-1) [22,23]. This finding reveals that p53 acts selectively on a subset of TGF-beta target promoters, rather than as general enhancer of TGF-beta responses. Interestingly, biochemical experiments unveiled that the mechanism of this selectivity is dual: p53 associates with Smad2 and Smad3 in vivo in a TGF-beta-dependent manner but, at the same time, p53 must contact its own cognate DNA site on a promoter to facilitate a robust TGF-beta induced transcription [22]. This model is supported by two observations. First, the Mix.2 and PAI-1 promoters, two paradigms of TGF-betainduced transcription, contain a p53-binding element in proximity of the TGF-beta enhancer. Mutation of these p53-elements—or depletion of endogenous p53 by siRNA—quantitatively inhibits the inducibility of these reporters by TGF-beta. Conversely, overexpression of p53 leads to a synergistic increase in TGF-beta-induced transcription of the same reporters. Second, point-mutations disrupting p53 DNA binding activity do block its biological activity in Xenopus assays [22]. In other words, p53 is unable to enter into a Smad-activated transcriptional complex unless it can bind to neighboring cognate DNA sequences. Perhaps, the affinity of the interaction between p53 and Smad is low and can occur efficiently, at physiological concentrations, only when the two proteins are brought close on promoter DNA. Alternatively, only ‘activated’/DNA-bound p53 may be competent to interact with the Smads. Whatever the mechanisms, it is the concomitant presence of a Smad-regulated enhancer and of a p53 binding site that ultimately defines a gene under joined-control of p53 and TGF-beta.
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leading to the assembly of a more stable and specific multifactorial complex [29] (Fig. 1). Although the domains of p53 required for interacting with the Smads are unknown, an interesting conclusion emerges from the published studies: the C-terminus of p53 plays an inhibitory function in TGF-beta-gene responses. Indeed, in mesoderm
4. A new twist for the p53 C-terminus? p53 binds the N-terminal MH1 domain of Smad2, that includes the Smads’ DNA-binding site and also contains the binding domain for other transcription factors, including JunD, LEF-1 and FoxO [15, 22,27,28]. This raises the intriguing possibility that these other proteins may modulate the interaction between p53 and Smads. In the p53/Smad2 complex, the C-terminal MH2 domain of Smad2 is free to interact with FAST-1 and Smad4. This suggests that Smad2 may bridge the DNA-bound p53 and FAST-1
Fig. 1. A model depicting the convergence of p53 and Smads in TGF-beta-mediated gene transcription. Upon TGF-beta stimulation, Smad2 moves to the nucleus where it associates with Smad4 and specific DNA binding co-factors, such as FAST1, and the resulting complex binds to a specific promoter sequence. p53 and the activated Smad complex bind to each other and to their cognate sites on DNA, leading to a synergistic transcriptional activation. Other components of the TGF-beta signaling cascade, transcriptional cofactors and the basal transcriptional machinery were omitted for simplicity.
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induction assays, injection of mRNA encoding either for a deletion of the last 30 aminoacids of Xp53 or for mouse p53AS (a naturally occurring alternatively spliced RNA generating a C-terminal variant) display a substantially higher efficiency than injection of comparable amounts of wild-type/regularly spliced p53 (p53R) [22,23]. This finding is intriguing as this represents the first clear indication on the physiological function of the p53 C-terminus in vivo [30]. Early reports showed that deletion of C-terminal residues had a dramatic increase in p53 ability to bind to naked DNA, in gel-shift assays [31]. In contrast, recent results found that these C-terminally deleted variants of p53 are actually much weaker than intact p53 in binding to chromatinized promoters in vitro and in vivo [32]. Moreover, in vivo experiments showed that p53AS and C-terminally deleted p53 have growthinhibiting ability comparable to regular-p53 [33] and display weaker effects on apoptosis [34,35]. Finding a role for the p53 C-terminus in the Xenopus model system paves the way for addressing the regulatory function of many post-translational modifications impinging on p53 in the context of TGF-beta gene responses.
5. A developmental role for p53 p53 is not only instrumental for fostering TGF-beta gene responses but it is also required for correct TGF-beta responsiveness. Indeed, Xenopus cells depleted of p53 become refractive to activin, and p53 knock-down in frog embryos inhibits mesoderm formation and patterning [22]. These studies suggest that the endogenous TGF-beta signals are by themselves insufficient, but require the concomitant presence of p53, for turning-on effectively several of their targets. Finding a developmental role for p53 is obviously surprising, given that p53 knock-out mice develop normally [36]. One possible explanation for this discrepancy is offered by the finding that different p53 family members are expressed—and remarkably abundant—in early mouse embryos and may compensate for loss of p53 [22]. This is not the case in lower vertebrates where p53 is the only member of its family expressed during early embryogenesis [37–40]. Of note, p63 and p73 are critical for embryonic development [41]. Recent work demonstrated that
p53-related proteins may converge to regulate common biological processes and, in so doing, one may receive assistance from its siblings [42,43]. One shared biological property may be the ability to bind and to cooperate with Smads. Indeed, p73 can also bind the Smads and different isoforms of p63/p73 can synergize with Smads in promoting a very robust increase in TGF-beta induced transcription of cloned reporter constructs [22]. In addition to redundancy, it has been proposed that the different developmental phenotypes caused by lack of p53 in frog and mammals may also reflect distinct developmental strategies among vertebrate embryos [29]. Indeed, amphibian embryos develop rapidly, whereas viviparity imposes that gastrulation is a late event in mammals, taking place days after fertilization. Thus, mammals might have evolved systems that bypass the requirement of p53 function. However, this would contrast not only with the high level of expression of all p53 family members at early stages, but also with the phylogenetic conservation of the p53 binding elements in frog, human and mouse Mix.2-related genes at the 3 0 of their Smad enhancer (Cordenonsi and Piccolo, unpublished). A particularly intriguing issue is whether a cooperation between p53 family members and TGF-beta signals may underlie some of the developmental roles of p63 and p73. p63 is essential for the maintenance of stem cells in several epithelial tissues, such as skin, prostate and breast, and for the stratification of the skin [44,45] whereas p73 is essential for neurogenesis, pheromone signaling and inflammation [46]. Clearly, genetic interaction studies and biochemical experiments will be required to understand precisely the developmental links between p63 and p73 with TGF-beta/Smad signaling.
6. Interplay of p53 and TGF-beta in the control of cell growth Irrespective of its conservation, one attractive hypothesis is that the link between p53 and TGF-beta in frog development may have served as a prelude for its tumor suppressing roles in longer-living vertebrates [29]. Indeed, it has been shown that p53 can be crucial for TGF-beta-induced growth arrest in mammalian cells. Lack of p53, either transient
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(upon siRNA transfection) or genetic (using p53K/K MEFs), involves a defective cytostatic response to TGF-beta and the concomitant incapacity to turn-on efficiently the expression of the CDK inhibitor p21WAF1 in some cell types [22]. Interestingly, this may have consequences for some slowly-proliferating adult stem cells, as shown for mouse hematopoietic progenitors, whose expansion in vitro is antagonized by TGF-beta in a p53-dependent manner [22]. The finding of an involvement of p53 in TGF-beta induced growth arrest is intriguing but it is apparently at odd with the observation that HaCaT keratinocytes, carrying inactivated p53 alleles, do respond to TGFbeta-promoted cytostasis [47]; yet, siRNA against p63 in HaCaT keratinocytes leads to an attenuated induction of p21 by TGF-beta [22]. This finding suggests that p53 may be the prominent regulator of TGF-beta induced growth inhibition only in some cells, such as those expressing mainly p53, or comparable amounts of the three family members. In contrast, it has been calculated that p53 is a fraction of p63/p73 in several epithelial cells, suggesting that p63/p73 isoforms may easily take-over in case of p53 loss [48]. In this context, it would be important to systematically correlate TGF-beta responsiveness with the expression levels and genetic status of p53 family members in primary cancers and cell lines. Finally, it must be recalled that the cell’s genetic make-up has a pivotal role in regulating TGF-betagene responses [6] and therefore mechanisms other than redundancy might be taken into account to compensate for p53 deficiencies.
7. How many jointly-controlled genes? The link between p53 and TGF-beta in development and somatic cell-growth is intriguing but opens many new issues that need to be addressed. A crucial one is: what are the genes under joint control of p53 and TGF-beta? As shown in Fig. 1, the cooperating effects of p53 toward TGF-beta responses are delivered through a p53-binding element in the vicinity of a Smad enhancer on a target promoter. Unfortunately, a TGF-beta responsive element has been characterized only in a very restricted number of promoters. p53 contributes to
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TGF-beta-mediated activation of the PAI-1 and Mix.2 genes, that bear a functional p53-binding element. In contrast, we could not identify a p53-binding element in the known regulatory regions of p53-independent TGF-beta targets, such as goosecoid and TIEG [22]. Despite the lack of systematic characterization of Smad enhancers, gene expression profiles of immediate TGF-beta target genes have been carried out for various cells by several laboratories [11–13]. Interestingly, in a bioinformatic cross-comparison between the regulatory sequences of these 800 TGF-beta targets and a genomic database of p53-targets [49], we identified about 200 genes putatively co-regulated. Strikingly, clustering these p53-positive genes suggests that only two cellular responses might be predicted to be under a global control of p53 family members and TGF-beta, namely, growth inhibition and, surprisingly, extracellular matrix remodelling and attachment (Cordenonsi and Piccolo, unpublished). It is temptive to speculate that these genes may be part of the pro-invasive phenotypes triggered by TGF-beta in advanced carcinomas, a context in which TGF-beta fosters migration, epithelial– mesenchymal transdifferentiation and induction of tissue specific metastasis [2,19,50,51]. Interestingly, p53 family members have been shown to be instrumental for cell movements and migration through a 3D collagen gel [52]. Although p53 itself is unlikely to be involved in these processes, several reports have indicated DeltaNp63 as a marker of malignant esophageal, breast and lung carcinomas [53–56]. Although, DeltaNp63 is an isoform of p63 that lacks the canonical N-terminal transactivation domain, it can display some transcriptional activation abilities [57,58]. DeltaNp63 can be oncogenic, as it antagonizes p53 activity, but it is still able to cooperate with the Smads [22,59]. Thus, it will be interesting to test whether overexpression of DeltaNp63 may constitute a step toward the gain of a proinvasive TGF-beta response in some tumors. In contrast to cytostasis and control of extracellular environment, processes such as TGF-beta-controlled apoptosis, metabolism and cytoskeletal remodeling are unlikely to be under global joined-control of p53 family members and TGF-beta (Cordenonsi and Piccolo, unpublished).
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8. p53, TGF-beta and cancer The p53/TGF-beta connection suggests that, during tumorigenesis, genetic or epigenetic modifications in p53 may contribute to the gain of resistance to TGF-beta induced growth inhibition. The role of p53 and TGF-beta has been studied only independently in the mouse skin model of chemical carcinogenesis, the best understood system for epithelial tumor progression [60]. In this system, rather than influencing the incidence of benign tumors, p53 acts to prevent transition from papilloma to carcinoma, such that in p53-null mice the frequency of malignant conversion is dramatically augmented [61]. A similar phenotype is observed in mice overexpressing a dominant negative form of type II TGF-beta receptor in the basal/follicular cells of the skin [62]. These results raise the tantalizing possibility that the selective loss of the cytostatic response to TGF-beta may coincide with loss of p53. Indeed, p53 mutations in chemically induced mouse skin tumors appear to occur at the benign-malignant transition [63,64]. An aspect related to cancer that merits further discussion is the possible role of mutant-p53. p53 mutations possess transforming activity and are found with very high frequency in common human malignancies [65]. Given the role of wild-type p53 in TGFbeta gene responses, one might predict that blockade of endogenous p53 functions with a mutant p53 allele would interfere with TGF-beta sensitivity. Indeed, this hypothesis is supported by experiments performed in the early 1990s, where introduction of a p53G132F allele in nonneoplastic BALB/MK keratinocytes triggered an attenuated response to TGF-beta [66]. However, the biological functions and the operating principles of mutant p53 remain poorly understood. Indeed, during cancer progression, there is a strong selective pressure for maintaining a mutant-p53 expression at high levels and losing the second p53 wild-type allele [67]. This suggests that mutant-p53 is endowed with dominant gain-of function properties going above and beyond its antagonist activity over the wild-type protein. For instance, it will be very important to establish whether mutant p53 does inhibit Smad activity directly or, oppositely, by acting downstream/in parallel to TGFbeta induced cytostasis.
As originally shown by Vize and co-workers, the developing frog embryo may provide useful insights into the study of mutant p53, as localized expression of p53R175H mRNA leads to an impaired terminal differentiation and the growth of ‘tumor-like’ masses that fail to be incorporated in normal tissues [68]. Once again, this result parallels with the dramatic effects of p53-deficiency on cancer cell differentiation [61,69]. Why the role of p53 in differentiation becomes apparent only in the context of developing tumors—as well as the involvement of TGF-beta in these processes—remains a mystery.
9. Regulation of p53 activity and TGF-beta gene responses: hints and challenges from embryonic development Distinct intracellular and extracellular stimuli, such as several types of stress, mitogens, and activation of oncogenes, converge on p53 to promote its transcriptional activity [65]. p53 is therefore an important element of the cellular context, able to rapidly integrate distinct inputs in TGF-beta gene responses. Precedents for this type of combinatorial control have been described in Drosophila embryos. During specification of mesoderm precursors of the fly, transcription of even-skipped requires local activators to support the effect of dpp/TGF-beta/ MAD signaling, being the latter insufficient by itself [70]. Therefore, in light of our results, integration of context-dependent activators and TGF-beta signals may be a general and conserved strategy to restrict the expression of key downstream effectors of TGF-beta only to specific-competent cells. There is no direct study on the spatial/temporal distribution and activity of p53 during embryogenesis. One possibility is that p53 may be equally active in all the cells of the early embryo. In this case, p53 may contribute to the TGF-beta response by broadening the effectiveness of the endogenous Nodal signals. p53 might be envisioned also as a competence factor, because its absence would render the Smad complex insufficient to reach the minimal threshold for promoter activation in vivo. Alternatively, p53 protein/mRNA stability may be patterned during embryogenesis; this is attractive because it may provide a mechanism to lock the activation of certain
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Fig. 2. Simplified model illustrating the regulatory interactions between molecules involved in the G1/S-phase transition.
genes only to restricted zones of competence. Consistent with this notion is the intriguing finding that p53-dependent transcription from a cloned p53-reporter is higher in the more vegetal cells, i.e. those devoted to form endoderm or mesoderm in response to TGF-beta, than in animal cells, the prospective ectoderm [23]. A prominent modulator of p53 is the E3-ubiquitin ligase mdm2, which promotes p53 degradation and cytoplasmic retention [71]. In the frog, mdm2 is expressed early during embryogenesis and its RNA decreases at the time of mesoderm specification, suggesting that it may play a role in regulating the timing of p53 activity in the embryo [72]. An additional way to regulate p53 may occur via E2F proteins, in keeping with the notion that loss of Rb or overexpression of E2F1 result in increased levels of p53 [73–75] (Fig. 2). It is unclear at present whether this pathway exists in early embryos to regulate p53 activity, as p19/14ARF was found only in mammals. However, a recent report has challenged this notion, suggesting that ARF may have been present in the common ancestors of mammals and birds [76]. Interestingly, E2F has been isolated in an expression cloning screen for genes able to activate posterior development, an approach very similar to that one used to isolate p53, and p53
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and E2F do share the ability to activate posterior homeobox genes [23,77]. Perhaps the most attractive scenario is that p53 activity itself may be regulated during embryogenesis. In recent years, a great emphasis has been placed on the potential role of post-translational modifications as ‘activators’ of p53 from a latent to a more active status. Exciting biochemical studies have shown that activation of p53 is not a single event, but rather entails a series of coordinated/sequential events whose nature is governed by the type and intensity of the activating input [78]. These studies provide a mechanistic basis for the capability of p53 to act as sensor of multiple pathways [65]. It is very unlikely that DNA damage, radiations and oxidative stress can play a major physiological role in regulating p53 activity in early embryos. More appealing is the idea that more developmentally relevant signaling cascades may regulate p53 activation in vivo. Notably, while the relevance of p53’s covalent modifications remain scattered in the case of the response to DNA damage, it is plausible that post-translational modifications may be indeed critical for other p53 responses, also in light of the evolutionary conservation of the p53 activation machinery. In conclusion, the merging of p53 and TGF-beta signaling networks unveils a new way to link TGF-beta with other pathways within the cell. Integrating information on growth, stress and cellular signaling within TGF-beta gene responses may provide useful insights for cancer therapy. Loss of p53 may have a causal relationship with the selective loss of TGF-beta tumor suppressive effects in some cancers; conversely, p53 family members may be attractive targets for therapeutic intervention to selectively restore or invalidate some of the pleiotropic roles of TGF-beta in malignancies.
Acknowledgements We apologize to those researchers whose work has not been cited because of space constraints. We thank G. Bressan for comments. Our work is supported by AIRC, Telethon, ISS and MIUR. SD is recipient of a post-doctoral fellowship from University of Padua.
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