Human Molecular Genetics Advance Access originally published online on August 19, 2003
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Human Molecular Genetics, 2003, Vol. 12, Review Issue 2 R239-R248
DOI: 10.1093/hmg/ddg288
© 2003 Oxford University Press
PTEN: tumour suppressor, multifunctional growth regulator and more
Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK
Received June 30, 2003; Accepted August 14, 2003
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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTIONThe tumour suppressor gene PTEN is mutated in a wide range of human cancers at a frequency roughly comparable with p53. In addition, germline PTEN mutations are associated with several dominant growth disorders. The molecular and cellular basis of these disorders has been elucidated by detailed in vivo genetic analysis in model organisms, in particular the fruit fly and mouse. Studies in the fly have shown that PTEN's growth regulatory functions are primarily mediated via its lipid phosphatase activity, which specifically reduces the cellular levels of phosphatidylinositol 3,4,5-trisphosphate. This activity antagonizes the effects of activated PI3-kinase in the nutritionally controlled insulin receptor pathway, thereby reducing protein synthesis and restraining cell and organismal growth, while also regulating other biological processes, such as fertility and ageing. Remarkably, this range of functions appears to be conserved in all higher organisms. PTEN also plays a role as a specialized cytoskeletal regulator, which, for example, is involved in directional movement of some migratory cells and may be important in metastasis. Furthermore, conditional knockouts in the mouse have recently revealed functions for PTEN in other processes, such as cell type specification and cardiac muscle contractility. Genetic approaches have therefore revealed a surprising diversity of global and cell type-specific PTEN-regulated functions that appear to be primarily controlled by modulation of a single phosphoinositide. Together with evidence from studies in cell culture that suggests links between PTEN and other growth regulatory genes such as p53, these studies provide new insights into PTEN-linked disorders and are beginning to suggest potential clinical strategies to combat these and other diseases.
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INTRODUCTION |
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Discovered less than a decade ago, the tumour suppressor gene PTEN (phosphatase and tensin homologue on chromosome 10) has already proven itself to be a remarkable molecule. Cloned by association with the human cancer susceptibility locus at 10q23 (1–3), this gene immediately aroused much excitement, because it encoded a protein phosphatase (4). However, it soon became apparent that PTEN's major action does not involve the suppression of one or more of the numerous overactive kinases identified in various tumours. In fact, PTEN has a fundamental role as a lipid phosphatase in suppressing growth (5,6).
Mutations in PTEN are commonly found in a wide range of human tumours, including endometrial and prostate carcinomas, glioblastomas and melanomas (7) at a frequency on a par with the DNA damage sensor P53. While in endometrial cancer PTEN may have a role in the onset of tumorigenesis, in glioblastoma and melanoma it frequently appears to act later and may be linked to metastatic behaviour (reviewed in 8). Germline mutations in PTEN have also been detected in humans with a number of dominant growth disorders such as Cowden's disease and Bannayan–Zonana syndrome (9–11). Affected patients typically present with benign hyperplastic outgrowths called hamartomas and increased susceptibility to certain cancers. Proteus syndrome (elephant man disease) and Proteus-like syndrome may also be caused by mutations in PTEN, although this is still a matter of some debate (12–15).
Although the structure of PTEN is consistent with it being a dual-specificity protein phosphatase, in vitro studies revealed that it has an unusual preference for acidic substrates (4,6). PTEN was shown to specifically dephosphorylate the phospholipid phosphatidylinositol 3,4,5-trisphosphate (or PI[3,4,5]P3) to produce phosphatidylinositol 4,5-bisphosphate (or PI[4,5]P2). Soon after this important breakthrough in our understanding of PTEN's biochemical role, the lipid phosphatase activity of PTEN was shown to be physiologically relevant in flies (16,17). These in vivo studies, involving both mutant and overexpression analyses, uncovered an essential role for PTEN as an antagonist of cell, tissue and animal growth. Similar functions have now been demonstrated for mammalian PTEN, using conditional tissue-specific knockouts in mice (reviewed in 18,19).
In this review, we will discuss the most recent developments concerning the in vivo functions of PTEN in model organisms. These will be related to studies in cell culture that have suggested additional targets and regulators for PTEN. These combined approaches have placed this multifunctional molecule at the centre of many key biological processes in the cell, and also suggested routes by which the clinical manifestations of PTEN deficiency might be alleviated in the future.
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THE STRUCTURE OF PTEN |
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Although PTEN mutations in human tumours tend to cluster around the phosphatase domain, there are some mutations distributed along the entire length of the molecule, suggesting that most regions in the protein are critical for its proper growth-suppressing function (7,20). Adjacent to the phosphatase domain, which is located in the N-terminal half of the molecule, there is a lipid-binding C2 domain and a tail region that is typically flanked at its C-terminal end by a PDZ binding domain (Fig. 1A). The PTEN protein is conserved from yeast to mammals, although aspects of its structure differ (7). For example, the C2 domain is absent in simple eukaryotes like yeast and slime moulds.
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PTEN's protein phosphatase domain contains three basic residues around the phosphatase signature motif and an enlarged active site, consistent with its preference for acidic phosphoinositide substrates (20). A specific mutation in the binding pocket, G129E, has been shown to block PTEN's lipid phosphatase activity, but not its protein phosphatase activity in vitro (6). This mutation inactivates PTEN's tumour suppressing functions, highlighting the importance of the molecule's lipid substrates in normal growth control. However, overexpression of the G129E mutant form of PTEN can have effects on proliferation, cell adhesion and spreading in some cell types (21–23), indicating a potential role for PTEN's protein phosphatase activity in these processes. Indeed, PTEN can dephosphorylate the signal transduction molecules focal adhesion kinase (FAK) and Shc, potentially inhibiting integrin-mediated cell spreading (24–26). However, there is no strong in vivo evidence to date, which supports this alternative mode of action for PTEN and, in many cases, the lipid phosphatase activity clearly plays a completely dominant role in tumour suppression.
The lipid-binding C2 domain of PTEN is required for membrane binding in vitro (20) and to correctly orient the phosphatase domain relative to its lipid substrate (27). At the C-terminal end of the molecule is a PDZ-binding domain, which allows PTEN to interact with PDZ domain-containing proteins that are typically associated with the cell surface cytoskeleton. This motif is not present in some isoforms of Drosophila PTEN and, surprisingly, it is not required for viability in the fly (16).
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TARGET PATHWAYS FOR PTEN IN CULTURED CELLS |
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As discussed above, some studies in cell culture have suggested that PTEN's protein phosphatase activity can modulate cell behaviour (26). However, most investigators have focused on the effects of its lipid phosphatase activity. Their findings have been discussed in numerous reviews (7,28).
In brief, PTEN's primary biochemical function is to directly antagonize the effects of Class I PI3-kinases (PI3-Ks) by converting PI[3,4,5]P3 back to the PI3-K substrate PI[4,5]P2. PI[3,4,5]P3 activates the protein kinase Akt (otherwise known as PKB) and its regulator PDK1. In collaboration with an unidentified kinase, PDK2, PDK1 appears to stimulate the activity of Akt, which then controls a range of downstream cellular events (Fig. 1B). Akt has been shown to be a negative regulator of many molecules that inhibit cell proliferation and survival, including several pro-apoptotic proteins and the cell cycle regulator p27 (discussed in 28). Akt has been shown to be activated in numerous human cell lines carrying PTEN mutations (7), and overexpression of wild type PTEN in these cells suppresses Akt, typically reducing cell proliferation and viability.
Studies in cell culture have been particularly helpful in highlighting an array of candidate signalling targets downstream of PTEN (Fig. 1B). Indeed, since some of these targets may only be regulated by PTEN in tumorigenic cells, it is conceivable that they will not be recognized by standard genetic approaches in model organisms. However, particularly in cases where PTEN has an involvement in the onset of tumorigenesis or plays a critical role in advancing the tumorigenic state, it is likely these roles and the signalling pathways involved will be highlighted in animals that specifically lack the PTEN gene. As we will see, genetic analysis has emphasized the importance of translational control and increased cell growth in tumorigenesis and revealed that PTEN normally plays a vital role in antagonizing the growth- promoting effects of insulin-like molecules in the developing animal.
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HIGHLIGHTING TRANSLATIONAL AND CELL GROWTH CONTROL IN THE LIVING FLY |
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The first and probably still the clearest indication that PTEN plays an essential role in regulating cell growth came from the generation of clonal patches of cells lacking PTEN in the compound eye of the fruitfly, Drosophila melanogaster (Fig. 2B and C) (16,17). The resulting mutant PTEN cells are up to three times the size of their wild type neighbours. In addition to controlling cell size, PTEN has been shown to restrict the size of a range of organs and indeed to control the size of the entire animal. Mutant combinations that only partially remove PTEN function produce viable, but ‘giant-sized’, flies that are up to 50% bigger than normal flies and contain larger cells (Fig. 2D) (30), (D.C.I. Goberdhan, unpublished data). However, enlarged cell size is not the only growth defect in mutant cells. Cell proliferation rate is also elevated by accelerating the G1/S transition, although this effect clearly does not keep pace with the increased growth rate in these cells (16,17,29).
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Further genetic analysis has shown that PTEN achieves its growth regulatory effects by antagonizing the DrosophilaPI3-K called Dp110 (16,17,29) and acting as a biological brake on nutritionally regulated insulin receptor (InR) signalling. Work on this pathway has proceeded at a dramatic pace over the last few years (Fig. 3) (reviewed in 31). Perhaps the most exciting discoveries involve the new links that have been forged between InR signalling, PTEN, another nutritionally regulated pathway involving Tor/S6-kinase (S6K) and previously unconnected molecules, such as the tuberous sclerosis complex genes and Rheb (32–37). Not only are the phenotypes associated with overexpression or mutation of all these proteins remarkably similar to one another, but elegant genetic epistasis experiments in which multiple genes are mutated or overexpressed in a single tissue have shown that these molecules regulate growth by signalling through a surprisingly linear pathway (Fig. 3) (31,38,39). Although biochemical analysis of mutant cells and cell lines has suggested that other parallel and feedback pathways may exist (40,41), the Tor pathway is almost certainly an important target for PTEN's growth regulatory activity in higher eukaryotic cells. Tor and S6K are known to regulate several molecules involved in translation, and these are believed to stimulate accumulation of macromolecules and induce cell growth (31). In addition, it is postulated that changes in the synthesis of selected proteins, such as Cyclin E, may be involved in growth-controlled modulation of cell proliferation (42), but this has yet to be confirmed experimentally in multicellular animals.
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One important property of tumorigenic cells is that they are relatively resistant to growth inhibitory signals from neighbouring tissues. Does activating the InR pathway by removing PTEN produce an overgrowth phenotype that is unaffected by surrounding structures? It has been possible to look at this question in flies by analysing PTEN-mediated overgrowth in the eyes of animals with very different body sizes. Remarkably the absolute level of growth in a tissue is not significantly changed, even when the eye is more than 5-fold enlarged relative to the rest of the animal (43). If Proteus syndrome in humans is linked to PTEN mutation, this disorder may represent a milder case of dominant PTEN-mediated tissue overgrowth in the head.
While genetic analysis has highlighted a fundamental role for PTEN and the InR in Drosophila growth control, it has also indicated other global and cell-type specific functions for these molecules (31). In particular, inhibition of the InR pathway reduces fertility, and slows development and adult ageing, producing flies that can live nearly twice as long as wild type animals (44,45). It also has a global effect on metabolism. These long-lived flies have elevated circulating sugars, as might be expected in animals with reduced insulin signalling (46). Neuroendocrine cells in the brain secrete insulin-like molecules during development in response to nutritional status. These cells have been shown to play a key role in modulating global InR functions (31,46,47). Developmental rate, fertility and ageing also appear to be regulated by insulin-like molecules in both nematodes and mammals (48,49), defining PTEN as an evolutionarily conserved inhibitor of these nutritionally regulated processes.
As discussed earlier, experiments in mammalian cell culture have indicated an important role for PTEN in stimulating apoptosis. Although PTEN has surprisingly little effect on cell survival in growing Drosophila tissues, overexpression in embryonic tissues, where no growth is taking place, does induce apoptosis, as does loss of Akt in the embryo (50). One possible interpretation of this result is that PTEN only reduces cell survival in certain cell types during normal development, but this function becomes increasingly important in cells as they become tumorigenic, since they may not receive the normal range of survival signals.
Other cell-type-specific phenotypes affecting the cytoskeleton and cell differentiation have been identified in tissues lacking components of the Drosophila InR signalling cascade, including PTEN. These will be discussed later in the context of related phenotypes found in other model organisms.
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MAMMALIAN PTEN: GROWTH CONTROL AND MORE |
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With studies in cell culture pointing to a link between PTEN and the regulation of cell proliferation and survival, and genetic analysis in flies revealing a primary role in cell growth, it has been necessary to turn to the genetically amenable mouse to determine the in vivo functions of this molecule in mammals. As with flies, mice homozygous for strong PTEN alleles die as embryos, at a stage too early to assess tissue growth phenotypes in detail (about day 10) (51–53). Adult mice heterozygous for PTEN, however, show an increased tendency to form tumours in a range of tissues comparable to those affected in humans (51–54).
As in flies, the effects of PTEN on cell size and growth in mammalian systems have been most clearly demonstrated by removing PTEN in only part of the animal using the Cre–Lox system, allowing mice to develop to post-embryonic stages and making comparative cell size analysis possible (19). These analyses have primarily focussed on tissues affected by PTEN-related human diseases.
For example, in light of the ubiquitous expression of PTEN in the central nervous system, the prevalence of PTEN-associated tumours in brain and the neurological effects of the PTEN-linked growth disorder, Lhermitte–Duclos disease (LDD), workers have analysed PTEN's role in neuronal development in some detail. Interestingly, different effects have been observed dependent on the developmental stage at which PTEN mutant cells are generated. Deletion of PTEN during later development produces mice with enlarged brains (55,56). Akt is hyperactivated in mutant cells (56) and the surface area of individual mutant granule cells in the cerebellum is approximately twice that of normal cells (55), consistent with the cerebellar overgrowth defects found in LDD.
By contrast, knocking out PTEN in neuronal precursor cells earlier in development causes an increase in cell proliferation, in addition to affecting the size of cells (57–59). CNS stem/progenitor cell numbers appear to be increased in these animals, suggesting that PTEN plays a role in amplifying these stem cells as well as stimulating the growth of their progeny (57). Interestingly, some cell migratory events are also affected in the brain (58,59), consistent with other observations that implicate PTEN in cell movement (see below).
Cells in other tissues that also normally grow via hypertrophic (non-proliferative) mechanisms, like skeletal and cardiac muscle, also enlarge in response to activation of PI3-K/Akt (60,61). However, in other organs where cells divide during growth, the pathway stimulates more co-ordinated increases in cell growth and proliferation (reviewed in 31). It is possible that the hyperplastic growth seen in these latter tissues is achieved by different mechanisms to those that stimulate PI3-K/Akt-mediated hypertrophic growth in growing tissues. However, we favour a model in which the signalling pathway outlined in Figure 3 plays an important role in all cells, but where the link between cell growth and proliferation is more tightly coordinated in many mammalian tissues. A critical requirement for this model is that the downstream growth-regulating targets of Akt, like Tor and S6K, should also be able to regulate mammalian cell size. This has recently been shown to be the case (62). It will now be important to extend this work, by performing experiments in which at least two of the molecules in this pathway are simultaneously mutated or overexpressed in vivo to test whether there are simple epistatic relationships between these components in multiple tissues, as has been found in flies. It will also be interesting to investigate whether insulin-like molecules are the primary activators of this pathway, as is found in Drosophila, or whether other growth factors are also involved (discussed in 31).
In addition to the size and growth phenotypes observed in PTEN mutant mice, other tissue-specific effects on apoptosis, cell type specification and migration have also been observed (reviewed in 19). For example, heterozygous PTEN mutant mice have autoimmune defects and immune cells show an impaired reaction to Fas-induced apoptosis (63). This has led several groups to generate PTEN knockouts in T and B cell lineages (64–66). As well as inducing malignant growth in these cells, several other phenotypes are observed, including a bias for T cells to differentiate with a CD4+ versus CD8+ phenotype. Roles for PTEN in cell differentiation events may not be unique to mammals. In Drosophila, for example, PTEN's downstream target Akt has been implicated in development of the tracheal system (67). Such cell-type-specific effects may merely reflect the presence in a particular cell subset of one or more targets for Akt and/or other PI[3,4,5]P3-regulated kinases, which then go on to have an important role in differentiation of these cells.
Studies of PTEN mutant heart muscle have revealed another example of PTEN's biological versatility. The mutant heart is not only larger, but unexpectedly muscle contractility is dramatically decreased (68). As in other tissues, PTEN antagonizes the well-characterized PI3-K
isoform to restrict organ size via effects on cell size. However, to modulate contractility, PTEN antagonizes the effects of an alternative PI3-K isoform, PI3-K
, which is normally activated by G protein-coupled and not tyrosine kinase receptors. Once activated, PI3-K inhibits cAMP production and thus reduces contractility. This result opens up the possibility that PTEN can independently modulate multiple phosphoinositide-mediated processes controlled by different molecules in the same cell. Whether different phospholipids or distinct spatial/temporal localization of the same substrate is involved in these processes remains to be elucidated.
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PTEN AND CELL MIGRATION |
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Studies in several organisms have demonstrated a role for PI3-K and in some cases, PTEN, in the regulation of cell movement and migration. This is best documented in the slime mould Dictyostelium discoideum, where PTEN is involved in sensing chemical gradients and controlling directional cell migration (69,70). Although PTEN in Dictyostelium lacks the lipid-binding C2 domain found in higher organisms, a functionally important membrane-binding domain, which may interact with PI[4,5]P2, has been identified at its N-terminus. In response to a chemotactic signal, this is involved in targeting PTEN to the trailing edge of the unicellular Dictyostelium. Coordinately, PI3-K is targeted to the leading edge of the cell. The response to the shallow chemoattractant gradient is subsequently amplified by two separate feedback loops at these two edges of the cell, producing a steep cellular PI[3,4,5]P3 gradient, which enables the cell to migrate in a targeted manner towards the chemoattractant.
Interestingly, at least some features of this mechanism seem to be conserved in certain mammalian migratory cells. Neutrophils moving towards their targets have been shown to have localized concentrations of PI[3,4,5]P3 at their leading edge (71) and neutrophils from PI3K mutant mice show impaired chemotaxis (72). More recent work has demonstrated that interplay at the leading edge between localized PI[3,4,5]P3 and cytoskeletal elements, several of which are known to be regulated by PIPs, is essential to maintain cell polarity and directed motility (73,74). However, it has yet to be shown in this case that exclusion of PTEN from the leading edge is an important part of the process.
The link between PI3-K, PTEN and cell polarity has now also been observed in the nervous system. In cultured hippocampal neurons, PI3-K is necessary for the specification of a single axon from a group of apparently identical neurites (75). It is proposed that this process is driven by the amplification of an initial PI[3,4,5]P3 bias in one neurite, which is then maintained by a form of positive feedback regulation probably related to the mechanism used by neutrophils. Overexpression of PTEN has been shown to antagonise this effect. Interestingly, mutant Tsc2 peripheral neurons in flies often overshoot and show excessive branching (76), perhaps indicating a role for downstream components of the InR pathway and translational regulation in neurite formation and outgrowth (reviewed in 31).
It therefore seems that, by antagonizing the effects of PI3-K isoforms in a localized fashion, PTEN modulates cell polarity and directional movement in a number of cell types. Indeed, other PTEN mutant phenotypes in Drosophila and mice are also consistent with a role in regulating cytoskeletal rearrangements (16,58,59). However, this is almost certainly not a universally important mechanism, since some migratory processes do not appear to have an obvious requirement for localised PIPs (77). Indeed, there are probably other regulatory processes that act in parallel to this system in higher eukaryotic cells, so that, even when PIPs are involved, the cytoskeletal phenotypes produced by deleting genes controlling PIP metabolism may be quite subtle (72). This redundancy may also help to explain why on the one hand, Leslie et al. (78) have found that overexpressed PTEN inhibits cell spreading in culture via a process that is significantly enhanced by its PDZ binding domain, while on the other hand, an isoform of PTEN that lacks this same domain can rescue strong loss-of-function PTEN mutations in flies (16).
It may seem surprising that a signalling pathway which acts as a global modulator of nutritionally regulated events, is also involved in processes such as migration and differentiation. However, it is important to recognize that this pathway is probably active in all cells of the body and even in extreme nutritional conditions, it will probably not be fully ‘on’ or ‘off’. Since PTEN seems to affect migration and polarity by modulating relative subcellular levels of PI[3,4,5]P3 across the cell, fluctuations in overall PI[3,4,5]P3 concentrations may therefore not significantly alter this process. We also do not know which differentiation events controlled by PTEN/Akt are differentially regulated by levels of PI[3,4,5]P3 that fall within the normal range. Such processes could be nutritionally regulated. By contrast, those events which are only affected when PTEN/Akt are deleted or highly overexpressed in experiments or in the disease state could not.
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PTEN REGULATION |
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In higher eukaryotes, PTEN clearly plays a critical and universal role in PI[3,4,5]P3 metabolism and growth control, but is it a ubiquitously expressed inhibitor with similar effects in all cells, or is its activity selectively regulated? Despite considerable progress in several other areas, we have very few answers to this question, although the case for regulation is becoming increasingly strong.
One significant piece of circumstantial evidence is that, while PTEN's phosphoinositide substrate is primarily located at the plasma membrane, only a small proportion of PTEN is typically found there. In fact, PTEN's subcellular localization pattern is quite variable. Particularly in quiescent cells, it is often preferentially localized in the nucleus (79–81). However, in neoplastic cells, there is often an increase in levels of cytoplasmic PTEN relative to corresponding normal cells derived from the same tissue (80,81). PIPs are known to be present in the nuclear envelope, where they may, for example, be involved in processes such as splicing (82,83). Therefore, these observations could indicate a novel role for PTEN in the nucleus. Alternatively, nuclear localization may simply provide a means of completely isolating PTEN from its target molecules in the plasma membrane.
Even when PTEN is primarily localized in the cytoplasm, only a small proportion binds to the plasma membrane, despite the presence of lipid-binding phosphatase and C2 domains in the molecule. It seems very likely that some form of regulation is involved in partitioning PTEN between plasma membrane and cytosol. Interestingly, phosphorylation events at the C-terminal tail of the molecule, some of which appear to modify the bulk of PTEN in cultured cells, have been shown to co-ordinately stabilize and reduce the activity of the protein (84,85). Some studies have indicated that phosphorylation modulates interactions between PTEN and other important proteins and complexes (84). However, more recent data suggest there is a direct electrostatic effect of phosphorylation on PTEN's membrane binding affinity (86). Casein kinase 2 (CK2) can catalyse these phosphorylation events in cultured cells (87,88), but it is unknown whether this molecule has an in vivo role.
There is also evidence that activated Akt can regulate phosphorylation of PTEN's C-terminal tail and PTEN stability (89), providing a potential negative feedback mechanism for the PI3-K/Akt signalling pathway. Assuming CK2, a ubiquitously expressed molecule with broad specificity, is the critical kinase in this phosphorylation process, Akt may regulate an as yet unidentified phosphatase that acts on PTEN or make PTEN a poorer substrate for CK2.
If the stability and activity of PTEN is highly regulated, it may provide a route for other pathways involved in oncogenesis and patterned growth to modulate PI[3,4,5]P3-mediated signalling. In support of this idea, BMP2, a member of the transforming growth factor-ß superfamily, has recently been shown to decrease PTEN degradation in a breast cancer cell line (90).
Is the regulation of PTEN activity, stability and protein–protein interactions a quirk of cultured cells or an important aspect of the in vivo growth control process? This is a question begging to be addressed in model organisms. In one example of protein–protein interactions, discussed below, where both in vivo and cell culture evidence is available, studies have revealed an additional and critical role for PTEN in tumorigenesis.
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PTEN AND P53: PARTNERS IN CRIME PREVENTION? |
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Although p53 and PTEN are the two most commonly mutated tumour suppressor genes in human cancer, in several forms of cancer it is rare to find them both mutated in the same tumour (91). Furthermore, mice heterozygous for both PTEN and p53 have a greatly increased likelihood of forming tumours compared with mice heterozygous for only one mutation. Significantly, some of these tumours still appear to carry a normal copy of both PTEN and p53 (92). Recent data from analysis in cell culture now suggest that the functions of these two genes are intimately linked (reviewed in 93).
In mammalian cells, the P53 protein protects against tumorigenic growth induced by DNA damage, in some instances by arresting the cell cycle to provide an opportunity for DNA repair, or in more extreme cases, by inducing programmed cell death (94). In normal cells, P53 levels are typically kept low by rapidly targeting the protein for degradation via E3 ubiquitin ligases, such as Mdm2. It is now clear that this process is regulated by PI[3,4,5]P3 signalling. Mdm2 translocates to the nucleus when phosphorylated by Akt and ubiquitinates P53, leading to its nuclear export and degradation (95,96). PTEN, as a key negative regulator of Akt, inhibits this process. Interestingly, nuclear P53 binds to sequences upstream of the PTEN gene to promote its transcription (97), providing a positive feedback loop in the P53 activation process.
In an even more surprising development, PTEN has also recently been shown to physically interact with P53 to modulate its activity. Intriguingly, binding is mediated via PTEN's C2 domain, and is independent of PTEN's phosphatase activity (92). This interaction increases the transcriptional activity of P53 and blocks its degradation. These complex interactions between PTEN and P53 are likely to have important implications in the treatment of tumours. Indeed, expression of PTEN in cancer cells has already been shown to sensitise cells to chemotherapeutic agents that act through P53 (98).
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CLINICAL PROSPECTS AND THE ROAD AHEAD |
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In this review, we have discussed the recent developments in our understanding of PTEN function in the whole organism and how they relate to studies that have dissected the roles of this molecule in cultured cells. In linking PTEN to Tor/S6K signalling and translational control, the most immediate clinical impact of these in vivo genetic studies has been to highlight analogues of the well-characterized inhibitor of Tor, rapamycin, as important and perhaps relatively broad specificity anti-cancer agents. Indeed, a rapamycin analogue has already been successfully used in phase I trials for treatment of solid tumours (99). With our new-found knowledge of the molecules involved in regulating Tor signalling, it might well be possible to predict from genetic analysis of tumour cells which patients are most likely to benefit from such drugs.
In the future, it may also be possible to develop strategies for treating some cancers by upregulating PTEN expression in tumour cells. Since PTEN has a multiplicity of effects, not only inhibiting growth and the cell cycle, but also stimulating P53-mediated apoptotic death, this may be a particularly powerful anti-cancer approach. However, to develop such treatments, a much more detailed analysis of PTEN's post-translational regulation will be required both in culture and in vivo, as well as a better characterisation of its transcriptional control (97,100). In fact, strategies designed to modulate the expression of PTEN may also have several other clinical applications. For example, it has been suggested that PTEN may have therapeutic value in treatments involving balloon angioplasty, a procedure that involves the insertion of a physical support to widen occluded arteries. The patient's response to this intervention, known as restinosis, involves reactivation of the normally quiescent muscle cells, which proliferate and migrate to form a ‘neointimal hyperplasia’. Inducing PTEN production in these cells might help to alleviate this complication, since overexpression of PTEN has been found to block vascular smooth muscle cell proliferation and migration, and reduce the activity of survival factors thought to be associated with restenosis (101). PTEN also has a vital role to play in the effects of insulin on metabolism, and a recent study in a diabetic mouse model has suggested that global reduction in PTEN activity can provide a means to normalise sugar levels and the response to insulin (102). In the longer term, it is even conceivable that up-regulating PTEN expression could become an option for extending lifespan, at least in the context of certain diseases of ageing.
At a more fundamental level, advances in our understanding of PTEN function have thrown up a whole series of new questions, which must now be addressed using both in vivo and cell culture approaches. On the one hand, we need a much clearer picture of the significance of cell growth control in regulating tissue growth and proliferation, particularly since several other homologues of oncogenes have recently been shown to play fundamental roles in growth control in the fly (reviewed in 31). On the other hand, if PTEN expression levels are to be modulated in the clinic, we must define the mechanisms by which PTEN controls processes other than growth in the living animal, and investigate the cellular conditions that lead PTEN to interact with other pathways regulating the cell cycle and apoptosis in cultured cells.
Although experiments in mouse models will make an important contribution to these studies, the fly also has much to offer as a test-bed to dissect out the complex web of molecular and biological interactions that PTEN makes in vivo. In addition, simple genetic screens in flies are continuing to reveal new players in growth regulation (35,36,103–105) and it seems very likely that some of these will have a conserved role to play in controlling PTEN expression and activity. While it will be important for fly workers to react to new developments emerging from studies in cell culture, it will be equally essential for oncologists to respond to the results of genetic studies in simpler organisms, and use these results in assessing the biological significance of molecular networks identified in tumour cell lines.
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ACKNOWLEDGEMENTS |
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D.C.I.G. is supported by a project grant from the Biotechnology and Biological Sciences Research Council, UK. Work in the C.W. laboratory is also supported by further BBSRC funding and a grant from Diabetes UK (ref. BDA:RD02/0002540).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 1865282661; Fax: +44 1865272420; Email: deborah.goberdhan{at}anat.ox.ac.uk
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