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ibility syndromes, such as Cowden syndrome, in which over 80% of patients have mutations of PTEN. Homo- zygous deletion of Pten causes embryonic lethality,.
Oncogene (2008) 27, 5443–5453

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REVIEW

PTEN: a new guardian of the genome Y Yin and WH Shen Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY, USA

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is a phosphatase that antagonizes the phosphoinositol-3-kinase/AKT signaling pathway and suppresses cell survival as well as cell proliferation. PTEN is the second most frequently mutated gene in human cancer after p53. Germline mutations of PTEN have been found in cancer susceptibility syndromes, such as Cowden syndrome, in which over 80% of patients have mutations of PTEN. Homozygous deletion of Pten causes embryonic lethality, suggesting that PTEN is essential for embryonic development. Mice heterozygous for Pten develop spontaneous tumors in a variety of organs comparable with the spectrum of its mutations in human cancer. The mechanisms of PTEN functions in tumor suppression are currently under intense investigation. Recent studies demonstrate that PTEN plays an essential role in the maintenance of chromosomal stability and that loss of PTEN leads to massive alterations of chromosomes. The tumor suppressor p53 is known as a guardian of the genome that mediates the cellular response to environmental stress, leading to cell cycle arrest or cell death. Through completely different mechanisms, PTEN also protects the genome from instability. Thus, we propose that PTEN is a new guardian of the genome. In this review, we will discuss new discoveries on the role of PTEN in tumor suppression and explore mechanisms by which PTEN maintains genomic stability. Oncogene (2008) 27, 5443–5453; doi:10.1038/onc.2008.241

and Peacocke, 1998). Hallmark features of Cowden disease are hamartomatous overgrowths of various tissues, including skin, breast, intestine and brain, as well as a predisposition to the development of breast and thyroid tumors (Liaw et al., 1997). Homozygous deletion of Pten causes embryonic lethality, suggesting that PTEN is essential for embryonic development (Di Cristofano et al., 1998). Mice heterozygous for Pten deletion develop tumors in multiple tissues comparable with those affected in humans (Di Cristofano et al., 1998; Suzuki et al., 1998; Podsypanina et al., 1999; Stambolic et al., 2000). These in vivo observations indicate that the homeostatic balance has been disrupted and shifted toward oncogenesis, which is likely due to lack of PTEN function in maintaining normal cellular processes. Indeed, studies in cell culture reveal a plethora of phenotypic changes in PTEN-deficient cells, such as increased proliferation (Sun et al., 1999; Backman et al., 2001), reduced apoptosis (Stambolic et al., 1998) and enhanced migration (Tamura et al., 1998; Liliental et al., 2000). In contrast, overexpression of wild-type PTEN in cancer cells induces apoptosis and blocks cell-cycle progression, colony formation and cell migration (Gu et al., 1998; Li et al., 1998; Tamura et al., 1998; Weng et al., 2001a, b). Intensive efforts have been focused on how PTEN acts to regulate such a broad spectrum of phenotypic features, answers to which may come from a better understanding of its biochemical features, structure, subcellular localization and signaling targets.

Keywords: PTEN; p53; tumor suppressor; genome guardian; genomic instability; chromosome instability PTEN: structure and functional domains PTEN as a powerful tumor suppressor Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/MMAC1 was cloned in 1997 through its association with the human cancer susceptibility locus at 10q23 (Li and Sun, 1997; Li et al., 1997; Steck et al., 1997). PTEN is one of the most frequently mutated genes in human cancers (Cantley and Neel, 1999; Simpson and Parsons, 2001). Germline PTEN mutations are primarily found in the autosomal dominant hamartoma syndrome Cowden disease (Eng Correspondence: Dr Y Yin, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, VC11-213, 630 West 168th Street, New York, NY 10032, USA. E-mail: [email protected]

There are two major domains of PTEN, the N-terminal phosphatase domain and the C-terminal domain (Lee et al., 1999). Although the role of PTEN in tumor suppression has been mostly attributed to its N-terminal lipid phosphatase activity (Cantley and Neel, 1999), more than 40% of PTEN tumorigenic mutations occur in the C-terminal domain (Waite and Eng, 2002). This fact indicates that PTEN may have other functions through its C terminal domain, which is significant in tumor suppression. The C-terminal domain contains both a C2 domain and a tail region that may be related to PTEN stability (Georgescu et al., 1999) and protein– protein interaction (Fanning and Anderson, 1999). The C2 domain has been implicated in PTEN stability (Georgescu et al., 2000) and its recruitment to phospholipid membranes (Das et al., 2003). Crystal structure

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analysis of this domain revealed a b-sandwich structure (Lee et al., 1999), suggesting a basis for its interaction with DNA and other proteins. It was reported that PTEN uses the C2 domain to interact with the centromere (Shen et al., 2007). PTEN as a dual lipid and protein phosphatatse PTEN is a lipid and protein phosphatase. PTEN protein phosphatase can dephosphorylate protein substrates on serine, threonine and tyrosine residues (Myers et al., 1997). The lipid phosphatase activity of PTEN was reported for the substrate phosphoinositol 3,4,5triphosphate (PIP3), a key signaling component of the phosphoinositol-3-kinase (PI3-kinase) pathway (Maehama and Dixon, 1998). The PI3-kinase/Akt pathway is regarded as the primary physiological target of PTEN (Sulis and Parsons, 2003; Leslie and Downes, 2004; Sansal and Sellers, 2004). Phosphatase activity, particularly lipid phosphatase activity, is believed to be the most pertinent property of PTEN as a tumor suppressor. The role of PTEN as a lipid phosphatase in growth suppression can be distinguished by a germline missense mutation, G129E, which abrogates lipid phosphatase but not protein phosphatase activity (Liaw et al., 1997). Most naturally occurring mutations, however, are both lipid and protein phosphatase inactive, such as C124S (Maier et al., 1999). Double phosphatase-deficient PTEN (C124S) (Maier et al., 1999) and lipid phosphatase-deficient PTEN (G129E) (Gildea et al., 2004) have been shown to inhibit cell invasion similar to wild type. These results imply that PTEN may suppress tumors independent of its phosphatase activity. PTEN phosphatase also targets different proteins. Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, has been identified as a direct protein target of PTEN (Tamura et al., 1998). Similarly, PTEN also reduces the tyrosine phosphorylation of p130Cas, a FAK downstream effector (Tamura et al., 1998). By targeting and dephosphorylating FAK and p130Cas, PTEN regulates dynamic cell surface interactions and inhibits cell migration and invasion. The lipid phosphatase activity of PTEN is dispensable for tyrosine dephosphorylation of FAK, as the lipid phosphatasedeficient G129E mutant retains this activity. In comparison with PIP3 as a ubiquitous target of PTEN lipid phosphatase, FAK/p130Cas as targets of PTEN protein phosphatase appear to be conditional. Although overexpression of PTEN dephosphorylates FAK/p130Cas (Tamura et al., 1998), their phosphorylation status remains unaffected in Pten/ cells (Liliental et al., 2000), suggesting that PTEN is not a sole regulator of FAK/p130Cas. Interestingly, Pten/ cells indeed exhibit increased cell motility, which is accompanied by induced activities of two small GTPases, Rac1 and Cdc42. In this case, however, PTEN lipid phosphatase activity seems to be involved in the inhibition of cell motility and phosphorylation of both Rac1 and Cdc42 (Liliental et al., 2000). In addition to FAK/p130Cas, Rac1 and Oncogene

Cdc42, PTEN can also regulate cell motility by directly targeting and dephosphorylating Shc kinase, thereby inhibiting the mitogen-activated protein (MAP) kinase signaling pathway (Gu et al., 1998; Tamura et al., 1998). As an important intracellular signaling pathway, the MAP kinase cascade provides multiple potential targets for PTEN. PTEN can inactivate multiple membraneproximal proteins upstream of MAP kinase such as Ras and IRS-1 (Gu et al., 1998; Weng et al., 2001b). The prototypical MAP kinase, extracellular signal-regulated kinase, is often affected by PTEN status. However, PTEN is unable to directly dephosphorylate extracellular signal-regulated kinase (Myers et al., 1997), indicating that extracellular signal-regulated kinase is not a direct substrate of PTEN. The protein phosphatase activity of PTEN has also been demonstrated to inhibit FAK and extracellular signal-regulated kinase and subsequently block the expression and secretion of matrix metalloproteinase-9, which may contribute to the suppression of glioblastoma invasion (Park et al., 2002). A recent report reveals that JNK is a functional target of PTEN (Vivanco et al., 2007), in which protein phosphatase activity of PTEN may not be directly involved. PTEN can also inhibit phosphorylation of proteins downstream of MAP kinases. One prominent example is ETS-2, a nuclear target of the MAP kinase pathway and a transcription factor whose DNA-binding ability is controlled by phosphorylation. PTEN may use its protein phosphatase activity to block ETS-2 phosphorylation in response to insulin-induced MAP kinase activation, in a PI3-kinase-independent manner (Weng et al., 2002). PTEN can also regulate phosphorylation of another transcription factor Sp1, likely through its protein phosphatase activity (Kang-Park et al., 2003). In addition to intracellular signaling molecules, receptor tyrosine kinases may also serve as direct protein targets of PTEN. PTEN physically associates with the receptor of platelet-derived growth factor and directly dephosphorylates the receptor, whereas the phosphatase-deficient PTEN mutant (C124S) not only fails to dephosphorylate the platelet-derived growth factor receptor but also acts in a dominant-negative fashion to increase its phosphorylation (Mahimainathan and Choudhury, 2004). Collectively, potential protein targets of PTEN range from membrane-bound receptor tyrosine kinases and cytoplasmic signaling molecules to transcription factors in the nucleus. Nevertheless, the currently recognized candidates are mostly identified from in vitro studies and many of them are found in some but not other cell systems. Therefore, the identification of more direct functional protein substrates of the PTEN phosphatase deserves further investigation. PTEN mutations in human cancers: a database to be updated The cloning of the PTEN gene was accompanied by detection of various types of mutations including homozygous deletion, frameshift, inframe deletion,

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truncation, point mutation and so on (Li et al., 1997; Shin et al., 2002). Over the last decade following the discovery of this powerful tumor suppressor, numerous PTEN mutations have been identified in a wide range of sporadic malignancies and at a high frequency in cancersusceptibility syndromes. PTEN mutations occur most frequently in three types of human cancer: glioblastoma, endometrial and prostate cancer (Supplementary Table 1). The first report on PTEN mutation in primary glioblastoma revealed a high mutational frequency (44%) among 34 cases of glioblastoma multiforme (Wang et al., 1997). Subsequent mutational analyses using different sample numbers also indicate glioblastoma as one of the cancer types bearing frequent PTEN mutations (Table 1). Owing to limited sample sizes in most of these studies, the frequencies of PTEN mutations vary from 9 to 44%. A summary of all these studies gives rise to a frequency of 28.8% with PTEN mutation in a total of 605 glioblastomas. Similarly, information about the frequencies of PTEN mutation in other types of human cancers has also been summarized in Table 1. In addition to the frequency, consolidation of information on position and type of each detected mutation would help in identifying hot spots in different cancers, and understanding how PTEN functions in the etiology and molecular pathogenesis of human cancer. It has been over 10 years since PTEN was discovered. A mutational database for this second most frequently mutated tumor suppressor is warranted. Mutational studies have indeed revealed some critical residues for PTEN function. The most prominent mutation, G129E, was found in Cowden disease kindred (Liaw et al., 1997). This famous mutation, in fact, led to the milestone finding that PTEN has a lipid phosphatase activity because the mutant is deficient specifically in its ability to dephosphorylate PIP3 (Myers et al., 1998). Interestingly, this mutation loses its specificity when the same glycine residue is mutated to arginine (G129R) instead of glutamate (G129E) (Myers et al., 1997, 1998). G129R is also a naturally occurring mutation in glioblastoma (Li et al., 1997). Whereas G129E retains the activity of dephosphorylating protein substrates, G129R loses both lipid phosphatase and protein phosphatase activity (Myers et al., 1997, 1998). In addition to G129R, many missense point mutations of PTEN found in primary human cancers have similar functional deficiencies, including H123Y (detected in endometrial cancer), L57W (glioblastoma), G165R (glioblastoma), T167P (breast cancer), S70R (Banna-

yan–Zonana syndrome) and C124S. The affected residues are located either in the catalytic motif (H123, C124 and G129) or a conserved a-helix that is critical for maintaining secondary structure of the phosphatase (L57, G165, T167 and S70) (Myers et al., 1997). Unfortunately, there are no reported PTEN mutations that are only deficient for protein phosphatase activity. Nevertheless, these naturally occurring PTEN mutations have contributed significantly to our understanding of PTEN function. Germline PTEN mutations are associated with 80% of Cowden syndrome cases and 60% of Bannayan–Riley– Ruvalcaba syndrome cases (Eng, 2003). Both of these cancer susceptibility syndromes are now referred to collectively as the PTEN hamartoma tumor syndrome. The above-mentioned PTEN mutations are mostly located in the N-terminal region of the PTEN molecule, which conveys its phosphatase catalytic activity. However, over 40% of the PTEN mutations are found in the C-terminal region (Waite and Eng, 2002). Moreover, many C-terminal truncated forms of PTEN have been found in Cowden syndrome, due to the generation of a stop codon from a single base substitution (Iida et al., 1998, 2000; Kanaseki et al., 2002; McGarrity et al., 2003; Agrawal et al., 2005). These C-terminal truncation PTEN mutations are particularly important for functional characterization of the C-terminal domain of PTEN. Recent studies have revealed the essential role of the C-terminal region of PTEN in determining subcellular PTEN localization (Trotman et al., 2007; Wang et al., 2007) and maintaining genomic stability (Shen et al., 2007). The C-terminal region is required for PTEN function to stabilize centromeres, although the phosphatase activity of PTEN is dispensable (Shen et al., 2007). Interestingly, phosphatase-deficient PTEN mutants, C124S and G129E, retain the ability to localize in the nucleus and physically associate with the kinetochore component CENP-C, whereas a C-terminal truncated PTEN mutant, PTEN189, fails to do so (Shen et al., 2007). Similarly, a C-terminal point mutation, K289E, confers a nuclear import defect, although it does not affect phosphatase activity (Trotman et al., 2007). These important C-terminal mutations have been very helpful in identifying the new functions of PTEN, particularly of nuclear PTEN. Identification and characterization of these PTEN mutations in various types of human cancer further supports the need for a PTEN mutational database. This database could greatly contribute to the continuing

Table 1 PTEN mutations in different tumor types Tumor types Glioblastoma Endometrial carcinoma Melanoma Prostate cancer Breast cancer

Mutation frequency (%)

Specimens with PTEN mutation

Specimens analyzed

28.8 34.6 12.1 11.8 3.5

174 148 17 18 9

605 428 141 152 254

Mutation frequencies summarized in Supplementary Table 1 are on the basis of currently available reports cited in Supplementary information. Oncogene

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discoveries of novel functions of PTEN and to a comprehensive understanding of how this versatile tumor suppressor safeguards important cellular machineries against tumorigenesis. Maintenance of genomic stability: novel functions of PTEN Genome or chromosome instability is a hallmark of cancers. Tumor suppressors play roles in maintaining genome stability, and loss of function of these tumor suppressors therefore leads to genomic instability. Genetic instability represents an inevitable consequence of the loss of tumor suppressors. Indeed, the frequent occurrence of PTEN mutation and genetic instability is found in a large range of PTEN-deficient human cancers (Cantley and Neel, 1999; Simpson and Parsons, 2001). However, the function of PTEN in maintaining genetic stability had not been recognized and demonstrated until recently. The first link between PTEN deficiency and genetic instability was revealed by the pioneer work of the Parsons’ group (Puc et al., 2005). Pten-null embryonic stem cells were shown to exhibit DNA repair checkpoint defects in response to ionizing radiation, which results in the accumulation of unrepaired chromosomes with DNA double-strand gaps and breaks. Further mechanistic study suggested that the observed G2 checkpoint defects may have resulted from functional impairment of an important checkpoint protein, CHK1, due to lack of PTEN. PTEN deficiency directly elevates AKT kinase activity, which triggers CHK1 phosphorylation (Figure 1). Phosphorylated CHK1 undergoes ubiquitination, which prevents its entry into the nucleus. Sequestering CHK1 in the cytoplasm impairs its normal function in initiating a DNA repair checkpoint. In addition to G2 checkpoint defects, CHK1 inactivation in Pten-deficient cells can lead to the accumulation of DNA double-strand breaks (Puc and Parsons, 2005). These in vitro observations have also been demonstrated in vivo. Examination of CHK1 localization in a large panel of primary human breast carcinomas indicates an increased cytoplasmic level of CHK1 in tumor cells with lower expression of PTEN and elevated AKT phosphorylation. Furthermore, aneuploidy was frequently observed in both human breast carcinomas with low expression of PTEN and prostatic intraepithelial neoplasia from Pten þ / mice (Puc and Parsons, 2005). These consistent in vitro and in vivo observations suggest that PTEN deficiency may initiate an oncogenic signaling process by causing dysfunction of important checkpoint proteins. The essential role of nuclear PTEN in the maintenance of chromosomal stability has been demonstrated recently in both mouse and human systems (Shen et al., 2007). As shown in Figure 1, nuclear PTEN may utilize two novel mechanisms to maintain chromosome integrity. First, PTEN interacts with centromeres and maintains their stability. It is believed that PTEN does so through its C2 domain, as mutant PTEN without this C2 domain loses the capability to interact Oncogene

with centromeres. Second, PTEN may be necessary for DNA repair because loss of PTEN results in a high frequency of double-strand breaks. PTEN affects double-strand breaks through regulation of Rad51, a key component for homologous recombination repair of DNA double-strand breaks. Consistently, regulation of PTEN nuclear localization has also been reported (Trotman et al., 2007; Wang et al., 2007). It has been demonstrated that PTEN physically associates with an integral component of centromeres in the nucleus, disruption of which causes centromere breakage and massive chromosomal aberrations (Shen et al., 2007). These new findings revealed the novel function of PTEN in guarding the genome and propelled PTEN research into the ‘nuclear age’ (Baker, 2007). The cytoplasm has been considered as the primary site for PTEN to elicit its tumor-suppressive function, and the ability of PTEN to block the PI3-kinase pathway through its phosphatase activity has been regarded as the key mechanism by which PTEN suppresses carcinogenesis (Parsons, 2004; Cully et al., 2006). However, the discoveries of PTEN localization and novel functions in the nucleus have expanded its role in tumor suppression. Although the cellular distribution of PTEN varies in different tissues, endogenous PTEN in neurons, gliomas and cells of the thyroid, pancreas and skin is found mostly in the nuclear compartment (Sano et al., 1999; Gimm et al., 2000; Lachyankar et al., 2000; Perren et al., 2000; Whiteman et al., 2002). Growing evidence has suggested that malignancies may be accompanied by translocation of PTEN from the nucleus to the cytoplasm (Gimm et al., 2000; Perren et al., 2000; Whiteman et al., 2002). The function of nuclear PTEN may deviate from its role in regulating PIP3 at the plasma membrane, but is consistent with an alternative role in controlling genetic stability and involving chromatin function. In addition, high expression levels of nuclear PTEN have recently been associated with cellcycle arrest at the G0/G1 phase (Ginn-Pease and Eng, 2003), indicating a likely role of nuclear PTEN in cell growth inhibition. Similarly, multiple aspects of the anti-oncogenic function of PTEN, such as regulation of cell growth and migration, are found independent of its phosphatase activity (Freeman et al., 2003; Raftopoulou et al., 2004; Okumura et al., 2005; Liu et al., 2005b; Tang and Eng, 2006; Leslie et al., 2007). Indeed, somatic PTEN mutations do occur outside the phosphatase domain (Eng, 2003), implying that PTEN can function in tumor suppression through additional activities besides antagonism of the PI3-kinase pathway. These earlier observations form a growing pool of evidence that supports the idea that PTEN may possess other functions beyond those exhibited in the cytoplasm and those dependent upon its phosphatase activity. As shown in Figure 1, PTEN localizes at centromeres and interacts with CENP-C, an integral kinetochore component, which is crucial for protection against chromosome instability. Importantly, it is found that physical association between PTEN and centromeres/kinetochores does not require the phosphatase activity of PTEN, because a phosphatase-deficient PTEN mutant

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Figure 1 A model of PTEN as a guardian of the genome. PTEN maintains genomic stability through multiple mechanisms. In the cytoplasm, PTEN antagonizes phosphoinositol-3-kinase by dephosphorylating PIP3 and prevents Akt activation and Chk1 phosphorylation, releasing Chk1 into the nucleus for DNA repair. PTEN can shuttle between the cytoplasm and the nucleus through an ubiquitin-dependent mechanism, in which NEDD-4-mediated monoubiquitination of PTEN promotes its nuclear import. In the nucleus, PTEN physically associates with centromeres through CENP-C and maintains chromosome integrity. PTEN also acts on chromatin to coordinate with E2F-1 for the transcriptional regulation of Rad51, thus resulting in the control of DNA double-strand break repair.

(C124S) can still interact with CENP-C. Interestingly, a few C-terminal PTEN mutants lacking association with centromeres still cause centromere instability, even though they retain phosphatase activity (Shen et al., 2007). These data suggest that the physical association of nuclear PTEN with centromeres, rather than its phosphatase activity, is crucial for its fundamental role in maintaining chromosomal integrity. Nuclear PTEN at the center of the stage The nuclear localization of PTEN has now obtained mechanistic support. Although PTEN lacks a typical nuclear localization sequence (NLS), ubiquitination of specific sites in its C-terminal region enhances transport of PTEN into the nucleus (Trotman et al., 2007). Consistent with our data, the nuclear import process seems to be unrelated to the phosphatase activity of PTEN because mutation of a critical ubiquitination site, K289, impairs nuclear import but does not affect catalytic activity (Trotman et al., 2007). Besides the ubiquitin-mediated mechanism, nuclear import of PTEN may involve other mechanisms, such as passive transport by diffusion (Liu et al., 2005a) and active transport through NLS-like signals (Chung et al., 2005). PTEN subcellular localization can exhibit a cellcycle-dependent pattern (Liu et al., 2005b) and nuclear-cytoplasmic partitioning of PTEN differentially

regulates cell cycle and apoptosis (Chung et al., 2005). Moreover, cell cycle-dependent nuclear PTEN localization and function can be regulated by its interaction with the major vault protein mediated by Ca2 þ signaling (Minaguchi et al., 2006). A recent study suggests that oxidative stress can also regulate PTEN nuclear localization in a manner independent of phosphatase activity but dependent upon phosphorylation of PTEN (Chang et al., 2008). Taken together, it appears that multiple mechanisms may direct PTEN nuclear localization and affect its nuclear function. Although nuclear transport of PTEN may be a phosphatase-independent process, PTEN could still need its phosphatase activity to exert multiple functions in the nucleus. It is known that PTEN controls a DNA repair process by regulating expression of Rad51 (Shen et al., 2007). Consistently, lack of PTEN also impairs CHK1 function and results in the accumulation of double-stranded DNA breaks (Puc and Parsons, 2005). Transcriptional modulation is obviously another novel function of PTEN in the nucleus, although the detailed mechanism remains to be elucidated. The function of PTEN in transcriptional regulation is found to be phosphatase-dependent, because the phosphatase-deficient PTEN mutant (C124S) is unable to induce Rad51 in PTEN-null cancer cells, whereas a catalytically active C-terminal mutant can mimic full-length PTEN in regulation of Rad51 (Shen and Yin, unpublished data). Although the C-terminal truncated PTEN mutant Oncogene

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retains the ability to induce Rad51, it can no longer associate with centromeres, causing chromosomal instability. As illustrated in Figure 1, these results suggest that different mechanisms may be responsible for the distinct functions of nuclear PTEN: maintenance of centromere stability (phosphatase-independent, through CENP-C) and control of DNA repair (phosphatasedependent, through Rad51). Although the PI3-kinase pathway may continue to serve as an important target for PTEN in the cytoplasm, identification of more nuclear-specific targets of PTEN will advance the field to a better understanding of PTEN function in tumor suppression. Interestingly, major elements of the PTENtargeted PI3-kinase pathway in the cytoplasm, including the PI3-kinase itself and AKT, have now been found in the nucleus (Trotman et al., 2006). Whether or not its phosphatase activity is indispensable and whether the PI3-kinase/AKT serves as a target pathway, the important and exciting news is that ‘PTEN enters the nuclear age’ (Baker, 2007). The nuclear PTEN and cytoplasmic PTEN may function complementarily in multiple pathways stabilizing the genome. Previous studies using cDNA microarray revealed global changes in gene expression profiles associated with alternate PTEN status (Hong et al., 2000; Matsushima-Nishiu et al., 2001), suggesting that PTEN may be involved in gene transcription regulation. Indeed, PTEN can negatively modulate the transcriptional activity of the androgen receptor (Nan et al., 2003), c-Met (Abounader et al., 2004), nuclear factor-kB (Mayo et al., 2002; Moon et al., 2004), CRE binding protein (Mayo and Donner, 2002) and AP-1 (Koul et al., 2007). PTEN also downregulates the promoter activity of MDM2 (Chang et al., 2004), insulin-like growth factor II (Kang-Park and Lee, 2003) and matrix metalloproteinase-9 (Moon et al., 2004). The transcription activity of hypoxia-inducible factor 1 is also regulated by PTEN in the nucleus (Emerling et al., 2008). A direct role for PTEN in transcriptional regulation has been recognized just recently in a report showing that PTEN regulates the TFIIIB-centered basic transcription machinery by interrupting formation of functional TFIIIB complexes (Woiwode et al., 2008). PTEN is physically associated with the promoter of Rad51 and cooperates with the E2F-1 transcriptional factor in synergistic regulation of Rad51 (Shen et al., 2007). These data suggest that PTEN acts on chromatin and regulates transcription of important genes involved in critical cellular processes such as cell cycle control and DNA repair. As one of the most frequently mutated genes associated with cancer, PTEN becomes the victim of defective DNA repair due to the mutation of other DNA repair genes, such as BRCA1. Loss of BRCA1 leads to recurrent gross mutation of PTEN (Saal et al., 2008). Various structural mutations on chromosome 10q23.31 at the PTEN locus were frequently found in a very aggressive type of breast cancer, basal-like breast cancer. More importantly, gross mutations of PTEN in this type of breast tumor are highly correlated with BRCA1 mutations. These results suggest that the PTEN Oncogene

gene and its pathway can be directly targeted by BRCA1 dysfunction, which may explain why basal-like breast cancer often develops due to BRCA1 deficiency. Given the critical roles of both PTEN and BRCA1 in maintaining chromosomal stability and proper DNA repair, successive hits on these important tumor suppressors give rise to an increasing chance of tumor development and may interpret the aggressiveness and genetically unstable feature of this specific type of breast cancer. Interestingly, the p53 gene, in addition to BRCA1 and PTEN, is also frequently mutated in basal-like breast cancer (Borresen-Dale, 2003; Langerod et al., 2007), which further intrigues the need to exploit how tumor suppressors coordinate with each other and form a complementary and cooperative network in protecting against tumorigenesis. Tumor suppressor network: p53, PTEN and beyond p53 is the most frequently mutated gene in human cancers (Vogelstein et al., 2000; Levine et al., 2004). In terms of overall frequency, PTEN is the second most commonly mutated gene in human cancers (Cantley and Neel, 1999; Simpson and Parsons, 2001). The mutation spectrums of p53 and PTEN in human cancers, however, are different. Mutations of p53 occur at high frequencies in lung, colon and breast cancers, whereas PTEN mutations are mostly found in glioblastoma, endometrial cancer, malignant melanoma and prostate cancer. There are germline p53 mutations in a familial syndrome of breast cancer, sarcomas and other neoplasms (Malkin et al., 1990). p53 is mutated in Li–Fraumeni syndrome, which is a familial cancer susceptibility syndrome that develops disease at an early onset (Bischoff et al., 1991; Strong et al., 1992; Yin et al., 1992). Germline mutations of PTEN are associated with some inherited hamartoma-cancer syndromes (Eng and Peacocke, 1998). Mice null for p53 develop normally and exhibit spontaneous tumors, mainly including lymphoma and sarcoma (Donehower et al., 1992). In contrast, homozygous deletion of Pten results in mouse embryonic lethality, suggesting that PTEN is necessary for embryonic development (Di Cristofano et al., 1998; Suzuki et al., 1998). Mice heterozygous for Pten develop spontaneous tumors and conditional tissue-specific disruption of Pten leads to different tumors in the affected tissues (Di Cristofano and Pandolfi, 2000; Stambolic et al., 2000; Kimura et al., 2003; Backman et al., 2004). Therefore, PTEN is a powerful and distinct tumor suppressor. Regulation of p53 protein stability is a major mechanism to control levels of the p53 protein (Shmueli and Oren, 2005). Mdm2 is a mediator for p53 ubiquitination, and MDM2 promotes the shuttle of p53 toward nuclear export, which leads to proteasomemediated degradation of p53 (Shmueli and Oren, 2004). p53 functions as a transcription factor that regulates target genes (Levine, 1997). p53 controls cellcycle progression, apoptosis and genomic stability (Yonish-Rouach et al., 1991; Kastan et al., 1992; Livingstone et al., 1992; Yin et al., 1992; Lowe et al.,

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1993; Tlsty et al., 1994). p53 is the first to be proposed as a guardian of the genome (Lane, 1992). PTEN functions as a phosphatase that inhibits PI-3 kinase/AKT signaling by converting PIP3 to PIP2. PTEN exerts tumor suppressor function by regulation of proliferation and survival, cell migration, invasion and angiogenesis. As a guardian of the genome, PTEN maintains genomic stability through multiple pathways. As an autonomous surveillance and defense system against genomic instability and tumorigenesis, tumor suppressors act in coordination with each other to form a regulatory network. The relationship between PTEN and p53 has attracted extensive interest and enthusiasm. Comparison of their spectra of mutations revealed that somatic mutations of PTEN and p53 are usually independent and even mutually exclusive (Fujisawa et al., 2000; Kato et al., 2000; Koul et al., 2002; Kurose et al., 2002), suggesting that these two guardians of the genome evolved to guard distinct aspects of homeostasis and cellular processes in a complementary mode. A similar complementary mode can also be implicated from their distinct patterns of expression and action. The p53 protein is maintained at low levels in normal cells and its induction requires stimulation upon cellular stress. Therefore, the sensitivity of p53 activation in response to stress signals, rather than its steady expression levels, is crucial for its action against tumorigenesis. In comparison, PTEN expression levels are steady and high in normal cells and the maintenance of its abundance is important to sustain its function to safeguard the genome. Besides PTEN mutation, loss or reduction of PTEN expression are often found in human cancers (Leslie and Downes, 2004), suggesting that PTEN function depends on its abundant expression and that insufficient PTEN expression may permit tumorigenesis. p53 action is dynamic and impulsive, whereas PTEN functions in a static and continuous fashion. PTEN was reported as a downstream transcriptional target of p53 in mediating apoptosis (Backman et al., 2001). Other studies suggest that PTEN may act upstream of p53 to regulate its expression levels and activity (Mayo and Donner, 2002; Freeman et al., 2003). PTEN can increase p53 stability and its DNA binding activity through physical association with p53 (Freeman et al., 2003). Furthermore, this cooperative interaction between PTEN and p53 was applied to an animal model in which loss of one allele of Pten dramatically accelerated tumor onset in p53 þ / mice (Freeman et al., 2003). The physical association between PTEN and p53 was confirmed by other studies, which give further evidence of their functional cross talk (Tang and Eng, 2006; Flores-Delgado et al., 2007). Recently, PTEN was found to physically associate with p300 in the nucleus, which maintains p53 in a highly acetylated state in response to DNA damage (Bilbao et al., 2006). All these data support a positive regulatory relationship between PTEN and p53. However, p53 and PTEN may have other types of interactions. For example, deletion of Pten resulted in enhanced expression of p53 as well as p21, a direct transcription target of p53 (Xiong et al., 1993). The

activation of the p53 pathway is consistent with a cellular senescence phenotype upon Pten deletion, and interestingly, combined deletion of p53 led to increased cell proliferation and escape from senescence (Chen et al., 2005). Consistently, combined inactivation of Pten and p53 greatly accelerated tumor development, which can be interpreted by deterioration of Pten deficiencyinduced tumorigenesis due to lack of p53-dependent cellular senescence (Chen et al., 2005). These data support a ‘one-by-one’ hit model for tumor development due to sequential loss of major tumor suppressor genes. In agreement, upregulation of p53 and senescence-like growth arrest in different human cells containing inactivated PTEN were observed (Trotman et al., 2007). These data suggest a coordinate and complementary pattern of interaction between PTEN and the p53 pathway and would have therapeutic as well as prognostic significance. Besides p53, PTEN may coordinate with another tumor suppressor, Rb, in the prevention of tumorigenesis. Rb plays an important role in controlling cell cycle progression (Friend et al., 1986; Weinberg, 1992). PTEN controls the cell cycle (Li and Sun, 1998; Ramaswamy et al., 1999) and Rb has been reported to be essential for PTEN-mediated cell cycle arrest and growth suppression (Paramio et al., 1999). There are two forms of Rb: phosphorylated Rb, which is an inactive form, and dephosphorylated Rb, which is activated. Rb is phosphorylated and inactivated by cyclin D-dependent kinases CDK4 and CDK6. Rb can be activated through dephosphorylation by CDK inhibitors, such as p16 and p18, thus retaining its growth-suppressive state (Guan et al., 1994; Medema et al., 1995; Quelle et al., 1995). Interestingly, knockout of p18Ink4c in the Pten þ / background accelerates tumor development with a wider spectrum than Pten þ / single deletion (Bai et al., 2006), indicating a functional collaboration between these two tumor suppressors. Protection against tumorigenesis is maintained by an innate defense system comprised of a number of tumor suppressors, each with special properties in controlling one or more cellular processes through their specific pathways. It is crucial that a network exists to integrate signals and to coordinate molecular events in response to environmental challenges. As p53 and PTEN are both guardians of the genome, interaction and cooperation between their respective pathways are necessary for proper actions in protection of the genome. As p53 and PTEN are distinct in many ways as discussed above, they have different roles in guarding the genome. Because PTEN expression is high in cells and tissues, PTEN may be on duty during normal peaceful times, acting like a police force. However, p53 is mainly responsible for dealing with emergency, such as DNA damage, as p53 levels are usually extremely low but highly increased following DNA damage and genotoxic stress. Consistently, p53 mediates cell cycle checkpoints and cell death, depending upon the type and nature of damage incurred. However, our preliminary studies indicate that PTEN mainly functions in mitosis, which is always active during the normal growth stage. ThereOncogene

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fore, PTEN and p53 function in different aspects of life, but together they protect cells against damage all the time. Given that frequent mutations often give rise to functional deficiencies of important tumor suppressors, a failsafe mechanism should exist among these tumor suppressors and their downstream effectors to maintain genomic integrity and to initiate protective responses. Understanding how they coordinate with each other will reveal the mechanism of tumor suppression and

contribute to the development of new therapeutic strategies for cancer treatment. Acknowledgements We are grateful to Drs C Walsh and HB Lieberman for their critical comments on the manuscript. This work was supported by a grant from the National Institutes of Health (R01 CA102447 to YY).

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