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Review

PTEN Mutation: Many Birds with One Stone in Tumorigenesis WEIJIN LIU 1, YONGGANG ZHOU 2, SVEN N. RESKE 3 and CHANGXIAN SHEN 4

1Department of Life Sciences, Huaihua University, Huaihua, P. R. of China; 2German Research Center, Heidelberg; 3Department of Nuclear Medicine, University of Ulm, Ulm, Germany; 4Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN, U.S.A.

Abstract. The PTEN ( and tensin homolog combination with the loss of certain other tumor suppressor deleted on chromosome ten) tumor suppressor gene is genes or with genetically engineered reduction of PTEN mutated in a wide range of malignancies and recent studies expression just below heterozygosity (3-7). have demonstrated that PTEN prevents tumorigenesis PTEN is a multiple-domain polypeptide of 403 amino acids. through multiple mechanisms. PTEN functions as a plasma- It contains an amino-terminal phosphatase domain homologous membrane lipid phosphatase that antagonizes the PI3K to chicken tensin and a C2 domain, which mediates the (phosphoinositide 3 )-AKT pathway. PTEN physically association of signal proteins to plasma membranes. The C- and genetically interacts with the central genome guardian terminal PDZ (PSD95, DlgA and Zo-1) domain-binding . PTEN also associates with the centromeric protein sequence and PEST (rich in proline, glutamate, serine, and CENP-C to maintain centromere integrity and suppresses threonine residues) domain regulate protein stability via the chromosomal instability from DNA double-strand breaks ubiquitin-proteasome pathway (2). PTEN has two canonical (DSBs) through transcriptional regulation of Rad51 PEST domains, which is a signature in many short-lived (radiosensitive mutant 51). Moreover PTEN controls proteins degraded by the ubiquitin pathway. Given that the the growth and proliferation of haematopoietic stem cells protein level of PTEN is highly correlated with carcinogenesis (HSC) and restrains cells from leukemia in an mTOR in a wide range of cancer cases, it should be tightly controlled. (mammalian target of rapamycin) dependent manner. Thus, Indeed, PTEN is regulated by ubiquitin-mediated proteasomal restoring PTEN functions in cancer cells directly or degradation (8, 9). PTEN can be polyubiquitinated and indirectly holds great promise for cancer therapy. monoubiquitined by the same ubiquitin ligase NEDD4-1 (neural precursor cell expressed, developmentally down- PTEN (phosphatase and tensin homolog deleted on regulated gene 4-1), a potential proto- (10). However, chromosome ten) was first identified as a tumor suppressor only the polyubiquitinated PTEN is targeted to proteasomes for gene in 1997 (1). PTEN is mutated in a wide range of degradation in the cytoplasm through the C-terminal PEST malignancies, especially solid tumors and it is, second only domain, whereas monoubiquitinated PTEN is required for to p53, the most frequently affected tumor suppressor in nuclear import. Once in the nucleus, the ubiquitin moiety of human carcinomas. Somatic mutations of PTEN occur in PTEN is removed by an unidentified deubiquitinase (11). multiple sporadic malignancies while germline mutations of Another negative regulator of PTEN protein is DJ-1 (also PTEN lead to inherited hamartoma and Cowden syndrome called PARK7, Parkinson’s disease gene) but its role in (2). Importantly, PTEN is haploinsufficient because a single suppressing PTEN is unclear (12). Additionally, PTEN copy was unable to prevent prostate cancer either in enzymatic activity can be strictly regulated by protein phosphorylation (8, 13-15).

PTEN Antagonizes PI3K-AKT Pathway in Correspondence to: Changxian Shen, Ph.D., Department of Carcinogenesis Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 N Lauderdale St., Memphis, TN 38105, U.S.A. Tel: +1 9014952111, Fax: +1 9014954290, e-mail: [email protected] The current understanding of the molecular mechanisms of PTEN in carcinogenesis comes primarily from the study of Key Words: PTEN, mTOR, p53, AKT, genome stability, , PTEN as a central negative regulatory factor of the PI3K review. (phosphoinositide 3 kinase)-AKT (AKT8 virus oncogene

0250-7005/2008 $2.00+.40 3613 ANTICANCER RESEARCH 28 : 3613-3620 (2008) cellular homolog) pathway (Figure 1). PI3K-AKT is one malignant transformation and its overexpression is found in of the major signal transduction pathways that promote cell a wide range of cancer cells (26, 27). Furthermore, AKT growth, survival and proliferation. It contributes to the phosphorylates and inactivates GSK3 kinase, which inhibits evasion of , loss of cell cycle control and genomic Myc (myelocytomatosis oncogene cellular homolog) and instability during tumorigenesis through numerous D1 as well (16). Myc is a proto-oncogene and is mechanisms (16, 17). In response to ligand binding to mutated in numerous carcinomas. It is an important receptor tyrosine kinase (RTK) or G-protein coupled transcription factor for cell cycle progression, particular receptor (GPCR), PI3K is activated and converts the during S-phase. Thus PTEN suppresses malignant phosphatidylinositol 4,5 phosphate (PIP2) to transformation by controlling proper cell cycle progression phosphatidylinositol 3,4,5 trisphosphate (PIP3), a major and DNA damage checkpoint responses through regulating second messenger in cellular signaling. PIP3 then recruits the key checkpoint proteins Chk1, p27 kip1 , p130 Rb2 , cyclin and binds to proteins with pleckstrin homology domain D1 and Myc (Figure s 1 and 2). (PH) such as phosphatidylinositide-dependent kinase 1 Taken together, PTEN suppresses tumorigenesis at least in (PDK1) and AKT (also called protein kinase B, PKB) (18). part by antagonizing PI3K-AKT signaling events that After membrane enrichment, PDK1, together with contribute to the resistance to the growth inhibitory signal, mTORC2 (mammalian target of rapamycin complex 2) the evasion of apoptosis and cell cycle progression during phosphorylates and activates AKT (19). Activated AKT the multistep process of tumorigenesis. then promotes proliferation and survival through the inhibition of numerous tumor suppressor-like signal PTEN and p53 molecules such as Bad (Bcl-XL/Bcl-2-associated death promoter), FOXO3 (forkhead box O3), GSK3 (glycogen Mutations of PTEN and p53 tumor suppressor genes are the synthase kinase 3) and TSC2 (tuberous sclerosis complex most common mutations found in carcinomas and 2), and activation of many such as MDM2 interestingly these mutations are often mutually exclusive (murine double minute 2), PDK1 and IKK (I κB kinase) by suggesting a functional connection underlying these two phosphorylation. Finally activated AKT inhibits apoptosis tumor suppressors. Indeed, PTEN null mice that die during by decreasing proapoptotic proteins Bad, Fas L (Fas embryonic development can be rescued by simultaneous p53 ligand), Bim (bcl-2-interacting mediator of cell death) and mutation and PTEN null cells show p53-dependent cellular p53 while increasing anti-apoptotic nuclear factor- κB (NF- senescence (28). The molecular mechanistic investigation κB) signaling (16-18). Therefore PTEN functions as the reveals that PTEN regulates p53 stability in a phosphatase- central negative regulator of the PI3K/AKT pathway in dependent manner by counteracting the PI3K-AKT-MDM2 controlling apoptosis during tumorigenesis. signaling pathway, which targets p53 to the proteasome for It has been established that the deregulation of cell cycle degradation (29, 30). Furthermore, PTEN interacts with and control results in tumorigenesis (20). Activated AKT also maintains the highly acetylated p53 in a phosphatase- phosphorylates and sequesters Chk1 (checkpoint kinase 1), independent way that enhances p53 DNA binding ability. On an essential factor for DNA replication and cell cycle the other hand, p53 positively controls the transcription of checkpoints in G 1, S and G 2-phase (21), in the cytoplasm. PTEN (31-33) (Figure 2). This functional interdependence This mechanism prevents Chk1 from entering the nucleus as may be the reason why mutations in PTEN and p53 are so a guardian for genome integrity (22) (Figure 2). Moreover frequent and gives an explanation for the role of PTEN in AKT inactivates FOXO3, which activates the transcription of carcinogenesis through p53. p27 kip1 and p130 Rb2 , and represses the expression of (16-18). p27 kip1 is one of the most important inhibitors PTEN and Chromosomal Integrity of the cyclin-dependent Cdk2 and Cdk1, masters of the propeller of cell cycle progression (23, 24). p130 Rb2 is a PTEN is found not only in the plasma membrane, but also member of the Rb (retinoblastoma) family, which is a in the nucleus of different normal and cancer cells, indicating negative regulator of G 1/S-phase transition through inhibition a functional role in the nucleus (33-35). Nuclear PTEN has of the (transcription factor family including E2F and been found to be required for the maintenance of genome DP-like subunits) that mediate gene transcription essential stability. In PTEN null mouse embryonic fibroblast (MEF) for G 1/S-phase transition and S-phase progression. Both cells there is a high frequency of centromere breakage. p27 kip1 and p130 Rb2 are tumor suppressors, mutated in many Further data have shown that PTEN localizes at the carcinomas (25). Cyclin D1 plays a vital role in the activity centromere via physical interaction with an essential of G 1-phase cyclin-dependent kinases Cdk4 and Cdk6 which centromeric protein CENP-C which targets PTEN to the are essential for the initiation of DNA replication and G 1- to inner core of the kinetochore. The association of PTEN with S-phase transition. Up-regulation of cyclin D1 leads to the centromere depends on the C-terminus of PTEN but not

3614 Liu et al : PTEN Mutation in Tumorigenesis ( Review )

Figure 1. PTEN antagonizes the PI3K-AKT signal transduction pathway. Following growth factor binding, activated receptor tyrosine kinase (RTK) phosphorylates a number of substrates which lead to the activation of the signal molecules Ras and IRS (insulin receptor substrate), either of which can activate PI3K, converting phosphatidylinositol 4,5 phosphate (PIP2) to phosphatidylinositol 3,4,5 trisphosphate (PIP3) in the plasma membrane. PIP3 recruits PDK1 and AKT for membrane enrichment. PDK1 together with mTORC2 phosphorylates and fully activates AKT. By phosphorylation, AKT then promotes , proliferation and survival through inhibition of numerous tumor suppressor-like signal molecules Bad, FOXO3, GSK3 and TSC2, and activation of oncogenes MDM2 and IKK. AKT promotes cell survival by decreasing proapoptotic proteins Bad, Fas L, Bim and p53, and at the same time increasing the antiapoptotic NF- κB signaling. AKT enhances cell proliferation by controlling p27 kip1 , p130 Rb2 , Cyclin D1 and Myc by inhibiting GSK3 and FOXO3. Another key function of AKT is to promote cell growth (increase of macromolecule synthesis) by increasing mTORC1 signaling. The downstream targets of mTORC1, eIF-4E (eukaryotic initiation factor 4E), enhances cap-dependent protein translation, while S6K1 promotes ribosome biogenesis. S6K1 also forms a positive feedback (S6K1-mTORC1) and a negative feedback (S6K1-IRS) loops to balance the signal transduction emitted from RTK. PTEN antagonizes PI3K-AKT mediated cellular metabolism via reversing PIP3 to PIP2.

the phosphatase activities. Disruption of this interaction leads PTEN, Cancer Stem Cells and mTOR to chromosome instability. Another obvious phenotype of PTEN null MEF is the high frequency of chromosome It has been suggested that cancer cells arise from cancer stem translocations (36), a hallmark of cancer cells (20), and cells, therefore targeting cancer stem cells should be the most mainly resulting from defects in DNA double-strand break efficient and specific means of cancer therapy (38). The (DSB) repair. Microarray screening data have shown that similarity between cancer stem cells and normal stem cells PTEN null MEF cells appeared to have noticeably low levels hinders the therapeutic strategies that selectively kill cancer of Rad51 (radiosensitive yeast mutant 51), an essential factor stem cells while sparing normal stem cells. Recently, it has involved in DSB repair. Further investigation revealed that been demonstrated that the loss of PTEN in mice resulted in PTEN directly controls the transcription of the Rad51 gene. the rapid expansion of hematopoietic stem cells (HSC). PTEN binds to the Rad51 promoter and synergizes with However, with time this leads to the depletion of HSC via a E2F1 to activate Rad51 transcription. Therefore, nuclear cell-autonomous mechanism and an increase of leukemia- PTEN maintains chromosomal integrity through physical initiating cells, suggesting the role of PTEN in maintaining the interaction with CENP-C and by controlling DSB repair (36, HSC and suppressing the leukemia-initiating cells. 37) (Figure 2). Furthermore, HSC development and leukemia stem cells are

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Figure 2. PTEN is interwoven in PI3K-AKT-mTOR and ATM-Chk2-p53 signaling networks. PTEN negatively controls mTORC1, the central regulator of cell growth and proliferation and the critical downstream effector of PI3K-AKT signaling. mTORC1 regulates cellular processes mostly through its control of protein translation via phosphorylation of 4E-BP1 and S6K1. mTORC1 marks the convergence of multiple signal transduction pathways such as Ras-ERK (extracellular signal-regulated kinase), LKB1/AMPK, oxygen and amino acids, most, if not all, of which impact on mTORC1 via the upstream TSC1/2 complex. TSC1/2 complex is a guanine nucleotide exchange factor for the Ras-like GTPase, Rheb, which is essential for mTORC1 activation. PTEN is also an important player in the DNA damage responses via p53, Rad51 and CENP-C. In response to DNA damage, a signal cascade is produced and amplified through the signal transducer ATM /ATR ( ataxia telangiectasia-mutated protein kinase/ATM and Rad3-related protein kinase) and Chk1/Chk2 to prevent cell cycle progression via phosphorylation and inhibition of the phosphatase cdc25 ( cycle protein 25). cdc25 is required for G 2/M-phase transition and Cdk2-dependent delivery of cdc45 for DNA replication origin firing. Chk1/Chk2-mediated phosphorylation of MDM2 and p53 leads to p53-dependent increase of which then delays cell cycle progression by preventing E2F-dependent expression of genes required for G 1/S transition and S-phase progression such as , , Myc and E2F through activating Rb by inhibiting Cdk2. The DNA damage checkpoint both promotes cell death of irreparably damaged cells via p53-dependent apoptosis and repairs DNA damage via Chk1/Chk2-mediated activation of BRCA1 (breast cancer growth suppressor protein 1), NBS1 (Nijimegen breakage syndrome 1) and c-Abl (Ableson leukemia concogene cellular homolog). Another key function of the checkpoint is to maintain the stability of the DNA replication fork and kinetochore. PTEN contributes to this genome stability both by promoting Rad51 expression to enhance homologous recombination (HR) and by physically interacting with CENP-C to maintain centromere integrity. Interestingly, the PI3K-AKT-mTOR and ATM-Chk2-p53 pathways are functionally linked. AKT decreases the checkpoint functions by inhibiting Chk1, p27 kip1 and apoptosis, while the checkpoint dampens the AKT-mTORC1 pathway via p53 dependent increase of PTEN, TSC2 and AMPK1. However, AKT-mTORC1 may also help the checkpoint responses by increasing the translation of the proteins required for repair and cell cycle arrest. The discovery of the complex linkage of these two pathways will greatly impact on the development of combinatorial anticancer therapies targeting PI3K-AKT-mTORC1 signaling in combination with cytotoxic .

distinctly rescued by rapamycin inhibition of mammalian regulator of cell growth and proliferation (41). mTOR marks target of rapamcyin (mTOR) in PTEN null cells (39, 40). the convergence of multiple signal transduction pathways, Indeed, mTOR is positively regulated by PI3K-AKT through such as Ras-MAPK (mitogen-activated protein kinase), LKB1 a cascade of phosphorylation events of several signal proteins (also called STK11, germline mutation of which leads to (41, 42) and PTEN probably controls mTOR by antagonizing Peutz–Jeghers syndrome)-AMPK (AMP-activated protein the PI3K-AKT axis. mTOR has emerged as the central kinase), oxygen and amino acids (Figure 2). mTOR controls

3616 Liu et al : PTEN Mutation in Tumorigenesis ( Review )

Table I. Restoring PTEN function as cancer therapy.

Effect on PTEN Target Examples References

Direct PTEN Adenovirus delivered PTEN (46) Non-viral delivered PTEN (47) Recombinant TAT-PTEN (48) Indirect p53 Adenovirus delivered WT p53 (49, 50) MDM2-p53 interaction nutlin (51) Growth factor receptors EGFR Cetuximab (53) HER2 Herceptin (52) IGF-IR SCH 717454 (57) PI3K LY294002 (55) Wortmannin Pan-inhibitor AKT N10-substituted phenoxazines (58) mTOR Rapamycin (55) CCI-779 Rad001 AP23573

many of its downstream cell processes through its control of viral vector-mediated delivery such as urocanic acid- protein translation, via the phosphorylation of 4E-BP1 modified chitosan-mediated PTEN delivery via aerosol (47). (eukaryotic initiation factor 4E binding protein) and S6K1 Another direct strategy is to deliver cell permeable (small subunit ribosomal protein S6 kinase 1). Dysregulation recombinant wild-type PTEN into cells via fusing PTEN of the mTOR pathway has been found in a number of with a cell permeable protein transduction domain (PTD) carcinomas (42, 43). Cancer cells with PTEN mutation are such as TAT (transactivator of transcription: human hypersensitive to rapamycin inhibition of mTOR (44). immunodeficiency virus protein transduction domain) (48). Therefore, according to the ‘oncogene addiction’ hypothesis This PTD-mediated cancer macromolecular therapy is very (45) the maintenance of the survival of PTEN mutation cancer promising as current biotechnolog ical advances make it easy cells probably depends on mTOR signaling (Figure 2). The to produce a high quality and quantity of recombinant rescue of HSC and the depletion of leukemia cells of PTEN proteins. There are numerous indirect ways to restore PTEN null mice by rapamcyin provides the possibility of killing function. For example the reintroduction of wild-type p53 cancer stem cells while sparing normal stem cells in leukemia. may partially complement the loss of PTEN function (49, 50). More recently, nutlin has been found to potently prevent Restoring PTEN for Cancer Therapy MDM2-mediated p53 degradation (51). A widely applied strategy is to decrease the activities of antagonists upstream As reviewed above, PTEN resides at several key nodes in of PTEN such as the growth factor receptor tyrosine kinases intracellular signal transduction pathways that are essential (GF-RTK). The common avenue is to produce GF-RTK for normal cell growth, proliferation and genome integrity specific monoclonal antibodies. Several of these GF-RTK TM (Figure 2). Aberration of the regulation of PTEN gene antibodies such as Herceptin for HER2 (human epidermal expression, or PTEN protein degradation or relocalization growth factor receptor 2) (52) and cetuximab for EGFR would disrupt the homeostasis of the signal network and tip (epidermal growth factor receptor) are in clinical use (53). the balance towards tumorigenesis. The mutation of PTEN A more recent widely recognized strategy to restore PTEN in a wide range of cancer cells reflects its importance in the is to target its downstream intracellular kinases such as by maintenance of cancer cell survival. Thus, restoring PTEN small molecular inhibitors of PI3K, AKT and mTOR (54, would break down the PTEN mutation-dependent cancer cell 55). It has been demonstrated that PTEN-deficient tumors growth (oncogene addiction) and might induce cell death were hypersensitive to the inhibitors of mTOR (44). under certain conditions. Indeed, in recent years increasing However, given that tumors are composed of multiple genetic interest in restoring PTEN function for cancer therapy has mutations, but not a single gene aberration (56), in the near been witnessed, some examples classified into two future a cocktail or combinatory cancer therapy protocols categories, direct and indirect strategies, are listed in Table such as IGF-1R (insulin-like growth factor 1 receptor) I. As a direct method, wild-type PTEN could be introduced antibody in combination with rapamycin for the treatment of into cells by viral vectors such as adenovirus (46) or by non- PTEN-deficient carcinomas will be available.

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Conclusion 10 Wang X, Trotman LC, Koppie T, Alimonti A, Chen Z, Gao Z, Wang J, Erdjument-Bromage H, Tempst P, Cordon-Cardo C, Great progress has been made over recent years in Pandolfi PP and Jiang X: NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128 : 129-139, 2007. elucidating the functions of PTEN in cell growth and 11 Trotman LC, Wang X, Alimonti A, Chen Z, Teruya-Feldstein J, tumorigenesis. As PTEN suppresses tumorigenesis through Yang H, Pavletich NP, Carver BS, Cordon-Cardo C, Erdjument- numerous mechanisms, loss of PTEN leads to centromere Bromage H, Tempst P, Chi SG, Kim HJ, Misteli T, Jiang X and instability, DSB repair defects, failure, Pandolfi PP: Ubiquitination regulates PTEN nuclear import and resistance to apoptosis and sustained cell growth, all of tumor suppression. Cell 128 : 141-156, 2007. which are the traits of cancer cells (20). 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