PTEN: Life as a Tumor Suppressor

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Experimental Cell Research 264, 29–41 (2001)doi:10.1006/excr.2000.5130, available online at http://www.idealibrary.com on

PTEN: Life as a Tumor SuppressorLaura Simpson and Ramon Parsons1

Institute of Cancer Genetics, College of Physicians and Surgeons, Columbia University, 1150 St. Nicholas Avenue,
Russ Berrie Pavilion Room 302, New York, New York 10032
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PTEN, a tumor suppressor located at chromosome10q23, is mutated in a variety of sporadic cancers andin two autosomal dominant hamartoma syndromes.PTEN is a phosphatase which dephosphorylates phos-phatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3),an important intracellular second messanger, lower-ing its level within the cell. By dephosphorylatingPtdIns-3,4,5-P3, PTEN acts in opposition to phosphati-dylinositol 3-kinase (PI3K), which has a pivotal role inthe creation of PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 is nec-essary for the activation of Akt, a serine/threoninekinase involved in cell growth and survival. By block-ing the activation of Akt, PTEN regulates cellular pro-cesses such as cell cycling, translation, and apoptosis.In this review, we will discuss the identification ofPTEN, its mutational status in cancer, its role as aregulator of PI3K, and its domain structure. © 2001

cademic Press

Key Words: tumor suppressor; PTEN; phosphatidyl-inositol 3-kinase; Akt; apoptosis.

INTRODUCTION

In his landmark epidemiological study, Knudsonproposed that two inactivating mutations in the reti-noblastoma susceptibility locus were required for thedevelopment of the disease [40]. Over 10 years later,the cloning of the retinoblastoma gene, RB1, reinforcedthis hypothesis and laid the groundwork for the dis-covery of other tumor suppressors [16]. Dozens of tu-mor suppressors have been identified to date and thesearch for others still continues. The investigation intothe function of tumor suppressors provides researcherswith invaluable knowledge indispensable to the under-standing and treatment of cancer.

THE DISCOVERY OF PTEN ON CHROMOSOME 10q23

For many years, genetic evidence pointed to the ex-istence of at least one important tumor suppressor onchromosome 10. Cytogenetic and molecular analysis

1 To whom correspondence should be addressed. Fax: (212) 304-
D5511. E-mail: [email protected].
29

revealed partial or entire loss of chromosome 10 inbrain, bladder, and prostate cancer [2, 20, 55]. Later,LOH analysis identified region 10q23 as the most com-mon region of loss on chromosome 10 in prostate cancer[36]. These studies implicated 10q23 as a chromosomalregion likely to contain one or more important tumor-suppressor genes. When wild-type chromosome 10 wasreintroduced into glioblastoma cell lines, it reduced theability of these cell lines to form tumors in nude mice inpart due to inhibition of angiogenesis [34]. During thesame year, a linkage analysis report of a cancer pre-disposition syndrome, Cowden disease (CD), deter-mined that a CD locus was present on chromosome10q23 [73]. These findings greatly bolstered the ideathat chromosome 10q23 contains a novel tumor-sup-pressor gene whose loss is key in the formation ofseveral types of cancer.

In 1997, Li et al. [51] identified a putative tumoruppressor at 10q23 through the use of representa-ional difference analysis (RDA). RDA was performedn 12 primary breast tumors and a probe derived fromhis analysis mapped to chromosome 10q23. To maphe location of the probe more precisely, yeast artificialhromosomes (YACs) that contain the probe and thatere present on the sequence-tagged site (STS)-basedap of the human genome were isolated. Two homozy-

ous deletions were identified in breast xenografts byalking away from the RDA marker on the STS-basedAC map. Using exon-trapping analysis, two exonsere identified and clones containing expressed se-uence tags (EST) which matched the sequences of thewo exons were used to assemble an open readingrame (ORF) of 403 amino acids. Sequence analysis ofhe ORF revealed a protein tyrosine phosphatase do-ain and a large region of homology to chicken tensin

nd bovine auxilin. The gene was, therefore, desig-ated PTEN for phosphatase and tensin homolog de-

eted on chromosome 10.Steck et al. [94] independently reported the identifi-

ation of the same candidate tumor-suppressor gene on0q23, which was termed MMAC1 for mutated in mul-iple advanced cancers. They performed a high-densitycan between microsatellite markers D10S191 and
10S221 in 21 glioma cell lines and primary cultures
0014-4827/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

30 SIMPSON AND PARSONS

to narrow the critical region on 10q. Homozygous de-letions were detected in four cell lines with the criticalregion of loss bordered by D10S541 and AFM280. Us-ing fragments from a BAC containing a portion of theregion that was homozygously deleted, exon trappingwas performed to identify exons of the tumor-suppres-sor gene. The gene discovered proved to be identical toPTEN.

Finally, in a search for novel human protein tyrosinephosphatases, a gene termed TEP1 (TGF-regulatedand epithelial cell-enriched phosphatase) was identi-fied [49]. This search was conducted using a methodentirely different from those previously mentioned.PCR using degenerate primer pairs that correspond tothe phosphatase catalytic domain was used to screenhuman cDNA libraries for clones. Also, conserved phos-phatase sequence motifs were used to search the Gen-Bank cDNA database. Through a combination of thesetwo approaches TEP1 was identified. TEP1 also provedto be identical to PTEN. The purified TEP1 protein wascapable of dephosphorylating phosphotyrosyl RCML,an in vitro substrate for many tyrosine phosphatases.When the essential cysteine residue in the tyrosinephosphatase signature motif was mutated to serine(C124S) the phosphatase activity was abolished, show-ing that PTEN was indeed a phosphatase.

PTEN IS FREQUENTLY MUTATEDIN SPORADIC CANCERS

Glioblastoma multiforme is the most aggressive formof glioma, and patients diagnosed with the diseaseusually survive less than 2 years. Loss of chromosome10q occurs in the vast majority of glioblastomas. Theinitial reports identifying PTEN as a candidate tumorsuppressor examined several cell lines and primarytumors for mutations in PTEN [51, 94]. These reportssuggested that glioblastoma cell lines and primary tu-mors had a high frequency of PTEN mutations accom-panied by LOH. Subsequently, other reports whichexamined glioblastomas for mutations in PTEN alsodetermined the mutation rate of PTEN to be high (20–44%) [7, 14, 54, 100, 108]. In lower grade glioma andglioneuronal tumors, however, PTEN mutations arerare [14]. One study examining the level of expressionof PTEN in glioblastomas versus lower grades of glio-mas by reverse transcription polymerase chain reac-tion found a significant difference between the twogroups [90]. Furthermore, immunostaining of glioblas-tomas revealed little or no PTEN expression in abouttwo-thirds of the tumors examined [90]. The reductionor loss of PTEN appears to be very important in theprogression of gliomas to glioblastoma multiforme.

Another tumor type that has a high frequency ofmutations in PTEN is endometrial carcinoma. Studies
examining endometrial carcinomas reported signifi-
cant LOH on chromosome 10 with two commonly de-leted regions, 10q22–24 and 10q25–26 [69, 79]. Thefrequency of PTEN mutations in endometrial carcino-mas of the endometrioid type is approximately 50%[87, 99]. In addition, endometrioid endometrial carci-nomas showed complete loss of PTEN protein expres-sion in 61% of cases and reduced expression in 97%[66]. Endometrial carcinomas of the less common se-rous type appear to be very rare [99]. Examination ofother gynecological malignancies revealed that endo-metrioid ovarian tumors had frequent mutations inPTEN (21%), while mutations of PTEN in serous andmucinous epithelial ovarian tumors were absent [74,99]. PTEN mutations in cervical cancer were also de-termined to be rare [95, 99].

LOH studies revealed that chromosome 10q is fre-quently lost in prostate cancer [26, 36, 43, 102]. Themost common region of loss (50% of tumors studied)spans region 10q23. One study examining clinicallylocalized prostate carcinoma determined inactivationof PTEN by homozygous deletion in 10–15% of thecases. In a study that examined both clinically local-ized and metastatic prostate carcinomas, researchersfound PTEN mutations in approximately 10% of thecases [6]. They also found that mutations occurredmore frequently in metastatic disease. Together, thesereports suggest that the inactivation of PTEN by mu-tation occurs predominately in advanced prostate can-cer. In addition to examining the mutational status ofPTEN in prostate cancer, one group analyzed the ex-pression of PTEN in xenografts derived from meta-static prostate cancer [112]. Expression of PTEN at themRNA and protein level was reduced in at least 50% ofthe cases. In a study of 109 cases of prostate cancer,PTEN expression was assessed by immunohistochem-istry and the loss of PTEN protein was correlated withpathological markers of poor prognosis [62]. Inactiva-tion or loss of expression of PTEN appears to be impor-tant in the development of advanced prostate cancer.

Another cancer to exhibit a high frequency of loss of10q is melanoma. Sporadic tumors have alterations inthe region of 10q22–10qter and LOH studies showfrequent early loss of 10q [31–33, 78, 86]. Although theloss or mutation of PTEN is high in malignant mela-noma cell lines (40%) [29], the frequency of PTENaberrations in primary tumors appears to be muchlower, with the majority of the mutations in patientswith metastatic disease [84, 103]. As with several othertumors, PTEN mutations appear to be associated withlate stage disease. Examination of 4 primary melano-mas and 30 metastases found little to no expression ofPTEN in 65% of the cases [119]. Reduction of expres-sion, therefore, may also play a role in the developmentof metastatic melanoma. One study examined the mu-tational status of both PTEN and NRAS in 53 cutane-
ous melanoma cell lines. They found 16 cell lines (30%)

31PTEN: LIFE AS A TUMOR SUPPRESSOR

with alterations in PTEN and 11 cell lines (21%) withactivating NRAS mutations with only 1 cell line havingmutations in both [104]. This suggests that PTEN andNRAS may function in the same pathway and thatperturbation of this pathway is necessary for the de-velopment of cutaneous melanomas.

Breast cancer has a high frequency of LOH at chro-mosome 10q. PTEN, however, is mutated in only asmall fraction of breast cancer cases (5%) [3, 15, 85,105]. Recent immunohistochemical analysis of 33 spo-radic primary breast carcinomas had either no or de-creased expression in 33% of these tumors. Loss ofPTEN may therefore play a more important role in thedevelopment of sporadic breast cancer than previouslythought [80]. PTEN mutations have also been found toa lesser extent in cancers of the bladder, lung, andlymphatic systems [5, 27, 38, 42, 116].

PTEN LOSS IS DETECTED IN TWO HAMARTOMASYNDROMES

Cowden disease is a rare, autosomal dominant familialcancer syndrome associated with a high risk for the de-velopment of breast cancer. The hallmark of this diseaseis the presence of hamartomas of the skin, breast, thy-roid, oral mucosa, and intestinal epithelium. In 1996, thegene responsible for CD was localized to chromosome10q22–23 [73]. When PTEN was discovered less than ayear later, researchers were eager to see if PTEN was thegene involved in this cancer syndrome. The first studyinvolving 5 families with CD identified mutations inPTEN in 4 of the families [52]. They found missense andnonsense mutations that disrupted the phosphatase do-main. A follow-up study involving 37 CD families foundPTEN mutations in 81% of the families. These includedmissense, nonsense, insertions, deletions, and splice-sitemutations [58]. Mutations in the phosphatase domaincomprised 43% of all mutations. The remaining muta-tions were scattered over the entire length of PTEN. In astudy of 10 families with CD in which 8 had germ-linemutations in PTEN, the importance of PTEN in the de-velopment of the characteristic hamartomas was investi-gated [59]. Twenty hamartomas were examined for LOHof markers flanking and within PTEN. LOH within theCD interval and including PTEN was identified in twofibroadenomas of the breast, a thyroid adenoma, and apulmonary hamartoma. The wild allele was lost in thesehamartomas. Reduced levels of PTEN RNA were foundin hamartomas from different tissues in a CD patient.From the above evidence, it appears that PTEN functionsas a tumor suppressor in CD.

Bannayan–Zonana syndrome (BZS) is another auto-somal dominant hamartomous disease. Some of thecharacteristic features of the disease are polyposis,macrocephaly, cutaneous lipomas, high birth weight,
and, in males, a speckled penis. Unlike CD, BZS is not
associated with an increased risk of malignancy. Theclinical overlap between CD and BZS led researchers toexamine BZS for possible germ-line mutations inPTEN. One study examined two families for germ-linemutations in PTEN and found mutations in both fam-ilies [60]. These mutations segregated with affectedfamily members but were absent in unaffected mem-bers and 100 control alleles. Interestingly, one muta-tion was identical to a mutation found in an unrelatedfamily with CD. Another study of seven BZS familiesdiscovered germ-line mutations in 57% of the cases[58]. They also observed a mutation that was commonto two unrelated CD families and one BZS family. CDand BZS may represent variable penetrance of thesame disorder. It is also possible to speculate that thereare modifying genetic or epigenetic factors that accountfor the varying manifestations of the two syndromes.Whatever the case, it is clear that PTEN plays a sig-nificant role in the development of both diseases andfulfills the criteria for a tumor suppressor.

PTEN INDUCES CELL CYCLE ARREST AND APOPTOSISIN SEVERAL SYSTEMS

To examine PTEN’s affect on cellular processes suchas cell cycle and apoptosis, several groups expressedexogenous PTEN in different cell lines. Expression ofPTEN in PTEN-null glioma cell lines caused growthsuppression [17, 48]. Apoptotic and cell cycle analysisshowed that PTEN-mediated growth suppression wasdue to G1 arrest. Phosphatase catalytically inactivemutants of PTEN did not suppress cellular growth orinduce cell cycle arrest. One group found that expres-sion of PTEN made a glioma cell line more susceptibleto anoikis, apoptosis initiated by disruption of cells’interaction with the extracellular matrix [10]. Expres-sion of PTEN in a variety of breast cancer cell linescaused growth suppression, but in this case the mech-anism was apoptosis [50]. Another study using a tet-racycline-inducible system to express PTEN in aPTEN-positive breast cancer cell line found that PTENinduced a G1 arrest followed by apoptosis [111]. Simi-lar to the studies in glioblastoma cell lines, a catalyti-cally inactive mutant of PTEN did not induce G1 arrestor apoptosis. Clearly, PTEN’s phosphatase domain isnecessary for its role in cell cycle arrest and apoptosis.

Mice which are homozygously and heterozygouslydeficient for PTEN were created to examine the role ofPTEN in development and tumor suppression. PTENwas shown to be essential in embryonic development.PTEN2/2 mice did not survive beyond day 9.5 of em-bryonic development and embryos appeared disorga-nized [12, 82, 93]. One group found abnormal pattern-ing in the PTEN2/2 embryo and overproliferation of thecephalic and caudal regions with no alterations in ap-
optosis [93]. PTEN heterozygous mice develop tumors

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in multiple tissues, such as endometrium, prostate,thyroid, and colon [12, 82, 93]. Some, but not all, of thetumors are associated with LOH. Another interestingphenotype of PTEN1/2 mice is the development of non-neoplastic hyperplasia of the lymph nodes. An inher-ited defect in apoptosis in B cells and macrophages wasobserved in the hyperplastic lymph nodes by Podsypa-nina et al. [82]. The lymphoadenopathy developed bysome PTEN1/2 mice was associated with autoimmuneglomerulopathy [11]. Fas-mediated apoptosis was im-paired in these mice and T lymphocytes showed re-duced activation-induced cell death and increasedproliferation upon activation. Immortalized mouse em-bryonic fibroblasts (PTEN1/2 and PTEN2/2) were gen-rated to analyze PTEN’s role in cellular processesuch as growth arrest and apoptosis [93]. PTEN2/2 cellsid not have an increased rate of proliferation com-ared to PTEN1/2 cells. However, PTEN2/2 cells exhib-

ited a decrease in sensitivity to a number of apoptoticstimuli, and reintroduction of PTEN into these cellsrestored the cells’ sensitivity to apoptosis. These mousestudies reveal the importance of PTEN in embryonicdevelopment and its role in the development of tumorspossibly due to a defect in apoptosis.

The Drosophila counterpart to mammalian PTENhas been recently cloned and characterized [18, 24, 35,91]. The amino-terminal half of the dPTEN protein ishighly conserved, sharing about 65% identity with thehuman PTEN in this region. As seen in the mouse,Drosophila dPTEN mutants die during early develop-ment. To study the function of PTEN, somaticdPTEN2/2 cells in both the eye and the wing werecreated [18, 24, 35]. Analysis in both the eye and thewing revealed that dPTEN2/2 clones were larger in sizethan their wild-type counterpart. This phenotype iscell autonomous since the mutant cells had no affect onwild-type neighbors. One group showed that overex-pression of dPTEN during eye development was shownto inhibit cell proliferation [35]. This overexpression ofPTEN causes a cell cycle arrest in the G2 or G2/Mphase in contrast to human tissue culture experiments,which show that PTEN expression arrests cells in theG1 phase. This difference could be due to stage ofdevelopment, cell type, and/or species-specific factors.Interestingly, overexpression of dPTEN in differentiat-ing cells during eye development induces apoptosis,showing that PTEN may act in a different fashiondepending on the context of its expression.

PTEN’s PHOSPHATASE DOMAIN IS ESSENTIAL FORITS ACTIVITY AS A TUMOR SUPPRESSOR

PTEN impacts embryonic development, tumor forma-tion, cell cycle, and apoptosis. Mammalian tissue culturestudies show that the phosphatase region is critical for
PTEN’s role in cell cycle arrest and apoptosis. Efforts
were made to determine the substrate of PTEN’s phos-phatase domain. Recombinant PTEN was shown to de-phosphorylate protein and peptide substrates phosphor-ylated on serine, threonine, and tyrosine residues, whichindicated that PTEN is a dual-specificity phosphatase[68]. PTEN also exhibited a high degree of substratespecificity for acidic substrates in vivo. The finding that aPTEN mutant, G129E, found in two CD kindreds, stillexhibited wild-type protein phosphatase activity towardpoly(Glu4Tyr1), however, suggested that the actual sub-strate in vivo had not yet been identified [68]. To addressthis question, Maehama et al. [56] transfected PTEN into93 cells and analyzed the changes in cellular phospho-ipids. Overexpression of PTEN reduced insulin-inducedtdIns-3,4,5-P3 levels without effecting PI3K activity.xpression of a catalytically inactive mutant caused Pt-Ins-3,4,5-P3 accumulation in the absence of insulintimulation. In vitro, purified PTEN catalyzed the de-hosphorylation of PtdIns-3,4,5-P3 on the D3 position ofhe inositol ring, whereas the mutant PTEN could not.his study revealed that PTEN is a lipid phosphatase initro and in vivo. Studies examining the PTEN-G129Eutant revealed the importance of the lipid phosphatase

ctivity of PTEN for its function as a tumor suppressor.his mutation in PTEN specifically ablates the lipidhosphatase activity of PTEN while retaining proteinhosphatase activity [67]. Expression of the PTEN-129E mutant in a PTEN-null glioblastoma cell line didot induce the growth suppression seen when expressingild-type PTEN, proving that the lipid phosphatase ac-

ivity is necessary for this effect [17].

PTEN REGULATES THE PI3K PATHWAY

The formation of PtdIns-3,4,5-P3 by PI3K is essen-tial for the activation of Akt. Akt is a serine/threoninekinase that is activated by a variety of growth factors,including insulin and IGF-1 [9]. PI3K activationthrough addition of growth factors results in an in-crease in PtdIns-3,4,5-P3. This leads to the transloca-tion of Akt to the membrane and a conformationalchange that allows PDK1 and PDK1/PRK-2 to phos-phorylate Ser-473 and Thr-308, activating Akt [9]. IfPTEN desphosphorylates PtdIns-3,4,5-P3 in vivo, thenPTEN should act as a negative regulator of the PI3K/Akt pathway (Fig. 1). Genetic studies involving theCaenorhabditis elegans model system have been in-strumental in the formation of our current understand-ing of the PI3K/Akt pathway and its role in insulinsignaling. DAF-2 is the C. elegans homolog of the mam-malian insulin/insulin-growth factor receptor [39].DAF-2 mutant animals constitutively form dauers, adiapausing larval stage in which their metabolism isshifted toward energy storage [25, 39, 45, 107]. DAF-2has been shown to reside upstream of AGE-1, a ho-
molog of the mammalian p110 catalytic subunit of

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PI3K, since DAF-2 and AGE-1 mutant animals displaysimilar phenotypes which include increased life span[65]. AKT-1 and AKT-2, two C. elegans homologs ofAkt, act downstream of AGE-1 in transducing signalsfrom DAF-2 [77]. Recently, researchers studying insu-lin signaling in C. elegans discovered DAF-18, a PTEN

omolog [21, 76, 89]. Studies of DAF-18 have estab-ished that it functions as a regulator of insulin signal-ng. Animals with mutations inactivating DAF-18 failo form dauers under appropriate conditions and mu-ations of DAF-18 suppress the AGE-1 constitutiveauer phenotype and suppress a similar phenotype inAF-2 mutants [21, 76]. Inactivation of DAF-18 usingNA interference also suppresses the dauer phenotypef DAF-2 and AGE-1 while introduction of a wild-typeAF-18 transgene rescues the dauer defect [76, 89].

nhibition of AKT-1 and AKT-2 by RNA interferenceauses dauer formation in both wild-type and DAF-18utants showing that DAF-18 is upstream of AKT-1

nd AKT-2 [76].Studies with Drosophila also place dPTEN in the

nsulin signaling pathway. Homozygous dPTEN mu-ant cells have growth phenotypes similar to those ofells expressing a constitutively active form of Dp110,PI3K homolog [47]. Overexpression of dPTEN com-

letely suppresses the growth-promoting activity ofverexpressed Dp110 [24]. Conversely, the small eyehenotype of dPTEN overexpression is suppressed byp110 and enhanced by a dominant negative Dp110

35]. As in mammals, dPTEN appears to act as anntagonist to the PI3K homolog. Further confirmationhat dPTEN is in the insulin signaling pathway inrosophila is seen in studies involving chico, the ho-olog of IRS-1-4. Eye clones mutant for chico usually

isplay a reduced growth phenotype, but in cells mu-ant for both chico and dPTEN, this phenotype isasked by the overgrowth phenotype associated with

oss of dPTEN function [24]. Additionally, removal ofne copy of chico enhances the small eye phenotype ofPTEN overexpression [35]. This strongly suggestshat dPTEN is downstream of chico in a negative reg-latory role in insulin signaling. Finally, one studyeported that Dakt1, an Akt homolog, could suppresshe dPTEN overexpression phenotype [18]. Studiesith C. elegans and Drosophila revealed that PTEN isvolutionarily conserved and have placed it in the in-ulin signaling pathway. Since PTEN is highly con-erved, these model systems provide researchers withn excellent way in which to study the function ofTEN.The effects of PTEN on Akt in mammals has also

een studied. Expression of PTEN in the cell leads tohe downregulation of Akt activity as evidenced byecreased levels of phospho-Akt [10, 48, 50, 83, 111,14]. Additionally, constitutively active Akt, but not
ild-type Akt, can rescue cells from PTEN-mediated
G1 arrest and apoptosis [50, 83, 114]. PI3K, the up-stream activator of Akt, cannot rescue cells from apo-ptosis, showing that PTEN acts below PI3K but aboveAkt in this signaling pathway. In agreement with thesefindings, tumor cell lines that are PTEN deficientshowed elevated levels of phospho-Akt compared toPTEN-positive cell lines [8, 114]. Studies with PTEN2/2

mouse fibroblasts have yielded similar results. Thelevels of Akt were elevated in PTEN2/2 cells comparedto PTEN1/2 cells. Pretreatment of serum-starved cellswith PI3K inhibitors eliminated basal as well asPDGF-dependent increase in Akt phosphorylation inboth PTEN2/2 and PTEN1/2 cells. Also, the expressionof PTEN in PTEN2/2 cells reduced Akt phosphoryla-tion. Finally, PTEN2/2 cells have elevated levels ofPtdIns-3,4,5-P3 compared to PTEN1/2 cells.

Recently, studies have focused on the role of PTENas a regulator of the insulin signaling pathway inmammals. Expression of PTEN in 3T3-L1 adipocytesinhibited insulin-induced 2-deoxyglucose, GLUT4translocation, and membrane ruffling [72]. Microinjec-tion of an anti-PTEN antibody led to increased PtdIns-3,4,5-P3 levels and subsequent upregulation of GLUT4translocation. Both Akt and p70S6 kinase were signif-icantly inhibited by PTEN overexpression.

Expression of PTEN in a PTEN-null breast cancercell line has been shown to cause upregulation of insu-lin receptor substrate-2 (IRS-2), at both the RNA andthe protein levels (L.S. and R.P., submitted for publi-cation). IRS-2 is a docking protein which is recruited byreceptor tyrosine kinases such as the insulin receptor.In turn, IRS-2 recruits PI3K to allow for signal trans-duction through this pathway. The IRS-2 produced bythe expression of PTEN is tyrosine phosphorylated andassociated with PI3K. These data suggest that PTENcan elicit a feedback upregulation of PI3K signaling.

The PI3K/Akt pathway has an established role inregulating translation and apoptosis within the cell.Overexpression of a constitutively active, membrane-targeted form of Akt induces phosphorylation of 4E-BP1, a repressor of mRNA translation, inactivating theprotein [23, 41]. When PTEN is transfected into cells,levels of active, phosphorylated Akt are diminishedand 4E-BP1 phosphorylation is reduced [114]. Akt hasalso been shown to phosphorylate Bad, a Bcl-2 familymember. Dephosphorylated Bad inactivates prosur-vival factors such as Bcl-XL, precipitating apoptosis.Expression of PTEN into PTEN-null cells resulted indecreased levels of phosphorylated Akt and phosphor-ylated BAD [10]. Reciprocally, PTEN-null ES cellsshowed increased levels of phosphorylated Akt andphosphorylated BAD [96]. In C. elegans, DAF-16 hasbeen shown to be antagonized by the insulin signalingpathway and to encode a Forkhead transcription fac-tor, proving that insulin signaling ultimately alters
transcription [13, 25, 37, 45, 53, 75, 77, 107]. The

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mammalian counterparts of DAF-16, AFX, FKHR, andFKHRL1, have recently been shown to be involved intranscriptional control and to be negatively regulatedthrough phosphorylation by Akt [4, 30, 44, 70, 98].

FIG. 1. Schematic representation of PTEN’s role as a regulatPtdIns-3,4,5-P3 (PIP3,4,5), lowering its levels in the cell. This actionthe most closely scrutinized. Akt, a serine/threonine kinase, is invol

FIG. 2. Protein domains of PTEN. PTEN is a 403-amino-acid p-terminal region with the phosphatase motif (HCXXGXXR; residuC2 domain (residues 186–351), which allows for the binding of PTEembrane. PEST sequences (degradation motifs) are located betwe

tripes). The tail region contains CK2 phosphorylation sites importahich allows PTEN to bind MAGI proteins, which may enhance th

omplexes at the membrane.

Overexpression of Forkhead transcription factors in-duces cell cycle arrest and apoptosis [4, 98]. AFX hasbeen shown to mediate cell cycle arrest by transcrip-tionally activating p27kip1 [63] and similarly FKHRL1

f the PI3K pathway. PTEN dephosphorylates the D3 position ofacts many different cellular processes but its effect on Akt has beenin the regulation of transcription, translation, and apoptosis.

ein which contains a phosphatase domain (residues 1–185) in the23–130) essential for its tumor-suppressor activity. PTEN containso phospholipids, perhaps for the effective positioning of PTEN at theresidues 350–375 and 379–396 within the tail region (indicated byfor the stability and activity of PTEN. There is also a PDZ domainfficiency of PTEN signaling through the formation of PTEN/MAGI

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induces transcriptional upregulation of Fas ligand [4].Therefore, PTEN may mediate cell cycle arrest andapoptosis through these transcription factors. Indeed,a recent report shows that expression of PTEN inPTEN-deficient cells induces the nuclear localizationand transcriptional activity of FKHR [71]. The resultsof these studies suggest that PTEN can control trans-lation thereby regulating the cell’s entry into G1 andits progression through the cell cycle. In addition, thesestudies reveal the mechanisms by which PTEN mayinduce apoptosis.

PTEN’s emerging role as an antagonist of the PI3Kpathway led to studies examining PTEN’s role in an-giogenesis since hypoxia, insulin, and IGF-1 induceangiogenic gene expression which may be regulatedthrough the PI3K/Akt pathway [61, 110, 117]. Reintro-duction of wild-type PTEN into PTEN-null glioblas-toma cell lines substantially reduced hypoxia andIGF-1 induction of HIF-1-regulated genes such as vas-cular endothelial growth factor (VEGF) [120]. PTENsuppresses the stabilization of hypoxia-mediatedHIF-1, which when stabilized through the PI3K/Aktpathway upregulates VEGF expression. Similar re-sults were seen in epidermal growth factor (EGF)-stim-ulated prostate cancer cell lines [118]. Stimulation ofcells with EGF leads to increased levels of HIF-1,HIF-1 transcriptional activity, and VEGF protein ex-pression, which is blocked by expression of PTEN. Thissuggests a possible role for PTEN in angiogenesis.

Although PTEN’s ability to inhibit the PI3K path-way has been examined mainly in glial and epithelialcell lines, it is possible that PTEN may play a similarrole in other cell types. When PTEN is exogenouslyexpressed in Jurkat T cells, it induces apoptosis [109].Apoptosis could be prevented by coexpression of a con-stitutively active, membrane-bound form of Akt. PTENalso decreased T cell receptor (TCR)-induced activationof mitogen-activated protein kinase, extracellular sig-nal-related kinase (ERK2), which is dependent uponPI3K activity. A recent report found that Jurkat T cellsexhibit constitutive phosphorylation of Akt, indicatinghigh basal levels of PtdIns-3,4,5-P3 [92]. In addition,there was constitutive membrane association of induc-ible T cell kinase (Itk), a pleckstrin homology-contain-ing tyrosine kinase downstream of PI3K. This kinasefacilitates TCR-dependent calcium influxes and in-creases in extracellularly regulated kinase activity.Expression of PTEN or treatment of the cells withPI3K inhibitors blocked constitutive phophorylation ofAkt and redistributed Itk to the cytosol. The PTEN-deficient Jurkat cells were hyperresponsive to TCRstimulation as measured by Itk activity, tyrosine phos-phorylation of phospholipase C-1, and activation ofERK. These studies provide evidence that PTEN mayhave an important role in TCR signaling in T cells. The
above-mentioned studies clearly provide evidence that
PTEN acts antagonistically to PI3K by dephosphoryla-tion of PtdIns-3,4,5-P3 and by doing so may affect avariety of cellular processes.

DOMAIN STRUCTURE OF PTEN

The initial study of PTEN showed that it is a 403-amino-acid protein that contains an amino-terminalregion with homology to auxilin and tensin and a pro-tein phosphatase domain [51]. Solving the crystalstructure of PTEN revealed a phosphatase domain thatis similar to protein phosphatases but has a enlargedactive site necessary for the accommodation of a phos-phoinositide substrate, which accounts for its ability todephosphorylate lipid substrates [46]. PTEN’s phos-phatase domain has been the focus of numerous stud-ies and its function is essential in PTEN’s tumor-sup-pressor function.

More recently, other domains of PTEN have beendiscovered and examined to determine their function(Fig. 2). The crystal structure of PTEN revealed theexistence a C2 domain that has affinity for phospho-lipid membranes in vitro [46]. Mutation of the solvent-exposed residues in the CBR3 and C2 elements of theC2 domain has reduced affinity for membranes in vitrocompared to wild-type PTEN. These mutations re-duced the growth suppression activity of PTEN in aglioma cell line. Cells expressing these mutants alsoproliferated better than those expressing wild-typePTEN and demonstrated anchorage-independentgrowth. In addition, the structure revealed that thephosphatase domain and C2 domains associate acrossan extensive surface, which suggests that the C2 do-main may have a role in positioning the catalytic do-main on the membrane. Georgescu et al. in a previousstudy had found that the expression of various C-ter-minal mutants in glioma cell lines allowed for anchor-age-independent growth that is usually suppressed bywild-type PTEN [19]. Various mutations created werein predicted b-strands (now known to be in the C2domain) and the level of phosphatase activity of thesemutants depended on the degree to which the mutationdisrupted these structural elements. The mutantswere unstable and subject to rapid degradation.

PTEN also contains two PEST sequences and a PDZmotif in the carboxy-terminal region and these ele-ments were found to be dispensable for tumor-suppres-sor function [19]. The function of PEST sequences is totarget proteins with short intracellular half-life forprotein degradation. Deletion of these sequences inPTEN, however, led to decreased protein expression,perhaps by impaired protein folding. From the resultsof these studies, the C-terminal region appears to beimportant in PTEN stability and enzymatic activity. Inanother study focusing on the C-terminal region,
Vazquez et al. [106] examined PTEN in which the


50-amino-acid tail was removed and found that it wasnecessary for maintaining protein stability. Surpris-ingly, the protein had increased activity as measuredby the ability of the mutant to induce G1 arrest andFKHR transcriptional activity. The tail was also shownto be phosphorylated on serine 370 and one or moresites of a serine/threonine cluster (Ser380, Thr382,Thr383, and Ser385). Mutation of Ser380, Thr382, andThr383 each reduced both the steady-state protein lev-els and the protein half-life. These mutants were moreactive in inducing G1 arrest and FKHR transcriptionalactivation. Phosphorylation of the tail, therefore, maymediate the regulation of PTEN function.

To determine what kinase phosphorylates the tail ofPTEN, researchers examined the amino acid sequenceof the tail region and found that CK2 phosphorylationsites were located within a C-terminal cluster of serine/threonine residues (Ser370, Ser380, Thr383, andSer385) [101]. CK2 is a serine/threonine kinase that isubiquitously expressed and phosphorylates a variety ofsubstrates involved in cell cycle and cell growth [1, 64,81]. PTEN was shown to be constitutively phosphory-lated by CK2 in vivo and PTEN phosphorylation-defec-tive mutants showed decreased stability in comparisonto wild-type PTEN. In addition, these mutants showedaccelerated proteasome-mediated degradation. Treat-ment with a CK2 inhibitor also resulted in a reductionin PTEN protein levels, but only in the absence of aproteasome inhibitor. These data suggest that levels ofPTEN in the cell could be regulated by its phosphory-lation status.

Since PTEN contains a PDZ domain, this raised thepossibility that PTEN may interact with other proteinsthat may be essential in PTEN’s function. PDZ do-mains are protein–protein interaction domains thatbind to consensus motifs (S/TXV) in the C-terminus ofpartner proteins although other more amino-terminalresidues may be involved in the specificity of PDZdomain binding. Using yeast two-hybrid systems,PTEN was found to associate with members of themembrane-associated guanylate kinase family withmultiple PDZ domains called MAGI for (membrane-associated guanylate kinase inverted) [113, 115].There are three distinct MAGI proteins (MAGI1, 2, and3) with a high degree of conservation of the functionaldomains, including the PTEN-binding PDZ domains.These proteins localize to epithelial cell tight junctions.PTEN was found to associate with both MAGI2 andMAGI3 in two separate studies. The ability of lowlevels of PTEN to inhibit Akt activity was enhanced bythe expression of either MAGI2 or MAGI3 compared tolow levels of PTEN alone. Removal of the PDZ domainfrom PTEN greatly reduced its ability to inhibit Aktactivity, which points to the potential importance ofthe interaction of PTEN and MAGI proteins in facili-
tating PTEN’s function as a phosphatase. A catalyti-
cally inactive mutant of PTEN (C124S), which containsamino acids 1–377, was subject to rapid degradation,and the addition of the PDZ domain back to this pro-tein conferred a greater degree of stability to the pro-tein [113]. This suggests that the interaction of PTENand MAGI may enhance the stability of PTEN thoughthis interaction is probably not the only stabilizingfactor needed by PTEN. Given these data, it is possiblethat the interaction between PTEN and MAGI proteinsimproves the efficiency of PTEN signaling by the for-mation of a complex at the cell membrane.

PTEN MAY BE MORE THAN A LIPID PHOSPHATASE

Although PTEN clearly acts as a lipid phosphatase,it has been shown to dephosphorylate protein sub-strates as well and may have roles in the cell otherthan antagonizing PI3K. Tamura et al. [97] overex-pressed PTEN in both fibroblasts and glioblastoma celllines and observed that PTEN inhibited cell migration.Integrin-mediated cell spreading and the formation offocal adhesions were downregulated by wild-type butnot by PTEN with an inactive phosphatase domain. Inaddition, PTEN interacted with focal adhesion kinase(FAK) and reduced its tyrosine phosphorylation. A mu-tant of PTEN, G129E, which had lost its lipid phospha-tase activity but retained its tyrosine phosphataseactivity, still inhibited cell spreading and dephospho-rylated FAK. The tyrosine phosphatase activity ofPTEN appears to be essential in regulating cell inter-actions with the cellular matrix. Further examinationof the role of PTEN in focal contact formation and cellspreading implicated PTEN in the dephosphorylationof Shc [28]. The dephosphorylation of Shc by PTENinhibited the recruitment of Grb2, which leads todownregulation of the MAP kinase pathway. The abil-ity of PTEN to dephosphorylate FAK is not universal,however. There appears to be no difference in the phos-phorylation status of FAK in PTEN2/2 versus PTEN1/1

ES cells [96]. Another group studying the role of PTENin invasion found that introduction of wild-type PTENor phosphatase-deficient (C124S) PTEN both inhibitedinvasion in glioma cell lines [57]. Reduction of FAKphosphorylation was not observed in conjunction withinhibition of invasion. This raises the possibility thatdomains other than the phosphatase domain may playa role in inhibiting invasion. Regarding PTEN’s abilityto regulate the MAP kinase pathway, it is possible thatPTEN can inhibit it without direct dephosphorylationof Shc. Expression of PTEN can inhibit the movementof Gab1 to the plasma membrane by dephosphorylationof PtdIns-3,4,5-P3 since the pleckstrin homology do-main of Gab1 binds this phospholipid in its transloca-tion to the membrane [88]. This inhibition would effec-tively block signaling through the MAP kinase
pathway. The Drosophila model also gives us a tanta-

hawPafspiltsictlc

b[


lizing clue that the role of PTEN may be broader thanjust the dephosphorylation of PtdIns-3,4,5-P3. Re-searchers have noted that the bristle, hair, and rhab-domere phenotypes observed in dPTEN mutants havenot been reported in Dp110 or insulin-signaling mu-tants, which raises the possibility that these effects arenot caused simply by an increase in PtdIns-3,4,5-P3levels [24]. In dPTEN mutants, the subcellular regula-tion of the assembly of actin microfilaments appears tobe abnormal, which may account for the structuraldefects observed. Goberdhan et al. [24] postulated thatthe loss of dPTEN function may alter communicationbetween the peripheral actin cytoskeleton, the plasmamembrane, and the extracellular matrix. The study ofhow PTEN may function in capacities other than thatof a lipid phosphatase still remains in its infancy.

FUTURE DIRECTIONS

Since the discovery of PTEN as a putative tumorsuppressor in 1997, its importance as a tumor suppres-sor has been validated by its mutation and/or loss ofexpression in a variety of sporadic cancers and itsassociation with Cowden disease, an autosomal domi-nant cancer syndrome. Reintroduction of PTEN intocells causes G1 cell cycle arrest and/or apoptosis de-pending on the cell type, revealing how PTEN mayfunction as a tumor suppressor. Studies examiningPTEN’s role as a lipid phosphatase have shown that itdephosphorylates the D3 position of PtdIns-3,4,5-P3,acting in opposition to PI3K, which allows it to affect anumber of cellular processes. Through the use of mousemodels, PTEN has been shown to be important inembryonic development, tumor formation, and apopto-sis. PTEN homologs have been identified in both C.elegans and Drosophila. Studies with these models

ave placed PTEN in the insulin signaling pathway asnegative regulator of a PI3K homolog in concordanceith studies in mammals. Many vital questions aboutTEN’s role in cellular processes still remain to benswered. Studies have shown that PTEN is essentialor development [12, 24, 35, 82, 93], but why? There isome evidence to support the idea that it regulates cellroliferation, possibly through affecting differentiationn the murine model. During human development highevels of PTEN expression were observed in the skin,hyroid, central nervous system, autonomic nervousystem, and upper gastrointestinal tract [22]. PTEN’snvolvement in the development of these tissues isurrently unknown. Regulation of PTEN levelshrough transcriptional, translational, and posttrans-ational mechanisms is another area that remains un-harted.

Phosphorylation of residues in the PTEN tail haseen shown to affect protein stability and activity
106]. A recent report identified Casein Kinase II as the
kinase that constitutively phosphorylates the tail re-gion of PTEN. This phosphorylation confers stability toPTEN and phosphorylation-defective mutants are sub-ject to rapid turnover via the proteasome. Identifica-tion of a phosphatase that can dephosphorylate PTENstill remains to be discovered. The nature of this phos-phatase will provide us with clues as to when PTENcan be dephosphorylated and under what conditionsthis can take place. It is possible that the selectivedephosphorylation of PTEN may be crucial in regulat-ing its specific activity under particular circumstancesin the cell. Finally, how PTEN regulates the expressionof other genes is an area of burgeoning interest. Oligo-nucleotide chip technology has revealed that IRS-2, acomponent of the insulin signaling pathway, is upregu-lated by PTEN. PTEN affects transcription, transla-tion, and apoptosis. Which of these processes is impor-tant for tumor suppression? The continuinginvestigation into the many aspects of PTEN’s functionwill give us a better understanding of how the loss ofPTEN affects tumor development and progression.

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eceived December 4, 2000ublished online February 9, 2001

PTEN: Life as a Tumor Suppressor

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