Proc. Natl. Acad. Sci. USA
`Vol. 96, pp. 4240–4245, April 1999
`Review
`
`Review
`
`New insights into tumor suppression: PTEN suppresses tumor
`formation by restraining the phosphoinositide 3-kinaseyAKT pathway
`LEWIS C. CANTLEY*†‡ AND BENJAMIN G. NEEL§
`*Department of Cell Biology, Harvard Medical School, Boston, MA 02115; †Division of Signal Transduction, Department of Medicine; Beth Israel Deaconess
`Medical Center, Boston, MA 02215; and §Cancer Biology Program, Division of Hematology-Oncology, Department of Medicine, Beth Israel Deaconess Medical
`Center, Boston, MA 02215
`
`The most recently discovered PTEN tumor
`ABSTRACT
`suppressor gene has been found to be defective in a large
`number of human cancers. In addition, germ-line mutations
`in PTEN result in the dominantly inherited disease Cowden
`syndrome, which is characterized by multiple hamartomas
`and a high proclivity for developing cancer. A series of
`publications over the past year now suggest a mechanism by
`which PTEN loss of function results in tumors. PTEN appears
`to negatively control the phosphoinositide 3-kinase signaling
`pathway for regulation of cell growth and survival by dephos-
`phorylating the 3 position of phosphoinositides.
`
`Cancer cells escape normal growth control mechanisms as a
`consequence of activating (i.e., gain-of-function) mutations
`andyor increased expression of one or more cellular protoon-
`cogenes andyor inactivating (i.e., loss-of function) mutations
`andyor decreased expression of one or more tumor suppressor
`genes. Most oncogene and tumor suppressor gene products are
`components of signal transduction pathways that control cell
`cycle entry or exit, promote differentiation, sense DNA dam-
`age and initiate repair mechanisms, andyor regulate cell death
`programs. Several oncogenes and tumor suppressor genes
`belong to the same signaling pathway. Perhaps the best-
`characterized pathway includes D-type cyclinycdk complexes,
`which can be oncogenes, and two tumor suppressor genes, the
`p16 cyclinycdk inhibitor and the retinoblastoma gene product
`(reviewed in ref. 1). Nearly all tumors have mutations in
`multiple oncogenes and tumor suppressor genes, indicating
`that cells employ multiple parallel mechanisms to regulate cell
`growth, differentiation, DNA damage control, and death. A
`rational approach to understanding cancer pathogenesis and
`developing novel, mechanism-based therapies requires iden-
`tifying the components of these signaling pathways and deter-
`mining how mutations in oncogenes and tumor suppressor
`genes disrupt them. Two papers appearing recently in the
`Proceedings, as well as papers appearing elsewhere, elucidate
`a novel mechanism of cell growth control mediated by the
`PTENyMMAC tumor suppressor gene and involving the cel-
`lular counterparts of at least two oncogenes [the genes en-
`coding phosphoinositide 3-kinase (PI3K) and the protein-Ser/
`Thr kinase (AKT)].
`PTEN (phosphatase and tensin homolog deleted on chro-
`mosome ten)/MMAC (mutated in multiple advanced cancers)
`was identified virtually simultaneously by two groups (2, 3) as
`a candidate tumor suppressor gene located at 10q23; another
`group (4) identified the same gene in a search for new
`dual-specificity phosphatases (see below) and named it TEP-1
`(TGF-b-regulated and epithelial cell-enriched phosphatase).
`Deletions in 10q22–25 occur in multiple tumor types, most
`prominently advanced glial tumors (glioblastoma multiformey
`anaplastic astrocytoma), but also prostate, endometrial, renal
`
`PNAS is available online at www.pnas.org.
`
`and small cell lung carcinoma, melanoma, and meningioma
`(see references in refs. 2 and 3). Early studies indicated that
`10q abnormalities are more common in advanced tumors
`(hence the appellation MMAC). The initial cloning studies
`reported PTENyMMACyTEP-1 (hereafter, PTEN) mutations
`in a large fraction of glioblastoma multiforme cell
`lines,
`xenografts, and primary tumors, as well as in smaller samples
`of breast and prostate cancers (2, 3). Subsequent analyses
`confirmed that homozygotic inactivation of PTEN occurs in a
`large fraction of glioblastomas (at least 30% of primary tumors
`and 50–60% of cell lines) but not in lower-grade (i.e., less
`advanced) glial tumors (5–8). PTEN mutations also are ex-
`tremely common in melanoma cell lines (.50%) (9), advanced
`prostate cancers (10, 11), and endometrial carcinomas (30–
`50%) (12, 13). Although PTEN mutations are found predom-
`inantly in advanced glial and prostate tumors, mutations occur
`with equal frequency at all stages of endometrial cancer (12),
`suggesting that PTEN activation is an early event in endome-
`trial carcinogenesis. A significant percentage (’10%) of breast
`cancer cell lines have inactivated PTEN (2, 3, 14). PTEN
`mutations are rare in sporadic breast tumors, independent of
`severity (15). Also, whereas germ-line PTEN mutations lead to
`increased breast cancer incidence (see below), PTEN muta-
`tions are not a frequent cause of familial breast cancer (16).
`Occasional PTEN mutations are reported in head and neck
`(17) and thyroid (18) cancers, but not in other tumors asso-
`ciated with 10q abnormalities, including meningioma (8) and
`lung cancer (17); these studies raise the possibility of another
`tumor suppressor gene distal to PTEN on chromosome 10.
`Overall, PTEN is one of the most common targets of mutation
`in human cancer, with a mutation frequency approaching that
`of p53.
`Germ-line mutations in PTEN cause three rare autosomal
`dominant inherited cancer syndromes with overlapping clinical
`features: Cowden disease (19–21), Lhermitte–Duclos disease
`(19), and Bannayan–Zonana syndrome (21, 22). These syn-
`dromes are notable for hamartomas, benign tumors in which
`differentiation is normal, but cells are highly disorganized.
`Cowden disease is characterized by hamartomas in multiple
`sites, including the skin, thyroid, breast, oral mucosa, and
`intestine. In addition, about a third of patients will have
`macrocephaly. Affected females have a 30–50% incidence of
`breast cancer, and Cowden disease patients have increased risk
`of thyroid carcinoma (’10% incidence) and meningiomas (see
`references in ref. 19). Lhermitte–Duclos patients have multi-
`ple hamartomas, together with macrocephaly, ataxia, and
`seizures, caused by cerebellar glial tumors. Besides their
`
`Abbreviations: PI3K, phosphoinositide 3-kinase; PTP, protein-
`tyrosine phosphatase; PtdIns, phosphatidylinositol; PH domain, pleck-
`strin homology domain; PDGF, platelet-derived growth factor; GSK3,
`glycogen synthase kinase 3.
`‡To whom reprint requests should be addressed at: Harvard Institutes
`of Medicine, 77 Avenue Louis Pasteur, Room 1024, Boston, MA
`02115. e-mail: cantley@helix.mgh.harvard.edu.
`
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`hamartomas, Bannayan–Zonana patients exhibit macroceph-
`aly, retardation, and unusual pigmentation of the penis (see
`references in ref. 21). Hamartomas from Cowden disease
`patients exhibit loss of heterozygosity around the PTEN locus,
`indicating that homozygotic loss of PTEN function probably is
`required for hamartoma formation (19, 23). Whether the type
`of mutation in PTEN contributes to the distinct features of
`these three hamartomatous syndromes remains unclear, but
`other (i.e., modifying) loci probably play the primary role in
`determining the spectrum of abnormalities evoked by a given
`mutation. Indeed, recent analyses of mutant mice (see below)
`suggest that genetic background can significantly affect the
`PTEN-deficient phenotype. However, in Cowden disease pa-
`tients, the type of PTEN mutation may affect the number of
`affected sites andyor the presence of breast disease (21).
`These genetic data strongly suggest that PTEN function is
`required for normal development and that loss of PTEN
`function contributes to carcinogenesis. Gene transfer and
`knockout studies have confirmed these ideas. Restoration of
`PTEN expression in PTEN2 mutant glioblastoma multiforme
`cells causes growth suppression (24, 25), whereas increasing
`expression in glioblastoma multiforme lines that retain normal
`PTEN expression does not inhibit cell growth (24). Subsequent
`studies showed that PTEN reconstitution inhibits the growth
`of PTEN2 prostate (26), melanoma (27), and breast cancer
`(28) cell lines. Interestingly, PTEN appears to suppress cell
`growth by distinct mechanisms in different types of tumors,
`producing G1 cell cycle arrest in glioblastoma cells (29), but
`inducing apoptosis in carcinomas (26, 28).
`Three different PTEN mutations have been introduced into
`mice (30–32). Homozygotic mutants from all three lines
`exhibit early embryonic lethality, consistent with an essential
`role for PTEN in normal development. Heterozygotes show
`increased tumor incidence, consistent with its identification as
`a tumor suppressor gene. Although the different mutant mice
`share these general features, some details of the phenotype
`differ substantially. The timing of embryonic death ranges
`from day 7.5 or earlier (30, 32) to day 9.5 (31). Hyperprolif-
`erative lesions of the intestine are common to all three lines,
`but the spectrum of other hyperproliferative and neoplastic
`disorders differs dramatically. Chimeric and heterozygotic
`PTEN mutant mice generated by Di Cristofano et al. (30)
`showed a high incidence of tumor formation, with colonic,
`testicular, thyroid, germ-cell, and hematopoietic (acute my-
`eloid leukemia) neoplasms observed. Suzuki et al. (31) and
`Podsypanina et al. (32) observed a high incidence of T cell
`lymphomasyleukemia; moreover, T cell lymphomagenesis in
`PTEN1/2 mice is markedly potentiated by irradiation (31).
`Other tumors, including prostate, early endometrial, thyroid,
`liver, and germ-cell neoplasms, were also observed by these
`workers. Finally, Podsypanina et al. (32) noted a high incidence
`of lymph node hyperplasia in PTEN heterozygotes, with
`consequent disruption of lymphoid architecture.
`Notably, none of the groups reported brain tumors, despite
`the frequent mutation of PTEN in glioblastoma. Conceivably,
`the role of PTEN in glioblastomagenesis may differ in mice and
`humans. A more likely explanation, consistent with the occur-
`rence of PTEN mutations late in glioblastomagenesis, is that
`generation of a glioblastoma requires more mutations than the
`other tumors found in PTEN heterozygotes, leaving insuffi-
`cient time for brain tumors to develop before these mice die
`from other diseases. Aside from the high incidence of intestinal
`hyperproliferation and polyposis, and the report of acanthotic
`skin lesions in one line of mice (but not the others) (30), the
`other features of Cowden disease and related syndromes,
`particularly hamartomas, are absent in PTEN1/2 mice. Further
`study will be required to determine the reasons for the
`differences in phenotype between the different strains of
`PTEN mutant mice and the discrepancy between the effects of
`PTEN deficiency in mice and humans.
`
`The PTEN cDNA sequence strongly suggested that is was a
`member of the protein-tyrosine phosphatase (PTP) gene
`superfamily (2, 3). PTPs consist of conserved catalytic do-
`mains, flanked by noncatalytic, regulatory sequences (re-
`viewed in ref. 33). All PTP catalytic domains contain the
`canonical sequence HCXXGXXRSyT, known as the PTP
`‘‘signature motif’’ (reviewed in ref. 33); the presence of this
`motif within any protein makes it a virtual certainty that it has
`PTP activity. Structural and enzymatic analyses have shown
`that signature motif residues constitute the catalytic center of
`each enzyme (reviewed in ref. 33). PTP superfamily members
`can be further subdivided into ‘‘classic’’ PTP and dual-
`specificity phosphatase families. The former are exquisitely
`selective for phosphotyrosine residues in vitro and in vivo.
`Dual-specificity phosphatases, as their name implies, typically
`dephosphorylate phosphotyrosine, phosphoserine, andyor
`phosphothreonine in vitro. Most known dual-specificity phos-
`phatases (e.g., MAP kinase phosphatases, cdc25 family mem-
`bers) dephosphorylate phosphothreonine and phosphoty-
`rosine in specific sequence contexts in vivo, although others
`may target specific tyrosine- or serineythreonine-phosphory-
`lated cellular proteins (reviewed in refs. 33–35).
`The PTEN sequence suggested that it is a dual-specificity
`phosphatase. Moreover, sporadic and germ-line mutations in
`PTEN cluster within the presumptive catalytic domain, with
`many mutations altering residues (e.g., the essential cysteine or
`arginine residues within the signature motif) required for
`enzymatic activity (see references cited above; reviewed in ref.
`36). These results strongly suggested that PTEN catalytic
`activity was vital for its biological function, an idea strength-
`ened by the inability of signature motif cysteine mutants of
`PTEN to restore growth suppression to PTEN-deficient tumor
`cell lines (24, 28, 29). Besides its catalytic domain, PTEN has
`a potential binding site for PDZ domain-containing proteins at
`its C terminus; PDZ proteins have been shown to direct the
`assembly of multiprotein complexes, often at membraney
`cytoskeletal
`interfaces such as synapses (37). PTEN also
`contains a stretch of sequence that overlaps the catalytic
`domain and is similar to a domain within the cytoskeletal
`proteins tensin and auxilin (hence the name PTEN; see above).
`This led to the suggestion that PTEN might participate in
`regulating cytoskeletal phosphorylation events. The biological
`significance of this tensin similarity is unclear, however, be-
`cause the catalytic domains of many PTPs contain a similar
`region (38). Most likely, this represents convergent evolution
`of tensin-like proteins and PTPs, rather than implying a
`specific role for PTEN (and other PTPs with tensin similarity)
`in cytoskeletal regulation. Indeed, in view of the recent finding
`that PTEN actually dephosphorylates phosphatidylinositol
`(PtdIns) phosphates (see below), one intriguing possibility is
`that this ‘‘tensin domain’’ actually constitutes a binding site for
`such lipids.
`Despite the clear sequence similarity between PTEN and
`other dual-specificity phosphatases, it was unexpectedly diffi-
`cult to detect significant catalytic activity in recombinant
`preparations of PTEN when conventional artificial substrates
`were used (4, 39, 40). This finding was surprising because PTPs
`are generally quite robust and remarkably efficient catalysts.
`The first convincing demonstration of dual-specificity phos-
`phatase activity in PTEN was provided by Myers et al. (40).
`Although these workers found that PTEN could dephosphor-
`ylate some serineythreonine-phosphorylated proteins, it dis-
`played maximal activity against poly(Glu-pTyr), which sug-
`gested that it prefers extremely acidic substrates. Many, al-
`though importantly, not all, PTEN mutants derived from
`patient samples were inactive in this assay. Of particular
`interest, however, two mutants derived from unrelated Cow-
`den disease families retained full activity against poly(Glu-
`pTyr). Moreover, even measured against poly(Glu-pTyr),
`PTEN was markedly less active than most other PTPs. These
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`findings suggested that PTEN might be highly selective for a
`particular phosphorylated protein. Alternatively, PTEN might
`target a nonproteinaceous phospho-substrate; a precedent for
`the latter had been established with the demonstration that a
`PTP family member serves as an RNA 5-triphosphatase (41).
`Work by Tamura et al. (42) suggested that PTEN might,
`indeed, be highly selective for a particular tyrosine-
`phosphorylated target. These workers found that over-
`expression of PTEN inhibited, whereas PTEN antisense oli-
`gonucleotides stimulated, fibroblast migration. They also re-
`ported that PTEN associated with and could dephosphorylate
`focal adhesion kinase (FAK) in vivo and in vitro, leading them
`to suggest that PTEN regulated focal adhesion structure, cell
`spreading, and motility, by controlling FAK activity. However,
`extremely high (stoichiometric, rather than catalytic) amounts
`of PTEN were required to dephosphorylate FAK in these
`experiments. Moreover, although PTEN mutated at its cata-
`lytic cysteine residue was unable to dephosphorylate FAK or
`affect migration and focal adhesion structure, a Cowden
`disease-derived mutation (G129E) that Myers et al. (40) had
`shown retained activity against poly(Glu-pTyr) was active in
`these experiments. Because Cowden disease-derived PTEN
`must, by definition, be biologically inactive (or at least less
`active than wild-type PTEN), these results imply that PTEN
`must have targets other than FAK in vivo.
`Recent studies suggest that the biologically relevant targets
`probably are not phosphoproteins at all, but rather a subset of
`inositol phospholipids. The key initial observation was pro-
`vided by Maehama et al. (43), who showed that PTEN could
`dephosphorylate the 3 position of PtdIns phosphates both in
`vitro and in vivo. Now, a recent paper from Myers et al. (26) in
`the Proceedings, as well as work by Furnari et al. (29), indicates
`that several independent patient-derived PTEN mutants re-
`
`tain protein [i.e., poly(Glu-pTyr)] phosphatase activity but lose
`the ability to dephosphorylate PtdIns phosphates. Since the
`mutations analyzed by both groups occur in sporadic tumors as
`well as Cowden disease patients, their results provide strong
`genetic evidence that the lipid phosphatase activity of PTEN
`is required for its tumor suppressor activity and for its role(s)
`in normal development. Consistent with this idea, PTEN-
`deficient tumor cell lines (26, 44), as well as immortalized
`fibroblasts from PTEN2/2 mice (45), have elevated concen-
`trations of PtdIns with phosphate at the 3 position.
`The finding that PTEN dephosphorylates PtdIns phosphates
`leads to a model for how PTEN acts as a tumor suppressor, a
`model linking PTEN to control of at least two known cellular
`protooncogenes, PI3K (46) and Akt (47). PTEN inhibits PI3K-
`dependent activation of AKT (also called PKB), a seriney
`threonine-kinase, and deletion or inactivation of PTEN results
`in constitutive AKT activation.
`PI3K was discovered more than 10 years ago as a PtdIns
`kinase activity copurifying with the oncoproteins pp60v-src and
`polyoma middle T antigen (48, 49). Although initially thought
`to be an enzyme in the pathway for synthesis of phosphatidyl-
`inositol 4,5-bisphosphate (PtdIns-4,5-P2), chemical analysis of
`the product revealed that this enzyme phosphorylates the 3
`position of the inositol ring, thus defining a new lipid signaling
`pathway (50).
`Interest in PI3K increased with evidence that the transform-
`ing ability of several oncoproteins correlated with their ability
`to associate with PI3K and elevate in vivo levels of specific lipid
`products of this enzyme (reviewed in ref. 51). When presented
`with appropriate substrates in vitro, oncoprotein-associated
`PI3K can catalyze the production of PtdIns-3-P, PtdIns-3,4-P2,
`or PtdIns-3,4,5-P3 (52, 53) (Fig. 1). However, growth factor
`stimulation and oncoprotein transformation correlate with
`
`FIG. 1. Reactions catalyzed by PI3K and PTEN. (A) PtdIns contains a myo-inositol headgroup connected to diacylglycerol by a phosphodiester
`linkage. The numbering system of the inositol ring is indicated. (B) The class I PI3K enzymes can phosphorylate the 3 position of PtdIns, PtdIns-4-P,
`or PtdIns-4,5-P2 to produce PtdIns-3-P, PtdIns-3,4-P2, or PtdIns-3,4,5-P3, respectively. PtdIns-3,4-P2 can also be produced by dephosphorylating
`the 5 position of PtdIns-3,4,5-P3, and one enzyme that does this is an SH2-containing 5-phosphatase called SHIP. In addition, PtdIns-3,4-P2 can
`be produced by phosphorylating the 4 position of PtdIns-3-P [reviewed by Fruman et al. (76)]. PTEN has been shown to dephosphorylate the 3
`position of both PtdIns-3,4,5-P3 (26, 43) and PtdIns-3,4-P2 (44) to reverse the reactions catalyzed by PI3K.
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`increases in the in vivo levels of PtdIns-3,4-P2 and PtdIns-
`3,4,5-P3 (52, 54). These lipids are nominally absent from
`quiescent cells but appear within seconds to minutes of
`stimulation with growthysurvival factors such as platelet-
`derived growth factor (PDGF), nerve growth factor (NGF), or
`insulin-like growth factor 1 (IGF-1).
`The acute production of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 in
`response to cell stimulation suggested that these lipids act as
`membrane-embedded second messengers, analogous to dia-
`cylglycerol. It now is clear that a variety of cytosolic signaling
`proteins have evolved the ability to bind to one or both of these
`lipids as a mechanism of recruitment to the membrane (for
`review, see ref. 55). Several proteins bind PtdIns-3-P lipids by
`pleckstrin homology (PH) domains that specifically recognize
`the phosphoinositide headgroup. These include a PH domain-
`containing protein-tyrosine kinase called BTK (56, 57), an
`exchange factor for ARF family members called GRP-1 (58),
`and, of particular interest for this story, two protein-seriney
`threonine kinases called AKT (59, 60) and PDK1 (61, 62).
`AKT was discovered as the product of a retrovirus-encoded
`oncogene that transforms lymphoid cells (47). The protein
`product has a catalytic domain with high similarity to the PKA
`and PKC family of protein kinases, and it was independently
`cloned as a PKC homologue and termed PKB (63). The first
`evidence that AKT is regulated via a pathway involving PI3K
`came from studies of PDGF-dependent AKT activation in
`fibroblasts (64). Further studies from several
`laboratories
`(reviewed in ref. 65) led to the model indicated in Fig. 2. In this
`model, the N-terminal PH domain of AKT acts as an autoin-
`hibitory domain. This domain has a high affinity for PtdIns-
`3,4-P2 and somewhat lower, but significant, affinity for PtdIns-
`3,4,5-P3. Binding to these phosphoinositides localizes AKT to
`the membrane and opens up the catalytic site. A second
`protein-serineythreonine kinase, PDK1, also has a PH domain
`
`FIG. 2. Cell survival and proliferation by means of PI3K and AKT
`and suppression by PTEN. Growth factors and survival factors activate
`receptors that recruit PI3K to the membrane. Phosphorylation of the
`membrane lipids PtdIns-4-P and PtdIns-4,5-P2 [PI4P, PI4,5P2] by PI3K
`produces the second messengers PtdIns-3,4-P2 and PtdIns-3,4,5-P3
`[PI3,4P2, PI3,4,5P3]. These lipids recruit the protein-serineythreonine
`kinases AKT and PDK1 to the membrane and induce a conformational
`change in AKT, exposing the activation loop. Phosphorylation of AKT
`at Thr-308 of the activation loop by PDK1 turns on the protein kinase
`activity. Phosphorylation of AKT at a C-terminal site (not shown)
`causes further activation. AKT phosphorylates and compromises the
`function of BAD and caspase-9, proteins involved in cell death
`pathways. AKT also phosphorylates and inhibits glycogen synthase
`kinase 3 (GSK3). GSK3 phosphorylates cyclin D, targeting it for
`proteolysis; thus, AKT may promote cyclin D accumulation. PDK1
`also phosphorylates and enables activation of p70S6kinase and protein
`kinase C (PKC) family members. PTEN turns off the pathway by
`dephosphorylating the 3 position of PtdIns-3,4-P2 and PtdIns-3,4,5-P3.
`
`that binds PtdIns-3,4-P2 and PtdIns-3,4,5-P3 tightly, allowing it
`to colocalize with AKT and phosphorylate the activation loop
`of the exposed catalytic domain. Full activation of AKT
`requires phosphorylation at a second C-terminal site by a
`distinct kinase.
`Although other signaling pathways are activated down-
`stream of PI3K, the AKT pathway has attracted much atten-
`tion because of its role in cell survival. The ability to evade cell
`death pathways is a critical event in cancer progression, and the
`PI3KyAKT pathway provides such a mechanism. Activation of
`PI3K and AKT has been shown to provide a survival signal in
`response to NGF, IGF-1, PDGF, IL-3 and the extracellular
`matrix (reviewed in refs. 65 and 66). AKT is likely to send
`survival signals by phosphorylating multiple targets, including
`the BCL-2 family member BAD (67) and the cell death
`pathway enzyme caspase-9 (68). In addition to promoting cell
`survival, AKT may regulate cell proliferation by means of the
`phosphorylation of other targets. For example, AKT-catalyzed
`phosphorylation of another serineythreonine kinase, glycogen
`synthase kinase 3 (GSK3), results in GSK3 inhibition (69).
`Recent studies indicate that GSK3 promotes cyclin D prote-
`olysis; thus by catalyzing GSK3 inhibition, AKT may contrib-
`ute to cyclin D accumulation and cell cycle entry (70). The
`protein-serineythreonine kinase p70S6 kinase is also activated by
`the PI3K pathway (Fig. 2), and this enzyme could also con-
`tribute to cell growth by regulating translation of key mRNAs
`(71).
`Several papers, including those by Myers et al. (26) and Wu
`et al. (72) in the Proceedings, now establish a link between the
`PI3KyAKT pathway and human cancers via defects in PTEN.
`Normally, AKT activity is low in the absence of growth factor
`stimulation. However, PTEN-deficient tumor cell lines (26, 28,
`29, 44, 72), as well as immortalized fibroblasts (45) and tumors
`(31) derived from PTEN-deficient mice, exhibit high basal
`levels of AKT phosphorylation. Consistent with the anti-apo-
`ptotic actions of AKT, PTEN2/2 fibroblasts are resistant to
`multiple pro-apoptotic stimuli. Reconstitution of wild-type
`PTEN expression restores normal AKT regulation and sensi-
`tivity to these stimuli (45). Moreover, PTEN action can be
`overcome by expression of constitutively active forms of AKT.
`Together, these results indicate that deregulation of the PI3Ky
`AKT signal transduction pathway may contribute to a large
`fraction of human cancers. These studies in mammalian cell
`systems are buttressed by very recent genetic analyses of the
`Caenorhabditis elegans homolog of PTEN, which emphasize
`the evolutionary importance of this conserved pathway (73).
`The prevailing paradigm of molecular oncology holds that
`multiple, at least partially redundant, pathways control cell
`growth, differentiation, and apoptosis, and, accordingly, mu-
`tations in many of these pathways are required to generate the
`fully neoplastic state. Available genetic evidence strongly
`suggests that the PTENyPI3KyAKT pathway almost certainly
`constitutes a new and important regulatory pathway. Analysis
`of melanomas indicates that PTEN and p16 mutations coexist
`within the same tumors, arguing that loss of PTEN confers an
`additional selective advantage over loss of the retinoblastoma
`regulatory pathway (the main target of p16; see ref. 1).
`Likewise, there is no apparent correlation between epidermal
`growth factor receptor (EGFR) amplification and PTEN
`mutation in glioblastomas (5), suggesting that these two on-
`cogenic events also lie in parallel regulatory pathways.
`Although it now seems clear that the main role of PTEN is
`to regulate the PI3KyAKT pathway, many important issues
`remain unresolved. PTEN expression prevents basal activation
`of AKT (i.e., activation in the absence of growth factor
`stimulation), yet in PTEN1 cells, growth factors remain able
`to activate AKT via a PI3K-dependent pathway (26, 44, 45).
`This observation suggests that PTEN is inactivated upon
`growth factor stimulation or that PTEN is activated by growth
`factor depletion. It will be important to identify components
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`of such a regulatory pathway(s), as they represent potential
`targets for oncogenic mutation as well. One attractive target
`for a PTEN regulator may be LKB1ySTK1 (74, 75), the
`recently cloned gene for Peutz–Jeghers syndrome, a condition
`that shares a number of clinical features with Cowden disease.
`Alternatively, PTEN regulation could be effected by altering
`its intracellular localization, perhaps by means of its C-
`terminal PDZ- binding domain. However, C-terminal hemag-
`glutinin epitope-tagged PTEN is able to rescue growth defects
`in glioblastoma cell lines, suggesting that its ability to bind
`PDZ-containing proteins is not absolutely required for PTEN
`function (24). It remains to be determined why PTEN recon-
`stitution into prostate (26, 72) and breast (28) carcinoma cells
`causes apoptosis, whereas it induces G1 cell cycle arrest in
`glioblastoma cells (24, 26, 28, 29). Altered regulation of AKT
`could affect both apoptosis (via BAD andyor caspase-9 phos-
`phorylation) and cell cycle entry (via GSK3, p70S6kinase, and
`possibly other pathways). However, why both pathways are not
`affected in any given tumor cell remains to be elucidated; most
`likely, other oncogenic mutations are responsible. The role of
`PTEN protein phosphatase activity, if any, also remains to be
`clarified. Although it now is clear that the lipid phosphatase
`activity of PTEN is required for its biological effects, these
`data do not rigorously establish that PTEN protein phospha-
`tase activity is dispensable. Proof of the latter would require
`the generation of PTEN mutants that retain lipid phosphatase
`but lose protein phosphatase activity and the demonstration
`that such mutants retain full biological activity. In the absence
`of such data, it remains possible that PTEN acts as both a lipid
`and a protein phosphatase in vivo, and that both activities are
`necessary for at least some of its biological roles.
`The elucidation of the PTENyPI3KyAKT pathway stands as
`a potentially important advance in molecular oncology. In view
`of the high frequency of such mutations in human tumors, new
`andyor existing pharmacological agents directed against com-
`ponents of this pathway may have therapeutic benefit.
`
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