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ONCOGENIC PI3K DEREGULATES TRANSCRIPTION AND TRANSLATION

Andreas G. Bader, Sohye Kang, Li Zhao and Peter K. Vogt
Abstract | There have long been indications of a role for PI3K (phosphatidylinositol 3-kinase) in cancer pathogenesis. Experimental data document a requirement for deregulation of both transcription and translation in PI3K-mediated oncogenic transformation. The recent discoveries of cancer-specific mutations in PIK3CA, the gene that encodes the catalytic subunit p110 of PI3K, have heightened the interest in the oncogenic potential of this lipid kinase and have made p110 an ideal drug target.
SH2 DOMAIN

A protein domain, homologous to an equivalent domain in the SRC protein, that facilitates proteinprotein interactions by binding to tyrosinephosphorylated protein sequences.
PLECKSTRIN HOMOLOGY DOMAIN

A protein domain that was originally identified in the protein pleckstrin that forms a phospholipid-binding pocket and in particular binds to PIP3.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Correspondence to P.K.V. e-mail: pkvogt@scripps.edu
doi:10.1038/nrc1753

Phosphatidylinositol 3-kinases (PI3Ks) phosphorylate phosphatidylinositol and its phosphorylated derivatives at the 3 position of the inositol ring, generating second messengers that control cellular activities and properties including proliferation, survival, motility and morphology mutations in PI3K function disrupt these processes110. PI3K proteins form a family that is divided into three classes that differ in structure, substrate preference, tissue distribution, mechanism of activation and, ultimately, in function1115 (FIG. 1). In regulating proliferation and in tumorigenesis, the most important PI3K proteins are those that belong to class IA the catalytic subunit p110 and its associated regulatory subunit p85. In quiescent cells, p85 binds to p110, stabilizing p110 and inactivating its kinase activity. Upon growth factor stimulation, the SH2 DOMAINS (Roussarcoma-oncogene homology-2 domains) of p85 bind to phosphorylated tyrosine in YxxM motifs in receptor tyrosine kinases or their substrate adaptor proteins. This binding relieves the inhibition of p110 and mediates recruitment of the catalytic subunit to the plasma membrane16. PI3K activity is further augmented by the interaction between p110 and the GTP-bound form of the RAS oncoprotein17,18. The high-affinity substrate of p110 is phosphatidylinositol 4,5-bisphosphate (PIP 2 ). This is phosphorylated to become phosphatidylinositol

3,4,5-trisphosphate (PIP 3 ), which then recruits proteins that contain a PLECKSTRIN HOMOLOGY DOMAIN to cellular membranes 19 . Among these are the serine-threonine kinase AKT, as well as its activating kinases PDK1 (3-phosphoinositide-dependent kinase 1) and possibly PDK2. The identity of the latter has remained controversial. Potential candidates for PDK2 include PDK1, AKT, integrin-linked kinase (ILK), DNA-dependent protein kinase (DNA-PK) and the RictorTOR (target of rapamycin) complex2024. Recruitment of these kinases by PIP3 results in the activation of AKT by phosphorylation at T308 and S473. Activated AKT is the predominant and essential mediator for the regulation of both growth and proliferation by PI3K. PIP3 is also a substrate of the phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), which dephosphorylates PIP 3 to generate PIP 2. PTEN is a negative regulator of PI3K signalling and functions as a tumour suppressor25,26. Genetic inactivation of PTEN occurs in numerous human tumours and results in a constitutive upregulation of PI3K signals2730. Wild-type p110 is not oncogenic, but its growth-regulatory functions confer a potential for oncogenicity that can be activated by simple genetic changes. These include retroviral transduction, point mutations, gene amplification, overexpression and

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constitutive activation of PI3KAKT signalling36,3840. Expression of mutant p85 in mice induces a lymphoproliferative disorder and solid tumours36,37. The gain of function in PI3KAKT signalling, which is characteristic of experimental transformation in cell culture and of many human tumours, affects the synthetic activities of the cell at the transcriptional and translational levels (FIG. 2).
PI3K and transcription

Summary Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that generate second messengers that govern cellular activities and properties including proliferation, survival, motility and morphology. PIK3CA, the gene that encodes the catalytic subunit p110 of PI3K, is frequently mutated in human solid tumours. Cancer-specific mutations are clustered in the helical and the kinase domains of p110. The amino-acid residues E542, E545 and H1047 are prominent mutational hot spots. The mutations in the p110 hot spots induce a gain of enzymatic function and confer oncogenic activity both in vitro and in vivo. The oncogenicity of PI3K is the result of interactions with transcriptional and translational controls.

the acquisition of sequences that localize the protein to cellular membranes. The avian PIK3CA gene and the mouse Akt gene have both been identified as oncogenes in retroviruses3133. Oncogenic forms of p110 and AKT are constitutively present in the cell membrane where they have high levels of kinase activity34,35. Mutations in the regulatory subunit of PI3K, p85, have been identified in transformed cell lines and in human tumour samples3640. The mutant forms of p85 induce

PI3KAKT signals control several growth-regulatory transcription factors. Two prominent examples are the forkhead box (FOXO) proteins and nuclear factor (NFB). The FOXO transcription factors, which are a subclass of the much larger FOX protein family, encompass FOXO1 (FKHR), FOXO3A (FKHRL1), FOXO4 (AFX) and FOXO6 REF. 41. FOX proteins bind to DNA as monomers with a highly conserved domain that is referred to as the forkhead or winged helix domain that also serves as the defining structural feature of the protein family. The FOXO proteins control the pace of the cell cycle, as well as apoptosis, DNA repair and protection from oxidative damage42,43. Their activities are regulated by post-translational modifications that include phosphorylation by several kinases, acetylation and ubiquitylation. These

Subunits Class I Structural features of catalytic subunits Adaptorbinding domain A RBD Helical domain p110 p101 p84 Catalytic p110,, Adaptor p85 p55 p50 p85 p55 Regulation Receptor tyrosine kinases and RAS (p110 is also regulated by G-protein-coupled receptors) G-protein-coupled receptors and RAS Lipid substrate specificity PIP2 PIP PI

C2 domain

Catalytic domain

B Coiled-coil domain II Proline-rich domain III C2 domain PX domain

PIP2 PIP PI

PI3K-C2,,

Receptor tyrosine kinases? G-protein-coupled receptors? Constitutive?

PIP2 PI

Vps34p analogues

p150

PI

Figure 1 | PI3Ks constitute a family of enzymes that is divided into three classes. The figure summarizes the domain organization and defining criteria of the three phosphatidylinositol 3-kinase (PI3K) classes. The catalytic subunit of class I enzymes (p110, , and ) contains an adaptor-binding domain, a RAS-binding domain (RBD), a C2 (protein-kinase-C homology-2) domain, a helical domain and a catalytic domain. Class IA enzymes are constitutively associated with an adaptor subunit to form a heterodimeric complex. There are three genes that encode at least five different adaptor subunits, all of which can bind to phosphorylated tyrosine in YxxM motifs through their SH2 (Rous-sarcoma-oncoprotein homology-2) domains. The only member of class IB is p110, which associates with the p101 regulatory subunit or the recently identified p84 adaptor subunit. Class I enzymes are regulated by receptor tyrosine kinases and RAS, and have lipid substrates that include phosphatidylinositol-bisphosphates (PIP2), phosphatidylinositol phosphate (PIP) and phosphatidylinositol (PI). Class II enzymes are comprised of a PI3K-C2, and domains. They lack an adaptor-binding site but possess carboxy-terminal phox (PX) and C2 domains that could mediate binding to phosphoinositides that have been phosphorylated at position D3 and other membrane lipids. Class II enzyme substrates include PIP2, PIP and PI. Although their adaptor subunits have not been identified, class II enzymes are believed to be regulated by receptor tyrosine kinases and G-protein coupled receptors. Class III enzymes comprise analogues of the yeast Vps34p PI3K subunit and a p150 adaptor subunit, and accept only PI as a substrate. They are believed to be constitutively active, and all isoforms of class I PI3K also possess an intrinsic protein kinase activity.

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modifications determine the cellular localization and, ultimately, the fate and functions of FOXO proteins nuclear FOXO proteins act as transcriptional regulators whereas cytoplasmic FOXO proteins are transcriptionally silent and are subject to proteasomal degradation. Nuclear FOXO proteins activate several distinct transcriptional programmes. One of these leads to G1 arrest through increased expression of the cyclin-dependent kinase inhibitors p27kip1 and p21cip1, and reduced expression of cyclin D1 and cyclin D2 REFS 4447. AKT terminates these activities by initiating nuclear export of FOXO proteins. It phosphorylates FOXO at three conserved residues4852, thereby masking the nuclear localization signal, interfering with DNA-binding, disrupting the association with the co-activator p300CBP (CREB-binding protein) and mediating the interaction with 14-3-3 proteins and the exportin CRM1 REFS 49,5358. The result is inhibition of FOXO nuclear functions and removal of FOXO from the nucleus. FOXO that has been exported from the nucleus as a result of AKT-dependent phosphorylation is rapidly degraded5962. The phosphorylation by AKT not only triggers nuclear export of FOXO but it also creates a binding site for the ubiquitin ligase S-phase kinase-associated protein 2 (SKP2, a suppressor of a cyclin-dependent-kinase-inhibitor proteolysis defect), which then marks FOXO for proteasomal degradation60. This radical elimination of transcriptionally active FOXO is a key feature of cells that have been transformed by oncogenic PI3K or AKT in these cells, levels of FOXO are dramatically reduced and the growth-regulatory activity of FOXO is suspended59. FOXO activity also influences other signalling pathways in a complex with SMAD, FOXO regulates p21cip1 gene expression during TGF signalling, and FOXO3a is inactivated by the NFB pathway45, 63. Whereas FOXO is inactivated by AKT signalling, the transcription factor NFB is activated. In human cancer cells, NFB positively regulates cell survival, proliferation, chemoresistance, angiogenesis, cellular invasion and oncogenesis6466. NFB is negatively regulated by the inhibitor IB, which anchors NFB in the cytoplasm. Positive regulation of NFB occurs through IB kinases (IKKs), which phosphorylate IB and mark it for proteasomal degradation. Released from IB, NFB travels into the nucleus and forms a functional transcriptional unit with its dimerization partner, REL-A, which is also known as p65 REFS 64,65. AKT stimulates the transcriptional activity of NFB by three distinct modes, all of which require IKK function. First, AKT interacts with the IKK complex, phosphorylates the subunit and increases IKK activity67,68. Next, AKT indirectly stimulates IKK activity through the mitogen-activated protein kinase kinase kinase COT69. Finally, AKT targets the transactivation domain of REL-A, increasing NFB activity7072. In specific cellular settings, the transcriptional activity of NFB is required for oncogenic transformation by PI3KAKT signalling. For example, disruption of the nuclear activity of NFB with peptide antagonists of its nuclear localization signal, or with non-degradable forms of IB, inhibits oncogenic transformation and cell proliferation, and induces apoptosis in pancreatic cancer cells73,74. NFB activation by PI3KAKT can also
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PIP2

PI3K

PIP3

PIP3 AKT

PIP3 PDK

PTEN

TSC MDM2 RHEB IKK TOR S6K 4EBP S6 4E Translation Transcription HIF1 MIZ1 FOXO NFB p53

GSK3

c-MYC AP1 -catenin

Figure 2 | PI3KAKT signalling affects translation and transcription. The lipid kinase phosphatidylinositol 3-kinase (PI3K) phosphorylates phosphatidylinositol-bisphosphates (PIP2), generating phosphatidylinositol-trisphosphates (PIP3), which recruit the serine-threonine kinase AKT and 3-phosphoinositide-dependent kinase (PDK) to the plasma membrane. Phosphorylation by PDK activates AKT. The signal branches at the level of AKT, regulating downstream proteins that control translation and transcription. During translation, AKT signalling stimulates protein synthesis by activating the ribosomal protein S6 and the translational initiation factor 4E. Stimulation of these translational regulators is indirect and involves the signalling molecules TSC (tuberous sclerosis complex), RHEB (RAS homologue enriched in brain) and TOR (target of rapamycin). TSC is a heterodimeric complex consisting of TSC1 (also known as hamartin) and TSC2 (also known as tuberin) and functions as a GTPase-activating protein for the small GTPase RHEB155159. TSC2 is directly phosphorylated and inactivated by AKT, which leads to increased RHEB activity155,160. In the GTP-bound state, RHEB activates TOR, presumably by direct interaction161. TOR then mediates phosphorylation of S6K and 4EBP (4E-binding protein), thereby stimulating the former and inactivating the latter, leading to increased S6 and 4E activity. During transcription, AKT affects numerous transcription factors, the control of which is either direct or indirect. Direct targets are the forkhead box proteins, FOXO, and the cell cycle inhibitor, MIZ1, both of which are inhibited upon AKT-mediated phosphorylation. AKT-dependent regulation of p53, nuclear factor B (NFB), c-MYC, activator protein 1 (AP1) and -catenin is indirect and also independent of TOR. However, TOR does regulate the transcriptional activity of the hypoxiainducible factor HIF1162,163. GSK3, glycogen synthase kinase 3; IKK, IB kinase; MDM2, murine double minute; PDK, 3-phosphoinositide-dependent kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10.

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previously proposed models in which S6K controls the translation of mRNAs that contain a 5-terminal oligopyrimidine tract adjacent to the mRNA CAP STRUC 108,109 . The 4EBP proteins are negative regulators of TURE protein synthesis. They interact with the cap-binding protein 4E and thereby prevent the formation of the 4F translational initiation complex, which depends on the availability of 4E110. The phosphorylation of 4EBP by TOR breaks the attachment to 4E, allowing 4E to build a functional initiation complex and recruit the scaffolding protein 4G and the RNA helicase 4A111113. TOR signals to its targets, S6K and 4EBP, through interaction with a binding partner, Raptor114,115. The function of Raptor in this process depends on a specific motif in the target proteins, named the TOS (TOR signalling) motif116118 (FIG. 2). The oncogenic activities of PI3K and of AKT are exquisitely sensitive to rapamycin. Low concentrations of the drug completely prevent transformation that is induced by oncogenic versions of PI3K and of AKT in cell culture119. However, rapamycin does not inhibit transformation that is induced by other oncoproteins, although some, such as SRC, are sensitive to rapamycin in combination with inhibitors of MAP kinase signalling119,120. Sensitivity of tumours to rapamycin is also seen in animal model systems121,122. PTEN-deficient mice develop spontaneous cancers that show constitutive gain of function in PI3KAKT signalling. The tumours respond to treatment with the rapamycin derivative CCI-779, which shows that TOR activity is essential for PI3K-dependent oncogenesis. In PI3Ktransformed or AKT-transformed cells, S6K and 4EBP are constitutively phosphorylated, indicating activation of the former and inactivation of the latter119. This constitutive phosphorylation is sensitive to rapamycin, and is therefore TOR-dependent119. The correlation between TOR-dependent intervention in protein synthesis and PI3K-induced oncogenic transformation is complemented by data on another regulator of protein synthesis, the mRNAbinding protein YB-1 (Y box-binding protein 1)123126. YB-1 is transcriptionally downregulated in PI3Ktransformed and AKT-transformed cells127. Vectormediated expression of YB-1 in normal cells induces specific resistance to transformation by PI3K or AKT but does not affect transforming activities of other oncoproteins127. YB-1 acts downstream of TOR, as the phosphorylation levels of the TOR targets S6K and 4EBP are not changed in YB-1-overexpressing cells127. A deletion analysis of YB-1 shows that interference with PI3K-induced and AKT-induced transformation is correlated with the ability of YB-1 to inhibit cap-dependent protein synthesis128. All interfering YB-1 mutants also bind to mRNA, including the cap structure, and are localized in the cytoplasm128. The data on YB-1 provide an independent line of evidence for an essential role of protein synthesis in PI3Kinduced and AKT-induced transformation. It is likely that this role does not extend to protein synthesis in general but is restricted to the efficient translation of specific mRNAs.

Table 1 | Aberrant PI3K-signalling in human cancers

Component p110 p85 PTEN AKT Type of alteration Gain of function by amplification, overexpression or somatic mutation Gain of function by mutation Loss of function by mutation or transcriptional downregulation Gain of function by amplification, overexpression or increased phosphorylation References 110,143,144,164166 3640 2729,158,167 144,167170

AKT, serine-threonine kinase that is a homologue of thymoma viral oncoprotein; p110, catalytic subunit of phosphotidylinositol 3-kinase (PI3K); p85, regulatory subunit of PI3K; PTEN, phosphatase and tensin homologue deleted on chromosome 10.

result in a positive auto-feedback loop NFB signalling indirectly represses transcription of the PTEN gene and therefore amplifies the PI3K signal75,76. Other transcriptional regulators whose activities are affected by PI3KAKT signalling include MIZ1 (Mycinteracting zinc-finger protein 1), p53, AP1 (activator protein 1), c-MYC, -catenin and HIF1 (hypoxia inducible factor 1). The exact roles of these proteins during PI3K-mediated oncogenesis are currently unknown, but they have all been linked to oncogenic transformation. AKT phosphorylates and thereby inactivates the cell-cycle inhibitor MIZ1 REF. 77, and also suppresses p53 activity by a mechanism that involves MDM2 (murine double minute)78,79. By contrast, the activities of AP1, c-MYC and -catenin are increased by AKT. These targets are negatively controlled by GSK3 (glycogen synthase kinase 3), which is inactivated by AKT-mediated phosphorylation8087. In cancer cell lines that upregulate PI3K signalling, levels of HIF1 are increased, and the gene targets of this transcriptional regulator (for example, vascular endothelial growth factor) are activated88.
PI3K and protein synthesis

MACROLIDE

A group of antibiotics that are produced by various strains of Streptomyces and have a complex macrocyclic structure.
POL I AND POL IIIDEPENDENT TRANSCRIPTION

Class I RNA polymerases synthesize all ribosomal RNAs except the 5S ribosomal RNA; class III RNA polymerases transcribe transfer RNA genes and the gene that encodes the 5S ribosomal RNA.
mRNA CAP STRUCTURE

The 5 end of virtually all mRNAs is protected by a cap with the chemical structure m7GpppN (where m7G represents 7-methylguanylate, p represents a phosphate group and N represents any base).

The key evidence that links protein synthesis to PI3K-mediated growth signals comes from studies with rapamycin and its target, TOR. Rapamycin is a MACROLIDE antibiotic that interacts with a small cellular protein FKBP12 (FK506-binding protein 12)89,90. The rapamycinFKBP12 complex binds to the kinase TOR, interfering with the TOR-Raptor (regulatory associated protein of TOR) connection and blocking the phosphorylation of TORRaptor targets9196. TOR has multiple and diverse functions, including the control of protein synthesis97. It stimulates POL I AND POL IIIDEPENDENT TRANSCRIPTION, thereby regulating the abundance of the components that make up the protein-synthesizing machinery of the cell98102. The activities of Pol I and of Pol III have long-term effects, but TOR also regulates protein synthesis on a shorter timescale by inducing the phosphorylation of S6K (p70 S6 kinase) and of the 4EBP (translation initiation factor 4E-binding protein) family of proteins97,103. S6K is a positive regulator of protein synthesis and cell size104107. Its mechanism of action is not fully understood; recent experiments have challenged

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Microarray analysis of polysomal RNA samples have shown a differential effect of PI3K, AKT and rapamycin treatment on the expression of specific mRNAs129 131 . These included mRNAs with lower translational fitness, presumably due to highly structured, complex 5-untranslated regions. Efficient translation of such mRNAs is dependent on high levels of active
p110 Mutated residue R38 Q60 R88 P104 G106 R108 E110 K111 G118 G122 P124 N345 D350 C378 S405 E418 C420 E453 P539 E542 E545 Helical Q546 Colon 1.1 0.4 0.4 0.4 0.4 0.2 0.2 0.4 0.4 0.4 0.4 0.3 0.1 0.1 0.2 0.2 1.2 0.2 0.3 3.6 6.2 0.2 0.5 3.3 0.2 0.2 0.2 0.1 Frequency of mutation (%) Breast Others Total 0.13 0.04 0.13 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.13 0.04 0.04 0.04 0.04 0.39 0.09 0.13 1.63 4.76 0.47

p85

RBD

C2

4E, which recruits an RNA helicase that can unwind 5-mRNA structures to facilitate ribosome scanning132. Numerous mRNAs that code for growth factors, oncoproteins and cell cycle regulators have highly structured 5-untranslated regions, making their efficient translation dependent on the abundance of 4E132. We conclude that deregulation of protein synthesis is necessary for PI3K- induced and AKT-induced transformation, but it might not be sufficient, as there are no reports that show oncogenic transformation by TOR or the TOR activator, RHEB (RAS homologue enriched in brain). The effects of AKT on transcriptional regulators are also essential features of the transformation process transcriptional and translational deregulation are both necessary and complement each other. In addition to deregulating transcription and protein synthesis, oncogenic PI3K also affects cell replication and survival by post-translational modification of proteins. Examples of these effects that are outside the scope of this review are the AKT-induced phosphorylation and inactivation of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, as well as of the pro-apoptotic proteins BAD (BCL2-anatagonist of cell death) and caspase 9 REFS 133140.
PI3K as a drug target

0.8 0.4 0.4 3.8 9.8 2.6

Q661 H701 K733 C901 F909 S1008 P1011 Y1021 T1025 E1035 M1043 N1044 A1046 H1047 G1049 H1065

0.4 0.3 0.4 0.4 0.4 0.4 0.1 0.2 0.1 0.2 0.1 0.1 0.1 0.1

0.04 0.13 0.04 0.09 0.09 0.04 0.04 0.13 0.17 0.04 0.21 0.04 0.04 5.48 0.09 0.04 15.1

Kinase

0.8 0.8

0.2 0.2 0.2 0.2 14.8 0.2

7.1

1.5 0.1 0.1 6.5

Total

32.3

29.3

Figure 3 | Point mutations in PIK3CA observed in human tumours. Analyses of the phosphatidylinositol 3-kinase PIK3CA gene in human tumour samples have identified somatic point mutations that affect a total of 38 residues110. Mutations other than point mutations have not been included in the figure. The mutations localize to various domains of the p110 primary structure as indicated. Hot-spot mutations that occur at high frequency are confined to residues E542, E545 and H1047 and are highlighted in orange. Overall frequencies of mutation have been determined for cancers of the colon (266 samples)1,3, breast (580 samples)13,5,6,8 and others, which include liver (73 samples)5, brain (382 samples)1,4,10, stomach (291 samples)1,5,7, lung (253 samples)1,5 and ovary (489 samples)3,6,9, with a total of 2,334 tumour samples. Protein domains have been determined using NCBI tools. C2, protein-kinase-C homology-2 domain; p85, p85-binding domain; RBD, RAS-binding domain.

Genetic aberrations that lead to a gain in PI3K signalling are commonly observed in human cancers TABLE 1. The recent sequencing of cancer cell genomes has revealed point mutations in PIK3CA110. These mutations occur at significant frequencies in several types of solid tumours (FIG. 3). They are observed in colon as well as in breast and hepatocellular carcinomas. The mutations in PIK3CA in human tumours are somatic, cancer-specific and heterozygous. They are mostly missense mutations; no truncating or nonsense mutations have been identified, but a few cases of in-frame deletions and insertions have been detected1,3,5. Rare cases of double mutations, in which two amino-acid residues are altered, have also been reported5,8. In breast carcinoma, mutations in PIK3CA and loss of PTEN function are almost always mutually exclusive 8 . However, mutations in PIK3CA are often correlated with overexpression of ERBB2 (also known as HER2/ NEU), indicating that gains of function in these two signalling components could have a synergistic effect in counteracting PTEN8. The most important attribute of the cancerspecific mutations in PIK3CA is a non-random distribution along the coding sequence (FIG. 3). This non-randomness is indicative of selection for mutations that confer a proliferative advantage, which act as dominant oncoproteins. The majority of the mutations map to three sites E542 and E545 in the helical domain and H1047 in the kinase domain of p110 (FIG. 3). E542 and E545 are commonly changed to lysine, whereas H1047 is frequently substituted with arginine. At all three sites, the mutations cause a gain of enzymatic function1,141. The three mutant

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proteins also induce oncogenic transformation when expressed in primary chicken-embryo fibroblasts and in NIH 3T3 cells141,142. AKT signalling is constitutively activated in the mutant-transformed cells, as indicated by high levels of phosphorylated AKT and of its downstream targets S6K and 4EBP141,142. The oncogenic transformation that is induced by the three mutant p110 proteins is inhibited by rapamycin, indicating that TOR and TOR-dependent protein synthesis are essential components of the transformation process141. Mutations in p110 and other mechanisms of activating PI3K signalling appear to be mutually exclusive. In ovarian cancer, mutations in p110 are confined almost exclusively to tumours that do not show amplification of the PIK3CA gene3. Since amplification of PIK3CA is common in ovarian cancers, mutations in p110 are rare in these tumours3, 9, 143. Likewise, in gastric tumours, the incidence of PIK3CA amplification far exceeds that of mutations in PIK3CA5,7,144. By contrast, tumours that lack PIK3CA amplification, such as in breast cancer or colorectal cancer, more frequently harbour mutations in p1103, 6. The mechanisms by which the mutations in p110 induce a gain of function remain to be determined. In the absence of structural data for p110, it is nevertheless possible to model the catalytic and helical domains of the molecule using the published crystal structure of the homologous p110 isoform145 (FIG. 1). In this model, the two most prevalent mutations in the helical domain, E542K and E545K, map to the surface of the protein6,146. Both mutations cause an electrostatic charge reversal from negative to positive that could change the ability of the protein to interact with regulatory proteins. Although deletion mapping has defined separate binding regions for the regulatory subunit p85 and for RAS that lie in the amino-terminus of p110, the data do not rule out an extended interaction of p85 or RAS with this helical domain. The p85 subunit can function as a negative regulator of p110, and reduction of p85 binding by the E542K or E545K mutations could increase p110 enzymatic activity147. There is also the possibility that these mutations disrupt the ability of p110 to interact with a still unknown regulatory protein. Another possible explanation for the gain of function that is induced by the mutations in the helical domain is that these cause a change in enzyme conformation. These mutations could affect the C2 (protein-kinase-C homology-2) domain and thereby increase the affinity of that domain for lipid membranes. The third of the prevalent mutations, H1047R, is located near the activation loop in the catalytic domain. It is likely that it affects specificity or affinity of p110 towards the lipid substrate of PI3K. Mutated kinases are ideal drug targets especially when mutated forms of these proteins are only expressed in tumour cells and result in a gain of function. Studies of patients with non-small-cell lung cancer have shown that tumours that express mutant forms of the receptor tyrosine kinase EGFR (epidermal growth factor receptor) are more responsive to EGFR inhibitors such as erlotinib and gefitinib, compared with tumours that express wild-type EGFR148150. Therapy with these inhibitors increased survival times of patients with tumours that express EGFR151. A rough estimate that is based on the published frequencies of tumour-specific mutations in PI3K indicates that the number of newly diagnosed patients with cancer who carry one of the hot spot mutations in PI3K (described in FIG. 3), is in excess of 10,000 per year in the United States and there are many more worldwide. Specific inhibitors of mutant forms of PI3K could therefore have a significant therapeutic effect. The feasibility of generating mutant-specific PI3K inhibitors has not been established. However, recent advances in the design and assembly of bivalent enzyme inhibitors indicate that compounds with the requisite specificity and potency could be within reach152154.

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2.

3.

4.

5.

6.

7. 8.

9.

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Competing interests statement

The authors declare no competing financial interests.

ERBB2 | FKBP12 | FOXO1 | FOXO3A | FOXO4 | FOXO6 | GSK3 | HIF1 | IKK | ILK | MDM2 | MIZ1 | NFB | p21cip1 | p27kip1 | p53 | PDK1 | PTEN | REL-A | RHEB | S6K | SKP2 | SMAD | YB-1 FURTHER INFORMATION Atlas of Genetics and Cytogenetics in Oncology and Haematology (PIK3CA): http://www.infobiogen.fr/services/ chromcancer/Genes_gc/GC_PIK3CA.html Peter K. Vogts homepage: http://www.scripps.edu/research/ faculty.php?tsri_id=1847 The PI3KPTENAKT signalling pathway: www.humpath. com/article.php3?id_article=890 Access to this interactive links box is free online.

Online links
DATABASES The following terms in this article are linked online to: National Cancer Institute: http://www.cancer.gov breast cancer | colorectal cancer| ovarian cancer | pancreatic cancer Swiss-Prot: http://www.expasy.org/sprot 4EBP | AKT | AP1 | BAD | -catenin | caspase 9 | c-MYC | COT | CRM1 | cyclin D1 | cyclin D2 | DNA-PK | EGFR |

Acknowledgements
The work of the authors is supported by grants from the National Cancer Institute.

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