Activation of AKT Kinases in Cancer: Implications for Therapeutic Targeting

Activation of AKT Kinases in Cancer: Implications for Therapeutic Targeting Alfonso Bellacosa,* C. Chandra Kumar,{ Antonio Di Cristofano,* and Joseph Robert Testa* *Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; { Department of Tumor Biology, Schering Plough Research Institute, Kenilworth, New Jersey 07033

I. II. III. IV. V. VI.

VII.

VIII.

IX. X.

XI.

Introduction Historical Perspective Structure of AKTs AKT Activation During Signal Transduction Crystal Structure of AKT Kinases Substrates of AKT Mediating its Cellular Functions A. Cell Proliferation B. Cell Survival C. Metabolism D. Cell Growth/Translation/Response to Nutrients E. Other Oncogenic Functions of AKT Signaling AKT Alterations in Human Cancers A. AKT Amplification and Overexpression B. AKT Activation in Human Tumors Alterations of Other Components of the PI3K/AKT Pathway in Human Cancers A. PI3K B. PTEN C. TSC2 D. eIF4E In Vivo Models of AKT Activation Implications of AKT Pathway Activation for Therapeutic Targeting A. Rationale for Targeting the AKT Pathway for New Drug Discovery Efforts B. Role of AKT in the Therapeutic Response of Tumor Cells C. Molecular Targets in the AKT Signaling Pathway D. Targeting AKT Kinases E. Targeting mTOR F. Liabilities Associated with Targeting the AKT Signaling Pathway Conclusions Acknowledgments References

Advances in CANCER RESEARCH Copyright 2005, Elsevier Inc. All rights reserved.

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0065-230X/05 $35.00 DOI: 10.1016/S0065-230X(04)94002-X

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The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention. # 2005 Elsevier Inc.

I. INTRODUCTION During the past decade, the field of cancer biology has witnessed an enormous upsurge of research activity concerning the AKT kinases, their role in tumorigenesis, and the possibility of targeting them therapeutically and/or as a chemoprevention strategy. The three AKT kinases are now known to represent central nodes in a variety of signaling cascades that regulate normal cellular process such as cell size/growth, proliferation, survival, glucose metabolism, genome stability, and neo-vascularization (reviewed in Bellacosa et al., 2004). In recent years, however, a burgeoning literature attests to the frequent hyperactivation of AKT kinases in a broad array of human solid tumors and hematological malignancies (reviewed in Cantley, 2002; Testa and Bellacosa, 2001), and a series of elegant studies using animal models has demonstrated that aberrant signaling involving the AKT pathway can, either alone or by cooperating with certain other genetic perturbations, induce malignancy or contribute to a more malignant phenotype (reviewed in Bellacosa et al., 2004; Bjornsti and Houghton, 2004; Di Cristofano and Pandolfi, 2000; Luo et al., 2003). It is now evident that AKT is a central player in a signaling pathway of which many components, including the upstream phosphatidylinositol 3-kinase (PI3K) (Philp et al., 2001; Samuels et al., 2004; Shayesteh et al., 1999), PTEN (phosphatase and tensin homologue deleted on chromosome ten) (reviewed in Cantley and Neel, 1999; Di Cristofano and Pandolfi, 2000), and LKB1 (Boudeau et al., 2003), and the downstream tuberous sclerosis complex

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2 (TSC2) (reviewed in Kwiatkowski, 2003; Manning and Cantley, 2003) and eukaryotic initiation factor 4E (eIF4E) (Avdulov et al., 2004; Bjornsti and Houghton, 2004; Mamane et al., 2004; Ruggero et al., 2004; Wendel et al., 2004), have been linked to tumorigenesis. Some of these proteins, such as the p110
catalytic and p85
regulatory subunits of PI3K, AKT, and eIF4E, are encoded by (proto)oncogenes, whereas others (PTEN, LKB1, and TSC2) are tumor suppressor gene products. Interestingly, in addition to sporadic genetic changes of these genes in many common human cancers, germ line mutations in PTEN, LKB1, and TSC2 result in three distinct, dominantly inherited cancer syndromes characterized by multiple hamartomas and predisposition to certain malignancies (reviewed in Boudeau et al., 2003; Eng, 2003; Kwiatkowski, 2003). Collectively, these signaling proteins are components of a PI3K-AKT-mTOR (mammalian target of rapamycin) axis that, when deregulated, leads to disruptions in the translation of various cancer-related mRNAs that are involved in such processes as cell cycle progression, autocrine growth stimulation, cell survival, invasion, and communication with the extracellular environment (Mamane et al., 2004). Because the AKT signaling cascade is frequently disrupted in many human cancers, and in light of the wide-ranging biologic consequences described above, this pathway is considered a key determinant of tumor aggressiveness and an attractive target for therapeutic intervention (Mitsiades et al., 2004). On the other hand, the fact that AKT signaling affects many important downstream pathways, such as glucose metabolism, means that the potential liabilities of such a molecularly targeted approach must be carefully addressed. The biochemical mechanisms involved in AKT kinase activation have been well delineated (Alessi and Cohen, 1998; Brazil et al., 2004; Brunet et al., 1999; Chan et al., 1999; Coffer et al., 1998; Datta et al., 1999; Downward, 1998; Franke et al., 1997; Hanada et al., 2004; Hemmings, 1997; Kops et al., 2002; Scheid and Woodgett, 2003; Simpson and Parsons, 2001; Testa and Bellacosa, 2001; Vivanco and Sawyers, 2002), and new substrates continue to be validated in vivo. It is currently less clear, however, whether AKT1, AKT2, and AKT3 are functionally redundant or whether each carries out a specific functional role (Bellacosa et al., 2004). In this review, we summarize current knowledge regarding the signaling properties and specificities of the various AKT kinases emerging from recent studies of human cancers and rodent models, as well as the status of current efforts to specifically target individual AKT family members and other upstream and downstreamcomponents of this pathway to maximize therapeutic efficacy. While AKT kinases are promising targets for pharmacological intervention, increased understanding of the distinct roles of each AKT family member could lead to improved design of highly specific targeted therapies having reduced toxicities and improved efficacy.

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II. HISTORICAL PERSPECTIVE Research on AKT function is currently proceeding at an extremely accelerated pace, with hundreds of publications added every year to the already long Medline list (at the time of this review, there are more than 5000 articles on AKT). This rapidly increasing pace of discovery is a reflection of both the recognized central role of AKT kinases in normal physiology and disease and the availability of effective and specific reagents, namely, phospho-specific antibodies and active/inactive mutants, for the rapid assay of AKT activation and its consequences. However, as is often the case in science, the early history of AKT had a relatively slow start and followed a convoluted path. The initial work on AKT was conducted by Steve Staal in the laboratory of Wally P. Rowe at the National Institutes of Health (NIH). Staal had isolated a retrovirus from a T-cell lymphoma localized in the thymus (improperly called thymoma) of the susceptible mouse strain AKR and called it AKT8, for AKR Thymoma #8 (Staal et al., 1977). He demonstrated that this virus formed peculiar foci in mink cells and had obtained partial clones of the AKT8 provirus. Staal showed that this provirus contained sequences of cellular origin, and the putative oncogene was called Akt. He isolated two human hybridizing sequences that he dubbed AKT1 and AKT2 (Staal, 1987; Staal and Hartley, 1988). One of us (J. R. Testa), in collaboration with Staal, mapped the AKT1 probe to chromosome 14, confirming its human origin (Staal et al., 1988). When Testa joined the Fox Chase Cancer Center, he initiated a collaboration on AKT with Philip Tsichlis and his post-doc Alfonso Bellacosa. The full-length AKT8 provirus was cloned from the original transformed mink cells (obtained from Janet Hartley at NIH). Sequence analysis revealed that the viral oncogene v-Akt encodes a protein with a high degree of homology to protein kinase C (PKC). The sequence was reported at the 1990 Oncogene Meeting in Frederick, MD, and published the following year (Bellacosa et al., 1991). v-Akt is one of the select oncogenes encoding a serine-threonine protein kinase, which immediately suggested a critical role of AKT in transformation, downstream of the more common oncogenic tyrosine kinases. In 1991, using homology-based approaches to identify genes for cellular kinases related to PKA and PKC, Paul Coffer and James Woodgett and the group of Brian Hemmings independently reported on the cloning of AKT1 and called it PKB
or RAC-PK
, for protein kinase related to PKA and PKC (Coffer and Woodgett, 1991; Jones et al., 1991) (the name RACPK has since been abandoned to avoid confusion with the small Ras-family GTPase Rac). While these two studies suggested an important role of AKT/ PKB in signal transduction pathways, our initial publication linked v-Akt, and therefore, its cellular homologue AKT1, to transformation and cancer.

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In subsequent collaborative work between Testa’s and Tsichlis’ laboratories, together with ovarian cancer biologists Andy Godwin and Tom Hamilton, we confirmed the cancer connection by showing that the highly related AKT2 gene (also called PKB) is frequently amplified and overexpressed in human ovarian carcinomas (see Section VII.A). Mammals contain a third AKT homologue, called AKT3 or PKB, that may also be linked to some forms of human cancer, such as estrogen receptor-negative breast carcinomas.

III. STRUCTURE OF AKTS All three AKT kinases belong to the more general class of AGC kinases (related to AMP/GMP kinase and protein kinase C) and consist of two conserved domains: an N-terminal pleckstrin homology (PH) domain, followed by a kinase domain that terminates in a regulatory hydrophobic motif (Fig. 1). This hydrophobic motif is a characteristic feature of all AGC kinases that

Fig. 1 Domain structure of the AKT family members and homology models of the ATP binding regions. All AKT family members contain an N-terminal pleckstrin homology domain, a catalytic kinase domain, and a C-terminal regulatory hydrophobic region. Computer-derived homology models of the ATP binding regions of the three AKTs are shown. ATP binding site is shown in blue. Residues unique to the AKT family members are colored in magenta and labeled. Staurosporine is in yellow.

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include PKA, PKC, PDK1 (for 30 -phosphoinositide-dependent kinase), as well as p70 and p90 ribosomal protein S6 kinases (p70 S6K and p90 RSK) (Coffer and Woodgett, 1991). The PH domain interacts with lipid products such as phosphatidylinositol (3,4,5) triphosphate (PIP3) and phosphatidylinositol (3,4) diphosphate (PIP2) with similar affinity. The crystal structure of the PH domain of AKT has recently been solved (Thomas et al., 2002). The kinase domain of AKT shares high similarity with other members of the AGC family of kinases such as PKA, PKC, p70 S6K, and p90 RSK. The sequence identities among the three AKTs in the kinase domain exceed 87% (Kumar et al., 2001). Homology models for the catalytic domains of the three AKT kinases were derived based on the complex structure of PKA/staurosporine (Knighton et al., 1991). As expected for these highly homologous sequences, the structural models of the three kinase domains are very similar (Fig. 1). The three AKT kinases are identical in the ATP binding region, except for one residue: Ala 230 of AKT1 is conserved in AKT2 (Ala 232) but switches to Val 228 in AKT3. In addition, each of the three AKT kinases has a C-terminal extension of about 40 amino acids. This region possesses the FXX-F/Y-S/T-Y/F hydrophobic motif (where X is any amino acid) that is characteristic of the AGC family of protein kinases.

IV. AKT ACTIVATION DURING SIGNAL TRANSDUCTION Activation of AKT is a multistep process that involves both membrane translocation and phosphorylation (Bellacosa et al., 1998) and is triggered by engagement of receptor tyrosine kinases by peptide growth factors and cytokines. The critical step in the signal transduction cascade leading to AKT activation is stimulation of the growth factor receptor-associated PI3K that forms a direct axis with AKT. PI3K generates 30 -phosphorylated phosphoinositides PIP3 and PIP2 at the plasma membrane. Both phospholipids bind with high affinity to the PH domain, mediating membrane translocation of AKT (Fig. 2). At the membrane, AKT is phosphorylated at two sites that effect its activation, threonine (Thr) 308 in AKT1 (309 in AKT2 and 305 in AKT3) in the activation loop, or T-loop, and serine (Ser) 473 (474 in AKT2 and 472 in AKT3) in the hydrophobic motif of the C-terminal tail. T-loop phosphorylation is absolutely required for AKT activation, while C-terminal phosphorylation potentiates AKT activity by promoting a conformational change in the T-loop (see Section V.). T-loop phosphorylation is mediated by another PH domain-containing AGC kinase, PDK1, also activated by PIP3 and PIP2 (Alessi et al., 1997). The identity of the kinase(s) responsible for hydrophobic motif phosphorylation, dubbed

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Fig. 2 Schematic depicting the conformational changes accompanying AKT activation and the role of the phosphorylated hydrophobic motif in the activation cycle. (A) AKT in its unphosphorylated state remains cytosolic and is inactive. In unphosphorylated AKT2, the activation (or T-loop),
B, and
C helices and the hydrophobic motif (HM) remain disordered. PDK2 phosphorylates AKT2 on Ser 474 in the HM. (B) PI3K-generated PIP3 and PIP2 recruit AKT and PDK1 to the plasma membrane. Phosphorylated HM stabilizes and activates PDK1, which then phosphorylates AKT2 on Thr 309. (C) The HM of AKT2 associates with and stabilizes the kinase domain, leading to ordered structures for
B and
C helices and the activation (T-loop). PH indicates the plekstrin homology domain; red circles denote phosphate groups.

30 -phosphoinositide-dependent kinase 2 (PDK2), is quite controversial (Chan and Tsichlis, 2001): candidates include the integrin-linked kinase ILK, mitogen-activated protein kinase-activated kinase 2 (MAPKAPK2), and AKT and PDK1 themselves. Recently, the PI3K-related DNA-dependent protein kinase has been identified as a prominent Ser 473 kinase in membrane extracts from HEK293 cells, suggesting a potential intersection of transmembrane signal transduction and DNA damage response pathways (Feng et al., 2004), which may be relevant to the role of AKT in the response of tumor cells to chemotherapeutic agents (see Section X.). Although less characterized in comparison to AKT activation, several mechanisms of AKT inactivation are emerging. The PI3K/AKT axis is directly antagonized by the 30 -lipid phosphatase activity of the tumor suppressor

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Role of AKT in Cancer

PTEN that converts 30 -phosphorylated phosphoinositides to their 30 -unphosphorylated form (Di Cristofano and Pandolfi, 2000). In addition, several serine-threonine phosphatases, including protein phosphatase 2A, may be involved in the inactivation of AKT in vivo. Negative regulation of AKT is also caused by binding to the Drosophila melanogaster tribbles homologue TRB3 and to C-terminal modulator protein (CTMP), which reduce phosphorylation of the AKT T-loop and hydrophobic motif (Du et al., 2003; Maira et al., 2001).

V. CRYSTAL STRUCTURE OF AKT KINASES The first structure of a protein kinase that was reported was that of PKA, a member of the AGC family of protein kinases (Knighton et al., 1991). The structure of PKA has been the hallmark for the entire protein kinase family. The crystal structures of AKT2 kinase domain in its inactive and active states were determined in 2002 by Barford’s group at the ICRF in England (Yang et al., 2002a,b). A second group at the Amgen Cambridge Research Center also determined the structure of an inactive AKT2 kinase domain independently (Huang et al., 2003). Generally, protein kinases such as AKT2 and PKA consist of an N-terminal small lobe of about 100 amino acids and a C-terminal large lobe that gives the catalytic core a characteristic “bean-like” or “taco-like” structure. ATP binds between the two lobes, directing the -phosphate outward, while the adenine ring lies in the cleft between the two lobes. The N terminus folds into an
/ structure with three helices, A, B, and C (only B and C for AKT2). The C-terminal domain is largely helical and contains most of the catalytic residues. All members of the AGC family of protein kinases require phosphorylation of a conserved Ser/Thr residue within their activation segment located within the C-lobe of the kinase domain for catalytic activity (Vanhaesebroeck and Alessi, 2000). In PKA, this is a constitutive autophosphorylation event at Thr 197, whereas in other AGC kinases it is reversible and confers a critical regulatory mechanism. In addition, most AGC kinases are phosphorylated on a second Ser/Thr residue within the conserved C-terminal hydrophobic motif FXXFS/TY. In some atypical PKCs and PKCrelated kinase 2 (PRK2), the phosphorylatable residue in the hydrophobic motif is replaced by an aspartic acid or glutamic acid residue, so that a permanent negative charge affords constitutive activation of the hydrophobic motif (Balendran et al., 1999). As indicated above, AKT1 is activated mainly by phosphorylation at Thr 308 within the activation T loop and at Ser 473 within the C-terminal hydrophobic motif. Phosphorylation of AKT1 at Thr 308 induces a change in conformation that facilitates substrate

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binding and a greatly elevated rate of catalysis. AKT is one example of the AGC family of kinases for which there is low activation loop phosphorylation in resting cells and rapid increase in phosphorylation upon growth factor stimulation. The phosphorylation at Thr 308 stimulates enzymatic activity by at least 100-fold, whereas phosphorylation of Ser 473 augments AKT activity 7- to 10-fold (Alessi et al., 1996). Hence, phosphorylation at both residues results in about a 1000-fold increase in protein kinase activity. Recently, the structures of the inactive (unphosphorylated) and partially activated (Thr 309 monophosphorylated) AKT2 ternary complex with glycogen synthase kinase-3 (GSK3) peptide and 50 -adenylylimidodiphosphate (AMP-PNP) were described (Yang et al., 2002a). AKT2 monophosphorylated at Thr 309 is only 10% as active as the biphosphorylated form, and in the crystal structure it adopts the predominant inactive conformation. A comparison of the crystal structures of PKA and AKT2 shows how Ser 474 (hydrophobic motif) phosphorylation promotes the fully activated conformation of AKT2. In the inactive AKT2 structure, interdependent regions of the kinase domains—comprising the B and C helices of the N-lobe, the activation segment or T-loop including Thr 309, and the C-terminal hydrophobic motif—are disordered (Yang et al., 2002a). The hydrophobic motif of PKA is unusual, because it includes only the first four residues of the conserved motif FTEF. Therefore, the enzyme does not require a hydrophobic motif phosphorylation for activity. In AKT2, phosphorylation of Ser 474 in the hydrophobic motif converts it into an intramolecular allosteric effector that associates with the N-lobe, thereby stabilizing the
C helix. An ordered
C helix is critical for promoting an active kinase structure (Fig. 2). In PKA, His 87 of the
C helix interacts with pThr 197 of the activation segment. Because the equivalent residue (His 196) of AKT2 is disordered, the AKT2 activation segment is also disordered, which, combined with the disordered
C helix, results in a different conformation of the conserved DFG motif located at the N terminus of the activation segment. This conformational difference results in disruption of the ATP and peptide substrate binding sites. The hydrophobic motif binding pocket is referred to as the PIF (PRK2 interacting fragment) pocket because it was initially determined to be the binding site for the hydrophobic motif fragment of PRK2 (Balendran et al., 1999). Accordingly, phosphorylated but not unphosphorylated hydrophobic motif peptides activated AKT2. The most potent activating peptide tested was PIFtide, a peptide corresponding to the C terminus of PRK2. PIFtide bound to AKT2 with a 1000-fold higher affinity than to its own hydrophobic motif (Yang et al., 2002a). PDK1 harbors a hydrophobic and phosphate binding pocket similar to other AGC kinases but lacks a complementary hydrophobic motif. It is proposed that in the absence of its own hydrophobic motif, PDK1 compensates by utilizing the hydrophobic motif of its substrates. Thus, the hydrophobic motif of AKT provides PDK1 with

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a docking and activation mechanism. It is thought that PDK1 interacts with the Ser 473-phosphorylated hydrophobic motif of AKT1, bringing PDK1 into proximity to Thr 308 (Fig. 2). The stabilizing effect of hydrophobic motif binding increases the specific activity of PDK1 (Pearl and Barford, 2002; Scheid and Woodgett, 2003). In the activated AKT2 structure, the
B and
C helices of the N-lobe, together with the activation segment and phosphorylated hydrophobic motif, become ordered. The ordered
C helix in activated AKT2 facilitates the adoption of an active kinase structure by maintaining the nucleotide binding site and activation segment in a catalytically competent state. Thus, the conserved hydrophobic groove of AGC kinases serves the dual purpose of binding a phosphorylated hydrophobic motif via an intramolecular mechanism to stimulate the kinase catalytic activity and, in some instances, functions as a docking site to mediate protein–protein interactions conferring specificity on kinase signaling cascades (Fig. 2).

VI. SUBSTRATES OF AKT MEDIATING ITS CELLULAR FUNCTIONS AKT proteins mediate a large spectrum of cellular functions, ranging from control of cell proliferation and survival to modulation of intermediary metabolism and angiogenesis. Such pleiotropic effects are the consequence of phosphorylation of an ever-increasing list of substrates (Fig. 3). With a few exceptions, most substrates share the consensus sequence for AKT phosphorylation, RXRXXS/T.

A. Cell Proliferation Identification of v-Akt as the retroviral oncogene of the AKT8 retrovirus (Bellacosa et al., 1991; Staal, 1987) immediately underscored the role of AKT in the regulation of cell proliferation and survival. Phosphorylation of multiple substrates results in the proliferative effect of AKT. Phosphorylation and consequent inhibition of GSK3 prevents degradation of cyclin D1 (Diehl et al., 1998), an effect that is coupled to promotion by AKT/mTOR pathways (see Section VI.D) of increased translation of cyclin D1 and D3 transcripts (Muise-Helmericks et al., 1998). AKT directly antagonizes the action of the cell cycle inhibitors p21WAF1 and p27Kip1: phosphorylation by AKT at a site located near their nuclear localization signal induces cytoplasmic retention of the cell cycle inhibitors (reviewed in Testa and

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Fig. 3 Downstream signaling: AKT substrates and functions. Schematic depicting AKT substrates and associated cellular functions. Continuous lines imply direct phosphorylation by AKT, leading to activation (arrow end) or inhibition (blunt end). Broken lines indicate indirect or unknown mechanisms of activation/inhibition.

Bellacosa, 2001). In the case of p27Kip1, cytoplasmic retention is promoted by the binding of the AKT-phosphorylated protein to the 14.3.3 scaffold protein (Viglietto et al., 2002).

B. Cell Survival AKT provides survival signals that prevent programmed cell death by several independent mechanisms that impinge both on the caspase cascade and on the transcriptional control of apoptosis (reviewed in Franke et al., 2003; Testa and Bellacosa, 2001). AKT is one of the kinases that phosphorylates the pro-apoptotic factor BAD, preventing release of cytochrome c from the mitochondria, the triggering event of the caspase cascade (Creagh and Martin, 2001). In parallel, AKT directly inhibits the caspase cascade by phosphorylating (pro)caspase-9 and by phosphorylating and stabilizing PED/PEA15, a cytosolic inhibitor of caspase-3 (Trencia et al., 2003). As mentioned above, AKT phosphorylation restricts nuclear entry of p21WAF1: cytoplasmic p21WAF1 binds to the apoptosis signal-regulating kinase (ASK1), inhibiting apoptosis (Zhou et al., 2001). In addition, AKT directly phosphorylates ASK1. Equally complex are the positive and negative transcriptional mechanisms by which AKT delivers anti-apoptotic signals. AKT phosphorylation restricts nuclear entry of transcription factors

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Role of AKT in Cancer

of the forkhead family by a mechanism similar to the effect on p27Kip1, namely, phosphorylation near the nuclear localization signal and binding to 14.3.3. Cytoplasmic retention of forkhead family factors prevents transcription of the pro-apoptotic genes Fas ligand, BIM, TRAIL, and TRADD. To the contrary, AKT promotes nuclear translocation of NF-B by phosphorylating and activating IB kinase (IKK), with consequent degradation of IB. NF-B transcribes the anti-apoptotic genes BFL1, cIAP1, and cIAP2. Recent evidence suggests that mTOR mediates some of the anti-apoptotic effects of AKT (see Section VI.D). While the mechanistic details of the AKT/mTOR pro-survival axis are presently unknown, they are likely linked to the mTOR-regulated translation of pro- and anti-apoptotic mRNAs (Wendel et al., 2004). Finally, AKT can also antagonize p53-mediated cell cycle checkpoints impinging on apoptosis by modulating subcellular localization of Mdm2. Phosphorylation of Mdm2 by AKT is necessary for localization to the nucleus, where Mdm2 can complex with p53 to promote its ubiquitin/proteasome-mediated degradation (Mayo and Donner, 2001).

C. Metabolism A primary function of AKT kinases closely linked to their pro-survival role (Gottlob et al., 2001; Plas and Thompson, 2002) is regulation of intermediary metabolism, in particular, glucose metabolism. It has become apparent that many of the effects of insulin on glucose metabolism are mediated by AKT. This line of investigation was heralded by the identification of the first AKT substrate, GSK3, which when inactivated by phosphorylation leads to increased glycogen synthesis (Cross et al., 1995). Insulin is known to stimulate glucose transport, which is mediated by AKT phosphorylation and membrane translocation of the glucose transporters GLUT1 and GLUT4 (Kohn et al., 1996). AKT stimulates glycolysis via phosphorylation of phosphofructokinase 2 (Deprez et al., 1997) and transcriptional activation of glycolytic enzymes (Majumder et al., 2004) (see Section VI.D). Recently, it has been proposed that AKT activation is directly responsible for the elevated rate of glycolysis of tumor cells under aerobic conditions (Elstrom et al., 2004), the so-called “Warburg effect” (reviewed in Warburg, 1956). In cancer cells expressing constitutively active AKT, only the rate of glycolysis is elevated, while oxidative phosphorylation, as measured indirectly by oxygen consumption, is unchanged. Since the rate of glucose uptake and utilization is in excess of cellular demand, lactate production is increased (Elstrom et al., 2004) and the NAD(P)H/NAD(P) ratio is elevated (Elstrom et al., 2004). It remains to be determined whether the aerobic glycolysis of tumor cells is a by-product

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of AKT activation frequently occurring in human cancer or whether it is selected for during tumorigenesis, for instance, to ensure high levels of NADPH as a protection from oxidative stress (Elstrom et al., 2004).

D. Cell Growth/Translation/Response to Nutrients AKT also participates in the control of cell growth defined as an increase in cell size, not cell number. Cell growth is the cellular response to increased availability of nutrients, energy, and mitogens. Cell growth pathways are modulated by mTOR, a kinase that stimulates protein synthesis. The role of AKT in cell growth was first noticed in animal models. Indeed, the Drosophila AKT homologue Dakt1 regulates apoptosis in early embryogenesis (Staveley et al., 1998), but in late embryogenesis it modulates insulin receptor pathways that stimulate cell growth and increase organ size, independently of any effect on apoptosis or cell proliferation (Verdu et al., 1999). The topics of cell and organ size control and their implications for tumorigenesis will be discussed in greater detail by Keyong Du and Phil Tsichlis in an upcoming issue of Advances in Cancer Research; here we focus on the signaling aspects of nutrient sensing, cell growth, and translation. mTOR is emerging as a major downstream target of AKT in the regulation of the cellular response to nutrients, and its role is akin to a gatekeeper of a cellular checkpoint controlled by nutrient and energy availability that restricts cell cycle progression in the presence of suboptimal growth conditions (Bjornsti and Houghton, 2004). The mechanisms of mTOR activation downstream of AKT are actively being characterized and involve the tumor suppressor proteins TSC1 and TSC2 that are defective in the hereditary cancer syndrome, tuberous sclerosis, and the Ras family small GTPase Rheb. The TSC1 and TSC2 proteins, also known as hamartin and tuberin, respectively, form a complex that restrains Rheb by virtue of the GTPaseactivating protein (GAP) activity of TSC2. AKT phosphorylates TSC2, destabilizing it and disrupting its interaction with TSC1 (Inoki et al., 2002; Potter et al., 2002). This leads to increased levels of active, GTPbound Rheb, which, by unknown mechanisms, results in the activation of mTOR kinase activity (Li et al., 2004). mTOR stimulates protein synthesis by phosphorylating two targets that have an immediate impact on translation: p70 S6K and eukaryotic initiation factor 4E binding protein 1, 2, and 3 (4E-BPs) (reviewed in Gingras and Sonenberg, 1997; Kim and Sabatini, 2004; Long et al., 2004; Martin and Blenis, 2002; Proud, 2004). p70 S6K phosphorylates the ribosomal protein S6, which leads to increased translation of mRNAs containing 50 -terminal oligopolypyrimidine (50 TOP) tracts, including ribosomal proteins and other proteins involved in ribosome biogenesis. mTOR phosphorylation of

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Role of AKT in Cancer

4E-BPs relieves inhibition on the initiation factor eIF4E, which results in more efficient cap-dependent translation of messages such as those for cyclin D1, c-Myc, and vascular endothelial growth factor (VEGF) (Bjornsti and Houghton, 2004; Gingras and Sonenberg, 1997; Ruggero and Pandolfi, 2003). A recently identified in vivo target of mTOR is the heterodimeric hypoxiainducible transcription factor HIF1. In a murine model of prostate intraepithelial neoplasia (PIN) induced by tissue-specific expression of constitutively active AKT, the PIN phenotype is completely reversed by treatment with the rapamycin analogue RAD001 (Majumder et al., 2004). Microarray analysis identified the HIF1
subunit as an mTOR target: both mRNA and protein levels of HIF1
were upregulated in PIN and decreased by mTOR inhibition with RAD001 (Majumder et al., 2004). In this study, genes for 9 of the 10 glycolytic enzymes involved in glucose conversion to pyruvate, plus the genes for lactate dehydrogenase and GLUT1, were identified as HIF1 transcriptional targets (Majumder et al., 2004), emphasizing the link between AKT activation and aerobic glycolysis described above. How is the AKT/TSC2/mTOR pathway linked to nutrient and energy levels? Recent findings indicate that tuberin is at the crossroads of both positive and negative signals. AKT phosphorylates and inactivates tuberin, delivering positive signals of growth factor stimulation. On the other hand, negative signals that indicate growth factor deprivation, stress, hypoxia, and low levels of energy and nutrients activate tuberin via its phosphorylation by the AMP-activated protein kinase (AMPK) (Inoki et al., 2003). AMPK is a master regulator of metabolism that redistributes energy expenditure and energy intake in response to a variety of stressors that lead to depletion of ATP and increased AMP/ATP ratio, such as hypoxia, low energy, and reduced nutrient (glucose, amino acids, etc.) availability (Carling, 2004; Kyriakis, 2003). Under stress conditions, AMPK is activated and phosphorylates several key targets, leading to enhanced fatty acid oxidation and glycolysis. In addition to enhanced catabolic reactions, AMPK activation reduces anabolic pathways, such as lipogenesis, cholesterol biosynthesis, and protein synthesis (Carling, 2004; Kyriakis, 2003). The latter effect appears to be mediated in large part by phosphorylation of tuberin that enhances its inhibitory effect on mTOR (Inoki et al., 2003; Shaw et al., 2004a) (Figs. 3 and 4). Interestingly, the heterotrimeric AMPK is activated by phosphorylation in the activation loop of its kinase (
) subunit. Recently, the most abundant (if not only) AMPK kinase has been identified as LKB1 (Hawley et al., 2003; Shaw et al., 2004b; Woods et al., 2003), which is encoded by the gene defective in the cancer-prone Peutz-Jeghers syndrome, characterized by gastrointestinal hamartomatous polyps. LKB1 tumor-suppressing activity is at least in part due to its ability to phosphorylate and activate AMPK, and in both Lkb1-null mouse embryo fibroblasts and Peutz-Jeghers polyps, mTOR

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Fig. 4 Alterations of the AKT pathway in human cancer and opportunities for therapeutic intervention: schematic depicting the AKT pathway. Molecular alterations are listed on the right, while pharmacological inhibitors are shown on the left.

signaling is elevated (Shaw et al., 2004a). Thus, signaling both upstream (i.e., via LKB1) and downstream of AMPK (i.e., via TSC2) and the intersection with the PI3K/PTEN/AKT/mTOR pathway establish a connection between control of cell proliferation by oncogenes and tumor suppressor genes and metabolic regulation by classical biosynthetic and catabolic enzymes. This is likely to become an area of intense investigation in the near future. The mTOR/eIF4E pathway is often activated in human tumors (see Section VIII.C, D), but, surprisingly, tumor cells rarely display increased size in comparison to their normal counterpart. It is possible that activation of this pathway is selected in human cancer because deregulation of the mTOR checkpoint and the consequent increase in global translation efficiency are permissive for enhanced cell proliferation. Increased translation of specific mRNAs (cyclins D1, D3, and E) (Muise-Helmericks et al., 1998) may be important in tumorigenesis. Moreover, recent studies indicate that,

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Role of AKT in Cancer

in addition to a role in control of cell size and cell proliferation, the mTOR/ eIF4E pathway can provide anti-apoptotic signals (Avdulov et al., 2004; Majumder et al., 2004; Ruggero et al., 2004; Wendel et al., 2004).

E. Other Oncogenic Functions of AKT Signaling AKT plays a role in additional processes that are considered hallmarks of cancer, such as sustained angiogenesis, unlimited replicative potential, and tissue invasion and metastasis (Hanahan and Weinberg, 2000). AKT promotes angiogenesis via increased nitric oxide production by phosphorylating endothelial nitric oxide synthase (eNOS) (Dimmeler et al., 1999; Fulton et al., 1999). The reverse transcriptase subunit of telomerase that stimulates unlimited replication is also an AKT substrate (Kang et al., 1999). Finally, AKT has a role in tumor invasion/metastasis by stimulating secretion of matrix metalloproteinases (Thant et al., 2000) and inducing epithelialmesenchymal transition (EMT) (Grille et al., 2003). In addition, recent work with colon cancer cells has shown that AKT activation mediates chromosomal instability by increasing anaphase bridge index and chromosomal aberrations (Aoki et al., 2003). Moreover, mounting evidence over the past dozen years indicates that AKT alterations are common in many forms of human cancer (see Section VII).

VII. AKT ALTERATIONS IN HUMAN CANCERS It is now apparent that hyperactivation of AKT kinases is one of the most common molecular perturbations in human malignancy. The AKT signaling pathway is activated in human cancer by an assortment of mechanisms, including amplification, overexpression or point mutation of the genes encoding AKT kinases and their upstream activators, overexpression of the downstream target eIF4E, and deletion or inactivation of tumor suppressors responsible for downregulation of the pathway (Fig. 4). An overview of the changes reported in human cancers is summarized in the following sections.

A. AKT Amplification and Overexpression In 1992, we reported the first recurrent involvement of an AKT gene in a human cancer, demonstrating amplification and overexpression of AKT2 in a subset of ovarian carcinomas (Cheng et al., 1992). AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. In the two ovarian carcinoma cell lines

Alfonso Bellacosa et al.

45

exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. An ensuing multicenter study confirmed and extended these findings, demonstrating AKT2 amplification in 16 of 132 (12%) ovarian carcinomas but in only 3 of 106 (3%) breast carcinomas (Bellacosa et al., 1995). Northern blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas that were negative for AKT2 amplification. Amplification of AKT2 was especially frequent in undifferentiated ovarian tumors (4 of 8, p < 0.02), suggesting that AKT2 alterations may be associated with tumor aggressiveness. Such amplification/overexpression of AKT2 could contribute to the malignant phenotype by permitting a tumor cell to become overly responsive to ambient levels of growth factors that normally would not provoke proliferation (Hanahan and Weinberg, 2000; Testa and Bellacosa, 2001). Subsequent studies by us and others documented amplification and/or overexpression of AKT2 in 10–20% of primary pancreatic carcinomas and pancreatic cancer cell lines (Cheng et al., 1996; Miwa et al., 1996; Ruggeri et al., 1998). Two cell lines with altered AKT2, PANC1 and ASPC1, exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein (Cheng et al., 1996). As an early indication of the potential importance of molecularly targeting the AKT pathway, J. Cheng in Testa’s laboratory found that AKT2 expression and tumorigenicity of PANC1 cells in nude mice was markedly inhibited by transfection with an antisense AKT2 construct but not with a control AKT2 construct in the sense orientation (Cheng et al., 1996). Furthermore, PANC1 and ASPC1 cells, as well as pancreatic carcinoma cells in which AKT2 is neither amplified nor overexpressed (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized using a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective untransfected cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between control and antisense-treated COLO 357 cells. Taken together, these data suggest that overexpression of AKT2 contributes to the growth and invasiveness of a subset of human ductal pancreatic cancers and that selective targeting of AKT2 could have significant therapeutic implications. Notably, amplification of the chromosome region 19q13.1–q13.2, the native location of the AKT2 gene, has also been reported in other ovarian tumors and cell lines, and amplification and overexpression of AKT2 were demonstrated in ovarian cancer cell lines (Thompson et al., 1996). In addition, AKT2 amplification has also been reported in a non-Hodgkin’s lymphoma in which a homogeneously staining region was located on chromosome 19 (Arranz et al., 1996).

46

Role of AKT in Cancer

In hepatocellular carcinoma, high expression of AKT2 protein was detected in nearly 40% of tumors, whereas AKT1 expression was moderate or less in all cases (Xu et al., 2004). Furthermore, AKT2 but not AKT1 overexpression was an independent prognostic marker. In an immunohistochemical analysis of human colorectal tissues conducted with a pan-AKT antibody, normal colonic mucosa and hyperplastic polyps expressed low levels of AKT, while intense AKT immunoreactivity was seen in 57% of colorectal cancers (Roy et al., 2002). AKT was also detected in 57% of the adenomas examined, implicating overexpression of AKT as an early event during colon tumorigenesis. Staining with an antibody specific for AKT2 appeared to duplicate the results seen with the pan-AKT antibody, suggesting that AKT2 was the predominant AKT family member involved in this particular malignancy (Xu et al., 2004). Unlike AKT2, amplification of AKT1 has not been reported as a recurrent change in any tumor type. AKT1 amplification was initially detected in a single gastric carcinoma (Staal, 1987). More recently, an investigation of 103 malignant glial tumors revealed a single case (a gliosarcoma) with amplification and overexpression of AKT1 (Knobbe and Reifenberger, 2003). While the gene is rarely amplified, AKT1 protein levels have been reported to be elevated in some types of cancer. For example, an immunohistochemical analysis of a large series of breast cancers revealed marked staining in 24% of the tumors for AKT1 but in only 4% of the tumors for AKT2 (Stal et al., 2003). It is noteworthy, however, that in another series of breast cancers HER-2/neu expression was found to correlate with elevated expression of AKT2, but not AKT1, and that AKT2 protein was upregulated in a breast cancer cell line by ectopic expression of HER-2/neu (Bacus et al., 2002). Moreover, in vitro experiments with human breast and ovarian cancer cells have demonstrated that overexpression of AKT2, but not AKT1 or AKT3, is associated with increased invasion and metastasis (Arboleda et al., 2003). To date, high-level amplification of AKT3 has not been described in any human cancers, although low-level increases in AKT3 copy number would be expected to be common in some tumor types, since an extra copy of the long arm of chromosome 1 (1q), the site of the native AKT3 locus, is a frequent event in many tumor types (Knuutila et al., 1998). For example, increased copy number of several cancer-related genes located in 1q, including AKT3, has been observed in hepatitis C-related hepatocellular carcinoma (Hashimoto et al., 2004). While evidence in support of amplification specifically targeting the AKT3 locus at chromosome 1q44 is lacking, AKT3 mRNA has been shown to be upregulated in estrogen receptor-negative breast carcinomas (Nakatani et al., 1999). We previously showed that overexpression of wild-type AKT2 can transform NIH3T3 fibroblasts (Cheng et al., 1997). However, wild-type AKT1 is

Alfonso Bellacosa et al.

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unable to transform NIH3T3 cells (Ahmed et al., 1993; Cheng et al., 1997), although NIH3T3 cells stably expressing constitutively activated AKT1 (MyrAkt) exhibit a malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice (Sun et al., 2001). Expression of v-Akt and MyrAkt in squamous cell carcinomas of the tongue was associated with EMT and increased invasiveness in a rat tracheal xenotransplant assay (Grille et al., 2003). In vitro studies with human breast and ovarian cancer cells have demonstrated that overexpression of AKT2 upregulates 1 integrins and increases invasiveness/metastasis (Arboleda et al., 2003). Unlike AKT1, AKT2 protein localized predominantly adjacent to the collagen IV matrix during cellular attachment. Overexpression of AKT2, but not AKT1 or AKT3, was sufficient to duplicate the invasive effects of PI3K-transfected breast cancer cells. Furthermore, expression of kinasedead AKT2, and not kinase-dead AKT1 or AKT3, prevented invasion induced by either activation of PI3K or overexpression of HER-2/neu. Collectively, these experimental data suggest that among members of the AKT family, AKT2 may have particular importance in mediating PI3K-dependent effects on adhesion, motility, invasion, and metastasis.

B. AKT Activation in Human Tumors Frequent activation of AKT has been reported in a broad range of human cancers, including various carcinomas, glioblastoma multiforme, and hematological malignancies (Table I). In some of these tumor types, AKT activation has been shown to correlate with advanced disease and/or poor prognosis (see comments in Table I). Among the specific AKT family members, increased AKT1 kinase activity was reported in about 40% of breast and ovarian cancers and in more than 50% of prostate carcinomas (Sun et al., 2001). In this series, 78% of all tumors with activated AKT1 were high grade and stage III/IV carcinomas. Activation of the AKT2 kinase has been observed in 30–40% of ovarian and pancreatic cancers (Altomare et al., 2003; Yuan et al., 2000). Increased AKT3 enzymatic activity was found in estrogen receptor-deficient breast cancer and androgen-insensitive prostate cancer cell lines (Nakatani et al., 1999), suggesting that AKT3 may contribute to the aggressiveness of steroid hormone-insensitive cancers. An assortment of mechanisms can lead to AKT activation in human tumors, including loss or downregulation of PTEN (Eng, 2003), amplification/upregulation of the PIK3CA gene (Shayesteh et al., 1999), mutation of PIK3CA or PIK3R1 (Philp et al., 2001; Samuels et al., 2004), overexpression of growth factor receptors such as HER-2/neu in breast cancer (Bacus et al., 2002; Stal et al., 2003; Zhou and Hung, 2003) and epidermal growth

48

Table I AKT Activation in Human Cancers Tumors with active AKT (%)

No. cases

Glioma

54

92

Western

Thyroid carcinoma

83

46

IHC

Thyroid carcinoma

83

6

Western

Papillary thyroid carcinoma

100

7

Western

Tumor type

Technique used

Comments Phospho-PI3K (p-PI3K) detected in 57% of cases and phospho-S6K in 39%. Phosphorylation of all three PI3K pathway members was significantly more frequent in glioblastoma multiforme than in non-glioblastoma multiforme tumors. Activation of PI3K pathway was significantly associated with reduced survival. Phospho-AKT (p-AKT) staining most intense in regions of capsular invasion. Staining was localized to nucleus in follicular cancers and cytoplasm in papillary cancers, except for invasive regions of papillary cancers where it localized to both compartments. Immunoactive AKT1, but not AKT2 or AKT3, correlated with p-AKT localization. p-AKT levels significantly higher in all three follicular cancers and in two of three papillary cancers. p-AKT levels significantly correlated with p-BAD and p-S6K levels.

Reference Chakravarti et al., 2004

Vasko et al., 2004

Ringel et al., 2001

Miyakawa et al., 2003

Breast carcinoma

27

274

IHC

Breast carcinoma

22

78

IHC

Breast carcinoma

54

93

IHC

Breast carcinoma

38 (AKT1)

50

Kinase assay, confirmed by IHC, Western

Small cell lung carcinoma Non-small cell lung carcinoma

62

42

IHC

51

110

IHC

49

p-AKT staining showed stronger correlation with AKT1 than AKT2 staining. Rate of locoregional tumor recurrence was significantly decreased with radiotherapy for AKT-negative patients. Significant inverse relationship observed between reduced PTEN expression (36% of cases) and increased p-AKT expression. p-AKT significantly associated with lower S-phase fraction and presence of heregulin beta 1-expressing stromal cells. p-AKTpositive patients more prone to relapse with distant metastasis, independently of S-phase fraction and nodal status. Most (15 of 19) AKT1-activated tumors were high grade and stage III/IV. Elevated PI3K activity observed in 7 of 19 breast tumors that exhibited AKT1 activation. All tumors with AKT1 activation expressed PTEN. Phosphorylated MAPK, but not p-AKT, predictive of survival. Positive staining for p-mTOR and p-FKHR were detected in 74 and 68% of tumors, respectively, and was significantly associated with activation of AKT. Incidence of p-AKT staining similar in low-stage and high-stage tumors, suggesting that activation occurs early in tumor progression. Metaplastic/dysplastic areas from 8 of 25 bronchial epithelial lesions from patients at high risk of lung cancer showed AKT activity.

Stal et al., 2003

Shi et al., 2003

Perez-Tenorio and Stal, 2002

Sun et al., 2001

Blackhall et al., 2003 Balsara et al., 2004

(continues)

50

Table I (continued)

Tumor type

Tumors with active AKT (%)

No. cases

Technique used

Non-small cell lung carcinoma

67 (47% with strong to moderate staining)

43

IHC

Non-small cell lung carcinoma

33

76

IHC

Non-small cell lung carcinoma (stage I) Gastric carcinoma Gastrointestinal stromal tumors

73

91

IHC

78

311

IHC

27

15

Western

Comments Incidence of p-AKT staining not different between primary and metastatic lesions, suggesting that AKT activation may play a role in NSCLC development rather than in disease progression. Among patients with resected early-stage or locally advanced NSCLC, p-AKT expression had no effect on tumor stage, histology, or survival. Of the histological groups examined, bronchial dysplasia specimens expressed p-AKT most frequently (88%), suggesting AKT activation is an early event in lung cancer progression and a potential target in future lung cancer prevention studies. Significant correlations were found between EGFR, TGF-alpha, and p-AKT expression, but none of these proteins had an impact on relapse-free survival. AKT activation was shown to correlate positively with APC and Smad4 expression. AKT phosphorylation high in 4 of 9 tumors with KIT exon 11 mutations but low in all 6 tumors with mutations of other KIT exons.

Reference Lee et al., 2002

Tsao et al., 2003

Mukohara et al., 2004

Nam et al., 2003 Duensing et al., 2004

Pancreatic carcinoma Pancreatic carcinoma

59

78

IHC

67

36

IHC

Pancreatic carcinoma

32 high AKT2 activity; 11 moderate

37

Kinase assay

Bile duct carcinoma

84

19

IHC

Ovarian carcinoma

68

31

IHC

Ovarian carcinoma

39 (AKT1)

28

Kinase assay, confirmed by IHC, Western

Ovarian carcinoma

57

49

IHC

AKT activation correlated with HER-2/neu overexpression and higher tumor grade. Similar frequency of AKT activation in intraductal papillary-mucinous tumors and invasive ductal adenocarcinomas. AKT phosphorylation closely correlated with Ki-67 immunoreactivity. Western blot analysis revealed loss of PTEN protein expression in 2 of 12 tumors with activated AKT2. In vitro PI3K assay showed high levels of PI3K activity in 7 of 9 available tumors with AKT2 activation. In vitro studies demonstrated that AKT activation in bile duct cancer cells is associated with radioresistance. p-AKT staining significantly associated with p-mTOR staining, with 17 (55%) tumors showing elevated expression of p-mTOR. Most (8 of 11) AKT1-activated ovarian tumors were high grade and stage III/IV. Elevated PI3K activity observed in 5 of 11 tumors with AKT1 activation. IHC revealed no PTEN expression in 2 of 11 ovarian tumors with elevated AKT1 activity. Significant inverse correlation observed between p-AKT expression and PTEN expression, but PTEN or AKT status not significantly associated with p27 or cyclin D1 expression.

Schlieman et al., 2003 Semba et al., 2003

Altomare et al., 2003

Tanno et al., 2004

Altomare et al., 2004 Sun et al., 2001

Kurose et al., 2001

51

(continues)

52

Table I (continued)

Tumor type

Tumors with active AKT (%)

No. cases

Technique used

Prostate carcinoma

45

74

IHC

Prostate carcinoma

53 (AKT1)

30

Kinase assay, confirmed by IHC, Western

Renal cell carcinoma

38

48

IHC

Endometrial carcinoma

36 (absence of PTEN expression)

103

IHC

Comments The staining intensity for p-Akt significantly greater in Gleason grades 8–10 (92% of such cases staining strongly) compared with prostatic intraepithelial neoplasia and all other grades of prostate cancer (only 10% of these cases staining strongly). Most (13 of 16) AKT1-activated prostate tumors were high grade and stage III/IV. Elevated PI3K activity observed in none of 16 prostate tumors with AKT1 activation. IHC revealed no PTEN expression in 10 of 16 tumors with elevated AKT1 activity. Elevated p-AKT staining significantly associated with tumor grade and metastatic disease, but not associated with tumor stage or histological subtype. Another 15% of cases showed both staining and non-staining tumor cells. Western blotting demonstrated significant inverse correlation between expression of PTEN and expression of phosphorylated AKT.

Reference Malik et al., 2002

Sun et al., 2001

Horiguchi et al., 2003

Terakawa et al., 2003

Anaplastic large cell lymphoma

100

4

Western

Multiple myeloma

89

18

IHC

Acute myeloid leukemia

72

61

Western

Samples were lymph node specimens infiltrated with lymphoma cells expressing oncogenic NPM/ALK fusion protein possessing constitutive tyrosine kinase activity. Most cases showed marked nuclear expression and weaker cytoplasmic reactivity in plasma cells. Phosphorylation of AKT significantly associated with phosphorylation of GSK3-beta, FKHR, and C-terminal regulatory domain of PTEN as well as with unfavorable prognosis.

Slupianek et al., 2001

Alkan and Izban, 2002 Cheong et al., 2003; Min et al., 2003

53

54

Role of AKT in Cancer

factor (EGF) receptor in glioblastoma multiforme (Schlegel et al., 2002), activation of PI3K due to autocrine or paracrine stimulation of receptor tyrosine kinases (Altomare et al., 2003; Eng, 2003; Nakatani et al., 1999; Sun and Steinberg, 2002; Tanno et al., 2001; Yuan et al., 2000), and/or Ras activation (Liu et al., 1998). Moreover, in some hematological malignancies, AKT can be constitutively activated due to a chromosomal translocation that triggers permanent activation of an upstream tyrosine kinase. Two such examples include the BCR-ABL protein, encoded by a chimeric (fusion) gene formed by a (9;22) translocation in chronic myeloid leukemia (CML) (Skorski et al., 1997) and the NPM-ALK protein, encoded by a fusion gene formed by a (2;5) translocation seen in some anaplastic large cell lymphomas (Slupianek et al., 2001). Such upstream alterations would be expected to have a significant effect on any member of the AKT family that is expressed in a given tumor. For example, we found that the pattern of AKT2 kinase activity was very similar to that observed for AKT1 and AKT3 in a series of pancreatic carcinomas, although in a few cases activation of a given AKT family member may not have been obvious because the expression of that particular protein was low (S. Tanno and J. Testa, unpublished data). It is also possible that some tumors may show activation of a single AKT family member due to a point mutation, e.g., in the kinase domain, although AKT mutations have not been reported at the time of this review. For example, DNA sequence analysis revealed no mutations in the regions encoding the AKT1 PH domain or the AKT1 activation-associated phosphorylation sites at codons 308 and 473 in various types of skin cancer (Waldmann and Wacker, 2001; Waldmann et al., 2001, 2002).

VIII. ALTERATIONS OF OTHER COMPONENTS OF THE PI3K/AKT PATHWAY IN HUMAN CANCERS A. PI3K It is now well documented that AKT belongs to a signaling pathway of which many components have been linked to tumorigenesis (Fig. 4). As noted earlier, c-Akt is the cellular homologue of a viral oncogene (Bellacosa et al., 1991; Staal, 1987; Staal et al., 1977). In addition, avian sarcoma virus 16 contains a potent transforming gene that is derived from the cellular gene for the catalytic subunit of PI3K (Chang et al., 1997), and its human homologue, PIK3CA, has been implicated as an oncogene in some human cancers. In ovarian carcinomas, for example, the PIK3CA gene has been reported to be frequently increased in copy number in association with increased PIK3CA transcription and p110
protein expression, as well as with increased PI3K

Alfonso Bellacosa et al.

55

activity (Shayesteh et al., 1999). Elevated PIK3CA gene copy number has also been observed in 20 of 55 (36%) primary gastric carcinomas and was detected primarily in tumors without PTEN loss of expression, suggesting that PIK3CA and PTEN alterations are mutually exclusive events in gastric tumorigenesis (Byun et al., 2003). As with ovarian cancer cell lines, increased PIK3CA copy number in gastric cell lines was strongly associated with increased expression of PIK3CA transcript and elevated levels of phosphorylated AKT. More recently, somatic missense mutations of the PIK3CA gene have been reported in several human cancer types, particularly colorectal carcinomas (32%), glioblastomas (27%), and gastric cancers (25%) (Samuels et al., 2004). Mutations were observed in only 2 of 76 premalignant colorectal tumors, both of which were very advanced adenomas, indicating that PIK3CA mutations generally arise late in tumorigenesis, just prior to or coincident with invasion. Not unexpectedly, the positions of the mutations within PIK3CA implied that they are likely to increase PI3K activity, and expression of a “hot spot” p110
mutant in NIH3T3 cells conferred more lipid kinase activity than did expression of the wild-type protein. A mutated form of the gene encoding the p85
regulatory subunit of PI3K (PIK3R1) was reported in a human T-cell lymphoma cell line derived from a patient with Hodgkin’s disease (Jucker et al., 2002). The mutant protein lacked most of the C-terminal SH2 domain but retained the inter-SH2 domain and was associated with a constitutively active form of PI3K. DNA analysis has also demonstrated the presence of somatic mutations of the PIK3R1 gene in human primary colorectal and ovarian tumors and cancer cell lines (Philp et al., 2001). Mutations were found in 3 of 12 colon cancer cell lines and in 1 of 2 ovarian cancer cell lines. Somatic mutations were also identified in 3 of 80 ovarian carcinomas and 1 of 60 colon carcinomas. Notably, the affected ovarian carcinoma cell line, OVCAR3, also exhibits amplification and overexpression of AKT2 (Cheng et al., 1992) and elevated kinase activity (Yang et al., 2004). All of these PIK3R1 mutations led to deletions in the inter-SH2 region of the molecule proximal to the Ser 608 autoregulatory site (Philp et al., 2001). Furthermore, expression of a mutant protein, consisting of a 23-amino-acid deletion, resulted in constitutive activation of PI3K, implicating p85
as yet another component of the PI3K/AKT pathway that, when mutated, behaves as an oncoprotein involved in human tumorigenesis.

B. PTEN The negative regulator of the PI3K/AKT pathway, PTEN, is a tumor suppressor. PTEN normally inhibits AKT activation by dephosphorylating the phosphoinositides PIP3 and PIP2, thus suppressing tumor formation by

56

Role of AKT in Cancer

restraining PI3K/AKT signaling (Cantley and Neel, 1999; Di Cristofano and Pandolfi, 2000; Di Cristofano et al., 1998; Myers et al., 1998; Stambolic et al., 1998; Wu et al., 1998). Germ line mutations of the PTEN tumor suppressor gene are present in Cowden disease and in Bannayan-Zonana syndrome, two related hereditary cancer predisposition syndromes associated with elevated risk of breast and thyroid cancer (Liaw et al., 1997; Marsh et al., 1997). Somatic mutation and biallelic inactivation of PTEN are frequently observed in high-grade glioblastoma, melanoma, and cancers of the prostate and endometrium, among others (reviewed in Sansal and Sellers, 2004). Loss of PTEN function leads to increased concentration of PIP3, the main in vivo substrate for PTEN, resulting in constitutive activation of downstream components of the PI3K pathway, including the AKT and mTOR kinases (Di Cristofano and Pandolfi, 2000). For example, in a recent report of 103 endometrial cancers, 37 (36%) showed negative immunohistochemical staining for PTEN, and Western blot analysis revealed a significant inverse correlation between expression of PTEN and phosphorylated AKT (Terakawa et al., 2003). In addition to its lipid phosphatase function, PTEN has protein phosphatase activity. The latter biochemical function is thought to be less central to its role in tumorigenesis and, instead, is involved in the inhibition of focal adhesion formation, cell spreading and migration, as well as the inhibition of growth factorstimulated MAPK signaling (reviewed in Wu et al., 2003). To better understand the function of PTEN in vivo, Pten knockout mouse models have been generated. Heterozygous Pten (þ/
) mice develop spontaneous tumors of various histologic origins (Di Cristofano et al., 1998; Stambolic et al., 1998). Moreover, Pten inactivation dramatically enhanced the ability of embryonic stem (ES) cells to generate tumors in nude and syngeneic mice.

C. TSC2 Other exciting recent work has demonstrated that perturbations of downstream effectors of PI3K/AKT signaling can recapitulate at least some aspects of AKT’s action in tumorigenesis. A prime example involves TSC2 inactivation in patients with tuberous sclerosis syndrome. As mentioned above, AKT phosphorylates and inhibits TSC2 (Inoki et al., 2002; Potter et al., 2002), resulting in the activation of the mTOR/p70 S6K/eIF4E pathway (Li et al., 2004). Germ line mutations in either TSC2 or another tumor suppressor gene, TSC1, are the cause of this syndrome, and hamartomas developing in these individuals usually exhibit loss of the remaining normal allele. In TSC tumor cells, biallelic inactivation of TSC2 or TSC1 results in constitutive mTOR activity independent of AKT activation. In fact, experiments with mouse models have demonstrated a marked

Alfonso Bellacosa et al.

57

reduction in Akt activation in cells lacking Tsc1 or Tsc2 in response to growth factor stimulation (Zhang et al., 2003). Primary tumors from TSC patients and the Eker rat model of TSC have been shown to express elevated levels of phosphorylated mTOR and its effectors p70 S6K, S6 ribosomal protein, 4E-BP1, and eIF4G (Kenerson et al., 2002). Moreover, in the Eker rat, short-term inhibition of mTOR by the drug rapamycin was associated with a significant tumor response, including induction of apoptosis and reduction in cell proliferation.

D. eIF4E The initiation factor of translation eIF4E, a downstream effector of mTOR, also has oncogenic effects in vivo and cooperates with other cancer genes to induce tumor formation. For example, eIF4E has been shown to cooperate with c-Myc in B-cell lymphomagenesis (Ruggero et al., 2004; Wendel et al., 2004). In a transgenic mouse model in which eIF4E expression is driven by the ubiquitous -actin promoter, c-Myc was found to override eIF4E-induced cellular senescence, whereas eIF4E antagonized c-Myc-dependent apoptosis (Ruggero et al., 2004). It is noteworthy that many of the tumor types observed in these transgenic mice paralleled those observed in human cancers (e.g., lung adenocarcinomas, lymphomas) characterized by eIF4E overexpression (Seki et al., 2002; Wang et al., 1999). These and other data implicate activation of eIF4E as a key event in oncogenic transformation involving the PI3K-AKT-mTOR signaling axis.

IX. IN VIVO MODELS OF AKT ACTIVATION The generation of mouse models in which AKT is selectively overexpressed in a variety of tissues has enabled investigators to genetically define the role that this kinase family plays in vivo during neoplastic transformation. While these approaches have been instrumental in validating, in a physiological context, a plethora of molecular data and pathways derived from in vitro approaches, they have also produced a few surprising results. Moreover, they have underscored the existence of several unresolved issues, thus warranting further in-depth examination. In order to achieve constitutive activation of AKT, most groups have generated transgenic mice employing either a myristylated form of AKT1 (MyrAKT1) or a mutant in which the two AKT1 activation sites (Thr 308 and Ser 473) are mutated to aspartic acid (AKT1-DD), thus mimicking a phosphorylated residue. Both these mutants bypass the need for PIP3 for

58

Role of AKT in Cancer

activation and, thus, cannot be inhibited by PTEN. A parallel approach has been to conditionally delete Pten, thanks to the increasing availability of mouse strains expressing the Cre recombinase in a tissue-specific manner, thus circumventing the embryonic lethality associated with the whole-organism ablation of this tumor suppressor (Di Cristofano et al., 1998; Podsypanina et al., 1999; Suzuki et al., 1998) and, at the same time, obtaining the unrestrained activation of Akt. The first general conclusion that can be drawn from the analysis of these transgenic and knockout mouse models is that constitutive activation of AKT1 alone is not sufficient to produce a fully transformed phenotype in epithelial cells but can potently cooperate with other induced genetic lesions in inducing or accelerating tumor development. Complete ablation of Pten in the same tissues, however, does not always perfectly phenocopy AKT activation: in some cases, it results in a more severe phenotype and tumor development (see below). The causes of such a discrepancy are not clear. While it is possible (and likely) that the genetic background in which the mice are bred (usually FVB for transgenic mice and BL/6 or 129Sv for knockouts) affects tumor susceptibility, Pten inactivation is anticipated to affect the three endogenous Akt kinases. In addition, the possibility that loss of Pten has an effect on molecules and pathways other than Akt alone cannot be completely excluded. Side-by-side comparison of the phenotypes of syngeneic AKT transgenic mice and Pten mutants would be useful to clarify the contribution of strain differences to this phenotypic variability. A second conclusion is that some of the in vivo effects of AKT activation, in particular, those involving metabolism, protein synthesis, and cell growth, are common to most tissues in which they have been analyzed, suggesting a general role of AKT in the control of these coordinated processes. Conversely, the extent to which proliferation, apoptosis, and other cellular functions are actually affected seems to be strictly determined by the cell type and by their relative relevance in the normal organ homeostasis (see below). The mouse mammary tumor virus (MMTV) long terminal repeat (LTR) has been utilized to drive AKT1 (Ackler et al., 2002) or AKT1-DD (Hutchinson et al., 2001) expression in the mouse mammary epithelium. In both cases, analysis for up to 1 year of multiparous females (which have undergone several rounds of high transgene expression) failed to reveal tumors. However, when AKT1-DD mice were crossed to transgenic mice expressing either a Polyoma virus middle T mutant decoupled from PI3K signaling (Hutchinson et al., 2001) or a mutant ErbB-2 receptor (Hutchinson et al., 2004), tumor development was strikingly accelerated, thus underlining the importance of AKT signaling in cooperative oncogenesis. Common features in the AKT-expressing mice were a strong delay in gland involution upon cessation of lactation due to a decrease in apoptosis, an increase in the expression of differentiation markers, and an increase in proliferation associated with elevated cyclin

Alfonso Bellacosa et al.

59

D1 protein levels. Interestingly, although the expression of AKT1-DD could efficiently complement, in terms of tumor incidence and onset, the mutation in middle T that uncouples it from PI3K activation, it could not restore the metastatic ability of fully functional middle T, thus implying the existence of PI3K-dependent, AKT-independent metastatic pathways (Hutchinson et al., 2001). However, more recently, tumors developing in double transgenic mice expressing mutant ErbB-2 and AKT1-DD were also severely impaired in their metastatic potential, suggesting that the actual origin of such an effect might be the increased differentiation associated with AKT1 activation (Hutchinson et al., 2004). When MMTV-Cre mice were crossed to Pten conditional mutants, several aspects of the phenotype of AKT1-overexpressing mice were reproduced. These mutants displayed precocious gland development and differentiation, increased proliferation, and delayed involution with a dramatic reduction in apoptosis (Li et al., 2002). However, contrary to the transgenic AKT1 model, these mice developed mammary tumors between 2 and 12 months. One possible explanation for this feature is that in the transgenic mice, AKT1 is cyclically active in a time window dictated by the MMTV promoter (i.e., during lactation), while in the knockout mice, Akt is constitutively active due to Pten absence. In any case, the considerable amount of time preceding tumor formation strongly argues against the sufficiency of Pten deletion/Akt activation for tumor development. To model AKT contribution to glioblastoma pathogenesis, MyrAKT1 has been expressed via the RCAS/tv-a system in neural/glial progenitors as well as in differentiated astrocytes. This system is based on the transgenic tissue-specific expression of the avian virus receptor Tva, which has no mammalian homologues. As a consequence, receptor-expressing cells can be specifically infected by replication-competent avian leucosis virusderived vectors (RCAS) expressing the oncogene of choice. Infection of the neural/glial progenitors did not result in tumor development unless an activated Kras allele was simultaneously coexpressed (Holland et al., 2000), again underlining the fact that AKT activation is not sufficient for tumorigenesis. Multinucleated giant MyrAKT-positive cells were commonly found in these tumors. Interestingly, this combination of oncogenes was unable to transform terminally differentiated astrocytes. These cells gave rise instead to tumors with a sarcomatous phenotype after concomitant activation of Kras and deletion of Ink4a-Arf (Uhrbom et al., 2002). Notably, while coexpression of MyrAKT1 did not alter tumor frequency, it shifted the tumor phenotype toward an astrocytic one. Deletion of Pten in the brain has been achieved using mice expressing Cre in differentiated cell types such as the granule neurons of cerebellum and dentate gyrus (Backman et al., 2001; Kwon et al., 2001) and Purkinje cells (Marino et al., 2002), as well as in the whole developing brain (Groszer

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et al., 2001) and in the developing cerebellum (Marino et al., 2002). These knockout models have confirmed that loss of Pten and Akt activation (1) do not suffice in promoting tumorigenesis in these cell types, (2) cause an increase in cell size, particularly in postmitotic cells, and (3) cause an increase in proliferation and a decrease in apoptosis in actively proliferating precursor cells, but not in terminally differentiated cells. Strikingly, the neuronal hypertrophy was reversed by treatment with the mTOR inhibitor CCI-779, consistent with the Akt/mTor pathway being directly responsible for the soma size increase in the mutant neurons (Kwon et al., 2003). Transgenic mice expressing MyrAKT1 in the ventral prostate under the control of the rat probasin promoter developed PIN-like lesions by 8 weeks of age (Majumder et al., 2003). These lesions resulted from both an increase in cell number and an increase in cell size. The increase in cell number must be the consequence of the modest proliferation increase, as spontaneous apoptosis levels were similar in transgenic and control mice (Majumder et al., 2004). Although the complete penetrance of this phenotype and the early age at which it could be detected support the notion that AKT1 activation is sufficient for PIN development, these mice, however, never developed invasive cancer, even when analyzed at 1.5 years of age. In this model, most of the consequences of bearing an activated AKT1 allele depend on the activation of mTor, because its inhibition could completely reverse the hyperplastic phenotype, not only reducing cell size and proliferation but also actively inducing apoptosis in the cell population that had lost contact with the basement membrane (Majumder et al., 2004). Tissuespecific deletion of Pten, instead, resulted not only in the appearance of PIN by 6 weeks of age, but also in the development of invasive and metastatic cancer starting at about 9 weeks (Backman et al., 2004; Trotman et al., 2003; Wang et al., 2003). Again, as in the transgenic model, an increase in both cell size and proliferation characterized the mutant cells. It is not clear why the knockout model developed tumors and the transgenic did not. In addition to the possibility of insufficient levels of AKT1 activation in the transgenic prostates, it is conceivable that loss of Pten may affect Akt-independent pathways. Finally, the issue of genetic background should not be underestimated, as initial crosses of the transgenic model (in the FVB strain) with BL/6 mice resulted in increased proliferation of the prostatic epithelium (Majumder et al., 2003). Injection of an adenoviral construct expressing MyrAKT1 into the mouse tail vein resulted, after only 4 days, in massive hepatocyte infection, hepatomegaly, and fatty degeneration of the liver (Ono et al., 2003). Similarly, tissue-specific deletion of Pten resulted in hyperplastic, fatty degeneration of the liver and increased proliferation (Horie et al., 2004). By 40 weeks, 50% of the mutant mice showed microscopic adenomas, and by 78 weeks all the mice had developed adenomas and carcinomas. In the pancreas, overexpression of MyrAKT1 under the control of the rat insulin II promoter resulted in

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islet hyperplasia but not in transformation of the -cells (Tuttle et al., 2001). When the RCAS/tv-a approach was directed to the mouse ovarian surface epithelium, no tumors were detected unless the expression of MyrAKT1 was accompanied by the expression of activated Kras or c-myc, in a Tp53-null background (Orsulic et al., 2002). Once more, these in vivo models support the hypothesis that AKT activation is not sufficient for tumor development, at least in the context of epithelial tumorigenesis that may require multiple, independent mutations of oncogenes and tumor suppressor genes (Bellacosa, 2003). Indeed, the non-epithelial model of transgenic mice in which MyrAKT1 expression was driven by the lck promoter and directed to the early stages of thymocyte development represents the only example of AKT transgenic mutants that developed tumors spontaneously. Transgenic lines expressing high levels of MyrAKT1 developed aggressive lymphomas within 10–20 weeks (Malstrom et al., 2001; Rathmell et al., 2003), while lines with lower levels of expression developed autoimmune features followed by lymphoma (Malstrom et al., 2001; Rathmell et al., 2003). The transgenic T-cells were characterized by increased size, increased proliferation, and resistance to apoptosis. Similarly, 70% of transgenic mice expressing a constitutively active version of AKT2 (MyrAKT2) under the control of the lck promoter were reported to develop lymphomas in a 600-day follow-up (Mende et al., 2001). Along the same lines, both T-cell-specific complete Pten ablation (Suzuki et al., 2001) and, to a certain extent, whole-body heterozygous Pten mutation (Di Cristofano et al., 1999) resulted in defective thymic-negative selection, reduced apoptosis, and increased proliferation as well as development of autoimmunity and lymphomas. It is conceivable that, due to their high proliferation rates and their unique dependence on strictly controlled apoptosis, as well as the lack of complex architectural features typical of epithelial tissues (Bellacosa, 2003), T-cells represent a more “favorable” environment for the oncogenic activity of activated AKT. Nevertheless, the time required for tumor development still suggests that additional genetic events are required for full transformation.

X. IMPLICATIONS OF AKT PATHWAY ACTIVATION FOR THERAPEUTIC TARGETING A. Rationale for Targeting the AKT Pathway for New Drug Discovery Efforts Because AKT and its upstream regulators are activated or deregulated in a wide range of tumors and play critical roles in many processes that are considered hallmarks of cancer (e.g., abnormal proliferation, evading

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apoptosis, invasion, and angiogenesis), approaches to target the AKT signaling pathway have been the subject of intense research efforts in major pharmaceutical and academic institutions. Components of the AKT signaling pathway are attractive targets for therapeutic intervention for the following reasons: (1) since AKT signaling promotes cell survival, proliferation, and invasion, blocking this pathway could inhibit the proliferation of tumor cells and either induce an apoptotic response or sensitize tumors to undergo apoptosis in response to other cytotoxic agents; (2) many components of this pathway are kinases, one of the “druggable” classes of targets; and (3) as activation of this pathway is seen in a wide variety of tumors, drugs targeting this pathway are likely to have wide therapeutic utility. Successful characterization or identification of tumors exhibiting hyperactive AKT signaling is a prerequisite to the targeting of this pathway. The development of high-quality antibodies against Ser 473-phosphorylated AKT1, which closely parallels AKT phosphorylation/activation, has made it possible to measure activation of this pathway using in situ histochemical staining methods (Fig. 5). The rationale for targeting the AKT signaling pathway for development of anticancer therapeutics comes from a number of studies: (1) expression of

Fig. 5 Immunohistochemistry showing activation of the AKT pathway in human lung tissues. (A) phospho-AKT/phospho-mTOR/phospho-FKHR staining of serial sections of a human nonsmall cell lung carcinoma, using phospho-specific antibodies. Note intense staining of tumor cells but little staining of non-malignant stroma. (B) Immunostaining of precancerous lung lesions using a phospho-specific pan-AKT antibody. Normal bronchial epithelium (left panel) shows no staining, whereas staining is observed in areas corresponding to metaplasia (middle) and dysplasia (right).

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AKT was shown to enhance IGF-1-mediated neuronal survival, whereas expression of a dominant negative allele of AKT enhanced apoptosis and blocked the antiapoptotic effect of IGF-1 (Dudek et al., 1997; Kulik et al., 1997); (2) constitutively active AKT constructs have been shown to protect cells from PTEN-mediated apoptosis and also reduce the sensitivity of tumor cells to proapoptotic and/or cytotoxic agents (Li et al., 1998; Whang et al., 2004); (3) reintroduction of PTEN into tumor cells that are mutant for PTEN deactivated AKT, leading to either cell cycle arrest (glioblastomas, renal cell carcinoma lines) (Lu et al., 1999) or apoptosis (breast and prostate cancer cell lines) (Di Cristofano and Pandolfi, 2000; Saito et al., 2003; Stambolic et al., 1998; Xu et al., 1999); (4) expression of antisense AKT2 RNA in PANC1 cells significantly reduced tumorigenicity in nude mice (Cheng et al., 1996); (5) expression of a dominant negative mutant of AKT, using an adenoviral vector system, induced apoptosis selectively in tumor cells expressing activated AKT but not in normal cells or other tumor cells expressing low levels of activated AKT (Jetzt et al., 2003); and (6) similarly, expression of PTEN induced selective apoptosis in tumor cell lines in which PTEN is inactivated but not in tumor cells that are wild type for PTEN expression (Jetzt et al., 2003; Xu et al., 1999). In addition, the growth of tumor cells in a mouse model was also significantly inhibited by intratumoral injection of a virus expressing dominant negative AKT (Jetzt et al., 2003). These studies show that tumor cells, unlike normal cells, are dependent on activated AKT for survival and are sensitive to inhibition of its activity. This specificity suggests that inhibition of AKT signaling may not be toxic to normal cells. These studies also validate the therapeutic concept that AKT inhibition elicits a selective antitumor effect and provide support for the development of small molecule inhibitors.

B. Role of AKT in the Therapeutic Response of Tumor Cells A special area of application of AKT inhibition is that of therapeutic response. In medical oncology, chemoresistance is a major hurdle for successful cancer therapy. AKT is a major mediator of survival signals that protect cells from undergoing apoptosis and, thus, is a potentially important therapeutic target. For example, transfection of constitutively active AKT into human cancer cells has been shown to inhibit the cytotoxic effects of the topoisomerase I inhibitor topotecan (Nakashio et al., 2000). Furthermore, ovarian cancer cell lines with either constitutive AKT1 activity or AKT2 gene amplification have been shown to be highly resistant to paclitaxel compared to cells with low AKT levels (Page et al., 2000). In vitro and

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in vivo ovarian cancer models that combined the PI3K inhibitor LY294002 with paclitaxel increased the efficacy of chemotherapy on tumor growth and dissemination compared to either agent alone (Hu et al., 2002). Moreover, PI3K inhibition in combination with paclitaxel markedly reduced ascites formation, which is often associated with ovarian carcinomas (Hu et al., 2002; Page et al., 2000). PI3K inhibitors selectively increased apoptosis in tumor cells expressing high levels of activated AKT but not in tumor cells with low levels of activated AKT (Altomare et al., 2004; Brognard et al., 2001; Clark et al., 2002). Proof of the principle that agents that target the AKT signaling pathway may have significant antitumor activity comes from studies using rapamycin and its analogues, which target mTOR kinase (see Section X.C).

C. Molecular Targets in the AKT Signaling Pathway Approaches for targeting the AKT signaling pathway include targeting receptor tyrosine kinases as well as PI3K, PDK1, AKT, and mTOR kinases. Receptor tyrosine kinases, which are upstream of the AKT signaling cascade, are promising targets to block this pathway. For example, tumors overexpressing Her-2/neu display constitutive activation of AKT (Bacus et al., 2002). It is likely that receptor tyrosine kinase inhibitors such as Herceptin, which blocks the HER-2/neu receptor, Iressa and Tarceva, which inhibit the EGF receptor tyrosine kinase, and Imatinib (Gleevec), an inhibitor of BCR-ABL, KIT, and PDGF receptor, achieve their antitumor effects at least in part by shutting off upstream signaling to the PI3K/AKT pathway. However, the efficacy of receptor tyrosine kinase inhibitors could be hampered by activating mutations or gene amplifications affecting downstream signaling components or by the loss of the PTEN tumor suppressor. Indeed, it has been shown that activation of AKT through loss of PTEN can confer resistance to Iressa by setting a higher threshold of AKT activity, and sensitivity can be achieved by restoring PTEN function (She et al., 2003). One approach to blocking the AKT signaling pathway is to target PI3K itself. The PI3K inhibitors LY2940002 and wortmannin have been used extensively as research tools. Both have demonstrated marked antitumor activity, particularly in PTEN-null cells or in cells overexpressing PI3K (Hu et al., 2002). Since PI3K is highly pleiotropic, it is likely that administration of PI3K inhibitors would likely be associated with significant toxicities. In addition, these compounds inhibit a host of kinases related to PI3K, including ATM and ATR (Sarkaria et al., 1998). Recent elucidation of the crystal structure of the PI3K catalytic subunit p110
should aid in the development of isoform-specific inhibitors that will spare other PI3K isoforms, such as

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class I PI3K, downstream of G protein-coupled receptors and, thus, affect fewer cellular processes (Djordjevic and Driscoll, 2002). Kinase inhibitors that target PDK1 would also be effective in blocking AKT activation in tumor cells. PDK1 is required for normal embryonic development, as mice embryos lacking Pdk1 die at day E9.5 (Lawlor et al., 2002). Pdk1 hypomorphic mice, in which a neomycin resistance gene was inserted into an intron of the Pdk1 gene that results in a 90% reduction of Pdk1 expression in all tissues, are viable (Lawlor et al., 2002). These mice display no obvious harmful phenotype, suggesting that an inhibitor of human PDK1 would not be highly toxic or harmful. Antisense-mediated depletion of PDK1 in human glioblastoma cells lacking expression of PTEN was shown to markedly reduce their proliferation and survival (Flynn et al., 2000). Moreover, overexpression of PDK1 in mammary epithelial cells induced their transformation by permitting their anchorage-independent growth in soft agar (Xie et al., 2003). Taken together, these observations suggest that an inhibitor of PDK1 might be beneficial for treatment of cancer cells possessing activation of the AKT pathway. One advantage of targeting PDK1 is that there is only one isoform of PDK1 to target for small molecule drug discovery efforts. The most potent PDK1 inhibitor is 7-hydroxystaurosporine (UCN-01), which inhibits PDK1 with an IC50 of 5 nM (Sato et al., 2002). UCN-01 inhibits the growth and induces apoptosis of many cancer cell lines and is currently in clinical trials for cancer patients, with positive results reported in phase I. Unfortunately, UCN-01 is a non-specific kinase inhibitor that inhibits many other kinases with a potency similar to that for PDK1 (Graves et al., 2000). Recently, the high-resolution crystal structures of UCN-01 and staurosporine in complex with the kinase domain of PDK1 have been reported (Biondi et al., 2002). UCN-01 and staurosporine bind to PDK1 in a similar fashion, but the 7-hydroxy group present in UCN-01 and absent in staurosporine generates additional hydrophobic contacts with active site residues (Komander et al., 2003). The elucidation of the binding mode of these inhibitors with PDK1 may be useful for the design of specific inhibitors. However, thus far no specific inhibitor of PDK1 has been reported.

D. Targeting AKT Kinases AKT family members are expressed differentially in different tissues, and different AKTs are overexpressed in different tumors (Vivanco and Sawyers, 2002). There is relatively little evidence for a tumor type-specific pattern of expression for a given AKT family member, and there is no evidence that proliferative and/or anti-apoptotic effects of AKT signaling are mediated

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by one or more AKT kinases. Optimal suppression of AKT signaling may require simultaneous blockade of all three AKT family members. In addition, recent studies have compared the regulatory and catalytic properties of the three AKT family members. AKT1, AKT2, and AKT3 were phosphorylated and activated by PDK1 at similar rates and to similar degrees (Walker et al., 1998). After activation, the specific activities of each AKT family member toward a variety of synthetic peptide substrates were very similar. Comparison of ATP binding regions of all three AKTs by homology modeling suggests that designing an inhibitor that targets the ATP binding site of a specific AKT family member will be challenging. Moreover, such a selective AKT inhibitor may have narrow therapeutic utility, being limited to tumors that exhibit upregulation of only that AKT family member. Therefore, pan-AKT inhibitors would likely have wider therapeutic utility. A number of non-selective small molecule inhibitors that block AKT signaling by unknown mechanisms have been described. Curcumin, a plant-derived pigment, was shown to inhibit prostate tumor cell growth by blocking activation of AKT (Chaudhary and Hruska, 2003). In addition, a compound synthesized from the natural plant compound rotenone (degeulin) has been shown to inhibit activation of AKT and inhibit malignant human bronchial epithelial cell proliferation. Rotenone was shown to induce cell cycle arrest in G2/M and apoptosis in malignant but not normal bronchial epithelial cells, suggesting a potential therapeutic window (Chun et al., 2003). Another recent study identified a small molecule inhibitor of AKT activation in tumor cells, referred to as API-2 (AKT/PKB signaling inhibitor2) (Yang et al., 2004). This compound was identified by screening the National Cancer Institute (NCI) diversity set, using a cell-based assay. API-2 treatment induced apoptosis selectively in tumor cells expressing activated Akt and potently inhibited tumor growth in nude mice. Another strategy for targeting AKT kinase is to disrupt the binding of its PH domain to PIP3 and prevent its membrane translocation and activation by PDK1. Novel analogues of the PIP3 phosphoinositide ring have been shown to be effective inhibitors in cell culture (Kozikowski et al., 2003). These inhibitors are also likely to block binding of PIP3 to other PH domain-containing proteins. Perifosin, a novel alkylphospholipid, was shown to inhibit AKT activation by interfering with membrane localization (Kondapaka et al., 2003). Recently, another AKT inhibitor, 1L-6-hydroxy-methylchiro-inositol 2(R)-2O-methyl-3-O-octadecylcarbonate, was described and shown to reduce resistance of leukemic cells to chemotherapeutic agents and ionizing radiation (Martelli et al., 2003). All major pharmaceutical companies have preclinical drug discovery efforts targeting Akt kinase. To date, no compound has entered clinical trials.

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E. Targeting mTOR A number of pharmaceutical companies are pursuing the clinical development of inhibitors of mTOR. Rapamycin, a bacterially derived natural product known to inhibit mTOR, is approved for preventing allograft rejection in organ transplantation due to its potent inhibition of T-cell activation (Vezina et al., 1975). The natural products program at the NCI identified rapamycin as a potential anticancer agent (Douros and Suffness, 1981). Rapamycin exerts its action by first binding to the immunophilin FK506 binding protein, FKBP12, which then binds to mTOR and thereby prevents phosphorylation of downstream targets such as S6K and 4E-BP1 (Bjornsti and Houghton, 2004; Sansal and Sellers, 2004). Rapamycin was shown to induce G1 arrest in various tumor cell lines at low nanomolar concentrations that closely matched that required for biochemical inhibition of mTOR in cells (Neshat et al., 2001). There are also examples in which rapamycin induced apoptotic responses in several tumor cell lines in vitro and in vivo (Huang et al., 2004; Majumder et al., 2003; Wendel et al., 2004). As noted earlier, rapamycin and its analogues CCI-779 and RAD001 have been shown to reduce tumor growth in vivo in Pten heterozygous mice and in mice carrying xenografted human tumor cells (Neshat et al., 2001; Podsypanina et al., 2001); furthermore, RAD001 treatment for 2 weeks eradicated all signs of PIN lesions in a transgenic mouse model (Majumder et al., 2003). RAD001 induced programmed cell death in the transgenic mice, whereas in xenograft models, this compound only inhibited proliferation (Mellinghoff and Sawyers, 2004). The studies with the transgenic PIN model suggest that mTOR mediates critical aspects of Akt-driven tumorigenesis and cell survival. Given the number of AKT-regulated signals that prevent apoptosis independent of mTOR, this prominent role of mTOR in mediating antiapoptotic signals was a surprise. In another recent study, investigators used a mouse model of B-cell lymphoma, a cancer of antibody-producing B-cells, to explore the role of Akt and mTor in cell survival and drug resistance (Wendel et al., 2004). To determine how Akt signaling influences tumorigenesis and treatment responses in vivo, the effects of a constitutively activated Akt mutant were compared to that of the antiapoptotic regulator Bcl-2 in a E-Myc model of B-cell lymphoma. E-Myc hematopoietic stem cells were transduced with Akt- or Bcl-2-overexpressing retroviruses and transplanted into lethally irradiated mice, and the recipients were monitored for lymphoma onset and pathology. Both Akt and Bcl-2 had an identical effect on c-Myc-induced lymphomagenesis in that they caused rapid onset of aggressive, multidrugresistant lymphomas. Tumors that overexpressed Akt to survive were refractory to conventional chemotherapeutic agents such as doxorubicin

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or cytoxan when these agents were used alone. Similarly, rapamycin alone had little effect on lymphomas of any genotype. However, in combination with chemotherapy, rapamycin induced massive apoptosis and lasting remissions without increased toxicity. In contrast, tumors that depended on Bcl-2 were resistant to chemotherapy-induced death by rapamycin. The fact that rapamycin, a specific inhibitor of mTOR, potently reversed Akt survival signaling suggested that control of mRNA translation may be important for Akt-mediated survival. These results show that rapamycin combined with chemotherapy can be synergistic in eliminating tumor cells exhibiting activation of the AKT pathway. Rapamycin has also been shown to synergize with Gleevec against BCR-ABL-transformed myeloid and lymphoid cells and increase survival in a murine CML model (Mohi et al., 2004). Rapamycin/Gleevec combinations also inhibited Gleevec-resistant mutants of BCR-ABL, and rapamycin plus the protein kinase inhibitor PKC412 similarly synergistically inhibited cells expressing PKC412-sensitive or -resistant leukemogenic FLT3 mutants (Mohi et al., 2004). Addition of a mitogen-activated protein kinase inhibitor to rapamycin or rapamycin plus protein kinase inhibitor further increased efficacy. These results suggested that simultaneous targeting of more than one signaling pathway activated by leukemogenic protein tyrosine kinases may improve the treatment of primary and relapsed CML and/or acute myelogenous leukemia caused by FLT3 mutations. These results suggest that similar strategies may be useful for treating solid tumors associated with mutant and/or overexpressed protein kinases. Clinical development of rapamycin was slow because of stability and solubility problems. Synthesis of analogues with superior solubility and stability properties has led to clinical trials with CCI-779 (Wyeth Research), RAD001 (Novartis), and AP 25373 (Ariad). Rapamycin and CCI-779 have also shown preliminary evidence of clinical antitumor activity as a monotherapy, with minor antitumor responses and/or prolonged (>4 months) disease stabilization in several drug-refractory cancers, including breast cancer, soft-tissue sarcoma, and cervical, uterine, and renal carcinomas (Dancey, 2002; Hidalgo, 2004; Sawyers, 2003). Phase II results have shown that CCI-779 is effective against renal cell carcinoma with a 7% objective response rate, minor responses in 29%, and stable disease in 40% of treated patients (Atkins et al., 2004). Based on these data, a phase III trial comparing CCI-779 with interferon-
or the combination of these two agents has been initiated. The preliminary evidence from these studies is that CCI-779 is well tolerated and active in renal cell carcinoma. Phase II trials are also under way in glioma, prostate, and metastatic breast cancers, renal carcinoma, lymphoma, melanoma, and small cell lung cancer. Pharmacodynamic markers to monitor drug-target inhibition have been developed. Inhibition of S6K1 activity in peripheral-blood mononuclear cells has been shown

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to correlate well with tumor inhibition in preclinical animal models (Boulay et al., 2004). The preliminary clinical results indicate that rapamycin analogues have promising antitumor activity even as monotherapy in a range of doses associated with relatively minor toxicities. These studies have yet to report the expression status of PTEN or the activation status of AKT or mTOR in the patients participating in these trials. A reasonable prediction, based on preclinical animal models and cell-based assays, is that rigorous patient selection might lead to greater efficacy with lower toxicity. In summary, the AKT signaling pathway is a key player in mediating tumor cell survival and escape from apoptosis, and components of this pathway have emerged as promising new targets for the development of cancer therapeutics. AKT activation is also linked to drug resistance in many cancers, and targeting this pathway can restore drug sensitivity. Preliminary results indicate that inhibitors targeting this pathway may synergize in vivo with other cytotoxic agents and targeted therapeutics under development. The future holds great promise for the development of selective novel anticancer agents specifically targeting components of this pathway.

F. Liabilities Associated with Targeting the AKT Signaling Pathway As AKT signaling is activated by a number of growth factors and is involved in proliferation and survival of normal cells, inhibition of AKT signaling may also affect normal cellular functions. Hence, for agents targeting this pathway to be effective as therapeutic agents, a reasonable therapeutic window would depend on tumors being more sensitive to inhibitors of this pathway than normal tissues. At this time, there are no reports of specific AKT inhibitors that are active against tumors in vivo and devoid of major toxicities to normal tissues. However, our recent studies using an AKT kinase-dead mutant in an adenoviral vector system indicated that blocking AKT signaling induced selective apoptosis in tumor cells expressing activated AKT and had very little effect on normal and tumor cells expressing low levels of activated AKT (Boulay et al., 2004). A similar lack of significant toxicity was observed in in vivo studies using the AKT inhibitor API-2 (Yang et al., 2004). This specificity suggests that AKT inhibition may not be toxic to normal cells, an important consideration for the development of inhibitors of this pathway. Thus, there is reasonable optimism that blocking AKT signaling may not have severe toxic side effects in normal tissues. Results from knockout animal models suggest that complete inhibition of individual AKT family members may not have severe side effects to normal tissues. Thus, Akt1- and Akt2-null mice are viable, suggesting that inhibition of individual AKT family members is achievable without severe toxic effects

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(Chen et al., 2001; Cho et al., 2001). However, mice lacking Akt1 and Akt2 show extreme growth deficiency and die shortly after birth (Peng et al., 2003). These mice have a defect in cell proliferation, suggesting a crucial threshold of Akt activity for normal cell growth and offspring viability. It should be emphasized that the phenotypes of Akt-null mice represent the cumulative impact of the absence of Akt function throughout embryonic development, in contrast to the setting of pharmacological inhibition in cancer patients where AKT function may be redundant for the survival and function of normal tissues. It is likely that AKT inhibitors will have less severe side effects than those suggested by the double knockout animal models. An important clinical precedent in support of this hypothesis is the fact that Gleevec, which blocks the activity of three different kinases (BCR-ABL, KIT, and PDGF receptor), has a highly favorable profile of manageable side effects (Reith et al., 1991; Soriano, 1997; Tybulewicz et al., 1991). However, the targeted inactivation of each one of the respective genes resulted in lethal phenotypes, suggesting that knockout mouse models may overestimate the severity of clinical side effects of specific inhibitors. In addition, clinical use of small molecule inhibitors of AKT may allow more flexibility in terms of the degree of AKT inhibition. It is likely that complete and sustained inhibition of AKT activity may not be required to achieve efficacy, and inhibitors can be applied intermittently to alleviate the severity of treatment-related side effects. Due to the critical role of AKT in insulin signaling and maintenance of glucose homeostasis, AKT inhibitors may have potential liabilities with regard to glucose metabolism (Whiteman et al., 2002). The expression and translocation of glucose transporters in insulin-responsive tissues, as well as activation of glycogen synthesis via inhibition of GSK3, are regulated by AKT (Brazil et al., 2004). Studies with rodent cells using siRNAs showed that ablation of Akt2 dramatically reduced insulin-stimulated glucose uptake, whereas siRNAs targeted to Akt1 and Akt2 are equipotent in reducing insulin-stimulated glycogen synthesis (Jiang et al., 2003; Katome et al., 2003). Results using knockout animal models suggest that the Akt2 kinase specifically is critical for the maintenance of glucose homeostasis. Akt2-null mice have defects in glucose homeostasis and exhibit insulin resistance and diabetes in addition to age-dependent loss of adipose tissue (Cho et al., 2001). Recent studies have identified a kindred that exhibits early-onset diabetes due to a mutation in AKT2 that renders it devoid of kinase activity (George et al., 2004). While heterozygous Akt2 mice show little alteration in metabolic phenotype and Akt2-null mice exhibit only a moderate degree of insulin resistance, humans heterozygous for the AKT2 mutation exhibit extreme hyperinsulinemia and insulin resistance. This may result from the dominant negative effect of the kinase-dead mutant on endogenous AKT family members. These results suggest that

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inhibition of AKT activity in humans may be associated with the development of a diabetic phenotype. Hence, clinical development of AKT inhibitors must include close monitoring of glucose levels, glucose tolerance, and hyperinsulinemia in patients undergoing treatment.

XI. CONCLUSIONS Knowledge about the AKT kinases has accumulated at an increasing pace over the past 15 years, a reflection of both the availability of critical research reagents and the recognized central role played by these molecules in a variety of physiological and pathological states. The exponential increase in knowledge is the consequence of an impressive array of methodological approaches, ranging from basic biochemistry and structural biology to cell biology, animal models, and human cancer. From this standpoint, it is safe to say that the AKT pathway is one of the signaling modules most characterized to date. There is now a wealth of information from studies of many cancer types indicating that AKT activation is one of the most common molecular alterations associated with human malignancy. New observations continue to emerge from the analysis of this pathway. Two examples are the convergence of tumor suppressors involved in three hereditary hamartomatous syndromes (PTEN, TSC1/TSC2, LKB1) with the oncogenic PI3K/AKT/mTOR axis and the intersection of cell proliferation and metabolic control via AMPK. The critical knowledge of the complex interactions within the AKT pathway along with detailed information on the structural features of individual AKT kinases and other associated signaling molecules are the best guarantee for the development of inhibitors/modulators for cancer therapy. The large-scale efforts at numerous pharmaceutical and academic institutions will face the problems of toxicity associated with AKT inhibition, a consequence of the several cellular functions modulated by these kinases. A potential approach to overcome or limit liabilities associated with AKT inhibition will involve the specific targeting of each AKT family member. Alternatively, it may be necessary to develop optimized combination regimens that target components of the AKT pathway, including specific downstream effectors of AKT, in a tailored fashion reflecting the altered circuitry in a given cancer. On the other hand, it is possible to envision novel therapeutic strategies that may target the metabolic dysregulation brought about by AKT activation. While it is difficult to anticipate which of these “working hypotheses” for AKT-based therapy will have the most merit and prove successful in the long term, it is not difficult to predict that many basic discoveries will

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continue to be made by investigators aiming to elucidate the mechanistic intricacies of the AKT pathway and its role in cancer.

ACKNOWLEDGMENTS The authors thank Drs. Vince Madison, William Windsor, and Philip Tsichlis for helpful discussions and critical review of the manuscript, Dr. Xiao Li for providing figures of homology models of AKT family members, Drs. Andres Klein-Szanto and Binaifer Balsara for preparing immunohistochemistry figures, and Kathryn Ireton and Rose Sonlin for secretarial assistance. This work was supported by NIH Grants CA105008, CA77429, CA83638 (SPORE in Ovarian Cancer), CA105008, CA097097, and CA06927 and by an appropriation from the Commonwealth of Pennsylvania to the Fox Chase Cancer Center.

REFERENCES Ackler, S., Ahmad, S., Tobias, C., Johnson, M. D., and Glazer, R. I. (2002). Delayed mammary gland involution in MMTV-AKT1 transgenic mice. Oncogene 21, 198–206. Ahmed, N. N., Franke, T. F., Bellacosa, A., Datta, K., Gonzalez-Portal, M. E., Taguchi, T., Testa, J. R., and Tsichlis, P. N. (1993). The proteins encoded by c-akt and v-akt differ in posttranslational modification, subcellular localization and oncogenic potential. Oncogene 8, 1957–1963. Alessi, D. R., and Cohen, P. (1998). Mechanism of activation and function of protein kinase B. Curr. Opin. Genet. Dev. 8, 55–62. Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings, B. A. (1996). Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15, 6541–6551. Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol. 7, 261–269. Alkan, S., and Izban, K. F. (2002). Immunohistochemical localization of phosphorylated AKT in multiple myeloma. Blood 99, 2278–2279. Altomare, D. A., Tanno, S., De Rienzo, A., Klein-Szanto, A., Skele, K. L., Hoffman, J. P., and Testa, J. R. (2003). Frequent activation of AKT2 kinase in human pancreatic carcinomas. J. Cell. Biochem. 87, 470–476. Altomare, D. A., Wang, H. Q., Skele, K. L., Rienzo, A. D., Klein-Szanto, A. J., Godwin, A. K., and Testa, J. R. (2004). AKT and mTOR phosphorylation is frequently detected in ovarian cancer and can be targeted to disrupt ovarian tumor cell growth. Oncogene 23, 5853–5857. Aoki, K., Tamai, Y., Horiike, S., Oshima, M., and Taketo, M. M. (2003). Colonic polyposis caused by mTOR-mediated chromosomal instability in Apcþ/Delta716 Cdx2þ/- compound mutant mice. Nat. Genet. 35, 323–330. Arboleda, M. J., Lyons, J. F., Kabbinavar, F. F., Bray, M. R., Snow, B. E., Ayala, R., Danino, M., Karlan, B. Y., and Slamon, D. J. (2003). Overexpression of AKT2/protein kinase Bbeta leads

Alfonso Bellacosa et al.

73

to up-regulation of beta1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res. 63, 196–206. Arranz, E., Robledo, M., Martinez, B., Gallego, J., Roman, A., Rivas, C., and Benitez, J. (1996). Incidence of homogeneously staining regions in non-Hodgkin lymphomas. Cancer Genet. Cytogenet. 87, 1–3. Atkins, M. B., Hidalgo, M., Stadler, W. M., Logan, T. F., Dutcher, J. P., Hudes, G. R., Park, Y., Liou, S. H., Marshall, B., Boni, J. P., Dukart, G., and Sherman, M. L. (2004). Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J. Clin. Oncol. 22, 909–918. Avdulov, S., Li, S., Michalek, V., Burrichter, D., Peterson, M., Perlman, D. M., Manivel, J. C., Sonenberg, N., Yee, D., Bitterman, P. B., and Polunovsky, V. A. (2004). Activation of translation complex eIF4F is essential for the genesis and maintenance of the malignant phenotype in human mammary epithelial cells. Cancer Cell 5, 553–563. Backman, S. A., Stambolic, V., Suzuki, A., Haight, J., Elia, A., Pretorius, J., Tsao, M. S., Shannon, P., Bolon, B., Ivy, G. O., and Mak, T. W. (2001). Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease. Nat. Genet. 29, 396–403. Backman, S. A., Ghazarian, D., So, K., Sanchez, O., Wagner, K. U., Hennighausen, L., Suzuki, A., Tsao, M. S., Chapman, W. B., Stambolic, V., and Mak, T. W. (2004). Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten. Proc. Natl. Acad. Sci. USA 101, 1725–1730. Bacus, S. S., Altomare, D. A., Lyass, L., Chin, D. M., Farrell, M. P., Gurova, K., Gudkov, A., and Testa, J. R. (2002). AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival. Oncogene 21, 3532–3540. Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney, P., Currie, R., Downes, C. P., and Alessi, D. R. (1999). PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr. Biol. 9, 393–404. Balsara, B. R., Pei, J., Mitsuuchi, Y., Page, R., Klein-Szanto, A., Wang, H., Unger, M., and Testa, J. R. (2004). Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis 25, 2053–2059. Bellacosa, A. (2003). Genetic hits and mutation rate in colorectal tumorigenesis: Versatility of Knudson’s theory and implications for cancer prevention. Genes Chromosomes Cancer 38, 382–388. Bellacosa, A., Testa, J. R., Staal, S. P., and Tsichlis, P. N. (1991). A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254, 274–277. Bellacosa, A., de Feo, D., Godwin, A. K., Bell, D. W., Cheng, J. Q., Altomare, D. A., Wan, M., Dubeau, L., Scambia, G., Masciullo, V., Ferrandina, G., Benedetti Panici, P., Mancuso, S., Neri, G., and Testa, J. R. (1995). Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int. J. Cancer 64, 280–285. Bellacosa, A., Chan, T. O., Ahmed, N. N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J., and Tsichlis, P. (1998). Akt activation by growth factors is a multiple-step process: The role of the PH domain. Oncogene 17, 313–325. Bellacosa, A., Testa, J. R., Moore, R., and Larue, L. (2004). A portrait of AKT kinases: Human cancer and animal models depict a family with strong individualities. Cancer Biol. Ther. 3, 267–275. Biondi, R. M., Komander, D., Thomas, C. C., Lizcano, J. M., Deak, M., Alessi, D. R., and van Aalten, D. M. (2002). High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site. EMBO J. 21, 4219–4228.

74

Role of AKT in Cancer

Bjornsti, M. A., and Houghton, P. J. (2004). Lost in translation: Dysregulation of cap-dependent translation and cancer. Cancer Cell 5, 519–523. Blackhall, F. H., Pintilie, M., Michael, M., Leighl, N., Feld, R., Tsao, M. S., and Shepherd, F. A. (2003). Expression and prognostic significance of kit, protein kinase B, and mitogen-activated protein kinase in patients with small cell lung cancer. Clin. Cancer Res. 9, 2241–2247. Boudeau, J., Sapkota, G., and Alessi, D. R. (2003). LKB1, a protein kinase regulating cell proliferation and polarity. FEBS Lett. 546, 159–165. Boulay, A., Zumstein-Mecker, S., Stephan, C., Beuvink, I., Zilbermann, F., Haller, R., Tobler, S., Heusser, C., O’Reilly, T., Stolz, B., Marti, A., Thomas, G., and Lane, H. A. (2004). Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells. Cancer Res. 64, 252–261. Brazil, D. P., Yang, Z. Z., and Hemmings, B. A. (2004). Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem. Sci. 29, 233–242. Brognard, J., Clark, A. S., Ni, Y., and Dennis, P. A. (2001). Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res. 61, 3986–3997. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999). Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868. Byun, D. S., Cho, K., Ryu, B. K., Lee, M. G., Park, J. I., Chae, K. S., Kim, H. J., and Chi, S. G. (2003). Frequent monoallelic deletion of PTEN and its reciprocal association with PIK3CA amplification in gastric carcinoma. Int. J. Cancer 104, 318–327. Cantley, L. C. (2002). The phosphoinositide 3-kinase pathway. Science 296, 1655–1657. Cantley, L. C., and Neel, B. G. (1999). New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA 96, 4240–4245. Carling, D. (2004). The AMP-activated protein kinase cascade–a unifying system for energy control. Trends Biochem. Sci. 29, 18–24. Chakravarti, A., Zhai, G., Suzuki, Y., Sarkesh, S., Black, P. M., Muzikansky, A., and Loeffler, J. S. (2004). The prognostic significance of phosphatidylinositol 3-kinase pathway activation in human gliomas. J. Clin. Oncol. 22, 1926–1933. Chan, T. O., and Tsichlis, P. N. (2001). PDK2: A complex tail in one Akt. Sci STKE 2001, PE1. Chan, T. O., Rittenhouse, S. E., and Tsichlis, P. N. (1999). AKT/PKB and other D3 phosphoinositide-regulated kinases: Kinase activation by phosphoinositide-dependent phosphorylation. Annu. Rev. Biochem. 68, 965–1014. Chang, H. W., Aoki, M., Fruman, D., Auger, K. R., Bellacosa, A., Tsichlis, P. N., Cantley, L. C., Roberts, T. M., and Vogt, P. K. (1997). Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 276, 1848–1850. Chaudhary, L. R., and Hruska, K. A. (2003). Inhibition of cell survival signal protein kinase B/ Akt by curcumin in human prostate cancer cells. J. Cell. Biochem. 89, 1–5. Chen, W. S., Xu, P. Z., Gottlob, K., Chen, M. L., Sokol, K., Shiyanova, T., Roninson, I., Weng, W., Suzuki, R., Tobe, K., Kadowaki, T., Hay, N., Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., 3rd, Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001). Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Genes Dev. 15, 2203–2208. Cheng, J. Q., Godwin, A. K., Bellacosa, A., Taguchi, T., Franke, T. F., Hamilton, T. C., Tsichlis, P. N., and Testa, J. R. (1992). AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc. Natl. Acad. Sci. USA 89, 9267–9271.

Alfonso Bellacosa et al.

75

Cheng, J. Q., Ruggeri, B., Klein, W. M., Sonoda, G., Altomare, D. A., Watson, D. K., and Testa, J. R. (1996). Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc. Natl. Acad. Sci. USA 93, 3636–3641. Cheng, J. Q., Altomare, D. A., Klein, M. A., Lee, W.-C., Kruh, G. D., Lissy, N. A., and Testa, J. R. (1997). Transforming activity and mitosis-related expression of the AKT2 oncogene: Evidence suggesting a link between cell cycle regulation and oncogenesis. Oncogene 14, 2793–2801. Cheong, J. W., Eom, J. I., Maeng, H. Y., Lee, S. T., Hahn, J. S., Ko, Y. W., and Min, Y. H. (2003). Phosphatase and tensin homologue phosphorylation in the C-terminal regulatory domain is frequently observed in acute myeloid leukaemia and associated with poor clinical outcome. Br. J. Haematol. 122, 454–456. Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001). Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science 292, 1728–1731. Chun, K. H., Kosmeder, J. W., Sun, S. H., Pezzuto, J. M., Lotan, R., Hong, W. K., and Lee, H. Y. (2003). Effects of deguelin on the phosphatidylinositol 3-kinase/Akt pathway and apoptosis in premalignant human bronchial epithelial cells. J. Natl. Cancer Inst. 95, 291–302. Clark, A. S., West, K., Streicher, S., and Dennis, P. A. (2002). Constitutive and inducible Akt activity promotes resistance to chemotherapy, Trastuzumab, or Tamoxifen in breast cancer cells. Mol. Cancer Ther. 1, 707–717. Coffer, P. J., and Woodgett, J. R. (1991). Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur. J. Biochem. 201, 475–481. Coffer, P. J., Jin, J., and Woodgett, J. R. (1998). Protein kinase B (c-Akt): A multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem. J. 335, 1–13. Creagh, E. M., and Martin, S. J. (2001). Caspases: Cellular demolition experts. Biochem. Soc. Trans. 29, 696–702. Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785–789. Dancey, J. E. (2002). Clinical development of mammalian target of rapamycin inhibitors. Hematol. Oncol. Clin. North Am. 5, 1101–1114. Datta, S. R., Brunet, A., and Greenberg, M. E. (1999). Cellular survival: A play in three Akts. Genes Dev. 13, 2905–2927. Deprez, J., Vertommen, D., Alessi, D. R., Hue, L., and Rider, M. H. (1997). Phosphorylation and activation of heart 6-phosphofructo-2-kinase by protein kinase B and other protein kinases of the insulin signaling cascades. J. Biol. Chem. 272, 17269–17275. Di Cristofano, A., and Pandolfi, P. P. (2000). The multiple roles of PTEN in tumor suppression. Cell 100, 387–390. Di Cristofano, A., Pesce, B., Cordon-Cardo, C., and Pandolfi, P. P. (1998). Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19, 348–355. Di Cristofano, A., Kotsi, P., Peng, Y. F., Cordon-Cardo, C., Elkon, K. B., and Pandolfi, P. P. (1999). Impaired Fas response and autoimmunity in Ptenþ/
mice. Science 285, 2122–2125. Diehl, J. A., Cheng, M., Roussel, M. F., and Sherr, C. J. (1998). Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. 12, 3499–3511. Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A. M. (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601–605.

76

Role of AKT in Cancer

Djordjevic, S., and Driscoll, P. C. (2002). Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases. Trends Biochem. Sci. 8, 426–432. Douros, J., and Suffness, M. (1981). New antitumor substances of natural origin. Cancer Treat. Rev. 8, 63–87. Downward, J. (1998). Mechanisms and consequences of activation of protein kinase B/Akt. Curr. Opin. Cell Biol. 10, 262–267. Du, K., Herzig, S., Kulkarni, R. N., and Montminy, M. (2003). TRB3: A tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science 300, 1574–1577. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997). Regulation of neuronal survival by the serinethreonine protein kinase Akt. Science 275, 661–665. Duensing, A., Medeiros, F., McConarty, B., Joseph, N. E., Panigrahy, D., Singer, S., Fletcher, C. D., Demetri, G. D., and Fletcher, J. A. (2004). Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene 23, 3999–4006. Elstrom, R. L., Bauer, D. E., Buzzai, M., Karnauskas, R., Harris, M. H., Plas, D. R., Zhuang, H., Cinalli, R. M., Alavi, A., Rudin, C. M., and Thompson, C. B. (2004). Akt stimulates aerobic glycolysis in cancer cells. Cancer Res. 64, 3892–3899. Eng, C. (2003). PTEN: One gene, many syndromes. Hum. Mutat. 22, 183–198. Feng, J., Park, J., Cron, P., Hess, D., and Hemmings, B. A. (2004). Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J. Biol. Chem. 279, 41189–41196. Flynn, P., Wongdagger, M., Zavar, M., Dean, N. M., and Stokoe, D. (2000). Inhibition of PDK-1 activity causes a reduction in cell proliferation and survival. Curr. Biol. 10, 1439–1442. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997). PI3K: Downstream AKTion blocks apoptosis. Cell 88, 435–437. Franke, T. F., Hornik, C. P., Segev, L., Shostak, G. A., and Sugimoto, C. (2003). PI3K/Akt and apoptosis: Size matters. Oncogene 22, 8983–8998. Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597–601. George, S., Rochford, J. J., Wolfrum, C., Gray, S. L., Schinner, S., Wilson, J. C., Soos, M. A., Murgatroyd, P. R., Williams, R. M., Acerini, C. L., Dunger, D. B., Barford, D., Umpleby, A. M., Wareham, N. J., Davies, H. A., Schafer, A. J., Stoffel, M., O’Rahilly, S., and Barroso, I. (2004). A family with severe insulin resistance and diabetes due to a mutation in AKT2. Science 304, 1325–1328. Gingras, A. C., and Sonenberg, N. (1997). Adenovirus infection inactivates the translational inhibitors 4E-BP1 and 4E-BP2. Virology 237, 182–186. Gottlob, K., Majewski, N., Kennedy, S., Kandel, E., Robey, R. B., and Hay, N. (2001). Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 15, 1406–1418. Graves, P. R., Yu, L., Schwarz, J. K., Gales, J., Sausville, E. A., O’Connor, P. M., and PiwnicaWorms, H. (2000). The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J. Biol. Chem. 275, 5600–5605. Grille, S. J., Bellacosa, A., Upson, J., Klein-Szanto, A. J., van Roy, F., Lee-Kwon, W., Donowitz, M., Tsichlis, P. N., and Larue, L. (2003). The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines. Cancer Res. 63, 2172–2178. Groszer, M., Erickson, R., Scripture-Adams, D. D., Lesche, R., Trumpp, A., Zack, J. A., Kornblum, H. I., Liu, X., and Wu, H. (2001). Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 294, 2186–2189.

Alfonso Bellacosa et al.

77

Hanada, M., Feng, J., and Hemmings, B. A. (2004). Structure, regulation and function of PKB/ AKT–a major therapeutic target. Biochim. Biophys. Acta 1697, 3–16. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57–70. Hashimoto, K., Mori, N., Tamesa, T., Okada, T., Kawauchi, S., Oga, A., Furuya, T., Tangoku, A., Oka, M., and Sasaki, K. (2004). Analysis of DNA copy number aberrations in hepatitis C virus-associated hepatocellular carcinomas by conventional CGH and array CGH. Mod. Pathol. 17, 617–622. Hawley, S. A., Boudeau, J., Reid, J. L., Mustard, K. J., Udd, L., Makela, T. P., Alessi, D. R., and Hardie, D. G. (2003). Complexes between the LKB1 tumor suppressor, STRADalpha/beta and MO25alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J. Biol. 2, 28. Hemmings, B. A. (1997). Akt signaling: Linking membrane events to life and death decisions. Science 275, 628–630. Hidalgo, M. (2004). New target, new drug, old paradigm. J. Clin. Oncol. 22, 2270–2272. Holland, E. C., Celestino, J., Dai, C., Schaefer, L., Sawaya, R. E., and Fuller, G. N. (2000). Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat. Genet. 25, 55–57. Horie, Y., Suzuki, A., Kataoka, E., Sasaki, T., Hamada, K., Sasaki, J., Mizuno, K., Hasegawa, G., Kishimoto, H., Iizuka, M., Naito, M., Enomoto, K., Watanabe, S., Mak, T. W., and Nakano, T. (2004). Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas. J. Clin. Invest. 113, 1774–1783. Horiguchi, A., Oya, M., Uchida, A., Marumo, K., and Murai, M. (2003). Elevated Akt activation and its impact on clinicopathological features of renal cell carcinoma. J. Urol. 169, 710–713. Hu, L., Hofmann, J., Lu, Y., Mills, G. B., and Jaffe, R. B. (2002). Inhibition of phosphatidylinositol 30 -kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Cancer Res. 62, 1087–1092. Huang, S., Shu, L., Easton, J., Harwood, F. C., Germain, G. S., Ichijo, H., and Houghton, P. J. (2004). Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity. J. Biol. Chem. 279, 36490–36496. Huang, X., Begley, M., Morgenstern, K. A., Gu, Y., Rose, P., Zhao, H. L., and Zhu, X. T. (2003). Crystal structure of an inactive Akt2 kinase domain. Structure 11, 21–30. Hutchinson, J., Jin, J., Cardiff, R. D., Woodgett, J. R., and Muller, W. J. (2001). Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression. Mol. Cell. Biol. 21, 2203–2212. Hutchinson, J. N., Jin, J., Cardiff, R. D., Woodgett, J. R., and Muller, W. J. (2004). Activation of Akt-1 (PKB-alpha) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res. 64, 3171–3178. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat. Cell. Biol. 4, 648–657. Inoki, K., Zhu, T., and Guan, K. L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590. Jetzt, A., Howe, J. A., Horn, M. T., Maxwell, E., Yin, Z., Johnson, D., and Kumar, C. C. (2003). Adenoviral-mediated expression of a kinase-dead mutant of Akt induces apoptosis selectively in tumor cells and suppresses tumor growth in mice. Cancer Res. 63, 6697–6706. Jiang, Z. Y., Zhou, Q. L., Coleman, K. A., Chouinard, M., Boese, Q., and Czech, M. P. (2003). Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc. Natl. Acad. Sci. USA 100, 7569–7574.

78

Role of AKT in Cancer

Jones, P. F., Jakubowicz, T., Pitossi, F. J., Maurer, F., and Hemmings, B. A. (1991). Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc. Natl. Acad. Sci. USA 88, 4171–4175. Jucker, M., Sudel, K., Horn, S., Sickel, M., Wegner, W., Fiedler, W., and Feldman, R. A. (2002). Expression of a mutated form of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase in a Hodgkin’s lymphoma-derived cell line (CO). Leukemia 16, 894–901. Kang, S. S., Kwon, T., Kwon, D. Y., and Do, S. I. (1999). Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit. J. Biol. Chem. 274, 13085–13090. Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L. C., Gotoh, Y., Kishi, K., Shiota, H., and Ebina, Y. (2003). Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions. J. Biol. Chem. 278, 28312–32823. Kenerson, H. L., Aicher, L. D., True, L. D., and Yeung, R. S. (2002). Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res. 62, 5645–5650. Kim, D. H., and Sabatini, D. M. (2004). Raptor and mTOR: Subunits of a nutrient-sensitive complex. Curr. Top. Microbiol. Immunol. 279, 259–270. Knighton, D. R., Zheng, J. H., Ten Eyck, L. F., Ashford, V. A., Xuong, N. H., Taylor, S. S., and Sowadski, J. M. (1991). Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 407–414. Knobbe, C. B., and Reifenberger, G. (2003). Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3 0 -kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol. 13, 507–518. Knuutila, S., Bjorkqvist, A. M., Autio, K., Tarkkanen, M., Wolf, M., Monni, O., Szymanska, J., Larramendy, M. L., Tapper, J., Pere, H., El-Rifai, W., Hemmer, S., Wasenius, V. M., Vidgren, V., and Zhu, Y. (1998). DNA copy number amplifications in human neoplasms: Review of comparative genomic hybridization studies. Am. J. Pathol. 152, 1107–1123. Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996). Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J. Biol. Chem. 271, 31372–31378. Komander, D., Kular, G. S., Bain, J., Elliott, M., Alessi, D. R., and Van Aalten, D. M. (2003). Structural basis for UCN-01 (7-hydroxystaurosporine) specificity and PDK1 (3phosphoinositide-dependent protein kinase-1) inhibition. Biochem. J. 375, 255–262. Kondapaka, S. B., Singh, S. S., Dasmahapatra, G. P., Sausville, E. A., and Roy, K. K. (2003). Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation. Mol. Cancer Ther. 2, 1093–1103. Kops, G. J., Medema, R. H., Glassford, J., Essers, M. A., Dijkers, P. F., Coffer, P. J., Lam, E. W., and Burgering, B. M. (2002). Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol. Cell. Biol. 22, 2025–2036. Kozikowski, A. P., Sun, H., Brognard, J., and Dennis, P. A. (2003). Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt. J. Am. Chem. Soc. 125, 1144–1145. Kulik, G., Klippel, A., and Weber, M. J. (1997). Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol. Cell Biol. 3, 1595–1606. Kumar, C. C., Diao, R., Yin, Z., Liu, Y., Samatar, A. A., Madison, V., and Xiao, L. (2001). Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling. Biochim. Biophys. Acta 1526, 257–268.

Alfonso Bellacosa et al.

79

Kurose, K., Zhou, X. P., Araki, T., Cannistra, S. A., Maher, E. R., and Eng, C. (2001). Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am. J. Pathol. 158, 2097–2106. Kwiatkowski, D. J. (2003). Tuberous sclerosis: From tubers to mTOR. Ann. Hum. Genet. 67, 87–96. Kwon, C. H., Zhu, X., Zhang, J., Knoop, L. L., Tharp, R., Smeyne, R. J., Eberhart, C. G., Burger, P. C., and Baker, S. J. (2001). Pten regulates neuronal soma size: A mouse model of Lhermitte-Duclos disease. Nat. Genet. 29, 404–411. Kwon, C. H., Zhu, X., Zhang, J., and Baker, S. J. (2003). mTor is required for hypertrophy of Pten-deficient neuronal soma in vivo. Proc. Natl. Acad. Sci. USA 100, 12923–12928. Kyriakis, J. M. (2003). At the crossroads: AMP-activated kinase and the LKB1 tumor suppressor link cell proliferation to metabolic regulation. J. Biol. 2, 26. Lawlor, M. A., Mora, A., Ashby, P. R., Williams, M. R., Murray-Tait, V., Malone, L., Prescott, A. R., Lucocq, J. M., and Alessi, D. R. (2002). Essential role of PDK1 in regulating cell size and development in mice. EMBO J. 21, 3728–3738. Lee, S. H., Kim, H. S., Park, W. S., Kim, S. Y., Lee, K. Y., Kim, S. H., Lee, J. Y., and Yoo, N. J. (2002). Non-small cell lung cancers frequently express phosphorylated Akt; an immunohistochemical study. APMIS 110, 587–592. Li, G., Robinson, G. W., Lesche, R., Martinez-Diaz, H., Jiang, Z., Rozengurt, N., Wagner, K. U., Wu, D. C., Lane, T. F., Liu, X., Hennighausen, L., and Wu, H. (2002). Conditional loss of PTEN leads to precocious development and neoplasia in the mammary gland. Development 129, 4159–4170. Li, J., Simpson, L., Takahashi, M., Miliaresis, C., Myers, M. P., Tonks, N., and Parsons, R. (1998). The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/ protein kinase B oncogene. Cancer Res. 58, 5667–5672. Li, Y., Corradetti, M. N., Inoki, K., and Guan, K. L. (2004). TSC2: Filling the GAP in the mTOR signaling pathway. Trends Biochem. Sci. 29, 32–38. Liaw, D., Marsh, D. J., Li, J., Dahia, P. L., Wang, S. I., Zheng, Z., Bose, S., Call, K. M., Tsou, H. C., Peacocke, M., Eng, C., and Parsons, R. (1997). Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet. 16, 64–67. Liu, A. X., Testa, J. R., Hamilton, T. C., Jove, R., Nicosia, S. V., and Cheng, J. Q. (1998). AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells. Cancer Res. 58, 2973–2977. Long, X., Muller, F., and Avruch, J. (2004). TOR action in mammalian cells and in Caenorhabditis elegans. Curr. Top. Microbiol. Immunol. 279, 115–138. Lu, Y., Lin, Y. Z, La Pushin, R., Cuevas, B., Fang, X., Yu, S. X., Davies, M. A., Khan, H., Furui, T., Mao, M., Zinner, R., Hung, M. C., Steck, P., Siminovitch, K., and Mills, G. B. (1999). The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 18, 7034–7045. Luo, J., Manning, B. D., and Cantley, L. C. (2003). Targeting the PI3K-Akt pathway in human cancer: Rationale and promise. Cancer Cell 4, 257–262. Maira, S. M., Galetic, I., Brazil, D. P., Kaech, S., Ingley, E., Thelen, M., and Hemmings, B. A. (2001). Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 294, 374–380. Majumder, P. K., Yeh, J. J., George, D. J., Febbo, P. G., Kum, J., Xue, Q., Bikoff, R., Ma, H., Kantoff, P. W., Golub, T. R., Loda, M., and Sellers, W. R. (2003). Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: The MPAKT model. Proc. Natl. Acad. Sci. USA 100, 7841–7846.

80

Role of AKT in Cancer

Majumder, P. K., Febbo, P. G., Bikoff, R., Berger, R., Xue, Q., McMahon, L. M., Manola, J., Brugarolas, J., McDonnell, T. J., Golub, T. R., Loda, M., Lane, H. A., and Sellers, W. R. (2004). mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat. Med. 10, 594–601. Malik, S. N., Brattain, M., Ghosh, P. M., Troyer, D. A., Prihoda, T., Bedolla, R., and Kreisberg, J. I. (2002). Immunohistochemical demonstration of phospho-Akt in high Gleason grade prostate cancer. Clin. Cancer Res. 8, 1168–1171. Malstrom, S., Tili, E., Kappes, D., Ceci, J. D., and Tsichlis, P. N. (2001). Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus. Proc. Natl. Acad. Sci. USA 98, 14967–14972. Mamane, Y., Petroulakis, E., Rong, L., Yoshida, K., Ler, L. W., and Sonenberg, N. (2004). eIF4E–from translation to transformation. Oncogene 23, 3172–3179. Manning, B. D., and Cantley, L. C. (2003). Rheb fills a GAP between TSC and TOR. Trends Biochem. Sci. 28, 573–576. Marino, S., Krimpenfort, P., Leung, C., van der Korput, H. A., Trapman, J., Camenisch, I., Berns, A., and Brandner, S. (2002). PTEN is essential for cell migration but not for fate determination and tumourigenesis in the cerebellum. Development 129, 3513–3522. Marsh, D. J., Dahia, P. L., Zheng, Z., Liaw, D., Parsons, R., Gorlin, R. J., and Eng, C. (1997). Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat. Genet. 16, 333–334. Martelli, A. M., Tazzari, P. L., Tabellini, G., Bortul, R., Billi, A. M., Manzoli, L., Ruggeri, A., Conte, R., and Cocco, L. (2003). A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells. Leukemia 17, 1794–1805. Martin, K. A., and Blenis, J. (2002). Coordinate regulation of translation by the PI 3-kinase and mTOR pathways. Adv. Cancer Res. 86, 1–39. Mayo, L. D., and Donner, D. B. (2001). A phosphatidylinositol 3-kinaseAkt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA 98, 11598–11603. Mellinghoff, I. K., and Sawyers, C. L. (2004). TORward AKTually useful mouse models. Nat. Med. 10, 579–580. Mende, I., Malstrom, S., Tsichlis, P. N., Vogt, P. K., and Aoki, M. (2001). Oncogenic transformation induced by membrane-targeted Akt2 and Akt3. Oncogene 20, 4419–4423. Min, Y. H., Eom, J. I., Cheong, J. W., Maeng, H. O., Kim, J. Y., Jeung, H. K., Lee, S. T., Lee, M. H., Hahn, J. S., and Ko, Y. W. (2003). Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: Its significance as a prognostic variable. Leukemia 17, 995–997. Mitsiades, C. S., Mitsiades, N., and Koutsilieris, M. (2004). The Akt pathway: Molecular targets for anti-cancer drug development. Curr. Cancer Drug Targets 4, 235–256. Miwa, W., Yasuda, J., Murakami, Y., Yashima, K., Sugano, K., Sekine, T., Kono, A., Egawa, S., Yamaguchi, K., Hayashizaki, Y., and Sekiya, T. (1996). Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer. Biochem. Biophys. Res. Commun. 225, 968–974. Miyakawa, M., Tsushima, T., Murakami, H., Wakai, K., Isozaki, O., and Takano, K. (2003). Increased expression of phosphorylated p70S6 kinase and akt in papillary thyroid cancer tissues. Endocr. J. 50, 77–83. Mohi, M. G., Boulton, C., Gu, T. L., Sternberg, D. W., Neuberg, D., Griffin, J. D., Gilliland, D. G., and Neel, B. G. (2004). Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs. Proc. Natl. Acad. Sci. USA 101, 3130–3135.

Alfonso Bellacosa et al.

81

Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E., Tsichlis, P. N., and Rosen, N. (1998). Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 273, 29864–29872. Mukohara, T., Kudoh, S., Matsuura, K., Yamauchi, S., Kimura, T., Yoshimura, N., Kanazawa, H., Hirata, K., Inoue, K., Wanibuchi, H., Fukushima, S., and Yoshikawa, J. (2004). Activated Akt expression has significant correlation with EGFR and TGF-alpha expressions in stage I NSCLC. Anticancer Res. 24, 11–17. Myers, M. P., Pass, I., Batty, I. H., Van der Kaay, J., Stolarov, J. P., Hemmings, B. A., Wigler, M. H., Downes, C. P., and Tonks, N. K. (1998). The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc. Natl. Acad. Sci. USA 95, 13513–13518. Nakashio, A., Fujita, N., Rokudai, S., Sato, S., and Tsuruo, T. (2000). Prevention of phosphatidylinositol 30 -kinase-Akt survival signaling pathway during topotecan-induced apoptosis. Cancer Res. 60, 5303–5309. Nakatani, K., Thompson, D. A., Barthel, A., Sakaue, H., Liu, W., Weigel, R. J., and Roth, R. A. (1999). Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgenindependent prostate cancer lines. J. Biol. Chem. 274, 21528–21532. Nam, S. Y., Lee, H. S., Jung, G. A., Choi, J., Cho, S. J., Kim, M. K., Kim, W. H., and Lee, B. L. (2003). Akt/PKB activation in gastric carcinomas correlates with clinicopathologic variables and prognosis. APMIS 111, 1105–1113. Neshat, M. S., Mellinghoff, I. K., Tran, C., Stiles, B., Thomas, G., Petersen, R., Frost, P., Gibbons, J. J., Wu, H., and Sawyers, C. L. (2001). Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA 98, 10314–10319. Ono, H., Shimano, H., Katagiri, H., Yahagi, N., Sakoda, H., Onishi, Y., Anai, M., Ogihara, T., Fujishiro, M., Viana, A. Y., Fukushima, Y., Abe, M., Shojima, N., Kikuchi, M., Yamada, N., Oka, Y., and Asano, T. (2003). Hepatic Akt activation induces marked hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement. Diabetes 52, 2905–2913. Orsulic, S., Li, Y., Soslow, R. A., Vitale-Cross, L. A., Gutkind, J. S., and Varmus, H. E. (2002). Induction of ovarian cancer by defined multiple genetic changes in a mouse model system. Cancer Cell 1, 53–62. Page, C., Lin, H. J., Jin, Y., Castle, V. P., Nunez, G., Huang, M., and Lin, J. (2000). Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis. Anticancer Res. 20, 407–416. Pearl, L. H., and Barford, D. (2002). Regulation of protein kinases in insulin, growth factor and Wnt signalling. Curr. Opin. Struct. Biol. 12, 761–767. Peng, X. D., Xu, P. Z., Chen, M. L., Hahn-Windgassen, A., Skeen, J., Jacobs, J., Sundararajan, D., Chen, W. S., Crawford, S. E., Coleman, K. G., and Hay, N. (2003). Dwarfism, impaired skin development, skeletal muscle atrophy, delayed bone development, and impeded adipogenesis in mice lacking Akt1 and Akt2. Genes Dev. 17, 1352–1365. Perez-Tenorio, G., and Stal, O. (2002). Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients. Br. J. Cancer 86, 540–545. Philp, A. J., Campbell, I. G., Leet, C., Vincan, E., Rockman, S. P., Whitehead, R. H., Thomas, R. J., and Phillips, W. A. (2001). The phosphatidylinositol 30 -kinase p85alpha gene is an oncogene in human ovarian and colon tumors. Cancer Res. 61, 7426–7429. Plas, D. R., and Thompson, C. B. (2002). Cell metabolism in the regulation of programmed cell death. Trends Endocrinol. Metab. 13, 75–78. Podsypanina, K., Ellenson, L. H., Nemes, A., Gu, J., Tamura, M., Yamada, K. M., CordonCardo, C., Catoretti, G., Fisher, P. E., and Parsons, R. (1999). Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. USA 96, 1563–1568. Podsypanina, K., Lee, R. T., Politis, C., Hennessy, I., Crane, A., Puc, J., Neshat, M., Wang, H., Yang, L., Gibbons, J., Frost, P., Dreisbach, V., Blenis, J., Gaciong, Z., Fisher, P., Sawyers, C.,

82

Role of AKT in Cancer

Hedrick-Ellenson, L., and Parsons, R. (2001). An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Ptenþ/
mice. Proc. Natl. Acad. Sci. USA 98, 10320–10325. Potter, C. J., Pedraza, L. G., and Xu, T. (2002). Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4, 658–665. Proud, C. G. (2004). Role of mTOR signalling in the control of translation initiation and elongation by nutrients. Curr. Top. Microbiol. Immunol. 279, 215–244. Rathmell, J. C., Elstrom, R. L., Cinalli, R. M., and Thompson, C. B. (2003). Activated Akt promotes increased resting T cell size, CD28-independent T cell growth, and development of autoimmunity and lymphoma. Eur. J. Immunol. 33, 2223–2232. Reith, A. D., Ellis, C., Lyman, S. D., Anderson, D. M., Williams, D. E., Bernstein, A., and Pawson, T. (1991). Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase. EMBO J. 10, 2451–2459. Ringel, M. D., Hayre, N., Saito, J., Saunier, B., Schuppert, F., Burch, H., Bernet, V., Burman, K. D., Kohn, L. D., and Saji, M. (2001). Overexpression and overactivation of Akt in thyroid carcinoma. Cancer Res. 61, 6105–6111. Roy, H. K., Olusola, B. F., Clemens, D. L., Karolski, W. J., Ratashak, A., Lynch, H. T., and Smyrk, T. C. (2002). AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis 23, 201–205. Ruggeri, B. A., Huang, L., Wood, M., Cheng, J. Q., and Testa, J. R. (1998). Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Mol. Carc. 21, 81–86. Ruggero, D., and Pandolfi, P. P. (2003). Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179–192. Ruggero, D., Montanaro, L., Ma, L., Xu, W., Londei, P., Cordon-Cardo, C., and Pandolfi, P. P. (2004). The translation factor eIF-4E promotes tumor formation and cooperates with c-Myc in lymphomagenesis. Nat. Med. 10, 484–486. Saito, Y., Swanson, X., Mhashilkar, A. M., Oida, Y., Schrock, R., Branch, C. D., Chada, S., Zumstein, L., and Ramesh, R. (2003). Adenovirus-mediated transfer of the PTEN gene inhibits human colorectal cancer growth in vitro and in vivo. Gene Ther. 10, 1961–1969. Samuels, Y., Wang, Z., Bardelli, A., Silliman, N., Ptak, J., Szabo, S., Yan, H., Gazdar, A., Powell, S. M., Riggins, G. J., Willson, J. K., Markowitz, S., Kinzler, K. W., Vogelstein, B., and Velculescu, V. E. (2004). High frequency of mutations of the PIK3CA gene in human cancers. Science 304, 554. Sansal, I., and Sellers, W. R. (2004). The biology and clinical relevance of the PTEN tumor suppressor pathway. J. Clin. Oncol. 22, 2954–2963. Sarkaria, J. N., Tibbetts, R. S., Busby, E. C., Kennedy, A. P., Hill, D. E., and Abraham, R. T. (1998). Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res. 58, 4375–4382. Sato, S., Fujita, N., and Tsuruo, T. (2002). Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). Oncogene 11, 1727–1738. Sawyers, C. L. (2003). Will mTOR inhibitors make it as cancer drugs? Cancer Cell 5, 343–348. Scheid, M. P., and Woodgett, J. R. (2003). Unravelling the activation mechanisms of protein kinase B/Akt. FEBS Lett. 546, 108–112. Schlegel, J., Piontek, G., and Mennel, H. D. (2002). Activation of the anti-apoptotic Akt/ protein kinase B pathway in human malignant gliomas in vivo. Anticancer Res. 22, 2837–2840. Schlieman, M. G., Fahy, B. N., Ramsamooj, R., Beckett, L., and Bold, R. J. (2003). Incidence, mechanism and prognostic value of activated AKT in pancreas cancer. Br. J. Cancer 89, 2110–2115.

Alfonso Bellacosa et al.

83

Seki, N., Takasu, T., Mandai, K., Nakata, M., Saeki, H., Heike, Y., Takata, I., Segawa, Y., Hanafusa, T., and Eguchi, K. (2002). Expression of eukaryotic initiation factor 4E in atypical adenomatous hyperplasia and adenocarcinoma of the human peripheral lung. Clin. Cancer Res. 8, 3046–3053. Semba, S., Moriya, T., Kimura, W., and Yamakawa, M. (2003). Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreas. Pancreas 26, 250–257. Shaw, R. J., Bardeesy, N., Manning, B. D., Lopez, L., Kosmatka, M., DePinho, R. A., and Cantley, L. C. (2004a). The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6, 91–99. Shaw, R. J., Kosmatka, M., Bardeesy, N., Hurley, R. L., Witters, L. A., DePinho, R. A., and Cantley, L. C. (2004b). The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. USA 101, 3329–3335. Shayesteh, L., Lu, Y., Kuo, W. L., Baldocchi, R., Godfrey, T., Collins, C., Pinkel, D., Powell, B., Mills, G. B., and Gray, J. W. (1999). PIK3CA is implicated as an oncogene in ovarian cancer. Nat. Genet. 21, 99–102. She, Q. B., Solit, D., Basso, A., and Moasser, M. M. (2003). Resistance to gefitinib in PTENnull HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 30 -kinase/Akt pathway signaling. Clin. Cancer Res. 12, 4340–4346. Shi, W., Zhang, X., Pintilie, M., Ma, N., Miller, N., Banerjee, D., Tsao, M. S., Mak, T., Fyles, A., and Liu, F. F. (2003). Dysregulated PTEN-PKB and negative receptor status in human breast cancer. Int. J. Cancer 104, 195–203. Simpson, L., and Parsons, R. (2001). PTEN: Life as a tumor suppressor. Exp. Cell Res. 264, 29–41. Skorski, T., Bellacosa, A., Nieborowska-Skorska, M., Majewski, M., Martinez, R., Choi, J. K., Trotta, R., Wlodarski, P., Perrotti, D., Chan, T. O., Wasik, M. A., Tsichlis, P. N., and Calabretta, B. (1997). Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway. EMBO J. 16, 6151–6161. Slupianek, A., Nieborowska-Skorska, M., Hoser, G., Morrione, A., Majewski, M., Xue, L., Morris, S. W., Wasik, M. A., and Skorski, T. (2001). Role of phosphatidylinositol 3-kinaseAkt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis. Cancer Res. 61, 2194–2199. Soriano, P. (1997). The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124, 2691–2700. Staal, S. P. (1987). Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: Amplification of AKT1 in a primary human gastric adenocarcinoma. Proc. Natl. Acad. Sci. USA 84, 5034–5037. Staal, S. P., and Hartley, J. W. (1988). Thymic lymphoma induction by the AKT8 murine retrovirus. J. Exp. Med. 167, 1259–1264. Staal, S. P., Hartley, J. W., and Rowe, W. P. (1977). Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc. Natl. Acad. Sci. USA 74, 3065–3067. Staal, S. P., Huebner, K., Croce, C. M., Parsa, N. Z., and Testa, J. R. (1988). The AKT1 protooncogene maps to human chromosome 14, band q32. Genomics 2, 96–98. Stal, O., Perez-Tenorio, G., Akerberg, L., Olsson, B., Nordenskjold, B., Skoog, L., and Rutqvist, L. E. (2003). Akt kinases in breast cancer and the results of adjuvant therapy. Breast Cancer Res. 5, R37–R44.

84

Role of AKT in Cancer

Stambolic, V., Suzuki, A., de la Pompa, J. L., Brothers, G. M., Mirtsos, C., Sasaki, T., Ruland, J., Penninger, J. M., Siderovski, D. P., and Mak, T. W. (1998). Negative regulation of PKB/ Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29–39. Staveley, B. E., Ruel, L., Jin, J., Stambolic, V., Mastronardi, F. G., Heitzler, P., Woodgett, J. R., and Manoukian, A. S. (1998). Genetic analysis of protein kinase B (AKT) in Drosophila. Curr. Biol. 8, 599–602. Sun, M., Wang, G., Paciga, J. E., Feldman, R. I., Yuan, Z. Q., Ma, X. L., Shelley, S. A., Jove, R., Tsichlis, P. N., Nicosia, S. V., and Cheng, J. Q. (2001). AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am. J. Pathol. 159, 431–437. Sun, S., and Steinberg, B. M. (2002). PTEN is a negative regulator of STAT3 activation in human papillomavirus-infected cells. J. Gen. Virol. 83, 1651–1658. Suzuki, A., de la Pompa, J. L., Stambolic, V., Elia, A. J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., Fukumoto, M., and Mak, T. W. (1998). High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8, 1169–1178. Suzuki, A., Yamaguchi, M. T., Ohteki, T., Sasaki, T., Kaisho, T., Kimura, Y., Yoshida, R., Wakeham, A., Higuchi, T., Fukumoto, M., Tsubata, T., Ohashi, P. S., Koyasu, S., Penninger, J. M., Nakano, T., and Mak, T. W. (2001). T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity 14, 523–534. Tanno, S., Tanno, S., Mitsuuchi, Y., Altomare, D. A., Xiao, G. H., and Testa, J. R. (2001). AKT activation up-regulates insulin-like growth factor-I receptor expression and promotes invasiveness of human pancreatic cancer cells. Cancer Res. 61, 589–593. Tanno, S., Yanagawa, N., Habiro, A., Koizumi, K., Nakano, Y., Osanai, M., Mizukami, Y., Okumura, T., Testa, J. R., and Kohgo, Y. (2004). Serine/threonine kinase AKT is frequently activated in human bile duct cancer and is associated with increased radioresistance. Cancer Res. 64, 3486–3490. Terakawa, N., Kanamori, Y., and Yoshida, S. (2003). Loss of PTEN expression followed by Akt phosphorylation is a poor prognostic factor for patients with endometrial cancer. Endocr. Relat. Cancer 10, 203–208. Testa, J. R., and Bellacosa, A. (2001). AKT plays a central role in tumorigenesis. Proc. Natl. Acad. Sci. USA 98, 10983–10985. Thant, A. A., Nawa, A., Kikkawa, F., Ichigotani, Y., Zhang, Y., Sein, T. T., Amin, A. R., and Hamaguchi, M. (2000). Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells. Clin. Exp. Metastasis 18, 423–428. Thomas, C., Deak, M., Alessi, D., and van Aalten, D. (2002). High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)trisphosphate. Curr. Biol. 12, 1256. Thompson, F. H., Nelson, M. A., Trent, J. M., Guan, X. Y., Liu, Y., Yang, J. M., Emerson, J., Adair, L., Wymer, J., Balfour, C., Massey, K., Weinstein, R., Alberts, D. S., and Taetle, R. (1996). Amplification of 19q13.1-q13.2 sequences in ovarian cancer. G-band, FISH, and molecular studies. Cancer Genet. Cytogenet. 87, 55–62. Trencia, A., Perfetti, A., Cassese, A., Vigliotta, G., Miele, C., Oriente, F., Santopietro, S., Giacco, F., Condorelli, G., Formisano, P., and Beguinot, F. (2003). Protein kinase B/Akt binds and phosphorylates PED/PEA-15, stabilizing its antiapoptotic action. Mol. Cell. Biol. 23, 4511–4521. Trotman, L. C., Niki, M., Dotan, Z. A., Koutcher, J. A., Di Cristofano, A., Xiao, A., Khoo, A. S., Roy-Burman, P., Greenberg, N. M., Dyke, T. V., Cordon-Cardo, C., and Pandolfi, P. (2003). Pten dose dictates cancer progression in the prostate. PLoS Biol. 1, E59.

Alfonso Bellacosa et al.

85

Tsao, A. S., McDonnell, T., Lam, S., Putnam, J. B., Bekele, N., Hong, W. K., and Kurie, J. M. (2003). Increased phospho-AKT (Ser(473) ) expression in bronchial dysplasia: Implications for lung cancer prevention studies. Cancer Epidemiol. Biomarkers Prev. 12, 660–664. Tuttle, R. L., Gill, N. S., Pugh, W., Lee, J. P., Koeberlein, B., Furth, E. E., Polonsky, K. S., Naji, A., and Birnbaum, M. J. (2001). Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. Nat. Med. 7, 1133–1137. Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163. Uhrbom, L., Dai, C., Celestino, J. C., Rosenblum, M. K., Fuller, G. N., and Holland, E. C. (2002). Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt. Cancer Res. 62, 5551–5558. Vanhaesebroeck, B., and Alessi, D. R. (2000). The PI3K-PDK1 connection: More than just a road to PKB. Biochem. J. 346, 561–576. Vasko, V., Saji, M., Hardy, E., Kruhlak, M., Larin, A., Savchenko, V., Miyakawa, M., Isozaki, O., Murakami, H., Tsushima, T., Burman, K. D., De Micco, C., and Ringel, M. D. (2004). Akt activation and localisation correlate with tumour invasion and oncogene expression in thyroid cancer. J. Med. Genet. 41, 161–170. Verdu, J., Buratovich, M. A., Wilder, E. L., and Birnbaum, M. J. (1999). Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat. Cell Biol. 1, 500–506. Vezina, C., Kudelski, A., and Sehgal, S. N. (1975). Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiot. (Tokyo) 28, 721–726. Viglietto, G., Motti, M. L., Bruni, P., Melillo, R. M., D’Alessio, A., Califano, D., Vinci, F., Chiappetta, G., Tsichlis, P., Bellacosa, A., Fusco, A., and Santoro, M. (2002). Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Aktmediated phosphorylation in breast cancer. Nat. Med. 8, 1136–1144. Vivanco, I., and Sawyers, C. L. (2002). The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat. Rev. Cancer 2, 489–501. Waldmann, V., and Wacker, J. (2001). Mutations of the PH domain of protein kinase B (PKB/ AKT) are absent in human epidermal skin tumors. Dermatology 203, 284–288. Waldmann, V., Wacker, J., and Deichmann, M. (2001). Mutations of the activation-associated phosphorylation sites at codons 308 and 473 of protein kinase B are absent in human melanoma. Arch. Dermatol. Res. 293, 368–372. Waldmann, V., Wacker, J., and Deichmann, M. (2002). Absence of mutations in the pleckstrin homology (PH) domain of protein kinase B (PKB/Akt) in malignant melanoma. Melanoma Res. 12, 45–50. Walker, K. S., Deak, M., Paterson, A., Hudson, K., Cohen, P., and Alessi, D. R. (1998). Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3phosphoinositide-dependent protein kinase-1 in vitro: Comparison with protein kinase B alpha. Biochem. J. 331, 299–308. Wang, S., Rosenwald, I. B., Hutzler, M. J., Pihan, G. A., Savas, L., Chen, J. J., and Woda, B. A. (1999). Expression of the eukaryotic translation initiation factors 4E and 2alpha in nonHodgkin’s lymphomas. Am. J. Pathol. 155, 247–255. Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas, G. V., Li, G., RoyBurman, P., Nelson, P. S., Liu, X., and Wu, H. (2003). Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4, 209–221. Warburg, O. (1956). On the origin of cancer cells. Science 123, 309–314.

86

Role of AKT in Cancer

Wendel, H. G., De Stanchina, E., Fridman, J. S., Malina, A., Ray, S., Kogan, S., Cordon-Cardo, C., Pelletier, J., and Lowe, S. W. (2004). Survival signalling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 428, 332–337. Whang, Y. E., Yuan, X. J., Liu, Y., Majumder, S., and Lewis, T. D. (2004). Regulation of sensitivity to TRAIL by the PTEN tumor suppressor. Vitam. Horm. 67, 409–426. Whiteman, E. L., Cho, H., and Birnbaum, M. J. (2002). Role of Akt/protein kinase B in metabolism. Trends Endocrinol. Metab. 13, 444–451. Woods, A., Johnstone, S. R., Dickerson, K., Leiper, F. C., Fryer, L. G., Neumann, D., Schlattner, U., Wallimann, T., Carlson, M., and Carling, D. (2003). LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 13, 2004–2008. Wu, H., Goel, V., and Haluska, F. G. (2003). PTEN signaling pathways in melanoma. Oncogene 22, 3113–3122. Wu, X., Senechal, K., Neshat, M. S., Whang, Y. E., and Sawyers, C. L. (1998). The PTEN/ MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc. Natl. Acad. Sci. USA 95, 15587–15591. Xie, Z., Zeng, X., Waldman, T., and Glazer, R. I. (2003). Transformation of mammary epithelial cells by 3-phosphoinositide-dependent protein kinase-1 activates beta-catenin and c-Myc, and down-regulates caveolin-1. Cancer Res. 17, 5370–5375. Xu, W., Harrison, S. C., and Eck, M. J. (1999). Three-dimensional structure of of the tyrosine kinase c-Src. Nature 385, 595–602. Xu, X., Sakon, M., Nagano, H., Hiraoka, N., Yamamoto, H., Hayashi, N., Dono, K., Nakamori, S., Umeshita, K., Ito, Y., Matsuura, N., and Monden, M. (2004). Akt2 expression correlates with prognosis of human hepatocellular carcinoma. Oncol. Rep. 11, 25–32. Yang, J., Cron, P., Good, V. M., Thompson, V., Hemmings, B. A., and Barford, D. (2002a). Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP. Nat. Struct. Biol. 9, 940–944. Yang, J., Cron, P., Thompson, V., Good, V. M., Hess, D., Hemmings, B. A., and Barford, D. (2002b). Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation. Mol. Cell 9, 1227–1240. Yang, L., Dan, H. C., Sun, M., Liu, Q., Sun, X. M., Feldman, R. I., Hamilton, A. D., Polokoff, M., Nicosia, S. V., Herlyn, M., Sebti, S. M., and Cheng, J. Q. (2004). Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res. 64, 4394 – 4399. Yuan, Z. Q., Sun, M., Feldman, R. I., Wang, G., Ma, X., Jiang, C., Coppola, D., Nicosia, S. V., and Cheng, J. Q. (2000). Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 19, 2324 –2330. Zhang, H., Cicchetti, G., Onda, H., Koon, H. B., Asrican, K., Bajraszewski, N., Vazquez, F., Carpenter, C. L., and Kwiatkowski, D. J. (2003). Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233. Zhou, B. P., and Hung, M. C. (2003). Dysregulation of cellular signaling by HER2/neu in breast cancer. Semin. Oncol. 30, 38–48. Zhou, B. P., Liao, Y., Xia, W., Spohn, B., Lee, M. H., and Hung, M. C. (2001). Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat. Cell Biol. 3, 245–252.