[Frontiers in Bioscience 10, 975-987, January 1, 2005]

AKT/PKB SIGNALING MECHANISMS IN CANCER AND CHEMORESISTANCE

Donghwa Kim, Han C. Dan, Sungman Park, Lin Yang, Qiyuan Liu, Satoshi Kaneko, Jinying Ning, Lili He, Hua Yang, Mei Sun, Santo V. Nicosia, Jin Q. Cheng

Departments of Pathology and Interdisciplinary Oncology, University of South Florida College of Medicine and H. Lee Moffitt Cancer Center, Tampa, Florida 33612

TABLE CONTENTS

1. Abstract
2. Background
3. Comparison of Akt isoforms
3.1. Sequence comparison and expression pattern
3.2. Phenotype of the knock-out mouse
4. Akt oncogenic activity and its role in chemoresistance
4.1. Mechanism of Akt activation
4.2. Transforming activity of Akt
4.3. Alterations of Akt pathway in human cancer
4.4. Akt and chemoresistance
5. Normal cellular function of Akt
5.1. Anti-apoptosis
5.1.1. Apoptotic proteins
5.1.2. Other molecules related cell survival
5.2. Cell cycle progression
5.2.1. Cell cycle regulators
5.2.2. mTOR and TSC2
5.2.3. Others
6. Akt pathway as potential therapeutic target for cancer intervention
6.1. Biological approaches
6.2. Pharmacological approaches for inhibition of upstream Akt
6.3. Akt inhibitors
7. Perspective
8. Acknowledgments
9. References

1. ABSTRACT

During the past decade, Akt (also known as protein kinase B, PKB) has been extensively studied. It regulates a variety of cellular processes by mediating extracellular (mitogenic growth factor, insulin and stress) and intracellular (altered tyrosine receptor kinases, Ras and Src) signals. Activation of Akt by these signals is via its pleckstrin homology (PH) domain binding to products of phosphatidylinositol 3-kinase (PI3K). This process is negatively regulated by a dual phosphatase PTEN tumor suppressor. Today, more than 30 Akt substrates have been identified. These phosphorylation events mediate the effects of Akt on cell survival, growth, differentiation, angiogenesis, migration and metabolism. Further, PI3K/PTEN/Akt pathway is frequently altered in many human malignancies and overexpression of Akt induces malignant transformation and chemoresistance. Thus, the Akt pathway is a major target for anti-cancer drug development. This review focuses on Akt signaling mechanism in oncogenesis and chemoresistance, and ongoing translational efforts to therapeutically target Akt.

2. BACKGROUND

Akt was originally identified as the oncogene transduced by AKT8 acute transforming retrovirus. The AKT8 retrovirus was isolated from an AKR mouse thymoma in 1977. This virus induces malignant transformation in the mink lung epithelial cell line CCL-64 and tumor formation, specifically thymic lymphoma, in nude mice (1). A decade later, its defective retrovirus was identified from mink lung epithelial cells infected with AKT8 virus, and was shown to contain a cell-derived oncogenic sequence, which was termed Akt (2, Figure 1).

In early 1990, sequence analysis of the Akt viral oncogene and its cellular homolog revealed that it encodes a serine-threonine protein kinase, composed of a carboxy-terminal kinase domain very similar to that of PKC and PKA and an amino terminal PH domain (3). Akt was also cloned based on its homology with PKC or PKA by two additional groups, who named it RAC or PKB (4, 5). To date, the protein is most commonly referred to as Akt/PKB.

3. COMPARISON OF AKT ISOFORMS

3.1. Sequence and expression pattern

Three major isoforms of Akt encoded by three separate genes have been identified in mammalian cells. Akt1/PKBa and Akt2/PKBb were the first isolated isoforms (3-7). Akt3/PKBg was subsequently cloned through homology screening (8, 9). While Akt1 is the true human homologue of v-akt (98% identity at the amino acid level), Akt2 and Akt3 are v-akt closely related kinases (6, 9). The three isoforms of Akt/PKB are highly homologous to v-akt. The overall homology between these three isoforms is >85%. They share a very similar structure, which contains an N-terminal PH domain, a central kinase domain, and a serine/threonine-rich C-terminal region. All three Akt/PKB isoforms possess conserved threonine and serine residues (T308/S473 in Akt1, T309/S474 in Akt2 and T305/S472 in Akt3) that together with the PH domain are critical for Akt/PKB activation. The C-terminal regions between these three isoforms are more diverse (homology 73%~84%) as compared to the kinase domain (homology 90%~95%), suggesting that C-terminal regions may represent functional difference between Akt1, Akt2 and Akt3 (Figure 1).

Although Akt1, Akt2, and Akt3 display high sequence homology, there are clear differences between them in terms of biological and physiological function: 1) overexpression of wild type (WT)-Akt2, but not Akt1 and Akt3, transforms NIH 3T3 cells and induces invasion and metastasis in human breast and ovarian cancer cells (10, 11); 2) Akt2, but not Akt1 and Akt3, is frequently amplified in certain types of human cancer even though alterations of three isoforms of Akt have been detected at kinase and protein levels in human malignancies (6, 12-19); 3) Akt1 expression is relatively uniform in various normal organs whereas high levels of Akt2 and Akt3 mRNA are detected in skeletal muscle, heart, placenta and brain (6, 9, 20, 21); 4) Akt2 but not Akt1 plays un unique role in muscle differentiation (22, 23) and 5) Akt1-, Akt2- and Akt3-deficient mice displayed different phenotypes (24-27).

3.2. Phenotype of the Akt knock-out mouse

A knockout study demonstrated that mice deficient in Akt2 are impaired in the ability of insulin to lower blood glucose because of defects in the action of the hormone on skeletal muscle and liver. Akt2-/- mice are born without apparent defects, but develop peripheral insulin resistance and nonsuppressible hepatic glucose production, resulting in hyperglycemia accompanied by inadequate compensatory hyperinsulinemia (24), similar in some important features to type 2 diabetes in human. These phenotypic characteristics are not compensated by the presence of Akt1 and Akt3, reflecting differences of substrate specificity in insulin-responsive tissues. In contrast, Akt1-deficient mice do not display a diabetic phenotype. The mice are viable but display impairment in organismal growth with smaller organs than wild type littermates (25, 26). Such relatively subtle phenotypic changes in Akt1-/- mice suggest that Akt2 and Akt3 may substitute to some extent for Akt1 (26). In contrast, a recent report shows that Akt3 knock out mice exhibit a uniformly reduced brain size, affecting all major brain regions, suggesting a central role of Akt3 in postnatal development of the brain (27). Nevertheless, these data indicate that there are non-redundant functions between 3 isoforms of Akt in certain tissues and/or cell types.

4. AKT ONCOGENIC ACTIVITY AND ITS ROLE IN CHEMORESISTANCE

4.1. Mechanism of Akt activation

Akt is activated by a variety of stimuli, including growth factors, protein phosphatase inhibitors, and cellular stress in a PI3K-dependent manner (28-31). Activation of Akt depends on the integrity of the PH domain, which binds to PI3K products PtdIns-3,4-P2 and PtdIns-3,4,5-P3, and on the phosphorylation of Thr308 (Thr309 in Akt2 and Thr305 in Akt3) in the activation loop and Ser473 (Ser474 in Akt2 and Ser472 in Akt3) in the C-terminal activation domain by PDK1 and ILK or DNA-PK (Figure 2, ref. 32-34). The activity of Akt is negatively regulated by PTEN, a tumor suppressor gene that is mutated in a number of human malignancies. PTEN encodes a dual-specificity protein and lipid phosphatase that reduces intracellular levels of PtdIns-3,4-P2 and PtdIns-3,4,5-P3 in cells by converting them to PtdIns-4-P1 and PtdIns-4,5-P2, respectively, thereby inhibiting the PI3K/Akt pathway (35, 36).

4.2. Transforming activity of Akt

Previous studies demonstrated that overexpression of WT-Akt2, but not WT-Akt1, in NIH 3T3 cells resulted in malignant transformation (10). Ahmed et al. also showed that Akt1 is not tumorigenic when overexpressed in the nontumorigenic rat T cell lymphoma cell line 5675. In contrast, v-akt-expressing 5675 cells and active forms of Akt-expressing chicken embryo fibroblasts were highly tumorigenic (37, 38). Since v-akt arose by way of an in-frame fusion of the viral Gag and Akt, the oncogenic difference between v-akt and Akt1 may be due to myristoylation of the amino-terminus of v-akt (3, 20, 38). Several lines of evidence show that the PH domain of Akt is required for its membrane translocation and activation, and that attachment of a membrane-targeting sequence (myristoylation/palmitoylation) to the amino-terminus of Akt is sufficient to induce its maximal activation (39). Recent data also show that overexpression of constitutively active Akt1 and Akt3, but not kinase-dead Akt1 (Myr-Akt1-K179M) and Akt3, in NIH 3T3 cells leads to oncogenic transformation (14 and unpublished data). These results suggest that the kinase activity of Akt contributes to the control of cell transformation.

4.3. Alterations of Akt pathway in human cancer

Akt2 locates at chromosome 19q13, which it is frequently overpresented in human cancers. Amplification of the Akt2 has been observed in 15% of human ovarian carcinomas and 20% of human pancreatic cancers (6, 12, 17, 19). In contrast to Akt2, Akt1 has been reported to be amplified in only a single human gastric carcinoma (2). Because of its location at chromosome band 14q32, proximal to the IGH locus, Akt1 had been proposed as a candidate gene targeted by 14q32 chromosome rearrangements in human T-cell malignancies, prolymphocytic leukemias, and mixed lineage childhood leukemia. However, no such alteration of Akt1 was detected in more than 30 hematologic specimens examined (unpublished data). Accumulated studies have shown frequent overexpression and/or activation of Akt in different human malignancies (12-19, 40-42). Alterations of Akt were predominantly observed in late stage and high-grade tumors, suggesting that Akt plays an important role in tumor progression rather than initiation.

4.4. Akt and chemoresistance

Recent studies indicate that overexpression of HER-2/neu or Xiap renders tumor cells resistant to TNFa or to chemotherapeutic agents through activation of the PI3K/Akt pathway (43-45). Cancer cells either expressing constitutively active Akt or containing Akt gene amplification are also far more resistant to paclitaxel than cancer cells expressing low levels of Akt (46). We have recently observed that cisplatin-sensitive ovarian cancer cells (A2780s and OV2008) transfected with constitutively active Akt2 become resistant to cisplatin, whereas overexpression of dominant-negative (DN) Akt2 renders cisplatin-resistant ovarian cancer cells (A2780cp and C13) susceptible to cisplatin-induced apoptosis (47, 48). In addition, we previously reported inhibition of tumorigenecity and invasiveness of pancreatic cancer cell lines by antisense Akt2 (12) and recently demonstrated that PI3K/Akt is a critical target for farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase I inhibitor (GGTI) -induced apoptosis. Constitutively active Akt overcame FTI-277 and GGTI298-induced programmed cell death (49, 50).Taken together, these data indicate that the Akt pathway is a critical target for cancer intervention and that activation of this pathway is associated with chemoresistance in human cancer.

The molecular mechanism by which Akt induces transformation and drug resistance is still not fully understood. It is believed that Akt anti-apoptotic activity and induction of cell cycle progression largely contribute to these processes (Figure 3).

5. NORMAL CELLULAR FUNCTION OF AKT

5.1. Anti-apoptosis

5.1.1 . Apoptotic proteins

In numerous cell types, it has been shown that Akt induces cell survival and suppresses apoptotic death induced by a variety of stimuli. A major identified target of Akt is BAD, which is a BH3 domain-containing proapoptotic protein that binds Bcl-2 and Bcl-XL and inhibits their anti-apoptotic potential (51). When BAD is phosphorylated on Ser1-36 by Akt, it does not exhibit proapoptotic activity in cells. It has also been shown that Akt activates PAK1, which in turn phosphorylates BAD at Ser-112 resulting in its release from Bcl-xL complex (52). Once phosphorylated, BAD is released from a complex with Bcl-2/Bcl-xL that is localized on the mitochondrial membrane, and forms a complex with 14-3-3 proteins (51-53).

BAX is a 21-kDa protein that is important in controlling cell death, particularly in hematopoietic cells. Cells that overexpress BAX show enhanced apoptosis (54), whereas BAX-null cells display resistance to both spontaneous and induced apoptosis. The BAX protein is normally found in the cytoplasm heterodimerized to anti-apoptotic Bcl-2 family members such as Mcl-1 and Bcl-xL; however, once the cell is exposed to an apoptotic stimulus, BAX translocates to the mitochondria (55-57), where it is thought to form oligomers. These promote apoptosis by forming large transmembranous pores, resulting in the loss of mitochondrial membrane potential and the release of cytochrome c (58, 59). We and others have shown that ectopic expression of Akt inhibits BAX conformational change and mitochondrial translocation induced by chemotherapeutic reagents. A recent study suggests that Akt might phosphorylate BAX at Ser-184 (60).

A previous study shows Akt inhibition of apoptosis at the postmitochondrial level (61). An X-linked inhibitor of apoptosis protein (XIAP) has been recognized as an important antiapoptotic protein by direct interaction and inhibition of activated caspases 9, 3 and 7 at postmitochondrial level (62-68). It is known that a number of chemotherapeutic reagents induce XIAP degradation leading to programmed cell death (69-71). Elevated level of XIAP rendered cells resistant to cisplatin whereas knockdown XIAP sensitized cells to apoptosis induced by cisplatin and trail (70, 72, 73). We have recently demonstrated that XIAP is a direct substrate of Akt. Akt phosphorylates XIAP and inhibits XIAP ubiquitination/degradation induced by cisplatin (74). Knockdown XIAP by RNA interference or antisense XIAP largely abrogates Akt-induced cisplatin resistance (74). Therefore, XIAP is a major target of Akt at postmitochondrial level.

Human caspase-9 has been reported to be phosphorylated by Akt, resulting in attenuation of its activity (75). However, the phosphorylation site is not conserved in other mammalian species, suggesting that this regulation of Akt is not likely to be a major physiological regulatory pathway.

5.1.2. Other cell survival- related molecules

Accumulated evidence has shown that JNK is activated by a number of chemotherapeutic drugs and plays an essential role in anti-tumor reagents-induced programmed cell death (76, 77). Knockout JNK renders cells resistant to DNA damage-stimulated apoptosis (78). It has been shown that JNK mediates chemotherapeutic drug-induced apoptosis by phosphorylation of Bim, Bmf and Bid (79). We and others have previously demonstrated that Akt inhibited JNK activation induced by cisplatin through phosphorylation of apoptosis signal regulating kinase ASK1. The phosphorylation of ASK1 by Akt inhibited its kinase activity and failed to stimulate JNK activation (59, 80, 81).

Forkhead transcription factor (FoxO1, FoxO3, FoxO4, previously known as FKHR, FKHRL1 and AFX) is important in the induction of apoptosis (82, 83). Their target genes include FasL and Bim, which plays a pivotal role in death receptor and mitochondrial pathways. Akt phosphorylates FoxO at three serine/threonine sites (84-86). Upon phosphorylation of FoxO proteins by Akt, FoxO binds to 14-3-3 proteins which results in translocation of FoxO to the cytosol from the nucleus and consequently inactivation of its function as a transcription factor (84, 85).

Akt has been demonstrated to phosphorylate Yes-associated protein (YAP) and induce its association with 14-3-3 proteins. As is the case for FoxO, this results in the localization of YAP into the cytosol (87). YAP is a transcriptional co-activator which binds to p73 and promotes the transcription of its target genes. As p73 is a member of p53 family that plays an important role in the induction of apoptosis, Akt phosphorylation of YAP impairs the transcriptional activity of p73 and attenuates the induction of pro-apoptotic gene expression in response to DNA damaging agents.

There have been some indications that Akt can induce the expression of pro-survival genes, including IAPs and Bcl2. This may be due to positive cross-talk between the Akt and NFkB pathways. Activation of NFkB is dependent on the phosphorylation and degradation of IkB, an inhibitor of NFkB, by the IkB kinase (IKK) complex. Akt has been shown to regulate IKK activity in both direct and indirect manner. It has been shown that Akt interacts with and phosphorylates IKKa on Thr-23 (88, 89). Several studies have also provided evidence that Akt phosphorylates Ser/Thr kinase Tpl-2 (or Cot) on Ser-400, resulting in IKK complex activation (90, 91).

5.2. Cell cycle progression

5.2.1. Cell cycle regulators

Akt targets several key cell cycle regulators including p21cip1/waf1, p27kip1, and MDM2. Akt phosphorylates p21cip1/waf1 on residues Thr-145 and Ser-146 (92-94). The phosphorylation of Thr-145 inhibits p21 nuclear localization and affinity to Cdk2, Cdk4 and PCNA leading to activation of cyclin/CDK and DNA replication. However, phosphorylation of Ser-146 enhances protein stability of p21 that may result in cell survival (93, 94). Human p27kip1, another major cyclin/CDK inhibitor, is also phsophorylated by Akt on Thr-157, even though this site is not conserved in other species (95-97). As Thr-157 resides within a nuclear localization signal (NLS) region, Akt phosphorylation of Thr-157 leads to p27 exclusion from the nucleus. Our laboratory has shown that Akt decreases TSC1/TSC2 tumor complex by phosphorylation of TSC2 resulting in the destabilization of p27 (98). In addition, Akt phosphrylation of forkhead protein inhibits p27 at transcriptional level (99). Taken collectively, Akt downregulates p27 at different levels leading to activation of cyclin/Cdk and cell cycle progression (95-99)

MDM2 is a major negative regulator of p53 tumor suppressor. Loss of p53 function has been thought to be a major mechanism of chemoresistance (100). MDM2 has ubiquitin E3 ligase activity, directly binds to p53 and targets it for ubiquitination and proteasome degradation (101, 102). Akt has been shown to phosphorylate MDM2 on Ser-166 and Ser-186 and induce MDM2-mediated p53 ubiquitination, even though there is still controversial regarding subcellular localization of Akt phosphorylated MDM2 (103-105).

5.2.2. mTOR and TSC2

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and is best known as a regulator of cell cycle progression and cell proliferation by integrating signals from nutrients (amino acids and energy) and growth factors (106-108). The best known biochemical function of mTOR is to regulate protein translation by initiation of mRNA translation and ribosome synthesis leading to an increased rate of cell growth (an increase in cell mass and size), which is required for supporting the rapid proliferation (an increase in cell number). A model for mTOR function suggests that mTOR regulates primarily the rate of cell growth and secondarily cell cycle progression. The mTOR-dependent downstream effectors, S6K1, 4E-BP1, and eIF4E, also regulate the rate of cell cycle progression. When quiescent U2OS cells are stimulated with serum to enter G1 phase from G0, overexpression of S6K1 and eIF4E accelerates S phase entry, while reduced expression of S6K1 with RNAi or overexpression of a dominant mutant of 4E-BP1 inhibits the rate of S phase entry (109, 110). Moreover, overexpression of rapamycin-resistant mutants of S6K1 or overexpression of eIF4E partially rescues the rapamycin-induced delay in G1 progression to S phase, indicating that S6K1 and eIF4E are downstream mediators of mTOR-dependent cell division (109, 110).

It has been shown that Akt mediates insulin and nutrient activation of mTOR pathway through both direct and indirect mechanism. Akt phosphorylates mTOR at serine-2448 and possible serine-2446, which fit the Akt phosphorylation consensus motif (RXRXXS/T) in 3T3-L1 adipocytes, HEK293 cells and intact skeletal muscle (111-115). Although one might expect that Akt phosophorylation of serine-2448 would activate mTOR, there is no direct evidence to support such an effect. In fact, when expressed in HEK293 cells, nonphosphorylatable mTOR-S2448A (converting serine to alanine) or mTOR-S2446/2448A exhibit the same effect as overexpressed wild type mTOR in mediating insulin-stimulated phosphorylation of S6K1 and 4E-BP1. These data suggest that mTOR is not a direct target of Akt. We and others have demonstrated that Akt interacts with and phosphorylates tuberin, a product of tumor suppressor tuberous sclerosis complex (TSC) 2 gene. A possible mechanism for Akt activation of mTOR has been proposed by finding of tumor suppressor TSC2 linking Akt to mTOR.

Tuberous sclerosis is an autosomal dominant disorder developing hamartomas in multiple organs and is caused by mutation of either the TSC1 or the TSC2 tumor suppressor gene. TSC1 and TSC2 function as a complex to inhibit cell growth. Overexpression of TSC2 and TSC1 inhibited mTOR activity and blocked the increase in phosphorylation of S6K1 and 4E-BP1 in response to nutrients or growth factor stimulation (116-118). We and others have shown that Akt phosphorylates TSC2 at multiple serine/threonine sites and causes to degradation of TSC2 and TSC1 and disruption of TSC1/TSC2 complex (98, 11-123), resulting in release of its inhibition of mTOR. Further, expression of nonphosphorylatable TSC2 mutants with alanine substitutions at Akt phosphorylation sites blocks growth factor-induced S6K1 activation. TSC2 has been shown to interact with overexpressed TOR in Drosophila but this interaction does not occur in mammalian cells (124).

Several studies have shown TSC2/TSC1 inhibition of mTOR through TSC2 GAP activity to hydrolyze Rheb-GTP to inactive Rheb-GDP form. Rheb is a GTP-binding protein and overexpresing Rheb increases S6K1 and 4E-BP1 phosphorylation but does not induce the activity of a rapamycin-resistant form of S6K1, suggesting that Rheb signaling to S6K1 is through mTOR and not through a parallel pathway. However, no evidence shows that Rheb directly activates mTOR. Disruption of Rheb in S. cerevisiae leads to an increase in the uptake of arginine and lysine by the amino acid permease Can 1p (125-128). This implies that Rheb may control mTOR indirectly by changing amino acid level. Recent studies have demonstrated that mTOR functions as part of a larger signaling complex. Two mTOR-associated proteins, Raptor and GbL, have been identified by sequencing proteins that coimmunoprecipitated with mTOR. The GbL binds to kinase domain of mTOR whereas Raptor links mTOR to S6K1 and 4E-BP1 by binding to their TOR signaling (TOS) motifs, leading to mTOR-dependent phosphorylation of S6K and 4E-BP1 in response to nutrients or growth factors (129-134). However, there is no evidence that these two proteins are involved in Akt regulation of mTOR.

In addition, a number of transcriptional factors that associate with cell cycle control are regulated by Akt. Cyclic AMP (cAMP)-response element binding protein (CREB), is phosphorylated by Akt on Ser-133. This process results in increased affinity of CREB to its co-activator CRB, leading to transcriptional upregulation of cell cycle associated genes such as cyclin D1 (135). Estrogen receptor (ER)a is also phosphorylated by Akt (136). The phosphorylated ERa will induce its target gene expression which is thought to involve anti-estrogen resistance (136, 137).

Finally, angiogenesis induced by Akt may also associate with its transforming activity and chemoresistance. Accumulated evidence shows that Akt plays a central role in the sprouting of new blood vessels by mediating many angiogenic growth factors and regulating downstream target molecules that are potentially involved in blood vessel growth. It is known that VEGF has various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation by selectively binding to the Flk-1/KDR receptor and subsequent activation of Akt pathway (138). Constitutively active Akt also induces VEGF mRNA expression by stabilization (139) and enhanced translation (140) of HIF1a through regulation of mTOR pathway. Moreover, Akt phosphorylates eNOS on Ser-1177, resulting in enzymatic activation of eNOS (141), which leads to production of NO and angiogenesis.

6. AKT PATHWAY AS A POTENTIAL THERAPEUTIC TARGET FOR CANCER INTERVENTION

Although cytotoxic chemotherapeutic drugs are first-line agents for cancer, chemoresistance remains a major therapeutic hurdle. The prospect of gene targeted anti-tumor agents as a therapeutic approach for cancer, particularly for the chemoresistant disease, has generated considerable excitement.

As described above, Akt pathway is essential for cell survival, cell cycle progression and angiogenesis. Amplification/overexpression/activation of PIK3CA (p110a) enzymatic subunit of PI3K and Akt as well as somatic mutation of gene encoding p110a are frequently detected in human malignancy (142-146). Inhibition of PI3K and/or Akt induces programmed cell death in cancer cells (144). Expression of constitutively active Akt results in cancer cells resistance to cisplatin and taxol-induced apoptosis, whereas dominant negative Akt sensitizes the cells to chemotherapeutic drugs (147, 148). Thus, PI3K/Akt pathway is a critical target for cancer intervention and inhibition of PI3K and/or Akt could overcome a subset of chemoresistant cancers.

6.1. Biological approaches

Biological approaches include antisense, dominant-negative, antibody of PI3K and Akt as well as peptides to mimic and compete pleckstrin-homology (PH) domain of Akt binding to PI3K products, PtdIns-3,4-P2 and PtdIns-3,4,5-P3. We and other have previously demonstrated that the introduction of antisense Akt2 or DN-Akt into several Akt-overexpressing cancer cell lines abrogates endogenous Akt expression and diminishes their invasiveness and tumor formation in nude mice (12, 149). Antisense oligonucleotides of Akt can inhibit Akt pathway and induce apoptosis in different cell lines141 and cell growth and survival can also be inhibited by the expression of dominant negative (DN) forms of PI3K and Akt (150, 151). Our recent data show that expression of DN-Akt in NIH3T3 cells remarkably reduces v-H-ras-induced colony formation and tumor formation (unpublished data). Moreover, consistent with the tumor-inhibitory effects of DN-PI3K and DN-Akt is the demonstration of the inhibition of Ras and BCR/ABL malignant transformation with p85d iSH2 and DN-Akt, respectively (152, 153). Microinjection of AKT2 antibody into myoblasts can also specifically block their function, i.e., induction of myotube (154). Further studies are required to investigate the effects of antibodies of PI3K and Akt on human cancer cell growth.

6.2. Pharmacological approaches for upstream inhibition of Akt

Although wortmanin and LY294002 efficiently abrogate PI3K activity and have been widely used in the cell culture system (155, 156), they have not been applied for clinical trails due to either toxicity (LY294002) or a short of half-live (wortmanin). We have demonstrated that farnesyltransferase inhibitor (FTI)-277, originally designed to block Ras oncoprotein, inhibits PI3K/Akt pathway and induces apoptosis in a number of human cancer cell lines (49, 157). FTIs are highly effective at inhibiting tumor growth without toxicity to normal cells. However, the mechanism by which they inhibit tumor growth is not well understood (158-160). FTIs are unable to induce apoptosis in Raf transformed NIH 3T3 cells even though MAPK pathway is inhibited by FTIs (158, 159), indicating that FTIs may target other cell survival pathway(s) regulated by Ras or other farnesylated proteins. Interestingly, our data showed that FTI-277 induces apoptosis only in Akt2-overexpressing human cancer cell lines. Furthermore, overexpression of Akt2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277; and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not Akt2-transformed NIH 3T3 cells (49, 157). These data suggest that FTIs specifically target the PI3K/Akt pathway to inhibit tumor cell growth and may be candidate agents for reversing resistance of human cancer to cytotoxic chemotherapeutic drugs.

6.3. Akt inhibitors

The importance of Akt in cell survival, growth, cell transformation and human malignancy has prompted the search for specific and safe pharmacological inhibitors for Akt. Five recent reports including ours have identified the compounds as potential Akt inhibitors that reduce Akt kinase activity in a number of cancer cell lines (161-165).

Hu et al. synthesized a phosphatidylinositol analogue (1L-6-hydroxy-methyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate) and showed that it inhibited Akt by compete with phosphatidylinositol (161). This compound reduces the resistance of human leukemia cells to chemotherapeutic drugs and ionizing radiation (161). Chaudhary et al. demonstrated that the plant-derived pigment curcumin reduces Akt activity resulting in cell growth arrest in several prostate cancer cell lines (162). A compound synthesized from the natural plant compound rotenone (degeulin) has also been identified as a potential Akt and PI3K inhibitor in malignant human bronchial epithelial cells (163). Meuillet et al. have used a novel strategy to identify a group of D-3-deoxy-phosphatidyl-myo-inositols that bind to the PH domain of Akt, trapping it in the cytosol and preventing its activation in response to growth factors (164). We have recently identified a small molecule inhibitor of Akt, API (Akt/PKB signaling inhibitor)-2/TCN, by screening the National Cancer Institute Diversity Set (165). API-2 inhibited the kinase activity of Akt resulting in suppression of cell growth and induction of apoptosis in human cancer cells harboring constitutively activated Akt. API-2 is highly selective for Akt and does not inhibit the activation of PI3K, PDK1, PKC, SGK , PKA, STAT3, Erk-1/2, or JNK. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells where Akt is aberrantly expressed/activated but not of those cancer cells where it is not. These findings suggest that API-2 exerts anti-tumor activity largely by inhibition of Akt (165).

7. PERSPECTIVE

In the past decade, the mechanism for Akt activation, lipid second messenger-mediated phosphorylation of Akt, has been well characterized. However, the physiological and pathological difference of three isoforms of Akt remains elusive. While Akt is a key molecule in cell survival, downstream targets that mediate this action are still obscure. As Akt plays a pivotal role in human cancer development and chemoresistance, it is essential that future work is aimed at developing pharmacological reagents as well as genetic and biochemical approaches that not only identify novel roles for Akt but also verify the physiological functions previously ascribed. The generation of a potent and specific Akt inhibitors, especially isoform-specific Akt inhibitors, would certainly revolutionize the study of the processes mediated by Akt in the same way inhibitors of MAP kinase kinase 1 activation (e.g., PD98059, PD184352, U0126) have on our understanding of processes regulated by the classical MAP kinase pathway. More importantly, such drugs or in combination with conventional chemotherapeutic agents would reasonably improve the outcome of human cancer.

8. ACKNOWLEDGEMENT

This work was supported by grants from National Cancer Institute Grants and Department of Defense (J.Q.C.)

9. REFERENCES

1. Staal S. P., J. W. Hartley, & W. P. Rowe: Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc Natl Acad Sci U. S. A. 74, 3065-3067 (1977)

2. Staal S. P: Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary gastric adenocarcinoma. Proc Natl Acad Sci USA. 84, 5034-5037 (1987)

3. Bellacosa A, J. R. Testa, S. P. Staal, & P. N. Tsichlis: A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254, 274-277 (1991)

4. Jones P. F., T. Jakubowicz, F. J. Pitossi, F. Maurer, & B. A. Hemmings: Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci USA. 88, 4171-4175 (1991)

5. Coffer P. J. & J. R. Woodgett: 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 (1991)

6. Cheng J. Q., A. K. Godwin, A. Bellacosa, T. Taguchi, T. F. Franke, T. C. Hamilton, P. N. Tsichlis, & J. R. Testa: 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 (1992)

7. Jones P. F., T. Jakubowicz, & B. A. Hemmings: Molecular cloning of a second form of rac protein kinase. Cell Regul 2, 1001-1009 (1991)

8. Konishi H., S. Kuroda, M. Tanaca, Y. Ono, K. Kameyama, T. Haga, & U. Kikkawa: Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. Biochem Biophys Res Commun 216, 526-534 (1995)

9. Nakatani K, H. Sakaue, D. A. Thompson, R. J. Weigel & R. A. Roth: Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site. Biochem Biophys Res Commun 257, 906-910 (1999)

10. Cheng J. Q., D. A.Altomare, W. M. Klein, W-C. Lee, G. D. Kruh, N. A. Lissy, & J. R. Testa: Transforming activity and mitosis-dependent expression of the AKT2 oncogene: evidence suggesting a link between cell cycle regulation and oncogenesis. Oncogene 14, 2793-2801 (1997)

11. Arboleda M. J., J. F. Lyons, F. F. Kabbinavar, M. R. Bray, B. E. Snow, R. Ayala, M. Danino, B. Y. Karlan, & D. J. Slamon: Overexpression of AKT2/protein kinase Bb leads to up-regulation of b1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res 63, 196-206 (2003)

12. Cheng J. Q., B. Ruggeri, W. M. Klein, G. Sonoda, D. A. Altomare, D. K. Watson, & J. R. Testa: Amplification of AKT2 in human pancreatic cancer cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci USA 93, 3636-3641 (1996)

13. Yuan Z., M. Sun, R. I. Feldman, G. Wang, X. Ma, D. Coppola, S. V. Nicosia, & J. Q. Cheng: 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 (2000)

14. Sun M., G. Wang, J. E. Paciga, R. I. Feldman, Z. Yuan, X. Ma, S. A. Shelley, R. Jove, P. N. Tsichlis, S. V. Nicosia, & J. Q. Cheng: AKT1/PKBa kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathology 159, 431-437 (2001)

15. Sun M., J. E. Paciga, R. I. Feldman, Z. Yuan, S. A. Shelley, S. V. Nicosia, & J. Q. Cheng: Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor a (ERa) via interaction between ERa and PI3K. Cancer Res 61, 5985-5991 (2001)

16. Bellacosa A., D. DeFeo, A. K. Godwin, D. W. Bell, J. Q. Cheng, D. A. Altomare, M. Wan, L. Dubeau, G. Scambia, V. Masciullo, G. Ferrandina, P. Bennedetti Panici, S. Mancuso, G. Neri, & J. R. Testa: Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 64, 280-285 (1995)

17. Ruggeri B., L. Huang, M. Wood, J. Q. Cheng, & J. R. Testa: Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinoma. Molecular Carcinogenesis 21, 81-86 (1998)

18. Nakatani K., D. A. Thompson, A. Barthel, H. Sakaue, W. Liu, R. J. Weigel, & R. A. Roth: Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem 274, 21528-21532 (1999)

19. Miwa W., J. Yasuda, Y. Murakami, K. Yashima, K. Sugano, T. Sekine, A. Kono, S. Egawa, K. Yamaguchi, Y. Hayashizaki, & T. Sekiya: 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 (1996)

20. Bellacosa A., T. F. Franke, M. E. Gonzalez-Portal, K. Datta, T. Taguchi, J. Gardner, J. Q. Cheng, J. R. Testa, & P. N. Tsichlis: Structure, expression and chromosomal mapping of c-akt: relationship to v-akt and its implications. Oncogene 8, 745-754 (1993)

21. Altomare D. A., K. Guo, J. Q. Cheng, G. Sonoda, K. Walsh, & J. R. Testa: Cloning, chromosomal localization and expression analysis of the mouse Akt2 oncogene. Oncogene 11, 1055-1060 (1995)

22. Kaneko S., R. I. Feldman, L. Yu, Z. Wu, T. Gritsko, S. A. Shelley, S. V. Nicosia, T. Nobori, & J. Q. Cheng: Positive feedback regulation between Akt2 and MyoD during muscle differentiation: Cloning of Akt2 promoter. J Biol Chem 277, 23230-23235 (2002)

23. Vandromme M., A. Rochat, R. Meier, G. Carnac, D. Besser, B. A. Hemmings, A. Fernandez, & N. J. Lamb: Protein kinase Bb/Akt2 plays a specific role in muscle differentiation. J Biol Chem 276, 8173-8179 (2001)

24. Cho H., J. Mu, J. K. Kim, J. L. Thorvaldsen, Q. Chu, E. B. 3rd. Crenshaw, K. H. Kaestner, M. S. Bartolomei, G. I. Shulman, & M. J. Birnbaum: Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBb). Science 292, 1728-1731 (2001)

25. Chen W. S., P. Z. Xu, K. Gottlob, M. L. Chen, K. Sokol, T. Shiyanova, I. Roninson, W. Weng, R. Suzuki, K. Tobe, T. Kadowaki, & N. Hay: Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev 15, 2203-2208 (2001)

26. Cho H., J. L. Thorvaldsen, Q. Chu, F. Feng, & M. J. Birnbaum: Akt1/PKBa is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276, 38349-38352 (2001)

27. Yang Z. Z., O. Tschopp, A. Baudry, B. Dummler, D. Hynx, & B. A.Hemmings: Physiological functions of protein kinase B/Akt. Biochem Soc Trans 32, 350-354 (2004)

28. Franke T. F., S. L. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, D. R. Kaplan, & P. N. Tsichlis: The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81, 727-736 (1995)

29. Burgering B. M. T., & P. J. Coffer: Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599-602 (1995)

30. Meier R., D. R. Alessi, P. Cron, M. Andjelkovic, & B. A. Hemmings: Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bb. J Biol Chem 272, 30491-30497 (1997)

31. Liu A. X., J. R. Testa, T. C. Hamilton, R. Jove, S. V. Nicosia, & J. Q. Cheng: 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 (1998)

32. Chan T. O., S. E. Rittenhouse, & P. N.Tsichlis: AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 68, 965-1014 (1999)

33. Persad S., S. Attwell, V. Gray, N. Mawji, J.T. Deng, D. Leung, J. Yan, J. Sanghera, M.P. Walsh, & S. Dedhar. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem 276, 27462-27469 (2001)

34. Feng J., J. Park, P. Cron, D. Hess, & B.A. Hemmings. Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase. J Biol Chem 279, 41189-41196 (2004)

35. Li J., L. Simpson, M. Takahashi, C. Miliaresis, M. P. Myers, N. Tonks, & R. Parsons: The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene. Cancer Res 58:5667-5672 (1998)

36. Stambolic V., A. Suzuki, J. L. de la Pompa, G. M. Brothers, C. Mirtsos, T. Sasaki, J. Ruland, J. M. Penninger, D. P. Siderovski, & T. W. Mak: Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 95, 29-39 (1998)

37. Ahmed N. N., T. F. Franke, A. Bellacosa, K. Datta, M.-E. Gonzalez-Portal, T. Taguchi, J. R. Testa, & P. N. Tsichlis: The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene 8, 1957-1963 (1993)

38. Aoki M., O. Batista, A. Bellacosa, P. N. Tsichlis, & P. K. Vogt: The akt kinase: molecular determinants of oncogenicity. Proc Natl Acad Sci USA 95, 14950-14955 (1998)

39. Alessi D. R., & P. Cohen: Mechanism of activation and function of protein kinase B. Curr. Opin Genet Dev 8, 55-62 (1998)

40. J. Brognard, A. S. Clark, Y. Ni, & P. A. Dennis: 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 (2001)

41. Sekharam M., H. Zhao, M. Sun, Q. Fang, Q. Zhang, Z. Yuan, H. C. Dan, D. Boulware, J. Q, Cheng, & D. Coppola: Insulin-like growth factor 1 receptor enhances invasion and induces resistance to apoptosis of colon cancer cells through the Akt/Bcl-x(L) pathway. Cancer Res 63, 7708-7716 (2003)

42. Nakayama H, T. Ikebe, M Beppu, K Shirasuna: High expression levels of NFkB, IKKa and Akt kinase in squamous cell carcinoma of the oral cavity. Cancer 92, 3037-3044 (2001)

43. Knuefermann C., Y. Lu, B. Liu, W. Jin, K. Liang, L. Wu, M. Schmidt, G. B. Mills, J. Mendelsohn, & Z. Fan: HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells. Oncogene 22, 3205-3212 (2003)

44. Fraser M., B. M. Leung, X. Yan, H. C. Dan, J. Q. Cheng, & B. K. Tsang: p53 is a determinant of X-linked inhibitor of apoptosis protein/Akt-mediated chemoresistance in human ovarian cancer cells. Cancer Res 63, 7081-7088 (2003)

45. Cheng J. Q., X. Jiang, M. Fraser, M. Li, H. C. Dan, M. Sun, & B. K.Tsang: Role of X-linked inhibitor of apoptosis protein in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase/Akt pathway. Drug Resist Updat 5, 131-146 (2002).

46. Li J., Q. Feng, J. M. Kim, D. Schneiderman, P. Liston, M. Li, B. Vanderhyden, W. Faught, M. F. Fung, M. Senterman, R. G. Korneluk, & B. K. Tsang: Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins. Endocrinology 142, 370-380 (2001)

47. Dan H. C., M. Sun, S. Kaneko, R. I. Feldman, S. V. Nicosia, H-G. Wang, B. K. Tsang, & J. Q. Cheng: Akt Phosphorylation and Stabilization of XIAP in Human Ovarian Cancer Cells. J Biol Chem. 279, 5405-5412 (2004)

48. Yuan Z., R. I. Feldman, G. E. Sussman, D. Coppola, S. V. Nicosia, & J. Q. Cheng: AKT2 inhibition of cisplatin-induced JNK/p38 and BAX activation by phosphorylation of ASK1: Implication of AKT2 in chemoresistance. J Biol Chem 278, 23432-23440 (2003)

49. Jiang K., D. Coppola, N. C. Crespo, S. V. Nicosia, A. D. Hamilton, S. M. Sebti, & J. Q. Cheng: The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20, 139-148 (2000)

50. Dan H. C., K. Jinag, D. Coppola, A. D. Hamilton, S. V. Nicosia, S. M. Sebti, & J. Q. Cheng: Phosphatidylinositol-3-OH Kinase/Akt and Survivin Pathways as Critical Targets for Geranylgeranyltransferase I Inhibitors Induced Apoptosis. Oncogene 23, 706-715 (2004)

51. Datta S. R., H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, & M. E. Greenberg: Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241 (1997)

52. Tang Y., H. Zhou, A. Chen, R. N. Pittman, & J. Field: The Akt proto-oncogene links Ras to Pak and cell survival signals. J Biol Chem 275, 9106-9109 (2000)

53. Datta S. R., A. Katsov, L. Hu, A. Petros, S. W. Fesik, M. B. Yaffe, & M. E. Greenberg: 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 6, 41-51 (2000)

54. Shinoura N., Y. Yoshida, A. Asai, T. Kirino, & H. Hamada: Relative level of expression of BAX and Bcl-XL determines the cellular fate of apoptosis/necrosis induced by the overexpression of BAX. Oncogene 18, 5703-5713 (1999)

55. Granville D. J., J. R. Shaw, S. Leong, C. M. Carthy, P. Margaron, D. W. Hunt, & B. M. McManus: Release of cytochrome c, BAX migration, Bid cleavage, and activation of caspases 2, 3, 6, 7, 8, and 9 during endothelial cell apoptosis. Am J Pathol 155, 1021-1025 (1999)

56. Wolter K. G., Y. T. Hsu, C. L. Smith, A. Nechushtan, X. G. Xi, & R. J. Youle: Movement of BAX from the cytosol to mitochondria during apoptosis. J Cell Biol 139, 1281-1292 (1997)

57. Murphy K. M., V. Ranganathan, M. L. Farnsworth, M. Kavallaris, & R. B. Lock: Bcl-2 inhibits BAX translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death & Differ 7, 102-111 (2000)

58. Eskes R., B. Antonsson, A. Osen-Sand, S. Montessuit, C. Richter, R. Sadoul, G. Mazzei, A. Nichols, & J. C. Martinou: BAX-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J Cell Biol 143, 217-224 (1998)

59. Yuan Z. Q., R. I. Feldman, G. E. Sussman, D. Coppola, S. V. Nicosia, & J. Q. Cheng: AKT2 inhibition of cisplatin-induced JNK/p38 and BAX activation by phosphorylation of ASK1: implication of AKT2 in chemoresistance. J Biol Chem 278, 23432-23440 (2003)

60. Gardai S. J., D. A. Hildeman, S. K. Frankel, B. B. Whitlock, S. C. Frasch, N. Borregaard, P. Marrack, D. L. Bratton, & P. M. Henson: Phosphorylation of BAX Ser184 by Akt regulates its activity and apoptosis in neutrophils. J Biol Chem 279, 21085-21095 (2004)

61. Zhou H., X. M. Li, J. Meinkoth, & R. N. Pittman: Akt regulates cell survivaland apoptosis at a postmitochondrial level. J Cell Biol 151, 483-494 (2000)

62. Deveraux Q. L., & J. C. Reed: IAP family proteins-suppressors of apoptosis. Genes Dev 13:239-252 (1999)

63. Miller L.K.: An exegesis of IAPs: salvation and surprises from BIR motifs. Trends Cell Biol 9, 323-328 (1999)

64. Deveraux Q. L., R.Takahashi, G. S. Salvesen, & J. C. Reed: X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388, 300-304 (1997)

65. Roy N., Q. L. Deveraux, R. Takahashi, G. S. Salvesen, & J. C. Reed: The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16, 6914-6925 (1997)

66. Deveraux Q. L., N. Roy, H. R. Stennicke, T. Van Arsdale, Q. Zhou, S. M. Srinivasula, E. S. Alnemri, G. S. Salvesen, & J. C. Reed: IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17, 2215-2223 (1998)

67. Deveraux Q. L., E. Leo, H. R. Stennicke, K. Welsh, G. S. Salvesen, & J. C. Reed: Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J 18, 5242-5251 (1999)

68. Huang Q., Q. L. Deveraux, S. Maeda, G. S. Salvesen, H. R. Stennicke, B. D. Hammock, & J. C. Reed: Evolutionary conservation of apoptosis mechanisms: lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9. Proc Natl Acad Sci USA 97, 1427-1432 (2000)

69. Sasaki H, Y Sheng, F Kotsuji, B. K. Tsang: Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells. Cancer Res 60, 5659-5666 (2000)

70. Cheng J. Q., X. Jiang, M. Fraser, M. Li, H. C. Dan, M. Sun, & B. K. Tsang: Role of X-linked inhibitor of apoptosis protein in chemoresistance in ovarian cancer: possible involvement of the phosphoinositide-3 kinase/Akt pathway. Drug Resist Updat. 5, 131-146 (2002)

71. Yang Y., S. Fang, J.P. Jensen, A. M. Weissman, & J. D. Ashwell: Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288, 874-877 (2000)

72. Kinoshita H., H. Yoshikawa, K. Shiiki, Y. Hamada, Y. Nakajima, & K. Tasaka: Cisplatin (CDDP) sensitizes human osteosarcoma cell to Fas/CD95-mediated apoptosis by down-regulating FLIP-L expression. Int J Cancer 88, 986-991 (2000)

73. Li J., Q. Feng, J. M. Kim, D. Schneiderman, P. Liston, M. Li, B. Vanderhyden, W. Faught, M. F. Fung, M. Senterman, R. G. Korneluk, & B. K. Tsang: Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins. Endocrinology 142, 370-380 (2001)

74. Dan H.C., M. Sun, S. Kaneko, R. I. Feldman, S. V. Nicosia, H. G. Wang, B .K. Tsang, & J. Q. Cheng: Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J Biol Chem. 279, 5405-5412 (2004)

75. Cardone M. H., N. Roy, H. R. Stennicke, G. S. Salvesen, T. F. Franke, E. Stanbridge, S. Frisch, & J. C. Reed: Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318-1321 (1998)

76. Benhar M., I. Dalyot, D. Engelberg, & A. Levitzki: Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress. Mol Cell Biol 21, 6913-6926 (2001)

77. Sanchez-Perez I., J. R. Murguia, & R. Perona: Cisplatin induces a persistent activation of JNK that is related to cell death. Oncogene 16, 533-540 (1998)

78. Tibbles L. A., & J. R. Woodgett: The stress-activated protein kinase pathways. Mol Life Sci 55, 1230-1254 (1999)

79. Yamamoto K., H. Ichijo, & S. J. Korsmeyer: BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Mol Cell Biol 19, 8469-8478 (1999)

80. Yoon S. O., M. M Kim, S. J. Park, D. Kim, J. Chung, & A. S. Chung: Selenite suppresses hydrogen peroxide-induced cell apoptosis through inhibition of ASK1/JNK and activation of PI3-K/Akt pathways. FASEB J 16, 111-113 (2002)

81. Kim A. H., G. Khursigara, X. Sun, T. F. Franke, & M.V. Chao: Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 21, 893-901 (2001)

82. Lin K., J. B. Dorman, A. Rodan & C. Kenyon: daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278, 1319-1322 (1997)

83. Ogg S., S. Paradis, S. Gottlieb, G.I. Patterson, L. Lee, H.A. Tissenbaum & G. Ruvkun: The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994-999 (1997)

84. Brunet A., A. Bonni, M.j. Zigmond, M. Z. Lin, P. Juo, L. S. Hu, M. J. Anderson, K. C. Arden, J. Blenis & M. E. Greenberg: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857-868 (1999)

85. Kops G.J., N. D. de Ruiter, A. M. De Vries-Smits, D. R. Powell, J. L. Bos & B. M. Burgering: Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398, 630-634 (1999)

86. Biggs W. H. I., J. Meisenhelder, T. Hunter, W. K. Cavenee & K. C. Arden: Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96, 7421-7426 (1999)

87. Basu S., N. F. Totty, M. S. Irwin, M. Sudol, & J. Downward: Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol Cell 11, 11-23 (2003)

88. Ozes O. N., L. D. Mayo, J. A. Gustin, S. R. Pfeffer, L. M. Pfeffer, & D. B. Donner: NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401, 82-85 (1999)

89. Romashkova J. A., & S. S. Makarov: NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86-90 (1999)

90. Kane L P., M. N. Mollenauer, Z. Xu, C. W. Turck, & A. Weiss: Akt-dependent phosphorylation specifically regulates Cot induction of NF-kappa B-dependent transcription. Mol Cell Biol 22, 5962-5974 (2002)

91. Mattioli I., A. Sebald, C. Bucher, R.P. Charles, H. Nakano, T. Doi, M. Kracht, ML. Schmitz: Transient and selective NF-kappaB p65 serine 536 phosphorylation induced by T cell costimulation is mediated by IkappaB kinase beta and controls the kinetics of p65 Nuclear Import. J Immunol 172, 6336-6344 (2004)

92. Zhou B. P., Y. Liao, W. Xia, B. Spohn, M. H. Lee, & M.C. Hung: Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol 3, 245-252 (2001)

93. Rossig L., A. S. Jadidi, C. Urbich, C. Badorff, A.M. Zeiher, & S. Dimmeler: Akt-dependent phosphorylation of p21 (Cip1) regulates PCNA binding and proliferation of endothelial cells. Mol Cell Biol 21, 5644-5657 (2001)

94. Li Y., D. Dowbenko, & L. A. Lasky: AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J Biol Chem 277, 11352-11361 (2002)

95. Viglietto G., M. L. Motti, P. Bruni, R.M. Melillo, A. D'Alessio, D. Califano, F. Vinci, G. Chiappetta, P. Tsichlis, A. Bellacosa, A. Fusco, & M. Santoro: Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer. Nat Med 8, 1136-1144 (2002)

96. Liang J., J. Zubovitz, T. Petrocelli, R. Kotchetkov, M. K. Connor, K. Han, J.H.Lee, S., Ciarallo, C. Catzavelos, R. Beniston, E. Franssen, & J.M. Slingerland: PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 8, 1153-1160 (2002)

97. Shin I., F. M. Yakes, F. Rojo, N.Y. Shin, A.V. Bakin, J. Baselga, & C.L. Arteaga: PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 8, 1145-1152 (2002)

98. Dan H.C., M. Sun, L. Yang, R. I. Feldman, X. M. Sui, C. C. Ou, M. Nellist, R. S. Yeung, D. J. Halley, S. V. Nicosia, & J. Q. Cheng: Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin. J Biol Chem 277, 35364-35370 (2002)

99. Nakamura N., S. Ramaswamy, F. Vazquez, S. Signoretti, M. Loda, & W. R. Sellers: Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN. Mol Cell Biol 20, 8969-8982 (2000)

100. Vousden K. H., & X. Lu: Live or let die: the cell's response to p53. Nat Rev 2, 594-604. (2002)

101. Shimizu H., & T.R. HuppIntrasteric: Regulation of MDM2. Trends Biochem Sci 28, 346-349 (2003)

102. Mayo L. D., & D.B. Donner: A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci USA. 98, 11598-11603 (2001)

103. Zhou B. P., Y. Liao, W. Xia, Y. Zou, B. Spohn, & M.C. Hung: HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat. Cell Biol 3, 973-982 (2001)

104. Ashcroft M., R. L. Ludwig, D. B. Woods, T. D. Copeland, H. O. Weber, E. J. MacRae, & K. H. Vousden: Phosphorylation of HDM2 by Akt. Oncogene 21, 1955-1962 (2002)

105. Ogawara Y., S. Kishishita, T. Obata, Y. Isazawa, T. Suzuki, K. Tanaka, N. Masuyama, & Y. Gotoh: Akt enhances Mdm2-mediated ubiquitination and degradation of p53. J Biol Chem 277, 21843-21850 (2002)

106. Heitman J., N. R. Movva, & M. N. Hall: Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905-909 (1991)

107. Kunz J., R. Henriquez, U. Schneider, M. Deuter-Reinhard, N.R. Movva, & M.N. Hall: Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell 73, 585-596 (1993)

108. Helliwell S.B., P. Wagner, J. Kunz, M. Deuter-Reinhard, R. Henriquezand M.N. Hall: TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell 5, 105-118 (1994)

109. Abraham R.T.: Identification of TOR signaling complexes: more TORC for the cell growth engine. Cell 111, 9-12 (2002)

110. T. Schmelzle, & M.N. Hall: TOR, a central controller of cell growth. Cell 103, 253-262 (2000)

111. Nave B.T., M. Ouwens, D. J. Withers, D. R. Alessi, & P. R. Shepherd: Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344, 427-431 (1999)

112. Sekulic A., C. C. Hudson, J. L. Homme, P. Yin, D. M. Otterness, L. M. Karnitz, R. T. Abraham: A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60, 3504-3513 (2000)

113. Reynolds T. H., S. C. Bodine, & J. C. Jr. Lawrence: Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J Biol Chem. 277, 17657-17662 (2002)

114. Alessi D. R., F. B. Caudwell, M. Andjelkovic, B. A. Hemmings, & P. Cohen: Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett 99, 333-338 (1996)

115. Bolster D. R., N. Kubica, S. J. Crozier, D. L. Williamson, P. A. Farrell, S. R. Kimball, & L. S. Jefferson: Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 15, 213-220 (2003)

116. Yeung R. S.: Multiple roles of the tuberous sclerosis complex genes. Genes Chromosomes Cancer. 38, 368-375 (2003)

117. Krymskaya V. P.: Tumour suppressors hamartin and tuberin: intracellular signalling. Cell Signal 15, 729-739 (2003)

118. Potter C. J., L. G. Pedraza, H. Huang, T. Xu: The tuberous sclerosis complex (TSC) pathway and mechanism of size control. Biochem Soc Trans 31, 584-586 (2003)

119. Inoki K., Y. Li, T. Zhu, J. Wu, & K.L. Guan: TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 4, 648-657 (2002)

120. Plas D.R., & C.B. Thompson: Akt activation promotes degradation of tuberin and FOXO3a via the proteasome. J Biol Chem. 278, 12361-12366 (2003)

121. Potter C. J., L. G. Pedraza, & T. Xu: Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol. 4, 658-65 (2002)

122. Manning B.D., A.R. Tee, M.N. Logsdon, J. Blenis, & L.C. Cantley: Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 10, 151-162 (2002)

123. Tee A. R., R. Anjum, & J. Blenis: Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin. J Biol Chem. 26; 278, 37288-37296 (2003)

124. Jaeschke A., J. Hartkamp, M. Saitoh, W. Roworth, T. Nobukuni, A. Hodges, J. Sampson, G. Thomas, & R. Lamb: Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J Cell Bio. 159, 217-224 (2002)

125. Garami A., F. J. Zwartkruis, T. Nobukuni, M. Joaquin, M. Roccio, H. Stocker, S. Kozma, E. Hafen, J. L. Bos, & G. Thomas: Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol Cell 1, 1457-1466 (2003)

126. Castro A. F., J. F. Rebhun, G. J. Clark, & L. A. Quilliam: Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem 278, 32493-32496 (2003)

127. Inoki K., Y. Li, T. Xu, & K. L. Guan. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 17, 1829-1834 (2003)

128. Tee A. R., B. D. Manning, P. P. Roux, L. C. Cantley, & J. Blenis: Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb. Curr Biol 3, 1259-1268 (2003)

129. Kim D. H., D. D. Sarbassov, S. M. Ali, J. E. King, R. R. Latek, H. Erdjument-Bromage, P. Tempst, & D.M. Sabatini: mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 10, 163-175 (2002)

130. Hara K., Y. Maruki, X. Long, K. Yoshino, N. Oshiro, S. Hidayat, C. Tokunaga, J. Avruch, & K. Yonezawa: Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177-189 (2002)

131. Nojima H., C. Tokunaga, S. Eguchi, N. Oshiro, S. Hidayat, K. Yoshino, K. Hara, N. Tanaka, J. Avruch, & K. Yonezawa: The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif. J Biol Chem 278, 15461-15464 (2003)

132. Choi K. M., L. P. McMahon, J. C. Jr. Lawrence: Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor. J Biol Chem. 278, 19667-19673 (2003)

133. Kim D. H., D. Sarbassov dos, S. M. Ali, R. R. Latek, K. V. Guntur, H. Erdjument-Bromage, P. Tempst, & D. M. Sabatini: GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell 1, 895-904 (2003)

134. Schalm S. S., D.C. Fingar, D. M. Sabatini, & J. Blenis: TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr Biol 13, 797-806 (2003)

135. Pradeep A., C. Sharma, P. Sathyanarayana, C. Albanese, J.V. Fleming, T.C. Wang, M.M. Wolfe, K.M. Baker, R.G. Pestell, & B. Rana: Gastrin-mediated activation of cyclin D1 transcription involves beta-catenin and CREB pathways in gastric cancer cells. Oncogene 23, 3689-3699 (2004)

136. Campbell R. A., P. Bhat-Nakshatri, N. M. Patel, D. Constantinidou, S. Ali, & H. Nakshatri: Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J Biol Chem 276, 9817-9824 (2001)

137. Dupont J., D. & Le Roith: Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects. Mol Pathol 54, 149-154 (2001)

138. Gerber H. P., A. McMurtrey, J. Kowalski, M. Yan, B. A. Keyt, V. Dixit, & N. Ferrara: Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem 273, 30336-30343 (1998)

139. Zhong H., K. Chiles, D. Feldser, E. Laughner, C. Hanrahan, M.M. Georgescu, J.W. Simons, & G. L. Semenza: Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 60, 541-545 (2000)

140. Laughner E., P. Taghavi, K. Chiles, P. C. Mahon, & G. L. Semenza: HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 21, 3995-4004 (2001)

141. Dimmeler S., I. Fleming, B. Fisslthaler, C. Hermann, R. Busse, & A.M. Zeiher: Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601-605 (1999)

142. Shayesteh L., Y.L. Lu, W.L. Kuo, R. Baldocchi, T. Godfrey, C. Collins, D. Pinkel, B. Powell, G.B. Mills, & J.W. Gray: PIK3CA is implicated as an oncogene in ovarian cancer. Nature Genet 21, 99-102 (1999)

143. Cheng J. Q., A. K. Godwin, A. Bellacosa, T. Taguchi, T. F. Franke, T. C. Hamilton, P. N. Tsichlis, & J. R. Testa: AKT2, a putative oncogene encoding a member of a novel subfamily of serine-threonine protein kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci USA 89, 9267-9271 (1992)

144. Yuan Z., M. Sun, R. I. Feldman, G. Wang, X. Ma, D. Coppola, S. V. Nicosia, & J. Q. Cheng: 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 (2000)

145. Sun M., G. Wang, J. E. Paciga, R. I. Feldman, Z. Yuan, X. Ma, S.A. Shelley, R. Jove, P.N. Tsichlis, S. V. Nicosia & J. Q. Cheng: AKT1/PKBa kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathology 159, 431-437 (2001)

146. Philp A. J., I. G. Campbell, C. Leet, E. Vincan, S. Rockman, R.H. Whitehead, R. J. Thomas, & W. A. Phillips: The phosphatidylinositol 3'-kinase p85a gene is an oncogene in human ovarian and colon tumors. Cancer Res 61, 7426-7429 (2001)

147. Herod J. O., A. G. Eliopoulos, J. Warwick, G. Niedobitek, L. S. Young, & D. J. Kerr: The prognostic significance of Bcl-2 and p53 expression in ovarian carcinoma. Cancer Res 56, 2178-2184 (1996)

148. Datta S. R., H. Dudek, X. Tao, S. Masters, H. Fu., Y. Gotoh, & M. E. Greenberg: Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231-241 (1997)

149. Jetzt A., J. A. Howe, M. T. Horn, E. Maxwell, Z. Yin, D. Johnson, & C. C. Kumar. 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 (2003)

150. Sato S., N. Fujita, & T. Tsuruo: Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 97, 10832-10837 (2000)

151. Orike N., G. Middleton, E. Borthwick, V. Buchman, T. Cowen, & A.M. Davies: Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons. J Cell Biol 154, 995-1005 (2001)

152. Rodriguez-Viciana P., P. H. Warne, A. Khwaja, B.M. Marte, D. Pappin, P. Das, M. D. Waterfield, A. Ridley, & J. Downward: Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 89, 457-67 (1997)

153. Skorski T., A. Bellacosa, M. Nieborowska-Skorska, M. Majewski, R. Martinez, J. K. Choi, R. Trotta, P. Wlodarski, D. Perrotti, T. O Chan, M. A. Wasik, P. N. Tsichlis, & B. Calabretta: Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway. EMBO J 16, 6151-6161 (1997)

154. Vandromme M., A. Rochat, R. Meier, G. Carnac, D. Besser, B. A. Hemmings, A. Fernandez, & N. J. Lamb: Protein kinase B b/Akt2 plays a specific role in muscle differentiation. J Biol Chem. 276, 8173-8179 (2001)

155. Dong Z., C. Huang, & W. Y. Ma: PI-3 kinase in signal transduction, cell transformation, and as a target for chemoprevention of cancer. Anticancer Res 19, 3743-3747 (1999)

156. Mills G. B, Y. Lu, X. Fang, H. Wang, A. Eder, M. Mao, R. Swaby, K. W. Cheng, D. Stokoe, K. Siminovitch, R. Jaffe, & J. Gray: The role of genetic abnormalities of PTEN and the phosphatidylinositol 3- kinase pathway in breast and ovarian tumorigenesis, prognosis, and therapy. Semin Oncol 28, 125-141 (2001)

157. Sebti S. M., & A. D. Hamilton: Farnesyltransferase and geranylgeranyltransferase I inhibitors in cancer therapy: important mechanistic and bench to bedside issues. Expert Opin Investig Drugs 9, 2767-2782 (2000)

158. Cox A. D., & C. J. Der: Farnesyltransferase inhibitors and cancer treatment: targeting simply Ras? Biochim. Biophys Acta 1333, F51-71 (1997)

159. Sebti S. M., & A. D. Hamilton: Inhibition of Ras prenylation: a novel approach to cancer chemotherapy. Pharmacol. Ther. 74, 103-114 (1997)

160. Lebowitz P. F., & G. C. Prendergast: Non-Ras targets of farnesyltransferase inhibitors: focus on Rho. Oncogene 17, 1439-45 (1998)

161. Hu Y., L. Qiao, S. Wang, S. B. Rong, E. J. Meuillet, M. Berggren, A. Gallegos, G. Powis, & A. P. Kozikowski: 3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth. J Med Chem. 43, 3045-3051 (2000)

162. Chaudhary L. R., & K. A. Hruska: Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells. J Cell Biochem 89, 1-5 (2003)

163. Chun K. H., J. W. 2nd Kosmeder, S. Sun, J. M. Pezzuto, R. Lotan, W. K. Hong, H. Y. Lee: 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 (2003)

164. Meuillet E.J., D. Mahadevan, H. Vankayalapati, M. Berggren, R. Williams, A. Coon, A.P. Kozikowski, & G. Powis. Specific inhibition of the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo-inositol analogues. Mol Cancer Ther. 2, 389-399 (2003)

165. Yang L., H. C. Dan, M. Sun, Q. Liu, X.M. Sun, R. I. Feldman, A. D. Hamilton, M. Polokoff, S. V. Nicosia, M. Herlyn, S. M. Sebti, & J. Q. Cheng: 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 (2004)

Key Words: Akt/PKB, Apoptosis, Cell Growth, Inhibitor, Chemoresistance, Review

Send correspondence to: Dr Jin Q. Cheng, Department of Pathology, University of South Florida College of Medicine and H. Lee Moffitt Cancer Center, 12901 Bruce B. Downs Blvd., MDC Box 11, Tampa, Florida 33612, Tel.: 813-974-8595; Fax: 813-974-5536; E-mail: jcheng@hsc.usf.edu