Involvement of PI3K/Akt Pathway in Cell Cycle Progression

MOLECULAR TARGETS FOR THERAPY (MTT)

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy F Chang1,4, JT Lee1,4, PM Navolanic1, LS Steelman1, JG Shelton1, WL Blalock2, RA Franklin1,3 and JA McCubrey1,3 1Department of Microbiology & Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA; 2Shands Cancer Center, University of Florida, Gainesville, FL, USA; and 3Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, NC, USA

The PI3K/Akt signal transduction cascade has been investi- Aberrant PI3K activity and oncogenic transformation gated extensively for its roles in oncogenic transformation. Initial studies implicated both PI3K and Akt in prevention of apoptosis. However, more recent evidence has also associated The relation between dysregulated PI3K activity and cancer is this pathway with regulation of cell cycle progression. Un- well documented. For example, a mutated form of the p85 covering the signaling network spanning from extracellular subunit of PI3K has recently been isolated in a Hodgkin’s environment to the nucleus should illuminate biochemical lymphoma-derived cell line (CO).12 The PI3K/Akt pathway has events contributing to malignant transformation. Here, we been shown to be the predominant growth-factor-activated discuss PI3K/Akt-mediated signal transduction including its pathway in LNCaP human prostate carcinoma cells.13 PI3K mechanisms of activation, signal transducing molecules, and activity has been linked to a variety of human tumors including effects on gene expression that contribute to tumorigenesis. 14 15 16 17 Effects of PI3K/Akt signaling on important proteins controlling breast cancer, lung cancer, melanomas, and leukemia cellular proliferation are emphasized. These targets include among others. Further evidence has shown that Akt (protein cyclins, cyclin-dependent kinases, and cyclin-dependent ki- kinase B, PKB), a downstream kinase of PI3K, is also involved in nase inhibitors. Furthermore, strategies used to inhibit the malignant transformation.18 Inhibition of this pathway is a PI3K/Akt pathway are presented. The potential for cancer promising approach for novel chemotherapeutic agents. treatment with agents inhibiting this pathway is also addressed. Leukemia (2003) 17, 590–603. doi:10.1038/sj.leu.2402824 Keywords: PI3K; PTEN; Akt; cell cycle; apoptosis; targeted Cytokine activation of PI3K/Akt pathway therapy; inhibition

Introduction After cytokine activation of a specific receptor, PI3K can be activated by two mechanisms. First, a phosphorylated tyrosine (Y) residue on the receptor serves as a docking site for the p85 An understanding of normal cellular function has been gained 1 from research characterizing various signal transduction path- regulatory subunit of PI3K. This recruits the catalytic subunit of ways. Despite these advances, processes responsible for PI3K, p110, to this complex. Alternatively, upon activation of the receptor by the cognate cytokine, Shc binds the receptor to neoplastic transformation of normal cells remain largely 19–22 undefined. For this reason, many chemotherapeutic regimens enable Grb-2 and Sos to form a complex that activates Ras. currently available are relatively nonspecific and have many Ras can then induce membrane localization and activation of the p110 subunit of PI3K, as determined by crystal structure toxic side effects. The ultimate goal of signal-transduction-based 23 research is to develop chemotherapeutic drugs that target studies. Specifically, this interaction occurs between regions cancerous cells without affecting nontransformed cells. on Ras, known as switch I (residues 33–41) and switch II (residues 63, 64, and 73), which bind to a sequence on PI3K-g Signaling through the Raf/MEK/ERK cascade is relatively well 24 understood1–9 and much progress has been achieved in the referred to as the Ras-binding domain (RBD). It was recently inhibition of this signaling pathway.10,11 In many cases, shown that the PI3K regulatory subunit, p85, contains two however, activation of the Raf/MEK/ERK pathway alone is not additional regulatory domains: a GTPase-responsive domain responsible for oncogenic transformation. Recent evidence has (GRD) that can mediate PI3K activation through binding small suggested that the PI3K/Akt pathway is a viable target for novel GTPases (Ras, Rac1), and an inhibitory domain that can block antineoplastic drugs. This review will describe PI3K/Akt path- these binding events. PI3K inhibition can be lifted when certain tyrosine kinases such as Src, Lck, and Abl phosphorylate the way signaling and its effects on cell cycle progression, 25 prevention of apoptosis, and malignant transformation. Lastly, S668 residue contained within the inhibitory domain. several potential anticancer therapies targeting this cascade are Different Ras isoforms have also been shown to have varying discussed. abilities to activate PI3K. Although Ras molecules are commonly believed to activate both PI3K/Akt and Raf/MEK/ERK cascades, Ha-Ras preferentially activates PI3K and Ki-Ras preferentially Correspondence: JA McCubrey, Department of Microbiology & 26 Immunology, Brody School of Medicine at East Carolina University, activates Raf. Greenville, NC 27858, USA; Fax: 1 252 816 3104 Activated PI3K converts phosphatidylinositol (4,5)-phos- 4 The first two authors contributed equally to this work. phate [PI(4,5)P2] into phosphatidylinositol (3,4,5)-phosphate Received 3 September 2002; accepted 23 January 2002 [PI(3,4,5)P3], which results in membrane localization of P13K/Akt pathway as a target for cancer chemotherapy F Chang et al 591

Figure 1 Activation of the PI3K/Akt pathway. Two mechanisms by which cytokine receptor activation can result in activation of the PI3K/Akt pathway are indicated. Note that signiﬁcant activation of this pathway occurs in close proximity to the cell membrane. SH2: Src homology-2 domain; PH: pleckstrin homology domain.

phosphotidylinositol-dependent kinase-1 (PDK1) via its pleck- active serine/threonine kinase monomers that become activated strin homology (PH) domain. Akt is also recruited to the lipid- when bound to specific cyclins. Cyclins are nuclear proteins that rich plasma membrane by its PH domain and is phosphorylated are transiently expressed in order to activate its cognate Cdk. at residues T308 and S473 by PDK1 and an unidentified kinase, These proteins form complexes that facilitate cell cycle respectively (Figure 1). progression through various cell cycle stages (Figure 3). Cdk Akt is the primary mediator of PI3K-initiated signaling and has activities are inhibited by two families of Cdk inhibitors (CKIs). a number of downstream substrates that may contribute to The first CKI family is composed of inhibitors of Cdk4 (INK4), malignant transformation. Some of these substrates are Bad, which are p16INK4a, p15INK5b, p18INK4c, and p19INK4d. INK4 procaspase-9, I-kB kinase (IKK), CREB, the forkhead family of family CKIs inhibit kinase activity of Cdk4/cyclin D and Cdk6/ transcription factors (FKHR/AFX/FOX), glycogen synthase ki- cyclin D complexes.32 The second family is Cip/Kip CKIs, which nase-3 (GSK-3), p21Cip1, and Raf.18 IKK and CREB are activated includes p21Cip1, p27Kip1, and p57Kip2. Cip/Kip family CKIs by Akt phosphorylation, whereas Raf, Bad, procaspase-9, FKHR, inhibit a broader set of Cdks, including Cdk2, Cdk4, and and GSK-3 are inactivated. Mechanisms by which Akt modu- Cdk6.33,34 The roles that these regulatory proteins play in cell lates activity of these proteins to promote cell survival and cell cycle progression can be modulated by PI3K signaling. cycle progression will be discussed in the following sections. Activity of the PI3K/Akt pathway is negatively regulated by p21Cip1 and PI3K/Akt: cell cycle regulatory effects phosphatases. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), which is also known as mutated in As previously discussed, the PI3K/Akt pathway is important for multiple advanced cancers-1 (MMAC1), as well as Src the regulation of cell cycle progression.35–40 For instance, the homology 2 (SH2) containing phosphatases 1 and 2 (SHIP-1 PI3K/Akt pathway is crucial for DNA synthesis in mesenchymal 27–31 35 and SHIP-2) remove phosphates from PI(3,4,5)P3. Muta- cells and expression of dominant negative (DN) Akt blocks tions in these phosphatases that eliminate their activity can lead platelet-derived-growth-factor (PDGF)-induced DNA synthesis to tumor progression. Consequently, genes encoding these and cell proliferation.40 p21Cip1 was initially thought to be a CKI phosphatases are referred to as antioncogenes or tumor because it inhibited cell proliferation when overexpressed in suppressor genes. An overview of the PI3K/Akt pathway is human fibroblasts. Evidence gathered by in vitro reconstitution presented in Figure 2. studies showed that p21Cip1 inhibited activity of most Cdk isoforms.41,42 Further investigation has suggested that p21Cip1 has a more complex role in regulating Cdk activity. Under Cell cycle overview physiological conditions, p21Cip1 can bind to Cdk4/cyclin D and Cdk6/cyclin D complexes, and induce their kinase activities 43 Cip1 Uncontrolled cell division can lead to tumorigenesis. Cell from early G1 until the middle of S phase. In contrast, p21 division is coordinated by a number of proteins including can bind to and inhibit kinase activities of Cdk2/cyclin A and 44,45 cyclins and cyclin-dependent kinases (Cdks). Cdks exist as in- Cdk2/cyclin E complexes from the late G1 until the S phase.

Leukemia P13K/Akt pathway as a target for cancer chemotherapy F Chang et al 592

Figure 2 Overview of the PI3K/Akt pathway. The PI3K/Akt pathway is activated either by the p85 PI3K regulatory subunit binding to an activated tyrosine residue on the activated IL-3 receptor or by the PI3K p110 subunit binding to activated Ras. Both result in membrane localization of PI3K. Some downstream effects of PI3K activation pathway are shown. PI(4,5)P2 is phosphorylated by PI3K to yield PI(3,4,5)P3, which promotes membrane localization of PDK1 through its PH domain. PDK1 then phosphorylates and activates Akt. Note that, depending on the residue phosphorylated, proteins can either be activated or inactivated by phosphorylation. Proteins activated by S/T or Y phosphorylation are indicated with a black P and a clear circle. Proteins inactivated by S/T or Y phosphorylation are shown with a white P in a black circle. Transcription factors are indicated by diamonds. Phosphatases are indicated by rectangles. Phosphatases that inhibit activity, such as PTEN and SHIP, are indicated in black rectangles. The PP2A phosphatase, which activates Raf, is shown in a white rectangle. Open arrows indicate phosphate groups hydrolyzed by phosphatases.

The function of p21Cip1 during cell cycle progression appears to enzyme activity.53,54 Phosphorylation of T145 by Akt causes ensure appropriate Cdk activation. PCNA release from the PCNA/p21Cip1/Cdk/cyclin quaternary p21Cip1 may also promote cell survival. Insulin-like growth complex (Figure 4). PCNA can then form a complex with the factor-I (IGF-I) improved survival of C3 myoblasts by inducing DNA polymerase d holoenzyme and activator protein-1 (AP-1) p21Cip1 through an Akt-dependent mechanism.46 IGF-I induc- to induce DNA synthesis.49,52 Cytoplasmic relocalization of tion of cell survival was significantly reduced by expression of p21Cip1 was observed in fibroblasts upon phosphorylation by antisense p21Cip1 cDNA. In summary, p21Cip1 has both positive Akt;49 however, this relocalization was not observed in and negative regulatory effects on cell cycle progression, and is endothelial cells.52 also a regulator of cell survival. Furthermore, T145 phosphorylation also decreased binding The PI3K/Akt pathway is required for p21Cip1 induction in affinity of p21Cip1 for Cdk2 by more than 50% in endothelial human ovarian carcinoma cells.47 Inactivation of this pathway cells. As a result, inhibition of Cdk2 by p21Cip1 decreased.52 by drug treatment or DN Akt expression decreased levels of Interestingly, it appears that modulation of Cdk activity by Akt- p21Cip1.47,48 In contrast, expression of a constitutively active Akt induced p21Cip1 phosphorylation at T145 was specific for Cdk2 mutant increased p21Cip1 expression.47 Mechanisms by which because it had little effect on Cdk4 activity. the PI3K/Akt pathway regulates p21Cip1 protein expression were Akt can also phosphorylate S146 on p21Cip1 (Figure 4)50,51. further explored by several groups. It was shown that Akt could Phosphorylation at this site is associated with increased p21Cip1 directly phosphorylate both T145 and S146 near the carboxyl protein stability. p21Cip1 is normally a short-lived protein with a terminus of p21Cip1.49–52 As a result of these phosphorylation high proteosomal degradation rate.55 Phosphorylation of S146 events, DNA synthesis and Cdk activity were stimulated, and in glioblastoma cell lines significantly prolonged the half-life of cellular proliferation increased. p21Cip1.50 Since Akt increases protein levels of both p21Cip1 and Cip1 Cip1 In normal cells, p21 exists as a complex with a cyclin, a cyclin D1, it consequently induces assembly of p21 /Cdk4/ Cdk, and proliferating cell nuclear antigen (PCNA) (Figure 4). cyclin D and p21Cip1/Cdk6/cyclin D complexes as well as their p21Cip1 binds PCNA directly and inhibits DNA pol d holo- associated kinase activities.50

Leukemia P13K/Akt pathway as a target for cancer chemotherapy F Chang et al 593 p21Cip1 on T57 (Figure 5). This phosphorylation event is associated with a higher degradation rate of p21Cip1. Further- more, if GSK-3-induced T57 phosphorylation is inhibited by PI3K/Akt activation, the half-life of p21Cip1 protein is signiﬁcantly increased.51 Interestingly, GSK-3 does not phosphorylate p27Kip1, which suggests functional diversity in regulation of these two Cip/Kip family CKIs.64

PI3K/Akt pathway and p27Kip1

The PI3K/Akt pathway has also been implicated in altering p27KIP1 activity. Expression of DN Akt in mesenchymal cells caused transcriptional induction of p27Kip1, which led to the inhibition of both Cdk2 activity and DNA synthesis.65 However, 48 Figure 3 Cell cycle overview. The cell cycle is divided into G1 these effects were not observed in an endothelial cell model. Kip1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis) stages. The Therefore, the effects of PI3K/Akt on p27 expression may be point after which cellular division must occur is referred to as the cell type speciﬁc. restriction point (R) and is located at the latter portion of G . 1 The forkhead family of transcription factors (TFs) has recently Progression through the cell cycle is promoted by formation of Cdk/ 66 cyclin complexes. The relative activity of each complex is represented been categorized into the forkhead box factor (FOX) family. by the thickness of gray arcs. Regulators of Cdk/cyclin complexes are The FOX family includes AFX/FOXO4, FKHR/FOXO1, and shown in black. FKHR-L1/FOXO3a. Akt can inactivate FOX TFs through direct phosphorylation of multiple S/T residues38,67,68 (Figure 6). As a result of these phosphorylation events, proapoptotic genes induced by FOX family transcription factors are downregulated, including both p27Kip1 and Fas. Thus, the PI3K/Akt pathway prevents cell death by both inducing and repressing gene expression. Recently, three laboratories almost simultaneously demon- strated that Akt can directly phosphorylate p27Kip1 on T157 and subsequently abolish its inhibitory activity against Cdk-2.69–71 In order to inhibit Cdk-2 kinase activity, p27Kip1 requires translocation from cytosol to the nucleus so as to bind with Cdk-2. Wild-type p27Kip1 was observed in both the cytosol and nucleus. A T157A p27Kip1 mutant, however, exclusively localized in the nucleus was found to be associated with cell cycle arrest. This suggested that T157 of p27Kip1 is important for nuclear localization and Cdk-2 inhibition. Furthermore, T157 is located within the nuclear localization sequence of p27Kip1 Cip1 (amino acids 151–166) and is contained in a putative Akt Figure 4 Phosphorylation of p21 by Akt regulates cell cycle 157 progression. Phosphorylation of p21Cip1 at T145 and S14b by Akt can consensus phosphorylation site (RXXRXXT D). Using breast affect both DNA synthesis and cell cycle progression. cell lines as a model, it was shown that Akt could phosphorylate p27Kip1 on T157 both in vitro and in vivo. Akt-induced T157 Kip1 PI3K and GSK-3 phosphorylation caused retention of p27 in the cytoplasm and thus, prevention of cell cycle arrest induced by p27Kip1. GSK-3 is a substrate of the PI3K/Akt pathway that is constitutively active in unstimulated cells. GSK-3 phosphorylates numerous proteins, such as glycogen synthase, cyclin D, and PI3K/Akt pathway and cyclin D1 c-Myc, which maintains these proteins in an inactive state.56 57 Activation of PI3K or Akt can lead to GSK-3 inactivation. The PI3K/Akt pathway is also a strong activator of cyclin D1,a Many kinases, including protein kinase C (PKC), p90RSK, p70SbK, critical player in cell cycle progression. In a manner similar to Cip1 and Akt, phosphorylate GSK-3 in vitro. However, Akt appears that described for p21 , cyclin D1 protein levels are also to be the kinase primarily responsible for phosphorylation of regulated by GSK-3. Cyclin D1 degradation occurs mainly GSK-3 at S9 in vivo (Figure 5).57,58 through the ubiquitin-dependent proteolysis pathway triggered Recently, it has been shown that like Akt, GSK-3 also by phosphorylation36 (Figure 7). In order to be recognized by regulates p21Cip1 protein stability (see Figure 5). p21Cip1 can ubiquitin-conjugating enzymes E1 and E2 for proteolysis, cyclin 59 36 be degraded through ubiquitin-mediated or ubiquitin-inde- D1 must be phosphorylated at T286 near its carboxyl terminus. 60,61 pendent pathways. The C8 a subunit of the 20S proteosome GSK-3 is the primary kinase that phosphorylates cyclin D1 at this Cip1 36 directly binds to the carboxy terminus of p21 , which is residue. Moreover, cyclin D1 phosphorylation at T286 by 61 necessary for proteosomal degradation. GSK-3, in its consti- GSK-3 is also associated with the translocation of cyclin D1 from Cip1 tutively active state, causes decreased p21 protein levels in the nucleus to the cytoplasm. Rapid cyclin D1 degradation endothelial cells.51 Moreover, speciﬁc inhibition of GSK-3 with induced by GSK-3 is inhibited by activation of PI3K/Akt lithium chloride62,63 prevents p21Cip1 degradation and increases pathway because Akt directly phosphorylates and inactivates its protein level 10-fold.51 GSK-3 can directly phosphorylate GSK-3.

Figure 5 Regulation of p21Cip1 protein stability by the PI3K/Akt pathway. Effects of Akt on GSK-3 and stability of p21Cip1 are indicated. Akt can induce GSK-3 phosphorylation, which renders it inactive. Akt can phosphorylate p21Cip1, which stabilizes the protein.

Figure 6 Effects of the PI3K/Akt pathway on FOX transcription factors. Akt can phosphorylate and inactivate FOX transcription factors. DN Akt can block this effect to increase expression of p27Kip1 and Fas, which inhibit cell cycle progression and cause apoptosis, respectively.

Figure 7 Regulation of cyclin D1 protein stability by the PI3K/Akt pathway. Cyclin D1 is phosphorylated by GSK-3, which can be prevented by phosphorylation of GSK-3 by Akt. Phosphorylated cyclin D is translocated from the nucleus to the cytoplasm and targeted for degradation by ubiquitination.

PI3K/Akt pathway and c-Fos transfection of rat mesenchymal cells with a DN Akt mutant decreased c-Fos transcription.67 Induction of c-Fos transcription c-Fos is a transcriptional regulator that can elevate expression of by PI3K or Akt was mediated by Elk-1.67 These data suggest that many proliferatory genes. Consequently, its ability to enhance the PI3K/Akt pathway executes some of its antiapoptotic effects transcription of these genes is strictly controlled. Transfection of through transcription factors such as Elk-1 and c-Fos. These NIH-3T3 mouse ﬁbroblast cells with a constitutively active PI3K results are intriguing because ERK also phosphorylates Elk-1, p110 mutant induced transcription from the c-Fos promoter, which places this transcription factor downstream of both Raf/ which was blocked by cotransfection with DN Ras.65 Moreover, MEK/ERK and PI3K/Akt pathways (Figure 8).

Figure 8 Effects of the PI3K/Akt signaling on Elk-1 and c-Fos Figure 9 PI3K/Akt and Raf/MEK/ERK pathways cooperate to transcription factors. PI3K/Akt and Raf/MEK/ERK pathways can regulate bad phosphorylation. Phosphorylation and inactivation of cooperate to induce c-Fos transcription. the proapoptotic Bad protein by cooperation between PI3K/Akt and Raf/MEK/ERK pathways. PI3K/Akt pathway and NF-jB

NF-kB is a transcription factor that can be induced by a variety phosphorylating Bad at S136100 (Figure 9). Mitochondrial Raf-1 of signals. Inhibitor of kB (I-kB) binds to NF-kB and inactivates it is unable to activate ERK1 or ERK2 and appears to inactivate Bad by localizing NF-kB to the cytosol where it is unable to regulate by a mechanism dependent upon MEK and Akt. In rat transcription. Phosphorylation of I-kB targets it for ubiquitina- hepatocytes, A-Raf has been found to be localized to inner tion and degradation. In the absence of I-kB, NF-kB’s nuclear and outer mitochondrial membrane protein import receptors localization signal is exposed and NF-kB localizes to the hTOM and hTIM, which suggests that A-Raf may also exert nucleus where it is able to induce transcription. The role of antiapoptotic effects.101 NF-kB was originally described as a factor associated with Akt can modulate Bad activity by phosphorylating S112.102 apoptosis. Many agents, which induce apoptosis, such as TNF-a, 72,73 Phosphorylated Bad then dissociates from Bcl-2 to form a also induce NF-kB activation. However, it was subse- complex with the 14-3-3 adaptor protein. Dissociation of Bad quently shown that inhibition of NF-kB activation potentiates 74–77 from Bcl-2 is associated with cell survival. Recent studies have apoptosis. In addition, inhibition of I-kB expression by S6K 78 also shown that p70 , which is also a target of the PI3K/Akt oligonucleotides led to cell transformation. The data now pathway, can also phosphorylate Bad at S112.103–105 Further- support the concept that NF-kB is an antiapoptotic molecule. more, Raf/MEK/ERK and PI3K/Akt pathways can synergize to Consistent with this role, NF-kB has been shown to induce 5,6 79–82 induce cell survival and cellular transformation. This may transcription of antiapoptotic proteins. IKK is known to result from phosphorylation of proapoptotic Bad at these two phosphorylate and induce the degradation of I-kB.83 Akt is 84,85 serine residues. It is worth noting that a third residue of Bad is reported to phosphorylate and activate IKK. Activation of phosphorylated by the PKA pathway.105 IKK causes phosphorylation and degradation of I-kB, which leads to localization of NF-kB to the nucleus, where it can 85 induce transcription of antiapoptotic genes. Thus, Akt can Interactions between Raf/MEK/ERK and PI3K/Akt pathways inhibit apoptosis by activating NF-kB. It has been shown that PI3K/Akt and Raf/MEK/ERK pathways can interact in multiple ways. In some cells, Akt can contribute to PI3K/Akt signaling and the Bcl-2 family of proteins Raf-1 inactivation (see Figures 2 and 10). However, phosphorylation of Raf-1 by Akt has not been universally observed and The Bcl-2 family of proteins are structurally related molecules complete inactivation of Raf-1 also requires phosphorylation at that play an instrumental role in the regulation of apoptosis. This other residues such as S621. Since Raf can regulate p21Cip1 family includes both proapoptotic proteins such as Bad and Bax activity, inhibition of Raf by Akt would be expected to reduce Cip1 as well as antiapoptotic proteins such as Bcl-2 and Bcl-XL. The p21 activity, which could affect cell cycle progres- balance of antiapoptotic to proapototic proteins is believed to sion64,65,67,106–109 (see Figure 10). dictate whether a cell will survive or undergo apoptosis. This The Raf/MEK/ERK cascade can also activate the PI3K/Akt concept has been termed the rheostat theory.86 pathway. We have observed that Raf can activate Akt activity in Raf has been shown to directly phosphorylate Bad and Bcl-2 hematopoietic cells transformed to grow in response to activated to exert antiapoptotic effects.87,88 However, the role of Raf in Raf in the absence of exogenous cytokines (unpublished data). apoptosis regulation remains controversial. Depending on cell Others have observed that some antiapoptotic effects of the type as well as apoptotic stimulus, Raf can inhibit89–96 or PI3K/Akt pathway are dependent upon the Raf/MEK/ERK path- promote97–99 apoptosis. Mitochondrial Raf-1 inactivates Bad by way. Even though both pathways can be regulated by Ras, they

Figure 10 Interactions between PI3K/Akt and Raf/MEK/ERK pathways. Potential sites of interaction between these two pathways are indicated. These pathways interact and cross-regulate each other by both direct and indirect mechanisms. These pathways regulate gene expression by transcriptional, post-transcriptional, and post-translational mechanisms.

cross-regulate one another to control both apoptosis and cell Akt, SGK can be activated by PI3K120–122 even though it lacks a cycle progression. PH domain. Secondly, both Akt and SGK are directly Ectopic overexpression of Raf and MEK oncoproteins led to phosphorylated and activated by PDK1.119,122 Interestingly, abrogation of cytokine dependence of murine FDC-P1 and SGK and Akt phosphorylate different residues of FKHR-L1 and human TF-1 hematopoietic cells.1,93–96,110–112 It has been synergistically inactivate B-Raf. postulated that Raf/MEK/ERK signaling can activate the PI3K/ Using murine FDC-P1 hematopoietic cells as a model, we Akt pathway to induce cell survival. The PI3K inhibitor have determined that Raf activation led to G1 progression in LY294002 and the downstream p70S6K inhibitor rapamycin are association with downregulation of p27Kip1 and upregulation of potent inhibitors of Raf-mediated proliferation, suggesting that p21Cip1 protein levels.64 Although Cdk4/cyclin D/p21Cip1 some effects of Raf on cell growth are mediated by the PI3K/Akt/ complex formation was detected, no binding was observed p70S6K pathway as discussed later. The issue is complicated between p21Cip1 and Cdk2/cyclin E or between p27Kip1 and because many Raf-transformed cells express autocrine cytokines Cdk4/cyclin D or Cdk2/cyclin E. The increases in p21Cip1 that activate the PI3K/Akt pathway. However, we have observed protein levels and binding of p21Cip1 with Cdk4/cyclin D that Raf could induce PI3K acivity in cytokine-dependent cells complexes induced by Raf were correlated with increased Cdk4 that do not secrete autocrine growth factors. kinase activity.64 Our results suggest that, in hematopoietic Crosstalk between these two pathways has been demon- cells, Raf signaling increases levels of p21Cip1 protein, which strated. It has been shown that Akt can directly phosphorylate then specifically binds to and activates the Cdk4/cyclin D Raf isoforms within their CR2 amino-terminal regulatory complex. The fact that p27Kip1 was found not to associate with domain. These sites include S259 on Raf-1 and both S364 and Cdk2 or Cdk4 suggested that, at least in our model system, S428 on B-Raf. Serine-phosphorylated Raf molecules then p27Kip1 has little effect on Cdk2 or Cdk4 kinase activity. This become inactivated by binding to the 14-3-3 adaptor pro- finding is interesting because it suggests that Raf activation tein.113–115 The inhibitory effect of Akt on Raf activity appears to differentially regulates p21Cip1 and p27Kip1 activities. Thus, be dependent upon cell type and stage of differentiation116,117 p21Cip1 and p27Kip1 appear to have similar, yet unique because some investigators have not observed this effect. functions. Certain differentially expressed mediators may be essential for association of Akt and Raf. Recently, a negative feedback loop has been identified Involvement of the PI3K/Akt pathway in neoplasia involving Raf/MEK/ERK and PI3K/SGK pathways.118 Serum and glucocorticoid-inducible kinase (SGK) shares a high homology The PI3K pathway is negatively regulated by phosphatases. with Akt.119 Even though physiological targets of SGK and their PTEN is considered a tumor suppressor gene.123 PTEN is a dual function(s) remain largely unknown, regulation of SGK has been specificity lipid and protein phosphatase that removes the 3- documented. SGK shares a regulatory pathway with Akt. Like phosphate from the PI3K lipid product PI(3,4,5)P3 to yield

Leukemia P13K/Akt pathway as a target for cancer chemotherapy F Chang et al 597 124–127 PI(4,5)P2, which prevents Akt activation. Recently, SHIP- 2, has been shown to remove the 5-phosphate from PI(3,4,5)P3 128,129 to yield PI(3,4)P2. Aberrant proliferative and antiapoptotic signals transduced by the PI3K/Akt pathway have been linked to malignant transformation.130 Ras can activate PI3K and some Ras mutations cause deregulated activation of PI3K and Akt.131–133 Since PTEN and SHIP-2 negatively regulate PI3K activity, loss of their phosphatase activity may also induce Akt activation.134–136 Mutations and hemizygous deletions of PTEN have been detected in primary acute leukemia and non-Hodgkin’s lymphoma. In addition, many hematopoietic cell lines lack or have low PTEN protein expression.134,135 Increased Akt expression has also been linked with tumor progression. Akt-2 gene ampliﬁcation has been observed in some cervical, ovarian, and pancreatic cancers as well as non-Hodgkin’s lymphomas.136–139 SHIP-1 may also affect Akt activity by controlling levels of PI(3,4,5)P3 and PI(3,4)P2. If SHIP-1 activitiy is lost, PI(3,4,5)P3 levels would be elevated, leading to Akt phosphorylation at T308 by PDK1. Conversely, PI(3,4)P2 levels would be decreased, which would prevent activation of the unidentiﬁed kinase responsible for Akt phosphorylation at S473.140 Thus, this pathway is regulated such that cetain mutations would cause partial, but not complete, Akt activation.

PI3K/Akt pathway as a target for chemotherapy

Inhibition of the PI3K/Akt pathway could prevent uncontrolled Figure 11 Signal transduction inhibitors targeting the PI3K/Akt cellular proliferation. Consequently, antiapoptotic signals trans- pathway. Structures of some small molecular weight membrane permeable drugs targeting this pathway are indicated: (a) LY294002 duced by PI3K and downstream molecules have become a focus (PI3K inhibitor), (b) wortmannin (PI3K inhibitor), (c) 1L-6-hydroxy- of recent drug discovery research. This section will discuss methyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Akt strategies that have been used to inhibit this pathway. Inhibitor), (d) rapamycin (p70S6K inhibitor), and (e) quercetin (PI3K Small molecule inhibitors (SMIs) have been discovered for inhibitor). many kinases involved in various signal transduction pathways (Figure 11). For example, quercetin is marketed by Calbiochem (San Diego, CA, USA) as an inhibitor of PI3K. However, its p70S6K at sites different from those associated with activation.147 specificity has been questioned because it has also been As a consequence, phosphorylation of the S6 ribosomal protein found to inhibit other serine/threonine kinases such as AMPK, necessary for cell growth does not occur. CK2, p90RSK2, and p70S6K1. 1412-(4-Morpholinyl)-8-phenyl-4H- Transfection with plasmid-based antisense vectors or treat- 1-benzopyran-4-one (LY294002) and wortmannin are two ment with synthetic antisense oligonucleotides have also been widely used PI3K inhibitors. Of these two, LY294002 is more used to disrupt signaling by the PI3K/Akt pathway. Antisense specific and is therefore more commonly used than wortman- nucleic acids inhibit translation by hybridizing to complemen- 141 nin. LY294002 is a quercetin derivative with a reported IC50 tary mRNA transcripts to prevent ribosome binding. Skorski value of 1.4 mm.142 Wortmannin is a fungal metabolite with an et al148 showed that expression of antisense cDNA or treatment 143 IC50 value of 5 nm. with antisense oligonucleotides downregulated p85 gene Akt inhibitors have been recently discovered. One is 1L-6- expression and abrogated PI3K signaling. Antisense RNAs hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecyl- were found to drastically reduce tumorgenicity of PANC1 carbonate, which is marketed by Calbiochem. It has been cells, which overexpress Akt2, in nude mice.149 Antisense reported to inhibit selectively Akt with an IC50 value of oligonucleotides targeted to Akt1 had several effects on a variety approximately 5 mm, which is much less than its IC50 value of of cancer cell lines, including reduced ability to grow on soft 90 mm for PI3K inhibition.144 Additionally, it was recently agar, induction of apoptosis, and enhanced susceptibility to reported that the tyrosine phosphorylation inhibitor (tyrphostin) various chemotherapeutic agents.150 Similar results were AG957, which is also named NSC 654705, caused dephos- obtained from studies of antisense oligonucleotides targeted to phorylation of Akt.145 This report is intriguing because AG957 PDK1.151 was previously described as an inhibitor specific for the Bcr-Abl Earlier in this review, it was discussed that PI3K is a known chromosomal translocation gene product implicated in devel- downstream substrate of the GTPase Ras. Disruption of Ras opment of chronic myelogenous leukemia (CML). activity may also be effective for inhibition of the PI3K/Akt p70S6K is another viable target for chemotherapeutic inter- pathway. Three approaches that have been taken to inhibit Ras vention within the PI3K/Akt pathway. Rapamycin is commonly signaling include prevention of Ras membrane localization, used to inhibit p70S6K activity. It is a macrolide antibiotic antisense strategies, and ectopic expression of DN proteins. produced by the filamentous bacterium Streptomyces hygro- Membrane localization of Ras has been inhibited with scopicus.146 Rapamycin is used as a powerful immunosuppres- farnesyltransferase inhibitors (FTIs). Ras becomes active only sant, which has been effective in a variety of clinical settings after post-translational addition of farnesyl or geranylgeranyl such as organ transplantation. Rapamycin dephosphorylates isoprenoid moieties to its carboxy terminus. Without such

Leukemia P13K/Akt pathway as a target for cancer chemotherapy F Chang et al 598 modiﬁcation, Ras remains in the cytosol where it cannot described use of a DN form of p110, the catalytic subunit of activate PI3K. Enzymes catalyzing transfer of these lipid PI3K.165 Furthermore, Mabuchi et al166 reported that ovarian moieties to Ras include farnesyltransferase (FT), geranylgeranyl- cancer cells become sensitized to paclitaxel by DN Akt transferase type I (GGTI), and geranylgeranyltransferase type II expression. (GGTII). An abundance of FTIs are currently available for Lastly, PI3K signaling can be abrogated through the activity of laboratory use. Chemical structures of some FTIs are shown in two known phosphatases. SHIP-2 overexpression has been Figure 12. For example, arglabin-DMA is a recently discovered observed to cause Akt inactivation and cell cycle arrest in FTI that is derived from a species of wormwood endemic to glioblastoma cells.167 Overexpression of PTEN inhibits prolif- central Asia.152 There are currently four FTIs undergoing clinical eration and malignant progression of prostate cancer cells.168 trials: R115777, SCH66336, L-778,123, and BMS- Taken together, these data indicate that phosphatases may have 214662.153,154 Jiang et al155 reported that apoptosis caused by therapeutic promise by abrogating signaling transduced through FTI-277 could be rescued through expression of a constitutively PI3K. Strategies for inhibiting the PI3K/Akt pathway are outlined active form of Akt, suggesting that FTIs may mediate some in Figure 13. inhibitory effects not only through the Ras/Raf/MEK/ERK pathway, but also the PI3K/Akt signaling cascade. Antisense strategies have also been used to disrupt Ras. Summary Expression of antisense K-Ras cDNA suppressed the malignant phenotype of large cell lung carcinomas156 and inhibited growth In this review, we have discussed the effects of the PI3K/Akt of H460a lung cancer cells nearly three-fold.157 ISIS 2503 is a pathway on cell cycle progression and apoptosis. This pathway 20-base antisense phosphorothioate oligodeoxyribonucleotide plays critical roles in cell proliferation, oncogenesis, and its targeting H-Ras that is currently in phase 2 clinical trials.158,159 activity is regulated positively by Ras and negatively by PTEN. Lastly, expression of DN proteins has been used to inhibit Ras The PI3K/Akt pathway can induce cell cycle progression by signaling. DN proteins bind and inhibit function of endogenous modulating the protein stability of cyclin D and p21Cip1. Akt can proteins. Two DN forms of Ras have been described. The ﬁrst, directly phosphorylate p21Cip1 on S146 and increase its protein Ras17N, blocks endogenous Ras GTPase activity, which is stability. Alternatively, Akt can also prolong the half-life of both essential for signal transduction.160,161 Ras17N has been p21Cip1 and cyclin D through inactivation of GSK-3, which described to inhibit Akt activation elicited by the v-Crk phosphorylates p21Cip1 and cyclin D to induce their degrada- oncogene.162 Another DN mutant binds endogenous Ras to tion. Akt facilitates this inactivation event via phosphorylation of form inactive complexes localized to the cytoplasm. Signals GSK-3 on residue S9. The PI3K/Akt pathway can also induce from the plasma membrane are not transduced by Ras because DNA synthesis. When T145 of p21Cip1 is phosphorylated by Akt, its localization to the plasma membrane is disrupted.163 DNA pol d is released from p21Cip1 and binds with AP-1 to Inhibition of both PI3K and Akt has also been achieved with induce DNA synthesis. Recently, Akt has also been shown to DN proteins. Expression of a DN form of p85, Dp85, decreased directly phosphorylate p27Kip1 on T157 to cause its retention in cell growth in Bcr-Abl-transformed cells.164 Another report the cytosol. As a result, p27Kip1 loses its inhibitory effect on

Figure 12 Farnesyltransferase inhibitors. Chemical structures of commonly used FTIs are shown.

Figure 13 Sites of inhibition of the PI3K/Akt pathway. Schematic overview of the potential negative regulators of PI3K/Akt signaling. Black boxes indicate mechanisms of inhibition.

Cdk2. Moreover, phosphorylation of FKHR-L1 by Akt also leads tion (9930099N), and the North Carolina Biotechnology Center to reduced p27Kip1 transcription. The PI3K/Akt pathway also (9705-ARG-0009). regulates activity of Bcl-2 family members. Both Akt and p70S6K can phosphorylate proapoptotic Bad, which promotes associa- References tion of Bad with 14-3-3 adaptor protein and allows excess Bcl-2 or Bcl-XL to out compete Bax, there by preventing apoptosis. 1 Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, It is becoming increasingly evident that interactions between Wang XY, Algate PA et al. Signal transduction, cell cycle the PI3K/Akt and Raf/MEK/ERK pathwys are important for the regulatory, and anti-apoptotic pathways regulated by IL-3 in regulation of cell cycle progression and apoptosis. Some hematopoietic cells: possible sites for intervention with anti- scientists have observed that Akt phosphorylates Raf-1 on neoplastic drugs. Leukemia 1999; 13: 1109–1166. S259 to inhibit its kinase activity, and these inhibitory effects 2 Franklin RA, McCubrey JA. Kinases: positive and negative regulators of apoptosis. Leukemia 2000; 14: 2019–2034. may be released by removal of this phosphate by protein 3 White MK, McCubrey JA. Suppression of apoptosis: role in cell phosphatase 2A. However, these interactions remain contro- growth and neoplasia. Leukemia 2001; 15: 1011–1021. versial because they have not been unequivocably conﬁrmed by 4 McCubrey JA, Steelman LS, Blalock WL, Lee JT, Moye PW, Chang other investigators. Future studies into these types of biomole- F et al. Synergistic effects of PI3K/Akt on abrogation of cytokine- cular interactions are, therefore, warranted. dependency induced by oncogenic Raf. Adv Enzyme Regul 2001; Despite the tremendous amount of research that remains 41: 289–323. 5 McCubrey JA, Lee JT, Steelman LS, Blalock WL, Moye PW, Chang uncompleted, it is evident that signal transduction pathways F et al. Interactions between the PI3K and Raf signaling pathways interact to regulate cell cycle progression and apoptosis. An can result in the transformation of hematopoietic cells. Cancer understanding of how these different cascades control expres- Detect Prev 2001; 25: 375–393. sion of cell cycle regulatory genes by transcriptional and post- 6 McCubrey JA, Blalock WL, Chang F, Steelman LS, Pohnert SC, transcriptional mechanisms may reveal novel strategies to Navolanic PM et al. Signal transduction pathways: cytokine prevent their aberrant activation in transformed cells. It is model. In: Blagosklonny M. (ed). Cell Cycle Checkpoints and Cancer. Austin, TX: Landes Bioscience Press, 2001, pp 17–51. hopeful that further characterization of pathways regulating 7 McCubrey JA, May WS, Duronio V, Mufson A. Serine/threonine cell cycle progression and apoptosis will facilitate develop- phosphorylation in cytokine signal transduction. Leukemia 2000; ment of chemotherapeutic inhibitors that will suppress hyper- 14: 9–21. activity of these cascades and thus prove effective for cancer 8 McCubrey JA, Blalock WL, Saleh O, Pearce M, Burrows C, treatment. Steelman LS et al. Enhanced ability of daniplestim and myelopoietin-1 to suppress apoptosis in human hematopoietic cells. Leukemia 2001; 15: 1203–1216. 9 Srinivasa SP, Doshi PD. Extracellular signal-regulated kinase and Acknowledgements p38 mitogen-activated protein kinase pathways cooperate in mediating cytokine-induced proliferation of a leukemic cell line. We thank Ms Catherine Spruill for the excellent artwork. This Leukemia 2002; 16: 244–253. work was supported by grants from the United States National 10 Lee Jr JT, McCubrey JA. The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic intervention in Institutes of Health (R01CA51025) and grants from the North leukemia. Leukemia 2002; 16: 486–507. Carolina Biotechnology Center (9805-ARG-0006, 2000-ARG- 11 Lee Jr JT, McCubrey JA. Targeting the Raf kinase cascade in 0003) to JAM. RAF was supported in part by grants from the cancer therapyFnovel molecular targets and therapeutic strate- American Cancer Society (IRG-97-149), American Heart Associa- gies. Expert Opin Ther Targets 2002 6: 659–678.

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