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Prostate and Prostatic Diseases (2005) 8, 108–118 & 2005 Nature Publishing Group All rights reserved 1365-7852/05 $30.00 www.nature.com/pcan Review The emerging role of the PI3-K-Akt pathway in progression

LLi1, MM Ittmann2, G Ayala1,2, M-J Tsai3, RJ Amato1, TM Wheeler2, BJ Miles1, D Kadmon1 & TC Thompson1,3,4* 1Scott Department of Urology, Baylor College of Medicine, Houston, Texas, USA; 2Department of Pathology, Baylor College of Medicine, Houston, Texas, USA; 3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA; and 4Department of Radiology, Baylor College of Medicine, Houston, Texas, USA

The PI3-K-Akt pathway plays a central role in the development and progression of prostate cancer and other malignancies. We review original studies and summarize relevant sections of previous reviews concerning the relationships between abnormalities in the PI3-K-Akt pathway and prostate cancer progression. We discuss laboratory and clinical data that indicate perturbation and dysregulation of PI3-K-Akt pathway is common in prostate cancer and other malignancies. We further discuss the critical role of the PI3-K-Akt pathway in the oncogenic signaling network and provide examples that establish the PI3-K-Akt pathway as a focal point for the future development of informative and effective therapies for prostate cancer. Prostate Cancer and Prostatic Diseases (2005) 8, 108–118. doi:10.1038/sj.pcan.4500776 Published online 22 February 2005

Keywords: Akt; prostate cancer; oncogenic signaling; ; therapeutic target; caveolin-1

Introduction mechanisms that underlie the ultimate androgen-resis- tant state of prostate cancer and to develop informative Prostate cancer remains the second leading cause of biomarkers and effective therapies for this condition. cancer mortality among American males. The predomi- Recent studies indicate that phosphatidylinositol nant reason for such high and persistent mortality is the 3-kinase (PI3-K)-Akt pathway is a major survival path- lack of curative therapies for androgen-resistant meta- way in human cancer and is central to the develop- static disease. An important observation from many ment and progression of specific malignancies (reviewed prostate cancer studies is that disease progression is in Blume-Jensen and Hunter, 2001;1 Cantley, 2002;2 linked with the development of resistance. The Nicholson and Anderson, 2002;3 Paez and Sellers, establishment and the continued growth of prostate 2003;4 Testa and Bellacosa, 2001;5 Vivanco and Sawyers, cancer metastases involve overcoming multiple endo- 20026). Akt was initially identified as an oncogene genous physiological barriers and therapeutic chal- harbored within the AKT8 isolated from a lenges, which normally induce an apoptotic response. murine T-cell lymphoma7 and was subsequently shown It is critical to elucidate the survival to be a proto-oncogene with a pleckstrin homology (PH) and serine/threonine kinase domains. Akt is also referred to as kinase B (PKB) since its catalytic *Correspondence: TC Thompson, Scott Department of Urology, domain is closely related to those of protein kinase A Baylor College of Medicine, 6560 Fannin, Suite 2100, Houston, (PKA) and protein kinase C (PKC) family members. The TX 77030, USA. E-mail: [email protected] kinase activity of Akt is stimulated by a variety of Received 29 July 2004; revised 17 September 2004; accepted 19 extracellular stimuli, such as growth factors, cytokines, October 2004; published online 22 February 2005 chemokines, integrin engagement and T-cell Akt pathway in prostate cancer LLiet al 109 activation. The activation of Akt leads to the phosphor- of several PI-3,4,5-P3/PI-3,4-P2 phosphatases. Included ylation and regulation of a wide spectrum of its substrates in these phosphatases is the tumor-suppressor phospha- involved in multiple cellular processes, including cell tase and tensin homologue deleted on 10 survival, , cell differentiation, (PTEN), a 3-position lipid phosphatase, which converts progression, cell proliferation and cellular metabolism. PI-3,4,5-P3 to PI-4,5-P2. The SHIP phosphatases (SHIP1 In this review, we will highlight the mechanisms of Akt and SHIP2) also dephosphorylate PI-3,4,5-P3, but remove regulation, the role of Akt in , the phosphate from the 5-position rather than the 3-position, genetic perturbations and dysregulation of the PI3-K-Akt creating PI-3,4-P2, which can still function as a second pathway in prostate cancer and other malignancies. We messenger to recruit PH-domain-containing , will also discuss the potential for the development of including Akt, to the membrane leading to their informative molecular biomarkers for prostate cancer subsequent activation. Therefore, although both PTEN based on clinical correlates of the expression patterns of and SHIP dephosphorylate PI-3,4,5-P3, PTEN apparently Akt and its downstream targets and the therapeutic functions as the primary negative regulator of the potential of targeting the PI3-K-Akt pathway. PI3-K-Akt pathway.13–16 On the other hand, phosphorylation of Akt can also be controlled by serine/threonine protein phosphatases.17–19 PP1 and PP2A are two major classes of serine/threonine Mechanisms of Akt regulation protein phosphatases that are involved in many different cellular processes, including glycogen metabolism, cell Akt is a family of serine/threonine kinases. The analysis cycle regulation, protein synthesis and intracellular of the domain structure revealed an N-terminal PH transport, RNA splicing, and signal transduction. Speci- domain, a catalytic domain and a C-terminal regulatory fically, many important signal transduction molecules, domain for this protein family. In humans, the Akt family including Akt, are substrates of PP1 and PP2A.18,20,21 is composed of three highly homologous members Through dephosphorylation of these signal transduction named Akt1, Akt2, and Akt3. While expression of Akt3 regulators, PP1 and PP2A positively or negatively is relatively tissue restricted, Akt1 and Akt 2 expression regulate multiple cellular signaling pathways. Several are widely distributed among various tissues. In un- recent studies support an important role for PP2A in the stimulated cells, Akt resides mainly in the cytosol. regulation of the PI3-K-Akt pathway. PP2A was identi- Following the activation of PI3-K by fied as the main phosphatase involved in the regulation receptors or other cell surface receptors in response to of Akt in rat adipocytes.22 It can be coprecipitated with ligands such as insulin, platelet-derived Akt and coexpression of the catalytic subunit of PP2A (PDGF), epidermal growth factor (EGF), or fibroblast with Akt inhibited Akt activity.23 The activation of PP2A growth factors (FGFs), the resulting second messenger, by N-ethylmaleimide, ceramide, palmitate or integrin phospholipids phosphatidylinositol 3,4,5-trisphosphate a2b1 inhibits Akt phosphorylation.23–26 Conversely, the (PI-3,4,5-P3) or phosphatidylinositol 3,4-bisphosphate inhibition of PP2A by okadaic acid increases Akt 21,25,26 (PI-3,4-P2), recruits Akt to the plasma membrane and activities. binds to its PH domain. This binding leads to a Previously, we and others found that expression of conformational change of Akt, which is favorable for caveolin-1 (cav-1), the major component of caveolae, is its phosphorylation at Thr308 in the kinase activation elevated in prostate cancer and other malignancies.27–40 loop by 3-phosphoinositide-dependent kinase 1 (PDK1) To address the molecular mechanism of cav-1-mediated and at Ser473 in the regulatory domain of the carboxyl- cell survival, we explored a possible link between cav-1 terminus by a yet unidentified kinase. A range of overexpression and Akt activation in prostate cancer different candidates including integrin-linked kinase cells. When we compared PI3-K-Akt signaling activities (ILK), MAPKAP kinase 2, conventional PKC isoforms, in cav-1-expressing LNCaP cells that had undergone PDK1, and Akt itself have been proposed for this Ser473 adenoviral vector-mediated cav-1 gene transduction kinase, but no single candidate seems to be indisputa- with PI3-K-Akt signaling activities in cav-1-negative ble.8 Although phosphorylation at Thr-308 partially vector control LNCaP cells, we found that Akt, but not activates Akt, full activation of Akt requires phosphor- PI3-K activities, were significantly higher in the former. ylation on the second site (Ser473). Phosphorylated Akt The cells expressing cav-1 were also more resistant to (P-Akt) then dissociates from the plasma membrane and after treatment with thapsigar- translocates to nucleus and other subcellular compart- gin, which is known to induce apoptosis. This PI3-K- ments to phosphorylate and regulate the function of independent Akt activation in cav-1 expressing cells many cellular proteins involved in cell proliferation, was further confirmed by the determination of P-Akt survival, motility and angiogenesis processes that are half-life using the PI3-K-specific inhibitor LY294002.41 critical for tumorigenesis and .3,5,9,10 These data suggested that cav-1 participates in the The primary consequence of PI3-K activation is the regulation of Akt by maintaining Akt in the activated generation of phospholipids at the membrane, which (phosphorylated) form. function as second messengers to activate downstream Since Akt and its direct upstream kinase PDK1 are kinases, including PDK1 and ILK. It is not clear whether substrates for serine/threonine protein phosphatases ILK is an immediate upstream kinase of Akt. However, it PP1 and PP2A, we investigated the possible involve- is generally accepted that ILK functions as a regulator of ment of PP1 and PP2A in cav-1-mediated maintenance Akt in response to integrin activation or growth factor of P-Akt. Interestingly, the catalytic subunits of PP1 stimulation.11,12 In normal mammalian cells the levels of and PP2A possess a previously described consensus 42 PI-3,4,5-P3/PI-3,4-P2 are tightly controlled by the com- cav-1 binding site. Immunoprecipitation (IP) experi- bined effects of stringent PI3-K regulation and the action ments demonstrated that cav-1 was present in the PP1 or

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al

110 PP2A IP complex and PP1 or PP2A was also present in Akt at T308. Overall, our data indicate that both PP1 and the cav-1 IP complex,41 suggesting that cav-1 associates PP2A can regulate the phosphorylation status of Akt, with PP1 and PP2A. In vitro IP experiments using however, PP2A appears to be the predominant Akt purified His-V5-human cav-1, PP1 (catalytic subunit) phosphatase. and PP2A (core ) further demonstrated that cav-1 In summary, oncogenic signaling that stimulates Akt could bind to PP1 and PP2A outside of an intact cell activities often involves extracellular growth or survival context. Importantly, serine/threonine protein phos- stimuli, such as hormones, growth factors or integrin phatase activity assays using PP1 or PP2A IP complex activation. Subsequently, tyrosine receptor protein kinase or purified recombinant enzyme (PP1 catalytic subunit activation can trigger the activity of PI3-K, which leads to or PP2A core enzyme) indicated that interaction with PDK1 activation (Figure 1). PDK1, and potentially other cav-1 leads to the inhibition of PP1 and PP2A activities.41 kinases, directly phosphorylate and activate Akt. PTEN Reduced overall activities of PP1 and PP2A favor functions as a major negative regulator in the PI3-K-Akt the maintenance of Akt phosphorylation and increased pathway through dephosphorylation of the PI3-K pro- Akt activities. duct PI-3,4,5-P3. Serine/threonine protein phosphatases To further demonstrate the roles of PP1 and PP2A in PP1 and PP2A participate in the regulation of this the regulation of Akt in prostate cancer, we incubated pathway through dephosphorylation of Akt. In prostate lysates from cav-1-expressing, LY294002-treated cells cancer, cav-1 maintains activated Akt interaction with with various concentrations of purified PP1 or PP2A and inhibition, of PP1 and PP2A. enzyme for 30 min at 301C in a protein phosphatase assay buffer with or without 1 mM MnCl2. Analysis of the phosphorylation status of Akt in cell lysates from cav-1 expressing cells in phosphatase assay buffer without The role of Akt in signal transduction MnCl2 revealed that PP2A effectively dephosphorylates Akt at both S473 and T308 whereas PP1 is significantly Recent studies have revealed that Akt regulates a variety less effective under the same conditions. Interestingly, in of cellular functions including cell survival, cell growth, the presence of MnCl2 PP1 efficiently dephosphorylates cell differentiation, cell proliferation, cell cycle progres-

Figure 1 Mechanisms of regulation of Akt. Stimulation of cells by extracellular stimuli such as hormones/growth factors results in the activation of PI3- K, triggering the production of PI-3,4,5-P3 at the plasma membrane, which leads to activation of PDK1 and recruitment of Akt to the plasma membrane where it binds to PI-3,4,5-P3 through its pleckstrin homology (PH) domain. This binding leads to a conformation change of Akt, that allows phosphorylation of Akt at Thr308 by PDK1 and at Ser473 by a yet unidentified kinase (putative candidates including ILK, MAPKAP kinase 2, conventional PKC isoforms, PDK1, and Akt itself). PTEN functions as a major negative regulator of the Akt pathway by converting PI-3,4,5-P3 to PI-4,5- P2. PP1 and PP2A are also involved in the regulation of Akt activities through dephosphorylation of Akt. In prostate cancer cells, cav-1 maintains activated Akt through interaction with and inhibition of PP1 and PP2A.

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al sion and cellular metabolism (reviewed in Blume-Jensen survival. Akt can also influence the activity of tumor- 111 and Hunter, 2001;1 Testa and Bellacosa, 2001;5 Vivanco suppressor through phosphorylation of the onco- and Sawyers, 2002;6 Brazil and Hemmings, 2001;10 Luo protein . MDM2 is a p53 target gene; however, the et al, 200343). Peptide and protein library screening MDM2 protein, in turn, binds the p53 defined an optimal substrate motif for Akt as Arg-Xaa- domain and promotes p53 degradation, leading to Arg-Xaa-Xaa-Ser/Thr, where Xaa is any amino acid.44 inhibition of transcription of p53 target involved Thus far, at least 24 Akt substrates containing such a in cell cycle arrest and apoptosis. Phosphorylation and motif have been identified. activation of MDM2 by Akt promotes translocation of One of the major functions of Akt in malignancy is MDM2 to the nucleus, where it binds and inhibits p53.54 promoting cancer cell survival. Cancer cells develop Moreover, Akt was reported to upregulate the expression specific mechanisms to overcome apoptosis, or pro- of insulin-like growth factor I receptor (IGF-IR), leading grammed cell death, which is a normal cellular function to enhanced cell survival activities and invasiveness in involved in the elimination of ‘unnecessary’ or damaged human pancreatic cancer cells.55 Akt activation may also cells. Akt plays a central role in antiapoptotic pathways contribute to tumor and metastasis by stimulat- through phosphorylation and regulation of a variety of ing secretion of matrix metalloproteinase 9.56 It is of cell survival-related downstream targets (Figure 2). For interest that Akt also mediates VEGF-stimulated en- example, FKHR is a member of the Forkhead family of dothelial cell proliferation.57 transcription factors that mediates transcription of Importantly, our studies in prostate cancer cells specific proapoptotic genes such as BIM and FAS . showed that cav-1-mediated maintenance of activated Phosphorylation of FKHR by Akt disrupts FKHR- Akt leads to increased phosphorylation of multiple mediated transcription by promoting export of FKHR downstream targets, including GSK3a/b, FKHRL, and from nucleus to cytosol where it is sequestrated by the MDM2.41 All of these three proteins play important roles 14-3-3 protein.45 BAD is a proapoptotic member of BCL-2 in cancer cell survival as discussed above. family proteins that promotes cell death by forming a Another interesting finding from our studies in nonfunctional heterodimer with the prosurvival mole- prostate cancer cells is that the expression of cav-1 in cule BCL-XL. Phosphorylation of BAD by Akt prevents cav-1-negative LNCaP cells led to significantly increased the binding of BAD to BCL-XL, restoring BCL-XL’s nuclear translocation of phosphorylated androgen re- antiapoptotic function.46 Akt can also inhibit the con- ceptor (AR) at both ablation (5 pM) and physiological formational change and mitochondrial translocation of (5 nM) androgen concentrations, suggesting a potential BAX, another proapoptotic member of BCL-2 family role for cav-1 in regulation of AR nuclear translocation. proteins, thus preventing the disruption of mitochon- AR has been one of major focuses in studies of normal drial membrane potential and the activation of caspase and abnormal prostate growth.58,59 Although the general 3.47 Caspase 9 is an important effector caspase in the molecular mechanisms of androgen action have been mitochondrial death pathway. Phosphorylation of pro- established, the discovery of specific molecular pathways caspase 9 by Akt abrogates cytochrome c-induced that lead to precise regulation of AR in either androgen- proteolytic processing of procaspase 9, which in turn dependent or -independent manners is still a critical area prevents the activation of downstream executor cas- of research in normal prostate function and in prostate pases.48 CREB is a group of transcription factors that cancer. Interestingly, two recent reports indicated that modulate the expression of genes containing promoters AR contains two Akt phosphorylation consensus se- with cyclic AMP response elements. Activation of CREB quences (RXRXXS/T),9 located at S213 (RAREAS) and by Akt phosphorylation triggers recruitment of the S791 (RMRHLS), and both were shown to be phosphory- coactivator CREB-binding protein (CBP)49 and promotes lated by Akt in vitro.60,61 However, a screen of phosphor- transcription of specific prosurvival genes such as BCL- ylation sites on AR in COS-1 cells using a combination of 2.50 The NF-kB promotes cell peptide mapping, Edman degradation, and mass spec- survival in response to several apoptotic stimuli. trometry led to the identification of seven other Phosphorylation and activation of its positive regulator phosphorylation sites on AR, but not Akt phosphoryla- IkB kinase by Akt leads to phosphorylation and tion sites at S213 and S791.62 Although the question of degradation of IkB, an inhibitor of NF-kB, promoting whether AR is a physiological substrate for Akt remains nuclear translocation of NF-kB and activation of its target unresolved, our results showing increased nuclear genes.51 GSK3b is known not only as a kinase for translocation of phosphorylated AR by cav-1 provides glycogen synthesis but also as a regulator for several new insight into the role of cav-1 in hormone-resistant signaling pathways because of its kinase activities and progressive prostate malignancies. In an indepen- for a variety of downstream targets including c-, dent study, physical interaction of cav-1 and AR was b-catenin, CREB, and NF-kB that are often found to be observed.63 One explanation for the increased nuclear overexpressed in multiple types of . Inactivation translocation of AR is that the interaction with cav-1 may of GSK3b by Akt phosphorylation favors increased result in an AR conformational change that favors ligand protein levels or kinase activities of these gene products binding and/or nuclear translocation. that affect cell survival through diverse mechanisms.52 A AR is also regulated by specific nuclear coactivator more recently identified Akt substrate is apoptosis- proteins.64–67 Recent studies have shown that in a signal-regulating kinase (ASK1), which stimulates the significant number of prostate cancer tissues (47%), activation of MAP kinase that activates p38 MAP kinase nuclear receptor coactivator SRC-3 (PCIP, ACTR, RAC3, and JNK. Phosphorylation and inhibition of ASK1 results AIB1, and TRAM-1) is overexpressed compared to in reduced JNK activity.53 In some systems JNK normal prostate tissues68 (and Tsai unpublished results). promotes apoptosis, and therefore, in this context, Furthermore, overexpression of SRC-3 positively corre- inactivation of ASK1 by Akt phosphorylation favors cell lates with the combined Gleason score or clinical stage

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al

112 (Tsai unpublished results). Experimental manipulation of growth (size) and invasive activities, yet suppresses SRC-3 expression in prostate cancer cells has demon- apoptosis in prostate cancer cells69 (and Tsai unpub- strated that SRC-3 stimulates cell proliferation, cellular lished results). The underlying mechanism of SRC-3-

a

b

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al induced cell proliferation and growth was elucidated consistent with increased angiogenesis. Activated Akt in 113 and the results indicate that SRC-3 induces Akt expres- prostate cancer cells can increase expression of HIF-1a sion and activation.69 This conclusion is further sup- and its transcriptional targets such as VEGF. In addition, ported by analysis of SRC-3 null mutant mice, which are Akt can have actions within endothelial cells that smaller in size and demonstrate lower levels of Akt promote angiogenesis. Endothelial nitric oxide synthase expression and activity compared to wild-type litter- (eNOS) is the nitric oxide synthase isoform responsible mates.69 Thus SRC-3 overexpression in human prostate for maintaining systemic blood pressure, vascular cancer is likely to lead to higher levels of Akt activation. remodeling and angiogenesis. Phosphorylation of eNOS Akt exerts its role in cancer cell proliferation/tumor by Akt leads to production of NO and enhances growth through phosphorylation and regulation of angiogenesis.76,77 ILK appears to be important in numerous signaling molecules involved in cell cycle regulating angiogenesis via Akt activation through both regulation and angiogenesis (Figure 2). The tumor- of these mechanisms. Thus, inhibition of Akt activity is a suppressor p21WAF1 is a p53 target gene that functions potential antiangiogenic therapeutic strategy. as a cell cycle inhibitor following induction by p53 in In summary, through phosphorylation and regula- response to specific cell stress signals such as DNA tion of a wide spectrum of substrates such as FKHR, damage. Phosphorylation by Akt prevents nuclear GSK3, MDM2, BAD, caspase 9, p27, , mTOR, IKKa/b, localization of p21WAF1, separating it from its cyclin- and eNOS, Akt promotes tumor cell survival, proli- dependent kinase targets. Thus, Akt activation abrogates feration, angiogenesis, and metastasis (Figure 2). Inter- p21WAF1-mediated cell cycle arrest.70 Moreover, cytoplas- estingly, recent studies of prostate cancer have revealed mic p21WAF1 binds to ASK1 and blocks its apoptotic common molecular regulators of AR and Akt, indicating function. Similarly, phosphorylation of p27Kip1 by Akt that an expanding number of signaling pathways leads to cytoplasmic retention of this cell cycle inhibitor involved in tumorigenesis and cancer progression and its sequestration by 14.3.3 scaffold protein.71 The involve Akt. Akt-mediated reduction of free p27Kip1 protein levels is associated with growth acceleration of prostate xenograft tumors.72 Phosphorylation and inhibition of GSK3b by Akt abolishes phosphorylation of cytoplasmic signaling Genetic perturbation and dysregulation molecule b-catenin, causing its stabilization and nuclear of the PI3-K-Akt pathway translocation, where it associates with T-cell factor (TCF)/lymphocyte enhancing-binding factor-1 (LEF-1) Akt is constitutively active in many human cancers due to induce the transcription of several genes, including to amplification of the Akt gene or as a result of cyclin D1, that promote cell cycle progression through amplification/ in components of the signaling hyperphosphorylation and inactivation of the retinoblas- pathway that regulates Akt activities (reviewed in Testa toma (Rb) tumor-suppressor protein.73 Phosphorylation and Bellacosa, 2001;5 Vivanco and Sawyers, 2002;6 Luo of MDM2 promotes its translocation to the nucleus et al, 200343). Akt gene amplification has been observed in where it binds and inhibits p53 activities towards cell prostate cancer78,79 and other malignancies including cycle regulation.54 mTOR (the mammalian target of ovarian cancer,80,81 pancreatic cancer,82 gastric cancer,83 rapamycin) is a serine/threonine kinase that regulates thyroid cancer,84 and .80,85 Elevated Akt cell growth by activating p70 ribosomal S6 kinase (RSK), expression and kinase activities are associated with a which promotes the translation of mRNA with 50 hormone-resistant phenotype and correlates with a poor polypyrimidine tracts, and by inhibiting 4E-BP1, which prognosis in human prostate cancer and breast can- acts as a translational inhibitor of mRNAs bearing a 50 cer.78,79,85,86 CAP structure.6 mTOR also regulates transcription of c- Upstream of Akt, amplification of PI3-K has been Myc and activation of Star 3, PKCa, and PKCd, that are reported for prostate,79 ovarian,87,88 colon,88 and breast involved in tumorigenesis.1,74 Phosphorylation by Akt cancer.85 Several protein–tyrosine kinases (PTKs), which activates mTOR and thus enhances cell growth. The act upstream of the PI3-K and are important regulators of breast cancer susceptibility-1 (BRCA1) gene encodes a the PI3-K-Akt pathway, have been found overexpressed nuclear phosphoprotein that plays a role in DNA repair. in various cancers because of activating or Phosphorylation by Akt impairs its nuclear localization other genetic alterations. For example, FGFR1 is over- and tumor-suppressor function.75 expressed in localized prostate cancer and amplified in Activation of Akt can also enhance tumor angiogen- hormone-resistant prostate cancers;79 ErbB2/HER2/Neu is esis. Loss of PTEN in prostate cancer is associated with overexpressed in prostate, breast, ovarian, gastric, increased microvessel density in prostate cancer tissue, nonsmall lung and colon cancers;89–92 IGF-1R expression

Figure 2 Akt plays a central role in an oncogenic signaling network through phosphorylation and regulation of a variety of cell survival-, cell proliferation-, and angiogenesis-related downstream targets. (a) When Akt is inactive, proapoptotic transcription factors of the FKHR family and cell cycle inhibitors p21 and p27 localize in the nucleus, while oncoproteins MDM2, mTOR, and NFkB are restrained in the cytoplasm. In addition, BAD, a proapoptotic member of BCL-2 family proteins, forms a nonfunctional heterodimer with prosurvival molecule BCL-XL. Caspase 9 forms complexes with Apaf-1 and cytochrome c, catalyzing the activation of downstream executor caspases. (b) After growth/survival factor stimulation and phosphorylation by Akt, the subcellular localization and activities of Akt substrates are dramatically altered, i.e., Akt phosphorylation promotes nuclear translocation of oncoproteins and prevents nuclear localization of proapoptotic transcription factors and cell cycle inhibitors. In addition to affecting its compartmentalization, phosphorylation of BAD by Akt prevents the binding of BAD to BCL-XL, restoring BCL-XL’s antiapoptotic function. Phosphorylation of procaspase 9 by Akt abrogates cytochrome c-induced proteolytic processing of pro-caspase 9, which in turn prevents the activation of downstream executor caspases. Akt may also phosphorylate AR (unresolved) and promote its translocation to nucleus, where it activates transcription of genes important to normal prostate growth and prostate cancer.

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al

114 is elevated in prostate, cervical and other PP2A.109,111,112 In addition, expression of the PP2A (reviewed in Blume-Jensen and Hunter, 20011). subunit Bg gene was found to be suppressed in human A most striking discovery is the association of .113 All of these findings support the notion functional loss of PTEN with advanced human cancer, that PP2A may function as a tumor-suppressor gene. including prostate cancer.13,15,93–95 Comprehensive sur- Although the role of PP1 in tumorigenesis is not as veys of various human cancers for PTEN deletion or evident as PP2A, it has been reported that PP1 interacts mutation reveal that functional loss of PTEN frequently with and dephosphorylates the Rb tumor-suppressor occurs in a wide spectrum of human cancers (reviewed protein returning Rb to its growth suppressive state in Vivanco and Sawyers, 20026). In prostate cancer, at (reviewed in Tamrakar et al, 2000114), and genetic least three mechanisms have been identified for func- alterations in PP1 have been identified in human tional loss of PTEN: chromosome deletion or loss of cancers.115,116 In prostate cancer, especially in advanced heterozygosity (LOH),96–101 somatic mutations95,99–103 prostate cancer, overexpression of cav-1 is frequently and epigenetic aberrations.104,105 The majority of human observed.29,37–40 Our finding of cav-1 interaction with prostate cancer cell lines and xenografts that have been and inhibition of two major serine/threonine protein evaluated show inactivation of the PTEN tumor-sup- phosphatases, PP1 and PP2A, establishes an important pressor gene. The extent to which PTEN is inactivated in link between cav-1 and Akt, that is, cav-1 compromises human prostate cancer in vivo is not completely certain PP1 and PP2A activities and thus favors the maintenance since a simultaneous, comprehensive, large-scale analy- of Akt in its active (phosphorylated) form. sis of PTEN LOH, homozygous deletion, mutation, and In summary, increased Akt activities in prostate cancer protein expression in the various clinical stages of and other malignancies are often due to amplification of prostate cancer has not been reported. The PTEN the Akt gene or its upstream kinases, such as PI3-K and tumor-suppressor gene maps to human chromosome protein tyrosine kinases, or deletion/mutation of nega- 10q23.3 and LOH at this locus has been detected in 25– tive regulators of this pathway, such as PTEN, PP2A, and 30% of clinically localized human prostate cancers, PP1. In prostate cancer, especially in advanced prostate although mutation or homozygous deletion of the PTEN cancer, elevated levels of cav-1 may play an important gene is detected in approximately 10% of these same role in limiting PP2A and PP1 activities, leading to the cases (for review, see Ittmann, 199894). Immunohisto- maintenance of activated Akt. chemical studies reveal complete loss of PTEN protein expression in 15–20% of clinically localized prostate cancers, with a substantial fraction of cases showing decreased expression. Thus, although it cannot be known Molecular biomarkers based on Akt with certainty due to the fragmentary nature of these activities studies, at least 20% of localized cancers have complete loss of PTEN activity due to loss of protein and/or The active phosphorylated form of Akt is an excellent inactivating point mutations. An additional 10–20% had prognostic marker for prostate cancer. Using tissue LOH at the PTEN locus without complete inactivation. microarray technology in a large cohort of patients, we Such LOH without biallelic inactivation is probably have identified that a high level of P-Akt in prostate clinically significant, since it has been shown in the cancer was predictive of a higher probability of TRAMP mouse model of prostate cancer that loss of a recurrence on univariate and multivariate analysis with single PTEN allele is associated with more rapid cancer hazard ratios considered high on semiquantitative progression, metastasis, and death. Similarly, LOH at analysis.117 Since most patients fall within the Gleason 10q23 is present in more than 50% of metastatic prostate 6 and 7 categories, and because of the known limitations cancers while biallelic PTEN inactivation is present in of Gleason scoring, we tested P-Akt for its predictive 30% of such cases.94 Given that a significant fraction of capacity within the subgroup. We found that P-Akt level men either present with or ultimately develop metastatic is also an independent prognostic indicator of survival prostate cancer, it is likely that complete or partial loss of when Gleason 6 and 7 patients were analyzed separately. the PTEN gene plays a role in approximately 40–50% of Surprisingly, a high level of P-Akt in non-neoplastic human prostate cancers overall. Loss of PTEN has been tissues is also an independent predictor of biochemical found associated with hormone-refractory,106 and ad- recurrence. vanced stage prostate cancer.98,107,108 Numerous targets of P-Akt have also been tested in In the past few years, the results of multiple studies this same patient cohort, including GSK3, FHKR and have shown that PP2A or PP1 can participate in the p27. Proliferation and apoptosis end points were regulation of PI3-K-Akt pathway through dephosphor- analyzed using Ki67 and TUNEL. Other markers that ylation of activated Akt and that these proteins can were related to, or potentially related to, Akt signaling function as the major phosphatases for Akt in some including PTEN, NF-kB, VEGFR3, ASCT2, ERa, AR, systems.21–26 Recent studies also show that alteration of DAX-1, TIF, SRC-3, SRC-1, and Sprouty were also tested. PP2A and PP1 is associated with Cox modeling was used to identify the best combination multiple human cancers. The PP2R1B gene that encodes of markers to predict PSA recurrence. We identified a the b isoform of the A subunit of PP2A is located in a number of significant predictors for PSA recurrence-free region showing high-frequency LOH.109 Mutations in the survival in prostate cancer, which were used to construct gene encoding the A subunit of PP2A have been found in a multivariate predictive model. The set of tissue human breast, lung, colon and colorectal carcinomas, biomarkers significantly (Po0.001) improved the com- and .109,110 These mutations display defective monly used clinical–pathological model of prediction or decreased binding to C subunit or B subunit of PP2A and mostly included P-Akt and its targets. Overall, we and therefore impair the normal functions of have demonstrated an incremental trend of improved

Prostate Cancer and Prostatic Diseases Akt pathway in prostate cancer LLiet al

PSA recurrence prediction through the use of a new provides an alternative approach to suppressing Akt 115 systems approach combining clinico-pathologic features activity in prostate cancer cells. Since cav-1 is secreted by together with immunohistochemical tissue analysis of P- prostate cancer cells and can be detected in the serum of Akt and its downstream117 (Ayala unpublished data). prostate cancer patients,123,124 using a specific antibody In summary, tissue microarray studies and immuno- to neutralize cav-1 may be therapeutically useful. In a histochemical studies of prostate cancer have established mouse model of metastatic prostate cancer, cav-1 anti- a positive correlation between P-Akt and clinical markers body therapy was shown to suppress orthotopic tumor of progression. Importantly, P-Akt is also an independent growth and metastasis.124 In addition to cav-1 antibody, predictor of biochemical recurrence. The development of cav-1 SiRNA may also serve as a useful tool to disrupt molecular biomarkers based on Akt activities will cav-1 function in cancer cells. provide insight into tumor progression, and clinically Drug targets can also be selected from specific Akt useful information for prostate cancer treatment. substrates that are involved in tumorigenesis, such as FKHR, BAD, and mTOR. The binding of 14-3-3 protein to FKHR or BAD could potentially be disrupted by peptidomimetics or small molecules, thus favoring Therapeutic potential of targeting the proapoptotic functions of these proteins. Targeting PI3-K-Akt pathway mTOR has been a focus over the past few years.6,43,125 Rapamycin, an inhibitor of mTOR kinase, and its analogs P-Akt is upregulated in a significant number of different CCI-779 and RAD001 have been shown to have types of malignancies including prostate cancer. The significant in vitro and in vivo antiproliferative activity results from intensive studies of the PI3-K-Akt pathway against a broad range of human cancers.126–129 Several have firmly established a central role for Akt in rapamycin analogs have already been approved for use tumorigenesis and cancer progression and have exposed in transplant patients as immunosuppressants.130 the PI3-K-Akt pathway as a promising therapeutic target. RAD001 and CCI-779 are currently in phase I and II Efforts have been made to develop small-molecule trials, respectively, as anticancer agents.125 kinase inhibitors targeting PI3-K. The first generation The PI3-K-Akt pathway appears to be a promising compounds, such as wortmannin and LY294002, which therapeutic target for cancer treatment. However, as this inhibit the p110 catalytic subunit of PI3-K, have been pathway is also involved in multiple cellular processes of widely used in vitro for years, but little progress has been normal cells, a reasonable therapeutic index is a made in clinical application. Wortmannin, which has a significant challenge for future drug development. short half-life, is not an acceptable pharmaceutical Hopefully, the continued search for new inhibitors of candidate. The administration of LY294002 in mouse this pathway will lead to the discovery of a new tumor models has been shown to confer antitumor generation of inhibitors that eliminate the pitfalls of the activity and to enhance the efficacy of the chemother- first generation inhibitors. Maximum antitumor efficacy apeutic agent paclitaxel with modest side effects.118 and minimum side effects may ultimately be achieved However, a concern that has arisen is the specificity of through the combination of specific PI3-K-Akt pathway this inhibitor, since it apparently acts against all the p110 inhibitor with others different types of cancer therapeutic isoforms as well as more distant PI3-K-like kinases such agents. as ATM and ATR.6 An alternative and potentially more In summary, because of its central role in tumorigen- specific approach is to inhibit the phosphotyrosine esis, the PI3-K-Akt pathway represents a promising binding of Src homology 2 (SH2) domain in p85 source of therapeutic targets for cancer treatment. regulatory subunit of PI3-K, preventing the recruitment Significant progress has been made in developing and activation of PI3-K by receptor tyrosine kinases. It small-molecule kinase inhibitors targeting PI3-K, the was demonstrated that small peptidomimetics derived PH domain of Akt, or specific Akt downstream targets, from the primary sequence of the phosphotyrosine motif such as FKHR, BAD, and mTOR. In prostate cancer, in the platelet-derived growth factor-b (PDGF-b) receptor targeting cav-1 through specific antibodies cav-1 or blocks the association of the C-terminal SH2 domain of SiRNA may prove to be useful in cancer treatment. the p85 subunit of PI3-K to PDGF-b receptor.119 Akt itself is an attractive therapeutic target. A potentially powerful strategy is to block the binding of PH domain of Akt to PI-3,4,5-P3 or PI-3,4-P2, thus Future perspective preventing its membrane translocation and activation by PDK1. Novel PI analogs have been shown to inhibit The results from more than a decade of research focused Akt effectively in cell culture.120 The 3D structural on the PI3-K-Akt pathway have firmly established a determination of the Akt PH domain,121 also provides central role of Akt in tumorigenesis and cancer progres- valuable information for researchers to design specific sion. For prostate cancer, significant progress has been inhibitors of this kinase as potential antitumor agents. made in the development of biomarkers based on P-Akt This type of drug may be more specific since different and Akt downstream targets and in the exposure of the PH domains among AGC family kinases normally share PI3-K-Akt pathway as a source of therapeutic targets. o20% sequence identity, allowing for the development Looking forward, final identification, purification, and of drugs that bind specifically to the PH domain of cloning of the elusive Ser473 kinase will provide the Akt.122 On the other hand, the effectors that regulate and missing piece of PDK2 in the Akt activation puzzle. The activate Akt activity may be also ‘drugable’. For example, development of proteomics technology will hopefully the elevated cav-1 in prostate cancer favors the main- allow the unambiguous identification and clarification of tenance of activated Akt,41 and therefore targeting cav-1 Akt substrates and new information regarding the

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