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¹ Brain Tumor Center, Department of Neuro-Oncology and ² Department of Molecular Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Dimpy Koul, Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Unit 1002, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-834-6202; Fax: 713-834-6230. E-mail: Dkoul{at}mdanderson.org.

	Abstract

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Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions, including cell cycle progression; cell migration, invasion, and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene, which is located downstream of PI3K, have been shown in a variety of cancers, including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways, including inhibitors of the PI3K pathway, have shown antitumor activity, but inhibitors of Akt have not been examined. In this study, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors, KP-372-1 and KP-372-2 (herein called KP-1 and KP-2), effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor–induced phosphorylation of Akt Ser⁴⁷³ at 125 and 250 nmol/L, which, in turn, reduced the activation of intracellular downstream targets of Akt, including GSK-3ß and p70s6k. Furthermore, the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively, these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas. [Mol Cancer Ther 2006;5(3):637–44]

	Introduction

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Malignant gliomas are the most common primary brain tumor in adults, but the prognosis for patients with these tumors remains poor despite advances in diagnosis and standard therapies, such as surgery, radiation therapy, and chemotherapy. Progress in the treatment of gliomas now depends to a great extent on an increased understanding of the biology of these tumors. Recent insights into the biology of gliomas include the finding that tyrosine kinase receptors and signal transduction pathways play a role in tumor initiation and maintenance. The epidermal growth factor receptor, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, Ras/Raf/mitogen-activated protein kinase, and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathways have all been found to be important for cell survival. The Akt pathway is of particular interest because it regulates several critical cellular functions, including cell cycle progression, migration, invasion, and survival as well as angiogenesis. In addition, the activated PI3K/Akt pathway provides major survival signals to glioblastoma multiforme cells and many other cancer cells (1–4). Furthermore, the ectopic expression of Akt induces cell survival and malignant transformation, whereas the inhibition of Akt activity stimulates apoptosis in a range of mammalian cells (5–8). Highlighting its potential as a therapeutic target is the fact that Akt is activated in 70% of gliomas (9–12). Recent studies have identified the substrates of Akt that are involved in the pro-cell survival effects, which thus far include glycogen synthase kinase-3, mTOR, FKHR, MDM2, p21, HIF-1, IKK, Bad, and caspase-9 (13, 14). Akt has also been implicated as an important target of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as the reintroduction of PTEN into PTEN-deficient glial, breast, and prostate cancer cells resulted in a decrease in activated Akt (15–17). Thus, the dysregulation of Akt seems to be an important consequence of the loss of PTEN function.

PTEN is a phospholipid phosphatase that dephosphorylates phosphatidylinositol 3,4,5-triphosphate (18, 19) and inhibits PI3K-dependent activation of Akt. The mutation or loss of PTEN leads to constitutively activated Akt. Consistent with this, Akt activity is elevated in about 70% of glioblastoma multiforme cells expressing mutant forms of PTEN (10). Activated Akt phosphorylates and inactivates Bad, caspase-9, and Forkhead transcription factors, which not only suppresses apoptosis but also promotes cell survival (13, 14, 20). Furthermore, Akt must be activated for glioblastoma multiformes to form from genetically modified neural progenitors and normal human astrocytes, suggesting that the activation of Akt plays an important role in glioma formation and progression (11, 21). These observations establish Akt as an attractive target for cancer therapy, both alone and in conjunction with standard cancer chemotherapies, as a means of reducing the apoptotic threshold and preferentially killing cancer cells. Attention is thus focused on the development of potent small-molecule Akt inhibitors, but the potential of this strategy to inhibit glioma cell growth has not been studied.

In the study we report on here, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We found that the small-molecule inhibitors of Akt, KP-1 and KP-2, inhibited Akt signaling in glioma cells, which inhibited cell growth and induced apoptosis.

	Materials and Methods

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Cell Lines
U87, U251, LN229, SNB19, U373, and D54 human glioblastoma cells were maintained at 37°C in culture medium (DMEM/F12, 10% fetal bovine serum) in a humidified atmosphere containing 5% CO₂. The normal human lung fibroblast line CCD32-Lu was obtained from the American Type Culture Collection (Manassas, VA). Normal human astrocytes were purchased from Clonetics/Bio Whittaker (Walkersville, MD). Normal human astrocyte cultures were maintained in astrocyte growth medium from an AGM-Astroctye Medium Bullet kit obtained from Clonetics/Bio Whittaker.

Drugs
QLT, Inc. (formerly Kinetek, Vancouver, British Columbia, Canada) has provided us with a series of KP compounds [KP-372-1 (KP-1) to KP-372-5 (KP-5)] that target the Akt component of the PI3K pathway. KP compounds used in this study have at least 10-fold selectivity against a limited number of kinase targets, including CDK, CK2, CSK, DNAPK, ERK1, GSK3b, LCK, MEK1, PAK3, PIM, PKA, PKC, and S6K.³ A stock solution of 5 mmol/L was prepared in DMSO and stored at –20°C. Dilutions for all assays were made before use. DMSO has been used to dissolve these drugs and as the vehicle control for all the experiments on these agents.

Cell Proliferation Assays
Five thousand cells were plated into 38-mm² wells of 96-well tissue culture plates. Cells were incubated with KP-1 and KP-2 (0.0625–2 µmol/L); control cells were incubated with either medium or DMSO alone. After a 2-day incubation, the number of metabolically viable cells was determined in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells was measured by an MR-5000 96-well microtiter plate reader (Dynatech, Inc., Chantilly, VA) at an absorbance of 570 nm. Growth inhibition was calculated by the following formula: cytostasis (%) = [1 – (A/B)] x 100, where A is the absorbance of treated cells, and B is the absorbance of control cells.

Colony-Forming Assay
Cells (1 x 10⁶) were incubated with KP-1 and KP-2, and control cells were incubated with medium or DMSO alone. After a 24-hour incubation, cells were trypsinized and plated at a density of either 100 or 500 per well in a six-well plate containing 1 mL of DMEM/F12/10% fetal bovine serum. Cells in culture were then incubated at 37°C in a humidified atmosphere containing 5% CO₂. Colony growth was assessed by the size and number of colonies after 28 days. Colonies exceeding the minimum diameter of 80 µm were counted in triplicate wells, and the fraction of treated surviving cells was calculated relative to the fraction of control surviving cells, which was taken as 1. Experiments were done in triplicate and repeated thrice.

Antibodies and Western Blotting
Subconfluent monolayers of cells were treated with KP-1 and KP-2 at the indicated doses in serum-free medium. Six hours later, cells were harvested, either with or without stimulation with epidermal growth factor (50 ng/mL) for 10 minutes. Cells were harvested in lysis solution containing 50 mmol/L HEPES (pH 7), 150 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 100 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mmol/L Na₃VO₄, 10 µmol/L pepstatin, 10 µg/mL aprotinin, 5 mmol/L iodoacetic acid, and 2 µg/mL leupeptin. Proteins equal to 50 µg from U87 cells, either untreated or treated with KP-1 and KP-2 for 48 hours, were resolved by SDS-PAGE, electroblotted to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and then probed with the following primary antibodies: phospho-specific Akt (Ser⁴⁷³), phospho-specific GSK-3ß (Ser⁹), phospho-specific mTOR (S2448), phospho-specific p70S6 kinase, and phospho-specific S6 kinase (Cell Signaling, Boston, MA), as well as poly(ADP-ribose) polymerase (PARP), Bcl-2, and Bax (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-ß-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

LIVE/DEAD Assay
To measure the cytotoxic effect of KP-1 and KP-2 on glioma cell lines, we did the LIVE/DEAD assay (Molecular Probes, Invitrogen, Carlsbad, CA), which determines intracellular esterase activity and plasma membrane integrity. This assay employs calcein, a polyanionic dye that is retained within live cells and produces green fluorescence. It also employs the ethidium bromide homodimer dye (red fluorescence), which can enter the cells through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of live cells. Briefly, 1 x 10⁵ cells were treated with KP-1 and KP-2 at the indicated doses for 48 hours at 37°C. Cells were stained with the LIVE/DEAD reagent (5 µmol/L ethidium bromide homodimer, 5 µmol/L calcein-AM) and then incubated at 37°C for 30 minutes. Cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).

Apoptosis Analysis
Cells (1 x 10⁶) were seeded on a 10-cm tissue culture dish and incubated overnight with serum-free medium followed by treatment with KP-1 and KP-2 at the indicated doses for 48 hours. Cells were maintained in medium supplemented with 10% FCS. Cells were harvested at various times thereafter. Cells for DNA analysis were fixed in 1% paraformaldehyde, stored in 70% ethanol, and analyzed for apoptosis by flow cytometry using the Apo-Bromodeoxyuridine kit (Phoenix Flow Systems, San Diego, CA) according to the manufacturer's instructions.

We also investigated the effect of the KP-1 and KP-2 on the expression of apoptotic proteins. In particular, we determined the effect of inhibitor treatment on PARP, a well-known substrate for caspase-3, caspase-6, and caspase-7, to determine whether inhibitor-induced apoptosis was mediated through caspase activation. In this experiment, U251 and U87 cells were treated with KP-1 or KP-2 for 48 hours, after which whole-cell extracts were prepared and analyzed for cleavage of PARP. Immunoblot analysis was then done to determine the activation status of the downstream caspases.

Terminal Deoxynucleotidyl Transferase–Mediated Nick End-Labeling Assay
We also assayed cytotoxicity using the terminal deoxynucleotidyl transferase–mediated nick end-labeling method, which examines DNA strand breaks during apoptosis. We used the Roche in situ cell death detection reagent (Roche Applied Science, Indianapolis, IN). Briefly, 1 x 10⁵ cells were treated with KP-1 and KP-2 at the indicated doses for 48 hours at 37°C. Thereafter, cells were washed with PBS, air-dried, fixed with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After being washed, cells were incubated with reaction mixture for 60 minutes at 37°C. Stained cells were mounted and analyzed under a fluorescence microscope (Labophot-2). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph version 4.6.5 software (Universal Imaging, Downingtown, PA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Akt Inhibitor Induces Cell Growth Inhibition
We first tested the growth-inhibitory effect of KP-1 and KP-2 on a set of glioma cell lines using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Treatment of cells with KP-1 and KP-2 for 2 days resulted in dose-dependent growth inhibition (Fig. 1A and B ), but the magnitude of the growth inhibition varied among the different cell lines, with the IC₅₀ ranging between 125 and 250 nmol/L. We also tested these drugs on normal human astrocytes and normal human fibroblasts to check the toxicity on these cells. Treatment of these cells with KP-1 and KP-2 had no growth inhibitory effect on normal astrocytes and fibroblasts (Fig. 1C), showing the cancer specific effect of the drugs. We then did a colony-forming assay using two well-characterized and drug-sensitive glioma cell lines, U251 and U87, to confirm the growth-inhibitory effects of these Akt inhibitors. Treatment of the U251 and U87 cells with different concentrations of KP-1 and KP-2 abrogated the ability of the cells to form colonies, confirming the growth-inhibitory and antitumorigenic effects of KP-1 and KP-2 (Fig. 1D). In contrast, untreated U251 and U87 cells efficiently formed colonies reflecting their transformed phenotype.

View larger version (36K): [in this window] [in a new window]   Figure 1. Effect of Akt inhibitors on proliferation of human glioma cell lines. Six glioma cell lines were plated in 96-well plates at a density of 5,000 per well. Cells were treated with increasing concentrations of KP-1 (A) or KP-2 (B) in triplicate wells for 48 h under serum-fed conditions, and cell viability was assessed in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay described in Materials and Methods. The value for DMSO-treated cells was set to 100% (Y axis), and the number of cells for all drug doses were normalized as percentage of the number of cells in DMSO. There was a dose-dependent decrease in cell viability in all cell lines tested. C, effect of Akt inhibitor on proliferation of normal human astrocytes and normal human fibroblasts treated similar way as indicated above. D, U251 and U87 cells, either untreated or treated with different concentrations of the Akt inhibitors, were cultured for 28 d. The efficiency of colony formation was determined as described in Materials and Methods. Anchorage-dependent cell growth was abrogated by Akt inhibition.

View larger version (56K): [in this window] [in a new window]   Figure 2. A, expression profile of phosphorylated Akt (pAkt) and downstream targets in glioblastoma cell lines, as determined in whole-cell lysates from exponentially growing cells analyzed by immunoblotting with antibodies against the indicated proteins. U87 (B) and U251 (C) cells were treated with the vehicle DMSO or IC50 concentrations of the Akt inhibitors (125–250 nmol/L) for 6 h. Cells were then stimulated with epidermal growth factor (50 ng/mL) for 10 min before harvesting. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with different antibodies, as indicated. KP compounds inhibited Akt phosphorylation and hence the phosphorylation of some of the downstream targets.

KP Compounds Induce Cytotoxicity in Glioma Cells
This ability of KP compounds to selectively inhibit the Akt pathway suggested that they should inhibit cell proliferation and/or induce apoptosis. To investigate this possibility, we did a LIVE/DEAD assay and terminal deoxynucleotidyl transferase–mediated nick end-labeling and propidium iodide staining to determine the effects of KP-1 and KP-2 on both the cell cycle profile and apoptosis. In keeping with this, the LIVE/DEAD assay showed that 250 nmol/L KP-1 and KP-2 were cytotoxic to 81% and 72% of U87 cells and 89% and 75% of U251 cells, respectively (Fig. 3A ). Furthermore, flow cytometry using the Apo-Bromodeoxyuridine kit showed that the Akt inhibitors induced apoptosis in both U87 and U251 glioma cells. Specifically, 22% and 52% of U87 cells treated with 125 and 250 nmol/L KP-1 and 19% and 49% of U87 cells treated with 125 and 250 nmol/L KP-2 showed apoptosis, respectively. Similarly, 38% and 70% of U251 cells treated with 125 and 250 nmol/L KP-1 and 25% and 55% of U251 cells treated with 125 and 250 nmol/L showed apoptosis, respectively. These data indicate that KP-1 is more potent than KP-2 in its ability to induce apoptosis (Fig. 3B). Terminal deoxynucleotidyl transferase–mediated nick end-labeling staining confirmed that the KP compounds induced apoptosis in U87 and U251 cells (Fig. 3C).

View larger version (36K): [in this window] [in a new window]   Figure 3. Analysis of apoptosis induced by Akt inhibitors in U87 and U251 glioma cells. A, U87 and U251 cells were pretreated with Akt inhibitors at a concentration of 125 and 250 nmol/L for 48 h. Cells were stained with LIVE/DEAD assay reagent for 30 min and then analyzed under a fluorescence microscope, as described in Materials and Methods. The KP compounds were cytotoxic in both cell lines. B, U87 and U251 cells were treated with KP-1 (left) or KP-2 (right) for 48 h at concentrations of 125 and 250 nmol/L. The percentages of apoptotic cells were determined by terminal deoxynucleotidyl transferase–mediated nick end-labeling followed by flow cytometric analysis. Columns, mean of triplicate determinations; bars, SE. Apoptosis was induced by the KP compounds in both cell lines. C, U87 and U251 cells were pretreated with Akt inhibitors for 48 h. Cells were fixed, stained with terminal deoxynucleotidyl transferase–mediated nick end-labeling assay reagent and analyzed under a fluorescence microscope as described in Materials and Methods. Original magnification, x200. D, effect of Akt inhibitors on apoptotic proteins, as shown by immunoblotting.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we showed that small-molecule inhibitors of Akt can inhibit the growth of glioma cell lines. In particular, the Akt inhibitors KP-1 and KP-2 blocked both the basal and epidermal growth factor–induced phosphorylation of Akt. They also reduced the activation of downstream targets of Akt, including p70s6k and GSK-3ß, consistent with the selective inhibition of Akt, and this was accompanied by inhibition of glioma proliferation. Indeed, KP-1 and KP-2 induced apoptosis enough to significantly inhibit cellular proliferation, although other cellular effects, such as cell cycle arrest, and the possibility of different findings in other cell systems cannot be ruled out.

PI3K is a lipid kinase (22) that promotes diverse biological functions, including cell proliferation, survival, and motility (23). Not surprisingly, therefore, the PI3K signaling pathway is frequently aberrantly activated in glioblastomas as a result of a mutation or loss of the tumor suppressor PTEN. Such PTEN abnormalities result in inappropriate signaling to downstream molecules, including Akt and mTOR. This, plus the fact that Akt is activated in 70% gliomas, makes the deregulation of the PI3K signaling pathway an attractive target for therapy (24, 25). Therefore, elucidating the molecular events associated with activation of this pathway represents an important goal because of the important use this knowledge can be put to in the development and clinical testing of PI3K pathway inhibitors.

The principle that kinase inhibitors can be effective treatments for some types of cancer has already been shown (24, 26), and PI3K pathway inhibitors are already in early clinical trials (27–30). The loss of PTEN expression is correlated with high levels of phosphorylated Akt in glioblastoma. Akt is considered an attractive target for chemotherapy, and it has been postulated that the inhibition of Akt alone or in combination with standard cancer chemotherapy will reduce the apoptotic threshold and preferentially kill cancer cells.

Several agents interfering with intracellular targets that are components of key oncogenic signaling pathways, such as the RAF kinase, PI3K/Akt, and Src kinases, are in preclinical and early clinical development. Two commonly used PI3K inhibitors, wortmannin and LY294002, may have limited clinical use, however, because they lack specificity and have potential adverse side effects, poor pharmacologic properties, low stability, and poor solubility (31). Indeed, most small-molecule inhibitors in this nascent field are classic ATP-competitive inhibitors that have little specificity. Nonetheless, there are several advantages to small-molecule drugs that make them worth pursuing; these include good delivery properties, good in vivo stability, a low probability of inducing an immune response, and low cost. Recently, novel allosteric inhibitors have been reported that are pleckstrin homology domain dependent and exhibit selectivity for the Akt isozyme.

Similarly, in this report, we describe potential small-molecule inhibitors of the Akt pathway. Specifically, our data have shown that KP compounds can inhibit the proliferation of glioma cells by down-regulating Akt, p70s6k, and GSK-3ß phosphorylation. They also induced apoptosis in glioma cell lines expressing high levels of activated Akt. Because we observed more extensive apoptosis in U87 cells (with wild-type p53) than in U251 cells (with mutant p53), we used a dominant-negative p53 variant of the U87 (32) cell line to show the role of p53 in inhibitor-induced apoptosis. Treatment of this cell line with an Akt inhibitor still induced apoptosis irrespective of the p53 status (data not shown). This suggests that KP compounds do not induce apoptosis through a p53-dependent pathway.

In addition to glioblastoma, PTEN is frequently mutated in endometrial and prostate cancer (33, 34), whereas Akt activity is also elevated in many ovarian and breast cancers. In these tumors, however, Akt activation is mainly due to amplification of the Akt oncogene or to activation by upstream regulators (35–37). It will therefore be of interest to determine whether KP compounds can inhibit other types of cancer that display high Akt kinase activity. In this regard, KP compounds have already been shown to inhibit Akt kinase activity and induce apoptosis in thyroid cancer cells with high Akt activity (38). These studies plus our study represent the first critical steps towards developing a small-molecule therapy that targets the Akt oncogenic pathway. This approach may prove effective not only against glioblastomas but also against prostate cancer, endometrial cancer, and other tumors in which PTEN and Akt disruption is common.

	Acknowledgments

 
We thank QLT for providing Akt inhibitors and Betty P. Notzon (Department of Scientific publications, The University of Texas M.D. Anderson Cancer Center) for reading and editing of the article.

	Footnotes

 
Grant support: National Cancer Institute/NIH grant RO1 CA56041 (W.K. Alfred Yung), Common Wealth Cancer Foundation, and ABC² (W.K. Alfred Yung).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: The current address for S. Shishodia is Department of Biology, Texas Southern University, 3100 Cleburne Street, Houston, TX 77004.

³ QLT, Inc., personal communication.

Received 11/ 1/05; revised 12/14/05; accepted 1/10/06.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999;13:2905–27.[Free Full Text]
2. Holland EC. Glioblastoma multiforme: the terminator. Proc Natl Acad Sci U S A 2000;97:6242–4.[Free Full Text]
3. Maher EA, Furnari FB, Bachoo RM, et al. Malignant glioma: genetics and biology of a grave matter. Genes Dev 2001;15:1311–33.[Free Full Text]
4. Wechsler-Reya R, Scott MP. The developmental biology of brain tumors. Annu Rev Neurosci 2001;24:385–428.[CrossRef][Medline]
5. Ahn JY, Hu Y, Kroll TG, Allard P, Ye K. PIKE-A is amplified in human cancers and prevents apoptosis by up-regulating Akt. Proc Natl Acad Sci U S A 2004;18:6993–8.
6. Cheng JQ, Nicosia SV. AKT signal transduction pathway in oncogenesis. Schwab D, editor. Encyclopedic reference of cancer. Berlin (Germany): Springer; 2001. p. 35–7.
7. Sun M, Wang G, Paciga JE, et al. AKT1/PKB kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 2001;159:431–7.[Abstract/Free Full Text]
8. Cheng JQ, Altomare DA, Klein MA, et al. Transforming activity and cell cycle-dependent expression of the AKT2 oncogene: evidence for a link between cell cycle regulation and oncogenesis. Oncogene 1997;14:2793–801.[CrossRef][Medline]
9. Alessi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen P. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett 1996;399:333–8.[CrossRef][Medline]
10. Haas-Kogan D, Shalev N, Wong M, Mills G, Yount G, Stokoe D. Protein kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC. Curr Biol 1998;8:1195–8.[CrossRef][Medline]
11. Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 2000;25:55–7.[CrossRef][Medline]
12. Rajasekhar VK, Viale A, Socci ND, Wiedmann M, Hu X, Holland EC. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol Cell 2003;12:889–901.[CrossRef][Medline]
13. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997;91:231–41.[CrossRef][Medline]
14. Cardone MH, Roy N, Stennicke HR, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318–21.[Abstract/Free Full Text]
15. Davies MA, Lu Y, Sano T, et al. Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis. Cancer Res 1998;58:5285–90.[Abstract/Free Full Text]
16. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997;275:1943–7.[Abstract/Free Full Text]
17. Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL. PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 1998;95:15587–91.[Abstract/Free Full Text]
18. Myers MP, Pass I, Batty IH, et al. The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc Natl Acad Sci U S A 1998;95:13513–8.[Abstract/Free Full Text]
19. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998;273:13375–8.[Abstract/Free Full Text]
20. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857–68.[CrossRef][Medline]
21. Sonoda Y, Ozawa T, Aldape KD, Deen DF, Berger MS, Pieper RO. Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res 2001;61:6674–8.[Abstract/Free Full Text]
22. Hidalgo M, Rowinsky EK. The rapamycin-sensitive signal transduction cascade as a target for cancer therapy. Oncogene 2000;19:6680–6.[CrossRef][Medline]
23. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489–501.[CrossRef][Medline]
24. Sawyers CL. Rational therapeutic intervention in cancer: kinases as drug targets. Curr Opin Genet Dev 2002;12:111–5.[CrossRef][Medline]
25. Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A 2001;98:10314–9.[Abstract/Free Full Text]
26. Druker BJ. Perspectives on the development of a molecularly targeted agent. Cancer Cell 2002;1:31–6.[CrossRef][Medline]
27. Hu L, Zaloudek C, Mills G, Gray J, Jaffe R. In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002). Clin Cancer Res 2000;6:880–6.[Abstract/Free Full Text]
28. Ballif B, Shimamura A, Pae E, Blenis J. Disruption of 3-phosphoinositide-dependent kinase 1 (PDK1) signaling by the anti-tumorigenic and anti-proliferative agent n-alpha-tosyl-L-phenylalanyl chloromethyl ketone. J Biol Chem 2001;276:12466–75.[Abstract/Free Full Text]
29. Kozikowski A, Sun H, Brognard J, Dennis P. Novel PI analogues selectively block activation of the pro-survival serine/threonine kinase Akt. J Am Chem Soc 2003;125:1144–5.[CrossRef][Medline]
30. Meuillet E, Mahadevan D, Vankayalapati H, et al. Specific inhibition of the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo-inositol analogues. Mol Cancer Ther 2003;2:389–99.[Abstract/Free Full Text]
31. West KA, Castillo SS, Dennis PA. Activation of the PI3K/Akt pathway and chemotherapeutic resistance. Drug Resist Updat 2002;5:234–48.[CrossRef][Medline]
32. Cerrato JA, Khan T, Koul D, et al. Differential activation of the Fas/CD95 pathway by Ad-p53 in human gliomas. Int J Oncol 2004;2:409–17.
33. Cairns P, Okami K, Halachmi S, et al. Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res 1997;57:4997–5000.[Abstract/Free Full Text]
34. Li D, Sun H. PTEN/MMAC1/TEP1 suppresses the tumorigenicity and induces G₁ cell cycle arrest in human glioblastoma cells. Proc Natl Acad Sci U S A 1998;95:15406–11.[Abstract/Free Full Text]
35. Cheng J, Godwin A, Bellacosa A, et al. 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 U S A 1992;89:9267–71.[Abstract/Free Full Text]
36. Bellacosa A, de Feo D, Godwin A, et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer 1995;64:280–5.[Medline]
37. Tashiro H, Blazes M, Wu R, et al. Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res 1997;57:3935–40.[Abstract/Free Full Text]
38. Mandal M, Kim S, Younes MN, et al. The Akt inhibitor KP372–1 suppresses Akt activity and cell proliferation and induces apoptosis in thyroid cancer cells. Br J Cancer 2005;23:1899–905.

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