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[Cancer Research 61, 6674-6678, September 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Akt Pathway Activation Converts Anaplastic Astrocytoma to Glioblastoma Multiforme in a Human Astrocyte Model of Glioma1

Yukihiko Sonoda, Tomoko Ozawa, Ken D. Aldape, Dennis F. Deen, Mitchel S. Berger and Russell O. Pieper2

Brain Tumor Research Center, Departments of Neurological Surgery, [Y. S., T. O., D. F. D., M. S. B., R. O. P.] and Pathology [K. D. A.], University of California-San Francisco, San Francisco, California 94115


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Human malignant gliomas are thought to develop as the result of stepwise accumulations of multiple genetic alterations. Recently, we showed that E6/E7-mediated inactivation of p53/pRb, ras pathway activation (initiated by expression of mutant H-Ras), and expression of human telomerase reverse transcriptase (hTERT) in combination converted normal human astrocytes into cells that formed intracranial tumors resembling human anaplastic astrocytoma (AA). In this study, we created human astrocytes that, in addition to expressing E6/E7, hTERT, and Ras, also expressed a constitutive activated form of Akt intended to mimic the Akt activation noted in grade IV glioblastoma multiforme (GBM). Although these cells grew no differently than astrocytes expressing E6, E7, and H-Ras in vitro or in the first 28 days following s.c. implantation, they ultimately formed tumors four to six times larger than those formed by the E6/E7/hTERT/Ras cells. Unlike the poorly vascularized, necrosis-free AA formed by E6/E7/hTERT/Ras cells, the tumors formed by s.c. or intracranial injection of Akt-expressing cells had large areas of necrosis surrounded by neovascularization and were consistent in appearance with grade IV human GBM. These results show that activation of the Akt pathway is sufficient to allow conversion of human AA to human GBM.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Gliomas are the most common human brain tumors and are divided into four stages by WHO classification scheme (1) . Less malignant gliomas are defined as grades I and II, whereas grade III (AA)3 and grade IV (GBM) are malignant. Although both grades III and IV are malignant, the prognoses for these tumors are quite different. Whereas the 2-year survival rate for grade III gliomas is ~50%, that for grade IV is <20% (2) . An understanding of the mechanism of progression from grade III to grade IV glioma could therefore be of importance in halting the advance of the disease and prolonging survival.

The malignant gliomas are believed to develop as the result of stepwise accumulations of genetic lesions (1) . AA typically exhibits loss of a functional p53 pathway, usually by p53 mutation; loss of a functional p16/pRb pathway, typically by deletion of the p16/ARF locus; ras pathway activation by means other than ras mutation, which is rare in any grade of glioma; and telomerase reactivation, which is rarely seen in NHAs or grade II glioma (1 , 3 , 4) . GBM, in addition to alterations in the p53 pathway and the p16/pRb pathway noted in AA, also frequently contain alterations in PTEN that lead to activation of the Akt pathway (5 , 6) . Akt in turn has been shown to inactivate/repress several targets, including Bad and Forkhead transcription factors (7, 8, 9) . Inactivation of Bad by Akt suppresses normal apoptotic response, whereas suppression of AFX/Forkhead transcription factor activity leads to reduced levels of a variety of proteins, including the cell cycle inhibitor p27 (10 , 11) . Additionally, Akt has been shown to increase vascular endothelial growth factor levels under hypoxic conditions (12) . Because Akt has the potential to suppress apoptosis, deregulate cell cycle, and alter angiogenic potential, and because up to 80% of all GBM expresses elevated levels of Akt, activation of the Akt pathway is strongly implicated in the development of human GBM (5 , 6) .

Recently, we developed a model by which individual genetic alterations could be assessed for their contribution to the transformation of NHAs and to the formation of human gliomas (13) . Using this model we showed that tumors resembling human AA could be created by s.c. or intracranial implantation of NHAs modified to express E6, E7, hTERT, and H-Ras (13) . Although substituting Akt for Ras did not allow for the formation of AA, it remained possible that the actions of Akt were important, but only at a later point in glioma development. To address this possibility, we created human astrocytes that, in addition to expressing E6, E7, hTERT, and Ras, also expressed a constitutively activated form of Akt. In this study, we show that additional expression of Akt allows the formation of GBM-like tumors.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cells.
NHAs (Clonetics, Walkersville, MD) were maintained in Astrocyte Growth Medium (Clonetics). The creation of NHAs expressing E6/E7, hTERT, and H-RasV12 (Ras astrocytes) has been described previously (13) . To create astrocytes expressing E6/E7, hTERT, H-Ras, and a constitutively activated myristylated form of Akt (Ras+Akt astrocytes), Phoenix A cells were first transfected with pWZL-hygro-myrAkt{Delta}4-129 by lipofection (14) . The myrAkt{Delta}4-129 retroviral construct was then introduced into NHAs expressing E6/E7, hTERT, and H-RasV12 with selection by hygromycin B (300 µg/ml for 5 days).

Soft Agar and Tumorigenicity Assays.
Soft agar cloning assays, s.c. implantation of modified astrocytes into immunodeficient mice, and intracranial implantation of modified astrocytes into immunodeficient rats were performed as described previously (13) .

Immunohistochemistry.
Pimonidazole hydrochloride (Hypoxyprobe; Natural Pharmacia International, Inc., Research Triangle Park, NC) was administered by i.p. infusion 2 h before sacrifice at a whole-body dose of 60 mg/kg. Mice were then perfused with 10% formalin. Tumors were kept in 10% formalin overnight and embedded in paraffin. Five-µm paraffin sections were stained with H&E. Unstained sections were deparaffinized and subjected to immunohistochemical analysis. For immunostaining of pimonidazole adducts, the primary antibody was diluted 1:50 for mouse antihyproxyprobe-1 (Natural Pharmacia International, Inc.). Incubation with primary antibody was for 40 min at room temperature. Following incubation with a biotin-SP-conjugated antimouse F(ab')2 (Accurate Chemical & Scientific Corp., Westbury, NY) for 10 min at room temperature, antigens were revealed with horseradish peroxidase and diaminobenzidine (DAKO, Carpinteria, CA). Sections were counterstained in methyl green.

In Situ End Labeling of DNA Fragmentation.
To detect apoptotic cells within tumor tissues, we performed in situ end labeling using the Tumor TACS kit (R&D Systems, Minneapolis, MN). Briefly, deparaffinized sections were preincubated with proteinase K for 15 min, and endogenous peroxidase activity was then blocked with 0.3% H2O2 in methanol for 20 min at room temperature. Slides were immersed in TdT labeling buffer for 5 min, followed by reaction with TdT enzyme, Mn2+, and TdT deoxynucleotide triphosphate mixture for 1 h at 37°C. The reaction was stopped by immersing the slides in TdT stop buffer for 5 min. Staining was visualized with streptavidin-horseradish peroxidase and diaminobenzidine. Sections were counterstained in methyl green. For quantitative analysis, the ratio of positive cells to the total number of cells was calculated. Positive cells within necrotic areas were not counted because the technique used could not distinguish DNA fragmentation in apoptotic cells from that which occurs in the late stages of necrosis (15) .

Western Blotting.
Analysis was as described previously (13) with the following primary antibodies: Actin (C-11; Santa Cruz Biotechnology, Santa Cruz, CA), Bad (Transduction Laboratories, Lexington, KY), and phospho-Bad (Ser136; Upstate Technology, Lake Placid, NY). Proteins were extracted from two independent regions of the tumors analyzed. All analyses were performed in triplicate, using actin expression as a control.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Akt Expression Does Not Influence the Frequency at Which Genetically Modified Astrocytes Form Colonies in Soft Agar or Tumors in Nude Animals.
As described previously, astrocytes expressing E6/E7 and hTERT or E6/E7, hTERT, and Akt did not grow in soft agar and were not transformed. However, astrocytes expressing E6/E7, hTERT, and Ras (Ras astrocytes) formed colonies in soft agar and formed tumors in >80% of the mice into which they were injected (Table 1)Citation . The astrocytes expressing E6/E7, hTERT, Ras, and Akt (Ras+Akt astrocytes) also formed colonies in soft agar and tumors in animals at rates indistinguishable from those of Ras astrocytes (Table 1)Citation .


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  		Table 1 Akt does not affect colony numbers and tumor incidence

 
Akt Contributes to Conversion of Grade III AA to Grade IV GBM.
Although the astrocytes expressing H-Ras and myrAkt were no more likely to grow in soft agar or in animals than astrocytes expressing H-Ras alone, the s.c. tumors formed by the Ras+Akt astrocytes were four to six times larger than those formed by Ras astrocytes (Fig. 1A)Citation . GFAP staining showed that, like the Ras tumors, the Ras+Akt tumors expressed GFAP and were of human glial origin (data not shown). H&E staining, however, showed that although both tumor types were highly cellular, contained numerous mitotic figures, and were consistent in appearance with malignant (AA or GBM) human gliomas, tumors formed by Ras astrocytes were devoid of necrosis and contained cells of uniform size and shape (Fig. 1B)Citation . In contrast, Akt-expressing tumors contained cells that were highly diverse in size and shape and displayed numerous areas of necrosis (Fig. 1B)Citation . To verify that these findings were not an artifact of site of injection, Ras and Ras+Akt astrocytes were implanted intracranially into rats and the resultant tumors were analyzed. As in the s.c. setting, intracranially implanted Ras astrocytes formed relatively small tumors that at 28 days after implantation did not affect the health of the animals (Fig. 1C)Citation . In contrast, 28 days after intracranial implantation, the Ras+Akt astrocytes formed tumors that occupied nearly the entire brain hemisphere and necessitated sacrifice of the animals (Fig. 1C)Citation . Histological examination of the tumors showed that, as described previously, intracranial tumors formed by Ras astrocytes were highly mitotic but lacked areas of necrosis and were phenotypically consistent with human grade III AA (Fig. 1D)Citation . In contrast, intracranial tumors formed by Ras+Akt astrocytes, in addition to being highly cellular and highly mitotic, were also pleomorphic and had numerous areas of necrosis surrounded by neovascularization (Fig. 1D)Citation . Necrosis and the multiforme phenotype are defining characteristics of GBM (1) , suggesting that although expression of E6/E7, hTERT, and Ras allowed formation of tumors consistent with AA, additional expression of Akt allowed for the formation of s.c. and intracranial tumors with histological characteristics of human GBM.



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  		Fig. 1. Subcutaneous and intracranial tumors derived from NHAs expressing E6/E7, hTERT, and Ras (left) or E6/E7, hTERT, Ras, and Akt (right). A, tumors formed 42 days after s.c. injection. B, H&E staining of s.c. tumors shown in A (magnification, x200). Arrowheads mark a region of necrosis. C, cross-sectional gross images of rat brains 24 days after implantation of Ras or Ras+Akt astrocytes. Arrows mark tumor borders. D, H&E staining of intracranial tumors shown in C (magnification, x200). Arrowheads and arrows mark necrosis and vessels, respectively.

 
Akt Function in the Formation of GBM.
In culture, the growth rate of Ras+Akt astrocytes was no different from that of Ras astrocytes (Fig. 2A)Citation . Similarly, in the first 24 days after s.c. implantation, Ras and Ras+Akt tumors grew at comparable rates (Fig. 2B)Citation . Ras+Akt astrocytes, however, ultimately formed tumors four to six times larger than those formed by Ras astrocytes, with the bulk of this growth difference occurring after the tumors reached a size of ~100 mm3. Because the growth of the Ras tumors lagged behind that of the Ras+Akt tumors in vivo and because the Ras tumors appeared to be less vascular than Ras+Akt tumors, we considered the possibility that the growth of the non-Akt tumors might be limited by hypoxia. To determine the oxygenation of the Ras and Ras+Akt tumors in vivo, we performed immunohistochemical analysis based on the hypoxia marker pimonidazole. Pimonidazole is a 2-nitroimidazole compound that forms protein adducts in mammalian cells at oxygen partial pressures <10 mm Hg (16) . Adduct formation, and therefore extent of hypoxia, can be detected by immunohistochemical analysis using pimonidazole-specific antibodies. Surprisingly, the results of this analysis showed that although Ras+Akt tumors appeared to be more vascular than Ras tumors, both tumors contained numerous diffuse regions of pimonidazole adducts and hypoxia (Fig. 2C)Citation . The differential ability of Ras+Akt astrocytes to proliferate was therefore apparent only under certain in vivo conditions associated with hypoxia.



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  		Fig. 2. Growth of Ras and Ras+Akt astrocytes in vitro and in vivo, and oxygenation of resultant tumors. A, in vitro growth history of NHAs expressing E6/E7, hTERT, and Ras ({blacksquare}) or Ras+Akt ({square}). B, in vivo growth history of E6/E7/hTERT-expressing astrocytes additionally expressing Ras ({blacksquare}) or Ras+Akt ({square}). Results are the means ± SD (bars) for five animals. C, immunostaining for pimonidazole binding of tumor sections derived from Ras (top) or Ras+Akt (middle) astrocytes. Tumor sections derived from Ras+Akt astrocytes without pimonidazole injection were used as negative control (bottom; magnification, x400). Sections are representative of five fields.

 
Because Akt is known to phosphorylate and suppress the activity of the proapoptotic protein Bad, we also addressed the possibility that Akt contributed to GBM formation by effects on apoptosis (7) . Although levels of Bad were similar in parental, Ras, and Ras+Akt astrocytes in culture (Fig. 3ACitation , Lanes 1–4), and in Ras and Ras+Akt tumors derived from these astrocytes (Fig. 3ACitation , Lanes 5 and 6 and 7 and 8, respectively), phosphorylated, inactivated Bad was detected only in Ras+Akt astrocytes and tumors (Fig. 3ACitation , Lanes 4 and 7 and 8, respectively). Similarly, analysis of in vivo apoptosis by in situ end labeling of DNA fragmentation showed that 4.5% of cells in sections from Ras tumors were apoptotic, whereas in sections from Ras+Akt tumors <1% of the cells were apoptotic, even in regions surrounding areas of necrosis (Fig. 3B)Citation . Akt expression therefore appears to exert a small, but perhaps biologically relevant effect on apoptotic pathways in transformed astrocytes.



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  		Fig. 3. Effects of Akt expression on apoptosis and apoptotic pathways in genetically modified astrocytes. Akt function in the formation of GBM. A, representative Western blot analysis of Bad, phosphorylated Bad, and actin in NHAs (Lane 1), nontransformed E6/E7/hTERT astrocytes (Lane 2), Ras astrocytes (Lane 3), Ras+Akt astrocytes (Lane 4), tumors induced by Ras cells (Lanes 5 and 6), and tumors induced by Ras+Akt cells (Lanes 7 and 8). B, in situ end labeling of DNA fragmentation of tumor sections derived from NHAs expressing Ras (left) or Ras+Akt (right) astrocytes. (magnification, x400). Both panels are representative of three analyses.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
In our previous studies, we showed that a constitutively activated form of Akt could not substitute for H-Ras in astrocytic transformation (13) . In the present study, however, we showed that Akt expression was sufficient to convert cells that formed malignant AA into cells that formed tumors that grew faster, exhibited areas of necrosis and vascularity, and closely resembled human GBM. These studies support the idea that the Ras and Akt pathways work cooperatively to drive GBM formation and at the same time clearly delineate the role of the Ras pathway in transformation and the Akt pathway in progression.

Although Akt activation has obvious effects in the system used, the basis for these effects remains incompletely defined. Ras+Akt astrocytes grew at a rate similar to that of Ras astrocytes in culture and in vivo, at least to a tumor size of ~100 mm3. Therefore, although Akt-expressing astrocytes do not inherently grow more readily than Ras astrocytes in vitro or in vivo, they do grow and/or survive more efficiently under certain conditions. The present studies suggest that hypoxia may be a key component of such conditions, but in a manner somewhat different from might be expected. Although we suspected that the slow-growing Ras tumors might be more hypoxic than the rapidly growing, vascularized Ras+Akt tumors, the Ras+Akt tumors were at least as hypoxic, if not more so. These findings are consistent with the persistent hypoxia noted in both AA and GBM in situ (17) . One possible explanation for the formation of GBM-like tumors by Ras+Akt astrocytes is that the growth of Ras astrocytes in vivo may have been limited by blood supply and oxygenation, such that once the Ras tumors outstripped their limited blood supply, cells proliferated slowly or underwent apoptosis. In contrast, in Ras+Akt tumors, Akt activation may have bypassed hypoxia-induced limits placed on cell cycle proliferation and/or suppressed Bad-induced apoptosis, resulting in tumor growth and, in some cases, death by necrosis. The present model therefore suggests that gliomas lacking activation of the Akt pathway may be limited in their ability to proliferate by their microenvironment, whereas gliomas that either have inactivated PTEN (40% of GBM) or have activated the Akt pathway (80% of GBM) grow and take on the characteristics of GBM (5 , 6) . It should be noted, however, that although the proposed model is consistent with the behavior of the tumors studied, the linkages between hypoxia, Akt pathway activation, cell cycle progression, apoptosis, and necrosis remain poorly defined. Furthermore, although tumors formed by Akt astrocytes resembled GBM, they lacked some GBM characteristics, including extensive endothelial cell proliferation and the ability to grow in an invasive fashion. The genetically modified astrocytes made and characterized in the present study should, however, provide the framework for a more complete understanding of the formation of AA and GBM.


   ACKNOWLEDGMENTS
 
We thank the University of California-San Francisco BTRC Tissue Bank for sectioning the tumors and the University of California-San Francisco Histopathology Laboratory for GFAP staining.


   FOOTNOTES
 
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.

1 Supported by NIH Grant CA78546 and funds from the Pediatric Brain Tumor Foundation of America and The Farber Foundation. Back

2 To whom requests for reprints should addressed, at Department of Neurological Surgery, University of California San Francisco, 2340 Sutter Street, Room N-261, Box 0875, San Francisco, CA 94115. Phone: (415) 502-7132; Fax: (415) 502-6779; E-mail: rpieper{at}cc.ucsf.edu Back

3 The abbreviations used are: AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; NHA, normal human astrocyte; hTERT, human telomerase reverse transcriptase; TdT, terminal deoxynucleotidyltransferase; GFAP, glial fibrillary acidic protein. Back

Received 5/29/01. Accepted 7/27/01.


   REFERENCES
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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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M. Kanamori, S. R. V. Berg, G. Bergers, M. S. Berger, and R. O. Pieper
Integrin {beta}3 Overexpression Suppresses Tumor Growth in a Human Model of Gliomagenesis: Implications for the Role of {beta}3 Overexpression in Glioblastoma Multiforme
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Hypermethylation and Transcriptional Downregulation of the Carboxyl-Terminal Modulator Protein Gene in Glioblastomas
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D. Xie, D. Yin, X. Tong, J. O'Kelly, A. Mori, C. Miller, K. Black, D. Gui, J. W. Said, and H. P. Koeffler
Cyr61 Is Overexpressed in Gliomas and Involved in Integrin-Linked Kinase-Mediated Akt and {beta}-Catenin-TCF/Lef Signaling Pathways
Cancer Res., March 15, 2004; 64(6): 1987 - 1996.
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G. S. Kapoor, Y. Zhan, G. R. Johnson, and D. M. O'Rourke
Distinct Domains in the SHP-2 Phosphatase Differentially Regulate Epidermal Growth Factor Receptor/NF-{kappa}B Activation through Gab1 in Glioblastoma Cells
Mol. Cell. Biol., January 15, 2004; 24(2): 823 - 836.
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Y. Sonoda, M. Kanamori, D. F. Deen, S.-Y. Cheng, M. S. Berger, and R. O. Pieper
Overexpression of Vascular Endothelial Growth Factor Isoforms Drives Oxygenation and Growth but not Progression to Glioblastoma Multiforme in a Human Model of Gliomagenesis
Cancer Res., April 15, 2003; 63(8): 1962 - 1968.
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S. Barbero, R. Bonavia, A. Bajetto, C. Porcile, P. Pirani, J. L. Ravetti, G. L. Zona, R. Spaziante, T. Florio, and G. Schettini
Stromal Cell-derived Factor 1{alpha} Stimulates Human Glioblastoma Cell Growth through the Activation of Both Extracellular Signal-regulated Kinases 1/2 and Akt
Cancer Res., April 15, 2003; 63(8): 1969 - 1974.
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P. Dhawan, A. B. Singh, D. L. Ellis, and A. Richmond
Constitutive Activation of Akt/Protein Kinase B in Melanoma Leads to Up-Regulation of Nuclear Factor-{kappa}B and Tumor Progression
Cancer Res., December 15, 2002; 62(24): 7335 - 7342.
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Y. Narita, M. Nagane, K. Mishima, H-J. S. Huang, F. B. Furnari, and W. K. Cavenee
Mutant Epidermal Growth Factor Receptor Signaling Down-Regulates p27 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway in Glioblastomas
Cancer Res., November 15, 2002; 62(22): 6764 - 6769.
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 Neuro Oncol DukeHome page
F. F. Lang, M. R. Gilbert, V. K. Puduvalli, J. Weinberg, V. A. Levin, W.K. A. Yung, R. Sawaya, G. N. Fuller, and C. A. Conrad
Toward better early-phase brain tumor clinical trials: A reappraisal of current methods and proposals for future strategies
Neuro-oncol, October 1, 2002; 4(4): 268 - 277.
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C. B. Knobbe, A. Merlo, and G. Reifenberger
Pten signaling in gliomas
Neuro-oncol, July 1, 2002; 4(3): 196 - 211.
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