This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Choe, G. Articles by Mischel, P. S. Search for Related Content PubMed PubMed Citation Articles by Choe, G. Articles by Mischel, P. S.

Analysis of the Phosphatidylinositol 3'-Kinase Signaling Pathway in Glioblastoma Patients in Vivo¹

Departments of Pathology and Laboratory Medicine [G. C., D. S., A. P., L. I., P. S. M.], Human Genetics and Biostatistics [S. H.] Henry E. Singleton Brain Cancer Research Program [T. F. C., P. S. M.], and The Howard Hughes Medical Institute [C. L. S.], David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California 90095; Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland FIN-00014 [A. P.]; and Cell Signaling Technologies, Beverly, Massachusetts 01915 [K. C., B. S.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Deregulated signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is common in many types of cancer, including glioblastoma. Dissecting the molecular events associated with activation of this pathway in glioblastoma patients in vivo presents an important challenge that has implications for the development and clinical testing of PI3K pathway inhibitors. Using an immunohistochemical analysis applied to a tissue microarray, we performed hierarchical clustering and multidimensional scaling, as well as univariate and multivariate analyses, to dissect the PI3K pathway in vivo. We demonstrate that loss of the tumor suppressor protein PTEN, which antagonizes PI3K pathway activation, is highly correlated with activation of the main PI3K effector Akt in vivo. We also show that Akt activation is significantly correlated with phosphorylation of mammalian target of rapamycin (mTOR), the family of forkhead transcription factors (FOXO1, FOXO3a, and FOXO4), and S6, which are thought to promote its effects. Expression of the mutant epidermal growth factor receptor vIII is also tightly correlated with phosphorylation of these effectors, demonstrating an additional route to PI3K pathway activation in glioblastomas in vivo. These results provide the first dissection of the PI3K pathway in glioblastoma in vivo and suggest an approach to stratifying patients for targeted kinase inhibitor therapy.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
PI3K⁴ is a lipid kinase that promotes diverse biological functions including cellular proliferation, survival, and motility. Considerable evidence demonstrates a role for PI3K signaling in oncogenic transformation and cancer progression (1) . Deregulated PI3K signaling also provides an attractive target for therapy (2, 3, 4) . The principle that kinase inhibitors can be effective treatments for some types of cancer has been demonstrated (2 , 5) , and PI3K pathway inhibitors are already in early clinical trials (4) . Currently, our understanding of the signaling events that regulate and are regulated by PI3K signaling derives primarily from in vitro models (1 , 6) . Mouse genetic studies further demonstrate a key role for this pathway in cancer development and progression (1) . Dissecting the molecular events associated with PI3K deregulation in cancer patients in vivo represents a critical extension of this work and has important implications for the design of "smart" clinical trials with PI3K inhibitors. Glioblastoma, the most common primary brain tumor of adults, is highly suited for this approach (7) . Glioblastomas commonly contain mutations and deletions of the PTENtumor suppressor gene, which negatively regulates PI3K signaling (8 , 9) . Up to now, assessment of multiple nodes in this pathway in routinely processed patient biopsy samples has not been possible. However, development of phosphorylation-specific antibodies that allow for detection of activated signaling molecules in paraffin-embedded biopsy tissues has enabled us to analyze the PI3K pathway in glioblastoma biopsies.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Tissue Microarray/Immunohistochemistry
We constructed a tissue array consisting of three representative 0.6-mm cores from formalin-fixed, paraffin-embedded tissue blocks from each of 45 glioblastomas. Sections were stained with monoclonal antibodies to PTEN (clone 6H2.1; Cascade Bioscience, Winchester, MA), EGFR (clone 31G7; Zymed, San Francisco, CA), and EGFRvIII (clone L8A4; a generous gift from Dr. Darrell Bigner) and phosphorylation-specific antibodies p-Akt (Ser-473) and p-FKHR (Thr-24)/p-FKHRL1 (Thr-32) that recognize phosphorylated forms of the forkhead family of transcription factors FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4), p-mTOR (Ser-2481), p-S6 ribosomal protein (Ser-235/236), and p-44/42 mitogen-activated protein kinase (p-Erk; Thr-202/Tyr-204; Cell Signaling Technology, Beverly, MA). We performed antigen retrieval with a 0.01 M citrate buffer (pH 6) for 30 min in a pressure cooker (phosphorylation-specific antibodies), 16 min in a microwave oven (PTEN), and 25 min in a vegetable steamer (EGFRvIII). For EGFR, we used Pronase [0.03 g/ml in 0.05 M Tris buffer (pH 7.4)] at 37°C for 8 min. Peroxidase activity was quenched with 3% hydrogen peroxide in water. Primary antibodies [PTEN at 1:400, EGFR at 1:150, EGFRvIII at 1:400, p-Akt at 1:50, p-mTOR at 1:50; p-FKHR/FKHRL1/AFX (FOXO1/FOXO3a/FOXO4) at 1:50, pS6 at 1:50, and p-Erk at 1:50)] were diluted in Tris-buffered saline with 0.1% Tween and applied for 16 h at 4°C, followed by biotinylated secondary antibodies (Vector) at 1:1000 dilution for 1 h and avidin-biotin complex (Elite ABC; Vector) for 1 h. Negative control slides received normal mouse serum (DAKO) as the primary antibody. Diaminobenzidine tetrahydrochloride was used as the enzyme substrate to visualize specific antibody localization for PTEN, EGFR, and EGFRvIII; Vector NovaRed (Vector) was used for phosphorylation-specific antibodies. Slides were counterstained with Harris hematoxylin.

Scoring and Interpretation of Immunohistochemistry
PTEN.
PTEN staining was scored according to a previously established scale of 0–2, which has been shown to be highly consistent (10, 11, 12, 13) . Tumor cells are graded as 2 if their staining intensity is equal to that of the vascular endothelium, graded as 1 if their staining intensity is diminished relative to the endothelium, and graded as 0 if staining intensity is undetectable in the tumor cells and present in the vascular endothelium (10, 11, 12, 13) . Two neuropathologists (G. C. and P. S. M.) scored the tumors independently. In addition, tumors were scored by one of the neuropathologists (P. S. M.) on two independent occasions. Both the inter-rater and the intra-rater agreement were >90%.

EGFR and EGFRvIII.
Tumors demonstrating strong EGFR immunopositivity in >20% of tumor cells were considered to be positive; tumors demonstrating at least focal moderate to strong immunoreactivity for EGFRvIII were considered positive. The inter-rater and intra-rater agreements for EGFR and EGFRvIII were >90%. The anti-EGFRvIII L8A4 monoclonal antibody was generated against the fusion junction of the mutant EGFRvIII and shown to be specific for EGFRvIII, with no detectable cross-reactivity for wild-type EGFRs expressed in normal tissues or at high levels in cancer cell lines (14) . However, we cannot entirely exclude the possibility that there may be some cross-reactivity with highly overexpressed wild-type EGFRs in glioblastoma tissue samples.

Phosphorylation-specific Antibodies.
p-Akt, p-mTOR, p-S6, and p-FKHR were scored on a scale of 0–2 (0⁺, no staining; 1⁺, mild intensity cytoplasmic staining; and 2⁺, strong cytoplasmic staining). For p-Akt, p-mTOR, and p-FKHR, staining of 1⁺ and 2⁺ was considered positive; for p-S6, staining of 2⁺ was considered positive. The agreement between reviewers, as well as for the same reviewer on independent reviews, was 80% for p-Akt. It was higher for p-mTOR, p-S6, and p-FKHR, ranging from 87% for p-mTOR to 100% for p-S6. For p-Erk, tumors that focally contained greater than 5% positive nuclear staining were considered positive, as reported previously. The agreement between reviewers and for the same reviewer on independent reviews was >85%.

Statistical Analysis
We fit two multivariate logistic regression models (Table 2) . First, we regressed PTEN status on p-Akt, p-mTOR, p-FKHR, p-S6, and p-Erk. In the multivariate model, only p-Akt turned out to be a significant (P = 0.0043) predictor of PTEN status; the odds of a case being PTEN positive when p-Akt is positive are only 0.13 (95% CI, 0.030–0.52) that of a p-Akt-negative case. Second, we regressed p-Akt status on p-FKHR family, p-S6, p-Erk, and EGFRvIII. We did not include mTOR in the model because it was highly correlated (Pearson correlation = 0.56; P = 0.00019) with p-FKHR and would have led to adverse multi-colinearity effects. In the multivariate model, only p-FKHR family turned out to be a significant predictor of p-Akt status (P = 0.015). The odds of a case being p-Akt positive when FKHR transcription factors are phosphorylated are 3.8 times (95% CI, 1.3–11.0) that of a case with p-FKHR negative and Akt (positive versus negative).

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Demonstration of the inter-relationships between signaling molecules in vivo. A, MDS analysis shows that PTEN protein and Akt phosphorylation are tightly linked and that FKHR family transcription factors, mTOR, and S6 phosphorylation are linked to Akt and associated with each other. The location of S6 between Erk and mTOR suggests dual activation. B, conventional PI3K signaling pathway diagram derived from in vitro studies. C, hierarchical clustering of glioblastoma patients based on pathway signatures. Two main subsets (branches) emerge from this analysis based on PTEN expression (P < 0.0000001). There are also two subsets of PTEN-expressing glioblastomas; one is enriched for EGFR expression (P = 0.00001). The PTEN-expressing, non-EGFR-expressing tumors generally lack PI3K and Erk signaling. EGFRvIII is coexpressed with EGFR in nearly all of the PTEN-expressing tumors but in only 26% of tumors lacking PTEN (P = 0.003).

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

View larger version (81K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Immunohistochemical analysis of the PI3K pathway. A, PTEN protein is lost in a subset of glioblastomas, as seen by retention of PTEN expression in the vascular endothelium with loss of PTEN in tumor cells. The PTEN-deficient tumors demonstrated phosphorylation of Akt, mTOR, FKHR, and S6. B, EGFR, EGFRvIII, and p-Erk were also detected by immunohistochemistry.

Because our data suggested that there are additional inputs into activation of mTOR, the family of FKHR transcription factors and S6, independent of PTEN loss, we analyzed the expression of EGFR and its constitutively active mutant, EGFRvIII, which are thought to activate the PI3K pathway (18) . EGFR overexpression/amplification is common in glioblastomas, and a mutant ligand independent variant (EGFRvIII) is coexpressed in approximately one-half of EGFR-amplified glioblastomas (19 , 20) . In our data set, EGFRvIII expression was correlated with EGFR expression (r = 0.31; P = 0.04), being seen in slightly more than one-half of the EGFR-positive tumors. EGFR expression was correlated with Erk (r = 0.34; P = 0.03) and S6 activation (r = 0.31; P = 0.06), but not with p-Akt, p-mTOR, or p-FKHR. In contrast, EGFRvIII had a strong association with phosphorylation of FKHR transcription factors, mTOR and S6. These results potentially suggest a role for EGFRvIII in activation of the PI3K pathway in the absence of PTEN protein loss and suggest an association between EGFR expression and Erk activation in vivo.

The results from the univariate and multivariate analyses are consistent with the presumed relationships between PTEN and other known components of the PI3K signaling pathway. Next, we asked if our data set might be used to uncover these relationships without prior knowledge of the connectivity between these proteins using MDS analysis, a form of principle component analysis. MDS is an unsupervised data analysis method that allows examination of potential relationships between variables without assuming previous knowledge of their interactions. Using this approach, the signaling molecules are plotted in two dimensions, and the distance between two molecules provides a measure of their inter-relatedness (21) . PTEN protein expression and Akt activation are closely related (Fig. 2B) , as detected by the short distance between them. In contrast, p-Erk is distant from PTEN and p-Akt. Activated Akt is positioned between PTEN and p-FKHR, p-mTOR, and p-S6, suggesting that Akt is intermediate between PTEN and this pathway. Furthermore, the proximity of p-S6 to p-mTOR and p-Erk suggests potential dual activation. This unbiased approach demonstrates a pattern of inter-relationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experimental studies and raises the possibility that this form of analysis might be applied to other data sets where the connectivity between the variables is less defined (Fig. 2C) .

Lastly, we asked whether these markers could provide meaningful "signatures" that can be used to stratify patients for targeted therapy. We performed hierarchical clustering of the tumors based on expression of EGFR, EGFRvIII, p-Erk, PTEN, p-AKT, p-mTOR, p-FKHR, and p-S6 to look for the emergence of molecular subsets. Two main subsets of glioblastomas were identified (main branches of the dendrogram) based on expression of PTEN protein (P < 0.0000001). We also identified two additional subsets of PTEN-expressing glioblastomas, based on expression of EGFR (P = 0.00001). One subset (branch) of the PTEN-expressing, non-EGFR-expressing tumors lacked either Erk or the PI3K pathway activation. In contrast, the EGFR-expressing subset coexpressed EGFRvIII in 14 of 16 (88%) cases, whereas, EGFRvIII was coexpressed in only 5 of 19 (26%) of the PTEN-deficient EGFR-expressing tumors (P = 0.003). This suggests that EGFRvIII may be selected for in the absence of PTEN loss (as a way to activate PI3K) and is line with a mouse genetic model demonstrating a key role for combined deregulation of the Ras and PI3K pathways in the development of glioblastomas (22) . This approach may provide a potential design for stratifying patients for therapy with PI3K pathway inhibitors, alone or in combination with Ras/Erk and EGFR inhibitors.

In conclusion, we have demonstrated an approach to analyzing the PI3K pathway in glioblastoma patients in vivo. We have shown that this pathway can be assessed in routinely processed patient samples, and we have demonstrated that the signaling relationships suggested in vitro are reflected in glioblastoma patients in vivo. We have shown that loss of the tumor suppressor protein PTEN is highly correlated with activation of Akt, which is significantly correlated with phosphorylation of downstream effectors mTOR, the FKHR family of transcription factors, and S6. We have also presented data suggesting that the mutant EGFR, EGFRvIII, may also potentially activate this pathway in glioblastomas with no loss of PTEN protein. We have used MDS to analyze the relationship between signaling molecules in vivo, suggesting that MDS might have utility in the analysis of data sets where pathway connectivity is less defined. Finally, we have provided an approach to identify subsets of glioblastoma patients based on these "pathway signatures" that may potentially be useful for stratifying patients for targeted kinase inhibitor therapy. This approach can be applied to routinely processed patient biopsies; therefore, it may potentially have wide clinical utility. As we move from tissue arrays to patient biopsy slides, we will need to begin to assess how intratumor molecular heterogeneity impacts pathway activation, and we will need to develop controls and standards that can be applied across diagnostic laboratories. Because the key challenge for cancer therapy will be to match the right pathway inhibitor or combination of inhibitors to the right patient, this approach may have important clinical implications.

	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 This work was supported by NIH Grants U01 CA88127 and K08 NS43147 (to P. S. M.), Accelerate Brain Cancer Cure Awards (to P. S. M. and C. L. S.), and the Doris Duke Duke Charitable Foundation. Dr. Sawyers is an Investigator of the Howard Hughes Medical Institute. This work was also supported by a generous donation from the Kevin Riley family to UCLA Comprehensive Brain Tumor Program, the Harry Allgauer Foundation through The Doris R. Ullmann Fund for Brain Tumor Research Technologies, a Henry E. Singleton Brain Tumor Fellowship and a STOP Cancer Award (to P. S. M.), and a generous donation from the Ziering Family Foundation in memory of Sigi Ziering. C. L. S. is an investigator of the Howard Hughes Medical Institute and a Doris Duke Distinguished Clinical Investigator. G. C. was supported by a postdoctoral fellowship from Korea Science & Engineering Foundation.

² Both authors contributed equally to this work.

³ To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1732. Phone: (310) 794-5223; Fax: (310) 206-0657; E-mail: pmischel{at}mednet.ucla.edu

⁴ The abbreviations used are: PI3K, phosphatidylinositol 3'-kinase; EGFR, epidermal growth factor receptor; p-, phosphorylated; Erk, extracellular signal-regulated kinase; CI, confidence interval; MDS, multidimensional scaling.

⁵ http://cran.r-project.org/.

⁶ Stanford University website.

Received 1/10/03. Accepted 4/14/03.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Vivanco I., Sawyers C. L. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat. Rev. Cancer, 2: 489-501, 2002.[Medline]
2. Sawyers C. L. Rational therapeutic intervention in cancer: kinases as drug targets. Curr. Opin. Genet. Dev., 12: 111-115, 2002.[Medline]
3. Neshat M. S., Mellinghoff I. K., Tran C., Stiles B., Thomas G., Petersen R., Frost P., Gibbons J. J., Wu H., Sawyers C. L. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA, 98: 10314-10319, 2001.[Abstract/Free Full Text]
4. Hidalgo M., Rowinsky E. K. The rapamycin-sensitive signal transduction cascade as a target for cancer therapy. Oncogene, 19: 6680-6686, 2000.[Medline]
5. Druker B. J. Perspectives on the development of a molecularly targeted agent. Cancer Cell, 1: 31-36, 2002.[Medline]
6. Blume-Jensen P., Hunter T. Oncogenic kinase signaling. Nature (Lond.), 411: 355-365, 2001.[Medline]
7. Mischel P. S., Cloughesy T. F. Targeted molecular therapy of glioblastoma. Brain Pathol., 13: 52-61, 2003.[Medline]
8. Ermoian R. P., Furniss C. S., Lamborn K. R., Basila D., Berger M. S., Gottschalk A. R., Nicholas M. K., Stokoe D., Haas-Kogan D. A. Dysregulation of PTEN and protein kinase B is associated with glioma histology and patient survival. Clin. Cancer Res., 8: 1100-1106, 2002.[Abstract/Free Full Text]
9. Davies M. A., Lu Y., Sano T., Fang X., Tang P., LaPushin R., Koul D., Bookstein R., Stokoe D., Yung W. K., Mills G. B., Steck P. A. Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis. Cancer Res., 58: 5285-5290, 1998.[Abstract/Free Full Text]
10. Gimm O., Perren A., Weng L. P., Marsh D. J., Yeh J. J., Ziebold U., Gil E., Hinze R., Delbridge L., Lees J. A., Mutter G. L., Robinson B. G., Komminoth P., Dralle H., Eng C. Differential nuclear and cytoplasmic expression of PTEN in normal thyroid tissue, and benign and malignant epithelial thyroid tumors. Am. J. Pathol., 156: 1693-1700, 2000.[Abstract/Free Full Text]
11. Perren A., Komminoth P., Saremaslani P., Matter C., Feurer S., Lees J. A., Heitz P. U., Eng C. Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells. Am. J. Pathol., 157: 1097-1103, 2000.[Abstract/Free Full Text]
12. Perren A., Weng L. P., Boag A. H., Ziebold U., Thakore K., Dahia P. L., Komminoth P., Lees J. A., Mulligan L. M., Mutter G. L., Eng C. Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast. Am. J. Pathol., 155: 1253-1260, 1999.[Abstract/Free Full Text]
13. Zhou X. P., Gimm O., Hampel H., Niemann T., Walker M. J., Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation. Am. J. Pathol., 157: 1123-1128, 2000.[Abstract/Free Full Text]
14. Wikstrand C. J., Hale L. P., Batra S. K., Hill M. L., Humphrey P. A., Kurpad S. N., McLendon R. E., Moscatello D., Pegram C. N., Reist C. J., et al Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res., 55: 3140-3148, 1995.[Abstract/Free Full Text]
15. Ihaka R., Gentleman R. R: a language for data analysis and graphics. J. Comput. Graphical Statistics, 5: 299-314, 1996.
16. Cox T. F., Cox M. A. A. . Multidimensional Scaling., CRC Press United Kingdom 2001.
17. Burgering B. M., Kops G. J. Cell cycle and death control: long live Forkheads. Trends Biochem. Sci., 27: 352-360, 2002.[Medline]
18. Nagane M., Lin H., Cavenee W. K., Huang H. J. Aberrant receptor signaling in human malignant gliomas: mechanisms and therapeutic implications. Cancer Lett., 162 (Suppl.): S17-S21, 2001.
19. Ekstrand A. J., Sugawa N., James C. D., Collins V. P. Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails. Proc. Natl. Acad. Sci. USA, 89: 4309-4313, 1992.[Abstract/Free Full Text]
20. Liu W., James C. D., Frederick L., Alderete B. E., Jenkins R. B. PTEN/MMAC1 mutations and EGFR amplification in glioblastomas. Cancer Res., 57: 5254-5257, 1997.[Abstract/Free Full Text]
21. Venables W. N., Ripley B. D. . Modern Applied Statistic with S-Plus., Springer New York 1999.
22. Holland E. C., Celestino J., Dai C., Schaefer L., Sawaya R. E., Fuller G. N. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat. Genet., 25: 55-57, 2000.[Medline]

This article has been cited by other articles:

				M. W. Ronellenfitsch, D. P. Brucker, M. C. Burger, S. Wolking, F. Tritschler, J. Rieger, W. Wick, M. Weller, and J. P. Steinbach Antagonism of the mammalian target of rapamycin selectively mediates metabolic effects of epidermal growth factor receptor inhibition and protects human malignant glioma cells from hypoxia-induced cell death Brain, June 1, 2009; 132(6): 1509 - 1522. [Abstract] [Full Text] [PDF]

				J.-S. Guillamo, S. de Bouard, S. Valable, L. Marteau, P. Leuraud, Y. Marie, M.-F. Poupon, J.-J. Parienti, E. Raymond, and M. Peschanski Molecular Mechanisms Underlying Effects of Epidermal Growth Factor Receptor Inhibition on Invasion, Proliferation, and Angiogenesis in Experimental Glioma Clin. Cancer Res., June 1, 2009; 15(11): 3697 - 3704. [Abstract] [Full Text] [PDF]

				W. A. Tyler, N. Gangoli, P. Gokina, H. A. Kim, M. Covey, S. W. Levison, and T. L. Wood Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation J. Neurosci., May 13, 2009; 29(19): 6367 - 6378. [Abstract] [Full Text] [PDF]

				M. J. van den Bent, A. A. Brandes, R. Rampling, M. C.M. Kouwenhoven, J. M. Kros, A. F. Carpentier, P. M. Clement, M. Frenay, M. Campone, J.-F. Baurain, et al. Randomized Phase II Trial of Erlotinib Versus Temozolomide or Carmustine in Recurrent Glioblastoma: EORTC Brain Tumor Group Study 26034 J. Clin. Oncol., March 10, 2009; 27(8): 1268 - 1274. [Abstract] [Full Text] [PDF]

				T. Klatte, D. B. Seligson, J. LaRochelle, B. Shuch, J. W. Said, S. B. Riggs, N. Zomorodian, F. F. Kabbinavar, A. J. Pantuck, and A. S. Belldegrun Molecular Signatures of Localized Clear Cell Renal Cell Carcinoma to Predict Disease-Free Survival after Nephrectomy Cancer Epidemiol. Biomarkers Prev., March 1, 2009; 18(3): 894 - 900. [Abstract] [Full Text] [PDF]

				L. Li, A. Dutra, E. Pak, J. E. Labrie III, R. M. Gerstein, P. P. Pandolfi, L. D. Recht, and A. H. Ross EGFRvIII expression and PTEN loss synergistically induce chromosomal instability and glial tumors Neuro-oncol, January 1, 2009; 11(1): 9 - 21. [Abstract] [Full Text] [PDF]

				J. Marsh, P. Mukherjee, and T. N. Seyfried Akt-Dependent Proapoptotic Effects of Dietary Restriction on Late-Stage Management of a Phosphatase and Tensin Homologue/Tuberous Sclerosis Complex 2-Deficient Mouse Astrocytoma Clin. Cancer Res., December 1, 2008; 14(23): 7751 - 7762. [Abstract] [Full Text] [PDF]

				Q. Xu, X. Yuan, G. Liu, K. L. Black, and J. S. Yu Hedgehog Signaling Regulates Brain Tumor-Initiating Cell Proliferation and Portends Shorter Survival for Patients with PTEN-Coexpressing Glioblastomas Stem Cells, December 1, 2008; 26(12): 3018 - 3026. [Abstract] [Full Text] [PDF]

				R. Williams Paul Mischel: All about brains J. Cell Biol., October 22, 2008; 181(7): 1044 - 1045. [Full Text] [PDF]

				T. Martens, Y. Laabs, H. S. Gunther, D. Kemming, Z. Zhu, L. Witte, C. Hagel, M. Westphal, and K. Lamszus Inhibition of Glioblastoma Growth in a Highly Invasive Nude Mouse Model Can Be Achieved by Targeting Epidermal Growth Factor Receptor but not Vascular Endothelial Growth Factor Receptor-2 Clin. Cancer Res., September 1, 2008; 14(17): 5447 - 5458. [Abstract] [Full Text] [PDF]

				L. Yang, M. J. Clarke, B. L. Carlson, A. C. Mladek, M. A. Schroeder, P. Decker, W. Wu, G. J. Kitange, P. T. Grogan, J. M. Goble, et al. PTEN Loss Does Not Predict for Response to RAD001 (Everolimus) in a Glioblastoma Orthotopic Xenograft Test Panel Clin. Cancer Res., June 15, 2008; 14(12): 3993 - 4001. [Abstract] [Full Text] [PDF]

				H. K. Thorarinsdottir, M. Santi, R. McCarter, E. J. Rushing, R. Cornelison, A. Jales, and T. J. MacDonald Protein Expression of Platelet-Derived Growth Factor Receptor Correlates with Malignant Histology and PTEN with Survival in Childhood Gliomas Clin. Cancer Res., June 1, 2008; 14(11): 3386 - 3394. [Abstract] [Full Text] [PDF]

				E. C. Brantley and E. N. Benveniste Signal Transducer and Activator of Transcription-3: A Molecular Hub for Signaling Pathways in Gliomas Mol. Cancer Res., May 1, 2008; 6(5): 675 - 684. [Abstract] [Full Text] [PDF]

				P. A Dennis Targeting Akt in Cancer: Promise, Progress, and Potential Pitfalls Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 25 - 35. [Abstract] [Full Text] [PDF]

				P. T. Jindra, A. Hsueh, L. Hong, D. Gjertson, X.-D. Shen, F. Gao, J. Dang, P. S. Mischel, W. M. Baldwin III, M. C. Fishbein, et al. Anti-MHC Class I Antibody Activation of Proliferation and Survival Signaling in Murine Cardiac Allografts J. Immunol., February 15, 2008; 180(4): 2214 - 2224. [Abstract] [Full Text] [PDF]

				N. de la Iglesia, G. Konopka, S. V. Puram, J. A. Chan, R. M. Bachoo, M. J. You, D. E. Levy, R. A. DePinho, and A. Bonni Identification of a PTEN-regulated STAT3 brain tumor suppressor pathway Genes & Dev., February 15, 2008; 22(4): 449 - 462. [Abstract] [Full Text] [PDF]

				A. Yaba, V. Bianchi, A. Borini, and J. Johnson A Putative Mitotic Checkpoint Dependent on mTOR Function Controls Cell Proliferation and Survival in Ovarian Granulosa Cells Reproductive Sciences, February 1, 2008; 15(2): 128 - 138. [Abstract] [PDF]

				K. Yoshimoto, J. Dang, S. Zhu, D. Nathanson, T. Huang, R. Dumont, D. B. Seligson, W. H. Yong, Z. Xiong, N. Rao, et al. Development of a Real-time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples Clin. Cancer Res., January 15, 2008; 14(2): 488 - 493. [Abstract] [Full Text] [PDF]

				S. Sathornsumetee, Y. Cao, J. E. Marcello, J. E. Herndon II, R. E. McLendon, A. Desjardins, H. S. Friedman, M. W. Dewhirst, J. J. Vredenburgh, and J. N. Rich Tumor Angiogenic and Hypoxic Profiles Predict Radiographic Response and Survival in Malignant Astrocytoma Patients Treated With Bevacizumab and Irinotecan J. Clin. Oncol., January 10, 2008; 26(2): 271 - 278. [Abstract] [Full Text] [PDF]

				I. S. Sarkaria, M. F. Zakowski, D. Pham, M. Hezel, M. I. Ebright, S. Chuai, E. S. Venkatraman, M. G. Kris, V. W. Rusch, and B. Singh Epidermal Growth Factor Receptor Signaling in Adenocarcinomas With Bronchioloalveolar Components Ann. Thorac. Surg., January 1, 2008; 85(1): 216 - 223. [Abstract] [Full Text] [PDF]

				L. Zhang, J. Yu, H. Pan, P. Hu, Y. Hao, W. Cai, H. Zhu, A. D. Yu, X. Xie, D. Ma, et al. Small molecule regulators of autophagy identified by an image-based high-throughput screen PNAS, November 27, 2007; 104(48): 19023 - 19028. [Abstract] [Full Text] [PDF]

				F. B. Furnari, T. Fenton, R. M. Bachoo, A. Mukasa, J. M. Stommel, A. Stegh, W. C. Hahn, K. L. Ligon, D. N. Louis, C. Brennan, et al. Malignant astrocytic glioma: genetics, biology, and paths to treatment Genes & Dev., November 1, 2007; 21(21): 2683 - 2710. [Abstract] [Full Text] [PDF]

				S. Ishiuchi, Y. Yoshida, K. Sugawara, M. Aihara, T. Ohtani, T. Watanabe, N. Saito, K. Tsuzuki, H. Okado, A. Miwa, et al. Ca2+-Permeable AMPA Receptors Regulate Growth of Human Glioblastoma via Akt Activation J. Neurosci., July 25, 2007; 27(30): 7987 - 8001. [Abstract] [Full Text] [PDF]

				G. D. Kao, Z. Jiang, A. M. Fernandes, A. K. Gupta, and A. Maity Inhibition of Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing Radiation J. Biol. Chem., July 20, 2007; 282(29): 21206 - 21212. [Abstract] [Full Text] [PDF]

				C. E. Pelloski, K. V. Ballman, A. F. Furth, L. Zhang, E. Lin, E. P. Sulman, K. Bhat, J. M. McDonald, W.K. A. Yung, H. Colman, et al. Epidermal Growth Factor Receptor Variant III Status Defines Clinically Distinct Subtypes of Glioblastoma J. Clin. Oncol., June 1, 2007; 25(16): 2288 - 2294. [Abstract] [Full Text] [PDF]

				Z. Jiang, N. Pore, G. J. Cerniglia, R. Mick, M.-M. Georgescu, E. J. Bernhard, S. M. Hahn, A. K. Gupta, and A. Maity Phosphatase and Tensin Homologue Deficiency in Glioblastoma Confers Resistance to Radiation and Temozolomide that Is Reversed by the Protease Inhibitor Nelfinavir Cancer Res., May 1, 2007; 67(9): 4467 - 4473. [Abstract] [Full Text] [PDF]

				H. Zhou, R. Miki, M. Eeva, F. M. Fike, D. Seligson, L. Yang, A. Yoshimura, M. A. Teitell, C. A.M. Jamieson, and N. A. Cacalano Reciprocal Regulation of SOCS 1 and SOCS3 Enhances Resistance to Ionizing Radiation in Glioblastoma Multiforme Clin. Cancer Res., April 15, 2007; 13(8): 2344 - 2353. [Abstract] [Full Text] [PDF]

				D. Faury, A. Nantel, S. E. Dunn, M.-C. Guiot, T. Haque, P. Hauser, M. Garami, L. Bognar, Z. Hanzely, P. P. Liberski, et al. Molecular Profiling Identifies Prognostic Subgroups of Pediatric Glioblastoma and Shows Increased YB-1 Expression in Tumors J. Clin. Oncol., April 1, 2007; 25(10): 1196 - 1208. [Abstract] [Full Text] [PDF]

				M. Failly, S. Korur, V. Egler, J.-L. Boulay, M. M. Lino, R. Imber, and A. Merlo Combination of sublethal concentrations of epidermal growth factor receptor inhibitor and microtubule stabilizer induces apoptosis of glioblastoma cells Mol. Cancer Ther., February 1, 2007; 6(2): 773 - 781. [Abstract] [Full Text] [PDF]

				D. Opel, C. Poremba, T. Simon, K.-M. Debatin, and S. Fulda Activation of Akt Predicts Poor Outcome in Neuroblastoma Cancer Res., January 15, 2007; 67(2): 735 - 745. [Abstract] [Full Text] [PDF]

				I. K. Mellinghoff, T. F. Cloughesy, and P. S. Mischel PTEN-Mediated Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors Clin. Cancer Res., January 15, 2007; 13(2): 378 - 381. [Abstract] [Full Text] [PDF]

				J. S. Lam, A. J. Pantuck, A. S. Belldegrun, and R. A. Figlin Protein Expression Profiles in Renal Cell Carcinoma: Staging, Prognosis, and Patient Selection for Clinical Trials Clin. Cancer Res., January 15, 2007; 13(2): 703s - 708s. [Abstract] [Full Text] [PDF]

				S. Horvath, B. Zhang, M. Carlson, K. V. Lu, S. Zhu, R. M. Felciano, M. F. Laurance, W. Zhao, S. Qi, Z. Chen, et al. Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target PNAS, November 14, 2006; 103(46): 17402 - 17407. [Abstract] [Full Text] [PDF]

				D. C. Birle and D. W. Hedley Signaling interactions of rapamycin combined with erlotinib in cervical carcinoma xenografts. Mol. Cancer Ther., October 1, 2006; 5(10): 2494 - 2502. [Abstract] [Full Text] [PDF]

				M. Y. Wang, K. V. Lu, S. Zhu, E. Q. Dia, I. Vivanco, G. M. Shackleford, W. K. Cavenee, I. K. Mellinghoff, T. F. Cloughesy, C. L. Sawyers, et al. Mammalian Target of Rapamycin Inhibition Promotes Response to Epidermal Growth Factor Receptor Kinase Inhibitors in PTEN-Deficient and PTEN-Intact Glioblastoma Cells Cancer Res., August 15, 2006; 66(16): 7864 - 7869. [Abstract] [Full Text] [PDF]

				T. F. Cloughesy, P. Y. Wen, H. I. Robins, S. M. Chang, M. D. Groves, K. L. Fink, L. Junck, D. Schiff, L. Abrey, M. R. Gilbert, et al. Phase II Trial of Tipifarnib in Patients With Recurrent Malignant Glioma Either Receiving or Not Receiving Enzyme-Inducing Antiepileptic Drugs: A North American Brain Tumor Consortium Study J. Clin. Oncol., August 1, 2006; 24(22): 3651 - 3656. [Abstract] [Full Text] [PDF]

				C. E. Pelloski, E Lin, L. Zhang, W.K. A. Yung, H. Colman, J.-L. Liu, S. Y. Woo, A. B. Heimberger, D. Suki, M. Prados, et al. Prognostic Associations of Activated Mitogen-Activated Protein Kinase and Akt Pathways in Glioblastoma. Clin. Cancer Res., July 1, 2006; 12(13): 3935 - 3941. [Abstract] [Full Text] [PDF]

				M. J. Riemenschneider, R. A. Betensky, S. M. Pasedag, and D. N. Louis AKT Activation in Human Glioblastomas Enhances Proliferation via TSC2 and S6 Kinase Signaling Cancer Res., June 1, 2006; 66(11): 5618 - 5623. [Abstract] [Full Text] [PDF]

				L. de la Pena, W. E. Burgan, D. J. Carter, M. G. Hollingshead, M. Satyamitra, K. Camphausen, and P. J. Tofilon Inhibition of Akt by the alkylphospholipid perifosine does not enhance the radiosensitivity of human glioma cells. Mol. Cancer Ther., June 1, 2006; 5(6): 1504 - 1510. [Abstract] [Full Text] [PDF]

				G. Powis, N. Ihle, and D. L. Kirkpatrick Practicalities of drugging the phosphatidylinositol-3-kinase/akt cell survival signaling pathway. Clin. Cancer Res., May 15, 2006; 12(10): 2964 - 2966. [Full Text] [PDF]

				D. A. Reardon, J. N. Rich, H. S. Friedman, and D. D. Bigner Recent Advances in the Treatment of Malignant Astrocytoma J. Clin. Oncol., March 10, 2006; 24(8): 1253 - 1265. [Abstract] [Full Text] [PDF]

				D. A. Reardon, J. A. Quinn, J. J. Vredenburgh, S. Gururangan, A. H. Friedman, A. Desjardins, S. Sathornsumetee, J. E. Herndon II, J. M. Dowell, R. E. McLendon, et al. Phase 1 Trial of Gefitinib Plus Sirolimus in Adults with Recurrent Malignant Glioma Clin. Cancer Res., February 1, 2006; 12(3): 860 - 868. [Abstract] [Full Text] [PDF]

				D. B. Ramnarain, S. Park, D. Y. Lee, K. J. Hatanpaa, S. O. Scoggin, H. Otu, T. A. Libermann, J. M. Raisanen, R. Ashfaq, E. T. Wong, et al. Differential Gene Expression Analysis Reveals Generation of an Autocrine Loop by a Mutant Epidermal Growth Factor Receptor in Glioma Cells Cancer Res., January 15, 2006; 66(2): 867 - 874. [Abstract] [Full Text] [PDF]

				C.-L. Tso, W. A. Freije, A. Day, Z. Chen, B. Merriman, A. Perlina, Y. Lee, E. Q. Dia, K. Yoshimoto, P. S. Mischel, et al. Distinct Transcription Profiles of Primary and Secondary Glioblastoma Subgroups Cancer Res., January 1, 2006; 66(1): 159 - 167. [Abstract] [Full Text] [PDF]

				C. M. Lee, C. B. Fuhrman, V. Planelles, M. R. Peltier, D. K. Gaffney, A. P. Soisson, M. K. Dodson, H. D. Tolley, C. L. Green, and K. A. Zempolich Phosphatidylinositol 3-Kinase Inhibition by LY294002 Radiosensitizes Human Cervical Cancer Cell Lines Clin. Cancer Res., January 1, 2006; 12(1): 250 - 256. [Abstract] [Full Text] [PDF]

				K. Oda, D. Stokoe, Y. Taketani, and F. McCormick High Frequency of Coexistent Mutations of PIK3CA and PTEN Genes in Endometrial Carcinoma Cancer Res., December 1, 2005; 65(23): 10669 - 10673. [Abstract] [Full Text] [PDF]

				J. N. Rich, S. Sathornsumetee, S. T. Keir, M. W. Kieran, A. Laforme, A. Kaipainen, R. E. McLendon, M. W. Graner, B.K. A. Rasheed, L. Wang, et al. ZD6474, a Novel Tyrosine Kinase Inhibitor of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor, Inhibits Tumor Growth of Multiple Nervous System Tumors Clin. Cancer Res., November 15, 2005; 11(22): 8145 - 8157. [Abstract] [Full Text] [PDF]

				I. K. Mellinghoff, M. Y. Wang, I. Vivanco, D. A. Haas-Kogan, S. Zhu, E. Q. Dia, K. V. Lu, K. Yoshimoto, J. H.Y. Huang, D. J. Chute, et al. Molecular Determinants of the Response of Glioblastomas to EGFR Kinase Inhibitors. N. Engl. J. Med., November 10, 2005; 353(19): 2012 - 2024. [Abstract] [Full Text] [PDF]

				K. V. Lu, K. A. Jong, G. Y. Kim, J. Singh, E. Q. Dia, K. Yoshimoto, M. Y. Wang, T. F. Cloughesy, S. F. Nelson, and P. S. Mischel Differential Induction of Glioblastoma Migration and Growth by Two Forms of Pleiotrophin J. Biol. Chem., July 22, 2005; 280(29): 26953 - 26964. [Abstract] [Full Text] [PDF]

				G. G. Chiang and R. T. Abraham Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 Is Mediated by p70S6 Kinase J. Biol. Chem., July 8, 2005; 280(27): 25485 - 25490. [Abstract] [Full Text] [PDF]

				T. A. Rege, C. Y. Fears, and C. L. Gladson Endogenous inhibitors of angiogenesis in malignant gliomas: Nature's antiangiogenic therapy Neuro-oncol, April 1, 2005; 7(2): 106 - 121. [Abstract] [PDF]

				F. Lefranc, J. Brotchi, and R. Kiss Possible Future Issues in the Treatment of Glioblastomas: Special Emphasis on Cell Migration and the Resistance of Migrating Glioblastoma Cells to Apoptosis J. Clin. Oncol., April 1, 2005; 23(10): 2411 - 2422. [Abstract] [Full Text] [PDF]

				L. Wu, D. C. Birle, and I. F. Tannock Effects of the Mammalian Target of Rapamycin Inhibitor CCI-779 Used Alone or with Chemotherapy on Human Prostate Cancer Cells and Xenografts Cancer Res., April 1, 2005; 65(7): 2825 - 2831. [Abstract] [Full Text] [PDF]

				S. Vignot, S. Faivre, D. Aguirre, and E. Raymond mTOR-targeted therapy of cancer with rapamycin derivatives Ann. Onc., April 1, 2005; 16(4): 525 - 537. [Abstract] [Full Text] [PDF]

				C.L. SAWYERS Making Progress through Molecular Attacks on Cancer Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 479 - 482. [Abstract] [PDF]

				R. K. Goudar, Q. Shi, M. D. Hjelmeland, S. T. Keir, R. E. McLendon, C. J. Wikstrand, E. D. Reese, C. A. Conrad, P. Traxler, H. A. Lane, et al. Combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition Mol. Cancer Ther., January 1, 2005; 4(1): 101 - 112. [Abstract] [Full Text] [PDF]

				G. V. Thomas, S. Horvath, B. L. Smith, K. Crosby, L. A. Lebel, M. Schrage, J. Said, J. De Kernion, R. E. Reiter, and C. L. Sawyers Antibody-Based Profiling of the Phosphoinositide 3-Kinase Pathway in Clinical Prostate Cancer Clin. Cancer Res., December 15, 2004; 10(24): 8351 - 8356. [Abstract] [Full Text] [PDF]

				Q. Shi, S. Bao, J. A. Maxwell, E. D. Reese, H. S. Friedman, D. D. Bigner, X.-F. Wang, and J. N. Rich Secreted Protein Acidic, Rich in Cysteine (SPARC), Mediates Cellular Survival of Gliomas through AKT Activation J. Biol. Chem., December 10, 2004; 279(50): 52200 - 52209. [Abstract] [Full Text] [PDF]

				D. K. Broderick, C. Di, T. J. Parrett, Y. R. Samuels, J. M. Cummins, R. E. McLendon, D. W. Fults, V. E. Velculescu, D. D. Bigner, and H. Yan Mutations of PIK3CA in Anaplastic Oligodendrogliomas, High-Grade Astrocytomas, and Medulloblastomas Cancer Res., August 1, 2004; 64(15): 5048 - 5050. [Abstract] [Full Text] [PDF]

				A. Chakravarti, G. Zhai, Y. Suzuki, S. Sarkesh, P. M. Black, A. Muzikansky, and J. S. Loeffler The Prognostic Significance of Phosphatidylinositol 3-Kinase Pathway Activation in Human Gliomas J. Clin. Oncol., May 15, 2004; 22(10): 1926 - 1933. [Abstract] [Full Text] [PDF]

				C. B. Knobbe, J. Reifenberger, B. Blaschke, and G. Reifenberger Hypermethylation and Transcriptional Downregulation of the Carboxyl-Terminal Modulator Protein Gene in Glioblastomas J Natl Cancer Inst, March 17, 2004; 96(6): 483 - 486. [Abstract] [Full Text] [PDF]

				C. L. Sawyers Opportunities and challenges in the development of kinase inhibitor therapy for cancer Genes & Dev., December 15, 2003; 17(24): 2998 - 3010. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Choe, G. Articles by Mischel, P. S. Search for Related Content PubMed PubMed Citation Articles by Choe, G. Articles by Mischel, P. S.