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Cancer Research 67, 7960, September 1, 2007. doi: 10.1158/0008-5472.CAN-07-2154
© 2007 American Association for Cancer Research
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Priority Reports

A Dual Phosphoinositide-3-Kinase {alpha}/mTOR Inhibitor Cooperates with Blockade of Epidermal Growth Factor Receptor in PTEN-Mutant Glioma

Qi-Wen Fan1,2,3,4, Christine K. Cheng1,2,3,4, Theodore P. Nicolaides1,2,3,4, Christopher S. Hackett5, Zachary A. Knight6, Kevan M. Shokat6,7,8 and William A. Weiss1,2,3,4

Departments of 1 Neurology, 2 Pediatrics, 3 Neurological Surgery and Brain Tumor Research Center, 4 Comprehensive Cancer Center, 5 Biomedical Sciences Graduate Program, 6 Program in Chemistry and Chemical Biology, 7 Cellular and Molecular Pharmacology, and 8 Howard Hughes Medical Institute, University of California, San Francisco, California

Requests for reprints: Qi-Wen Fan, Neurology, Room U441K, University of California at San Francisco, 533 Parnassus Avenue, San Francisco, CA 94143. Phone: 415-502-1695; Fax: 415-476-0133; E-mail: QiWen.fan{at}ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have shown previously that blockade of epidermal growth factor receptor (EGFR) cooperates with a pan-selective inhibitor of phosphoinositide-3-kinase (PI3K) in EGFR-driven glioma. In this communication, we tested EGFR-driven glioma differing in PTEN status, treating with the EGFR inhibitor erlotinib and a novel dual inhibitor of PI3K{alpha} and mTOR (PI-103). Erlotinib blocked proliferation only in PTENwt cells expressing EGFR. Although erlotinib monotherapy showed little effect in PTENmt glioma, PI-103 greatly augmented the antiproliferative efficacy of erlotinib in this setting. To address the importance of PI3K blockade, we showed in PTENmt glioma that combining PI-103 and erlotinib was superior to either monotherapy or to therapy combining erlotinib with either rapamycin (an inhibitor of mTOR) or PIK-90 (an inhibitor of PI3K{alpha}). These experiments show that a dual inhibitor of PI3K{alpha} and mTOR augments the activity of EGFR blockade, offering a mechanistic rationale for targeting EGFR, PI3K{alpha}, and mTOR in the treatment of EGFR-driven, PTEN-mutant glioma. [Cancer Res 2007;67(17):7960–5]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Both amplification of epidermal growth factor receptor (EGFR) and activation of phosphoinositide-3-kinase (PI3K) feature prominently in glioma (1). Activation of PI3K that occurs as a consequence of EGFR amplification should respond to inhibitors of EGFR. PI3K may also be activated independently of EGFR through gain-of-function mutations in PI3K or from inactivation of the lipid phosphatase PTEN, a negative regulator of PI3K (2–4). Because the activation of PI3K is uncoupled from the upstream amplification of EGFR in PTEN-mutant glioma, we previously tested inhibitors of PI3K and of EGFR in combination, demonstrating the efficacy for this approach in EGFR-driven, PTEN-mutant glioma (5). The clinical relevance of this work was recently corroborated. In two retrospective studies, tumors in which PI3K was activated independently of EGFR responded poorly to EGFR inhibition (6, 7). Collectively, these reports suggest that in tumors with EGFR amplification and PTEN inactivation (comprising half of EGFR-amplified glioma), combined inhibition of EGFR and PI3K represents a promising therapy.

Toxicity associated with pan-selective inhibition of PI3Ks is a major hurdle preventing the translation of this approach to patients (8). To assess the impact of inhibiting individual PI3Ks, we recently synthesized a series of isoform-selective inhibitors of PI3Ks and enumerated their biochemical targets (9, 10). Using this chemical array, we linked increased specificity to decreased toxicity for agents targeting within the PI3K family and identified PI3K{alpha} as critical for proliferation in malignant glioma (11). We noted particular efficacy in combining inhibitors of PI3K{alpha} with inhibitors of mTOR, a serine-threonine kinase complex critical for cell growth (12). This result was at first somewhat surprising because mTOR is activated in response to PI3K. PI3Ks activate the pleckstrin homology domain containing serine-threonine kinase Akt. Akt, in turn, signals through effectors, including mTOR, to promote viability (13). Importantly, inhibitors of mTORC1 actually activate signaling through PI3K (11, 14, 15). Thus, the efficacy of mTOR blockade is achieved at the cost of driving other outputs of Akt signaling, contributing to the overall disappointing results observed using inhibitors of mTOR clinically. Combined blockade of mTOR and PI3K{alpha} shuts down mTOR and, in addition, abrogates the activation of PI3K observed using mTOR inhibitors alone. We recently validated this dual-inhibitor approach by combining rapamycin, an inhibitor of mTOR, with PIK-90, an inhibitor of PI3K{alpha}, and also through testing a dual inhibitor of PI3K{alpha} and of mTOR (PI-103) that was well tolerated and highly effective against glioma xenografts (11).

These preclinical studies support the use of combination therapy directed against EGFR and PI3K and, in parallel, show that inhibitors of PI3K{alpha} cooperate with inhibitors of mTOR in glioma. The goal of our current work is to test inhibitors of EGFR in combination with inhibitors of PI3K{alpha} and mTOR. In this communication, we show that PI-103 cooperates with erlotinib in PTEN-mutant glioma, establishing a mechanistic rationale for the blockade of EGFR, PI3K{alpha}, and mTOR in the treatment of EGFR-driven, PTEN mutant glioma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. LN229 and U87 cells transduced with EGFRvIII or EGFR as described (16) were grown in media containing 10% fetal bovine serum (FBS). Erlotinib tablets (Genentech) were ground to powder and dissolved in aqueous HCl, and aqueous phase was extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate and concentrated to yield pure erlotinib. Rapamycin was purchased from Cell Signaling; EGF was purchased from Roche Molecular Biochemicals; and PIK-90 and PI-103 were synthesized as described (9).

Cell proliferation assay and flow cytometry. For viability, 105 cells were seeded in 12-well plates in the presence of erlotinib, PI-103, PIK-90, rapamycin, erlotinib plus PIK-90, erlotinib plus rapamycin, erlotinib plus PIK-90, and rapamycin or erlotinib plus PI-103 for 72 h. Cell viability was determined using a WST-1 assay (Roche Molecular Biochemicals). Flow cytometry was as previously described (11).

Small interfering RNA transfection and retroviral infection. Synthetic control small interfering RNA (siRNA) and siRNA against PTEN were purchased from Santa Cruz Biotechnology and transfected using LipofectAMINE 2000 (Invitrogen) as previously described (16). pBabe puroL PTEN plasmid 10785 was purchased from Addgene. To generate the retrovirus, the packaging cell line 293T was cotransfected with plasmids expressing gag/pol and VSVg, using Effectene-transfected reagent (Qiagen). High-titer viruses were collected 48 h later and used to infect U87:EGFR cells as previously described (16).

Immunoblot analysis. Membranes were blotted with antisera to p-Akt (Ser473), Akt, p-Erk (Ser202/204), p-S6 ribosomal protein (Ser235/236), and S6 ribosomal protein (Cell Signaling Technology), 4G10, ß-tubulin (Upstate Biotechnology), Erk2, or EGFR (Santa Cruz Biotechnology). Immunoblotting and detection were as previously described (11).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTEN status and efficacy: erlotinib versus PI-103. To clarify the role of PTEN as a determinant of response to inhibitors of EGFR/PI3K/mTOR signaling, we transduced EGFR into glioma cell lines LN229 and U87 and treated these with erlotinib or PI-103. In contrast to the PTENmt cell line U87:EGFR, LN229:EGFR cells (PTENwt) showed a prominent response to erlotinib (Fig. 1A ). Flow-cytometric analysis showed G0-G1 arrest in LN229:EGFR cells (Fig. 1B). In comparison, U87:EGFR cells showed little response (Fig. 1B). These data are consistent with earlier studies that PTEN status represents an important determinant of response to EGFR inhibitors (6, 7).


Figure 1
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  		Figure 1. EGFR inhibitor erlotinib inhibits cell proliferation and induces G0-G1 arrest dependent on PTEN status. In contrast, antiproliferative effects of the dual PI3 kinase/mTOR inhibitor PI-103 were not dependent on PTEN status. LN229:EGFR (PTENwt) and U87:EGFR (PTENmt) cells were treated with erlotinib or PI-103. A, viability was determined using WST-1 assay after treatment with erlotinib or PI-103 in 12 wells for 72 h at dosages indicated. Bars, error between triplicate measurements. B, cells were treated with 5 µmol/L erlotinib or 0.5 µmol/L PI-103 for 24 h. Cells were trypsinized and prepared for flow-cytometric analysis of cell cycle distribution. Percentage of cells in G0-G1, S, and G2-M phases of the cell cycle is indicated. C, the EGFR inhibitor erlotinib inhibits signaling through Akt and mTOR in a PTEN-dependent manner, and the dual PI3K{alpha}/mTOR inhibitor PI-103 blocks both Akt and mTOR irrespective of PTEN status. LN229:EGFR (PTENwt) and U87:EGFR (PTENmt) cells were treated with erlotinib or PI-103 at dosages shown for 24 h. EGF (50 ng/mL) was added 15 min before harvest, and lysates were immunoblotted as indicated. Erlotinib therapy led to decreased Erk signaling in both cell lines, but impacted levels of p-Akt and the mTOR target p-rpS6 kinase only in cells wild-type for PTEN. Although U87:EGFR cells had higher baseline signaling through p-Akt, treatment with PI-103 led to dose-dependent blockade of both p-Akt and p-rpS6, without appreciably impacting levels of p-Erk.

 
Although mutation of PTEN would be expected to upregulate PI3K, and downstream signaling through Akt to mTOR [and subsequent feedback to PI3K (14–16)], we reasoned that PTEN status would not affect the ability of PI-103, a potent inhibitor of both PI3K and mTOR, to block the outputs of this signaling pathway and induce cell cycle arrest. Consistent with this model, PI-103 was equipotent against PTENwt and PTENmt cells, leading to arrest at G0-G1 (Fig. 1A and B). The response to this compound contrasted with the clear PTEN dependence observed using erlotinib and suggests that PTEN status is not a critical determinant of response to PI-103.

Erlotinib blocks mTOR in PTENwt cells. To explore downstream targets mediating the response of glioma cells to EGFR blockade, we did immunoblotting of PTENwt and PTENmt cells in response to erlotinib using phosphospecific antibodies. Treatment of cells with EGF led to equivalent responses in mitogen-activated protein (MAP) kinase signaling irrespective of PTEN status, as indicated by levels of p-Erk (Fig. 1C). Inhibition of EGFR impacted p-Erk similarly in both cell lines, consistent with pathways linking EGFR to MAP kinase signaling that were not impacted by PTEN status (Fig. 1C). In contrast, although treatment with EGF led to activation of p-Akt in PTENwt cells, EGF showed little impact on p-Akt in PTENmt cells (Fig. 1C). PTENmt cells had high levels of p-Akt at baseline, consistent with the activation of PI3K through the loss of PTEN. These high levels of p-Akt were only modestly affected by the treatment of PTENmt cells with erlotinib (Fig. 1C) and stood in contrast to comparable experiments treating PTENwt cells with this compound (Fig. 1C), again supporting a model in which the loss of PTEN effectively uncouples the activation of PI3K/Akt from upstream signaling through EGFR.

To provide mechanistic insights with regard to molecular alterations occurring as a result of PTEN mutation in combination with EGFR overexpression, we did similar experiments in LN229 cells and U87 cells expressing endogenous levels of EGFR or expressing the constitutively active tumor-derived EGFRvIII allele. LN229 (PTENwt) cells expressing endogenous levels of EGFR again responded to erlotinib, whereas U87 (PTENmt), cells were resistant (Supplemental Fig. S1A). In contrast, cells transduced with EGFRvIII were moderately resistant to erlotinib even in the setting of wild-type PTEN (Supplemental Fig. S1A). EGFR blockade only minimally impacted phosphorylation of both EGFRvIII and Akt in LN229:EGFRvIII and U87:EGFRvIII cells (Supplemental Fig. S1B), consistent with observations that the EGFRvIII allele is resistant to tyrosine kinase inhibition (17).

To evaluate the response of mTOR signaling, we analyzed the mTOR target ribosomal protein S6 kinase (rpS6). At baseline, levels of p-rpS6 were prominent in both lines, apparently unaffected by PTEN status. In contrast, PTEN status was important in determining whether the blockade of EGFR affected p-rpS6. In PTENwt cells, treatment with erlotinib led to decreased p-rpS6, which changed in parallel with p-Akt (Fig. 1C). Importantly, treatment of PTENmt cells with erlotinib did not impact p-rpS6, even at doses sufficiently high to block signaling through p-Akt (Fig. 1C). These data show that PTEN links EGFR to mTOR, that inhibition of EGFR can block signaling through mTOR in PTENwt but not in PTENmt cells, and suggest that blockade of mTOR correlates with the efficacy of EGFR inhibitors.

We next asked whether PTEN status was a determinant of the biochemical response to PI-103. Both PTENwt and PTENmt cells showed qualitatively similar blockade of p-Akt and p-rpS6 in response to PI-103 (Fig. 1C), whereas levels of p-Erk were only minimally affected. These experiments are consistent with results in Fig. 1A and B, demonstrating that PI-103 was equipotent in blocking proliferation in both PTENwt and PTENmt cells.

Erlotinib cooperates with PI-103 to arrest cells. Results in Fig. 1 argue that mutation at PTEN uncouples EGFR from downstream signaling through PI3K and mTOR, suggesting that the blockade of PI3K and/or mTOR could enhance the efficacy of EGFR inhibition in PTENmt glioma. To address the efficacy of this combination with regard to molecular alterations occurring as a result of PTEN mutation in combination with EGFR overexpression, we treated cells with erlotinib in combination with PI-103 in the settings of endogenous levels of EGFR and in cells transduced with either the tumor-derived EGFRvIII allele or with wild-type EGFR in both LN229 PTENwt and U87 PTENmt cells (Fig. 2 ). As expected, erlotinib led to G0-G1 arrest in PTENwt LN229:vector cells or LN229:EGFR cells and, with a 5-µmol/L dose, was minimally augmented by PI-103 (Fig. 2A and C). In contrast, erlotinib with a dose of 5 µmol/L had little impact as monotherapy in PTENmt cells and PTENwt LN229:EGFRvIII cells. However, erlotinib did show efficacy when combined with low-dose (100 nmol/L) PI-103 in this setting, with combination therapy effectively promoting arrest at G0-G1 (Fig. 2).


Figure 2
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  		Figure 2. Inhibitors of EGFR cooperate with the dual PI3K{alpha}/mTOR inhibitor PI-103 to arrest growth of PTEN mutant human glioma cells. LN229 (PTENwt) and U87 (PTENmt) cells transduced with vector (A), EGFRvIII (B), or wild-type EGFR (C) were treated with erlotinib, PI-103, or erlotinib plus PI-103 at indicated dosages. Flow cytometry analysis shows effects on cell cycle after treatment for 24 h. Percentage of cells in G0-G1, S, and G2-M phases of the cell cycle is indicated.

 
Immunoblotting experiments further reinforced results in Fig. 2, demonstrating that PTEN status correlated with the ability of erlotinib monotherapy to impact signaling through mTOR, regardless of EGFR levels, and was consistent in cells transduced with wild-type EGFR and EGFRvIII (Fig. 3A–C ). As monotherapy, erlotinib could block signaling through mTOR most effectively in PTENwt cells. Addition of PI-103 to erlotinib in these cells further blocked signaling through mTOR. In contrast, mutation at PTEN was an important and negative determinant of erlotinib's ability to impact the activation of mTOR. Whereas treatment of PTENmt cells with erlotinib did not impact the mTOR target p-rpS6, addition of PI-103 to erlotinib in these cells led to blockade of p-rpS6 (Fig. 3) and effected arrest at G0-G1 (Fig. 2). These observations again suggest a model in which PTEN status correlates with the ability of EGFR inhibitors to impact signaling through mTOR and supports combining PI-103 with erlotinib in EGFR-driven PTENmt tumors.


Figure 3
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  		Figure 3. PTEN status correlates with the ability of erlotinib monotherapy to impact signaling through mTOR. A–C, cells were treated as in Fig. 2. EGF (50 ng/mL) was added in (C) 15 min before harvest. Cell lysates were subjected to immunoblot analysis with antisera indicated. D, LN229:EGFR (PTENwt) cells were transfected with control siRNA or siRNA against PTEN for 24 h and then treated with inhibitors indicated for 24 h. Viability was measured by WST-1 assay, and levels of signaling molecules were measured by Western blot. In comparison with Fig. 1A, erlotinib treatment of PTEN knockdown cells led to increased viability (left, fourth column), correlating with increased levels of p-Akt and p-rps6 (right, fifth lane).

 
To verify the importance of PTEN in mediating relative sensitivity and resistance to erlotinib, we used siRNA to knock down PTEN in PTENwt cells (Fig. 3D). Knockdown of PTEN in LN229:EGFR cells led to increased viability in the absence of treatment and mediated relative resistance to erlotinib [45.1% viability in LN229:EGFR cells treated with 5 µmol/L erlotinib (Fig. 1A), compared with 77% viability with PTEN knockdown (Fig. 3D)]. Immunoblotting revealed that erlotinib loses potency against p-Akt and p-rpS6 as a result of PTEN knockdown (Fig. 3D). We also transduced wild-type PTEN into PTENmt U87:EGFR cells (Supplementary Fig. S2). This manipulation led to decreased viability in the absence of treatment and increased sensitivity to erlotinib [84.2% viability in U87:EGFR cells treated with 5 µmol/L erlotinib (Supplementary Fig. S3A), compared with 55.3% viability with transduced wild-type PTEN (Supplementary Fig. S2A)], associated with more efficient blockade of p-Akt. Interestingly, however, levels of p-rpS6 were unchanged (Supplementary Fig. S2B), an observation also reported by others (18).

Blockade of EGFR, PI3K{alpha}, and mTOR in glioma. Having shown that that PI-103 can augment the response to erlotinib in PTENmt cells, we next asked whether all three targets of these agents (EGFR, PI3K{alpha}, and mTOR) were critical to maximal proliferative blockade. We treated PTENmt U87:EGFR cells with erlotinib in combination with the pure PI3K{alpha} inhibitor PIK-90, the mTOR inhibitor rapamycin, combination therapy using both PIK-90 and rapamycin, or the dual PI3K{alpha}/mTOR inhibitor PI-103 (Supplementary Fig. S3). We also treated PTENmt U87:EGFRvIII cells and did a comparable but more extensive analysis analyzing each inhibitor individually and assessing the impact of paired inhibitors, comparing erlotinib plus PI-103 to triple therapy using erlotinib, rapamycin, and PIK-90 (Fig. 4 ). Measures of overall viability and of proliferation were consistent, showing that blockade of mTOR cooperated with inhibition of EGFR, and that further blockade of PI3K{alpha} led to maximal proliferation block (Fig. 4 compare column 7 to 8 or 9; Supplementary Fig. S3A, compare columns 7 and 9, P < 0.0001 Student's t test). Terminal nucleotidyl transferase–mediated nick end labeling and sub-G1 fractions showed no significant difference in apoptosis among these therapies (data not shown).


Figure 4
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  		Figure 4. Cooperative inhibition of EGFR, p110{alpha}, and mTOR are critical to arrest growth of PTENmt human glioma cells transduced with EGFRvIII. U87:EGFRvIII (PTENmt) cells were treated with erlotinib; PIK-90; rapamycin; PI-103; erlotinib plus PIK-90; erlotinib plus rapamycin; erlotinib plus PIK-90 and erlotinib triple therapy; or erlotinib plus PI-103 at dosages indicated. Flow cytometry analysis shows effects on cell cycle after treatment for 24 h. Percentage of cells in G0-G1, S, and G2-M phases of the cell cycle is indicated (top). Cell lysates were subjected to immunoblot analysis with antisera indicated (bottom).

 
Immunoblotting experiments (Fig. 4, Supplementary Fig. S3B) were aligned with flow cytometry. Cooperative inhibition of EGFR and of mTOR led to decreased levels of both p-EGFR and p-rpS6; however, rapamycin actually increased levels of p-Akt (Fig. 4, Supplementary Fig. S3B). Whereas erlotinib could efficiently block p-EGFR (Fig. 4, Supplementary Fig. S3B), combining erlotinib with either PIK-90 or rapamycin blocked either p-Akt or p-rpS6 (Fig. 4, Supplementary Fig. S3B). Further inhibition of PI3K{alpha} was required to simultaneously block p-Akt and p-rpS6 (Fig. 4, Supplementary Fig. S3B). These results show that blockade of EGFR and of mTOR cooperate in the treatment of EGFR-driven, PTENmt glioma, and that further efficacy can be achieved through concomitant blockade of PI3K{alpha}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The malignant gliomas show intrinsic resistance to most medical therapies, contributing to their poor prognosis (1). The association of EGFR amplification with high-grade glioblastoma multiforme tumors therefore led to early optimism that EGFR inhibition would be beneficial. This initial optimism was mitigated by observations that only a subset of patients with EGFR-amplified glioma actually responded to the blockade of EGFR (19). The failure of this approach in the majority of patients could stem from inefficient blockade of EGFR or from the inability to reverse signaling abnormalities associated with EGFR amplification, even in the setting of adequate blockade. Loss of PTEN contributes to this failure because loss of PTEN blocks the ability of EGFR inhibitors to impact downstream signaling through PI3K and mTOR.

In this communication, we present a preclinical approach aimed at reversing signaling abnormalities associated with EGFR amplification, offering a mechanistic rationale to combine inhibitors of EGFR and of mTOR to effect proliferation blockade in patients with EGFR-amplified, PTENmt glioma. We showed the efficacy for inhibitors of EGFR as monotherapy in glioma cells wild-type for PTEN, and that the antiproliferative effect of EGFR inhibitors correlated with the ability of these agents to impact levels of mTOR.

In contrast to PTENwt cells, erlotinib treatment of PTENmt cells did not appreciably impact proliferation and specifically did not impact mTOR, even when inhibitors of EGFR were used at doses sufficiently high to block p-Akt (Fig. 1C). Although erlotinib had little measurable activity as monotherapy in PTENmt cells, erlotinib clearly augmented the efficacy of PI-103 as measured both by blockade of mTOR and of proliferation (Fig. 4). Intriguingly, the ability of PI-103 and erlotinib to impact mTOR again was observed in a setting where combination therapy did not appreciably alter levels of p-Akt in comparison with PI-103 alone (Fig. 4). The dissociation of Akt from mTOR in PTENmt glioma has also been observed by others (18) and suggests the presence of Akt-independent regulators of mTOR.

The failure of inhibitors of EGFR to impact mTOR signaling in PTENmt glioma also provides a rationale to combine inhibitors of EGFR and mTOR. While targeting both kinases simultaneously led to decreased proliferation in comparison with targeting either EGFR or mTOR alone (Fig. 4), blockade of mTOR by rapamycin actually led to increased levels of p-Akt (Fig. 4), a phenomenon previously described by others (14, 15).

In response to the failure of EGFR inhibitors to block PI3K, Akt, or mTOR in PTENmt glioma, and because mTOR inhibitors actually activate the PI3K/Akt axis, we tested inhibitors of EGFR and of mTOR in combination with inhibitors of PI3K{alpha}. The combinatorial inhibition of three targets effectively blocked signaling and was more effective than any two targeted therapies in combination as measured both biochemically and through flow-cytometric analyses (Fig. 4). Even using approaches that blocked EGFR, PI3K{alpha}, and mTOR in combination, and in the setting of efficient inhibition of Akt, an important mediator of antiapoptotic signaling (20), we failed to observe any appreciable apoptosis in any glioma cell lines tested. Thus, whereas the translation of our findings to patients awaits clinical development of isoform-specific inhibitors of PI3K, the ability to induce cytotoxic rather than cytostatic responses in the malignant gliomas represents a more formidable challenge and one that may be critical to the long-term efficacy of these approaches in patients.


   Acknowledgments
 
Grant support: This work, dedicated to the late Jeffrey P. Weiss, was supported by grants from the Burroughs Wellcome Fund, The Brain Tumor Society, The National Brain Tumor and Samuel G. Waxman Foundations, The Sandler Family, and the National Cancer Institute Specialized Programs of Research Excellence Program.

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.

We are grateful to Russ Pieper and Cynthia Cowdry for cell lines, William Sellers for the pBabe puroL PTEN plasmid, and Lou Chesler and Matt Grimmer for critical review.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/11/07. Revised 6/29/07. Accepted 7/17/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Persson A, Fan QW, Phillips J, Weiss WA. Glioma. San Diego: Elsevier; 2006. p. 433–44.
  2. Vogt PK, Bader AG, Kang S. PI 3-kinases: hidden potentials revealed. Cell Cycle 2006;5:946–9.[Medline]
  3. Zhao JJ, Roberts TM. PI3 kinases in cancer: from oncogene artifact to leading cancer target. Sci STKE 2006;(365):pe52.
  4. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006;7:606–19.[CrossRef][Medline]
  5. Fan QW, Specht KM, Zhang C, Goldenberg DD, Shokat KM, Weiss WA. Combinatorial efficacy achieved through two-point blockade within a signaling pathway—a chemical genetic approach. Cancer Res 2003;63:8930–8.[Abstract/Free Full Text]
  6. Haas-Kogan DA, Prados MD, Tihan T, et al. Epidermal growth factor receptor, protein kinase B/Akt, and glioma response to erlotinib. J Natl Cancer Inst 2005;97:880–7.[Abstract/Free Full Text]
  7. Mellinghoff IK, Wang MY, Vivanco I, et al. Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J Med 2005;353:2012–24.[Abstract/Free Full Text]
  8. Workman P, Clarke PA, Guillard S, Raynaud FI. Drugging the PI3 kinome. Nat Biotechnol 2006;24:794–6.[CrossRef][Medline]
  9. Knight ZA, Gonzalez B, Feldman ME, et al. A pharmacological map of the PI3-K family defines a role for p110{alpha} in insulin signaling. Cell 2006;125:733–47.[CrossRef][Medline]
  10. Knight ZA, Chiang GG, Alaimo PJ, et al. Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold. Bioorg Med Chem 2004;12:4749–59.[CrossRef][Medline]
  11. Fan QW, Knight ZA, Goldenberg DD, et al. A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 2006;9:341–9.[CrossRef][Medline]
  12. Sabatini DM. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 2006;6:729–34.[CrossRef][Medline]
  13. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006;441:424–30.[CrossRef][Medline]
  14. Sun SY, Rosenberg LM, Wang X, et al. Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res 2005;65:7052–8.[Abstract/Free Full Text]
  15. O'Reilly KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006;66:1500–8.[Abstract/Free Full Text]
  16. Fan QW, Weiss WA. RNA interference against a glioma-derived allele of EGFR induces blockade at G2M. Oncogene 2005;24:829–37.[CrossRef][Medline]
  17. Learn CA, Hartzell TL, Wikstrand CJ, et al. Resistance to tyrosine kinase inhibition by mutant epidermal growth factor receptor variant III contributes to the neoplastic phenotype of glioblastoma multiforme. Clin Cancer Res 2004;10:3216–24.[Abstract/Free Full Text]
  18. Wang MY, Lu KV, Zhu S, 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 2006;66:7864–9.[Abstract/Free Full Text]
  19. Nicholas MK, Lukas RV, Jafri NF, Faoro L, Salgia R. Epidermal growth factor receptor-mediated signal transduction in the development and therapy of gliomas. Clin Cancer Res 2006;12:7261–70.[Abstract/Free Full Text]
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