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1 Department of Hematology and Oncology and Winship Cancer Institute and 2 Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia
Requests for reprints: Shi-Yong Sun, Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road, C3088, Atlanta, GA 30322. Phone: 404-778-2170; Fax: 404-778-5520; E-mail: shi-yong_sun{at}emoryhealthcare.org.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The phosphatidylinositol-3 kinase (PI3K)/Akt signaling represents a major cell survival pathway. Its activation has long been associated with malignant transformation and apoptotic resistance (4). It has been well documented that mTOR functions downstream of the PI3K/Akt pathway and is phosphorylated (or activated) in response to stimuli that activate the PI3K/Akt pathway (1, 3). Normally, the phosphatase PTEN counters the PI3K activity and thus negatively regulates PI3K/Akt survival pathway. However, PTEN activity is frequently inactivated in many human tumor types through deletion, mutation, or silencing, leading to increased activation of Akt (4, 5). Additional mechanisms have also been found to induce the activation of the PI3K/Akt pathway, including oncogene (e.g., Ras) amplification and mutations, active mutations in the p110 and p85 subunits of PI3K, and Akt overexpression. Thus, mTOR signaling pathways are constitutively activated in many types of human cancer (1, 6). Recent studies have shown that the mutation of tuberous sclerosis complex and overexpression of Rheb that work downstream of Akt in regulating the mTOR signaling also occur in human cancers, contributing to mTOR activation (7). Moreover, eIF4E is overexpressed or amplified in multiple human cancers, which is often oncogenic (1, 6, 8). Therefore, mTOR signaling has emerged as an important and attractive therapeutic target for cancer therapy (1, 2). The potential applications of mTOR inhibitors for treating various types of cancer have been actively studied both preclinically and clinically. A recent animal study has shown that mTOR inhibition induces apoptosis of epithelial cells and reverses Akt-dependent prostate intraepithelial neoplasia (9). In the United States, several phase II or III trials are ongoing to test the effects of mTOR inhibitors on various cancers, including renal cell carcinoma, prostate, breast, pancreatic, and small cell lung cancers, recurrent brain tumors, recurrent mantle-cell lymphoma, and melanoma (1, 10).
The intrinsic sensitivity to mTOR inhibition by rapamycin among different cancer cell lines may vary by several orders of magnitude ranging from 1 to 5,000 nmol/L (IC50; ref. 11), indicating that some cancer cell lines are actually resistant to mTOR inhibition. Therefore, understanding the mechanisms by which cells become resistant to mTOR inhibitors may guide the development of successful mTOR-targeted cancer therapy. Using human non–small cell lung cancer (NSCLC) cells, here we report that mTOR inhibition by rapamycin induces activation of survival pathways involving increase of Akt and eIF4E phosphorylation. We show that prevention or disruption of the activation of Akt and eIF4E enhanced rapamycin-mediated growth inhibition, indicating that the induced activation of Akt and eIF4E survival pathways counteracts the mTOR inhibitor's effect on the growth of human cancer cells.
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Materials and Methods |
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Cell lines and cell culture. Human NSCLC and other cancer cell lines used in this study were purchased from the American Type Culture Collection (Manassas, VA). They were grown in monolayer culture in RPMI 1640 supplemented with glutamine and 5% fetal bovine serum at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air.
Growth inhibition assay. Cells were seeded in 96-well cell culture plates and treated on the second day with rapamycin, LY294002, or rapamycin combined with LY294002. At the end of a 3-day treatment, cell number was estimated by the sulforhodamine B (SRB) assay as previously described (12). The percentage of growth inhibition was calculated by using the equation: % growth inhibition = (1 – At / Ac) x 100, where At and Ac represent the absorbance in treated and control cultures, respectively.
Colony formation assay. Cells (single-cell suspension) were plated in 12-well plates at a density of 200 to 300 cells per well. On the second day, cells were treated with rapamycin, LY294002, or rapamycin plus LY294002. Every 3 days, the medium was replaced with fresh medium containing the corresponding agents. After a 10-day treatment, the medium was removed and cell colonies were stained with SRB dye as described (12). Pictures were then taken using a digital camera to record the result.
Western blot analysis. The procedures for preparation of whole cell protein lysates and for Western blotting were described previously (13). Whole cell protein lysates (50 µg) were electrophoresed through 7.5%, 10%, or 12% denaturing polyacrylamide slab gels and transferred to a Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) by electroblotting. The blots were probed or reprobed with the primary antibodies and then antibody binding was detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) according to the manufacturer's protocol.
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Results |
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1 nmol/L was effective in inhibiting the growth of NSCLC cells, albeit with varying degrees. However, at concentrations ranging from 1 to 1,000 nmol/L, rapamycin did not seem to exhibit a dose-dependent growth-inhibitory effect. Rapamycin at concentrations <1 nmol/L dramatically decreased its efficacy against the growth of NSCLC cells. Interestingly, rapamycin at concentrations up to 1,000 nmol/L inhibited the growth of NSCLC cells only by 50% to 75%. Even when rapamycin's concentration was increased to 10 µmol/L, its growth-inhibitory effects were not further increased (data not shown). These results suggest that certain portions of cells in the population are resistant to rapamycin or cancer cells have some resistant mechanisms to bypass growth inhibition caused by mTOR inhibitors. Under the microscope, cells exposed to rapamycin remained attached on dishes and had normal morphology in comparison with control cells, suggesting that rapamycin inhibits the growth of human NSCLC cells without apparently inducing cell death.
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To correlate the dynamic changes in p-Akt and p-eIF4E with the inhibition of mTOR in response to rapamycin, we did a detailed time course analysis to examine rapamycin's effects on the alterations of p-p70S6K, p-4E-BP1, p-Akt, and p-eIF4E in two representative NSCLC cell lines. In both H157 and A549 cell lines, p-p70S6K and p-4E-BP1 levels decreased 3 hours after exposure to rapamycin; this decrease was sustained up to 24 hours. Concurrently, p-Akt and p-eIF4E levels increased soon after a 3-hour exposure to rapamycin; this increase was still evident up to 24 hours after treatment (Fig. 3A). We also examined the effects of rapamycin on the expression levels of p70S6K, Akt, and eIF4E and found that rapamycin did not markedly alter their expression (Fig. 3A). To get more information on the dynamic changes of p-p70S6K, p-Akt, and p-eIF4E in response to rapamycin, we further shortened the exposure time to rapamycin. As shown in Fig. 3B, suppression of p-p70S6K and increase of p-Akt were detected 15 minutes after the cells were exposed to rapamycin. The increase of p-eIF4E was also detected 30 minutes (A549) and 60 minutes (H157) after rapamycin treatment. Collectively, it seems that the decrease of p-p70S6K and the increase of p-Akt are rapid and concurrent events in cells treated with rapamycin.
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Phosphatidylinositol 3-kinase is required for rapamycin-induced phosphorylation of Akt and eIF4E. To understand the mechanism by which rapamycin induces Akt activation, we investigated the involvement of PI3K, an upstream regulator of Akt, in this process. PI3K catalyzes the production of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the cell membrane. PIP3 in turn recruits other pleckstrin homology domain–containing proteins, in particular Akt, to the membrane, where Akt is activated by PDK1 and a Ser473 kinase (4). If PI3K is involved in Akt activation induced by mTOR inhibition, the PI3K inhibitor LY294002 would block or suppress the Akt phosphorylation or activation by rapamycin. Therefore, we examined the effects of LY294002 on rapamycin-induced Akt activation and eIF4E phosphorylation. In the absence of LY294002, rapamycin at both 1 and 10 nmol/L increased p-Akt and p-GSK3ß levels. In the presence of LY294002, rapamycin failed to increase Akt and GSK3ß phosphorylation (Fig. 4A). These results suggest that rapamycin-induced Akt activation requires activated PI3K. Similarly, LY294002 also blocked the rapamycin-induced increase of eIF4E phosphorylation (Fig. 4A), suggesting that the rapamycin-induced increase of p-eIF4E is dependent on PI3K activation as well.
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Combination of rapamycin with LY294002 exhibits enhanced inhibitory effects on the growth and colony formation of non–small cell lung cancer cells. It is well documented that Akt is a major survival kinase (4). Recently, eIF4E has also been shown to be a tumor survival factor (8, 14). Our data clearly show that suppression of mTOR by rapamycin activates Akt and elF4E survival pathways. Thus, we speculated that PI3K/Akt and eIF4E activation would counteract mTOR inhibitors' anticancer effects. Because rapamycin-induced Akt activation requires its upstream regulator, PI3K, (Fig. 4A and B), blocking the PI3K/Akt survival pathway by LY294002 would be expected to enhance the rapamycin effect. Thus, we examined the effects of rapamycin combined with LY294002 on the growth of human NSCLC cells. As shown in Fig. 4C, the combination of rapamycin and LY294002 in a 3-day growth inhibition assay apparently exhibit growth-inhibitory effects that are greater than those caused by each single agent alone. For example, in H157 cells, rapamycin at 1 nmol/L inhibited cell growth by 30%, whereas LY294002 at 0.5, 1.0, 2.5, and 5.0 µmol/L caused 4.6%, 3.2%, 11%, and 17.9% growth inhibition, respectively. However, their combinations led to growth inhibition by 46.5%, 52.2%, 58.9%, and 67.3%, respectively. These effects are apparently greater than the sum of the inhibitory effects caused by each agent alone, indicating a more than additive or synergistic effect. In the long-term colony formation assay, we obtained similar results as determined from the 3-day assay. LY294002 and rapamycin alone did not decrease the number of colonies, although they reduced the sizes of the colonies. The combination of the two agents not only decreased the size of colonies but also reduced the number of colonies (Fig. 4D), indicating that the combination causes a greater growth-inhibitory effect than that of each single agent. Taken together, these results indicate that the combination of rapamycin and LY294002 results in an augmented growth-inhibitory effect.
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Discussion |
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Rapamycin at concentrations (1 nmol/L) that exhibited growth inhibition rapidly and effectively suppressed the phosphorylation of p70S6K and 4E-BP1, indicating that rapamycin indeed blocks mTOR signaling. This blockage may well be the molecular basis for rapamycin to inhibit the growth of cancer cells. Unexpectedly, rapamycin rapidly induced the activation of Akt as shown by phosphorylation at Ser473 and the phosphorylation of its substrate, GSK3ß, whereas suppressing the phosphorylation of p70SK and 4E-BP1. It seems that mTOR inhibition by rapamycin activates the Akt survival pathway. Even more surprisingly, cells treated with rapamycin within a concentration range that inhibits mTOR signaling increased eIF4E phosphorylation. To our knowledge, this is the first report to show that mTOR inhibition by rapamycin increases eIF4E phosphorylation.
Activation of Akt by mTOR inhibition was reported in Drosophila when studying the functional role of Rheb in regulation of p70S6K activity (24) and also in mammalian skeletal muscle cells, adipocytes, and fibroblasts when studying insulin signaling (25–27). Our study clearly shows that mTOR inhibition by rapamycin results in the activation of the Akt survival pathway in human NSCLC and other types of cancer cell lines. To the best of our knowledge, this is the first report to show mTOR inhibition–induced Akt activation in human cancer cells. Results from previous studies of insulin signaling suggest that the mTOR activation by insulin initiates a feedback inhibition of PI3K/Akt through p70S6K activation and its subsequent phosphorylation of insulin receptor substrate-1 (IRS-1). The phosphorylation of IRS-1 promotes IRS-1 degradation and reduces IRS-1 expression, leading to decreased activity of PI3K/Akt. Rapamycin suppresses p70S6K and thus relieves this negative feedback inhibition of Akt (28, 29). In our study, the presence of the PI3K inhibitor, LY294002, abrogated rapamycin-induced Akt activation (Akt and GSK3ß phosphorylation), suggesting that PI3K activity is required for Akt activation by rapamycin. However, we currently do not know whether the Akt activation induced by rapamycin in human cancer cells is mediated by p70S6K suppression through the stabilization of IRS-1. It has been shown that there are two mTOR complexes in mammalian cells: the rapamycin-sensitive mTOR-raptor complex and the rapamycin-insensitive mTOR-rictor complex (30). A recent study has shown that the mTOR-rictor complex can directly phosphorylate Akt at Ser473 (31). Reduction of mTOR expression using small interfering RNA decreased Akt phosphorylation (31). Our data seem to favor the model that rapamycin induces Akt phosphorylation in a PI3K-dependent mechanism. However, it remains possible that rapamycin may indirectly stimulate the mTOR-rictor kinase activity to phosphorylate Akt. Elucidation of these mechanisms requires further investigation.
eIF4E plays a critical role in the regulation of cap-dependent-protein translation and thus its activity is integral in determining global translation rates (32). Consistent with this role, eIF4E is required for cell cycle progression, exhibits antiapoptotic or survival activity, and when overexpressed, transforms cells (8, 14), largely due to its critical role in initiating translation of mRNAs that encode cell cycle regulators or oncogenic proteins such as cyclin D1, ornithine decarboxylase, c-Myc, hypoxia-inducible factor 1
, fibroblast growth factor, and vascular endothelial growth factor (1, 8, 24). Therefore, it is not surprising that elevated levels of eIF4E are found in a broad spectrum of transformed cells and human cancers, including lung cancers, and is often associated with aggressive, poorly differentiated tumors (1, 8). eIF4E is phosphorylated (usually at Ser209) in many systems in response to extracellular stimuli including growth factors, hormones, and mitogens (15, 16). Its phosphorylation increases its affinity for the cap and for mRNA and may also favor its entry into initiation complexes (15, 16, 33). Although mTOR inhibitors are expected to inhibit cap-dependent translation via activation of 4E-BP1 (i.e., promoting its dephosphorylation), we paradoxically found that cells treated with rapamycin exhibited increased eIF4E phosphorylation. Thus, it seems that rapamycin treatment generates conflicting signal to cap-dependent protein translation. Collectively, these findings suggest that rapamycin may promote cap-dependent protein translation, probably under certain conditions.
It has been documented that MEK/ERK and p38 MAPK signaling pathways activate eIF4E through Mnk1-mediated phosphorylation of eIF4E (15, 16). However, the MEK inhibitors U0126 and PD98059 or the p38 MAPK inhibitor SB203580 did not inhibit a rapamycin-induced elevation of p-eIF4E. Instead, LY294002 abolished the increase of p-eIF4E by rapamycin. Thus, it seems that the PI3K activity is required for mediating rapamycin-induced eIF4E phosphorylation. The investigation on the involvement of Mnk1 in rapamycin-induced eIF4E phosphorylation is ongoing.
Because Akt and eIF4E are often associated with cell survival and resistance to cancer therapy (4, 14), our findings imply that the activation of Akt and eIF4E through mTOR inhibition may counteract mTOR inhibitors' anticancer efficacy and confers resistance to mTOR-targeted cancer therapy. According to our results, Akt and eIF4E phosphorylation induced by rapamycin all occur downstream of PI3K. These findings may provide us the opportunity to interrupt or disrupt activation of the Akt and eIF4E survival pathways using a PI3K inhibitor or even an Akt inhibitor to enhance mTOR inhibitors' anticancer efficacy or mTOR-targeted cancer therapy. Our results indeed show that LY294002 in combination with rapamycin exhibits enhanced (synergistic) effects on the growth and colony formation of human NSCLC cells. Therefore, from a therapeutic point of view, our findings suggest a novel strategy to enhance mTOR-targeted cancer therapy through combining an mTOR inhibitor with an inhibitor of the PI3K/Akt pathway.
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Acknowledgments |
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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.
Received 3/18/05. Revised 5/16/05. Accepted 6/16/05.
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D. Zhong, X. Liu, K. Schafer-Hales, A. I. Marcus, F. R. Khuri, S.-Y. Sun, and W. Zhou 2-Deoxyglucose induces Akt phosphorylation via a mechanism independent of LKB1/AMP-activated protein kinase signaling activation or glycolysis inhibition Mol. Cancer Ther., April 1, 2008; 7(4): 809 - 817. [Abstract] [Full Text] [PDF]
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P. C. McDonald, A. Oloumi, J. Mills, I. Dobreva, M. Maidan, V. Gray, E. D. Wederell, M. B. Bally, L. J. Foster, and S. Dedhar Rictor and Integrin-Linked Kinase Interact and Regulate Akt Phosphorylation and Cancer Cell Survival Cancer Res., March 15, 2008; 68(6): 1618 - 1624. [Abstract] [Full Text] [PDF]
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D. Kong, S. Banerjee, W. Huang, Y. Li, Z. Wang, H.-R. C. Kim, and F. H. Sarkar Mammalian Target of Rapamycin Repression by 3,3'-Diindolylmethane Inhibits Invasion and Angiogenesis in Platelet-Derived Growth Factor-D-Overexpressing PC3 Cells Cancer Res., March 15, 2008; 68(6): 1927 - 1934. [Abstract] [Full Text] [PDF]
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 A. Moreno, A. Akcakanat, M. F Munsell, A. Soni, J. C Yao, and F. Meric-Bernstam Antitumor activity of rapamycin and octreotide as single agents or in combination in neuroendocrine tumors Endocr. Relat. Cancer, March 1, 2008; 15(1): 257 - 266. [Abstract] [Full Text] [PDF]
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 C. Gridelli, P. Maione, and A. Rossi The Potential Role of mTOR Inhibitors in Non-Small Cell Lung Cancer Oncologist, February 1, 2008; 13(2): 139 - 147. [Abstract] [Full Text] [PDF]
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 J. Mariotti, J. Foley, U. Jung, T. Borenstein, N. Kantardzic, S. Han, J. T. Hanson, E. Wong, N. Buxhoeveden, J. B. Trepel, et al. Ex Vivo Rapamycin Generates Apoptosis-Resistant Donor Th2 Cells That Persist In Vivo and Prevent Hemopoietic Stem Cell Graft Rejection J. Immunol., January 1, 2008; 180(1): 89 - 105. [Abstract] [Full Text] [PDF]
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B. T. Hennessy, Y. Lu, E. Poradosu, Q. Yu, S. Yu, H. Hall, M. S. Carey, M. Ravoori, A. M. Gonzalez-Angulo, R. Birch, et al. Pharmacodynamic Markers of Perifosine Efficacy Clin. Cancer Res., December 15, 2007; 13(24): 7421 - 7431. [Abstract] [Full Text] [PDF]
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H.-G. Wendel, R. L.A. Silva, A. Malina, J. R. Mills, H. Zhu, T. Ueda, R. Watanabe-Fukunaga, R. Fukunaga, J. Teruya-Feldstein, J. Pelletier, et al. Dissecting eIF4E action in tumorigenesis Genes & Dev., December 15, 2007; 21(24): 3232 - 3237. [Abstract] [Full Text] [PDF]
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X. Wang, P. Yue, C.-B. Chan, K. Ye, T. Ueda, R. Watanabe-Fukunaga, R. Fukunaga, H. Fu, F. R. Khuri, and S.-Y. Sun Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation Mol. Cell. Biol., November 1, 2007; 27(21): 7405 - 7413. [Abstract] [Full Text] [PDF]
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C. Bouchard, S. Lee, V. Paulus-Hock, C. Loddenkemper, M. Eilers, and C. A. Schmitt FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf Genes & Dev., November 1, 2007; 21(21): 2775 - 2787. [Abstract] [Full Text] [PDF]
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M. Fouladi, F. Laningham, J. Wu, M. A. O'Shaughnessy, K. Molina, A. Broniscer, S. L. Spunt, I. Luckett, C. F. Stewart, P. J. Houghton, et al. Phase I Study of Everolimus in Pediatric Patients With Refractory Solid Tumors J. Clin. Oncol., October 20, 2007; 25(30): 4806 - 4812. [Abstract] [Full Text] [PDF]
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G. A. Finlay, A. J. Malhowski, Y. Liu, B. L. Fanburg, D. J. Kwiatkowski, and D. Toksoz Selective Inhibition of Growth of Tuberous Sclerosis Complex 2 Null Cells by Atorvastatin Is Associated with Impaired Rheb and Rho GTPase Function and Reduced mTOR/S6 Kinase Activity Cancer Res., October 15, 2007; 67(20): 9878 - 9886. [Abstract] [Full Text] [PDF]
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C.-H. Lu, S. L. Wyszomierski, L.-M. Tseng, M.-H. Sun, K.-H. Lan, C. L. Neal, G. B. Mills, G. N. Hortobagyi, F. J. Esteva, and D. Yu Preclinical Testing of Clinically Applicable Strategies for Overcoming Trastuzumab Resistance Caused by PTEN Deficiency Clin. Cancer Res., October 1, 2007; 13(19): 5883 - 5888. [Abstract] [Full Text] [PDF]
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Q.-W. Fan, C. K. Cheng, T. P. Nicolaides, C. S. Hackett, Z. A. Knight, K. M. Shokat, and W. A. Weiss A Dual Phosphoinositide-3-Kinase {alpha}/mTOR Inhibitor Cooperates with Blockade of Epidermal Growth Factor Receptor in PTEN-Mutant Glioma Cancer Res., September 1, 2007; 67(17): 7960 - 7965. [Abstract] [Full Text] [PDF]
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B. E. Johnson, D. Jackman, and P. A. Janne Rationale for a Phase I Trial of Erlotinib and the Mammalian Target of Rapamycin Inhibitor Everolimus (RAD001) for Patients with Relapsed Non Small Cell Lung Cancer Clin. Cancer Res., August 1, 2007; 13(15): 4628s - 4631s. [Abstract] [Full Text] [PDF]
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X. Wan and L. J. Helman The Biology Behind mTOR Inhibition in Sarcoma Oncologist, August 1, 2007; 12(8): 1007 - 1018. [Abstract] [Full Text] [PDF]
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 L. Kopelovich, J. R. Fay, C. C. Sigman, and J. A. Crowell The Mammalian Target of Rapamycin Pathway as a Potential Target for Cancer Chemoprevention Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1330 - 1340. [Abstract] [Full Text] [PDF]
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R. T. Abraham and J. J. Gibbons The Mammalian Target of Rapamycin Signaling Pathway: Twists and Turns in the Road to Cancer Therapy Clin. Cancer Res., June 1, 2007; 13(11): 3109 - 3114. [Abstract] [Full Text] [PDF]
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T. Li, Y.-H. Ling, I. D. Goldman, and R. Perez-Soler Schedule-Dependent Cytotoxic Synergism of Pemetrexed and Erlotinib in Human Non-Small Cell Lung Cancer Cells Clin. Cancer Res., June 1, 2007; 13(11): 3413 - 3422. [Abstract] [Full Text] [PDF]
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E. F. Petricoin III, V. Espina, R. P. Araujo, B. Midura, C. Yeung, X. Wan, G. S. Eichler, D. J. Johann Jr., S. Qualman, M. Tsokos, et al. Phosphoprotein Pathway Mapping: Akt/Mammalian Target of Rapamycin Activation Is Negatively Associated with Childhood Rhabdomyosarcoma Survival Cancer Res., April 1, 2007; 67(7): 3431 - 3440. [Abstract] [Full Text] [PDF]
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C. A. Granville, N. Warfel, J. Tsurutani, M. C. Hollander, M. Robertson, S. D. Fox, T. D. Veenstra, H. J. Issaq, R. I. Linnoila, and P. A. Dennis Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen-Induced Murine Lung Tumors Clin. Cancer Res., April 1, 2007; 13(7): 2281 - 2289. [Abstract] [Full Text] [PDF]
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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]
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Z. Ma, S. L. Gibson, M. A. Byrne, J. Zhang, M. F. White, and L. M. Shaw Suppression of Insulin Receptor Substrate 1 (IRS-1) Promotes Mammary Tumor Metastasis Mol. Cell. Biol., December 15, 2006; 26(24): 9338 - 9351. [Abstract] [Full Text] [PDF]
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 E. Peponi, E. Drakos, G. Reyes, V. Leventaki, G. Z. Rassidakis, and L. J. Medeiros Activation of Mammalian Target of Rapamycin Signaling Promotes Cell Cycle Progression and Protects Cells from Apoptosis in Mantle Cell Lymphoma Am. J. Pathol., December 1, 2006; 169(6): 2171 - 2180. [Abstract] [Full Text] [PDF]
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K. W.L. Yee, Z. Zeng, M. Konopleva, S. Verstovsek, F. Ravandi, A. Ferrajoli, D. Thomas, W. Wierda, E. Apostolidou, M. Albitar, et al. Phase I/II Study of the Mammalian Target of Rapamycin Inhibitor Everolimus (RAD001) in Patients with Relapsed or Refractory Hematologic Malignancies Clin. Cancer Res., September 1, 2006; 12(17): 5165 - 5173. [Abstract] [Full Text] [PDF]
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D. Del Bufalo, L. Ciuffreda, D. Trisciuoglio, M. Desideri, F. Cognetti, G. Zupi, and M. Milella Antiangiogenic Potential of the Mammalian Target of Rapamycin Inhibitor Temsirolimus Cancer Res., June 1, 2006; 66(11): 5549 - 5554. [Abstract] [Full Text] [PDF]
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B. A. Jacobson, M. D. Alter, M. G. Kratzke, S. P. Frizelle, Y. Zhang, M. S. Peterson, S. Avdulov, R. P. Mohorn, B. A. Whitson, P. B. Bitterman, et al. Repression of Cap-Dependent Translation Attenuates the Transformed Phenotype in Non-Small Cell Lung Cancer Both In vitro and In vivo. Cancer Res., April 15, 2006; 66(8): 4256 - 4262. [Abstract] [Full Text] [PDF]
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P. Maione, C. Gridelli, T. Troiani, and F. Ciardiello Combining Targeted Therapies and Drugs with Multiple Targets in the Treatment of NSCLC. Oncologist, March 1, 2006; 11(3): 274 - 284. [Abstract] [Full Text] [PDF]
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K. S. Klos, S. L. Wyszomierski, M. Sun, M. Tan, X. Zhou, P. Li, W. Yang, G. Yin, W. N. Hittelman, and D. Yu ErbB2 Increases Vascular Endothelial Growth Factor Protein Synthesis via Activation of Mammalian Target of Rapamycin/p70S6K Leading to Increased Angiogenesis and Spontaneous Metastasis of Human Breast Cancer Cells Cancer Res., February 15, 2006; 66(4): 2028 - 2037. [Abstract] [Full Text] [PDF]
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K. E. O'Reilly, F. Rojo, Q.-B. She, D. Solit, G. B. Mills, D. Smith, H. Lane, F. Hofmann, D. J. Hicklin, D. L. Ludwig, et al. mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt Cancer Res., February 1, 2006; 66(3): 1500 - 1508. [Abstract] [Full Text] [PDF]
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250 cells per well were seeded in 12-well plates. On the second day, cells were treated with the indicated concentrations of LY294002 alone, 1 nmol/L rapamycin alone, and their respective combinations. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with SRB dye. The picture of the colonies was then taken using a digital camera.