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Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity

Linda A. deGraffenried, William E. Friedrichs, Douglas H. Russell, Elissa J. Donzis, Amanda K. Middleton, Jessica M. Silva, Richard A. Roth and Manuel Hidalgo
Linda A. deGraffenried
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William E. Friedrichs
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Douglas H. Russell
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Elissa J. Donzis
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Amanda K. Middleton
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Jessica M. Silva
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Richard A. Roth
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Manuel Hidalgo
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DOI: 10.1158/1078-0432.CCR-04-0035 Published December 2004
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    Fig. 1.

    Mitogen-independent activation of Akt. A, protein lysates from control and myristoylated Akt-transfected (Akt) MCF-7 cells before (Earle’s) and after (+ insulin) stimulation with 100 nmol/L insulin were subjected to immunodetection with antibodies to the phospho Ser473 residue of Akt on both the endogenous (Mr 64,000, endogenous phospho-Akt), as well as the myrAkt1 transgene product (Mr 46,000, phospho-myrAkt1), the ER, and actin. B, Akt kinase activity of the control and myrAkt1 MCF-7 cells (clones 13 and 29) under nonstimulated (Earle’s) and insulin-stimulated (Earle’s + insulin) conditions. Kinase activity is presented as relative-fold activity compared with that in the unstimulated control cells and is a combination of three independent experiments.

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    Fig. 2.

    Proliferation response to in vitro tamoxifen and rapamycin treatment. Control (▪), myrAkt1 clone 13 (□), and myrAkt1 clone 29 () MCF-7 cells were grown for 96 hours in the presence of increasing concentrations of tamoxifen (0, 10-9 to 10-6), either alone (left panel), or with 20 nmol/L rapamycin (right panel). Growth was assessed by MTT dye conversion and presented as the percentage of cell growth compared with vehicle-treated cells grown in the same culture plate at 96 hours. The data presented were the average of four replicate experiments.

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    Fig. 3.

    Rapamycin effects on ER-α. A, protein lysates from control (C) and myrAkt1 (A) MCF-7 cells that had been serum starved or placed in 10% FBS-containing media with vehicle (10% FBS), 10-7 mol/L tamoxifen, 20 nmol/L rapamycin, both tamoxifen and rapamycin, or 100 nmol/L insulin were subjected to immunodetection for expression of phosphorylated p70s6k, phosphorylated ER-α at the 118 serine residue, total ER-α, or actin as a loading control. B, ER-α activity was assessed in the control (▪) and myrAkt1 (□) cells with an ERE-tk-luciferase reporter. Activity was assessed under unstimulated (serum starved) or stimulated (10% FBS) conditions, as well as stimulated conditions with 10-7 mol/L tamoxifen, 20 nmol/L rapamycin, or a combination of tamoxifen and rapamycin. Results are the combination of three independent experiments done in triplicate and are presented as luciferase units normalized to Renilla.

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    Fig. 4.

    CCI-779 modulation of mTOR activity in vivo. Control (C) and clone 13 myrAkt1 (A) xenografts were harvested from nude mice 24 hours after treatment with vehicle alone (control), CCI-779, tamoxifen, or a combination of CCI-779 and tamoxifen (combination). Protein lysates from the tumors were subjected to immunodetection with antibodies to the phospho-Ser473 residue of Akt on both the endogenous (p-endogenous Akt), as well as the myrAkt1 transgene product (p-myrAkt1), total Akt, phosphorylated and total p70s6k (p- and total p70s6k, respectively), phosphorylated and total 4EBP1 (p- and total 4EBP1, respectively), the ER and actin. Figures presented are representative of at least three tumors from each treatment group per cell line.

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    Fig. 5.

    In vivo growth responses to tamoxifen and CCI-779. A. Control (solid line) and myrAkt1 (dashed line) xenograft tumor growth was recorded the day 17 of drug treatment initiation (baseline) and on days 3, 8, 10, 15, and 17. Growth of tumors in mice treated with vehicle (squares) were compared to those in mice treated with tamoxifen (top panel, triangles), CCI-779 (middle panel, circles) and the combination of CCI-779 and tamoxifen (bottom panel, diamonds) B. Control (solid line) and myrAkt1 (dashed line) xenografts were collected from nude mouse hosts after day 17 of treatment with vehicle alone (Control squares), tamoxifen (triangles), CCI-779 (circles), or a combination of tamoxifen and CCI-779 (Combination, diamond), and assessed for tumor volume. The data are presented as the mean and the SEM. C. Tumor growth inhibition on each treatment (CCI-779, tamoxifen, and the combination of CCI-779 and tamoxifen) was calculated as: 100% − (the percentage of tumor growth on treatment compared to vehicle-treated tumors).

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    Fig. 6.

    In vivo apoptosis assessment. Control (top panels, A–D) and myrAkt1 (bottom panel, E–H) tumors from animals treated with vehicle (A and E), CCI-779 (B and F), tamoxifen (C and G), or the combination of CCI-799 with tamoxifen (D and H) were harvested after 17 days treatment, processed for histology, and analyzed for terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Positive terminal deoxynucleotidyl transferase-mediated nick end labeling staining is highlighted with arrows. Magnification, ×200.

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  • Table 1

    Flow cytometry analysis of control and myrAkt1 MCF-7 cells exposed to different treatments

    ControlmyrAkt1
    ControlRapa (20 nmol/L)Tam (10−7)Rapa + TamControlRapa (20 nmol/L)Tam (10−7)Rapa + Tam
    G127 ± 2 *23 ± 325 ± 225 ± 145 ± 446 ± 344 ± 141 ± 2
    S54 ± 357 ± 257 ± 152 ± 236 ± 137 ± 340 ± 339 ± 2
    G218 ± 320 ± 118 ± 223 ± 319 ± 317 ± 116 ± 220 ± 1
    Sub-G118 ± 223 ± 335 ± 339 ± 316 ± 215 ± 114 ± 326 ± 1
    • NOTE. Control and myrAkt1 MCF-7 cells were exposed for 24 hours to either vehicle (control), 20 nmol/L rapamycin (Rapa), 10-7 mol/L tamoxifen (Tam) or the combination of the two, then assessed for cell cycle status, as well as apoptotic (sub-G1) status.

    • * Each value represents the average ± SD of three independent observations.

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Clinical Cancer Research: 10 (23)
December 2004
Volume 10, Issue 23
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Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity
Linda A. deGraffenried, William E. Friedrichs, Douglas H. Russell, Elissa J. Donzis, Amanda K. Middleton, Jessica M. Silva, Richard A. Roth and Manuel Hidalgo
Clin Cancer Res December 1 2004 (10) (23) 8059-8067; DOI: 10.1158/1078-0432.CCR-04-0035

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Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity
Linda A. deGraffenried, William E. Friedrichs, Douglas H. Russell, Elissa J. Donzis, Amanda K. Middleton, Jessica M. Silva, Richard A. Roth and Manuel Hidalgo
Clin Cancer Res December 1 2004 (10) (23) 8059-8067; DOI: 10.1158/1078-0432.CCR-04-0035
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