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, 22 (23), 8184-98

Rapamycin Potentiates Transforming Growth Factor Beta-Induced Growth Arrest in Nontransformed, Oncogene-Transformed, and Human Cancer Cells

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Rapamycin Potentiates Transforming Growth Factor Beta-Induced Growth Arrest in Nontransformed, Oncogene-Transformed, and Human Cancer Cells

Brian K Law et al. Mol Cell Biol.

Abstract

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.

Figures

FIG. 1.
FIG. 1.
TGF-β and rapamycin cooperate to induce G1 cell cycle arrest of Mv1Lu cells and NMuMG cells. (A) Nontransformed Mv1Lu cells were treated for 22 h with the indicated concentrations of rapamycin. The cells were pulsed for an additional 2 h with [3H]thymidine, and thymidine incorporation (Inc.) was quantitated as described in Materials and Methods. Results are presented as the average percent control incorporation from six replicate wells ± standard deviation. (B) Nontransformed Mv1Lu cells were treated for 24 h with the indicated concentrations of TGF-β for 24 h and were processed, and the results were presented as in panel A. The last bar (TGF-β + Rapa.) represents treatment for 24 h with both 10 ng of TGF-β/ml and 100 nM rapamycin. (C) Nontransformed Mv1Lu cells were treated for 24 h with normal growth medium (Control), 10 ng of TGF-β/ml, 100 nM rapamycin, or 10 ng of TGF-β/ml plus 100 nM rapamycin. The cells were stained with propidium iodide and subjected to flow cytometry as described in Materials and Methods. (D) NMuMG cells were treated with the indicated concentrations of TGF-β1 and rapamycin either alone or in combination for 24 h, and [3H]thymidine incorporation assays were performed as for panel A.
FIG. 2.
FIG. 2.
TGF-β and rapamycin cooperate to induce Rb dephosphorylation in NMuMG cells. (A) NMuMG cells were treated for 24 h with either normal growth medium (C), 10 ng of TGF-β/ml (T), 100 nM rapamycin (R), or 10 ng of TGF-β/ml and 100 nM rapamycin (T + R) and were subjected to [3H]thymidine incorporation analyses as for Fig. 1. (B) NMuMG cells were treated as for panel A, and cell extracts were prepared and subjected to immunoblot analyses as described in Materials and Methods using antibody specific for total Rb, Rb phosphorylated on Ser249/Thr252 [P-Rb(S249/T252)], Rb phosphorylated on Thr821 [P-Rb(821)], E2F1, c-Myc, or β-tubulin as a loading control.
FIG. 3.
FIG. 3.
Rapamycin cooperates with TGF-β to induce growth arrest of cells transformed by E2F1, c-Myc, and V12H-Ras. (A) Mv1Lu cells transformed with E2F1 (E2F1-13) were treated for 24 h with normal growth medium (C), 10 ng of TGF-β/ml (T), 100 nM rapamycin (R), or 10 ng of TGF-β/ml plus 100 nM rapamycin (T + R). The cells were then subjected to [3H]thymidine incorporation assays as for Fig. 1 (left panel), or cell extracts were subjected to immunoblot analyses using antibody to total Rb, Rb phosphorylated on Ser249/Thr252 [P-Rb(S249/T252)], Rb phosphorylated on Thr821[P-Rb(T821)], E2F1, or c-Myc or β-tubulin as a loading control (right panel). Results of [3H]thymidine incorporation assays are presented as the average percent control incorporation of six replicate determinations ± standard deviation. (B) Mv1Lu cells transformed with c-Myc (c-Myc-5) were subjected to [3H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A. (C) Mv1Lu cells transformed with c-Myc2(Ala58) [c-Myc(58A)-7] were subjected to [3H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A. (D) Mv1Lu cells transformed with V12H-Ras (Ras-9) were subjected to [3H]thymidine incorporation analyses (left panel) or immunoblot analyses (right panel) as for panel A.
FIG. 4.
FIG. 4.
Rapamycin potentiation of TGF-β-induced growth arrest is mediated by rapamycin binding to FK506 binding protein(s). (A) MDA-MB-231 cells were treated for 24 h with normal growth medium (C), dimethyl sulfoxide vehicle (V), 10 ng of TGF-β/ml (T), 100 nM rapamycin (R), or 10 ng of TGF-β/ml plus 100 nM rapamycin (T + R), and 125I-TGF-β affinity cross-linking experiments were performed as described in Materials and Methods. Puro. Cont. cells (B) or DU145 cells (C) were treated with 10 ng of TGF-β/ml and various combinations of rapamycin, the Novartis rapamycin derivative RAD001, or FK506 for 24 h as indicated, and [3H]thymidine incorporation assays were performed as for Fig. 2.
FIG. 5.
FIG. 5.
TGF-β and rapamycin cooperate to inhibit the proliferation of human carcinoma cells. MDA-MB-231 (A) or DU145 human carcinoma (B) cells were subjected to [3H]thymidine incorporation assays as for Fig. 2. The values are shown over the bars. MDA-MB-231 (C) or DU145 (D) cells were plated as described in Materials and Methods and treated as for Fig. 4A and B, and cells were counted on sequential days using a Coulter counter. Results are presented as the average of triplicate determinations ± standard deviation.
FIG. 6.
FIG. 6.
DU145 cells exhibit TGF-β-dependent activation of Smad signaling. (A) DU145 cells were treated for 24 h with normal growth medium (C), 10 ng of TGF-β1/ml (T), 100 nM rapamycin (R), or 10 ng of TGF-β1/ml plus 100 nM rapamycin (T + R), and immunoblotting experiments on whole-cell extracts were performed using antibodies specific for the indicated proteins. Total extracts from HEK 293 (293) cells were used as a positive control in Rb, p107, and p130 immunoblotting experiments. (B) DU145 cells were treated as for panel A, and (CAGA)12-luciferase assays were performed as described in Materials and Methods. Assays were performed in triplicate, and results are presented as relative luciferase units (RLU) normalized to micrograms of protein assayed. (C) Luciferase assays were performed using an E2F-luciferase reporter construct, and the results are presented as for panel B.
FIG. 7.
FIG. 7.
TGF-β and rapamycin cooperate to inhibit Cdk2 activity in DU145 cells. (A) DU145 cells were treated as for Fig. 6, and cell extracts were prepared and subjected to immune-complex kinase assays using Cdk2 or Cdk4 antibody as described in Materials and Methods. GST, glutathione transferase. (B) A portion of the kinase assays was subjected to immunoblot (IB) analysis using antibody specific for Cdk4, Cdk2, p21, or p27. IP, immunoprecipitation.
FIG. 8.
FIG. 8.
TGF-β and rapamycin cooperate to inhibit Cdk2 activity in NMuMG cells. (A) NMuMG cells were treated as in Fig. 7, and cell extracts were prepared and subjected to immune-complex kinase assays using Cdk2 or Cdk4 antibody or normal rabbit IgG (Rab. IgG) as a control (top panel). A portion of the kinase assays was subjected to immunoblot (IB) analysis using antibody to Cdk2, p21, or p27 (lower panels). IP, immunoprecipitation. (B) Total protein extracts from cells treated as for panel A were analyzed by immunoblot with antibody specific for p21 or p27 or actin as a loading control.
FIG. 9.
FIG. 9.
TGF-β plus rapamycin inhibit Cdk2 activity in DU145 cells in a time-dependent manner. (A) DU145 cells were treated with normal growth medium (C) or 10 ng of TGF-β1/ml plus 100 nM rapamycin (T + R) for the indicated periods, and cell extracts were prepared and subjected to immune-complex kinase assays as for Fig. 7. IP, immunoprecipitation; IB, immunoblotting. The results of kinase assays were quantitated and plotted (B) with the control Cdk2 activity at each time point normalized to 100%. (C) Total protein extracts from cells treated as for panel A were analyzed by immunoblotting with antibodies specific for the indicated proteins. (D) Cells treated as for panel A were pulsed with [3H]thymidine for the last 2 h of each time point, and [3H]thymidine incorporation into DNA was quantitated and presented as the average of six replicate determinations ± standard deviation.
FIG. 10.
FIG. 10.
TGF-β plus rapamycin alter the composition of Cdk2 complexes in a time-dependent manner. DU145 cells were treated as for Fig. 9, and cell extracts were subjected to immunoprecipitation (IP) using Cdk2 antibodies or normal rabbit IgG (Rab. IgG). Immunoprecipitates were analyzed by immunoblotting (IB) with antibody specific for Cdk2, Cdk2 phosphorylated on Thr160, E2F4, p130, p107, p21, or p27.

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