mTORC2 is required for proliferation and survival of TSC2-null cells

Abstract

Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

Fig. 1.

(A and B) Rheb, mTOR, and raptor, but not rictor, are required for S6 phosphorylation in TSC2-null cells. (A) Cells were comicroinjected with siRNA specific to Rheb, mTOR, raptor, or rictor or control scrambled siRNA and GFP; then, dual immunocytochemical analysis was performed with phospho-S6 antibody (red) and GFP (green) to detect microinjected cells (arrowheads). (B) The data represent the percentage of P-S6-positive cells per total number of microinjected cells (mean values ± SE) by ANOVA (Bonferroni-Dunn). *, P < 0.001 for control siRNA versus siRNA for Rheb, siRNA for mTOR, and siRNA for raptor. (C) TSC2-null cells were transfected with 50 ng/ml specific siRNAs (+) or scrambled control siRNA (−); 48 h posttransfection, protein levels were detected by immunoblot analysis with specific anti-Rheb, anti-mTOR, anti-rictor, and anti-raptor antibodies. The lanes separated by white lines were run on the same gel but were noncontiguous. (D) Rheb, mTOR, raptor, and rictor are required for TSC2-null cell proliferation. Cells were comicroinjected with siRNA for mTOR, siRNA for Rheb, siRNA for raptor, or siRNA for rictor or scrambled control siRNA, followed by a BrdU incorporation assay. The mitotic index represents the percentage of BrdU-positive microinjected cells compared to the total number of microinjected cells. The data represent mean values ± SE by ANOVA (Bonferroni-Dunn). *, P < 0.001 for control siRNA versus Rheb, mTOR, raptor, and rictor siRNAs.

Fig. 2.

(A) Reexpression of TSC2 inhibits RhoA GTPase activity in TSC2-null cells. Shown is a RhoA activation assay of serum-deprived cells transfected with GFP-TSC2 (+) or control GFP (−). Reexpression of TSC2 was confirmed by immunoblot analysis. The images are representative of three separate experiments. (B) siRNA for rictor inhibits P-Ser⁴⁷³-Akt in TSC2-null and LAMD cells. Serum-deprived cells, transfected with siRNA for rictor or control scrambled siRNA, were subjected to immunoblot analysis with specific antibodies to detect the indicated proteins. (C) siRNA for rictor inhibits RhoA activity in TSC2-null and LAMD cells. Serum-deprived cells were transfected with siRNA for rictor or control scrambled siRNA, and RhoA activity was measured using an active Rho pulldown assay followed by immunoblot analysis with anti-RhoA antibodies. Total RhoA was used as an internal control. rictor downregulation was confirmed by immunoblot analysis. (D) Statistical analysis of mTORC2-dependent inhibition of RhoA in TSC2-null and LAMD cells from two independent experiments (mean values ± SE) by ANOVA (Bonferroni-Dunn). RhoA-GTP/total RhoA ratios for serum-deprived cells transfected with control scrambled siRNA were taken as 1-fold.

Fig. 3.

(A to D) RhoA GTPase contributes to TSC2-null and LAMD cell proliferation. (A and B) C3 transferase inhibits DNA synthesis in TSC2-null and LAMD cells. Serum-deprived TSC2-null (A) or primary human LAMD (B) cells were incubated with different concentrations of C3 transferase or diluent for 18 h, followed by DNA synthesis analysis using a BrdU incorporation assay. The data represent the percentage of BrdU-positive cells per total number of cells (mean values and SE by ANOVA [Bonferroni-Dunn]); 200 cells per condition were analyzed in each experiment. (C) TSC2-null cells were transfected with siRNA for RhoA or control (scrambled) siRNA (contr) and serum deprived, and then immunoblot analysis with anti-RhoA, anti-total actin, anti-phospho-S6, and anti-total S6 antibodies was performed. (D) siRNA for RhoA inhibits TSC2-null and LAMD cell proliferation. Serum-deprived cells were transfected with siRNA for RhoA or control siRNA for 72 h, followed by DNA synthesis analysis using a BrdU incorporation assay. DNA synthesis in cells transfected with control siRNA was taken as 100%. The data represent mean values ± SE from three independent experiments by ANOVA (Bonferroni-Dunn); 200 cells per condition were analyzed in each experiment. (E and F) HA1077 attenuates TSC2-null and LAMD cell proliferation. Serum-deprived TSC2-null ELT3 (E) and LAMD (F) cells were incubated with diluent or different concentrations of HA1077 for 18 h, followed by DNA synthesis analysis using the BrdU incorporation assay. The data represent the percentage of BrdU-positive cells per total number of cells (mean values ± SE by ANOVA [Bonferroni-Dunn]); 200 cells per condition were analyzed in each experiment.

Fig. 4.

Constitutively active RhoA GTPase rescues rictor siRNA, TSC2-, and TSC1-siRNA induced inhibition of DNA synthesis in TSC2-null cells. Cells were transfected with siRNA for rictor (A), GFP-TSC2 (B), and siRNA for TSC1 (C) alone or in combination with constitutively active RhoA mutant GST-V14RhoA. GFP and GST plasmids were used as controls. Then, the cells were serum deprived for 24 h, followed by DNA synthesis analysis using the BrdU incorporation assay. The data represent the percentage of BrdU-positive cells per total number of cells (taken as 100%). The data are mean values ± SE from two independent experiments by ANOVA (Bonferroni-Dunn). A minimum of 200 cells per condition were counted in each experiment.

Fig. 5.

(A) RhoA is required for TSC2-null cell survival. Cells transfected with control (scrambled) or RhoA for siRNA for 48 and 72 h were resuspended in annexin V binding buffer, incubated with annexin V conjugate, and then plated on glass slides. Apoptotic cells were visualized using an Eclipse E400 microscope under ×200 magnification with appropriate filters. (Left) Representative images of three independent experiments. (Right) Statistical analysis. The data represent the percentage of apoptotic cells per total number of cells (taken as 100%) (mean values ± SE from three independent repetitions). A minimum of 200 cells were analyzed per experimental condition. *, P < 0.001 for siRNA for RhoA versus control siRNA by ANOVA (Bonferroni-Dunn). (B) C3 transferase promotes apoptosis in TSC2-null cells. (Left) Serum-deprived cells were incubated with different concentrations of C3 transferase or diluent for 18 h, followed by apoptosis analysis using the In Situ Cell Death Detection kit. (Right) The data represent the percentage of apoptotic cells per total number of cells (taken as 100%); 200 cells per condition were analyzed in each experiment. The data are mean values and SE. *, P < 0.001 for C3 transferase versus diluent by ANOVA (Bonferroni-Dunn). (C and D) C3 transferase modulates levels of Bcl2 family proteins. Serum-deprived TSC2-null (C) and LAMD (D) cells were treated with 1 μg/ml C3 transferase (C3 transf) (+) or diluent (−), followed by immunoblot analysis with specific antibodies to detect the indicated proteins. The images are representative of two separate experiments. (E) Tsc2^+/+ and Tsc2^−/− MEFs with p53 (top) and Tsc2^+/+ p53^−/− and Tsc2^−/− p53^−/− MEFs with p53 deleted (bottom) were maintained in serum-free medium with 1 μg/ml C3 transferase (+) or diluent (−) for 18 h, and then flow cytometry analysis with primary annexin V and secondary FITC-conjugated antibodies was performed. The negative control included diluent-treated cells incubated with matched IgG and secondary FITC-conjugated antibody. The data represent the percentage of annexin V-positive cells per total number of cells.

Fig. 6.

Simvastatin inhibits RhoA GTPase activity. Serum-deprived TSC2-null (A) and LAMD (B) cells were treated with the indicated concentrations of simvastatin (Simva) or diluent, followed by Rho activation assay. (Top) RhoA-GTP was pulled down with Rhotekin-RBD agarose, followed by immunoblot analysis with anti-RhoA antibodies. Total RhoA was used as an internal control. The images are representative of two independent experiments. (Bottom) Statistical analysis of experiments. The data represent mean values ± SE from two independent experiments. (A) *, P < 0.05 for 1 μM simvastatin versus diluent; **, P < 0.001 for 10 μM simvastatin versus diluent. (B) *, P < 0.001 for simvastatin versus diluent by ANOVA (Bonferroni-Dunn).

Fig. 7.

(A and B) Effects of simvastatin and rapamycin on DNA synthesis in TSC2-null and LAMD cells. TSC2-null (A) and LAMD (B) cells serum deprived for 24 h were treated with the indicated concentrations of simvastatin (Simva) or diluent alone or in the presence of 20 nM rapamycin (RAPA) for 18 h, followed by DNA synthesis analysis using the BrdU incorporation assay. The data represent the percentage of BrdU-positive cells per total number of cells (mean values ± SE from three independent experiments). A minimum of 200 cells were analyzed per condition in each experiment. (A) *, P < 0.05 for 1 μM simvastatin versus diluent; **, P < 0.001 for 10 μM simvastatin versus diluent; ***, P < 0.001 for simvastatin plus RAPA versus RAPA. (B) *, P < 0.001 for simvastatin versus diluent; **, P < 0.01 for simvastatin plus RAPA versus RAPA by ANOVA (Bonferroni-Dunn). (C and D) Simvastatin and rapamycin inhibit LAMD cell growth. Serum deprived for 24 h, cells were maintained for 14 days in serum-free medium containing the indicated concentrations of simvastatin (Simva) or diluent (C) or 0.5 μM simvastatin and 20 nM RAPA separately or in combination (D) that was changed daily. Cell counts were performed every second day with three repetitions for each condition. (C) *, P < 0.001 for diluent-treated cells on day 0 versus diluent-treated cells on days 2 to 14; **, P < 0.01 for 0.3 μM simvastatin versus diluent; ***, P < 0.001 for 0.5 μM simvastatin versus diluent. (D) *, P < 0.001 for diluent-treated cells on day 0 versus diluent-treated cells on days 2 to 14; **, P < 0.001 for simvastatin versus diluent; ***, P < 0.001 for RAPA versus diluent; ****, P < 0.01 for simvastatin plus RAPA versus simvastatin and for simvastatin plus RAPA versus RAPA by ANOVA (Bonferroni-Dunn).

Fig. 8.

(A and B) Simvastatin induces apoptosis in TSC2-null cells. Serum-deprived cells were treated with 0.1, 1, and 10 μM simvastatin (Simva) or diluent in the presence or absence of 20 nM rapamycin (RAPA) for 24 h, followed by apoptosis analysis using the In Situ Cell Death Detection Kit. (A) Representative images from three independent experiments were taken using an Eclipse TE2000-E microscope at ×400 magnification with appropriate filters. (B) Statistical analysis. The data represent the percentage of apoptotic cells per total number of cells (taken as 100%) (mean values ± SE from three independent experiments by ANOVA [Bonferroni-Dunn]). A minimum of 200 cells were analyzed per condition in each experiment. (C and D) Activated RhoA rescues simvastatin-induced apoptosis in TSC2-null cells. (C) Cells were transfected with GST-V14RhoA or mock transfected and treated with 1 μM simvastatin or diluent, and then dual immunocytochemical analysis to detect cleaved caspase 3 and GST was performed. (D) The data represent mean values ± SE by ANOVA (Bonferroni-Dunn). A minimum of 200 cells were examined per condition in each experiment.

Fig. 9.

(A) Schematic representation of the experimental design. NCRNU-M female athymic nude mice with TSC2-null ELT3 subcutaneous tumors were subjected to a 50-day treatment with rapamycin, simvastatin, and the combination of rapamycin and simvastatin. Untreated animals were used as controls. Upon treatment termination, mice were monitored for tumor recurrence either until the tumors reached 10% of the animal's body weight or for 9 months after treatment withdrawal. IHC, immunohistochemical; IB, immunoblotting. (B) Rapamycin and simvastatin improve the survival of mice bearing TSC2-null tumors. Survival analysis was performed using as the termination time the time when the tumor diameter reached 10 mm. Untreated mice were used as controls. A minimum of 18 mice were analyzed per experimental condition. (C) Effects of simvastatin and rapamycin on TSC2-null tumor growth in nude mice. Mice were treated as described above, and tumor growth was monitored using calipers. The data represent mean values ± SE by ANOVA (Bonferroni-Dunn). (D and E) Simvastatin prevents posttreatment tumor regrowth. After 50 days of treatment with simvastatin and rapamycin separately or in combination, five mice from each group with visible tumor disappearance were subjected to treatment withdrawal; the animals were monitored for tumor recurrence for 9 months or until the tumors reached 10% of total body weight. (D) The data represent mean values ± SE. *, P < 0.01 for rapamycin-pretreated mice versus simvastatin- or simvastatin-rapamycin-pretreated mice by ANOVA (Bonferroni-Dunn). (E) Representative images of mice at day 21 (top) and 4 months (bottom) after rapamycin and rapamycin-simvastatin treatment withdrawal. The circle indicates the recurrent tumor in the rapamycin-pretreated mouse.

Fig. 10.

(A and B) Simvastatin promotes apoptosis in TSC2-null tumors. Tumor tissues collected at days 0, 10, and 20 of the experiment were analyzed using the In Situ Cell Death Detection Kit (green); DAPI staining (blue) was performed to detect nuclei. (A) Representative images of tumors collected at day 20 of treatment were taken using an Eclipse TE2000-E microscope at ×200 magnification with appropriate filters. (B) Statistical analysis. The data (mean values ± SE; tumors from a minimum of five animals per treatment condition were analyzed) represent the percentage of apoptotic cells per total number of cells (taken as 100%). A minimum of 300 cells were analyzed per condition in each tumor. *, P < 0.001 for simvastatin on day 10 versus the control on day 10, for simvastatin on day 10 versus RAPA on day 10, for RAPA plus simvastatin on day 10 versus the control on day 10, for RAPA plus simvastatin on day 10 versus RAPA on day 10, for simvastatin on day 20 versus the control on day 20, for simvastatin on day 20 versus RAPA on day 20, for RAPA plus simvastatin on day 20 versus the control on day 20, and for RAPA plus simvastatin on day 20 versus RAPA on day 20 by ANOVA (Bonferroni-Dunn). (C and D) Simvastatin and rapamycin inhibit TSC2-null cell growth in vivo. Tumor tissues were collected on days 0, 10, and 20 of the experiment and subjected to immunohistochemical analysis with anti-Ki67 antibody (red). DAPI staining (blue) was performed to detect nuclei. (C) Representative images of tumors collected at day 20 of treatment were taken using an Eclipse E400 microscope at ×200 magnification with appropriate filters. (D) Statistical analysis. The data (mean values ± SE; tumors from a minimum of five animals per treatment condition were analyzed) represent the percentage of Ki67-positive cells per total number of cells (taken as 100%). A minimum of 300 cells were analyzed per condition in each tumor. *, P < 0.01 for RAPA on day 10 versus the control on day 10 and for RAPA on day 20 versus the control on day 20; **, P < 0.05 for simvastatin on day 20 versus the control on day 20; ***, P < 0.01 for RAPA plus simvastatin on day 10 versus RAPA on day 10, for RAPA plus simvastatin on day 10 versus simvastatin on day 10, for RAPA plus simvastatin on day 20 versus RAPA on day 20, and for RAPA plus simvastatin on day 20 versus simvastatin on day 20 by ANOVA (Bonferroni-Dunn).

Fig. 11.

TSC2-null tumors collected from control and rapamycin (RAPA)-, simvastatin-, and RAPA plus simvastatin-treated mice at days 45, 41, 34, and 32 of the experiment, respectively, were subjected to immunohistochemical analysis with anti-P-S6 (red) and anti-Ki67 (red) antibodies; apoptosis was examined using a TUNEL-based In Situ Death Detection Kit (green). DAPI staining (blue) was performed to detect nuclei. The images were taken using an Eclipse E400 microscope at ×200 magnification with appropriate filters.

Fig. 12.

Rapamycin, but not simvastatin, inhibits S6 phosphorylation in TSC2-null tumors. (A and B) Tumor tissues collected at days 0, 10, and 20 of the experiment were subjected to immunocytochemical analysis with anti-phospho-S6 antibody (red). DAPI staining (blue) was performed to detect nuclei. (A) Representative images of tumors collected at day 20 of treatment were taken using an Eclipse TE2000-E microscope at ×200 magnification with appropriate filters. (B) Statistical analysis. The data represent mean values ± SE; tumors from a minimum of five animals per treatment condition were analyzed. The P-S6 OD at day 0 was taken as 100%. *, P < 0.001 for rapamycin (RAPA) on day 10 versus the control on day 10 and for RAPA plus simvastatin on day 10 versus the control on day 10; **, P < 0.001 for RAPA on day 20 versus the control on day 20 and for RAPA plus simvastatin on day 20 versus the control on day 20 by ANOVA (Bonferroni-Dunn). (C) Immunoblot analysis of tumor tissues collected at days 15, 30, and 40 of the experiment with anti-phospho-S6, anti-S6, anti-phospho-Akt(Ser-473), and anti-Akt antibodies. Representative images from three independent experiments are shown. The arrowheads indicate tissues collected at day 30 of the experiment. (D) Statistical analysis of tissues collected at day 30 of the experiment. The data are mean values ± SE from three independent experiments by ANOVA (Bonferroni-Dunn).