Signal transducer and activator of transcription 3 is required for abnormal proliferation and survival of TSC2-deficient cells: relevance to pulmonary lymphangioleiomyomatosis

Abstract

Tumor suppressor complex TSC1/TSC2 represents a key negative regulator of mammalian target of rapamycin (mTOR)-S6 kinase 1 signaling. Mutational inactivation of TSC1 or TSC2, linked to a rare lung disease, lymphangioleiomyomatosis (LAM), manifests as neoplastic growth of smooth-muscle (SM)-like cells and cystic destruction of the lungs that induces loss of pulmonary function. However, the precise mechanisms of abnormal cell growth in LAM remain uncertain. Here, we demonstrate increased signal transducer and activator of transcription (STAT) 3 expression, phosphorylation, and nuclear localization in SM-like cells in LAM lungs and in TSC2-null xenographic tumors. Treatment of TSC2-null tumors with mTOR inhibitor rapamycin attenuated STAT3 expression and phosphorylation. Increased STAT3 level and activation were also observed in LAM-dissociated (LAMD) cell cultures compared with normal human bronchus fibroblasts (HBFs) from LAM patients. Although interferon (IFN)-gamma inhibited proliferation of HBFs, IFN-gamma treatment had little effect on proliferation of LAMD and TSC2-null cells. Re-expression of TSC2 or treatment with rapamycin inhibited IFN-gamma-induced STAT3 phosphorylation and synergized with IFN-gamma in inhibiting TSC2-null and LAMD cell proliferation. Reduction of STAT3 protein levels or activity using specific small interfering RNA or inhibitory peptide, respectively, decreased proliferation and induced apoptosis in TSC2-null and LAMD cells and sensitized cells to growth-inhibitory and proapoptotic effects of IFN-gamma. Collectively, our data demonstrate that STAT3 activation is required for proliferation and survival of cells with TSC2 dysfunction, that STAT3 impedes growth-inhibitory and proapoptotic effects of IFN-gamma, and that TSC2- and rapamycin-dependent inhibition of STAT3 restores antiproliferative effects of IFN-gamma. Thus, STAT3 may provide a novel therapeutic target for diseases associated with TSC1/TSC2 dysfunction.

Figures

Fig. 1.

IFN-γ has little effect on LAMD and TSC2-null ELT3 cell proliferation. A, serum-deprived ELT3 cells were treated with 10, 100, and 1000 U/ml rat IFN-γ or diluent for 18 h in the absence or presence 10 ng/ml PDGF followed by BrdU incorporation assay. Data represent mean values ± S.E. from three independent experiments by ANOVA (Bonferroni-Dunn). B and C, serum-deprived LAMD cells and HBFs were treated with 10, 100, and 1000 U/ml human IFN-γ or diluent for 18 h in the absence (B) or presence (C) of 10 ng/ml PDGF followed by [3H]thymidine incorporation assay. Data represent mean values ± S.E. from three independent experiments. *, p < 0.001 for HBFs treated with 100 U/ml IFN-γ + PDGF or 1000 U/ml IFN-γ + PDGF versus PDGF-treated HBFs by ANOVA (Bonferroni-Dunn).

Fig. 2.

STAT3 is activated in nonstimulated LAMD cells. A, immunocytochemical analysis of serum-deprived LAMD cells and HBFs with anti-STAT3 antibody. Representative images of three separate experiments were taken using a TE2000E microscope (Nikon) at 400× magnification. B, statistical analysis of three independent experiments. Data represent a percentage of cells with STAT3 nuclear localization per total number of cells. Data are mean values ± S.E. by ANOVA (Bonferroni-Dunn). At minimum, 60 cells per each condition were analyzed in each experiment. C, cytoplasmic (C) and nuclear (N) fractions of serum-deprived LAMD cells and HBFs were subjected to immunoblot analysis with anti-phospho-Tyr705STAT3, anti-total STAT3, and anti-β-actin antibodies. Top, representative images of two independent experiments. Middle and bottom, statistical analysis of two independent experiments. Phospho-STAT3 optical density (OD) in nuclear fraction of LAMD cells (middle) or total STAT3 OD in cytoplasmic fraction of LAMD cells (bottom) was taken as one-fold.

Fig. 3.

Increased STAT3 expression and phosphorylation in SM-like cells in LAM lungs (A and B) and in TSC2-null tumors (C and D). Tissue specimens from the lungs of LAM patients (LAM lungs), normal human lungs, and normal human trachea were subjected to dual immunohistochemical analysis with anti-phospho-Tyr705STAT3 (red) and anti-SM α-actin (green) (A); or anti-total STAT3 (red) and anti-phospho-S6 (green) antibodies (B). DAPI staining (blue) was performed to visualize the nuclei. Representative images from four LAM patients were taken using an Eclipse E400 microscope (Nikon) at 400× magnification. Arrows indicate phospho-STAT3 (A) or total STAT3 (B) nuclear localization. V, blood vessel. C and D, TSC2-null xenographic tumors in NCRNU-M nude female mice were subjected to immunohistochemical analysis with anti-phospho-Tyr705STAT3 (red) antibody (C) or dual immunostaining with anti-total STAT3 (red) and anti-phospho-S6 (green) antibodies (D). Arrows indicate cells with nuclear localization of phospho-STAT3 (C) or cells with colocalization of cytosolic total STAT3 and phospho-S6 (D). Images are representative of two independent experiments. Images were taken using an Eclipse E400 microscope at 400× magnification with appropriate filters.

Fig. 4.

A to C, STAT3 is required for abnormal proliferation of LAMD and ELT3 cells. A, LAMD cells were transfected with siRNA STAT3 or control siRNA GLO for 72 h, serum-deprived for 24 h, and then treated with 100 U/ml IFN-γ, 200 nM RAPA, separately or in combination, or diluent for 18 h followed by BrdU incorporation assay. Data represent a percentage of BrdU-positive cells per total number of cells. Data represent means + S.E. from three independent experiments. *, p < 0.001 for siRNA GLO + RAPA versus siRNA GLO; **, p < 0.001 for siRNA GLO + RAPA + IFN-γ versus siRNA GLO + RAPA; ***, p < 0.001 for siRNA STAT3 versus siRNA GLO; ****, p < 0.001 for siRNA STAT3 + IFN-γ versus siRNA STAT3; *****, p < 0.001 for siRNA STAT3 + RAPA versus siRNA STAT3; ******, p < 0.01 for siRNA STAT3 + IFN-γ + RAPA versus siRNA STAT3 + IFN-γ or siRNA STAT3 + RAPA by ANOVA (Bonferroni-Dunn). STAT3 knockdown by siRNA STAT3 was confirmed by immunoblot analysis with anti-STAT3 and anti-β-actin antibodies to detect equal loading (top). B and C, serum-deprived for 24 h LAMD (B) and ELT3 (C) cells were treated with 100 μM STAT3 inhibitory peptide, 100 U/ml IFN-γ, 200 nM RAPA, or diluent, separately or in the combination, for 18 h, and then BrdU incorporation assay was performed. Data represent a percentage of BrdU-positive cells per total number of cells. Data represent means + S.E. from three independent experiments. B, *, p < 0.001 for RAPA versus diluent; **, p < 0.01 for RAPA + IFN-γ versus RAPA; ***, p < 0.001 for STAT3 inhibitory peptide versus diluent; ****, p < 0.001 for STAT3 inhibitory peptide + IFN-γ versus STAT3 inhibitory peptide; *****, p < 0.001 for STAT3 inhibitory peptide + RAPA versus STAT3 inhibitory peptide; ******, p < 0.001 for STAT3 inhibitory peptide + IFN-γ + RAPA versus STAT3 inhibitory peptide + IFN-γ or STAT3 inhibitory peptide + RAPA. C, *, p < 0.001 for RAPA versus diluent; **, p < 0.05 for RAPA + IFN-γ versus RAPA; ***, p < 0.01 for STAT3 inhibitory peptide versus diluent; ****, p < 0.02 for STAT3 inhibitory peptide + IFN-γ versus STAT3 inhibitory peptide; *****, p < 0.001 for STAT3 inhibitory peptide + RAPA versus STAT3 inhibitory peptide; ******, p < 0.001 for STAT3 inhibitory peptide + IFN-γ + RAPA versus STAT3 inhibitory peptide + IFN-γ or STAT3 inhibitory peptide + RAPA by ANOVA (Bonferroni-Dunn). D, effect of IFN-γ on STAT3 phosphorylation. Serum-deprived LAMD cells and HBFs were treated with 100 U/ml IFN-γ or diluent for 30 min followed by immunoblot analysis with anti-phospho-Tyr705STAT3, anti-total STAT3, and anti-total actin antibodies. Top, representative images of three independent experiments. Bottom, statistical analysis of experiments. Phospho-STAT3 optical density (OD) per total STAT3 OD in diluent-treated LAMD cells was taken as one-fold. Data represent means + S.E. from three independent experiments by ANOVA (Bonferroni-Dunn). E, rapamycin inhibits IFN-γ-dependent STAT3 phosphorylation in TSC2-null ELT3 cells. Serum-deprived cells were preincubated with 20 and 200 nM RAPA, or diluent for 18 h, and then treated with 100 U/ml IFN-γ or vehicle for 30 min, and cell lysates were then subjected to immunoblot analysis with anti-phospho-Tyr705STAT3 or anti-STAT3 antibodies. Images are representative of two separate experiments.

Fig. 5.

STAT3 is required for LAMD cell survival. A, serum-deprived cells, transfected with siRNA STAT3 or control siRNA GLO for 72 h, were treated with 100 U/ml IFN-γ, 200 nM RAPA or diluent, separately or in combination, for 24 h followed by apoptosis analysis with In Situ Cell Death Detection kit. Data represent percentage of apoptotic cells per total number of cells. At minimum, 60 cells were analyzed in each experiment per each condition. Data represent mean values + S.E. from two independent experiments. *, p < 0.001 for siRNA STAT3 versus siRNA GLO; **, p < 0.01 for siRNA STAT3 + IFN-γ versus siRNA STAT3; ***, p < 0.001 for siRNA STAT3 + RAPA versus siRNA STAT3; ****, p < 0.05 for siRNA STAT3 + IFN-γ + RAPA versus siRNA STAT3 + IFN-γ or siRNA STAT3 + RAPA. B, serum-deprived cells were treated with 100 μM STAT3 inhibitory peptide, 100 U/ml IFN-γ, 200 nM RAPA, or diluent, separately or in combination, for 24 h, and then apoptosis analysis with In Situ Cell Death Detection kit was performed. *, p < 0.001 for STAT3 inhibitory peptide versus diluent; **, p < 0.001 for STAT3 inhibitory peptide + IFN-γ versus STAT3 inhibitory peptide; ***, p < 0.001 for STAT3 inhibitory peptide + RAPA versus STAT3 inhibitory peptide; ****, p < 0.01 for STAT3 inhibitory peptide + IFN-γ + RAPA versus STAT3 inhibitory peptide + IFN-γ or STAT3 inhibitory peptide + RAPA by ANOVA (Bonferroni-Dunn).

Fig. 6.

Rapamycin and IFN-γ have synergistic inhibitory effects on TSC2-null ELT3 and LAMD cell proliferation. A, serum-deprived ELT3 cells were preincubated with 200 nM RAPA, 100 U/ml rat IFN-γ, 200 nM RAPA + 100 U/ml IFN-γ, or diluent in the presence or absence of 10 ng/ml PDGF for 18 h followed by BrdU incorporation analysis. Data represent mean values + S.E. from three independent experiments. *, p < 0.001 for RAPA versus control and IFN-γ; **, p < 0.01 for IFN-γ + RAPA versus IFN-γ and RAPA; ***, p < 0.05 for RAPA + PDGF versus PDGF; ****, p < 0.001 for IFN-γ + RAPA + PDGF versus IFN-γ + PDGF and RAPA + PDGF. B and C, LAMD cells, serum-deprived (B) or treated with 10 ng/ml PDGF (C), were incubated with 1, 10, 100, and 1000 U/ml human IFN-γ or diluent in the presence or absence of 200 nM RAPA for 18 h, and then the [3H]thymidine incorporation assay was performed. Data represent mean values + S.E. from three independent experiments. Dotted lines indicate the levels of DNA synthesis in rapamycin-treated cells. *, p < 0.01 for 1000 U/ml IFN-γ + RAPA versus RAPA and IFN-γ; **, p < 0.001 for PDGF + IFN-γ + RAPA versus PDGF + RAPA and PDGF + IFN-γ by ANOVA (Bonferroni-Dunn).

Fig. 7.

Rapamycin abrogates STAT3 phosphorylation and attenuates total STAT3 protein levels in TSC2-null tumors. TSC2-null ELT3 cells (5 × 106) were injected in flanks of NCRNU-M female mice. When tumors reached 5 mm in diameter, mice were treated with diluent or rapamycin for 14 days. Then mice were sacrificed, and immunohistochemical analysis of tumors was performed with anti-phospho-Tyr705STAT3 antibody (red; A), or tumors were subjected to dual immunohistochemical analysis with anti-total STAT3 (red) and anti-phospho-S6 (green) antibodies (B). DAPI staining (blue) was performed to detect the nuclei. Images are representative of eight animals per each condition. Images were taken at 200× magnification using an Eclipse E400 microscope (Nikon).

Fig. 8.

A and B, re-expression of TSC2 restores inhibitory effect of IFN-γ on cell proliferation. TSC2-null ELT3 (A) or LAMD (B) cells, transfected with plasmids expressing GFP-TSC2 or control GFP, were treated with 100 U/ml IFN-γ, rat or human, respectively, or diluent in the presence or absence of 10 ng/ml PDGF for 18 h, and then BrdU incorporation assay was performed. Data represent a percentage of GFP- and BrdU-positive cells per total number of GFP-positive cells. Data represent mean values + S.E. from two independent experiments. A, *, p < 0.001 for GFP-TSC2-transfected cells versus GFP-transfected cells; **, p < 0.01 for GFP-TSC2 + PDGF versus GFP + PDGF and GFP + IFN-γ + PDGF; ***, p < 0.01 for GFP-TSC2 + IFN-γ + PDGF versus GFP-TSC2 + PDGF. B, *, p < 0.001 for GFP-TSC2-transfected cells versus GFP-transfected cells; **, p < 0.01 for GFP-TSC2 + IFN-γ versus GFP-TSC2 and GFP + IFN-γ by ANOVA (Bonferroni-Dunn). C and D, TSC2 inhibits IFN-γ-dependent STAT3 phosphorylation in TSC2-null ELT3 and LAMD cells. TSC2-null ELT3 (C) or LAMD (D) cells, transfected with plasmids expressing GFP-tagged TSC2 (GFP-TSC2) or control GFP were serum-deprived for 24 h and then treated with 100 U/ml IFN-β, 100 U/ml IFN-γ, or diluent for 30 min followed by immunoblot analysis with anti-phospho-Tyr705STAT3 and anti-STAT3 antibodies. Images are representative of two independent experiments (top). Dotted lines indicate the levels of DNA synthesis in GFP-TSC2-transfected cells treated with diluent. Quantitative analysis (bottom) was performed using Gel-Pro Analyzer software (Media Cybernetics, Inc., Bethesda, MD). Phospho-STAT3 optical density (OD) per total STAT3 OD in ELT3 (C) or LAMD (D) cells transfected with GFP was taken as one-fold. Data represent means + S.E. from two independent experiments by ANOVA (Bonferroni-Dunn).

Fig. 9.

Schematic representation of a potential mechanism of IFN-γ- and STAT3-dependent cell growth modulated by TSC2/mTOR signaling pathway. Top, in normal unstimulated cells, tumor suppressor TSC2 inhibits mTOR/S6K1 signaling pathway and cell proliferation. Treatment with IFN-γ activates canonical STAT1 signaling, induces modest phosphorylation of STAT3, and attenuates cell proliferation. Bottom, loss of TSC2 function leads to the constitutive activation of mTOR/S6K1 and increased STAT3 activity, which are both required for abnormal cell proliferation. IFN-γ induces STAT1 activation and increases STAT3 phosphorylation. Re-expression of TSC2 or inhibition of mTOR activity inhibits IFN-γ-dependent STAT3 phosphorylation and synergizes with IFN-γ in inhibition of cell proliferation.