Figure 1. Depletion of TACC3 causes premature senescence and upregulation of the Interleukin 6-Receptor. (A) MCF7 cells stably expressing a doxycycline-inducible shRNA targeting TACC3 or a control shRNA were seeded at day 0 in medium containing 5 µg/ml doxycycline (DOX). Cells were supplemented every 2 d with fresh medium containing doxycycline. Cells were stained for senescence-associated β-galactosidase (SA-βgal) activity at days 2, 4, and 6. Microscopic pictures are representative for 3 independent experiments. (B) IL-6R mRNA levels in MCF7 cells were measured on day 4. Cells expressing either control shRNA or TACC3 shRNA were treated with doxycycline and compared with untreated cells. The results show the mean ± s. d. of 3 independent experiments. (C) Cells were treated as described in (A) and harvested at days 0.5, 1, 2, 4, and 6. The amount of IL-6R in the cell lysate was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. (D) Cells were treated as described in (A) and harvested at days 0.5, 1, 2, 4, and 6. The amount of IL-6R in the cell supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. (E) MCF7 cells stably expressing a DOX-inducible TACC3-specific or a control shRNA were treated as described under panel (A). On day 6, cells were starved for 4 h in serum-free medium and stimulated with IL-6 (2 ng/ml). Cells were lysed at the time points indicated and phosphorylation of STAT3 assessed via western blotting. One of 2 experiments with similar outcome is shown. (F and G) MCF10a cells stably expressing a DOX-inducible shRNA either targeting human TACC3 or an unrelated control were seeded at day 0 in medium containing DOX. Cells were supplemented every 2 d with fresh medium containing DOX. Cells were harvested at days 4, 6, and 8. The amount of IL-6R (F) in the cell lysate or (G) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments.

Figure 2. Ectopic overexpression of oncogenic Ras causes senescence and transient IL-6R upregulation. (A) MCF7 cells stably expressing doxycycline-inducible HRAS^G12V were seeded on day 0 in medium containing 5 µg/ml DOX. Microscopic pictures were taken at days 2, 4, and 6 and are representative for 3 independent experiments. (B and C) MCF7 cells stably expressing a DOX-inducible HRAS^G12V were seeded on day 0 in medium containing 5 µg/ml DOX and harvested after the indicated time. The amount of IL-6R (B) in the cell lysate or (C) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments.

Figure 3. Inhibition of mTOR by rapamycin prevents IL-6R upregulation and premature senescence. (A) MCF7 cells stably expressing DOX-inducible TACC3-specific or control shRNAs were each seeded at day 0 in medium containing 5 µg/ml DOX and either 500 ng/ml rapamycin or the solvent DMSO. Medium was exchanged every 2 d and fresh DOX and 500 ng/ml rapamycin or DMSO added. Microscopic pictures were taken at days 2, 4, and 6 and are representative for 3 independent experiments. (B and C) Cells were treated as described under panel (A). Cells were harvested at the indicated time points and the amount of IL-6R (B) in the cell lysate or (C) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments.

Figure 4. The soluble IL-6R is partly generated through constitutive shedding by the metalloprotease ADAM10. (A) MCF7 cells stably expressing a DOX-inducible TACC3-specific or a control shRNA were seeded at day 0 in medium containing 5 µg/ml DOX. Medium was exchanged every 2 d and fresh DOX added. On day 6, medium was replaced with 1 ml serum-free medium and cells were stimulated with 1 µM ionomycin or DMSO as control for 60 min. Where indicated, cells were pre-incubated with the ADAM10-specific inhibitor GI254023X (GI) for 30 min. The amount of shIL-6R in the supernatant was quantified via ELISA. (B) Cells were treated as described under panel (A), but stimulated with PMA for 2 h and pre-incubated with the combined ADAM10/ADAM17-specific inhibitor GW280264X (GW) where indicated. All results show the mean ± s. d. of 3 independent experiments. (C and D) MCF7 cells stably expressing a DOX-inducible shRNA targeting human TACC3 were seeded at day 0 in medium containing 5µg/ml doxycycline and either 3 µM of the ADAM10-specific inhibitor GI, 3 µM of the combined ADAM10/ADAM17-specific inhibitor GW or the solvent DMSO. Medium was exchanged every 2 d and fresh DOX and 3 µM GI, 3 µM GW, or DMSO added. Cells were harvested at the indicated time points and the amount of IL-6R (C) in the cell lysate or (D) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. n.d., not detectable.

Figure 5. Activation of the glucocorticoid receptor causes IL-6R upregulation and increased sIL-6R generated by ADAM10. (A and B) MCF7 cells were seeded on day 0.1 µM dexamethasone or the solvent DMSO as control were added 24 h later, and the cells were harvested at the indicated time points. The amount of IL-6R (A) in the cell lysate or (B) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. (C) MCF7 cells were treated as described under panel (A). Besides 1 µM dexamethasone or DMSO as control, cells were incubated with the ADAM10-specific inhibitor GI254023X (GI) or the combined ADAM10/ADAM17-specific inhibitor GW280264X (GW) where indicated. Cells were harvested after 20 h, and the amount of sIL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. (D and E) MCF7 cells were seeded on day 0 and stimulated with either 1 µM dexamethasone, 1 µM dexamethasone together with 5 µM of the glucocorticoid receptor antagonist RU486, or 5 µM RU486 alone. Cells were harvested at the indicated time points and the amount of IL-6R (D) in the cell lysate or (E) of soluble IL-6R in the supernatant was measured via ELISA. The results show the mean ± s. d. of 3 independent experiments. (F) MCF7 cells stably expressing a doxycycline-inducible shRNA targeting TACC3 were seeded at day 0 in medium containing 5 µg/ml DOX. Medium was exchanged every 2 d, and fresh DOX was added. On day 6, medium was replaced by fresh medium containing doxycycline with either DMSO, 1 µM dexamethasone, 1 µM dexamethasone plus 5 µM RU486, or 5 µM RU486 alone. Cells were harvested 24 h after treatment on day 7. The results show the mean ± s. d. of 3 independent experiments.

Figure 6. Activation of mTOR by EGF drives IL-6R expression. (A and B) MCF7 (A) or HepG2 (B) cells were starved 24 h post-seeding for one day in serum-free medium. Medium was then replaced either with serum-free medium or medium supplemented with 10% serum. Recombinant EGF (20 ng/ml) and/or rapamycin (500 nM) were added when indicated. Cells were harvested 24 h later. (C) HepG2 cells were treated as described above and harvested after 24 h. The amounts of IL-6R mRNA were determined as described under “Materials and Methods”. The results show the mean ± s. d. of 3 independent experiments, each performed in triplicates. (D) MCF7 cells were treated as described under panel (B), serum starved for 3 h, and stimulated with 2 ng/ml recombinant IL-6 for 15 min. Stimulation with hyper-IL-6 served as positive control. Phosphorylation of STAT3 was assessed by western blotting. One of 2 performed experiments with similar outcome is shown. (E) Primary murine hepatocytes were stimulated with recombinant EGF (20 ng/ml) and/or rapamycin (500 nM) where indicated or left untreated. Cells were harvested 24 h later and IL-6R levels determined via ELISA. The amount of IL-6R in unstimulated cells was considered 100%, and the other samples were calculated accordingly. The results show the mean ± s.d. of 3 independent experiments. (F) The amount of smIL-6R in the serum of wild-type and PTEN^+/− mice (n = 3 each) was determined via ELISA (mean ± s. d.).