Table 1.
CCLE cell lines with reported nonsense mutations, frameshift deletions or insertions, or in-frame deletions in TSC1 and TSC2 [adapted from 27, 28].
Fig 1.
Identification and characterization of cell lines with TSC1 or TSC2 mutations.
Cells were serum starved for 24h (−) or had serum add back for 30 min (+) after starvation. MGH-U1, a bladder cancer cell line with normal mTOR signaling, was used as a control.
Fig 2.
Characterization of TSC1 and TSC2 deficient cell lines.
Immunoblots of TSC1 and TSC2 deficient cell lines were performed to examine multiple components of the mTORC1 signaling pathway. Cells were serum starved for 24h (−) or had serum add back for 30 min (+) after starvation. CAL-72 and PEER cells showed no TSC1 and reduced TSC2 expression. SNU-878, SNU-886, and SNU-398 showed an absence of TSC2 expression. pS6K (Thr389), pS6 (Ser235/236 and Ser240/244) p4E-BP1 (Thr37/46), and pEIF2 (Ser51) showed a strong expression in a serum starved condition in all TSC1 or TSC2 deficient cell lines, indicating constitutive mTORC1 activation. GAPDH and BIP were used as loading controls.
Table 2.
IC50 determination of the most effective drugs.
Fig 3.
Cell viability after ganetespib, ganetespib with Torin2 and ganetespib with rapamycin treatment.
Ganetespib was serially diluted three-fold from 10 μM to 1.5 nM. The Torin2 concentration was 5nM, and the rapamycin concentration 20 nM. Cell viability was determined 72h after treatment using CellTiter-Glo and XLfit4.0 software. Cell viability is shown in relative control activity, n = 2–4. IC50 was calculated as the drug concentration that reduced cell viability by 50% compared to untreated cells. All TSC1 or TSC2 deficient tumor cell lines showed a strongly decreased cell viability after treatment with ganetespib. Combined therapy of genetespib and rapamycin or genetespib and Torin2 showed an additive effect with even lower IC50 values. However, the significance of this additive effect was not clear.
Table 3.
IC50 determination of Torin2, Ganetespib and combination therapy.
Fig 4.
Expression and signaling effects of TSC2 add back to TSC2 deficient cells.
TSC2 cDNA or an empty vector was added to TSC2 deficient cell lines via retroviral transfection. Cells were serum starved for 24h (−) or had serum add back for 30 min (+). In the TSC2 add back cells there is a strong TSC2 expression, a decreased pS6 (S240/244) expression in the absence of serum, and increased pAKT (Ser473) expression following serum add back. No pAKT (Ser473) was seen in SNU-398 cells, with or without TSC2 addback.
Table 4.
IC50 determination of TSC2 deficient cells, cells with TSC2 add back and control vector.
Fig 5.
Impact of rapamycin and Torin1 on constitutively activated mTOR signaling pathway in TSC1 or TSC2 deficient cells.
Cells were treated with Torin1 (250 nM) or rapamycin (20 nM) for 24h. Untreated cells and MGH-U1 were used as controls. Cells were serum starved for 24h (−) or received after starvation serum add back for 30 min (+). Rapamycin and Torin1-treated cells show no expression of pS6K (Thr389) and pS6 (Ser 240/244) in serum absence or stimulated conditions compared to the upregulated expression in untreated cells. pAKT (Ser 473) levels are lower after Torin1 and higher after rapamycin treatment.
Fig 6.
Effect of ganetespib on protein expression and mTORC1 signaling in TSC-mutant cell lines.
Cells were treated with ganetespib in increasing concentrations (1, 10, 100 nM, and 1 μM) for 24h. Cells were serum starved for 24h (−) or after starvation had serum add back for 30 min (+). Effects on mTOR, AKT, and pS6 (Ser240/244) expression were seen at 0.1 and 1mM.
Fig 7.
Impact of rapamycin, Torin1, and ganetespib on constitutively activated mTOR signaling and cleaved caspase 3.
Cells were treated with rapamycin (20 nM), Torin1 (250 nM) or ganetespib (100 nM) for 24h. Cells were serum starved for 24h (−) or received after starvation serum add back for 30 min (+). Cells treated with rapamycin do not show expression of pS6 (Ser240/244) and expression of p4E-BP1 isoforms are reduced due to mTORC1 inhibition. Torin1-treated cells show no pS6 (Ser240/244), p4E-BP1, and a lowered pAKT (Ser473) expression, due to mTORC1/2 inhibition. Ganetespib-treated cells show a lowered AKT, S6K, and pS6 (Ser240/244) expression. The expression of cleaved caspase 3 is very distinctive among different cell lines.
Fig 8.
Xenograft tumor growth under vehicle, rapamycin, INK 128, ganetespib (a, b) combined ganetespib and INK 128 (c) and ganetespib and rapamycin (d) treatment.
Tumor volume is shown as normalized to tumor volume at day 1 of treatment. Tumor size is shown as mean and standard deviation. Normalized tumor size of different treatment groups was compared on day 22 using Wilcoxon Rank Sum test. P-values less than 0.05 were considered statistically significant. Mice were treated with rapamycin (3 mg/kg, 3x/week, i.p., n = 5), INK 128(1mg/kg, 5x/week, i.g., n = 5) or ganetespib (50mg/kg, 1x/week, i.v., n = 9) (a) or in combination of ganetespib and INK 128 (n = 5) (c) or ganetespib and rapamycin (n = 6) (d). Tumor size of treated mice is significantly smaller on day 22 compared to mice, which received vehicle (n = 8) (b).
Fig 9.
Immunohistochemical analysis of xenograft tumors of mice treated with vehicle, INK 128, ganetespib, or rapamycin for 21 days.
Xenograft tumors generated from SNU-398 cells were harvest 24h after ganetespib treatment, 6h after INK 128 and rapamycin treatment, and stained using H&E, pS6 (S235/236), TSC2, ApopTag, or PCNA antibodies. Images shown were 60X magnified, insets showed portions of the tumor at higher magnification (400X). Tumors showed a distinct vascularization. TSC2 was not expressed in any tumor. PS6 (S235/236) expression was stronger in vehicle- and ganetespib-treated mice and correlated with locations of higher proliferation.
Fig 10.
Effects of rapamycin, INK 128, ganetespib, and combination treatment on expression and mTOR signaling in SNU-398 xenograft tumor and liver cells.
Mice were treated with rapamycin (3 mg/kg, 3x/week, i.p.), INK 128 (1mg/kg, 5x/week, p. o.), ganetespib (50mg/kg, 1x/week, i.v.) or in combination. Tumors were harvest and lysed 24h after ganetespib treatment and 6h after INK 128 or rapamycin treatment. Rapamycin treatment inhibited pS6K (Thr 389) and pS6 (Ser240/244) expression in the tumors. INK 128 treatment reduced pS6K (Thr 389), pS6 (Ser240/244), and p4E- BP1 isoform expression. Ganetespib reduced pS6K (Thr 389) and pS6 (Ser240/244) expression in some tumors. Combined ganetespib and rapamycin or INK 128 treatment showed a stronger inhibition of the mTOR pathway than each drug by itself. Rapamycin even inhibited pS6 (Ser240/244) expression in the liver. Abbreviations: c: cells from cell culture, v: vehicle, G: Ganetespib, I: INK 128, R: Rapamycin.