1. Volume 67, Issue 3, Pages 512-527.e4 (August 2017)
Focal Adhesion- and IGF1R-Dependent Survival and Migratory Pathways Mediate Tumor Resistance to mTORC1/2 Inhibition 
Sang-Oh Yoon, Sejeong Shin, Florian A. Karreth, Gwen R. Buel, Mark P. Jedrychowski, David R. Plas, Shoukat Dedhar, Steven P. Gygi, Philippe P. Roux, Noah Dephoure, John Blenis 
Molecular Cell 
Volume 67, Issue 3, Pages e4 (August 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 512-527.e4DOI: (10.1016/j.molcel.2017.06.033)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Akt Re-phosphorylation at Hydrophobic Motif Following mTORC1/2 Inhibition Is mTORC2 Independent
(A) Cancer cell lines were grown in complete media with/without mTOR inhibitor, Torin1 (250 nM). Media and Torin1 were replaced every 2 days and cells were counted at the indicated time points. Data are the means ± SD of three separate experiments performed in triplicate.
(B and C) Breast cancer (B) or melanoma (C) cell lines were treated with/without Torin1 (250 nM) for 48 hr (B) or for 24 hr and 48 hr (C). Cells were lysed and immunoblot analysis was performed.
(D) Stably knocked down A375 cell lines with mTOR shRNAs were lysed and immunoblot analysis was performed.
(E) Tsc2 WT or Tsc2 null MEFs were treated with/without rapamycin (20 ng/mL) or Torin1 (250 nM) for 48 hr and immunoblot analysis was performed.
(F) WT MEFs were treated with Torin1 (250 nM) for the indicated time points. Cells were lysed and immunoblot analysis was performed.
(G) MEFs were treated with Torin1 for 48 hr. During this time, cells were treated with Torin1 again for the indicated time. Cells were lysed and immunoblot analysis was performed.
Data are representative of at least three independent experiments.
See also Figure S1.
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4. Figure 2 mTOR Inhibition Leads to PI3K-Dependent Activation of Akt
(A) MEFs and A375 melanoma cell line were treated with Torin1 (250 nM) for the indicated time. Cells were lysed and immunoblot analysis was performed.
(B) The same method was used as in (A).
(C) Control or Akt1 knockdown MEFs were treated with/without Torin1 for 48 hr and immunoblot analysis was performed.
(D) MEFs were treated with Torin1 for 48 hr. During this time, cells were treated with Torin1 again for the indicated time. Cells were lysed and immunoblot analysis was performed.
(E) MEFs and A375 were treated with Torin1 for 24 or 48 hr. Cells were lysed and immunoblot analysis was performed.
(F) Rictor control or KO MEFs were treated with Torin1 for 48 hr. Cells were lysed and immunoblot analysis was performed.
(G) MEFs were treated with Torin1 for 24 hr. After which, PI3K inhibitors, BKM120 (5 μM) or GDC0941 (3 μM) were treated for another 24 hr in the presence of Torin1 and immunoblot analysis was performed.
(H) IKKε, TBK1, or IKKε/TBK1 KO MEFs were treated with Torin1 for 48 hr and immunoblot analysis was performed.
Data are representative of at least three independent experiments.
See also Figure S2.
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5. Figure 3 mTOR Inhibition Results in Re-organization of Focal Adhesion
(A) MEFs were treated with Torin1 for 24 hr or 48 hr, and immunoblot analysis was performed using phospho-tyrosine antibodies.
(B) MEFs were treated with Torin1 for 36 hr, and immunoprecipitation was performed using phospho-tyrosine antibodies. Proteins were separated by SDS-PAGE and analyzed by mass spectrometry. The graph shows functional groups of proteins and numbers of tyrosine phosphorylated proteins changed by Torin1 treatment.
(C) A375 cells were treated with Torin1 for 48 hr and localization of proteins was visualized by confocal microscopy. Scale bar, 50 μm. Focal adhesion size was measured by Fiji program. The bar graphs show the average ± SE.
(D and E) A375 cells were grown in the presence or absence of Torin1 for 48 hr. Migration (D) or invasion (E) assay was performed with/without Torin1 in the upper and lower chambers as described in the STAR Methods in detail. Data are the means ± SD of three separate experiments performed in triplicate. Results were statistically significant (∗p < 0.01) as assessed by t test.
Data are representative of at least three independent experiments.
See also Figure S3.
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6. Figure 4
Integrin α2 and Focal Adhesion Kinase Are Involved in mTOR Inhibitor-Mediated Akt Phosphorylation
(A and B) A375 cells were treated with Torin1 for 48 hr, and total (A) or cell surface integrin levels (B) were measured by immunoblot analysis and flow cytometry, respectively.
(C) A375 cells were treated with Torin1 for 24 hr after which BTT3033 (20 μM, integrin α2 inhibitor) or SB (5 μM, integrin αV inhibitor) was treated for additional 24 hr in the presence of Torin1. Immunoblot analysis was performed.
(D) Integrin α2 knockdown A375 cells were treated with Torin1 for 48 hr, and immunoblot analysis was performed.
(E) The same method was used as in (C) except focal adhesion kinase inhibitors, PF (3 μM) or PF (10 μM), were used instead of integrin inhibitors.
(F) Focal adhesion kinase (FAK) knockdown A375 cells were treated with Torin1 for 48 hr and immunoblot analysis was performed.
(G and H) A375 cells were grown in the presence Torin1 for 48 hr. Migration (G) or invasion (H) assay was performed with DMSO, BTT3033, PF431396, or PF in the presence of Torin1. Data are the means ± SD of three separate experiments. Results were statistically significant (∗p < 0.01) as assessed by t test.
Data are representative of at least three independent experiments.
See also Figure S4.
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7. Figure 5 mTORC1/2 Inhibition Induces FAK/IGFR Signaling
(A) The graphs show changes of tyrosine phosphorylated IRS2 or IGF1R compared to control following Torin1 treatment.
(B) A375 cells were treated with Torin1 for 24 hr, after which BMS (10 μM, IGFR inhibitor), AZD8931 (10 μM, EGFR inhibitor), Erlotinib (5 μM, EGFR inhibitor), or PDGFR inhibitor III (10 μM) was treated for additional 24 hr in the presence of Torin1. Cells were lysed and immunoblot analysis was performed.
(C) A375 cells were treated with Torin1 for 24 hr, after which two different IGFR inhibitors, BMS (10 μM) and OSI-906 (5 μM), were treated for additional 24 hr in the presence of Torin1. After cell lysis, immunoblot analysis was performed.
(D) IGF1R knockdown A375 cells were treated with Torin1 for 48 hr and immunoblot analysis was performed.
(E) A375 cells were treated with Torin1 (250 nM) or KU (3 μM) for 48 hr and immunoblot analysis was performed.
(F) A375 cells were treated with Torin1 (250 nM) for the indicated time. Cells were lysed and immunoblot analysis was performed.
(G) Melanoma cell lines were treated with Torin1 for 24 hr, after which two different IGFR/IR inhibitors were treated for additional 24 hr in the presence of Torin1. Immunoblot analysis was performed.
(H–L) A375 cells were treated with Torin1 for 24 hr, after which inhibitors of integrin α2 (H), FAK (I), IGFR/IR (J), integrin α2 (K), or FAK (L) were treated for additional 24 hr in the presence of Torin1. Cells were lysed and immunoblot analysis was performed.
(M) Cells were treated with rapamycin (20 ng/mL) or Torin1 (250 nM) for 48 hr, and immunoblot analysis was performed.
Data are representative of at least three independent experiments.
See also Figure S5.
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8. Figure 6
ILK-Dependent Pathway Is Involved in Akt Phosphorylation at Hydrophobic Motif Following mTOR Inhibition
(A) HA-ILK was stably expressed in ILK knockdown A375 cells. The cells were treated with Torin1 for 48 hr and localization of HA (ILK) and vinculin was visualized by confocal microscopy. Scale bar, 50 μm.
(B) ILK control or KO MEFs were treated with Torin1 for 48 hr and immunoblot analysis was performed.
(C and D) A375 cells were treated with Torin1 for 24 hr, after which ILK inhibitors, QLT0267 (20 μM) (C) or Cpd22 (10 μM) (D), were treated for additional 24 hr in the presence of Torin1. Cells were lysed and immunoblot analysis was performed.
(E) The same method was used as in (C) and (D) except Rictor KO MEFs were used instead of A375 cells.
(F and G) A375 cells were grown in the presence Torin1 for 48 hr, after which migration (F) and invasion (G) assay was performed with DMSO, QLT0267, or Cpd22 in the presence of Torin1. Data are the means ± SD of three separate experiments. Results were statistically significant (∗p < 0.01) as assessed by t test.
Data are representative of at least three independent experiments.
See also Figure S6.
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9. Figure 7
Combinatorial Targeting of mTOR and IGFR/IR Efficiently Block Tumor Progression
(A) A375 cells were treated with Torin1 (250 nM for single treatment and 100 nM for combination) and/or BMS (10 μM for single treatment and 5 μM for combination) for 48 hr, and apoptosis rate was performed using flow cytometry. Data are the means ± SD of three separate experiments. Results were statistically significant (∗p < 0.01) as assessed by t test.
(B–D) Mice were injected with A375 cells and treated with drugs as described in the STAR Methods. Each group had four mice with eight tumor sites. Mouse weight (B) or tumor growth (C) was monitored (∗p < 0.01). Images (D) were taken with four representative tumors from each group. Scale bar, 1 cm.
Data are representative of at least three independent experiments.
See also Figure S7.
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