Fig. 5.

Acute inhibition of insulin/IGF1 signaling in KRAS-mutant lung cancer cells leads to in vivo growth suppression and dependence on autophagy. (A and C) Levels of total and phosphorylated IGF1R and IR, total and phosphorylated AKT, as well as LC3B-I and LC3B-II in murine Kras-mutant, p53-null lung cancer (KP) cells (A) or NSCLC A549 and Calu-1 cells (C) grown in 10% serum and treated with 2 μM NVP-AEW541 for 0, 4, or 24 h; β-ACTIN was used as a loading control. (B and D) Levels of amino acids in KP cells (B) or Calu-1 cells (D) cultured in the presence of 10% serum with or without 2 μM NVP-AEW541 for 24 h. Metabolite levels were first normalized to protein levels, and then to the median of all samples for each amino acid; n = 4 biological replicates per condition. (E) Proliferation curves of Calu-1 shCtrl and DKD cells, which were grown in 10% serum and treated with either vehicle control (DMSO), CQ, NVP-AEW541, or both drugs for 7 d; n = 4. Data represent the mean ± SEM. ****P < 0.0001 between single compound treatment and combinatorial treatment conditions; ns, nonsignificant. (F) Growth of individual s.c. xenograft tumors derived from A549 cells (Top) over 67 d (one mouse had to be killed at 51 d) and Calu-1 (Bottom) over 84 d (Left) with endpoint average tumor volume (Right); n = 12 (A549 sgCtrl); n = 11 (A549 DKO); n = 10 (Calu-1 shCtrl); n = 12 (Calu-1 DKD-1). (G) Percent growth of s.c. xenograft Calu-1 tumors with control or IRS1/IRS2 knockdown in mice treated with vehicle control (PBS) or chloroquine (CQ) (60 mg/kg), 5 d per week for 32 d after median tumor volume reached ∼150 mm³; n = 8 (shCtrl PBS); n = 10–12 (shCtrl CQ); n = 10 (DKD-1 PBS); n = 10 (DKD-1 CQ). In B, D, F, Right, and G, data represent the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.