Sorafenib synergizes with metformin in NSCLC through AMPK pathway activation

Abstract

The multikinase inhibitor sorafenib is under clinical investigation for the treatment of many solid tumors, but in most cases, the molecular target responsible for the clinical effect is unknown. Furthermore, enhancing the effectiveness of sorafenib using combination strategies is a major clinical challenge. Here, we identify sorafenib as an activator of AMP-activated protein kinase (AMPK), in a manner that involves either upstream LKB1 or CAMKK2. We further show in a phase II clinical trial in KRAS mutant advanced non-small cell lung cancer (NSCLC) with single agent sorafenib an improved disease control rate in patients using the antidiabetic drug metformin. Consistent with this, sorafenib and metformin act synergistically in inhibiting cellular proliferation in NSCLC in vitro and in vivo. A synergistic effect of both drugs is also seen on phosphorylation of the AMPKα activation site. Our results provide a rationale for the synergistic antiproliferative effects, given that AMPK inhibits downstream mTOR signaling. These data suggest that the combination of sorafenib with AMPK activators could have beneficial effects on tumor regression by AMPK pathway activation. The combination of metformin or other AMPK activators and sorafenib could be tested in prospective clinical trials.

Keywords: AMP-activated protein kinase; metformin; non-small cell lung cancer; salicylate; sorafenib.

Figure 1

Sorafenib synergizes with metformin and other AMPK activators in NSCLC cell lines. a–f, Colony formation assay of A549 (a, c, e) and H460 (b, d, f) NSCLC cells with increasing concentrations of sorafenib (0–4 μM) in the absence or presence of increasing concentrations of metformin (0–2 mM) (a, b); A-769662 (0–100 μM) (c, d); or salicylate (0–2 mM) (e, f). Cells were grown in six-well plates and refreshed every 3 days. The cells were fixed, stained and photographed after 10 days.

Figure 2

Sorafenib in combination with metformin or the AMPK activator salicylate enhances AMPK activation. a, b, AMPK activation with the combination of sorafenib and metformin in LKB1 mutant KRAS mutant (A549 and H460) NSCLC cells (a), LKB1 wild-type KRAS mutant (H358) (b, left panel) or LKB1 mutant KRAS wild-type (H838) NSCLC cells (b, right panel). Cells were treated for 48 hr with sorafenib (1–3 μM), metformin (1–1.5 mM) or the combination of sorafenib and metformin with the same concentrations as were used for the individual treatments. c, AMPK activation with the combination of sorafenib and salicylate in LKB1 mutant KRAS mutant (A549 and H460) or LKB1 mutant KRAS wild-type (H838) NSCLC cells. Cells were treated for 48 hr with sorafenib (1–3 μM), salicylate (1–1.5 mM) or the combination of sorafenib and salicylate with the same concentrations as were used for the individual treatments. Cell lysates were harvested for western blot analysis and probed with the indicated antibodies.

Figure 3

Sorafenib in combination with metformin or the AMPK activator salicylate represses the mTOR targets p-S6 and p-4E-BP1. a, Western blot analysis with the combination of sorafenib and metformin in LKB1 mutant KRAS mutant (A549), LKB1 wild-type KRAS mutant (H358) or LKB1 mutant KRAS wild-type (H838) NSCLC cells. b, Western blot analysis with the combination of sorafenib and salicylate in LKB1 mutant KRAS mutant NSCLC cells. Cells were treated for 48hrs with either sorafenib (1–3 μM), metformin (1–1.5 mM) (a) or salicylate (1–1.5 mM) (b) alone or the combination of sorafenib with metformin/salicylate, with the same concentrations as were used for the individual treatments. Cell lysates were harvested for western blot analysis and probed with the indicated antibodies.

Figure 4

Activation of AMPK by sorafenib is dependent on the AMPK kinases LKB1 and CAMKK2. a, Concentration dependent AMPK activation by sorafenib in LKB1 mutant (A549 and H460) and LKB1 wild-type (H358) NSCLC cells. Cells were treated for 6 hr with increasing concentrations of sorafenib (0–8 μM). b and c, AMPK activation by sorafenib is CAMKK2-dependent in LKB1 mutant (A549 and H460) and CAMKK2-independent LKB1 wild-type (H358) NSCLC cells. b, Cells expressing shctrl or shCAMKK2 were treated for 6 hr with 6 μM sorafenib. c, Cells were treated for 6 hr with increasing concentrations of the CAMKK inhibitor STO-609 (0–50 μM) in absence or presence of 6 μM sorafenib. d, AMPK activation by sorafenib is mainly LKB1-dependent in LKB1 wild-type H358 cells. H358 cells expressing shctrl (left panel) or shLKB1 (right panel) were treated with the CAMKK inhibitor STO-609 (0–50 μM) in absence or presence of 6 μM sorafenib for 6 hr. The p-AMPK blot in d is relatively high exposed to highlight to differences in AMPK phosphorylation between shctrl cells and shLKB1 H358 cells. e, AMPK activation by sorafenib is also seen in LKB1 wild-type hepatocellular carcinoma (HCC) Huh-7 cells, independent of CAMKK2. Huh-7 cells expressing shctrl or shCAMKK2 were treated for 6 hr with 6 μM sorafenib. f, AMPK activation by induced oxidative stress is mainly LKB1-dependent in LKB1 wild-type (H358) and completely CAMKK2-dependent in LKB1 mutant (H460) NSCLC cells. Cells were treated for 1-hr with 400 μM H₂O₂ in absence or presence of 50 μM of the CAMKK inhibitor STO-609. g, H358 cells expressing shLKB1 were treated for 1-hr with 400 μM H₂O₂ in absence or presence of 50 μM of the CAMKK inhibitor STO-609. Cells treated with the combination were pretreated for 30 minutes with the CAMKK inhibitor before adding H₂O₂. h, AMPK activation by induced oxidative stress is blocked with the ROS-scavenger N-acetyl-l-cysteine. H358 cells were treated for 1 hr with 400 μM H₂O₂ in absence or presence of 20 mM of the ROS-scavenger N-acetyl-l-cysteine (NAC). Cell lysates were harvested for western blot analysis and probed with the indicated antibodies.

Figure 5

Effect of the combination of sorafenib with metformin in a tumor xenograft model. a, The growth curves of A549 cells as tumor xenografts in nude mice treated with vehicle (black curve), sorafenib (blue curve), metformin (yellow curve) or the combination (purple curve). Error bars represent SD; n = 5–8. The black curve (vehicle) is shown until 25 days after start treatment, because of a bacterial infection outbreak in this group. b, Mean tumor volume (mm³) after 40 days treatment with sorafenib (blue dots; n = 5) or the combination of sorafenib with metformin (purple triangles; n = 8). A p-value < 0.05 was considered as statistically significant (*). c, Mean tumor growth rate (mm³/day), calculated by linear regression modeling of the individual growth curves, for the sorafenib treated tumor xenografts (blue dots; n = 5) and the tumor xenografts treated with the combination of sorafenib with metformin (purple triangles; n = 8). A p-value < 0.05 was considered as statistically significant (*). d, H-score of phospho-4E-BP1 (Thr37/46) immunostaining of A549 xenograft tumor sections of the different treatment groups. p-values < 0.05 were considered as statistically significant (*); ns = nonsignificant. e, Images of A549 xenograft tumor sections of vehicle (top) and combination-treated (bottom) tumors immunostained for phospho-4E-BP1 (400× magnification).