Fig 2: Blocking ATK activation largely abolishes glycolysis driven by LIF in breast cancer cells and the promoting effect of LIF on xenograft breast tumor growth.A, B Blocking AKT activation by MK2206 (1 µM for 12 hours) (A, left panel), Wortmannin (2 µM for 6 h) (A, right panel) or AKT-DN expression (B) largely abolished the promoting effect of LIF on glucose uptake in breast cancer cell lines. C, D. Treatment with MK2206 (C, left panel), Wortmannin (C, right panel) or AKT-DN expression (D) largely abolished the promoting effect of LIF on lactate production in breast cancer cell lines. E. Blocking AKT signaling by Wortmannin greatly inhibited the promoting effect of LIF on xenograft tumor growth. Mice with xenograft tumors (~50 mm3) were administered with Wortmannin (i.p., 1.5 mg/kg mice, daily for two weeks). F Wortmannin treatment largely abolished the promoting effect of LIF on glucose uptake in xenograft tumors. Glucose uptake was measured in xenograft tumors described in E. G Schematic illustration of the role of LIF in increasing glycolysis, which in turn promotes breast tumorigenesis. Data are present as mean ± SD. For A–D, F: n = 3/group; For E: n = 6/group. *p < 0.05; **p < 0.01; ***p < 0.001; unpaired Student’s t-test for A–D, F, and 2-way ANOVA followed by Student’s t-test for E.

Fig 3: LIF drives glycolysis in breast cancer cells.A Ectopic LIF expression (left panel) or treatment with rhLIF (right) enhanced lactate production of different breast cancer cell lines analyzed by measuring lactate levels in the medium using a lactate assay kit. B LIF neutralization antibody (LIF neu-ab) blocked the promoting effect of LIF on lactate production in MCF7, MDA-MB 231 and T47D cells. C Knockdown of endogenous LIF decreased lactate production in MDA-MB 231 cells. D, E Ectopic LIF expression enhanced glycolysis rates (D), whereas knockdown of endogenous LIF decreased glycolysis rates (E) in different breast cancer cell lines as calculated by ECAR measured by using a Seahorse analyzer. F The diagram of glycolysis. G–I The fold change of inter-metabolites in glycolysis resulted from ectopic LIF expression in MCF7 and MDA-MB 231 cells (G), rhLIF treatment in MCF7 cells (H) or LIF knockdown in MDA-MB 231 cells (I) as determined by LC/MS metabolomics analysis. J The fold change of inter-metabolites in glycolysis induced by ectopic LIF expression in MCF7 xenograft tumors determined by LC/MS metabolomics analysis. Data are presented as mean ± SD (n = 3/group). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired Student’s t-test.

Fig 4: LIF promotes Glut1 PM translocation through the activation of AKT signaling.A LIF activated the AKT signaling, which can be blocked by MK2206, an AKT inhibitor (upper panel), or Wortmannin (Wort), a PI3K inhibitor (lower panel), in MCF7, MDA-MB 231 and T47D cells. The levels of p-AKTSer473 (p-AKT), which reflect the activity of AKT were measured by Western-blot assays. B Blocking AKT signaling by MK2206 (left) or Wortmannin (right) largely abolished the promoting effect of LIF on Glut1 PM translocation in breast cancer cells. C, D Blocking AKT signaling by expression of dominant-negative AKT (AKT-DN) largely abolished the promoting effect of LIF on Glut1 PM translocation in breast cancer cells. Cells were transfected with vectors expressing AKT-DN. The levels of p-AKT and total AKT (C), as well as Glut1 PM translocation (D), were determined by Western-blot assays. Uncropped Western-blot images are shown in Supplementary Fig 3.

Fig 5: 2-DG, a glycolysis inhibitor, preferentially inhibits the proliferation of breast cancer cells and the growth of xenograft tumors with LIF overexpression.A 2-DG preferentially inhibited the proliferation of breast cancer cells with ectopic LIF expression compared with control cells transduced with control vectors. MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression were treated with 2-DG at different concentrations. B 2-DG treatment exhibited a more pronounced inhibitory effect on growth of xenograft tumors formed by MCF7 and MDA-MB 231 cells with ectopic LIF expression than those formed by control cells. Mice with xenograft tumors (~50 mm3) were administered with 2-DG (i.p., 1 g/kg mice, every two days for two weeks). Arrows represent the start of 2-DG treatment. Data are presented as mean ± SD. In A, n = 3/group; in B, n = 5/group. *p < 0.05; **p < 0.01; ***p < 0.001; 2-way ANOVA followed by Student’s t-test.