Fig 1: Inhibition of PI3K/AKT leads to STAT3 activation. (A) Western blot analysis of H460 and H2126 cell lysates using specific antibodies to phosphorylated or total proteins of STAT3, ERK1/2, AKT, S6 or GAPDH (loading control). The cells were treated with increasing concentrations of BKM120 for 24 h. (B) Western blot analysis of H460 cell lysates, following 24 h cell culture in the absence (DMSO) or presence of LY294002 (10 µM), MK2206 (10 µM) or BEZ235 (0.5 µM). (C) Western blotting analysis of H460 cell proteins following transfection with negative control siRNA (NC) or specific siRNAs for p110α (si-p110α) or AKT (si-AKT). (D) The STAT3 DNA-binding activity of nuclear extracts from H460 and H2126 cells treated with 0.1, 0.5 or 1 µM BKM120 for 12 h. (E) Reverse transcription quantitative-polymerase chain reaction analysis of STAT3 downstream target genes after 24 h of BKM120 treatment. *P<0.05, **P<0.01 and ***P<0.001, compared with the DMSO control group. PI3K, phosphoinositide 3-kinase; STAT3, signal transducer and activator of transcription 3; ERK, extracellular regulation kinase; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; MMP9, matrix metallopeptidase 9; bcl-2, B-cell lymphoma 2; HGF, hepatocyte growth factor; CRP, C-reactive protein; Mcl-1, BCL2 family apoptosis regulator.
Fig 2: MET/STAT3 inhibition promotes the pro-apoptotic and anti-proliferative effects of PI3K/AKT inhibitors. (A) The expression of phosphorylated or total forms of STAT3 and AKT were detected by western blotting following culture of H460 cells with DMSO (NC), BKM120 (1 µM), PF-2341066 (1 µM) or STAT3 inhibitor stattic (5 µM) for 60 h. (B) The rate of apoptosis in H460 and H2126 cells treated with singular or combined inhibitors was examined by flow cytometry. *P<0.05, **P<0.01 and ***P<0.001, compared with the NC group, (C) The number of colonies formed after treatment with singular or combines inhibitors. MET, MET proto-oncogene; STAT3, signal transducer and activator of transcription 3; PI3K, phosphoinositide 3-kinase; DMSO, dimethyl sulfoxide; NC, negative control (DMSO); B, BKM120; P, PF-2341066; S, static.
Fig 3: PI3K/AKT inhibition induces MET expression and activation. (A) An RTK array to determine the phosphorylation status of each RTK in duplicate wells after 24 h of starvation in H460 cells and 12 h treatment with DMSO or BKM120 (1 µM). (B) The expression of phosphorylated or total forms of MET protein was analyzed by western blotting. (C) The expression of phosphorylated or total forms of MET captured under fluorescence microscopy following a 12-h treatment with 1 µM BKM120. Scale bar, 50 µm. (D) Following transfection of H460 cells with si-MET in the absence (DMSO) or presence of BKM120 (1 µM) for 24 h, changes in protein expression were examined by western blotting using the indicated antibodies. PI3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase; DMSO, dimethyl sulfoxide; MET, proto-oncogene; si-MET, MET small interfering RNA; p, phosphorylated.
Fig 4: Targeting the MET/STAT3 pathway potentiates the antitumor efficacy of PI3K/AKT inhibitors in vivo. (A) BALB/c nude/nude mice (n=10 per group) bearing H460 xenografts were treated with DMSO, BKM120 (15 mg/kg), PF-2,341066 (25 mg/kg), or BKM120 and PF-2341066 and the tumor volumes were determined over time. (B) Western blot analysis of the changes in phosphorylated or total forms of MET, STAT3 and AKT protein expression in the xenograft tumors. (C) The rate of tumor growth of H460-derived xenografts treated with DMSO, BKM120 (15 mg/kg), stattic (3 mg/kg) or both. (D) Western blot analysis of protein expression of members of the STAT3/AKT pathway in mouse tumor samples. (E) Schematic representation of the PI3K/AKT and MET/STAT3 pathways. Inhibition of PI3K/AKT caused activation of the MET/STAT3 signaling pathway, leading to cell survival. Dual inhibition of the two pathways by combination targeted therapy enhanced the antitumor efficacy in non-small cell lung cancer. MET, MET proto-oncogene; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated; PI3K, phosphoinositide 3-kinase; DMSO, dimethyl sulfoxide; NC, negative control (DMSO); B, BKM120; P, PF-2341066; S, static; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PDK1, pyruvate dehydrogenase kinase isozyme 1; mTORC1, mammalian target of rapamycin complex-1; S6K, ribosomal protein S6 kinase; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1.
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