The NSCLC cell lines, H460 and H2126, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The 2 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all from HyClone; GE Healthcare, Chicago, IL, USA) at 37°C with 5% CO₂. BKM120 (a selective PI3K inhibitor; S2247), LY294002 (a pan-PI3K inhibitor; S1105), MK-2206 (an AKT allosteric inhibitor; S1078), BEZ235 [a dual PI3K/mechanistic target of rapamycin (mTOR) catalytic inhibitor; S1009], PF-2341066 (a MET inhibitor; S1068) and stattic (a STAT3 inhibitor; S7024) were obtained from Selleck Chemicals (Houston, TX, USA), and dissolved in DMSO. Specific small interfering RNAs (siRNAs) for p110α (si-p110α; 100 nM; cat. no. 6359S), AKT (si-AKT; 100 nM; cat. no. 6211S) and MET (si-MET; 100 nM; cat. no. 6618S) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Transfection was performed using Lipofectamine 2000^® in Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were transfected with an individual siRNA or treated with an inhibitor for 24 h prior to subsequent experimentation.

Cell lysates of H460 and H2126 cells were prepared using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and protease inhibitors. Total protein concentration was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). 12% Tricine-SDS-PAGE was used to separate 10 µg protein. Subsequent to transfer to a nitrocellulose membrane, the following primary antibodies were used: Phosphorylated-STAT3 (p-STAT3, Y705; cat. no. 9145; dilution 1:1,000), p-extracellular regulation kinase 1/2 (p-ERK1/2, T202/Y204; 4376; dilution 1:1,000), p-AKT (S473; cat. no. 4060; dilution 1:1,000), p-AKT (T308; cat. no. 13038; dilution 1:1,000), p-S6 (S240/244; cat. no. 5364; dilution 1:1,000), p-MET (Y1349; cat. no. 3133; dilution 1:1,000), STAT3 (cat. no. 12604; dilution 1:1,000), ERK1/2 (cat. no. 4695; dilution 1:1,000), AKT (catalog no., 4685; dilution, 1:1,000), S6 (cat. no. 2217; dilution 1:1,000) and MET (cat. no. 8198; dilution 1:1,000) were obtained from Cell Signaling Technology (Beverly, MA, USA), and incubation at 4°C with gentle shaking overnight. A 10% (w/v) solution of bovine serum albumin (Sigma-Aldrich; Merck KGaA) was used as blocking buffer (at 4°C with gentle shaking for 1 h). Blots were washed thrice in 1X TBS with 0.1% Tween-20. HRP-linked anti-rabbit IgG secondary antibody (Cell Signaling Technology; cat. no. 7074; dilution 1:2,000) incubated with the membranes at 4°C with gentle shaking for 1 h. Anti-GAPDH was purchased from Beijing CoWin Biotech Co., Ltd. (Beijing, China) and used as a loading control. Blots were washed thrice and exposed to the ECL Reagent substrate (Thermo Fisher Scientific, Inc.). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to compare the intensity of bands.

The adherent H460 cells were cultured on glass coverslips and treated with 1 µM BKM120 for 12 h. Cells were fixed with 2% buffered paraformaldehyde for 30 min on ice. The cells were stained using p-MET (cat. no. 3077; dilution 1:50) and MET antibodies (cat. no. 8198; dilution 1:50) for 1 h at 4°C. Anti-rabbit IgG F (ab')2 Fragment (Alexa Fluor^® 594 Conjugate; cat. no. 8889; Cell Signaling Technology) was incubated with the cells for 30 min at 4°C. Fluorescence microscopy was used to examine the expression of p-MET and MET.

H460 and H2126 cells were treated with 1 µM BKM120 for 12 h, and the Pierce LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Inc.) was used to confirm that BKM120 increased STAT3 DNA-binding activity, as described previously (19).

Total RNA was extracted from H460 and H2126 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) after 12 h of treatment with 0.1% dimethyl sulfoxide (DMSO), or 0.1 or 1 µM BKM120. The Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) was used to obtain cDNA, which was used in reaction with GoTaq qPCR Master Mix with SYBR-Green (Promega Corporation, Madison, WI, USA) and primers specific for matrix metallopeptidase 9 (MMP9), B-cell lymphoma 2 (bcl-2), survivin, cyclin-D1, hepatocyte growth factor (HGF) and C-reactive protein (CRP). PCR included a 12 min denaturation step at 94°C, followed by 38 cycles of 94°C for 10 sec, 60°C for 20 sec and 71°C for 10 sec. GAPDH was used as endogenous control. Primers for these genes were sourced from PrimerBank (http://pga.mgh.harvard.edu/primerbank/), and the sequences are as follows: MMP9 forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′; Bcl-2 forward, 5′-GGTGGGGTCATGTGTGTGG-3′ and reverse, 5′-CGGTTCAGGTACTCAGTCATCC-3′; surviving forward, 5′-AGGACCACCGCATCTCTACAT-3′ and reverse, 5′-AAGTCTGGCTCGTTCTCAGTG-3′; cyclin-D1 forward, 5′-GCTGCGAAGTGGAAACCATC-3′ and reverse, 5′-CCTCCTTCTGCACACATTTGAA-3′; HGF forward, 5′-GCTATCGGGGTAAAGACCTACA-3′ and reverse, 5′-CGTAGCGTACCTCTGGATTGC-3′; CRP forward, 5′-AACGAAGCCTCTCAAAGCCTT-3′ and reverse, 5′-CTCTTGGTGGCATACGAGAAAAT-3′. The ^2-∆∆Cq method was applied to present final results (20).

After 24 h of cell starvation in serum-free medium, the H460 cells were incubated for 12 h at 37°C in the absence (DMSO) or presence of BKM120 (1 µM). The mixture of proteins was prepared, and the phosphorylation status of each protein was examined by RTK array analysis. Array 001 (R&D Systems, Inc., Minneapolis, MN, USA) was used to detect tyrosine-phosphorylated RTKs, according to the manufacturer's instructions.

H460 and H2126 cells were seeded at 2×10⁵ cells/well in 24-well plates and left overnight prior to treatment with DMSO [negative control (NC)], BKM120 (1 µM), PF-2341066 (1 µM) or stattic (5 µM) for 60 h. Cells were harvested by centrifugation (250 × g, 5 min, 4°C), washed once with PBS and resuspended in 1X fluorescence-activated cell sorting buffer (BD Biosciences, Franklin Lakes, NJ, USA) at 1×10⁶ cells/ml. The frequency of apoptosis was detected using the Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

A total of 300 cells were seeded in 6-well plates overnight and treated with DMSO (NC), BKM120 (1 µM), PF-2341066 (1 µM) or stattic (5 µM) and cultured for a further 14 days. The cell clones were fixed and stained with 0.05% crystal violet at room temperature for 15 min, and the number of clones was counted under an optical microscope.

5-week-old female BALB/c mice (Weitonglihua Biotechnology, Beijing, China) weighing 17–20 g were maintained in the pathogen-free animal facility under controlled conditions (12:12 h light and dark cycle, 50% humidity and 22°C). These animals were provided rodent chow and water. A total of 2×10⁶ H460 cells were inoculated subcutaneously into the flank of the mice. The mice were randomly divided into 6 groups (n=10) when tumors reached a size of ~100 mm³. The mice were treated with control [0.5% (w/v) aqueous hydroxypropylmethylcellulose solution by oral gavage], BKM120 [15 mg/kg, in 0.5% (w/v) aqueous hydroxypropylmethylcellulose solution by oral gavage], PF-2341066 [25 mg/kg, in 0.5% (w/v) aqueous hydroxypropylmethylcellulose solution by oral gavage] or stattic [3 mg/kg, by intraperitoneal injection]. Each drug was administered daily. The tumors were measured using calipers and volume was calculated using the following formula: Length × width²/2. The procedures for care and use of animals were approved by the Ethics Committee of the Nanjing Jiangbei People's Hospital (approval no. IACUC/201606B03; Nanjing, China).

Data are presented as the mean ± standard error (SE). Multiple group comparisons of the means were performed by one-way analysis of variance and post-hoc Student-Newman-Keuls test. *P<0.05 was considered to indicate a statistically significant difference.

H460 and H2126 cells were treated with the selective PI3K inhibitor, BKM120. BKM120 markedly inhibited p-AKT at Ser473 and Thr308, and a dose-dependent increase in p-STAT3 was observed, whereas the phosphorylation of ERK1/2 remained unchanged (Fig. 1A). BKM120 also demonstrated mTORC1 inhibitory activity, which is evident from the reduction of p-S6 with the increasing concentration of BKM120.

A number of different PI3K/AKT inhibitors were used to confirm the activation of STAT3 by pharmacological inhibition of the PI3K/AKT pathway. Fig. 1B demonstrates the elevated expression of p-STAT3 with the treatment with all reagents, including LY294002 (pan-PI3K inhibitor), MK-2206 (AKT inhibitor) and BEZ235 (a dual p110/mTOR inhibitor).

To eliminate the possibility that these results were due to off-target pharmacological effects of the inhibitors, PI3K/AKT signaling was silenced via p110α or AKT using 2 different target-specific siRNAs. Silencing of p110α or AKT resulted in markedly enhanced activation of STAT3, and decreased AKT activity in the si-AKT group (Fig. 1C).

The STAT3 DNA-binding activity increased in H460 and H2126 cells with the concentration of BKM120 treatment (Fig. 1D). STAT3-dependent transcriptional activity was examined by detecting the expression of STAT3 downstream target genes, including MMP9, bcl-2, survivin, cyclin-D1, HGF, CRP and bcl-2 family apoptosis regulator (Mcl-1) (21). As demonstrated in Fig. 1E, treatment with BKM120 increased the expression of all genes, except Mcl-1. Taken together, these results suggest that the pharmacological or genetic inhibition of PI3K/AKT signaling triggered compensatory activation of STAT3 and altered its downstream signaling events.

The regulatory mechanism behind the compensatory activation of STAT3 induced by PI3K/AKT inhibition was further investigated. Since multiple RTKs were involved in the activation of STAT3 signaling, the phosphorylation level of RTKs was detected in BKM120-treated H460 cells using an RTK array. Elevated p-MET was observed in H460 cells with BKM120 treatment (Fig. 2A).

In consensus with these results, western blot analysis demonstrated that BKM120 treatment upregulated the expression levels of p-MET and total MET protein (Fig. 2B), which was further corroborated by IF (Fig. 2C). In order to elucidate the function of MET in regulating the activation of STAT3 signaling following PI3K/AKT inhibition, MET expression was silenced by transfection with specific siRNA (si-MET). The results demonstrate that the depletion of MET resulted in inhibition of p-STAT3, as well as a modest suppression of p-AKT (Fig. 2D). Taken together, these results suggest that PI3K/AKT inhibition may activate STAT3 signaling via upregulating the expression and activation of MET.

To investigate the compensatory activation of the MET/STAT3 pathway as a resistance mechanism to PI3K/AKT targeted therapy, the antitumor efficacy of PI3K/AKT inhibitors was evaluated when MET/STAT3 signaling was inhibited. As demonstrated in Fig. 3A, MET inhibition markedly abrogated STAT3 phosphorylation and AKT activity, and STAT3 inhibition completely abrogated BKM120-induced activation of STAT3. Antitumor efficacy was observed of BKM120 alone, and its potency increased in combination with PF-2341066 or stattic. This was indicated by increased apoptosis of tumor cells treated with the inhibitor combination compared with those treated with each inhibitor singly (Fig. 3B). A significant decrease in colony number was also observed when cells were treated with BKM120 in combination with PF-2341066 or stattic compared with cells treated with a single inhibitor (Fig. 3C).

To further verify the antitumor efficacy of the combination therapy in vivo, a subcutaneous xenotransplanted tumor model of H460 cells was established in mice. As demonstrated in Fig. 4A, combination treatment with BKM120 and PF-2341066 inhibited tumor cell growth effectively compared with treatment with each agent singly. The activity of MET, STAT3 and AKT in tumor tissues was further investigated by western blotting. In consensus with the in vitro experimental results, the protein expression levels of p-MET, p-STAT3 and p-AKT were all reduced with combination therapy compared with BKM120 or PF-2341066 treatment alone, which only inhibited AKT or MET/STAT3 phosphorylation respectively (Fig. 4B). Similar effects were achieved when BKM120 was applied in combination with stattic (Fig. 4C). The combined effect of the reagents was more effective than each single agent in repressing tumor growth and the expression of proteins of the STAT3 signaling pathway (Fig. 4D). Fig. 4E illustrates a schematic presentation of the potential molecular mechanism behind these effects.