Reagents
Gefitinib and lovastatin were purchased from Aladdin (Shanghai, China). Osimertinib was obtained from Glpbio (Montclair, CA, USA). XCT790, BAY 11-7082, WH-4-023 and Plicamycin were purchased from MCE (New Jersey, USA). Rapamycin, Y27632 and SCH772984 were obtained from AbMole (Houston, TX, USA). Lapatinib, AG-490 and mevalonate were obtained from Topscience (Shanghai, China). SIS3 was purchased from Selleck (Shanghai, China). Cholesterol and MβCD were obtained from Sigma-Aldrich (St Louis, MO, USA).
Cell culture
For all experiments, human NSCLC cell line PC-9 (BCRJ, Rio de Janeiro, Brazil), PC-9/GR, H1975 (Cell Bank of the Shanghai Academy of Life Sciences, Shanghai, China), and PC-9/OR were maintained in Dulbecco modified Eagle medium (KGM12800N-500, KeyGen BioTech, Nanjing, China) supplemented with 10% fetal bovine serum (086150, Wisent, St-Bruno, Quebec, Canada),100 μg/mL streptomycin and 100 U/mL penicillin (ST488, Beyotime Biotechnology, Shanghai, China) in a humidified cell culture incubator at 37 °C with an atmosphere of 5% CO2.
Transfection
For cell transfection, 100 nM siEGFR (the target sequence: 5′- GCAACAUGUCGAUGGACUUTT − 3′) (HanBio, Shanghai, China) and 2.5 μg pCDNA3.1 or pCDNA3.1-SP1 (Genomeditech, Shanghai, China) were transfected into NSCLC cells with Lipofectamine 2000 (11668-019, Invitrogen, Carlsbad, CA, USA) growing in serum-free opti-MEM media (51985-034, Gibco, Gaithersburg, MD, USA).
Western blot
For evaluating protein expression in cells or tumor tissues, proteins were extracted. The same amounts of proteins were separated through 10-15% SDS-PAGE and transferred to polyvinylidene difluoride membranes, following by blocking with 5% nonfat milk. Subsequently, the membranes were incubated with primary antibodies, then with secondary antibodies. Immunoblot signals were visualized by ECL detection and the expression of proteins was quantified by Image J software. Antibodies used for Western blot were: anti-p-EGFR, anti-EGFR, anti-Erk, anti-SP1 (Cell Signaling Technology, Boston, MA, USA); anti-GAPDH, anti-p-Erk (Proteintech, Wuhan, China); anti-p-Src (Affinity Biosciences, Changzhou, China); anti-Lamin B (Wanleibio, Shenyang, China); anti-Caveolin1 (HUABIO, Hangzhou, China); anti-ERRα, anti-Src, HRP-labeled goat anti-rabbit IgG(H + L) (Beyotime Biotechnology, Shanghai, China).
RT-qPCR
Trizol reagent (R401-01, Vazyme, Nanjing, China) was used to extract total RNAs from NSCLC cells as described by the manufacturer’s instructions. cDNA was synthesized using mRNA as a template with HiScript III RT SuperMix (R323-01, Vazyme, Nanjing, China). ChamQ SYBR qPCR Master Mix (Q341-02, Vazyme, Nanjing, China) was used for RT-qPCR analysis. GAPDH expression was used to normalize the data. The mRNA primer sequences used are shown as below: 5′-GTCTCCTCTGACTTCAACAGCG-3′ and 5′-ACCACCCTGTTGCTGTAGCCAA-3′ for GAPDH; 5′-CCTCTGTGACCTCTTTGACC-3′ and 5′-TACTGACATCTGGTCAGAC-3′ for ERRα [30].
Cell viability assay
Cell viability was determined by MTT assay. Cells were treated with indicated drugs following by adding 0.5 mg/mL MTT solution (3580GR001, Biofroxx, Guangzhou, China) at 37 °C for 4 h. Finally, the absorbance of each well was measured using a microplate reader.
Luciferase reporter assay
Cells were transfected with pRL-TK and pGMERRα-Lu (Genomeditech, Shanghai, China) and cultured in medium with indicated drugs for 36 h. Subsequently, cells were lysed with 1× cell lysis buffer at room temperature for 5 min and the luciferase activities were detected with Dual-Luciferase Reporter Assay System (DL101-01, Vazyme, Nanjing, China). Renilla luciferase activity was used to normalize the firefly luciferase activity.
Co-immunoprecipitation (co-IP)
Cells were treated with indicated drugs and collected, then lysed in RIPA lysis buffer (KGP703-100, KeyGen BioTech, Nanjing, China). Lysates were centrifuged at 14,000 g, 4 °C for 10 min and supernatant were transferred to another tube with EGFR primary antibody (1:100, 4267S, Cell Signaling Technology, Boston, MA, USA) incubated overnight at 4 °C. Then, the protein complexes were collected by incubation with 60 μL of Protein A + G Agarose (P2055, Beyotime Biotechnology, Shanghai, China) for 1 h. Finally, the protein complexes were washed with RIPA lysis buffer and analyzed by Western blot assay.
CHIP assay
Chromatin immunoprecipitation (CHIP) assay was conducted following the manufacturer’s instructions of the CHIP assay kit (P2078, Beyotime Biotechnology, Shanghai, China). Briefly, H1975 cells were fixed with 1% formaldehyde for 10 min, quenched in glycine for 5 min, washed and lysed in SDS lysis buffer. Cell lysates were sonicated until the DNA fragments were 200-1000 bp in size. Then the lysates were collected and pre-cleared with Protein A + G Agarose/Salmon Sperm DNA for 30 min. 2% percent of the chromatin was used as input control and the rest was incubated with antibody against IgG (1:100, A7016, Beyotime Biotechnology, Shanghai, China) and SP1 (1:100, 9389S, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. The chromatin complexes were then incubated with Protein A + G Agarose/Salmon Sperm DNA for 1 h at 4 °C, following by washed with low salt immune complex wash buffer, high salt immune complex wash buffer, LiCl immune complex wash buffer, TE buffer and elution buffer (1% SDS, 0.1 M NaHCO3). The DNA-protein complexes were incubated with 0.2 M NaCl overnight at 65 °C, RNase A for 30 min at 37 °C, proteinase K (ST532, Beyotime Biotechnology, Shanghai, China) for 1.5 h at 45 °C. The DNA was purified using a DNA Clean Up Kit (D0033, Beyotime Biotechnology, Shanghai, China). Finally, the precipitated DNA was quantified using qPCR. Primers used for ERRα were: 5′-TGACCCCATCCGAGTGGAATTTGAGT-3′ and 5′-AGAAAGCTCAAGGTCACTGCGGTG for-3′.
Immunofluorescence
For EGFR and Src fluorescence colocalization analysis, cells were seeded and exposed to the indicated drugs. Then, cells were fixed with 4% paraformaldehyde, blocked with goat serum and incubated with primary antibody against EGFR (1:100, 4267S, Cell Signaling Technology, Boston, MA, USA) overnight at 4 °C. CoraLite®594-conjugated c-SRC (1:100, CL594-60315, Proteintech, Wuhan, China) and Goat Anti-Rabbit IgG H&L (FITC) (1:100, ab97050, Abcam, Cambridge, MA, USA) were used to treated the cells for 2 h at 37 °C. nucleus was counterstained with 5 μg/mL DAPI (C1002, Beyotime Biotechnology, Shanghai, China) for 10 min at room temperature. All images were taken with Zeiss LSM800 and measured by Image Pro Plus software [31]. Pearson’s correlation coefficient was calculated by Image J software [32, 33].
IHC
One-Step IHC Assay kit (KGOS60, KeyGen BioTech, Nanjing, China) was used for immunohistochemical (IHC) staining. Tissue slides acquired from NSCLC xenografts were deparaffinized and rehydrated, following by antigen retrieval, permeabilizated and 3% H2O2 treatment. After being blocked with goat serum, slides were incubated with primary antibody against Ki67 (1:100, WL01384a, Wanleibio, Shenyang, China) or ERRα (1:100, sc-65715, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. On the second day, immune complexes were determined with horseradish peroxidase conjugates using DAB detection.
Nuclear protein extraction
The nuclear protein was extracted using Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells that have been treated with indicated drugs were harvested and dissociated in 200 μL Reagent A mixture containing 1 mM PMSF (KGP610, KeyGen BioTech, Nanjing, China). The cell suspension was incubated on ice for 15 min. Then, 10 μL Reagent B was added and the mixture was vortexed for 5 s with 1 min ice bath, following by centrifugation at 16,000 g, 4 °C for 5 min. The supernatant was collected as cytoplasmic protein and the precipitation was further resuspended in 50 μL nuclear protein extraction reagent containing 1 mM PMSF. After being vortexed and ice bath in turn for 30 min, the mixture was centrifugated at 16,000 g, 4 °C for 5 min and the supernatant was saved as nuclear protein. The subcellular fractions were analyzed by Western blot assay.
Isolation of lipid rafts [34]
Cells were seeded, treated with indicated drugs and collected. The lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100) was used to lyse cells for 30 min. Then, the lysates were centrifugated at 16,000 g, 4 °C for 30 min and supernatants were collected and referred to as the non-lipid rafts fractions. The rest insoluble pellets were added with 200 μL buffer (0.5% SDS and 2 mM DTT), re-suspended and incubated for 10 min. Finally, the samples were centrifugated at 16,000 g, 4 °C for 30 min and the supernatants were collected into another tubes, referring to as lipid rafts fractions.
Tumor xenograft model
BALB/c nude mice (4 weeks old) (Yangzhou University, Yangzhou, China) were housed under individual ventilated cages in compliance with the institutional guidelines. Mice were randomly assigned into four groups (n = 5), injected with PC-9, PC-9/GR, H1975 and PC-9/OR cells subcutaneously. We monitored the tumor volume every 3 days. In the second xenograft experiments, PC-9/GR cells were subcutaneously injected into the mice. After 1 week injection, mice were randomly assigned into three groups (n = 5) according to tumor volume as follows: gefitinib group (treat with 50 mg/kg gefitinib), gefitinib + lovastatin group (treat with 50 mg/kg gefitinib and 100 mg/kg lovastatin), and gefitinib + XCT790 group (treat with 50 mg/kg gefitinib and 8 mg/kg XCT790). In all experiments, gefitinib and lovastatin were administrated by oral gavage daily, while XCT790 was administrated by intraperitoneal injection. The tumor volume was measured every 3 days and calculated based on the formula: tumor volume (mm3) = (length×width2)/2. After approximately 3 weeks, mice were sacrificed and xenografts tissue were collected for further analysis.
Statistical analysis
Data were presented as mean ± SEM and statistical analysis were performed by GraphPad Prism 8.0 software (GraphPad Software Inc., CA, USA). Unpaired Student’s t test was used to compare differences between two groups, while analysis of variance (ANOVA) was used for comparisons among multiple groups. The Kaplan–Meier method was used for survival analysis, and the log-rank test was used to determine the statistical significance. P < 0.05 was considered statistically significant.