Cell culture
Human lung cancer cell lines A549 and H1299 were purchased from the American Type Culture Collection (ATCC, Washington, USA) and cultured in RIPM-1640 medium containing 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin and 100 U/mL penicillin. Cells were maintained at an atmosphere of 5% CO2% and 95% air at 37˚C. All cell lines were recently authenticated by cellular morphology and short tandem repeat profiling.
Cell line establishment
Control or HDAC1-targeting shRNA templates were inserted into the pLV-H1-EF1a-Puro vector (Biosettia, San Diego, CA, USA) (named shCtrl, and shHDAC1). H1299 cells were infected with lentivirus expressing shHDAC1, followed by clone selection using 2 μg/mL puromycin to establish HDAC1 knockdown H1299 cells. TGIF2-silenced and TGIF2-rescue (shTGIF2#2 + TGIF2resis) stable H1299 and A549 cells, TGIF2WT-, TGIF2DD- and TGIF2AA-overexpressing H1299 cells were previously established [19]. The sequences of shRNA-HDAC1was: 5’-GGTGCTGTACATTGACATT-3’.
Western blot
The western blot steps were described previously [20]. The blots were cut prior to hybridisation with antibodies during blotting. The original images of all blots are presented in the Supplementary Information. The proteins were detected using the following primary antibodies: anti-E-cadherin, HDAC1, Flag, vimentin and fibronectin (Cell Signaling Technology, Danvers, MA, USA). Anti-β-actin, TGIF2, ERK and p-ERK (Santa Cruz Biotechnology, Dallas, TX, USA).
Real-time PCR
Real-time PCR was carried out according to the established protocols described previously [21]. The primer sequences are shown in Supplementary table S1.
Immunohistochemistry (IHC) staining
The tissue microarrays were purchased from Alenabio Inc. (cat. #LC2081, Shanxi, China). The tissue sections were incubated with primary antibodies against TGIF2, E-cadherin, and Vimentin (1:200) overnight at 4 °C. The tissues were incubated with a peroxidase-conjugated secondary antibody at 37 °C for 1 h before the DAB Substrate Kit was used to reveal bound secondary antibody. The semiquantitative H score was used to estimate the immunoreactivity according to the established protocols described previously [22].
Immunofluorescence staining
LUAD cell lines were fixed with 4% paraformaldehyde and were incubated with 5% goat serum for 1 h to block nonspecific interactions. Then cells were labeled with primary antibodies against E-cadherin, Vimentin, TGIF2, HDAC1 or Flag overnight at 4 °C, followed by incubation with fluorescent-dye-labeled secondary antibodies (1:200) (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Nuclei were stained with DAPI. Finally, the images were captured with a confocal fluorescence microscope (Olympus, Tokyo, Japan).
Dual-luciferase reporter assay
The CDH1 gene promoter fragment was amplified from human genomic DNA and cloned into the firefly luciferase plasmid pGL3-basic-IRES. H1299 cells were transiently transfected with pGL3-CDH1 reporter plasmid and TGIF2WT or TGIF2AA expression plasmids using Lipofectamine Reagent (Invitrogen, Waltham, MA, USA). After transfection for 36 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase activity was normalized to Renilla luciferase activity. The primer sequences used in this assay are listed in Supplementary table S2.
Chromatin immunoprecipitation (ChIP)
ChIP was carried out using the EZ-Zyme Chromatin Prep Kit (Millipore, Billerica, MA, USA). Anti-TGIF2 antibody and Anti-HDAC1 antibody were used to precipitate DNA crosslinked with TGIF2 and HDAC1, anti-mouse IgG as a negative control. PCR and RT-PCR were performed to detect DNA fragments of the CDH1 promoter region. Primers used in this assay are summarized in Supplementary tables S3,S4.
Cell migration assays
For Transwell assays, 1 × 105 cells were re-suspended in 200 μL of RPMI 1640 medium supplemented with 1% FBS and seeded into 5-μm pore Transwell chamber (Millipore, Burlington, MA, USA). Medium containing 10% FBS was added to the lower chamber. After incubation for 8 h, cells that migrating to the membrane surface were stained with 0.1% crystal violet staining solution. Four random fields were counted per chamber.
For wound healing assays, 1 × 106 A549 or H1299 cells were seeded and grown to 90% confluence. A sterile pipette tip was used to create scratch wounds. Images were taken at 0 h, and 10 h (H1299) or 24 h (A549) with an IX71 inverted microscope (Olympus). Three independent experiments were performed.
Immunoprecipitation
Wild type and Flag-TGIF2AA-overexpressing H1299 cell protein lysates were incubated with protein A/G agarose beads (Life Technologies) and antibody overnight at 4 °C. Then, the samples were centrifuged at 2500 rpm for 30 s. The pellets were washed with prechilled phosphate-buffered saline (PBS). Finally, bound proteins were boiled and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Immunoprecipitates were analyzed by western blotting with anti-TGIF2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flag antibody ((Sigma-Aldrich, St. Louis, MO, USA)) and anti-HDAC1 antibody (Cell Signaling Technology, Danvers, MA, USA).
Animal studies
To confirm whether TGIF2 promotes metastasis in vivo. Six-week-old NOD/SCID male mice were purchased from SPF Biotechnology (Beijing, China). Mice were intravenously injected with 2 × 106 TGIF2-silenced, TGIF2-rescued or control H1299 cells. Twenty days after tumor cell inoculation, the mice underwent bioluminescence imaging and were sacrificed. The lung tissues were fixed with formalin, embedded in paraffin, and subjected to HE staining or IHC staining. The percentage of lung metastatic foci was calculated using the formula: (area of metastatic foci/ total area of five lung lobes) × 100%. The area was measured using Photoshop software (Adobe, San Jose, CA, USA).
To determine whether p-TGIF2 was necessary for TGIF2-enhanced metastasis. 1.5 × 106 H1299-vector, H1299-TGIF2WT or H1299-TGIF2AA cells were subcutaneously injected into each NOD/SCID mouse. Tumor volume was measured using calipers and calculated using the formula: (length × width2)/2. Thirty-five days after inoculation, the mice were sacrificed and subjected to lung metastatic analysis.
For ERK inhibitor and HDAC1 inhibitor treatments, 2 × 106 TGIF2WT-overexpressing H1299 cells were injected into NOD/SCID mice via the tail vein. Twelve days after injection, mice were randomized into four groups (PBS, ERK inhibitor, HDAC1 inhibitor, ERK inhibitor + HDAC1 inhibitor). The mice were intraperitoneally injected with 50 mg/kg ERK inhibitor (SCH772984) (Selleck, Houston, Texas, USA) every day for 14 days. The mice received 80 mg/kg HDAC1 inhibitor (MGCD0103) (Selleck, Houston, Texas, USA) by daily intragastric administration for 14 days, and PBS was used as a control. Thirty days after inoculation, the metastatic foci in the lung were subjected to HE staining or IHC staining.
Bioinformatics analysis
Gene expression profiles of 532 LUAD patients were downloaded from The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov). Gene set enrichment analysis (GSEA) was performed according to the authors' guidelines published on the Broad Institute web pages (http://www.broadinstitute.org/gsea/index.jsp) with cell adhesion molecules signature and epithelial-mesenchymal transition gene set (from the MSigDB C2 curated gene sets collection) using Signal2Noise ratios. Survival analyses were conducted with the online tool (http://kmplot.com/analysis/index.php?p=service&cancer=lung) using the 2015 version. Patients with lung adenocarcinoma were selected for the First Progression (FP) and Post Progression Survival (PPS) assays. Log-rank was automatically computed.
Statistical analysis
The data are presented as the mean ± standard deviation. A two-tailed Student’s t-test was performed to compare the differences between the two groups. A one-way ANOVA with post hoc Tukey’s Multiple Comparison Test was used to analyze the xenograft growth. A p-value of < 0.05 was used as the criterion for statistical significance. Spearman’s r test was used to determine the correlation between TGIF2 and E-cadherin. GraphPad Prism 8 was used to perform the statistical analyses.