Cell lines and drugs
Human colon cancer cells, SW480, SW620, HCT116, RKO, and FHs 74 Int (ATCC, Rockville, MD), were maintained in 1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2. The HCT116 highly invasive (HCT116 I8) cell line was screened by using repeated invasion assays as previously described . All the cell lines were authenticated by short tandem repeat profiling and tested for mycoplasma contamination. U0126, trichostatin A (TSA) and avobenzone were purchased from Selleck Chemicals (Houston, TX, USA). Respectively, U0126 (10mM, 10mg in 2.34mL dimethyl sulfoxide (DMSO)), TSA (10mM, 5mg in 1.65mL DMSO), and avobenzone (10mM, 10mg in 3.22mL DMSO) were dissolved for in vitro usage. Avobenzone (2mg/mL, 14mg) was dissolved in corn oil (7mL) for in vivo usage.
Mass spectrometry and bioinformatics analyses
Cells were lysed by lysis buffer (Cell Signaling Technology, Danvers, MA, USA), and the cell lysates were further digested by trypsin (Hualishi Scientific, Beijing, China) through filter-aided sample preparation (FASP). The peptide samples were then dried by vacuum freeze and redissolved in trifluoroacetic acid (TFA) (0.5% in Water, Optima LC/MS Grade, Fisher Chemical) solution. After further desalination through MonoTIPTM C18 Pipette Tips (GL Sciences, Tokyo, Japan), the peptide samples were loaded and analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Then, the raw data were analyzed through Spectronaut software (Omicsolution Co., Ltd., Shanghai, China), and the differentially expressed proteins were analyzed by Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Redwood City, CA, USA).
Plasmids, siRNAs and transfection
Flag-PTPLAD1 and Flag-PHB plasmids were generated by PCR amplification of sequences obtained from a colon cancer cDNA library and cloned into a pcDNA3.1 vector. The following primers were used to generate the PTPLAD1 and PHB plasmids: PTPLAD1: forward (5’-CGCGGATCCATGGAGAATCAGGTGTTGACGCCGC-3’) and reverse (5’-CGCGAATTCTTACTTGTCATCGTCGTCCTTGTAGTCGTGGATCTTTTTCTTTTTTTGTC-3’); PHB: forward (5’- CGCGGATCCCATGGCTGCCAAAGTGTTT -3’) and reverse (5’- CGCGAATTCTTACTTGTCATCGTCGTCCTTGTAGTCCTGGGGCAGCTGGAGGAGCACGGAC -3’). Transfections were performed using LipofectamineTM 3000 reagent (Thermo Fisher Scientific) according to the manufacturer’s recommendations. HCT116 cells stably expressing PTPLAD1, PHB, and PTPLAD1+PHB or GFP control genes were established with approximately 2 weeks of puromycin selection as described previously . The siRNA sequences and primers used to generate other plasmids are listed in Supplementary Tables S1 and S2.
The cell pellets were suspended in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and incubated on ice for 30 min. The cell lysates were subsequently centrifuged at 14000 g for 30 min at 4°C, after which supernatant was mixed with loading buffer and boiled for 10 min at 95°C before being loaded onto a sodium dodecyl sulfate (SDS) polyacrylamide gel for electrophoresis. The proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% fat-free milk in Tris-buffered saline-Tween 20 (TBST), the membranes were probed with the appropriate primary antibodies, followed by the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology). The signals were detected by Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and visualized by exposure to autoradiographic film. Antibodies against the following proteins were used for the experiment: PHB (#60092-1-Ig), ERK (#66192-1-Ig) and Raf1 (#51140-1-AP) were obtained from Proteintech Group (Chicago, IL, USA); phospho-c-Raf (Ser338, #9427) was obtained from Cell Signaling Technology (Danvers, MA, USA); and phospho-ERK 1/2 (#ab50011) and PTPLAD1 (#ab139063) were obtained from Abcam (Cambridge, MA, USA).
Boyden Chamber Invasion Assay
Cell invasion assays were performed with an 8-μm pore size invasion chamber coated with 5% Matrigel (BD Biosciences, San Jose, CA, USA) in serum-free 1640 medium. Cells were suspended in serum-free medium and seeded into the upper chamber, and the lower compartment was filled with complete medium. After incubation at 37°C and 5% CO2 for 36 h, the invaded cells adhering to the bottom surface of the chamber membrane were fixed with methanol and then stained with crystal violet (0.2% in methanol). Images of three different fields were captured from each membrane, and the number of invaded cells was counted.
Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed as described previously . In brief, total RNA was isolated using TRIzol reagent according to the manufacturer's protocol (Invitrogen). cDNA was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The mRNA expression levels of PTPLAD1 and the GAPDH control were detected by real-time PCR using SYBR® Premix Ex Taq™ II (Takara). The primers used were 5’-GCCTAAATAAACGCCGACT -3’ (forward) and 5’- AGAATCGCACAGTCAGGTT-3’ (reverse) for ANKRD1 and 5’-GAAGGTGAAGGTCGGAGTC-3’ (forward) and 5’-AAGATGGTGATGGGATTTC-3’ (reverse) for GAPDH.
The cell lysates were prewashed with IgG (Santa Cruz Biotechnology) and protein A/G Sepharose beads (Invitrogen, Gaithersburg, MD) for 1 h at 4°C, and the cell supernatants were incubated with the appropriate primary antibody overnight at 4°C before being incubated with protein A/G Sepharose beads for 4 h. The beads were washed thrice with lysis buffer and eluted in 2 × SDS/PAGE loading buffer for immunoblotting. For LC-MS/MS analysis, the lanes on the silver-stained gels were cut into several bands and digested in an in-gel, after which the peptide mixtures were analyzed by LC-MS/MS.
Cells were transiently co-transfected with PTPLAD1-mCherry and PHB-GFP plasmids for 48 h and then fixed in 4% paraformaldehyde. The cells were subsequently stained with 40,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA) and observed by laser scanning confocal microscopy (Carl Zeiss AG, Jena, Germany).
Chromatin immunoprecipitation (ChIP) assay
The chromatin immunoprecipitation (ChIP) assay was performed by using the simple ChIP enzymatic chromatin IP kit (Cell Signaling, Beverly, MA, USA) according to the manufacturer's manual. Briefly, in vivo protein and DNA crosslinking was performed using 37% formaldehyde, followed by sonication and chromatin digestion. The protein–DNA complexes were immunoprecipitated by using the acetylated H3K9 antibody or negative control IgG antibody. After elution and reversal of crosslinking with proteinase K, the purified DNA was subjected to qRT-PCR analysis (Bio-Rad).
Experimental metastasis assay in mice
Female NCD mice aged 5-8 weeks were maintained under standard conditions and cared for according to the institutional guidelines for animal care. PTPLAD1 stably expressing or knocked down cells, as well as the corresponding negative control cells, were constructed based on ectopic luciferase expression in HCT116 cells (i.e., PTPLAD1-overexpressing cells. HCT116-Luc). Approximately 1 × 106 HCT116 cells were suspended in phosphate buffered saline (PBS) and injected intravenously through the lateral tail vein of the mice. For pharmacological experiments, the animals were treated every three days with avobenzone (10 mg/kg) or vehicle through intragastric administration after injection of cancer cells. Metastatic activity was assessed by counting tumor nodes in main organs, such as the lung, liver and kidney. The animals were euthanized at the end of the experiment (21 days), and the main organs were collected for further analyses, such as histological analysis. All animal experiments were approved by the Ethics Committee for Animal Experiments of Jinan University.
Fifteen pairs of colon tumor tissues and adjacent nontumor tissues were used for qRT-PCR and ChIP assays. Ethical consent was granted from the Ethical Committee Review Board of Jinan University.
Purification of Raf1-His and PHB-GST fusion proteins
Protein purification was performed as described previously . The Raf gene was amplified with genomic DNA from HCT116 cells by PCR using the forward primer 5’- CGCGGATCCGCATCAATGGAGCACATACAGG -3’ and the reverse primer 5’- CGCGAATTCGAAGACAGGCAGCCTCGGGGAC -3’ and cloned into the pET-28b vector with a His tag. The PHB gene was amplified using the forward primer 5’- CGCGGATCCGAGGGTCCAGCAGAAGGAAAC -3’ and reverse primer 5’- CGCGAATTCGTGCAGGCAGGGTGGGCCCTCA -3’ and cloned into the pGEX-4T-1 vector with GST flag. Raf and PHB expression plasmids were transformed into E. coli BL21 star (DE3) cells for subsequent expression. The bacteria were grown to an optical density at 600 nm (OD600) of approximately 1.0 at 37°C, induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 6 h and lysed via sonication. Raf1-his and PHB-GST fusion proteins were isolated by loading whole cell lysate across His-binding and GST-binding columns according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China) and verified by SDS-PAGE and western blotting.
Enzyme-linked immunosorbent assay (ELISA) screening system
The experiment was performed as described previously . Briefly, the first layer antibody His-tag antibody (Proteintech) in coating buffer (1 ng/μL) was added to a 96-well plate and cultured overnight at 4°C. After washing with PBS and blocking with 5% bovine serum albumin (BSA), the purified fusion protein Raf-His (1 μg) was added, and the plate was softly rocked for 5 h at room temperature. The plate was then incubated with 1 μg purified PHB-GST protein for 3 h at 37°C, and 440 small molecular inhibitors (10 μM) from the FDA-approved Drug Library (Selleck Chemicals, USA) were added individually into each well. After incubation with GST-tagged antibody, the corresponding secondary antibody and tetramethylbenzidine (TMB), the absorbance was measured at wavelengths of 450 nm and 630 nm. The level of Raf-PHB interaction was determined using the formula O.D.450-O.D.630. Coating buffer, TMB and termination buffer were purchased from NeoBioscience (Shenzhen, China).
The results were analyzed by using GraphPad PRISM software (GraphPad Software Inc., San Diego, CA, USA), and the data from different experiments were compared by Student’s t-test and expressed as the mean ± SD. Sample size in animal experiments was chosen based on the literature documentation of similar well-characterized experiments, and no statistical method was used to predetermine sample size. Pearson’s chi-square test was performed to analyze the association between PTPLAD1 expression levels and categorical clinicopathological variables. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. P < 0.05 was considered statistically significant.