Protection of c-Fos from autophagic degradation by PRMT1-mediated methylation fosters gastric tumorigenesis

Both AP-1 and PRMT1 are vital molecules in variety of cellular progresssion, but the interaction between these proteins in the context of cellular functions is less clear. Gastric cancer (GC) is one of the pernicious diseases worldwide. An in-depth understanding of the molecular mode of action underlying gastric tumorigenesis is still elusive. In this study, we found that PRMT1 directly interacts with c-Fos and enhances AP-1 activation. PRMT1-mediated arginine methylation (mono- and dimethylation) of c-Fos synergistically enhances c-Fos-mediated AP-1 liveliness and consequently increases c-Fos protein stabilization. Consistent with this finding, PRMT1 knockdown decreases the protein level of c-Fos. We discovered that the c-Fos protein undergoes autophagic degradation and found that PRMT1-mediated methylation at R287 protects c-Fos from autophagosomal degradation and is linked to clinicopathologic variables as well as prognosis in stomach tumor. Together, our data demonstrate that PRMT1-mediated c-Fos protein stabilization promotes gastric tumorigenesis. We contend that targeting this modification could constitute a new therapeutic strategy in gastric cancer.


Preparation of whole-cell lysates, tissues, and immunoblotting
A total of 30 pairs of GC patient tumor tissues and normal adjacent tissues (NATs) was obtained from patients who underwent surgical resection of GC at Ajou University Hospital. Specimens were collected at Ajou Human Bio-Resource Bank and frozen at −80℃ until use. Written informed consent for the usage and storage of specimens was received from all patients. The present study was conducted in accordance with the ethics code of the World Medical Association (Declaration of Helsinki) and was approved by the Institutional Review Board of Ajou University Hospital (AJIRB-BMRKSP-19-059) as previously reported [30,33]. The GC patient tumor tissues and NATs specimens were ground with liquid nitrogen, while the cell lines were washed with phosphate-buffered saline (PBS) (B2814; Samchun Pure Chemical, Pyeongtaek, Korea), collected, and centrifuged at 12,000 rpm for 5 min at 4°C. They then were lysed with buffer (20 mM ], 2 mM of ethylenediaminetetraacetic acid [EDTA], 2 mM of ethyleneglycotetraacetic acid [EGTA], 1 mM of DTT, 50 mM of β-glycerol phosphate, 0.1 mM of sodium vanadate, 1.6 mM of pervanadate, 1% Triton X-100, 10% glycerol, 10 µg/mL of aprotinin, 10 μg/mL of leupeptin, 10 μg/mL of pepstatin, 1 mM of benzamide, and 50 µM of PMSF). Protein lysates were pelleted via centrifugation (12,000 rpm, 5 min, 4°C), and the supernatant was used for western blotting with antibodies against Myc, Flag, GFP, total c-Fos, LC3B, mme-R, adme-R, and β-actin [35].

Immunoprecipitation
Cell lysates containing equal amounts of protein (1,000 μg) were prepared, and samples were incubated with 5 µL of antibodies overnight at 4°C. Immune complexes were mixed with 40 μL of protein A-or G-coupled Sepharose beads (50% v/v) and rotated for 4 h at 4°C. Boiled immune complexes were immunoblotted, and the protein levels were determined as previously reported [36][37][38].
Immunofluorescence and image analysis HEK293T cells were plated on a glass coverslip overnight and transfected with Myc-c-Fos and EGFP-PRMT1 for 48 h. Cells were fixed in 3.7% paraformaldehyde in PBS and then permeabilized with 1% Triton X-100 in PBS at room temperature. After washing, cells were treated with 1% bovine serum albumin in PBS to minimize nonspecific staining. For staining, cells were incubated with anti-Myc, anti-Flag, or anti-adme-R (1:1000 dilution) for 1 h at room temperature. Secondary antibodies were conjugated with Alexa Fluor (1:000 dilution) for 1 h and then with Hoechst staining solution (1:1000 dilution) for 20 min at room temperature. DAPI was used for DNA counterstaining [39]. The coverslips were mounted on slide glasses using fluorescent mounting medium (DakoCytomation, Carpentaria, CA, USA). A confocal laser-scanning microscope (LSM 700; Zeiss, Jena, Germany) equipped with a C-Apochromat 63×/1.2 water immersion objective was used to acquire images [40,41]. mRNA analysis by semi-quantitative RT-PCR or quantitative real-time PCR To examine the mRNA expression level of c-Fos, HEK293T cells were transfected with Mycc-Fos and EGFP-PRMT1. The total RNA from the cells was isolated with TRI reagent (TR118; Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. The total RNA was stored at −70°C until use. Semi-quantitative RT-PCR was performed as previously reported [42]. The mRNA quantification was conducted via real-time RT-PCR with SYBR Premix according to the manufacturer's instructions (PCRBIOSYSTEMS, London, UK) using a real-time thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) as previously reported [43,44].

Protease protection assay
The protein level in proteasomes was evaluated by a protease protection assay according to a previous report [45]. c-Fos or c-Fos/PRMT1 plasmids were transfected (3 samples/group) for the indicated time, and the reaction was stopped by adding cold PBS. Cells were harvested and washed twice with PBS. Next, 6.5 µg/mL of digitonin solution (100 mM of potassium phosphate [pH: 6.7], 5 mM of MgCl2, and 250 mM of sucrose) was added to cells (2 samples/group) that then were washed with cold PBS. Cells were incubated with 1 µg/mL of proteinase K for 10 min with or without 0.1% Triton X-100, which dissolved all membranes. Buffer was removed, and sample buffer was added for protein analysis. β-actin was used as a protein control.

LC-MS/MS spectrometry
Myc-c-Fos-transfected cells were collected and immunoprecipitated with Myc. Immunoprecipitated samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue dye. The samples were subjected to tryptic digestion at 37°C overnight; followed by lyophilization, reconstitution, and fractionation; and then analyzed by strong cation exchange (SCX) liquid chromatography (LC) and mass spectrometry.
Wound healing assay MKN45 cells were transfected with empty vectors or the indicated plasmids (PRMT1 and c-Fos WT or c-Fos R287K) and seeded in a 12-well plate with 5.5 × 10 5 cells/well. Cells were incubated overnight at 37°C under 5% CO2 and then scratched using sterile P200 pipette tips in a straight line in the center of the wells, which then received new media. Cells were photographed after 0, 24, 48, and 72 h, and the wound closure rate was determined by ImageJ analysis (U.S. National Institutes of Health, Bethesda, MD, USA). Data are shown as the mean ± SD.
Colony formation assay MKN45 cells were transfected with empty vectors or the indicated plasmids (PRMT1 and c-Fos WT or c-Fos R287K) for 24 h using Lipofectamine 2000 (11668-019; Invitrogen). Cells were seeded (0.5 × 10 3 cells/well) in six-well plates. After 10 days of incubation (37°C, 5% CO2), the colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet (C3886; Sigma-Aldrich). The number of colonies was captured using a digital camera and counted using ImageJ. Data are shown as the mean ± SD.
Invasion assay PRMT1-overexpressing MKN45 cells or PRMT1-knockdown MKN45 cells were cotransfected with c-Fos-WT or mutants for 24 h using Lipofectamine 2000. Cells were plated (5 × 10 4 cells/well) in an upper Transwell chamber with membrane-permeable polycarbonate filters coated with Matrigel (356237; BD Biosciences, San Diego, CA, USA) in Opti-MEM (11058021; Gibco Laboratories). The bottom chamber of the 24-well plate was filled with RPMI 1640 as a chemoattractant. After 24 h incubation (37°C, 5% CO2), the upper chamber was removed, followed by fixation of cells on the lower side (invaded cells) using 4% paraformaldehyde. Invaded cells were stained with hematoxylin (ab220365; Abcam, Cambridge, US) and eosin y solution (HT110116; Sigma-Aldrich). The invaded cells were captured using a camera microscope and counted in three random fields per well. Data are shown as the mean ± SD.

Statistical analysis
Statistical analysis and data visualization were performed using SigmaPlot 11.0 (Systat Software, San Jose, CA, USA) or R (version 4.2.1). The results were analyzed by Student's t test or the Mann-Whitney U test. The survival curve was evaluated by Kaplan-Meier analysis. P < 0.05 was considered statistically significant.              201   ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKI  ILAAHRPACKM  ILAAHKP I CKI  ILAAHKPACKI   108   AGAYSRAGVVK  AGAYARAGMVK  TGAYARAGVVK  AGAYSRAGIVK  AGAYSRAGVVK  AGAYSRAGVMK