NUPR1 promotes the proliferation and metastasis of oral squamous cell carcinoma cells by activating TFE3-dependent autophagy

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy, and metastasis accounts for the poor prognosis of OSCC. Autophagy is considered to facilitate OSCC development by mitigating various cellular stresses; nevertheless, the mechanisms of autophagy in OSCC cell proliferation and metastasis remain unknown. In our study, high-sensitivity label-free quantitative proteomics analysis revealed nuclear protein 1 (NUPR1) as the most significantly upregulated protein in formalin-fixed paraffin-embedded tumour samples derived from OSCC patients with or without lymphatic metastasis. Moreover, NUPR1 is aberrantly expressed in the OSCC tissues and predicts low overall survival rates for OSCC patients. Notably, based on tandem mass tag-based quantitative proteomic analysis between stable NUPR1 knockdown OSCC cells and scrambled control OSCC cells, we confirmed that NUPR1 maintained autophagic flux and lysosomal functions by directly increasing transcription factor E3 (TFE3) activity, which promoted OSCC cell proliferation and metastasis in vitro and in vivo. Collectively, our data revealed that the NUPR1–TFE3 axis is a critical regulator of the autophagic machinery in OSCC progression, and this study may provide a potential therapeutic target for the treatment of OSCC.


Materials and Methods
Tissue microarray (TMA) For all included patients, TMA was taken. TMA analysis and IHC staining were performed by Wuhan Servicebio Technology Co., Ltd. Tissue sections were incubated with NUPR1 antibody (Abcam, ab234696). Two expert pathologists examined and scored the TMA independently without any sample information.
FFPE tissue preparation and high-sensitivity label-free quantitative proteomics analysis FFPE tumour tissue preparation and label-free were performed as previously reported. 1 FFPE biobank specimens (5 serial sections, 10 μM thick) were deparaffinized by xylene (50°C, 5 min) and washed in 1 ml absolute ethanol twice. Areas containing 70 % or more tumor were dissected from the slide according to a same tumor slide as reference which was performed by hematoxylin stained. Lysis was extracted in 4 % SDS at 99°C for 60 min and accompanied by 15 min sonication. Proteins in the cleared lysate were reduced with 10mM DTT for 30 min and alkylated with 55 mM iodoacetamide for an additional 30 min. 100 μg proteins were purified by acetone precipitation and the protein pellets resolved in 100 mL 6 M urea/2 M thiourea. Protein lysis was digested with LysC buffer for 3 h and 4 volumes of 50 mM ammonium bicarbonate buffer, 1 mg trypsin were added for tryptic digestion overnight. The next day, digestion was stopped by 1 % TFA. Peptides were finally desalted on C18 StageTips, suspended in 10 mL 2 % acetonitrile, 0.1 % TFA and kept at -20°C until LC-MS/MS analysis. Samples from the two patient groups were measured in random order. Peptide samples were diluted to 1 μg/μl on-board buffer, the sample volume was set to 5 μl, and the scanning mode was 120 min. Scan the peptides with a mass-to-charge ratio of 350-1500 in the sample. Prepare mobile phase A solution (98 % water, 2 % ACN, 0.1 % FA), B solution (98 % ACN, 2 % water, 0.1 % FA), pre-column (300 μm×0.5 mm, 3 μm), analytical column (3 μm, 75 μm×150 mm, Welch Materials, Inc), spray voltage 1.9 KV, peptides separated by liquid phase are ionized by nanoESI source and enter into tandem mass spectrometer Q-Exactive HFX (Thermo Fisher Scientific, San Jose , CA) detection. MS raw files were processed with the MaxQuant software. The integrated Andromeda search enginewas used for peptide and protein identification at an FDR of less than 1 %. The human UniProtKB database was used as forward database and the automatically generated reverse database for the decoy search. Label free protein quantification was performed using the MaxLFQ algorithm. The mass spectrometry proteomics data of FFPE tissues have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the iProX partner repository with the dataset identifier PXD030554.

Colony formation assay
The colony formation assay was performed as described in a previous report. 2 Briefly, 1 ml 0.8% agar solution in medium was added and solidified in six-well plates and then overlaid with 1 ml 0.3% agar solution in medium containing 2000 cells. After 10 days, the cell colonies were stained with 0.5 ml iodonitrotetrazolium violet (INT, 0.5 mg/ml; Merck, I7375-1G). The cell colonies were then counted and photographed.

Cell migration assay
After the cells were seeded in 6-well plates to 100 % confluency, a wound was induced by scratching the cell cultures with a 5 μl pipette tip. Following three rinses with phosphate-buffered saline (PBS) to remove detached cells, adherent cells were cultured in a medium without serum.
Four random fields of each well were imaged immediately after injury and 24 h later using a microscope (Nikon Corporation, Tokyo, Japan) at ×10 magnification. The distance between the wound edges was calculated using Nikon Application Suite software.

Cell invasion assay
Cell invasion assays were performed using Transwell chambers (Corning, 3422) as described in a previous report. 3 A total of 2 × 10 4 cells in serum-free medium were inoculated into Matrigelcoated upper chamber, and 600 μl medium with 10% FBS was inoculated into the lower chamber.
After crystal violet staining, the cells that crossed the membrane were counted and photographed.
TMT quantitative proteomic analysis Stable NUPR1 KD Cal27 cells and scrambled control were dissociated in SDT buffer (4 % SDS ， 100 mM Tris-HCl ， 1mM DTT ， pH 7.6). The proteomics analysis, including protein digestion, TMT labelling, fractionation, LC-MS/MS analysis, protein identification, and protein quantitation was performed by Applied Protein Technology (Shanghai, China) as described in our previous study. 4

Western blot analysis
The cells were dissociated in RIPA buffer (Beyotime, P0013B). The sample protein was electrophoresed by SDS-PAGE and transferred onto PVDF membranes. Membranes were subsequently probed with primary antibody at 4°C overnight. Then, the membranes were incubated with the secondary antibody for 1 h at room temperature, and the bands acquired from membranes were scanned with a ChemiDoc XRS + System. The signals of the bands were analysed with Image Lab (Bio-Rad). All the antibodies used are listed in Table S4.

Immunofluorescence analysis
Immunofluorescence analysis was performed according to our previous report. 6 The cells were fixed with 4% paraformaldehyde (Beyotime, P0099) and infiltrated with 0.25% Triton X-100 (Sangon Biotech, A110694-0100). The cells were blocked with 10% BSA in PBS and incubated with primary antibody at 4°C overnight, followed by fluorescent secondary antibodies based on the source of the primary antibody. The stained slides were examined using a confocal laser scanning microscope (Leica TCS SP8, Germany) equipped with a 63 × or 40 × oil objective. The colocalization coefficient was calculated using ImageJ software. At least 30 cells were counted for each experiment. All the antibodies used are listed in Table S5.

Real-Time PCR analysis
Total RNA was isolated with RNAiso Plus (TaKaRa, 9109) as previously described. 8 First-strand cDNA was synthesized from total RNA using SuperScript IV Reverse Transcriptases (Thermo Fisher, 18090050). Real-time PCR was detected by SYBR Green PCR Master Mix (Thermo Fisher, 4309155) using the LightCycler 96 System (Roche). The qPCR primers are listed in Table S6.
After 24 h, these cells were treated with rapamycin (0.1 µM) for another 24 h. The Secrete-Pair Dual Luminescence Assay kit (GeneCopoeia, SPDA-D010) was used to detect TFE3 promoter luciferase activity as previously reported. 9 BALB/c nude mice BALB/c nude mice (male, 4 weeks and approximately 20 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and bred in specific-pathogen-free (SPF) facilities at Shanghai Ninth People's Hospital.