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Global landscape of 2-hydroxyisobutyrylation in human pancreatic cancer

As a new type of post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) was firstly identified in histones and functioned as a regulator of transactivation in mammals. However, the role of Khib proteins remains to be investigated. Here, we firstly identified 10,367 Khib sites on 2,325 modified proteins in seven patients with pancreatic cancer by applying liquid chromatography with tandem mass spectrometry (LC-MS/MS) qualitative proteomics techniques. Among them, 27 Khib-modified sites were identified in histones. Bioinformatics analysis revealed that the Khib-modified proteins were mainly distributed in the cytoplasm and enhanced in metabolic pathways, including glycolysis/gluconeogenesis, the tricarboxylic acid cycle (TCA cycle), and fatty acid degradation. In an overlapping comparison of lysine 2-hydroxyisobutyrylation, succinylation, and acetylation in humans, 105 proteins with 80 sites were modified by all three PTMs, suggesting there may be a complex network among the different modified proteins and sites. Furthermore, MG149, which was identified as a Tip60 inhibitor, significantly decreased the total Khib modification level in pancreatic cancer (PC) and strongly suppressed PC’s proliferation, migration, and invasion ability. Overall, our study is the first profiling of lysine 2-hydroxyisobutyrylome and provides a new database for better investigating Khib in PC.


Introduction
Post-translational modifications (PTM) of proteins contribute to protein function by adding chemical groups to amino acid residues to alter the charge, structure, and molecular weight of the protein (1), thereby affecting protein activity, subcellular location, interaction chaperones, stability, and amplifying/ weakening protein function (2). Until now, more than 200 PTMs have been identified, including phosphorylation, ubiquitination, acetylation, succinylation, and 2-hydroxyisobutyrylation (3)(4)(5).
It is universally acknowledged that 2-hydroxybutyrate (2-HIBA) is a microbial metabolite detected at micromolar concentrations in human bio-fluids, including blood, saliva, and urine (6). Previous reports demonstrated that elevated 2-HIBA levels were associated with metabolic diseases, including diabetes (7) and obesity (8). 2hydroxyisobutyrate was a synthetic precursor of 2-hydroxyisobutyryl coenzyme A, which was regulated by carbon sources (9) and identified as the donor of lysine 2-hydroxyisobutyrylation (K hib ) (10). Lysine 2-hydroxyisobutyrylation is a novel PTM, which was first detected in histones in 2014 (4) and has been validated by following reports in animals, plants, and microorganisms (11)(12)(13)(14). Tip60 was identified as a K hib writer in mammalian cells, which could catalyze K hib modifications in vitro and in vivo. In contrast, histone deacetylase 2 (HDAC2) and histone deacetylase 3 (HDAC3), but not histone deacetylase 1 (HDAC1), acted as erasers to remove K hib modifications in vivo (14). Previous studies have confirmed that K hib is a widespread type of PTM with strict evolutionary conservation, and it could be up-regulated by carbon sources generated by the pentose phosphate pathway and glycolysis/ gluconeogenesis (12), which strongly suggestes a close correlation between K hib modification and metabolism. Recently, K hib was reported to be involved in cancer progression. K hib was identified in ENO1 and promoted the ability of glycolysis and proliferation in hepatoma cells (15). Also, K hib in oral squamous cell carcinoma (OSCC) was suggested to promote cancer progression (16).
Pancreatic cancer (PC) is one of the most malignant digestive tumors, with a five-year survival rate of only 9%. In the past decade, the mortality rate of men with PC has increased steadily, rather than decreasing. According to a statistical report, there were up to 420,000 new PC cases and 410,000 deaths worldwide in 2020 (17), and it is predicted that PC may become the second most deadly malignancy in 2030, just after lung cancer (18). Currently, the treatment of PC is mainly surgical resection described as pancreaticoduodenectomy; however, owing to its insidious onset, high malignancy, and rapid progression, the operable rate is far below 20% (19). Post-translational modifications have a vital role in epigenetics and signal transduction, numbers of studies have focused on the contribution of PTMs as diagnostic markers or therapeutic targets for cancer treatment (20). Although there have been many reports on lysine 2-hydroxyisobutyrylation in recent years, the role of K hib in PC development remained unclear.
In the present study, we first reported the K hib sites in human pancreatic cancer via proteomics. Totally, 10,367 lysine 2hydroxyisobutyrylation modification sites on 2325 proteins were identified, and they mainly enriched in protein synthesis and metabolic pathways. Moreover, MG149, a selective inhibitor of Tip60, was proved to enormously decrease the total Khib level in PC and suppress PC cell proliferation and metastasis abilities. The novel K hib sites and proteins identified in this research not only promote our insights into the functional role of K hib but also provide new perspectives and potential drug targets for future investigations in PC.

Result
The lysine 2-hydroxyisobutyrylation modification in pancreatic cancer To demonstrate the K hib modification stage in PC, the tissues from seven different patients (Table S1) were digested and lysed to extract proteins (Figure S1A), and then Western Blotting with pan-2-hydroxyisobutyryllysine antibody (pan-anti-K hib ) was used to prove the status of K hib in these proteins ( Figure 1A). Also, proteins obtained from different pancreatic cell lines (HPDE, SW1990, BXPC, ASPC, Mia PaCa-2, PANC-1) were examined by WB ( Figure 1B and Figure S1B). Our analysis showed that the level of K hib modification in different PC tissues or cell lines was slightly different, mainly concentrated at 35-75 kD, indicating Khib was widely distributed and conserved in PC.

Systematic analysis of lysine 2hydroxyisobutyrylation in pancreatic cancer
Based on the antibody enrichment and liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods, proteomic analysis was performed on seven pancreatic cancer tissue samples extracted from patients with PC ( Figure 2A). In this study, a total of 10,367 K hib -modified sites in 2,325 proteins were identified with a localization probability > 0.75. The details of all proteins and associated K hib sites identified in this study were listed in Table S2. Our study showed a great depth of detection in K hib proteins compared to previous studies and contributed to a more comprehensive K hib map in PC ( Figure 2B). To characterize the extent of K hib coverage in the substrate proteins, we calculated the number of every protein modification site. Most proteins showed 1-3 modification sites, 38%, 17%, and 11%, respectively. In comparison, the percentage of proteins with > 20 sites was 3% ( Figure 2C), demonstrating that K hib modifications always have more than one site in most of the proteins, suggesting us to pay attention to the position of modification and possible interrelationships in the subsequent protein modification studies.
The Motif-X program was performed to detect the sequence motifs in the K hib -modified peptides (  E). The motif enrichment heatmap showed a significant preference for negatively charged glutamate (E) and aspartate (D) around the modified lysine residue. In contrast to previous studies, alanine (A) and valine (V) showed great preference around the modified lysine residue ( Figure 2F), which indicated the complex nature of the potential PTM mechanism in PC.

Functional analysis of the lysine 2hydroxyisobutyrylated proteins in PC
To systematically describe the K hib modified proteins, Wolfpsort software was applied to analyze the subcellular location of the 2-hydroxyisobutyrylated proteins in PC. There were 37%, 23%, 13%, 13%, and 6% K hib proteins distributed in the cytoplasm, nucleus, mitochondria, extracellular, and plasma membrane, respectively ( Figure 3A), suggesting that the K hib modification was widespread in PC. Then, Gene Ontology (GO) analysis, including biological processes, cellular components, and molecular functions, was applied to demonstrate the potential functions and locations of the K hib proteins (Table S4). According to the cellular component data, K hib proteins were enriched in the cytoplasm (27%), organelles (16%), membrane-enclosed lumen (9%), and membrane (9%). In accordance with the molecular function analyses, the K hib proteins were mainly associated with binding (63%) and enzyme activity (21%), revealing that K hib modification may play a key role in protein function regulation, DNA transcription, and metabolic activity. The biological process analyses presented that the K hib proteins were mainly distributed in the metabolism (31%), biological process (13%), stimulation response (12%), and location (7%) ( Figure 3B and Figure S3A). Furthermore, to explore the characteristics and role of the K hib proteins in PC, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to show that proteins with K hib modification concentrated in metabolic pathways, including glycolysis/gluconeogenesis, TCA cycle, and fatty acid degradation, among others, in accordance with a previous study on Oryza sativa (11) and Ustilaginoidea virens (21) ( Figure S3C). Also, ribosomes, proteasomes, and proteins related to multiple diseases were enriched in the KEGG pathways analysis, indicating that the K hib proteins had a close interrelationship with protein synthesis, degradation, bio-metabolism, and disease development ( Figures 3C, B). The K hib proteins were observed intensively in the proteasome subunit, 14-3-3 protein, serpin (serine protease inhibitor), cofilin/tropomyosin-type actin-binding protein, thioredoxin-like domain, and Core histone H2A/H2B/H3/H4 in the protein domain enrichment analysis ( Figure 3D). The conservation and specificity of the lysine 2-hydroxyisobutyrylated proteins Lysine 2-hydroxyisobutyrylation was first reported in the histones and was critical for regulating chromatin function (4). To investigate the K hib modification status of histones in PC, we listed 27 K hib sites on the histones identified in this study (Table S5), and a comparison was performed with other species, including rice seed (22), mammals (14), and moss (23) ( Figure 4A), which revealed multiple overlapping K hib modification sites on the H3 and H4 proteins, and the specific sites H4K13 and H4K78 were identified. Among these, nine sites on H2B were observed, which were mainly distributed between K34-K120 in the globular structure, and it may affect the binding of H2B to DNA by shifting the charge state (4). Overall, different species have their unique K hib sites, suggesting that K hib is not only conserved but is also a unique PTM in histones. Genome-wide identity and profiling of the K hib proteins and sites in PC. (A) Workflow for the identification of K hib modification in PC. Proteins extracted from the PC tissues were digested into peptides, and then the specific K hib antibody enriched the K hib sites-containing peptides, followed by the liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. (B) Comparison of the K hib proteins and sites identified in our study and those in previous reports. (C) Numbers and ratios of K hib sites for each protein. (D) The top five K hib motifs. The size of each letter indicates the frequency of that amino acid residue at that position. (E) Frequency of occurrence of each motif among the peptides with K hib modifications. (F) The heatmap of the amino acid profile of the K hib sites, featuring the enrichment (red) and depletion (green) of the amino acids at every position (from -10 to +10) on both sides of the K hib site.
Simultaneously, the K hib modification in proteins excluding histones showed its features. By comparing the K hib proteins identified in mammalian (HeLa cells) (14), psoriasis skin (24), and PC detected in this study, 615 Khib proteins were observed in all the reports ( Figure 4B and Table S6). The KEGG enrichment analysis of these common K hib proteins showed a preferred distribution in the ribosome and metabolism, including glycolysis/gluconeogenesis, carbon metabolism, and TCA cycle ( Figure 4C), indicating that K hib modification was conserved among different species. Meanwhile, the K hib proteins, especially those populated in PC, were enriched in the metabolic, phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling, and tight junction pathways (Table S6 and Figure   S6), indicating that K hib modification was of great significance in the fields of bio-metabolism in PC

Overlap of lysine 2hydroxyisobutyrylation and other PTMs
Post-translational modifications played crucial roles in regulating protein functions. It has been reported that various types of PTMs can be observed in one protein or even in the same locus. Compared with the results of previous studies in humans (25,26), a total of 105 proteins with 80 sites were identified to be modified by K hib , lysine succinylation (K su ), and lysine acetylation (K ac ) ( Figure 4D and Table S6). In addition, 242 and 297 K hib proteins presented K su and K ac modifications, respectively. In addition, 463 and 308 K hib sites were shared with K su and K ac , respectively ( Figure 4E and Table S6). Among them, some lysine sites closely related to protein activity showed multiple modifications, suggesting that K hib might be biologically meaningful.

Lysine 2-hydroxyisobutyrylation in central metabolism
As described in the KEGG analysis, K hib proteins were concentrated in metabolic pathways in PC ( Figure 3C). Compared with other species ( Figure S3C) (11,21), glycolysis/gluconeogenesis was observed in the K hib proteins analysis, suggesting that K hib plays a critical role in regulating glucose metabolism. Further, many enzymes in central metabolism, including glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate metabolic pathways, were mapped to study the function of K hib in PC ( Figure 5). It is worth noting that some critical enzymes represented multiple modification sites, including PGK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase (LDH) (Table S7). Clearly, K hib modifications were prevalent in metabolic enzymes, and the modification status of specific sites affected enzyme activity and served a vital role in various metabolic pathways.

Inhibiting lysine 2-hydroxyisobutyrylation level suppressed PC proliferation, migration, and invasion
MG149 was identified as a selective inhibitor of Tip60 (27), which was known as a "writer" of K hib modification in mammalian cells (14). When treated with MG149, the level of K hib modification in PC cells was significantly decreased (Figures 6A, B and Figure  S5). In order to investigate the role of K hib in PC development, the CCK8, colony-forming, wounding, and transwell assays were performed with the SW1990 and ASPC-1 cells. The CCK8 and colony-forming assay results suggested that MG149 contributed to reduced viability in the SW1990 and ASPC-1 cells (Figures 7A, B).
Besides, the results of wounding and transwell assay demonstrated that the ability of migration and invasion was significantly suppressed when PC cells were treated with MG149 ( Figures 7C,  D). Thus, the results above strongly suggested that lysine 2hydroxyisobutyrylation affected the PC cells' proliferation, migration, and invasion ability in vitro.

Discussion
Post-translational modifications of proteins, including phosphorylation, acetylation, and lactylation, alter protein function by adding chemical groups. 2-hydroxyisobutyrylation FIGURE 5 The enrichment of the 2-hydroxyisobutyrylation (K hib ) proteins in the central metabolic pathways in pancreatic cancer (PC). The K hib enzymes are marked with red.
is a novel type of PTM identified in histones, and it played a crucial role in the regulatory function of chromatin (4) with operational differences from acetylation modifications (28). K hib is prevalent in several species, including plants, microorganisms, and humans. Recently, studies on K hib have been extended to non-histone proteins, and it is suggested to be involved in the development of human diseases. The K hib proteins identified in peripheral blood from patients with immunoglobulin A (IgA) nephropathy aggregate in the interleukin-17 (IL-17) signaling pathway and phagosome class (29), and they were also observed to be enriched in the PI3K-Akt signaling pathway in psoriasis skin lesions, which is associated to psoriasis progression (24). Conversely, K hib has rarely been studied in pancreatic cancer. To investigate the role of K hib in the progression of PC, we identified 10367 Khib sites on 2325 proteins in this study, which were more compared to that in previous studies, and expanded the databases of K hib proteins in PC.
In agreement with previous studies, 27 K hib sites in histones were identified in this study, of which the modification sites in H2B were mainly concentrated in the spherical structure of K34-A B FIGURE 6 The total 2-hydroxyisobutyrylation (K hib ) level in pancreatic cancer (PC) cells were suppressed by MG149. The SW1990 and ASPC-1 cells were treated with MG149 (74 mM), and then the immunofluorescence and Western blot assays were performed with pan-K hib antibody. (A) Immunofluorescence assay for PC cells treated with MG149. Different stains represent specific target genes, K hib (green) and DAPI (blue). Scale bar, 100 mm. (B) Western blotting analysis of the K hib proteins in the SW1990 and ASPC-1 cells treated with MG149. **P < 0.01, determined by independent Student's t tests. Data are derived from three independent experiments and presented as means ± SDs. K120, which may have affected the binding of H2B to DNA (4). Given that there are fewer studies on K hib in humans, for reasons of homology, we chose Khib data from mammalian cells and psoriasis skin in humans to perform a comparison with K hib proteins in PC. Notably, 615 K hib proteins were found in the above studies, and they were predicted to make great contributions to regulating metabolism pathways, including the TCA cycle, glycolysis, and carbon metabolism. In addition, 105 proteins and 80 sites were found to show three modifications by overlapping analysis with acetylation, succinylation, and 2hydroxyisobutyrylation. This may result from regulation by the transcriptional co-activator P300, which was suggested to catalyze various modifications of histones, including acetyla tion, propionylati on, butyrylation, and 2hydroxyisobutyrylation (6, 28, 30, 31). Therefore, it is reasonable to suppose that there is either synergistic or competitive crosstalk among different types of PTMs, suggesting the possibilities of multiple modifications of proteins should be fully considered in studies of proteins, highlighting the importance and necessity of protein modification mapping.
Pancreatic tumor is exposed to severe physical, oxidative, and inflammatory stress. Thus, cancer cells rewire intermediary metabolism to promote unlimited proliferation in a hypoxicischemic environment, evade immune surveillance, and develope distant metastases. Previous studies have demonstrated that metabolic reprogramming promotes pancreatic tumorigenesis and metastasis through epigenetic regulation (32,33). Notably, a total of 66 K hib proteins with 670 sites were observed in central metabolism (Table S7) KEGG analysis showed an apparent enrichment in metabolic pathways, including glycolysis/gluconeogenesis, TCA cycle, and fatty acid degradation ( Figure 3C). These results suggested that K hib may significantly influence PC metabolism and contribute to PC progression. MG149 was identified as a highly selective inhibitor of Tip60, which was known as a writer catalyzing K hib in mammalian cells (14). In order to explore the contribution of K hib to PC development, the SW1990 and ASPC-1 cells were treated with MG149 to decrease the total level of K hib modification. Then, we conducted the CCK8 and colonyforming assay, the results of which showed that MG149 strongly reduced the proliferation viability of the SW1990 and ASPC-1 cells compared to the control group. In addition, the results of the wounding and transwell assay suggested that MG149 suppressed the ability of migration and invasion in the SW1990 and ASPC-1 cells. The results above demonstrated that lysine 2-hydroxyisobutyrylation played an essential role in PC development and reminded us to pay more attention to 2hydroxyisobutyrylation modification in PC. PTMs impact protein stability and function, and PTM therapies targeting proteins have made considerable advances in cancer (20,34). It is promising that the inhibitors focusing on K hib proteins may be potential targets for pancreatic cancer therapy. Since K hib modification is a new type of protein modification, it is still unexplored and lacks mature sitespecific antibodies, making further in-depth studies challenging. Indeed, comparing data between cancerous and paraneoplastic tissues should be more convincing; however, given individual patient characteristics, a large number of samples are required. Based on the considerations above, we aimed to explore K hib protein profiles of PC with cancer tissue and provided a new perspective for further studies. In addition, although mass spectrometry has developed rapidly, the information on low-abundance proteins and amino acid residues with low recognition sensitivity was often missed, which made subsequent studies difficult. Bao et al. developed the 2-hydr_Ensemble residue identification algorithm with artificial intelligence approaches, which was more accurate than traditional algorithms in inferring K hib -modified residues (35) and it could be helpful for understanding the functions and further studying the K hib -specific modification sites. In the current study, we first described 10367 K hib -modified sites on 2325 modified proteins in seven persons with pancreatic cancer and then demonstrated that K hib modification played a significant role in PC development. Indeed, the potential mechanisms by which K hib promoted PC progression remained unclear, more samples and studies are needed to elucidate how lysine 2-hydroxyisobutyrylation affects PC development. Our work has undeniably helped to enhance our view of this new lysine PTM. The significant amount of K hib proteins identified in PC is expected to serve as an invaluable database for future pancreatic cancer-related research, providing new insights and potential targets for treating pancreatic cancer.

Conclusions
2-hydroxyisobutyrylation at lysine is a novel type of PTMs, first reported in the histones of HeLa cells. Recently, great studies about K hib modification identified in proteins have been reported in fields of diverse species. This study provides an indepth insight into the K hib protein in pancreatic carcinoma for the first time and found that the K hib proteins are mainly enriched in bio-metabolic pathways. Further, we have demonstrated that K hib may act as a tumor promoter in PC development and provided a new perspective to explore the multiple functions of 2-hydroxyisobutyrylation and potential treatment targets in PC.

Patients and clinical sample collection
Seven patients with PC who had not been treated with radiotherapy were recruited for our study, and all patients independently signed written informed consent and donated tumor tissues for scientific research. This study was approved by the Tongji Hospital Research Ethics Committee and the Institutional Review Board. The enrolled patients' demographic characteristics and clinical information are listed in Table S1. After surgery for PC, cancer tissues were rapidly dipped into MACS Tissue Storage Solution (Miltenyi Biotec GmbH, USA) and then stored at -80°C.

Protein extraction
Samples were ground to cell powder in liquid nitrogen, and then four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail, 3 mM TSA, and 50 mM NAM) were added, followed by sonication three times on ice, after centrifugation (12,000 g at 4°C for 10 min), and collection of the supernatant. Then, the protein concentration was determined by using the BCA kit.

Trypsin digestion
The protein solution was reduced with dithiothreitol (5 mM, 56°C, 30min) and then alkylated with 11 mM iodoacetamide (15 min, room temperature, dark conditions) and diluted with 100 mM TEAB in order to make the sample urea concentration less than 2 M. The first digestion was performed overnight at a trypsin-to-protein ratio of 1:50(trypsin: protein, weight to weight), followed by second digestion of 1:100 for 4 h.

Affinity enrichment of lysine 2hydroxyisobutyrylated peptides
Tryptic peptides dissolved in NETN buffer were incubated overnight with prewashed antibody beads(PTM-802, PTM Bio), followed by washing with NETN buffer and H 2 O. 0.1% trifluoroacetic acid was used to elute the bound peptides and then combined and vacuum-dried. Finally, all samples were desalted for LC-MS/MS analysis.

LC-MS/MS analysis
Tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a reversed-phase analytical column (15-cm length, 75 mm i.d.). As described previously (13), the gradient comprised of solvent B at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. Then, the peptides were subjected to the NSI source, followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC, with the following parameter: 2.0 kV electrospray voltage, 350 to 1800 m/z scan range, and resolution of 70,000 for detection. Selected peptides MS/MS and fragments were detected in the Orbitrap at a resolution of 17,500. Depending on the data, a program of 20 MS/MS scans was carried out alternating after one MS scan with 15.0s dynamic exclusion. Automatic gain control (AGC) was programmed at 5E4. The fixed first mass was set at 100 m/z.

Database search
The MS/MS data were manipulated using the MaxQuant search engine (v. 1.5.2.8). The detailed steps have been previously described (13).

Bioinformatics analysis
Gene Ontology annotation was performed using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). The KEGG database was applied to identify the protein pathway. The Motif-X algorithm was used to analyze the motif characteristics of the modification sites. Wolfpsort software was used to predict subcellular localization.

Inhibitor
MG149 was used with a concentration of 74 µM, which was reported as half maximal inhibitory concentration (IC 50 ) of Tip60 (27).

Western blot
The western blotting assay was performed to detect the expression of the Khib proteins in PC tissues and cell lines. Briefly, cells or tissues were lysed by pre-formulated lysis buffer (RIPA, 1% Protease Inhibitor Cocktail, 3 mM TSA, and 50 mM NAM). The protein concentrations were measured by BCA kit (P0012S; Beyotime Biotechnology, China), and then we analyzed 50mg protein of every sample on 10% SDS-PAGE gels. All blots were visualized using ECL Kit (RM00020P; ABclonal, Wuhan, China) and the intensity of the bands was assessed by Image Lab (Bio-Rad, California, USA). The antibodies used in this study were: pan-lysine 2-hydroxyisobutyrylation antibody (1:1000; PTM-802; PTM Bio, Hangzhou, China), FITC antibody (1:50; AS011; ABclonal, Wuhan, China), b-actin antibody (1:1000; AC026; ABclonal, Wuhan, China). b-actin was used as a loading control and the intensity of the entire bands was assessed with ImageJ2 (National Institute of Mental Health, Bethesda, MD, USA). All experiments were performed three times following the same procedure.

Immunofluorescence
Cells were spread evenly on coverslips and incubated for 24 hours, then fixed in 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.1% TX-100 for 10 min at room temperature. Then, the cells were blocked with 5% BSA in PBS for 1 hour and incubated overnight at 4°C with pan-Khib antibody (PTM-802; PTM Bio, Hangzhou, China). After washing three times with PBS, cells were incubated with the appropriate secondary antibody for 1 hour at 4°C. Coverslips were stained with DAPI and mounted. Immunofluorescence images were then observed and captured under a fluorescent microscope.

Cell proliferation and drug cytotoxic assay in vitro
The viability of the SW1990 and ASPC-1 was evaluated with the cell counting kit-8 (Dojindo Laboratories Co. Ltd, Kumamoto, Japan). In brief, both SW1990 and ASPC-1 cells were seeded in 96-well plates at a density of 3 × 10 3 cells per well. There were five replicates per group (n = 5). At the time points indicated, 10 mL of CCK-8 solution was added to each well and incubated at 37°C and 5% CO 2 for 3 hours. Then, the absorbance at 450 nm at different time points with the plate reader (Bio-Tek Elx 800, USA) to assess cell proliferation. All experiments were performed three times following the same procedure.

Colony formation assay
The SW1990 and ASPC-1 were seeded into 6-well plates with a density of 500 cells per well and cultured in a completed medium at 37°C and 5% CO 2 for 14 days. Then, the indicated cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystalline violet dye solution. After drying, the wells were photographed with a cell phone. All experiments were repeated three times.

Wound healing assay
The indicated PC cells were cultured in 6-well plates until the cell density approached 90%; we scratched the wound vertically across the center of the well with the tip of a 200 mL pipette gently. After being washed with PBS three times, the PC cells were cultured with a serum-free medium at 37°C, 5% CO2. The result of the assay was observed and captured with a microscope (Nikon, Japan) at 0 and 24 hours, respectively. All experiments were repeated three times.

Transwell assay
To examine cell migration capacity, the PC cells were suspended at a density of 1 × 10 5 /mL in a 24-well transwell plate (8 mm pore size; Corning) containing 200 mL of a serumfree medium in the upper transwell chamber, and 700 mL of a complete medium in the lower chamber. After co-culturing for 24 h, we fixed the PC cells on the sub-membrane surface with 4% paraformaldehyde and stained them with the crystalline violet solution. Counted the number of stained cells with a Nikon light microscope (Nikon, Japan). In the case of the invasion assay, 60 mL of Matrigel matrix gel is placed in the upper transwell chamber (BD Biosciences, America). Other operations performed were the same as cell migration assay described above. All experiments were carried out three times following the same procedure.

Statistical analyses
One-way analysis of variance (ANOVA) and Duncan's multiple range test were performed by SPSS v22.0. Quantitative data were analyzed by two-tailed independent Student's t tests.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material. The mass spectrometry proteomics data in this study were deposited in the ProteomeXchange Consortium via the PRIDE (36) partner repository with the dataset identifier PXD029817 (http://www.ebi.ac.uk/pride).

Ethics statement
The studies involving human participants were reviewed and approved by Tongji Hospital Research Ethics Committee and the Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.

Author contributions
YL conceived the idea and designed the study, analyzed data, and wrote the manuscript. XL, performed most of the experiment. KZ, PQ, ZD, provided help for data analysis. JW and WY supervised the entire project.

Funding
This study was supported by the National Natural Science Foundation of China (Nos.81874062 and 82072730), and the Youth Program of National Natural Science Foundation of China (81902439).