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Prostate cancer

N-glycosylation of GDF15 abolishes its inhibitory effect on EGFR in AR inhibitor-resistant prostate cancer cells

Cell culture

LNCaP (ATCC) cell and 22Rv1 (ATCC) cell were grown in phenol red free Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Scientific, CA, USA) containing 5% fetal bovine serum (FBS; ThermoFisher), or 5% charcoal-stripped FBS (c-FBS; ThermoFisher) and 1% streptomycin-penicillin at 37 °C with 5% CO2. The cell lines were authenticated by short tandem repeat analysis and mycoplasma contamination was tested by the PCR Mycoplasma Detection Set (Takara, Otsu, Japan). LNCaP cell was treated with the ENZ (10 µM) or EPI (8 µM) at concentrations as reported [41, 42]. ST cell was treated for 9 days, and LT cell was treated for 33 days according to the method of Sharma et al. [43].

Chemicals

Enzalutamide (MCE, HY-70002, NJ, US), EPI-001 (Selleck, S7955, TX, US), R1881 (Sigma, R0908, MO, US), Afatinib (Selleck, S1011, TX, US), Bosutinib (Selleck, S1014, TX, US), PD98059 (Selleck, S1177, TX, US), Cycloheximide (MCE, HY-12320, NJ, US), and Tunicamycin (MCE, HY-A0098, NJ, US) were stored as stock solutions in DMSO (Sigma, MO, US).

Cell viability analysis

Cell viability was assessed by Cell Counting Kit (CCK-8; MCE, HY-K0301, NJ, US) according to the manufacturer’s instructions. Briefly, cells were seeded at a concentration of 6000 cells/200 µL/well into 96-well plates, incubated overnight, changed to fresh medium with various inhibitors. Following treatment, 10 µL CCK-8 solution were added and cells were incubated for 4 h at 37 °C. Optical density (OD) value was measured at 450 nm by a microplate spectrophotometer (Thermo Fisher). IC50 concentrations of ENZ and EPI were used in all drug treatment experiments. All experiments were performed three times in triplicates.

Western blotting

Cells were treated as described and then lysed by boiling for 10 min in sample buffer (2% SDS, 10% glycerol, 10% β-mercaptoethanol, bromphenol blue and Tris-HCl, pH = 6.8). Lysates were fractionated on SDS-PAGE gels and transferred to PVDF membranes (Millipore, IPVH00010, NH, US). The blots were probed with specific antibodies followed by secondary antibody then membranes were detected by ECL (Sigma, WBULS0500, MO, US). AR (22089-1-AP; 1:1000), TMPRSS2 (14437-1-AP; 1:1000), PSA (60338-1-Ig; 1:2000), UBE2C (66087-1-Ig; 1:2000), CDC20 (10252-1-AP; 1:500), P21 (60214-1-Ig; 1:1000), Cyclin E1 (11554-1-AP; 1:1000), CDC6 (11640-1-AP; 1:2000), CDC2 (19532-1-AP; 1:1000), SRC (11097-1-AP; 1:500), GDF15 (27455-1-AP; 1: 2000), AKT (60203-2-Ig; 1: 5000), pAKT (66444-1-Ig; 4000), GAPDH (60004-1-Ig; 1:50000), β-actin (66009-1-Ig; 1:10000), β-tubulin (10068-1-AP; 1:4000) antibodies were purchased from Proteintech Group (IL, US). AR-V567es (ab200827; 1:1,000) antibodies were purchased from Abcam (MA, US). EGFR (4267; 1:2000), pEGFR (3777; 1:2000), ERK1/2 (9102; 1:2000), pERK1/2 (4376; 1:2000) antibodies were purchased from Cell Signaling Technology (MA, US). ConA (Canavalia ensiformis)-Biotin (C7401; 1:1000) antibody was purchased from Sigma (MO, US). AAL (Aleuria Aurantia Lectin)-Biotin (B-1395-1; 1:1000) antibody was purchased from Vector laboratories (CA, US).

Secondary antibodies were conjugated with HRP (Proteintech Group; SA00001-2, SA00001-1; 1:10000). Uncropped WB are shown in Figure S2.

Cell cycle analysis

LNCaP-NC, ST, and LT cells were harvested and washed, and then fixed with 70% ethanol solution (v/v) at 4 °C for more than 18 h. After washing with pre-cold PBS, cells were stained with propidium iodide (PI) containing RNase (PI/RNase Staining Solution, CST 4087, MA, US) for 15 min in the dark, and then subjected to cell cycle analysis on a flow cytometer. The cell cycle data were analyzed using ModFit LT 5.0 software (Verity, ME, US).

Proteomics study

LNCaP-NC, ST-EPI, ST-ENZ, LT-EPI, and LT-ENZ cells were used for quantitative proteomics analysis. Samples were lyzed with 8 M Urea (pH = 8.0) and concentration was quantified using BCA kit (Beyotime, P0012, Shanghai, China). Proteins were reduced with dithiothreitol (DTT) and then alkylated with iodoacetamide (IAM) in dark. Sequencing-grade trypsin (Promega, WI, USA) was added for overnight digestion. Peptides were desalted and reconstituted in 0.5 M tetraethyl-ammonium bromide (TEAB) and processed with TMT10 plexTM kit according to the manufacturer’s protocol (Thermo Scientific, CA, USA). Global-peptides were resuspended in 2% acetonitrile (ACN) and 0.1% formic acid (FA) solution and then analyzed using an EASY-nLC 1200 system (Thermo Scientific, CA, USA) coupled with a high-resolution Orbitrap Fusion Lumos mass spectrum (Thermo Scientific). Peptides were first separated with an RSLC C18 column (1.9 µm × 100 µm × 20 cm) packed in house, then selected for MS/MS using NCE setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.

Glycomics study

Intact glycopeptides (IGPs) were enriched as described previously [44]. IGPs were desalted, resuspended and then analyzed using nanoLC-MS system. LC conditions and MS parameters for IGPs were described previously [44]. Results were filtered based on the following criteria: (1) a false discovery rate (FDR) less than 1% for glycoproteins and (2) each peptide spectra matches (PSM) annotated by at least one N-linked glycan.

Glycoproteins and site analysis

The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against homo_Uniprot-organism database (https://www.uniprot.org/taxonomy/9606) concatenated with reverse decoy database. For IGPs identification, data were searched using GPQuest 2.0 [45]. Parameters for analysis were described previously [44].

Functional annotation and enrichment analysis

Gene Ontology (GO) annotation glycoproteome was derived from the UniProt-GOA database (https://www.uniprot.org/uniprot/A0A178W639). Firstly, identified protein IDs were converted to UniProt ID and then mapped to GO IDs by protein ID. If some identified proteins were not annotated by UniProt-GOA database, InterProScan (https://www.ebi.ac.uk/interpro/about/interproscan) would be used to annotated protein’s GO functional based on protein sequence alignment method. Then proteins were classified by Gene Ontology annotation based on three categories: biological process, cellular component, and molecular function. For enrichment of pathway analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/) was used to identify enriched pathways by a two-tailed Fisher’s exact test to detect the enrichment of differentially expressed protein against all quantitative glycoproteins. Pathways with a corrected FDR ≤ 0.05 were considered significant, and classified into hierarchical categories. Furthermore, proteins in selected pathways were visualized by a heat map using the “pheatmap” function from the “pheatmap” R-package (https://www.r-project.org/).

Quantitative real-time PCR

LNCaP Cells were cultured with c-FBS for 5 days before treatment with DMSO + ENZ/EPI or R1881 + ENZ/EPI for 12 h. Total RNA was extracted using TRIzol (Takara, Japan) according to the manufacturer’s instructions. One µg of total RNA was used for complementary DNA synthesis using a cDNA reverse transcription kit (Takara, Japan). Real-time PCR was performed in triplicate using gene-specific primers on a Bio-Rad CFX96 PCR system. The gene-specific primers are listed in Table S7.

Immunoprecipitation (IP)

Cells were lyzed in IP buffer (Beyotime, P0013, Shanghai, China). Protein samples were quantified using BCA kit (Beyotime, P0012, Shanghai, China) and precleared with A + G magnetic beads (MCE, HY-K0202, NJ, US). Immunoprecipitation was performed with GDF15 antibody (Abcam, ab206414, MA, US) overnight at 4 °C. Protein A + G magnetic beads were added, mixed for 4 h at 4 °C, captured with magnetic frame, washed 3 times, and then boiled in sample buffer. Supernatants were used for WB detection using GDF15, anti-Con A (Sigma, C7401) and AAL antibodies (Vector laboratories, B-1395-1).

Enzyme-linked immunosorbent assay (ELISA)

GDF15 were quantified using human cell culture supernatant with ELISA kit from Mlbio (ml024335-2, Shanghai, China), according to the protocols provided by the manufacturers.

Small-interfering RNA (siRNA)- mediated gene knockdown

EGFR siRNA (si-EGFR; 5′-CGCAAAGUGUGUAACGGAAUATTUAUUCCGUUACACACUUUGCGTT-3′), SRC siRNA (si-SRC; 5′-GACAGACCUGUCCUUCAAGAATTUUCUUGAAGGACAGGUCUGUCTT-3′), MAPK3 siRNA (si-ERK1; 5′-ACCUGCUGGACCGGAUGUUAATTUUAACAUCCGGUCCAGCAGGUTT-3′), and GDF15 siRNA (si-GDF15; 5′-CUAUGAUGACUUGUUAGCCAATTUUGGCUAACAAGUCAUCAUAGTT-3′) were made by Jiangsu Saisofi Biotechnology Co., Ltd (Wuxi, China), and Negative/GAPDH siRNA (si-neg; 5′-UUCUCCGAACGUGUCACG UTTACGUGACACGUUCGGAGAATT-3′/si-pos; 5′-UGACCUCAACUACAUGGUUTTAACCAUGUAGUUGAGGUCATT-3′) were used as negative/positive control. The siRNAs were transfected into Parental, ST, and LT cells using Polyplus-transfection (jetPRIME, NY, US) according to the manufacturer’s instructions. Successful knockdown was verified by quantitative real-time PCR (qRT-PCR) and WB.

Plasmid- mediated overexpressing and gene mutation

Overexpression plasmids of pcDNA3.1(+)-GDF15 and pcDNA3.1(+)-SRC plasmids were constructed. Plasmids of pcDNA3.1(+)-GDF15/N70Q and dominant negative SRC (DCsrc)/K296R/Y528F were constructed. All plasmids were transiently transfected using Fugene HD transfection reagent (Promega, WI, USA).

Animal study

For hypodermic CRPC xenografts, male BALB/c-nude mice (age of 8–9 weeks) were anaesthetized and then 1*106 22Rv1-WT or 22Rv1-N70Q stably transfected cells suspended in 30 µL 50% matrigel were surgically injected. One week after injection, the tumor-bearing mice were castrated and the tumor size was monitored weekly using vernier caliper. After 21 days, the mice were euthanized, tumors were dissected, photographed, and weighed. The experimental protocol was approved by the Animal Ethics Committee of Jiangnan University, China (JN.No20190630b2120101[190]).

Hi-Myc transgenic prostate cancer mice (gifted from George V. Thomas laboratory) [32] were used and all experimental protocols were approved by the Animal Ethics Committee of Jiangnan University, China (JN.No20190630t1360101[191]). To define temporal development of castration resistance, four-month-old male mice were randomly assigned to control (NC) and ENZ (10 mg/Kg) group, treated with drug by intragastric administration (i.g.) every 3 days for required months. The mice were then euthanized every month, and the prostate (anterior lobes, dorsal lateral lobes, and ventral lobes) were dissected, photographed, and weighed. After an initial decrease in prostate weight, regaining of prostate growth was considered as the emergence of castration resistance. Six-month old (4 months plus 2 months treatment) mice were randomly assigned to NC (saline solution i.g.), ENZ (10 mg/Kg i.g.), Bosutinib (10 mg/Kg i.g.), and Bosutinib + ENZ groups. Mice were treated every 3 days, euthanized every month. Prostate were dissected, photographed, weighed and then subjected to histopathological and WB analysis. Blood samples were collected from the orbital sinus and sera were separated, filtered, and stored at −80 °C until use.

Tissue staining

Briefly, after deparaffinization and rehydration, 5 µm thick longitudinal sections were stained with hematoxylin solution for 22 s, dipped in 1% hydrochloric acid ethanol, rinsed with distilled water, stained with eosin solution for 30 s, dehydrated with graded alcohol and cleared in xylene. Mounted slides were scanned using an Pannoramic Scanner (3DHISTECH, Budapest, Hungary).

Clinical study

Human peripheral blood samples were collected from the Affiliated Hospital of Jiangnan University. All patients signed informed consent. The study was approved and authorized by the Ethics Committee of the Affiliated Hospital of Jiangnan University (Approval document number: LS202128). The patients’ information is listed in Table S6.

Statistical analysis

Student’s t-test was used to compare means of two groups. One-way ANOVA was used to compare means of 3 or more groups (GraphPad, CA, USA). Turkey test was used to perform multiple comparison (IBM SPSS, NY, USA). Data were presented as mean ± std of biological repetition. P < 0.05 was considered as significant in all of the tests.

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