Introduction

PTEN encodes a membrane-bound protein phosphatase with potent tumor suppressor activity.1,2,3 Loss of PTEN function leads to excessive activation of the PI3K/Akt oncogenic pathway, which stimulates cell growth and survival.4,5,6 Recent studies have suggested that nuclear PTEN exhibits a complementary mechanism with cytoplasmic PTEN to suppress tumor development.7 Nuclear PTEN interacts with centromeres to maintain their stability,8 is involved in DNA repair,9 and regulates gene transcription.10,11,12,13 Mono-ubiquitination of PTEN by Nedd4 and deubiquitination by HAUSP/USP7 contribute to the regulation of the nuclear import and export of PTEN.14,15 SUMOylation of PTEN also controls its nuclear localization. In cells exposed to genotoxic stress, SUMOylated PTEN was rapidly exported from the nucleus, a process that was dependent on the protein kinase ATM.16

Nedd8 is a ubiquitin-like protein that is covalently conjugated to substrates in a manner similar to ubiquitin.17 The neddylation system consists of an activating enzyme (E1, a heterologous dimer composed of UBA3 and NAE1/APP-BP1), two conjugating enzymes (E2s, UBE2M/Ubc12 and UBE2F) and a variety of E3 ligases.18 Neddylation primarily targets Cullin–RING ubiquitin ligases for their activation, and an increasing number of non-Cullin targets of Nedd8 have been identified. Neddylation can be removed by deneddylases, including NEDP1 and JAB1/CSN5.19,20 An inhibitor of E1, named MLN4924 (also known as pevonedistat), has been evaluated in a series of phase I/II/III clinical trials both alone and in combination with chemo-/radiotherapies.21,1. MMTV-PyMT mice (PyMT/WT mice, FVB/N background) were kindly gifted by Professor Mingyao Liu from East China Normal University. The db/db mice were purchased from Shanghai Model Organisms Center, Inc. The NAE inhibitor MLN4924 (HY-70062), proteasome inhibitor MG132 (HY-13259), XIAP inhibitor Embelin (HY-17473), p300/CBP inhibitor ICBP112 (HY-19541), 2-Deoxy-d-glucose (a glucose analog and a competitive inhibitor of glucose metabolism) (2-DG, HY-13966) were purchased from MCE. Cisplatin (P4394) and methyl methane sulfonate (MMS) (129925) were purchased from Sigma Aldrich. SUMOylation inhibitor 2-D08 (S8696) was purchased from Selleck. The antibody details were listed in Supplementary information, Table S2.

Cell culture and transfections

MCF-7, MDA-MB-231, MDA-MB-468, 4T1, HEK293T cell lines were purchased from ATCC, and authenticated by STR profiling and tested for mycoplasma contamination by GENEWIZ. MCF-10A (a human non-tumorigenic breast epithelial cell line) cells were kindly gifted by Qinong Ye from Bei**g Institute of Biotechnology. HEK293T NEDP1+/+ and NEDP1–/– cells were kindly gifted by Professor **aofeng Zheng from Peking University. MCF-7, MDB-MA-468, HEK293T, HEK293T NEDP1+/+ and NEDP1–/– cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). MDA-MB-231 cells were cultured in Leibovit’s L-15 medium supplemented with 10% FBS. MCF-10A cells were cultured in mammary epithelial cell growth medium (Promo Cell, C-39115) with 10% horse serum, New Zealand Origin (Invitrogen, 16050-114). Mouse 4T1 cells were cultured with MEM medium supplemented with 10% FBS. Cells were transfected with various plasmids using TuboFect (Thermo Fisher Scientific, R0531), Sage LipoPlusTM, Lipofectamine 3000 (Invitrogen, L3000001) according to the manufacturer’s protocol.

Immunoprecipitation and immunoblotting

For immunoprecipitation assays, cells were lysed in EBC lysis buffer (0.5% NP-40, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF and 1 mM β-mercaptoethanol) supplemented with protease inhibitor cocktail (Roche, 11836170001). Immunoprecipitations were performed using the indicated primary antibody and protein A/G agarose beads (Santa Cruz, sc-2003) at 4 °C. The immunoprecipitants were washed at least three times in NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH7.8, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.

RNA interference

siRNAs against Mdm2 (sc-29394), Roc1 (sc-44072), c-Cbl (sc-29242), XIAP (sc-37508), Nedd4-1 (sc-41079), WWP2 (sc-40362), WWP1 (sc-40366), NEDP1 (sc-44452) were purchased from Santa Cruz Biotechnology. Sequence information of the siRNA, shRNA and sgRNA is provided in Supplementary information, Table S3.

In vivo modification assays

Cells were solubilized in modified lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1% SDS, 1 mM Na3VO4, 1 mM DTT and 10 mM NaF) supplemented with a protease inhibitor cocktail. The cell lysate was incubated at 60 °C for 10 min. The lysate was then diluted 10 times with modified lysis buffer without SDS and incubated with the indicated antibody for 3 h at 4 °C before adding Protein A/G-plus Agarose (Santa Cruz, sc-2003). Then the lysate was rotated gently for 8 h at 4 °C. The immunoprecipitants were washed at least three times in wash buffer (50 mM Tris, pH7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1 mM DTT and 10 mM NaF). Proteins were analyzed by western blotting with indicated antibodies.

In vitro modification assays

GST-PTEN and His-XIAP were expressed in Escherichia coli BL21 (DE3). For the Neddylation assay, 0.5 μg of GST-PTEN, 0.5 μg of His-XIAP were incubated with 2 μg of Nedd8, 10 ng of E1 (APPBP1-UBA3) and 200 ng of E2 (Ubc12) in a total reaction volume of 20 μL Nedd8 conjugation Rxn Buffer Kit (BostonBiochem, SK-20). Nedd8 (UL-812), Nedd8 E1 (APPBP1/UBA3) (E-313), Ubc12 (A-655) were purchased from BostonBiochem. Samples were incubated at 30 °C for 1 h, and reactions were terminated with SDS-PAGE loading buffer before western blotting.

His pull-down by Ni-NTA columns

Cells were collected and resuspended in binding buffer (6 M guanidine HCl, 0.1 M Na2HPO4, 0.1 M NaH2PO4, 0.01 M Tris (pH8.0), 10 mM β-mercaptoethanol, 5 mM NEM, 5 mM imidazole) and incubated with Ni2+-NTA Superflow Cartridges (QIAGEN, 17-5318-02) at 4 °C for 12 h. Immunoblotting was performed using indicated antibodies.

Generation of knockout cells

The knockout cell lines were generated using the CRISPR–Cas9 method. CRISPR guide sequences targeting PTEN, UBA3, Ubc12, XIAP and NEDP1 were designed by software at http://crispr.mit.edu and cloned into lenti-Crispr pXPR_001. The sgRNA sequences were listed in Supplementary information, Table S3. MCF-7 and MCF-10A cells were co-transfected with the Lenti-Crispr vector and packaging plasmids pVSVg and psPAX2. Puromycin-resistant single cells were plated in a 96-well dish to screen for positive monoclonal cells.

Fluorescence microscopy

After fixation with 4% paraformaldehyde and permeabilization in 0.2% Triton X-100 (PBS), cells were incubated with the indicated antibodies for 12 h at 4 °C, followed by incubation with goat anti-rabbit IgG H&L Alexa Fluor 488 or 594 antibody for 1 h at 37 °C. The nuclei were stained with DAPI, and images were visualized with a Zeiss LSM 510 Meta inverted confocal microscope.

Cell fractionation

Nuclear fractions were prepared by using the Minute TM Nuclear and Cytoplasmic Extraction Reagents Kit (Invent biotech, SC-003) according to the manufacturer’s protocol.

Real-time PCR

Total RNA was isolated and converted to cDNA using the ReverTra Ace® (Toyobo, TRT-101). Quantitative PCR reactions were carried out using SYBR Green PCR master mix (Toyobo, QPK201). Primers were shown in Supplementary information, Table S4.

Cell proliferation assay

Cells were plated on 96-well plates (1000 cells per well). After adding Cell Counting Kit-8 (Do**do, CK04) to the wells for 1 h, cell numbers were measured at 450 nm. Each cell line was set up in 4 replicate wells, and the experiment was repeated three times.

Colony formation assay

Cells were seeded into 6-well tissue culture plate with 6000 cells per well. Plates were incubated at 37 °C and 5% CO2 and colonies were scored 21 days after preparation.

Cell migration assay

The assay was performed in an invasion chamber consisting of a 24-well tissue culture plate with 12 cell culture inserts (Becton-Dickinson). A cell suspension in serum-free culture medium (5 × 104 cells per well) was added to the inserts, and each insert was placed in the lower chamber containing 10% FBS culture medium. Cells were allowed to migrate for 24 h in a humidified chamber at 37 °C with 5% CO2. Then filter was removed and fixed with 4% formaldehyde for 20 min. Cells located in the lower filter were stained with 0.1% crystal violet for 15 min and photographed.

Tumor growth in mice

The experimental procedures in mice have been approved by the Animal Care and Use Committee of Academy of Military and Medical Sciences. BALB/c nude mice (6-week old, 18.0 ± 2.0 g) were obtained from Shanghai Laboratory Animal Center (SLAC, China). Diabetic db/db mice (6-week old, 35 ± 2 g) were obtained from Shanghai Model Organisms Center, Inc. (SMOC, China). Cells (5 × 106 per mouse) were inoculated subcutaneously into the right flank of the mice. Tumor size was measured every 3 days and converted to TV according to the following formula: TV (mm3) = (a × b2)/2, where a and b are the largest and smallest diameters, respectively. All animals were killed 4 weeks after injection, and the transplanted tumors were removed, weighed and fixed.

Cohort and immunohistochemistry

The breast tissue microarrays were purchased from Shanghai Biochip Company (HBre-Duc170Sur-01, HBre-Duc139Sur-01, HBre-Duc090Sur-01) and Shanghai Superbiotek Company (BRC1601). HBre-Duc170Sur-01 and HBre-Duc090Sur-01 were immunostained with NEDP1, UBA3 and PTEN (Supplementary information, Data S4 and S6). HBre-Duc139Sur-01 and HBre-Duc090Sur-01 were immunostained with Nedd8-PTEN K402 antibody (Supplementary information, Data S5). The patient’s pathological information in HBre-Duc139Sur-01 is fully included in the HBre-Duc170Sur-01 array. Breast cancer tumors and matched adjacent normal tissues were approved by the department of breast and thyroid Surgery at the Second People’s Hospital of Shenzhen (14 cases in Supplementary information, Data S7). BRC1601 were used for multicolor fluorescence immunohistochemistry with neddylated PTEN on K402, FASN, UBA3, XIAP and PTEN antibodies (Supplementary information, Data S8). All staining was assessed by a quantitative imaging method; the percentage of immunostaining and the staining intensity were recorded. An H-score was calculated using the following formula: H-score = Σ (PI × I) = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3). PI indicates the percentage of positive cells versus all cells.

ELISA assay

The quantitation of FASN level was performed using ELISA assay kit (Biomatik, EKU04054) according to the manufacturer’s instructions.

LC-MS/MS analysis

Proteins in IP samples were precipitated with IP buffer (0.5% NP-40, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF and 1 mM b-mercaptoethanol) and then washed three times with IP buffer. The samples from in-gel digestion were analyzed on a ThermoTM Q Exactive (Thermo Fisher Scientific) interfaced with an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific). The tryptic peptides were dissolved in 0.1% FA, directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo). Peptide separation was performed using a reversed phase analytical column (Acclaim PepMap RSLC, Thermo). The gradient was comprised of an increase from 6% to 25% solvent B (0.1% FA in 98% ACN) over 16 min, 25% to 40% over 6 min and climbing to 80% in 4 min then holding at 80% for the last 4 min, all at a constant flow rate of 320 nL/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM (Thermo) coupled online to the UPLC. Intact peptides were detected in the orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 28; ion 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 was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the orbitrap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. The resulting MS/MS data were processed using Mascot search engine (v.2.3.0). Tandem mass spectra were searched against Swissport Human database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys were specified as fixed modification and oxidation on Met, acetylation on Protein N-term were specified as variable modifications. Peptide ion score was set ≥ 20. Protein identification data (accession numbers, peptides observed, sequence coverage) are available in Supplementary information, Data S1 and S3, respectively. All raw data and search results have been deposited to the PRIDE database (http://www.iprox.org/index). The accession numbers are PXD011368 (Supplementary information, Data S1), PXD021544 (Supplementary information, Data S2) and PXD011395 (Supplementary information, Data S3).

GC-FID/MS analysis of fatty acids

Fatty acid composition was measured in the methylated forms. About 5 mg protein lysed from indicated cells was homogenized with 5% methanol sulfate and 0.2% BHT methanol at 90–95 °C water bath for 1.5 h. Then Saturated saline and N-hexane were added before centrifugation 5 min at 4 °C. Methyl-19-carbonate was added and vortexed for 10 s. After methylated, fatty acid composition was analyzed using 7890B Gas Chromatograph (GC) 5977 A Mass Selective Detector (MSD) (Agilent Technologies, USA) which was equipped with a flame ionization detector (FID) and a mass spectrometer carrying an electron impact (EI) ion source. An Agilent DB-225 capillary GC column (10 m, 0.1 mm ID, 0.1 μm film thickness) was used with 1 μL sample injection volume and a splitter (1:30). The temperature of injection port and detector was set at 230 °C. The column temperature was programmed with 55 °C for 1 min, and then increased to 205 °C with a rate of 30 °C per min. Column temperature was kept at 205 °C for following 3 min and increased to 230 °C at 5 °C per min. For identification, the methylated fatty acids were compared with a chromatogram from a mixture of 37 known standards and then confirmed with their mass spectral data. Each fatty acid was quantified with the FID data by comparing its signal integrals with peak integrals of internal standards. These data were expressed as μmol of fatty acids per gram of tissue, which are available in Supplementary information, Data S9.

Micro-PET/CT

Before 18F-FDG administration, mice were deprived of food for 8–12 h. 18F-FDG (11.1 MBq (300 mCi) in 0.2 mL) was injected through the tail vein. Micro-PET/CT imaging was started 1 h after 18F-FDG injection. Micro-PET/CT images was performed on a Super Nova PET/CT scanner (PINGSENG Healthcare (Kunshan) Inc.). For micro-PET/CT imaging, all mice were subjected to isoflurane anesthesia (1%–2% in 100% oxygen) on a temperature self-adjust tube to maintain body temperature throughout the procedure. Mice were visually monitored for breathing and any other signs of distress throughout the full imaging period. The images were reconstructed using a three-dimensional ordered subsets expectation maximum algorithm (OSEM) without attenuation correction. For data analysis, the region of interest (ROI) was manually drawn to cover the whole tumor on fused images.

Further information of materials and methods is listed in Supplementary information, Data S10.

Ethics statement

All animals were handled in strict accordance to the “Guide for the Care and Use of Laboratory Animals” and the “Principles for the Utilization and Care of Vertebrate Animals”, and all animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at the Bei**g Institute of Lifeomics. The clinical samples were approved by the department of breast and thyroid surgery at the Second People’s Hospital of Shenzhen. Informed consent was obtained from all subjects or their relatives.

Statistical analysis

All data were representative of no less than three independent experiments. All data were presented as means ± SD. Data were analyzed using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). The differences between groups were assessed by Student’s t-test. The correlations were analyzed using Pearson correlation test. A value of P < 0.05 was considered to be statistically significant.