Suppression of G6PD induces the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin to block epithelial-mesenchymal transition and lymphatic metastasis

Background As the rate-limit enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) plays important roles in tumour progression, but the exact mechanism through which G6PD controls cancer metastasis remains unclear. Methods G6PD expression in resected oral squamous cell carcinoma (OSCC) samples was analysed by immunohistochemistry. The effects and mechanism of G6PD suppression on OSCC cell lines were measured by transwell assay, wound healing assay, western and lectin blot, mass spectrometer analysis, ChIP-PCR, and luciferase reporter assay. BALB/c-nude mice were used to establish orthotopic xenograft model. Results G6PD expression in the tumours of 105 OSCC patients was associated with lymphatic metastasis and prognosis. In vitro cellular study suggested that G6PD suppression impaired cell migration, invasion, and epithelial-mesenchymal transition. Furtherly, G6PD knockdown activated the JNK pathway, which then blocked the AKT/GSK-3β/Snail axis to induce E-Cadherin expression and transcriptionally regulated MGAT3 expression to promote bisecting GlcNAc-branched N-glycosylation of E-Cadherin. An orthotopic xenograft model further confirmed that dehydroepiandrosterone reduced lymphatic metastatic rate of OSCC, which was partially reversed by JNK inhibition. Conclusions Suppression of G6PD promoted the expression and bisecting GlcNAc-branched N-glycosylation of E-Cadherin via activating the JNK pathway, which thus acted on OSCC metastasis.


G6PD activity detection
G6PD activity detection was performed according to the manufacturer's instructions (Abcam, Cambridge, MA, USA). Briefly, the cells were seeded in a 6-well plate at a density of 4×10 5 cells per well. After treated with DHEA, total proteins were extracted from the cells using RIPA buffer (Huaxingbio, Beijing, China) containing a protease inhibitor. Protein concentration was measured by BCA method, and all samples were equalled to same concentration. 50 μL of standard dilutions (prepared with NADH standard) and samples were added into a 96-well plate. Each sample was added to four wells with three detection wells and one background well. Then, 50 μL of G6PD reaction mix was added into each standard and sample wells, and 50 μL of G6PD background mix was added into the background wells. The OD value was and measured at 450 nm on a microplate reader 10 min and 20 min later. The amount of NADH in sample well calculated from standard curve, and G6PD activity was calculated by dividing NADH amount with reaction time (10 min).

Immunoprecipitation of E-Cadherin
After treatment by siRNA, total protein was extracted from the cells following protein extraction protocol described in Western blot. E-Cadherin in each 100 μl of protein lysate was then immunoprecipitated by adding 1 μl of anti-E-Cadherin monoclonal antibodies (CST) and 15 μl of a Protein G Agarose Suspension (Millipore), followed by an overnight rotation at 4°C. Thereafter, the antibody-antigen-agarose complexes were separated by centrifuge, and washed by protein extracting solution for three times. 3×loading buffer (with SDS) was used to break the complex at 100℃ for 15min.

N-glycan structures analysis by mass spectrometry
Immunoprecipitated E-Cadherin were suspended in a reduced sample buffer, heated to 100°C for 10min, resolved by 10% (w/v) SDS-PAGE, and the gel was excised and cut into pieces. The gel pieces were distained and dehydrated with acetonitrile. Then the protein in gel was reduced and alkylated by the incubation with dithiothreitol and iodoacetamide. The protein was cut by trypsin at the concentration of 15 ng/μL, and the peptides were extracted by extraction solution (5% trifluoroacetic acid and 50% acetonitrile were diluted in water). LC/multistage MS (MS n ) was carried out on an Elite Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, USA). The eluents were 0.1% acetate, 2% acetonitrile (phase A) and 0.1% acetate, 80% acetonitrile (phase B). The peptides were separated on a capillary high-performance liquid chromatography (Thermo Fisher Scientific, USA) with a linear gradient of 6-95% phase B in 60 min. A full MS 1 scan (m/z 350-1550) followed by data dependent MS 2 for the most abundant ions was performed in HCD modes. The data were analysed by Byonic Software (Protein Metrics).

Metabolites associated with PPP analysis by mass spectrometry
After treated by siRNA, the cells were digested by 0.25% trypsin, followed by threetime PBS washing. Liquid Chromatography was performed by means of a UHPLC system (Shimadzu, Kyoto, Japan) equipped a LC-30AD solvent delivery system, a SIL-30AC autosampler, a CTO-30A column oven, a DGU-20A3 degasser and a CBM-20A controller. The separation of the compounds was carried out on Agilent XDB C18 (3.5 um, 3.0 X 100 mm) operated at 40°C. The eluents were 10 mM Dihexylammonium Acetate (DHAA) in water, pH=5.0 (phase A) and 10 mM DHAA in acetonitrile (phase B). They were delivered at a flow rate of 0.5 mL/min under a gradient program. The gradient system was 0-1.0min, 2% B; 1.0-8.4 min, 2-60% B; 8.4-8.5 min, 60-95% B; 8.5-12.0 min, 95-2% B. The mass spectra were acquired using a TripleTOF™ 5500 system with a Duo Spray source (SCIEX, CA, USA) in negative ESI mosde. The data were analysed by Peak View Software 2.2 (SCIEX).

ROS detection
For the measurement of ROS, we harvested the cells treated with siRNA for 48 h and then washed the treated cells twice with PBS. Then, 1×10 6 cells were suspended in 1 mL PBS and incubated with 10 μM DCFH-DA (Sigma, St. Louis, MO, USA), a cell membrane permeable fluorescence probe. Next, the cells were washed and suspended in 1 mL of PBS for measurements. Fluorescence levels were measured using flow cytometry (Beckman CytoFLEX) with excitation and emission wavelengths set at 480 nm and 530 nm, respectively.

Construction of luciferase reporter plasmids
The pGL3-basic plasmid (Promega) was used as the backbone. The sequence of the 2000 bp ahead of the transcriptional start site of MGAT3 was searched in the NCBI database (Gene ID: 4248, Transcript ID: NM_002409.5). The sequence was synthesised by Shanghai Shenggong Co., Ltd. in the pUC57 backbone. Kpn I and Nhe I (New England Biolabs, UK) were used for re-establishing the pGL3-MGAT3promoter plasmid. The Fast Site-Directed Mutagenesis Kit (Tiangen biotech, Beijing, CN) was used for point mutation on the plasmid. The mutation scheme is shown in Supplemental Figure S5B. All the plasmids were amplified in DH5α competent cells (Tiangen biotech). The EndoFree Mini Plasmid Kit II (Tiangen biotech) was used for plasmid extraction.

Immunofluorescence and lectin fluorescence
The tissue sections were prepared as described above. For immunofluorescence, antigen retrieval and endogenous peroxidase activity blockage were performed in the same manner as used for IHC. The tissue sections were then incubated with rabbit antihuman E-cadherin antibody (1:200, CST) overnight at 4℃, followed by a FITClabelled secondary antibody (Zhongshan Biosciences Inc.). For lectin fluorescence, the tissue sections were blocked with 1× Carbo Free Blocking Solution (Vector Laboratories) after deparaffinisation and rehydration and were incubated with Biotinylated Phaseolus Vulgaris Erythroagglutinin (PHA-E, 1:200, Vector Laboratories) overnight at 4℃. This was followed by staining with DyLight 594 Streptavidin (Vector Laboratories) for 1 h at RT. Nuclear staining was performed by incubation with DAPI (Zhongshan Biosciences Inc.). The images were then scanned with an optimal microscope (Olympus).

Name of antibody Dilution rate Brand
Rabbit  Table 2 The sequence of the primers used in qPCR  Wound healing assays in CAL27 cells treated with siNC, siG6PD, or siG6PD with SP600125 (10 μM). Five different fields were recorded and measured. (E) Transwell assays in CAL27 cells treated with siNC, siG6PD, or siG6PD with SP600125 (10 μM). Five different fields were recorded and measured. (F) Western blot analysis of Ecadherin and G6PD in CAL27 cells treated with siNC, siG6PD, or siG6PD with SP600125 (10 μM). Densitometric analysis of protein expression relative to RPS18 levels (n=2 per group). (G) Lectin blot analysis of PHA-E in CAL27 cells treated with siNC, siG6PD, or siG6PD with SP600125 (10 μM). Densitometric analysis of protein expression relative to E-cadherin levels (n=2 per group). The data in the graph are presented as the mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001.