Cell lines and antibodies
Human breast cancer (MCF-7, MDA-MB-231) and embryonic kidney 293T (HEK293T) cell lines were cultured as previously reported . The HRD1 antibody for western blotting was obtained from Sigma Aldrich (St. Louis, MO, USA). PFKP antibody was obtained from Cell Signaling Technology. HRD1 antibody for IHC was purchased from Abgent (San Diego, California, USA). Antibodies against β-Actin, myc-Tag, and HA-Tag were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
The extracellular acidiﬁcation rate (ECAR) and oxygen consumption rate (OCR) were measured using a Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts, USA). MCF-7 and MDA-MB-231 cells (2×104/well) were seeded into an XF96-well plate and allowed to attach overnight. For ECAR assay, cells were pre-incubated with unbuffered media for 1 h, followed by a sequential injection of 10 mmol/L glucose, 1 mmol/L oligomycin and 80 mmol/L 2-deoxy-D-glucose (2-DG). OCR was assessed under basal conditions and after sequential injection of 1 μmom/L oligomycin, 1 μmol/L fluoro-carbonyl cyanide phenylhydrazone (FCCP) and 2 mmol/L antimycin A and rotenone.
(1) Cellular glucose uptake was determined by flow cytometry analysis of uptake of 2-[N-(7-nitrobenz-2-oxa-1, 3-diazo-l-4-yl) amino] -2-deoxy-D-glucose (2-NBDG). Breast cancer cells were incubated with 50μmol/L of 2-NBDG for 30 min at 37 °C in a humidified atmosphere containing 5% CO2/95% air. The fluorescence intensity of 2-NBDG taken up by the cells was analyzed on an EasyCyte Plus Flow Cytometry System (Millipore, Chicago, IL, USA). (2) The glucose concentration in the medium was measured using an Amplex Red Glucose/Glucose oxidase assay kit (Invitrogen). The absorbance of the samples was measured using a Varioskan multimode microplate spectrophotometer (Thermo, MA) and used to calculate the concentration of glucose in the medium.
Measurement of lactate and ATP levels
Lactate levels in the extracellular medium and the intracellular lactate levels (determined from cell lysates) were measured using a lactate assay kit (Abcam, Cambridge, UK). Data were normalized to the final cell counts. ATP content was determined by collecting the cells and lysing them in ATP lysis buffer, followed by ATP concentration determinations using an ATP assay kit (Beyotime Biotechnology, Shanghai, China) and following the manufacturer's protocol.
PFKP enzyme activity
Cell lysates were added into to a reaction mixture containing 50 mmol/L Tris-HCl (pH=8.0), 50 mmom/L potassium fluoride and 2.5 mmol/L EDTA, 100 mmol/L KCl, 5 mmol/l MgCl2, 1 mmol/L ATP, 0.2 mmol/lL NADH, 5 mmol/L Na2HPO4, 0.1 mmol/L AMP, 1 mmol/L NH4Cl, 5 mmol/L fructose-6-phosphate, 5 units triose phosphate isomerase, 1 unit aldolase, and 1 unit α-glycerophosphate dehydrogenase. The absorbance at 340 nm was measured at room temperature using a spectrophotometer. PFKP activity was represented as the percent loss of DPNH in the reaction mixture.
WT-PFKP or PFKP mutants (S386D-PFKP, S386A-PFKP, and K10R-PFKP) were generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, California) according to the manufacturer’s instructions.
The in vitro ubiquitylation assay was conducted by incubating purified WT HA-HRD1 (2 μg) or HA-C291S (a HRD1 ligase-dead mutant) (2 μg) with purified His-PFKP and 100 nmol/L E1, 2 μmol/L His-E2 (Ubc4), 10 μmol/L GST-Ub, and 2 mmol/L ATP in a reaction buffer (50 mmol/L Tris-HCl [pH=7.5], 2.5 mmol/L MgCl2, and 0.5 mmol/L DTT) for 90 min at room temperature.
The in vivo ubiquitylation assay was run by transfecting MDA-MB-231 cells with ubiquitin-HA for 48 h, followed lysis in the denaturing buffer (6 mol/L guanidine-HCl [pH=8.0], 0.1 mol/L Na2HPO4/NaH2PO4, and 10 mmol/L imidazole) containing 5 mmol/L N-ethylmaleimide to prevent de-ubiquitylation. The cell lysates were immunoprecipitated using the appropriate antibodies and the precipitates were washed and subjected to immunoblotting analysis.
Co-immunoprecipitation (Co-IP) and immunofluorescence (IF) staining
MDA-MB-231 cells were grown to confluence and processed for Co-IP by standard procedures. Briefly, the cells (5 × 107) were lysed in cold lysis buffer, centrifuged, and 10% of the supernatants were used as inputs. The remaining supernatants were immunoprecipitated with protein A/G agarose beads, IgG (negative controls), or antibody for target proteins at 4°C overnight. After washing, the pellets were suspended in 2 × SDS and subjected to western blotting.
The IF staining assay was performed using standard protocols. The MDA-MB-231 cells were fixed and incubated with primary antibodies (anti-HRD1 or Anti-PFKP) at a dilution of 1:100, fluorescent dye-conjugated secondary antibodies, and 4’, 6’-diamidino-2-phenylindole (DAPI). Images were acquired with a laser scanning microscope (Olympus).
Migration, invasion, cell proliferation, and colony-formation assays
Stable cell lines of MCF-7 and MB231 cells overexpressing HRD1 were constructed and cultured as previously reported . Stable cell lines were treated with 5mmol/L 2-deoxy-D-glucose (2-DG) for 72 h and then cell proliferation was measured using CCK-8 assays. The migration and invasion assays were conducted on stable cell lines treated with 5mmol/L 2-DG for 48 h and then placed in serum-free media. The colony-formation assays were performed by seeding stable cell lines in 6-well plates at a density of 200 cells per well and treating them with 5mmol/L 2-DG for 3 weeks.
Cells were washed twice in ice-cold PBS and then solubilized in radioimmunoprecipitation assay (RIPA) lysis buffer (Vazyme, Nanjing, China). Samples containing equal amounts of protein were analyzed by western blotting as previously reported .
Real-time PCR assay
Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The mRNA was quantified by real-time PCR using a LightCycler480 II Sequence Detection System (Roche, Basel, Switzerland). The following primers were used to identify PFKP: forward, 5′-CGG AAG TTC CTG GAG CAC CTC TC-3′ and reverse, 5′-AAG TAC ACC TTG GCC CCC ACG TA-3′. GAPDH was used as an internal control: forward, 5′-CCC CTT CAT TGA CCT TCA ACT A-3′ and reverse, 5′-GAG TCC TTC CAC GAT ACC AAA G-3′. All reactions were performed in triplicate, and the relative PFKP mRNA level was calculated using the 2-ΔΔCT method.
Animal tumor model
All mouse experiments were approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University. MDA-MB-231 cells (1× 107 in 100 µL PBS) stably expressing HRD1 with or without overexpression of PFKP were injected into the flank region of 6- to 8-week-old female BALB/c nude mice. The tumor size was measured every 4 days, beginning one week after the implantation, and the tumor volume was analyzed using the formula V = 0.5 ×length ×width2.
Cells were injected into tail vein of the mice to generate the lung metastasis model. The mice (ten mice per group) were sacrificed 6 weeks after the injection. The size and weight of the lungs were assessed, and visible tumors on the lung surface were counted.
Immunohistochemistry (IHC) staining
Tumor tissues used for IHC staining were measured as described previously . The primary antibody against HRD1 (1:200) anti-PFKP (1:200), and secondary antibody (1:1000) were used. Diaminobenzidine (DAB) substrate solution was applied before hematoxylin counterstaining. Normal IgG was used for negative control assays. Images were captured and evaluated with a laser scanning microscope (Olympus).
Statistical analyses were performed using SPSS 19.0 statistical analysis software. Data were expressed as the mean ± SD. Analysis of variance (ANOVA) was used to determine the statistical differences among the groups. A multivariate analysis of the independent prognostic factors was conducted using the Cox proportional hazards model. A P value < 0.05 was considered statistically significant.