The p52-ZER6/G6PD axis alters aerobic glycolysis and promotes tumor progression by activating the pentose phosphate pathway

Abnormal glucose metabolism is a highlight of tumor metabolic reprogramming and is closely related to the development of malignancies. p52-ZER6, a C2H2-type zinc finger protein, promotes cell proliferation and tumorigenesis. However, its role in the regulation of biological and pathological functions remains poorly understood. Here, we examined the role of p52-ZER6 in tumor cell metabolic reprogramming. Specifically, we demonstrated that p52-ZER6 promotes tumor glucose metabolic reprogramming by positively regulating the transcription of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway (PPP). By activating the PPP, p52-ZER6 was found to enhance the production of nucleotides and nicotinamide adenine dinucleotide phosphate, thereby providing tumor cells with the building blocks of ribonucleic acids and cellular reductants for reactive oxygen species scavenging, which subsequently promotes tumor cell proliferation and viability. Importantly, p52-ZER6 promoted PPP-mediated tumorigenesis in a p53-independent manner. Taken together, these findings reveal a novel role for p52-ZER6 in regulating G6PD transcription via a p53-independent process, ultimately resulting in tumor cell metabolic reprogramming and tumorigenesis. Our results suggest that p52-ZER6 is a potential target for the diagnosis and treatment of tumors and metabolic disorders.


ChIP assay
ChIP analysis was performed using the ChIP-IT Express (Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, to crosslink proteins to DNA, formaldehyde (final concentration 1%) was added to the culture medium of HCT116 cells overexpressing p52-ZER6. Cells were then collected, and the pellets were treated with Lysis Buffer prior to sonication using ultrasonic crusher (Chongqing Medical Equipment Factory, Chongqing, China) to shear DNA into 0.2-1.0 kb fragments. Sonication was performed for 10 s (power: 150 W) followed by 10 s interval on ice, and was repeated 10 times. After the cellular debris was removed, the chromatins were immunoprecipitated using protein A+G Agarose/Salmon Sperm DNA (Beyotime Biotechnology) and anti-ZER6 antibody, anti-Histone H3 antibody or normal rabbit IgG. Chromatin was then de-crosslinked for 4 h at 65°C, and treated with 5 M NaCl, 0.5 M EDTA, 1 M Tris (pH 6.5), and 20 mg/ml proteinase K. Immunoprecipitated chromatin was then subjected to PCR analysis using PrimeSTAR Max (Takara Bio). The sequence of the forward primer used was 5'-TGT CTT TGG GGA AAA GGA CCA C-3'; while that of the reverse primer was 5'-GGC CGG CGT GCT TAT CAT TA -3'.

RNA extraction and quantitative real time PCR (qRT-PCR) analysis
Total RNA was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer's instruction. Total RNA (1 g) was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio), and qRT-PCR was performed with SYBR Premix Ex Taq (Takara Bio). The sequences of the primers used for qRT-PCR were shown in Supplementary Table S1. -actin was used to normalize sample amplifications. The results are shown as relative to the expression level in the corresponding controls, which were assumed as 1.

Western blotting
For cell culture experiments, cells were collected and lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail; Roche Applied Science, Mannheim, Germany). For clinical specimens and samples from xenografted tumors, frozen specimens were homogenized with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail to obtain protein extracts. Equal amounts of total protein (20 g) were electrophoresed on sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The antibodies used are listed in Supplementary Table S2 and immunoblotting with anti--actin antibody was conducted to ensure equal protein loading. The signals were detected by using SuperSignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific, Waltham, MA). Quantification was performed using Quantity One, and the result was normalized using -actin.

Immunohistochemistry
Tissue sections were obtained from fresh colon carcinoma and adjacent tissues or xenografted tumors at 4 m thickness using a cryostat and subjected to immunohistochemistry. Briefly, the tissue sections were incubated with primary antibodies for 1 h. The specimens were then incubated with corresponding second antibodies conjugated with horse-radish peroxidase.
Visualization was performed using a DAB Kit (DAKO, Denmark) under microscope. The nuclei were then counterstained with hematoxylin, followed by dehydration and coverslip mounting. The antibodies used were listed in Table S2. Images were taken by using Pannoramic Midi (3DHistech).
Glucose consumption, lactate production, G6PD enzyme activity, and intracellular

NADPH level
Cells were transfected with indicated vectors and selected using puromycin as indicated above.
For measuring glucose consumption and lactate production, the medium was replaced after selection, and the cells were incubated for an additional 24 h. Glucose and lactate levels in the culture medium were determined using the Glucose Colorimetric Assay Kit (BioVision,

5-ethynyl-2′-deoxyuridine (EdU) incorporation assay
Cells were transfected with indicated vectors and selected using puromycin as indicated above prior being re-seeded in 24-well plate (1×10 5 cells/well). EdU incorporation assay was performed using BeyoClick TM EdU Cell Proliferation Kit with Alexa Flour 488 (Beyotime Biotechnology) according to the manufacturer's instruction. Hoechst was used to stain the nuclei. Images were taken with DMI6000B (Leica, Heidelberg, Germany). The ratio of de novo DNA synthesis was calculated as the ratio of EdU-positive cells to Hoechst-positive cells.

Dual luciferase reporter assay
Cells were co-transfected with indicated vectors, reporter vector bringing the firefly luciferase, and Renilla luciferase expression vector pRL-SV40 (Promega). After 24 h, luciferase activities were analyzed by using Dual Luciferase Reporter Assay (Promega). The activities of the firefly luciferase reporters were normalized using those of Renilla luciferase.

Colony formation assay
Cells were transfected with indicated shRNA expression vectors or overexpression vector and screened using puromycin as indicated above. After being re-seeded into 6-well plates at a density of 300 cells/well and cultured for 12 days, cells were then fixed with 4% paraformaldehyde and stained with Crystal Violet Staining Solution (Beyotime Biotechnology), then the numbers of the colonies formed were counted. The investigator was blinded during the assessment.

Cell cycle analysis
Cells were transfected with indicated shRNA expression vectors or overexpression vector.
Transfected cells were selected using puromycin as described above and subjected to starvation for 24 h before being incubated further for 24 h in medium containing 10% FBS. Cells were then harvested and stained with propidium iodide (Solarbio, Beijing, China). The percentages of the cells in each cell cycle phase were determined by flow cytometry.     Cells transfected with shCon or pcCon were used as controls. Total protein was used for normalization. Quantification data are expressed as mean ± SD (n = 3). shp52: shRNA expression vector targeting p52-ZER6; pcCon: pcEF9-Puro; pcp52: p52-ZER6 overexpression vector; **P < 0.01; ***P < 0.001.