UHRF1 Regulates The Glucose Metabolism Of Breast Cancer Cells In Aerobic And Hypoxic Conditions By Two Interacting Proteins

Part Ⅰ:UHRFl binds to PGKl and increases the pentose phosphate pathway of glucose metabolism in human breast cancer cells under aerobic conditionsObjective:To identify unknown proteins which can interact with UHRF1 by using Immunoprecipitation and Mass Spectroscopy and to further study the function of UHRF1 in breast cancer.Methods:N-terminal FLAG-tagged UHRF1 lentiviral vector was constructed and packed into HEK 293T cells to get lentivirus. Then the lentivirus containing FLAG-UHRF1 was infected to MCF-7 human breast cancer cells. After puromycin selection, the stably FLAG-UHRF1 over-expressing MCF-7 cells were obtained. Using Immunoprecipitation and Mass Spectroscopy, we got the UHRF1 interacting candidates. The human phosphoglycerate kinase 1 (PGK1) was selected and then proved to be an UHRF1-interacting protein by Co-Immunoprecipitation. Then, we investigated whether the interaction between UHRF1 and PGKl was involved in the metabolism of glucose. After stable interference of UHRF1, we examined the change of glucose uptake, NADPH production, and Lactate accumulation of human breast cancer cell under aerobic or hypoxic conditions.Results:The stable FLAG-UHRF1 over-expressing MCF-7 cells were obtained. Using Immunoprecipitation and Mass Spectroscopy, fattyacid synthase、Myosin-9、 UHRF1BP1, OGT1, DDX5, PGK1,Actin-alpha, Actin-beta, PRDX1 and PRDX2 were identified as UHRF1 interacting candidates. The key glycolytic enzyme PGK1 was selected for the further study for its important role in glucose metabolism. Co-Immunoprecipitation confirmed the interaction between UHRF1 and PGK1.In MCF-7 and MDA-MB-231 cells,interference of UHRF1 increased the Lactate accumulation and reduced NADPH production but without any significant change in glucose uptake. Differently, down-expression of UHRF1 did not affect the glucose uptake, NADPH production, and Lactate accumulation under hypoxic conditions.Conclusions:We found a new UHRFl interacting protein PGK1. The interaction between UHRF1 and PGK1 in breast cancer cells enhanced the pentose phosphate pathway under aerobic conditions.Part Ⅱ:The interaction between prolyl hydroxylase-EGLN2 and UHRF1 promotes the glycolytic metabolism of glucose under the hypoxic conditionsObjective:To investigate the mechanism by which UHRF1 loses the capacity to interact with PGK1 and affects the metabolism of glucose under hypoxic conditions.Methods:To identify the hypoxic-specific UHRF1-binding protein, Immunoprecipitation combined with Mass Spectrometry was performed with FLAG-UHRF1 stable over-expressing MCF-7 cells under hypoxic conditions. The candidate protein EGLN2 was selected for its function as an oxygen sensor. Co-Immunoprecipitation was used to comfirm the interaction under hypoxic conditions. Interference of EGLN2 was done to examine its role in the metabolism of glucose and its affect to the interaction between UHRF1 and PGK1 under hypoxic conditions. Addtionally, UHRF1 may undergo phosphorylation modification. Thus, we treated the cell lysate with Alkaline Phosphatase (CIP) to check the possible phosphorylation of UHRF1. CIP was also used to investigate whether phosphorylation of UHRF1 might disturb the interaction with EGLN2 under aerobic conditions.Results:EGLN2 (egl nine homolog 2) was confirmed as a hypoxic-specific UHRF1-binding protein with Co-Immunoprecipitation followed by the examination with Immunoprecipitation and Mass Spectrometry. In MCF-7 and MDA-MB-231 cells, interference of EGLN2 decreased the Lactate accumulation and increased NADPH production but without any significant change in glucose uptake under hypoxic conditions. Meanwhile, the interaction between UHRF1 and PGK1 was restored. Dephosphorylation assay with CIP also showed a decrease of the upper band of UHRF1 in the immunoblotting assay and a restoration of the interaction between UHRF1 and EGLN2 under aerobic conditions.Conclusions:We found an interaction between UHRFl and EGLN2 under hypoxic conditions.This binding disturbed the interaction between UHRF1 and PGK1 and thus inhibited the pentose phosphate pathway under hypoxic conditions. Addtionally, the phosphorylation of UHRF1 under aerobic conditions might disturb its binding to EGLN2.