Fig 1: OVOL2 inhibits glycolytic gene expression and glycolysis. A) Heatmap of transcription factors and known hypoxia‐inducible glycolytic genes identified by RNA‐seq using MCF7 cells treated with hypoxia (1% O2) for indicated times. B) RT‐qPCR analysis of transcription factors and some known hypoxia‐inducible glycolytic genes identified in (A) in MCF7 cells treated with hypoxia for indicated times. C) Glucose uptake and lactate production in MCF7 cells transfected with the indicated FLAG‐tagged expression vectors or empty vector (EV). LDHA was used as a positive regulator of glucose uptake and lactate production, and p53 as a negative regulator of glucose uptake and lactate production. Typical immunoblot shows the expression of FLAG‐tagged proteins. α‐Tubulin was used as a loading control. D) Measurement of glucose uptake and lactate production in ZR75‐1 and MDA‐MB231 cells transfected with FLAG‐tagged OVOL2 (FLAG‐OVOL2) or EV. Representative immunoblot shows FLAG‐OVOL2 expression. E) Heatmap of glycolytic genes and known OVOL2 downstream target genes identified by RNA‐seq using OVOL2 wild‐type (WT) or knockout (KO) MCF7 cells. F) KEGG pathway analysis of genes differentially expressed between OVOL2 WT and KO MCF7 cells as in (E). G) RT‐qPCR and immunoblot analysis of glycolytic gene expression in OVOL2 WT or KO MCF7 or MDA‐MB‐231 cells. To avoid potential off‐target effects of KO, the KO cells were stably infected with lentivirus carrying OVOL2. α‐Tubulin was used as a loading control for analyzing glycolytic gene expression. A schematic diagram of the aerobic glycolysis pathway is indicated on the left. H) RT‐qPCR and immunoblot analysis of glycolytic gene expression in OVOL2 WT or KO MEFs isolated from corresponding mouse. WT mice were littermates of the KO mice (n = 3). I) Representative immunoblot analysis of glycolytic gene expression in OVOL2 WT and KO mouse mammary tissues isolated from mammary‐specific OVOL2 KO mice generated by mating conditional OVOL2 KO mice with MMTV‐Cre transgenic mice. J) Measurement of glucose uptake and lactate production in OVOL2 WT or KO MEFs from (H). K,L) Cell proliferation curves of OVOL2 WT and KO MEFs (n = 3) cultured in media containing 25 mm glucose (K) or 10 mm galactose (L). Data shown are mean ± standard deviation (SD) of triplicate measurements that have been repeated 3 times with similar results (B,C,G,H,K,L). Data shown are mean ± SD of quintuplicate measurements that have been repeated 3 times with similar results (D,J). Two‐sided Student's t‐test was used to compare the means of two groups. When more than two groups were compared, one‐way ANOVA was performed. *p < 0.05, **p < 0.01 versus control cells or EV.
Fig 2: OVOL2 binds the OVOL2‐responsive element to inhibit glycolytic gene promoter activity. A) Luciferase activity of different glycolytic gene promoter reporters in MDA‐MB‐231 cells transfected with MYC‐tagged OVOL2 or EV. Filled circles indicate the position of putative OVOL2‐binding sites, and ‘‘ × ’’ indicates the mutated OVOL2‐binding sites. Red letters indicate the putative or mutated OVOL2‐binding sequences. WT, wild‐type; Mut, mutant. B) ChIP analysis of OVOL2 occupancy on promoters of glycolytic genes in MDA‐MB‐231 cells. IgG (immunoglobulin G): normal serum. The different number after each gene represents regions containing different putative OVOL2‐binding sites from left to right, as shown in (A). Data shown are mean ± SD of triplicate measurements that have been repeated 3 times with similar results. Data were analyzed using a two‐tailed Student's t‐test. *p < 0.05, **p < 0.01 versus respective promoter reporter without OVOL2 (A). **p < 0.01 versus respective normal IgG (B).
Fig 3: Aerobic glycolysis is responsible for OVOL2 modulation of cancer cell proliferation, invasion, and metastasis. A) MDA‐MB‐231 cells stably expressing shNCoR or shCtrl were stably transfected with MYC‐OVOL2 or NCoR‐R as indicated, and the cell proliferation curve was then determined. B) Cell invasion assay of MDA‐MB‐231 cells as described in (A). The relative cell invasions are shown in the lower panel. C) OVOL2 WT or KO MDA‐MB‐231 cells were stably transfected with PKM2 shRNA (shPKM2) or shCtrl as indicated. The cells were treated with or without 2.5 mm 2‐DG, and the cell proliferation curve was then assessed. Representative immunoblot reveals expression of PKM2 and OVOL2. D) Cell invasion assay of MDA‐MB‐231 cells as described in (C). The relative cell invasions are shown in the right panel. E) Tumor growth curve of MDA‐MB‐231 cells stably expressing MYC‐OVOL2, shNCoR, or MYC‐OVOL2 plus shNCoR as indicated. Images of xenograft tumors are shown in the right panel. The column indicates the designated number of each mouse, with each column corresponding to different mice in different groups. The lactate level of representative tumor tissues (the fourth column) was determined. Representative immunoblot shows expression of PKM2 and LDHA in representative tumor tissues (the fourth column). F) Representative bioluminescence image, lung tissues and H&E‐stained sections of the lung tissues at 30 days from nude mice injected by tail vein with MDA‐MB‐231 cells expressing firefly luciferase and the indicated constructs as described in (E) (n = 6). The luminescence signal is represented by an overlaid false‐color image with the signal intensity indicated by the scale (middle panel) and the number of tumor nodules shown (right panel). G) Tumor growth curve of OVOL2 WT or KO MDA‐MB‐231 cells stably expressing shPKM2 or shCtrl and treated with or without 2‐DG as indicated. The lactate level of representative tumor tissues (the fourth column) was examined. Representative immunoblot indicates expression of PKM2 and LDHA in representative tumor tissues (the fourth column). H) Representative bioluminescence image, lung tissues and H&E‐stained sections of the lung tissues at 30 days from nude mice injected by tail vein with MDA‐MB‐231 cells expressing firefly luciferase and the indicated constructs as described in (G) (n = 6). The luminescence signals and the number of tumor nodules are shown (middle and right panels). Statistical significance was assessed by one‐way ANOVA. **p <0.01.
Fig 4: OVOL2 inhibits glycolytic gene expression by interacting with the NCoR/HDAC3 co‐repressor complex. A) Cellular lysates from MCF7 cells stably expressing FLAG (control) or FLAG‐OVOL2 were purified with anti‐FLAG affinity columns and eluted with FLAG peptide. The eluates were resolved by SDS‐PAGE and stained with silver. The differential protein bands were retrieved and analyzed by mass spectrometry. B) MCF7 or MDA‐MB‐231 cells were immunoprecipitated with anti‐OVOL2 or normal IgG, and precipitates were analyzed by immunoblotting (IB) with indicated antibodies. IP, immunoprecipitation. C) Co‐IP analysis of MDA‐MB‐231 cells treated with or without DNase I. DNA agarose gel electrophoresis serves as a control for DNase I activity. D) MDA‐MB‐231 cells transfected with indicated plasmids were immunoprecipitated with anti‐FLAG agarose beads. The immune complexes were eluted with FLAG peptide and reimmunoprecipitated (Re‐IP) with anti‐MYC or normal IgG. The resulting precipitates were analyzed by IB with indicated antibodies. E) MDA‐MB‐231 cells stably infected with lentivirus carrying NCoR short hairpin RNA (shRNA) or control shRNA (shNCoR or shCtrl) cells were immunoprecipitated with anti‐OVOL2 or normal IgG, and precipitates were analyzed by IB with indicated antibodies. F,G) MDA‐MB‐231 cells stably infected with lentivirus carrying shNCoR or shCtrl were stably transfected with MYC‐tagged OVOL2 or shRNA‐resistant NCoR (NCoR‐R) or EV as indicated. Glycolytic gene expression was examined using RT‐qPCR (F) and IB (G). H) ChIP analysis of OVOL2, NCoR, and HDAC1/3 occupancy on glycolytic gene promoters in MDA‐MB‐231 cells. Promoter regions of each gene represent the region containing the first OVOL2 binding site shown in Figure 2B unless only one OVOL2 binding site is present within the gene promoter analyzed. I) Re‐ChIP analysis of the occupancy of OVOL2 and NCoR or HDAC3 on the indicated glycolytic gene promoters in MDA‐MB‐231 cells. Re‐ChIP was performed after ChIP with anti‐OVOL2. J) ChIP analysis of OVOL2, NCoR, HDAC3, and histone H3 acetylation (H3K27ac and H3K4ac) occupancy on indicated glycolytic gene promoters in OVOL2 WT or KO MDA‐MB‐231 cells or MDA‐MB‐231 cells stably infected with lentivirus carrying shNCoR or HDAC3 shRNA (shHDAC3). Representative immunoblot shows the expression of OVOL2, NCoR and HDAC3. Data shown are mean ± SD of triplicate measurements that have been repeated 3 times with similar results. Statistical significance was assessed by a two‐tailed Student's t‐test. **p < 0.01 versus respective MDA‐MB‐231 cells transfected with EV and shCtrl (F). **p < 0.01 versus respective normal IgG (H–J).
Fig 5: OVOL2 inhibits aerobic glycolysis through the NCoR co‐repressor. A) Glucose uptake, pyruvate level, lactate production, and ATP level in OVOL2 WT or KO MDA‐MB231 cells or OVOL2 KO MDA‐MB‐231 cells stably transfected with OVOL2. B) ECAR and OCR in the cells from (A). C) HK, PFK, ALDO, PKM, and LDH activities in the cells from (A). D) MDA‐MB‐231 cells stably expressing shNCoR or shCtrl were stably transfected with MYC‐OVOL2 or shRNA‐resistant NCoR as indicated, and glucose uptake, pyruvate level, lactate production and ATP level were then examined. E) ECAR and OCR were examined in the cells from (D). Data shown are mean ± SD of quintuplicate measurements and have been repeated 3 times with similar results. Statistical significance was assessed by one‐way ANOVA. ** p < 0.01 versus OVOL2 WT MDA‐MB‐231 cells (A–C). * p < 0.05, ** p < 0.01 versus MDA‐MB‐231 cells stably expressing EV and shCtrl (D,E).
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