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  • br We next determined whether BACH is functionally involved

    2020-08-07


    We next determined whether BACH1 is functionally involved in the increased glycolysis. Using the CRISPR-SAM approach, we
    (E) Schematic showing intratracheal administration of pSECC lentiviruses encoding Cre recombinase, CAS9, and the gRNAs sgTom or sgBach1.
    (F) Metastasis incidence in KP mice 8 months after intratracheal administration of pSECC-sgTom or -sgBach1 lentiviruses; vitamin E (VitE, 0.5 mg/kg) was administered in the chow diet 1 week after the lentiviral infection.
    (G) Schematic of the CRISPR/sgBach1-SAM strategy. sgRNAs target a CAS9-VP64 fusion to the Bach1 promoter, stimulating transcription of the endoge-nous gene.
    (H) Left, western blots showing amounts of BACH1 in mTC E-64-c transduced with SAM-sgBach1; control cells received a nontargeting construct (SAM-sgTom).
    H3 and ACTIN were loading controls. Right, amounts of BACH1 determined by densitometry in two experiments.
    (I) Transwell migration of mTC-SAM-sgBach1 and mTC-SAM-sgTom cells. Right, representative photos of migrated cells.
    (J) Transwell invasion assay of mTC-SAM-sgBach1 and mTC-SAM-sgTom cells.
    Figure 4. Combined Genome Occupancy and Transcriptomic Analyses Identify Hk2 and Gadph as BACH1 Target Genes
    (A) Left, Venn-diagram showing overlap of 240 genes bound by BACH1 in ChIP-seq analyses (green) and genes regulated in RNA-seq analyses (blue). Right,
    STRING analysis of protein-protein interactions (PPI) identified three clusters among the 240 genes: one centered around metabolic processes (circled), one around RHO proteins, and a MYC-centered network. PPI enrichment, p = 4.31e–06.
    (B) Plot of the 20 ‘‘metabolic process’’ genes. x axis, gene expression from RNA-seq data; y axis, level of BACH1 binding identified in the ChIP-seq data.
    (C) qPCR analyses of RNA from mTC and mTN cells (n = 3). Values were normalized to Rplp0 expression and then to mTC.
    (D) Top, western blots showing amounts of HK2 and GAPDH in mTC and mTN cells. Bottom, protein amounts determined by densitometry data from 3 and 1 experiments. respectively.
    (E) Left, identification of BACH1-binding sites 300 bp upstream of transcriptional start sites (TSSs) of Hk2 and Gapdh. Red arrows show primers used for ChIP-qPCR. Right, 400–500 bp of promoter sequences containing the wild-type BACH1 motifs (blue) or mutated motifs (red) were cloned into the pGL3 luciferase reporter vector.
    (F) BACH1 enrichment in promoter regions of Hk2 (compared with control IgG binding). Values are the mean of two experiments with two biological replicates/condition.
    (G) BACH1 enrichment in promoter regions of Gapdh (compared with IgG binding). Values are the mean of two experiments with two biological replicates/condition.
    (H) Luciferase activity of vectors reporting Hk2 promoter activity (shown in E) and transfected into mTC and mTN cells with and without BACH1 expression.
    (I) Luciferase activity of vectors reporting Gapdh promoter activity.
    found that increasing BACH1 expression was sufficient to in-crease glycolysis in naive lung cancer cells (Figures 5F, 5G, and S6B). We also tested the effect of inhibiting BACH1 on glycolysis with three strategies. First, knockout of Bach1 reduced Gapdh and Hk2 expression and abolished the 
    increased glycolysis rates, glucose uptake, pyruvate levels, and lactate secretion of mTN cells (Figures 5H–5K, S6C, and S6D). Similarly, knockout of BACH1 in A549, H1975, and H838 cells prevented antioxidant-induced glycolysis (Figures S6E– S6H). Second, BACH1 suppression with shRNAs reduced
    Figure 5. Antioxidants Stimulate Glycolysis in a BACH1-Dependent Fashion
    (A) Glycolysis rates in mTC and mTN cells (n = 3), as judged by Seahorse analyses. Values were normalized to mTC.
    (C) Glucose uptake and lactate secretion (n = 3/cell type).
    (D) Heatmap showing levels of transcripts for glycolytic genes identified in the RNA-seq analysis.
    (E) Co-expression of BACH1 and HK2, PFKFB3, and SLC16A1 in human lung cancer (173 datasets). Data are from the SEEK co-expression database.
    (F) Glycolysis rates of mTC-SAM-sgBach1 and control mTC-SAM-sgTom cells. Values are the mean of two experiments with two cell lines/condition normalized to control.
    (G) Lactate secretion by cells in (F).
    (H) Glycolysis rates of mTC cells and control (sgTom) and Bach1-deficient mTN cells (n = 2/condition). Values are normalized to mTC.
    (K) Lactate secretion by cells in (H).
    (M) Hk2 and Gapdh expression determined by qPCR of RNA isolated from mTC and mTC-sgBach1 cells incubated for 24 h with KI-696.
    (N) Glucose uptake by cells shown in (M).