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Figure 3. BACH1 Is Required for Antioxidant-Induced Metastasis and Can Induce Metastasis in the Absence of Antioxidants
(A) Left, Transwell migration assay of mTC and mTN DCFH-DA incubated with 10 mM hemin for 24 h. Right, representative photos of migrated cells.
(B) Western blots showing amounts of BACH1 in mTC and mTN cells transduced with CAS9 and sgRNAs targeting Bach1; sgRNA targeting dTomato (Tom) was used as control. HISTONE 3 (H3) was the loading control.
(C) Left, Transwell migration assay of mTN-sgBach1, mTN-sgTom, and control mTC cells (n = 2 biological replicates/condition). Right, representative photos of migrated cells.
(D) Left, lung tumor burden in NSG recipient mice 3 weeks after i.v. injection of control and Bach1-deficient mTC and mTN cells (0.5 3 105 cells/mouse; n = 5–8 mice/condition). Right, representative lung sections.
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addition of hemin, which degrades BACH1, reduced the high migration rates of mTN cells but did not affect basal migration of mTC cells (Figure 3A). CRISPR/CAS9-mediated knockout of Bach1 did not affect the proliferation of mTC and mTN cells but normalized the high migration rates of mTN cells (Figures 3B and 3C; Figure S4A). Bach1 knockout also prevented the increased migration induced by NRF2 activation in mTC cells (Figure S4B) and by antioxidant treatment in human lung cancer cell lines (Figure S4C–S4F). In NSG mice, Bach1 knockout also prevented mTN cells from metastasizing to the lung after i.v. injection (Figure 3D), but it did not alter the growth of primary tumors when the cells were injected subcutaneously (Fig-ure S4G). We also used the pSECC lentivirus (Sa´nchez-Rivera et al., 2014) for in vivo CRISPR/CAS9-mediated knockout of Bach1 in KP tumors (Figure 3E). In line with the previous exper-iment, BACH1 depletion in vivo prevented vitamin E-induced authentic metastasis in KP mice without affecting the growth of primary tumors (Figures 3F and S4H–S4J).
To determine whether BACH1 is sufficient to stimulate migra-tion and metastasis we used the CRISPR-SAM (synergistic activa-tion mediator) strategy to increase the expression of endogenous BACH1 (Figure 3G). In naive mTC cell lines transduced with SAM-sgBach1 constructs, BACH1 protein levels increased 4- to 6-fold, which increased cell migration and invasion (Figures 3H–3J). The ability of SAM-sgBach1 mTC cells to metastasize to the lung after i.v. injection in NSG mice was 2.5-fold higher than in controls and similar to that of mTN cells (Figure 3K). Moreover, transducing A549 and H1975 human lung cancer cells with SAM-sgBACH1 increased endogenous BACH1 levels, stimulated migration, and increased lung metastases by 3- to 4-fold (Figures S4K–S4N).
Genes Encoding the Glycolytic Enzymes HK2 and GAPDH Are BACH1 Targets
To identify BACH1 target genes responsible for the increased metastasis, we immunoprecipitated chromatin bound to BACH1 in mTC and mTN cells and sequenced the associated DNA (chromatin immunoprecipitation sequencing [ChIP-seq]). The known BACH1 binding motif (from GEO: GSE31477) was present in half of the identified target genes, as was a de novo BACH1-like motif containing the ARE consensus motif (Fig-ure S5A). Known BACH1 target genes were also identified (Fig-ure S5B). BACH1 bound to several regions of the Hmox-1 pro-moter in mTC and mTN cells, but binding levels did not differ significantly (Figures S5B and S5C). Integrating ChIP-seq and RNA-seq data revealed that BACH1 had bound to 240 differen-tially expressed genes (Figure 4A).
Further analysis of these genes in the STRING database of protein-protein interactions identified ‘‘metabolic process’’ as one of three enriched networks (Figure 4A). Among the ‘‘meta-bolic process’’ genes that showed higher BACH1 binding in mTN than mTC cells, Hexokinase 2 (Hk2) was the most signifi-cantly upregulated in the RNA-seq analysis (Figure 4B). We confirmed that expression of Hk2 and also Gapdh, but not Hk1, was 2- to 3-fold higher in mTN than mTC cells (Figures 4C and 4D). Analyses of Hk2 and Gapdh promoters revealed that BACH1-binding sequences were present 300 base pairs (bp) upstream of the annotated transcriptional start site (Fig-ure 4E). ChIP-qPCR analyses confirmed the increased BACH1 occupancy on Hk2 and Gapdh promoters in mTN cells (Figures 4F, 4G, S5D and S5E).