br O Lactate secretion by cells
(O) Lactate secretion by (±)-Baclofen shown in (M).
Figure 6. HK2-Stimulated Glycolysis Mediates the Antioxidant- and BACH1-Induced Metastatic and Invasive Phenotype
(A) Top, western blot showing amounts of HK2 and BACH1 in mTC cells transduced with a control plasmid or a plasmid encoding human HK2 and their glycolysis rates (bottom).
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Bach1 expression by 40%–60% and reduced Hk2 expression, glycolysis rates, and migration of mTN cells to the levels in mTC cells (Figures S6I–S6K). Third, hemin administration reduced glycolysis rates specifically in mTN cells and reduced migration and glycolysis in antioxidant-treated human NSCLC cells (Figures S6L–S6Q).
Interestingly, KI-696-induced activation of NRF2, which stabi-lized BACH1 and increased migration of mTC cells (Figures S3M and S3N), increased glycolysis in mTC but not mTN cells (Figures 5L and S6R). NRF2-induced glycolysis was accompanied by Bach1-dependent increases in Hk2 and Gapdh expression, glucose uptake, and lactate secretion (Figures 5M–5O). We conclude that BACH1 can stimulate glycolysis both in the pres-ence and absence of exogenous antioxidants and in response to NRF2 activation.
Glycolysis Drives Antioxidant- and BACH1-Dependent and -Independent Migration and Metastasis
To determine whether the invasion and glycolysis phenotypes are connected, we used genetic constructs to manipulate glycolysis rates. Overexpressing human HK2 increased glycol-ysis and migration of mTC and A549 cells without affecting BACH1 levels (Figures 6A, 6B, S7A, and S7B). After i.v. injection into NSG mice, mTC cells overexpressing HK2 produced 2.5-fold more lung metastases than control cells (Figure 6C), and, after s.c. transplantation, they produced more and larger lymph node metastases, despite producing slightly smaller pri-mary tumors (Figures 6D–6F). Conversely, overexpressing the catalytic subunit of human glucose-6-phosphatase reduced glycolysis rates and migration of mTN cells and significantly reduced lung metastases after i.v. injection into NSG mice (Fig-ures 6G–6I). Furthermore, short hairpin RNA (shRNA)-mediated suppression of Hk2 reversed BACH1-induced glycolysis and migration of mTC-SAM-sgBach1 cells (Figures 6J–6L). One advantage of a high glycolysis rate is that it can increase ATP production rates and total ATP levels, which fuel cell motility (Cairns et al., 2011; Liberti and Locasale, 2016; Pollard and Borisy, 2003; Shiraishi et al., 2015). Indeed, mTN cells and anti-oxidant-treated A549 cells had higher glycolysis-derived and total ATP production rates than controls and higher steady-state ATP levels (Figures 6M–6O, S7C, and S7D). The increased
ATP availability of mTN cells was normalized in Bach1-knockout cells (Figure 6P).
Targeting Glycolysis Prevents Antioxidant- and BACH1-Stimulated Metastasis
Next, we evaluated how drugs that activate or inhibit enzymes associated with glycolysis affect antioxidant- and BACH1-induced migration (Figure 7A). In response to high doses of 2-deoxyglucose (2-DG), mTN cells had 50% lower viability, indi-cating greater dependence on glycolysis, than mTC cells (Fig-ure 7B). Nontoxic inhibition of glycolysis around the hexokinase step with 2-DG and the HK2 inhibitor lonidamine reduced glycol-ysis rates and the antioxidant-induced migration of mTN cells and also the basal migration of mTC cells (Figures 7C–7E). 2-DG and lonidamine also reversed BACH1-induced migration of mTC-SAM-sgBach1 cells (Figure S7E). Inhibiting GAPDH with 3-bromopyruvate (3-BP) specifically prevented antioxi-dant-induced migration of mTN cells (Figures 7F and 7G), as did dicholoroacetate, a pyruvate dehydrogenase kinase inhibitor that inhibits lactate production (Figure 7H) (Michelakis et al., 2008). Inhibiting mitochondrial import of pyruvate with the mito-chondrial pyruvate carrier (MPC) inhibitor UK5099 did not influ-ence migration (Figure S7F); however, inhibiting lactate transport out from the cell with the MCT-1 inhibitor AZD3965 specifically prevented mTN migration (Figure 7I, S7G, and S7H). Inhibiting lactate secretion also prevented antioxidant-induced migration of A549 cells (Figure S7I). The GAPDH and lactate secretion steps are important in human LUAD as high expression of GAPDH and SLC16A1 is associated with reduced survival (Fig-ures S7J and S7K). Last, the pentose-phosphate pathway
(PPP) provides reducing power in the form of NADP(H) and can contribute to the increased fitness of cells performing aerobic glycolysis (Vander Heiden et al., 2009). However, incubating cells with the PPP inhibitor 6-aminonicotinamide (6-AN) did not influence the migration of mTN or mTC cells, despite inhibition of NADPH production (Figures S7L and S7M). Thus, the HK2, GAPDH, and MCT-1 steps of glycolysis are required for antioxi-dant-induced migration, whereas the PPP and MPC steps are not. To determine whether glycolysis inhibition would influence antioxidant-induced metastasis in vivo, we chose 3-BP and AZD3965 as they specifically blocked migration of mTN cells.