Loss of glucose 6-phosphate dehydrogenase function increases oxidative stress and glutaminolysis in metastasizing melanoma cells

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To address this, we mutated the substrate binding site of glucose 6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors, but when these tumors spontaneously metastasized, the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species, decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing malic enzyme activity and glutamine consumption. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutaminase than control melanomas, both for oxidative stress management and anaplerosis. The oxidative pentose phosphate pathway, malic enzyme, and glutaminolysis thus confer layered protection against oxidative stress during metastasis.

The pentose phosphate pathway is an important source of NADPH for oxidative stress resistance (1–5). The oxidative branch of the pentose phosphate pathway contains two enzymes that generate NADPH from NADP⁺, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD) (SI Appendix, Fig. S1). NADPH is an important source of reducing equivalents for oxidative stress resistance because it is used by cells to convert oxidized glutathione (GSSG) to glutathione (GSH), an abundant redox buffer. Complete deficiency for G6PD is embryonic-lethal in mice (2, 6, 7) but hypomorphic G6PD mutations are common in certain human populations, perhaps because they protect against malaria (8, 9). These partial loss-of-function G6PD mutations are well tolerated in adults, though they sensitize red blood cells to hemolysis from oxidative stress under certain circumstances (10).Several studies have reported a lower incidence and mortality for certain cancers in people with hypomorphic mutations in G6PD (11–14), suggesting that cancer cells depend upon G6PD to manage oxidative stress. Cells experience high levels of oxidative stress during certain phases of cancer development and progression, including during metastasis (15–17). Antioxidant mechanisms thus promote the survival of cells during oncogenic transformation (18, 19) as well as during metastasis (15, 16). For example, relative to primary cutaneous tumors, metastasizing melanoma cells exhibit increased dependence upon the folate pathway (15), monocarboxylate transporter-1 (MCT1) (20), and glutathione peroxidase-4 (GPX4) (21), each of which directly or indirectly attenuate oxidative stress. By better understanding the mechanisms that confer oxidative stress resistance in cancer cells, it may be possible to develop pro-oxidant therapies that inhibit cancer progression by exacerbating the oxidative stress experienced by cancer cells.G6PD (22) or PGD deficiency (23–25) reduce the growth of some cancers, including melanoma, but G6PD deficiency has little effect on primary tumor formation by K-Ras–driven epithelial cancers (26). This is at least partly because loss of G6PD in these cancers leads to compensatory increases in the function of other NADPH-generating enzymes, including malic enzyme and isocitrate dehydrogenase (1, 27). Nonetheless, pentose phosphate pathway function may increase during metastasis (20, 28–30) and higher G6PD expression is associated with worse outcomes in several cancers (31–33), raising the question of whether metastasizing cells are particularly dependent upon G6PD. G6PD is not essential for metastasis in a breast cancer cell line but it reduces their capacity to form metastatic tumors (26).Melanoma cells show little evidence of oxidative stress in established primary tumors but exhibit increased levels of reactive oxygen species (ROS) and dependence upon antioxidant mechanisms during metastasis (15, 20, 21). To test if these cells are more dependent upon the pentose phosphate pathway during metastasis, we generated three G6PD mutant melanomas, including two patient-derived xenografts and one human melanoma cell line. Reduced G6PD function had little effect on the formation or growth of primary subcutaneous tumors but significantly increased ROS levels and reduced spontaneous metastasis. G6PD mutant melanomas compensated by increasing malic enzyme activity and glutamine consumption, both to increase oxidative stress resistance and to replenish tricarboxylic acid (TCA) cycle intermediates through anaplerosis. Melanoma cells thus have redundant layers of protection against oxidative stress during metastasis, including the abilities to alter fuel consumption and antioxidant pathway utilization.