Santoro Valeria, Kovalenko Ilya, Vriens Kim, Christen Stefan, Bernthaler Andreas, Haegebarth Andrea, Fendt Sarah-Maria, Christian Sven
Bayer AG, Drug Discovery, Berlin 13353, Germany.
Current address: University of Michigan, Cancer Center, Ann Arbor, MI 48108, USA.
Oncotarget. 2020 Feb 25;11(8):801-812. doi: 10.18632/oncotarget.27486.
SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored. Our results indicate that the gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.
SLC25A32是线粒体转运体溶质载体25家族的成员。SLC25A32将四氢叶酸（THF）以及黄素腺嘌呤二核苷酸（FAD）转运到线粒体中，并调节线粒体的一碳代谢和氧化还原平衡。虽然已知癌细胞需要一碳和FAD依赖的线粒体代谢来维持细胞增殖，但SLC25A32在癌细胞生长中的作用仍未得到探索。我们的结果表明，该基因在不同肿瘤中高度扩增，且扩增与mRNA表达增加和患者生存率降低相关。在不同来源的癌细胞中，通过小干扰RNA（siRNA）介导的SLC25A32敲低和CRISPR介导的敲除，鉴定出了对SLC25A32抑制敏感和耐药的细胞系。从机制上讲，氘代丝氨酸追踪显示，通过液相色谱-质谱联用（LC-MS）测量，SLC25A32敲低并不影响线粒体/胞质叶酸通量。相反，SLC25A32抑制导致FAD依赖的复合体II酶处的呼吸链功能障碍，活性氧（ROS）的诱导以及还原型谷胱甘肽（GSH）的消耗，从而损害癌细胞增殖。此外，丁硫氨酸亚砜胺（BSO）处理进一步使细胞对SLC25A32敲低后ROS介导的细胞增殖抑制敏感。用FAD前体核黄素和GSH处理细胞可挽救SLC25A32下调后的癌细胞增殖。我们的结果表明，通过靶向SLC25A32降低线粒体FAD浓度作为单一药物或与批准的癌症药物联合使用具有潜在的临床应用价值，可导致氧化应激增加和肿瘤生长减少。
