SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism

Abstract

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 SLC25A32 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 knockout 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.

Epistasis-driven identification of SLC25A51 as a regulator of human mitochondrial NAD import

“…Importantly, we further observed SLC25A51specific negative interactions with two additional members of the SLC25 family, the paralog SLC25A53 and the gene for the putative folate/FAD transporter SLC25A32 (Fig. 1b) 39 . Although no function has been assigned to SLC25A53 yet, the interaction to SLC25A32 pointed to a possible involvement of SLC25A51 in mitochondrial cofactor transport.…”

Riboflavin metabolism: role in mitochondrial function

“…SLC25A32 has recently been revealed to be a novel regulator of cancer cell proliferation and mitochondrial FAD metabolism. SLC25A32 knock-down in sensitive tumor cells resulted in inhibition of the FADdependent Complex II, increased succinate levels, and reduced oxygen consumption rate [32] . These findings corroborate evidence of decreased Complex II protein levels and OXPHOS activity, which is a marker for mitochondrial FAD in muscle of patients with severe neuromuscular phenotype and novel variants in SLC25A32 [30,31] .…”

“…These findings corroborate evidence of decreased Complex II protein levels and OXPHOS activity, which is a marker for mitochondrial FAD in muscle of patients with severe neuromuscular phenotype and novel variants in SLC25A32 [30,31] . Reduction of mitochondrial FAD concentrations by inhibition of SLC25A32 is antiproliferative in a subset of tumor cell lines and has potential clinical applications as a novel cancer target by increasing oxidative stress and reducing tumor growth [32] .…”

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy

“…In biochemical and cellular alterations induced by altered mitochondrial FAD transporter, a central role is played by the flavoprotein subunit of SDH. This is true not only in yeast, but also in human cell model recently introduced [ 91 ].…”

“…The role of FLX1 in catalyzing intra-mitochondrial FAD efflux to the cytosol was proposed by our group [ 88 ]. S. cerevisiae flx1 mutant strain was used to identify, by complementation, the human orthologue SLC25A32A [ 87 , 91 ].…”