Mitochondrial Uncoupling Inhibits Reductive Carboxylation in Cancer Cells

Abstract

When the electron transport chain (ETC) function is impaired, cancer cells rely on reductive carboxylation (RC) to convert α-ketoglutarate (αKG) to citrate for macromolecular synthesis, thereby promoting tumor growth. Currently, there is no viable therapy to inhibit RC for cancer treatment. In this study, we demonstrate that the mitochondrial uncoupler treatment effectively inhibits RC in cancer cells. Mitochondrial uncoupler treatment activates the ETC and increases the NAD+/NADH ratio. Using U-13C-glutamine and 1-13C-glutamine tracers, we show that mitochondrial uncoupling accelerates the oxidative tricarboxylic acid (TCA) cycle and blocks RC under hypoxia, in von Hippel-Lindau (VHL) tumor suppressor-deficient kidney cancer cells, or under anchorage-independent growth condition. Together, these data demonstrate that mitochondrial uncoupling redirects α-KG from RC back to the oxidative TCA cycle, highlighting that the NAD+/NADH ratio is one key switch that determines the metabolic fate of α-KG. Inhibiting RC could be a key mechanism by which mitochondrial uncouplers inhibit tumor growth.

Implications: Mitochondrial uncoupling is a novel strategy to target RC in cancer.

Figure 1.. Mitochondrial uncoupling inhibits reductive carboxylation induced by hypoxia.

(A) Schematic of ¹³C-labeling patterns of TCA cycle metabolites from U-¹³C-glutamine and 1-¹³C-glutamine. Blue circles indicate ¹³C from U-¹³C-glutamine, yellow circles labelled “1” indicate ¹³C from 1-¹³C-glutamine, and hollow circles indicate ¹²C. (B, C) SK-N-BE(2) and NB16 cells were pretreated under normoxia or hypoxia (0.5% O₂) for 16 hours, followed by DMSO or 1 μM NEN for 3 hours, then labeled with U-¹³C-glutamine for 2 hours. The relative isotopic labeling fraction of metabolites were measured using LC-MS (n=3). (D) SK-N-BE(2) and NB16 cells were plated and treated by different concentration of NEN under normoxia and hypoxia, followed by cell counting by Cytation5 (n=6).

Figure 2.. Mitochondrial uncoupling inhibits reductive carboxylation generation in VHL-null ccRCC cells.

(A, B) The ccRCC cells were pretreated with DMSO or 1 μM NEN for 3 hours, then labeled with U-¹³C-glutamine for 3 hours. Relative isotopic labeling fractions or ratios of metabolites were measured using LC-MS. (C) The RCC4 and 786-O cells were treated by DMSO or NEN for 5 or24 hours, followed by immunoblot to examine the protein level.

Figure 3. Mitochondrial uncoupling inhibits reductive carboxylation during anchorage-independent growth.

(A, B) SK-N-BE(2) and NB16 cells in monolayer culture, or spheroid culture condition treated with DMSO or 1 μM NEN for 3 hours, were labeled with U-13C-glutamine for 3 hours. Relative isotopic labeling fractions or ratios of metabolites were measured using LC-MS. (C) Morphology of SK-N-BE(2) and NB16 cells treated with DMSO or 1 μM NEN for 12 days in low attachment dishes. (Scale bar: 500μm) (D) Quantitation of spheroid and cell number from samples shown in (C).