Research ArticleOriginal Article
Open Access

Mitochondrial uncoupling inhibits serine catabolism via FTO activation in metastatic breast cancer

Xin Jin, Albert M. Li, Man Zhao, Haowen Jiang, Yiren Xiao, Michaela Yip, Stavros Melemenidis, Scott Jackson, Yanan Yang, Cathyrin Simmermaker, Meng-Ning Zhou, Subarna Sinha, Daniel J. Cuthbertson, Erinn B. Rankin and Jiangbin Ye
Cancer Biology & Medicine September 2026, 23 (9) 1303-1319; DOI: https://doi.org/10.20892/j.issn.2095-3941.2025.0444

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  • The study was structured in three parts to systematically investigate the therapeutic potential of mitochondrial uncoupling in metastatic breast cancer. Part I used a U-13C-serine tracing approach, which revealed that mitochondrial uncoupling suppresses the MSCP. Part II delineated the underlying mechanism, combining bioinformatic analysis, immunoblotting, MeRIP-qPCR, and ELISA to demonstrate that mitochondrial uncoupling downregulates key MSCP enzymes via FTO-dependent RNA demethylation. Part III evaluated the clinical relevance of FTO expression as a prognostic biomarker and explored the therapeutic efficacy of mitochondrial uncouplers in metastatic breast cancer models. Collectively, this work established mitochondrial uncoupling as a novel strategy to promote m6A demethylation and inhibit the MSCP, offering a promising therapeutic avenue for metastatic breast cancer. MSCP, mitochondrial serine catabolic pathway; MBC, metastatic breast cancer; NEN, niclosamide ethanolamine; LC-MS, liquid chromatography-mass spectrometry; IB, immunoblotting; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; FTO, fat mass and obesity-associated protein; MeRIP, methylated RNA immunoprecipitation; ELISA, enzyme-linked immunosorbent assay.
    Study Flowchart

    The study was structured in three parts to systematically investigate the therapeutic potential of mitochondrial uncoupling in metastatic breast cancer. Part I used a U-13C-serine tracing approach, which revealed that mitochondrial uncoupling suppresses the MSCP. Part II delineated the underlying mechanism, combining bioinformatic analysis, immunoblotting, MeRIP-qPCR, and ELISA to demonstrate that mitochondrial uncoupling downregulates key MSCP enzymes via FTO-dependent RNA demethylation. Part III evaluated the clinical relevance of FTO expression as a prognostic biomarker and explored the therapeutic efficacy of mitochondrial uncouplers in metastatic breast cancer models. Collectively, this work established mitochondrial uncoupling as a novel strategy to promote m6A demethylation and inhibit the MSCP, offering a promising therapeutic avenue for metastatic breast cancer. MSCP, mitochondrial serine catabolic pathway; MBC, metastatic breast cancer; NEN, niclosamide ethanolamine; LC-MS, liquid chromatography-mass spectrometry; IB, immunoblotting; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; FTO, fat mass and obesity-associated protein; MeRIP, methylated RNA immunoprecipitation; ELISA, enzyme-linked immunosorbent assay.

  • Mitochondrial uncoupling inhibits serine catabolism and de novo purine synthesis. (A) A schematic of the SCP pathway with U-13C-serine tracing. The dark cycle indicates 13C labeling in glycine, while the light cycle represents 13C labeling in one-carbon units. (B and C) 4175-LM2 and MDA-MB-468 cells were treated with DMSO control or 1 μM NEN and labeled with U-13C-serine for 24 h. The isotope labeling fractions of metabolites were measured using LC-MS (mean ± SD, n = 3, **P < 0.01 ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t-test). (D) 4175-LM2 and MDA-MB-468 cells were treated with DMSO control, 1 μM NEN, or 1 μM rotenone + antimycin A (Rot/AA). The oxygen consumption rate (OCR) was measured continuously on Seahorse XF Analyzer (n = 4). Red arrow indicates when treatment started. (E) 4175-LM2 cells were treated with 1 μM NEN or 10 μM BAM15 for the indicated times and the NAD+:NADH ratio was determined by LC-MS (mean ± SD, n = 3).
    Figure 1

    Mitochondrial uncoupling inhibits serine catabolism and de novo purine synthesis. (A) A schematic of the SCP pathway with U-13C-serine tracing. The dark cycle indicates 13C labeling in glycine, while the light cycle represents 13C labeling in one-carbon units. (B and C) 4175-LM2 and MDA-MB-468 cells were treated with DMSO control or 1 μM NEN and labeled with U-13C-serine for 24 h. The isotope labeling fractions of metabolites were measured using LC-MS (mean ± SD, n = 3, **P < 0.01 ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t-test). (D) 4175-LM2 and MDA-MB-468 cells were treated with DMSO control, 1 μM NEN, or 1 μM rotenone + antimycin A (Rot/AA). The oxygen consumption rate (OCR) was measured continuously on Seahorse XF Analyzer (n = 4). Red arrow indicates when treatment started. (E) 4175-LM2 cells were treated with 1 μM NEN or 10 μM BAM15 for the indicated times and the NAD+:NADH ratio was determined by LC-MS (mean ± SD, n = 3).

  • Mitochondrial uncoupling downregulates mitochondrial serine catabolic pathway (MSCP) enzymes. (A) Immunoblots of the MSCP enzymes in 4175-LM2 and MDA-MB-468 cells treated with different concentrations of NEN for 48 or 72 h. (B) RT-qPCR analysis of SCP gene expression in 4175-LM2 cells treated with 1 μM NEN for 24 or 48 h (mean ± SD, n = 4, *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t-test).
    Figure 2

    Mitochondrial uncoupling downregulates mitochondrial serine catabolic pathway (MSCP) enzymes. (A) Immunoblots of the MSCP enzymes in 4175-LM2 and MDA-MB-468 cells treated with different concentrations of NEN for 48 or 72 h. (B) RT-qPCR analysis of SCP gene expression in 4175-LM2 cells treated with 1 μM NEN for 24 or 48 h (mean ± SD, n = 4, *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t-test).

  • Mitochondrial uncoupling downregulates mitochondrial serine catabolic pathway (MSCP) enzyme expression through FTO. (A) Heatmap showing the expression of RNA-binding proteins in breast cancer and normal tissues. (B) Box plots depicting the average level (transcripts per million) of FTO or ALKBH5 expression in normal tissue and different subtypes of breast cancer. (C) Immunoblot analysis of SCP enzymes in 4175-LM2 cells treated with DMSO control or NEN (1 μM) with or without the FTO inhibitor, FB23-2 (5 μM), for 48 h. (D) Immunoblot analysis of SCP enzymes in 4175-LM2 cells with doxycycline (Dox)-induced FTO knockdown (72 h) and NEN (1 μM) treatment for 48 h. (E) Gene-specific m6A qPCR analysis in SHMT2 exon I and MTHFD1L 3′UTR in 4175-LM2 shCtrl and shFTO cells after m6A IP (MeRIP). Data are presented as the mean ± SEM, n = 3. Statistical significance was determined using an unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).
    Mitochondrial uncoupling downregulates mitochondrial serine catabolic pathway (MSCP) enzyme expression through FTO. (A) Heatmap showing the expression of RNA-binding proteins in breast cancer and normal tissues. (B) Box plots depicting the average level (transcripts per million) of FTO or ALKBH5 expression in normal tissue and different subtypes of breast cancer. (C) Immunoblot analysis of SCP enzymes in 4175-LM2 cells treated with DMSO control or NEN (1 μM) with or without the FTO inhibitor, FB23-2 (5 μM), for 48 h. (D) Immunoblot analysis of SCP enzymes in 4175-LM2 cells with doxycycline (Dox)-induced FTO knockdown (72 h) and NEN (1 μM) treatment for 48 h. (E) Gene-specific m6A qPCR analysis in SHMT2 exon I and MTHFD1L 3′UTR in 4175-LM2 shCtrl and shFTO cells after m6A IP (MeRIP). Data are presented as the mean ± SEM, n = 3. Statistical significance was determined using an unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).
    Figure 3

    Mitochondrial uncoupling downregulates mitochondrial serine catabolic pathway (MSCP) enzyme expression through FTO. (A) Heatmap showing the expression of RNA-binding proteins in breast cancer and normal tissues. (B) Box plots depicting the average level (transcripts per million) of FTO or ALKBH5 expression in normal tissue and different subtypes of breast cancer. (C) Immunoblot analysis of SCP enzymes in 4175-LM2 cells treated with DMSO control or NEN (1 μM) with or without the FTO inhibitor, FB23-2 (5 μM), for 48 h. (D) Immunoblot analysis of SCP enzymes in 4175-LM2 cells with doxycycline (Dox)-induced FTO knockdown (72 h) and NEN (1 μM) treatment for 48 h. (E) Gene-specific m6A qPCR analysis in SHMT2 exon I and MTHFD1L 3′UTR in 4175-LM2 shCtrl and shFTO cells after m6A IP (MeRIP). Data are presented as the mean ± SEM, n = 3. Statistical significance was determined using an unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).

  • Reduction of 2-HG mediates the effects of mitochondrial uncoupling on MSCP enzyme regulation. (A) 4175-LM2 cells were treated with 1 μM NEN for 24 h. 2-HG levels were determined using LC-MS. (B) 4175-LM2 cells were treated with 0.5 μM or 1 μM NEN for 8 or 24 h and m6A levels were determined using an EpiQuik m6A RNA Methylation Quantification Kit. (C) 4175-LM2 cells were treated with 0.2 mM octyl-(S)-2-HG or 2.5 mM dimethyl α-KG for 24 h and m6A levels were determined with an EpiQuik m6A RNA Methylation Quantification Kit. For (A)–(C), data represent the mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. (D and E) Immunoblot analysis of SCP enzymes in 4175-LM2 cells treated with 0.2 mM octyl-(S)-2-HG or 2.5 mM dimethyl α-KG for the time indicated.
    Figure 4

    Reduction of 2-HG mediates the effects of mitochondrial uncoupling on MSCP enzyme regulation. (A) 4175-LM2 cells were treated with 1 μM NEN for 24 h. 2-HG levels were determined using LC-MS. (B) 4175-LM2 cells were treated with 0.5 μM or 1 μM NEN for 8 or 24 h and m6A levels were determined using an EpiQuik m6A RNA Methylation Quantification Kit. (C) 4175-LM2 cells were treated with 0.2 mM octyl-(S)-2-HG or 2.5 mM dimethyl α-KG for 24 h and m6A levels were determined with an EpiQuik m6A RNA Methylation Quantification Kit. For (A)–(C), data represent the mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t-test. (D and E) Immunoblot analysis of SCP enzymes in 4175-LM2 cells treated with 0.2 mM octyl-(S)-2-HG or 2.5 mM dimethyl α-KG for the time indicated.

  • High FTO expression is associated with increased relapse-free survival in breast cancer patients. (A) and (B) Kaplan–Meier survival curves comparing FTO-high and -low patients in two patient cohorts (GSE42568 and GSE31448). (C) Levels of FTO and SHMT2 RNA expression across different stages of breast cancer. UQN; Upper Quartile Normalization.
    Figure 5

    High FTO expression is associated with increased relapse-free survival in breast cancer patients. (A) and (B) Kaplan–Meier survival curves comparing FTO-high and -low patients in two patient cohorts (GSE42568 and GSE31448). (C) Levels of FTO and SHMT2 RNA expression across different stages of breast cancer. UQN; Upper Quartile Normalization.

  • Mitochondrial uncoupling inhibits breast cancer metastasis. (A) Representative images of tumor burden and (B) quantification of luminescence signal in the lungs of control diet- or NEN diet-fed mice injected with 4175-LM cells (mean ± SEM; *P < 0.05 by two-tailed Student’s t-test, ctrl: n = 8; NEN: n = 9). (C) Quantification of human tumor burden in mouse lungs from control and NEN-treated groups. Human tumor content was measured by qPCR using the ratio of human GAPDH-to-human and mouse 18S. Data are presented as the mean ± SEM. Ctrl: n = 8; NEN: n = 9.
    Figure 6

    Mitochondrial uncoupling inhibits breast cancer metastasis. (A) Representative images of tumor burden and (B) quantification of luminescence signal in the lungs of control diet- or NEN diet-fed mice injected with 4175-LM cells (mean ± SEM; *P < 0.05 by two-tailed Student’s t-test, ctrl: n = 8; NEN: n = 9). (C) Quantification of human tumor burden in mouse lungs from control and NEN-treated groups. Human tumor content was measured by qPCR using the ratio of human GAPDH-to-human and mouse 18S. Data are presented as the mean ± SEM. Ctrl: n = 8; NEN: n = 9.

  • Mitochondrial uncoupling inhibits serine catabolism through FTO activation in metastatic breast cancer. Left: The m6A RNA demethylase, FTO, utilizes α-KG as a substrate. Under the Warburg effect, the reduced NAD+:NADH ratio leads to conversion of α-KG to 2-HG. 2-HG, which is structurally similar to α-KG, acts as a competitive inhibitor of FTO. This inhibition results in the accumulation of m6A on target RNAs, including target RNAs encoding the MSCP pathway enzymes, SHMT2, MTHFD2, and MTHFD1L. The resulting upregulation of these enzymes, which are essential for de novo purine biosynthesis, thereby fulfills the metabolic demands of metastatic breast cancer cell proliferation. Right: Mitochondrial uncouplers, a class of compounds that dissipate the proton gradient across the inner mitochondrial membrane, can activate electron transport chain activity and alleviate reductive stress. This activation of the electron transport chain restores the NAD+:NADH ratio, reverses the Warburg effect, and lowers 2-HG levels. Consequently, FTO activity is recovered, leading to decreased m6A methylation on MSCP gene transcripts, reduced expression of the corresponding enzymes, and suppressed metastasis. α-KG, α-ketoglutarate; 2-HG, 2-hydroxyglutarate; m6A, N6-methyladenosine; FTO, fat mass and obesity-associated protein; SHMT2, serine hydroxymethyltransferase 2; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; MTHFD1L, methylenetetrahydrofolate dehydrogenase 1-like.
    Figure 7

    Mitochondrial uncoupling inhibits serine catabolism through FTO activation in metastatic breast cancer. Left: The m6A RNA demethylase, FTO, utilizes α-KG as a substrate. Under the Warburg effect, the reduced NAD+:NADH ratio leads to conversion of α-KG to 2-HG. 2-HG, which is structurally similar to α-KG, acts as a competitive inhibitor of FTO. This inhibition results in the accumulation of m6A on target RNAs, including target RNAs encoding the MSCP pathway enzymes, SHMT2, MTHFD2, and MTHFD1L. The resulting upregulation of these enzymes, which are essential for de novo purine biosynthesis, thereby fulfills the metabolic demands of metastatic breast cancer cell proliferation. Right: Mitochondrial uncouplers, a class of compounds that dissipate the proton gradient across the inner mitochondrial membrane, can activate electron transport chain activity and alleviate reductive stress. This activation of the electron transport chain restores the NAD+:NADH ratio, reverses the Warburg effect, and lowers 2-HG levels. Consequently, FTO activity is recovered, leading to decreased m6A methylation on MSCP gene transcripts, reduced expression of the corresponding enzymes, and suppressed metastasis. α-KG, α-ketoglutarate; 2-HG, 2-hydroxyglutarate; m6A, N6-methyladenosine; FTO, fat mass and obesity-associated protein; SHMT2, serine hydroxymethyltransferase 2; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; MTHFD1L, methylenetetrahydrofolate dehydrogenase 1-like.

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