bims-mibica 2025-06-01 papers

bims-mibica

Biomed News

on Mitochondrial bioenergetics in cancer

Issue of 2025–06–01
fifteen papers selected by
Kelsey Fisher-Wellman, Wake Forest University

Impact of physiological media on acute myeloid leukemia bioenergetics and cell proliferation.
Design, Synthesis and Biological Evaluation of POLRMT Inhibitors for the Treatment of Acute Myeloid Leukemia.
CoQ imbalance drives reverse electron transport to disrupt liver metabolism.
Mitochondrial DNA transfer between malignant cells and T lymphocytes shapes the cancer-immunity dialogue.
BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress.
The Metabolic Landscape of Cancer Stem Cells: Insights and Implications for Therapy.
P5CS deacetylation mediated by SIRT2 facilitates tumor growth by enhancing mitochondrial respiration in hepatocellular carcinoma.
Acetylation-induced degradation of ECHS1 enhances BCAA accumulation and proliferation in KRAS-mutant colorectal cancer.
CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis.
Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid.
Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities.
Oncogenic role of fumarate hydratase in breast cancer: metabolic reprogramming and mechanistic insights.
Mitochondrial calcium signaling regulates branched-chain amino acid catabolism in fibrolamellar carcinoma.
Stable 13C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.
Butyrate confers colorectal cancer cell resistance to anti-PD-1 therapy by promoting CPT1A-mediated fatty acid oxidation.

Cancer Metab. 2025 May 26. 13(1): 25

Impact of physiological media on acute myeloid leukemia bioenergetics and cell proliferation.

Brett R Chrest, McLane M Montgomery, Raphael T Aruleba, Polina Krassovskaia, Emely A Pacheco, James T Hagen, Kayla J Vandiver, Kang Tung, Molly K Alexander, Nicholas C Williamson, Joshua G Taylor, Riley N Bessetti, Heather A Belcher, Filip Jevtovic, Zoe S Terwilliger, Everett C Minchew, Tonya N Zeczycki, Linda May, Nicholas T Broskey, Christopher B Geyer, Karen Litwa, Espen E Spangenburg, Johanna L Hannan, Jessica M Ellis, Joseph M McClung, P Darrell Neufer, Kelsey H Fisher-Wellman.

Increasing emphasis has been placed on improving the physiological relevance of cell culture media with formulations such as Human Plasma-Like Medium (HPLM). Given that shifts in mitochondrial metabolism and nutrient use are emerging as anti-cancer targets, the present study sought to investigate the impact of culture media formulation on mitochondrial bioenergetics and cancer cell growth. To do this, we used acute myeloid leukemia (AML) cells and compared acute and chronic effects of HPLM versus different supraphysiological medias. The AML mitochondrial phenotype was largely unaffected by exposure to either physiological or supraphysiological medias, establishing that the key features of AML mitochondria remain phenotypically stable under diverse nutrient conditions and proliferation rates. Both acute and chronic culturing in HPLM slowed AML cell proliferation. However, merely identifying and supplementing single nutrients that were deficient in HPLM did not improve proliferation and was not sufficient to pinpoint actionable fuel preferences. Transferring cells back to native Iscove's Modified Dulbecco's Medium (IMDM) media immediately restored the proliferative phenotype, suggesting responsiveness to the entirety of the nutrient environment. Supraphysiological culture medias other than IMDM were all characterized by slower proliferation; however, none were associated with changes in cell viability, demonstrating that the native culture medium is optimal if the experimental aim is maximal growth. Despite Dulbecco's Modified Eagle Medium (DMEM) being similar in nutrient composition to IMDM and categorized as supraphysiological, both acute and chronic culturing in DMEM resulted in slower growth, akin to what was observed with HPLM. Altogether, independent of growth, AML mitochondria remain largely unperturbed by changes in the culture media, and rather than specific nutrients or physiological relevance, AML cell proliferation is influenced by the complete nutrient profile.

DOI: https://doi.org/10.1186/s40170-025-00395-1
Chem Biol Drug Des. 2025 May;105(5): e70127

Design, Synthesis and Biological Evaluation of POLRMT Inhibitors for the Treatment of Acute Myeloid Leukemia.

Tianli Liu, Xiaoling Cheng, Yupeng Wang, Wenli Hao, Hangyu Wang, Ke Zhang, Jinhui Wang.

  The metabolic dependence of acute myeloid leukemia (AML) cells on mitochondrial oxidative phosphorylation (OXPHOS) has become a cutting-edge area in cancer energy metabolism research, playing a pivotal role in cell survival and drug resistance. Consequently, targeted inhibition of human mitochondrial RNA polymerase (POLRMT) to block mitochondrial gene expression emerges as a novel potential strategy for treating AML through OXPHOS modulation. In this study, based on the previously reported crystal structure of the POLRMT inhibitor IMT1B, we employed a scaffold hopping strategy to design and synthesize a series of derivatives featuring additional hydrophobic occupying groups. A new potent POLRMT inhibitor (10a) was discovered, which displayed potent antiproliferative activity and could disrupt mitochondrial function and induce apoptosis in MOLM-13 cells. Together, these results demonstrate that 10a is a new POLRMT inhibitor, which may provide a candidate lead for AML treatment.

Keywords:  AML; OXPHOS; POLRMT inhibitor

DOI:  https://doi.org/10.1111/cbdd.70127
Nature. 2025 May 28.

CoQ imbalance drives reverse electron transport to disrupt liver metabolism.

Renata L S Goncalves, Zeqiu Branden Wang, Jillian K Riveros, Güneş Parlakgül, Karen E Inouye, Grace Yankun Lee, Xiaorong Fu, Jani Saksi, Clement Rosique, Sheng Tony Hui, Mar Coll, Ana Paula Arruda, Shawn C Burgess, Isabel Graupera, Gökhan S Hotamışlıgil.

Mitochondrial reactive oxygen species (mROS) are central to physiology1,2. Excess mROS production has been associated with several disease states2,3; however, the precise sources, regulation and mechanism of generation in vivo remain unclear, which limits translational efforts. Here we show that in obesity, hepatic coenzyme Q (CoQ) synthesis is impaired, which increases the CoQH2 to CoQ (CoQH2/CoQ) ratio and drives excessive mROS production through reverse electron transport (RET) from site IQ in complex I. Using multiple complementary genetic and pharmacological models in vivo, we demonstrate that RET is crucial for metabolic health. In patients with steatosis, the hepatic CoQ biosynthetic program is also suppressed, and the CoQH2/CoQ ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

DOI: https://doi.org/10.1038/s41586-025-09072-1
Oncoimmunology. 2025 Dec;14(1): 2512109

Mitochondrial DNA transfer between malignant cells and T lymphocytes shapes the cancer-immunity dialogue.

Liwei Zhao, Peng Liu, Oliver Kepp, Guido Kroemer.

  Nonmutated mitochondrial DNA (mtDNA) from T lymphocytes can be incorporated into cancer cells bearing mutated mtDNA to repair their bioenergetic deficiency. However, a recent paper by Ikeda et al. indicates that mutated mtDNA from malignant cells can also be transferred into tumor-infiltrating T lymphocytes to subvert their function in cancer immunosurveillance.

Keywords:  Immune checkpoint inhibition; Immunotherapy; immunosuppression

DOI:  https://doi.org/10.1080/2162402X.2025.2512109
Nat Commun. 2025 May 28. 16(1): 4931

BH3 mimetics targeting BCL-XL have efficacy in solid tumors with RB1 loss and replication stress.

Andreas Varkaris, Keshan Wang, Mannan Nouri, Nina Kozlova, Daniel R Schmidt, Anastasia Stavridi, Seiji Arai, Nicholas Ambrosio, Larysa Poluben, Juan M Jimenez-Vacas, Daniel Westaby, Juliet Carmichael, Fang Xie, Ines Figueiredo, Lorenzo Buroni, Antje Neeb, Bora Gurel, Nicholas Chevalier, Lisha Brown, Olga Voznesensky, Shao-Yong Chen, Joshua W Russo, Xin Yuan, Dejan Juric, Himisha Beltran, Johann S De Bono, Matthew G Vander Heiden, David J Einstein, Taru Muranen, Eva Corey, Adam Sharp, Steven P Balk.

BH3 mimetic drugs that inhibit BCL-2, BCL-XL, or MCL-1 have limited activity in solid tumors. Through assessment of xenograft-derived 3D prostate cancer models and cell lines we find that tumors with RB1 loss are sensitive to BCL-XL inhibition. In parallel, drug screening demonstrates that disruption of nucleotide pools by agents including thymidylate synthase inhibitors sensitizes to BCL-XL inhibition, together indicating that replication stress increases dependence on BCL-XL. Mechanistically we establish that replication stress sensitizes to BCL-XL inhibition through TP53/CDKN1A-dependent suppression of BIRC5 expression. Therapy with a BCL-2/BCL-XL inhibitor (navitoclax) in combination with thymidylate synthase inhibitors (raltitrexed or capecitabine) causes marked and prolonged tumor regression in prostate and breast cancer xenograft models. These findings indicate that BCL-XL inhibitors may be effective as single agents in a subset of solid tumors with RB1 loss, and that pharmacological induction of replication stress may be a broadly applicable approach for sensitizing to BCL-XL inhibitors.

DOI: https://doi.org/10.1038/s41467-025-60238-x
Cells. 2025 05 15. pii: 717. [Epub ahead of print]14(10):

The Metabolic Landscape of Cancer Stem Cells: Insights and Implications for Therapy.

Martina Milella, Monica Rutigliano, Savio Domenico Pandolfo, Achille Aveta, Felice Crocetto, Matteo Ferro, Antonio d'Amati, Pasquale Ditonno, Giuseppe Lucarelli, Francesco Lasorsa.

  Cancer stem cells (CSCs) are a subpopulation with self-renewal and differentiation capacities believed to be responsible for tumor initiation, progression, and recurrence. These cells exhibit unique metabolic features that contribute to their stemness and survival in hostile tumor microenvironments. Like non-stem cancer cells, CSCs primarily rely on glycolysis for ATP production, akin to the Warburg effect. However, CSCs also show increased dependence on alternative metabolic pathways, such as oxidative phosphorylation (OXPHOS) and fatty acid metabolism, which provide necessary energy and building blocks for self-renewal and therapy resistance. The metabolic plasticity of CSCs enables them to adapt to fluctuating nutrient availability and hypoxic conditions within the tumor. Recent studies highlight the importance of these metabolic shifts in maintaining the CSC phenotype and promoting cancer progression. The CSC model suggests that a small, metabolically adaptable subpopulation drives tumor growth and therapy resistance. CSCs can switch between glycolysis and mitochondrial metabolism, enhancing their survival under stress and dormant states. Targeting CSC metabolism offers a promising therapeutic strategy; however, their adaptability complicates eradication. A multi-targeted approach addressing various metabolic pathways is essential for effective CSC elimination, underscoring the need for further research into specific CSC markers and mechanisms that distinguish their metabolism from normal stem cells for successful therapeutic intervention.

Keywords:  cancer; markers; stem cells; treatment

DOI:  https://doi.org/10.3390/cells14100717
Oncogene. 2025 May 27.

P5CS deacetylation mediated by SIRT2 facilitates tumor growth by enhancing mitochondrial respiration in hepatocellular carcinoma.

Xiaofang Geng, Mengyao Li, Lu Zhang, Yihan Cai, Xin Chen, Xiayue Mu, Jie Wang, Bowen Liu.

Cancer cells typically exhibit enhanced mitochondrial metabolism to fulfill their energy and biosynthetic demands for growth. The mitochondrial response to fluctuations in cellular energy demand is essential for cellular adaptation and proper organ function. The mitochondrial delta-1-pyrroline-5-carboxylate synthase (P5CS) encoded by the ALDH18A1 gene, the key enzyme for proline synthesis, is frequently up-regulated during tumor development. However, the regulatory mechanisms governing P5CS activity in the occurrence and development of hepatocellular carcinoma (HCC) remain largely unknown. In this study, we observe that P5CS is highly expressed in HCC tissues, and elevated levels of P5CS expression are associated with poor prognosis in HCC patients. Notably, the knockdown of P5CS inhibits the proliferation, migratory and invasive capabilities of HCC cells by reducing mitochondrial respiration. Furthermore, we demonstrate that SIRT2 interacts with P5CS and mediates the deacetylation of P5CS at lysines K311 and K347, thereby activating its enzymatic activity. Activated P5CS significantly enhances mitochondrial respiration, which supports the proliferation and tumorigenesis of HCC cells. In addition, SIRT2 knockdown inhibits the proliferation, migratory and invasive capabilities of HCC cells. These observations suggest that SIRT2-mediated P5CS deacetylation is a crucial signaling event through which cancer cells sustain mitochondrial respiration and promote HCC progression. This finding offers the potential for targeting SIRT2-mediated P5CS deacetylation as a therapeutic strategy for HCC.

DOI: https://doi.org/10.1038/s41388-025-03456-3
J Exp Clin Cancer Res. 2025 05 28. 44(1): 164

Acetylation-induced degradation of ECHS1 enhances BCAA accumulation and proliferation in KRAS-mutant colorectal cancer.

Zhenkang Li, Zhengyu Liu, Mingdao Lin, Huayang Pan, Yuechen Liu, Yang Liu, Yuwen Xie, Jinchao Zhang, Shenyuan Guan, Yongsheng Li, Mulan Zhu, Yuan Fang, Zhiyong Shen, Haijun Deng.

   BACKGROUND: Branched-chain amino acid (BCAA) metabolism is dysregulated in colorectal cancer (CRC), with elevated plasma BCAA levels significantly associated with an increased risk of developing the disease. However, whether BCAAs directly promote CRC progression and their underlying mechanisms remain unclear.
METHODS: In this study, we investigated the metabolic alterations in KRAS-mutant CRC. We examined the effects of restricting BCAA supply on the proliferation and metastasis of KRAS-mutant CRC cells both in vitro and in vivo.
RESULTS: We found that in KRAS-mutant CRC, BCAAs and their metabolic products accumulate markedly. Restricting the BCAA supply specifically inhibits the proliferation of KRAS-mutant CRC cells but does not affect metastasis. In these cancer cells, enoyl-CoA hydratase-1 (ECHS1), a key enzyme in BCAA metabolism, is downregulated. Furthermore, BCAAs enhance the acetylation of lysine 204 on ECHS1, impairing its ability to bind enoyl-CoA and reducing its catalytic activity. This modification triggers the ubiquitination of ECHS1 and its subsequent degradation, diminishing BCAA catabolism and leading to its cellular accumulation. This accumulation activates the mTORC1 signaling pathway, which induces the transcriptional activation of downstream target proteins and promotes the malignant progression of CRC.
CONCLUSIONS: Limiting BCAA intake not only suppresses tumor growth in KRAS-mutant CRC but also enhances the efficacy of the KRAS G12D inhibitor MRTX1133 and the monoclonal antibody bevacizumab. Our findings reveal a previously unknown regulatory mechanism of ECHS1 in CRC and offer new potential therapeutic targets.

Keywords:  Acetylation; BCAAs; Colorectal cancer; KRAS

DOI:  https://doi.org/10.1186/s13046-025-03399-3
Proc Natl Acad Sci U S A. 2025 Jun 03. 122(22): e2502876122

CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis.

Sophia M Lamperis, Kaylin M McMahon, Andrea E Calvert, Jonathan S Rink, Karthik Vasan, Madhura R Pandkar, Eliana U Crentsil, Zachary R Chalmers, Natalie R McDonald, Cameron J Kosmala, Marcelo G Bonini, Daniela Matei, Leo I Gordon, Navdeep S Chandel, C Shad Thaxton.

  Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.

Keywords:  cancer; cell death; ferroptosis; lipids; nanoparticles

DOI:  https://doi.org/10.1073/pnas.2502876122
Elife. 2025 May 30. pii: RP93621. [Epub ahead of print]13

Inhibition of mitochondrial protein import and proteostasis by a pro-apoptotic lipid.

Josep Fita-Torró, José Luis Garrido-Huarte, Lucía López-Gil, Agnès H Michel, Benoit Kornmann, Amparo Pascual-Ahuir, Markus Proft.

  Mitochondria-mediated cell death is critically regulated by bioactive lipids derived from sphingolipid metabolism. The lipid aldehyde trans-2-hexadecenal (t-2-hex) induces mitochondrial dysfunction from yeast to humans. Here, we apply unbiased transcriptomic, functional genomics, and chemoproteomic approaches in the yeast model to uncover the principal mechanisms and biological targets underlying this lipid-induced mitochondrial inhibition. We find that loss of Hfd1 fatty aldehyde dehydrogenase function efficiently sensitizes cells for t-2-hex inhibition and apoptotic cell death. Excess of t-2-hex causes a profound transcriptomic response with characteristic hallmarks of impaired mitochondrial protein import, like activation of mitochondrial and cytosolic chaperones or proteasomal function and severe repression of translation. We confirm that t-2-hex stress induces rapid accumulation of mitochondrial pre-proteins and protein aggregates and subsequent activation of Hsf1- and Rpn4-dependent gene expression. By saturated transposon mutagenesis, we find that t-2-hex tolerance requires an efficient heat shock response and specific mitochondrial and ER functions and that mutations in ribosome, protein, and amino acid biogenesis are beneficial upon t-2-hex stress. We further show that genetic and pharmacological inhibition of protein translation causes t-2-hex resistance, indicating that loss of proteostasis is the predominant consequence of the pro-apoptotic lipid. Several TOM subunits, including the central Tom40 channel, are lipidated by t-2-hex in vitro and mutation of accessory subunits Tom20 or Tom70 confers t-2-hex tolerance. Moreover, the Hfd1 gene dose determines the strength of t-2-hex mediated inhibition of mitochondrial protein import, and Hfd1 co-purifies with Tom70. Our results indicate that the transport of mitochondrial precursor proteins through the outer mitochondrial membrane is sensitively inhibited by the pro-apoptotic lipid and thus represents a hotspot for pro- and anti-apoptotic signaling.

Keywords:  S. cerevisiae; apoptosis; biochemistry; chemical biology; genetics; genomics; lipid signaling; mitochondrial protein import; proteostasis; sphingolipid metabolism; yeast

DOI:  https://doi.org/10.7554/eLife.93621
Redox Biol. 2025 May 13. pii: S2213-2317(25)00190-9. [Epub ahead of print]84 103677

Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities.

Fiorella Faienza, Claudio Laquatra, Matteo Castelli, Gianmarco Matrullo, Salvatore Rizza, Federica Guarra, Azam Roshani Dashtmian, Alessia Magro, Paola Giglio, Chiara Pecorari, Lavinia Ferrone, Elisabetta Moroni, Francesca Pacello, Andrea Battistoni, Giorgio Colombo, Andrea Rasola, Giuseppe Filomeni.

  The mitochondrial chaperone TRAP1 exerts protective functions under diverse stress conditions. It induces metabolic rewiring and safeguards cancer cells from oxidative insults, thereby contributing to neoplastic progression. TRAP1 works as a homodimer, but recent evidence indicated that it forms tetramers whose effects remain elusive. Here, we find that TRAP1 generates redox-sensitive tetramers via disulfide bonds involving cysteines 261 and 573. TRAP1 tetramerization is elicited by oxidative stress and abrogated upon expression of the double C261S/C573R mutant. In cancer cells, the TRAP1 C261S/C573R mutant is unable to inhibit the activity of its client succinate dehydrogenase and to confer protection against oxidative insults, thus hampering the invasiveness of aggressive sarcoma cells. Overall, our findings indicate that TRAP1 undergoes tetramerization in response to oxidative stress and identify C261 and C573 as critical for TRAP1 structural rearrangement and functions.

Keywords:  Cysteine; Metabolism; Mitochondria; Oxidative stress; Tumorigenesis

DOI:  https://doi.org/10.1016/j.redox.2025.103677
Cancer Metab. 2025 May 29. 13(1): 26

Oncogenic role of fumarate hydratase in breast cancer: metabolic reprogramming and mechanistic insights.

Shyng-Shiou F Yuan, Anupama Vadhan, Hieu D H Nguyen, Pang-Yu Chen, Chih-Huang Tseng, Ching-Hu Wu, Yu-Chieh Chen, Yi-Chia Wu, Stephen Chu-Sung Hu, Steven Lo, Ming-Feng Hou, Yen-Yun Wang.

Breast cancer remains the most prevalent malignancy among women globally, with its complexity linked to genetic variations and metabolic alterations within tumor cells. This study investigates the role of fumarate hydratase (FH), a key enzyme in the tricarboxylic acid (TCA) cycle, in breast cancer progression. Our findings reveal that FH mRNA and protein levels are significantly upregulated in breast cancer tissues and correlate with poor patient prognosis and aggressive tumor characteristics. Using in vitro and in vivo models, we demonstrate that FH overexpression enhances breast cancer cell proliferation, migration, and invasion through metabolic reprogramming and by increasing reactive oxygen species (ROS) production. Furthermore, we identify matrix metalloproteinase 1 (MMP1) as a downstream effector of FH, linked to p21 downregulation, elucidating a novel regulatory pathway influencing tumor behavior. Interestingly, unlike its tumor-suppressing role in other cancer types, this study highlights FH's oncogenic potential in breast cancer. Our results suggest that FH enhances cancer cell viability and aggressiveness via both catalytic and non-catalytic mechanisms. This work not only underscores the metabolic adaptations of breast cancer cells but also proposes FH as a potential biomarker and therapeutic target for breast cancer management.

DOI: https://doi.org/10.1186/s40170-025-00397-z
Sci Adv. 2025 May 30. 11(22): eadu9512

Mitochondrial calcium signaling regulates branched-chain amino acid catabolism in fibrolamellar carcinoma.

Nicole M Marsh, Melissa J S MacEwen, Jane Chea, Heidi L Kenerson, Albert A Kwong, Timothy M Locke, Francisco Javier Miralles, Tanmay Sapre, Natasha Gozali, Madeleine L Hart, Theo K Bammler, James W MacDonald, Lucas B Sullivan, G Ekin Atilla-Gokcumen, Shao-En Ong, John D Scott, Raymond S Yeung, Yasemin Sancak.

Metabolic adaptations are essential for survival. The mitochondrial calcium uniporter plays a key role in coordinating metabolic homeostasis by regulating mitochondrial metabolic pathways and calcium signaling. However, a comprehensive analysis of uniporter-regulated mitochondrial pathways has remained unexplored. Here, we investigate consequences of uniporter loss and gain of function using uniporter knockout cells and fibrolamellar carcinoma (FLC), which we demonstrate to have elevated mitochondrial calcium levels. We find that branched-chain amino acid (BCAA) catabolism and the urea cycle are uniporter-regulated pathways. Reduced uniporter function boosts expression of BCAA catabolism genes and the urea cycle enzyme ornithine transcarbamylase. In contrast, high uniporter activity in FLC suppresses their expression. This suppression is mediated by the transcription factor KLF15, a master regulator of liver metabolism. Thus, the uniporter plays a central role in FLC-associated metabolic changes, including hyperammonemia. Our study identifies an important role for the uniporter in metabolic adaptation through transcriptional regulation of metabolism and elucidates its importance for BCAA and ammonia metabolism.

DOI: https://doi.org/10.1126/sciadv.adu9512
Bio Protoc. 2025 May 20. 15(10): e5322

Stable 13C-glutamine Tracing Resolved Metabolomics for Cancer Metabolism Study.

Yaogang Zhong, Liqing He, Xinmin Yin, Logan Mazik, Xiang Zhang, Deliang Guo.

  Stable isotopes have frequently been used to study metabolic processes in live cells both in vitro and in vivo. Glutamine, the most abundant amino acid in human blood, plays multiple roles in cellular metabolism by contributing to the production of nucleotides, lipids, glutathione, and other amino acids. It also supports energy production via anaplerosis of tricarboxylic acid cycle intermediates. While 13C-glutamine has been extensively employed to study glutamine metabolism in various cell types, detailed analyses of specific lipids derived from 13C-glutamine via the reductive carboxylation pathway are limited. In this protocol, we present a detailed procedure to investigate glutamine metabolism in human glioblastoma (GBM) cells by conducting 13C-glutamine tracing coupled with untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS/MS). The method includes step-by-step instructions for the extraction and detection of polar metabolites and long-chain fatty acids (LCFAs) derived from 13C-glutamine in GBM cells. Notably, this approach enables the distinction between isomers of two monounsaturated FAs with identical masses: palmitoleic acid (16:1n-7) (cis-9-hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-hexadecenoic acid) derived from 13C-glutamine through the reductive carboxylation process. In addition, using this protocol, we also unveil previously unknown metabolic alterations in GBM cells following lysosome inhibition by the antipsychotic drug pimozide. Key features • Methods for analyzing the flux of the stable isotope 13C-glutamine in cancer cells and identifying its derived polar metabolites and long-chain fatty acids (LCFAs). • Distinguishes isomers of long-chain fatty acids, such as palmitoleic acid (16:1n-7) (cis-9-Hexadecenoic acid) and palmitelaidic acid (16:1n-7) (trans-9-Hexadecenoic acid), which share the exact same mass. • The method is utilized to investigate glutamine metabolism reprogramming in cancer cells following lysosome inhibition.

Keywords:  13C-glutamine; GBM cells; LC–MS/MS; Long-chain fatty acids; Lysosome; Pimozide; Polar metabolites

DOI:  https://doi.org/10.21769/BioProtoc.5322
Discov Oncol. 2025 May 27. 16(1): 935

Butyrate confers colorectal cancer cell resistance to anti-PD-1 therapy by promoting CPT1A-mediated fatty acid oxidation.

Ran Zhu, Shujiang Gu, Yuan Tao, Yan Zhang.

  Immunotherapy including anti-PD-1 demonstrated therapeutic promise to colorectal cancer (CRC) patients, but tumor cell resistance limits their efficacy. Butyrate may influence therapeutic outcomes by modulating tumor metabolism, but it remains unclear whether butyrate influences CRC cell resistance to anti-PD-1 therapy. We aimed to investigate whether butyrate promotes resistance to anti-PD-1 therapy in CRC and underlying metabolic and immunologic mechanisms. CRC murine models were established by subcutaneously inoculating MC38 cells or butyrate/anti-PD-1-administered tumor cells of mice, followed by treatment with butyrate, anti-PD-1, or a combination. Therapeutic efficacy was assessed by tumor growth and survival outcomes. In vitro, HCT116 cells were exposed to monotherapy or co-therapy regimens. Carnitine Palmitoyltransferase 1A (CPT1A) knockdown was conducted by shRNA transfection both in vivo and in vitro. Fatty acid oxidation (FAO) was determined by oxygen consumption rate and CPT1A expression. CD8+ T cell cytotoxicity assays and CD8 expression in tumors were performed to evaluate immune cell infiltration. The addition of butyrate into anti-PD-1 treatment combination did not improve survival or reduce tumor volume compared to anti-PD-1 alone, with a marked activation of CPT1A observed in treated tumor tissues. Butyrate significantly elevated FAO, contributing to elevated oxygen consumption rate and reduced CD8+ T cell cytotoxicity. However, in sh-CPT1A models, the combination therapy significantly improved antitumor efficacy and restored CD8+ T cell infiltration. Furthermore, CRC patient samples resistant to anti-PD-1 therapy exhibited elevated CPT1A levels. Butyrate-induced CPT1A-mediated FAO promotes resistance to anti-PD-1 therapy in CRC, suggesting that targeting CPT1A might enhance the efficacy of immunotherapy.

Keywords:  Anti-PD-1 therapy; Butyrate; CPT1A; Colorectal cancer; Fatty acid oxidation; Immune resistance

DOI:  https://doi.org/10.1007/s12672-025-02686-x