bims-midysc Biomed News
on Mitochondria dysfunction in cancer
Issue of 2025–03–16
fourteen papers selected by
Papachristodoulou Lab



  1. Cell. 2025 Mar 05. pii: S0092-8674(25)00194-1. [Epub ahead of print]
      Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ∼75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.
    Keywords:  DOGMA-seq; end joining; mitochondrial pathologies; mtDNA; mtDNA deletion
    DOI:  https://doi.org/10.1016/j.cell.2025.02.009
  2. Mol Nutr Food Res. 2025 Mar 09. e70008
      Cruciferous and allium vegetables contain the sulfur compound S-methyl-L-cysteine-sulfoxide (SMCSO). Considering SMCSO is found at a higher abundance compared to the glucosinolates, there are limited reports on its effect on health, with the majority of the evidence on the beneficial effects on glucose metabolism in rodent models. In the current study, we investigated the metabolic effects of SMCSO and its metabolite, S-methyl methanethiosulfonate (MMTSO), on prostate cancer metabolism. DU145 prostate cancer cells were cultured in 5.5 mM (basal), 10 mM (intermediate) and 25 mM (high) glucose concentrations in the presence of SMCSO or MMTSO (100 µM). Using Seahorse technology, MMTSO but not SMCSO reduced mitochondrial metabolism, mitochondrial ATP, and the percentage of oxidative phosphorylation and increased the fatty acid dependency of DU145 cells. Transcriptomic and metabolomic analyses observed cellular and energy metabolism pathways and immune response changes. These data show that MMTSO alters several features of energy metabolism in DU145 prostate cancer cells, shifting them towards a non-cancerous phenotype. These data are consistent with the notion that MMTSO may contribute to the beneficial effects of a broccoli-rich diet and metabolic effects of prostate cancer.
    Keywords:  DU145 prostate cancer cells; MMTSO; SMCSO; broccoli; energy metabolism
    DOI:  https://doi.org/10.1002/mnfr.70008
  3. Biochim Biophys Acta Mol Cell Res. 2025 Mar 05. pii: S0167-4889(25)00034-5. [Epub ahead of print] 119929
      To investigate the effects of varying NAT1 activity in different cell-lines, mitochondrial oxidative phosphorylation, aerobic glycolysis and mitochondrial fuel usage was quantified in a panel of human cell-lines. As NAT1 activity increased, mitochondrial reserve respiratory capacity increased while aerobic glycolysis decreased. In addition, phosphorylation of PDH-E1α in these cells limited their ability to use glucose as a primary fuel source. Those cells with high NAT1 activity exhibited a quiescent metabolic phenotype and proliferated more slowly. This might explain, in part, why some cancer patients with low NAT1 expression in their tumour tissue show poorer survival outcomes compared to those with high NAT1 expression. The current study demonstrated that NAT1 enzymatic activity is important for metabolism in cancer cell-lines and increasing NAT1 activity may better equip cells to survive under stressed conditions by increasing reserve respiratory capacity.
    Keywords:  Acetyl-coenzyme A; Aerobic glycolysis; Arylamine N-acetyltransferase; Fuel usage; Mitochondrial respiration; Pyruvate dehydrogenase
    DOI:  https://doi.org/10.1016/j.bbamcr.2025.119929
  4. Nat Cell Biol. 2025 Mar;27(3): 393-407
      Mitochondria are multifaceted organelles with crucial roles in energy generation, cellular signalling and a range of synthesis pathways. The study of mitochondrial biology is complicated by its own small genome, which is matrilineally inherited and not subject to recombination, and present in multiple, possibly different, copies. Recent methodological developments have enabled the analysis of mitochondrial DNA (mtDNA) in large-scale cohorts and highlight the far-reaching impact of mitochondrial genetic variation. Genome-editing techniques have been adapted to target mtDNA, further propelling the functional analysis of mitochondrial genes. Mitochondria are finely tuned signalling hubs, a concept that has been expanded by advances in methodologies for studying the function of mitochondrial proteins and protein complexes. Mitochondrial respiratory complexes are of dual genetic origin, requiring close coordination between mitochondrial and nuclear gene-expression systems (transcription and translation) for proper assembly and function, and recent findings highlight the importance of the mitochondria in this bidirectional signalling.
    DOI:  https://doi.org/10.1038/s41556-025-01625-w
  5. J Cell Sci. 2025 Mar 13. pii: jcs.263693. [Epub ahead of print]
      Mitochondria perform diverse functions, such as producing ATP through oxidative phosphorylation, synthesizing macromolecule precursors, maintaining redox balance, and many others. Given this diversity of functions, we and others have hypothesized that cells maintain specialized subpopulations of mitochondria. To begin addressing this hypothesis, we developed a new dual-purification system to isolate subpopulations of mitochondria for chemical and biochemical analyses. We used APEX2 proximity labeling such that mitochondria were biotinylated based on proximity to another organelle. All mitochondria were isolated by an elutable MitoTag-based affinity precipitation system. Biotinylated mitochondria were then purified using immobilized avidin. We used this system to compare the proteomes of endosome- and lipid droplet-associated mitochondria in U-2 OS cells, which demonstrated that these subpopulations were indistinguishable from one another but were distinct from the global mitochondria proteome. Our results suggest that this purification system could aid in describing subpopulations that contribute to intracellular mitochondrial heterogeneity, and that this heterogeneity might be more substantial than previously imagined.
    Keywords:  Biochemistry; Mitochondria; Proximity Labeling; Purification
    DOI:  https://doi.org/10.1242/jcs.263693
  6. Methods Mol Biol. 2025 ;2906 229-242
      Mitochondria are multifunctional organelles that play a crucial role in numerous cellular processes, including oncogene-induced senescence. Recent studies have demonstrated that mitochondria undergo notable morphological and functional changes during senescence, with mitochondria dysregulation being a critical factor contributing to the induction of this state. To elucidate the intricate and dynamic structure of these organelles, high-resolution visualization techniques are imperative. Electron microscopy offers nanometer-scale resolution images, enabling the comprehensive study of organelles' architecture. This chapter provides a detailed guide for preparing fixed samples from cultured cells for electron microscopy imaging. It also describes various quantification methods to accurately assess organellar parameters, including morphometric measurements of mitochondrial shape, cristae structure, and mitochondria-endoplasmic reticulum contact sites. These analyses yield valuable insights into the status of subcellular organelles, advancing our understanding of their involvement in cellular senescence and disease.
    Keywords:  EM sample preparation; Electron microscopy; MERCs; Mitochondria visualization; Mitochondrial structure
    DOI:  https://doi.org/10.1007/978-1-0716-4426-3_13
  7. Cell Death Discov. 2025 Mar 07. 11(1): 91
      A one-two punch sequential regimen of senescence-inducing agents followed by senolytic drugs has emerged as a novel therapeutic strategy in cancer. Unfortunately, cancer cells undergoing therapy-induced senescence (TIS) vary widely in their sensitivity to senotherapeutics, and companion diagnostics to predict the response of TIS cancer cells to a specific senolytic drug are lacking. Here, we hypothesized that the ability of the BH3 profiling assay to functionally measure the mitochondrial priming state-the proximity to the apoptotic threshold-and the dependencies on pro-survival BCL-2 family proteins can be exploited to inform the sensitivity of TIS cancer cells to BH3-mimetics. Replicative, mitotic, oxidative, and genotoxic forms of TIS were induced in p16-null/p53-proficient, BAX-deficient, and BRCA1-mutant cancer cells using mechanistically distinct TIS-inducing cancer therapeutics, including palbociclib, alisertib, doxorubicin, bleomycin, and olaparib. When the overall state of mitochondrial priming and competence was determined using activator peptides, the expected increase in overall mitochondrial priming was an exception rather than a generalizable feature across TIS phenotypes. A higher level of overall priming paralleled a higher sensitivity of competent TIS cancer cells to BCL-2/BCL-xL- and BCL-xL-targeted inhibitors when comparing TIS phenotypes among themselves. Unexpectedly, however, TIS cancer cells remained equally or even less overally primed than their proliferative counterparts. When sensitizing peptides were used to map dependencies on anti-apoptotic BCL-2 family proteins, competent TIS cancer cells appeared to share a dependency on BCL-xL. Furthermore, regardless of senescence-inducing therapeutic, stable/transient senescence acquisition, or genetic context, all TIS phenotypes shared a variable but significant senolytic response to the BCL-xL-selective BH3 mimetic A1331852. These findings may help to rethink the traditional assumption of the primed apoptotic landscape of TIS cancer cells. BCL-xL is a conserved anti-apoptotic effector of the TIS BCL2/BH3 interactome that can be exploited to maximize the efficacy of "one-two punch" senogenic-senolytic strategies.
    DOI:  https://doi.org/10.1038/s41420-025-02379-y
  8. Biotechnol Bioeng. 2025 Mar 13.
      Cancer-associated fibroblasts are increasingly recognized to have a high impact on prostate tumor growth and drug resistance. Here, we bioengineered organotypic prostate cancer 3D in vitro models to better understand tumor-stroma interplay, the metabolomic profile underlying such interactions, and their impact on standard-of-care therapeutics performance. The assembly of robust and uniform spheroids was evaluated and compared in monotypic PC-3 and heterotypic microtumors comprised of either a healthy or malignant stroma and prostate cancer cells. Our findings demonstrate that the precise inclusion of prostate cancer stromal elements is crucial to generating robust PC-3 prostate cancer spheroids with reproducible morphology and size. The inclusion of cancer-associated fibroblasts promoted the establishment of more compact microtumors exhibiting characteristic expression of major proteins. Exometabolomic profile analysis also highlighted the impact of stromal cells on tumor models metabolism. The optimized heterotypic spheroids were additionally exploited for screening standard-of-care therapeutics, exhibiting a higher resistance when compared to their monotypic counterparts. Our findings demonstrate that including stromal elements in PC-3 prostate cancer models is crucial for their use as increasingly organotypic testing platforms, being relevant for screening candidate anti-cancer therapeutics and for the discovery of potential combinations with emerging anti-stroma therapies.
    Keywords:  3D in vitro models; preclinical screening; prostate cancer; spheroids; tumor–stroma interactions
    DOI:  https://doi.org/10.1002/bit.28971
  9. Anal Chem. 2025 Mar 13.
      Single-cell multi-omics has transformed our understanding of cellular heterogeneity, yet incorporating mitochondrial DNA (mtDNA) remains challenging. Recent studies underscore the critical role of fixation and permeabilization techniques in preserving sample integrity, optimizing Tn5 tagmentation, and retaining mtDNA. In this Perspective, we review the chemical principles underlying fixation and permeabilization methods and highlight new single-cell multiomics technologies leveraging these approaches. We also explore future directions, particularly workflows designed to incorporate mtDNA mutations, enabling simultaneous analysis of mitochondrial genotypes and cellular states. These advances promise to deepen insights derived from single-cell multi-omics, broadening its impact on biological research and clinical applications.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06777
  10. Nat Commun. 2025 Mar 08. 16(1): 2338
      During mitochondrial damage, information is relayed between the mitochondria and nucleus to coordinate precise responses to preserve cellular health. One such pathway is the mitochondrial integrated stress response (mtISR), which is known to be activated by mitochondrial DNA (mtDNA) damage. However, the causal molecular signals responsible for activation of the mtISR remain mostly unknown. A gene often associated with mtDNA mutations/deletions is Polg1, which encodes the mitochondrial DNA Polymerase γ (PolG). Here, we describe an inducible, tissue specific model of PolG mutation, which in muscle specific animals leads to rapid development of mitochondrial dysfunction and muscular degeneration in male animals from ~5 months of age. Detailed molecular profiling demonstrated robust activation of the mtISR in muscles from these animals. This was accompanied by striking alterations to enzymes in the mitochondrial folate cycle that was likely driven by a specific depletion in the folate cycle metabolite 5,10 methenyl-THF, strongly implying imbalanced folate intermediates as a previously unrecognised pathology linking the mtISR and mitochondrial disease.
    DOI:  https://doi.org/10.1038/s41467-025-57299-3
  11. Anal Chem. 2025 Mar 13.
      5-Methylcytosine (5mC) functions as a well-characterized epigenetic DNA mark in nuclear DNA, but its presence in mitochondrial DNA (mtDNA) remains elusive. Here, we report a new and rigorous nuclear DNA (nDNA)-excluding mass spectrometry assay enabling the reliable and accurate identification of 5mC in mtDNA for the first time. First, circular mtDNA is enriched over 809-946-fold by combining alkaline lysis and linear DNA-specific RecBCD cutting; nDNA accounts for ∼12-19% of the DNA remaining after this step. Second, assisted by the restrictive endonucleases BbsI (for human mtDNA) and EcoRV (for mouse mtDNA), circular mtDNA was cut into only one or two linearized mtDNA fragments, while the residual nuclear DNA was efficiently degraded into shorter fragments; thus, the linearized mtDNA fragment(s) could be well isolated from the residual degraded nDNA via gel electrophoresis. Finally, the linearized mtDNA bands are excised and subjected to in-gel digestion followed by precise stable isotope-diluted LC-MS/MS analysis. With this sensitive and accurate method, we demonstrated that mtDNA is hypomethylated in a normal mouse cell line, which is rationally attributed to de novo methylation. Overall, we provide a powerful, gold-standard mass spectrometry assay for screening and identifying mtDNA 5mC in diverse scenarios.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06090
  12. bioRxiv. 2025 Mar 01. pii: 2025.02.26.640389. [Epub ahead of print]
      The de novo purine synthesis pathway is fundamental for nucleic acid production and cellular energetics, yet the role of mitochondrial metabolism in modulating this process remains underexplored. In many cancers, metabolic reprogramming supports rapid proliferation and survival, but the specific contributions of the tricarboxylic acid (TCA) cycle enzymes to nucleotide biosynthesis are not fully understood. Here, we demonstrate that the TCA cycle enzyme succinate dehydrogenase (SDH) is essential for maintaining optimal de novo purine synthesis in normal and cancer cells. Genetic or pharmacological inhibition of SDH markedly attenuates purine synthesis, leading to a significant reduction in cell proliferation. Mechanistically, SDH inhibition causes an accumulation of succinate, which directly impairs the purine biosynthetic pathway. In response, cancer cells compensate by upregulating the purine salvage pathway, a metabolic adaptation that represents a potential therapeutic vulnerability. Notably, co-inhibition of SDH and the purine salvage pathway induces pronounced antiproliferative and antitumoral effects in preclinical models. These findings not only reveal a signaling role for mitochondrial succinate in regulating nucleotide metabolism but also provide a promising therapeutic strategy for targeting metabolic dependencies in cancer.
    DOI:  https://doi.org/10.1101/2025.02.26.640389
  13. Bioorg Chem. 2025 Mar 04. pii: S0045-2068(25)00197-X. [Epub ahead of print]158 108317
      Androgen deprivation therapy (ADT) is currently the primary treatment regime for Prostate cancer patients for advanced and local tumors. However, 70 % of the patients develop resistance to ADT due to various underlying mechanisms over the years. Researchers have identified the involvement of Tousled-like kinase 1 (TLK1) as a primary reason for ADT resistance and metastatic tumor development, representing TLK1 as an effective druggable target for prostate cancer. To date, phenothiazines-which are known antipsychotic drugs, are the only class of inhibitors reported against TLK1. In this study, we focus on developing a new class of TLK1 inhibitors to broaden the spectrum of understanding TLK1 inhibition. As an approach, we designed, synthesized, and validated pyridazinone-fused indole molecules with potent TLK1 inhibition with the concept of ligand-based drug discovery. The inhibition studies and biochemical assays identified a molecule 5n with better inhibition potential than reported J54. Also, the synthesized inhibitors are toxic to androgen-sensitive LNCaP prostate cancer cell lines in sub-micromolar levels and inhibit the TLK1 pathway in cells. Additionally, the combination of anti-androgens and 5n reduces the clonogenicity of cells, causes an accumulation of DNA damage, and induces apoptosis cell death in the LNCaP cells. We anticipate that our step towards exploring a new class of potent TLK1 inhibitors would aid in elevating the therapeutics to existing prostate cancer therapy and provide strong validation for future drug design for more potent and specific TLK1 inhibitors.
    Keywords:  ADT; Apoptosis; Ligand-based drug discovery; mCRPC
    DOI:  https://doi.org/10.1016/j.bioorg.2025.108317
  14. bioRxiv. 2025 Feb 27. pii: 2025.02.26.640157. [Epub ahead of print]
      One of the hallmarks of aging is a decline in the function of mitochondria, which is often accompanied by altered morphology and dynamics. In some cases, these changes may reflect macromolecular damage to mitochondria that occurs with aging and stress, while in other cases they may be part of a programmed, adaptive response. In this study, we report that mitochondria undergo dramatic morphological changes in chronologically aged yeast cells. These changes are characterized by a large, rounded morphology, decreased co-localization of outer membrane and matrix markers, and decreased mitochondrial membrane potential. Notably, these transitions are prevented by pharmacological or genetic interventions that perturb sphingolipid biosynthesis, indicating that sphingolipids are required for these mitochondrial transitions in aging cells. Consistent with these findings, we observe that overexpression of inositol phospholipid phospholipase (Isc1) prevents these alterations to mitochondria morphology in aging cells. We also report that mitochondria exhibit similar sphingolipid-dependent morphological transitions following acute exposure to oxidative stress. These findings suggest that sphingolipid metabolism contributes to mitochondrial remodeling in aging cells and during oxidative stress, perhaps as a result of damaged sphingolipids that localize to mitochondrial membranes. These findings underscore the complex relationship between mitochondria function and sphingolipid metabolism, particularly in the context of aging and stress.
    DOI:  https://doi.org/10.1101/2025.02.26.640157