COPD is the third leading cause of death worldwide, a progressive disease in which chronic inflammation and tissue damage produce irreversible airflow limitation. Cigarette smoke, the dominant risk factor, contains more than 7,000 compounds, including reactive oxygen species such as superoxide and hydroxyl radicals. Although the gaseous phase of smoke cannot easily cross cell membranes, the lipophilic components captured in cigarette smoke extract, including aldehydes and polycyclic aromatic hydrocarbons, can penetrate cells and reduce mitochondrial membrane potential and ATP production. The resulting damage activates immune cells, amplifies lung tissue destruction, and promotes COPD in susceptible individuals. Yet not all smokers develop the disease, and mounting evidence points to epigenetic mechanisms, including DNA methylation, as key players in determining who succumbs.

Mitochondria carry their own small genome, separate from the DNA housed in the nucleus, and this circular genome lacks the protective histone proteins that organize nuclear DNA. That absence has led researchers to focus on methylation of the mitochondrial DNA itself as a potential regulatory layer. Mitochondrial gene expression changes have already been documented in smokers: a comparative analysis found that 32 of 37 mitochondrial genes were upregulated in smokers with normal lung function, and lung epithelial cells from smokers show increased mitochondrial DNA copy number correlated with nuclear DNA methylation. Mitochondrial DNA methylation differences have also been reported in aging, metabolic disorders, neurodegeneration, and cardiovascular disease, making the mitochondrial epigenome an attractive suspect in COPD pathogenesis.

To test that suspicion, the Groningen team, led by Lin Liang and Irene H. Heijink, exposed human bronchial epithelial BEAS-2B cells to gradually increasing concentrations of cigarette smoke extract over periods ranging from four weeks to 25 weeks. Using Seahorse extracellular flux analysis, they measured the oxygen consumption rate, the gold-standard readout of mitochondrial respiration. After ten weeks of exposure reaching 5% smoke extract, the cells displayed a striking loss of respiratory capacity: basal respiration, ATP-linked respiration, and maximal respiration all fell significantly compared with unexposed controls, confirming that prolonged smoke exposure genuinely cripples mitochondrial energy production.

The damaged mitochondria were accompanied by a transcriptional response. Expression of the protein-coding mitochondrial genes MT-CYB and MT-CO2, which encode components of respiratory chain complexes III and IV, rose significantly in cells exposed to 5% and 6% smoke extract, along with the rRNA gene MT-RNR1 at the 5% time point. Because mitochondrial DNA copy number remained stable throughout the 25-week exposure, the researchers interpret this upregulation as a compensatory boost in transcriptional activity as the cells struggle to maintain energy output. Intriguingly, the elevation vanished by the 25-week mark at 10% smoke extract, suggesting that once dysfunction becomes severe enough, the compensatory capacity of mitochondrial gene regulation is itself overwhelmed.

The critical question was whether these changes were written into the mitochondrial epigenome. Using pyrosequencing targeted to the D-loop, the non-coding region harboring the mitochondrial promoters, and to the MT-CYB and MT-CO2 genes, the team measured methylation at individual cytosines, including the non-CpG sites that can be methylated in mitochondria. They complemented this with liquid chromatography tandem mass spectrometry to quantify global mitochondrial DNA methylation. The result was resoundingly negative: despite dramatic functional impairment and gene expression shifts, methylation in the long-term exposed BEAS-2B cells showed no statistically significant changes at any assessed site or across the whole mitochondrial genome. Some cytosines shifted consistently, but by less than 1%, a magnitude the authors consider biologically negligible.

Short-term exposure told a similar story. A 24-hour bath in 10% smoke extract triggered a massive surge in the oxidative stress marker HMOX1, upregulated more than 200-fold, and significantly reduced mitochondrial DNA copy number in BEAS-2B cells, possibly reflecting smoke-damaged mitochondria being cleared by mitophagy. Yet mitochondrial gene expression and methylation held steady. In the 16HBE bronchial epithelial cell line, which more faithfully mirrors primary airway cells, neither copy number nor gene expression budged after 24-hour exposures, although mean methylation of MT-CYB and MT-CO2 rose modestly at the 15% dose, hinting at a dose-dependent and cell-type-specific response that disappeared at 20%, where cellular tolerance may be exceeded.

The team then extended the work to primary airway epithelial cells isolated from six COPD patients, all with severe GOLD stage IV disease, and six non-COPD donors. Here the findings were equally unambiguous: mitochondrial gene expression, copy number, and methylation were indistinguishable between COPD and control cells, and 24-hour smoke extract exposure failed to alter any of these measures in either group. The authors caution that the modest donor numbers and the lack of information on the control donors’ sex, age, and smoking status could obscure subtler differences, and that the submerged culture conditions used here cannot capture the effects of the gaseous phase of smoke, which air-liquid interface cultures would expose.

Taken together, the study delivers a sobering message for the mitochondrial epigenetics field: mitochondrial DNA methylation appears not to be a dominant driver of the pathological changes that cigarette smoke inflicts on airway epithelium. The smoke-induced mitochondrial dysfunction seen in COPD likely arises through other mechanisms, while mild shifts in mitochondrial gene expression may be consequences rather than causes. The authors point toward promising future directions, including the emerging role of N6-methyladenine, a different DNA modification shown to regulate mitochondrial transcription and replication and to accumulate in mitochondrial DNA during aging across species. As researchers refine methods for measuring mitochondrial methylation, accounting for artifacts such as incomplete bisulfite conversion, the Groningen results provide a rigorous negative control that will help steer COPD research toward the molecular mechanisms that truly matter.

Subject of Research: Effects of cigarette smoke extract on mitochondrial function, gene expression, and mitochondrial DNA methylation in airway epithelial cells in relation to COPD

Article Title: The effects of cigarette smoke extract on mitochondrial function, mitochondrial gene expression and mitochondrial DNA methylation in airway epithelial cells

Article References: Liang, L., Wang, L., Jonker, M. R., Kosse, W., Jellema, P. G., Rots, M. G., & Heijink, I. H. (2025). The effects of cigarette smoke extract on mitochondrial function, mitochondrial gene expression and mitochondrial DNA methylation in airway epithelial cells. Epigenetics Communications, 6(1), Article 1. https://doi.org/10.1186/s43682-025-00040-4

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00040-4

Keywords: mitochondrial DNA methylation, mitochondrial dysfunction, COPD, cigarette smoke extract, airway epithelial cells, mitochondrial gene expression, epigenetics, D-loop, oxygen consumption rate, oxidative stress, MT-CYB, MT-CO2

The logic of the field has shifted because metabolites themselves turn out to be messengers. S-adenosylmethionine, or SAM, a product of one-carbon metabolism, serves as the methyl donor for chemical marks placed on mitochondrial DNA, including 5-methylcytosine and the bacteria-like N6-methyladenine. The methyltransferase METTL4 installs 6-methyladenine on mitochondrial DNA, which disrupts binding of the transcription factor TFAM and suppresses mitochondrial transcription. Hypoxia strongly induces METTL4 in liver and breast cancer cells, throttling mitochondrial metabolism precisely when tumors want it restrained. Methylation of the mitochondrial genome appears to protect it from oxidative damage, and DNA methyltransferases DNMT3A and DNMT3B mediate this in embryonic cells — enzymes whose aberrant expression or mutation drives cancer initiation. The picture is complicated, however: the true abundance of 5-methylcytosine on mitochondrial DNA remains debated, with optimized protocols reporting levels of only 0.19 to 0.67 percent in mouse oocytes and human lung carcinoma cells, and technical artifacts such as inefficient bisulfite conversion and mis-mapping of nuclear-integrated mitochondrial sequences continuing to muddy clinical analyses.

The replication machinery itself is equally metabolically entangled. DNA polymerase gamma, POLG, together with its accessory subunit POLG2, copies the mitochondrial genome, and its overactivation has been linked to elevated risk of prostate cancer and acute myeloid leukemia. Proteomic surveys have revealed a dense landscape of post-translational modifications on POLG and POLG2 that couple cellular fuel state to genome maintenance. The acetyltransferase GCN5 acetylates POLG at lysine 1039 using acetyl-CoA as donor, while the NAD+-dependent deacetylase SIRT3 reverses the mark — a push-pull system that reads acetyl-CoA and NAD+ availability directly. In parallel, poly(ADP-ribose) polymerase 1 uses NAD+ to attach ADP-ribose groups to POLG, triggering ubiquitination and degradation. Through these routes, the cell’s energy currency and redox state literally decide how much replication enzyme survives, and cancer cells exploit these metabolite-sensing nodes to rewire mitochondrial function to their advantage.

Transcription is governed by the same logic. The mitochondrial RNA polymerase POLRMT is succinylated at lysine 622, a modification that impairs its DNA-binding activity. The metabolic enzyme SUCLG1, part of the succinyl-CoA ligase complex, restricts mitochondrial succinyl-CoA levels to keep POLRMT hypo-succinylated, sustaining mitochondrial respiration and driving leukemia progression. Succinyl-CoA thus functions as a key metabolic signal bridging mitochondrial metabolism and transcription in both leukemia and solid tumors. TFAM, the packaging factor that recruits POLRMT to promoters, is itself phosphorylated, acetylated, and — most recently — arginine-methylated at position 82 by PRMT5, a modification essential for DNA binding and mitochondrial genome integrity. Loss of PRMT5 leaves breast carcinoma cells vulnerable to mitochondrial DNA damage and oxidative stress, and several PRMT5 inhibitors are already in clinical trials, raising the prospect of attacking tumor bioenergetics through the transcription apparatus itself.

Downstream, RNA processing and ribosome assembly consume enormous metabolic resources. The 12S and 16S ribosomal RNAs of the mitoribosome are studded with SAM-dependent methylation marks: TFB1M dimethylates the 3′ end of 12S rRNA to stabilize the small subunit, NSUN4 methylates cytosine 911, METTL15 installs N4-methylcytidine at position 839, and TRMT61B places N1-methyladenosine at position 947 of 16S rRNA, a modification conserved across all vertebrates that stabilizes local ribosome structure. Strikingly, when SAM is depleted, unprocessed pre-rRNA accumulates and mitoribosome assembly grinds to a halt. The same SAM pool feeds transfer RNA modifications, including the taurine-and-GTP-dependent wobble modification 5-taurinomethyluridine installed by GTPBP3 and MTO1, which is essential for translation fidelity — and, notably, MTO1 upregulation in melanoma sustains oxidative phosphorylation and confers resistance to vemurafenib. The dioxygenase ALKBH7, which depends on alpha-ketoglutarate and oxygen to erase methylation marks that otherwise delay transfer RNA maturation, adds another metabolite-gated checkpoint to the pipeline.

At the level of protein synthesis, the review highlights an increasingly granular view of mitoribosome regulation. Human mitoribosomes contain three iron-sulfur clusters installed by GLRX5 and BOLA3, enzymes with prognostic value in bladder cancer and lung adenocarcinoma, while METTL17 carries a [4Fe-4S] cluster that confers redox-sensing activity to protein synthesis. Lipid metabolism matters too: cardiolipin, enriched in cristae membranes where mitochondrial protein synthesis is spatially confined, promotes the inner-membrane docking of ribosomes, and the remodeling enzyme tafazzin maintains the membrane curvature this requires. Perhaps the most striking recent discovery is lactylation — the attachment of lactate-derived lactyl groups to proteins — now documented on TFAM and multiple ribosomal proteins in clinical tumor tissues, and linked to chemoresistance and metastasis in breast, kidney and lung cancers. Lactate, once dismissed as waste, is actively consumed by mitochondria as fuel and now appears to write epigenetic marks on the very machinery that builds the respiratory chain.

The mitochondrial genome also hides a second layer of coding information. Beyond its thirteen canonical proteins, it produces microproteins with potent biological activity. Humanin, a 24-amino-acid peptide, suppresses apoptosis by interfering with Bax activation; it is elevated in the serum of breast cancer patients and accelerates tumor growth, lung metastasis, glioblastoma invasion and temozolomide resistance in experimental models. MOTS-c, a 16-amino-acid peptide from the 12S rRNA open reading frame, activates AMPK and shows context-dependent effects — circulating levels rise in prostatic precancerous lesions and hepatocellular carcinoma yet fall in adrenal tumors, while in liver cancer MOTS-c promotes apoptosis under hypoxia and suppresses tumor growth. Additional peptides such as the SHLP family, SHMOOSE and CYTB-187AA underscore that the mitochondrial central dogma generates unanticipated regulators whose roles in cancer are only beginning to be mapped.

The translational payoff is substantial. The allosteric POLRMT inhibitor IMT1B blocks mitochondrial transcription with minimal toxicity in normal tissues and suppresses leukemia, endometrial carcinoma and other tumors in preclinical models. The thymidine analog alovudine inhibits POLG, impairing oxidative phosphorylation in acute myeloid leukemia, while small-molecule activators such as PZL-A allosterically restore mutant polymerase activity. FDA-approved antibiotics quinupristin and dalfopristin inhibit mitochondrial translation and reduce glioblastoma stem cell clonogenicity, and linezolid suppresses T cell metabolism through the same route, with hematological toxicities limiting its use. Inhibition of dihydroorotate dehydrogenase by ML390 starves leukemia cells of pyrimidines needed for mitochondrial biogenesis, and the iron chelator deferoxamine disrupts OXPHOS super-complexes in breast cancer with encouraging selectivity. Genome-wide sequencing has also revealed that colorectal cancer mitochondrial DNA mutations carry prognostic weight, with MT-CYB mutations significantly correlating with poor patient survival.

Perhaps the most futuristic front is mitochondrial genome editing itself. DddA-derived cytosine base editors and TALE-linked deaminases now allow precise CG-to-TA and A-to-G conversions in mitochondrial DNA, and adeno-associated virus vectors have delivered these editors to post-mitotic mouse tissue, while engineered mitoARCUS nucleases have cleared mutant mitochondrial DNA systemically in animal models. Off-target nuclear editing — reported at rates up to 17.5 percent at some sites — and unintended heteroplasmy shifts remain serious concerns, spurring strategies such as nuclear export signals, strand-selective editors like CyDENT, and tumor-specific promoters to restrict editing to cancer cells. The review’s authors close with a call to integrate high-resolution structural biology, single-cell metabolomics and in vivo lineage tracing to decode the spatiotemporal choreography of mitochondrial gene expression. If they are right, the organelle’s second genome — long overshadowed by the nucleus — may prove to be one of the most exploitable weaknesses a tumor carries.

evolutionary backdrop helps explain why this control system is so intricate. Mitochondria descend from a free-living prokaryotic ancestor engulfed more than 1.45 billion years ago, and the long history of metabolic trade-offs with the host cell left the organelle semi-autonomous: most of its roughly 1,500 proteins are nuclear-encoded, yet the thirteen genes retained in the compact circular genome encode core respiratory chain subunits whose expression must be tuned to local energy demand. Because mtDNA copy number varies naturally across tissues, its dysregulation is directly tied to cancer risk, and tumor cells dynamically adjust both genome abundance and gene output to match proliferative and metastatic needs.

Structural organization adds another layer of control. The mitochondrial genome packs into nucleoids within the matrix, with most protein- and rRNA-coding regions flanked by transfer RNA genes, and regulatory marks such as 5-methylcytosine concentrate in the displacement loop, the non-coding region where replication and transcription originate. Recent structural work has also complicated the classic picture of the replicase: POLG2 can contact DNA independently of POLG without altering polymerase activity, hinting at versatile functions beyond processive elongation.

Methodological caveats deserve emphasis for readers weighing the evidence. Detecting low-frequency mitochondrial modifications demands careful technique; antibody-based capture, bisulfite conversion, isotope labelling, HPLC–mass spectrometry and next-generation sequencing each carry distinct biases, and incomplete light-strand coverage can distort tumor measurements. As optimised protocols spread and quantitative data accumulate across tumor types, the field should be better placed to separate genuine metabolic regulation from technical noise, sharpening the therapeutic opportunities this review maps out.

Subject of Research: Metabolic regulation of the mitochondrial central dogma in cancer

Article Title: Exploring mitochondrial central dogma in cancer from a metabolic perspective

Article References: Zhang, S., Han, B., Chen, L., Chen, L., Wang, T., Lin, M., Liu, J., & Wang, Y. (2026). Exploring mitochondrial central dogma in cancer from a metabolic perspective. Clinical Cancer Bulletin, 5(1), Article 19. https://doi.org/10.1007/s44272-026-00071-5

Image Credits: AI Generated

DOI: 10.1007/s44272-026-00071-5

Keywords: mitochondria, mitochondrial DNA, mtDNA replication, POLRMT, TFAM, mitoribosome, RNA modifications, lactylation, mitochondrial translation, humanin, MOTS-c, mitochondrial genome editing