Tatsunori Nishimura

One-carbon (1C) metabolism is frequently upregulated in cancer to support anabolic growth and nucleotide biosynthesis. However, the contribution of mitochondrial 1C metabolism to cancer stemness and metastatic progression remains incompletely understood. Here, we identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), a mitochondrial 1C metabolic enzyme, as a critical regulator of breast cancer stemness, tumor initiation, and lung metastasis. Genetic depletion of MTHFD1L in triple-negative breast cancer (TNBC) cell lines and patient-derived breast cancer models markedly impaired proliferation, sphere formation, tumorigenicity, and lung colonization. Metabolomic profiling revealed extensive metabolic rewiring following MTHFD1L loss, characterized by accumulation of the purine biosynthetic intermediates SAICAR and AICAR together with perturbations in glycolytic and pentose phosphate pathways. Importantly, suppression of lung metastasis was accompanied by reduced expression of the stemness-associated transcription factor SOX2 and decreased proliferative activity in metastatic lesions. Collectively, our findings establish MTHFD1L as a key metabolic dependency linking mitochondrial 1C metabolism to stem-like properties and metastatic progression in breast cancer, and highlight MTHFD1L as a promising therapeutic target in metastatic TNBC.

The heterogeneity of cancer stem cells (CSCs) within tumors presents a challenge in therapeutic targeting. To decipher the cellular plasticity that fuels phenotypic heterogeneity, we undertook single-cell transcriptomics analysis in triple-negative breast cancer (TNBC) to identify subpopulations in CSCs. We found a subpopulation of CSCs with ancestral features that is marked by FXYD domain–containing ion transport regulator 3 (FXYD3), a component of the Na⁺/K⁺ pump. Accordingly, FXYD3⁺ CSCs evolve and proliferate, while displaying traits of alveolar progenitors that are normally induced during pregnancy. Clinically, FXYD3⁺ CSCs were persistent during neoadjuvant chemotherapy, hence linking them to drug-tolerant persisters (DTPs) and identifying them as crucial therapeutic targets. Importantly, FXYD3⁺ CSCs were sensitive to senolytic Na⁺/K⁺ pump inhibitors, such as cardiac glycosides. Together, our data indicate that FXYD3⁺ CSCs with ancestral features are drivers of plasticity and chemoresistance in TNBC. Targeting the Na⁺/K⁺ pump could be an effective strategy to eliminate CSCs with ancestral and DTP features that could improve TNBC prognosis.