Metformin-restricted motility of an NRF2-activated lung cancer cell line involves NAD+ depletion, rather than AMPK- or BACH1 signaling.

NRF2 is a redox-sensitive and cytoprotective transcription factor. Cancer cells exploit mutations in the NRF2-inhibitor KEAP1 and subsequent NRF2 overactivation for increased therapy resistance and malignancy. AMP-activated protein kinase (AMPK) leads to NRF2 phosphorylation and enhanced βTrCP-mediated degradation in KEAP1-deficient contexts. This study examined whether pharmacological activation of AMPK by metformin decreased levels of NRF2 and stress resilience in KEAP1-deficient A549 lung adenocarcinoma cells. Metformin treatment did not alter NRF2 half-life, expression of selected canonical NRF2 target genes, or sensitivity to cisplatin or taxol. Instead, metformin reduced abundance of BACH1, a transcription factor not only competing with NRF2 but also favoring migration of cancer cells. Testing for a potential AMPK-BACH1 motility axis in A549 cells, we observed reduced cell migration in wound closure and transwell assays upon treatment with metformin. However, depletion or overexpression of BACH1, or AMPK knockdown negated that metformin impaired cell migration primarily via BACH1 or AMPK. Metformin, as mitochondrial complex I inhibitor, increased the cellular NADH/NAD+ ratio. Supplementation with nicotinamide mononucleotide fueling NAD+ synthesis restored motility in metformin-treated cells. Thus, metformin administration does not seem to alleviate NRF2 activity but suppresses migration in KEAP1-deficient A549 cells. The latter is linked to NAD+ depletion, rather than AMPK activation or BACH1 downregulation.