Tumor-educated macrophages promote ferroptosis inhibition in pancreatic cancer via activating the p62-KEAP1-NRF2 pathway

Background Pancreatic cancer is one of the most aggressive and lethal gastrointestinal malignancies, and ferroptosis resistance contributes to chemoresistance. Tumor-educated macrophages(TAMs) are key components of the tumor microenvironment, but whether and how they regulate ferroptosis in pancreatic cancer cells remains largely unknown. This study aimed to investigate the regulatory role of tumor-educated macrophages (TEMs) in pancreatic cancer cell ferroptosis and the underlying molecular mechanism. Methods TEMs were generated by coculturing THP-1 cells with the pancreatic cancer cell lines PANC1 and CFPAC, and TEM-conditioned medium (TEM-CM) was collected to treat pancreatic cancer cells. Ferroptosis was induced by erastin, and ferroptotic events were assessed by lipid ROS, MDA, iron accumulation, GSH levels, and transmission electron microscopy. RNA sequencing was performed to identify differentially expressed genes. The expression and activity of VEGFR2, C-Raf, ERK1/2, mTOR, LC3, p62, KEAP1, and NRF2 were investigated by treating cells with pharmacological inhibitors (Ki8751, LY3009120, chloroquine, and ferrostatin-1), performing gene knockdown and overexpression, and conducting Western blot co-immunoprecipitation, ubiquitination, and cycloheximide chase assays. In vivo experiments were conducted using an orthotopic mouse model of pancreatic cancer (n = 5 per group). Results TEM-CM reduced the sensitivity of PANC1 and CFPAC cells to erastin-induced ferroptosis, as evidenced by decreased lipid ROS, MDA, and iron accumulation and increased GSH levels. Neutralization of VEGF, but not IL-10 or TGFβ, significantly reversed the ferroptosis-inhibiting effect of TEM-CM. RNA sequencing identified TM7SF2 as a key mediator. TEM-CM upregulated TM7SF2 mRNA and protein levels, activated mTOR, and suppressed autophagy, leading to p62 accumulation. Accumulated p62 competitively bound KEAP1, disrupted the KEAP1–NRF2 interaction, inhibited NRF2 ubiquitination and degradation, and promoted NRF2 nuclear translocation, thereby upregulating GPX4, HMOX1, and GCLM. Mechanistically, TEM-derived VEGF bound VEGFR2 and activated the C-Raf/ERK1/2 pathway by upregulating TM7SF2, which in turn promoted mTOR phosphorylation. These effects were reversed by TM7SF2 knockdown, VEGF neutralization, VEGFR2 inhibition, or C-Raf inhibition. Conclusions These findings suggest that TEM-derived VEGF promotes ferroptosis resistance in pancreatic cancer cells through the VEGFR2–TM7SF2–C-Raf/ERK1/2–mTOR–autophagy–p62–NRF2 axis. This axis may represent a potential therapeutic target for overcoming ferroptosis resistance in pancreatic cancer. However, our conclusions are mainly based on in vitro cell models, and further validation in additional cell lines, patient-derived organoids, and in vivo models is warranted.