Mitophagy-Competent Cancer-Associated Fibroblasts Fuel Chemoresistance by Rewiring Pyrimidine Metabolism in Pancreatic Cancer

Pancreatic cancer remains one of the deadliest malignancies, with gemcitabine-based chemotherapy as the mainstay treatment for most patients, yet resistance emerges almost universally. A defining feature of pancreatic cancer is its dense, fibroblast-rich stroma, in which heterogeneous cancer-associated fibroblasts (CAF) actively shape tumor biology and therapeutic response. Here, we elucidated a stromal-metabolic mechanism through which chemoresistant CAFs confer gemcitabine resistance. We identified a subset of mitophagy-competent CAFs that enhanced pancreatic cancer gemcitabine resistance. The epithelial-mesenchymal transition transcription factor ZEB1 acts as a master regulator of this CAF-driven chemoresistance program, being upregulated and epigenetically activated via SETD1A-mediated H3K4 methylation in gemcitabine-resistant CAFs. ZEB1 promotes BNIP3-mediated mitophagy in CAFs, leading to increased nucleotides secretion, which competitively inhibited gemcitabine incorporation into cancer cells while simultaneously supplying pyrimidine metabolism substrates for pyrimidine metabolism. Concurrently, ZEB1 transcriptionally activated CXCL8, engaging the CXCR1/2-MEK/ERK pathway in tumor cells and further augmenting pyrimidine metabolism via the RRM1/E2F1/G6PD axis, collectively diminishing gemcitabine cytotoxicity. Notably, combined inhibition of CXCR1/2 or G6PD with gemcitabine robustly suppressed tumor growth and restored chemosensitivity both in vitro and in vivo. These findings uncover a key stromal-metabolic axis in pancreatic cancer, linking mitophagy CAF activity to metabolic remodeling in tumor cells and identifying ZEB1 and its downstream network as actionable targets to overcome chemoresistance. SIGNIFICANCE: ZEB1 reprograms mitophagy-competent CAFs to promote gemcitabine resistance by enhancing nucleotide supply and activating tumor pyrimidine metabolism, supporting therapeutic targeting of the ZEB1-CXCL8-pyrimidine axis in pancreatic cancer.