Cancer-associated fibroblasts promote doxorubicin resistance in triple-negative breast cancer through enhancing ZFP64 histone lactylation to regulate ferroptosis.

["KeJing Zhang", "Lei Guo", "Xin Li", "Yu Hu", "Na Luo"]

Journal of translational medicine 2025

BACKGROUND: Cancer-associated fibroblasts (CAFs) have been identified to drive chemotherapy resistance in triple-negative breast cancer (TNBC). This study evaluated the functions of CAFs-mediated suppressive ferroptosis in doxorubicin (DOX) resistance in TNBC and its detailed molecular mechanisms. METHODS: TNBC cell lines were co-cultured with CAFs isolated from DOX-sensitive (CAF/S) or DOX-resistant (CAF/R) breast cancer tissues. Cell viability and death were assessed by cell counting Kit-8 (CCK-8) and propidium iodide (PI) staining. Ferroptosis was evaluated by detection of Fe2+, malondialdehyde (MDA), glutathione (GSH), and lipid reactive oxygen species (ROS) levels. Histone lactylation was determined by lactate production, pan-Kla and H3K18la expression. Molecular mechanism was determined by chromatin immunoprecipitation (ChIP) and dual luciferase reporter system. Molecule and protein expression was detected by quantitative Real-Time PCR (RT-qPCR), Western blotting, immunofluorescence and immunohistochemical staining. TNBC cells were injected into the mammary fat pad of nude mice to investigate DOX sensitivity in vivo. RESULTS: CAFs-derived lactate repressed ferroptosis to confer resistance of TNBC cells to DOX. Moreover, zinc finger protein 64 (ZFP64) expression was elevated in DOX-resistant TNBC and was associated with high histone lactylation level. CAFs facilitated histone lactylation to enhance ZFP64 expression, which triggered ferroptosis inhibition and DOX resistance. In addition, ZFP64 bound to the promoters of GTP cyclohydrolase-1 (GCH1) and ferritin heavy chain 1 (FTH1), thereby promoting their expression. Rescue experiments indicated that ZFP64 silencing-induced ferroptosis and high sensitivity of TNBC cells to DOX could be counteracted by GCH1 or FTH1 overexpression. CONCLUSION: CAFs acted as a ferroptosis inhibitor to cause DOX resistance of TNBC via histone lactylation-mediated ZFP64 up-regulation and subsequent promotion of GCH1-induced lipid peroxidation inhibition and FTH1-induced intracellular Fe2+ consumption.

6 Figures Extracted

Fig. 1 PMC

Up-regulation of ZFP64 in DOX-resistant TNBC tissues was positively associated with high histone lactylation level. ( A & B ) Pan-Kla level in DOX...

Fig. 2 PMC

ZFP64 expression was increased by CAFs-mediated histone lactylation. ( A & B ) ZFP64 expression was measured by RT-qPCR and Western blotting in TN...

Fig. 3 PMC

ZFP64 knockdown counteracted CAFs-induced DOX insensitivity in TNBC cells. ( A ) CCK-8 determined the viability of DOX-treated TNBC cells co-cultured ...

Fig. 4 PMC

ZFP64 directly bound to the promoters of GCH1 and FTH1 to promote their transcription. ( A ) Expression levels of ferroptosis-related molecules in TNB...

Fig. 5 PMC

FTH1 or GCH1 overexpression abolished shZFP64-mediated promotion in ferroptosis and DOX sensitivity in TNBC cells. ( A - D ) TNBC cells were transfect...

Fig. 6 PMC

CAFs up-regulated ZFP64 to repress ferroptosis, thereby leading to DOX resistance in vivo. BT-549 and MDA-MB-231 cells (5 × 10 6 ) were stably transfe...