Analyses of amino acids metabolic patterns in trastuzumab primary resistant and sensitive HER2 positive breast cancer patients. (A) Workflow of analyses performed in this study. (B) Volcano plot of different circulating metabolites in the comparison between trastuzumab primary resistant and sensitive patients. (C) Enrichment analysis of circulating metabolites downregulated in trastuzumab primary resistant patients. (D) Enrichment analysis of circulating metabolites upregulated in trastuzumab primary resistant patients. (E-F) Volcano plot (E) and violin plots (F) of different circulating protein-construction amino acids in the comparison between trastuzumab primary resistant and sensitive patients. (G) Volcano plot of different genes between non-pCR and pCR patients in trastuzumab-based neoadjuvant treatment (I-SPY2, GSE181574). (H) Amino acids metabolic pathway analysis of non-pCR and pCR patients. (I) Relations between cysteine metabolic genes and trastuzumab treatment outcomes.

Heterogeneity of cysteine metabolism characteristics in HER2 positive breast cancer with different trastuzumab response. (A) Volcano plot of hydrophilic metabolites in JIMT1 and SKBR3. (B) Upregulated metabolic processes in JIMT1 based on enrichment analysis of metabolites. (C) Downregulated metabolic processes in JIMT1 based on enrichment analysis of metabolites. (D) Different metabolic activities between JIMT1 and SKBR3 based on transcriptomics data. (E) Joint pathway analysis based on metabolomics and transcriptomics data. (F) Map of essential genes involved with cystine/cysteine metabolism. Upregulated genes in JIMT1 are highlighted in red and downregulated genes in JIMT1 are highlighted in blue. (G) Expression of essential proteins participating in cysteine and glutathione metabolism. (H) The comparison of cystine uptake ability between JIMT1 and SKBR3. (I) The comparison of intracellular cysteine abundance between JIMT1 and SKBR3. (J) The comparison of GSH:GSSG ratio between JIMT1 and SKBR3.

Ferroptosis sensitivity of HER2 positive breast cancer with different trastuzumab response. (A-B) Knockdown of SLC7A11 (A) and GPX4 (B) induced increased lipid peroxidation (BODIPY-C11) in JIMT1. (C-D) Treatment with erastin, RSL3 and cysteine starvation for 12 hours induced lipid peroxidation (BODIPY-C11) in JIMT1 (C) and SKBR3 (D). (E-F) JIMT1 and SKBR3 featured different lipid peroxidation sensitivity to erastin by measuring BODIPY-C11 (E) and DCFH-DA (F). (G-H) JIMT1 and SKBR3 featured different lipid peroxidation sensitivity to RSL3 (G) and cysteine starvation (H). (I) SLC7A11 expression under different trastuzumab treatment time. (J) Cystine uptake ability under different trastuzumab treatment time. (K) The combination of erastin or cysteine starvation and trastuzumab increased lipid peroxidation in JIMT1. H, trastuzumab; Cys, cysteine starvation.

Targeting cysteine metabolism synergizes with trastuzumab to induce ferroptosis. (A) Schematic outline of treatment on cysteine metabolism in trastuzumab resistant breast cancer. JIMT1 cells were injected into the mammary fat pads of BALB/c nude mice at day -7. Treatment with DMSO, erastin, cysteine starvation, trastuzumab combined with or without erastin and cysteine starvation started at day 0 for 21 days. Tumor volume and mice weight were measured. (B) Tumor of different treatment groups. (C) Fold change of tumor volume in different treatment groups. (D) Mice weight of different treatment groups. (E-H) Abundance of CD49b+ (E), NKp46+ (F), Granzyme B+ (G) and different development status (H) tumor infiltrating NK cells. (I) Representing immunohistochemistry images of SLC7A11, GPX4, MDA and 4-HNE in different treatment groups. Scale bar, 50 μm. H, trastuzumab; Cys, cysteine starvation.

Increased H3K4me3 modifies SLC7A11 transcription and regulated cystine uptake. (A-B) Alterations of H3K4me3 (A) and H3K27me3 (B) peaks between JIMT1 and SKBR3. (C) Different abundance of H3K4me3 and H3K27me3 at SLC7A11 promoter regions in JIMT1 and SKBR3. (D) Decreased ASH2L inhibited total H3K4me3 expression. (E) Decreased ASH2L inhibited SLC7A11 expression. (F) Decreased ASH2L suppressed H3K4me3 expression at SLC7A11 promoter regions. (G) Decreased ASH2L suppressed cystine uptake ability. (H) Decreased ASH2L induced lipid peroxidation and ferroptosis.

Decreased 5-mC modifications on SLC7A11 promoter relate with enhanced cystine uptake. (A) Alterations of 5-mC peaks between JIMT1 and SKBR3. (B) Different abundance of 5-mC at SLC7A11 promoter regions in JIMT1 and SKBR3. (C) Schematic outline of CRISPR-based epigenetic editing. dCas9-DNMT3A increased methylation of specific CpG islands in SLC7A11 promoter regions and inhibited gene transcription. (D) dCas9-DNMT3A increased 5-mC levels at SLC7A11 promoter regions. (E) dCas9-DNMT3A inhibited SLC7A11 expression. (F) dCas9-DNMT3A suppressed cystine uptake ability. (G) dCas9-DNMT3A induced lipid peroxidation and ferroptosis.

Summary of targeting cysteine metabolism in trastuzumab resistant HER2 positive breast cancer.