Figure 1. Transcriptomic and metabolomic analyses uncover distinct metabolic alterations in doxorubicin and epirubicin resistant breast cancer cells, compared to anthracycline-sensitive Control cells.

(a) Model detailing stepwise generation of Control, DoxR, and EpiR breast cancer cells, as previously described (Hembruff et al., 2008). (b) Viable cell number of anthracycline-resistant cells in the presence of drugs and anthracycline-treated Control cells compared to untreated Control (DMSO). N = 4, ***p<0.001 Control vs Control +Dox, ##p<0.01 ###p<0.001 DoxR vs Control +Dox, †††p<0.001 Control vs Control +Epi, ‡p<0.05 ‡‡‡p<0.001 EpiR vs Control +Epi (two-way ANOVA, Tukey’s multiple comparison test). (c) Viability of anthracycline-resistant cells and treated or untreated (DMSO) Control cells after 7 days growth. N = 4, ***p<0.001 (paired Student’s t-test). (d) Venn diagram of differentially expressed genes in DoxR or EpiR cells compared to Control cells. Legend: green (differentially expressed in DoxR only), red (differentially expressed in EpiR only), and white (differentially expressed in both). N = 3. (e) Principal component analysis of global gene expression profiling between Control, DoxR, and EpiR cells. N = 3. (f) Gene set enrichment analysis (GSEA) of DoxR and EpiR cells compared to Control cells (KEGG database, N = 3). Data are shown as normalized enrichment score (NES) for DoxR and EpiR, where color designates the p-value associated with each enrichment (from yellow at p=0.1 to red at p=0.0001) and where white bars designate non-significant enrichments (n/s, p>0.1). (g) Partial least squares discriminant analysis of metabolite profile data between Control, DoxR, and EpiR cells. N = 6, p=0.008 (1000 permutations). (h) Volcano plot of metabolite profile of DoxR and EpiR cells compared to Control cells (N = 6). Significant features (p<0.05, paired Student’s t-test) highlighted. (i) Integrated metabolic network analysis of DoxR (left) and EpiR (right) cells compared to Control cells. Metabolites are represented by nodes, with p-value represented by node size. Enzymes are represented by edges, with p-value of gene expression represented by edge thickness. Direction and magnitude of fold changes in gene expression and metabolite abundance are represented on a yellow (depleted in resistant) to red (enriched in resistant) color scale. Major pathways are highlighted in shaded areas. (j) Model detailing shRNA screen targeting 1215 drug target genes. Changes in shRNA barcode abundance were measured after 7 days, and viable gene targets were ranked by p-value and fold change of target shRNA abundance. Depleted shRNA were considered cytotoxic or cytostatic to resistant cells in the presence of drug, while enriched barcodes were considered to promote cell proliferation or survival. (k) (left) Top five metabolic pathways identified by Gene Set Enrichment Analysis (KEGG database) of ranked shRNA gene targets depleted in DoxR (top) and EpiR (bottom) cells. (right) GSEA plots detailing enrichment of key metabolic pathways identified in DoxR cells (Glutathione Metabolism, top) or EpiR (Oxidative Phosphorylation, bottom). All data presented as averages ± S.E.M. (b-c).

Figure 1—figure supplement 1. Common and distinct pathways modulated in DoxR and EpiR cells compared to Control cells.

(a) Viable cell counts in DoxR and EpiR cells treated for 3 days with 98.1 nM doxorubicin or 852 nM epirubicin respectively, compared to DoxR and EpiR cells treated with DMSO control, after a 7-week (49 days) drug holiday. N = 3. Data presented as averages ± S.E.M. (b) Overlap of differentially expressed genes in DoxR (compared to Control) cells and patient biopsies post-chemotherapy (compared to pre-chemotherapy). Overlap size is drawn as a step function over respective ranks. Top ranks correspond to upregulated and Bottom ranks to downregulated genes. The yellow lines represent the expected overlap ±95% empirical confidence intervals derived by random shuffling strategy. (c) Overlap of differentially expressed genes in EpiR (compared to Control) cells and patient biopsies post-chemotherapy (compared to pre-chemotherapy), as in (b). (d) Gene Set Enrichment Analysis (GSEA) plots of over-represented and under-represented KEGG pathways in global gene expression of DoxR and EpiR cells compared to Control cells. (e) Fold change in abundance of shRNA targeting genes of the glutathione metabolism, oxidative phosphorylation, and methionine metabolism pathways, after 7 days in Control, DoxR, or EpiR cells. *p<0.05 **p<0.01 ***p<0.001 day 7 vs day 0 (MAGeCK analysis). Data are shown as box plots representing the average of three to seven independent shRNA barcodes per gene target ± S.E.M.