The Differential Metabolic Signature of Breast Cancer Cellular Response to Olaparib Treatment

Abstract

Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line-dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose-dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations.

Keywords: DNA damage; breast cancer; metabolic reprogramming; oncometabolites; precision medicine; triple-negative.

Figure 1

Corresponding MTS dose–response curves for MCF7, HCC1937, and MDA-MB-231 cells treated with ascending doses of olaparib (0.1–500 µM) for seven days. The corresponding R² values for fitted dose–response curves in MCF7 (IC₅₀ = 10 µM), MDA-MB-231 (IC₅₀ = 14 µM), and HCC1937 (IC₅₀ = 150 µM) cells were 0.89, 0.91, and 0.85, respectively.

Figure 2

The formation of p53BP1 foci in response to treatment with either growth medium or medium containing olaparib at the IC₅₀ dose. Representative images of immunolabelled P53BP1 foci (red), DAPI (blue) nuclear counterstain and composite (p53BP1 (red) and DAPI (blue)) in MCF-7, MDA-MB-231, and HCC1937 cells treated with olaparib for seven days (a,c,e). Corresponding p53BP1 foci counts determined using Cell Profiler (b,d,f). 9 repeats with, on average, >100 cells per each sample. p-values have been determined through ANOVA test. Dunnett’s multiple comparison test was used as a follow up to ANOVA test and the p-values were represented as: ns, non-significant; *, 0.05; **, 0.005; ****, >0.00005.

Figure 3

The formation of γH2AX foci formation in response to treatment with either growth medium or medium containing olaparib at the IC₅₀ dose. Representative images of immunolabelled γH2AX foci (green), DAPI (blue) nuclear counterstain and composite (γH2AX and DAPI) in MCF-7, MDA-MB-231, and HCC1937 cells treated with for seven days (a,c,e). Corresponding γH2AX foci counts determined using Cell Profiler (b,d,f). (>100 cells per sample). Dunnett’s multiple comparison test was used as a follow up to ANOVA and corresponding p-values were represented as: ns, non-significant; **, 0.005; ****, >0.00005.

Figure 4

Statistical analyses of global metabolic features identified in MCF7, MDA-MB-231, and HCC1937 upon exposure to the IC₅₀ olaparib dose for seven days acquired in positive and negative ionization mode. For each treatment group, five replicates were used. Data points in the two-dimensional PCA score plot were central scaled. (a) PCA pairwise analysis and differential analysis of metabolites altered in IC₅₀-treated cells, (b) Volcano plots displaying enriched (blue) and depleted (grey) metabolic features by representing the log2 fold change in altered features and the −log10 adjusted p-values with cut off values selected at >1.5 and <0.05, respectively. Upward arrows represent enrichment of features, while downward arrows represent depleted features.

Figure 5

Pathway enrichment analysis of MCF7 (10 µM), MDA-MB-231 (14 µM), and HCC1937 (150 µM) cells following a seven-day exposure to olaparib. Enrichment analysis was based on the hypergeometric test. Topological analysis was based on betweenness centrality. The tight integration method was used by combining genes and metabolites into a single query. A p < 0.05, and pathway impact >0.1 were deemed significant.

Figure 6

Heatmap cluster analysis of relevant metabolites associated with the pathways altered upon exposure to olaparib in MCF7 (10 µM), MDA-MB-231 (14 µM), and HCC1937 (150 µM) cells for seven days. Clustering and distance function are Ward and Euclidean, respectively. Normalized areas indicate chromatographic peaks areas that have been normalized based on the QC samples to compensate for batch effects.

Figure 7

A summary of putatively identified metabolic pathways altered in response to olaparib exposure at IC₅₀ doses. Significantly altered features with a Log2 fold change of >1.5 (blue-enriched and grey-depleted). Fitness effect score of metabolic enzymes (light-blue boxes) in relation to PARP expression in each cell line. Positive and negative scores are in green and red, respectively. MCF-7 (), MDA-MB-231 (), and HCC1937 (). Fitness effect score is based on the Chronos algorithm.