Molecular Atlas of HER2+ Breast Cancer Cells Stimulated with EGF and HRG: Temporal Insights into Mechanisms in Trastuzumab Resistance [ATAC-Seq]
Trastuzumab therapy in HER2+ breast cancer patients have mixed success owing to acquired resistance to therapy. In this study, we investigate the cellular mechanisms underlying acquired resistance using -sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG, across time. Our measurements probe early receptor organization through microscopy, signaling events through multi-omic measurements and assess the cellular energetic state through mitochondrial measurements. Our integrative analyses of these multimodal measurements highlight differential mechanisms in both cell lines in response to ligands. In BT474, an active PI3K-AKT-mTORC1 signaling contributes to an active mitochondrial bioenergetic state (glycolysis and lipid metabolism) for both ligands EGF and HRG. A HIF1A mediated increase in cellular bioenergetics is also seen. In BT474R, there is a ligand-dependent activation of signaling cascades. In EGF treated BT474R, an EGFR driven IRF1/STAT1/STAT2 activation with likely impact on cellular bioenergetics is seen, whilst an AR mediated alteration of lipid metabolism is pronounced after HRG treatment. Overall design: We performed qSMLM, RPPA, RNA and ATAC sequencing to dynamically interrogate the cell state as defined by HER2 receptor localization, downstream protein signaling, its transcriptomic landscape and the accessibility of chromatin in response to ligands in BT474 and BT474R cultured cell lines. To capture the temporal dynamics of ligand treatments, samples were sequenced at four timepoints, T0 (baseline, pretreatment only), at 1, 12 and 24hours.  Vehicle/water treated cells at each of the same timepoints were used as their respective controls (Figure 1a). Protein lysates were additionally collected at 30 minutes for RPPA measurements, to capture the phosphorylated protein dynamics All samples for the above measurements were obtained in duplicates (n=2) and differential analyses performed with respect to controls at a given timepoint.  Given the time complexity of qSMLM experiments, T0 was used as baseline control (n=2-3). Sample preparation, extraction, and imaging/sequencing protocols for each of the aforementioned are presented in detail within the Methods sections. Ligand concentrations were based on published studies7–11 and by titration experiments (data not shown).