pp. this lipotoxicity is most likely due to a combination of genetic alterations that exist in HER2/neu-positive breast cancer cells, considering that the HER2 amplicon has been shown to comprise several genes in a large section on chromosome 17 [3, 4, 15C19] and other genes have been shown to be co-overexpressed and required for breast cancer cell survival [6]. This sensitivity to lipotoxicity may have consequences in patient populations in light of a recent epidemiological study. The investigators followed 337,327 women for 11.5 years and evaluated fat intake as a predictor of breast cancer development and found that a diet high in saturated fatty acids was positively associated with the development of HER2/neu-negative disease, but not HER2/neu-positive disease [20]. In this study, we have used global metabolite profiling and a multi-omics network analysis approach to identify the metabolic changes that result from stressing the Warburg-like physiology of HER2/neu-positive breast A-1210477 cancer cells with exogenous palmitate. The work provides insights into the molecular basis of the lipotoxic phenotype and its relevance to disease prevention and treatment. RESULTS Supplementation of culture media with saturated fatty acids induces distinct responses in breast cancer cells HER2/neu-positive breast cancer cells contain high levels of endogenous saturated fatty acids and neutral lipids and generally exhibit a pro-lipogenic phenotype. Our previous studies have established that BT474 (luminal B; ER+, HER2+), MDA-MB-361 (luminal B; ER+, HER2+), SKBR3 (HER2 enriched; ER-, HER2+) but not MCF-7 (luminal A; ER-, HER2wt) or human mammary epithelial cells exhibit this Warburg-like physiology which relies on active fatty acid synthesis for survival and aggressive behavior [6, 8-10, 14, 21]. Additionally, molecular profiling experiments from this work have shown that the MCF7 cell line (HER2-normal) and the SKBR3 cell line (HER2/neu-positive) are representative lines to investigate the differential effects of fatty acids as a model of increased dietary fat intake. The use of MCF7 cells as a control is preferable since they can be grown in the same culture medium as SKBR3 cells and previous studies have shown that the response to exogenous A-1210477 fatty acids in MCF7 cells is comparable to Oaz1 that of non-tumorigenic MCF10A mammary epithelial cells or normal human mammary epithelial cells (HMECs) [8, 22]. We cultured MCF7 and SKBR3 cells in the presence of either 250 M palmitate A-1210477 (C16), stearate (C18), oleate (C18:1) or palmitate and oleate in combination (250 M and 150 M, respectively) and monitored cell count as well as levels of intracellular neutral fat stores compared to vehicle control. Supplementing the growth A-1210477 media with the saturated fatty acids palmitate and stearate significantly reduces the number of SKBR3 cells, but not MCF7 cells, indicating the induction of distinct responses to saturated fat in the two cell lines. These effects are mediated by the effects of palmitate on cellular physiology and not as effects on cellular integrity which are not seen at concentrations in this range [14]. This distinction is further evidenced through observed changes in lipid content. While A-1210477 SKBR3 cells show higher basal levels of stored neutral fats that do not change with palmitate or stearate treatment, MCF7 cells display low basal neutral fat content which increases significantly upon saturated fatty acid exposure (Figure 1A, 1B and Supplementary Figure 1). In SKBR3 cells, palmitate has been shown to induce a partial ER-stress response and CHOP-dependent apoptosis [14]. Supplementation with.