Abstract P5-05-05: Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells

Abstract

Abstract Introduction. With the goal of defining the in-breast environment that facilitates the development of estrogen receptor negative (ER-) breast cancer (BC), we have previously identified a set of genes involved in lipid metabolism with increased expression in the contralateral unaffected breast of women with ER- BC (PMID: 28263391). MCF-10A cells exposed to lipids in vitro display profound changes in gene expression, chromatin packing density, chromatin accessibility and histone posttranslational modifications (PTMs). In addition, flux through thirty-eight metabolic reactions was significantly increased by this exposure, including those of the electron transport chain (ETC). However, the mechanisms by which lipids induce molecular changes that result in epigenomic reprogramming, and potentially promote malignant transformation remain to be elucidated. We hypothesized that dysfunction of the ETC and increased serine, glycine, one carbon (SGOC) metabolism pathway activity play a role in the modulation of epigenetics. Methods. To interrogate potential mechanisms that link lipids to ER- BC genesis, estrogen and progesterone receptor negative MCF-10A cells were cultured in MEBM/MEGM and exposed to vehicle, octanoic acid (C8) or linoleic acid (C18) for 24 hours. Histone PTMs were assayed by liquid chromatography-mass spectrometry. Gene expression was measured by RNA-seq and differentially expressed genes between vehicle and lipid treated groups were identified using the DESeq2 R package. ATAC-seq was performed to study chromatin accessibility. MCF-10A cells were exposed to C8 in presence or absence of inhibitors of Complex I (Metformin) and Complex III (Antimycin A) of the ETC. Real-time qPCR was performed to measure epigenetic modulators, Unfolded Protein Response (UPR) pathway and SGOC metabolism genes. Results. Histone PTMs changed significantly following 24 hours exposure to C8 or C18. Exposure to C8 resulted in the modulation of the expression of genes involved in the UPR/recognition of mitochondrial dysfunction (ATF4), de novo serine biosynthesis (PHGDH, PSAT1, SPH) and one-carbon metabolism (SHMT2, MTHFD2) pathways as well as numerous genes coding for epigenetic modulators. Upon C18 exposure, the binding motif for ATF4 was among the most significantly enriched motifs in the open chromatin regions and p53 was among the motifs with significantly decreased accessibility. Interestingly ATF4 actives PHGDH expression while p53 suppresses it. Exposure to C8 in presence of ETC inhibitors affected gene expression in the same direction as lipid exposure and co-treatment had an additive effect. Fatty acid oxidation and inhibition of the ETC increased the expression of PHGDH: C8, 2.6-fold (p-value = 0.005); Metformin, 2.3-fold (p-value = 0.0052); Antimycin A, 5.1-fold (p-value < 0.0001); C8 plus Metformin, 5.3-fold (p-value < 0.0001), and C8 plus Antimycin A, 5.2-fold (p-value < 0.0001). Conclusions. The modulation of epigenetic effectors does not appear to be a consequence of increased flux through the ETC but rather a consequence of increased SGOC metabolism, and the methionine cycle. Our in vitro model shows that lipid-induced stress increases ATF4 expression. ATF4 controls the expression of PHGDH, PSAT1, SPH, SHMT2 and MTHFD2 which influences epigenetics through S-adenosyl methionine production, the major methyl donor. PHGDH overexpression appears to be the key in our model as it has been associated with ER- BC and its engineered overexpression in non-transformed breast cells causes alterations associated with malignant transformation. The increase in PDGDH expression is hypothesized to compensate for mitochondrial dysfunction and to restore redox homeostasis by producing precursors that can be used for the production of antioxidants, such as glutathione. Citation Format: Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-05.