Abstract
Background:
There are no targeted pharmacologic interventions currently available for the prevention of hormone receptor negative breast cancer. Primary prevention with endocrine agents decreases the risk of ER positive disease with no effect against ER negative (ER-) disease. Thus, there is a compelling need to identify women at high risk for ER-negative breast cancer and to uncover the molecular mechanisms involved in its genesis. Our recent observation that a set of lipid metabolism (LiMe) genes are over-expressed in the contralateral unaffected breasts of women with unilateral ER- breast cancer suggests the novel hypothesis that specific lipid metabolism pathways in the breast produce a physiological milieu favoring the development of ER- breast cancer. We are now testing the specific hypothesis that lipids are the source of the acetyl-coA that is utilized to acetylate histones, an epigenetic modification that reprograms transcription.
Methods:
We developed an in-vitro model that relies on octanoic acid, a medium chain fatty acid that freely diffuses though the plasma and mitochondrial membranes. MCF-10A cells were plated and allowed to adhere overnight and then exposed to an increasing dose of sodium octanoate for 24 hours in complete media. Acetylation of Lysine 9 of Histone 3 (H3K9) was analyzed by Western blot and RNA was extracted for qPCR and RNA-seq. Chromatin packing density at the nanoscale was quantified bypartial wave spectroscopic (PWS) microscopy. Mammary organoids were prepared from breast tissue by collagenase digestion and similarly treated.
Results:
We found a striking, dose-dependent increase of H3K9 acetylation in octanoate treated MCF-10A cells. The acetylation is specific to the lipids as no acetylation was observed in cells treated with the same concentration of the alcohol 1,4-Cyclohexanedimethanol. RNA-Seq revealed the differential expression of LiMe genes together with a significant upregulation of Hedgehog and Notch signaling pathways. Individual genes from various pathways were further verified by qPCR which revealed, for example, a four-fold increase in SHH expression and 25-fold increase in DLL4. The expression of two of the previously identified LiMe genes, HMGCS2 and ACSL3, was increased four-fold in the octanoate treated MCF10A cells. We repeated the octanoate treatment in organoids and found similar effects. PWS in live cells showed a dose-dependent increase in chromatin packing scaling (D) in cells exposed to octanoate, suggesting that accessibility of chromatin to transcription factors is increased upon fatty acid treatment.
Conclusion:
A lipid rich microenvironment affects metabolism in ER- MCF10A cells and stimulates pro-neoplastic signaling via histone modifications. This supports our hypothesis that perturbed lipid metabolism plays an important role in the development of ER- breast cancer. Further mechanistic studies will determine if the genes differentially expressed in cell culture are also differentially expressed in antecedent benign breast biopsies from women eventually diagnosed with ER+ and ER- cancer.
Citation Format: Yadav S, Choi M, VanDerway D, Bauer G, Backman V, Khan SA, Clare SE. Deregulated lipid metabolism fuels the genesis of estrogen receptor negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-06.
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