Abstract P1-05-02: Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage

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Abstract Introduction: We have investigated the breast microenvironment to identify factors that promote Estrogen Receptor Negative Breast Cancer (ERneg BC) and that may be disrupted for prevention. To that end, we have identified a lipid metabolism gene signature associated with the risk of ERneg BC. To better understand lipid metabolism in the breast, we studied the effect of fatty acids (FA) on non-transformed breast epithelial cells and tissues. FA exposure alters histone methylation, affecting gene expression and increase flux through serine, one-carbon, glycine (SOG) and methionine pathways. The association of the serine pathway and ERneg BC was first observed over a decade ago. A SOG pathway gene signature is significantly correlated with ERneg status. We hypothesized that the metabolism of FA results in a metabolic shift toward the de novo serine synthesis pathway (SSP), which ultimately increases S-adenosylmethionine (SAM), altering histone methylation, profoundly changing gene expression and fostering ERneg oncogenesis. Methods: Non-transformed MCF-10A cells were used for in vitro metabolic and epigenomic analyses. Cells exposed to the medium-chain FA octanoic acid (OA) were utilized for proteomics and U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± blockade of the serine pathway. Reactive Oxygen Species (ROS)-induced redox changes were monitored live cells. Comet assay was performed to detect DNA damage. CUT&RUN was performed for H3K4me3. Human breast tissue derived microstructures were utilized for genomic analysis. Single-cell RNA-seq (scRNAseq) was performed in microstructures exposed to ± OA. Metabolic flux analyses was performed using Compass. Results: 13C flux analysis revealed that OA led to increased flux to methylation. OA significantly increased the main methyl donor SAM, the antioxidant GSH via the transsulfuration pathway and the oncometabolite 2-HG after 15 min exposure. Blocking the first and rate limiting enzyme in the SSP, PHGDH, prevented these increases. Proteomics revealed the overexpression of PHGDH following OA exposure. Upon exposure to OA, scRNAseq analysis revealed increased expression of the SSP transcription factor (TF) ATF3 and the SSP genes PHGDH and PSAT1 in epithelial and non-epithelial clusters. Upon OA the proportion of three subtypes within the epithelial compartment increased: basal BSL1, Hormone sensing HS1 and luminal progenitor LP3. Compass, an algorithm to characterize cellular metabolic states, revealed flux greatly increased through the three enzymes of the SSP: PHGDH, PSAT1 and PSPH secondary to OA exposure in BSL1, LP3 and HS1 cells. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. Motif analysis revealed an overrepresentation of binding sites for SSP TFs ATF3/4 (p < 0.05). After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.01), peaking at 15 min. OA exposure triggered DNA damage likely due to ROS increase in the nucleus. Compass predicted an increase in GSH metabolism and ROS detoxification in BSL1. Conclusions: Protein levels of PHGDH are elevated in 70% of ERneg BCs. This cannot be explained by gene amplification alone as PHGDH gene amplification is observed in only approximately 6% of all breast cancers. This suggests that there are mechanisms other than gene amplification that contribute to PHGDH dysregulation. One of those mechanisms may be the lipid induced metabolic shift toward the SOG and methionine pathways that we have identified. The increased SAM and 2-HG foster epigenetic phenotypic plasticity via altered histone methylation. ROS increase shortly after OA exposure and are controlled by antioxidant defenses (e.g. GSH), which favors the survival of specific cell subtypes with acquired DNA damage which likely facilitates malignant transformation. Citation Format: Mariana Bustamante Eduardo, Curtis W. McCloskey, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Jason Locasale, Marcelo G. Bonini, Maxim V. Frolov, Elizaveta V. Benevolenskaya, Rama Khokha, Navdeep S. Chandel, Seema A. Khan, Susan E. Clare. Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-02.

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