Abstract 2019: Comparison of non-metastatic and metastatic breast cancer cell and lipid droplet proteomes.

Abstract Metastatic breast cancer cells often have increased cellular storage of neutral lipids, such as triacylglycerol (TAG), in cytoplasmic lipid droplets (LDs). Although LD accumulation is associated with increased cancer aggressiveness, the role of these organelles in contributing to metastatic disease remains unclear. Aside from TAG storage within the core of the LD, the phospholipid monolayer is also decorated with a variety of proteins. Given that LD-associated proteins reflect the organelle’s function within a given physiological context, we characterized the proteome of LDs from a human metastatic compared to non-metastatic breast cancer cell line to assess potential candidate protein pathways that may associate with LDs to promote metastasis. Untargeted shotgun proteomics of LDs isolated via sucrose density gradient ultracentrifugation from non-metastatic MCF10CA1h (less LDs) and metastatic MCF10CA1a (more LDs) cell lines was performed. The LD proteomes, as well as the proteome of each cell line’s whole cell lysate (WCL), were compared utilizing MaxQuant, MassDynamics, Metascape, and STRING. We identified that LDs from metastatic cells are highly enriched in proteins related to LD organization and protein localization compared to LDs from non-metastatic cells. Interestingly, proteins involved in the sequestering of NFκB were the most highly enriched lipid-related protein category in LDs of metastatic compared to non-metastatic cells. Further, NFκB was similarly enriched in the WCLs of both cell lines; however, was only identified in the isolated LD fraction from metastatic cells. Additionally, the negative regulator of LD catabolism, hypoxia inducible lipid droplet associated protein (HILPDA), was only identified in the metastatic cell LD fractions. Given the role of NFκB and HILPDA in tumor progression and therapy resistance, results from this study provide testable hypotheses that can elucidate how LDs contribute to breast cancer metastasis. For example, LDs may act as scaffolds for signaling to coordinate lipid metabolism and metastatic processes or as a stress-adaptive platform to enable rapid lipid-mediated signaling under hypoxia inducible factor (HIF) activation. Additionally, given that HILPDA stabilizes LDs which store precursors for eicosanoid synthesis, which in turn modulate NFκB activity and subsequent inflammatory response, the interactions of these mediators may integrate into a metabolic-inflammatory axis to promote tumor progression and immune evasion. Together, our results indicate that though the global proteome of non-metastatic and metastatic breast cells derived from the same parental line are similar, the proteins associated with LDs vary greatly. This study provides a list of candidate proteins for additional experimentation to determine their role in sustaining LD accumulation and metastatic progression. Citation Format: Chaylen Andolino, Kimberly K. Buhman, Dorothy Teegarden. Comparison of non-metastatic and metastatic breast cancer cell and lipid droplet proteomes abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2019.