Abstract 2562: Causes and consequences of adiposomogenesis in breast cancer cells

Abstract

Abstract Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and thrive and to out-compete the stroma into which they invade. Acid adaptation has been associated with chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. In addition to these survival mechanisms, tumor cells under acidic conditions accumulate cytoplasmic lipid droplets (adiposomes); dynamic organelles that store neutral lipids surrounded by a shell of perilipin (PLIN) proteins and a phospholipid monolayer. Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins (PLIN2) and a phospholipid monolayer. High expression of PLIN2 was observed to be strongly associated with poor overall survival in breast cancer patients. In vitro, breast cancer cells rapidly and robustly accumulated adiposomes when grown in acidic media revealed by nile red and PLIN-2 staining. The acid-induced lipogenic phenotype persists even when the cells are grown in de-lipidated serum, indicating that the source of lipids is de-novo and endogenous. Adiposome formation at low pH was attenuated when cells were treated with inhibitors of fatty acid synthesis, FAS; such as TOFA or C75. Further, these inhibitors were selectively cytotoxic under acidic conditions indicating that adiposomogenesis is a survival mechanism. Consistent with increased FAS we also observed using 13C isotopomer analysis a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, which results in increased production of NADPH, necessary for de novo lipid synthesis. In addition, we observed that cells at low pH had higher rates of oxygen consumption (OCR) compared to controls using Seahorse profiling. The major source of the lipid precursors was identified to be autophagy-derived ketogenic amino acids using LC-LS/MS following 13C tracer pre-incubation. Further, we tested the hypothesis that adiposomogenesis is induced by acid signal and this signaling is mediated by one (or more) acid sensing G-protein coupled receptors. Both OGR1 and TDAG8 were strongly expressed in our systems. CRISPR/Cas9 mediated depletion of these receptors showed that TDAG8 KO had no effect, but that KO of OGR1 abrogated adiposome accumulation under acidic conditions in MCF7 and T47D cells. Further, OGR1 knockout cells were defective in acid induced autophagy. In xenograft models, OGR1 knockout MCF7 cells formed significantly smaller tumors compared to control cells. Hence, accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. This increased dependence on lipid metabolism revealed novel therapeutic vulnerabilities Citation Format: Smitha R. Pillai, Iqbal Mahmud, Michael Langsen, Jonathan Wojtkowiak, Jonathan Nguyen, Marylin Bui, Robert Gatenby, Timothy Garrett, Robert Gillies. Causes and consequences of adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2562.