Abstract 2395: Acid-induced autophagic protein products are stored as adiposomes in breast cancer cells
Authors: Pillai Smitha R. , Wojtkowiak Jonathan W. , Nguyen Jonathan , Damaghi Mehdi , Bui Marilyn M. , Garrett Timothy , Gillies Robert J. 
Published: August 2018

Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in an acidic extracellular microenvironment. Adaptation to acidic conditions is a pre-requisite for tumor cells to survive and to out-compete the stroma into which they invade. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats lipid droplets (adiposomes). Adiposomes are dynamic organelles that store neutral lipids surrounded by a shell of proteins and phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Fatty acid synthesis inhibition was selectively toxic under acidic conditions as compared to neutral pH and attenuated acid-induced adiposome accumulation. Using 13C isotopomer analysis, we observed a major shift in glucose metabolism from Embden Meyerhof to the Pentose Phosphate Pathway, resulting in increased production of NADPH, necessary for de novo lipid synthesis. To identify the carbon source of the lipid precursors in adiposomes, we employed 13C tracer analysis using [UL] 13C Glucose, 13C3 Leucine (labeled to steady-state) 13C2 Acetate and [UL] 13C glutamine as sources of lipids in the media. 13C label incorporation was determined by LC-MS/MS from adiposomes isolated from cells grown in media containing delipidated serum at pH 6.5. Label incorporation to glycerol backbone and acyl chains of lipids was observed when 13C glucose was used as the source. We could detect (M+1 ~6% of total) labeled 13C in triglycerides from adiposomes isolated from 13C3 leucine labeled MCF7, T47D and ZR75.1 cells. These data indicate that ketogenic amino acids arising from the autophagic breakdown of proteins are the major source of carbons in adiposomal lipids. Additionally, adiposome accumulation was significantly inhibited when cells were treated with autophagy inhibitors indicating that acid induced adiposomogenesis depends on autophagy. Further, we investigated the role of various acid-sensing GPCRs such as OGR1 and TDAG8 in transducing the acid signal. CRISPR/Cas9 mediated depletion of these receptors demonstrated that only OGR1 depletion abrogated acid induced adiposome accumulation and were defective in autophagy. In addition, acid-induced adiposomogenesis required PI3K and pAkt signaling. Hence, accumulation of adiposomes is a highly regulated metabolic process related to storing autophagic products, and appears to be important in cell survival under acidosis. This increased dependence on lipid metabolism reveals novel therapeutic vulnerabilities.

Citation Format: Smitha R. Pillai, Jonathan W. Wojtkowiak, Jonathan Nguyen, Mehdi Damaghi, Marilyn M. Bui, Timothy Garrett, Robert J. Gillies. Acid-induced autophagic protein products are stored as adiposomes in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2395.