Investigating lipid phenotype as a metabolic adaptation to microenvironmental stress

Breast cancers emerge through a years-long process of somatic evolution within the physical constraint of milk ducts, characterized by accumulation of heritable genetic and epigenetic changes. While these genetic alterations have been extensively studied, less attention has been paid to the microenvironmental conditions

that select for these genotypes. We and others have demonstrated that evolving pre-cancerous hyperplastic cells are subjected to spatio-temporal variations in oxygenation due to poor vasculature, acidosis due to fermentative metabolism, and nutrient scarcity due to competition among cancer cells and between cancer and

stromal compartments. Adaptations to these microenvironmental stressors are prerequisite for survival and eventually results in selection of cells with more aggressive phenotypes. To model the eco-evolutionary dynamics of tumor microenvironment (TME) in vitro, we subjected benign breast cancer cells (eg. MCF7 and

MCF10AT) to harsh conditions encountered in TME (ie nutrient deprivation, hypoxia and acidosis resulting from metabolism) and we observed that the cells that survive these selection pressures exhibited a lipogenic phenotype characterized by robust accumulation of lipid droplets (LDs). We selected three nutrient stress

adapted clones (NSA) to further characterize the distinct metabolic phenotypes and to determine if this phenotype switch contributes to aggressive tumor behavior. The selected clones maintained the lipid phenotype under nutrient replete, normoxic and normal pH culture conditions implying that this is a hard-wired metabolic

phenotype. Indeed, the nutrient stress adapted cells (NSA) were more migratory in vitro and highly metastatic in orthotopic xenograft models, however, grew smaller primary tumors compared to control cells. We will further characterize lipid phenotype of the NSA cells by assessing differential gene expression patterns that define

distinct metabolic pathways. Further, we will determine if these clones engage in alternate nutrient acquisition pathways such as macropinocytosis, receptor mediated endocytosis and entosis to scavenge nutrients and use them to generate lipids. We will employ stable isotope tracing as well as image-based assays (live confocal high

content microscopy using Opera Phenix) to define the mechanism of lipid accumulation in adapted cells. Finally, we will perform metabolic profiling to measure oxygen consumption rate and proton production rate and determine their ability to metabolize alternate fuels including lipids. Combining these approaches, we propose to

decipher the mechanism whereby microenvironmental stress-induced evolution results in hard-wired phenotypic adaptations, represented by a lipogenic phenotype. At the end this study, we expect to have a more complete and comprehensive understanding of the microenvironmentally-induced gene expression changes that occur

during carcinogenesis, and how these relate to hard-wired phenotypic profiles, such as the lipogenic phenotype that contribute to cancer progression. A deeper understanding of this metabolic switch has potential to identify novel therapeutic targets.