Lipid droplets (LDs) are recently recognized organelles that have an essential role in lipid storage for energy production, but are also involved in the regulation of cellular stress. LDs can rapidly form or break down in response to various environmental factors and cellular energy levels. Recent research has shown that LDs are also engaged in a complex relationship with autophagy, a major cellular stress response mechanism involving lysosomal degradation of cytoplasmic organelles and various cytosolic material. Some studies reveal that autophagy can provide fatty acids for LD biogenesis, while other suggest that it is also involved in the breakdown of LDs via a selective form of autophagy called “lipophagy”. It has also been shown that LDs can be sources of lipids for autophagosome membrane assembly and thus enable the initiation of autophagy. It is not yet entirely clear how this complex interplay is regulated and which molecular pathways are responsible for each particular outcome. This relationship is particularly important in cancer cells, which are often exposed to nutrient imbalances in the tumour microenvironment and use both autophagy and LDs for protection against stress. The aim of the present master thesis was to investigate some basic connections between autophagy/lipophagy and LDs in highly invasive breast cancer cells exposed to nutrient deficiency. To achieve this goal, we optimized some already established and tested some new methods for visualization and quantification of the interactions between autophagosomal structures and LDs by live-cell confocal microscopy. We found that autophagy is active in breast cancer cells not only during severe starvation in the absence of amino acids and serum, but also during mild starvation in serum-free medium. We show that inhibition of late stages of autophagy with bafilomycin A1 or chloroquine leads to a decrease in LD content during severe starvation, suggesting the possibility that autophagy is involved in LD biogenesis under these conditions. On the other hand, live-cell confocal microscopy experiments revealed that autophagosomal and lysosomal structures colocalize with LDs during mild but not during severe starvation, suggesting that lipophagic breakdown of LDs is active only in the milder, serum-free starvation conditions. Namely, serum-starved, but not amino acid- and serum-starved breast cancer cells, displayed a marked overlap of autophagosomal (mRFP-LC3) and lysosomal (LAMP1-RFP) fluorescent protein markers and LDs. We also found that late-stage autophagy inhibitors do not affect breast cancer cell survival, at least within the relatively short 24-hour starvation period tested here. In summary, our research work points to a dual, starvation conditions-dependent role of autophagy in the biogenesis and breakdown of LDs in breast cancer cells and offers new clues that will contribute to the explanation of the dynamics of cancer cell stress responses.
|
