Lipid Storage and Autophagy in Melanoma Cancer Cells

Abstract

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology.

Keywords: autophagy; lipids; melanoma; stem cells.

Figure 1

CD133 antigen (also known as prominin-1) expression and lipid-storing phenotype in melanosphere-derived cells. (A) CD133 marker is up-regulated in melanosphere-derived cancer stem cells (MSC). MSC and differentiating melanosphere-derived melanoma cells Early and Late (PC-E and PC-L) were lysed; protein samples were subjected to Western blot analyses and CD133 was detected. Western blot panel is representative of three independent experiments and reveal CD133 progressive down-regulation in melanoma cells undergoing progressive differentiation. β-Actin was used as a loading control. Densitometric analyses of CD133 relative to β-Actin in three independent experiments are presented in the graph as the mean ± s.d. expressed as Arbitrary Units (AU). * indicates p ≤ 0.05 vs. MSC; (B) Lipid Droplets (LD) staining is higher in MSC. MSC, PC-E and PC-L were stained with Oil Red O and then observed by bright-field microscopy. The images shown are representative of three independent experiments and reveal reduction in LD color intensity with melanoma cell differentiation. Bar corresponds to 35 μm; (C) MSC display increased lipid up-take. Increase of Nile Red (NR)-stained neutral lipids was evaluated by flow-cytometry after oleic acid treatment in MSC, PC-E and PC-L and reported in the graph as FL2 fluorescence fold increase vs. untreated cells. The data shown are the mean ± s.d. of three independent experiments. * indicates p ≤ 0.05 vs. MSC.

Figure 2

MSC express higher PPAR-γ and SREBP-1 levels as compared to PC-E and PC-L. MSC, PC-E and PC-L were lysed and protein samples were subjected to Western blot analyses using Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) as a loading control. Representative panels of: PPAR-γ (A); and SREBP-1 (C) are shown. Densitometric analyses of: PPAR-γ (B); and SREBP-1 (D) relative to GAPDH in three independent experiments are presented in the graph as the mean ± s.d. expressed as Arbitrary Units (AU). PPAR-γ and SREBP-1 show down-regulation in melanoma cells undergoing progressive differentiation. * indicates p ≤ 0.05 in PC vs. MSC.

Figure 3

LC3-II increases in melanoma cells undergoing progressive differentiation revealing autophagic flux up-regulation. (A) MSC, PC-E and PC-L were cultured in the presence or absence of the lysosomal degradation inhibitor bafilomycin A1 (BAF). They were lysed and protein samples were subjected to Western blot analysis. LC3-II increase in differentiated melanoma cells, although evident in either case, is more evident after BAF treatment, thus demonstrating autophagic flux up-regulation as function of time of differentiation. Western blot panel is representative of three independent experiments. β-Actin was used as a loading control; (B) densitometric analysis of LC3-II relative to β-Actin in three independent experiments is presented in the graph as the mean ± s.d. expressed as AU. BAF-treated MSC vs. untreated MSC display significant increase (p ≤ 0.05). Similarly, ΒΑF treated PC-E vs. untreated PC-E display significant increase (p ≤ 0.05). ** indicates p ≤ 0.05 in BAF-treated PC vs. BAF-treated MSC. * indicates p ≤ 0.05 in untreated PC vs. untreated MSC.

Figure 4

AMPK and mTOR phosphorylation levels change with melanoma cell differentiation. MSC, PC-E and PC-L were lysed and protein samples were subjected to Western blot using β-Actin or GAPDH as a loading control. Representative panels of P-AMPK and AMPK (A); and P-mTOR and m-TOR (C) are shown; (B,D) densitometric analyses of P-AMPK/AMPK and P-mTOR relative to the corresponding loading control, respectively, reveal progressive P-AMPK/AMPK increase and progressive P-mTOR decrease as function of time of differentiation. Data are presented as the mean ± s.d. expressed as AU of three independent experiments. * indicates p ≤ 0.05 in PC vs. MSC.

Figure 5

LC3-stained autophagosome spots co-localize with perilipin-stained LD suggesting LD clearance through autophagy. Immunofluorescence for perilipin (green) or LC3 (red) was performed on MSC, PC-E and PC-L, cultured in the absence (CTR) or presence of the lysosomal degradation inhibitor bafilomycin A1 (BAF). Yellow spots, indicating LC3-perilipin co-localization, increase as function of differentiation and are more evident in BAF-treated cells. Representative images of three experiments. All images were taken at the same magnification Bar corresponds to 60 μm. Insets correspond to further 5× enlargement.