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. 2021 Jun 1:11:576326.
doi: 10.3389/fonc.2021.576326. eCollection 2021.

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells

Alyssa S Zembroski  1 , Chaylen Andolino  1 , Kimberly K Buhman  1 , Dorothy Teegarden  1
Affiliations

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells

Alyssa S Zembroski et al. Front Oncol. .

Abstract

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer.

Keywords: breast cancer; cytoplasmic lipid droplets; metastasis; proteomics; triacylglycerol.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cytoplasmic lipid droplets (CLDs) are present in MCF10CA1a cells. (A) Representative transmission electron microscopy (TEM) image of a MCF10CA1a cell containing CLDs (boxed region), scale bar 2 μm. (B) Magnified image of the CLDs present in (A) scale bar 1 μm.
Figure 2
Figure 2
Cytoplasmic lipid droplet (CLD) size distribution. Percentage of CLDs analyzed in Table 1 within the indicated size range. 50 cells were counted and used for the analysis. CLD diameter was measured using ImageJ.
Figure 3
Figure 3
Validation of cytoplasmic lipid droplet (CLD) isolation. (A) Triacylglycerol (TAG) to protein ratio of each isolated fraction. CLDs were isolated from MCF10CA1a cells using sucrose density gradient ultracentrifugation. Fractions were removed sequentially from the top of the gradient to the bottom. Floating fraction (FF): isolated CLDs, 1-10: soluble fractions, P: pellet. (B) Western blot of isolated fractions and whole cell lysate (WCL). Fractions were loaded by volume: 10 μL FF-10, 5 μL P and WCL. Membrane was probed for markers of CLDs (PLIN3), cytosol (GAPDH), and endoplasmic reticulum (CANX). Approximate molecular weight markers for each protein are listed. See Supplementary Figure 1 for a representative Ponceau stain reflecting the relative levels of protein in each fraction.
Figure 4
Figure 4
PLIN3 surrounds cytoplasmic lipid droplets (CLDs) in MCF10CA1a cells. Representative immunofluorescence images of MCF10CA1a cells. Cells were stained with Alexa Fluor 633 to visualize PLIN3, BODIPY to visualize CLDs, and DAPI to visualize nuclei. Signals from all three channels were merged for the final image.
Figure 5
Figure 5
General functions of identified proteins and Gene Ontology (GO) term enrichment. (A) Identified proteins grouped into general categories. Data shown as a percent of total proteins identified. Categories with the highest to lowest percent of proteins listed from top to bottom and are read clockwise around the pie chart. (B) Chart of the top 10 most enriched Gene Ontology Biological Process (GO_BP) terms. Most to least enriched term listed from top to bottom. Data shown as -log10 (p-value). Enrichment scores/p-values calculated in DAVID. See Supplementary Table 2 for full list of enriched GO terms and enrichment scores.
Figure 6
Figure 6
STRING analysis of identified proteins involved in lipid metabolism. Proteins with known functions in lipid metabolism and those associated with lipid-related Gene Ontology Biological Process (GO_BP) terms. Red: cholesterol biosynthetic process; green: fatty-acyl-CoA metabolic process; purple: phospholipid metabolic process; yellow: lipid droplet organization.
Figure 7
Figure 7
SQLE and NSDHL localize to cytoplasmic lipid droplets (CLDs) in MCF10CA1a cells. Representative immunofluorescence images of MCF10CA1a cells. Cells were stained with Alexa Fluor 633 to visualize SQLE (A) and NSDHL (B), BODIPY to visualize CLDs, and DAPI to visualize nuclei. Signals from all three channels were merged for the final image in (A, B).
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