Abstract
Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.
Highlights
Abbreviations acyl-CoA cholesterol acyltransferase (ACAT) Acyl-CoA cholesterol acyltransferase Anx Annexin Ca2+ Calcium CHO Chinese hamster ovary epidermal growth factor (EGF) Epidermal growth factor epidermal growth factor receptor (EGFR) Epidermal growth factor receptor endoplasmic reticulum (ER) Endoplasmic reticulum focal adhesions (FA) Focal adhesion focal adhesion kinase (FAK) Focal adhesion kinase Low-Density lipoproteins (LDL) Low density lipoprotein late endosomes (LE) Late endosomes
annexin A6 (AnxA6) depletion in Niemann Pick Type C1 (NPC1) mutant cells upregulated Rab[7] activity, rescuing late endosomal cholesterol (LE-Chol) export for cholesteryl ester storage in lipid droplets via transport routes involving the StAR-related lipid transfer domain-3 (StARD3) protein[28,29]. These findings indicated that AnxA6 controls LE-Chol export routes that operate in the absence of N PC128,29
In A431 cell line stably expressing AnxA6 (A431-A6) cells, which already exhibit an NPC1-like phenotype in serum-containing m edia[23], this inhibitory effect of U18666A on cell migration was less pronounced (Fig. 1b)
Summary
Abbreviations ACAT Acyl-CoA cholesterol acyltransferase Anx Annexin Ca2+ Calcium CHO Chinese hamster ovary EGF Epidermal growth factor EGFR Epidermal growth factor receptor ER Endoplasmic reticulum FA Focal adhesion FAK Focal adhesion kinase LDL Low density lipoprotein LE Late endosomes. Metabolic adaptations in cancer include an increased demand for membrane lipids[1]. This includes cholesterol, which supports growth and motility, in particular the formation of focal adhesions (FA), enabling cells to move forward[2,3]. Earlier studies identified methyl-β-cyclodextrin, which triggers a robust cholesterol depletion at the plasma membrane, to disrupt the functional integrity of FAs, suggesting cholesterol being vital for proper FA structure and a ssembly[2,7,8]
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