Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells

Abstract

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10–35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells

“…TEM was carried out as described previously [3,20,48]. A 400-mesh copper grid coated with formvar/carbon films was hydrophilically treated.…”

“…For EV diameter distribution analysis, 40 µL of EV fraction solved in PBS (-) was analyzed in a range between 0 and 10,000 nano-diameters in Zetasizer nano ZSP (Malvern Panalytical, UK) as described previously [3,20]. Alternatively, the diameter distribution of EVs was analyzed using ELS-8000 (Otsuka Electronics, Hirakawa, Japan) for a range of 1-10,000 nm with a setting of sidecut L20 as described previously [48].…”

“…For single or double knockdown, cells were cultured in a 6-cm dish and 27 pmol siRNA was transfected with 8 µL of Lipofectamine RNAi MAX per dish for 24 h. The medium was replaced with serum-free fresh medium and the EV and whole cell lysate (WCL) were prepared at 48 h post-transfection. For triple knockdown, electroporation-mediated transfection was optimized and performed as described previously [3,60]. To optimize electroporation for each cell type, cells (1 × 10 5 to 1 × 10 6 cells), siRNA (40 pmol total), and serum-free medium were mixed to 100 µL total in a green cuvette with a 1-mm gap (NepaGene, Ichikawa, Tokyo, Japan) and set to NEPA21 Super Electroporator (NepaGene).…”

“…Tumor cells are often exposed to various stresses such as immune/inflammatory stress, therapeutics [1], hypoxia [2][3][4], acidification, oxidative stress [5,6], starvation [7], nutrient stress [8], heat and cold [9,10], thermal stress, replication stress [11], endoplasmic reticulum (ER) stress, neurotoxic stress [12], genotoxic (DNA damage) [13] and proteotoxic stress [14,15]. Heat shock proteins (HSPs) were originally found to be induced upon heat shock stress (HSS) [9].…”

CDC37 and HSP90 are Essential for Stressome Release and Tumor Progression in Resistant Prostate Cancer

“…A 400-mesh copper grid coated with formvar/carbon films was hydrophilically treated. The EV suspension (5 to 10 μl) was placed on parafilm, and the grid was floated on the EV liquid For triple knockdown, electroporation-mediated transfection was optimized and performed as described previously [57,76]. To optimize electroporation for each cell type, cells (1 × 10 5 to 1 × 10 6 cells), siRNA (40 pmol total), and serum-free medium were mixed to 100 µl total in a green cuvette with a 1-mm Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 11 February 2020 doi:10.20944/preprints202002.0148.v1 gap (NepaGene, Ichikawa, Tokyo) and set to NEPA21 Super Electroporator (NepaGene).…”