LSD1 deficiency in breast cancer cells promotes the formation of pre-metastatic niches

Abstract

Lysine-specific demethylase 1 (LSD1), a histone demethylating enzyme, plays a crucial role in cancer metastasis. Studies show LSD1 knockout promotes breast cancer lung metastasis, but it's unknown if it alters the lung microenvironment for metastasis. In this study, we investigated the effects of exosomes from LSD1-knockdown (LSD1 KD) breast cancer cells on pre-metastatic niche formation. Injecting exosomes from LSD1 KD cells in mice resulted in a substantial increase in lung colonization by breast cancer cells, while treatment with exosomes derived from LSD1 KD cells decreased the expression of the ZO-1 and occludin, leading to increased vascular permeability. The LSD1 KD reduced the expression of circDOCK1, which augmented the levels of miR-1270 in exosomes. And miR-1270 inhibited ZO-1 expression in human endothelial cells, which enhanced their permeability. Our study uncovered a novel mechanism in which the LSD1 promotes the formation of pre-metastatic niches via the regulation of exosomal miRNA.

Fig. 1. Exosomes from LSD1 knockdown cells promote lung colonization of MCF7.

a Western blot analysis of LSD1 expression in MCF7 cells transfected with the control construct (Control), MCF7 cells transfected with LSD1 shRNA (LSD1 KD), and LSD1 KD MCF7 cells in which LSD1 expression is restored (Rescue). b qRT-PCR analysis of LSD1 expression in Control, LSD1 KD, and Rescue cells. Data are presented as means ± SEM (n = 3). ***p < 0.001. c Western blot analysis of the exosomal markers CD63 and TSG101 in purified exosomes from Control, LSD1 KD, and Rescue cells. d Transmission electron micrographs of exosomes from Control, LSD1 KD, and Rescue cells. Scale bars: 100 nm. e Nanoparticle tracking analysis (NTA) of the diameter and concentration of exosomes from Control, LSD1 KD, and Rescue cells. f Schematic representation (created with BioRender.com) of exosome injection via tail vein and the intracardiac injection of luciferase-labeled MCF7 cells into BALB/c-nu/nu mice. g, h Bioluminescence imaging of the whole body and lungs of mice injected with luciferase-labeled MCF7 cells and treated with the indicated exosomes. i The tumor burden in lungs was quantified by measuring the total flux (photons/s) in bioluminescence imaging. Data are presented as means ± SEM (n = 5). *p < 0.05. j Immunohistochemical (IHC) staining for Ki67 in lung sections from mice treated with the indicated exosomes.

Fig. 2. Exosomes from LSD1 knockdown cells increase vascular permeability.

a Schematic representation (created with BioRender.com) of the administration of the indicated exosomes and rhodamine-dextran via tail vein injection. b–e Immunofluorescence (IF) staining of lung (b, c) and brain (d, e) sections for ZO-1 (b, d) and occludin (c, e). CD31 (red) and nuclei (blue) are also stained. Tissues positive for CD31 are indicated by arrowheads. Scale bars: 20 μm. f IF analysis of rhodamine-dextran (red) in lung and liver sections of mice treated with the indicated exosomes. Nuclei are stained with DAPI (blue). LPS, Lipopolysaccharide. Scale bars: 100 μm. g, h Quantification of rhodamine in the lungs and liver (n = 3). Data are presented as means ± SEM. ***p < 0.001.

Fig. 3. Exosomes from LSD1 knockdown cells disrupt barrier function in endothelial cells.

a, b Immunofluorescence (IF) analysis of human lung microvascular endothelial cells (HPMECs) a and human umbilical vein endothelial cells (HUVECs) b incubated with PKH67-labeled exosomes (green). Scale bars: 10 μm. c, d Western blotting analysis of ZO-1 and occludin expression in HPMECs and HUVECs treated with the indicated exosomes. e, f Transwell assays for the determination of the amount of rhodamine-dextran that passes through monolayers formed by HPMECs (n = 4) and HUVECs (n = 3) treated with the indicated exosomes. g, h Analysis of trans-endothelial electrical resistance (TEER) in monolayers formed by HPMECs (n = 6) and HUVECs (n = 4) treated with the indicated exosomes. i, j Transwell assays for the determination of the number of GFP-labeled MCF7 cells that pass through monolayers formed by HPMECs (n = 5) and HUVECs (n = 5) treated with the indicated exosomes, with quantification of the numbers of invading MCF7 cells. Scale bars: 50 μm.

Fig. 4. LSD1 knockdown alters the expression of exosomal miRNAs in MCF7 cells.

a Western blot analysis of LSD1 expression in Control, LSD1 knockdown (KD), and Rescue cells in three independent batches of samples. b Venn diagram showing the number of differentially expressed miRNAs between Control and LSD1 KD exosomes and between LSD1 KD and Rescue exosomes. c Heat map of the exosomal miRNAs that are significantly upregulated in LSD1 KD exosomes and restored in Rescue exosomes. d qRT-PCR analysis of the expression levels of the indicated miRNAs in Control, LSD1 KD, and Rescue exosomes. Data are presented as means ± SEM (n = 3 unless otherwise indicated). *p < 0.05, **p < 0.01.

Fig. 5. MiR-1270 disrupts the barrier function of endothelial monolayers by decreasing the expression of ZO-1.

a, b Transwell assays for the amount of rhodamine-dextran that passes through monolayers formed by HPMECs and HUVECs transfected with Control or miR-1270 mimics. c, d Analysis of trans-endothelial electrical resistance (TEER) in HPMECs (n = 6) and HUVECs (n = 9) transfected with Control or miR-1270 mimics. e, f Transwell assays for the numbers of GFP-labeled MCF7 cells that pass through monolayers formed by HPMECs and HUVECs transfected with Control, miR-1270 mimics, or miR-1270 inhibitor. Scale bars: 50 μm. g Venn diagram showing the number of miR-1270 target genes predicted by TargetScan, miRDB, miRWalk, and mirDIP. h KEGG pathway analysis of the 162 miR-1270 target genes predicted by the four databases. i, j Western blot analysis of ZO-1 expression in HPMECs and HUVECs transfected with Control, miR-1270 mimics, or miR-1270 inhibitor. k The sequences of the predicted miR-1270 binding sites in the 3′-UTR of ZO-1. Firefly luciferase reporter plasmids containing wild-type (ZO-1 WT) or mutant (ZO-1 MUT) binding sites for miR-1270 were co-transfected with miR-1270 mimics or into HEK293T cells, and the relative luciferase activities were analyzed. Data are presented as means ± SEM (n = 3 unless otherwise indicated). ***p < 0.001.

Fig. 6. LSD1 knockdown decreases the expression of circRNAs.

a Venn diagram showing the number of differentially expressed circRNAs between Control and LSD1 knockdown (KD) cells and between LSD1 KD and Rescue cells. b Heat map of the circRNAs that are both significantly changed in LSD1 KD cells and restored in Rescue cells. c The relative expression of circDOCK1 and circSEMA3A in Control, LSD1 KD, and Rescue cells (n = 4). d Venn diagram showing the number of circDOCK1 target miRNAs predicted by circBank and Circinteractome and the number of miRNAs upregulated in LSD1 KD exosomes. e Schematic diagram showing the binding sites of miR-1270, miR-1246, and miR-1265 in circDOCK1. f PCR analysis of circDOCK1 and its linear isoform DOCK1 after treatment with RNase R. g qRT-PCR analysis of circDOCK1 and its linear DOCK1 after treatment with RNase R. h Schematic diagram showing the genomic locus of the DOCK1 gene and the back-splicing of circDOCK1. The red arrow indicates the back-splicing site of DOCK1, which was confirmed by Sanger sequencing. i qRT-PCR analysis of the nuclear-cytoplasmic fractionation of circDOCK1. j Fluorescence in situ hybridization (FISH) analysis of the subcellular localization of circDOCK1 in MCF7 cells. Data are presented as means ± SEM (n = 3 unless otherwise indicated). ***p < 0.001.

Fig. 7. CircDOCK1 suppresses the functions of miR-1270.

a The sequences of si-circDOCK1#1 and si-circDOCK1#2, which were designed to target the back-splicing junction site of circDOCK1. b qRT-PCR analysis showing that si-circDOCK1#1 and si-circDOCK1#2 specifically target circDOCK1. c qRT-PCR analysis of miR-1270 expression in MCF7 cells and exosomes after transfection with si-circDOCK1#1 and si-circDOCK1#2. d qRT-PCR analysis of the expression of circDOCK1 and linear DOCK1 in MCF7 cells transfected with Control or circDOCK1 expression constructs. e qRT-PCR analysis of miR-1270 expression in MCF7 cells transfected with Control or circDOCK1 expression constructs. f Pulldown assay for circDOCK1 using the negative control (NC) probe or the circDOCK1 probe. g Pulldown assay for miR-1270 using the NC probe or the circRNA probe. h Pulldown assay for circDOCK1 using the NC probe or the miR-1270 probe. i Shown are the sequences of wild-type and mutant miR-1270 binding sites that were cloned into the DOCK1 luciferase reporter plasmid. Wild-type (circDOCK1 WT) or mutant (circDOCK1 MUT) luciferase reporter plasmids and miR-1270 mimics or Control were transfected into HEK293T cells, and the relative luciferase activities were analyzed. j Fluorescence in situ hybridization (FISH) analysis of the colocalization of circDOCK1 and miR-1270 in MCF7 cells. Scale bars: 10 μm. Data are presented as means ± SEM (n = 3 unless otherwise indicated). ns not significant, *p <0.05, **p < 0.01, ***p < 0.001.

Fig. 8. The LSD1/circDOCK1/miR-1270 axis regulates the formation of pre-metastatic niches.

In breast cancer cells, LSD1 deficiency decreases the expression of circDOCK1, resulting in increased levels of miR-1270 in exosomes. Vascular endothelial cells in distant organs uptake these breast cancer cell-derived exosomes with increased miR-1270 expression, leading to the downregulation of the expression of ZO-1 and other tight junction proteins. Vascular permeability is subsequently enhanced, leading to the formation of pre-metastatic niches and the promotion of lung metastasis. Created with BioRender.com.