Fig. 3
ApoA1 inhibit metastasis of TNBC trough downregulating KRT14. (a) KRT14 expression in tumor samples from patients with TNBC (n = 299) and other types of breast cancers (non-TNBC, n = 1605). Data collected from TCGA. (b) The KRT14 knockdown cell line, 4T1KRT14 − KD, was generated using lentivirus containing KRT14-specific shRNA, and KRT14 expression was confirmed by western blotting. (c) and (d) Survival curve and lung metastasis of mice orthotopically inoculated with 4T1KRT14 − KD or 4T1Ctr. (e) and (f) mRNA expression of KRT14 in 4T1ApoA1 and 4T1 cells infected with pApoA1 plasmid, tested using qPCR. (g) Volcano plots of transcriptome comparison between 4T1ApoA1 and 4T1WT cells. (h) and (i) GSEA and expression pattern of desmosome-associated genes in 4T1WT and 4T1ApoA1 cells. (j) and k) Expression of KRT14 in 4T1, MDA-MB-231, and HCC1937 cells transfected with pApoA1 plasmid, tested using western blotting. l) Expression of KRT14 in 4T1ApoA1 cells. m) Orthotopically injected 4T1WT and 4T1ApoA1 cells into BALB/c mice. Tumors were collected on day 7 for immunofluorescence analysis of KRT14. n) Expression of KRT14 in 4T1 cells in the presence of LXR623. o) Expression of the precursor of sterol regulatory element-binding transcription factor 2 (SREBP2, 125 kDa) in 4T1ApoA1 and 4T1WT cells. p) mRNA levels of cholesterol uptake or de novo synthesis-related genes. HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR: low-density lipoprotein receptor; SQLE: squalene epoxidase. q) Expression of LDLR and KRT14 in 4T1 cells transfected with LDLR-encoding plasmid pLDLR. r) 4T1ApoA1 cells were treated with or without 1mM of DIDS, and KRT14 expression was determined 24 h later using western blotting. s) Expression of KRT14 in ABCA1 knockdown 4T1 cells (4T1sh-ABCA1) transfected with pApoA1 plasmid. Cells were collected 48 h after pApoA1 transfection. t) Expression of KRT14 in 4T1ApoA1 cells in the presence of 2 μm of cholesterol-saturated methyl-b-cyclodextrin. u) mRNA expression of KRT14 in 4T1ApoA1 + KRT14 cells generated using a KRT14-encoding lentivirus. v) Lung metastasis of mice orthotopically inoculated with 4T1ApoA1 + KRT14 or 4T1ApoA1. w-y) The migration, invasion, and colony formation abilities of 4T1ApoA1 cells transfected with either KRT14 expressing plasmid (pKRT14) or control plasmid (pCtr). w) Cell mgration was tested using wound healing assays. x) Cell invasion was evaluated using transwell assays and stained with crystal violet. y) Colony formation of cells was analyzed using colony formation assays in Matrigel and imaged at 24, 48, and 72 h. **p < 0.01; ***p < 0.001; ****p < 0.0001