JAG1 enhances angiogenesis in triple-negative breast cancer through promoting the secretion of exosomal lncRNA MALAT1

Abstract

Despite significant improvements in five-year survival rates due to early diagnosis and combination therapy, triple-negative breast cancer (TNBC) treatment remains a major challenge. Finding new and effective targets for diagnosis and drug therapy is urgent for TNBC patients. Jagged-1 (JAG1), one of the canonical ligands of the Notch signaling pathway, is involved in vascular budding and is a poor prognostic factor of TNBC. In this study, combined with quantitative real-time PCR, database analysis, animal experiments, and other means, JAG1 was confirmed to be related to the poor prognosis of TNBC patients. JAG1 was highly expressed in MDA-MB-231 Bone (231B) cells, with stronger invasion and metastasis ability than MDA-MB-231 (231) cells. Treatment of human vascular endothelial cells (HUVEC) with TNBC conditioned medium showed that TNBC JAG1 promoted the angiogenesis of HUVEC. Next, we detected the exosomes extracted from TNBC conditioned medium and found that JAG1 promoted the exosome secretion from 231 cells via ALIX-RAB11A/RAB35. In addition, we also found that the exosomes from JAG1 overexpressed TNBC cells contained more long non-coding RNA (lncRNA) MALAT1, and MALAT1 promoted angiogenesis of HUVEC by targeting miR-140-5p. Finally, the angiogenesis-promoting effect of JAG1 in TNBC was further investigated by matrix gel assay. In conclusion, we reveal that JAG1 has a pro-invasion effect on TNBC and is involved in microenvironment angiogenesis by promoting exosome secretion and the MALAT1-miR-140-5p-JAG1/VEGFA pathway.

Keywords: Angiogenesis; Exosome; JAG1; Triple-negative breast cancer; lncRNAMALAT1.

Figure 1

JAG1 was closely related to the malignant phenotype of TNBC. (A) The expression of the Notch signaling pathway in different breast cancer cell lines was detected by the qRT-PCR assay. (B) The expression of JAG1 in different breast cancer subtypes was analyzed by UALCAN database. (C) The correlation between JAG1 and the survival of breast cancer patients was analyzed by the Kaplan–Meier Plotter database. (D) The tumor volume in nude mice with injection of 231 and 231B into the mammary pad. (E) Tumors in situ and liver metastasis in nude mice. (F) HE staining for tumors in situ and liver metastasis. ns means “Not statistically significant” (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001).

Figure 2

JAG1 promoted angiogenesis in the TNBC microenvironment. (A–C) GeneMANIA database was used to analyze the relationship of JAG1, VEGFA, and DLL4. (D) Analysis of the correlation of JAG1 and VEGFA via the GEPIA database. (E) CCK-8 assay was performed to detect the effect of TNBC conditioned medium on HUVEC proliferation. (F) Matrigel plug assays to detect the effect of TNBC conditioned medium on HUVEC tubulogenesis ability. (G) qRT-PCR was conducted to detect the expression of VEGFA in HUVEC treated with TNBC conditioned medium (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001).

Figure 3

rJAG1 treatment promoted the exosome secretion of 231 cells. (A) Schematic diagram showed the extraction procedure of exosomes from 231-Blank, 231 treated with DAPT (10 μM), 231 treated with rJAG1 (100 ng/mL), and 231B. (B) Representative images of transmission electron microscope (TEM) analysis. (C, D) Particle size analysis to determine the size distribution (C) and particle concentration (D). (E) Western blotting to determine ALIX and CD63 protein levels of exosomes from indicated cells (^∗P < 0.05, ^∗∗P < 0.01).

Figure 4

JAG1 promotes exosome biogenesis and release through RAB11A and ALIX. (A) qRT-PCR to test RAB5, RAB7, RAB11A, RAB27A, RAB27B, and RAB35 expression in 231-Blank, 231 treated with DAPT, and 231 treated with rJAG1. (B) Western blotting was used to test ALIX and RAB11A protein expression in 231 treated by DAPT and rJAG1. Quantification of ALIX (C) and RAB11A (D) protein expression in (B). qRT-PCR to test lncRNA expression of SNHG16, MALAT1, and DANCR in cell (E) and exosomes (F) of 231-Blank, 231-DAPT, 231-rJAG1. (G) The uptake of tumor exosomes (Red) by HUVEC (Green) was detected by immunofluorescence assay (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001).

Figure 5

Exosomal MALAT1 promotes the angiogenesis of HUVEC through a cascade of miR-140-5p/JAG1/VEGFA signals. (A) Screening MALAT1-related miRNAs in breast cancer by Venn diagram. (B) qRT-PCR to verify the knockdown efficiency of the three interference fragments. (C) qRT-PCR to detect the expression of miR-140-5p in HUVEC. (D) KEGG pathway analysis of miR-140-5p. (E, F) qRT-PCR to detect JAG1 (E) and VEGFA (F) expression (^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001).

Figure 6

Experiments in vivo showed that JAG1 was correlated with angiogenesis in the TNBC microenvironment. (A) The procedures of animal experiments. (B) Images of plugs and immunohistochemical staining to detect CD31 protein expression level (white arrows showing typical microvessels). (C) Immunohistochemical staining to detect protein expression levels of CD31 and VEGF. (D) JAG1-mediated angiogenesis in the breast cancer microenvironment.

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