ACE2 inhibits breast cancer angiogenesis via suppressing the VEGFa/VEGFR2/ERK pathway

Abstract

Background: Breast cancer angiogenesis is key for metastasis and predicts a poor prognosis. Angiotensin-converting enzyme 2 (ACE2), as a member of the renin-angiotensin system (RAS), was reported to restrain the progression of hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) through inhibiting angiogenesis. However, the relationship between ACE2 and breast cancer angiogenesis remains unclear.

Methods: The prognosis and relative gene selection were analysed using the GEPIA, GEO, TCGA and STRING databases. ACE2 expression in breast cancer tissue was estimated by reverse transcription-quantitative polymerase chain reaction (qPCR). Breast cancer cell migration, proliferation and angiogenesis were assessed by Transwell migration, proliferation, tube formation, and wound healing assays. The expression of vascular endothelial growth factor A (VEGFa) was detected by qPCR and Western blotting. The phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), mitogen-activated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated protein kinase 1/2 (ERK1/2) was examined by Western blotting. Breast cancer metastasis and angiogenesis in vivo were measured using a zebrafish model.

Results: ACE2 was downregulated in breast cancer patients. Patients with higher ACE2 expression had longer relapse-free survival (RFS). In vitro, ACE2 inhibited breast cancer migration. Meanwhile, ACE2 in breast cancer cells inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, tube formation and migration. In the zebrafish model, ACE2 inhibited breast cancer cell metastasis, as demonstrated by analyses of the number of disseminated foci and the metastatic distance. Neo-angiogenesis was also decreased by ACE2. ACE2 downregulated the expression of VEGFa in breast cancer cells. Furthermore, ACE2 in breast cancer cells inactivated the phosphorylation of VEGFR2, MEK1/2, and ERK1/2 in HUVECs.

Conclusions: Our findings suggest that ACE2, as a potential resister to breast cancer, might inhibit breast cancer angiogenesis through the VEGFa/VEGFR2/ERK pathway.

Trial registration: Retrospectively registered.

Keywords: ACE2; Angiogenesis; Breast cancer; ERK; VEGFR2; VEGFa.

Fig. 1

ACE2 is downregulated in breast cancer tissue, and this downregulation is associated with worse prognosis. (a) Expression of ACE2 in breast cancer and normal tissues in the GEPIA database (n = 1197). (b) Expression of ACE2 mRNA in breast cancer and paired normal tissues (n = 29). (c) Kaplan-Meier curve of RFS stratified by the ACE2 level using the GEO database (n = 3951)

Fig. 2

ACE2 inhibits breast cancer cell migration. (a) Basal expression of ACE2 in breast cancer cell lines. (b) mRNA level of ACE2 in MDA-MB-231 cells overexpressing ACE2 and ACE2-knockdown MCF-7 cells. (c) Protein levels of ACE2 in MDA-MB-231 cells overexpressing ACE2 and ACE2-knockdown MCF-7 cells. (d) Transwell migration assay of transfected MDA-MB-231 and MCF-7 cells. (e) CCK8 proliferation assay of transfected MDA-MB-231 and MCF-7 cells. Microscope magnification: 50×. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

ACE2 inhibits breast cancer angiogenesis in vitro. (a) Bubble plot of ACE2 and breast cancer angiogenesis obtained from the GO enrichment analysis. (b) Proliferation of HUVECs after 72 h in the TCM from transfected MDA-MB-231 and MCF-7 cells. (c) Tube formation ability of HUVECs cultivated for 6 h in the TCM from transfected MDA-MB-231 and MCF-7 cells. (d) Wound healing ability of HUVECs after 48 h in the TCM from transfected MDA-MB-231 and MCF-7 cells. Microscope magnification: 50×. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 4

ACE2 inhibits breast cancer angiogenesis in vivo. (a) Equal number of Dil-labelled transfected MDA-MB-231 and MCF-7 cells were microinjected into the perivitelline space of embryos at 48 h post-fertilization. Thick arrows point to the injection sites. BF: bright field; RF: rhodamine fluorescence. (b) Dissemination and metastasis of the tumour cells in the zebrafish at 24 h post-injection (observed under a fluorescence microscope) and quantification of the number of disseminated foci and maximal metastatic distance. Small arrowheads point to the disseminated and metastatic tumour foci (MDA-MB-231, n = 9; MCF-7, n = 6). (c) Vasculature and neo-angiogenesis surrounding the primary tumour and metastasis sites in the zebrafish at 24 h post-injection (observed under a confocal microscope). Thin arrows point to newly formed tumour vessels. Fluorescence microscope magnification: 50×. Confocal microscope magnification: 200×. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5

ACE2 inhibits the VEGFa/VEGFR2/ERK pathway to suppress breast cancer angiogenesis. (a) Heat map of the correlation of ACE2 with genes participating in breast cancer angiogenesis. (b) UpSet plot of the intersection of angiogenetic cytokines and ACE2 in breast cancer. (c) PPI plot of the correlation of ACE2 and potentially related genes. (d) KEGG pathway enrichment of ACE2 and VEGFa. (e) mRNA level of VEGFa in transfected MDA-MB-231 and MCF-7 cells. (f) Protein levels of VEGFa in transfected MDA-MB-231 and MCF-7 cells. (g) Phosphorylation level of ERK1/2 in transfected MDA-MB-231 and MCF-7 cells determined by Western blot analysis. (h) Western blot analysis of the phosphorylation level of VEGFR2, MEK1/2, and ERK1/2 in HUVECs cultivated for 24 h in the TCM of the transfected tumour cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6

Schematic diagram summarizing the signalling pathway through which ACE2 inhibits breast cancer angiogenesis