Abstract
MicroRNAs (miRNAs) are small endogenously produced RNAs, which regulate growth and development, and oncogenic miRNA regulate tumor growth and metastasis. Tumour-associated angiogenesis and lymphangiogenesis are processes involving the release of growth factors from tumour cells into the microenvioronemnt to communicate with endothelial cells to induce vascular propagation. Here, we examined the roles of cyclo-oxygenase (COX)-2 induced miR526b and miR655 in tumour-associated angiogenesis and lymphangiogenesis. Ectopic overexpression of miR526b and miR655 in poorly metastatic estrogen receptor (ER) positive MCF7 breast cancer cells resulted in upregulation of angiogenesis and lymphangiogenesis markers vascular endothelial growth factor A (VEGFA); VEGFC; VEGFD; COX-2; lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1); and receptors VEGFR1, VEGFR2, and EP4. Further, miRNA-high cell free conditioned media promoted migration and tube formation by human umbilical vein endothelial cells (HUVECs), and upregulated VEGFR1, VEGFR2, and EP4 expression, showing paracrine stimulation of miRNA in the tumor microenvironment. The miRNA-induced migration and tube formation phenotypes were abrogated with EP4 antagonist or PI3K/Akt inhibitor treatments, confirming the involvement of the EP4 and PI3K/Akt pathway. Tumour supressor gene PTEN was found to be downregulated in miRNA high cells, confirming that it is a target of both miRNAs. PTEN inhibits hypoxia-inducible factor-1 (HIF1α) and the PI3K/Akt pathway, and loss of regulation of these pathways through PTEN results in upregulation of VEGF expression. Moreover, in breast tumors, angiogenesis marker VEGFA and lymphangiogenesis marker VEGFD expression was found to be significantly higher compared with non-adjacent control, and expression of miR526b and miR655 was positively correlated with VEGFA, VEGFC, VEGFD, CD31, and LYVE1 expression in breast tumour samples. These findings further strengthen the role of miRNAs as breast cancer biomarkers and EP4 as a potential therapeutic target to abrogate miRNA-induced angiogenesis and lymphangiogenesis in breast cancer.
Highlights
Breast cancer is the deadliest and most prevalent cancer among women, being responsible for the greatest number of cancer-related deaths among women worldwide [1]
In breast tumors, angiogenesis marker vascular endothelial growth factor A (VEGFA) and lymphangiogenesis marker VEGFD expression was found to be significantly higher compared with non-adjacent control, and expression of miR526b and miR655 was positively correlated with VEGFA, VEGFC, VEGFD, CD31, and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1) expression in breast tumour samples
It was found that lymphangiogenesis marker, VEGFD, was significantly upregulated in MCF7-miR526b and MCF7-miR655 cell lines, while VEGFC and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) were marginally upregulated in MCF7-miR526b and significantly upregulated in MCF7-miR655 (Figure 1A)
Summary
Breast cancer is the deadliest and most prevalent cancer among women, being responsible for the greatest number of cancer-related deaths among women worldwide [1]. Upregulation of COX-2 is correlated with breast cancer disease progression, metastasis, and poor patient survival [3,4]. Production of PGE2 by COX-2 results in binding of PGE2 to four G-protein coupled PGE receptors, EP1-4, each of which have distinct signalling pathways [5]. Overproduction of PGE2 and activation of EP4 receptor results in many tumourigenesis-promoting phenotypes such as inactivation of host anti-tumour immune cells, enhanced tumour cell migration and invasion, stem-like cell (SLC) induction, and tumour-associated angiogenesis and lymphangiogenesis [6,8].
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