Overexpression of the miR-141/200c cluster promotes the migratory and invasive ability of triple-negative breast cancer cells through the activation of the FAK and PI3K/AKT signaling pathways by secreting VEGF-A

Abstract

BackgroundThe role of microRNA-200 (miR-200) family members in the migration and invasion of breast cancer is controversial. This study investigated the mechanisms by which the miR-200 family members modulated the migratory and invasive abilities of an aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231.MethodsThe miR-200 family (miR-200b/200a/429 and miR-141/200c clusters) and green fluorescence protein (GFP) were transduced into MDA-MB-231 cells using a lentiviral system. Stable cells highly expressing the miR-200 family and GFP were isolated by puromycin selection and fluorescence-activated cell sorting. Gene expression was evaluated using real-time polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR). The migratory and invasive abilities were assessed using trans-well and wound-healing assays. The secreted cytokines and growth factors in cultured media were quantified using a Bio-Plex200 multiplex array system. Western blot assays and immunofluorescence staining were conducted to investigate miR-200 family-regulated signaling pathways. The entire dataset obtained in this study was statistically evaluated using a one-way ANOVA followed by a t-test.ResultsThe stable overexpression of the miR-200b/200a/429 or miR-141/200c cluster suppressed cell growth and significantly increased migration and invasion of MDA-MB-231 cells. miR-141/200c overexpression was more effective in decreasing cell growth and promoting migration and invasion of MDA-MB-231 cells than was miR-200b/200a/429 overexpression. In addition, the overexpression of the miR-200b/200a/429 or miR-141/200c cluster led to an increase in the phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT). Chemical inhibitors of FAK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT suppressed the migration and invasion of MDA-MB-231 cells that was enhanced by the overexpression of the miR-200b/200a/429 or miR-141/200c cluster. Compared to the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells, the miR-141/200c cluster-transduced MDA-MB-231 cells exhibited a significant increase in vascular endothelial growth factor (VEGF)-A secretion and integrin-alphaV (integrin-αV) expression. Treatment with an anti-VEGF-A-neutralizing antibody inhibited the increase in migration and invasion in both the miR-200b/200a/429- and miR-141/200c-transduced MDA-MB-231 cells but significantly reduced the phosphorylation of FAK and AKT in only the miR-141/200c cluster-transduced MDA-MB-231 cells.ConclusionsTaken together, our data demonstrate a mechanism in which the miR-141/200c cluster, through FAK- and PI3K/AKT-mediated signaling by means of increased VEGF-A secretion, promotes the migratory and invasive abilities of MDA-MB-231 cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2620-7) contains supplementary material, which is available to authorized users.

TBCRC 002: a phase II, randomized, open-label trial of preoperative letrozole with or without bevacizumab in postmenopausal women with newly diagnosed stage 2/3 hormone receptor-positive and HER2-negative breast cancer

“…The hsa-miR-141-5p is the less abundant product from the same pre-miRNA with hsa-miR-141-3p and may in fact represent a surrogate measurement of hsa-miR-141-3p. Overexpression of hsa-miR-141-3p and its family member hsa-miR-200c-3p in MDA-MB-231 breast cancer cells promotes significant migration and invasion and enhances VEGF-A secretion [50]. VEGF-A neutralizing antibodies abrogate these phenotypic consequences conferred by hsa-miR-141-3p and hsa-miR-200c-3p overexpression [50].…”

“…Overexpression of hsa-miR-141-3p and its family member hsa-miR-200c-3p in MDA-MB-231 breast cancer cells promotes significant migration and invasion and enhances VEGF-A secretion [50]. VEGF-A neutralizing antibodies abrogate these phenotypic consequences conferred by hsa-miR-141-3p and hsa-miR-200c-3p overexpression [50]. hsa-miR-449a targets cysteine-rich intestinal protein 2 (CRIP2) mRNA, a transcription factor and a tumor suppressor [51].…”

Effect of miR-146a-5p on proliferation and metastasis of triple-negative breast cancer via regulation of SOX5

“…Jia et al (31) reported that miR-490-3p could suppress the growth and invasion of TNBC by inhibiting tankyrase-2 expression. Choi et al (32) indicated that miR-141/200c was involved in TNBC migration and invasion via activating the focal adhesion kinase and phosphoinositide 3-kinase/AKT signaling pathways. miRNA-454 was revealed to be associated with poor prognosis in TNBC (17) and miRNA-200b could suppress TNBC metastasis by regulating protein kinase C α (19).…”

microRNA-200c/141 upregulates SerpinB2 to promote breast cancer cell metastasis and reduce patient survival

“…Our preliminary studies showed that miR-200c/141 cluster overexpression in a triple-negative (TN) human BCC line, MDA-MB-231, which lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal receptor 2 (HER2) expression, enhances the migration and invasion abilities [12], upregulates SerpinB2 and promotes lymph node (LN) and lung metastasis in mouse models. SerpinB2, also known as plasminogen activator inhibitor-2 (PAI-2), inhibits urokinase plasminogen activator (uPA) activity [13–15].…”