Angiogenic role of miR-20a in breast cancer

<div><p>Background</p><p>Angiogenesis is a key process for tumor progression and a target for treatment. However, the regulation of breast cancer angiogenesis and its relevance for clinical resistance to antiangiogenic drugs is still incompletely understood. Recent developments on the contribution of microRNA to tumor angiogenesis and on the oncogenic effects of miR-17-92, a miRNA cluster, point to their potential role on breast cancer angiogenesis. The aim of this work was to establish the contribution of miR-20a, a member of miR-17-92 cluster, to tumor angiogenesis in patients with invasive breast carcinoma.</p><p>Methods</p><p>Tube-formation in vitro assays with conditioned medium from MCF7 and MDA-MB-231 breast cancer cell lines were performed after transfection with miR-20a and anti-miR20a. For clinical validation of the experimental findings, we performed a retrospective analysis of a series of consecutive breast cancer patients (n = 108) treated with neoadjuvant chemotherapy and with a full characterization of their vessel pattern and expression of angiogenic markers in pre-treatment biopsies. Expression of members of the cluster miR-17-92 and of angiogenic markers was determined by RT-qPCR after RNA purification from FFPE samples.</p><p>Results</p><p><i>In vitro</i> angiogenesis assays with endothelial cells and conditioned media from breast cancer cell lines showed that transfection with anti-miR20a in MDA-MB-231 significantly decreased mean mesh size and total mesh area, while transfection with miR-20a in MCF7 cells increased mean mesh size. MiR-20a angiogenic effects were abrogated by treatment with aflibercept, a VEGF trap. These results were supported by clinical data showing that mir-20a expression was higher in tumors with no estrogen receptor or with more extensive nodal involvement (cN2-3). A higher miR-20a expression was associated with higher mean vessel size (<i>p</i> = 0.015) and with an angiogenic pattern consisting in larger vessels, higher VEGFA expression and presence of glomeruloid microvascular proliferations (<i>p</i><0.001). This association was independent of tumor subtype and VEGFA expression.</p><p>Conclusions</p><p>Transfection of breast cancer cells with miR-20a induces vascular changes in endothelial tube-formation assays. Expression of miR-20a in breast invasive carcinomas is associated with a distinctive angiogenic pattern consisting in large vessels, anomalous glomeruloid microvascular proliferations and high VEGFA expression. Our results suggest a role for miR-20a in the regulation of breast cancer angiogenesis, and raise the possibility of its use as an angiogenic biomarker.</p></div

Angiogenic <i>in vitro</i> effects of miR-20a in breast cancer cell lines (MCF7 and MDA-MB-231).

<p>Angiogenic <i>in vitro</i> effects of miR-20a in breast cancer cell lines (MCF7 and MDA-MB-231).</p

Association of miR-20a expression with vascular characteristics of breast cancer.

<p>(A) Association between high (over the median value) MVS and miR-20a expression in breast cancer (U Mann-Whitney; p = 0.013). (B) Association of high miR-20a (<i>p</i><0.001) and VEGFA (<i>p</i> = 0.002) expression with a high-risk angiogenic profile (GMP+/MVS high).</p

Effect of VEGFA exposure on miR-20a expression by MCF7 cells.

<p>No significant change of MCF7 miR-20a expression (relative to snU6 expression) after exposure to low (0.5 ng/mL) or high (10 ng/mL) recombinant human VEGFA concentrations (24 and 48 hours).</p

Comparison of angiogenic effects of conditioned media from MDA-MB-231 and MCF7 on endothelial cells.

<p>(A) Higher total tubule length induced by MCF-7 conditioned media (<i>p</i> = 0.001). (B) No significant differences in total mesh area. (C) Significantly larger mesh size (<i>p</i> = 0.005) induced by conditioned media from MDA-MB-231 breast carcinoma cells. All experiments were repeated three times (with eight biological replicates per condition). Units of length (tubule length and mesh size) and area (total mesh area) correspond to x10⁴ pixels.</p

Effect of mir-20a changes on EA.hy926 capillary-like structure formation.

<p>(A) Total mesh area not modified by miR-20a overexpression in MCF7 and MDA-MB-231 breast carcinoma cells. (B) Transfection of MDA-MB-231 with anti-miR-20a induced a significant decrease in total mesh area (<i>p</i> = 0.001). (C) Mean mesh size of capillary-like structures formed by EA.hy926 was significantly increased by miR-20a transfection of MCF7 cells (<i>p</i> = 0.046). (D) A decrease of mean mesh size induced by anti-miR-20a transfection of MDA-MB-231 (<i>p</i> = 0.005). (E) No significant changes in total tubule length induced by miR-20a transfection. (F) Anti-miR20a transfection of MDA-MB-231 induced a slight, although significant (<i>p</i> = 0.049), increase of total tubule length. All experiments were repeated three times (with eight biological replicates per condition). Units of length (tubule length and mesh size) and area (total mesh area) correspond to x10⁴ pixels.</p

Correlation between miR-20a expression and the angiogenic characteristics of breast carcinomas.

<p>Correlation between miR-20a expression and the angiogenic characteristics of breast carcinomas.</p

Association of miR-20a expression and clinical-pathological characteristics of breast cancer patients (n = 95).

<p>Association of miR-20a expression and clinical-pathological characteristics of breast cancer patients (n = 95).</p