Breast cancer-secreted miR-939 downregulates VE-cadherin and destroys the barrier function of endothelial monolayers

Abstract

Exosomes-secreted microRNAs play an important role in metastatic spread. During this process breast cancer cells acquire the ability to transmigrate through blood vessels by inducing changes in the endothelial barrier. We focused on miR-939 that is predicted to target VE-cadherin, a component of adherens junction involved in vessel permeability. By in silico analysis miR-939 was found highly expressed in the basal-like tumor subtypes and in our cohort of 63 triple-negative breast cancers (TNBCs) its expression significantly interacted with lymph node status in predicting disease-free survival probability. We demonstrated, in vitro, that miR-939 directly targets VE-cadherin leading to an increase in HUVECs monolayer permeability. MDA-MB-231 cells transfected with a miR-939 mimic, released miR-939 in exosomes that, once internalized in endothelial cells, favored trans-endothelial migration of MDA-MB-231-GFP cells by the disruption of the endothelial barrier. Notably, when up taken in endothelial cells exosomes caused VE-cadherin down-regulation specifically through miR-939 as we demonstrated by inhibiting miR-939 expression in exosomes-releasing TNBC cells. Together, our data indentify an extracellular pro-tumorigenic role for tumor-derived, exosome-associated miR-939 that can explain its association with worse prognosis in TNBCs.

Exosome: emerging biomarker in breast cancer

“…Several miRNAs have been proved to be associated with breast tumor subtype and stage. Exosomal MiR-939 was found highly expressed in the basal-like tumor subtypes and associated with worse prognosis in triple-negative breast cancers [127]. One research has revealed that decreased several levels of exosomal miR-101 concentrations are associated with lymph-node positive cancer, and miR-373 levels were significantly upregulated in triple negative and ER and PR negative breast cancer patients [128].…”

Tumor‐derived exosomes enhance invasion and metastasis of salivary adenoid cystic carcinoma cells

“…Zhou et al demonstrated that breast cancer cell line MDA‐MB‐231‐derived exosomes could downregulate the expression of ZO‐1 in HMVECs, which could destroy the endothelial barrier and promote tumor metastasis . Di Modica M et al found that exosomal miR‐939 secreted in breast cancer downregulated VE‐cadherin and destroyed the barrier function of endothelial monolayers . Sukma Dewi et al identified a novel mechanism that miR‐142‐3p, a microRNA enriched in exosomes during acute cellular rejection, was transferred to endothelial cells and compromised endothelial barrier function via downregulation of RAB11FIP2 .…”

Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

“…Averaging more than one HKP as a loading control in order to lower the probability of error due to the afore‐stated issues (similar to quantitative RNA analysis). Consequential issues : The experimental time and costs are further extended and the above‐mentioned saturation issue is not resolved. Thoroughly searching and characterizing the appropriate HKPs prior to performing experiments Spiking‐in equal amounts of a standard protein into each sample (similar to quantitative RNA analysis). Consequential issues : Spike‐in protein(s) must be chosen, acquired, and tested, which is time consuming. Visualizing the total protein of the gel before transfer onto the blotting membrane Visualizing the total protein of a parallel gel, either separated by molecular weight or stacked into a single band …”

“…Spiking‐in equal amounts of a standard protein into each sample (similar to quantitative RNA analysis).…”