Breast cancer-derived exosomes regulate cell invasion and metastasis in breast cancer via miR-146a to activate cancer associated fibroblasts in tumor microenvironment

Abstract

Objective: To explore the effects of breast cancer (BC)-derived exosomes on invasion and migration of BC cells.

Methods: Exosomes (Exo-MA, Exo-M7, Exo-M1) were extracted from normal breast epithelial cells (MCF-10A), BC cells (MCF-7/MDA-MB-231) and BC cells with miR-146a overexpression or knockdown using multi-step differential centrifugation. Morphologies and sizes of exosomes were observed by transmission electron microscope (TEM) and particle size analysis respectively. BC mouse models were injected with DIR labeled Exo-MA, Exo-M7 or Exo-M1. The epithelial-mesenchymal transition (EMT) in BC cells was determined by PCR and Western blot. PKH67 labeled Exo-MA, Exo-M7 and Exo-M1 were incubated with NFs or MCF-7 to measure the activation of CAFs. Cell invasion and migration abilities were determined by scratch test and Transwell assay.

Results: Exo-MA, Exo-M7, Exo-M1 were successfully extracted with positive expressions of Alix, CD63 and TSG101. Contents of Ki67, N-cadherin, Vimentin and Snail-1 were increased but E-cadherin was decreased, compared to Exo-MA group. Exo-M7 or Exo-M1 could increase BC cell proliferation and enhance EMT in nude mouse. Exo-M7 and Exo-M1 could accelerate the transformation of NFs into CAFs and promote the recruitment of CAFs in MCF-7. Transfection of miR-146a could promote the transformation of NFs into CAFs and promote cell invasion and migration of MCF-7 cells. As a target gene of miR-146a, TXNIP could inhibit the activation of CAFs. miR-146a overexpression or TXNIP silence enhance the activation of Wnt signal pathway.

Conclusion: BC-derived exosomes promote the activation of CAFs through miR-146a/TXNIP axis to activate Wnt pathway, which in turn enhances invasion and metastasis of BC cells.

Keywords: Breast cancer-derived exosome; CAFs; Invasion and metastasis; TXNIP; Wnt pathway; miR-146a.