MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3’-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer.

Effectively targeting cancer stem cells, a subpopulation of tumorigenic, aggressive, and radioresistant cells, holds therapeutic promise. However, the effects of the microRNA miR-142-3p, a small endogenous regulator of gene expression on breast cancer stem cells, have not been investigated. This study identifies the influence of miR-142-3p on mammary stemness properties and breast cancer radioresistance to establish its role in this setting. miR-142-3p precursor transfection was performed in MDA-MB-468, HCC1806, and MCF-7 cells, and stem cell markers CD44, CD133, ALDH1 activity and mammosphere formation were measured. β-catenin, the canonical wnt signaling effector protein, was quantified by Western blots and cell fluorescence assays both in miR-142-3p-overexpressing and anti-miR-142-3p-treated cells. Radiation response was investigated by colony formation assays. Levels of BRCA1, BRCA2, and Bod1 in miR-142-3p-overexpressing cells as well as expression of miR-142-3p, Bod1, KLF4, and Oct4 in sorted CD44/CD24 cells were determined by quantitative polymerase chain reaction. miR-142-3p overexpression resulted in a strong decline in breast cancer stem cell characteristics with a decrease in CD44, CD133, ALDH1, Bod1, BRCA2, and mammosphere formation as well as reduced survival after irradiation. miR-142-3p expression was strongly reduced in sorted CD44/CD24 stem cells, while Bod1, Oct4, and KLF4 were overexpressed. β-catenin levels strongly decreased after miR-142-3p overexpression, but not after anti-miR-142-3p treatment. We conclude that miR-142-3p downregulates cancer stem cell characteristics and radioresistance in breast cancer, mediated by a reduced role of β-catenin in miR-142-3p-overexpressing cells. miR-142-3p might therefore help to target cancer stem cells.

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