miR-142-3p attenuates breast cancer stem cell characteristics and decreases radioresistance in vitro.

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. ß-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-/low 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-/low stem cells, while Bod1, Oct4, and KLF4 were overexpressed. ß-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 ß-catenin in miR-142-3p-overexpressing cells. miR-142-3p might therefore help to target cancer stem cells.

Increased replication origin firing links replication stress to whole chromosomal instability in human cancer.

Chromosomal instability (CIN) is a hallmark of cancer and comprises structural CIN (S-CIN) and numerical or whole chromosomal CIN (W-CIN). Recent work indicated that replication stress (RS), known to contribute to S-CIN, also affects mitotic chromosome segregation, possibly explaining the common co-existence of S-CIN and W-CIN in human cancer. Here, we show that RS-induced increased origin firing is sufficient to trigger W-CIN in human cancer cells. We discovered that overexpression of origin firing genes, including GINS1 and CDC45, correlates with W-CIN in human cancer specimens and causes W-CIN in otherwise chromosomally stable human cells. Furthermore, modulation of the ATR-CDK1-RIF1 axis increases the number of firing origins and leads to W-CIN. Importantly, chromosome missegregation upon additional origin firing is mediated by increased mitotic microtubule growth rates, a mitotic defect prevalent in chromosomally unstable cancer cells. Thus, our study identifies increased replication origin firing as a cancer-relevant trigger for chromosomal instability.

Mild replication stress causes aneuploidy by deregulating microtubule dynamics in mitosis.

Chromosomal instability (CIN) causes structural and numerical chromosome aberrations and represents a hallmark of cancer. Replication stress (RS) has emerged as a driver for structural chromosome aberrations while mitotic defects can cause whole chromosome missegregation and aneuploidy. Recently, first evidence indicated that RS can also influence chromosome segregation in cancer cells exhibiting CIN, but the underlying mechanisms remain unknown. Here, we show that chromosomally unstable cancer cells suffer from very mild RS, which allows efficient proliferation and which can be mimicked by treatment with very low concentrations of aphidicolin. Both, endogenous RS and aphidicolin-induced very mild RS cause chromosome missegregation during mitosis leading to the induction of aneuploidy. Moreover, RS triggers an increase in microtubule plus end growth rates in mitosis, an abnormality previously identified to cause chromosome missegregation in cancer cells. In fact, RS-induced chromosome missegregation is mediated by increased mitotic microtubule growth rates and is suppressed after restoration of proper microtubule growth rates and upon rescue of replication stress. Hence, very mild and cancer-relevant RS triggers aneuploidy by deregulating microtubule dynamics in mitosis.