Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis. It has been shown to occur in resistance to a range of cancer therapies, including tamoxifen. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. To investigate the role of miRNAs in the development of resistance to tamoxifen as well as accompanying EMT-like properties, we established a tamoxifen-resistant (TamR) model by continually exposing MCF-7 breast cancer cells to tamoxifen. In addition to the molecular changes known to be involved in acquired tamoxifen resistance, TamR cells displayed mesenchymal features and had increased invasiveness. Genome-wide miRNA microarray analysis revealed that miRNA-375 was among the top downregulated miRNAs in resistant cells. Re-expression of miR-375 was sufficient to sensitize TamR cells to tamoxifen and partly reversed EMT. A combination of mRNA profiling, bioinformatics analysis and experimental validation identified metadherin (MTDH) as a direct target of miR-375. Knockdown of MTDH partially phenocopied the effects of miR-375 on the sensitivity to tamoxifen and the reversal of EMT. We observed an inverse correlation between the expression of miR-375 and its target MTDH in primary breast cancer samples, implying the pathological relevance of targeting. Finally, tamoxifen-treated patients with higher expression of MTDH had a shorter disease-free survival and higher risk of relapse. As most cancer-related deaths occur because of resistance to standard therapies and metastasis, re-expression of miR-375 or targeting MTDH might serve as potential therapeutic approaches for the treatment of TamR breast cancer.

No abstract

Tamoxifen is an endocrine therapy which is administered to up to 70% of all breast cancer patients with oestrogen receptor alpha (ERα) expression. Despite the initial response, most patients eventually acquire resistance to the drug. MicroRNAs (miRNAs) are a class of small non-coding RNAs which have the ability to post-transcriptionally regulate genes. Although the role of a few miRNAs has been described in tamoxifen resistance at the single gene/target level, little is known about how concerted actions of miRNAs targeting biological networks contribute to resistance. Here we identified the miRNA cluster, C19MC, which harbours around 50 mature miRNAs, to be up-regulated in resistant cells, with miRNA-519a being the most highly up-regulated. We could demonstrate that miRNA-519a regulates tamoxifen resistance using gain- and loss-of-function testing. By combining functional enrichment analysis and prediction algorithms, we identified three central tumour-suppressor genes (TSGs) in PI3K signalling and the cell cycle network as direct target genes of miR-519a. Combined expression of these target genes correlated with disease-specific survival in a cohort of tamoxifen-treated patients. We identified miRNA-519a as a novel oncomir in ER+ breast cancer cells as it increased cell viability and cell cycle progression as well as resistance to tamoxifen-induced apoptosis. Finally, we could show that elevated miRNA-519a levels were inversely correlated with the target genes' expression and that higher expression of this miRNA correlated with poorer survival in ER+ breast cancer patients. Hence we have identified miRNA-519a as a novel oncomir, co-regulating a network of TSGs in breast cancer and conferring resistance to tamoxifen. Using inhibitors of such miRNAs may serve as a novel therapeutic approach to combat resistance to therapy as well as proliferation and evasion of apoptosis in breast cancer. Published by John Wiley & Sons, Ltd. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.