Epithelial cancers such as breast carcinomas and their metastatic progression are mainly directed by a phenomenon referred to as epithelial to mesenchymal transition (EMT) [1, 2]. As well described in several reviews, EMT is supported by the same transcription factors (TFs) including ZEB factors and the Snail family of zinc finger proteins both during embryonic development and the metastatic cascade [1, 3–5]. In addition, specific microRNAs (miRNAs) including miR-206, miR-221/222, miR-200, miR-141, miR-203, miR-130a, have been shown to regulate EMT [6–11].
Mounting evidence indicates that the acquisition of an aggressive cancer phenotype through EMT, as well as other cellular events, may be understood by evaluating the regulatory interplay between TFs and miRNAs [12, 13]. Therefore, recent studies have investigated the interactions among specific miRNAs, TFs and target genes associated with this phenomenon. Direct evidence of these circuits in EMT is still little. Some specific networks have been described including miR-203 – Snai1 , a self-reinforcing loop miR-1/miR-200 via Slug , miR-200/miR-192 – p53 , miR-221/222 – TRPS1 , p53/miR-34 axis , and ZEB/miR-200 .
To investigate the key regulatory networks underlying EMT in breast cancer, we evaluated a potential correlation between Slug (SNAI2) transcription factor and miR-221. The ability of miR-221 and Slug to promote EMT and induce invasiveness in breast cancer cell lines has been documented, but crosstalk between these molecules has not been characterized [3, 17, 20].
Slug is a member of the Snail family of zinc-finger transcription factors, and, together with Snail (SNAI1), acts as a master regulator of EMT. Various studies over the past several years have documented the involvement of Slug in human cancers including leukemias , osteosarcoma , esophageal carcinomas , and breast cancers [3, 24], where Slug expression is strongly correlated with the loss of E-cadherin. Multiple lines of evidence suggest that Slug can be considered a marker of malignancy as well as an attractive target for therapeutic modulation of invasiveness in the treatment of specific cancers [25–28].
miR-221 is often overexpressed in aggressive cancers, increases cell proliferation and protects cancer cells against different apoptotic stimuli [29–31]. Recently, the expression level of miR-221 has been significantly associated with Estrogen Receptor alpha (ERα) status in breast cancer, and several studies have demonstrated that miR-221 directly targets ERα [9, 32, 33]. Breast tumors from patients with high miR-221 plasma levels tend to be ERα-negative, more aggressive and show poorer clinical outcomes than ERα positive cancers . In addition, ERα signaling has been correlated with Slug, and at least two different mechanisms showed that ERα decreases Slug expression [35–37].
In this study, we knocked down Slug and miR-221 in ERα-negative breast cancer cells, MDA-MB-231. We determined a functional correlation between these two molecules demonstrating “in vivo” interaction between Slug and miR-221. Rescue experiments with ectopic expression of miR-221, analysis of the expression of genes involved in breast cancer phenotype, and wound healing assay, suggested that the largest contribution to the invasion ability of the cells and their aggressive phenotype comes from Slug rather than miR-221.