MicroRNAs miR-221 and miR-222: a new level of regulation in aggressive breast cancer

MicroRNAs (miRNAs) are crucial in the initiation and progression of tumors. A recent study has reported that the miRNAs miR-221 and miR-222 are involved in the promotion of an aggressive basal-like phenotype in breast cancer, functioning downstream of the RAS pathway and triggering epithelial-to-mesenchymal transition. These new insights into the roles of miR-221/222 in breast cancer metastasis, drug resistance and RAS pathways could potentially have applications in medical practice.


MicroRNAs and their role in aggressive cancers
The past decade has shown remarkable advances in our knowledge of microRNAs (miRNAs) and their functional importance in a wide range of developmental and physio logical processes. These small endogenous noncoding RNAs, which posttranscriptionally regulate the expres sion of several hundred genes, have recently been shown to have an important role in various human cancers [1]. There is growing evidence that miRNAs are critical in cancer predisposition, initiation and progression, with several miRNAs demonstrating oncogenic and/or tumor suppressor properties. In the past decade, several studies have focused on exploring the connection of miRNAs with tumor proliferation, invasion, migration and stem cell behavior. Because of the widespread dysregulation of miRNAs in all types of tumors, their expression profiles are useful diagnostic and prognostic signature classifiers. miRNA expression analysis has confirmed that specific miRNAs show differential expression patterns among normal and tumor tissues. For example, the miRNA miR 10b is specifically overexpressed only in metastatic breast cancers but downregulated in primary breast tumors [2], and miR335 and miR126 are lost in relapsed breast cancers with distal metastasis [3]. miRNAs are also found to be specifically overexpressed in resistant tumors with poor prognosis. Thus, specific miRNAs are involved in different phenotypes of aggressive cancers, providing an important rationale for their investigation as therapeutic targets.

New insights into the role of miR-221 and miR-222 as regulators in basal-like breast cancer
A recent study published in Science Signaling by Stinson et al. [4] identified miR221 and miR222 as regulators of epithelialtomesenchymal transition (EMT) using high throughput analysis and a human in vitro cell culture model. The study [4] reported miR221/222 to be specifically expressed in basallike breast cancer and to act downstream of the oncogenic RASRAFMEK path way. These miRNAs mediate metastasis through increased invasion and migration by targeted repression of tricho rhinophalangeal syndrome type 1 protein (TRPS1), which in turn increases the abundance of the EMT promoting protein zinc finger Eboxbinding homeobox 2 (ZEB2). Stinson et al. [4] further explore the involve ment of miRNAs in promotion of clinically aggressive meta static breast cancer ( Figure 1).
The study by Stinson and colleagues [4] is significant because it substantiates the concept that specific miRNAs can promote transformation to more aggressive cancer phenotypes with poor prognosis. The authors performed miRNA microarray screening using multiple cell lines representing luminallike and basallike subtypes of breast cancer, and identified miR221/222 to be differen tially expressed. The overexpression of miR221/222 in estrogen receptor (ER)negative breast cancer has been reported previously [5], implying a role of miR221/222 in predisposition of ER status in aggressive breast cancers. Transfection of miR221/222 synthetic mimetics into an immortalized and nontransformed mammary cell line (MCF10A) resulted in a significant enrichment of genes involved in EMT and the RAS pathway. miR221/222 induced EMTlike characteristics, with enhanced invasion and migration, increased levels of the mesenchymal marker vimentin and reduced levels of the Abstract MicroRNAs (miRNAs) are crucial in the initiation and progression of tumors. A recent study has reported that the miRNAs miR-221 and miR-222 are involved in the promotion of an aggressive basal-like phenotype in breast cancer, functioning downstream of the RAS pathway and triggering epithelial-to-mesenchymal transition. These new insights into the roles of miR-221/222 in breast cancer metastasis, drug resistance and RAS pathways could potentially have applications in medical practice. epithelial marker Ecadherin. Consequently, treatment of MDAMB231 cells (a metastatic basallike breast cancer cell line) with miR221/222 inhibitors induced a reverse phenotype with mesenchymaltoepithelial transition (MET)like characteristics [4]. The reversal to MET by inhibiting miR221/222 is an important finding that can be therapeutically exploited, but more in vivo studies will be required before any significance can be established.
In addition, the authors [4] confirmed the correlation between increased miR221/222 levels and vimentin and Ecadherin expression in primary breast cancer samples. Previous reports [6] have established that miR200 family members (miR200a, miR200b, miR200c, miR141 and miR429) are downregulated in aggressive breast cancers. Thus, a combination of a miR221/222 and miR200 signa ture with other EMT markers such as vimentin and Ecadherin can provide a clinical predictive model for identifying patients with poor prognosis.
The authors [4] further studied the regulation of miR221/222 and reported a novel association between miRNA and the EMTpromoting RASRAFMEK signal ing pathway. They identified an upstream binding site for FOSL1, a member of the FOS family of transcription factors and, using chromatin immunoprecipitation and luciferase assays, they showed that the transcription of miR221/222 is regulated by FOSL1. They identified miR221/222 as downstream of the epidermal growth factor receptor (EGFR)RASRAFMEK pathway by show ing that treatment with EGFR and MEK inhibitors knocked down the expression of both the miRNAs and FOSL1. TRPS1, a transcription factor with decreased abun dance in clinically aggressive breast cancers, was identified as the direct target of miR221/222; and in turn, TRPS1 targeted the EMTsuppressor protein ZEB2. Thus, repression of TRPS1 by miR221/222 caused an increase in the levels of ZEB2, which directly inhibits E cadherin and upregulates vimentin, promoting EMT. These results together reveal an intricate association between miR221/222 and the EGFRRASRAFMEK cascade ( Figure 1).
In addition, the paper [4] highlights the functional significance of miRNAs in the process of EMT and stem cell formation in breast cancer [7]. The role of miR335, miR206 and miR126 in suppressing breast cancer meta stasis was initially described by Tavazoie et al. [3], who reported that selective loss of miRNA expression induced metastatic potential. Other studies reported increased expression of miR10b, miR21, miR9 and miR29b in malignant breast cancers [8], whereas the miR200 family is downregulated in metastatic cancers [6]. These miRNAs function at different stages of the multistep metastatic cascade, by acting as direct downstream effectors of oncogenes, sensitizing tumor cells to signals from the tumor microenvironment, or altering expression of EMTrelated markers, such as Ecadherin and vimentin [7,8]. The study by Stinson et al. [4] elucidates a further layer of regulation that controls the EMTdriven cellular phenotype.
The study provides a logical association between the earlier studies demonstrating the importance of miR221/ 222 in metastatic breast cancers and drug resistance. An initial study by Zhao et al. [5] showed that miR221/222 directly targeted ERα and that their overexpression in breast cancer contributed to progression of the more aggressive ERnegative basal phenotype in a subset of breast cancers. miR221/222 have also been shown to act as oncogenes by repressing cell cycle inhibitor proteins p27/Kip1 and p57 and thus facilitating cell proliferation and selfrenewal [9]. These functions of miR221/222 both contribute to tamoxifen resistance in basaltype MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; p27 Kip1 , cyclin-dependent kinase inhibitor p27 Kip1 ; SOS, Son of sevenless homolog; TRPS1, tricho-rhino-phalangeal syndrome type 1; ZEB, zinc finger E-box-binding homeobox.  [4] adds an additional aspect to the story and links the basalsubtypespecific expression of miR221/222 with increased abundance of mesenchymal markers and an enhanced EMT phenotype. It positions miR221/222 downstream of the RAS path way, targeting TRPS1 and thus increasing the abundance of EMTpromoter ZEB2; this provides a clearer picture of the mechanism of miR221/222 in promoting aggressive breast cancer phenotypes with chemoresistance and poor prognosis.
Finally, another important finding [4] is the involve ment of different miRNAs in different phenotypes of breast cancer. Previous studies have demonstrated that miR200 family miRNAs act as EMT repressors by inhibiting ZEB1 protein; their expression levels are greatly reduced in metastatic breast cancers compared with normal tissues [6], and a negative feedback loop connects miR200 family expression, ZEB1 expression and EMT occurrence [10]. Stinson et al. [4] discuss possible involvement of the miR221/222 family in this feedback inhibition of miR200 family members: they may act by relieving the TRPS1mediated repression of ZEB2 protein and committing cells to EMT. Thus, regulation of the miR200 and miR221/222 families reflects a fine balance linking miRNAs as stimulators and inhibitors of metastasis and identifies important molecular targets that could potentially control a specific tumor behavior.

The challenges ahead
In conclusion, these new findings [4] add yet another significant dimension to the regulation of the EMT phenomenon. The report presents a critical link between the EMTrelated miRNAs miR221 and miR222 and the process of EMT and metastasis in aggressive breast cancers. The importance of Stinson et al.'s paper [4] rests in the identification of clinically useful miRNAs that act as regulators of EMT and represent not only a novel therapeutic strategy for the treatment of advanced breast cancer, but also potential use as biomarkers for clinically aggressive breast cancers with poor prognosis. Direct functional screens of global miRNA expression profiles in transformed tumor cells might help illuminate involvement of other miRNAs in EMT. Furthermore, the in vivo phenotypic consequences of miR221/222 gain and loss of function in mouse models will help evaluate the practicality of miR221/222 expression manipulation for therapy of human breast cancers. Identification of the as yet unknown targets for miR221/222 also remains a key objective for the design of novelmiRNAbased therapy. Overexpressed miRNAs can be effectively inhibited in vivo using antisense oligonucleotides with specific modifications, such as cholesterolconjugated antagomiRs, locked nucleic acid oligonucleotides or 2'Omethyl modifications. However, several challenges regarding the stability and delivery strategies of these antimiRs still need to be answered before they can be useful as therapeutic agents. More experimental evidence establishing that correction of a specific miRNA altera tion can reestablish homeostasis and stabilize tumori genesis is required. In this regard, the potential conse quences of inducing MET in normal tissues during the course of miR221/222 therapy also need to be deter mined. Thus, miRNAs hold great potential as therapeutic targets for new drug development; however, more insights into the functional consequences and efficiency of miRNA inhibition for patient survival need to be addressed.