From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment

MicroRNAs (miRNAs or miRs) are a family of small non-coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) or tumor suppressor miRNAs and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancers; ii) the oncosuppressor p27 Kip1 , a validated miR-221 target, is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27 Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the down-regulation of Slug and the upregulation of p27 Kip1 .


Introduction
MicroRNAs (miRNAs or miRs) (Fig. 1) are a family of small (19 to 25 nucleotides in length) non-coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences (1)(2)(3)(4)(5). Since their discovery and first characterization, the number of miRNA sequences deposited in the miRBase databases is increasing (6)(7)(8)(9)(10). Considering that a single miRNA can target several mRNAs and a single mRNA may contain several signals for miRNA recognition in the 3'UTR sequence, it has been calculated that at least 10-40% of human mRNAs are a target for miRNAs (11)(12)(13). Hence, the identification of validated targets of miRNAs is of great importance.
This specific field of miRNA research has confirmed that the complex networks constituted by miRNAs and mRNA targets coding for structural and regulatory proteins lead to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis (14)(15)(16). The low expression of a given miRNA is expected to be linked with a potential expression of target mRNAs. Conversely, the high expression of miRNAs is expected to negatively affect the biological functions of target mRNAs (1)(2)(3)(4)(5).
A similar tumor-promoting activity has been exhibited by miR-221 and miR-222, which can stimulate the proliferation of human prostate carcinoma cell lines following the inhibition of the expression of the tumor suppressor p27 Kip1 (41).
An opposite effect on tumor development has been displayed by other miRNAs; for instance miR-31 expression levels inversely correlate with the metastatic ability of breast tumor cell lines and the inhibition of miR-31 promotes metastasis. Another study revealed that miR-31 blocks several steps of metastasis, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization (42). Taken together, these data demonstrate that miRNAs play a double role in cancer, behaving both as oncogenes or tumor suppressor genes.
Another very interesting feature of miRNAs has been found by studying cancer-associated miRNAs in different experimental model systems; cancer-specific miRNAs are present in extracellular body fluids and may play a crucial role in the cross-talk between cancer cells and surrounding normal cells (47)(48)(49)(50)(51)(52). Of note, evidence of the presence of miRNAs in serum, plasma and saliva supports their potential as an additional set of biomarkers for cancer. Extracellular miRNAs are protected by exosome-like structures, small intraluminal vesicles shed from a variety of cells (including cancer cells), with a biogenesis connected with the endosomal sorting complex required for transport machinery in multivesicular bodies. These extracellular structures, originally considered as a 'garbage bag' devoted to discarding degraded proteins, are now considered to play an important role as an intercellular communication tool. It is still unclear as to whether these exosome-associated miRNAs occur as a result of tumor cell death and lyses, or are actively excreted from tumor cells into the microenvironment. However, this novel secretory machinery of miRNAs may be involved in tumor-associated features, such as the enhancement of angiogenesis, the increase of cytokine secretion and migration to pre-metastatic niche. Table I illustrates a summarized list of oncomiRs and metastamiRs.

Oncosuppressor microRNAs
In addition to oncogenic activities, miRNAs exhibit, as has already been pointed out, oncosuppressor properties by targeting mRNAs encoding oncoproteins (29-37). Piovan et al recently explored the interaction between certain miRNAs and transcriptional factors involved in determining cell fate, including the well known 'genome guardian', p53 (53). They demonstrated that miR-205, an oncosuppressive miRNA lost in breast cancer, is directly transactivated by the oncosuppressor p53. Moreover, evaluating miR-205 expression in a panel of cell lines belonging to the highly aggressive triplenegative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] breast cancer subtype, which still lacks an effective targeted therapy and is characterized by an extremely undifferentiated mesenchymal phenotype, the authors demonstrated that this miRNA is critically downregulated compared with a normal cell line. The re-expression of miR-205 strongly reduced cell proliferation, cell cycle progression and clonogenic potential in vitro, and inhibited tumor growth in vivo. The tumor suppressor activity of miR-205 is partially exerted by targeting of E2F1, one of the master regulators of cell cycle progression, and LAMC1, a component of the extracellular matrix involved in cell adhesion, proliferation and migration. In another study, Lee et al (54), demonstrated that an estrogen-downregulated miRNA, miR-34b, acts as an oncosuppressor that targets cyclin D1 and Jagged-1 (JAG1) in an ERα-positive/wild-type p53 breast cancer cell line (MCF-7), as well as in ovarian and endometrial cells, but not in ERα-negative or mutant p53 breast cancer cell lines (T47D, MBA-MB-361 and MDA-MB-435). The negative association between ERα and miR-34b expression levels has also been found in ERα-positive breast cancer patients. In addition, the overexpression of miR-34b has been shown to inhibit ERα-positive breast tumor growth in an orthotopic mammary fat pad xenograft mouse model. Table II illustrates a summarized list of oncosuppressor miRNAs (29-37,55-58).

MicroRNAs in breast tumors
As already presented in the previous chapters, miRNAs play a crucial role in breast tumors (59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72)(73). Several studies have been undertaken with the objective of determining the correlation between the expression profile of oncomiRs and tumor suppressor miRNAs, and, in particular, the tumorigenic potential of triple-negative primary breast cancers. In the study by Radojicic et al (68) 49 primary triple-negative breast cancer cases, along with 34 matched tumor-associated normal samples were investigated for the expression of 9 miRNAs using qRT-PCR. Correlations between the expression of miR-10b, miR-21, miR-122a, miR-145, miR-205, miR-210, miR-221, miR-222 and miR-296 and the pathological features of the tumors were examined, as well as the effects of miRNA expression on patient overall and cancer-specific survival. miR-21, miR-210 and miR-221 were significantly overexpressed, whereas miR-10b, miR-145, miR-205 and miR-122a were significantly underexpressed in the triple-negative primary breast cancers. Significant correlations among all the studied miRNAs were scored both in the breast cancer and control tissues. The expression of miR-222 and miR-296 did not exhibit any significant difference between the breast cancer and normal tissue.
A number of studies (61)(62)(63)(64)(65)(66)(67)74) have identified miR-221 and miR-222, as basal-like subtype-specific miRNAs that decrease the expression of epithelial-specific genes and increase the expression of mesenchymal-specific genes. In addition, the expression of these miRNAs increases cell migration and invasion; collectively, these are characteristics of epithelial-tomesenchymal transition (EMT). The basal-like transcription factor, FOSL1 (also known as Fra-1), directly stimulates the transcription of miR-221/222, and the abundance of these miRNAs decreases with the inhibition of MEK (mitogenactivated or extracellular signal-regulated protein kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222-mediated reduction in E-cadherin abundance is dependent on their targeting of the 3'UTR of trichorhinophalangeal syndrome type 1 (TRPS1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibits EMT by directly repressing the expression of Zinc finger E-box-binding homeobox 2 (ZEB2). Therefore, these molecular data support the hypothesis that miR-221/222 contribute to the aggressive clinical behavior of basal-like breast cancer.
Furthermore, the expression of miR-221 is clearly involved in chemoresistence. Studies have revealed an elevated expression of miR-221 in adriamycin-resistant MCF-7/ADR cells. In conclusion, several studies have indicated that miR-221 is one of

Plasma miR-221 as a diagnostic marker in breast cancer
In consideration of the importance of miR-221 in the tumor phenotype of breast cancer, several studies have been performed with the objective of analyzing miR-221 in biological fluids as a marker of breast cancer. Zhao et al (75) demonstrated that plasma miR-221 can be considered as a predictive biomarker for chemoresistance in breast cancer patients who have previously received neoadjuvant chemotherapy. The expression levels of circulating miR-221 were assessed in the plasma of 93 breast cancer patients who had previously received neoadjuvant chemotherapy (NAC), as well as in 32 healthy individuals. The correlation between miR-221 and clinicopathological features and chemosensitivity was also analyzed. The expression level of miR-221 was significantly associated with the hormone receptor (HR) status. Patients with higher plasma miR-221 levels tended to be HR-negative. Patients with varying miR-221 levels had significant differences in the overall response rate but not in the pathological complete response rate. These results indicate that plasma miR-221 may be a predictive biomarker for sensitivity to NAC in breast cancer patients.

Connecting miRNA-221 with the expression of cellular genes altered in breast cancer cells: the Slug/miR-221 network
This issue has great impact on the design of novel therapeutic approaches. On the one hand, it is very important to determine whether the expression of the miR-221/miR-222 cluster is under the transcriptional regulation of cellular proteins (for instance tumor-associated transcription factors). On the other hand, it is imperative to determine which mRNAs are specifically targeted by miR-221 (for instance tumor-suppressor mRNAs) determining the tumorigenic potential of this miRNA. Finally, it should be verified whether mRNAs regulated by miR-221 encode proteins able to regulate upstream miR-221 modifiers, therefore activating a 'vicious intracellular cycle'. As regards these issues, a number of studies have been published.
Lambertini et al (74) recently demonstrated that the Slug transcription factor binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. In order to investigate the possible correlation between the Slug transcription factor and miR-221, they performed Slug gene silencing in MDA-MB-231 breast cancer cells and evaluated the expression of genes involved in supporting the breast cancer phenotype by qRT-PCR and western blot analysis. Chromatin immunoprecipitation and wound healing assays were employed to determine a functional link between these two molecules. The results of their study (Fig. 3) revealed that Slug silencing significantly decreased the level of miR-221 and vimentin, reactivated ERα and increased E-cadherin and TRPS1 expression (74). It was demonstrated  that miR-221 is a Slug target gene, and the authors identified a specific region of the miR-221 promoter that is transcriptionally active and binds the transcription factor Slug in vivo. In addition, they observed a more potent inhibiton of cell migration in the Slug-silenced cells, which retained residual miR-221 (approximately 38%), compared with antagomiR-221-treated cells with a complete knockdown of miR-221. As a whole, their study reported for the first time evidence of a correlation between the Slug transcription factor and miR-221 in breast cancer cells, suggesting that miR-221 expression is, at least in part, dependent on Slug, which is more effective than miR-221 in sustaining cell migration and invasion.

Effects of antagomiRs targeting oncomiRs
In miRNA therapeutics, by targeting oncomiRs and metasta-miRs, several strategies have been performed to inhibit the functions of oncomiRs and metastamiRs. One of the most common approaches involves the use of antisense miRNAs (antagomiRs) capable of knocking down miRNAs. Velu et al (76) demonstrated the efficacy of the knockdown of miR-21, which is involved in myelopoiesis, using antagomiRs in primary murine bone marrow stem/progenitor cells. This approach has a clear potential impact in anticancer therapy, as demonstrated in a very recent study by Poltronieri et al (77), who hypothesized that, as oncomiRs promote the growth of cancer cells and support survival during chemotherapy, thus miRNA-silencing therapies may be a valuable approach in conjunction with anticancer drugs and chemotherapy treatments. Specifically, they focused on miR-155, which they found overexpressed in different types of cancer. Of particular interest was the finding that GABA-A receptor downregulation was found to correlate with the glioma grade, with decreasing levels being associated with a higher grade of malignancy. The demonstration that the knockdown of miR-155 involves the re-expression of GABRA 1 protein in vivo has a great implication on the effectiveness of RNA-silencing approaches against miR-155, with the aim to control proliferation and signalling pathways regulated by the GABA-A receptor.
Another study also focused on potential anticancer therapy based on miRNA knockdown. Ma et al aimed to control mammary tumor metastasis (78). They demonstrated that the systemic treatment of tumor-bearing mice with miR-10b antagomiRs suppresses breast cancer metastasis, both in vitro and in vivo. The silencing of miR-10b with antagomiRs significantly decreased miR-10b levels and increased the levels of a functionally important miR-10b target, Hoxd10. Of note, the administration of miR-10b antagomiRs to mice bearing highly metastatic cells did not reduce primary mammary tumor growth but markedly suppressed the formation of lung metastases in a sequence-specific manner. The miR-10b antagomiR, which is well tolerated by healthy animals, appears to be a promising candidate for the development of novel anti-metastatic agents.

Novel drugs in miRNA therapeutics: peptide nucleic acid (PNA)
Peptide nucleic acid (PNA) (Fig. 4) is a DNA analogue in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl) glycine units (79)(80)(81)(82)(83)(84). These molecules efficiently hybridize with complementary DNA and RNA, forming a double helix with Watson-Crick base pairs (79,80). Accordingly, PNA has been suggested for use in antisense and anti-gene therapy in a number of studies (83)(84)(85)(86). PNA is promising for RNA recognition, since it has a higher affinity for RNA than for DNA, is more specific, and is resistant to DNases and proteases (80). PNA can be modified in order to achieve a better performance in terms of cellular permeation, higher affinity and specificity for the target DNA and RNA sequences (87)(88)(89)(90)(91)(92)(93).
In the case of the development of PNA-based miRNA therapeutics for altering gene expression in breast cancer cells, PNA targeting miR-221 has shown to specifically interact with miR-221 expressed in aggressive breast cancer cell lines (94). In order to maximize uptake in target cells, a polyarginine-peptide (R8) was conjugated, generating an anti-miR-221 PNA (R8-PNA-a221) displaying very high affinity for RNA and efficient uptake within target cells without the need of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only R8-PNA-a221 markedly inhibited miR-221 in MDA-MB-231 breast cancer cells. This is illustrated in Fig. 5A, describing the effects of two PNA-based antagomiRs, R8-PNA-a210 and R8-PNAa221, targeting miR-210 and miR-221, respectively. As it is clearly evident, R8-PNA-a210 inhibits miR-210 but not miR-221 and vice-versa, R8-PNAa221 inhibits miR-221 but not miR-210. Therefore, targeting miR-221 with R8-PNAa221 resulted in i) a specific decrease in the hybridization levels of miR-221 measured by qRT-PCR; and ii) the upregulation of p27 Kip1 , mRNA and protein, measured by qRT-PCR and western blot analysis ( Fig. 5B and C).
While research on anti-miR PNA has just begun (95-100), pre-clinical results are expected in the near future to sustain the hypothesis that miRNA-targeted molecules based on PNA can be successfully applied to treat human diseases. An example was recently reported by Yan et al (101), who addressed the potential effects of PNA-anti-miR-21 in vivo on the growth of breast cancer cells. In their experiments, MCF-7

Conclusion
The large number of studies on miRNAs in different types of in vitro and in vivo experimental models, both in basic and applied research, demonstrate the high prognostic, diagnostic and therapeutic value of these recently discovered molecules. The demonstration of the functions of these molecules has not only led to the discovery of a new system of regulation of gene expression that renews the concepts of molecular biology known to date, but has also allowed the development of novel clinical tools. The close collaboration between researchers and clinicians will be required in the near future to enhance the knowledge on the biology of miRNAs and exploit their potential to improve human health. In this context, miR-221 plays a crucial role in breast cancer for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancers; ii) the oncosuppressor p27 Kip1 , a validated target of miR-221, is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network is thus proposed, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27 Kip1 downregulation (Fig. 6). Interference with this process can be achieved using antagomiR molecules targeting miR-221, inducing the downregulation of Slug and the upregulation of p27 Kip1 .  Targeting miR-221 with DNA analogues may be clinically relevant in antagomiR therapeutics.