A potential role of miR-206 in suppressing breast cancer was first identified by employing miRNA microarrays to compare miRNA expression between normal and breast cancer tissues 28. miR-206 was upregulated in estrogen receptor (ER)α-negative breast cancers, suggesting a role of miR-206 in regulation of the estrogen receptor gene ERα (ESR1). Indeed, miR-206 was recently shown to inhibit the expression of ESR1 mRNA through two binding sites in the ESR1 3' UTR 33. The latter study also showed that miR-206 expression was strongly repressed by ERα agonists, but not by an ERβ agonist or progesterone, suggesting the existence of a feedback loop. Another study showed that miR-206 expression decreased in ERα-positive human breast cancer tissues and that miR-206 suppresses ESR1 expression and inhibits growth of MCF7 breast cancer cells. In addition to miR-206, the authors found that ESR1 mRNA was a direct target of miR-18a, miR-18b, miR-193b and miR-302c in breast cancer cells 34. Moreover, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c were also shown to induce cell cycle arrest and to inhibit estrogen-induced proliferation, with inhibition levels comparable to those achieved by ERα small interfering RNA (siRNA). Since dysregulation of ERα expression is a hallmark of most human breast cancers, these studies provide important insights into the molecular mechanisms that lead to breast cancer pathogenesis. The tumor suppressive role for miR-206 in breast cancer was further substantiated by the recent demonstration that miR-206, miR-335 and miR-126 are potently downregulated in metastatic breast cancer cells compared to parental cells 35. Importantly, restoring the expression of these three miRNAs reduced their invasive capacity. The authors identified four direct targets of miR-335, PTPRN2, MERTK, TNC and SOX4; among them, TNC (encoding an extracellular matrix component) and SOX4 (encoding a transcription factor that is involved in tumorigenesis) were shown to be functional targets implicated in metastasis. Restoring miR-206 expression in metastatic cells did not influence their proliferation or sensitivity to apoptosis, but altered cellular morphology, possibly contributing to a decrease in cell motility that could limit the migration of metastatic cells 35. These findings suggest that miR-206 could be a novel candidate for breast cancer therapy.

miR-17-5p, also known as miR-91, is located on chromosome 13q31, a genomic region that undergoes loss of heterozygosity in multiple cancers, including breast cancer 36. The oncogene AIB1 (amplified in breast cancer) is a direct target of miR-17-5p 37. The protein encoded by the AIB1 gene is a steroid receptor co-activator that enhances the transcriptional activity of ERα, E2F1 (which is also directly regulated by miR-17-5p) and other transcription factor genes. miR-17-5p represses the translation of AIB1 mRNA, thereby inhibiting the function of E2F1 and ERα. Downregulation of AIB1 by miR-17-5p results in the suppression of estrogen-stimulated proliferation and estrogen/ER-independent breast cancer cell proliferation 37. In breast cancer cells, the gene cyclin D1 (CCND1), which is overexpressed in approximately 50% of human cancers, was recently identified as a direct target of miR-17-5p 38. miR-17-5p inhibits the proliferation of breast cancer cells by suppressing cyclin D1 protein synthesis and this effect was abrogated by a CCND1 siRNA in cyclin D1-deficient breast cancer cells. This study also identified a regulatory mechanism in which cyclin D1 induces miR-17-5p expression, thus providing a negative feedback loop that limits cyclin D1 expression.

The HER2 (ERBB2) gene encodes a member of the epidermal growth factor receptor family of tyrosine kinases. This protein has no ligand-binding domain of its own and cannot, therefore, bind growth factors. However, it binds tightly to other ligand-bound epidermal growth factor receptor family members to form a heterodimer, stabilizing ligand-binding activity and enhancing kinase-mediated activation of downstream signaling pathways, such as those involving mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase 39. Amplification and/or over expression of this gene has been reported in numerous cancers, including breast tumors. miR-125a and miR-125b are downregulated in HER2-amplified and HER2-overexpressing breast cancers 40. These two miRNAs are potential tumor suppressors and their overexpression in SKBR3 cells (a HER2-dependent human breast cancer cell line) suppresses HER2 and HER3 mRNA and protein levels, leading to a reduction in anchorage-dependent growth, cell motility, and invasiveness 41. However, this influence was subtle in non-transformed HER2-independent breast cancer cells (MCF10A).

Invasion and metastasis are the hallmarks of malignant tumor progression. Increasing evidence indicates that activation of the embryonic program 'epithelial-mesenchymal transition' (EMT) promotes these processes by allowing detachment of cells from each other, thereby increasing tumor cell mobility and dissemination 42. In recent years, it has become evident that aberrant activation of EMT is responsible for the malignant transformation of many human cancers. EMT is activated by EMT-inducing transcriptional repressors, including members of the Snail family and the ZFH family of transcription factors 43. These proteins inhibit the transcription of epithelial genes, such as that encoding E-cadherin. Recently, ZEB1, a ZFH-family member, was shown to be a crucial EMT activator in human cancers, including breast cancer 4445, and to promote metastasis of tumor cells in a mouse xenograft model 46. The miR-200 family of miRNAs has been shown to be crucial inducers of an epithelial phenotype by suppressing the expression of the EMT inducers ZEB1 and ZEB2. Gregory et al. 47 induced EMT in MDCK cells (human Madin Darby canine kidney epithelial cells) either by transforming growth factor (TGF)-β or by stable expression of the tyrosine phosphatase Pez. The authors found that all five members of the miR-200 family, which are clustered in two genomic locations, were downregulated upon TGF-β-induced EMT. Functional studies showed that inhibition of miR-200a, b, and c in MDCK cells triggered TGF-β-independent EMT that was associated with increased expression of vimentin, fibronectin and N-cadherin, and downregulation of E-cadherin. Conversely, overexpression of miR-200 family members in mesenchymal cells initiated mesenchymal-to-epithelial transition. Luciferase reporter assays under the control of ZEB1 and ZEB2 3' UTR verified that these genes are direct targets of the miR-200 family. The clinical relevance of these findings was further suggested by additional analyses of human breast cancer tissues showing that a decrease in miRNA-200 family miRNAs was associated with highly aggressive, metaplastic breast tumors. Their finding that ZEB1 and ZEB2 expression is controlled by the miR-200 family suggests that downregulation of these miRNAs is an essential early step in tumor metastasis.

Similar overlapping results were obtained in another study, which employed the NCI-60 panel of cell lines, representing 60 cell lines of different human tumor types 48. By using two markers, the authors divided the panel into two major groups, characterized either by an epithelial phenotype (marked by E-cadherin) or a mesenchymal phenotype (marked by vimentin) and found that the miR-200 family was strongly associated with the epithelial phenotype. Overexpression of miR-200 induced epithelial differentiation in undifferentiated breast cancer cells (MDA-MB231) whereas antagonizing it induced an EMT phenotype in the colorectal cancer cell line HCT116. ZEB1 and ZEB2 were again detected as targets of the miR-200 family members. These and other recent reports on the miR-200 family 4950 add important miRNAs to the growing list of tumor-associated miRNAs. Furthermore, a recent study showed that the expression of the miR-200 family was decreased by Akt2, suggesting that, in many cases, breast cancer metastasis may be under the control of the Akt-miR-200-E-cadherin pathway 51. Interestingly, a recent study showed that expression of miR-200 unexpectedly enhanced macroscopic metastases in mouse breast cancer cell lines 52. Their results suggest that, for some tumors, tumor colonization at metastatic sites might be enhanced by mesenchymal-to-epithelial transition and that the epithelial nature of a tumor may not predict metastatic outcome. Although this paper is the first to show the direct enhancement of metastasis by the miR-200 family, changes in miR-200 family levels have been associated with enhanced tumorigenesis. For instance, the miR-200 family is upregulated in human ovarian cancers compared to normal ovarian tissue 29. Moreover, overexpression of the miR-200 family significantly correlates with decreased survival. Patients with ovarian tumors with high miR-200a expression have been shown to have approximately 50% decrease in median survival time compared to those lacking significant miR-200a expression. Furthermore, the region on chromosome 1 that encodes the miR-200 cluster was also found to be amplified in several epithelial cancers - ovarian cancer, breast cancer and melanoma 53. A new study 54 revealed the roles of three miRNA clusters (miR-200c-141, miR-200b-200a-429 and miR-183-96-182) in the regulation of self-renewal in cancer stem cells and normal stem cells. These miRNAs were downregulated in human breast cancer stem cells, normal human and mouse mammary stem/progenitor cells as well as embryonal carcinoma cells. The authors provided evidence that BMI1 (a gene that promotes stem cell self-renewal) is specifically inhibited by these miRNAs and showed that ectopic overexpression of miR-200c in embryonal carcinoma cells resulted in growth retardation and neural differentiation and suppressed tumorigenicity of breast cancer stem cells in vivo 55. Taken together, these studies suggest that miRNAs of the miR-200 family play important roles in regulating tumor progression and metastasis.

let-7, one of the founding members of the miRNA family, was originally discovered to play a role in the developmental timing of C. elegans 56 and is conserved throughout the animal phyla. let-7 is poorly expressed or deleted in many human cancers. Recent data from both hematologic malignancies and solid tumors suggest that each includes minor populations of cells that are capable of tumor initiation 57. These tumor-initiating cells (T-ICs) have properties of tumor stem cells and can undergo asymmetric cell division and self renewal. T-ICs comprise only a minor fraction among the bulk of the more differentiated cells in the tumor. Based on the cancer stem cell hypothesis, T-ICs are responsible for the initiation, progression, metastasis and resistance to therapy. Breast T-ICs (BT-ICs) can be enriched by purifying CD44+CD24^-/low cells by sorting or by purifying spherical clusters of self-replicating cells (mammospheres) from cell suspensions.

A recent study compared miRNA expression in self-renewinzand differentiated cells from breast cancer lines and found that the expression of let-7 was strongly reduced in BT-ICs and increased with differentiation 58. Introducing let-7 in BT-ICs reduced their proliferative capacity, their ability to form mammospheres and tumor formation and metastasis in vivo. Conversely, knocking down let-7 enhanced self-renewal of non-T-ICs in vitro. Known oncogenic targets of let-7, such as H-RAS and HMGA2, were downregulated by let-7 overexpression. Silencing H-RAS in a BT-IC-enriched cell line reduced self-renewal but had no effect on differentiation, while knocking down HMGA2 enhanced differentiation but did not affect self-renewal. Their results suggest that let-7 regulates multiple BT-IC stem cell-like properties and that let-7 may offer a unique opportunity to attack tumor stem cells using therapeutic RNA. Delivery of the let-7 miRNA to tumors could potentially deplete stem cells by inducing cellular differentiation.

A recent report linked the protein RKIP (Raf kinase inhibitory protein) to let-7 and breast cancer metastasis. RKIP (also called PEBP1) inhibits MAPK, G protein-coupled receptor kinase-2, and NF-κB signaling cascades 59. The authors found that RKIP inhibited invasion by metastatic breast cancer cells and repressed breast tumor cell intravasation and bone metastasis in a mouse model. Inhibition of MAPK decreased transcription of LIN28 by MYC; in turn, downregulation of LIN28, an inhibitor of let-7 biogenesis, enhanced let-7 expression in breast cancer cells leading to decreased expression of HMGA2, a chromatin-remodeling protein that activates proinvasive and prometastatic genes, including Snail. Collectively, their findings indicate that RKIP suppresses invasion and metastasis via a signaling cascade that involves MAPK, MYC, LIN28, let-7, and downstream let-7 targets such as HMGA2.

miR-34a is one of several miRNAs that are downregulated in multiple cancers 26 and has been shown to be transcriptionally regulated by p53. In the context of breast cancer, only one study 60 has shown that miR-34a levels were lower in triple negative and mesenchymal breast cancer cell lines compared with normal epithelial lines and HER-2+ lines. The authors propose that p53 mutations in these subtypes of breast cancer might contribute to low miR-34a expression. To elucidate the relationship between miR-34a and radiosensitivity in these cells, the authors compared the sensitivity of a normal breast epithelial line (HMEC), a HER-2+ line (UACC812) and a mesenchymal line (MDA-MB-231) and found that the MDA-MB-231 cell line (with low miR-34a levels) was significantly more sensitive to radiation than the HMECs or UACC-812 (with high miR-34a levels). Increasing the levels of miR-34a protected the MDA-MB-231 cells from radiation-induced cell death, and downregulating it had the converse effect. These results show that miR-34a is necessary for the survival of MDA-MB-231 cells from non-apoptotic cell death and suggest that miR-34a may have therapeutic potential in breast cancers, since antagonizing miR-34a increases the sensitivity of breast cancer cells towards radiation.

miR-31 was recently shown to prevent metastasis at multiple steps by inhibiting the expression of prometastatic genes 61. miR-31 is expressed in normal breast cells and its abundance was shown to be dependent on the metastatic state of the tumor. It is moderately decreased in non-metastatic breast cancer cell lines and is almost undetectable in metastatic mouse and human breast cancer cell lines. Importantly, the authors demonstrated that introducing miR-31 in metastatic breast cancer cells suppressed metastasis-related functions (motility, invasion and resistance to anoikis) in vitro and metastasis in vivo. Notably, they found that although miR-31 over-expressing breast cancer cells formed larger and more proliferative tumors, these tumors were well encapsulated and thereby less invasive, suggesting that miR-31 inhibits metastasis in the early stages of the metastasis cascade. Injecting miR-31 overexpressing cells directly into the circulation impeded the ability of the cells to survive and form secondary tumors in the lung, suggesting that miR-31 inhibits metastasis at multiple steps of the metastatic cascade. Conversely, inhibition of miR-31 function increased invasiveness and promoted metastasis in vivo. To identify functionally relevant mRNA targets of miR-31 through which it might exert its antimetastatic effects, the authors employed Gene Ontology analysis on the putative targets of miR-31 predicted by the miRNA-target prediction algorithms TargetScan and PicTar. Using this approach, they validated six targets of miR-31 in human breast cancer cells: frizzled3 (Fzd3), integrin α-5 (ITGA5), myosin phosphatase-Rho-interacting protein (M-RIP), matrix metallopeptidase 16 (MMP16), radixin (RDX) and the ras homolog gene family member A (RhoA). Interestingly, they showed that re-expression of three of these genes, ITGA5, RDX and RhoA, in metastatic breast cancer cells abrogated the motility defects and reversed the impaired invasion and anoikis resistance defects conferred by miR-31, suggesting that these three genes are functionally important targets of miR-31. Taken together, their findings demonstrate that miR-31 may also be an attractive therapeutic target for breast cancer as it exerts its antimetastatic effect by targeting multiple prometastatic genes of the metastasis cascade.

miR-21 has been found to be consistently overexpressed in many tumors, including breast cancer 2862. Consistent with these findings, miR-21 was also shown to be highly upregulated in breast tumors compared to the matched normal breast tissues among 157 human miRNAs analyzed by real-time RT-PCR arrays 63, suggesting that miR-21 may function as an oncogene. The latter study also examined the oncogenic role of miR-21 in cell culture as well as in mouse xenografts. Although the influence of knocking down miR-21 on cell survival was marginal (25% decrease), the authors found that this growth inhibition increased when the transfected MCF-7 cells were treated with the anticancer drug topotecan, suggesting that suppression of miR-21 can sensitize tumor cells to anticancer agents. To further investigate whether suppression of miR-21 alone influences tumorigenesis, they antagonized miR-21 in MCF-7 cells and then injected them into mammary pads of female nude mice. Tumors derived from MCF-7 cells transfected with anti-miR-21 were one-half the size of those derived from the cells transfected with the negative control, while anti-Ki-67 indicated that the reduced tumor growth was likely due to a lower proliferation caused by anti-miR-21. These results show that miR-21 is an oncogenic miRNA that plays an important role in tumorigenesis. The authors also found that antagonizing miR-21 caused apoptosis in MCF-7 cells, which was associated with lower expression of Bcl-2 protein in the anti-miR-21-transfected MCF-7 cells and also in tumors derived from these cells, suggesting that Bcl-2 may be an indirect target of miR-21. To further elucidate the molecular mechanisms that regulate the function of miR-21, proteomic analysis of the above-mentioned xenograft tumors revealed that the tumor suppressor protein tropomyosin 1 (TPM1), which is known to be downregulated in breast cancer epithelial cell lines, was a target of miR-21 64. In the context of breast cancer, miR-21 has also been shown to inhibit the expression of the tumor suppressor PDCD4 (programmed cell death-4). Although depletion of PDCD4 protein in MCF-7 cells by PDCD4 siRNA had no effect on cellular proliferation, it significantly alleviated the anti-proliferative effect of miR-21 inhibition, underscoring an essential role for PDCD4 as a mediator of the biological effects of miR-21 in breast cancer cells 65. Another important target of miR-21 is the tumor suppressor gene phosphatase and tensin homolog (PTEN) 66. In addition to TPM1 and PDCD4, one study also identified Maspin as a direct target of miR-21 67. These three genes were shown to reduce invasiveness of a metastatic breast cancer cell line 64. Their findings further establish miR-21 as an oncogenic miRNA and suggest that miR-21 has a role not only in tumor growth but also in invasion and tumor metastasis by targeting multiple anti-metastatic genes.

miR-155 is over-expressed in a number of human malignancies, including breast cancer 2862. A recent study has shown that miR-155 is upregulated in normal mouse mammary gland epithelial cells (NMuMG cells) by the TGF-β/Smad4 pathway and mediates TGF-β-induced EMT and cell invasion 68. To investigate the role of miR-155 in TGF-β-induced EMT, the authors found that ectopic expression of miR-155 in NMuMG cells disrupted proper tight junction formation and promoted cell migration and invasion. Conversely, antagonizing miR-155 in NMuMG cells reduced the occurrence of TGF-β-induced EMT and cell migration and invasion. miR-155 directly inhibited the expression of RhoA, a gene that regulates many cellular processes, including cell adhesion, motility, and polarity, and is an important modulator of cell junction formation and stability. Importantly, miR-155-induced phenotypes were restored by expressing a miR-155 insensitive version of RhoA in miR-155 overexpressing cells. These findings suggest that miR-155 is regulated by the TGF-β/Smad4 pathway and downregulates RhoA protein expression to drive EMT progression. They further demonstrated that miR-155 is associated with cancer invasiveness in human primary breast carcinoma by showing that miR-155 is highly expressed in invasive tumors but not in noninvasive cancer tissues. These results implicate miR-155 in EMT and invasion as observed in NMuMG cells and suggest that miR-155 may play a critical role in breast cancer metastasis.

miR-10b was the first miRNA found to influence the metastatic potential of human cancer cells 69. Unlike miR-155, which is overexpressed in many breast tumors, miR-10b was highly expressed only in metastatic cancer cells and was found to promote cell migration and invasion in vitro and initiate tumor invasion and metastasis in vivo. Expression of miR-10b was found to be induced by the transcription factor Twist; in turn, miR-10b inhibits the translation of the transcription factor homeobox D10 (HOXD10), resulting in a cascade of cellular alterations that include expression of the prometastatic gene RHOC (ras homologue gene family member C), a gene that promotes cancer cell migration and invasion.

miRNAs of the miR-373/520c family were identified 60 as prometastatic using a forward genetic screen involving overexpression of almost 450 miRNAs in a nonmetastatic human breast cancer cell line (MCF-7 cells). MCF-7 cells were transduced with these miRNAs and subjected to a trans-well cell migration assay to identify miRNAs that stimulate cell migration. The authors found that miR-373 and miR-520c promoted cancer cell migration and invasion in vitro and in vivo. They also showed that some cancer cell lines required miR-373 for migration and that these miRNAs elicited a migratory phenotype by inhibiting expression of the metastasis repressor CD44. Importantly, ectopic overexpression of CD44 without its 3' UTR significantly reduced the migration of MCF-7 cells that express miR-373 or miR-520c, suggesting that downregulation of CD44 is required for the migration phenotype of MCF-7 cells expressing miR-373 and miR-520c. Finally, the authors showed that miR-373 was upregulated whereas CD44 was decreased in clinical breast cancer metastasis samples, further implicating its role in breast cancer metastasis.