Complications from metastatic disease are the major reason for cancer mortality. Metastasis is multi-step process involving local invasion of cells from the primary tumor, entry into the circulation, survival in the circulation, invasion into other tissues, the establishment of micrometastases, and growth of secondary tumors. Analogous to oncomiRs and tumor suppressor miRNAs, miRNAs can also promote or suppress these steps in migration and metastasis without influencing primary tumor development. Welch and colleagues recently coined the term “metastamir” to refer to these metastasis regulatory miRNAs [97
]. These metastamirs regulate key steps in the metastatic program and processes such as EMT, apoptosis, and angiogenesis. Furthermore, as recently discussed and reviewed by Calin and colleagues, there is an emerging theme that many of the same miRNAs are involved both in CSC regulation and in steering metastasis [98
The process of EMT has been associated with tumor cell invasion and metastasis. As mentioned above, the miR-200 family, as well as miR-205, regulate EMT through targeting Zeb1 and Zeb2. While miR-205 was shown to suppress cell growth in MCF7 cells, it was shown to inhibit invasion and metastasis in MDA-MB-231 cells [81
]. Most of these studies have used in vitro
assays to show that miR-200 family members can suppress the invasive properties of cells in culture [60
]. Unexpectedly, over-expression of miR-200 in the non-metastatic murine mammary tumor cell line 4TO7 enables these cells to metastasize to the lung and liver [99
]. This may be due to the fact that some tumors may have the requirement of a mesenchymal to epithelial transition (MET) occurring for efficient tumor colonization at the metastatic site. Further studies using in vivo
metastasis models are surely warranted to elucidate the role of the miR-200 family in metastasis. Furthermore, miR-200c targets class III β-tubulin (TUBB3), and restoration of miR-200c expression in cells with low miR-200 family levels results in increased chemosensitivity to microtubule-directed agents [100
]. As a pro-metastatic miRNA, miR-9 targets E-cadherin, loss of which results in loss of epithelial characteristics, cell migration and invasion, ultimately leading to an EMT [101
]. Further studies on breast cancer cells demonstrated miR-9 is directly regulated by MYC and MYCN, and over-expression of miR-9 resulted in an EMT, increased proliferation, enhanced metastasis, and induced angiogenesis when transplanted [101
Breast cancer metastasis suppressor 1 (BRMS1) is a protein that suppresses metastasis in multiple tumor types. By analyzing the miRNA expression profiles of metastatic MDA-MB-231 and MDA-MB-435 cells compared to BRMS1-transduced non-metastatic counterparts, Welch and colleagues found that BRMS1 coordinately regulates a number of these metastamirs [102
]. BRMS1 decreased metastasis-promoting miRNAs: miR-10b, miR-373, and miR-520c. Metastasis suppressing miRNAs: miR-146a, miR-146b, and miR-335 were up-regulated following BRMS1 overexpression. This shows that BRMS1-containing SIN3/HDAC complexes may be recruited to and regulate miRNA promoters in addition to coding genes involved in regulating cancer metastasis.
miR-10b, which is highly expressed in metastatic breast cancer, is also involved in breast cancer metastasis [103
]. This control is due to a feedback loop where the transcription factor Twist regulates transcription of miR-10b via direct E-box binding, which in turn represses translation of HOXD10 via a highly conserved miR-10b binding site in the 3′UTR. Decreased levels of HOXD10 then resulted in increased expression the pro-metastatic gene, RHOC, although a direct connection here has not been established. miR-10b has also been shown to directly repress Tiam1 expression in breast carcinoma cells [104
]. This inhibits Tiam1-mediated Rac activation suppressing migration and invasion. Furthermore, over-expression of miR-10b in non-metastatic breast cancer cells promoted metastasis, while inhibition of miR-10b in metastatic breast cancers decreased metastatic phenotypes of cells in vitro
. However, systemic administration of miR-10b antagomirs to mice bearing highly metastatic 4T1 cells was not able to reduce growth of the primary lesion, but was shown to markedly suppress formation of lung metastases, representing a promising approach for an anti-metastasis agent [105
By comparing miRNA expression profiles between MDA-MB-231 parental cells and derivatives with higher metastatic ability, Tavazoie, et al.
, identified miR-335 and miR-126 as low in more metastatic lines. Both miR-126 and miR-335 were significantly down-regulated in the metastatic breast cancers, and this decreased expression was correlated with poor prognosis. Induction of miR-335 expression is sufficient to suppress lung and bone metastasis in a metastatic breast cancer cell line. SOX4, which is critical in cell migration, was shown to be a functional target of miR-335, as well as the extracellular matrix component tenascin C [106
]. A miR-335 six-gene target signature (SOX4, PTPRN2, MERTK, PLCB1, COL1A1 and TNC) is associated with poor metastasis-free survival in a large patient dataset. Additionally, miR-126 expression reduced overall tumor growth and proliferation. This inhibition of cell cycle progression was shown to be possibly through inhibition of Insulin Receptor Substrate-1 (IRS-1) [107
]. Other studies of the metastatic MDA-MB-231 cell line indicated that miR-146 inhibits invasion and metastasis of cells by downregulating NFkB, in addition to targeting IRAK1 and TRAF6 [108
Valastyan et al.
demonstrated that miR-31, which is expressed in normal mammary cells, is specifically lost in metastatic breast cancer cell lines. Overexpression of miR-31 expression in metastatic cells can inhibit multiple steps in metastasis, and in non-metastatic cell lines, deletion of miR-31 is sufficient to confer metastatic properties. [109
]. In a subsequent study, the authors demonstrate that while miR-31 is predicted to modulate the expression of >200 mRNAs, its regulation of metastasis can be accounted for primarily by three targets, integrin α5 (ITGA5), radixin (RDX), and RhoA [110
]. Interestingly, each of these targets affects distinct steps in the metastatic process and re-expression of these three genes concurrently abrogates metastasis suppression by miR-31.
miR-373 and miR-520c were identified as promoters of tumor invasion and metastasis through a genetic screen using MCF7 cells subjected to a trans-well migration assay. These miRNAs can stimulate the normally non-metastatic MCF7 cell line to display metastatic behavior of cell invasion and migration, interestingly by targeting CD44 [111
In MDA-MB-231 cells, unlike MCF7 cells, miR-21 does not affect growth of the primary tumor. It was however shown that miR-21 promotes cell invasion and lung metastasis in this model [73
]. This is likely through the down-regulation of multiple targets including several other tumor suppressor genes.
miR-145 was recently shown to suppress invasion and experimental metastasis assays using metastatic breast cancer cell lines MDA-MB-231 and the LM2-4142 lung metastatic subline [112
]. This was in part due to miR-145 directly targeting MUC1, which in turn down-regulates β-catenin, Cyclin D, and Cadherin 11.
Liu et al.
found that the miR-17–92 cluster was expressed at higher levels in metastatic breast cancer cell lines. The expression of the cluster was diminished by treatment with a ROCK inhibitor (Y27632). They further showed that blockade of miR-17 using anti-miR-17 molecules decreased breast cancer cell invasion in vitro
and lung metastasis from the orthotopic site [113
]. In contrast, another group found that miR-17/20 (miR-17-5p and miR-20a) were reduced in highly invasive breast cancer cell lines and node-positive breast tumors, and in vitro
miR-17/20 could inhibit migration and invasion of neighboring cells via a unique mechanism of heterotypic secreted signaling [114
Recently, Baffa et al
. performed a miRNA microarray analysis of 43 matched primary tumors (13 breast, 10 lung, 10 bladder, and 10 colon cancers) and corresponding lymph node metastasis to identify deregulated miRNAs in metastasis. They identified 32 differentially expressed miRNA [115
]. These included a number of the above mentioned metastasis-regulating miRNAs including the upregulation of miR-10b and miR-21 and the down-regulation of miR-141, miR-200b, miR-200c, and miR-205 in metastatic samples.
miRNAs that regulate the process of metastasis may become useful prognostic markers and/or targets for anti-metastatic therapy. Inhibition of metastasis promoting miRNAs and/or expression of metastasis suppressing miRNAs using tools we review later in the article may, therefore, provide therapeutic strategies for metastatic breast cancer.