As previously discussed, though miRNAs are generally considered to have suppressive properties, multiple miRNAs have been implicated as oncomiRs in breast cancer, most of which function through the suppression of tumor suppressors. One such oncomiR is miR-155, a miRNA which targets the tumor suppressor SOCS1 directly, thereby indirectly activating JAK-STAT signaling [49]. Similarly, miR-181 directly targets the tumor suppressor ATM and has been shown to support malignant transformation and tumorigenesis in both cell culture and mouse models, respectively [50]. Other potential oncomiRs have been identified as biomarkers. miR-21 was initially identified as a biomarker in breast cancer and subsequently shown to target a number of tumor suppressors to promote breast tumorigenesis and metastasis [51–53]. See Table 13.1 for a partial list of miRNAs identified as oncomiRs in breast cancer.

In line with the suppressive functions of miRNAs, a larger number have been identified as potential tumor suppressors in breast cancer. One of the first miRNAs ever identified and one of the first to be designated a bona fide tumor suppressor in breast cancer was let-7 [54]. Let-7 targets the oncogene RAS and is a regulator of the mammary stem cell population [54, 55]. Upregulation of this miRNA forces stem cells to exit the self-renewing population thought to harbor cancer-initiating cells [55]. Another regulator of the stem cell population is miR-200 which functions in a similar manner [56]. miR-200 also suppresses tumorigenesis by promoting mesenchymal–epithelial transition (MET) and targeting the AKT prosurvival pathway [57–60]. Another identified tumor suppressor is miR-125 which was shown to directly target HER2 in this aggressive subtype of breast carcinoma [61]. See Table 13.1 for a partial list of miRNAs identified as tumor suppressors in breast cancer. See Fig. 13.3 for a list of miRNAs involved in breast carcinogenesis and disease progression.

In addition, to the context-dependent miRNAs discussed previously, many miRNAs have been shown to have varied and conflicting roles in breast cancer. A notable example is the miR-17–92 cluster, originally identified as a tumor suppressor in breast cancer by targeting cyclin D1 and IL-8 to inhibit the aggressive features of cancer, including proliferation, motility, and invasion [62, 63]. However, more recent studies have found an oncogenic role for this cluster, suggesting that it may have tumor suppressive or oncogenic effects depending on cellular context [64]. It is likely that many miRNAs will ultimately be determined to have multiple and conflicting roles in breast cancer in a context-dependent manner.

Many miRNAs with no identified role in breast tumorigenesis have been implicated specifically in disease progression and metastasis. Best studied is miR-10b which, though absent or downregulated in primary tumors, is upregulated in metastatic lesions as it is induced by the epithelial–mesenchymal transition (EMT) transcription factor Twist. Homeobox D10 (HOXD10) is targeted by miR-10b, indirectly inducing RAS homolog C (RHOC) to promote invasion and motility [65]. Other miRNAs such as miR-335 and miR-126 have been identified which are suppressed in metastatic lesions and likely inhibit disease progression by targeting genes involved in invasion and motility [66]. In experimental models, reconstituting expression of these miRNAs can suppress carcinoma cell migration and inhibit metastasis in mice [66]. A partial list of miRNAs involved in disease progression can be found in Table 13.1.

Though there is currently little evidence associating particular miRNAs with the various morphologies of breast carcinoma, many miRNAs have been associated more broadly with histopathologic phenotypes. For example, a number of miRNAs have been associated with hormone receptor status, an immensely important clinicopathologic feature in predicting prognosis and guiding treatment. For example, miR-191 is upregulated in estrogen receptor (ER) positive cancer compared to ER negative breast cancer [67, 68]. Other miRNAs have been associated with HER2 status, molecular or intrinsic subtypes (to be discussed further in the next section), and broad morphologic categories (i.e., ductal vs. lobular). Many of these are mentioned in Table 13.1.

Though analysis of particular miRNAs or miRNA signatures in breast tumor samples will be useful in patient management once validated, detection and accurate diagnosis of breast cancer remains an invasive process involving biopsy and/or lumpectomy. Much recent work in diagnostics has focused on noninvasive means of detection in order to detect breast cancers early and avoid unnecessary invasive procedures. The miRNAs expressed in normal and malignant tissues are frequently detectable in the body fluids of patients. As miRNAs are remarkably stable in the serum and detection methodologies advancing, it is possible that a simple blood test could detect cancer-associated miRNAs with high sensitivity and specificity [74–76]. In particular, miR-21 has been used in several studies to differentiate patients with and without breast cancer, suggesting this may be a promising candidate serum biomarker [77–81]. A variety of other miRNAs have been identified in the serum whose expression levels correlate to various clinicopathologic features [82–88].

Great advances in molecular pathology have revolutionized breast cancer medicine, identifying particular biomarkers with prognostic significance such as ER/PR and HER2. However, the management dilemma remains that prediction of relapse-free and overall survival is not perfect, and many patients with low risk cancer based on ER and PR positivity, for example, will experience local or distant relapse at various timepoints after the initial diagnosis. New diagnostic molecular assays such as Oncotype DX and Mammaprint attempt to bridge that gap with some success. miRNA and integrated miRNA/mRNA analysis to identify certain prognostic signatures as discussed previously show great promise as alternative or additional tests to determine which patients are most likely to relapse and require more aggressive treatment. Such tests may become commercially available in the near future.

Resistance to standard chemotherapy is currently one of the major areas of focus in breast cancer research. Though certain markers are known to associate with sensitivity to particular therapies, such as HER2 for Herceptin or ER for tamoxifen, we do not currently have markers of resistance in widespread clinical use. A number of large studies have identified genetic signatures associated with treatment response, including a study that identified miRNA signatures associated with sensitivity to several commonly used chemotherapeutics [89]. A variety of other studies have used miRNA profiling to identify particular miRNAs which are up- and downregulated in drug-sensitive versus drug-resistant cell lines [90–95]. Many of these are mentioned in Table 1. In the study of treatment resistance, miRNAs have become attractive targets, as more therapeutic options exist for overcoming resistance associated with these genetic elements [96]. Current studies have not used this information in clinical decision-making, and further characterization of identified miRNAs and miRNA signatures will be required to apply this data in patient studies.

As investigation continues to develop new therapeutics for resistant and difficult-to-treat cancer (i.e., triple negative), miRNAs have recently become attractive targets for therapy. These molecules are minimally antigenic compared to protein and carbohydrate biological drugs and small enough to penetrate a cell as a nanoparticle, liposome, or exosome without the need for potentially dangerous viral vectors [47]. However, tumor-specific drug delivery is a major concern, as systemic exposure to a miRNA or antagomiR (antisense oligonucleotide specific to a particular miRNA) could have unforeseen consequences in other organ systems. Tumor-specific antibody- and ligand-mediated systems will likely help to solve this major challenge [47].

Though miRNA therapeutics have not yet been studied in human patients, a number of potential therapies have been successfully tested in animal models. AntagomiR-10b has been used by Weinberg’s group to inhibit motility and invasiveness of a mouse mammary tumor cell line in a mouse model without adverse effect [97]. AntagomiR-21 has also been used to inhibit angiogenesis and induce apoptosis in a murine breast cancer model [98]. Other groups have used antagomiRs to treat neuroblastoma and cardiac hypertrophy, among others [99–101]. Though only one study has explored miRNA replacement therapy in breast cancer cells in vitro for radio-sensitization, miRNA replacement therapy has been tested with some success in cancers of the liver, colon, and lung using adenoviral, nanoparticle, or lipid-based delivery systems [102–106]. Unfortunately, tumor-specific delivery was not possible in these studies. However, successful treatment without major adverse effects is promising for the development of safe, effective new miRNA-based therapeutics in the near future.

The analysis of miRNA expression requires isolation of RNA from clinical specimens, including plasma samples, fine needle aspirates, and formalin-fixed tissues. There is a remarkable stability of small RNAs in the blood, particularly in the exosomal fraction, and miRNA can be isolated from the plasma with relative ease [107]. Most commonly, RNA is isolated using commercial kits and reagents, though the underlying methodology is the same. In brief, organic components are isolated through fluid phase separation by centrifugation. Chloroform and phenol in an aqueous buffer are added to the sample prior to centrifugation. A denaturing reagent, often guanidinium isothiocyanate, is also added to denature ribonucleoproteins which may complex with RNA, as well as RNAases. Following centrifugation, nucleic acids (DNA and RNA) will be found in the aqueous phase, lipids in the chloroform interphase, and other organic molecules in the phenol organic phase. The aqueous phase is then mixed with ethanol or isopropanol to precipitate nucleic acids. This mixture can then be applied to a silica-based column (i.e., RNeasy) to isolate total RNA (see Fig. 13.4) [74].

Though isolation of RNA from plasma is relatively rapid and simple, miRNA analysis of a tumor often requires fine needle aspirates of the lesion or formalin-fixed paraffin-embedded (FFPE) tissue blocks, as fresh tissue is not frequently available in the clinical setting. Fortunately, the small size of miRNAs is relatively protective against the damaging effects of formalin. Tissue is isolated from paraffin tissue curls treated with xylene by centrifugation. The resulting tissue pellet is treated with the broad specificity enzyme proteinase K to degrade contaminating proteins in the preparation. Nucleic acids are then precipitated in ethanol and total RNA isolated as previously discussed, usually using a commercially available silica-based column (see Fig. 13.4) [74].

As these techniques separate total RNA from the tissue specimen, miRNAs of interest must then be specifically detected. This is normally accomplished in one of two ways: (1) Northern blot or (2) real-time reverse transcriptase polymerase chain reaction (RT-PCR). In Northern blotting, total RNA extracts are separated by charge and size in an agarose or polyacrylamide gel, transferred to a nylon membrane, hybridized to sequence-specific probes, and detected radiographically or by chemiluminescence. While blotting is semiquantitative and best for determining qualitatively whether a miRNA is expressed, RT-PCR is truly quantitative. First, extracted RNA is reverse transcribed by the enzyme reverse transcriptase in a thermal cycler to produced complementary DNA (cDNA). Next, traditional PCR is used to amplify the target miRNA sequence using specific primers. A variety of fluorescent dyes and sequence-specific oligonucleotide probes are commercially available for detection of amplified miRNA sequences which utilize straight fluorescence or Förster resonance energy transfer (FRET), respectively. Direct detection of miRNAs in situ is also possible using locked nucleic acid (LNA) or Morpholino oligonucleotide probes (see Fig. 13.4) [74, 108].

Though the ability to detect specific miRNAs has allowed great advances in our understanding of these molecules in human disease, microarray analysis has allowed the simultaneous investigation of hundreds of miRNAs. Briefly, extracted RNA from a clinical sample is applied to a chip containing hundreds of specific oligonucleotide probes to which miRNAs in the sample will hybridize, allowing detection and quantitation of many miRNAs at once in a single sample. By utilizing probes for housekeeping genes in the microarray, the resulting data can be normalized, allowing comparison of multiple clinical samples. For example, it is possible to determine which miRNAs are up- or downregulated in tumor samples compared to normal tissue or identify miRNA signatures associated with a particular tumor subtype, hinting at potential biomarkers and pharmacological targets (see Fig. 13.4) [74].