Keywords

1 Introduction

Despite worldwide research on detection and therapy, breast cancer remains the leading cause of cancer death in women. Early detection of breast cancer is essential for survival and efficient treatment, yet current methods of detection, such as mammography, lack the sensitivity to sufficiently detect occult cancer and differentiate indolent from aggressive breast cancer. This deficiency may result in mortality due to a missed diagnosis or require additional invasive testing, which can result in unnecessary distress or over-treatment. Biomarkers that could function as an adjunct to mammography to detect breast cancer at an early stage, to identify aggressive disease or predict metastasis, could have a major impact on the management and outcome of this disease.

Currently, standard clinical parameters such as tumor size, grade, lymph node involvement and tumor-node-metastasis staging correlate with outcome and serve to stratify patients with respect to (neo)adjuvant chemotherapy and/or radiotherapy. Furthermore, molecular breast cancer markers of which the best known are estrogen receptor overexpression or HER2 amplification are used to predict the response to hormone therapy. However, stage-matched tumors grouped by histological or molecular subtypes can respond differently to the same treatment, so there is an additional need for tumor classifying molecular biomarkers. Emerging players in genetics are classes of non coding RNA molecules (like miRNAs and lncRNAs), due to their key roles in almost every developmental and cellular process, there for the possibility they could function as biomarkers in (breast) cancer is not unthinkable.

2 MicroRNAs

The first microRNAs (miRNAs) were characterized in the early 1990s [1]. However, miRNAs were not recognized as a distinct class of biological regulators with conserved functions until the early 2000s. MicroRNAs are small (ca. 22 nucleotides) non-coding RNA molecules found in plants and animals, which function in transcriptional and post-transcriptional regulation of gene expression [2]. Encoded by eukaryotic nuclear DNA, a miRNA will translationally repress or degrade his target through base-pairing with complementary sequences within mRNA molecules [3, 4]. The human genome may encode over 1000 miRNAs, which may target about 60 % of mammalian genes and are present in many human cell types [5–8].

Most microRNA genes are found in intergenic regions or in anti-sense orientation to certain genes and hence contain their own miRNA gene promoter and regulatory units [9–12]. However, probably 40 % is situated in introns of protein and non-protein coding genes or even rarely in exons. These are usually, though not exclusively, found in a sense orientation and thus show a concurrent transcription and regulation expression profile originating from a common promoter with their host genes [13, 14].

Mature miRNAs are produced through a multistage process that starts in the nucleus, where primary (pri-)miRNAs (several hundred to a thousand nucleotides in length) are transcribed by RNA polymerase II [12, 15, 16]. Pri-miRNAs are then processed to shorter (70–85 nucleotide) precursor (pre-)miRNAs mediated by Drosha, an RNase III enzyme, and its cofactor DGCR8 [17, 18]. Subsequently, pre-miRNAs are exported to the cytoplasm by exportin 5 [18, 19] and then cleaved by Dicer, another RNase III enzyme, to produce a ∼22 nucleotide double-stranded miRNA duplex [20–22]. The strand containing less stable hydrogen-bonding at its 5′ end is a mature miRNA and is integrated into the RNA-induced silencing complex, while the other strand is degraded [23, 24]. The microRNA /RISC complex attaches to the messenger RNA (mRNA) in one of two ways: when the sequences are perfectly complementary, the microRNA/ RISC complex binds tightly to the mRNA and, with the help of the enzyme Ago2, the mRNA is degraded [23, 25]. More commonly, when the sequences are imperfectly complementary, the microRNA/RISC complex binds and inhibits translation of the mRNA without degradation. The final outcome of either of these pathways is a decrease in the protein level of the target gene.

miRNAs are thought to have a key role in gene regulation although mostly they exhibit only partial complementarity to their mRNA targets [26, 27]. A ‘seed region’ of about 6–8 nucleotides long at the 5′ end of a miRNA is an important determinant of target specificity [8, 28]. It has been shown that gene regulation by miRNAs is a complex network, a given miRNA may have multiple different mRNA targets and a given target might similarly be targeted by multiple miRNAs [29, 30].

2.1 MiRNAS and Breast Cancer

Three important observations in the early history of miRNAs suggested a potential role in human cancer:

  1. 1.

    The miRNAs discovered in C. elegans and Drosophila were shown to be involved in cell proliferation and apoptosis, hence their deregulation may therefore contribute to proliferative diseases such as cancer [1, 31].

  2. 2.

    When human miRNAs were discovered, it was noticed that many miRNA genes were located at loci in the genome that are frequently amplified or deleted in human cancer [32]. A specific example of this is the polycistron cluster miR-17–92 at the c13orf25 locus on chromosome 13q31. This locus is known to undergo loss of heterozygosity in a number of different cancer types, including breast cancer [33]. Similarly, miR-125b, which is underexpressed in breast cancer, is located at chromosome 11q23-24, one of the regions most frequently deleted in breast, ovarian, and lung tumors [33].

  3. 3.

    Malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues [34–36]. The question remains whether the altered miRNA expression observed in cancer is a cause or consequence of malignant transformation.

In 2002, Calin et al. [37] reported the first direct evidence of miRNAs playing a role in human cancer; they found that miR-15 and miR-16 contribute to chronic lymphocytic leukemia. They discovered that this specific miRNA cluster was deleted in a significant portion of chronic lymphocytic leukemia cases. It was found that normally these miRNAs had a direct repressive effect on Bcl-2 , a well-characterized anti-apoptotic protein. In these CLL cases with low miR-15a/16-1 expression, it was found that Bcl-2 levels were increased, leading to an increased ability to avoid apoptosis, cell-death and tumor suppressor mechanism.

Subsequently, more examples of miRNAs involved in human cancer were discovered. Iorio and colleagues [38] first demonstrated miRNA deregulation in human breast cancer by miRNA microarray analysis; they identified a set of 15 miRNAs that was able to correctly predict the nature of the sample analyzed (i.e., tumor or normal breast tissue) with 100 % accuracy. These results leave few doubts that aberrant expression of miRNA is indeed involved in human breast cancer. They found that miR-10b , miR-125b, and miR-145 were down regulated, while miR-21 and miR-155 were up regulated, suggesting that these miRNAs may have potential tumor suppressor genes or oncogenes as targets. In addition, miRNA expression was correlated with biopathologic features such as estrogen receptor (ER), progesterone receptor status and tumor stage [38]. Measurement of miRNA levels demonstrates a global decrease in miRNA expression in breast tumours compared to adjacent normal tissue and a gradual decline with increased tumour grade [39].

The differential expression of miRNAs in breast tumor compared with normal breast tissue, and the indication of associations between miRNAs and tumor subtypes, suggest a potential role for such molecules in diagnostic biomarker panels.

2.2 miRNAs and Breast Tumor Tissue Profiling

To date, commonly three markers are established in the routine evaluation of breast tumors: estrogen and progesterone receptors (ER/PR: for predicting response to endocrine therapies) and HER2/neu (for predicting response to Trastuzumab) [40]. The most commonly employed technique to evaluate the hormone receptor status of breast tumors is immunohistochemistry, which relies on recognition of the receptor protein by specific antibodies. Although technically easy to perform and cost effective, this method is subjective and time consuming. If miRNAs prove useful for clinical diagnosis, their key advantage might be their high stability. In contrast to most messenger RNAs, they are long-lived in vivo and very stable in vitro, which might allow analysis of paraffin-embedded samples for routine diagnostic applications [24, 41].

miRNA profiling can be used to cluster cancer types with the cell of origin [42], so miRNA profiling may provide useful information to classify and diagnose metastases of unknown origin. This type of classification represents an important application in the diagnosis of patients with metastases (late-stage disease) without an established primary tumor (i.e., a site where a therapeutically curative or palliative intervention can be performed).

miRNA expression profiles have been used to differentiate tumor tissue from surrounding normal tissue for tumor classification and for prognostication. The capacity of miRNA expression profiles to classify breast tumors according to clinicopathologic variables currently used to predict disease progression highlights the potential of miRNA signatures as novel prognostic indicators which may contribute to the improved selection of patients for adjuvant therapy.

There are large-scale molecular differences between estrogen receptor (ER) α-positive and ERα-negative breast cancers [43, 44]. Endocrine therapy has become the most important treatment option for women with ERα-positive breast cancer, and approximately 70 % of primary breast cancers express ERα. ERα is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ERα-positive breast cancer [45–47]. Of all histopathological clinical parameters, ER status has the largest effect on the miRNA expression profiles (Dvinge et al. 2013). Multiple studies now have shown that ERα-expression is regulated by miRNAs. For example, miRNAs, miR-18a, miR-18b, miR-22, miR-193b, miR-302c, and miR-221/222, as well as miR-206, directly target ERα in 3’UTR reporter assays. Adams et al. [48] investigated the relationship between miR-206, and the expression of ERα. The authors identified and verified two specific miR-206 binding sites in the ERα 3′-UTR. Transfection of MCF-7 breast cancer cells with synthetic pre–miR-206 induced a dose-dependent repression of ERα mRNA levels. Conversely, MCF-7 cell transfection with antagomiR-206 resulted in increased ERα mRNA, indicating that miR-206 regulates endogenous ERα mRNA levels. Furthermore, treatment with ERα-selective agonists decreased miR-206 levels within MCF-7 cells. Notably, this study detected at least 65 putative miRNA target sites in the 3′-UTR of the ERα transcript, confirming that multiple miRNAs may play a role in regulation of ERα expression. The existence of a feedback loop between miR-206 and estradiol has considerable implications for our understanding of the endocrine influence on breast cancer, and the mechanisms involved in hormonal therapy resistance. Another interesting observation is that the expression of various let-7 miRNA isoforms is associated with features like progesterone receptor status, lymph node metastasis, or high proliferation index in breast tumor samples. The human let-7 miRNA family consists of 13 members located in 8 genomic locations frequently deleted in human cancers [49]. Nine distinct mature let-7 miRNAs with identical seed sequences are produced from 12 precursor sequences [50]. A reduced expression of let-7a in breast cancer was associated with larger tumor size and higher proliferative status, indicative that reduced let-7a expression may contribute to tumor growth.

mRNA profiles have identified distinct molecular subclasses of breast cancer, predictive of prognosis, based on their ER and Her2neu/ERBB2 classification (luminal A, luminal B, basal-like, Her2-overexpressing, normal-like) [44]. A comprehensive study of the breast cancer subclasses through miRNA expression profiling could probably further characterize the molecular basis underlying these subtypes, perhaps define more precise subsets of breast cancer. Blenkiron et al. [51] performed an integrated analysis of miRNA expression, mRNA expression and genomic changes in breast cancer and found that many miRNAs were differentially expressed between the different molecular subclasses of breast tumors. They identified a miRNA signature that differentiated basal from luminal subtypes and found nine miRNAs that were differentially expressed between luminal A and luminal B tumors (miR-100, miR-9 9a, miR-130a, miR-126, miR-136, miR-146b, miR-15b, miR-107 and miR-103). Similar to mRNA profiling, Mattie et al. [52] could show clustering of breast cancer tumors according to Her2/neu/ErBB2 status or ER/progesterone receptor status by miRNA profiling, Her-2neu/ERBB2–overexpressing breast tumors exhibit aggressive growth and unpredictable response to therapy; enhanced understanding of the regulation of ERBB2 expression has the potential to greatly improve the management of these aggressive tumors and miRNA profiles may have superior accuracy to mRNA profiling in this regard [35].

Dvinge et al. found that the mRNA-miRNA landscape is dominated by positive associations, suggesting that downregulation of target mRNA levels by miRNAs across the global breast tumour cohort is minor. They postulated that rather than acting as on-off switches of particular mRNAs, most miRNAs exert their effect by modulating the relationship between effector and target mRNAs, conceptually acting as co-repressors or.

2.3 Circulating miRNAs

A number of circulating tumor markers (for example: carcinoembryonic antigen and carbohydrate antigen 15–3) can be used in the management of breast cancer, but the sensitivity of these markers is low [53–55]. Therefore, they are not well suited to monitor disease progression or recurrence. Current challenges in the management of breast cancer include an ongoing search for sensitive and specific biomarkers that can be used to detect early neoplastic changes to facilitate the detection of breast cancer at an early stage. Furthermore, biomarkers are needed to monitor the progress of patients with breast cancer and their response to treatments. Existing diagnostic tools and biomarkers for breast cancer have many deficiencies. Mammography is currently the golden standard as diagnostic tool however it is not without limitations, including its use of ionizing radiation and a false positive rate of 8–10 % [56].

MiRNA presence in serum was described in patients with diffuse large B-cell lymphoma a few years ago [57]. Subsequently, a number of studies have reported similar findings on the presence of circulating miRNAs and have illustrated the potential of these miRNAs as novel biomarkers for diseases [58, 59]. Circulating miRNAs have many of the essential characteristics of good biomarkers. They are stable in the circulation and resistant to storage handling. Serum miRNAs are resistant to RNase digestion and other harsh conditions such as extreme pH, boiling, extended storage, and multiple freeze-thaw cycles. Further, most miRNAs sequences are conserved across species and third in some cases, changes in miRNA levels in circulation have been associated with different diseases as well as certain biological or pathological stages [60–63].

Although the exact mechanisms on how the small RNAs enter the plasma/serum and whether or not they are biologically functional need further investigations, it is possible that circulating miRNAs, compared to ‘tissue’ miRNAs are a unique diagnostic system. There is little doubt that plasma/serum miRNAs are cancer related, but the releasing mechanisms may be complicated. While the majority of miRNAs are found intracellularly, a significant number of miRNAs have been observed outside of cells, including various body fluids [64–68]. Given the instability of most RNA molecules in the extracellular environment, the presence and apparent stability of miRNAs here is surprising. Serum and other body fluids are known to contain ribonuclease, which suggests that secreted miRNAs are likely packaged in some manner to protect them against RNase digestion [69]. Tumor-derived microvesicles/exosomes are probably involved in the way miRNAs enter the circulation, rather than a simply leaking from cancer cells. One may hypothesize that at least some of the exported miRNAs are used for cell-to-cell communication, although further investigations are needed to determine how miRNAs are specifically targeted for secretion, recognized for uptake, and what information can be transmitted via this process [61].

Before this relatively new source of biomarkers can make it to the clinic, certain points remain to be explored. An important issue is the suitability of different sample types for miRNA detection. While Mitchell et al. [70] found no significant differences when comparing serum and plasma levels of miRNAs this result was limited to only four miRNAs and might not reflect the general image [71]. Studies have been performed in non-enriched or enriched whole blood, serum and plasma, without clear data being available on the distribution of miRNAs in these different blood compartments. It is acceptable that only a selection of miRNAs is, actively or passively, shed from circulating tumor cells.

A variety of independent studies have successfully proved the use of circulating miRNAs as diagnostic tools. Wu and colleagues [72] found that miR-21 and miR-29 were significantly up regulated in the serum of breast cancer patients and may be useful biomarkers for breast cancer detection [72, 73]. Heneghan et al. [74] surveyed a panel of 7 candidate miRNAs in whole blood RNAs from 148 breast cancer patients and 44 age-matched and disease free controls. They found that the expression of miR-195 was significantly elevated in breast cancer patients. Additionally, they observed a significant reduction in miR-195 in post-operative whole blood compared to the pre-operative samples of the same patients. However, Zhao et al. [75] could not confirm differential expression of miR-195 between cases and controls. The discrepancy between two studies might be due to different study materials. The first study used whole blood for detection of miRNAs, while the second one used plasma. Whole blood contains different types of cells, so the detected miRNAs may be circulating miRNAs as well as cellular miRNAs from additional cell types. Another explanation is that the discrepancy may reflect the heterogeneity of breast cancer. Different molecular pathways are involved in different subtypes of breast cancer, with different molecular characteristics between luminal A, luminal B, and basal like subtypes. In the Heneghan study, 59 % of breast cancers were stage I and II, 71 % were invasive ductal cancer, and 82 % were ER positive versus all stage I and II invasive ductal cancers, and only 55 % ER positive in the second study.

Ng et al. [76] identified significant increase of miR-16 , miR-21 , and miR-451 and significant decrease of miR-145 in the plasma of breast cancer patients. Intriguingly, the combination of plasma miR-145 and miR-451 levels provided the best markers for breast cancer prediction. The optimal sensitivity was 90 % and optimal specificity was 92 % in discriminating breast cancer from control subjects including all other types of cancers recruited in their study. The odds ratio for the cases with combined miR-145 and miR-451 level being associated with breast cancer was 44.2. In the blind validation, the positive predictive value was 88 % and the negative predictive value was 92 %.

Another drawback on the breakthrough of circulating miRNAs as biomarkers is the scarcity of data on the occurrence and expression levels of circulating miRNAs in healthy individuals. Expansion of this data set can be done either by testing selected panels of miRNAs in a large cohort of gender- and otherwise matched healthy controls in parallel with cancer patients and will help in the quest to determine new circulating biomarkers.

2.4 miRNA and Metastasis

A major complication of breast cancer is its metastatic potential. Metastasis is a process characterized by local invasion, intravasion, transport of tumor cells to the parenchyma of other organs, extravasation and establishment of secondary lesions [77]. There is evidence that metastasis can originate from genetic and epigenetic alterations in the molecular profile of a subpopulation of cells within the primary tumor, whose behavior is modulated towards a more aggressive phenotype [78]. Mutations occur primarily in the DNA sequence whereas epigenetic changes are related to the structure of the chromatin and might involve DNA methylation, histone modifications and non-coding RNAs [79]. Hence many studies have focused on identifying the critical regulatory molecules involved in the malign transformation of cells, and both proteins and miRNAs are believed to play a key role [80, 81].

miRNAs can function as suppressors or promoters of metastasis according to their mRNA targets [81]. In the next paragraphs we provide a brief overview of a handful of well-known examples of miRNAs believed to suppress or promote metastasis. Many more miRNAs are hypothesized to be involved in the metastatic process.

One of the first miRNAs identified as playing a role in metastasis, despite some conflicting evidence, was miR-10b . Functional studies have demonstrated that miR-10b overexpression promotes cell migration and invasion in vitro, and initiates tumor invasion and metastasis in vivo [82]. Upstream of mir-10b activation, the transcription factor Twist was found, which has been previously shown to be associated with invasive lobular carcinoma [82]. Downstream, it appears miR-10b inhibits translation of homeobox D10 (HOXD10) ensuring increased expression of ras homolog gene family, member C, a gene that promotes cell migration and invasion. In the same study breast carcinomas from metastasis-free patients showed low levels of miR-10b expression, whereas high levels of miR-10b expression were detected in 50 % of metastasis-positive patients. Gee et al. [83] studied miR-10b expression in patients with primary tumors and nodal metastases versus primary tumors without nodal metastases, but could not confirm a significant association between miR-10b levels and metastasis or prognosis. Added to these findings, miR-10b expression has been associated with the prognostically favorable luminal A subtype [51]. Further investigations and large scale studies will be required to fully elucidate the role of miR-10b in breast cancer metastasis.

Huang et al. [84] found a negative correlation between miR-21 and the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which suggests PTEN is a potential target of miR-21. In the same study, the authors compared miR-21 expression to markers of aggressive phenotype. They found a correlation between increased expression and lymph node positivity, higher proliferation index and advanced TNM stage. Since the tumor suppressor gene PTEN is also implicated in cell migration and invasion [85, 86], mir-21 may also have a role in invasion and metastasis. This supports the results of two other studies, which also identified a correlation between increased miR-21 expression and poor disease-free survival in early-stage patients and advanced clinical stage, lymph node metastases and shortened survival [87, 88]. According to Zhu et al. [89] miR-21 may promote tumor invasion and metastasis by simultaneously down regulating multiple metastasis-related tumor suppressor genes operating at distinct steps of tumor progression. Given that a miRNA can target over 100 genes [25], additional miR-21 targets which have a role in invasion and metastasis may be identified.

The miR-200 family contains 5 members (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) clustered in 2 genomic loci (200b-200a-429 and 200c-141) which target members of the Zeb family of transcriptional repressors [90]. The miR-200 family is believed to play an essential role in tumor suppression by inhibiting epithelial-mesenchymal transition (EMT), the initiating step of metastasis [91]. During EMT, cells lose adhesion and increase in motility [92]. Epithelial cells typically have normal cell-to-cell junction and adhesion, while mesenchymal cells have weaker cell wall adhesion, making them more motile and likely to enhance invasive characteristics. EMT has been found to play an essential role in tumor invasion, metastatic dissemination, and the acquisition of resistance to current cancer therapies (reviewed in [77]). Studies indicate that the miRNA-200 family could regulate the EMT process by targeting specific molecular markers of EMT [93]. In the pioneering work by Gregory et al. [94], it was suggested that the entire miR-200 family is down regulated upon exposure to transforming growth factor-β (TGF-β). TGF-β is a cytokine that is known to induce the EMT phenotype [95–97]. Re-expression of the miR-200 family significantly inhibited EMT that was induced by TGF-β, while inhibition of the miR-200 family resulted in the induction of EMT phenotype. Furthermore, there were increased levels of ZEB1 and ZEB2 following the induction of EMT, which suggests that the miR-200 family is a negative regulator of the mesenchymal markers, ZEB1 and ZEB2. Down regulation of miR-200b and miR-200c has been demonstrated to be associated with loss of E-cadherin expression in breast cancer cells with mesenchymal phenotype, as a result of a consequential up regulation of the E-cadherin transcriptional repressor, ZEB1 [98]. Conversely, miR-200b or miR-200c restoration induced E-cadherin expression, therefore inhibiting EMT and causing a less aggressive phenotype in the cancer cells. Another study [99] focused on the miRNAs suppressed by ZEB1 and showed that the affected miRNAs were members of the miR-200 family. It was revealed that ZEB1 can bind to highly conserved sites in the promoter and directly suppress transcription of the complete cluster of the miR-200 family. Furthermore, ZEB1 is also a target of miR-200c (as mentioned above), which indicates that there is an EMT-inducing feed-forward cycle. Thus, evidence from all these studies suggests that the miR-200 family acts as a central regulator in tumorigenesis, metastasis and aggressiveness.

miR-31 has been identified as an inhibitor of multiple steps of the invasion-metastasis cascade in breast cancer [100, 101]. miR-31 is encoded by a single genomic locus and is expressed in a variety of human tissues [102, 103] and this miRNA is one of the pleiotropically cancer-relevant miRNAs. Valastyan et al. [101] identified miR-31 as a regulator of metastatic progression in human breast cancer. The authors demonstrated an inverse correlation between miR-31 expression and the invasive capability in 15 different breast epithelial cell lines. Additionally, miR-31 levels in primary human breast tumors were revealed to be inversely associated with the propensity of clinically detectable distant metastases.

miR-335 was found to inhibit metastasis through the targeting of a set of metastasis genes, including the transcription factor SOX4 and the extracellular matrix protein Tenascin-C, however, its expression is down regulated in the majority of primary breast tumors from patients who subsequently relapse [104]. The combined genetic and epigenetic targeting of the miR-335 locus in all metastatic derivatives obtained from distinct patients highlights the significance of this molecule as a barrier to metastatic progression in breast cancer. The observation that miR-335 is often silenced in the primary tumor has led to the identification of this miRNA as an inhibitor of tumor re-initiation in addition to its established role as a suppressor of invasion and metastatic colonization [105]. This miRNA, like let-7, can suppress tumor initiation in breast cancer. Interestingly, while let-7 also suppresses proliferation and tumor growth, miR-335 selectively abolishes tumor re-initiation without inhibiting proliferation or tumor growth [106].

miR-9 is up regulated in breast cancer cells through activation by MYC and MYCN, and it directly targets E-cadherin-encoding mRNA and CDH1, leading to increased cell motility and invasiveness [107]. miR-9 levels correlate with grade in primary breast tumors and are significantly elevated in those patients with metastases compared with those without. These findings are consistent with the observation that miR-9 expression is higher in breast cancer patients with local relapse. The higher expression of miR-9 in cancer cells may indicate a more aggressive tumor, also suggested by the association with higher stage in the study of Zhou et al. [108].

Many examples of a correlation of miRNA expression and metastasis are present in literature. The challenge of selecting the ones usable in the clinic remains.

3 Long Non Coding RNA Molecules

Long non-coding RNAs (lncRNAs) are a heterogeneous group of non-coding transcripts longer than 200 nucleotides that are involved in many biological processes. This class of ncRNA makes up the largest portion of the mammalian non-coding transcriptome [109]. Long non-coding RNAs provide a new opportunity to identify both functional drivers and cancer-type-specific biomarkers. As the knowledge about lncRNAs grows, studies concluded that lncRNAs tend to show more tissue-specific expression than protein-coding genes [110]. This property of makes them highly attractive as tissue-specific biomarkers.

Very little is known about lncRNA biogenesis, in contrast with miRNAs, pre-processing mechanisms are not necessary. Until recently, very few lncRNAs were annotated within the human genome. Now, various groups have developed independent catalogs of human lncRNAs [110–112]. Despite the thousands of human lncRNAs now predicted, only a handful of lncRNAs have been well characterized to date, little is known about the expression patterns of most lncRNAs in different cell types.

Various mechanisms of transcriptional regulation of gene expression by lncRNAs have been suggested. lncRNAs utilize a large arsenal of mechanisms to regulate gene expression. lncRNAs act as co-activators, binding to transcription factors and enhancing their transcriptional activity [113–115]. Another mechanism is transcriptional interference, where the act of transcribing a lncRNA interferes with transcription initiation, elongation or termination of another gene [116]. lncRNAs can also affect transcription by binding to transcription factors and shuttling them into the cytoplasm to keep them away from their nuclear targets [117]. Recent evidence also suggests that some lncRNAs may have enhancer-like function [118], activating expression of nearby genes by an unknown mechanism.

While the study of lncRNA function is still in its infancy, a role for a number of these transcripts has recently been established in cancer, in general, as well as specifically in breast cancer. Like protein-coding genes and miRNAs, lncRNAs play key roles in tumorigenesis. They have been shown to play a functional role in a number of fundamental processes associated with cancer including cell cycle regulation, apoptosis, the DNA damage response, and metastasis. Similar to mRNA profiling and miRNA profiling one may hypothesize that lncRNA profiling could serve as a signature to divide breast cancer tumors in clinically relevant subtypes.

Since the functions of most lncRNA still need to be unraveled or confirmed (although it is clear they can function, like miRNAs, as oncogenes or tumor suppressor genes) the focus in this chapter lies on two well known examples (HOTAIR and BC-200), both with great potential as breast cancer biomarkers.

Gupta et al. [119] found that lncRNA in the HOX loci become deregulated during breast cancer progression. This study identified a distinct set of HOX lncRNA to be overexpressed in primary tumors and very frequently overexpressed in metastases. One such lncRNA, HOTAIR, was increased in primary tumors and metastases and its expression level in primary tumors was a predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb Repressive Complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity [119, 120]. Chisholm et al. [121] provided evidence that expression levels of lncRNA in the HOX loci do tend to cluster with some clinicopathologic data, such that increased ncHoxA1 trends with proliferation rate, and ncHoxD4 trends with positive PR receptor status.

BC200 , also known as BCYRN1 (brain cytoplasmic RNA 1), is a 200 nucleotide long ncRNA selectively expressed in the nervous system and usually not detected in somatic cells other than neurons. It is, however, overexpressed in several solid cancers including breast cancer [122]. BC200 RNA is expressed at high levels in invasive breast carcinomas, but is barely detectable in normal tissue or in benign tumors [123]. Interestingly, in ductal carcinomas in situ (DCIS), significant BC200 expression is associated with high nuclear grade. This suggests that BC200 may be a useful marker for early detection of breast cancer and that the presence of BC200 RNA in early lesions might have utility as a prognostic indicator of tumor progression.

In conclusion, this overview clearly suggest that non coding RNAs have great potential to be used as biomarkers in breast cancer. Various applications (tumor profiling, risk of relapse, detection of early stage breast cancer) are possible but many aspects still need to be explored before these RNA molecules will be transferred from bench to bedside.