Micro-RNAs and Breast Cancer

MicroRNAs [miRNA] are a class of naturally occurring non-coding RNA molecules similar to interfering RNAs which control expression of genes via the targeting of mRNAs and triggering degradation of RNA or translation of repression. It has been established 133 miRNAs are expressed in normal and primary human breast cancers (Blenkiron et al 2007). Medical research also indicates miRNAs are a major factor in the initiation and progression of cancers (Negrini, Calin 2008). The discovery of this class of molecules have prompted the identification of new gene regulatory mechanisms which play a major role in developmental processes and cellular processes such as differentiation, cell growth and death. Therefore it has been suggested miRNAs are involved in human diseases such as cancer more specifically breast cancer [BC] due to their aberrant expression. Aberrant expression or mutation of miRNAs has also been noted in other diseases such as chronic lymphocytic leukaemia’s [CLL] resulting miRNA deletions such as mir-15a and mir-16-1 which are associated in specific clinical and biological features of the disease. It was also established that the genes were affected via chromosomal translocations and was down-regulated in 50%-60% of all CLL patients (Iorio et al 2005). The most prominent deregulated miRNAs include mir-125b, mir-145, mir-21 and mir-155 which were confirmed via techniques such as northern blot analysis and microarray analysis. These expressions were associated with pathophysiological features of BC, such as oestrogen and progesterone receptor expression, vascular invasion, and tumour stage or proliferation index [PI]. Current research states miRNAs are contributing factors to oncogenesis via components of up-regulated tumour suppressions, such as the case in let-7 family targeting the RAS oncogenes in lung cancer or miR-15a and mi-R-16-1 targeting the BCL2 oncogenes of CLL. MiRNAs function as over expressed oncogenes, such examples include miR17-92 cluster targeting the E2F1 oncogene in lymphomas or miR-21 which is a regulator of PTEN tumour suppressor in hepatocellular carcinomas and BC. Furthermore it was highlighted that the TP53 gene is a regulator of transcription of the miR-34 family and is a mediator of apoptosis, cell cycle arrest and senescence (Negrini, Calin 2008). Down regulation of let-7 miRNAs expression has also been observed in some BC samples consisting of lymph node metastasis or high PI (Iorio et al 2005).

MiR-10 has been established as the initiator of invasion and metastasis of BC, while miR-335 was the suppresser of metastasis and migration by targeting of SOX4 and tenascin C [extracellular component]. It has also been reported miR-373 and miR-520c stimulate the migration and invasion of cancer cells due to suppression of CD44. The miRNAs with increased and decreased expression in malignancy for BC include miR-21, miR-29b-2, miR-125b, miR-145, miR-10b, miR-155, miR-17-5p and miR-27b respectively. The polycistron cluster miR-72-92 located at c13 or f25 locus on chromosome 13q31undergoes loss of heterozygosity in BC, while a number of other miRNAs such miR-10a and miR-196 are located within the homeobox cluster which are involved in the development of BC. This is further described by current expression studies of miRNAs in 76 breast tumours and 34 normal specimens indicating the differential expression of 29 miRNAs and a further 15 miRNAs that may discriminate between normal and tumour (Tavazoie et al 2008).

It has also been noted miRNAs bind to mRNAs via irregular complementarities resulting in events such as upregulate translation of tumour necrosis factor- α [TNF-α] and the activation or inactivation of miRNAs regulating the expression of protein coding genes [PCGs] in tumourigenesis. Pre-miRNA precursors consisting of 70-100 nucleotides with a hairpin structure are cleaved by cytoplasmic RNase III dicer, producing miRNA duplex of 22 nucleotides, composed of 2 strands of which 1 serves as the mature miRNA (Negrini, Calin 2008). The binding of single stranded miRNAs to the untranslated regions of the 3’ end via partial sequence homology of target mRNAs, results in inhibition of translation or degradation of mRNA although this step is less frequent. Hormones such as oestrogen and progesterone regulate certain miRNAs such as mir-30 which are down regulated in both ER- and progesterone receptor negative tumours (Cheng et al 2009).

Human tumours are classified on a molecular level using mRNA microarray profiling and a number of classifiers which are currently used for human breast tumours such as expression signatures that are used as prognostic tools (Fassan et al 2009). Some classifiers are used as a single sample predictors [SSP] to assign specific samples using any one of the BC subtypes; luminal B [LB], luminal A [LA], basal-like [BL], HER2+ and normal breast like [NBL]. Differential expression of miRNAs in breast tumour biopsies and correlation of miRNAs with HER2+ and oestrogen receptor +/- [ER+/ER-] status has become common and has been noted by current studies. It is indicated the breast tumour subtypes display an array of clinicopathological features such as varying survival rates. It was also established BL and HER2+ have low level differentiation but with aggressive features while LA and LB tumours are mainly ER+ with good and poor clinical outcomes respectively. Differential expression amongst LA and LB samples consisted of 9 different miRNAs belonging to 7 various miRNA families. Associations between 31 miRNAs representing 20 distinct miRNA families, molecular tumour subtypes and clinicopathalogical factors have also been assessed using statistical associations with tumour characteristics such as molecular subtype, grade, stage, vascular invasion, ER status, Nottingham Prognostic Index [NPI] and TP53 status which was determined using mutation screening and HER2+ status, which in turn determined by immunohistochemistry . Aggressive associations were verified between grade 3/ER- tumours, with grade 1/ER- associations displaying less aggressive expressions. Less strong associations have been found amongst NPI, TP53, HER2+, stage or vascular invasion (Blenkiron et al, 2007). It has been established mir-145 is progressively down regulated from normal breast to cancer consisting of high PI while mir-21 is progressively up regulated from normal breast to cancer composed of high tumour stage (Iorino et al 2005). Therefore the evidence suggests miRNAs have a regulatory role. The regulatory role of miRNAs has been portrayed by regulatory effect score [RE-score], where the inhibitory effect of a miRNA is measured using the average difference in expression of its targets versus non-targets in the form of a mathematical expression (Fig.1). According to RE-scores miRNA repression is higher in ER- than ER+ BC sub-types, suggesting a higher inhibitory effect of miRNAs involving ER-. However, miRNA processing genes such as Ago2, Ago1, TRBP and Dicer consisted of differential expression between ER- and ER+BCs, suggesting the difference in the regulatory effects of each sub-type. However, Ago1 and Ago2 are up-regulated in ER- while Dicer and TRBP are significantly down-regulated (Fig. 2) (Cheng et al 2009).

Since miRNAs act as gene regulators of BC and half of all mature miRNAs are within the cancer associated area of the genome, they are novel candidates for indicators of prognosis and diagnosis and therapeutic targets, and may play a significant role in the future. The challenges of management of BC include the search for sensitive markers to detect neoplasmic changes within the initial stages of the disease, monitor progression of the disease and response to therapeutics. Although mammography is the current gold standard for the detection of BC, miRNAs has potential to serve as biomarkers due to their aberrant expression. These expressions are tissue specific and miRNAs are known to be stable molecules. Recent studies also indicated miRNAs are preservable, detectable and quantifiable in circulation and bodily fluids providing promising prospects in therapeutics. The ability of aberrant expression of miRNA proves it is feasible in therapeutic interventions such as anti-miRNA 2-O-methyl or locked nucleic acid oligonucleotides used in the inactivation of oncomirs, such as miR-21 in breast tumours which may reduce growth. Therefore, tumour reduction associate with anti-miR-21 may be potentiated by the addition of the chemotherapeutic drug topotecan which is an inhibitor of DNA topoisomerase I, suggesting suppression of oncogene miR-21would sensitize tumour cells to anti-cancer therapy (Heneghan et al 2009). In general, miRNAs have an array of positive and negative roles in breast cancer making them dynamic molecule of interest.