MicroRNAs in breast cancer and breast cancer stem cells and their potential for breast cancer therapy (2013)

The discovery of the first miRNA, lin-4, in Caenorhabditis elegans initiated a new era of miRNA biology. Since then, thousands of miRNAs have been identified and annotated, many of which have been shown to play roles in a variety of biological processes, including development, differentiation, apoptosis, proliferation, and cell death.1 Furthermore, growing evidence indicates that miRNA deregulation is a critical cause of cancer formation. The biogenesis, function, and potential application of miRNAs have become active areas of research. With the development of molecular biological technologies, such as northern blotting with radio-labeled probes, cloning, quantitative PCR, serial analysis of gene expression (SAGE)-based techniques, bead-based profiling methods, and oligonucleotide microarrays,2 it is possible to conduct miRNA research precisely and comprehensively. BIOGENESIS OF MICRORNAS MicroRNAs are derived from introns or exons of protein-coding and non-coding genes,3,4 and are either transcribed by polymerase II as a long primary transcript (primary miRNA) or originate from the introns of mRNAs. Primary miRNAs are further processed by the Drosha microprocessor complex, which recognizes stem-looped secondary structures within primary miRNAs, resulting in the excision and release of ˜70 nucleotide hairpin precursors termed pre-miRNAs (precursor microRNAs).5 The mirtron subclass of miRNAs, which are encoded in the introns of genes, generate pre-miRNAs directly from byproducts of intron splicing and disbranching events in the nucleus with the assistance of a “debranching enzyme”.6 After being exported from the nucleus by exportin-5, the pre-miRNAs are subsequently cleaved by Dicer to release a 22-nucleaotide miRNA-miRNA duplex. One strand of this duplex is incorporated into the RNA-induced silencing complex (miRISC), and eventually serves as a mature microRNA, while the other strand is degraded. The “seed” region of the mature microRNA (nucleotides 2-8 at the 5′ end) can bind partially or completely to the 3′UTR of specific protein-coding gene mRNAs.7,8 MicroRNAs regulate their targets by directly cleaving mRNAs or inhibiting protein synthesis, depending on the degree of complementarity with the 3′UTRs of their targets.4 Even though the 3′UTR is generally accepted as the target site of miRNAs, new discoveries have demonstrated that miRNA target interactions are not restricted to the 3′UTR. Chi et al9 immunoprecipitated the RISC complex and removed all mRNAs by RNase treatment except for the portion that was bound to and protected by the RISC complex. In addition to the many 3′UTR sites (32% of all identified sites), they identified a substantial portion of binding sites (8%) in regions that were annotated to be downstream of 3'UTRs. This possibly reflected unannotated 3′UTR sequences. Many sites were found in the coding regions of genes (25%). Similar results have been described in several other studies,10-12 indicating that coding regions of genes are probably also important target sites that are yet to be described in detail. In addition, another study13 showed that miR-10a interacts with the 5′UTRs of mRNAs encoding ribosomal proteins to enhance their translation, suggesting that 5′UTRs are another possible functional site for miRNAs. THE MECHANISMS OF MICRORNA DYSREGULATION IN CANCER Several studies have investigated miRNA expression in cancer patients and found that miRNAs are differentially expressed between normal and tumor tissues.14-18 There are several explanations regarding the mechanisms of miRNA dysregulation. A widely accepted mechanism is genetic; chromosomal abnormalities could lead to the deletion, amplification, or translocation of miRNAs.19 Many common chromosomal amplifications and deletions in cancer contain miRNA-coding sequences.20 For instance, copy number alteration of genomic loci containing miRNA genes correlates with altered miRNA expression; 72.8% of breast cancers exhibited miRNA gene number abnormality. In addition to aberrant expression due to altered miRNA gene copy number, a high frequency of copy number abnormalities are seen in Dicer1, Argonaute2, and other miRNA-associated genes that may result in miRNA deregulation.21 Furthermore, miRNAs are often located inside or close to fragile sites, in minimal regions of loss of heterozygosity, in minimal regions of amplification, or in common breakpoints associated with cancer.22 These data support the causal relationship between genetic abnormality and anomalous expression of microRNAs and provide evidence for the genetic abnormality theory. Considering the complexity of miRNA biogenesis, any disturbance is likely to contribute to aberrant expression of miRNAs at the transcriptional and post-transcriptional regulation level. MiRNAs are regulated at the levels of miRNA promoter transcription, methylation, miRNA processing, RNA editing, and miRNA target interactions.5 First of all, miRNA transcription is positively and negatively regulated at the promoter level. Transcription factors c-Myc and p53 can bind to distinct miRNA promoter elements to modulate miRNA gene expression.5 Tumor suppressor protein, P53, is well known to be tightly suppressed in many tumors, and down-regulation of P53 reduces miRNA promoter activity, leading to transcriptional disturbance of miRNAs. MicroRNA deregulation in cancer might also be attributable to the impairment of miRNA-processing steps.23-26 The two key steps of miRNA processing include Drosha-mediated nuclear processing of primiRNAs and Dicer-mediated cytoplasmic processing of pre-miRNAs. For instance, many tumors seem to have a reduced capacity to process pri-miRNAs into their mature forms. The level of primary miRNA gene transcripts (pri-miRNAs) does not correlate with the level of mature miRNAs in tumors, whereas it does correlate in normal tissues. The possible mechanism is a Drosha processing block.24 Studies5 have demonstrated that several factors are involved in the regulation and control of Drosha-mediated nuclear processing, including transcription factor activators, such as SMAD1, 3 and 5, the RNA-binding proteins, hnRNA A1 and KSRP (the KH-type splicing regulatory protein), the inhibitor of adenosine to inosine (A to I) editing, and the RNA-binding protein, LIN-28. Any subtle change of post-transcriptional factors would therefore activate or inhibit Drosha-mediated processing. Likewise, A to I editing of precursor sequences, or LIN-28 and KSRP complexes could block the cytoplasmic processing of pre-miRNAs by Dicer,5 implying that miRNA deregulation would eventually result in tumor occurrence. In addition to miRNA transcription disruption at the promoter level, RNA editing and miRNA-target interactions partially account for miRNA deregulation in cancer cells.5 Finally, aberrant hypermethylation is an additional mechanism for miRNA gene inactivation in human breast cancer. Extensive down-regulation of mir-9-1 correlates closely with its promoter hypermethylation in breast cancer.27 When miR-34a expression is silenced by aberrant CpG methylation of its promoter, ectopic miR-34a induces a G1 cell cycle arrest, senescence, and apoptosis, eventually causing several types of cancer, including breast cancer.28 MiR-335, as a robust inhibitor of tumor reinitiation, undergoes genetic deletion and epigenetic promoter hypermethylation in metastatic derivatives from independent patients' malignant cell populations.29 Similar results were found for other candidates, such as miR-9-330 and let-7a-3 in human lung cancer,31 and the miR-124a family in various human tumor types,32 indicating that epigenetic methylation plays an important role in miRNA dysregulation. MICRORNAS IN BREAST CANCER MicroRNAs play a wide variety of roles in the tumorigenesis and progression of breast cancer, including acting as oncogenes, tumor suppressor genes and modulators of tumor proliferation, invasion, apoptosis, and therapy resistance. To fully understand the roles that miRNAs play in breast cancer, newly identified miRNAs and their functions in breast cancer are summarized as follows (Tables 1-4).Table 1: miRNAs and invasion and metastasis in breast cancerTable 2: MicroRNAs and proliferation in breast cancerTable 3: MicroRNAs and therapy resistance in breast cancerTable 4: microRNAs and apoptosis in breast cancerMICRORNAS AND BREAST CANCER STEM CELLS According to cancer stem cell theory, cancer initiating stem cells are responsible for the formation, progression, metastasis, therapy resistance, and recurrence of malignant tumors. Easy invasion, strong cancer-causing capability, and chemotherapy and radiotherapy resistance are features of this class of cells, which can contribute to cancer relapse. Recent research has focused on the relationship between miRNAs and cancer stem cells. Aberrant expression of miRNAs was identified in cancer stem cells. Sun et al used microarray technology to test for differences in expression of miRNAs between ESA+CD44+CD24-/low breast cancer stem cells and the MCF-7 breast cancer cell line; they reported that 19 miRNAs were over-expressed, with levels four times higher in BCSCs than in MCF-7 cells. These 19 miRNAs included, miR-122a, miR-152, miR-212, miR-224, miR-296, miR-31, miR-373, miR-489, PRED_MIR127, PRE-D_MIR154, PRED_MIR157, PRED_MIR162, PRE-D_MIR165, PRED_MIR191, PRED_MIR207, PRED_MIR219, PRED_MIR246, PRED_MIR88, and PRE-D_MIR90. Furthermore, miR-200a, miR-301, miR-188, miR-21, miR-181d, and miR-29b showed four times lower expression levels in BCSCs compared to MCF-7 cells.72 Likewise, Shimono et al73 found 37 microRNAs that were differentially expressed between human BCSCs and non-tumorigenic cancer cells. More importantly, among them, three miRNA clusters in breast cancer stem cells were significantly down-regulated, including miRNA-200c-141 located on chromosome 12p13, miR-200b-200a-429 located on chromosome 1p36 and miR-183-96-182 on chromosome 7q32. Transfecting miR-200c-expressing lentivirus into MMTV-Wnt-1 murine breast cancer cells leads to a 96% reduction of colony formation compared with control lentivirus. To evaluate the effect of miR-200c on human BCSCs, human BCSCs were infected with the miR-200c-expressing lentivirus. Then, 5000 to 10 000 infected BCSCs were injected into the mammary fat pad of NOD/SCID mice. Human BCSCs infected with control lentivirus formed six tumors out of 13 injections, whereas miR-200c-expressing BCSCs formed only one tumor out of 13 injections. BMI-1, involved in regulating self-renewal and differentiation of various stem cells, was shown to be the target of miR-200c. In addition to suppressing the expression of BMI1, it has recently been shown that miR-200 family miRNAs prevent EMT (epithelial-to-mesenchymal transition) by suppressing expression of ZEB1 and ZEB2, two transcriptional repressors of E-cadherin.45,74 EMT is a stem cell property that has been linked to both normal and cancer stem cells.75 Therefore, miR-200c has the capability to modulate self-renewal, proliferation and tumorigenesis of breast cancer stem cells. Yu et al76 showed that let-7 was markedly reduced in tumor-initiating cells (BT-ICs). Infecting BT-ICs with let-7-lentivirus reduced proliferation, mammosphere formation, the proportion of undifferentiated cells in vitro, and tumor formation and metastasis in NOD/SCID mice. With regards to the underlying mechanism, H-RAS and HMGA2 were demonstrated to be two functional targets. Silencing H-RAS in a BTIC-enriched cell line reduced self renewal but had no effect on differentiation, while silencing HMGA2 enhanced differentiation but did not affect self renewal. Likewise, enforced constitutive expression of miR-30 in BT-ICs inhibited their self-renewal capacity by reducing Ubc9, and induced apoptosis through silencing ITGB3. In contrast, blocking miRNA with a specific antisense oligonucleotide (ASO) in differentiated breast cancer cells revived their self-renewal capacity. Furthermore, ectopic expression of miR-30 in BT-IC xenografts reduced tumorigenesis and lung metastasis in nonobese diabetic/severe combined immunodeficient mice, while blocking miR-30 expression enhanced tumorigenesis and metastasis.77 Taken together, it has been shown that miR-200c, miR-let7, and miR-30 are involved in the proliferation, self-renewal, differentiation, and tumor formation of breast cancer stem cells. Regulating the expression of one specific miRNA could decrease the tumor-initiating, self-renewal ability of BCSCs or promote differentiation, which provides a promising new direction for clinical cancer therapy. POTENTIAL THERAPEUTIC APPLICATION OF MICRORNAS FOR BREAST CANCER It is generally accepted that aberrant miRNA expression is an important causal mechanism of tumorigenesis. As summarized above, miRNAs function as oncogenes and tumor suppressors and are involved in the regulation of cancer metastasis, proliferation, apoptosis, and therapy resistance. More significantly, miRNAs have proved to be integral modulators of self-renewal, differentiation, and tumor formation of breast cancer stem cells. Therefore, developing new medicines that target miRNAs is a promising direction for treating breast cancer. When one specific miRNA is down-regulated and demonstrated to be a tumor suppressor in breast cancer, up-regulation of that miRNA can be effective in inhibiting or even eliminating cancer. First of all, delivering tumor suppressor miRNA mimics or miRNA-expressing lentivirus to a particular tissue or organ can increase the number of tumor suppressor miRNAs, and effectively inhibit tumor proliferation, development and metastasis. Liang et al78 designed and synthesized pre-miRNAs against CXCR4 (CXC chemokine receptor-4) and found that they could regulate tumor migratory and metastatic properties. This double-stranded miRNA gene was ligated into a miR-155-based Block-iT Pol II miR RNAi expression vector. Expression levels of CXCR4 in CXCR4-miRNA-transfected breast tumor cells declined significantly. These cells exhibited reduced migration and invasion in vitro. In addition to miRNA replacement therapy to modulate miRNA function in vivo, a new ‘sponge’ vector expressing miRNA target sites was designed to saturate endogenous miRNAs, thereby preventing regulation of their natural targets.79 In this case, miR-9 was identified as a pro-metastatic miRNA in breast cancer,80 and the miRNA sponge-mediated suppression of miR-9 in the highly metastatic 4T1 mouse mammary tumor cells reduced lung metastasis by 50%, although no effect was observed in the onset of the primary tumor. In contrast, some miRNAs are over-expressed in breast cancer and might act as oncogenes. Antisense oligonucleotides are being developed to utilize the intrinsic RNAi pathway for gene therapy. Oligonucleotides inhibit miRNA function by binding to complementary base-pairs of miRNAs.81 To improve oligonucleotide stability in vivo, a number of different chemically modified oligonucleotides have been developed, including locked nucleic acid, 2′-O-methyl,82 2′-O-methoxyethyl,83 morpholinos,84 cholesterol,85 and phosphorothioate modifications.86 Of these chemical modifications, the two most promising ones are locked nucleic acid (LNA) and 2′-O-methylation. LNA is a conformational RNA analogue that binds complementary RNA with unprecedented affinity and specificity.81 Simoes-Wust et al87 found that LNA-modified oligonucleotide inhibited Bcl-2 and Bcl-xL expression more effectively compared with a 2′-O-(2-methoxy)ethyl (2′-MOE) modified gapmer antisense oligonucleotide, thus facilitating tumor cell apoptosis in MDA-MB-231 breast and H125 lung cancer cells. LNA modifications enhanced the efficacy and favorably modulated the target specificity of antisense oligonucleotides. Besides high potency, LNA shows high affinity to RNA target molecules and adequate cellular uptake and biodistribution, which are mandatory for the development of safe and effective therapeutic drugs81 2'-O-methyl oligonucleotides bind efficiently and irreversibly to RISC by base-pairing with the small guide RNA. Injection of a 2′-O-methyl oligonucleotide complementary to the let-7 miRNA into C. elegans larvae phenocopied a let-7 loss-of-function mutation, demonstrating that 2'-O-methyl oligonucleotides can disrupt the function of a single miRNA in vivo. 2′-O-methyl oligonucleotides are potent and irreversible inhibitors of small RNA-directed RNA silencing in vivo and in vitro.82 One of the key challenges in the development of effective microRNA-based therapies is the delivery of stable and efficient agents into target cells88 To improve the efficiency of miRNA/anti-miRNA delivery in vivo, gene therapy vectors based on adeno-associated virus (AAV) have been investigated. Kota et al89 showed that AAV-mediated miR-26a delivery potently suppressed cancer cell proliferation and activated tumor-specific apoptosis in vivo, resulting in dramatic suppression of tumor progression. AAV has several advantages, such as running a minimal risk of insertional mutagenesis,90 and efficient targeting of many tissues of interest.91,92 Moreover, Xu et al developed tumor-specific, ligand-targeting, self-assembled, nanoparticle-DNA lipoplex systems designed for systemic gene therapy of cancer. These nanovector systems employ transferrin (Tf) or scFv against transferrin receptor (TfR), which is over-expressed in the majority of human cancers, as a tumor-targeting ligand.93 This nanovector system shows promising efficiency and specificity in targeted delivery of various genes and anti-sense oligonucleotides to cancer but not normal tissues in vivo.94 All these studies imply that AAV and nanoparticle-DNA lipoplex systems could be effective tools for delivering miRNA to cancer tissues for tumor treatment. In conclusion, miRNAs are involved in tumorigenesis and progression of breast cancer and they can act as oncogenes, tumor suppressors, or metastasis regulators. More significantly, miRNAs have the potential to be targets for tumor therapy, which has attracted the attention of researchers. However, obstacles still exist for miRNA therapy. For example, each miRNA may target several different mRNAs and may function in cancer through targeting a regulatory network of genes. Conversely, a single mRNA could be targeted by several miRNAs. Furthermore, miRNA targets include not only mRNA but also DNA. One of the advantages of multiple targets is that a miRNA can work on multiple cell signaling pathways at the same time, so that it might create united and potent efficacy. In contrast, altering a miRNA may affect many unexpected genes, and it may also induce unexpected side effects. As many miRNA targets remain undetermined, miRNA mimic or infection might degrade “innocent” mRNAs or inhibit indispensable protein synthesis; the outcome of this scenario would not be positive. It is also evident that miRNAs are important participants in the regulation of CSCs. Studies have already demonstrated that up-regulation of miRNAs has the capacity to inhibit or even eliminate CSCs.76,77 However, one problem is that normal stem cells and BCSCs share common molecular mechanisms that regulate stem cell functions, such as self-renewal, proliferation, and EMT.76 Whether artificial modulation of miRNAs would influence the important physiological functions of normal stem cells remains unclear. Finally, even though new technologies are emerging to improve the specificity, stability, and efficiency of miRNA delivery and therapy, the final outcomes remain uncertain. To date no miRNA technology is clinically applicable. To fully reveal the mechanisms of miRNA function and to clinically apply miRNAs to tumor therapy, a substantial amount of further work is needed.