MicroRNA in cancer: New hopes for antineoplastic chemotherapy

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that are evolutionarily conserved and widely distributed among species. Their major function is to negatively regulate mRNA target genes, and miRNA expression has been found to be deregulated in all human cancers, where miRNAs play critical roles in tumorigenesis, functioning either as tumor suppressors or as oncogenes. This review provides a current overview of the connection between miRNAs and cancer by covering the recent advances in miRNA involvement in human cancer including initiation, growth, invasion, and metastasis. We will also highlight the literature where application of miRNAs has created the foundation for the development of potential future miRNA therapy.


Introduction
MicroRNAs (miRNAs) are small non-coding RNAs belonging to a novel class of negative regulators that control gene expression by imperfect base pairing to the 3 untranslated region (3UTR) of a target mRNA, leading to a protein translation inhibition or mRNA degradation (1). MiRNAs are initially transcribed by RNA polymerase II as long primary precursors which are rapidly processed by an endonucleolytic multistep process into 18-22nt long mature miRNAs (2). Once produced, miRNAs are loaded into the RNA-induced silencing complex (RISC) and guide the degradation or translational repression of the target mRNA (3). MiRNAs are found in the majority of multicellular and complex eukaryotes; it is currently estimated that miRNAs represent 1% of the predicted genes and that up to 10%-30% of human transcriptome is a direct target of miRNAs, indicating a pivotal role for miRNAs in almost every biological process, including cell cycle regulation, cell growth, apoptosis, cell differentiation, and stress response (1). However, genetic analysis of Caenorhabditis elegans highlighted that only 10% of individual miRNA knock-outs have a distinct morphological and developmental defect (4), illustrating the existence of functional redundancy among miRNAs or miRNAregulated pathways. This redundancy shows that miR-NAs may have evolved to control and refine specific aspects of terminal differentiation programs. Indeed, another important aspect of microRNA activity is represented by their ability to target thousands of genes per single miRNA, based on the 5 end-restricted complementarity between animal miRNAs and mRNA targets (only five to eight nucleotides of perfect complementarity, termed 'seed sequence'). This target multiplicity may explain their ability to control several mRNAs that function in the same or similar processes. For example, miR-221 and miR-222 have been shown to activate cell proliferation by repressing a series of tumor suppressors, all of which are involved in cell cycle inhibition, like p27 (5), its activator FOXO3a (6), p57 (7), BMF (8), PTEN (9), and APAF1 (10).
Given their essential role in all the aspects of the cellular life, it is not surprising that miRNAs were found to cover important roles in tumorigenesis, and, since this discovery, over 5000 publications link miRNAs to cancer, including more than 1000 reviews.
In this review we will outline the biological and molecular roles of miRNAs in cancer and metastasis by highlighting the most promising advances for the use of miRNAs in the clinical setting, both as direct and adjuvant chemotherapeutic agents.

MiRNAs in cancer
Genome-wide miRNA expression profiling analyses have reported a general dysregulation of miRNA expression in all tumors (11). To date, over 1000 miRNAs have been reported in humans (miRbase: 1527 at November 2011), and hundreds of them map to chromosomic regions that are known to be altered in human cancer, such as loss of heterozygosity regions (LOH) (e.g. miR-15a/16-1), amplified regions (e.g. miR-17-92 cluster, miR-155), and breakpoint regions and fragile sites (FRA) (e.g. let-7 family members) (12). Published studies have defined that elevated expression of some miRNAs (oncogenes) as well as down-regulation of others (tumor-suppressors) accompanies carcinogenesis and correlates with the development of cancerassociated phenotypes.
The first evidence of miR involvement in cancer came from our laboratory in 2004. We identified that the miR-15 and miR-16 genomic locus is heterozygously deleted in 68% of all patients with B-cell chronic lymphocytic leukemia (CLL) (13). Loss of miR-15 and miR-16 reflects their tumorsuppressor function via uncontrolled expression of their anti-apoptotic target protein BCL2 in an in vitro model as well as in patient specimens of B-CLL (14). Furthermore, a sequencing-based screen for miRNAs dysregulated in familial CLL patients identified a germ-line mutation in the primary precursor of miR-15a/16-1 that impairs their processing, highlighting that not only deletions but also mutations may lead to miRNAloss of function (15). The importance of genetic lesions reducing miR-15/16-1 cluster expression in CLL was furthermore confirmed by the identification of a point mutation located in the 3 flanking region of miR-16 that reduces miR-16 expression in a naturally occurring CLL mouse model, the New Zealand black (NZB) mouse, that develops a hematological disorder similar to the human CLL (16). Even more definitive genetic evidence of the tumor-suppressive effects of miR-15 and miR-16 came recently from the Dalla-Favera lab where miR-15/16 knock-out mice were generated (17). Targeted deletion of miR-15 and miR-16 in mice at the age of 18 months recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5(+) monoclonal B-cell lymphocytosis, and CD5-non-Hodgkin lymphomas (17).
Specifically, miR-15a/16-1 deletion accelerates the proliferation of both human and mouse B-cells by modulating the expression of genes controlling cell cycle progression like cyclin D3, cyclin E, CDK6, CHK1, and MCM5. However, the same authors also demonstrated that the size of 13q14 deletions, where the miR-15/16-1 cluster is located, influences the phenotype of lymphoproliferative disorders and potentially the severity of disease, suggesting a tumor suppressor function for genetic elements besides miR-15a/16-1 and their host gene DLEU2 (18). In fact, an additional deletion of a 0.69 mb-large region telomeric to miR-15/16-1 increased the appearance of CLL, and mice seemed to succumb to their disease faster than miR-15/16-1-deleted mice. Deletion of the miR-15/16-1 cluster also occurs in other forms of tumors, such as multiple myeloma (19) and prostate cancer (20), indicating that the loss of these miRNAs could be relevant to other pathogenic events. In fact, the De Maria group demonstrated that, in prostate cancer, miR-15a/miR-16 levels are strongly downregulated in the vast majority of cases (up to 85% of the analyzed samples) (20). Interestingly, intraprostatic injection of miRNA antisense RNA oligonucleotide ('antagomirs') specific to miR-15a and miR-16 in 6-week-old male BALB/c mice resulted in marked hyperplasia, and knock-down of miR-15a and miR-16 promoted survival, proliferation, and invasiveness of untransformed prostate cells, which became tumorigenic in immunodeficient NOD-SCID mice (20). Furthermore, they also demonstrated that miR-15 and miR-16 are down-regulated in fibroblasts surrounding the prostate tumors. Increased miR-15 and miR-16 expression in cancer-associated fibroblasts impaired tumor growth and expansion of prostate tumors in xenograft models through the reduced post-transcriptional repression of Fgf-2 and its receptor Fgfr1 (21).
Similar to the down-regulation of the miR-15/16-1 cluster, another miRNA frequently lost in cancer is represented by the miR-34 family. These miRNAs, miR-34a on chromosome 1p36 and the miR-34b/c cluster on chromosome 11q23, are frequently deleted in neuroblastoma, breast, pancreas, hepatic, and colon carcinoma (22). Loss of expression of the miR-34b/ c cluster has also been associated with the hypermethylation of their CpG-associated promoter region in gastric cancer, while the same chromosomal region was not methylated in normal gastric mucosa (23). The miR-34 family has been shown to be transcriptionally activated by p53, and it represents one of the main effectors of p53-induced apoptosis, senescence, and cell cycle arrest. These effects are achieved by repressing the expression of multiple oncogenes such as Notch-1 (24), Notch-2 (24), HMGA2 (24), Myc (25), several cyclins and CDKs (26), CD44 (27), the anti-apoptotic factor BCL2 (28), and the p53 deacetylase SIRT1 (29). Recently, miR-34 family knock-out mice have been generated, and no obvious developmental or pathological abnormalities were observed at up to 12 months of age (30). However, the authors showed that miR-34-deficient mouse embryonic fibroblasts accelerate the reprogramming, not as expected through cell proliferation, but instead, at least partly, by post-transcriptional derepression of pluripotency genes, such as Sox2, N-Myc, and Nanog (30). Many other miRNAs have been reported to act as tumor suppressors (Table I) (38).
Although there seems to be a general trend towards down-regulation of microRNAs in cancer (39), many different miRNAs follow the opposite pattern and, based on their ability to initiate or accelerate cancer, are named 'oncomiRs'. The first evidence of an oncogenic miRNAappeared in 2005 when the Mendell lab demonstrated that Myc can directly induce the expression of the polycistronic cluster miR-17/92 (40). By using a reconstituted mouse model of Myc-induced lymphoma expressing a truncated version of the miR-17/92 cluster in hematopoietic cells, the authors showed an accelerated tumor development probably due to an anti-apoptotic mechanism associated with the suppression of their direct targets, such as inhibitors of proliferation CDKN1A (41), direct regulators of the apoptosis PTEN (42), and BCL2L11 (also known as BIM) (43). This cluster has been shown to be up-regulated in many solid tumors such as breast, stomach, prostate, lung, and pancreas tumors (44), and genetic deletion of the miR-17/92 cluster in mice highlighted their importance in B-cell development, as homozygous deletion of the cluster results in premature death of B-cells at the pro-B and pre-B stages, resulting in lymphopenia (45). Interestingly, functional dissection of the miR-17/92 cluster in the context of B-cell transformation in vivo revealed that miR-19a and b mediate the oncogenic activity of the entire cluster, at least in part, by targeting the tumor suppressor PTEN and thereby activating Akt-mTOR signaling to promote cell survival (42).
Recently, the Slack lab generated the first conditional knock-in of miR-21 overexpressing mice that showed a 15-30-fold induction of miR-21 (64). These mice developed a severe pre-B-cell lymphoma, but when miR-21 was reduced to endogenous levels the mouse tumors completely disappeared, defining the concept of 'oncomiR addition' (64). Interestingly, a 4-6-fold overexpression of miR-21 resulted in no obvious phenotype; however, miR-21 overexpression potentiates the lung tumorigenesis of a constitutively activated KRAS proto-oncogene (65). Another in vivo evidence that miR-21 exerts an oncogenic function came from the DMBA-TPA skin carcinogenesis model used by Ma et al. on miR-21-null mice (66). The miR-21 mice showed a significant reduction in papilloma formation compared with wild-type mice, with miR-21-null mice exhibiting an increase in cellular apoptosis and decrease in cell proliferation that is explained by the up-regulation of the miR-21 targets Spry1 (62), Pten (50), and Pdcd4 (56).
Many other miRNAs have been reported to act as oncogenes (Table II) by inducing tumor growth, aggressiveness, and resistance to chemotherapy when expressed ectopically in vitro or in vivo: miR-155, miR-221, and miR-222 cluster, miR-9 and miR-103/107 family.

MiRNAs in metastasis
Accumulating data have pointed to a central regulatory role of miRNAs in the acquisition of invasive abilities and generation of the malignant phenotype that consists of loss of cellular adhesion, acquisition of cell motility, intra-and extravasation, and finally proliferation at a distant site. The first example of a 'metastamiR' is represented by miR-10a (67). Weinberg's group demonstrated that miR-10a is overexpressed in about 50% of metastatic breast cancers and is transcriptionally activated by the pro-metastatic transcription factor TWIST1. Specifically, they demonstrated that miR-10a did not affect cell proliferation but instead promoted in vitro and in vivo migration, invasion, and metastasis of otherwise non-invasive breast cancer cells by repressing the homeobox protein D10, a transcription factor already known  for its roles in cell motility. Contrary to miR-10a, miR-31 is highly down-regulated in metastatic breast cancer, and its overexpression strongly reduces metastatic spreading without affecting primary tumor growth (37). MiR-31's actions on already-seeded lung metastases can be mediated via co-ordinate repression of a cohort of metastasis-promoting genes, including ITGA5, RDX, and RhoA. The invasion-metastasis cascade, the multistep process whereby tumor cells disseminate from their  (70). Another important study in the comprehension of miRNA involvement in metastatic spread came from the Massague lab, which has discovered that miR-335, miR-126, and miR-206 are lost in human lung and bone metastatic cells (71). Overexpression of all three miRs suppressed the metastasis to lungs and bone in mice. Although miR-126 inhibits tumor growth and cell proliferation, miR-335 specifically suppresses metastatic invasion by repressing the transcription factor SOX4 and the extracellular matrix Highlighted a hitherto unappreciated role of SA5 in lymphoma biology and defined a unique mechanism used by cancer cells to escape TGFb's growth-inhibitory effects protein tenascin C. A further involvement of miR-126 in the metastatic setting was recently defined by the Tavazoie group (72). They revealed that endogenous miR-126 non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. Specifically, it suppresses metastatic endothelial recruitment, metastatic angiogenesis, and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1, and MERTK-novel pro-angiogenic genes and biomarkers of human metastasis.

New therapeutic approaches using miRNAs
The strong involvement of miRNAs in cancer appearance, development, and progression provides a rationale for the therapeutic approach to miRNAs in cancer treatment. During the years of miRNA research, several strategies have been pursued, and they can be recapitulated essentially in miRNAbased therapy and miRNA-adjuvant therapy. The first approach will consist in reducing/increasing miRNA levels (oncomiRs/tumor suppressor) and/or interfering with the miRNA/mRNA interaction. As a therapeutic possibility, miRNAs may be used as single molecules or in a combinatorial way to target one or multiple genes. For example, if a mutation is altering the binding site for a miRNA, combining two or three miRNAs can overcome the therapy unresponsiveness. In addition, a combination of antagomiRs for onco-miRs and miRNA mimics for tumor-suppressors may represent a future approach. The first example to be cited in this area is the therapeutic use of let-7 molecules in lung cancer. Let-7 has been shown to be down-regulated in a large fraction of human cancers, and low levels of hsa-let-7a expression are correlated with poor survival of lung cancer patients (44,73). Intratumoral injection of liposome-pre-let-7 molecules in preformed lung tumor xenografts resulted in the strong inhibition of overall tumor growth (74). However, this delivery route might be inadequate in a clinical setting, as peripheral tumor cells remained present and showed limited knock-down of let-7 targets by immunohistochemistry. Another approach with potential for translation into clinical settings is the use of a neutral lipid emulsion (NLE). NLE has shown tumor-inhibitory effects of let-7 NLE formulations in a mouse model of lung cancer (a transgenic mouse with a constitutively activated KRAS) with an already established lung tumor (75). Another interesting approach of miRNA-based therapies came from Dr Mendell's lab. MiR-26a is strongly downregulated in hepatocellular carcinoma (HCC) as compared with paired non-cancerous tissues, and miR-26a's low expression is associated with a shorter overall survival but a better response to interferon therapy (76). Mendell's lab demonstrated that systemic delivery of a miR-26a-expressing adenoviral particle via tail intravenous injections into a MYCinduced hepatocellular carcinoma mouse model resulted in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression, without toxicity (77). However, the therapeutic approach of miR-26a needs to be carefully considered based on the fact that miR-26a has been found to be up-regulated in T-ALL patients and its expression enhanced leukemogenesis in a mouse model of T-ALL by suppressing the levels of PTEN and BIM tumor suppressor protein (77). A recent work by Hatziapostolou and co-workers defined a new player in hepatocarcinogenesis and new potential targets for the treatment of HCC (78). The authors identified the existence of a positive feedback loop involving miR-24/miR-629/HNF4alpha/ miR-124/STAT3/IL-6R, in which perturbations amplify and perpetuate the knock-down of HNF4alpha, thereby inducing liver cancer. To test the relevance of this novel inflammatory circuitry in vivo, they delivered miR-124 to the liver during diethylnitrosamine (DEN)-induced hepatocarcinogenesis by using tail injection of a miR-124 precursor liposome particle. Not onlydid miR-124 reduce the number of carcinogeninduced tumors by 90% when administered throughout the entire study, but it also led to a striking 80% decrease when treatment was started only 4 weeks before sacrifice. Even considering that therapeutic success in mouse models is not always reproducible in humans, this study demonstrated the efficacy of miR-124 in the treatment of liver cancer, and more importantly it opens the possibility of using miR-124 therapy in the advanced stages of HCC.
As previously stated, only the 'seed sequence' of a miRNA is required to identify the appropriate mRNA target, and miRNA'target multiplicity' is the natural consequence of the small number of bases required for miRNA function. Therefore, blocking the function of a single miRNA that is dysregulated in a neoplastic state can have a major effect on disease progression, because it will reactivate the expression of a large fraction of genes. However, this reactivation is not a simple task, as different miRNAs can target the same sequence and miRNAs have identical seeds but differ in the non-seed region. In fact, the traditional 15-mer antagomiR would target more than the seed sequence, thereby generating the potential for large, off-target inhibition of other miRNAs. The solution appears to be selectively inhibiting miRNAs using only the seed as a target. Sakari Kauppinen and colleagues described an important new strategy for silencing the function of miRNAs after investigating the functional consequences of targeting only the seed sequence (79). They found that targeting the seed region with a perfectly matched 8-base antimiR potently inhibited miR-21 function and resulted in up-regulation of a miR-21 target protein (79). Moreover, by targeting the seed region of let-7, they showed how it is possible to block the function of an entire family of microRNAs. A key feature of this approach for future therapy is the simple systemic delivery; in fact, intravenously injected tiny LNAs (locked nucleic acid: chemically modified ribonucleotides) are taken up by a range of mouse tissues, such as breast, lung, and liver, which leads to long-lasting silencing of miRNA function.
The second clinical application of miRNAs is related to a more therapeutic value of miRNAs as acute, rather than chronic, adjuvant targets. This application is essentially supported by the numerous studies describing the modulation of cell sensitivity to chemotherapeutic agents after miRNA expression modulation. A majority of these studies note the identification of modulated miRNAs in cells resistant to chemotherapeutic drugs. In this regard, the first story that we would like to illustrate came from our laboratory and describes the involvement of the miR-221 and miR-222 cluster in TNF-related apoptosis-inducing ligand (TRAIL)-and gefitinibresistance of non-small cell lung cancer (NSCLC) (10,80,9). In 2005, our lab addressed the implication of miRNAs in TRAIL-resistance of non-small cell lung carcinoma, and we found that miR-221 and miR-222 were markedly up-regulated in TRAILresistant and semi-resistant cells compared to TRAIL-sensitive NSCLC cells. Specifically, we demonstrated that silencing p27 kip1 but not Kit, both functional targets of miR-221 and miR-222, increased resistance to TRAIL (80). This result supports well the involvement of miR-221 and miR-222 in determining the TRAIL-resistant/sensitive phenotype in NSCLC cells mainly by miR-221 and miR-222 interfering with p27 kip1 expression and TRAIL-induced caspase machinery. Recently, we also reported that the hepatocyte growth factor receptor (MET) oncogene, through c-Jun transcriptional activation, upregulates miR-221 and miR-222 expression, which, in turn, by targeting PTEN and TIMP3, confers resistance to TRAIL-induced cell death and enhances tumorigenicity of lung and liver cancer cells (82). The results suggest that therapeutic intervention involving the use of miRNAs should not only sensitize tumor cells to drug-inducing apoptosis, but also inhibit their survival, proliferation, and invasion. In our most recent manuscript, we explored the involvement of MET-modulated miR-221 and miR-222 in the development of de novo and acquired resistance of lung cancer cells to tyrosine kinase inhibitors, such as gefitinib, the first line of treatment for EGFRpositive lung cancer patients (10). We demonstrated that these two miRNAs are regulated by both MET and EGFR receptors in lung cancer cells, and more importantly, that their expression levels are downregulated upon gefitinib treatment only in gefitinibsensitive cells but not in resistant cells because of MET overexpression. We also demonstrated that gefitinib resistance could be overcome by MET inhibitors, which down-regulate miR-221 and miR-222 and sensitize lung cancer cells to gefitinib, or by anti-miR-221 and anti-miR-222, which strongly increase gefitinib sensitivity in vitro and in xenograft mouse models in vivo (10). Another interesting application of miR-221 and miR-222 on drug resistance has been described in tamoxifen resistance in breast cancer. Tamoxifen is one of the most widely used selective estrogen receptor modulators in breast cancer therapy for estrogen receptor-positive patients. One of the main problems related to its use is the appearance of tamoxifen resistance in a large fraction of patients who initially respond to the therapy. Majumder's group demonstrated in 2008 that miR-221 and miR-222 are the most modulated miRNAs in cells with acquired resistance to tamoxifen (81). Interestingly, ectopic expression of miR-221 and miR-222 rendered the parental cells resistant to tamoxifen, and the effect was partially related to the down-regulation of the cell cycle inhibitor p27 kip1 , a known target of miR-221 and miR-222. Up-regulation of miR-221 and miR-222 has also been implicated in resistance to drugs such as fulvestrant (82) and cisplatin (83) in breast cancer, castration-resistant prostate cancer (84), TRAILresistant non-small cell lung cancer cells (80), and radiation-resistant gastric carcinoma cells (85).
Another interesting story that relates microRNA expression to chemoresistance is represented by the ability of miR-21 to modulate the sensitivity of cancer cells to 5-fluoruracil (5-FU). High levels of miR-21 were correlated with a very low response to therapy and poor outcome in colorectal cancer patients (86); the low response to the drug is partially associated to reduced levels of core mismatch repair mutator genes (63). Interestingly, inhibition of miR-21 expression was able to sensitize cells to 5-FU, suggesting that miR-21 inhibition may represent a future way to overcome cell resistance to traditional chemotherapy (63). Like miR-21, miR-34 has also been shown to modulate cancer cell sensitivity to 5-FU (87). In this case, ectopic expression of miR-34 in colorectal cancer cells inhibited cell proliferation and sensitized cancer cells to 5-FU. However, miR-34 inhibition has been shown to enhance the apoptotic response to bortezomib in Myc-transformed B-cells, clearly demonstrating that miRNA involvement in chemotherapy is context-dependent (88).

Conclusion
MiRNAs continue to reveal high diversity and complexity in their biological functions and involvement in disease. However, despite remarkable recent progress, the connection between cancer and miRNAs remains incompletely understood, and many open questions remain with regard to the possible application of miRNAs in therapy. First, based on the miRNA target multiplicity and tissue-dependent effects, one of the main goals of future miRNA research must be the identification of the widespread effects on the transcriptome of every single miRNA. To this aim, genome-wide analyses will need to delineate the global alterations in miRNA genes or copy number alterations in various human tumors in order to identify all of the putative tumor-suppressor and oncogenic miRNAs. At the same time, more sophisticated in vivo models are needed to identify and define miRNA functions. Because miRNAs can have widespread effects on the transcriptome, their full biological properties are unlikely to be explained by the suppression of a single or few proteins. In fact, the major challenge is to define all of the mRNA targets and cancer-related pathways that are controlled by the dysregulated miRNAs in neoplasia. Unmistakably, miRNAs have a role in cancer, and the major challenge is to recognize the impact on the networks rather than on a few isolated targets. With this aim, new sophisticated computational target prediction methods will be helpful. Although miRNAs only moderately suppress their targets, miRNAs could exert both strong and broad effects, largely because they suppress many genes and because they are implicated in multiple feedback loops with other regulators of gene expression. Exploring the roles of miRNAs in these intimate cross-talks will help us to understand the causes of cancer and other diseases and in creating new alternative therapeutics.

Declaration of interest:
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.