Measurable evidence of miRNAs as regulators of cancer networks and therapeutic targets

Keywords::

• cancer
• genetic regulation
• miRNAs
• networks
• therapeutic targets

Figure 1. Transcriptome–miRNA networks in cancer.

(A) In this figure, the relationship between vital oncogenic transcriptome networks and the miRNAome are graphically represented. Target mRNAs for each major pathway are represented by circles with a unique color. miRNAs are represented as hairpin structures in the centre. The arrows connecting miRNAs and mRNAs indicate validated mRNA–miRNA interactions. The small arrow in the circles indicates the biological effects on the pathway by the miRNA acting on its target. (B) Gene–protein network in normal tissues and in cancer. miRNAs are transcribed from miRNA noncoding genes, which have their own transcriptional unit, or from introns of protein-coding genes. In general, one gene is transcribed to one mRNA and translated to one protein. By contrast, miRNAs are transcribed from one, or in certain cases, two genes. miRNAs coordinately regulate multiple mRNAs (shown as a net of connections), thus affecting the output of many proteins. miRNAs have a crucial role in keeping the gene–protein network interconnected.

CDC42: Cell division cycle 42; CEBPB: CCAAT/enhancer binding protein β; DNMT: DNA methyltransferase; HDAC4: Histone deacetylase 4; HOXA1: Homeobox A1; HOXD10: Homeobox D10; KIT: v-kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homologue; MCL1: Myeloid cell leukemia sequence 1 (BCL-2-related); MLH1: MutL homologue 1, colon cancer, nonpolyposis type 2; MMP2: Matrix metalloproteinase 2; MSH: MutS homologue, colon cancer, nonpolyposis type 1; PDCD4: Programmed cell death 4; PTEN: Phosphatase and tensin homologue; SHIP1: Src homology 2 domain-containing inositol 5-phosphatase 1; SOX4: SRY (sex-determining region Y)-box 4; SP1: Sp1 transcription factor.

Reproduced with permission from [6].

miRNAs are small noncoding RNAs (ncRNAs) that contribute to gene expression regulation. In diseases such as cancer, deregulated miRNA expression can be measured by high-throughput technology. Restoring perturbed miRNA expression may result in novel therapeutic developments that, when added to current standard treatment, could improve the outcomes of patients with cancer. The advantage of using miRNA approaches is based on their ability to target multiple genes concurrently, resulting in homeostasis of deregulated signaling pathway networks. However, this impact on expression profiling of multiple genes simultaneously, several of which may regulate normal function of noncancer cells, can lead to substantial adverse effects. Apart from miRNAs, transcription factors, epigenetic changes and chromatin contribute to gene regulation in a complex functional regulatory network. Here I discuss the latest advances in translating miRNA research into the improved management of cancer, and how challenges might be overcome.

Gene expression & regulatory systems

When Lee and colleagues discovered miRNAs in the gene lin-14 in Caenorhabditis elegans in 1993 [1] they could not have predicted the impact that their experimental findings would have on the current explosion of biomedical translational medicine [2]. Approximately two decades later, as incredible as it might seem, not only has the genome annotation nearly been completed, but our understanding of the functional organization of the worm C. elegans[3] and another model organism, the fly Drosophila melanogaster[4], has also been dramatically improved using high-throughput screening technology and next-generation sequencing technology. These advances and future research on flies and worms will quite often provide the shortest and most efficient path to curing human disease [2]. High-quality experimental evidence now reveals that miRNAs, as ncRNAs, as well as transcription factors and epigenetic changes, regulate gene expression in space and time [3–5]. Silencing of tumor-suppressor genes and reactivation of oncogenic retroviruses are involved during cancer development and progression, and therefore miRNAs, epigenetic modifications and transcription factors provide exciting new avenues for cancer research [6–8]. Currently, miRNA-based anticancer therapies are being developed, with the goal to improve disease response and increase cure rates [6].

miRNA biogenesis

miRNAs are small evolutionarily conserved ncRNAs of 18–25 nucleotides in length that have become rising stars in cancer genetics. They act as expression regulators of genes involved in fundamental cell processes, such as development, differentiation, proliferation, survival and death [9].

miRNAs are mostly transcribed from intragenic or intergenic regions by RNA polymerase II into primary transcripts of variable length, called pri-miRNAs. The primary transcripts undergo further processing in the nucleus, thereby resulting in a hairpin intermediate of approximately 70–100 nucleotides, called pre-miRNA. The pre-miRNA is then transported out of the nucleus to the cytoplasm by exportin 5. In the cytoplasm, the pre-miRNA is processed by another ribonuclease, Dicer, into a mature double-stranded miRNA of variable length (∼18–25 nucleotides). After strand separation, the guide strand or mature miRNA is incorporated into an RNA-induced silencing complex (RISC) [10]. The RISC is the effector complex of the miRNA pathway and is comprised of miRNA, argonaute proteins (argonaute 1–argonaute 4) and other protein factors [10]. Argonaute proteins have a crucial role in miRNA biogenesis, maturation and miRNA effector functions [10]. The mature strand is important for target recognition and for the incorporation of specific target mRNAs into the RISC [10].

Targeting multiple genes

Each miRNA has the potential to target a large number of genes (on average ∼500 for each miRNA family) [11]. An interplay between mRNAs and miRNAs, as suggested by bioinformatical analysis and experimental validation, reveals that miRNAs might cooperate to regulate gene expression. Overall, the complexity and widespread regulation of gene expression by miRNAs should be taken into consideration when developing miRNA-based therapies [6].

Measurable evidence of miRNA deregulation in cancer

Aberrant expression of miRNAs is associated with human diseases, such as cancers, because miRNAs act as expression regulators of genes involved in fundamental cell processes, such as development, differentiation, proliferation, survival and death [9]. With the advent of high-throughput technologies, such as quantitative reverse transcriptase PCR and microarrays for the global measurement of miRNAs, numerous studies have explored associations between miRNAs and cancer features. For example, it was reported that selected groups of distinct miRNAs were commonly and concurrently upregulated or downregulated in distinct types of human neoplasia, and were often associated with distinct cytogenetic abnormalities [12]. miR-17 and miR-21 were identified to be consistently upregulated in colon, lung, stomach, prostate and pancreatic tumors, and miR-155 was discovered to be upregulated in breast, lung and colon cancer [12].

Rationale for targeting miRNAs

The fundamental principles for using miRNAs as anticancer drugs is based on two major findings: that miRNA expression is deregulated in cancer compared with normal tissues; and that the cancer phenotype can be changed by targeting miRNA expression [6,12–14]. One of the most profound effects of miRNAs as therapeutic agents is their ability to target multiple genes, frequently in the context of a network, making them extremely efficient in regulating distinct biological cell processes relevant to normal and malignant cell homeostasis (Figure 1)[6,15]. This capacity of miRNAs to regulate multiple genes in a molecular pathway makes them excellent candidates for novel molecular-targeting treatments.

Cancer networks & miRNAs

Somatic and inherited mutations and epigenetic modifications drive cancer initiation, progression and metastasis [16,17]. However, how these changes across the genome and epigenome lead to complex diseases, such as cancer, is poorly understood [18]. Beyond genome sequencing for the identification of genetic variation underlying disease, understanding how molecules regulate gene expression still remains a daunting challenge. The field of functional genomics using next-generation sequencing technology for exploring gene regulation has rapidly evolved over the last few years [18,19]. Molecular networks and regulatory systems are increasingly attracting scientific interest to explain how the structure and function of animals and humans are organized [20]. Although the concept of molecular networks that regulate cellular processes and biological systems is not new [21], this hypothesis was only recently supported by validated experimental evidence [3,4]. Gene expression, which is crucial in designing novel therapies, is orchestrated by three interacting systems that, apart from miRNAs and other ncRNAs, include transcription factors and epigenetics [5].

In cancer, as a result of mutations, deletion or epigenetic alterations in miRNA genes or in protein-coding genes, aberrant miRNA and mRNA expression occurs, resulting in the expression of oncogenic proteins that cause a certain cancer phenotype. Recent state-of-the-art studies, ranging from technological advances in miRNA detection and profiling, clinically oriented miRNA profiling in several human cancers, to a systems biology analysis of global patterns of miRNA regulation of signaling and metabolic pathways, now provide critical insight into interactions of miRNAs with transcription factor regulatory networks. Gene–gene and protein–protein networks and interactions with transcription factor regulatory networks and mathematical modeling of miRNA regulation are now investigated widely [22–24]. As miRNAs coordinate responses in a network by targeting multiple genes, the perturbation of the miRNA network has a vital function during carcinogenesis, causing aberrations in the transcription of large numbers of genes. The miRNA network is ‘hijacked’ to promote malignancy. By modifying the miRNA network, it may be possible to restore homeostasis in cancer [6].

Challenges of miRNA-based therapies

The challenges for developing miRNA-based therapeutics are the same as the challenges for small interfering RNA therapeutics, and include issues of delivery, potential off-target effects and safety [6]. One of the major problems for the use of miRNA therapeutics in vivo relates to tissue-specific delivery and to cellular uptake of sufficient amounts of synthetic oligonucleotides to achieve sustained target inhibition [25]. The first obstacle to overcome is the biological instability of these compounds in bodily fluids or tissues, as unmodified ‘naked’ oligonucleotides are rapidly degraded by cellular and serum nucleases [25]. The second obstacle is the poor cellular uptake of oligonucleotides owing to their size and negative charge, which could prevent them from crossing through cell membranes [25].

However, whilst awaiting the results of the first Phase I and II studies, the outcomes of this class of miRNA-based agents on safety and efficacy cannot currently be predicted. It is still not known whether the advantage of miRNAs to target multiple genes simultaneously can increase the efficacy by killing more cancer cells that are regulated by a set of genes; however, at the same time they may be associated with serious adverse effects because this set of genes may regulate important cellular processes. The second concern is that by only targeting miRNAs without considering other important regulatory systems, such as transcription factors, other ncRNAs and epigenetic modifications, may have limited clinical efficacy.

Conclusion

The discovery of miRNAs and the measurable evidence of their expression deregulation in cancer has revolutionized biomedical research, offering us a global understanding of cancer functional complexity. With the explosion in miRNA research, a new era in the development of anticancer drugs targeting miRNAs is beginning. Although it is still unclear whether the miRNA-based drugs discovery direction will be translated with success into oncological practice, it is clear that miRNA research provides an exciting opportunity to deeply investigate highly complex functional regulatory networks.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.