MicroRNAs and other non-coding RNAs as targets for anticancer drug development

Abstract

The first cancer-targeted microRNA (miRNA) drug - MRX34, a liposome-based miR-34 mimic - entered Phase I clinical trials in patients with advanced hepatocellular carcinoma in April 2013, and miRNA therapeutics are attracting special attention from both academia and biotechnology companies. Although miRNAs are the most studied non-coding RNAs (ncRNAs) to date, the importance of long non-coding RNAs (lncRNAs) is increasingly being recognized. Here, we summarize the roles of miRNAs and lncRNAs in cancer, with a focus on the recently identified novel mechanisms of action, and discuss the current strategies in designing ncRNA-targeting therapeutics, as well as the associated challenges.

Figure 1. Mechanisms of action of miRNAs and the use of therapeutic agents to block or activate their function

(A) The diverse mechanisms of miRNA activity are presented together with the related miRNA-targeting strategies. In blue are the agents that reduce the miRNA activity, while in red are the agents that increase miRNA activity. Initially transcribed as primary miRNA (pri-miRNA), processed into precursor miRNA (pre-miRNA) by a microprocessor complex composed of Drosha and DGCR8, transported from nucleus to the cytoplasm by Exportin 5 in the presence of the Rna-GTP cofactor, and further processed into mature form by Dicer, miRNA recruits on the RISC and regulates the output of protein-coding genes through diverse mechanisms. The interaction of miRNA with the 3′-untranslated region (3′-UTR) of protein-coding genes is considered as the main mechanism, which usually leads to a decrease in protein output by either mRNA degradation or translational repression. Recent studies also suggest that miRNA can interact with the 5′-untranslated region (5′-UTR) of protein-coding genes via complementarity, and cause translational repression or activation of the targeted proteins . Similarly, miRNA can also target the coding sequence, and repress the translation of targeted genes . Moreover, miRNAs can interact with regulatory protein complexes such as AGO2 and FXR1, and indirectly upregulate translation of a target gene . Whether the “non-canonical” mechanisms represent a general mechanism or just exceptions to the canonical one remains to be determined. Various means can be used to enhance (such as by Enoxacin) or block (by any defects in the biogenesis machinery) general miRNA production, however this approach is not specific. More specific regulation of miRNA activity can be achieved by using miRNA mimics or anti-miRs such as LNAs, antagomirs and miR sponges, which bind and thereby functionally block specific miRNAs. While most of the miRNA therapeutics are still in preclinical development, one LNA anti-miR (miravirsen) and one miRNA mimic (MRX34) have reached clinical trials. (B) miRNAs can also be packaged into multivesicular bodies (MVBs), released into the extracellular environment as exosomes, carried through the circulation system and act on recipient cells. This has been found to play a role in cancer development (see Box 2). Blocking of such secreted miRNAs can be achieved by interfering with their secretion from the cells of origin (e.g. cancer cells), for example with inhibitors of neutral sphingomyelinase such as GW4869 . Alternatively anti-miR strategies can be employed to interfere with the function of secreted miRNAs in the recipient cell. Conversely, miRNA mimics with suitable formulation, for example with lipid encapsulation, of secreted miRNAs can be employed to enhance their function.

Figure 1. Mechanisms of action of miRNAs and the use of therapeutic agents to block or activate their function

(A) The diverse mechanisms of miRNA activity are presented together with the related miRNA-targeting strategies. In blue are the agents that reduce the miRNA activity, while in red are the agents that increase miRNA activity. Initially transcribed as primary miRNA (pri-miRNA), processed into precursor miRNA (pre-miRNA) by a microprocessor complex composed of Drosha and DGCR8, transported from nucleus to the cytoplasm by Exportin 5 in the presence of the Rna-GTP cofactor, and further processed into mature form by Dicer, miRNA recruits on the RISC and regulates the output of protein-coding genes through diverse mechanisms. The interaction of miRNA with the 3′-untranslated region (3′-UTR) of protein-coding genes is considered as the main mechanism, which usually leads to a decrease in protein output by either mRNA degradation or translational repression. Recent studies also suggest that miRNA can interact with the 5′-untranslated region (5′-UTR) of protein-coding genes via complementarity, and cause translational repression or activation of the targeted proteins . Similarly, miRNA can also target the coding sequence, and repress the translation of targeted genes . Moreover, miRNAs can interact with regulatory protein complexes such as AGO2 and FXR1, and indirectly upregulate translation of a target gene . Whether the “non-canonical” mechanisms represent a general mechanism or just exceptions to the canonical one remains to be determined. Various means can be used to enhance (such as by Enoxacin) or block (by any defects in the biogenesis machinery) general miRNA production, however this approach is not specific. More specific regulation of miRNA activity can be achieved by using miRNA mimics or anti-miRs such as LNAs, antagomirs and miR sponges, which bind and thereby functionally block specific miRNAs. While most of the miRNA therapeutics are still in preclinical development, one LNA anti-miR (miravirsen) and one miRNA mimic (MRX34) have reached clinical trials. (B) miRNAs can also be packaged into multivesicular bodies (MVBs), released into the extracellular environment as exosomes, carried through the circulation system and act on recipient cells. This has been found to play a role in cancer development (see Box 2). Blocking of such secreted miRNAs can be achieved by interfering with their secretion from the cells of origin (e.g. cancer cells), for example with inhibitors of neutral sphingomyelinase such as GW4869 . Alternatively anti-miR strategies can be employed to interfere with the function of secreted miRNAs in the recipient cell. Conversely, miRNA mimics with suitable formulation, for example with lipid encapsulation, of secreted miRNAs can be employed to enhance their function.

Figure 2. Mechanisms of action of lncRNAs and use of therapeutic agents to regulate their function

The mechanisms of action of lncRNAs are more diversified than those of miRNAs, and several representative examples are shown. About 20% of long intergenic RNAs (lincRNAs) are bound to polycomb repressive complex 2 (PRC2) and inhibit transcriptional activity by trans regulation. Enhancer RNAs (eRNAs) associate with mediator proteins to modify chromatin structure and activate gene transcription in cis. Natural antisense transcripts (NATs) are another type of lncRNAs that are transcribed from either the same genomic site or distant from the gene locus where the sense transcript counterpart is produced. NATs repress, and in some cases can also activate, transcription of the targeted protein-coding genes via mechanisms such as DNA methylation and chromatin modification at the genomic loci of the targeted genes. Several methods including small interfering RNAs (siRNAs), antisense oligonucleotides and ribozymes can be used to block the lncRNA function. The double stranded siRNA duplex can be stably produced by vectors encoding short hairpin RNA (shRNA) or transiently transfected with synthetic double stranded short RNA. The antisense strand of the siRNA duplex loads on to the RNA-induced silencing complex (RISC) and degrades the targeted lncRNA. Antisense oligonucleotides are single stranded, chemically modified DNA-like molecules (13 to 25 nt in length) that are designed to be complementary to a targeted RNA. Antisense oligonucleotide forms a heteroduplex with the RNA, and RNase H recognizes the RNA-DNA heteroduplex and cleaves the RNA strand. Most of the current studies to target lncRNAs use siRNAs and antisense oligonucleotides as the main tools. However, unique features of hammerhead ribozymes, naturally occurring or artificially synthesized, including activity independence (not rely on the RISC, which mediates siRNA-induced degradation or RNase H, which is essential for activity of antisense oligonucleotides) and specificity in target recognition make it an ideal candidate for lncRNA therapeutics.