Targeting microRNAs in cancer: rationale, strategies and challenges

Abstract

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate gene expression. Early studies have shown that miRNA expression is deregulated in cancer and experimental data indicate that cancer phenotypes can be modified by targeting miRNA expression. Based on these observations, miRNA-based anticancer therapies are being developed, either alone or in combination with current targeted therapies, with the goal to improve disease response and increase cure rates. The advantage of using miRNA approaches is based on its ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation and survival. In this Review, we describe the role of miRNAs in tumorigenesis and critically discuss the rationale, the strategies and the challenges for the therapeutic targeting of miRNAs in cancer.

Figure 1. MicroRNA biogenesis and effectors pathways

(A) miRNAs are transcribed by RNA polymerase II (pol II) into long primary miRNA transcripts of variable size (pri-miRNA), which are recognized and cleaved in the nucleus by the RNase III enzyme Drosha, resulting in a hairpin precursor form called pre-miRNA^-. (B) Pre-miRNA is exported from the nucleus to the cytoplasm by exportin 5 and is further processed by another RNase enzyme called Dicer (C), which produces a transient 19–24-nt duplex^-. Only one strand of the miRNA duplex (mature miRNA) is incorporated into a large protein complex called RISC (RNA-induced silencing complex)^-. D) The mature miRNA leads RISC to cleave the mRNA or induce translational repression, depending on the degree of complementarity between the miRNA and its target^-. While the most frequent site of interaction is the 3' UTR of the target mRNA, miRNAs have been described that bind to the open reading frame sequences as well as the 5' UTR^-(E). This last interaction has been associated with activation rather repression. (F) MiRNAs can also bind directly to proteins, in particular RNA binding proteins in a sequence dependent manner and prevent these proteins to bind their RNA targets. These “decoy activities” of miRNAs are RISC independent. G) MiRNAs can also regulate gene transcription by binding directly or by modulating methylation patterns at the target gene promoter level^-.

Figure 2. MicroRNAs as oncogenes and tumor suppressors

(A) In this model, we propose that a miRNA that normally downregulates an oncogene can be defined as a tumor suppressor gene, and is often lost in tumor cells. The loss of function of this miRNA by mutation, deletion, promoter methylation or any abnormalities in the miRNA biogenesis might result in an abnormal expression of the target oncogene, which subsequently contributes to tumor formation by inducing cell proliferation, invasion, angiogenesis and decreased cell death. Some of the proposed mechanisms for inactivation of miRNAs in cancer are experimentally proven, such as the down-regulation of miR-15a/miR-16-1 expression in CLL patients that harbor homozygous and heterozygous deletions at 13q14.3, where the miR-15a/miR-16–1 cluster is located and the loss of miR-29b-1/miR-29a cluster in AML patients with 7q- (This cluster is located in 7q32). In addition, germ-line mutations were found in the miR-15a/miR-16–1 precursor that resulted in lower miR-15a and miR-16-1 expression levels. Overall, the loss of both miR-15a/miR-16-1 and miR-29b-1/miR-29a cluster results in up-regulation of target oncogenes like BCL-2, MCL-1, TCL-1, CDK6 and DNMT3a^,,,,. (B) The amplification or overexpression of a miRNA that downregulates a tumor suppressor or other important genes involved in differentiation might contribute to tumor formation by stimulating proliferation, angiogenesis and invasion and preventing apoptosis and increasing genetic instability. For example, amplifications of the oncogenic miRNAs, miR-17–92 cluster, miR-21 and miR-155 have been clearly associated with tumor initiation and progression by repressing the expression of tumor suppressor genes like PTEN, BIM and PDCD4^,-,. The impact of the aberrant miRNA expression on the transcriptome and proteome will result in increased cell proliferation, angiogenesis, invasion, anti-apoptosis and genomic instability, which in turn will damage further the genome, perpetuating a dangerous cycle. For example, increased genomic instability may predispose for more mutations that may induce cancer progression or refractoriness to treatment.

Figure 3. Transcriptome-miRNA networks in cancer

In thiscartoon we graphically represent the relationship between critical oncogenic transcriptome networks and the miRNome. Target mRNAs for each major pathway are represented by circles with a unique color. MiRNAs are represented as hairpins structures in the center. The arrows connecting miRNAs and mRNAs indicate validated mRNA-miRNA interactions. The small arrow next to the circles indicate the biological effects on the pathway by the miRNA action on its target (i.e. miR-15a induces apoptosis by targeting BCL-2 or miR-29b suppresses cell proliferation by blocking CDK6. Some miRNAs like miR-29b, coordinately regulate multiple targets within different pathways. As shown in the cartoon, miR-29b modulates target mRNAs involved in apoptosis, cell proliferation, DNA methylation, histone acetylation and cell adhesion.

Figure 3. Transcriptome-miRNA networks in cancer

In thiscartoon we graphically represent the relationship between critical oncogenic transcriptome networks and the miRNome. Target mRNAs for each major pathway are represented by circles with a unique color. MiRNAs are represented as hairpins structures in the center. The arrows connecting miRNAs and mRNAs indicate validated mRNA-miRNA interactions. The small arrow next to the circles indicate the biological effects on the pathway by the miRNA action on its target (i.e. miR-15a induces apoptosis by targeting BCL-2 or miR-29b suppresses cell proliferation by blocking CDK6. Some miRNAs like miR-29b, coordinately regulate multiple targets within different pathways. As shown in the cartoon, miR-29b modulates target mRNAs involved in apoptosis, cell proliferation, DNA methylation, histone acetylation and cell adhesion.

Figure 4. Strategies for miRNA-based therapies

Blocking oncogenic miRNAs can be achieved by the use of antisense oligonucleotides, miRNA sponges, miRNA-mask and small RNA inhibitors^-. (A) Antisense oligonucleotides can bind to the target miRNAs following the Watson and Crick complementarities and induces either degradation or duplex formation^,. The three most common oligonucleotide modification structures are shown; Locked nucleic acid (LNA), 2-0-methyl (2-0-ME) and phosphorothiolate (PS)^,. (B) The miR-mask oligonucleotides are synthetic oligonucleotides complementary to the 3' UTR target mRNA that compete with endogenous miRNAs for its target. Therefore, miR-mask is able to block oncogenic miRNA deleterious functions at the target level. (C) The miRNA sponges are oligonucleotide constructs with multiple complementary miRNA binding sites (in tandem) to the target miRNA. When introduced to the cell, sponges will “soak” endogenous miRNAs (Red oligos), decreasing the expression levels of an oncogenic miRNA. (D) Small molecule miRNA inhibitors regulate miRNA expression at transcriptional level. Restoring down-regulated miRNA expression could be achieved by (E) using synthetic miRNAs (miRNA mimics) or (F) by inserting genes coding for miRNAs into viral constructs, such as the adeno associated viral vectors ^-.