"MicroRNA reexpression as differentiation therapy in cancer".

Prasun J. Mishra and Glenn Merlino

Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Building 37, Room 5002, 37 Convent Drive, Bethesda, Maryland 20892-4264, USA. Phone: (301) 496-4270; Fax: (301) 480-7618; E-mail: gmerlino@helix.nih.gov

Commentary:

Since their discovery in the early 2000s, microRNAs (miRNAs) and their penchant for RNA interference have taken the scientific community by storm, working their way into virtually every corner of biological inquiry. The very nature of their action, the ability to simultaneously extinguish the expression of a multitude of genes and negate their functions, immediately suggested therapeutic promise. In this issue of the JCI, a step toward the realization of this promise is described. Taulli et al. demonstrate that the miRNAs miR-1/miR-206, which are routinely lost in advanced, poorly differentiated rhabdomyosarcoma
(RMS) but characteristically expressed in the mature skeletal muscle from which these tumors arise, restore the myogenic differentiation program and block the tumorigenic phenotype (see the related article beginning on page 2366). Their data support the notion that these small RNAs, effectively functioning as “micro-sheriffs” by restoring myogenic law and order, hold substantial clinical potential as differentiation therapy for RMS and perhaps other solid tumors. miRNA reexpression therapy constitutes a novel approach to handcuff oncogenes and arrest tumor development.

One of the largest, most intriguing scientific stories concerns the ability of developing organisms to orchestrate the expression of a vast multitude of genes to achieve the formation of the various tissues and organs required for life, despite the fact that every cell has the same complement of genetic material. One obvious explanation that has emerged is that every tissue has its own set of transcription factors that regulate tissue-specific gene expression and development. With the discovery of epigenetics,
we now know that promoters of many genes, including transcription factors, can be turned on and off early during development, through covalent chromatin modifications. Most recently, the discovery of
“tiny” tissue-specific microRNAs (miRNAs) has provided further complexity and coordination to the regulation of developmental gene sets, framing a broader picture in which epigenetic programming of specific transcription factors and miRNAs during development define tissue specificity.

miRNAs are small, noncoding RNAs (22–23 nucleotides long), shown to regulate gene expression at translational and posttranslational levels (1). Processed from longer transcripts by Drosha and Dicer, miRNAs mostly bind to the 3' untranslated regions of target genes and inhibit gene expression
translationally and/or by destabilizing the target mRNA (2). miRNAs, encoded in intergenic/intronic noncoding regions once thought to be “junk” DNA, contribute to differentiation when expressed in a tissuespecific manner (2) and even possess the power to shift global mRNA expression patterns with respect to differentiation signatures (3). Such miRNAs are responsible for tissue integrity and homeostasis, behaving functionally as “micro-sheriffs” (Table 1).

Tissue-specific/tissue-enriched miRNAs are often downregulated and play a role in cancer (shown in bold) (5, 7, 34). For example, brain-specific neuromiR-124 is downregulated in glioblastomas, and Taulli et al

Like a sheriff, miRNAs maintain law and order in specific tissues, by stringently regulating the expression of their target genes, especially oncogenes. Accumulating evidence now suggests that such miRNAs are assigned their working “zip codes” early during development by epigenetic modifications of miRNA promoters (4).

With respect to cancer, miRNAs are often located in genomic unstable regions and therefore are typically downregulated in tumors (reviewed in ref. 5); moreover, inhibiting miRNA biogenesis tends to enhance
tumorigenesis (6). Downregulation may be achieved through mutation or by epigenetic silencing of the miRNA, resulting in loss of tissue-specific miRNA synthesis and overexpression of pro-proliferation genes
(i.e., oncogenes); these miRNAs normally function as tumor suppressors (7). Of course, miRNAs can also act as oncogenes (8, 9). Many investigators have posited that reexpression of specific miRNAs may have
therapeutic anticancer value (10, 11). In a report published in the current issue of the JCI, Taulli et al. demonstrate that expression of specific miRNAs regulating skeletal muscle development, miR-1/miR-206 (also known as myomiRs), is reduced in rhabdomyosarcoma (RMS) (12). RMS tumors, the most common soft tissue sarcomas in pediatric patients and young adults, are thought to arise from the skeletal muscle
lineage, coexpressing markers of proliferation and myogenic differentiation (13). Reexpression of these myomiRs to physiological levels suppressed many aspects of the transformed phenotype and induced
myogenic differentiation (12), raising the possibility that miRNA reexpression may represent effective differentiation therapy for RMS and perhaps other cancer types.

miRNA reexpression as differentiation therapy in cancer

The discovery of miRNAs has added an entirely new dimension to antitumor therapeutic approaches; the potential of noncoding RNAs as drugs for cancer patients is both intriguing and compelling. As miRNA expression seems to be altered in many human diseases, including cancer, the miRNA revolution has already set the stage for “miRNA reexpression therapy” (Figure 1).

Reexpressing lost miRNA in a cell can deliver a dramatic effect, because miRNAs regulate a vast number of genes and pathways. Among the many genes that miRNAs can regulate are oncogenes and tumor suppressors, targets of drugs currently used in the clinic. Although a few miRNAs are overexpressed in cancer and seem to function as oncogenes themselves promote differentiation and inhibit tumor growth under a variety of conditions. The authors also used microarray analysis to show that reexpression of miR-206 can differentiate RD18 RMS cells by switching the global mRNA expression profiles back to their original myogenic phenotype. Upregulated genes included many musclespecific genes, such as those encoding titin, muscle creatine kinase, myosin light chain, troponin C, myomesin 2, and tropomyosin 2. Many of the detected downregulated genes have been associated with cancer, including those involved with the cell cycle, metabolism, and DNA repair. Thus, miR-206 was sufficient to force neoplastic cells into resuming and completing the full myogenic program.

miR-1/miR-206 are known to target MET (22, 23), which is overexpressed in many cancers including RMS. Notably, deregulated MET signaling in genetically engineered mice induces embryonal RMS (26). MET is known to be rapidly downregulated at the onset of myogenic differentiation (27). Taulli and colleagues show
that MET downregulation correlates with miR-206 upregulation in normal myogenic cells and, in fact, directly suppresses MET expression in RMS cells (12). This was confirmed through reexpression of
constitutively active oncogenic Tpr-Met, which lacks the MET 3' untranslated region, in miR-206–expressing RMS cells, abrogating myomiR-mediated differentiation. The authors correctly concluded that MET is a key target for the anticancer effect of miR-1/miR-206 in RMS, and their data suggest that reexpression of miR-1/miR-206 to its physiological levels may be useful as a therapeutic differentiation approach for RMS by restoring a normal muscle phenotype (12).

miR-1/miR-206 has been reported to possess activities that strongly influence other solid tumors. miR-206 was shown to target estrogen-responsive genes, including those encoding estrogen receptor, steroid receptor coactivators (SRC1, SRC3) and GATA binding protein 3 (GATA3), and has been suggested as a biomarker for basal-like breast cancers (28). Introduction of miR-206 into estrogen-dependent MCF-7 breast cancer cells inhibited cell growth in a dose-and time-dependent manner. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas (HCCs) compared with normal liver tissues. miR-1 reexpression in HCC cells downregulated its direct/indirect targets, including forkhead box P1 (FOXP1), histone deacetylase 4 (HDAC4), and MET, and inhibited cell growth and proliferation (23). Recently miR-1 has been implicated in lung cancer therapy (22). miR-1 is expressed in the normal bronchial epithelium but downregulated in human primary lung cancer tissues and cell lines, along with its activator C/EBPa (CCAAT/enhancer–binding protein). Notably, HDAC inhibitor treatment of lung cancer cells induced the expression of repressed miR-1, downregulating oncogenes such as MET, PIM1, FOXP1, and HDAC4, and reversing the tumorigenicity of lung cancer cells. Interestingly, miR-1 reexpression made a lung cancer cell line more sensitive to the chemotherapeutic doxorubicin by inducing apoptosis (22).

microRNA reexpression therapy: the pros, the cons, and the promise

Like siRNAs, miRNAs are easy to synthesize and can potentially target any gene, including otherwise non-druggable targets. However, miRNA therapy has many advantages over the originally envisioned RNA interference–based therapeutics (siRNA therapy). The major advantage of miRNA therapy is
that miRNA reexpression can influence the expression of hundreds of genes involved in many cellular pathways. While siRNA therapy is more gene-specific, miRNA therapy can target an array of different gene products, more closely resembling the action of the so-called “dirty drugs” used in the clinic today; in fact, both the sense and antisense strands of miRNAs might target different mRNAs. Moreover, dirty drugs typically target only a handful of gene products, whereas miRNAs can target hundreds of genes,
casting miRNAs as “super” dirty drugs. Of course, as with any such drug, the advantage of hitting a broad spectrum of targets is also a potential disadvantage. However, miRNAs are evolutionarily conserved, and
targeting the upregulation or downregulation of a tissue-specific tumor suppressor miRNA or oncogenic miRNA, respectively, to its “physiological level” may incite fewer of the nonspecific, off-target effects often associated with artificial siRNAs or currently available dirty drugs.

miRNA therapy shares many of the disadvantages of siRNA-therapy, including delivery limitations, instability, and off target effects. A major obstacle to effective miRNA-based therapy is the requirement
for successful delivery. Unlike many other drugs, miRNAs do not freely diffuse into cells; therefore, miRNAs may require special delivery approaches to achieve the desired effect. Moreover, small RNAs tend to be unstable and might be degraded upon entering a cell; new methods may be required to stabilize these small sequences. Another factor is that double-stranded RNA and unmethylated CpG sequences
are potentially immunogenic; their presence might increase IFN production and induce an immune response in patients. Moreover, in the case of miRNA reexpression therapy of cancer, preventing miRNA
expression from exceeding physiological levels also represents a therapeutic challenge. Ultimately, miRNA-based gene therapy (i.e., delivering miRNAs encoded in a vector) has many of the same disadvantages as gene therapy (29–31). However, developing smarter/safer ways to deliver stable miRNAs/miRNA inhibitors into cells should go a long way toward overcoming the majority of these limitations, and preclinical models will be extremely helpful in this regard.

Although successful use of small RNAs as therapeutic agents represents a substantial challenge, miRNA therapeutics also carry great expectations based on the ability of a single miRNA to specifically
target multiple oncogenes, many of which are being individually targeted by drugs already in the clinic or under preclinical development. But cancer remains a complex disease and patients with the same diagnosis will harbor gene mutations and polymorphic variants in a myriad of permutations; therefore, incorporation of personalized medicine approaches will continue to be advisable (reviewed in ref. 32). Cancer differentiation therapy has seen success in hematological cancers (e.g., acute promyelocytic leukemia) (5, 33) but not yet for solid tumors. The recent work of Taulli et al. and others (10, 11) on solid tumors demonstrates that coaxing the micro-sheriff out of retirement and back into town represents a promising and novel approach for arresting tumor growth and reestablishing cellular law and order.

Address correspondence to: Glenn Merlino, Laboratory of Cancer Biology and Genetics, National Cancer Institute, Building 37, Room 5002, 37 Convent Drive, Bethesda, Maryland 20892-4264, USA. Phone: (301)
496-4270; Fax: (301) 480-7618; E-mail: gmerlino@helix.nih.gov

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