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Commentary

MicroRNAs: regulators of oncogenesis and stemness

Thales=20 Papagiannakopoulos¹^,2 and = Kenneth S Kosik¹^,2

¹Neuroscience Research Institute, University of = California,=20 Santa Barbara, Santa Barbara, CA 93106, USA

²Department of Molecular, Cellular and Developmental = Biology, University of California, Santa Barbara, Santa Barbara, = CA 93106,=20 USA

=20 author email=20 corresponding author email

BMC Medicine 2008, = 6:15doi:10.1186/1741-7015-6-15

The electronic version of this article is the complete one and = can be=20 found online at: http://www.biomedcen= tral.com/1741-7015/6/15

Received:	19 March 2008
Accepted:	24 June 2008
Published:	24 June 2008

=C2=A9 2008 Papagiannakopoulos and Kosik; licensee BioMed = Central=20 Ltd.
This is an Open Access article distributed under the terms = of the=20 Creative Commons Attribution License (http://creativecommon= s.org/licenses/by/2.0),=20 which permits unrestricted use, distribution, and reproduction in = any=20 medium, provided the original work is properly cited.

Abstract

MicroRNAs (miRNAs) are essential post-transcriptional = regulators that=20 determine cell identity and fate. Aberrant expression of miRNAs = can lead=20 to diseases, including cancer. Expression of many miRNAs in the=20 de-differentiated brain tumor cancer stem cells resembles that of = neural=20 stem cells. In this issue of BMC Medicine, Silber et al = provide=20 evidence of the expression of such miRNAs and their potential to = mediate=20 differentiation in both stem cell populations. In this commentary, = we=20 discuss the known functions of miRNAs in cancer and stem cells, = their=20 therapeutic potential and how the findings of Silber et al provide = insight=20 into the role of miR-124/miR-137 dysregulation in = glioblastomas.

MicroRNA biogenesis and function

With over 500 microRNA (miRNA) genes identified experimentally = in the=20 human genome and a plethora of computationally predicted mRNA = targets, it=20 is believed that these small non-coding RNAs have a central role = in=20 diverse cellular and developmental processes. miRNAs function as=20 endogenous triggers of the RNA interference pathway. When mature = (active)=20 they are ~22 nt small RNAs, generated by two cleavage steps, which = are=20 catalyzed by two distinct complexes: Drosha, which is found in the = nucleus, and Dicer, which is found in the cytoplasm (Figure 1) [1]. = Following=20 Dicer cleavage, the mature form of the miRNA is incorporated into = the=20 RNA-induced silencing complex (RISC) where the 3' untranslated = region=20 (3'-UTR) of the target mRNA associates with the major component of = the=20 RISC complex, Argonaute (Figure 1) [1].

Figure 1. MicroRNA=20 biogenesis. Primary transcripts are transcribed from the = genome=20 and form a long hairpin loop, pri-miRNA, which is processed by = Drosha to=20 form pre-miRNA. Pre-miRNA is exported to the cytoplasm by Exportin = 5 where=20 Dicer cleaves off the hairpin loop to form a duplex that contains = the=20 mature 21=E2=80=9323 nucleotide microRNA (miRNA). The mature miRNA = is then=20 incorporated into the RNA-induced silencing complex to target the = 3'=20 untranslated region of the target mRNA.

miRNAs act on cellular transcripts by two different mechanisms. = The=20 first mechanism is cleavage-dependent RNA degradation of the = transcript at=20 the site where the miRNA binds. The other mechanism is = miRNA-mediated=20 translational repression of the target transcript, while the miRNA = binds=20 to the target 3'-UTR with several mismatches. This partial = complementarity=20 allows for miRNAs to target multiple 3'-UTRs [1]. A = key=20 question that arises from the targeting of multiple transcripts is = whether=20 there has been evolutionary enrichment in targets that are = involved in=20 similar cellular functions.

miRNAs in cancer

Aberrant expression of miRNA genes can lead to human diseases,=20 including cancer. In recent years, a number of informative = profiling=20 studies have determined the levels of miRNAs in various types of = cancer=20 cell lines and tumors [2]. = Most of=20 these studies have identified multiple dysregulated miRNAs, which = in many=20 cases can classify cancer types [3]. In = addition,=20 multiple functional studies on tumor suppressive or oncogenic = miRNAs were=20 identified from profiling, screens or due to their = cancer-associated=20 genomic loci [2,3].

The first tumor suppressive miRNAs identified were the=20 miR-15/miR-16 cluster, located in a genomic region of = chr13,=20 which is often deleted in chronic lymphocytic leukemias (CLLs). = Therefore,=20 these two miRNAs are not expressed in CLLs, leading to increased = levels of=20 an oncogenic target, the apoptotic inhibitor Bcl-2 [2].

The first functional evidence of a miRNA, or a non-coding RNA, = acting=20 as a mammalian 'oncogene' or 'onco-miR' was the miR-17=E2=80=9392 = polycistron.=20 Enforced expression of this 'onco-miR' was shown to act with c-Myc = to=20 accelerate tumor development in a mouse B-cell lymphoma model [4]. = c-Myc, which=20 is a well-studied oncogene, activates the expression of = miR-17=E2=80=9392=20 through its direct interaction with the putative promoter = region.=20 This finding could explain the high expression levels of the miRNA = cluster=20 in lung, breast, stomach, prostate, colon and pancreatic cancers = where it=20 potentially plays a similar oncogenic role [2,5]. = miRNAs from=20 this cluster target tumor suppressor genes such as E2F1, = PTEN=20 and TGF=CE=B2RII [2,5].

Gliomas are the most common primary tumors in the brain and are = divided=20 into four clinical grades on the basis of their histology and = prognosis.=20 Grade IV Glioblastomas are the most malignant of all brain tumors = and are=20 almost always fatal [6,7]. = Profiling of=20 glioblastoma tumors and cell lines has revealed increased levels = of miR-21=20 [8]. In = the same=20 study, inhibition of miR-21 in glioblastoma cells led to increased = cell=20 death, suggesting that miR-21 could be acting as an oncogene to = inhibit=20 cell death in glioblastomas. Subsequent cancer profiling studies = also=20 reported increased levels of miR-21 in most types of cancer where = miR-21=20 had the same anti-apoptotic role [2]. = Recently, a=20 study determined that miR-21 knockdown in glioblastoma cells = significantly=20 repressed the ability of these cells to form tumors in an in = vivo=20 mouse xenograft model [9].

In this issue of BMC Medicine, a study by Silber et al = demonstrates that two miRNAs, miR-124 and miR-137, are = significantly=20 decreased in anaplastic astrocytomas (AA, WHO grade III) and = glioblatoma=20 multiforme (GBM, WHO grade IV) relative to non-neoplastic brain = tissue. In=20 addition to the profiling performed in this study, in situ=20 hybridization experiments would be helpful to determine the = exact=20 cell populations in which miR-124 and miR-137 are dysregulated. = When these=20 miRNAs were overexpressed in glioblastoma cell lines, they led to = G1 cell=20 cycle arrest by repression of Retinoblastoma (RB) and CDK6. = Downregulation=20 of CDK6 occurs partially through direct targeting by miR-137. This = is a=20 significant tumor suppressive potential of these two miRNAs by = repressing=20 cell cycle progression and thus the growth of GBM cells.

microRNAs in cancer stem cells

Chen et al were the first to suggest that embryonic stem cells = (ESCs),=20 embryoid bodies, and day 11 mouse embryos exhibit a less complex = miRNA=20 profile than more mature somatic tissues. They hypothesized that = the=20 degree of cellular or tissue differentiation can be characterized = by a=20 particular miRNA signature [10]. = The=20 obvious parallel that can be drawn is that undifferentiated stem = cells=20 display miRNA expression profiles reminiscent of cancer cells. = Numerous=20 known oncogenic miRNAs are expressed in early stages of = development in=20 undifferentiated cells; however, their expression decreases in=20 differentiated tissue, whereas the opposite holds true for tumor=20 suppressive miRNAs.

In recent years, there has been a sudden increase in studies on = a=20 subpopulation of cells in tumors that have stem cell properties, = cancer=20 stem cells (CSCs). The CSC hypothesis proposes that cancers are = derived=20 from a small fraction of cancer cells that constitute a reservoir = of=20 self-sustaining cells with the exclusive ability to self-renew and = maintain the tumor. Small numbers of CSCs are sufficient to form=20 orthotopic tumors in vivo. Cells which could be described = as CSCs=20 have been isolated from hematological malignancies, brain, breast = and=20 colon tumors [6,7,11-13].

GBM-derived CSCs share the fundamental stem cell properties of=20 self-renewal and multi-potency with neural stem cells (NSCs). They = were=20 first isolated from tumors by virtue of the expression of CD133 = that marks=20 NSCs and neural progenitor cells (NPCs) [6,7]. = Microarray=20 studies have since revealed considerable overlap between the=20 gene-expression signatures of glioblastomas and progenitor cells = of the=20 developing forebrain. Forced differentiation of CD133^{+ =}cells=20 with BMP4 addition abrogates their stem cell phenotypes, making = this a=20 potential therapeutic approach for eliminating this subpopulation = of GBMs=20 [7].

In the study published by Silber et al, two miRNAs with low = expression=20 in GBMs and AAs, miR-124 and miR-137, increase in expression upon=20 differentiation of mouse NSCs with growth factor withdrawal = (Figure 2).=20 Interestingly, the expression of miR-124, which is a brain = enriched miRNA,=20 has been shown to correlate with the first appearance of = differentiated=20 neurons in culture from NPCs [14].=20 Overexpression of these two miRNAs in undifferentiated mNSCs, = mouse=20 oligodendroglioma-derived stem cells and CSCs termed human = GBM-derived=20 stem cells (hGSCs) led to morphological changes and expression of = markers=20 indicating neuronal differentiation (Figure 2). = The=20 forced differentiation of these CSCs which is mediated by these = two miRNAs=20 leads to loss of their self-renewal and oncogenic capacity. These = data are=20 in accordance with previous approaches where CSC oncogenicity was = severely=20 disrupted by differentiation. A challenge in this field is to = define the=20 functional, molecular, regulatory and antigenic features that = characterize=20 NPCs from CSCs and ultimately to devise therapeutic strategies = that=20 specifically target the tumor population, but leave normal stem = and=20 precursor cells unharmed.

Figure 2. mir-137 and = mir-124 are=20 essential for the canonical differentiation of neural stems cells = and=20 neural progenitor cells. Abrogation of their expression = could=20 lead to cancer stem cell (CSC) formation and tumorogenesis. = Upregulation=20 of these two microRNAs in CSCs could present a therapeutic = approach in=20 differentiating these cells, thus eliminating their stem cell=20 properties.

Treatment of GBM tumors with current methods has proven largely = unsuccessful. To a great extent this is because of the = ineffectiveness of=20 current therapeutics to target the CSC population of GBM tumors. = This=20 study indicates a new approach targeting the CSC subpopulation of = tumors=20 by overexpressing microRNAs which are normally expressed in the=20 surrounding differentiated tissue. Upregulation of miR-124 and = miR-137=20 could have therapeutic use in blocking this CSC subpopulation of = GBM=20 tumors. Modulation of GBM tumor dysregulated miRNAs such as miR-21 = and=20 miR-124/miR-137 by RNAi-based therapeutics, in conjunction with = current=20 treatments may prove a key approach in the fight against these = deadly=20 tumors.

Development of miRNA/RNAi-based therapeutics requires several = critical=20 experimental steps, which include: (1) miRNA profiling of cancer = versus=20 healthy tissue, (2) functional analysis of dyregulated miRNAs, and = (3)=20 in vivo studies with use of different RNAi-based = therapeutic=20 methods to dysregulate miRNAs. Silber et al have performed the two = initial=20 steps for miR-124/miR-137 as therapeutic targets in GBMs. A major = caveat=20 in RNAi-based therapeutics has been their in vivo = targeting to=20 the brain and particularly crossing of the blood brain barrier [15]. = This=20 problem can be overcome with the use of lipid encapsulation, = direct=20 administration of the therapeutics to the brain tumor or = development of=20 safe gene therapy approaches using viral delivery [15]. = In the=20 dawn of miRNA research, there is confidence that RNAi can be = successfully=20 maintained in vivo in a tumor-specific manner for = prolonged=20 clinical efficacy. The success of such strategies for gene therapy = will=20 provide clinicians with a larger repertoire that includes=20 miRNA-therapeutic agents, which can be used in parallel with = currently=20 available drugs.

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