Review Article MicroRNAs: A New Paradigm on Molecular Urological Oncology Leonardo Oliveira Reis, Tiago Campos Pereira, Iscia Lopes-Cendes, and Ubirajara Ferreira MicroRNAs (miRNA) are a class of naturally occurring, small, noncoding RNAs that function as specific repressors of protein-encoding genes, being pivotal regulators of development and cellular homeostasis. They have been functionally classified as proto-oncogenes or tumor suppressors and are aberrantly expressed in different cancer types. Deregulation of these so-called “cancerous” miRNAs can figure prominently in tumor initiation and progression. The Human miRNA profile is under development, and it is of concern that there is so little agreement on the miRNAs found in each of the studies. Bioinformatics progress and efficient delivery are essential to overcome barriers to miRNA use in clinical practice. UROLOGY 76: 521–527, 2010. © 2010 Elsevier Inc.
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ne hallmark of tumor cells is their aberrant gene expression profile. Recent advances in molecular genetics and high-throughput sequencing technologies have allowed a detailed and comprehensive understanding of the genetic alterations present in cancer cells. Genes found to be up- or downregulated in cancer tissues have been used as biological markers for diagnosis and/or prognostic evaluation and considered as promising targets for molecular therapies.1 MicroRNAs (miRNAs) are a new and distinct class of recently discovered genes that (1) do not code for proteins, but for regulatory RNAs, and (2) are very small, comprising only a few tens of base pairs, whereas the genome has approximately 30,000 genes, most of them coding for proteins (eg, enzymes, antibodies, structural components) and spanning from a few thousand up to a million base pairs. The first miRNA was discovered in 1993 by Lee et al,2 who observed that a 22-base RNA (lin-4) could bind to the 3-untranslated region (3-UTR) of lin-14 mRNA and repressed its translation in the animal model Caenorhabditis elegans. Seven years later, another 22-base RNA (let-7) was identified in C. elegans by an independent group.3 This time scientists immediately realized that these small RNA molecules were playing important roles in the regulation of gene expression. Identification and functional studies of miRNAs extended rapidly from C. elegans to other species, including human beings. Currently, nearly 500 human miRNAs
The first and second authors contributed equally to this work. From the Urologic Oncology Division, School of Medical Sciences, University of Campinas, UNICAMP, São Paulo, Brazil; Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, Brazil; and Medical Genetics Department, School of Medical Sciences, University of Campinas, UNICAMP, São Paulo, Brazil Reprint requests: Dr. Leonardo Oliveira Reis, M.D., M.Sc., R. Votorantim, 51, ap. 43, Campinas-SP, Brazil 13073-090. E-mail:
[email protected] Submitted: November 29, 2009, accepted (with revisions): March 1, 2010
© 2010 Elsevier Inc. All Rights Reserved
have been identified and it is postulated that up to 1000 miRNAs exist.4 Transcription of a miRNA yields a primary miRNA (pri-miRNA), which length varies from hundreds to thousands of nucleotides, depending on its origin. PrimiRNA is in turn processed by a nuclear enzyme named drosha into a premature miRNA (pre-miRNA), about 70-100 nt long. Pre-miRNA displays a hairpin shape and is exported to the cytoplasm by exporting-5, where it is processed and unwound by dicer into its final functional product: the mature miRNA,5,6 an 18-23 nt long singlestranded RNA (Fig. 1). miRNAs then enter a complex named RNA-induced silencing complex (RISC) and downregulate RNAs in one of the 2 ways: (1) by targeting cleavage if it presents full complementarity or (2) by inhibition of translation if it displays partial complementarity. Computational studies have shown that a single miRNA can control tens or even hundreds of different target genes. In addition, a single gene can be controlled by several miRNAs and it is hypothesized that up to one third of all human genes is controlled by miRNAs. Their high impact on developmental processes, pathophysiology, cancer biology, immunology, and many other fields was portrayed as a new paradigm in gene expression regulation7 and the breakthrough of the year in 2002 by the journal Science.8 As expected, miRNA expression profiles and processing steps are also altered in cancers, including urological. This link was demonstrated for the first time by the finding that the 2 miRNAs, miR-15 and miR-16, are lost in most cases of chronic lymphocytic leukemia.9 Considering that each miRNA can downregulate a myriad of targets at the same time, this class of genes is proposed to play important roles in tumorigenesis, but can also be useful for diagnosis, prognosis, and therapeutics. 0090-4295/10/$34.00 doi:10.1016/j.urology.2010.03.012
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tion. The principle of these methods is that complementary sequences, which are labeled for example with fluorescent dyes or radioactive beads, hybridize to the target RNA. The techniques of in situ hybridization and in situ RTPCR have been used to identify the subcellular distribution of miRNAs.11 Moreover, different miRNA cloning strategies are available, basically involving the addition of RNA fragments to both miRNA ends and thus enlongating it for reverse transcription for cDNA synthesis purposes.12 This longer cDNA can then be cloned into adapted vector for sequencing.
miRNA USEFULNESS IN DIAGNOSTICS AND PROGNOSTICS
Figure 1. Biogenesis and mode of action of miRNAs. miRNA genes are transcribed as small RNA molecules named primiRNAs. Within the nucleus, these molecules are processed by an enzyme named drosha into shorter hairpin-like molecules named pre-miRNAs. In the cytoplasm, dicer enzymes convert pre-miRNAs into miRNAs, and only the strand is loaded into the RNA-induced silencing complex, which may promote target RNA cleavage or translational inhibition.
MOLECULAR ANALYSES OF miRNAs Because of its small size, miRNA sequencing is a relatively simple task; in fact, high-throughput sequencing and deep sequencing strategies have provided a large amount of interesting data in recent years.10 However, cloning and high-throughput gene expression analysis demanded a set of novel molecular strategies. The cellular levels of miRNAs in clinical cancer samples can be measured using various RNA detection approaches. The most frequently used methods for miRNA detection have been microarrays and miRNA-adapted reverse transcription–polymerase chain reaction (RT-PCR), which uses stem-loop primers able to anneal and amplify these short RNA molecules.11 In microarray experiments, target RNA is labeled with fluorescent dyes and hybridized to immobilized oligonucleotides on a glass slide. Microarrays have the advantage of high-throughput capability. In real time RT-PCR, the target RNA is amplified and the product is monitored using intercalating dyes or fluorescent probes. This method is quantitative and displays high specificity and sensitivity because of the target amplification. Other methods are in situ hybridization, RNAse protection assay, Northern blotting, or bead-based detec522
Molecular analyses of miRNA expression in cancer samples invariably reveal alterations when compared with control cells. This altered profile can be a hope for diagnosis and stage classification purposes.13 A recent study on primary pigmented nodular adrenocortical disease (PPNAD), caused by mutations on protein kinase A regulatory subunit type 1A (PRKARIA), reported a deep investigation on miRNA expression signature and its relevance to tumorigenesis.14 The authors demonstrated that 33 miRNAs were upregulated and 11 were downregulated in this condition. One miRNA, let-7b presented a negative correlation with cortisol levels, whereas the upregulated miR-449 was shown to target WISP2 protein, a strong candidate to be the primary mediator of tumorigenesis in this syndrome. In addition, a recent report on expression profile in bladder tumors assessed 464 miRNAs and showed interesting results15 when analyzing 7 patients and comparing tumors with normal tissue form the same patients, as control. The number of upregulated miRNAs in each individual ranged from 18 to 50, whereas the group of genes that were downregulated ranged from 9 to 89. Changes in gene expression were not conserved among all patients, but 2 findings were very consistent. First, all T1 stage tumors presented an average 2-fold increase in 9 miRNAs (miRNA-129, miRNA-141, miRNA-494, miRNA498, miRNA-500, miRNA-513, and 3 unknown miRNAs) and a 2– to 3-fold decrease in expression of 13 miRNAs (miRlet-7a, 7b, 7c, 7d, miR-143, miR-199a*, miR-21, miR-24, miR-26a, miR-29c, miR-30a-5p, miR-30c, and miR-30e5p). Second, there were 4 miRNAs that were downregulated in all T2 stage tumors, and no common upregulated miRNAs were found. In addition, the common signature to all bladder tumors, regardless their stage was the downregulation of 4 miRNAs (miR-26a, miR-29c, miR-30c, and miR-30e-5p). In a similar study, Ichimi et al16 evaluated the expression profile of 156 miRNAs in 14 bladder cancer (BC) samples, 3 BC cell lines, and 5 normal bladder epithelium. As in the previous study, the common hallmark was the downregulation of a small set of miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b, UROLOGY 76 (3), 2010
and miR-199a*). If independently validated, both findings could represent the establishment of a miRNA profile in bladder cancers, which may be useful for diagnostic purposes. Yet an additional study examined the expression profile of 245 human and mouse miRNAs in kidney specimens (20 carcinomas, 4 benign renal tumors, and 3 normal parenchyma) revealing a set of 4 human miRNAs (miR-28, miR-185, miR-27, and let-7f-2) significantly upregulated in renal cell carcinoma compared with controls (normal tissue/benign lesion).17 The same study reported an upregulation of 10 miRNAs (miR-223, miR26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, and miR-205) in cancers compared with normal tissue. In addition to the use of tissue-based expression profiles, special interest is directed on the measurement of miRNAs in blood and urine. It can be assumed that either cancer cells with the corresponding typical miRNA pattern or just cancer-specific free miRNAs are released from the cancer into biological fluids. This could raise the possibility of the noninvasive detection of circulating tumor cells or free miRNAs characteristic of the corresponding cancer, avoiding the inconvenience of the heterogeneity of tissue samples, once they are very stable in both plasma and serum. It was recently reported that there is a 6.35-fold increase in miR-125b in the sera of patients with metastatic prostate cancer compared with sera from healthy men. In the case of miR-141, the differential expression was 46fold. This study involved 25 patients with metastatic disease. The authors reported 60% sensitivity and 100% specificity when using miR-141.18 Similarly, serum from patients with metastatic disease could potentially be tested before and after the initiation of therapy to estimate disease aggressiveness and therapy response. Clearly, there is reason to hope that a better understanding of the role played by microRNAs in this process may lead to the development of better biomarkers and new therapeutic strategies. To develop novel noninvasive diagnostics methods, Hanke et al describes a miRNA analysis in patients with urothelial bladder cancer using the urine as the potential source of molecular markers. In their study, they found that miR-126: miR152 ratio was able to detect bladder cancer in urine samples at a specificity of 82% and a sensitivity of 72%.19 More specific uses of miRNAs in determining the outcome of tumors was demonstrated in a study involving a series of bladder carcinoma cell lines (MGHU1, J82, HU456, EJ, UM-UC-3, KK47, T24, TCCSUP, BC16.1, CUBIII, 5637, PSI, RT4, and RT112). The rationale was to determine the differential miRNA expression between invasive and noninvasive cell lines. The expression profile of 343 miRNAs revealed that invasiveness could be associated to a 10-fold increase in miR-21:mir-205 expression ratio compared with noninvasive cell lines.20 UROLOGY 76 (3), 2010
Moreover, 53 bladder tumors (28 superficial and 25 invasive) were analyzed to assess miR-21:mir-205 ratio in terms of sensitivity and specificity in determining invasiveness. The respective values were 100% and 78%, suggesting that miRNA ratios may be useful as biological markers for prognosis regarding tumor invasiveness. Other promising miRNAs biomarkers in bladder cancer are miR-129, miR-133b, and miR-518c*.21 These genes were discovered in an expression profile study of 290 miRNAs performed in 106 bladder tumors. Of particular interest is miR-129, which induced cell death and growth inhibition upon transfection in bladder carcinoma cell lines (T24 and SW780) and was reported to be associated with poor outcome.
miRNA ROLES AND TARGETS IN CANCER miRNAs may be involved in tumorigenesis basically in 2 distinct ways: (1) in normal cells it may repress protoooncogenes and it would be downregulated in tumors (thus the miRNA i acts as a tumor suppressor gene); or (2) it may control tumor suppressor genes and it would be upregulated in tumors (thus the miRNA ii acts as a proto-oncogene).22 An example of the first scenario is shown by the EZH2 protein, which is directly involved in epigenetically silencing tumor suppressor genes in cancer. Friedman et al23 have recently shown that miRNA-101 targets EZH2 and that this microRNA is downregulated in human bladder transitional cell carcinoma (TCC) samples. Therefore, in cancer cells EZH2 protein is overexpressed and promotes silencing of tumor suppressor genes. Moreover, its tumor suppressor activity was demonstrated as miRNA-101 inhibited cell proliferation and colony formation in TCC cell line. Another tumor suppressor in bladder cancer is miR-143, which probably targets RAS protein and is downregulated in tumor tissues.24 In addition, miRNA143 transfection into EJ and T24 cells inhibited cell proliferation. By contrast, miRNA-186 and miR-150 were shown to be upregulated in cancer epithelial cells and that they target P2X7 receptor, a proapoptotic protein. Thus, both miRNAs act as protoo-oncogenes, silencing P2X7 and hindering apoptosis in cancer cells.25 In renal carcinomas miR-34a was shown to be overexpressed and associated with proliferation; inhibition of this miRNA reduced cell growth.26
THERAPEUTICS miRNAs found to be upregulated in urological tumors are the immediate targets for therapeutic application. miRNA inhibition has been shown to be a simple and straightforward process. The basic mechanism relies on antamirs (or antagomirs): short RNA molecules of nearly 20 nt with full complementarities to miRNA,27 (Fig. 2). Antamirs present an interesting approach, in that target523
Figure 2. Antamirs and miRNA sponges in therapeutics. Certain cancer types display upregulated miRNA expression. Control of these exceeding molecules can be done through antamirs (short complementary RNA molecules) or longer RNA molecules with multiple binding sites (miRNA sponges).
directed therapy is possible as it is based on Watson–Crick base pairing rules (A:U/C:G); however, as with all conventional drugs, direct delivery remains an obstacle. Another interesting approach is the miRNA sponge: RNA molecules that harbor several sites for the same (or different) miRNA(s).28 This approach is interesting when a huge increase in gene expression is observed for a specific miRNA, or when several key target miRNAs must be repressed at once to achieve a therapeutic effect. Moreover, the number of binding sites in the sponge may be conceived to modulate 2 miRNAs with different increase folds, thus promoting a rational control. As previously shown, cancer cells also display miRNA downregulation. In some instances this is caused by methylation of the promoter sequence, ie, the DNA region responsible for gene expression. Other alterations, such as histone deacetylation, may lead to the same effect. Therefore, drugs that are able to demethylate (eg, 5-aza-2=-deoxycytidine, 5-Aza-CdR) and/or inhibit histone acetylation (eg, 4-phenylbutyric acid, PBA) can be used to restore an miRNA normal expression pattern.29 This approach was successfully demonstrated in T24 bladder cancer cell line, which presents downregulation of the tumor suppressor miRNA miR-126.30 When this cell line was treated with each drug separately, it led to a slight increase in miR-126; however this induction was stronger (nearly 2-fold) when both drugs were added simultaneously, synergistically acting on miR-126 gene expression. Another possibility is the temporary expression of key downregulated miRNAs in tumors. Because those are small genes, cassettes expressing a few miRNAs could be tested. This strategy was demonstrated for miR-34a, which is downregulated in human prostate cancer cell line PC3 because of epigenetic silencing.31 Retroviral vector expressing pri-miR-34a leads this cell line to senescence (enlargement, -galactosidase activity at a pH 6.0 and permanent arrest).
DELIVERY OF SMALL NONCODING RNAs Small noncoding RNAs (ncRNAs) is a class of short RNA molecules which does not code for proteins, rather, 524
they regulate or modify other cellular RNAs. This class includes: (1) miRNAs, (2) small interfering RNAs (siRNAs, which are used for gene silencing in a post-transcriptional manner), and (3) small nucleolar RNAs (snoRNAs; which guide chemical modifications of other RNAs), among others. The efficient delivery of therapeutic ncRNAs is a major challenge in the establishment of its medical application. It relies on several conditions: (1) protection of the molecule from nucleolytic degradation by serum nucleases, preserving their efficacy, and maintaining their activity; (2) efficient cellular uptake and subsequent intracellular release into the cytoplasm; and (3) absence of intracellular immune responses.32 The strategies and technologies for delivering ncRNA into cells evolves in an incredible speed and also can be used in miRNA delivery with the advantage of 1-hit, multiple-targets effect. Viral vectors are the most efficient means to transfer DNA sequences in vivo. Their transfection efficacy results from the inherent ability of viruses to transport genetic material into cells. Associated side effects, however, limit their value in clinical applications. Iatrogenic-induced leukemia caused by insertional mutagenesis events warrants a thorough reassessment of the potential risk of virus-mediated gene therapy.33 Lipossome formulations present biocompatibility, biodegradability, low toxicity, and low immunogenicity. They have been developed for encapsulating both conventional anticancer drugs and new genetic drugs (eg, plasmid DNA containing therapeutic genes, antisense oligonucleotides, and siRNAs). Lipossome properties include (1) diameter ⬍100 nm, allowing a high drug-to-lipid ratio; (2) excellent retention of the encapsulated drug and long circulating lifetime; and (3) high concentration in tumors.34 Nanotechnology offers other attractive drug delivery systems, and the use of sustained-release polymeric nanoparticles for downregulation of annexin A2 expression in mice was demonstrated as an effective adjuvant treatment option for prostate cancer.35 UROLOGY 76 (3), 2010
Especially in urological oncology, the potential of local instillation in the case of superficial bladder cancer is one extraordinary example of a direct administration of nucleic acid inhibitors against a solid tumor type. The relatively low number of cellular layers of the normal human bladder represents a short barrier for penetration of exogenous RNAs into deep tissue layers compared with a tumor capsule or dense stromal tissues characteristic of other carcinoma entities.36 This evidence suggests that transurethral therapy can overcome the delivery problem and cause a breakthrough in clinical applications of nucleic acid therapeutics. Moreover, the pharmacologic activity can be controlled locally. Possible off-target effects, including nonspecific toxicity, should be limited to the urinary bladder, and the estimated side effect would be less than that of systemic administration. Nogawa et al37 published the first demonstration (to our knowledge) of inhibition of cancer growth in murine bladder by intravesical siRNA/cationic liposomes, which showed a low cytotoxicity. This complex attaches to the target cancer cells, allowing high efficacy of transfection during a limited time. In this way, intravesical administration seemed to achieve similar effects with a 50 – to 100-fold lower dose of the systemic complex administration. Local administration of siRNA was suggested to be superior to intravenous injection considering in vivo toxicity and time of metabolization/elimination by liver/ kidney, thus limiting silencing efficacy due to a shorter half-life of the RNAi complex in the target organ in the intravenous injection.38 In a similar strategy, the use of chitosan to allow drug-loaded microparticles to bind to the bladder wall and to release active drug directly at the target tissue, Hadaschik et al39 confirmed better efficacy in intravesical nucleic acid therapeutics compared with the same formulation without this carrier. Consequently, these therapeutics enforce the uptake by endocytotic pathways and release siRNAs within the cell. It is noteworthy that RNAs are biological molecules, presenting natural metabolism and pharmacokinetics. As smart cell-and-tissue–specific delivery strategies are constantly being developed, and the high-throughput analyses of “cancer genomes” and tumor-related mutations, it is possible that RNA-based antitumor drugs and cocktails may emerge in the near future to add the current therapeutic arsenal in clinical practice. Still, many interesting questions remain to be answered, among them: (1) the effects of using single versus combined RNAs (cocktail); (2) applicability right after surgical tumor resection; and (3) the possibility of implementation as a combination therapy into conservative treatment strategies.36 Translational clinical trials will reveal optimal carrier, dosing, pharmacokinetic behavior, and potential side effects, as well as the best targets in each human cancer. UROLOGY 76 (3), 2010
FUTURE CLINICAL APPLICATION OF miRNAs IN UROLOGICAL ONCOLOGY The miRNA signatures are tumor and tissue specific and often reflect the differentiation grade of the tumor. Thus, from a diagnostic point of view, tissue-based miRNA expression profiles may be helpful for the following: (1) to discriminate normal and cancer tissue as well as reactive lesions; (2) to classify poorly differentiated tumors that would otherwise be indeterminable by conventional histologic and immunohistochemical methods; and (3) to differentiate tumors from the same organ with different histology.40 miRNA profiles were shown to predict survival prognosis and therapeutic outcome of patients with colon carcinoma,41 clinical response to the drug gefitinib in lung cancer,42 or chemotherapy resistance of cancer cells.43 It was recently shown that, until now, unexplained methotrexate resistance is based on the miR-24 polymorphism.44 This polymorphism leads to a novel, miRNA-based mechanism of drug-resistance. It is the first example of the so-called “miRNA pharmacogenomics.” Thus the prediction of individual drug response allows for personalized therapy.
PERSPECTIVES AND CONCLUSIONS The human miRNA profile is under development and its impact on urological oncology is just emerging. MicroRNAs have come forward as a new paradigm in molecular medicine as they pose as central regulators of gene expression. Altered expression of a single miRNA can affect hundreds of targets at once; therefore its impact on tumor biology is highly relevant. Despite their recent discovery, miRNAs have proved to be very promising as molecular markers for diagnosis as well as disease prognosis because they are easily identified, sequenced, and analyzed. Concurrently, therapies targeting miRNA are promising, as drug design is based on simple rules of base pairing. Advantageous use of miRNAs might be the more direct connection between the miRNA regulation and protein expression compared with the mRNA expression, also lower technical and statistical interferences using microarrays with a few tens of miRNAs in comparison with those with tens of thousands of mRNAs. However, only a few of these differentially expressed miRNAs were validated in this way. Although the “classical model of action” of the miRNA is by directly binding its target and promoting its post-transcriptional silencing, there are probably more complex and less direct relationships. For example, it is possible that a certain miRNA modulates an intermediate miRNA that in turn leads to changes in the end target gene. To unveil such elusive mechanisms, more sophisticated computational or molecular approaches must be developed and used. A clear example of this is the recent finding that miRNAs can also regulate genes at the transcriptional level.45 525
Discrepancies between research groups are exemplified by the divergent expressions of miR-125b.46,47 Some of these inconsistencies are caused by the different discovery microarray platforms as well as the samples themselves (ie, degree of stroma and epithelia in cancer and normal samples). Analytic studies are necessary to solve the above-mentioned methodologic issues. The clinical validity of data has to be improved by multicenter studies and use of greater sample numbers. Future studies should address microRNA stability, issues between fresh and paraffin tissue, and issues relating to tissue-specific and cell cycle–associated alterations. Moreover, microdisssection procedures should be widely used in such high-throughput analyses to achieve reproducible results.48 Once new cancer-related miRNAs are identified, an important issue is to uncover their targets and to identify those that are biologically relevant. To elucidate the contribution of each miRNA to cancer pathogenesis, a large body of work is necessary, and bioinformatic tools are essential. Functional characterization of these miRNAs remains a great challenge, and more efficient biochemical and molecular strategies are necessary to tackle these issues.49 Single nucleotide polymorphism (SNPs) analyses in miRNAs and in genes of miRNA biogenesis comprise another promising field of investigation toward the identification of risk factors for urologic cancers.50 Continuing discoveries regarding the different aspects of miRNAs are necessary to shed light on this new frontier in cancer biology.
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