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MicroRNAs as regulators of signal transduction in urological tumors.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs that have been shown to play pivotal roles in carcinogenesis. In the past decade, miRNAs have been the focus of much research in oncology, and there are great expectations for their utility as cancer biomarkers and therapeutic targets.

CONTENT: In this review we examine how miRNAs can regulate signal transduction pathways in urological tumors. We performed in silico target prediction using TargetScan 5.1 to identify the signal transduction targets of miRNA, and we summarize the experimental evidence detailing miRNA regulation of pathways analyzed herein.

SUMMARY: miRNAs, which have been shown to be dysregulated in bladder, prostate, and renal cell cancer, are predicted to target key proteins in signal transduction. Because androgen receptor signaling is a major regulator of prostate cancer growth, its regulation by miRNAs has been well described. In addition, members of the phosphatidylinositol 3-kinase/Akt (RAC-alpha serine/threonine-protein kinase) signaling pathway have been shown to be susceptible to miRNA regulation. In contrast, there are very few studies on the impact of miRNA regulation on signaling by VHL (von Hippel-Lindau tumor suppressor) and vascular endothelial growth factor in renal cell carcinoma or by fibroblast growth factor receptor 3 and p53 in bladder cancer. Many miRNAs are predicted to target important signaling pathways in urological tumors and are dysregulated in their respective cancer types; a systematic overview of miRNA regulation of signal transduction in urological tumors is pending. The identification of these regulatory networks might lead to novel targeted cancer therapies. In general, the targeting of miRNAs is a valuable approach to cancer therapy, as has been shown recently for various types of cancer. MicroRNAs (miRNAs or miRs) [5] are short noncoding RNAs with lengths of approximately 19-22 nucleotides. The first discovery of an miRNA, which occurred in 1993, was of an miRNA in the nematode Caenorhabditis elegans. The second miRNA discovery, of let-7 in Drosophila, was at the beginning of this decade, and after several others were subsequently identified by sequencing it became apparent that these single-stranded RNAs comprise a distinct class of regulatory small RNAs. miRNAs are a class of small RNAs whose members have lengths of <300 nucleotides. miRNAs and other members of this class, namely small nucleolar RNAs, small nuclear RNAs, small interfering RNAs, and PIWI (P-element induced wimpy testis in Drosophila)-interacting RNAs, have important regulatory functions in physiological and pathological processes (1).

The research field of miRNAs is rapidly growing, as highlighted by the 7641 published works on miRNAs cited in the PubMed database by January 2011 compared with the 6700 works published by December 2009. As of January 2011, 1037 human miRNAs had been identified and listed in the continuously updated miRNA database (http://www.mirbase.org), a number that is already larger than the number predicted in 2005 (2). miRNA regulation has been described in various physiological and pathological processes, in particular developmental processes and cancer. The dysregulation of miRNA expression in cancer is well established. miRNAs are important regulators of all hallmarks of cancer, including cell growth and cell cycle control, evasion of apoptosis, tissue invasion and metastasis, angiogenesis, and unlimited replicative potential (3, 4). Several articles published in Clinical Chemistry have also highlighted the emerging importance of miRNAs as cancer biomarkers (5-7). We and other groups have recently summarized the progress in miRNA-related research in urological tumors (8-12).

miRNAs are key players in signal transduction and participate in various stages of the signal transduction process. A detailed description of how miRNAs affect signal transduction was recently provided (13). Signal transduction mediators respond to signals in a dose-dependent manner and must be robust against signaling noise. miRNAs can serve as mediators of signaling robustness either by amplifying a signal or by buffering and balancing the signal response. Furthermore, miRNAs can be important mediators of signal transduction crosstalk, and they can participate in feedback or feed-forward signaling loops.

In this review we aimed to (a) describe the role of miRNA regulation and function in prostate, renal cell, and bladder cancers, highlighting certain highly-deregulated signal transduction pathways; (b) perform in silico analyses of miRNA binding sites in the 3'-untranslated regions (3'-UTRs) of the members of each specific pathway and search for experimental evidence of the predicted interactions in PubMed; and (c) summarize recent progress in the development of miRNA-based therapies in cancer, which have evolved greatly after the discovery of the important regulatory function of miRNAs in signal transduction in many tumor types.

miRNA Function in Carcinogenesis

The biogenesis of miRNAs in animals has been described elsewhere (14); herein, we provide only a short overview. miRNAs are transcribed as large precursors with lengths of a few hundred to a few thousand nucleotides. These precursors, called preliminary miRNAs, contain a 5' cap structure with 7-methylguanosine and a3' poly(A) tail and are either mono- or polycistronic. The mature miRNA sequences are located in characteristic stem-loop structures. In the nucleus, a preliminary miRNA is processed by the endonuclease Drosha, [6] [encoded by the drosha, ribonuclease type III (DROSHA) gene] and its cofactor DGCR8 [encoded by DiGeorge syndrome critical region gene 8 (DGCR8)] to yield a smaller precursor miRNA. Thereafter, this precursor miRNA is exported to the cytoplasm by exportin 5. An additional processing enzyme in the cytoplasm, Dicer, excises the stem, which contains the mature miRNA duplex. The mature miRNA duplex is unwound by still-unknown helicases. Although the sense strand of the miRNA duplex is incorporated into the miRNA-induced silencing complex, the passenger strand is generally degraded.

miRNAs function in posttranscriptional gene regulation via binding to complementary sequences in the 3'-UTR of mRNAs. It has been predicted that approximately 30% of the transcriptome will be found to be regulated by miRNAs (15). Although perfect complementarity occurs in plants, imperfect binding of the miRNA to the 3' -UTR appears to be the dominant process in mammals. The seed sequence, which is comprised of nucleotides 2-8 of the miRNA, displays perfect complementarity to the 3'-UTR, whereas there is no complementarity in the central region of the miRNA. Various computer programs are available for predicting potential miRNA-mRNA interactions. In this review, we used the stringent TargetScan 5.1 (http://www.targetscan.org/) algorithm to identify potential mRNA targets for the dysregulated miRNAs. Because degradation by Ago2, a component of the miRNA-induced silencing complex, is possible only when perfect complementarity occurs, translational inhibition has been proposed to be the major mechanism of miRNA action. In recent years, a more complex model of miRNA function has been established, including not only translational inhibition but also destabilization and RNA decay (16). mRNAs and miRNAs as well as proteins involved in RNA decay can be enriched in so-called processing bodies. Processing bodies are thought to serve as storage compartments or places where mRNAs are finally degraded by processes independent of Ago2. These mRNAs are mainly degraded by decapping and deadenylation, which mark them for conventional mRNA decay pathways (17).

miRNAs can function as both oncogenes and tumor suppressors. Their modes of action are mainly-dependent on their target genes. Because of the multitude of regulated targets, it can be hypothesized that miRNAs exert tissue- or organ-specific functions. Although some miRNAs have been described preliminarily as oncogenes, they seem to have tumor-suppressive functions in other types of cancer. miR-184, for example, is overexpressed in squamous cell carcinoma, and its inhibition induces apoptosis and decreases proliferation of the cancerous cells (18). In contrast, overexpression of the same miRNA in neuroblastoma cell lines inhibits proliferation and reduces tumor growth in xenograft models (19).

miRNA genes are susceptible to mutations and frequentlyoccur in fragile sites, minimal regions of loss of heterozygosity, and minimal regions of amplification, which have been associated with cancer (20). Although miRNA oncogenes are located in regions of amplification, tumor-suppressive miRNAs are frequently deleted. Single nucleotide polymorphisms in miRNA genes can also be associated with cancer risk, recurrence, and survival, as has been shown in renal cell carcinoma (21).

The loci of miRNA genes can be intergenic, intronic, or located in exons of protein-coding genes (22). miRNAs that are located in introns or exons often share the promoter of the protein-coding gene and are therefore coexpressed with their host gene.

Changes in miRNA expression levels are not only due to genomic variation. The promoter regions of miRNA genes are targets of a variety of epigenetic modifications, such as promoter hypermethylation or histone modifications. Several miRNAs have been shown to be silenced by promoter hypermethylation in urological tumors (23, 24). In prostate cancer, results of a recently reported study demonstrated that binding of the vitamin D (1,25-dihydroxyvitamin D3) receptor [encoded by the vitamin D (1,25-dihydroxyvitamin D3) receptor (VDR) gene] to the MCM7 promoter [encoded by minichromosome maintenance complex component 7 (MCM7)] resulted in acetylation of histone H3 lysine 9 and, subsequently, enhanced transcription of miR-106b, which is located in an intron of the MCM7 gene (25).

Aberrant miRNA expression can also result from improper regulation by tumor-suppressor or oncogenic transcription factors. In prostate cancer, for example, miR-100 is regulated by the hypoxia-inducible factor (HIF) transcription factors (26), whereas miR34a, miR-34c, and miR-145 are under the control of the tumor suppressor p53 (27, 28). Transcriptional regulation by the androgen receptor (AR) is extremely important in prostate cancer, and its impact on miRNA expression is discussed in detail below.

miRNA Regulation of Important Cancer Signal Transduction Pathways

miRNAs have been reported in recent years to be deregulated in urological tumors and have been shown to be involved in their carcinogenesis [reviewed in (8)]. In this review, we focus on the miRNA regulation of highly dysregulated signaling pathways in prostate, bladder, and renal cell carcinoma. To identify putative miRNA binding sites in the 3'-UTR of members of these pathways, we performed in silico target prediction using the TargetScan 5.1 algorithm, which is a stringent tool for identification of miRNA binding sites. Furthermore, we analyzed the prediction for miRNAs that are dysregulated in these tumor entities as summarized previously by Schaefer et al. (8) and researched experimental evidence for miRNA regulation of signal transducers.

PROSTATE CANCER

A major signal transduction pathway in prostate cancer is the phosphatidylinositol 3-kinase/RAC-alpha serine/threonine-protein kinase (PI3K/Akt) signaling pathway, which is hyperactivated in approximately 30% to 50% of prostate cancers (29). Many prostate cancer patients display mutations in the phosphatase and tensin homolog (PTEN) gene. Active Akt signaling functions to inhibit apoptosis through phosphorylation of proapoptotic factors such as BAD [encoded by BCL2-associated agonist of cell death (BAD)], CASP9 [encoded by caspase 9, apoptosis-related cysteine peptidase (CASP9)], and FOXO1 [encoded by forkhead box O1 (FOXO1)]. Activated Akt also promotes cell cycle transition through the regulation of CDKN1B [encoded by cyclin-dependent kinase inhibitor 1B (CDKN1B)], GSK3B [encoded by glycogen synthase kinase 3 beta (GSK3B)], and mTOR [encoded by mammalian target of rapamycin (MTOR)] signaling. Fig. 1 (see also Supplemental Fig. 1 and Supplemental Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol57/issue7) shows the PI3K/Akt-signaling pathway and its putative regulation by miRNAs, as predicted by TargetScan. Many of the predicted miRNAs are differentially regulated in prostate cancer. Interestingly, for some miRNAs binding sites in the 3' -UTRs of multiple members have been predicted. For example, miR-128 has 6 predicted gene targets [pyruvate dehydrogenase kinase, isozyme 1 (PDK1); pyruvate dehydrogenase kinase isozyme 2 (PDK2); MTOR; phosphodiesterase 3A, cGMP-inhibited (PDE3A); phosphodiesterase 3B, cGMP-inhibited (PDE3B); FOXO1], and miR-141 is predicted to target 5 genes [PTEN; PDK2; cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A); CDKN1B;PDE3B]. Recent experimental evidence has shown that these dysregulated miRNAs can indeed inhibit members of the PI3K/Akt pathway. miR-21 has been described as an oncogene and is upregulated in prostate cancer (30). In prostate cancer cells, which ectopically express miR-21, phosphorylation of Akt is induced by interferon, whereas it is not induced in cells that do not express miR-21. In addition, knockdown of miR-21 in cells induces expression of PTEN that is enhanced compared with that found in control cells (31). Results of another recent study call into question the oncogenic role of miR-21 in prostate cancer as well as its inhibition of PTEN (32). The reasons for this discrepancy are not clear for now. Both groups studied expression of PTEN in the same cell line (DU145), so the discrepancies cannot be due to a cell-context specificity. However, different knockdown approaches were used in the 2 studies. Therefore, different knockdown efficiencies might account for the contradictory results.

The miR-106b-25 cluster has been suggested to be a novel oncogenic miRNA cluster in prostate cancer, and it is predicted to target PTEN (33). Members of this cluster (miR-22, miR-25, and miR-92) downregulate PTEN in prostate cancer cell lines. Furthermore, the MCM7 gene, which harbors the miR-106b~25 cluster, was shown to induce transformation in vivo, thus establishing the oncogenic function of this locus (33). miR-331-3p, which is frequently downregulated in prostate cancer patients, can block phosphorylation of Akt by regulating the ERBB2 (erythroblastosis oncogene B 2) tyrosine kinase receptor [encoded by the v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (ERBB2) gene], one of the upstream growth factor receptors that activates PI3K/Akt signaling (34). Another upstream transducer of this signaling pathway, E2F1 [encoded by E2F transcription factor1 (E2F1)] is inhibited by miR-330 and subsequently induces apoptosis (35).

In the available studies research has focused not only on Akt itself and its upstream regulators but also on the regulation of downstream Akt targets by miRNAs. The most studied regulation in this context is the regulation of CDKN1B by miR-221/-222 (12, 36-38). By inhibiting CDKN1B, miR-221 and miR-222 serve as regulators of the cell cycle. The correlation of miR-221 and CDKN1B transcript expression was studied in human prostate cancer, but no correlation was observed (39), a finding that contradicts the in vitro results. However, only expression of the transcript and not of the protein were investigated. Given the assumption that miR-221 acts mainly via translational inhibition, mRNA levels of CDKN1B might not be altered. Nevertheless, we must mention that in most studies miR-221 and miR-222 were observed to be downregulated in human prostate cancer tissue, which clearly contradicts its described oncogenic function in prostate cancer cell lines (8). miR-106b, which has been shown to be part of an oncogenic miRNA cluster that regulates PTEN, has been suggested to have a regulatory effect on CDKN1A (40). miR-106b is suppressed upon radiation of prostate cancer cell lines, and overexpression of this miRNA can inhibit radiation-mediated CDKN1A activation, thereby overcoming subsequent arrest of the cell cycle. Furthermore, it has been shown that binding of the vitamin D receptor to the MCM7 locus leads to an upregulation of miR-106b, resulting in enhanced p21 activation and cell cycle arrest (25).

The second pathway we discuss is the AR signaling pathway (41). Epithelial cells of the prostatic ducts depend on testosterone for growth. Testosterone is reverted in prostate cells to 5-[alpha]-dihydrotestosterone, which binds to the AR, thereby inducing the expression of multiple genes that regulate cell growth. Androgen ablation therapy is frequently used for treatment of recurrent prostate cancer. However, the majority of tumors eventually become independent of androgen for growth. Castration-resistant prostate cancer is always caused by hyperactive AR due to mutations, amplification, increased stability or sensitivity of the AR, or de novo androgen synthesis in prostate cancer cells (42). Because of the dominant role that AR signaling plays in prostate growth, the effects of miRNA regulation on AR signaling in prostate cancer have been well studied. Potential interactions between dysregulated miRNAs and members of the AR signaling pathway, as predicted with TargetScan, are provided in online Supplemental Table 2.

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In 4 recent studies investigators performed global comparisons of miRNA patterns in androgen-dependent vs androgen-independent cells and identified upregulated and downregulated miRNAs (12, 4245). A fifth profiling study focused on expression of specific oncogenic clusters and their host genes (miR17_5p~92, miR-106b~25, and miR-23b~24). Although the authors of this study found statistically significant differences between cell lines, they were not able to show an androgen status-specific pattern (46).

miR-125b, an androgen-sensitive miRNA in prostate cancer, has been reported to stimulate androgen-independent growth and to regulate apoptosis by inhibition of BAK1 [encoded by BCL2-antagonist/killer 1 (BAK1)] (12,47,48). Another recently identified androgen-sensitive miRNA, miR-101, was upregulated in cells from the human prostate cancer cell line LNCaP after treatment with the synthetic androgen methyltrienolone (R1881). In contrast, miR-101 has been shown to regulate the expression of EZH2 [encoded by enhancer of zeste homolog 2 (Drosophila) (EZH2)], thereby reducing invasion and inducing morphological changes in prostate cancer cells (26). miR-148 is another miRNA that is androgen inducible in LNCaP cells. Expression of this miRNA was shown to promote proliferation by regulation of CAND1 [encoded by cullin-associated and neddylation dissociated 1 (CAM51)] (49). miR-221 and miR-222 were strongly upregulated in androgen-independent cells. Ectopic expression of this cluster was demonstrated to induce androgen-independent growth and to lower the response of the prostate-specific antigen (PSA) promoter to testosterone treatment (36). miR146, which is expressed at lower levels in androgen-independent cell lines, targets ROCK1 [encoded by rho-associated, coiled-coil containing protein kinase 1 (ROCK1)], a key kinase in hyaluronan-mediated transformation to androgen independency (45). Changes in miRNA expression were also observed in androgen-dependent vs androgen-independent tumors, and let7c, miR-100, and miR-218 were identified as upregulated in metastatic cancer (50).

To gain a full picture of miRNA expression upon androgen treatment, a microarray analysis of androgen-dependent cell lines treated with the synthetic androgen R1881 identified 16 miRNAs that were subsequently induced (miR-594, miR-16, miR-21, miR-29b, miR-148a, miR-29c, miR-106a, miR-17-5p, miR20a, miR-20b, miR-29a, miR-19b, miR-93, let-7g, miR-15b, and let-7d). Several of these miRNAs are transcribed as clusters (30). The AR can bind directly to the miR-21 promoter. Overexpression of miR-21 alone is sufficient to overcome androgen-dependent growth inhibition in surgically castrated mice (30). The role of miR-21 in AR signaling was further established by the fact that this miRNA, together with miR330, is downregulated in prostate cancer cells upon treatment with epigallocatechin gallate, a green tea phenol, and blocks the ligand-binding domain of the AR (51). The functional relevance of miR-21 also makes it a putative marker of androgen status in prostate cancer. Serum miR-21 concentrations are increased in patients with castration-resistant prostate cancer compared to those with localized prostate cancer, and the concentrations of miR-21 are especially high in patients with docetaxel resistance (52).

Not all miRNAs that have been shown to be androgen responsive are directlyregulated by AR binding to their promoter, as was demonstrated for miR-21. For example, inhibition of the AR indirectly mediates miR-34 expression via p53 (28). miR-34 is induced by p53 upon doxorubicin treatment and subsequently induces apoptosis. On AR knockdown, the expression of kinases that phosphorylate p53 is inhibited; therefore, the expression of miR-34 and apoptosis are impaired (28).

As described above, miRNAs can be androgen inducible, but they can also in turn directly regulate the expression of the AR. miR-488* inhibits the AR in androgen-dependent and androgen-independent cell lines, thereby regulating the proliferative and apoptotic responses of these cells (53). Expression of miR331-3p blocks androgen signaling and reduces the activity of the PSA promoter, thereby decreasing expression of kallikrein-related peptidase 3 (KLK3, also known as PSA) (34).

RENAL CELL CANCER

The von Hippel-Lindau tumor suppressor (VHL) signaling pathway is the most important dysregulated pathway in clear cell renal cell carcinoma, the dominant subtype of kidney cancers (54). Mutations in the von Hippel-Lindau tumor suppressor (VHL) gene can be inherited in patients with von Hippel-Lindau syndrome. These patients have a higher risk of developing renal cell cancer. Furthermore, the VHL gene can be spontaneously deleted or hypermethylated. VHL is part of a protein complex that directs degradation of hypoxia inducible factor 1 alpha subunit (HIF1A) under normoxic conditions. Under hypoxic conditions, HIF1A is stabilized and forms a transcription complex. An overview of the VHL signaling pathway is presented in Fig. 2, and it includes regulatory miRNAs predicted by TargetScan 5.1. A number of miRNAs are predicted to target 6 genes each in this signaling pathway. The miR-17_5p cluster targets the genes VHL; hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) (HIF1A); egl nine homolog 1 (C. elegans) (EGLN1); egl nine homolog 3 (C. elegans) (EGLN3); E1A binding protein p300 (EP300); and CREB binding protein (CREBBP), whereas miR-200b, miR-200c, and miR-429 target VHL; transcription elongation factor B (SIII), polypeptide 1 (15 kDa, elongin C) (TCEB1); EGLN1/3; EP300; and CREBBP. The regulation of components of the VHL pathway by miRNAs has not been well studied in renal cell cancer. Although interactions have been hypothesized (55),a direct interaction between miRNAs and VHL or HIF1A has been only recently proven (unpublished data). Nevertheless, a subset of miRNAs has been identified that is regulated by the VHL pathway. miR-31, miR-21, and let-7i have been shown to be upregulated in cells with functional VHL; however, miR-155, miR-193b, miR-17, miR-18a, miR-20a, and miR-210 were downregulated. The knockdown of HIF1A or HIF1B [encoded by aryl hydrocarbon receptor nuclear translocator (ARNT)] also reduced miR-210 and miR-155 expression levels. miR-210 has further been shown to be expressed at significantly higher levels in tumors with either VHL mutations or methylation of the VHL promoter and to be correlated with the expression of CA9 [encoded by carbonic anhydrase IX (CA9)], a known transcriptional target of HIF transcription factors (56). Furthermore, it has been shown that miR-23b, which is upregulated in renal cancer specimens, can directly target PRODH [encoded by proline dehydrogenase (oxidase) 1 (PRODH)], a mitochondrial tumor suppressor that induces apoptosis through the generation of reactive oxygen species and a decrease in HIF signaling (57).

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A second commonly dysregulated signal transduction pathway is the vascular endothelial growth factor (VEGF) signaling pathway (58). VEGF [encoded by the genes vascular endothelial growth factor A (VEGFA), VEGFB, and VEGFC] is transcriptionally regulated by the HIF transcription complexupon hypoxia or due to loss of VHL in renal cell cancer. Binding of VEGF to its receptor on the surface of endothelial cells increasesvascular permeabilityas well as endothelial cell proliferation and migration that eventually leads to the formation of novel blood vessels in the tumor (59). The VEGF/VEGF receptor system is an attractive target for existing and novel therapies to treat renal cell cancer. miRNAs are promising new molecules that regulate VEGF signaling and thereby novel targets for therapy of this cancer. TargetScan predicts that the VEGF signal transduction pathway might be targeted by well-known dysregulated miRNAs in renal cancer, including miR-200b, miR-200c, miR-141, or the miR-17_5p~92 cluster (Fig. 3). Interactions between miRNAs and the VEGF system have not been well studied in renal cell cancer. Only 1 miRNA, miR29b, has been proven to indirectly regulate VEGF expression in renal cancer cells (60). This miRNA regulates angiogenesis in renal cancer cells by regulating VEGF expression via ZFP36L1 [encoded byzinc finger protein 36, C3H type-like 1 (ZFP36L1)] under normoxic conditions (60), an effect that is also dependent on VHL expression. A strong reverse correlation between the miR-200 family (miR-200a-c, miR-141) and VEGFA was recently observed, and it was hypothesized that VEGFA is a direct target of these miRNAs (61).

BLADDER CANCER

Mutation or overexpression of the fibroblast growth factor receptor 3 (FGFR3) gene occurs in approximately 80% of all patients with low-grade noninvasive urothelial carcinomas (62). FGFR3 and other growth factor receptors can activate the Ras kinase signaling pathway, leading to increased proliferation and motility. Despite the high FGFR3 mutation rate in noninvasive carcinomas, these mutations occur less frequently in muscle-invasive tumors. In contrast, these tumors mainly contain mutations in the TP53 gene (62). Thus, a well-established 2-pathway model of bladder cancer has been established.

An overview of FGFR3 signaling with its predicted miRNA regulators is shown in Fig. 4. Some miRNAs that have been proven to be dysregulated in bladder cancer are predicted by TargetScan 5.1 to have multiple target genes in the FGFR3 pathway. In particular, miR-129 has 16 predicted target genes [platelet-derived growth factor receptor, alpha polypeptide (PDGFRA); platelet-derived growth factor receptor, beta polypeptide (PDGFRB); epidermal growth factor receptor (EGFR); phospholipase C, gamma 1 (PLCG1), SHC (Src homology 2 domain containing) transforming protein 4 (SHC4); protein kinase C, alpha (PRKCA); protein kinase C, beta (PRKCB); protein kinase C, delta (PRKCD) protein kinase C, epsilon (PRKCE); protein kinase C, iota (PRKCI); growth factor receptor-bound protein 2 (GRB2); son of sevenless homolog 1 (SOS1); SOS2; v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS); death-associated protein kinase 1 (DAPK1); DAPK2; DAPK3; and ribosomal protein S6 kinase, 90kDa, polypeptide 5 (RPS6KA5) (also known as MSK1)]. In addition, some well-established dysregulated miRNAs in bladder cancer carcinogenesis are predicted to target the FGFR3 receptor; for example, miR-145, miR-101, miR-99a, and miR-100 are all predicted to target FGFR3. Indeed, the regulation of FGFR3 by miR-99a and miR-100 has been experimentally validated (63). FGFR3 and other growth factor receptors are susceptible to miRNA regulation during bladder cancer carcinogenesis. The miR-200 family is associated with an epithelial phenotype in bladder cancer cell lines. Ectopic expression of miR-200 in bladder cancer cell lines induces upregulation of epithelial markers and the downregulation of mesenchymal markers. The epithelial phenotype was accompanied by an increased sensitivity to EGFR inhibitors, which was explained by a direct regulation of ERRFI1 by miR-200 (64). Upregulation of miR-143 in bladder cancer specimens is accompanied by a downregulation of Ras. In addition, the forced overexpression of miR-143 in bladder cancer cell lines decreased Ras levels (65).

Although mutations in the tumor protein p53 (TP53) gene are characteristic of invasive urothelial carcinomas, functional studies to investigate the role of miRNA regulation on p53 signaling in bladder cancer are lacking. Several miRNAs that are dysregulated in bladder cancer are predicted by TargetScan to target p53 or its regulators (Fig. 5). miR-10, for example, is predicted to target Mdm2 p53 binding protein homolog (mouse) (MDM2), Mdm2 p53 binding protein homolog (mouse) (MDM4), and ataxia telangiectasia mutated (ATM), whereas miR-129 potentially targets the genes MDM4 and ATM in this pathway. Additionally, miR-125b, miR-143, miR-30a/c, and miR-223 are predicted to target p53 directly. Despite missing functional data, noninvasive and muscle-invasive carcinomas display specific miRNA patterns that enable investigators to pinpoint the roles of miRNAs in both pathways of bladder carcinogenesis (66). miR-7 was identified as an miRNA that is associated with the FGFR3 mutation status in bladder cancer (66). In the same study investigators also identified miR-10a, miR-125b, and miR-222 as predictors of muscle-invasive carcinomas.

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Targeting Signal Transduction Pathways Using miRNA Regulation in Cancer Therapy

Due to the central role of aberrantly activated signal transduction pathways in carcinogenesis, these pathways are attractive targets for cancer therapy. A suitable approach to targeting these pathways might be found in altering the expression of key miRNAs that regulate them. miRNAs target hundreds of mRNAs at the same time, because one mRNA 3'-UTR contains multiple miRNA binding sites. The effects of one miRNA on a single gene are moderate, because the inhibition of protein levels appears to be approximately 50% or less. Nevertheless, the complexity of miRNA regulation might result in the targeting of hundreds of downstream tumor suppressors or oncogenes, thereby multiplying the moderate effects on single genes. We have summarized above the prediction that some of the established dysregulated miRNAs in urological tumors target several members ofthe same signal transduction pathway. The miR-106b cluster is an example for which the recurrent inhibition of one pathway has been experimentally proven. Presumably, targeting of key miRNAs might exert strong effects on signal transduction and, therefore, provide an effective tool for cancer treatment.

To date, treatment options specifically for urological tumors are not well elucidated. However, several miRNA-based therapeutic options for cancer therapy in general have been discussed in recent years and are summarized in Table 1.

The most straightforward approach to target oncogenic miRNAs is to treat cancer cells with antisense miRNAs. The utility of these anti-miRNA oligonucleotides has been described (67). In recent years, so-called multitargeting anti-miRNA oligonucleotides have been designed to target several miRNAs at once (68). These oligonucleotides have the advantage of exerting a larger inhibitory effect on cancer cell growth, as treatment with anti-miRNA oligonucleotides directed against single miRNAs (69). Another approach to target a single miRNA is to use miRNA sponges. miRNA sponges are expressed from transgenes and carry multiple complementary binding sites for a specific miRNA, thereby increasing the inhibitory effect. As they "soak up" the miRNA, miRNA sponges relieve the inhibition on the target gene (70). Similar approaches use miRNA erasers, which are long tandem repeats of antisense miRNAs that inhibit expression of an oncogenic miRNA (71).

In certain studies investigators have focused on finding small molecules that can inhibit either miRNAs directly or components of the miRNA processing pathway. Watashi et al. (72) identified 2 small compounds that were able to target Dicer or inhibit the function of AGO2. For both compounds, inhibition of tumor growth was successfully shown. Methyl 2-cyano-3,11-dioxy-18beta-olean-1,12-dien-30-oate inhibits the oncogenic function of miR-27a in colon cancer (73), and retinoic acid receptor antagonists have been shown to block the function of miR-10a in pancreatic cancer (74).

It is also possible to restore the function of tumor-suppressor miRNAs, there by inhibiting the function of an oncogene. A recent study has shown that designing artificial miRNAs according to currently known target binding algorithms is sufficient to develop functional miRNAs to inhibit cancer cell growth in vitro (75). Another study has shown that a short hairpin-looped oligodesoxynucleotide, which targets the same 3'-UTR sequence of the v-raf-1 murine leukemia viral oncogene homolog 1 (RAF1) mRNA as miR-125b is able to inhibit protein expression, but not mRNA levels, of RAF1 (76).

We have discussed the fact that epigenetic silencing is one regulatory mechanism that controls miRNA expression. Therefore, epigenetic modulation of gene expression might be useful for modulating miRNA expression. Treatment of cancer cells with hydroxamic acid HDACi LAQ824 resulted in the upregulation of 22 miRNAs, whereas 5 miRNAs were downregulated (77). miR-126 and its host gene EGF-like-domain, multiple 7 (EGFL7) can be successfully reexpressed in prostate cancer cells upon treatment with inhibitors of DNA methylation or histone deacetylation (78).

A major problem of current research on miRNA-based therapies is the delivery of the miRNAs to the cancer site. Recent advancements in transfection agent formulation have improved in vitro delivery (79-81). Another option for miRNA delivery is via viral vectors (69, 82, 83), although these are not without serious problems, as discussed in a recent review (84).

Successful in vivo delivery has been demonstrated for several miRNAs, including miR-182 (85), miR-10b (86), let-7 (87), and miR-16 (88). These studies have shown that systemic treatment is suitable for detectable expression and functionality of the miRNA in the tumors and that delivery of miRNAs, which are highly expressed in normal tissues, results in minimal side effects.

In addition to being targets of therapeutic approaches, miRNAs can also help to improve existing approaches. miR-21 sensitizes glioma cells to treatment with 5-fluorouracil, and both can be simultaneously delivered into cancer cells (89). Transfection of cancer cells with an adenoviral vector expressing p53 and an artificial miRNA targeting CDKN1A increases chemosensitivity of cancer cells to doxorubicin. Increased expression of miR-521 can heighten sensitivity of cancer cells to radiation, probably by targeting DNA repair enzymes (90).

miRNAs can also be used to improve viral-based therapies. Two studies have introduced miRNA binding sites to the 3'-UTR of genes that regulate viral replications. In the first study, 4 miR-122 binding sites were introduced into the 3'-UTR of the EA1 binding cassettes of an adenovirus, which exhibits strong cytotoxicity in the liver (91). The new virus displays significantly lower cytoxicity in normal tissue, whereas cancer tissue was specifically targeted. In a second study, miR-143 and miR-145 binding sites were introduced downstream of the transcriptional regulator 1CP4 gene of an oncolytic herpes simplex virus. Those miRNAs are highly expressed in normal prostate tissue, but are no longer expressed in prostate cancer tissue. The modified virus showed strong oncolytic activity in the tumor cells but very few adverse effects on normal tissue (92).

Conclusion and Outlook

In recent years great progress has been made in miRNA research with regard to urological tumors. miRNAs have been shown to be dysregulated in these cancer types, and they also target important pathways in carcinogenesis. In this review, we summarized recent knowledge of how miRNAs can target signal transduction pathways. Although miRNA-related articles on urological tumors are increasing rapidly, there has not been a systematic overview detailing signal transduction regulation in these tumors. Although information for some pathways is available, such as for the AR signaling pathway, the regulation of other pathways has not yet been described. However, many miRNAs that have been reported to be dysregulated in urological tumors are predicted to target key pathways, which makes regulation of these pathways by miRNAs in carcinogenesis probable. Therapeutic strategies that target miRNA function are still in their infancy, but there has been progress in recent years. Therefore, studying the function of miRNA regulation in signal transduction can lead to novel strategies to target the pathways mentioned here for therapeutic purposes.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: A. Fendler, Foundation of Urologic Research, Berlin, Sonnenfeld-Stiftung, Berlin; K. Jung, Foundation of Urologic Research, Berlin, Sonnenfeld-Stiftung, Berlin. Expert Testimony: None declared.

Acknowledgments: Due to space limitations we could not reference every article in this review. We apologize for that incompleteness.

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Annika Fendler, [1,2,3] Carsten Stephan, [1] George M. Yousef, [4] and Klaus Jung [1,2] *

[1] Department of Urology, University Hospital Charite, Berlin, Germany; 2 Berlin Institute of Urologic Research, Berlin, Germany;3 Department of Biology, Chemistry and Pharmacy, Free University, Berlin, Germany, 4 Department of Laboratory Medicine, St. Michael's Hospital, University of Toronto, Toronto, Ontario, Canada.

* Address correspondence to this author at: Department of Urology, Research Division, University Hospital Charite, Schumannstrasse 20/21, 10117 Berlin, Germany. Fax +49-30-450515904; e-mail klaus.jung@charite.de.

Received February 7, 2011; accepted April 28, 2011.

Previously published online at DOI: 10.1373/clinchem.2010.157727

[5] Nonstandard abbreviations: miRNA or miR, microRNA; 3'-UTR, 3'-untranslated region; HIF, hypoxia-inducible factor; AR, androgen receptor; PI3K/Akt, phosphatidylinositol 3-kinase/RAC-alpha serine/threonine-protein kinase; CDK1B, cyclin-dependent kinase inhibitor 1B; GSK3B, glycogen synthase kinase 3 beta; PTEN, phosphatase and tensin homolog; PSA, prostate-specific antigen; VHL, von Hippel-Lindau tumor suppressor; HIF1A, hypoxia inducible factor 1; VEGF, vascular endothelial growth factor; FGFR3, fibroblast growth factor receptor 3.

[6] Human genes: AKT1, v-akt murine thymoma viral oncogene homolog 1; ARNT, aryl hydrocarbon receptor nuclear translocator (also known as HIF1B); ATM, ataxia telangiectasia mutated; ATRataxia telangiectasia and Rad3 related; BAD, BCL2-associated agonist of cell death; BAK1, BCL2-antagonist/killer 1; CA9, carbonic anhydrase IX; CALN1, calneuron 1; CAND1, cullin-associated and neddylation-dissociated 1; CASP9, caspase 9, apoptosis-related cysteine peptidase; CDC42, cell division cycle 42 (GTP binding protein, 25kDa), CDKN1A, cyclin-dependent kinase inhibitor 1A (p21, Cip1) (also known as P21CIP1); CDKN1B, cyclin-dependent kinase inhibitor 1B (p27, Kip1) (also known as P27KIP1); CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (also known as (p14ARF); CHEK1, CHK1 checkpoint homolog (S. pombe); CHEK2, CHK2 checkpoint homolog (S. pombe); CHUK, conserved helix-loop-helix ubiquitous kinase; CREBBP, CREB binding protein (also known as CBP); CUL2, cullin 2; DAPK1, death-associated protein kinase 1; DAPK2, death-associated protein kinase 2; DAPK3, death-associated protein kinase 3; DGCR8, DiGeorge syndrome critical region gene 8; DROSHA, drosha, ribonuclease type III; E2F1, E2F transcription factor 1; EGF, epidermal growth factor; EGFL7, EGF-like-domain, multiple 7; EGFR, epidermal growth factor receptor; EGLN1, egl nine homolog 1 (C. elegans); EGLN3, egl nine homolog 3 (C. elegans); EP300, E1A binding protein p300; ERBB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (also known as HER-2/neu); ERRFI-1, ERBB receptor feedback inhibitor 1; EZH2, enhancer of zeste homolog 2 (Drosophila); PTK2, PTK2 protein tyrosine kinase 2 (also known as FAK, FGF3, fibroblast growth factor 3; FGFR3, fibroblast growth factor receptor 3; FOXO1, forkhead box O1; GRB2, growth factor receptor-bound protein 2; GSK3B, glycogen synthase kinase 3 beta; HIF1A, hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor); HSPB2, heat shock 27kDa protein 2; ICP4, transcriptional regulator ICP4; KAT5, K(lysine) acetyltransferase 5; KLK3, kallikrein-related peptidase 3 (also known as PSA); KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; MAP2K1, mitogen-activated protein kinase kinase 1; MAP2K2, mitogen-activated protein kinase kinase 2; MAPK1, mitogen-activated protein kinase 1; MAPK14, mitogen-activated protein kinase 14; MAPKAPK2 mitogen-activated protein kinase-activated protein kinase 2; MCM7, minichromosome maintenance complex component 7; MDM2, Mdm2 p53 binding protein homolog (mouse), MDM4, Mdm4 p53 binding protein homolog (mouse); MSK1, antigen identified by monoclonal antibody AJ9; MTOR, mechanistic target of rapamycin (serine/threonine kinase); NFAT5, nuclear factor of activated T-cells 5, tonicity-responsive; NOS3, nitric oxide synthase 3 (endothelial cell); PDE3A, phosphodiesterase 3A, cGMP-inhibited; PDE3B, phosphodiesterase 3B, cGMP-inhibited; PDGFA, platelet derived growth factor A; PDGFB, platelet derived growth factor B; PDGFRA, platelet-derived growth factor receptor, alpha polypeptide; PDGFRB, platelet-derived growth factor receptor, beta polypeptide; PDK1, pyruvate dehydrogenase kinase, isozyme 1; PDK2, pyruvate dehydrogenase kinase, isozyme 2; PI3K, phosphoinositide-3-kinase; PLCG1, phospholipase C, gamma 1; PLCG2, phospholipase C, gamma 2; PRKCA, protein kinase C, alpha) (see online Supplemental Fig. 3); PRKCB, protein kinase C, beta (also known as PRKCB1); PRKCD, protein kinase C, delta; PRKCE, protein kinase C, epsilon; PRKCI, protein kinase C, iota; PRODH, proline dehydrogenase (oxidase) 1; PTEN, phosphatase and tensin homolog; PXN, paxillin; RAF1, v-raf-1 murine leukemia viral oncogene homolog 1; RASSF1, Ras association (RalGDS/AF-6) domain family member 1; RBX1, ring-box 1, E3 ubiquitin protein ligase; ROCK1, rho-associated, coiled-coil containing protein kinase 1; RPS6KA5, ribosomal protein S6 kinase, 90kDa, polypeptide 5 (also known as MSK1); SH2D2A, SH2 domain containing 2A; SHC1, SHC (Src homology 2 domain containing) transforming protein 1; SHC2, SHC (Src homology 2 domain containing) transforming protein 2; SHC3, SHC (Src homology 2 domain containing) transforming protein 3; SHC4, SHC (Src homology 2 domain containing) transforming protein 4; SOS1, son of sevenless homolog 1 (Drosophila); SOS2, son of sevenless homolog 2 (Drosophila); SPHK1, sphingosine kinase 1; SPHK1, sphingosine kinase 2; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian); TCEB1, transcription elongation factor B (SIII), polypeptide 1 (15 kDa, elongin C); TCEB2, transcription elongation factor B (SIII), polypeptide 2 (18 kDa, elongin B); TP53, tumor protein p53; VDR, vitamin D (1,25- dihydroxyvitamin D3) receptor; VEGFA, vascular endothelial growth factor A; VEGFB, vascular endothelial growth factor B; VEGFC, vascular endothelial growth factor C; VEGFR2, kinase insert domain receptor (a type III receptor tyrosine kinase) (also known as KDR); VHL, von Hippel-Lindau tumor suppressor; ZFP36L1, zinc finger protein 36, C3H type-like 1.
Table 1. Potential approaches to therapeutically target miRNAs
in urological carcinomas.

 Approach Description References

Inhibition of an
oncogenic miRNA

Anti-miRNA Targeting the Esau (67)
oligonucleotides function of a single
 miRNA with an
 antisense
 oligodesoxyribonucleotider

Multitargeting Targeting the Wang et al. (69)
anti-miRNA function of multiple
oligonucleotides miRNAs with a single
 antisense
 oligodesoxyribonucleotide

Sponges Targeting the Ebert et al. (70)
 function of a single
 miRNA by a transgene
 that carries
 multiple binding
 sites for the miRNA

Erasers Inhibition of a Sayed et al. (71)
 single miRNA by
 transfection of a
 double-stranded
 oligonucleotide with
 long tandem repeats
 of the antisense
 sequence

Small molecules Inhibition of a Watashi et al.
 single miRNA or (72), Chinthar-
 components of miRNA lapalli et al.
 processing by small (73
 molecules

Inhibition of an
oncogenic mRNA/
restoration of miRNA
function

Design of artificial Designing novel De et al. (75)
miRNAs miRNAs to target 3'-
 UTRs of oncogenic
 mRNAs

Design of Designing short DNA Hofmann et al.
hairpin-loop sequences that (7)6
oligodesoxi- target binding sites
nucleotides of known miRNA
 inhibition

Delivery by viral Reexpression of Wang et al. (69),
expression vectors miRNAs through viral Kota et al. (82),
 vector delivery Nair (83)

Epigenetic
modulation of miRNA
expression

Combined treatment Reexpression of Saito et al. (78)
with epigenetically
methyltransferase silenced miRNAs
and histone
deacetylases

Treatment with Reexpression of Scott et al. (77)
hydroxamic acid epigenetically
HDACi LAQ824 silenced miRNAs or
 inhibition of
 aberrantly expressed
 miRNAs

Improvement of
existing therapies

Sensitizing cells to Overexpression of Josson et al. (90)
radiation miR-106b or miR-521
 sensitizes cells to
 radiation

Sensitizing cells to miRNA overexpression Ren et al. (89)
chemotherapeuticals can sensitize cells
 to treatment with
 chemotherapeutic
 agents (5-
 fluorouracil or
 doxorubicin)

Improving Introduction of Cawood et al.
specificity of viral miRNA binding sites (91), Lee et
treatment downstream of viral al. (92)
 reproduction genes
 to restrict their
 activity to cancer
 cells
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Author:Fendler, Annika; Stephan, Carsten; Yousef, George M.; Jung, Klaus
Publication:Clinical Chemistry
Date:Jul 1, 2011
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