MicroRNAs as regulators and mediators of c-MYC function

Biochimica et Biophysica Acta 1849 (2015) 544–553

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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagrm

Review

MicroRNAs as regulators and mediators of c-MYC function☆ Rene Jackstadt, Heiko Hermeking ⁎ Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-Universität München, D-80337 Munich, Germany

a r t i c l e

i n f o

Article history: Received 5 December 2013 Received in revised form 27 March 2014 Accepted 4 April 2014 Available online 13 April 2014 Keywords: MYC Oncogene miRNA Cancer

a b s t r a c t In the past ten years microRNAs (miRNAs) have been widely implicated as components of tumor suppressive and oncogenic pathways. Also the proto-typic oncogene c-MYC has been connected to miRNAs. The c-MYC gene is activated in approximately half of all tumors, and its product, the c-MYC transcription factor, regulates numerous processes e.g. cell cycle progression, metabolism, epithelial–mesenchymal transition (EMT), metastasis, stemness, and angiogenesis, thereby facilitating tumor initiation and progression. c-MYC target-genes, which mediate these functions of c-MYC, represent a complex network of protein- and non-coding RNAs, including numerous miRNAs. For example, c-MYC directly regulates expression of the miR-17–92 cluster, miR-34a, miR15a/16-1 and miR-9. Moreover, the expression and activity of c-MYC itself are under the control of miRNAs. Here, we survey how these networks mediate and regulate c-MYC functions. In the future, miRNAs connected to cMYC may be used for diagnostic and therapeutic approaches. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The protein product of the proto-oncogene c-MYC (hereafter MYC) is a transcription factor that regulates a broad range of cellular processes, which may contribute to the initiation and progression of tumors [1,2]. In tumors MYC expression is often found to be activated constitutively via several mechanisms: e.g. chromosomal translocations [3], amplification [4], loss of auto-regulation [5], increased translation of MYC mRNA [5,6], stabilizing mutations [7,8], or activating mutations in components of upstream regulatory pathways, such as Wnt signaling [9–11]. In most cases these alterations lead to constitutive expression of the MYC gene, which is normally only expressed during certain phases of the cell cycle. Ectopic MYC is sufficient to induce cell cycle progression and proliferation. MYC has been shown to induce or repress the expression of hundreds of genes, which are thought to mediate its biological functions [12–14]. Cells have evolved a failsafe mechanism against aberrant proliferation caused by deregulation of MYC (and other mitogenic oncogenes), which involves activation of the transcription factor p53, which then mediates apoptosis and/or senescence to eliminate cells with deregulated MYC expression [15]. The activation of p53 by MYC may occur via activation of the ATM/ATR-pathway due to DNA damage

☆ This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. ⁎ Corresponding author at: Experimental and Molecular Pathology, Institute of Pathology, Ludwig-Maximilians-Universität München, Thalkirchner Strasse 36, D-80337 Munich, Germany. Tel.: +49 89 2180 73685; fax: +49 89 2180 73697. E-mail address: [email protected] (H. Hermeking).

http://dx.doi.org/10.1016/j.bbagrm.2014.04.003 1874-9399/© 2014 Elsevier B.V. All rights reserved.

caused by MYC-induced replication stress or by MYC-mediated upregulation of the ARF/MDM2 pathway [16,17]. MYC belongs to the basic-helix–loop–helix leucine-zipper (bHLH-LZ) transcription factor family. The C-terminus of MYC harbors a HLH-LZ motif, which mediates the heterodimerization with the bHLH-LZ transcription factor MAX. The basic regions of the MYC/MAX heterodimer mediate the DNA binding to the consensus E-Box sequence CA(C/T)GTG [17]. The N-terminus of MYC encompasses two evolutionary conserved regions, Myc-Box I and II, which act as transactivation domains. Upon DNA binding the MYC/MAX heterodimer recruits co-factors, which mediate multiple effects of MYC on gene expression in a context-dependent manner [18]. Several MYC-mediated chromatin modifications, which result in transcriptional silencing, promoter clearance [19] and transcriptional elongation [20] have been described as consequences of MYC occupancy at promoters. Besides with MYC, MAX may heterodimerize with MAD, MXI1 and MNT, which thereby antagonize MYC function [21,22]. Whereas direct E-Box binding of MYC/MAX heterodimers generally mediates the induction of MYC target gene transcription, repression by MYC is often mediated by binding of a MYC/MAX/MIZ1 complex to initiator (Inr) elements [23–26]. The Inr represents a 17 bp pyrimidine-rich motif, which mediates transcriptional initiation from TATA-less promoters [27,28]. miRNAs represent small non-coding RNAs, which bind to 3′-UTRs of target mRNAs and mediate translational repression or mRNA degradation [29]. Induction of miRNA-encoding genes represents an alternative mechanism by which MYC indirectly represses gene expression. Evidence described in this review suggests that this mode of repression by MYC is rather common. Genes which encode miRNAs are mostly transcribed by RNA polymerase II, resulting in a primary transcript miRNA (pri-miRNA) (Fig. 1A).

R. Jackstadt, H. Hermeking / Biochimica et Biophysica Acta 1849 (2015) 544–553

Subsequently, this transcript becomes processed by the double-stranded RNA (dsRNA)-specific RNase III endonuclease DROSHA into precursor miRNAs (pre-miRNAs). Then Exportin 5 (XPO5) binds these ~ 70 nucleotide-long molecules and shuttles them to the cytoplasm, where the mature miRNA is generated by the dsRNA-specific RNase III enzyme DICER1, followed by incorporation into the RNAinduced silencing complex (RISC). The guide strand of the duplex associates with an Argonaute (AGO) protein, while the opposite strand (miRNA*) is often degraded and therefore not detectable [29–31]. The association with target mRNAs occurs via ~ 7 nucleotide-long stretches, the so-called seed-sequence, present in the 5′-region of the miRNA. The complementary sequence is mostly located in the 3′-untranslated region (3′-UTR) of the respective target gene. Additional base pairings can occur through nucleotides in the central region and 3′-ends of the miRNA. As a consequence of the recruitment of the miRNA/Ago complex translation of the bound mRNA is inhibited and often secondary degradation of the mRNA is observed. This involves mechanisms reviewed in [32,33]. Since a relatively short seed region is important for target recognition, a single miRNA might regulate several target mRNAs [33]. The influence of miRNAs on gene expression is widespread, with N 60% of human protein-coding genes predicted to be subject to regulation by miRNAs [34]. The biological functions of miRNAs are highly dependent on cellular context, which may be due to the differential expression of their target mRNAs. Some miRNAs function either as

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oncomiRs or as tumor suppressive miRNAs, in a cell-type dependent manner. Besides regulating the expression of miRNA-encoding genes by directly binding to their promoters MYC regulates miRNA expression by alternative mechanisms. An example for this is represented by the transcriptional activation of the genes encoding the RNA binding proteins Lin28 and Lin28b by MYC [35] (Fig. 1B). Lin28/Lin28b interacts directly with let-7 pre-miRNA stem–loops and thereby blocks the processing of let-7 by DROSHA and DICER1 [35,36]. Furthermore, Lin28/Lin28b recruit the 3′ terminal uridylyl transferase TUT4 to pre-let-7, resulting in uridylation and subsequent decay of the pre-miRNA [37–39].MYC may also enhance pri-miRNA processing by directly inducing the expression of DROSHA [40] (Fig. 1C). Alterations in the miRNA processing machinery may affect MYCinduced tumorigenesis. For example, DICER1 was characterized as a haploinsufficient tumor suppressor gene in a mouse model of soft-tissue sarcoma [41], suggesting that global loss of miRNA expression is protumorigenic. In support of this conclusion, decreased expression of DICER1 correlates with poor prognosis in human lung cancer [42]. Surprisingly, a deletion of one DICER1 allele in B-cells failed to promote Bcell malignancy or accelerate MYC-induced B-cell lymphomagenesis in mice [43]. Moreover, deletion of both DICER1 alleles in B-cells of Eμ-myc mice significantly inhibited lymphomagenesis [43]. Therefore, the function of DICER1, and therefore general miRNA expression, seems to be either tumor suppressive or oncogenic in a context-dependent manner.

Fig. 1. Transcriptional and post-transcriptional influences of MYC on miRNA processing. (A) MYC binding to the promoter of miRNA genes, by recognition of E-boxes, regulates the transcription of pri-miRNAs transcripts. (B) MYC effect on miRNA processing (let-7), by transcriptional regulation of Lin28. (C) Inhibition of miRNA processing by transcriptional repression of DROSHA.

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2. MYC-induced miRNAs The first evidence, that cancer-related mechanisms, such as differentiation, apoptosis and proliferation are regulated by miRNAs, was obtained in Caenorhabditis elegans and Drosophila [44,45]. Since these processes are also relevant during carcinogenesis, an involvement of miRNA deregulation in tumor initiation and progression seemed plausible. The first identified, MYC-regulated miRNAcluster, was miR17–92 (oncomiR-1). The miR-17–92 miRNA cluster, encodes miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR92a-1 [46,47]. Two paralogs of the miR-17–92 cluster have been identified: the miR-106b-25 cluster is located in the 13th intron of the MCM7 gene, whereas the miR-106a-363 cluster is encoded by its own mRNA [48]. Both miR-17–92 and miR-106b-25 derived miRNAs are expressed at high levels in several adult tissues and during embryogenesis, whereas miR-106a-363 members are generally expressed at low levels [48,49]. The 15 miRNAs of the miR-17–92 family and its two paralogs can be subdivided into 4 groups with similar seed sequences, which show high conservation in vertebrates [49]. These 15 miRNAs are frequently activated in several solid tumors and B-cell lymphomas [47,50-53]. Furthermore, ectopic expression of the miR-17–92 cluster, under the control of an Eμenhancer and Ig heavy-chain promoter, leads to development of Bcell lymphomas in mice [54]. In addition, conditional knock-out of miR-17–92 in MYC-driven lymphomas leads to increased cell death and reduced tumorigenicity [55]. Furthermore, knock-out of single members of the miR-17–92 cluster revealed that the six miRNAs encoded by the cluster are not functionally equivalent during cancer development. Two members of the miR-19 seed subgroup were found to be required and sufficient to recapitulate the oncogenic activity of the full cluster [56]. Additionally, ectopic miR-17–92 expression in a murine retinoblastoma mouse model in which tumors are initiated by deletion of the RB family members RB and p107, as well as in colorectal colonocytes and granule neuron progenitors, enhances tumorigenesis [57–59]. Taken together, these results show that the miR-17–92 cluster represents a bona fide oncogene and a mediator of MYC-induced tumorigenesis. MYC activates miR-17–92 expression by binding to an E-box in its first intron [47]. Furthermore, several MYC binding sites have been characterized in the vicinity of the transcription start sites (TSS) of miR-106a-363 and miR-106b-25. Recently, the direct regulation of the miR-106a-363 cluster by MYC during trophoblast differentiation was described [60]. Besides MYC, the mitogenic transcription factor E2F was shown to regulate the expression of miR-17–92 [61,62]. Since the miRNAs miR-17 and miR-20 directly target E2F1 via identical seedmatching sequences, E2F1 and these miRNAs constitute a negative feedback loop (Fig. 2A) [63], which may represent an auto-regulatory failsafe mechanism to restrict E2F1 expression and thereby control the balance between proliferation and apoptosis [46,61,62]. Additionally, MCM7, the host gene of miR-106b-25, is regulated by the E2F family member E2F1 and its expression levels correlate with miR-106b-25 expression in primary gastric tumors and normal mucosa [64,65]. Interestingly, MYC also regulates the expression of MCM7 and therefore also activates the miR-106b-25 cluster [63]. Members of the miR-17–92 cluster target many different mRNAs, which in turn affect several MYCassociated cancer promoting functions e.g. cell proliferation, cell survival, angiogenesis, and metabolic reprogramming (see also Table 1). Another MYC-induced miRNA, namely miR-22, was recently shown to act as a potent proto-oncogenic miRNA via genome-wide deregulation of the epigenetic state through inhibition of methylcytosine dioxygenase TET (ten-11 translocation) proteins [66]. Thereby, miR-22 influences chromatin remodeling and silences miR-200 genes which promotes EMT and enhances stemness, breast cancer development and metastasis. In addition, miR-22 was characterized as a key regulator of self-renewal in the hematopoietic system [67]. miR-22 was found to reduce the global level of 5-hydroxymethylcytosine (5-hmC) modifications

in mouse hematopoietic stem cells (HSCs) and thereby triggers an increase in the self-renewal capability of HSC. The TET2 enzyme represents a critical target of miR-22 in this context. Furthermore, miR-22 was shown to repress the tumor-suppressor PTEN (phosphatase tensin and homolog) in prostate cancer and mouse models of cardiac hypertrophy [68]. However, further studies suggested, that miR-22 may also act as a tumor-suppressor, e.g. by targeting ERBB3 in lung cancer cells [68–70]. This is consistent with the notion that miRNAs often display a contextdependent function. miR-378 represents an additional, oncogenic MYC-induced miRNA [71]. Interestingly, miR-378 cooperates with activated RAS or HER2 to promote cellular transformation. miR-378 acts oncogenic by targeting TOB2, and tumor suppressive by repressing cyclin D1. As expected, the c-MYC homolog N-MYC also induces the miR-17–92 cluster and several other c-MYC-regulated miRNAs [72–75]. One of them is miR-9 (discussed below), a repressor of the epithelial marker E-cadherin, which is often downregulated during cancer progression [76]. miR-130a and miR-214 represent further miRNAs, which are induced by both, c-MYC and N-MYC [72]. 3. c-MYC-repressed miRNAs The first experimental evidence that MYC represses miRNAs was provided by Chang and co-workers [75], who showed that multiple miRNAs are negatively regulated by MYC. These miRNAs represent potent cellcycle inhibitors or apoptosis promoters [77–79] (Table 1). Furthermore, MYC activates several oncogenic factors by repression of miRNAs, e.g. MYC-mediated repression of let-7 results in up-regulation of RAS or HMGA2 expression [80]. Another tumor promoting activity of MYC is to increase mitochondrial glutaminase (GLS) activity by repressing miR23a and miR-23b, which both target the GLS transcript [81]. GLS is a key enzyme that converts glutamine to glutamate, which serves as a substrate in the TCA cycle for the production of ATP. In addition, MYC regulates the glutamine metabolism through several mechanisms, including the direct transcriptional activation of glutamine transporters [82]. Both glutamine and GLS are required for MYC-mediated cancer cell survival and proliferation. Besides enhancing the supply of cells with glutamine MYC activation also enhances the levels of glucose, the second major carbon source needed by proliferative cancer cells, and activates aerobic glycolysis via inducing several enzymes involved, such as LDH-A [1,83]. A miRNA-mediated enhancement of glycolysis by MYC was recently reported: by inducing miR-19 family members, which down-regulate PTEN, MYC enhances PI3K (phosphoinositide 3-kinase) activity [55], which strongly promotes glucose metabolism by increasing glucose transporter expression and enhancing glycolytic enzyme activity [84]. Therefore, MYC coordinates both glucose and glutamine metabolism through miRNA regulation. Furthermore, the miR-15a/16-1 host gene DLEU2 is directly repressed by MYC [85]. The miR-15a/16-1 cluster is located in an intron of the DLEU2 gene, which is frequently deleted or down-regulated in human tumors [86-88]. Zhang et al. demostrated the co-localization of MYC and HDAC3 at the two alternative promoters of the DLEU2 gene [89]. In fact, miR-15a/16-1 represent the first miRNAs, which were shown to be the target of cancer-specific deletions [90]. Later, a knock-out of DLEU2 or the miR-15a/16-1 bearing intron in mice revealed that loss of miR-15a/16-1 expression is sufficient to cause chronic lymphatic leukemia (CLL) [91]. Therefore, DLEU2 is presumably the tumor suppressor gene located in the 13q14 region. Importantly, this mouse knock-out study provided the first genetic proof of a miRNA to represent a bona fide tumor suppressor gene. miR-15a/16-1 targets multiple mRNAs, which encode oncogenic factors, such as BCL2 and cyclin D1, and thereby exerts its tumor suppressive function by inducing apoptosis or inhibiting cell cycle progression [92–95]. Interestingly, the DLEU2 gene does neither seem to encode a functional protein, nor a functional RNA besides miR-15a/16-1. We could recently show that the MYCinduced bHLH-LZ transcription-factor AP4 directly represses DLEU2

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Table 1 Compilation of miRNAs induced/repressed by MYC or targeting c-MYC. The miRNAs, the respective host genes and the methods by which the regulation was characterized are summarized. The respective references are given in the last column. The putative transcription start site of the miRNA transcript or of the host gene was analyzed, in order to determine the direct regulation by MYC via DNA binding (ChIP). The down-stream miRNA targets and processes of these regulations are indicated. Lin28 is the direct MYC target gene. Its protein product influences the processing of the let-7 miRNAs. miRNA

Host gene

Method of validation

Target mRNAs

Process

Reference

MYC-induced miRNAs miR-9 C1ORF61 miR-17 C13ORF25 miR-18a C13ORF25 miR-19a C13ORF25 miR-19b-1 C13ORF25 miR-20a C13ORF25 miR-22 MGC14276 miR-25 MCM7

ChIP-on-chip ChIP ChIP ChIP ChIP ChIP – ChIP

E-cadherin, LIFR, CREB, NF1 STAT3, MAPK14, p21, p57, TGFBR2, PTEN, BIM, E2F1–3 SMAD2/4, CTGF, TSP-1, ISL1, TBX1 CTGF, TSP1, PTEN, BIM CTGF, TSP1, PTEN, BIM STAT3, MAPK14, p21, p57, TGFBR2, PTEN, BIM PTEN, TET, TIAM, MMP2, MMP9 ISL1, TBX1, PTEN, BIM, RECK, SMAD7, WWP2, FBXW7, EZH2, EP300

[76] [46,47] [48,49] [46,47] [46,47] [46,47] [85] [60]

miR-92a-1 miR-93 miR-106b

C13ORF25 MCM7 MCM7

ChIP ChIP ChIP

ISL1, TBX1, PTEN, BIM, ITGA5, RECK STAT3, MAPK14, p21, p57, TGFBR2, PTEN, BIM, DAB2, LATS2, EP300 STAT3, MAPK14, p21, p57, TGFBR2, PTEN, BIM, EP300

miR-130a – miR-214 – miR-378 PPARGC1B MYC-repressed miRNAs Lin28/let-7 – miR-15a/ DLEU2 16-1 miR-23a – miR-23b C9orf3 miR-26a CTDSP1 miR-29a/b – miR-34a –

ChIP ChIP –

ATG2B, DICER1, c-MET, HOXA5, SMAD4 PTEN, BCL2L2, TFAP2C, EZH2, LTF TOB2, MAPK1, SOX2, SUFU, FUS1

EMT, metastasis Cell cycle, apoptosis Angiogenesis, apoptosis Survival, angiogenesis, metabolism, apoptosis Survival, angiogenesis, metabolism, apoptosis Cell cycle, apoptosis EMT, metastasis Apoptosis, heart development, reprogramming, EMT, metastasis Apoptosis, heart development, angiogenesis Cell cycle, apoptosis, angiogenesis, metastasis Apoptosis, heart development, reprogramming, EMT, metastasis Lung development, drug resistance, cell cycle Proliferation, invasion, migration, apoptosis Survival, proliferation angiogenesis

ChIP, luc-reporter HMAG, RAS, TWIST, CDC25A, CCND2 ChIP, luc-reporter AP4, BCL2, BMI1, VEGF, CARD10, CDC27

EMT, cell cycle proliferation, invasion, metastasis EMT, metastasis, stemness, angiogenesis, cell cycle

[34,74] [74,89]

– ChIP ChIP ChIP ChIP

GLS, E-cadherin PTEN, GLS, SRC, PYK2 PTEN, EZH2, Lin28B, ZCCHC11, FGF9, MCL1, HGM1, IL6 CDK6, IGF1R CDK6, MET, CD44, LDHA, SIRT1, SNAIL, ZNF281, c-KIT, Notch1, BCL2, BIRC5, IL6R MYC, CCKBR, BCL2, WNT1, DNMT1, ROCK1, CDC25B DNMT1, ATR, MYC, RHOA, CDC42

Metabolism, EMT Metabolism, cell cycle, invasion, migration Cell cycle, invasion, metastasis Cell cycle Cell cycle, apoptosis, senescence, stemness, EMT, metastasis EMT, metastasis, apoptosis, proliferation Cell cycle, metastasis

[80] [80] [85,97] [85,101] [85]

USP28, PDPN

Invasion, metastasis

[125]

miR-148a miR-185

– C22orf25

miR-363

–

ChIP ChIP; lucreporter ChIP

[46,47] [60] [60] [72] [72] [71]

[125] [124]

miRNA

Host gene

Method of validation

SMS

Reference

miRNAs targeting c-MYC let-7 miR-24 miR-34b/c miR-145 miR-148a miR-184 miR-185 miR-196b miR-449c miR-494

– C9ORF3 − − − − C22orf25 HOXA9 CDC20B –

Western blot, qPCR Western blot, qPCR Western blot, qPCR, luc-reporter Western Blot, luc-reporter GFP-reporter, Western Blot Western blot, qPCR, luc-reporter Western blot, qPCR, luc-reporter qPCR Western blot, qPCR, luc-reporter Western blot, qPCR, luc-reporter

+ − + + + + + + + +

[126] [135,136] [134] [132] [125] [126] [124] [127] [129] [100]

Abbreviations: SMS = seed-matching sequence, qPCR = quantitative polymerase chain reaction, luc = luciferase assay including MYC-binding site or SMS mutagenesis, GFP = green fluorescent protein, ChIP = chromatin immunoprecipitation.

expression [96]. Since AP4 also represents a target of miR-15a/16-1, AP4 and miR-15a/16-1 constitute a double-negative feedback loop, which controls EMT and metastasis (discussed below). In contrast to MYC, p53 induces expression of miR-15a/16-1 by several mechanisms. Initially, p53 was shown to enhance the post-transcriptional maturation of miR-15a/16-1 by directly interacting with DROSHA via the DEAD-box RNA helicase DDX5 and thereby influencing the processing from pri-miRNAs to premiRNAs [97]. Later, the DLEU2 gene was shown to be a transcriptional target of p53 in B-cells [98]. A repressive complex containing MYC, HDAC3, and EZH2 down-regulates the expression of the miR29 family in mantle cell lymphoma and other MYC-associated lymphomas [99]. Furthermore, MYC up-regulates EZH2 expression via repression of the EZH2-targeting miR-26a [100], and EZH2 induces MYC via inhibition of the MYC targeting miR-494 [99]. These regulations result in a double-positive feedback loop (Fig. 2B). Additional studies in cholangiocarcinoma cells showed repression of miR-29b by MYC, NF-κB and Hedgehog [101]. Since replacement of miRNAs repressed by MYC in tumors seemed to be an attractive

therapeutic option (discussed below), Kota et al. employed a MYC-driven mouse hepatocellular carcinoma model and an adenovirus associated virus (AAV) vector delivery system, which allowed the reintroduction of miR-26a and reversal of disease progression [102]. Notably, delivery of miR-26a into the liver led to regression of established liver tumors by induction of apoptosis in cancer cells but not in non-malignant hepatocytes. Among the p53-induced miRNAs, miR-34a displays the most pronounced induction by p53 [78,79,87,103-108]. Interestingly, miR-34a is repressed by MYC via binding in the vicinity of the miR-34a promoter, as shown by ChIP analyses in Epstein–Barr virus-immortalized human B-cells [75]. Therefore, the miR-34a regulation may serve as another platform for the antagonism between MYC and p53. In mammalians, the miR-34 family comprises at least six processed miRNAs that are encoded by three different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c are encoded by a common primary transcript. The miR-449 cluster, which encodes the highly conserved miR-449a, miR-449b and miR-449c, belongs to the miR34 family, due to the conservation of their seed sequences [109].

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A Proliferation

B E2F

MYC

Chromatin regulation MYC

C Auto-regulation

miR-26a

MYC

miR-148

EZH2

USP28

miR-363

miR-29 miR-106b-25

miR-17-92

miR-494

D p53 pathway

E

MYC

ARF

miR-145

Mitogenic signaling

SIRT1 MYC

miR-34b/c

MYC

MK5

FOXO3a

PTEN

miR-23b miR-26a

miR-34 miR-22 miR-19a; miR-106b miR-25; miR-93 miR-214

Cell cycle

H

MYC

Angiogenesis MYC

AP4

MIZ1

miR-17 miR-106b miR-20a

VEGF p21

I

F

p53

HDM2

G

Mitogenic signaling

miR-15a miR-16-1 miR-34a miR-106a/b miR-20a; miR-93 miR-17

Epithelial-mesenchymal transition Vimentin E-cadherin

MYC

miR-9

SNAIL

AP4

E-cadherin miR-15a/16-1

mesenchymal

ZNF281

miR-34a

p53

Vimentin E-cadherin

epithelial

Fig. 2. MYC involving miRNA networks. MYC participates in several miRNA networks, which regulate (A) proliferation, (B) chromatin modifications, (C) auto-regulation, (D) p53 pathway, (E and F) mitogenic signaling, (G) cell cycle and (H) angiogenesis. (I) Role of MYC and connected miRNAs in EMT. MYC positively regulates the indicated EMT inducing factors, thereby promoting a mesenchymal state allowing invasion and metastasis. The mesenchymal state is characterized e.g. by high expression of Vimentin and low expression of E-cadherin. In contrast p53 promotes the epithelial state characterized by high E-cadherin and low Vimentin expression. Factors acting oncogenic or representing oncogenes are indicated in orange, whereas tumor suppressive factors or tumor suppressor genes are indicated in green.

Therefore, the targets of miR-34 and miR-449 miRNA family members are presumably overlapping. However, the regulation of miR-449 differs from miR-34 since it is not induced by p53, but by E2F1 [109]. Since E2F1 is generally induced after MYC activation, miR-449 expression is presumably elevated by MYC. In mice, miR-34a is ubiquitously expressed with the highest expression in the brain, whereas miR-34b/ c are mainly expressed in lung tissues [107]. By repression of miR-34a MYC may antagonize several functions ascribed to miR-34a, such as inhibition of cell cycle progression [103] and stemness [110], as well as induction of senescence [108] and mesenchymal–epithelial transition [111] (MET; discussed below in detail; Table 1). More recently, we could show that genetic ablation of miR-34a allows invasion of colitisassociated colon cancer by activation of an IL6R/STAT3/miR-34a feedback loop [112]. Furthermore, loss of miR-34a/b/c cooperates with p53 loss in prostate cancer progression in mouse tumor models [113]. In addition, miR-34a/b/c deletion cooperates with deletion of one p53 allele in

a lung cancer model [114]. By inhibiting these new tumor-suppressive activities of miR-34 MYC may also contribute to tumor progression. Besides being repressed by MYC, the miR-34a/b/c genes are downregulated during tumor development and metastasis by promoter methylation [85,115–117]. Similar to the therapeutic application described above for miR-26, Sotillo and colleagues determined whether restoration of miR-34a levels in B-lymphoid cells with enhanced MYC expression would induce apoptosis [118]. Unexpectedly, delivery of miR-34a, which does not target p53 directly, severely decreased the steadystate p53 levels. This effect was preceded and mediated by downregulation of MYC, which enhances p53 protein levels via the ARF– HDM2 pathway. As a result, in the presence of MYC, miR-34a inhibited p53-dependent bortezomib-induced apoptosis as efficiently as p53specific knock-down. Conversely, inhibition of miR-34a using antagomirs sensitized lymphoma cells to apoptosis. Thus, in certain tumors with deregulated MYC expression, miR-34a confers drug

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resistance and could be considered as a therapeutic target [118]. However, reintroduction of miR-34a into neuroblastoma cells, which lacked the 1p36 region containing the miR-34a gene, or into cells, which express low levels of miR-34a, resulted in a pronounced growth inhibition, which was in part due to the direct regulation of N-MYC by miR-34a [119–121]. In addition, miR-34 prevented cancer initiation and progression in a K-RAS and p53-induced mouse model of lung adenocarcinoma [122]. Furthermore, tail vein injection of miR-34a oligonucleotide complexed with a lipid-based delivery agent led to reduced prostate cancer growth and metastasis in an orthotropic mouse model, by directly regulating the stemness marker CD44 [123]. Because of these promising results, treatment with miR-34 mimics entered a Phase I clinical trial in patients with advanced hepatocellular carcinoma (HCC) in April 2013 [124]. MYC plays an important role in normal development and tumorigenesis of the liver [125]. Besides the repression of miR-26 MYC may contribute to HCC through an additional miRNA-mediated feedback loop comprising of miR-148a, miR-363, and the ubiquitin-specific protease 28 (USP28) (Fig. 2C): MYC directly binds to conserved regions in the promoters of the host-genes of the two miRNAs and represses their expression. miR-148a directly targets and inhibits MYC, whereas miR-363 destabilizes MYC by directly targeting and inhibiting USP28 [125]. Inhibition of miR-148a or miR-363 induced hepatocellular tumorigenesis by promoting G1/S-phase transition, whereas activation of these miRNAs had the opposite effect [125]. In addition, another auto-regulatory mechanism involves miR-185, which directly represses MYC translation and is repressed by MYC [126]. 4. MYC regulating miRNAs Several regulatory mechanisms control MYC expression by influencing its transcription and translation, as well as mRNA and protein stability [1]. More recently, a number of miRNAs have been described that regulate MYC expression. Bioinformatics analyses predicted let-7a seed-matching sequences in the MYC 3′-UTR. Indeed, ectopic expression of let-7a down-regulated MYC on mRNA and protein level and reverted MYC-induced proliferation of Burkitt lymphoma cells [127]. Interestingly two different miRNAs, namely miR-196b and miR-184, both concomitantly regulate MYC and BCL2 to block cell proliferation and survival [128]. Interestingly, miR-184 is directly regulated by the tumor suppressor PDCD4, which inhibits cell proliferation, migration and invasion [129]. Ectopic miR-449c suppresses invasion and migration via direct repression of MYC [130]. Interestingly, miR-449 also indirectly targets the transcription factor E2F1 to achieve its tumor suppressive functions [109]. However, in non-small lung cancer (NSCLC) miR-449c is increased in tumor tissue compared to normal tissue, suggesting an oncogenic role of miR-449c in NSCLC. Therefore, miR449 displays both tumor suppressive and oncogenic properties in a context-dependent manner. In addition to the previously described transcriptional repression of MYC by p53 [131], the p53-mediated induction of miR-145, which targets MYC, provides an alternative mechanism for p53-mediated repression of MYC (Fig. 2D) [132]. Additionally, p53-induced miR-34a represses MYC [133,134]. Furthermore, miR-34b/c is involved in a negative feedback loop with the MK5/PRAK kinase and FOXO3a to regulate MYC expression (Fig. 2E) [135]. MK5 phosphorylates FOXO3a, which leads to nuclear localization of FOXO3a and transcriptional activation of miR-34b/c in colorectal cancer (CRC) cells. Subsequently, miR-34b/c bind to the 3′-UTR of MYC and thereby block its translation. Interestingly, the expression of MK5 in CRC tumors correlates with increased differentiation and therefore enhanced survival of colorectal cancer patients. The Lieberman lab showed that miR-24, which is up-regulated during terminal differentiation of multiple heamatopoetic lineages and inhibits the cell cycle can repress MYC expression via “seedless” 3′-UTR microRNA recognition elements [136]. Later the ribosomal protein L11 was shown

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to regulate the turnover of MYC mRNA [137]. A L11/AGO2/RISC/miR-24 complex binds to MYC mRNA 3′-UTR leading to mRNA degradation. Knock-down of L11 drastically increases the levels and stability of MYC mRNA. Knock-down of AGO2 abrogated the L11-mediated repression of MYC, whereas knockdown of L11 rescued miR-24-mediated mRNA decay. 5. Regulatory circuits controlled by MYC and miRNAs miRNA-target circuits have been described to stabilize or fine tune biological processes by reinforcing transcriptional programs, constituting bistable switches or providing robustness [138]. Since MYC is an important factor during normal and tumor development, MYC/miRNA circuits and their deregulation are likely to contribute to the oncogenic functions of MYC [139]. An intensively studied feedback loop involves the miR-17–92 family, E2Fs and MYC (Fig. 2A) [46,140]. The E2F transcription factors participate in the control of cell proliferation and apoptosis, and depending on their levels of expression they can act as oncogenes or tumor suppressors [140,141]. Positive feedback loops in the regulation of these factors may lead to bi-stability, which allows switch-like transitions in the concentrations of transcription factors in response to minor changes of extra-cellular signals [142]. Furthermore, post-translational inhibition of E2Fs by members of the miR-17–92 family and the induction of the miR-17–92 transcript by E2F1 and MYC represent negative feedback loops, which may serve to prevent overshooting inductions of the respective pathways [143]. Several additional complex feed-forward loops involving MYC are known: for example the MYC/PTEN/miR-106b, miR-93, miR-25, miR19a, miR-22, miR-26a, and miR-23b circuit (Fig. 2F). The mRNA encoded by the PTEN tumor suppressor gene is a target of several miRNAs, which are also regulated by MYC [144,145]. The regulation of the cell cycle inhibitor CDKN1A/p21 by MYC represents another coherent feed-forward loop (Fig. 2G). Initially, MYC was shown to bind to MIZ1 and thereby repress p21 expression [24]. More recently, the bHLH-LZ transcription factor AP4, was shown to be directly induced by MYC and repress p21 [19, 146]. The MYC-induced miR-17, miR-20a and miR-106b suppress p21 expression and therefore represent another mechanism of MYCmediated suppression of p21 [65,147]. A third circuitry involves MYC/ VEGF/miR-106b, miR-106a, miR-93, miR-34a, miR-20a, miR-17, miR16-1 and miR-15a, and controls several aspects of angiogenesis (Fig. 2H). All of these eight miRNAs are under the control of MYC and target VEGF [57,148–151]. Depending on the context, this loop can act as coherent or incoherent feed-forward loop. 6. MYC and miRNA networks in EMT and metastasis During cancer progression, metastasis represents the last and death causing step. The molecular mechanisms underlying the invasionmetastasis cascade are not fully understood. Although the precise role of MYC in this process is still elusive, multiple studies have shown that MYC controls and supports this complex multistep process at different stages [152]. Furthermore, elevated expression and amplification of MYC correlates with metastasis and poor survival in several tumor types [153,154]. In order to form metastases at distant sites tumor cells have to acquire migratory and invasive capacities, invade into the stroma surrounding the primary tumor, overcome the basement membrane, intravasate into blood vessels and disseminate via the circulation [155,156]. This is often achieved by undergoing an EMT [Fig. 2I], which is followed by an MET when cells have reached a metastatic niche in the target organ and resume proliferation and undergo partial differentiation to form micro- and macro-metastases [157,158]. The cellular EMT program has presumably evolved to facilitate multiple developmental processes, such as gastrulation and neural crest formation, that involve the migration of epithelial cells, which transiently acquire a mesenchymal state [159]. It was shown that MYC induces EMT through the

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GSK3β/SNAIL axis or via repression of the WNT inhibitors DKK1 and SFRP1 in mammary epithelial cells [160,161]. Furthermore, induction of SNAIL by TGF-β was abrogated by knock-down of MYC, indicating that MYC is central for this process [162]. Recent research on MYCregulated miRNA networks has shed light onto the regulation of the metastatic process [163]. It was shown that miR-9 is directly induced by MYC and N-MYC to promote metastasis in breast cancer [74]. miR9 achieved this by repressing the cell adhesion protein E-cadherin and thereby promoting migration and invasiveness of breast cancer cells. The repression of E-cadherin also stimulates Wnt signaling, which induces the expression of the vascular endothelial growth factor (VEGF) and tumor angiogenesis. Furthermore, the miR-9 levels correlate with N-MYC amplification and metastatic spread in patients with neuroblastoma [76]. In addition, miR-9 can also promote metastasis in an Ecadherin-independent manner via the repression of leukemia inhibitory factor receptor (LIFR). LIFR can inhibit metastasis through the Hippo– YAP pathway and in human breast cancer the loss of LIFR is associated with poor prognosis [164]. Interestingly, miR-9, which is secreted by tumors, promotes endothelial cell migration and angiogenesis by activating the JAK–STAT pathway [165]. Therefore, MYC-induced miR-9 may also act in a paracrine fashion. Surprisingly, recent evidence suggests a role of MYC in suppressing metastasis formation and invasion, which may also involve the induction of the miR-17–92 family members miR-17/20 [166,167]. It is therefore likely that the observed functions of MYC during the metastatic process are dependent on the tumor stage and type of tissue under investigation. We could recently show that MYC associates with and induces the Krüppel-type transcription factor ZNF281 [168,169]. Interestingly, ZNF281 itself represents a miR-34 target [169] and MYC-mediated repression of miR-34 may therefore mediate the induction of ZNF281 by MYC (Fig. 2I). Furthermore, ectopic expression of ZNF281 induced EMT, migration and invasion was associated with increased stemness and enhanced Wnt pathway activity. ZNF281-induced EMT was dependent on the induction of SNAIL and also SNAIL-induced EMT required ZNF281 expression. miR-34 may represent an important link between these two EMT-TFs, which allows a feed-forward regulation. During tumor progression this regulation may be shifted towards the mesenchymal state by down-regulation of miR-34, e.g. by p53 mutation, miR-34a and miR-34b/c promoter methylation or by activation of MYC [85,111,169]. Previously, we could show that the MYC-induced transcription factor AP4 induces EMT and is required for lung-metastasis formation of xenotransplanted CRC cell lines [170]. Furthermore, elevated AP4 expression is associated with distant metastasis and poor survival of colorectal cancer patients. Therefore, AP4 represents a new EMT-TF. In addition, AP4 directly down-regulates p16 and p21 to suppress senescence and mediate transformation [171]. Moreover, we recently found that AP4 forms a negative feedback loop with the p53-induced and MYC-repressed miRNA miR-15a/16-1 [Fig. 2I] [96]. This loop is similar to the SNAIL/miR-34 feedback-regulation. Similar to ectopic miR-34, miR-15a/16-1 also induces MET, albeit in an AP4-repression dependent manner [19,96]. Furthermore, miR-15a/16-1 inhibited lung-metastasis formation of xenotransplanted CRC cell lines and its expression in CRC patient samples is inversely correlated with AP4 and poor survival of patients, which is generally associated with metastasis. Since, both MYC and AP4 directly repress miR-15a/16-1, the down-regulation of these anti-metastatic miRNAs presumably represents an important step towards metastatic disease. MYC and SIRT1 form a positive feedback loop, which connects MYC to the metabolic state of cells, since SIRT1 is a NAD-dependent enzyme with deacetylating activity [172]. Interestingly, the MYC-repressed miR34a negatively regulates SIRT1 [173]. Besides, its impact on the regulation of chromatin modifications and states, this circuitry may also influence EMT-related processes, since SIRT1 interacts with the EMT-TF ZEB1 to enhance prostate cancer invasion and metastasis [174]. In addition, TGF-β driven EMT of mammary epithelial cells involves up-

regulation of SIRT1, which may silence the miR-200 promoter through histone deacetylation [175]. Notably, SIRT1 and miR-200 seem to form a negative feedback loop, as miR-200 targets the 3′-UTR of SIRT1 [176]. However, the role of SIRT1 in EMT is still controversial, since a gain of mesenchymal markers by inhibition of SIRT1 was observed, in epithelial mammary tumor cells. Furthermore, SIRT1 knock-down led to significantly reduced survival in a xenograft mouse model of metastasis formation [175]. 7. Therapeutic targeting of c-MYC-regulated miRNAs Targeting of MYC-induced miRNAs or restoring the expression of MYC-repressed miRNAs seems to be an obvious strategy to combat MYC-driven cancers [178]. Several MYC-driven mouse models are suitable for pre-clinical studies of targeting MYC-regulated miRNAs (reviewed in [179]). The biggest obstacle when using miRNA mimetics, is their delivery to the tissue of interest. Enhanced stability and increased uptake of miRNAs into tumor cells are critical points that need further improvement. A detailed discussion of the recent developments in this area can be found in [180,181]. In order to use MYC as a therapeutic target several strategies are conceivable. For example, silencing of MYC by RNA interference, modulation of MYC protein stability, disruption of MYC/MAX interaction by small molecules and drug-mediated inhibition of enzymes, which are downstream targets of MYC (discussed in detail in [177,182]). For the last approach miRNAs seem to be good tools. One possible strategy is to reintroduce MYC down-regulated miRNAs, which may be regarded as tumor suppressive. This was successful in the case of miR-26a, miR-34a and let-7, since it caused regression of already established tumors or reduction of tumor formation [102,122,183,184]. The alternative strategy is to down-regulate MYC-induced, oncogenic miRNAs by using e.g. antagomirs. This was tested in neuroblastoma, which frequently harbor N-MYC amplifications. Silencing of miR-17–92 members induced cell cycle arrest and apopotosis, and caused inhibition of tumor growth of xenografted neuroblastoma cells with N-MYC amplification [73,147]. Currently, several labs are developing small-molecule drugs that target specific miRNAs (SMIRs) and modulate their activities. SMIRs have several disadvantages as they display poor specificity, undesirable miRNA-independent effects and require a complicated design when compared with oligonucleotide-based therapeutics. The most beneficial concept seems to be the combinatorial usage of different miRNA agents with conventional chemotherapy [122]. This led to positive results in a clinical Phase II study and further recruitment of participants [124]. Furthermore, multiplex strategies may be advantageous. The combined use of multiple miRNAs, which inhibit the same pathway, may allow to achieve a superior effect in inducing e.g. apoptosis. One example, would be the combined delivery of miR-15a and miR-34a mimetics, which may reduce the expression of anti-apoptotic proteins such as MCL1 (by miR-15a), SIRT1 (by miR-34a) and BCL 2 (by miR15a and miR-34a). Furthermore, the combined restoration of miR-15a/ 16-1 and miR-34a could induce a MET and thereby prevent metastasis. However, it should be mentioned that the ectopic expression of the tumor-suppressive miR-200 increased the number of metastasis in a xenograft mouse model, since the formation of micro-metastasis often involves an MET process [184]. Obviously, this could be a hurdle for the use of MET-inducing miRNAs for tumor therapeutic purposes. 8. Conclusions and outlook Here, we have summarized the current state of the literature describing the influence of MYC/miRNA circuits on diverse oncogenic processes, such as cell cycle regulation, apoptosis, metabolism, angiogenesis, EMT and metastasis. miRNAs add a functionally relevant layer of complexity to the MYC network of cellular regulation. In the future, more publicly available datasets of miRNA expression in cancer patient cohorts which allow to determine correlations with mutations,

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