MicroRNAs -- the micro steering wheel of tumour metastases

Abstract

Recently, microRNAs (miRNAs) have been discovered to have a role in metastasis. Here we describe how miRNAs are involved in advanced stages of tumour progression, stressing their roles as metastasis activators or suppressors, and discuss their possible use in the clinic as predictive markers and as therapeutic strategies for patients with metastases. Furthermore, we develop the concept that the same miRNAs could be involved both in the cancer stem cell phenotype and in the ability of specific cancer cells to produce metastases, thus representing a mechanistic link between the initial and the final steps of tumorigenesis.

108. Weber, C., Zernecke, A. & Libby, P. The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nature Rev. Immunol. 8, 802815 (2008).

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Acknowledgements

The authors thank H. Peinado for his assistance with FIG. 1. B.P. received research support from a Kay Kendall Leukaemia Fund Travelling Fellowship and a Fulbright Scholarship in Cancer Research. D.L. receives grants from the National Cancer Institute (RO1CA098234); Susan G. Komen for the Cure; National Foundation for Cancer Research; Emerald Foundation; Malcolm Hewitt Wiener Foundation; Nancy C. and Daniel P. Paduano Foundation; American Hellenic Educational Progressive Association; Charles and Meryl Witmer Family Foundation; Butler Foundation and the Childrens Cancer and Blood Foundation.

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o p i n i o n

MicroRNAs the micro steering wheel of tumour metastases

Milena S. Nicoloso, Riccardo Spizzo, Masayoshi Shimizu, Simona Rossi and George A. Calin

Abstract | recently, microrNAs (mirNAs) have been discovered to have a role in metastasis. Here we describe how mirNAs are involved in advanced stages of tumour progression, stressing their roles as metastasis activators or suppressors, and discuss their possible use in the clinic as predictive markers and as therapeutic strategies for patients with metastases. Furthermore, we develop the concept that the same mirNAs could be involved both in the cancer stem cell phenotype and in the ability of specific cancer cells to produce metastases, thus representing a mechanistic link between the initial and the final steps of tumorigenesis.

The metastatic programme encompasses multiple sequential steps: cell motility, tissue invasion, intravasation, dissemination through the blood or lymph, extravasation and finally proliferation at a new site. However, the molecular pathways underlying each step are still obscure1,2. With the latest deciphering of roles for micrornAs (mirnAs) in the metastatic programme there are new hopes that this scenario will rapidly change. Since mirnAs were connected to cancer pathogenesis3, accumulating data have pointed to a central regulatory role for mirnAs and other noncoding rnAs (ncrnAs; rnAs that do not have an open reading frame and do not encode protein) in

the initiation and progression of most cancers analysed thus far. recent studies show that mirnAs may be members of the still elusive class of cancerpredisposing genes and that other types of ncrnAs also participate in the genetic puzzle giving rise to the malignant phenotype (for reviews see ReFs 47).

miRnAs and cancer

mirnAs were originally identified as small ncrnAs that control the timing of larval development in Caenorhabditis elegans8.

mirnAs are short singlestranded rnA molecules, which serve as master regulators of gene expression (BOX 1). Their

abnormal levels in tumours have important

nATurE rEvIEWS | CanCer voluME 9 | APrIl 2009 | 293

of mirnA genes in tumours and identified an mirnA hypermethylation profile characteristic of human metastasis20. These are only initial steps toward the understanding of the causes of mirnA deregulation during metastasis; other mechanisms, such as deletion or amplification due to the location of an mirnA in a cancerassociated genomic region and abnormal processing of precursor mirnAs to mature molecules, can also be postulated and will probably be identified soon. This Perspective discusses the many possible roles of mirnAs in metastasis, considering those that have confirmed roles and those that have activities that correlate with processes that contribute to metastasis.

miRnAs as metastasis activators

In the past 18 months, several mirnAs have been shown to activate metastasis by acting on multiple signalling pathways and targeting various proteins that are major players in this process.

The TWIST1miR-10bHOXD10 linkto metastasis. The advances in decodingthe roles of mirnAs in tumour metastasis started with the study of Ma and colleagues from Weinbergs group, which revealed that upregulation of miR-10b, although it did not affect cell proliferation, promoted in vitro and in vivo migration, invasion and meta stasis of otherwise noninvasive breast cancer cells18

(FIG. 1; TABLe 1). miR-10b was originally found

to be downregulated in a group of breast cancer specimens (unselected for metastasis presence) compared with normal breast tissue21

but, subsequently, Ma and colleagues showed that this mirnA is actually overexpressedin about 50% of metastatic breast cancers compared with metastasisfree tumours or normal breast tissues18. The finding that only half of metastasispositive tumours analysed overexpressed miR-10b is not in conflict with the previous finding that generally miR-10b is expressed at low levels in breast cancer. In fact, mirnA expression analysis was done on the bulk of the heterogeneous primary tumour, where the invasive and metastatic cells may be only a small fraction of the total. Additionally, confirming its relevance in the metastatic programme, miR-10b is transcriptionally activated by the pro metastatic transcription factor TWIST1

(ReF. 18) and is essential for TWIST1induced epithelialmesenchymal transition (EMT) that promotes cell motilityand invasiveness22. Similarly to TWIST1 overexpression, miR-10b expression is not an early event during tumour progression, and

pathogenetic consequences: mirnAs that are overexpressed in tumours contribute to oncogenesis by downregulating tumour suppressors (for example, the miR-17miR-92 cluster in lymphomas reduces tumorigenic levels of the transcription factor, http://www.uniprot.org/uniprot/Q01094

Web End =E2F1 and http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000077

Web End =miR-21 represses the tumour suppressor http://www.uniprot.org/uniprot/P60484

Web End =PTEn in hepatocellular carcinomas (HCCs)), whereas mirnAs lost by tumours generally participate in oncogene overexpression (for example, the let-7 family represses KrAS, nrAS, high mobility group A2 (http://www.uniprot.org/uniprot/P52926

Web End =HMGA2 ) and http://www.uniprot.org/uniprot/P01106

Web End =MYC in lung cancers, and the http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000069

Web End =miR-15a http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000070

Web End =miR-16-1 cluster downregulates http://www.uniprot.org/uniprot/P10415

Web End =BCl2 in chronic lymphocytic leukaemias and http://www.uniprot.org/uniprot/P24385

Web End =cyclin D1 in prostate cancer and mantle cell lymphoma)7.

notably, roles of mirnA in cancer are tissue and tumour specific: for example, in solid cancers of epithelial origin and in leukaemias and lymphomas, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000681

Web End =miR-155 is highly expressed and acts as an oncogene, whereas in endocrine tumours it is highly downregulated and possibly has suppressive functions3.

Mechanisms of miRnA deregulation

The widespread differential expressionof mirnA genes between malignant and normal cells is a complex phenomenon, in that it may require mirnA transcriptional control by oncogenes, tumour suppressor genes, epigenetic mechanisms9 and genomic abnormalities (mirnAs are frequently located at cancerassociated genomic

regions)10, all acting in concert for abnormal expression levels of mirnAs. In a representative example of this sophistication, the tumour suppressor http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000268

Web End =miR-34a is positively controlled by http://www.uniprot.org/uniprot/P04637

Web End =p53 (ReF. 11), is kept in check by MYC12, is silenced by aberrant CpG methylation13 and is located at 1p36 (ReF. 14), a chromosomal region that is frequently lost in neuroblastomas. Accordingly, numerous genetic studies allowed the identificationof mirnA abnormalities in human cancer by dissecting their transcriptional regulators12,14,15. Cancerassociated mirnAshave been located downstream of major oncogenes and tumour suppressors thatact as transcription factors: for example, p53 promotes the transcription of all the members of the miR-34 family12,14,15 and

MYC can both positively and negatively regulate transcription of different mirnAs (for example, the miR-17miR-92 cluster16

and let-7 family12, respectively) to promote tumorigenesis17. Similarly, transcription factors governing metastatic gene expression programmes have also been found to control mirnAs involved in metastasis, such as the pleiotropic transcription factor http://www.uniprot.org/uniprot/Q15672

Web End =TWIST1 (ReF. 18), which transactivates the prometastatic http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000267

Web End =miR-10b , and http://www.uniprot.org/uniprot/Q13485

Web End =SMAD4 , which activates miR-155 transcription downstream of transforming growth factor (TGF) signalling19. Additionally, a study by lujambio and colleagues from the Esteller group examined epigenetic changes

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its expression at early breast cancer stages does not correlate with distant recurrencefree survival23. The prometastatic effectsof miR-10b are exerted through translational repression of homeobox protein D10 (http://www.uniprot.org/uniprot/P28358

Web End =HoXD10 ), a transcription factor already known for its roles in cell motility24. In particular, among HoXD10restrained genes, the rho GTPase http://www.uniprot.org/uniprot/P08134

Web End =rHoC was held responsible for the miR-10binduced migratory and invasive phenotype. In short, during tumour progression, TWIST1 induces miR-10b, which in turn indirectly increases rHoC levels by direct downregulation of its transcriptional repressor HoXD10.

miR-373 at the crossroads between cancer progression and dissemination. In a collaborative effort between Huang and Agamis groups, through a forward genetic screen using an mirnA expression library, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000781

Web End =miR-373 and http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0003158

Web End =miR-520c were also identified as metastasispromoting genes25 (FIG. 1; TABLe 1).

miR-373 was previously identified by Agamis group as a potential oncogene in testicular germ cell tumours owing to its ability to overcome HrASinduced p53 responses and to participate in cell transformation26.

In MCF7 breast cancer cells, overexpression of miR-373 or miR-520c promoted an in vitro migratory and invasive phenotype. Furthermore, nude mice transplanted with these cells developed multiorgan metastatic nodules, whereas control cells did not. miR-373 and miR-520c were found to share a similar seed sequence (BOX 1) and

to have a common direct target. Their target is http://www.uniprot.org/uniprot/P16070

Web End =CD44 , which encodes a cell surface receptor for hyaluronan, is lost in breast cancer with high metastatic potential and acts as a metastatic suppressor in prostate and colon cancer. Accordingly, forced expression of CD44 curtailed migratory advantages dueto miR-373 or miR-520c overexpression. The clinical significance of the studies performed in breast cancer cell lines was strengthened by the observation that miR-373 was upregulated in primary breast carcinomas, especially in those with lymph node metastasis, and displayed an inverse correlation with CD44 expression26.

miR-21 as a master regulator of the meta-static programme. Alterations in cytoskeletal protein expression levels by mirnAs can affect cell morphology and motility, as has been shown for miR-21 (ReF. 27) (FIG. 1;

TABLe 1). Besides being the most commonly overexpressed oncogenic mirnA among solid tumours with different origin28,

miR-21 is involved in several aspects of the

metastatic programme. In a breast cancer model, miR-21 was first shown to downregulate the tumour suppressor tropo myosin 1 (http://www.uniprot.org/uniprot/P09493

Web End =TPM1 , an actinbinding protein that is able to inhibit anchorageindependent growth)29. owing to the relevance of TPM1 in cytoskeletal organization, it was further assessed whether, through this or other targets, miR-21 had any relevance in cell motility. In fact, miR-21 was found to stimulate cell invasion and metastasis in different tumour models (breast cancer, colon cancer and gliomas) using similar in vitro and in vivo assays27,30,31. This ability was partially explained by its direct repression of maspin (http://www.uniprot.org/uniprot/P36952

Web End =SErPInB5 )27 and http://www.uniprot.org/uniprot/Q53EL6

Web End =PDCD4 (programmed cell death 4)27,30, both regulators of the metastasispromoting factor urokinase

plasminogen activator surface receptor (http://www.uniprot.org/uniprot/Q03405

Web End =uPAr ). Clinical relevance of miR-21 to these biological targets was given by the finding that miR-21 expression levels are inversely correlated with those of PDCD4 or maspin in human breast cancer specimens27. Additionally, miR-21 was shown to potentiate the metastatic abilities of tumour cells through an overall increase of metalloproteinase (MMP) activity. miR-21 exerts this effect by direct repression of http://www.uniprot.org/uniprot/O95980

Web End =rECK and http://www.uniprot.org/uniprot/P35625

Web End =TIMP3 (tissue inhibitor of metalloproteinases 3)31, two MMP inhibitors and the phosphatase PTEn32. In fact, miR-21induced loss of PTEn increases focal adhesion kinase 1 (http://www.uniprot.org/uniprot/Q05397

Web End =FAK1 ) phosphorylation, which is responsible for increased http://www.uniprot.org/uniprot/P08253

Web End =MMP2 and http://www.uniprot.org/uniprot/P14780

Web End =MMP9 expression32.

a

c d

Migration, invasion and adhesion

b EMT and stem cell-like properties

miR-335

SOX4

TWIST1

miR-10b

miR-200 miR-205

ZEB1 ZEB2

E-cadherin

HOXD10

RHOC

ROCK1

miR-126

CRK

miR-101

miR-21

TPM1

TGF2

let-7

EZH2

HMGA2 HRAS

miR-373 miR-520

CD44

miR-146

miR-155

RHOA

ECM modification

Proliferation (at distant sites and primary tumour)

Micrometastasis Macrometastasis

RECK TIMP3

PTEN MMP2 MMP9

miR-221 miR-222

miR-18

CTGF

miR-21

let-7

HMGA2 HRAS

p27

Laminin-1 Collagens TNC

miR-29c

miR-126

Unknown

miR-335

Figure 1 | microrna (mirna)-regulated pathways in tumour metastasis. The figure shows the networks of mirNAs and protein-coding genes identified in the different steps of the metastatic program. increased cell motility in a tumour can be achieved in several ways: by loss of miR-335 and miR-126, which regulate at the transcriptional level (inhibiting sOX4 transcription factor) and at the effector level (inhibiting crK adaptor protein involved in actin remodelling) respectively different migratory steps; by upregulation of miR-373 and miR-520, which through suppression of cD44 favour loosening of cellextracellular matrix (ecM) connections (a); by loss of miR-146, which though rOcK1 suppression sustains nuclear factor-B (NF-B) pro-migratory signalling (a); and by transactivation through TWisT1 of miR-10b, which through indirect inhibition of rHOc can contribute to the epithelialmesenchymal transition (eMT) (a). in fact, mirNAs involved in eMT (miR-200 family, miR-205 and miR-155 at different levels) and in stemness maintenance (let-7 through HrAs and high mobility group A2 (HMGA2) suppression and miR-101 through eZH2 suppression) also contribute to a meta-static phenotype (b). High miR-21 levels, by indirectly upregulating matrix metalloproteinase (MMP) expression, together with low miR-29c and miR-335 levels, which determine an increase of pro- metastatic matrix proteins (laminin-1, collagens and tenascin (TNc), respectively) contribute to ecM remodelling (c). Loss of let-7 and miR-126 and increase of miR-221 and miR-222 contribute to cell proliferation (d) and/or cell motility at primary and distant sites. mirNAs commonly found upregulated are shown in red boxes and those downregulated during malignant progression are in grey boxes. Protein-coding genes that positively regulate eMT are in green boxes.

Nature Reviews | Cancer

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Table 1 | miRnAs involved in metastasis: functions and targets

mirna functions in metastasis targets* deregulation in tumours Molecular regulation refs

Activators of metastasismiR-373 Modulation of cellecM interactions and signalling

cD44 Overexpressed in seminoma and non-seminoma testicular tumors and in breast cancer, where expression correlates with invasive lymph node-positive tumors

ND 25,26

miR-520c Modulation of cellecM interactions and signalling

cD44 Overexpressed in breast cancer metastases compared with primary tumours

ND 26

miR-155 Tight junction dissolution, cell polarity and eMT regulation

rHOA Overexpressed in several solid tumours and leukaemias and lymphomas

TGF and sMAD4 positively regulate

19,28

miR-21 regulation of cell contractility by TPM1 and of ecM composition indirectly by PTeN (increase of MMPs) and directly by suppression of MMP inhibitors

serPiNB5 PDcD4 PTeN TPM1 recK TiMP3

27,28, 31,83, 99,100

Overexpressed in glioblastoma, breast, lung, prostate, colon, stomach, oesophageal and cervical carcinoma, uterine leiomyosarcoma and DLBcL; overexpression in pancreatic cancer correlates with liver metastasis, and in breast cancer with advanced clinical stage, lymph node metastasis and poor outcome

Transcriptionally induced by iL-6 through sTAT3

miR-10b indirect control of rHOc levels, favouring cell motility

HOXD10 Overexpressed in metastatic breast cancer Transcriptionally induced by TWisT1

18

Suppressors of metastasis miR-9family

ND ND cancer-specific cpG island hypermethylation associated with lymph node metastasis; reduced expression associated with vascular invasion and lymph node metastasis in breast cancer

Methylation 20,21

miR-340, miR-34b and miR34c

ND MYc cDK6 e2F3

cancer-specific cpG island hypermethylation associated with lymph node metastasis

Methylation transcriptionally induced by p53 and repressed by MYc; LOH

1113, 20

miR-148a ND TGiF2 cancer-specific cpG island hypermethylation associated with lymph node metastasis

Methylation 20

miR-126 in tumour cell, proliferationat distant sites, and actin remodeling and cell adhesion by crK repression; pro-angiogenic functions in endothelial cells

crK1

PiK3r2 sPreD1 vcAM1

Downregulated and associated with a shorter median time to metastatic relapse in breast cancer; downregulated in lung cancer

Methylation 33,37, 38,95, 101103

miR-200 family

Their repression during eMT sustains the process by releasing ZeB1, ZeB2 and TGF2

ZeB1 ZeB2 TGF2

Downregulated in metaplastic and basal breast cancer

Transcriptionally repressed by ZeB1 and ZeB2

76

miR-206 ND ND Downregulated and associated with a shorter median time to metastatic relapse in breast cancer

ND 33

miR-335 remodelling of ecM and control of migratory gene programmes by sOX4 regulation

sOX4 TNc

Downregulated and associated with a shorter median time to metastatic relapse in breast cancer

ND 34

miR-146a and miR146b

control of MAT by rOcK1 repression and of NF-B pro-migratory functions by suppression of regulators

rOcK1 irAK1 TrAF6

Downregulated in hormone-refractory prostate cancer and papillary thyroid carcinomas

Transcriptionally induced by NF-B and BrMs1

4446,

104,

105

miR-29c remodelling of ecM collagens

Laminin-1

Downregulated in nasopharyngeal carcinomas and cLL

Transcriptionally repressed by MYc

40,106

let-7 family

inhibition of anchorage-independent growth, of self-renewal and multipotent differentiation potential

HMGA2 HrAs, KrAs and NrAs

Downregulated in lung cancers, in which its expression correlates with poor survival and in breast cancer-derived tumour-initiating cells

ND 59,107,

108

miR-101 control of cell proliferation and motility epigenetic pathways

eZH2 Downregulated in prostate, breast, ovarian, lung and colon cancer and further reduced in metastasis

LOH 61

*Metastatic targets include both experimentally validated targets by in vitro assays (western blot and luciferase assays) and/or targets predicted by in silico programs and found to be inversely correlated with microrNA (mirNA) expresson levels in cell lines or patient samples. specific to colon, lung, breast, head and neck carcinomas, and melanomas. BrMs1, breast cancer metastasis-suppressor 1; cLL, chronic lymphocytic leukaemia; DLBcL, diffuse large B cell lymphoma; ecM, extracellular matrix; eMT, epithelialmesenchymal transition; HOXD10, homeobox D10; iL-6, interleukin 6; irAK1, iL-1 receptor-associated kinase 1; LOH, loss of heterozygosity; MAT, mesenchymalamoeboid transition; ND, not determined; NF-B, nuclear factor-B; PiK3r2, phosphoinositide 3-kinase regulatory, subunit 2; PDcD4, programmed cell death 4; rOcK1, rho-associated, coiled-coil containing protein kinase 1; sTAT3, signal transducer and activator of transcription; TGF2, transforming growth factor 2; TGiF2, TGF-induced factor homeobox 2; TiMP3, tissue inhibitor of metallopeptidase 3; TNc ,tenascin c; TPM1, tropomyosin 1; TrAF6, TNF receptor-associated factor 6; vcAM1, vascular cell adhesion molecule 1.

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miRnAs as metastasis suppressors

Additional evidence supports the idea that mirnAs can have opposite roles and act as inhibitors of various steps involved in metastasis.

Multifaceted miRNAs in metastasis: miR-335 and miR-126. Tavazoie and coworkers from Massagus group were the first to pinpoint the existence of metastasissuppressor mirnAs that is, mirnAsthat are downregulated during metastasis by analysing breast cancer cells as they acquired metastatic potential33. Among these, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000816

Web End =miR-335 , http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000471

Web End =miR-126 and http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000490

Web End =miR-206 (FIG. 1;

TABLe 1) were consistently downregulated

in metastatic foci and their restoration in breast cancer cells decreased the number of metastases in inoculated mice. Suitably, the authors found that low miR-335 or miR-126 expression in human primary tumours was significantly associated with poor metastasisfree survival, providing clinical relevance to their findings. The capacity of miR-335 to suppress the metastatic ability of breast cancer cells was in part explained by its power to directly repress the expression of http://www.uniprot.org/uniprot/Q06945

Web End =SoX4 . SoX4 is a transcription factor with a role in cell progenitor development and migration34

and has recently been demonstrated to potentiate metastasis in HCC by transcriptionally controlling genes such as http://www.uniprot.org/uniprot/O14786

Web End =neuro http://www.uniprot.org/uniprot/O14786

Web End =pilin 1 and semaphorin 3C (http://www.uniprot.org/uniprot/Q99985

Web End =SEMA3C )35.

Concomitantly, miR-335 was also shown to repress the expression of the glycoprotein tenascin C (http://www.uniprot.org/uniprot/P24821

Web End =TnC ), a protein that reduces cellextracellular matrix (ECM) interactions, that is highly expressed in the microenvironment of most solid tumours and that participates in multiple steps of malignant transformation, including metastasis36.

Hence, loss of miR-335 expression endows tumour cells with a metastatic advantage, upregulating both a transcription factor that activates metastatic genes and an ECM component that triggers tumour cell motility.

The progression toward metastasis formation also requires proliferation of tumour cells at distant sites. Consistently, Massagus group identified loss of a functionally distinct mirnA in human metastatic tumours: miR-126. This gene does not affect cell motility in vitro but instead specifically affects in vitro cell proliferation, in vivo primary tumour volume and colonization of distant sites, probably owing to inhibition of cell proliferation33. However, in an in vitro model of nonsmallcell lung carcinoma, miR-126 was found to inhibit cell adhesion, migration and invasion partially through

suppression of http://www.uniprot.org/uniprot/P46108

Web End =CrK 37, an adaptor signalling protein that participates in actin remodelling, focal adhesion formation and cell migration38.

Taken together, these two papers substantiate the supposition that mirnA functions can be context dependent: miR-126 in lung cancer models acts as an inhibitor of motility only, without any effects on proliferation, whereas in breast cancer models miR-126 is actually an inhibitor of cell proliferation. Still, it must be mentioned that in the lung cancer study of miR-126 no in vivo assays were performed, so it cannot be excluded that in nonsmallcell lung carcinoma miR-126 might exert noncellautonomous effects on tumour volume as well.

miR-29c: a modulator of tumour microenvironment. As exemplified by the finding that miR-335 regulates TnC33, mirnAs may contribute to the metastatic programme by modulating the structure of local tumour microenvironment, which is an important variable influencing the ability of cancer cells to properly move and/or survive (for more information on the role of the microenvironment in metastasis see ReF. 39

in this issue). The MYCrepressed http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000735

Web End =miR-29c 12

(FIG. 1; TABLe 1) is a further example of

mirnA abnormalities in tumours modifying the composition of ECM. In fact, miR-29c, significantly reduced in the highly invasive and metastatic nasopharyngeal carcinoma40,

reduces the mrnA levels and the expression

of a set of genes encoding for ECM proteins, such as collagens (up to six different collagen family members) or laminin1 G1 (ReF. 40).

Interestingly, these miR-29c target genes belong to a gene expression signature that predicts tumour metastatic probability41.

Although direct evidence of miR-29c metastasis suppression has not been presented yet, its strong association with high tumour invasiveness and its subset of targets cardinal for tumour milieu and cell motility40 indicate that miR-29c is probably a metastasis suppressor mirnA.

miR-146: an emerging motility knob. Cancer cells have the potential to adopt different types of motility to successfully metastasize to distant organs. The mesenchymal amoeboid transition42 allows tumour cells to engage in the highspeed propulsive migratory mechanism termed amoeboid motility and to successfully metastasize. During amoeboid motility cells become highly deformable and circumnavigate rather than degrade the ECM barriers (as occurs instead in proteasedependent mesenchymal motility)42. http://www.uniprot.org/uniprot/P61586

Web End =rHoA signalling through kinases http://www.uniprot.org/uniprot/Q13464

Web End =roCK1 (rhoassociated, coiledcoil containing protein kinase 1) and http://www.uniprot.org/uniprot/P70336

Web End =roCK2 is central to producing the actomyosin contractile forces required for the mesenchymal amoeboid transition43.

Interestingly, work on prostate cancer metastasis formation identified an mirnA, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000477

Web End =miR-146a , that is able to affect

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actin remodelling by rho pathways44 (FIG 1;

TABLe 1). miR-146a is commonly lost in

metastatic prostate cancer, in which the pattern of mirnA expression has been related to the extent of tumour cell differentiation. miR-146a was shown, in PC3 androgenindependent prostate cancer cells, to directly decrease the expression of the rhoactivated protein kinase roCK1. roCK1, besides being central to amoeboid motility, is also involved in in vivo hyaluronanmediated hormone refractory prostate cancer transformation and metastasis44. Accordingly, miR-146a overexpression in PC3 cells markedly reduced cell proliferation, invasion and metastasis to human bone marrow endothelial cell monolayers44, whereas itdid not exert any effect on differentiated androgendependent cells that still retain miR-146a expression. In fact, miR-146a loss is a late event in prostate cancer progression and its effect on cell proliferation may not be as relevant for the primary tumour as for the development of distant metastases.

Further confirming the role of miR-146a in metastasis, the miR-146 family was shown, through repression of http://www.uniprot.org/uniprot/P51617

Web End =IrAK1 (interleukin 1 receptorassociated kinase 1) and http://www.uniprot.org/uniprot/Q9Y4K3

Web End =TrAF6 (TnF receptorassociated factor 6) expression, to turn off nuclear factorB (nFB) activity and reduce the metastatic potential of MDAMB231 breast cancer cells45. nFB constitutive activation is common to different types of cancers and, besides having a crucial role in promoting malignant proliferation, evasion from apoptosis and angiogenesis, it has been shown to take part in EMT, inducinggenes such as Twist, http://www.uniprot.org/uniprot/P37275

Web End =ZEB1 , http://www.uniprot.org/uniprot/O60315

Web End =ZEB2 , http://www.uniprot.org/uniprot/P08670

Web End =vimen http://www.uniprot.org/uniprot/P08670

Web End =tin , MMPs, http://www.uniprot.org/uniprot/P07858

Web End =cathepsin B , http://www.uniprot.org/uniprot/Q9UBR2

Web End =cathepsin Z and TnC, and consequently bear promigratory and metastatic potential (for a reviewsee ReF. 46). Interestingly, inhibition of roCK1 by miR-146a could also be partof a roundabout mechanism for nFB repression, as it was shown that roCK1 can increase nFB transcriptional activity through http://www.uniprot.org/uniprot/P62745

Web End =rHoB 47. Given the complexity of signals that lead to the aberrant activation of nFB in cancer cells, it is promising that the miR-146 family can shut down nFB signalling pathways and demands further proof in cancer models other than breast cancer.

Epigentic signature for metastatic miRNAs. As mentioned earlier, mirnA expression can be regulated by specific epigenetic modifications. Among the mirnAs that were reactivated after DnA demethylating treatment, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000253

Web End =miR-148a , http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000742

Web End =miR-34b and http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000743

Web End =miR-34c

(TABLe 1) were able to inhibit in vitro cell

motility and in vivo tumour growth and metastasis formation, through downregulation of their oncogenic targets, such as MYC, http://www.uniprot.org/uniprot/O00716

Web End =E2F3 , cyclindependent kinase 6 (http://www.uniprot.org/uniprot/Q00534

Web End =CDK6 ), and http://www.uniprot.org/uniprot/Q9GZN2

Web End =TGIF2 . Most importantly, the silencing of miR-34b, miR-34c, miR-148 and http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000468

Web End =miR-9-3 by CpG island hypermethylation in primary tumours of various origins (such as colon, lung, breast, and head and neck carcinomas, and melanomas), was significantly associated with the presence of lymph node metastases20.

miRnAs, metastasis and cancer stem cells

We envision that the same set of specific mirnAs may be involved both in maintenance of the cancer stem cell (CSC) phenotype and in invasion and metastasis of tumour cells, possibly accounting for the thread of malignancy connecting primary to metastatic tumours.

miRNACSCEMT connection. CSCs seem to account for all steps of tumorigenesis (initiation, progression and dissemination) and their features throughout these processes are supposedly preserved by a common denominator that is still poorly understood48. mirnAdriven pathways are fundamental for cell stemness49. Embryonic stem cells are highly enriched with a specific repertoire of mirnAs50, which on one hand have power over embryonic stem cell gene

regulation and on the other are under the control of selfrenewal and pluripotency transcription factors51. Strikingly, the majority of mirnAs that are important in embryonic stem cells are also involved in cell cycle regulation and oncogenesis, fuelling the hypothesis that mirnA determinants of cell stemness may be recapitulated by expression abnormalities of mirnAs in tumours.

Increasing data suggest that mirnAs might affect and therefore connect stemness and metastasis through regulation of EMT, which is a genetic developmental programme shared by both phenomena. on one hand, the emerging intersection of EMT and cell stemness (for a review on this topic see ReF. 52 in this issue) has become tangible with the work of Mani and colleagues from Weinbergs laboratory depicting how tumour cells undergoing EMT resemble CSCs53. on

the other hand, multiple mirnAs have been reported to be involved in EMT (BOX 2). EMT

(and therefore mirnAs driving EMT) is not only a migratory escape strategy exploited during tumour progression54,55; EMT is also a process crucial for tumour initiation, as shown by its requirement to bypass oncogeneinduced cellular senescence56 and for tumour cells to maintain their selfrenewal capacity at distant sites53. only cells with a high replicative potential and the plasticity to survive at secondary sites, such as stem cells have57 by virtue of their EMT properties, will allow the complete execution of

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the metastatic programme and give riseto macroscopic metastasis. For example,a commonly overexpressed mirnA in tumours, miR-155 (ReF. 28), that is endowed

with tumourinitiating properties58 has been shown to participate in EMT and in tumour progression19, possibly hinting at hidden stem cell skills of tumours.

Linking tumour cells, stem cells and meta-static cells. Yu and colleagues from Songs group advanced a role for let-7 (FIG. 1;

TABLe 1) both in the expansion and in the

motility of CSCs59. let-7 expression was markedly reduced in breast CSCs, also called tumourinitiating cells, and was shown to be responsible for breast CSC properties including in vitro anchorageindependent growth and selfrenewal through HrAS suppression and multipotent differentiation potential through HMGA2 suppression. reduced let-7 in breast CSC also increased in vivo tumorigenic and metastatic capability when the cells were serially transplanted in noDSCID (nonobese diabetic severe combined immunodeficient) mice59. Another stemness mirnA miR-206, which is lost during differentiation of mesenchymal stem cellderived neuronal cells60 was recently inversely associated with breast cancer metastatic relapse and depicted as an active suppressor of cancer cell metastatic potential33. An additional example of the potential dual role of mirnAs in stemness and metastasis is represented by miR-101-1 (ReF. 61). Through

direct inhibition of http://www.uniprot.org/uniprot/Q15910

Web End =EZH2 , an epigenetic regulator of the polycomb group proteins with important functions in embryonic stem cell regulation, miR-101 can control tumour cell proliferation, but also tumour cell invasiveness and metastatic ability. miR-101, which is significantly underexpressed in prostate, breast, ovarian, lung and colon cancers, was further diminished in metastatic prostate cancers compared with localized disease, concomitant with a strong increase in EZH2 expression. Therefore, miR-101 reduction, which impinges on epigenetic pathways common to pluripotent embryonic stem cells, is a molecular lesion associated with tumour progression and metastasis61.

miRnAs as markers of metastases

The identification of markers that can predict the occurrence of metastases is one of the highest priorities for translational cancer research. mirnAs could represent such longawaited markers and the potential for this has been shown for several different cancer types.

miRNA metastasis signatures in breast cancers. In the study by Massagus group discussed above33, the authors identified a sixgene signature that was downregulated by miR-335. Analysis of published breast cancer gene expression datasets showed that this signature was significantly allied with poor metastasisfree survival in a cohort of 368 patients. Furthermore, reduced expressionof miR-335, as well as miR-126, which also correlated with poor metastasisfree survival, could be used as biomarkers in metastasis risk assessment. These results suggest the potential use of this sixgene signature together with miR-335 and miR-126 in prognostic stratification of breast cancer patients.

miRNA metastasis signatures in HCC. By analysing a large cohort of HCC patients, Budhu and colleagues from Wangs group identified a unique 20mirnA metastasis signature that could predict primary HCC tumours with venous metastases from metastasisfree tumours62. Two points of interest should be stressed regarding these results: first, none of these 20 mirnAs were previously identified in breast cancer studies, indicating that tissuespecific mirnA metastasis pathways could exist. Second,the mirnA signature consists of a smaller number of genes than proteincoding gene (PCG) metastasisspecific signatures, which usually comprise hundreds of members (for a review see ReF. 1).

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miRNA metastasis signatures in other cancers. Two studies in other cancer types further support the view that mirnAs might be useful biomarkers of metastasis. By using realtime quantitative reverse transcription PCr profiling, http://microrna.sanger.ac.uk/cgi-bin/sequences/mirna_entry.pl?acc=MI0000285

Web End =miR-205 was shown to have potential as a novel molecular marker for the detection of metastatic head and neck squamous cell carcinoma63. In addition, mir-29b, miR-9 and miR-9* (mirnA* denotes the small rnA processed from the opposite arm of the premirnA hairpin to the mature mirnA) are specifically expressed in brain primary tumours and can be used to differentiate primary from metastatic brain tumours64.

Deciphering cancer of unknown primary site (CUP) with miRNAs. Patients with CuP (35% of all cancers) carry metastases without an established primary tumour; therefore, the therapeutic interventions are limited and survival of such patients is short. Two large studies support the clinical applications of mirnA profiling for CuP patients. lu and colleagues, by analysing more than 350 tumour samples with a beadbased flow cytometric technique, showed that the small set of poorly differentiated tumours with nondiagnostic histological appearance is much better identified by usingthe mirnAbased classifier than the PCG mrnA classifier65. By using a combination of microarray and realtime PCr on 400 paraffinembedded and freshfrozen samples from 22 different tumour tissues and metastases including CuP cases, rosenfeld and colleagues showed that a classifier based on 48 mirnAs reached over 90% classification accuracy for most tissue classes, including 131 metastatic samples66. The implication that profilinga few tens of mirnAs has a much better predictive power for CuP diagnosis than profiling several hundreds of mrnAs for PCGs is encouraging for the clinical applications of these discoveries.

miRnAs and metastatic cancer therapy

The majority of cancer deaths are due to the dissemination of primary tumours and the development of metastases and therefore, along with a clearer understanding of the metastatic programme, novel modalities to identify early metastases and to better treat patients with metastatic disease remain a medical priority. The rationale for using mirnAs as potential therapeutic targets is offered by many studies that have shown that specific mirnA deregulations,

both overexpression and downregulation, in cancer cells have a pathogenic effect67.

Therefore, reducing the expression levels for miR-10b, miR-21, the miR-146 family, miR-155, miR-373 and miR-520c in solid cancers by locked nucleic acid antimirnAs or antagomirs, or reexpressing miR-126, miR-148a, miR-206, miR-335 and the miR-200 family by mimic mirnAs (BOX 3)

could be initially tested in preclinical settings and then, if successful, in Phase I trials for cancer patients with advanced disease in combination with existing regimens. Although promising, the use of mirnAbased therapy in metastatic patients has yet to show significant increases in patient survival and reduced toxic effects.

Future perspectives

It is likely that in the near future the link between ncrnAs and metastases will find further experimental support and clinical relevance. First, in the next few years we should witness more studies linking ncrnAs and metastases in any type of human cancer and the identification of a complex and interrelated network of interactions between known PCGs involved in metastases and mirnAs. other ncrnAs, such as noncoding ultraconserved genes (transcripts that arise from stretches of the genome with 100% conservation between human, rat and mouse genomes)68 or

other yet unknown noncoding transcripts could be identified as new players in metastasis, regulating the expression not only of PCGs but also of other ncrnAs, including mirnAs. Second, it is highly probable that a specific set of mirnAs may be found to have significant biological activities in both CSCs and cells that are initiating metastases. Another point that must be considered is that mirnAs might indirectly affect metastatic spread through effects on angiogenesis (BOX 4). Finally, and most important for the patients, mirnAs and ncrnAs in general should start to be incorporated in new panels of independent predictors for metastases, and specific methods for rnA inhibition or rnA mimicking must be developed as soon as possible, based on these promising discoveries.

Milena S. Nicoloso, Riccardo Spizzo, Masayoshi Shimizu, Simona Rossi and George A. Calin are at the

Experimental Therapeutics Department, MD Anderson Cancer Center, University of Texas,

Houston, Texas 77030, USA. e-mail: mailto:[email protected]

Web End [email protected]

doi:10.1038/nrc2619 Published online 5 March 2009

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Acknowledgements

G.A.C. is supported as a Fellow at The University of Texas M. D. Anderson Research Trust, and as a Fellow of The University of Texas System Regents Research Scholar and by the Ladjevardian Regents Research Scholar Fund. This study was supported in part by an Institutional Research Grant, by a Cancer Center Support Grant (New Faculty Award), by a CTT/3I-TD grant and by a Breast Cancer SPORE Developmental Award to G.A.C.

Two fundamental models of metastasis

The use of primary tumours to predict therapy response is based on one of the two models discussed here, referred to as the linear progression model. In this model, tumour ontogeny proceeds to full malignancy within the primary tumour microenvironment, after which tumour cell dissemination founds a metastasis. Therefore, the primary tumour prescribes the molecular characteristics of DTCs spread throughout the body. In the second model, parallel progression, tumour cells depart the primary lesion before the acquisition of fully malignant phenotypes to undergo somatic progression and metastatic growth at a distant site. The proposition of early dissemination and divergent progression of primary tumours and DTCs towards metastasis questions the role of the primary tumour for therapy prediction. It is important to note that, unfortunately, neither model is supported by direct and incontrovertible evidence and they have been derived indirectly.

The linear progression model. The linear progression model is based on leslie Foulds description of a stepwise progression of morphological abnormalities (reviewed in ReF. 6) accompanying cancer. Accumulation of genetic and epigenetic alterations was subsequently associated with this process7. At its simplest, the model states that cancer cells pass through multiple successive rounds of mutation and selection for competitive fitness8 inthe context of the primary tumour. After a significant number of such rounds the cells may be able to proliferate relatively autonomously at a competitive rate. These tumour cell clones expand and individual cancer cells leave the primary site to seed secondary growths (FIG.1). The final clonal expansion of fully malignant clones is linked to tumour size. For example, mutations ofthe tumour suppressor gene http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157

Web End =TP53 are rare in T1 stage (<2 cm; sizes refer to diameter throughout) breast cancers, and significantly more frequent in T3 stage (>5 cm) tumours. Clonal expansion of TP53 mutated cells therefore often occurs when tumours grow beyond 2 cm (ReF. 9). Such

observations and the wellknown association of tumour size with higher frequency of metastasis, which is the basis of the routinely used TnM classification system (see below), have promoted the concept that only tumour cells that are shed late in primary tumour progression have the possibility of eventually spawning macroscopic

o p i n i o n

Parallel progression of primary tumours and metastases

Christoph A. Klein

Abstract | systemic cancer progression is accounted for in two basic models. The prevailing archetype places the engine of cancer progression within the primary tumour before metastatic dissemination of fully malignant cells. The second posits parallel, independent progression of metastases arising from early disseminated tumour cells. This Perspective draws together data from disease courses, tumour growth rates, autopsy studies, clinical trials and molecular genetic analyses of primary and disseminated tumour cells in support of the parallel progression model. consideration of this model urges review of current diagnostic and therapeutic routines.

Although advances have been made in reducing mortality rates and improving survival, cancer is the leading cause of death among men and women under 85 years of age in the united States1. Most deaths from cancer are due to metastatic disease, and prevention of later arising metastasis has moved to the centre of clinical attention. Prevention of metastasis combines early surgery (or radiotherapy in some cases) with systemic therapy given before or after surgery (respectively neoadjuvant or adjuvant therapy). Systemic therapy mainly targets tumour cells that have detached from the primary lesion to lodge elsewhere, undetectable by clinical imaging and inaccessible to excision.once primary tumours are resected, metachronous metastases must arise from tumour cells that disseminated to ectopic

sites before surgery, called disseminated tumour cells (DTCs). The term DTC is used for any tumour cell that has left the primary lesion and travelled to an ectopic environment. However, it is not predictable a priori which DTCs will grow into overt metastases (the majority will not) and which molecular traits are required for that process; that is, the metastasis founder cells are unknown. In this situation, models of cancer progression become important, as they provide predictions about the target cells of systemic therapies. They generate therapeutic hypotheses, motivate preclinical research and contribute to the designof clinical trials. An example is the current practice of using molecular traits of the primary tumour to predict responses of DTCs to adjuvant therapies that target specific molecular mechanisms25.

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