post-transcriptional regulation in cancer

Review

Post-transcriptional regulation in cancer

Yann Audic a,*, Rebecca S. Hartley b,1

a Laboratoire de Génétique et de développement CNRS-UMR6061, Université de Rennes I, Faculté de Médecine, 2 Avenue du Professeur Léon Bernard,CS 34317, 35043 Rennes Cedex, France

b Cell Biology and Physiology, MSC08-4750 1 University of New Mexico, Albuquerque, NM 87131-0001 USA

Received 16 March 2004; accepted 19 May 2004

Available online 23 June 2004

Abstract

Deregulation of gene expression is a hallmark of the cancer cell. Acquiring a new profile of expressed proteins may enable the cell tore-enter the cell cycle, or give them a growth or motility advantage over “normal cells”. An efficient and rapid way to alter gene expression isvia regulation of mRNAs already transcribed. Modifications of mRNA stability and/or translational efficiency are increasingly reported incancer. mRNA stability and translation are controlled through a complex network of RNA/protein interactions involving recognition ofspecific target mRNAs by RNA-BPs. We review how alterations in regulatory sequences, RNA-BPs, or in upstream signalling pathways affectthe stability and/or translational efficiency of mRNAs encoding proto-oncogenes, cytokines, cell cycle regulators and other regulatory proteinsto promote tumorigenesis and cancer progression.

A more thorough understanding of post-transcriptional mechanisms such as these will enable the design and development of specifictherapies based on modulating the translation or stability of specific mRNAs.© 2004 Elsevier SAS. All rights reserved.

Keywords: Oncogenesis; Translation; Stability; mRNA; RNA binding proteins

1. Introduction

After a gene has been transcribed, many events are re-quired before the protein product is synthesized. These post-transcriptional events consist of mRNA processing, nucleo-cytoplasmic export, mRNA localization, mRNA stabilizationand translational regulation. The more we know about spe-cific post-transcriptional mechanisms, the more obvious itbecomes that defects in them are linked to disease processessuch as cancer.

Oncogenes are defined as genes able to induce specificcharacteristics of cancer cells in normal cells. Oncogenesencode many different effectors such as kinases, receptors,growth factors or transcription factors. In some tumors mR-NAs encoding oncogenes are stabilized, leading to proteinoverproduction and a predisposition towards unrestrainedcell division. This stabilization may be the consequence or

the cause of cellular transformation. In addition, numerousgenes not defined as oncogenes are overexpressed duringcellular transformation due to mRNA stabilization.

For example, the mRNAs of proto-oncogenes, cytokines,and cell cycle regulators are stabilized in cancer cells leadingto increased proliferation. The structure of the mRNA - a 5’untranslated region (5’UTR) with a 5’ cap structure, an openreading frame (ORF), a 3’ untranslated region (3’UTR) and3’ terminal poly (A) tail – dictates how different molecularmechanisms control mRNA stability and translational effi-ciency. Across the animal kingdom, numerous conservedsequence elements are located in the untranslated regions ofmRNAs, suggesting an important role for these elements(Duret et al., 1993).

Control at the 5’ UTR level involves internal ribosomeentry site, upstream open reading frames, 5’ terminal poly-pyrimidine sequences, RNA secondary structure, and themethylation state of the cap structure. In the ORF, a synthe-sized peptide may affect translation efficiency, while thepresence of rare codons will cause the ribosome to pauseduring translation. Sequence elements located in the ORFmay also target the mRNA for rapid degradation as can

* Corresponding author: Tel : (33) 2 2323 4475 ; Fax : (33) 2 2323 4478.E-mail addresses: [email protected] (Y. Audic),

[email protected] (R.S. Hartley).1 Tel: (1) 505-272-4009; Fax: (1) 505-272-9105.

Biology of the Cell 96 (2004) 479–498

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termination of translation (Hosoda et al., 2003). 3’ UTRs arewell known to contain regulatory sequences that bindmRNA-binding proteins (RNA-BPs) to target a specificmRNA for translational activation/repression orstabilization/destabilization.

Another crucial element is the 3’ terminal poly(A) tail. Itis primarily added in the nucleus after transcription by anon-template dependent mechanism. Once in the cytoplasmthe poly(A) tail is the substrate for a dynamic equilibriumbetween poly(A) addition (polyadenylation) and poly(A) re-moval (deadenylation). The adenylation state of the mRNAboth controls and is controlled by translation. An mRNAwith a long poly(A) tail is efficiently translated while anmRNA with a short poly(A) tail is not. Similarly, removal ofthe poly(A) tail is often the first step in the degradation of themRNA body; an mRNA with a long poly(A) tail is morestable than an mRNA with a short or no poly(A) tail. For amore detailed understanding of the mechanisms involved,please refer to the volume 95, Issue 3-4 of Biology of the Cell(Inge-Vechtomov et al., 2003; Kean, 2003; Linder, 2003;Osborne, 2003; Paillard and Osborne, 2003; Prevot et al.,2003; Schaeffer et al., 2003; Touriol et al., 2003). You couldalso refer to Bevilacqua et al., 2003.

mRNA stability and translation are controlled through acomplex network of RNA/protein interactions involving rec-ognition of specific target mRNAs by RNA-BPs. Mutationsin cis–regulatory elements or aberrant expression of RNA-BPs can modify the regulation of a given mRNA as canmodulation of signalling pathways that post-translationallymodify RNA-BPs or other associated proteins (Fig. 1). Thesealterations occur in diverse cancer types and are often corre-lated with advanced stage and grade of tumors, resulting inmisregulation of genes involved in cancer progression.

In this review we will focus on how modification ofmRNA stability and/or translational efficiency may be in-volved in cancer. We will discuss how alterations in regula-tory sequences, RNA-BPs, or in upstream signalling path-ways affect levels of mRNAs encoding proto-oncogenes,cytokines, cell cycle regulators, and other regulatory proteinsto promote tumorigenesis and cancer progression. Sections2 to 4 will describe regulatory elements in the 3′UTR, codingregion, and 5′UTR of genes mis-expressed in cancer. Section5 will review RNA-BPs and Section 6 will review the signal-ling pathways implicated in control of mRNA stability andtranslation.

2. Regulatory elements in the 3’UTRs

Although regulatory elements are found in all parts of themRNA, it is clear that the 3’ untranslated region is a place ofchoice for such elements. From a mechanistic point of view,it is likely that the 3’UTR is not scanned by the ribosomes(Poyry et al., 2004). Therefore, any protein/RNA interactionstaking place in this region persist through translation, en-abling regulation to take place at any time.

2.1. C-fos

C-fos is a transcription factor that heterodimerizes withmembers of the Jun family of transcription factors (Gloverand Harrison, 1995). The resulting AP-1 complexes are in-volved in a vast panel of physiological events, from bonedifferentiation to neuronal plasticity. The function of c-fos isalso critical for malignant tumor development. Saez andcolleagues (Saez et al., 1995) demonstrated that developmentof malignant skin tumors was impaired in c-fos null mice.Conversely, ectopic expression of c-fos in transgenic miceinduces formation of chondrogenic tumors (Wang et al.,1991). C-fos mRNA is very labile and its stability is con-trolled by at least two different mechanisms (Shyu et al.,1991). One pathway is driven by an AU-rich element (ARE)located in the 3’UTR while a coding region determinant(CRD) (Kabnick and Housman, 1988; Shyu et al., 1989)drives the other. Both elements direct the rapid removal of thepoly(A) tail of the mRNA and participate in its rapid turnover(Shyu et al., 1991).

AREs were originally identified as instability determi-nants in mRNAs encoding proteins involved in the inflam-matory response (Caput et al., 1986). These widespread ele-ments are found in the 3’ UTRs of unstable mRNAs andserve as binding sites for a variety of trans-acting factors thatmodulate mRNA half-life (for a database of ARE containingRNAs see Bakheet et al., 2001). The c-fos ARE targets themRNA for rapid deadenylation followed by rapid degrada-tion of the body of the transcript (Wilson and Treisman,1988). Mutational analysis identified two distinct domains inthe c-fos ARE: region I, containing canonical AUUUA re-peats (Class I ARE) able to drive both rapid deadenylationand degradation and region II, AU-rich but without AUUUA

Fig. 1. Mechanisms of post-transcriptional regulation and their alteration incancer.The numbers correspond to different ways in which the post-transcriptionalregulation of an mRNA can be changed.RE Regulatory Element; ORF Open Reading Frame; RNA-BP RNA Bin-ding Protein. A ribosome is shown in grey. P denotes a phosphorylationevent.

480 Y. Audic, R.S. Hartley / Biology of the Cell 96 (2004) 479–498

repeats (Class II ARE) that enhances region I-mediated dead-enylation (Chen and Shyu, 1994). This ARE is clearly criticalfor the control of c-fos expression as its deletion stabilizesc-fos mRNA and converts this gene into a transformingoncogene (Meijlink et al., 1985). Alteration of its 3′UTR alsoallows c-fos to induce bone tumors in mice (Ruther et al.,1987). V-fos, the viral counterpart of c-fos, is the transform-ing gene of the FBJ-murine osteosarcoma retrovirus. Thev-fos gene is missing a part of the 3′UTR that contains theARE and this may account in part for its oncogenic potential.

2.2. C-myc

V-myc is a retroviral-transforming gene. Alteration of itscellular counterpart, c-myc, is associated with numerousforms of cancer. For example, c-myc mRNA or protein isknown to be elevated in 66-80% of colorectal cancers. c-mycmRNA encodes a transcription factor that when dimerizedwith its partner binds to E-box DNA and transactivates alarge collection of genes (Levens, 2002; Menssen and Her-meking, 2002). Alteration of c-myc expression affects cellproliferation, growth, metabolism and differentiation. Thisbroad spectrum of effects is associated with its potent role asan oncogene (Hecht and Aster, 2000; Lewis et al., 2000;Marcu et al., 1992; Spencer and Groudine, 1991). c-mycmRNA is expressed from 4 different promoters and its ex-pression is regulated both at the transcriptional and post-transcriptional levels. C-myc mRNA is extremely labile innormal cells (Dani et al., 1984), with its translation andstability controlled by at least three different mechanisms:AREs in its 3′UTR (Herrick and Ross, 1994, and reviewed inChen and Shyu, 1995), a coding region determinant (Wisdomand Lee, 1991), and a 5’UTR internal ribosomal entry site.(IRES) (Nanbru et al., 1997; Stoneley et al., 1998).

The c-myc mRNA ARE specifies its instability (Jones andCole, 1987). C-myc mRNA is stabilized in both a humanT-cell leukaemia line missing a 61-nt ARE (Aghib et al.,1990) and in a human myeloma with a 3′UTR translocation(Hollis et al., 1988), suggesting that loss of this region stabi-lizes the mRNA in cancer cells. In contrast, deletion of thec-myc 3’UTR by homologous recombination does not affectits mRNA steady state level in murine ES Cells. When micecarrying the same deletion were produced, heterozygotesshowed no alteration in c-myc mRNA stability from theARE-deleted allele and homozygotes were healthy and gaverise to progeny (Langa et al., 2001). A previous study alsoshowed that deletion of the ARE in the c-myc 3’UTR doesnot stabilize the mRNA (Laird-Offringa et al., 1991). Thesecontrasting studies suggest that additional regulation, possi-bly via the coding region determinant or IRES is essential forcorrect mRNA behavior (see section 3.2).

2.3. C-jun

Along with c-fos, c-jun is a major component of the AP-1transcription factor complex that regulates expression ofgenes involved in proliferation, differentiation and apoptosis

(Hartl et al., 2003). C-jun is usually expressed at low levelsuntil its transcriptional activation by various growth factors(Karin et al., 1997). Following its transcription, c-jun mRNAlevels decrease rapidly due to high instability (Almendral etal., 1988; Lau and Nathans, 1987). C-jun overexpression hasbeen reported in Hodgkin’s lymphoma (Mathas et al., 2002)and its overexpression in the MCF-7 breast cancer cell linegives these cells a tumor-like phenotype with increased cel-lular motility, increased expression of the matrix metallo-protease 9 (MMP-9) and increased tumor formation in mice(Smith et al., 1999). C-jun mRNA stability is regulated by a3′UTR ARE.

The c-jun ARE has no canonical AUUUA repeats (classIII) and activates both poly(A) tail removal and mRNA desta-bilization (Chen and Shyu, 1994). The regulation of class IIIAREs has not been well studied but it was recently reportedthat the c-jun ARE binds the regulatory protein EDEN-BP/CUGBP (Paillard et al., 2002). This protein acts as adeadenylation factor and translational repressor (Paillard etal., 2002).

2.4. MMP-13

Malignant tumors spread and invade normal tissue bygiving rise to metastases, which contribute to the lethal out-come of cancer. Enzymes responsible for the reshaping of theextracellular matrix during cell migration, proliferation andmorphogenesis are also involved in metastasis. Among them,the matrix metalloproteases carry out most of the proteolysisof connective tissue. Matrix metalloprotease 13 (MMP-13,also known as collagenase-3) was first identified in breastcarcinoma (Freije et al., 1994) and is expressed in a numberof pathological conditions including ulcers, arthritis andwound repair (Borden et al., 1996; Reboul et al., 1996;Vaalamo et al., 1997). MMP-13 is expressed in a limited setof cancers, but cancers with MMP-13 overexpression areusually both aggressive and invasive (Brinckerhoff et al.,2000). mRNA stability and/or translation of MMP13 is con-trolled by its 3′UTR ( Yu et al., 2003a). Specific mechanismsof 3′UTR regulation and its potential involvement in cancerremain to be determined.

2.5. Cyclooxygenase-2

Arachidonic acid metabolites are very important both innormal and aberrant growth, including carcinogenesis. Cy-clooxygenase 2 (COX-2) is the key enzyme catalysing therate-limiting step in the production of prostaglandins, con-verting arachidonic acid to prostaglandin H2. COX-2 is ei-ther not present or present in very low amounts under basalconditions. Various growth factors, cytokines and tumor pro-moters activate its transcription. This, in turn, results inproduction of prostaglandins associated with inflammationor carcinogenesis.

In colon cancer, COX-2 overexpression favors tumor pro-gression while its overexpression in mammary tissue is asso-ciated with tumor formation (for a review on the pathway

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regulated by COX-2 see Cao and Prescott, 2002). COX-2expression promotes tumor formation by preventing apopto-sis. For example, COX-2 expression following exposure toUV irradiation prevents UV-induced apoptosis (for a reviewsee Brecher, 2002). COX-2 mRNA is regulated by an ARE inits 3′UTR. It is not known whether COX-2 overexpression incancer results in part from increased mRNA stability.

2.6. Cyclins

Cyclin/cyclin-dependant kinase (cdks) complexes are keyplayers in initiation of and progression through the cell cycle.The expression level of cyclins primarily controls the level ofcdk activity. Therefore, any signals that modify the level ofcyclin protein may alter cdk activity and perturb cell cycleprogression. mRNA stability critically regulates cyclin ex-pression. Cyclin mRNAs are stable only when their encodedprotein is needed and mRNA stabilization is seen in cancercells and primary tumors.

Keyomarsi and Pardee (1993) showed that human infil-trating ductal and metastatic breast carcinomas overex-pressed mRNAs for cyclins A and B and cyclin-dependentkinase 1 (cdk1). These investigators also showed that10 breast cancer cell lines had deranged expression of cyclinE. 9 out of 10 cell lines overexpressed the mRNAs andproteins for cyclins A and B and cdk1, and one cell line hadincreased stability of mRNAs for cyclins E, A, B, D1, and D3and their associated cdks, compared to normal breast epithe-lial cells.

2.6.1. Cyclin DD-type cyclins are expressed during G1 and bind to and

activate cdk4 and cdk6. The complexes then phosphorylateand inactivate the tumor suppressor pRB, enabling the E2Ffamily of transcription factors to enhance transcription ofS-phase specific target genes.

Both cyclins D1 and D3 are regulated by 3′UTR elements.3′ UTR-truncated cyclin D1 mRNA is overexpressed in twochronic lymphocytic leukaemia cell lines (Withers et al.,1991). The cyclin D1 3′UTR is rearranged in patients witheither mantle cell lymphomas or t(11q13)-associated leu-kaemia (Rimokh et al., 1994). In both cases the AU-richregion of the 3′UTR is missing, the mRNA is more stable,and cyclin D1 is overexpressed. A human breast cancer cellline (MDA MB-453) also contains a cyclin D1 mRNA miss-ing the AU-rich region. This short transcript is overexpressedand more stable compared to the full length mRNA (Lebwohlet al., 1994). The chemotherapeutic agent Prostaglandin A2(PGA2) causes growth arrest associated with decreased cy-clin D1 in several cancer cell lines. In human non-small celllung carcinoma cells (H1299 cells), PGA2 leads to the desta-bilization of cyclin D1 mRNA via a 3′UTR element thatbinds the ARE-binding protein AUF1 (Lin et al., 2000).

Glucocorticoids down regulate cyclin D3 in murine Tlymphoma cells (the principal G1 cyclin in these cells) andcause G0/G1 arrest (Reisman and Thompson, 1995). Twopyrimidine-rich 3′UTR elements in the cyclin D3 mRNA

interact with RNA-BPs and are necessary for glucocorticoiddestabilization. This region is predicted to form a stable stemloop and is conserved in human cyclin D3 (Garcia-Gras et al.,2000). Glucocorticoids inhibit lymphoid T cell proliferationand are used for treatment of leukaemia and other malignantand non-malignant lymphoproliferative diseases. The factthat PGA2 and glucocorticoids, two agents already in use forcancer treatment, work via mRNA destabilization highlightsthe potential importance of targeting cyclin mRNA stabilityin cancer therapy.

2.6.2. Cyclin ECyclin E is a G1 cyclin that forms complexes with Cdk2

and is crucial for entry into S-phase (Dulic et al., 1992).Keyomarsi and Pardee (1993) showed that a breast cancercell line overexpressing cyclin E had an 8-fold gene amplifi-cation, 64-fold overexpression of mRNA and altered proteinexpression. Cyclin E mRNA stability was increased in 10 celllines and correlated with increased protein. These investiga-tors also showed that cyclin E is present in constitutivelyactive cdk2 complexes in breast cancer cells (Keyomarsi etal., 1995).

The linkage between cyclin E overexpression and onco-genesis is strong. Cyclin E is overexpressed in breast, lung,skin, gastrointestinal, cervical, ovarian, and bladder cancer(Donnellan and Chetty, 1999). Although the majority ofstudies on cyclin E expression in cancer are correlative,breast cancer is the best characterized. High levels of cyclin Eprotein are found in most primary breast tumors and are oftencorrelated with low levels of p27, a cdk inhibitor that nor-mally inhibits cyclin E/cdk2 (Loden et al., 1999). Breastcancer patients with high cyclin E in their tumors or highcyclin E coupled with low p27, have a significantly increasedrisk of death or relapse. Constitutive overexpression of cyclinE specifically targeted to mammary epithelium of transgenicmice increases the incidence of breast hyperplasias and car-cinomas (Bortner and Rosenberg, 1997). Oda and colleagues(1995) reported that cyclin E mRNA is stabilized upon G1phase re-entry in Rat-1A cells. However, the molecular basisof this mechanism is unknown. Understanding the mecha-nisms of increased mRNA stability and differential transla-tion of cyclin E should have a profound effect on futurecancer treatments.

2.6.3. Cyclins A and BCyclins A and B bind to their cognate cdks to phosphory-

late targets required for DNA synthesis and mitosis, respec-tively. The stability of cyclin B1 mRNA in HeLa cells, acervical cancer cell line, is controlled throughout the cellcycle. The half-life increases from about 1 h in G1 to 13 h inG2/M when its mRNA level peaks. Gamma irradiation in Sphase reduces its half-life from 13 h to 2-3 h, but has no effecton cyclin A1 mRNA. Upon gamma irradiation, the decreasein cyclin B1 mRNA stability is coincident with a G2 arrestand a transient reduction in B1 mRNA levels (Maity et al.,1995). Presumably, the decreased stability of cyclin B1mRNA leads to the G2 arrest, giving the cell time to repair


DNA damage before undergoing mitosis. Similarly, cyclinA1 mRNA that is unstable in G1 becomes stable at the G1/Stransition (Maity et al., 1997). A recent study implicates theARE-BP HuR in binding the 3′UTR of cyclins A and B tostabilize their mRNAs (Wang et al, 2000a).

2.7. Cdk inhibitors

The activity of cdk/cyclin complexes is negatively regu-lated by their interaction with Cdk inhibitors (CKIs). CKIsbelong to two families: the INK4 family (p16Ink4A,p15ink4B, p18ink4C and p19ink4D), which inhibits cyclinD/cdk4-6 and the Cip/Kip family (p21, p27, p57), whichinhibits various cyclin/cdk complexes.

p21 mRNA is stabilized in human myeloid leukaemiacells in response to TNFa treatment (Shiohara et al., 1996),and in colorectal carcinoma cells by UV exposure (Gorospeet al., 1998). UVC induces p21 via binding of the ARE-BPHuR to its mRNA (Wang et al., 2000b). In human MDA-468breast cancer cells, both UVC and EGF up regulate p21mRNA and protein through increased mRNA stability (Gileset al., 2003). HuR and poly(C) binding protein bind the3′UTR to mediate UVC and EGF-mediated stabilization,respectively. This data implies that different stimuli can regu-late mRNA stability via different cis-elements and RNA-BPs.

2.8. DNA methyltransferase 1

Modulation of transcription is critical for the normal pro-gression of the cell cycle and is clearly altered in cancer.DNA methylation is inversely correlated with the pattern oftranscription. High methylation is associated with gene si-lencing while hypomethylation is associated with gene ex-pression. DNMT1 is the major DNA methyl transferasewhose expression is coordinated with the cell cycle, peakingin S phase (Szyf et al., 1991). This expression profile iscontrolled both at the transcriptional and post-transcriptionallevels (Bigey et al., 2000; Kishikawa et al., 2003; Peterson etal., 2003).

High DNMT1 is observed both in cancer cells in vitro aswell as in tumors (for a review see Baylin, 1997). It wasrecently shown that the DNMT1 gene is required to maintainmethylation and gene silencing of the tumor suppressorp16INK4A in human cancer cells. In HCT116 cells (a humancolon cancer cell line), the promoter of p16INK4A is hyper-methylated. Depletion of DNMT1 by antisense oligonucle-otides led to the time-dependent demethylation of the p16promoter and p16 re-expression (Robert et al., 2003). Fur-thermore, ectopic expression of DNMT1 results in cellulartransformation (Vertino et al., 1996; Wu et al., 1993a). Post-transcriptional regulation of DNMT1 is via the 3′UTR.

A 54-nt element in the 3’UTR of DNMT1 mRNA issufficient and necessary to confer growth-dependent expres-sion to a reporter mRNA. This element is conserved inseveral species including zebrafish, frog, chicken, mouse andhuman (Detich et al., 2001). This element confers rapid

decay to mRNAs in growth arrested cell extracts and is animportant potentiator of DNMT1-induced transformation, asDMT1 lacking the 3′UTR transforms 3T3 cells whereasinclusion of the 3′UTR prevents transformation. A 40-kDaprotein (p40) specifically binds the DNMT1 3’UTR ingrowth arrested cells (where DNMT1 is repressed) but not inserum-stimulated cells. Whether alterations of p40 expres-sion or mutations in its binding element are associated withtumorigenesis remains to be determined as does the identityof p40.

3. Regulatory elements in the coding region

The two best-characterized coding region determinants(CRDs) of mRNA instability are those of c-fos and c-mycmRNAs. Despite having identical names these are differentsequences and proteins, which function by distinct mecha-nisms.

3.1. C-fos

As for other early response genes, translational inhibitionincreases the stability of c-fos mRNA. Blocking the transla-tion of an unstable c-fos reporter mRNA by inserting a stemloop in its 5’UTR stabilizes it and blocks its deadenylation(Schiavi et al., 1994), suggesting that ongoing translationleads to deadenylation and degradation. Two functional in-stability determinants in the c-fos coding region work inconcert to deadenylate and destabilize the mRNA. This twopart CRD functions both in and out of frame (Wellington etal., 1993), showing that the sequence of the nascent polypep-tide is not involved in the regulation. The minimum CRD isan 87-nt region sufficient to target a reporter RNA for rapiddegradation if it is located sufficiently far away from thepoly(A) tail. This CRD is also sufficient for the specificbinding of a complex of proteins (Chen et al., 1992; Grossetet al., 2000).

A 22-nt stretch of purines within the 87-nt CRD was usedto purify a protein complex containing UNR, HnRNPD,PABP1, NSAP1, and PAIP1. This “bridging complex” isthought to link the CRD with both the 3’ and the 5’ ends ofthe mRNA (Fig. 2A). PABP1 (Poly(A) binding protein 1)interacts with both the poly(A) tail and the CRD, whilePAIP1 (Poly(A) Interacting Protein 1) interacts with bothPABP1 and eIF4A, a component of the translation initiationmachinery (Roy et al., 2002). The specificity of binding tothe purine stretch is attributed to the RNA-BP UNR (Trique-neaux et al., 1999). The interactions of the other partners ofthe complex are unknown. PABP1 is also known to interactdirectly with the translation initiation factor eIF4G (Imatakaet al., 1998), bridging the 5’ and 3’ UTRs (Fig. 2B). Thisinteraction is usually associated with protection of thepoly(A) tail and efficient translation of the mRNA. Thisexample shows that distinct complexes sharing commonpartners can be formed depending on the presence or absenceof the CRD.


The stability of the untranslated c-fos mRNA may be dueto the bridging complex maintaining its polyadenylationstate. When translated, the complex would be kicked off theCRD but still remain associated with the mRNA, tagging itfor rapid deadenylation and degradation. This system wouldensure that the RNA was translated at least once before beingdegraded and would render degradation of the c-fos mRNAtranslation-dependent. However, it is not clear how themRNA is targeted for deadenylation, or which poly(A) exori-bonuclease is involved. Uchida and colleagues (Uchida et al.,2004) identified a complex of two proteins, human poly(A)nuclease 2 and human poly(A) nuclease 3 (hPAN2, hPAN3)that interact with PABP1 and whose in vitro deadenylationactivity is enhanced by this interaction. It would be interest-ing to determine whether this nuclease complex associateswith any of the components of the CRD bridging complex.Overexpression of any of the proteins in the bridging com-plex leads to the stabilization of a reporter mRNA containingthe CRD (Grosset et al., 2000), suggesting that overexpres-sion of any of these proteins in a pathologic state couldfavour overexpression of c-fos or other target mRNAs.

3.2. C-myc

C-myc is also induced upon translational inhibition as aconsequence of mRNA stabilization. The c-myc mRNA con-tains a translation-dependent CRD in the sequence encodingamino acids 357 to 439 (Wisdom and Lee, 1991; Lee et al.,

1998). An RNA corresponding to amino acids 357-439 wasable to induce, in trans, the destabilization of c-myc RNA byendonucleolytic cleavage inside this region of the mRNA(Lee et al., 1998), suggesting that a protective factor wastitered off. This factor was later identified as the c-mycCRD-BP. Regulation of c-myc mRNA stability by the CRDrelies on a competition between an endoribonuclease andCRD-BP, both of which are associated with ribosomes(Bernstein et al., 1992; Lee et al., 1998; Prokipcak et al.,1994). A model of these interactions is shown in Fig. 3. Thec-myc CRD is bipartite, it contains rare codons in its 5’ partwhich favor translational pausing (Lemm and Ross, 2002)and an endonuclease site in its 3’ part. Translational pausingexposes the endonucleolytic site and enables the rapid deg-radation of the RNA. However, destabilization is abrogated ifthe CRD-BP binds to the RNA, shielding the cleavage site.This mechanism is dynamic in that after CRD-BP binds,translation resumes and CRD-BP is displaced. Therefore,each time a ribosome pauses, the race for the CRD betweenthe nuclease and CRD-BP begins anew.

C-myc CRD-BP was purified from K562 human erythro-leukæmia cells (Prokipcak et al., 1994) and shown to belongto a family of RNA binding proteins containing 4 HnRNPKhomology domains (KH domains) in their C-terminus and2 RNA recognition motifs in their N-terminus. CRD-BP(also termed IGF-II mRNA BP) is closely related to thehuman KOC protein, which is overexpressed in pancreaticcancer (KH-domain containing protein Overexpressed inCancer), and p62, a human hepatocellular carcinoma autoan-tigen (Lu et al., 2001). KOC is 84% identical to Vg1-RBP aXenopus RNA binding protein involved in the localization ofmRNA during development (Mueller-Pillasch et al., 1999).Arecent review (Yaniv andYisraeli, 2002) describes this familyof proteins in detail.

CRD-BP is expressed in fetal rat tissues and cancer cellsbut is normally not detectable in the adult. The CRD-BP gene

Fig. 2. (A) Ribonucleoprotein complex formation on the c-fos CRD and (B)General model of the interactions between the 5’ and the 3’ end of themRNA via PABP1.The proteins that are part of the CRD bridging complex are green. PAIP1bridges PABP1 to eIF4A. UnR interacts directly with the CRD, whilePABP1 interacts with the poly(A) tail. The interactions between the otherproteins of the CRD bridging complex are unknown.

Fig. 3. Model showing competition between the coding region determinant-binding protein (CRD-BP) and a nuclease for the c-myc mRNA.The nuclease is shown as a red lightening bolt and the ribosome is depictedby two circles. Once translation is initiated, the ribosome brings both thenuclease and CRD-BP to the CRD. Competition between CRD-BP and thenuclease ensues for binding to the CRD. If the nuclease associates first theRNA is cleaved (1), otherwise, CRD-BP protects the mRNA and the ribo-some can continue scanning (2). The RNA can thus be degraded at eachcycle of translation.


was amplified in approximately 1/3 of human breast cancerssampled (12 of 40) while not being amplified in adjacentnormal cells (Doyle et al., 2000). In another study (Ross etal., 2001), CRD-BP was expressed in 58.5% of 118 primarybreast carcinomas screened. CRD-BP was expressed in 81%of human colorectal cancers examined (17 of 21 adenocarci-nomas), but not in normal colon and inflammatory bowel.C-myc mRNA levels also appeared elevated in these tumors.

To address the significance of CRD-BP in oncogenesis,Tessier and colleagues (2004) generated transgenic miceexpressing CRD-BP in mammary tissue. CRD-BP transgenicmice developed mammary tumors in a CRD-BP dose-dependent fashion, but the expression of c-myc was notaltered. This may be because redundant post-transcriptionalmechanisms control c-myc mRNA. It is noteworthy that theeffects of the c-mycARE and CRD on c-myc mRNA stabilityare not additive (Herrick and Ross, 1994), suggesting thatthese elements act in different physiological conditions orcell types. Nevertheless, in CRD-BP transgenic mice twoknown binding targets of CRD-BP (H19 and IGF-II mRNAs)were overexpressed. Notably, H19 mRNA is untranslatedshowing that CRD-BP may bind and affect mRNA in theabsence of ongoing translation.

CRD-BP alters the localization of the H19 mRNA (Rungeet al., 2000) but it is not known whether stabilization of H19mRNA relies on CRD-BP. H19 is overexpressed in variouscancers and enhances the tumorigenic properties of breastcancer cell lines (Lottin et al., 2002 and reference therein).Determining whether CRD-BP dependent carcinogenesis re-lies on H19 overexpression could be accomplished by cross-ing CRD-BP transgenic mice with H19 null mice.

Antisense oligonucleotides complementary to part of theCRD were shown to limit CRD-BP binding to RNA in vitroand to decrease both c-myc mRNA and protein levels inK562 erythroleukæmia cell lines (Coulis et al., 2000). Thisresult demonstrates the therapeutic potential of targetingRNA/protein complexes.

4. Regulatory elements in the 5’UTR

Translational initiation can be either cap-dependent orcap-independent. In cap-dependent translation, the 5’ capstructure is recognized by a set of initiation factors thatenable binding of the 40S ribosomal subunit to the 5′UTR.The small subunit then migrates until it finds an initiationcodon, the 60S ribosomal subunit joins, and translation pro-ceeds. In cap-independent translation, mRNAs contain inter-nal ribosomal entry sites (IRES) within a long structured5’UTR. In this case, the 40S ribosomal subunit is recruited tothe vicinity of the initiation codon without influence of thecap (Vagner et al., 2001). The efficiencies of both mecha-nisms may be altered by the presence of specific cis-actingelements located in the 5’ untranslated region.

4.1. Internal ribosomal entry site

4.1.1. C-mycIn addition to being controlled at the level of mRNA

stability, c-myc mRNA is also translationally controlled.C-myc mRNA is transcribed from four different promoters.The two major promoters (P1 and P2) produce mRNAs thatcontain an extended 5’ UTR that contains an IRES (Nanbruet al., 1997; Stoneley et al., 1998). This IRES functionspredominantly during the G2/M transition of the cell cycle,when cap-dependent translation is inhibited. Several IREScontaining mRNAs are specifically translated at G2/M. Cap-independent translation may be enhanced at this time be-cause abrogation of cap-dependent translation results in theavailability of initiation factors, or it may be due to specificIRES activation through RNA-protein interactions. Thec-myc IRES binds hnRNPC (Kim et al., 2003). At mitosis,when the nuclear membrane is absent, hnRNPC becomescytoplasmic thereby activating c-myc translation. Mutationof the c-myc IRES correlates with enhanced c-myc expres-sion in multiple myeloma cell lines. When tested in variouscell lines, this mutated IRES drives translation more effi-ciently than the wild-type IRES. Furthermore, the mutatedIRES is able to bind hnRNPK in vitro more efficiently thanits wild type counterpart. The binding of hnRNPK to theIRES enhances c-myc translation in vitro (Evans et al.,2003). Therefore, IRES mutations may also account for thederegulation of c-myc expression observed during oncogen-esis (Chappell et al., 2000).

4.1.2. p27Translation of the cdk inhibitor p27 is also regulated by an

IRES. HeLa cells arrested in G1 with lovastatin have in-creased p27 protein due to increased translation (Hengst andReed, 1996). It must be noted that there is controversy aboutwhether lovastatin synchronizes cells (Cooper, 2002; Keyo-marsi et al., 1991). Nevertheless, in 293T cells, p27 transla-tion requires a U-rich sequence in the 5′UTR (Millard et al.,2000) that binds HuR, hnRNPC1 and C2. This U-rich ele-ment is part of a 5′UTR IRES through which HuR negativelyregulates p27 translation (Kullmann et al., 2002). Since p27-deficient animals develop tumors (Cordon-Cardo et al.,1998; Fero et al., 1996; Kiyokawa et al., 1996; Nakayama etal., 1996; Park et al., 1999), it will be interesting to analyzethe 5′UTRs from tumors or cancer cell lines that have re-duced p27 levels for mutations or deletions in the IRES.

4.2. Upstream Open reading frames

The presence of multiples reading frames upstream of themajor initiation codon has been known for several years. Awell known example is the one of the yeast GCN4 mRNA inwhich upstream ORF render the translation of the main ORFnutrient dependant (Hinnebusch, 1997). It is estimated that50% of the human mRNA contain upstream AUG or ORF(Wang and Rothnagel, 2004). Furthermore, leaky scanningenabling translation of downstream AUGs was always


present and significant protein level from dowstream AUGsmay be produced. This suggests that the first AUG rules oftenused to determine the ORF in genomic sequences has to bereexamined.

4.2.1. Mdm2The proto-oncogene mdm2 encodes an E3 ubiquitin ligase

that activates degradation of the transcription factor p53(Fang et al., 2000; Honda et al., 1997). In human and murinetissues, mdm2 and p53 form a regulatory feedback loop, withp53 activating mdm2 expression and mdm2 protein inhibit-ing p53 by activating its degradation (Wu et al., 1993b).

p53 is increased in a variety of proliferating cells and itsaltered expression is involved in more than 50% of cancers(Levine, 1997; for a recent review, see Iwakuma and Lozano,2003). Mdm2 protein is overexpressed in several Burkitt’slymphoma cell lines (Capoulade et al., 1998), in cutaneousmelanoma (Polsky et al., 2001) and in breast cancer cells(Okumura et al., 2002). Mdm2 is often overexpressed in theabsence of any gene amplification and its overexpression iscorrelated to enhanced translation of its mRNA. Its transla-tional efficiency is modulated by the presence of upstreamORFs (uORFs) in the 5’UTR and by BCR/ABL kinase sig-nalling.

Two mRNA isoforms with different 5’UTRs, L-mdm2(long 5’UTR) and S-mdm2 (short 5’UTR) are produced byalternative promoter usage. The long 5’UTR contains 2 uO-RFs that decrease the overall translational efficiency of themRNA (Bortner and Rosenberg, 1997). The short 5’UTRallows high translational efficiency. Thus, differential pro-moter usage causes translational activation of the mdm2mRNA. In particular, in some human breast cancer cells,S-mdm2 is predominantly produced leading to an overabun-dance of mdm2 protein that represses p53 by stimulating itsdegradation (Okumura et al., 2002).

4.2.2. p18INK4cp18INK4c, a member of the p16/INK4 cdk inhibitor fam-

ily is induced during myogenic differentiation, the proteinaccumulating to 50-fold its basal level. This expression iscoupled to a promoter switch that leads to the transcription ofa mRNA devoid of a long 5’UTR. The long 5’UTR containsseveral uORFs that control the efficiency of translation, simi-lar to the mdm2 mRNA. The overall p18INK4c mRNA levelis not changed in differentiated cells but the translationalefficiency of the shortened mRNA is greatly enhanced(Phelps et al., 1998).

4.3. Initiation codon switch

4.3.1. p16INK4AMutations affecting translational initiation efficiency are

seen in CDKN2A, the gene encoding the cdk inhibitorp16INK4A, whose deletion is frequently found in tumorcells. A point mutation in the 5′UTR of this gene has beenlinked to melanoma predisposition. This G to T mutation islocated 34-nt upstream from the initiation codon where it

introduces a new AUG out of the normal translation frame.This AUG is efficiently recognized as an initiator codon andimpedes translation. This mutation co-segregates with mela-noma and is not found in non-affected individuals (Liu et al.,1999).

4.3.2. C/EBPbThe transcription factors of the CCAAT/enhancer binding

protein (C/EBP) family play crucial roles in differentiationand regulation of cellular growth (for detailed reviews seeGrimm and Rosen, 2003; Ramji and Foka, 2002). BothC/EBPa and C/EBPb mRNAs give rise to multiple proteinsfrom a single unspliced mRNA. Alternative translation prod-ucts are primarily produced by differential translational ini-tiation (Descombes and Schilber, 1991; Ossipow et al.,1993). Specific proteolysis also aids in the production ofsome alternative products. Fig. 4 depicts the organisation ofhuman C/EBPb mRNA. Three in-frame initiation codons (A,B and C) produce three protein isoforms: codon A producesliver-enriched activating protein 1 (LAP-1), codon B pro-duces LAP-2 and codon C produces a small isoform, liverenriched inhibitory protein (LIP). A small out of frame ORF(D) is located between initiation codons A and B. LIP andLAP isoforms are overexpressed in breast cancer cell(Raught et al., 1996; Zahnow et al., 1997) and some trans-genic mices overexpressing the LIP isoform developed mam-mary intraepithelial neoplasias and carcinomas (Zahnow etal., 2001).

Calkhoven and colleagues (Calkhoven et al., 2000) clearlydemonstrated the importance of the small ORF in the relativeusage of the different initiation codons. Its deletion increasesthe usage of codon B and decreases the usage of codon Ctherefore altering protein isoform production. Conversely,making codon D a strong initiation site decreases the usageof B and increases that of C. If codon A is mutated into astrong initiation site then only LAP-1 is produced. Therefore,leaky scanning enables usage of three initiation sites (A, Band D) while reinitiation on codon C after translation of thesmall upstream ORF is key for production of LIP.

The relative usage of A, B and D depends on the availabil-ity of initiation factors, as increasing the activity of eIF2a or

Fig. 4. Alternative translation initiation in C/EBPb.The mRNA for C/EBPb is shown as a black line. The initiation codon arelabeled A to D. A to C are in frame initiation codon while D drives theexpression of a small out of frame reading frame (black box). The Stopcodon for A, B and C is shown as a star. The various translation products areindicated. The activation domains are shown as green boxes, repressiondomains are shown as red boxes, the basic region is shown as grey box andthe leucine as stripped boxes. This schematic is adapted from (Ramji andFoka, 2002)


eIF4E increases the usage of D, which in turn enhances LIPproduction. Proteins binding the 5’ UTR of C/EBPb includecalreticulin and CUGBP1. Both bind in the vicinity ofcodons A, B and D. Binding of these proteins may be linkedto the overall inhibition of C/EBPb translation (Timchenko etal., 1999; Timchenko et al. 2002; Baldwin et al. 2004).

5. RNA binding factors

Almost all of the elements that regulate mRNA translationor stability described above do so by binding proteins. RNA-BPs may act either directly to alter translational efficiency orindirectly by bridging proteins together on the mRNA. Theymay also serve to tag a mRNA for rapid deadenylation ordegradation or to protect it from nucleases. Competitionbetween protection or degradation factors or translationactivators/inhibitors will ultimately determine the overallamount of protein produced by one mRNA. It is very difficultto determine what mRNAs a given protein controls. Further-more, it is probable that different RNA-protein complexesare formed on different mRNAs. In the human genome thereare about 500 proteins containing known RNA binding do-mains (Anantharaman et al., 2002). Here we discuss thesubset of characterized RNA-BPs known to be altered incancer.

5.1. ARE binding proteins

AU-rich element binding proteins (ARE-BPs) bind AU-rich or U-rich sequences in the 3′UTR to regulate localiza-tion, translation and degradation of mRNAs encoding criticalgrowth-response genes, cytokines, and cell-cycle regulatoryproteins. At least 14 ARE-BPs have been identified (Brennanand Steitz, Cell Mol Life Sci 2001 58:266-77; Wilson andBrewer, Methods 1999 17:74-83), with the following impli-cated in cancer.

5.1.1. HuRHu RNA-BPs were first identified as tumor antigens in

lung carcinomas of individuals with paraneoplastic neuro-logical disorder (Dalmau et al., 1990; Szabo et al., 1991).Patients developed antibodies against Hu proteins, whichpenetrated the blood-brain barrier and led to neuronal degen-eration. The features of paraneoplastic diseases suggestedthat Hu proteins might play a pivotal role in controllingexpression of growth regulatory genes. HuR is now known tobe an ubiquitously expressed ARE-BP. The overexpressionof ARE-BPs such as HuR and others may contribute totumorigenesis by stabilizing mRNAs of cytokines and othergrowth regulators. A recent work performed in Gorospe labused immunoprecipitation of the HuR containingRNA/protein complex to identify globally the HuR mRNAtarget (Lopez de Silanes et al., 2004). About 15 % of9600 genes analysed showed specific immunoprecipitationwith anti-HuR antibody.

Specific analysis of HuR binding to mRNA target hasbeen performed in earlier works. HuR binding to the p53

3’UTR enhances its translation. The translational regulationof p53 was first reported in 1984 (Maltzman and Czyzyk,1984). It’s now known that the p53 mRNA contains apoly(U) translational repressor in its 3’UTR. When cells areexposed to gamma-irradiation, the poly(U) region mediatesan increase in p53 translation that correlates with enhancedHuR binding, with no change in mRNA stability (Mazan-Mamczarz et al., 2003). HuR’s function in direct mRNAtranslational activation remains elusive.

HuR binds an ARE in the COX-2 3′UTR (Cok and Mor-rison, 2001; Dixon et al., 2000). The ARE consists of AU-UUA repeats and is broadly conserved in the human andmouse COX-2 3’UTRs (76% identity over 116-nt) with astrict conservation of the AUUUA repeats. HuR binds theCOX-2 mRNA in the colon cancer cell lines HT-29 and LoVoand is expressed at high levels in HT-29 as compared to LoVocells. HT-29 cells have a high level of mRNA-bound HuRand increased COX-2 protein, suggesting that HuR stabilizesthe mRNA and increases protein levels. In support of this,overexpression of HuR in LoVo cells increases COX-2 pro-tein and enhances the expression of a reporter gene contain-ing the COX-2 3’UTR (Dixon et al., 2001).

COX-2 and the angiogenic and proliferative factors VEGFand IL-8 are overexpressed in colon cancer cells presumablydue to ARE-binding and mRNA stabilization by overex-pressed HuR (Dixon et al., 2001). Likewise, these factors areup regulated in malignant brain tumors that overexpress HuR(Nabors et al., 2001). HuR binds to these mRNAs in vitro andis known to bind them in other systems. These data, coupledto the co-localization of the highest levels of HuR withgrowth factor up regulation, suggests a role for mRNA stabi-lization in growth of malignant brain tumors and colon can-cer.

Wang and colleagues showed that the expression andhalf-life of cyclins A and B1 mRNAs could be reduced incolorectal carcinoma (RKO) cells by knocking down HuRexpression with antisense oligonucleotides (Wang et al.,2000a). Cells with decreased HuR have reduced growth,indicating a role for this RNA-binding protein in regulatingcell proliferation via cyclin mRNA stabilization. Impor-tantly, the cyclin A and B1 mRNAs bind HuR in vivo. Thisdata provides evidence that cell proliferation relies on cellcycle-dependent stabilization of specific cyclin mRNAs viaRNA-protein interactions. Whether disruption of these inter-actions in the context of cyclins A and B contribute to thepathogenesis of cancer remains to be determined.

5.1.2. TristetraprolinGM-CSF expression in a myc-induced monocytic tumor

results from constitutive stabilization of this normally short-lived mRNA. An ARE and a tumor specific trans-actingfactor (Schuler and Cole, 1988) mediate mRNA stabiliza-tion. More recently, the ARE-BP tristetraprolin (TTP) hasbeen shown to regulate GM-CSF mRNA stability via dead-enylation (Carballo et al., 2000). Another recent study sug-gests that ARE-mediated decay of GM-CSF mRNA requires


ubiquitination and proteasome activity. As proteasome activ-ity varies during the cell cycle, it will be interesting to see ifdifferential stability of this mRNA is linked to the protea-some activity cycle (Laroia et al., 2002).

TTP may also bind the COX-2 3’UTR. Sawaoka et al.identified two COX-2 mRNAs in a human colorectal carci-noma cell line (HCA-7) that are polyadenylation variants,differing by the length of their 3′UTRs. The longer one bindsTTP and is repressed more rapidly than the shorter one thatdoes not bind TTP. Thus, the shorter COX2 mRNA sustains astronger COX2 expression (Sawaoka et al., 2003).

5.1.3. TIARIn rat mesangial cells, the COX-2 ARE not only binds

HuR, but also the ARE-BPs TIA-1, TIAR and hnRNPU, aswell as other unidentified proteins (Cok et al., 2003). Thefunction of this multiprotein complex in post-transcriptionalcontrol of COX-2 remains to be determined. MMP-13 isunder the transcriptional control of a number of factors suchas TGFb1 (Ravanti et al., 1999) and IL-1b (Mengshol et al.,2000)). IL-1b induces transcription of MMP-13 both in Rat2cells and in human mesangial cells, but MMP-13 protein isdetected only in Rat2 cells, suggesting post-transcriptionalregulation (Yu et al., 2003a). When fused to the humanMMP-13 coding region, the 3′UTR of either rat or humanMMP-13 attenuates its protein level in human but not in rat-2cells, showing that a human-specific factor post-transcriptionally down regulates MMP-13. The ARE-binding protein TIAR may be this factor.

Two alternatively spliced forms of TIAR exist in humancells, TIAR-a and TIAR-b. They differ by a 17 amino acidinsertion into the first RNA recognition motif of TIAR-b(Beck et al., 1996). Co-transfection of a peptide consisting ofthese 17 amino acids along with a reporter construct contain-ing the MMP-13 ORF and 3’UTR, resulted in overexpressionof MMP-13 protein, suggesting that the peptide competeswith TIAR for binding to the 3′UTR and thus reverses itstranslational repression. Interestingly, overexpression ofTIAR-a in Rat2 cells (where it is normally absent) inhibitsMMP-13 accumulation. This study suggests that modulatedexpression of different isoforms of RNA-BPs could alter thefinal level of proteins that play crucial roles in cancer pro-gression. For example, one could envisage that repression ofTIAR-a favors the overexpression of MMP-13 thereby al-lowing remodelling of the extracellular matrix and metasta-sis.

5.1.4. CUGBPCUGBP-1 and CUGBP-2 are members of the CELF

(CUGBP and ETR3-like factors) family of RNA bindingproteins. Both proteins bind to CUG repeats found in themyotonin protein kinase gene (Laird-Offringa et al., 1991;Lu et al., 1999). CUGBP and CUGBP2 are more than 76%identical and are involved in splicing regulation and transla-tional control (Paillard et al., 2003). CUGBP-2 may bind theARE in the COX-2 mRNA. Upon binding, COX2 mRNA isstabilized but translationally repressed (Mukhopadhyay et

al., 2003). Despite its known functions in splicing, howCUGBP-2 alters COX-2 translation is unknown (Mukho-padhyay et al., 2003).

CUGBP1 is also known as EDEN-BP, an RNA bindingprotein characterized as a specific deadenylation-promotingfactor during early development (Paillard et al., 1998). Thisfactor has also been shown to regulate alternative splicing(Philips et al., 1998 ; Ladd et al., 2001). As previouslyindicated (Paillard et al., 2002), EDENBP/CUGBP can bindto the c-jun ARE and target it for deadenylation. However, Itis not known whether c-jun mRNA stability is regulated byCUGBP in mammalian cells.

5.1.5. AUF1AUF1 (also called hnRNP D) determines the fate of ARE-

containing mRNAs by altering their stability. AUF1 consistsof four isoforms of 37, 40, 42, and 45 kDa, produced byalternative splicing of a single mRNA (Wagner et al., 1998).The p37 AUF1 isoform has the highest affinity for AREs invitro and the highest destabilizing effect (Loflin et al., 1999).Transgenic mice highly expressing p37 AUF1 protein de-velop sarcomas (Gouble et al., 2002). The tumors in thesemice strongly express AUF1 transgenic protein and CyclinD1. These data show that AUF1 is a key regulatory factor ofgene expression in vivo and that its deregulation leads totumorigenesis. On the other hand, as noted in section 2.6.1,prostaglandin A2 destabilizes cyclin D1 via AUF1 binding innonsmall cell lung carcinoma (Lin et al., 2000). This appar-ent dual role of AUF1 to either promote or inhibit tumorigen-esis is consistent with reports that this protein can eitherstabilize or destabilize mRNAs (Wilson et al., 2003; Goubleet al., 2002). The mechanisms by which one RNA-BP canhave opposite effects probably depend on its post-translational modification, binding partners, or even bindingsequence.

5.2. Proteins that mediate alternative translationalinitiation

5.2.1. LaLa RNA-BP was originally identified as an antigen recog-

nized by autoantibodies from patients with Sjogren syn-dromes and lupus erythematosus. It is involved in a variety ofprocesses, including RNA polIII transcript maturation, stabi-lization of histone mRNA and as a positive regulator ofmRNA containing a 5’-terminal polypyrimidine sequence(TOP mRNA) (Crosio et al., 2000). La binds a sequence inexon 2 of the mdm2 mRNA. This element is upstream of theinitiator AUG and enhances mdm2 translation in chronicmyelogenic leukaemia (CML) and in some acute lympho-blastic leukaemias (ALL) (Goetz et al., 2001). Mdm2 over-expression requires BCR/ABL kinase activity as well asproteasome activity, and is associated with decreased p53activity. BCR/ABL is a chimeric protein resulting from areciprocal translocation (t9;22) that is found in CML andALL. BCR/ABL transforms haematopoietic cells and fibro-blasts.


In CML and ALL, enhanced mdm2 translation is indepen-dent of the uORFs but requires the exon 2 element. La siRNAknockdown experiments showed that La binding was re-quired for enhanced translation while expression of a domi-nant negative La devoid of the RNA binding motif, but stillable to dimerize, led to a decrease in mdm2 synthesis (Trottaet al., 2003).

La protein is increased in BCR/ABL expressing cellsrelative to nonexpressing cells. This increase is dependentupon BCR/ABL activity and seems to be functionally linkedto stabilization of La protein. La has been implicated in bothcap- and IRES-dependent translation of several viral andcellular mRNAs (Crosio et al., 2000; Meerovitch et al.,1993). However, the molecular basis and mechanics of trans-lational activation by La protein remain elusive. A model forLa enhancement of mdm2 translation in BCR/ABL express-ing cells is presented in Fig. 5.

5.2.2. Mdm2Mdm2 has been shown to interact with the ribosomal

protein L5 (Marechal et al., 1994) and with mRNA (Elenbaaset al., 1996), suggesting a role in translational regulation.While mdm2 targets p53 for degradation by the proteasome,it also induces translation of two distinct p53 isoforms, p53and p53/47, from two in-frame translation initiation sites.p53/47 lacks the mdm2-binding domain and the first tran-scription transactivation domain; p53/47 is therefore not tar-geted for ubiquitination and degradation and has a differentpotential for gene activation. Mdm2 functions in p53 proteindegradation and mRNA translation are separable as the ex-pression of the mdm2 inhibitor p14-ARF does not affecttranslational induction. Translational induction is dependentupon binding of mdm2 to the N terminal region of p53 (Yin etal., 2002). Mdm2 binding to the nascent p53 protein mayenhance the translation of both p53 and p53/47 protein in cis.Since p53/47 also lacks the first trans-activation domain of

p53, it has a different potential for trans-activation that leadsto imbalanced p53 target gene expression. At this time, thefunctional significance of p53/47 expression remains to befully elucidated.

5.3. VHL regulation of mRNAs

Von Hippel-Lindau disease is a cancer syndrome resultingfrom inactivation of the VHL tumor suppressor gene. Thisdisease is autosomal dominant and affected individuals de-velop highly vascularized tumors of multiple origins. Inparticular, patients may develop renal cell carcinomas (for areview see Kaelin, 2002). VHL is expressed as two proteinisoforms arising from differential initiation start site usage onthe same transcript. Both isoforms have a similar function aspart of an E3 ubiquitin ligase complex that ubiquitinates andtargets specific protein for proteasomal degradation. Onemain target of VHL is the transcription factor Hypoxia Induc-ible Factor 1 a (HIF1a) that is targeted to the proteasome in aVHL dependent manner under normal oxygen conditions. Inhypoxic conditons, HIF1a associates with HIF1b to activategenes that control angiogenesis and erythropoiesis.

VHL has recently been shown to regulate the translationand stability of various growth factor mRNAs includingTGF-a, TGF-b1, and VEGF (Knebelmann et al., 1998). AsVHL is directly involved in protein degradation, its effects ontranslation and mRNA stability is probably indirect. In renalcell carcinoma lines that lack wild type VHL (786-0 cells),TGFa mRNA is down regulated 5-fold when compared to786-0 cells stably transfected with VHL (786-0 +VHL cells).The half-life of TGFa is about 5h in 786-0 +VHL cells and25 h in 786-0 cells. TGFa supports growth of renal cellcarcinoma through an autocrine loop (Atlas et al., 1992)emphasizing the importance of its down-regulation by VHL,but the mechanism is still unknown. Similarly, TGFb1 isrepressed 3- to 4-fold by the expression of VHL in 786-0cells partly due to decreased stability of the TGFb mRNA(Ananth et al., 1999).

VEGF (Vascular endothelial cell growth factor) is an im-portant inducer of both normal and pathological angiogen-esis. It is expressed at low levels in normal cells but is highlyexpressed in some human tumors and tumor cell lines. Tu-mors with high VEGF levels show constitutive stabilizationof VEGF mRNA (White et al., 1995). The VEGF mRNA isinduced during hypoxia both transcriptionally and post-transcriptionally (Ikeda et al., 1995; Liu et al., 1995; Stein etal., 1995).

In 786-O cells, VEGF is expressed independently of oxy-gen conditions. Oxygen-dependency is restored when wildtype VHL is reintroduced, with VEGF release by cells in highoxygen conditions reduced and VEGF mRNA half-life de-creased by 4 times. A 600-nt element in the VEGF 3’UTRbinds an hypoxia-inducible protein complex composed ofthree proteins of 34, 28, and 17 kDa. (Levy et al., 1996).Protein binding correlates with increased stability of VEGFmRNA. To date several RNA-BPs have been implicated in

Fig. 5. BCR/ABL kinase activity enhances mdm2 expression.The BCR/ABL kinase inhibits La protein degradation by an unknownmechanism. The La protein binds to a target sequence (red box) in the5’UTR of the mdm2 mRNA. This binding enhances translation of mdm2mRNA and the quantity of mdm2 protein (Blue hexagon) increases. Thisincreased level of mdm2 may repress p53 activity. The STI 571 ABL kinaseinhibitor blocks BCR/ABL kinase activity, thereby enhancing La degrada-tion.


VEGF mRNA stability including HuR (Goldberg-Cohen etal., 2002; Levy et al., 1998), which could be the 34 kDaprotein.

In human M21 melanoma cells, 126-nt of the VEGF3′UTR confers hypoxia-dependent stability to a reportermRNA. Under hypoxic conditions, 88/90 kDa and 60 kDaprotein complexes bound this element. HnRNPL was identi-fied as the 60Kda protein. Disruption of the HnRNPL/126-ntelement interaction by oligonucleotides decreases VEGFmRNA stability (Claffey et al., 1998; Shih and Claffey,1999).

A comparative microarray survey of mRNAs associatedwith polysomes between VHL-expressing and deficient cellsidentified several mRNAs whose translation is affected byVHL (Galban et al., 2003a; Galban et al., 2003b). In thisstudy, VHL repressed translation of TNFa via its 3′UTR andenhanced the translation of p53. VHL enhanced the transla-tion of p53 by promoting the cytoplasmic localization ofHuR and thus its binding to p53.

These studies show that VHL protein profoundly alters thepanel of expressed genes at the post-transcriptional level(summarized in Fig. 6). VHL represses expression of growthfactors by destabilizing their mRNAs or by specifically in-hibiting their translation. VHL enhances the translation ofother mRNAs, such as the tumor suppressor p53, by promot-ing the nuclear export of HuR. The many and opposite effectsof VHL on post-transcriptional regulation is certainly theresult of VHL affecting multiple factors involved in regula-tion of mRNA translation or stability. Since one direct func-tion of VHL is an E3 ubiquitin ligase that targets specificproteins for proteolysis, some of the proteins targeted may beRNA-BPs. If so, the differing balance of RNA-BPs betweenVHL-expressing and VHL-deficient cells could affect thepost-transcriptional regulation of target mRNAs. Much workremains in order to establish the molecular mechanisms in-volved in VHL-mediated regulation of mRNA.

Considering that the relative proportion of RNA-BPs inthe cell may be crucial for correct post-transcriptional controland control of protein stability may affect the relative abun-dance of RNA-BPs, it is probable that other ubiquitinationpathway components will be linked to control of mRNA

translation and stability. Consistent with this, the ARE-mediated decay of GM-CSF mRNA requires ubiquitinationand proteasome activity (Laroia et al., 2002).

6. Signalling pathways

Cell signalling events may also alter translational regula-tion and mRNA stability. The MAPK pathway affects mRNAstability and translation via differential phosphorylation ofRNA-BPs (Chen et al., 1998; Esnault and Malter, 2002). TheWnt/b-catenin and the Notch extracellular signalling path-ways also affect the stability and translation of mRNAs. Wewill first focus on p38 MAPK-mediated stabilization of mR-NAs since it is the best characterized (Table 1) and thenbriefly discuss the other pathways.

6.1. p38 MAPK signalling

The p38 MAPK pathway is regulated by proinflammatorycytokines, growth factors and cellular stress. p38 MAPK isactivated by upstream MAPK kinases such as MKK6 andMKK3 and acts through downstream substrates includingprotein kinases and transcription factors (Fig. 7). Phos-phatases such as the dual specificity phosphatase 1 (DUSP1,

Fig. 6. Von Hippel Lindau protein (VHL) regulates gene expression viamultiple post-transcriptional mechanisms. VHL destabilizes growth factormRNAs, inhibits TNF-a translation, and promotes p53 translation via pro-moting the export of HuR into the cytoplasm.

Table 1mRNA substrates of p38-MAPK dependent post-transcriptional regulation

COX-2 (Dean et al., 1999; Lasa et al., 2000)TNF-a (Prichett et al., 1995)IL-6 (Miyazawa et al., 1998; Wang et al., 1999)MKK-6 (Ambrosino et al., 2003)IL-8 (Winzen et al., 1999; Yu et al., 2003b)GM-CSF (Winzen et al., 1999)c-fos (Winzen et al., 1999)UPA (Cao and Prescott, 2002; Chen et al., 2001)MIP1-alpha

(Miyazawa et al., 1998)

MIP-2 (Rousseau et al., 2002).

Fig. 7. The p38 MAPK signalling pathway controls mRNA translation anddegradation. Proinflammatory stimuli activate upstream activators ofp38MAPK including MKK6 and MKK3. Downstream effectors of p38include MK2, which phosphorylates several RNA-BPs including hnRNPA0, PABP1 and TTP. Their phosphorylation may inhibit mRNA deadenyla-tion, resulting in mRNA stabilization and increased translation.


also known as CL100 or MKP-1) negatively regulate p38MAPK.

One common motif for control of mRNA stability by thep38 pathway is the presence of AREs in the 3’UTR. How-ever, not all ARE-containing mRNAs are dependent on p38.For example, the well-characterized c-myc ARE is not p38dependent (Lasa et al., 2000). The RNA-BP targets of p38 areto a large extent unknown. TTP is directly phosphorylated byMK2, a kinase directly downstream of p38 (Mahtani et al.,2001). However, as pointed out by the authors, the phospho-rylation of TTP upon p38 activation followed rather thanpreceded the stabilization of the target mRNA and thus doesnot seem to be the cause of mRNA stabilization. Therefore,other RNA-BPs are certainly involved in mRNA stabilizationupon p38 activation. The following sections will discuss uPAand TNF-a, two mRNAs regulated by the p38 MAPK signal-ling pathway, and lastly downstream effectors of mRNAstabilization.

6.1.1. UPAUrokinase plasminogen activator (uPA) is overexpressed

in several invasive cancers, where it plays a key role ininvasion and metastasis. uPA degrades the proenzyme plas-minogen into plasmin; plasmin then degrades extracellularmatrix proteins and activates matrix metalloproteases. Ininvasive breast cancer cell lines, overexpression of uPA iscorrelated with invasive properties and an increased metasta-sis risk. uPA expression relies on stabilization of its mRNAthrough the p38 MAPK pathway. The RAC1-MKK3-p38-MK2 pathway is required for av integrin induced uPA over-expression via mRNA stabilization (Han et al., 2002).

6.1.2. TNF-�Tumor necrosis factor-a (TNF-a) is a highly potent me-

diator of inflammation and is expressed in reaction to injuryor infection. Controlled expression of TNF-a is crucial, asoverexpression can lead to autoimmune disease and tissuedamage. TNF-a production is controlled at multiple levelsfrom transcription to mRNA stability and translation. WhileTNF-a was originally named for its ability to induce necrosisof tumors (Carswell et al., 1975), it harbours numeroustumor-promoting activities such as induction of angiogenicfactors, activation of matrix metalloproteases (Leber andBalkwill, 1998; Rosen et al., 1991), induction of positive cellcycle regulators and repression of cdk inhibitors (Gaiotti etal., 2000). Up regulation of TNF-a by p38 MAPK activationis clearly established but whether this up regulation is due toincreased mRNA stability, enhanced translation of TNF-amRNA, or both is not clear.

In the human monocytic cell line THP-1, translation ofTNF-a mRNA is enhanced upon p38 activation and re-pressed by a p38 MAPK inhibitor without changes in mRNAlevels (Prichett et al., 1995). TNF-a mRNA stability is regu-lated by an ARE (Brook et al., 2000) and an adjacent Consti-tutive Decay Element (CDE) (Stoecklin et al., 2003). In celllines deficient in ARE decay, the CDE drives rapid degrada-tion of TNF-a mRNA. Lipopolysaccharide (LPS) stimula-

tion of macrophages expressing reporter constructs bearingthe TNF-a 3’UTR, the ARE, or the CDE showed that theexpression and stability of endogenous TNF-a mRNA paral-leled that of the reporter containing either the full length3’UTR or solely the CDE. These results show that TNF-amRNA degradation is ensured by a constitutive decay path-way distinct from ARE-mediated decay. This decay pathwaymight guarantee that even when translation of TNF-a isenhanced by activation of p38MAPK, it remains limited.

6.1.3. Downstream effectorsMore work is needed to clarify how p38 MAPK signalling

stabilizes mRNA. One discrepancy that exists is in the regu-lation of MAPK kinase 6 mRNA (MAPKK6, MKK6 orMEK6). MKK6 is an activator of p38 MAPK (Frevel et al.,2003). In THP-1 cells, MKK6 mRNA is destabilized uponpharmacological p38 inhibition, suggesting that p38 MAPKstabilizes MKK6 in a positive feedback loop. On the otherhand, transgenic mice lacking p38 up-regulated MKK6 bothat both the RNA and protein levels (Ambrosino et al., 2003).The expression level of the p38 activators MKK3 and MKK4were not affected in the same conditions. These studiesindicate that mRNA stability can be differentially controlleddepending on the cellular context.

The key effector of the p38 pathway seems to be MK2which, when activated, is sufficient to stabilize p38 MAPKresponsive mRNAs. This stabilization of mRNAs upon p38activation has recently been correlated to the inhibition ofARE–mediated deadenylation (Dean et al., 2003). Inhibitionof deadenylation may have two non-mutually exclusive ef-fects: inhibition of mRNA degradation and increase in trans-lational efficiency. These two effects would lead to increasedprotein production upon p38 MAPK activation. Interestingly,p38 MAPK has been reported to have effects both on mRNAstability and on translational efficiency; deadenylation func-tionally links these effects.

Multiple articles point to the role of MK2 in mRNAstabilization mediated by p38. In particular, targeted disrup-tion of MK2 in mice leads to a 90% reduction in the level ofTNF-a protein induced by LPS. However, secretion ofTNF-a was not affected and the level of TNF-a mRNAremained unchanged. The stability of TNF-a mRNA afteractinomycin D inhibition of transcription was unchangedbetween wild type and MK2-/- cells. Other cytokines weredown regulated in MK2-/- cells, but it was unclear whetherthis was a direct effect of MK2 disruption or if it was aconsequence of TNF-a down regulation. In order to restorenormal TNF-a levels, MK-/- mice were crossed with micebearing an ARE-deleted allele of TNF-a. The MK2-/-

TNFDARE/+ offspring expressed normal levels of TNF-a afterLPS stimulation. Despite normal TNF-a levels, IL-6 mRNAstability and IL-6 production were still reduced in these mice.Furthermore, the stability of IL-6 was dependent upon thepresence of an ARE in its 3’UTR. Thus, IL-6 mRNA is likelya target of MK2 in addition to being controlled by TNF-a.

Consistent with a role in deadenylation, MK2 phosphory-lates TTP (Chrestensen et al., 2003) and PABP1 (Bollig et al.,


2003). MK2 phosphorylation of TTP on serines 52 and178 has been shown to increase 14-3-3 binding to TTP(Chrestensen et al., 2003). 14-3-3 binding promotes the cyto-plasmic localization of TTP. To date, the role of a phospho-rylation of PABP1 has not been elucidated.

HnRNP A0 is also a substrate of MK-2 (Rousseau et al.,2002). HnRNP A0 was isolated, along with other proteins, byvirtue of its ability to bind the TNF-a ARE. Among theisolated proteins, it was shown that hnRNP A0 was phospho-rylated by MK2 in vitro. Immunoprecipitation of the ribo-nucleoprotein complex containing hnRNP A0 showed that itbound to MIP-2, COX2 and TNF-a mRNAs in vivo. HnRNPA0 binding to these mRNAs was abrogated when the p38MAPK pathway was pharmacologically inhibited. It is notknown how hnRNP A0 affects mRNA stability.

These results highlight the elaborate regulation of mRNAstability and translation by one signalling pathway, p38MAPK. Multiple signalling pathways may control the over-all efficacy of protein production by one given mRNA. Eachof these pathways may be differentially controlled and there-fore produce a unique profile of expression for each mRNAspecies.

6.2. Wnt/b-catenin

The Wnt/b-catenin pathway rapidly induces transcriptionof the cell type specific transcription factor Pitx2. Pitx2mediates cell proliferation during mammalian development.Activation of the Wnt/b-catenin pathway also stabilizesPitx2 mRNA as well as the mRNAs of its transcriptionaltargets c-jun and cyclins D1 and D2. Pitx2 mRNA stabiliza-tion is due to reduced interaction of its 3′UTR with thedestabilizing ARE-BPs KSRP and TTP and increased inter-action with the stabilizing ARE-BP HuR. (Briata et al.,2003). Pitx2 itself appears to be a mediator of Wnt/b-catenininduced mRNA stabilization. Since Wnt activation increasescytoplasmic HuR, it is speculated that Wnt effects mRNAstabilization via regulated and coordinated cytoplasmictranslocation of both stabilizing and destabilizing factors. AsWnt/b-catenin has been extensively linked to regulation ofgene transcription and to oncogenesis (for a review on thelink between Wnt pathway and cancer see Kikuchi, 2003), itis tempting to propose that the alteration of the stability ofsome mRNAs participate in the oncogenicity of b-catenin.

6.3. Notch signalling and Musashi

The Notch signalling pathway is involved in maintainingthe proliferative state of various cell types. Notch signallingpromotes cell cycle progression by inducing cyclin D1 andcdk2 expression and inhibiting p53 dependent apoptosis (fora review see Nickoloff et al., 2003). In mice, Notch signallingis attenuated by Numb (Guo et al., 1996), whose translationis inhibited by the RNA-BP Musashi. Therefore Musashipotentiates Notch signalling by translationally repressingNumb. Ectopic expression or misregulation of Musashi maygive a selective growth advantage to notch-activated cells

(Fig. 8). Musashi contains two RNA recognition motifs andis evolutionarily conserved from fly to humans. Selex experi-ments identified specific Musashi binding sites (Imai et al.,2001; Okabe et al., 2001) for Drosophila (GU3-6(G/AG)) andmouse ((G/A)UnAGU).

Musashi was originally described as being expressed inneuronal precursor cells with its expression gradually downregulated in the course of differentiation (Toda et al., 2001).In Drosophila, Musashi represses the translation oftramtrack, a transcriptional repressor that is necessary andsufficient to specify a non-neuronal cell identity (Jan and Jan,1998). Musashi is used as a marker of progenitor cells inintestinal epithelia, breast epithelia, and the central nervoussystem. Its expression is also found in cancer of these tissues.Its level of expression correlates with the grade of malig-nancy, with cells harbouring high Musashi expression havinga high proliferative index. It is not clear if Musashi is amarker of an undifferentiated proliferating state or if it isinvolved in maintaining this proliferative state.

7. Conclusions

Deregulation of gene expression is a hallmark of thecancer cell. Acquiring a new profile of expressed proteinsmay enable the cell to re-enter the cell cycle, or give them agrowth or motility advantage over “normal cells”. The over-expression of oncogenes such as c-myc or mdm2 may bedeleterious for an organism. Similarly, the level of cell cycleregulators such as cyclins must be tightly regulated so thatcells divide in a controlled fashion. This tight control is theresult of transcriptional, post-transcriptional and post-translational events. The above studies emphasize that regu-lation of mRNA stability and translation is crucial for correctregulation of gene expression.

Post-transcriptional regulation of gene expression is ageneral mechanism found in all living organisms. The stabil-ity of a mRNA will determine the level and kinetics of itsaccumulation following transcription while translational ef-ficiency will determine the quantity of protein produced fromone RNA. Specificity of the regulation is complex as thesame regulatory sequence may interact with different RNAbinding proteins that either promote or repress gene expres-sion. A given mRNA may also be controlled by severalpost-transcriptional mechanisms in order to achieve the cor-

Fig. 8. Musashi-1 control of the Notch pathway. Mushashi-1 binding to the3′UTR of Numb mRNA inhibits its translation, thereby activating Notchsignalling. One downstream effector of Notch signalling is the transcriptionfactor RBP-J (also called suppressor of hairless).


rect expression in both time and space. Accurate post-transcriptional control results from a dynamic equilibriumbetween regulatory sequences, RNA-BPs, and the signallingpathways that modify them in a particular cellular context.

Alteration of any of these factors may cause disequilib-rium in the expression level of a given protein. Obviously,alterations in RNA-BPs, including their post-translationalmodification, have a broader effect than alterations in theregulatory sequences, as multiple mRNA species can beaffected. There are a growing number of reports of the asso-ciation of specific factors with a given RNA. This informa-tion is crucial, but it gives only a fixed picture of post-transcriptional regulation. The dynamics of the complexes,their kinetics of association/dissociation and their relativeabundance must be addressed in order to understand how theexpression level of a given gene is achieved and how thisaffects oncogenesis. A more detailed understanding of thepost-transcriptional mechanisms will enable the design anddevelopment of specific therapies based on modulating thetranslation or stability of specific mRNAs.

Acknowledgements

RSH is supported by the American Cancer Society (RSG01-054-01-CCG) and the NIH/NCI (R01 CA095898-01A1).We would like to thank members of the CNRS-UMR 6061(Génétique et Développement) for critical reading of themanuscript.

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post-transcriptional regulation in cancer

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