a bicistronic cyclin d1-trop2 mrna chimera demonstrates a novel oncogenic mechanism in human cancer

A Bicistronic CYCLIN D1-TROP2 mRNA Chimera Demonstrates a

Novel Oncogenic Mechanism in Human Cancer

Emanuela Guerra,1,2Marco Trerotola,

1,2Roberta Dell’ Arciprete,

2Veronica Bonasera,

1

Barbara Palombo,1,2Tarek El-Sewedy,

2Tommaso Ciccimarra,

2Carlo Crescenzi,

2

Franco Lorenzini,2Cosmo Rossi,

1,3Giovanna Vacca,

1,2Rossano Lattanzio,

1

Mauro Piantelli,1and Saverio Alberti

1,2

1Unit of Cancer Pathology, Department of Oncology and Neurosciences and CeSI, University ‘‘G. d’ Annunzio’’ Foundation, Chieti Scalo,Italy; 2Laboratory of Experimental Oncology, Department of Cell Biology and Oncology; and 3Animal Care Unit and Experimental Models,Institute Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy

Abstract

A chimeric CYCLIN D1-TROP2 mRNA was isolated from humanovarian and mammary cancer cells. The CYCLIN D1-TROP2mRNA was shown to be a potent oncogene as it transformsnaıve, primary cells in vitro and induces aggressive tumorgrowth in vivo in cooperation with activated RAS . Silencing ofthe chimeric mRNA inhibits the growth of breast cancer cells.The CYCLIN D1-TROP2 mRNA was expressed by a largefraction of the human gastrointestinal, ovarian, and endome-trial tumors analyzed. It is most frequently detected inintestinal cell aneuploid cancers and it is coexpressed withactivated RAS oncogenes, consistent with a cooperativetransforming activity in human cancers. The chimeric mRNAis a bicistronic transcript of posttranscriptional origin thatindependently translates the Cyclin D1 and Trop-2 proteins.This is a novel mechanism of CYCLIN D1 activation thatachieves the truncation of the CYCLIN D1 mRNA in theabsence of chromosomal rearrangements. This leads to ahigher CYCLIN D1 mRNA stability, with inappropriate expres-sion during the cell cycle. The stabilized CYCLIN D1 mRNAcooperates with TROP2 in stimulating the growth of theexpressing cells. These findings show a novel epigenetic,oncogenic mechanism, which seems to be widespread inhuman cancers. [Cancer Res 2008;68(19):8113–21]

Introduction

The TROP2 (TACSTD2, GA733-1) gene is a functional retro-transposon of the TROP1 (TACSTD1, GA733-2) gene (1–3) thatencodes the Trop-2 cell surface calcium signal transducer (3–5).The TROP genes show highly conserved genomic structure (6) andexpression patterns (6, 7) across species, suggesting strongevolutionary pressure for conserved functional roles in theregulation of cell-cell adhesion (8).

Trop-1 is associated with epidermal cell proliferation, isinduced by cell transformation, and is frequently overexpressedby human carcinomas from the early stages of tumor development

(4, 6). Trop-1 is sufficient to stimulate the growth of expressingcells and is required for tumor growth in vivo (6). Overexpres-sion of TROP2 has been similarly observed in most humancancers (7, 9)4 and has been shown to potently stimulatetumor development.4 This stimulatory capacity depends on thepresence of an S303 protein kinase C phosphorylation site in thecytoplasmic region of Trop-2 and on intact signaling by proteinkinase C (10).4

CYCLIN D1 is a frequent site of chromosomal rearrangement inhuman B-cell tumors (11, 12). These chromosomal translocationsjuxtapose immunoglobulin gene enhancers to the CYCLIN D1 gene,thereby activating its transcription (13). Inversions of or deletionsin the CYCLIN D1 locus, e.g., in parathyroid adenomas (14), lead tothe prevalent transcription of a shorter mRNA. This 1.3-kb CYCLIND1 message contains the entire Cyclin D1 coding region but isdevoid of most of its 3¶ untranslated region (D3¶UT-CYCLIN D1).This D3¶UT-CYCLIN D1 mRNA has an increased half-life and showstransforming activity (15, 16). A third mechanism of activation ofCyclin D1 is the amplification of the CYCLIN D1 gene in epithelialcell tumors (breast, head and neck, bladder, ovarian, andesophageal cancers; refs. 17, 18).

Post-transcriptional RNA processing has a fundamental role incontrolling protein expression. Alternative splicing occurs in f70%of human genes (19) and results in the production of differentopen-reading frames (ORF) and/or in the modulation of expres-sion, e.g., if alternative exons include the 5¶ or 3¶ untranslatedregulatory regions. Canonical alternative splicing uses consensusdonor and acceptor sites (GT-AG). Aberrant alternative splicing,e.g., because of point mutations in cis-regulatory elements ordefects in the splicing machinery, has been shown to be frequentand is the basis of many inherited pathologies (20). It is alsofrequently found in cancer, where it may play a causative roleand can be associated with worse outcomes (21). Splicing can alsobe carried out by endonuclease complexes via spliceosome-independent mechanisms (22, 23).

Mature mRNA molecules can also be formed through intermo-lecular splicing, producing chimeric RNAs that are derived fromtwo unlinked loci in the absence of genomic recombination. Intrypanosomatids, a common 5¶ leader is spliced in trans onto gene-specific transcripts (24). Similar RNA processing has beendescribed in several other species (ref. 25 and references therein),including mammals (ref. 26 and references therein). Together withthe products of canonical trans-splicing, chimeric mRNAs have

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Current address for T. El-Sewedy: King Abdulaziz City for Science and Technology,Riyadh, Saudi Arabia.

Requests for reprints: Saverio Alberti, Unit of Cancer Pathology, Center forExcellence in Research on Aging, University ‘‘G. d’ Annunzio’’ Foundation, Via Colledell’ Ara, 66100 Chieti Scalo (Chieti), Italy. Phone/Fax: 39-0871-541-551; E-mail:[email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-6135 4 Manuscript in preparation.

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also been described that do not harbor conserved splice sites at thejunctions (27–29), raising the issue that the spliceosomalmachinery may not be involved in the generation of noncanonicalchimeric mRNAs.

In the present study, we show that the post-transcriptionaljoining of the TROP2 mRNA to CYCLIN D1 transcripts generates anovel, potent oncogene. The chimeric mRNA is shown to inducethe inappropriate overexpression of otherwise unaltered growth-regulatory proteins, such as Cyclin D1 and Trop-2. The CYCLIND1-TROP2 chimera induces the immortalization and transforma-tion of the expressing cells. We show that the chimera is frequentlyexpressed by human cancers and indicate that this novel oncogenicmechanism may be widespread in human tumors.

Materials and Methods

Cells. The OVCA-432, Ovcar-3, MCF-7, and ENAMI cell lines were grown

in RPMI 1640. The L, 293, and COS-7 cell lines were maintained in DMEM.

The medium was supplemented with 10% FCS (Life Technologies). Cellsfrom kidneys of 8-d-old Sprague-Dawley rats (baby-rat kidney cells; BRK

cells) were obtained and cultured as described (15). Peripheral blood

leukocytes were obtained by centrifugation of heparinized blood over Ficoll-

Hypaque density gradients. Keratinocytes were obtained from human skin(two cases) upon treatment with collagenase type IV immediately after

surgery. Keratinocyte RNA was extracted in Trizol (Sigma).

In vitro cell growth assays. MCF-7 cells transfected with the chimera

shRNA or control vector alone were seeded at 4 � 103 per well in 96-wellplates (six replica wells per data point). Cell numbers were quantified by

staining with crystal violet, as described (30).

Growth in soft agarose. Colony assays for growth of transformed cellsin soft agarose were done as described previously (31). Briefly, 3 � 104,

7 � 104, or 105 BRK transfectants or primary BRK cells were seeded in each

3.5-cm-diameter dish. Visible colonies of growing cells were scored weekly

in replica plates after staining with methylene blue.Tumorigenicity in nude mice. Tumorigenicity of transformed cells was

assayed by injecting 5 � 106 normal or RAS/chimera–transformed BRK cells

in the flank of 8-wk-old female C57/Bl6 nude mice (10 mice per group). The

longer and shorter diameters of each tumor were measured weekly and

used to calculate tumor volumes (32).Statistical analysis. The statistical significance of differences between

the numbers of foci in different experimental groups in the in vitro

transformation assays was assessed by m2 and Student’s t tests. The

statistical significance of the different percentages of expression of thechimera by different tumor histotypes was assessed by Fisher’s exact tests.

Results

Expression of a CYCLIN D1-TROP2 chimera by humancancer cells. A CYCLIN D1-TROP2 chimeric cDNA was isolatedfrom an OVCA-432 ovarian cancer cell library (SupplementaryFig. S1). RNase protection (Fig. 1A and B), reverse transcription-PCR (RT-PCR; Fig. 1C and D), sequencing of the amplified bands,and Northern blotting (see below) showed that this cDNAoriginated from a full-length hybrid CYCLIN D1-TROP2 mRNA thatis constitutively expressed by OVCA-432 cells (33). Consistently,CYCLIN D1-TROP2 mRNA chimeras could be obtained in vivo intransfected cells independently transcribing the two genes.5 On theother hand, amplification of reconstituted samples (in vitro mixingof RNA from cells separately expressing CYCLIN D1 and TROP2)did not lead to the generation of the chimera (Fig. 1D).

To determine if this CYCLIN D1-TROP2 hybrid mRNA wasexpressed by other cancer cells, MCF-7 cells, which express bothTROP2 and CYCLIN D1 (Fig. 1A), were analyzed for the presence ofthe CYCLIN D1-TROP2 chimera by RNase protection, RT-PCR, andsequencing. A hybrid mRNA was identified that was identical insequence to that from the ovarian OVCA-432 cells and that wasexpressed at similar levels (Fig. 1A). This chimeric CYCLIN D1-TROP2 message was not detected in Ovcar-3, ENAMI, and 293

Figure 1. Expression of the CYCLIN D1-TROP2 chimeraby human cancer cell lines, RNase protection (A and B ),or RT-PCR (C and D ). A and B, RNase protection.Arrows, the full 336-base protection by the chimera;arrowheads, protection by the canonical TROP2(219-base) or CYCLIN D1 (117-base) transcripts.B, COS-7/Chimera : COS-7 cells were transfected with afull-length CYCLIN D1-TROP2 chimera. This signalwas shown to exactly correspond to that of the nativeOVCA-432 and MCF-7 cancer cell lines. C, expressionof a full-length CYCLIN D1-TROP2 chimera in OVCA-432.Amplification with PRAD1.F2/T2.F5c (lane 1) andreamplification with PRAD1.F3/T2.F5tris (lane 2 ). Lane 3,control L-cell cDNA. CYCLIN D1 probe. The same bandwas highlighted by a TROP2 probe (not shown).Arrowheads, expected chimeric bands. D, lack ofgeneration of the CYCLIN D1-TROP2 chimera in in vitroreconstitution experiments. L/TROP2, L cells transfectedwith TROP2 ; mix, RNA from L cells transfected withTROP2 mixed with RNA from Ovcar-3 cells (thesenormally express CYCLIN D1 ); MCF-7 and OVCA-432,cell line RNA. The amplification products were hybridizedwith CYCLIN D1 or TROP2 probes. Arrowheads, theexpected chimeric bands.

5 Manuscript in preparation.

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Cancer Res 2008; 68: (19). October 1, 2008 8114 www.aacrjournals.org



human cancer cells, indicating a selective expression of thechimera by specific cancer cell types.

Sequencing of the RT-PCR–amplified regions did not reveal anysequence mutation or structural alteration of the CYCLIN D1 andTROP2 regions in the chimera (1, 34), at variance with canonical,somatically mutated oncogenes (35).

Lack of rearrangements between the CYCLIN D1 and TROP2loci. Chimeric transcripts in transformed cells are commonlygenerated by chromosomal translocations, and CYCLIN D1 is afrequent site of chromosomal rearrangement (14). Thus, we

investigated whether a translocation between the CYCLIN D1 andTROP2 loci was present in the cells expressing the chimera.

In the chimeric mRNA, the cleavage/poly-adenylation site inexon V of CYCLIN D1 (36) is joined to a full-length transcript fromthe intronless TROP2 gene (1). Hence, no introns were expected inthe putative chromosomal rearrangement region. We attempted aPCR amplification of a putative recombined chromosome usingprimers flanking the expected translocation site. No genomicCYCLIN D1-TROP2 fusion sequences were detected in either OVCA-432 or MCF-7 cells.

Figure 2. Transforming ability of the CYCLIN D1-TROP2 chimera in vitro. A, left, transfectants expressing the CYCLIN D1-TROP2 chimera at low levels(tet-repressed pUHD vector). Top, CYCLIN D1-TROP2 and activated Ha-RAS ; middle, vector-transfected cells; bottom, nontransfected cells. Arrows, clusters ofproliferating (top ) or single quiescent (middle, bottom ) rounded, refractile cells. Right, transfectants stably overexpressing the CYCLIN D1-TROP2 chimera. Top, fullytransformed, scattered epithelial cells; middle, epithelioid focus; bottom, fibroblastoid focus. Phase-contrast microphotographies, �40 objective. B, Northern blotanalysis of the levels of the CYCLIN D1-TROP2 chimera (3.1 kb) in pUHD in uninduced BRK transfectants. Constitutive 1/1,000 and 1/100: RNA from Cos-7 cellsexpressing the truncated CYCLIN D1-TROP2 cDNA (2.4 kb; ref. 39), diluted 1/1,000 or 1/100 with RNA from murine L cells; Tet-repressed: RNA from BRK cellstransfected with the full-length CYCLIN D1-TROP2 chimera in the pUHD vector and maintained in the presence of tetracycline. Filters were hybridized withthe PRAD1.F4/T2.F5bis probe at high stringency to prevent hybridization to the D3¶UT-CYCLIN D1 message. C, growth of nontransfected BRK cells and ofHa-RAS /D3¶UT-CYCLIN D1 or Ha-RAS/CYCLIN D1-TROP2 transfectants in soft agarose. D and E, tumors generated in nude mice by BRK cells transfected withactivated Ha-RAS and D3¶UT-CYCLIN D1 (left ) or CYCLIN D1-TROP2 (right ). D, individual growth curves of s.c. injected transformed BRK cells. E, BRK celltumor histopathology. High cellular densities with polymorphic fibrosarcomatous appearance and palisades of fusiform, transformed cells are observed.A pseudo-capsule is present.

The CYCLIN D1-TROP2 mRNA Chimera Is a Novel Oncogene




Southern blotting of genomic DNA after digestion at enzymesites close to the rearranged regions can efficiently reveal novelrearranged DNA fragments. However, no rearrangements weredetected by Southern blotting of OVCA-432 and MCF-7 cells(Supplementary Fig. S2A and B and data no shown).

However, a rearrangement could have occurred at a long distancefrom the CYCLIN D1 and TROP2 loci and could have generated afusion mRNA via the splicing of a long, intergenic transcript (33).This would escape detection by genomic PCR and Southernblotting. Thus, we performed interphase and metaphase fluorescentin situ hybridization (FISH) analysis of the CYCLIN D1 and TROP2loci (1, 14; Supplementary Fig. S2C and data not shown). FISHanalysis of metaphase spreads of OVCA-432 or MCF-7 cells localizedthe TROP2 (TACSTD2) gene to 1p32 and the CYCLIN D1 gene to11q13, i.e., at germ line sites (2, 14). No rearrangements between thetwo loci were detected in cancer cell metaphase spreads (notshown). FISH analysis of interphase spreads showed that OVCA-432cells possess three to four copies of CYCLIN D1 and two copies ofTROP2 per cell. MCF-7 cells were found to contain four copies ofCYCLIN D1 and two copies of TROP2 per cell. The controllymphocytes showed the expected two copies of CYCLIN D1 andof TROP2 per cell. No spots with overlapping hybridization to theCYCLIN D1 and TROP2 genes were detected in the cancer cellsexpressing the chimera (Supplementary Fig. S2C).

Taken together, these findings excluded a chromosomaltranslocation between the CYCLIN D1 and TROP2 loci in the cell

lines analyzed, indicating that the chimeric RNA originates fromintermolecular splicing, i.e., occurring in trans between twoindependently transcribed mRNAs.

Transformation of primary cells in culture. To assess thepossible functional relevance of low levels of expression of theCYCLIN D1-TROP2 chimera (i.e., those of cancer cell lines inculture), fresh BRK cells (15) were cotransfected with the pUHD-CYCLIN D1-TROP2 and mutated Ha-RAS and were continuouslymaintained in tetracycline (0.2–1 Ag/mL) to repress the conditionalexpression of the plasmids. Therefore, the transfected cells wereonly exposed to the minimal amounts of mRNA transcription thatescaped the tight tetracycline-mediated control (ref. 37; f1% ofthe levels of fully transcribing cells; Fig. 2). Strikingly, thesetransfectants were stimulated to proliferate and showed anextended life span (up to 3 months in culture; Fig. 2A). They alsoshowed a scattering activity and a characteristic refractile, roundmorphology that was typical of a subset of the CYCLIN D1-TROP2fully transformed epithelial BRK cells (Fig. 2A, top panels).

Human cancers show much higher levels of this hybrid CYCLIND1-TROP2 mRNA compared with cell lines (see below). Thus, weinvestigated whether a constitutive, high-level expression of theCYCLIN D1-TROP2 chimera could induce full cell transformation. Afull-length CYCLIN D1-TROP2 cDNA was transfected alone ortogether with an activated Ha-RAS and a retinoblastoma-binding-defective mutant E1-A (mE1-A) in fresh BRK cells (ref. 15; Table 1;Supplementary Table S1; Fig. 2). Effective transfection and

Table 1. Focus formation assays on transfected BRK cells

No. foci*

Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5

Chimerac

0 0 0 ND ND

D3¶UT-CYCLIN D1b

0 0 0 ND ND

TROP2x 0 0 0 ND ND

RASk 2 F 2 5 6 ND NDRAS/Chimera ND 14 F 2 20 F 6 28 F 9 10 F 5

RAS/D3¶UT-CYCLIN D1 ND 19 F 2 17 ND 14 F 6

RAS/CYCLIN D1 full length{ ND ND 0 0 NDRAS/TROP2 ND 0 0 ND ND

RAS/MUT1 ** ND ND ND ND 1 F 2

RAS/DcytTROP2cc

ND ND ND ND 3 F 2

RAS/Chimera-MUT1bb

ND ND ND ND 1 F 1RAS/D3¶UT-CYCLIN D1/TROP2 ND ND ND ND 10 F 5

RAS/D3¶UT-CYCLIN D1/MUT1 ND ND ND ND 4 F 1

RAS/D3¶UT-CYCLIN D1/DcytTROP2 ND ND ND ND 3 F 2

NOTE: The full table is presented in the Supplementary Table S1.

Abbreviation: ND, not determined.

*Number of foci F SD per 10 cm diameter plates seeded with 1 million BRK cells. Statistical significance was assessed by m2 (P < 0.01 for RAS/chimera

and RAS/D3¶UT-CYCLIN D1 versus all other groups in each experiment) and Student’s t tests (experiment 5: P < 0.05 for RAS/D3¶UT-CYCLIN D1 versusall other groups with mutated TROP2 and for RAS/chimera and RAS/D3¶UT-CYCLIN D1/TROP2 versus RAS/Chimera-MUT1 and RAS/MUT1).cCYCLIN D1-TROP2 chimera.bCYCLIN D1 , 1.3-kb message.xTROP2 .kActivated Ha-RAS .{CYCLIN D1 , 4.4-kb message.

**MUT1 , nonsense mutated TROP2 .ccTROP2 devoid of the cytoplasmic region.bbCYCLIN D1-TROP2 chimera with nonsense mutated TROP2 .

Cancer Research




expression of the constructs were determined by Southern,Northern, and Western blotting (Figs. 2B and 3). Two- to 3-foldhigher levels of Cyclin D1 and efficient expression of Trop-2 wereshown in these transfectants (Fig. 3). Nontransfected and vector-transfected BRK cells stopped growing and died after 3 to 4 weeksin culture, without generating foci. Only a few, short-lived, fociwere seen with mutated Ha-RAS alone (Table 1; SupplementaryTable S1). The wild-type Ha-RAS proved consistently ineffective(not shown). Only three groups of BRK transfectants generatedcontinuously growing foci: (a) CYCLIN D1-TROP2 and activated Ha-RAS , (b) D3¶UT-CYCLIN D1 and activated Ha-RAS , and (c) D3¶UT-CYCLIN D1 and TROP2 in trans and activated Ha-RAS . Theretinoblastoma-binding-defective mE1-A made no difference intransformation efficiency (Supplementary Table S1). The threegroups showed approximately the same efficiency of focusformation (Table 1; Supplementary S1). However, they showedevident differences at subsequent stages of tumor progression(in vivo growth) and distinct mechanisms of cell transformation(see below). Of interest, the focus-stimulatory activity of TROP2required intact signaling capacities, as the deletion of thecytoplasmic tail abrogated this effect (Table 1; Supplementary S1)as did a truncating mutation of the TROP2 ORF. On the other hand,cells transfected with the full-length CYCLIN D1 mRNA (thatincluded the 3¶UT) and Ha-RAS did not generate any foci.Correspondingly, they did not survive longer than nontransfected

or vector-transfected BRK cells, did not grow in soft agar, and didnot generate tumors in nude mice.

The pBJI-neo– or Rc/cytomegalovirus–driven expression con-structs induced foci with essentially identical efficiency. Cellstransfected with pBJI-neo expression constructs were selected ingeneticin-containing medium, generating stably expressing trans-fectants, as shown by flow cytometry and Western blotting.Notably, stable BRK transfectants showed the same number ofcolonies, corresponding transformed morphology, and similargrowth rates compared with ‘‘focus-forming cells’’ (i.e., BRKtransfectants only selected because of higher growth rate versusuntransformed cells). This excluded trivial expression artifacts(e.g., vector interference or influence of the selecting drug on cellgrowth) and confirmed that cell transformation was specificallycaused by the transfected genes.

Taken together, these findings show that low levels of theCYCLIN D1-TROP2 chimera induce growth and cause scattering ofnaıve primary cells in cooperation with Ha-RAS . Higher levels ofexpression of the hybrid mRNA (constitutive transcription from aviral promoter) together with Ha-RAS immortalize and transformprimary cells.

Growth in soft agarose. A reduced dependence of cell growthand survival from adhesion to a substrate is a hallmark ofcell transformation (31). Thus, we tested if CYCLIN D1-TROP2–transformed cells were able to grow in soft agarose. Nontransfected

Figure 3. Mechanisms of cell transformation by theCYCLIN D1-TROP2 chimera. A, expression levels ofTrop-2, Cyclin D1, and Ha-RAS in transfected BRK cells.Flow cytometry analysis for Trop-2. Inset, Western blotanalysis for Cyclin D1; Northern blot analysis for Ha-RAS.B, expression levels of Cyclin D1 in L cells transfectedwith the CYCLIN D1-TROP2 chimera. Western blotanalysis; the anti-Cyclin D1 antiserum is cross-reactivebetween mouse and man. Arrow, human Cyclin D1;arrowheads, ubiquitinated human Cyclin D1 (45); star,murine Cyclin D1. Molecular weight markers are indicatedon the left. C, CYCLIN D1 mRNA stability. TransfectedBRK cells were treated with actinomycin D. The RNAextracted at different time points after the treatment wasanalyzed by Northern blotting (inset ). Signal intensitieswere quantified and plotted over time.





and transformed BRK cell groups (Ha-RAS/chimera and Ha-RAS/D3¶UT-CYCLIN D1) were plated in soft agarose at different densitiesand the colonies were counted weekly (Fig. 2C ; Table 2). TheCYCLIN D1-TROP2 and D3¶UT-CYCLIN D1–expressing cells formednumerous, progressively growing colonies, whereas none weregenerated by nontransfected BRK cells.

These findings show that immortalized BRK cells expressing thechimera are truly transformed and grow under a reducedrequirement for adhesion to a substrate (31).

Tumorigenicity in nude mice. A pivotal characteristic of fullytransformed cells is their ability to grow as tumors in vivo . Thus, wetested the tumor-forming ability of CYCLIN D1-TROP2 by injectingtransformed BRK cells s.c. in nude mice. Tumors appeared in all ofthe animals injected with CYCLIN D1-TROP2 or D3¶UT-CYCLIN D1–expressing cells, whereas no tumors were generated by non-transfected BRK cells. Tumors expressing CYCLIN D1-TROP2 orD3¶UT-CYCLIN D1 showed similar take rates and latencies andreached similar maximum tumor volumes (Fig. 2D). However, theCYCLIN D1-TROP2–transformed cells showed a characteristicbiphasic growth, suggesting diversity in tumor growth controlmechanisms of the chimera versus D3¶UT-CYCLIN D1 . Tumorsappeared as malignant, polymorphic, and densely populatedfibrosarcomas in both groups (Fig. 2E). These findings show thatthe CYCLIN D1-TROP2–transformed cells are fully tumorigenicin vivo .

Inhibition of cell growth by a CYCLIN D1-TROP2 chimera–directed shRNA. A CYCLIN D1-TROP2 chimera–directed shRNAwas used to silence the CYCLIN D1-TROP2 chimera in MCF-7 cells.Transfection was performed on adherent cells and the growth oftransfectants was measured (Fig. 4). Marked inhibition of growthof MCF-7 cells was induced by the CYCLIN D1-TROP2 shRNA

(Fig. 4A). Death of shRNA transfected cells was also observed,suggesting that CYCLIN D1-TROP2 expression is essential for cellsurvival.

The levels of the CYCLIN D1, TROP2 , and chimeric mRNAwere quantified at 24 hours after transfection with the CYCLIN D1-TROP2 shRNA. No differences in the levels of bona fide CYCLIN D1and TROP2 mRNA were detected, whereas those of the CYCLIND1-TROP2 chimera were reduced essentially to zero (Fig. 4B ;Supplementary Fig. S3). Consistent with the absence of off-target effects, an shRNA for the CYCLIN D1-TROP2 chimeracontaining two mismatches was dramatically less effective in thesame assays.

Increased stability of the CYCLIN D1-TROP2 mRNA. TheCYCLIN D1 ORF in the chimeric RNA ends with its germ line stopcodon and is separated from the TROP2 ORF by an untranslatedregion (Supplementary Fig. S1). Thus, the chimera is a bicistronictranscript. As such, it was expected to generate bona fide CyclinD1 and Trop-2 proteins, rather than a fusion peptide, at variancewith most fusion oncogenes (13). Efficient translation of bothCyclin D1 and Trop-2 was, indeed, shown (Fig. 3A and B). Bothproteins showed correct molecular weights and subcellularlocation (Fig. 3A and B and data not shown), confirming thatthe CYCLIN D1-TROP2 chimera is a functional bicistronictranscript.

The 3¶UT of CYCLIN D1 regulates the half-life (t1/2) of thecorresponding mRNA (16, 36) through A/U-rich instabilitysequences (38). Thus, we argued that the chimeric mRNA mightbe more stable than the canonical CYCLIN D1 mRNA. Wemeasured the t1/2 of the CYCLIN D1-TROP2 chimera, the full-length CYCLIN D1 , and the D3¶UT-CYCLIN D1 mRNA inexpressing BRK cells (Fig. 3C). Remarkably, the t1/2 of the CYCLIND1-TROP2 chimera was 23 hours, compared with 15 minutesfor the full-length CYCLIN D1 and up to 5 hours for the D3¶UT-CYCLIN D1 mRNA. Notably, this 90-fold increase in stabilityversus the canonical, full-length CYCLIN D1 implies an inappro-priate expression of Cyclin D1 throughout the cell cycle (22 hoursin L cells). Consistent with the longer t1/2 of the chimeric mRNA,larger steady-state levels of mRNA and of Cyclin D1 protein werepresent in the CYCLIN D1-TROP2–transformed cells, comparedwith the D3¶UT-CYCLIN D1–transformed cells (Fig. 3A). The t1/2

of the TROP2 mRNA, as calculated by real-time PCR (Supple-mentary Fig. S4), was 19 hours (Supplementary Fig. S5), in goodagreement with the t1/2 of the CYCLIN D1-TROP2 chimera (Fig. 3).Together with the lack of early rebound of D3¶UT-CYCLIN D1mRNA levels (the paradoxical increase in mRNA levels observedshortly after the actinomycin D treatment), this suggested adominant stabilizing effect of the TROP2 sequences. Fourindependent stop codon mutants of TROP2 were generated togenerate non–membrane-anchored, inactive forms of Trop-2(Supplementary Table S2). All mutants showed lower stability/steady-state levels, possibly because of nonsense-mediated decay.Stop-codon–containing TROP2 sequences lost their stabilizingactivity versus the CYCLIN D1 mRNA (same t1/2 as the D3¶UT-CYCLIN D1 mRNA; Supplementary Fig. S6) and any transformingcapacity. However, as the Trop-2 protein was also disabled, acostimulatory capacity of the latter was left open. Hence, wegenerated a TROP2 deletion mutant devoid of the cytoplasmic tailand inactive in growth stimulation.6 The t1/2 of the deletion

Table 2. Colony formation assays of transformed BRKcells in soft agarose

No. colonies*

Cell numberc

Exp. 1 Exp. 2

BRKb

3 � 104 0 07 � 104 0 0

105 0 0

D3¶UT-CYCLIN D1/RAS/mE1-Ax 3 � 104 ND 140 F 14

7 � 104 210 F 30 258 F 21105 255 F 90 392 F 25

Chimera/RAS/mE1-Ak 3 � 104 76 F 10 163

7 � 104 133 F 10 179 F 26

105 216 F 22 329 F 40

Abbreviation: ND, not done.

*Number of colonies F SD per 3-cm-diameter plates visible 3 wk after

seeding.cNumber of cells seeded in each 3-cm-diameter plate.bNormal BRK cells.xBRK cells cotransformed with an activated Ha-RAS , mE1-A, and the

D3¶UT form of CYCLIN D1 .kBRK cells cotransformed with an activated Ha-RAS , mE1-A, and the

CYCLIN D1-TROP2 chimera.

6 Submitted for publication.

Cancer Research




mutant mRNA remained essentially that of the wild-type(Supplementary Fig. S6) but lost a costimulatory capacity(in trans with D3¶UT-CYCLIN D1 or full-length CYCLIN D1 ;Supplementary Table S1).

Together with the focus formation assays, in particular the lackof transforming activity by a short-lived CYCLIN D1 , these findingsindicate that a higher stability of the CYCLIN D1-TROP2 mRNAplays a key role in its oncogenic ability. They also indicate acostimulatory role of Trop-2.

Expression of the CYCLIN D1-TROP2 chimera by humantumors. Surgical specimens of human cancers were analyzedfor expression of the CYCLIN D1-TROP2 chimera by Northernblotting and RT-PCR (Supplementary Fig. S7 and S8; Supplemen-tary Tables S3 and S4).

The chimeric transcript was expressed at high levels by 17 ofthe 40 tumors analyzed (42%; Supplementary Fig. S7 and S8;Supplementary Tables S3 and S4). Marked heterogeneity in theexpression of the hybrid mRNA was seen. The highest frequency(71%) was observed in gastric cancers, whereas the lowestfrequency (9%) was observed in breast cancers (P < 0.0004). Sixty-two percent of the ovarian tumors and 50% of the colorectalcancers expressed the chimeric mRNA (P < 0.002 and P < 0.005versus breast cancers, respectively). Heterogeneity was observedamong tumor subtypes. A striking example is that of the

intestinal cell gastric cancers (Supplementary Fig. S7 and S8;Supplementary Tables S3 and S4), all of which expressed thechimera.

Interestingly, some tumors that expressed high levels of CYCLIND1 and TROP2 mRNAs did not express detectable levels of thechimera (1 colon, 1 stomach, 5 breast, and 2 ovarian cancers).Conversely, cells with low amounts of CYCLIN D1 and TROP2mRNA in several cases generated considerable amounts of thechimeric message (4 stomach, 1 breast, and 2 ovarian cancers;Supplementary Table S3; Supplementary Fig. S8B : as an example,compare tumor 30 with tumor 34 in Supplementary Fig. S7B). Anextreme case was that of breast cancers, where five tumors did notexpress the chimera despite high levels of CYCLIN D1 and TROP2mRNAs (P < 0.017 versus colon and P < 0.025 versus stomach). Onthe other hand, 4 of 7 stomach cancers generated chimeric mRNAin spite of barely detectable levels of CYCLIN D1 and TROP2mRNAs (P < 0.0025). These findings show that the expression ofthe CYCLIN D1-TROP2 chimeric mRNA is not solely dependent onthe levels of the CYCLIN D1 and TROP2 mRNAs and argue in favorof a regulated expression of the hybrid mRNA.

Of interest, 67% of the CYCLIN D1-TROP2 chimera–expressingtumors (which included 100% of the intestinal cell type gastriccancers) were aneuploid. On the other hand, no correlation wasdetected between the expression of the chimera and expression of

Figure 4. Inhibition of the growth of cancer cells by CYCLIN D1-TROP2 chimera shRNA. A, vector alone–transfected MCF-7 cells are in thin line/triangles. Cellstransfected with CYCLIN D1-TROP2 chimera shRNA are in thick line/squares. Bars, SE of cell number measurements. B, mRNA levels of CYCLIN D1, TROP2 , andCYCLIN D1-TROP2 chimera in the MCF7 shRNA transfectants, as measured by Sybr Green RT-PCR. Plots of emission intensity of the reporter (Rn ) versus PCRcycles are shown for the shRNA (y) and the vector-alone (�) MCF7 transfectants. 28S levels were used as an internal quantification standard.





Her-2, Bcl-2, and nuclear p53, nor between the fraction ofproliferating cells (percentage of cells in S phase and fraction ofcells expressing Ki-67), the neovasculature abundance or localinvasiveness (Supplementary Fig. S7), or expression of estrogen andprogesterone receptors (not shown).

As the chimeric CYCLIN D1-TROP2 mRNA cooperates withmutated RAS in inducing cell transformation, tumors wereanalyzed for activating mutations of Ha-RAS and Ki-RAS .Activating RAS mutations were found in gastric (1 of 7), colorectal(4 of 8), renal (1 of 3), and ovarian (1 of 8) cancers. No activatingRAS mutations were detected in breast cancers. Overall, about onethird of the tumors with activating RAS mutations also expressedthe CYCLIN D1-TROP2 chimera (data not shown).

Expression of the CYCLIN D1-TROP2 chimera by humannormal tissues. The expression of the CYCLIN D1-TROP2 chimerawas analyzed in normal colon, kidney, lung, pancreas, placenta,prostate, stomach, and uterus by real-time RT-PCR, and in freshkeratinocytes from human skin, fibroblasts, and peripheral bloodleukocytes by end-point PCR. No chimeric message was detected inany of the normal tissues analyzed, with the exception ofkeratinocytes, where it was highly expressed, as verified bysequencing of the amplified bands.

Discussion

In the present study, we have shown a novel mechanism oftransformation of human cells through the intermolecular splicingbetween independently transcribed CYCLIN D1 and TROP2 mRNAs(1, 14, 29, 39). At variance with most fusion oncogenes, the CYCLIND1-TROP2 chimera is not generated by a chromosomal transloca-tion, does not generate a chimeric protein, and functions asan efficient bicistronic transcript that independently translateswild-type Cyclin D1 and Trop-2 proteins. Consistently, no sequencemutations were revealed in either the CYCLIN D1 or TROP2moieties (1, 34), at variance with canonical, somatically mutatedoncogenes (35).

Minimal amounts of the CYCLIN D1-TROP2 chimera were shownsufficient to induce cell proliferation and to extend the life spanof senescent primary cells in culture. Higher levels of the CYCLIND1-TROP2 chimera fully transform primary, naıve rodent cells incooperation with activated RAS .CYCLIN D1 is a frequent site of chromosomal rearrangement

(11, 40). However, of interest, no evidence has been found to date ofa generation of chimeric proteins by the fusion of Cyclin D1 toheterologous partners on translocated chromosomes (12). Inver-sions of, or deletions in, the CYCLIN D1 locus have been observed(14) and lead to the prevalent transcription of a long-livedtranscript devoid of most of its 3¶UT (15, 16). Moreover, activationof Cyclin D1 can be driven by the amplification of the CYCLIN D1gene in epithelial tumors (17, 18). Our findings show a novelmechanism to achieve truncation and activation of the CYCLIN D1message by mRNA trans-splicing in the absence of chromosomalrearrangements.

An altered expression of a normal Cyclin D1, rather than thegeneration of an aberrant protein, leads to oncogenic activation(11). Indeed, overexpression of Cyclin D1 causes unrestrained cellgrowth in culture (41), full cell transformation (15), and inductionof tumors in transgenic animals (42). Consistently, our findingsindicate that overexpression of a wild-type Cyclin D1, as induced bythe CYCLIN D1-TROP2 chimera, plays a key role in inducingoncogenic transformation.

The higher stability of the mRNA and the functional role ofTROP2 appear both as important in this process. Indeed, thesplicing of the CYCLIN D1 mRNA to the TROP2 mRNA induces amarked stabilization of the former, much greater than thatcaused by the simple removal of the CYCLIN D1 3¶UT instabilitysequences. As the t1/2 of the CYCLIN D1-TROP2 mRNA isremarkably similar to that of the TROP2 mRNA, our findingssuggest a dominant stabilizing effect of TROP2 sequences. Thelong half-life of the CYCLIN D1-TROP2 mRNA is consistent withpersistence throughout the different phases of the cell cycle. Ofinterest, the loss of physiologic fluctuation of CYCLIN D1 levelsthrough the cell cycle is sufficient to cause cell transformation(41) and has been associated with malignancy in human tumors(18). The TROP2 mutagenesis and cotransfection assays, togetherwith the different characteristics of tumor growth in vivo , indicatethat the TROP2 moiety in the chimera also actively contributes tocell transformation. This contributing role of Trop-2 requires anintact structure of the molecule and of its signal transductionability.6

This CYCLIN D1-TROP2 mRNA is frequently expressed by humancancers. A striking example is that of intestinal cell type gastrictumors that expressed the chimera in all of the cases analyzed.Ovarian, endometrial, and renal tumors also frequently expressedthe chimeric mRNA. Several tumors, e.g., breast cancers, coex-pressed high amounts of the CYCLIN D1 and TROP2 mRNAs, butfailed to generate detectable amounts of the chimera. Othertumors, e.g., gastric cancers, generated high amounts of chimericmRNA in spite of barely detectable amounts of CYCLIN D1 andTROP2 mRNA, suggesting a regulated generation of the chimera.Chimeric mRNA was shown in a fraction of the tumors carryingRAS mutations. Thus, our findings are consistent with thepossibility of a functional interaction of the chimera with mutatedRAS in a subset of human tumors. A correlation between theexpression of the chimera and tumor aneuploidy was alsoobserved, suggesting a selective advantage at late phases of tumorprogression. This is consistent with the aggressive growth ofchimera-expressing BRK cell tumors in experimental animals. Ofinterest, normal tissues generally do not express the chimera,indicating a strong association between expression of the chimeraand cell transformation. A notable exception is that of freshkeratinocytes, which are the only normal tissue that was found todetectably express the CYCLIN D1-TROP2 chimera. Normalkeratinocytes are highly proliferating cells (43) that constitutivelyexpress TROP2 (44), suggesting these to be the fundamentalrequirements for the generation of the chimeric message, as intumor tissues. The role of TROP2 in the control of the growth andapoptosis of normal tissues is currently under investigation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 11/7/2007; revised 7/18/2008; accepted 7/30/2008.Grant support: Italian Association for Cancer Research (Italy), Fondazione of the

Cassa di Risparmio della Provincia di Chieti, Association for the Application ofBiotechnology in Oncology (ABO and ABO Project S.p.A., grant no. VE01D0019), andMarie Curie Transfer of Knowledge Fellowship-European Community’s SixthFramework Programme, contract number 014541.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank C.P. Berrie for the critical appraisal of the manuscript and L. Antolini forhelp with the statistical analysis.

Cancer Research






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2008;68:8113-8121. Cancer Res Emanuela Guerra, Marco Trerotola, Roberta Dell' Arciprete, et al. CancerDemonstrates a Novel Oncogenic Mechanism in Human

mRNA ChimeraCYCLIN D1-TROP2A Bicistronic

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a bicistronic cyclin d1-trop2 mrna chimera demonstrates a novel oncogenic mechanism in human cancer

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