Gene Expression Patterns Associated with the Metastatic ... · tor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones

[CANCER RESEARCH 61, 1569–1577, February 15, 2001]

Gene Expression Patterns Associated with the Metastatic Phenotype in Rodentand Human Tumors1

Andrea Nestl,2 Oliver D. Von Stein, Kurt Zatloukal, Wolf-Gerolf Thies, Peter Herrlich, Martin Hofmann, 3 andJonathan P. Sleeman4

Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics [A. N., O. D. V. S., W-G. T., P. H., M. H., J. P. S.] and University of Karlsruhe, Institute of Genetics [P. H.],D-76021 Karlsruhe, Germany, and University of Graz, Department of Pathology, Division of Experimental Cell Research and Oncology, A-8036 Graz, Austria [K. Z.]

ABSTRACT

Using subtractive technology, we have generated metastasis-associatedgene expression profiles for rat mammary and pancreatic adenocarcino-mas. Several genes whose expression is thought to be related to tumorprogression such as c-Met, urokinase-type plasminogen activator recep-tor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were therebyisolated. Half of the metastasis-associated clones showed no significanthomology to genes with known function. Notably, several of the metasta-sis-associated clones were also expressed in metastatic lines but not innonmetastatic lines of other tumor models. Furthermore,in situ hybrid-ization using selected clones documents the relevance of these results forhuman cancer because strong expression in tumor cells including metas-tases was detected in human colorectal cancer samples and, to a lesserextent, in mammary cancer samples. These data support the concept thattumors express a “metastatic program” of genes.

INTRODUCTION

A generally accepted principle is that tumor cells need to acquirealtered cellular properties to metastasize. The nature and number ofsuch properties will determine the range of secondary sites to whichthe tumor cells can spread. During progression to metastatic cancer,the development of heterogeneity within the primary tumor, drivenlargely by genomic instability (1), generates populations of tumorcells with the properties needed for invasion, migration, and meta-static colony formation in other organs (2). Not every tumor celldevelops all these properties because the ability to enter the circula-tory system or even to form micrometastases is insufficient for pro-gressive metastatic growth (reviewed in Refs. 2 and 3). The criticalfactors determining overt metastasis formation are whether dissemi-nated tumor cells die, survive at distant sites but remain dormant, orhave the properties required to grow progressively as metastases.Thus, tumor cells that have the necessary properties to metastasize areselected for during their metastatic journey.

The mechanisms leading to the dissemination of tumor cells, theroutes metastasizing tumor cells take through the organism, and theselection pressures tumor cells have to overcome are similar for manydifferent types of cancer (reviewed in Ref. 3). It is therefore reason-able to assume that tumor cells from different tumor types requirecommon properties and that they express similar metastasis-promot-ing genes. Attempts have been made to identify such genes usingdifferential screening techniques, each identifying one gene or a fewsuch genes (4–9). However, many outstanding fundamental questions

remain to be answered. How many genes are involved in the meta-static process? Are any of these genes absolutely required for metas-tasis formation? Conversely, can the same property required formetastasis be provided by several different genes or groups of genes?Due to the effects of genomic instability, to what extent are genes thatdo not play a role in the metastatic process up-regulated duringprogression to the metastatic phenotype? To begin to find answers tothese important questions, it would be necessary to describe andcompare the repertoire of genes specifically expressed in metastasiz-ing cells but not in their nonmetastasizing counterparts.

New technologies allow global descriptions of complex transcrip-tional changes associated with different tumor properties (10–12). Wehave used SSH5 coupled with a sensitive high-throughput screeningprotocol (13) to define the profile of genes whose expression isup-regulated during progression from a locally growing tumor tometastatic competence. Using two rat tumor progression model sys-tems, we have isolated and identified 268 different cDNAs represent-ing individual genes whose expression is exclusive to or up-regulatedin the metastatic phenotype (279 total clones, 11 of which were foundin both screens). These target cDNAs were further screened forexpression in other tumorigenic cell lines of both rat and humanorigin. In addition, human primary tumors and metastases of differenttissue origin were monitored for expression of selected cDNAs byinsitu hybridization studies. From our data, we conclude that subsets ofgenes are commonly up-regulated during metastatic progression andthat several of the genes we have newly identified as being up-regulated in metastasizing cells may be useful diagnostically or astherapeutic targets in the treatment of cancer.

MATERIALS AND METHODS

Tumor Samples and Cell Lines.The rat pancreatic cell lines BSp73-1AS,BSp73-10AS, and BSp73-ASML (14); the rat prostatic tumor cell lines G,AT-1, AT-3 AT-6, Mat-Lu, and Mat-LyLu (15); and the rat mammary carci-noma cell lines MTPa, MTC, MTLY, MTLN-2, and MTLN-3 (16) werecultured as described previously. NM081 and MT450, nonmetastasizing andmetastasizing rat mammary carcinoma cell lines, respectively (17), were bothmaintained in DMEM supplemented with 10% FCS and antibiotics. Twohuman cell lines, MDA-MB-231 and MDA-MB-468, were obtained fromAmerican Type Culture Collection (American Type Culture Collection num-bers: MDA-MB-231, HTB-26; MDA-MB-468, HTB-132). All human tumorsamples and corresponding normal tissue used for thein situ hybridizationwere obtained from the Institute of Pathology, University of Graz (Graz,Austria).

Subtractive Library Construction and Screening. The construction andscreening method of the subtracted mammary carcinoma library used in thisstudy was essentially identical to that used in Ref. 13, using the Clontech SSHkit (PCR-Select cDNA Subtraction Kit; Clontech Laboratories, Palo Alto, CA).Briefly, the subtracted cDNA library was amplified and TA cloned. Cloneswere picked, and their inserts were amplified by colony PCR. Equal amountsof the PCR reactions were electrophoresed on duplicate high-density gels. Gels

Received 7/31/00; accepted 12/15/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by a grant from the Mildred-Scheel Stiftung and Grant S7401-MOBfrom the Austrian Science Fund (to K. Z.)

2 Present address: LYNX Therapeutics GmbH, Im Neuenheimer Feld 515, D-69120Heidelberg, Germany.

3 Present address: LION Bioscience AG, Im Neuenheimer Feld 515, D-69120 Heidel-berg, Germany.

4 To whom requests for reprints should be addressed, at Forschungszentrum Karlsruhe,Institute of Toxicology and Genetics, P. O. Box 3640, D-76021 Karlsruhe, Germany.Phone: 49-7247-826069; Fax: 49-7247-823354; E-mail: [email protected].

5 The abbreviations used are: SSH, suppression subtractive hybridization; ML, mam-mary carcinoma-specific library; PL, pancreatic carcinoma-specific library; poly(A)1

RNA, polyadenylated RNA; EST, expressed sequence tag.; SAGE, serial analysis of geneexpression; uPAR, urokinase-type plasminogen activator receptor; ER, estrogen receptor.

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were blotted and individually probed with driver and tester cDNA to identifydifferentially expressed clones.

PCR Amplification. All clones were amplified by colony PCR using 96-wellmicrotiter plates as follows. Fiveml of each bacterial culture were added to 95mlof sterile water and denatured for 5 min at 94°C. Amplification of 5ml of thislysate was performed in a total volume of 100ml using standard PCR buffer(Eurobio), 250mM deoxynucleotide triphosphates, 10 pM each of primer PN1 andPN2, and 1 unit of Taq (Eurobio). The primers used were the inner primers of theClontech SSH-Kit: (a) PN1, 59-TCGAGCGGCCGCCCGGGCAGGT-39; and (b)PN2, 59-AGGGCGTGGTGCGGAGGGCGGT-39.

Cycle conditions were as follows: 30 cycles of 94°C for 20 s, 68°C for 12 s,and 72°C for 30 s. Products were purified from agarose gels using DNAeasy(Biozyme) and used for Northern analysis.

Northern Analysis. Poly(A)1 RNA was isolated from cell lines by stand-ard methods, and 2-mg aliquots were size-fractionated on a 1.0% formalde-hyde-agarose gel. Ethidium bromide staining was used to ensure equivalentloading in each lane. Gels were blotted onto Hybond N1 membrane filters(Amersham). The filters were then cross-linked (UV Stratalinker 2400; Strat-agene) and hybridized at 65°C in Church buffer [1 mM EDTA, 0.5M NaHPO4

(pH 7.2), and 7% SDS]. Probes were generated by PCR amplification of thetarget clones, followed by gel purification and32P-labeling of the cDNAfragment (ReadyPrime; Amersham). Unincorporated label was removed beforehybridization using an Elutip (Schleicher & Schull) according to the manu-facturer’s specifications. After hybridization with the labeled probes, mem-branes were washed twice in 23SSC and 0.1% SDS and twice in 13SSC and0.1% SDS at 64°C, after which they were exposed to film for between 2 h and8 days. Filters were stripped and subsequently probed with another gene to beanalyzed. Alternatively, stripped filters were finally hybridized with a glycer-aldehyde-3-phosphate dehydrogenase probe to ensure the presence of equiva-lent amounts of poly(A)1 RNA in each lane of a filter and to ensure that thehybridization patterns of different filters could be compared.

Computational Analysis. All computational analyses concerning the iden-tity of the clones obtained through the subtracted libraries were performedusing the blastn sequence similarity search.6 The clones were compared withthe sequences contained in the human and rodent public domain databases,including all of the nonredundant GenBank, European Molecular BiologyLaboratory, DDBJ, and PDB sequences, and the DDBJ EST divisions. Beforecomparative blast screens, we developed a program that automatically recog-nizes and removes the flanking nested primers to compare only the inserts andthus avoids false blastn results. In the first round of comparison, all of thesequences were compared with each other to identify fragments isolated

several times (18). In the next round, the sequences were compared withhuman and rodent sequences in the public domain databases (19).

In Situ Hybridization. In situ hybridization was performed to detect S7and CD24 expression in human tumor material. The inserts to be tested werecloned into the bidirectional TA vector pGEM-Teasy (Promega) and tran-scribedin vitro using SP6 or T7 RNA polymerase (Life Technologies, Inc.).Sections (4mm thick) of paraffin-embedded human tumor samples weremounted on silanized glass slides, deparaffinized, and postfixed in 4%paraformaldehyde in PBS for 20 min at room temperature. After rinsing withTBS [50 mM Tris-HCl (pH 7.5) and 150 mM NaCl], sections were treated with0.2 M HCl for 10 min, and, after washing in TBS, incubated in 20mg/mlproteinase K (Sigma, St. Louis, MO) in TBS containing 2 mM CaCl2 for 15min at 37°C. The reaction was stopped with TBS for 5 min at 4°C. Aftertreatment with 0.5% acetic anhydride in 100 mM Tris (pH 8.0) for 10 min,sections were rinsed with TBS, dehydrated in graded ethanol, and air dried.Hybridization with fluorescence-labeled (RNA color kit; Amersham Life Sci-ence) sense and antisense RNA probes was performed according to themanufacturer’s instructions. After H&E counterstaining, the sections wereanalyzed by light microscopy. Thein situ hybridizations were performed with10 mammary carcinomas (invasive ductal carcinomas including lymph nodemetastases from four of the tumors), 5 samples with benign fibrocystic disease,and 10 colorectal carcinomas (including the corresponding nonneoplasticmucosa from the resection margins and lymph node metastases from fourtumors). The carcinomas were staged according to UICC guidelines (20).

RESULTS

Choice of Experimental System.We set out to define sets ofgenes up-regulated or exclusively expressed in metastatic humantumors. Instead of starting directly from human tumor material, wechose to analyze defined clonal rodent tumor cell lines (14, 16). Thedisadvantage of this approach is that contributions to metastasis bystromal cells and transcripts induced by the interaction of tumor cellswith their environment are lost. This is outweighed by numerousadvantages. Tumor cell lines often exhibit a reproducible metastatic ornonmetastatic phenotype that can be retested at any stage of theanalysis (e.g.,Ref. 17). Moreover, tumor cell lines are accessible togenetic manipulation and functional tests in experimental animals. Rattumor cells have the advantage of being able to be passaged insyngeneic animals, whereas human tumor cells have to be passaged inthe rather artificial setting of an immunodeficient host. Furthermore,the expected high degrees of homology between rodent and humansequences should permit subsequent isolation of human homologuesof candidate tumor progression genes and evaluation of their expres-sion in primary human tumor material.

For the molecular comparisons, we used two rat carcinomamodels. The rat pancreatic adenocarcinoma model is comprised ofseveral clones that differ in their metastatic potentialin vivo andhave been derived from a common primary tumor (14). For exam-ple, BSp73-1AS cells form primary tumors but do not metastasize,whereas BSp73-ASML cells are highly metastatic and, after s.c.injection, disseminate via the lymphatics to finally colonize thelungs. The rat mammary adenocarcinoma cell system 13762NF(16) is composed of a number of cell lines derived from a parentalmammary tumor and its corresponding spontaneous lung andlymph node metastases. For example, the cell line MTPa has beenreported to be nonmetastaticin vivo in syngeneic animals, whereasMTLY is highly metastatic, giving rise to multiple metastases inthe lymph nodes and lungs (16).

Before subtractions, the phenotypes of the cell lines were verifiedby s.c. injection of each cell line into syngeneic animals (Table 1). Inall cases, 100% of animals developed tumors after injection of5 3 105 cells. MTPa and BSp73-1AS did not metastasize at all; theMTLN cell lines and BSp73-ASML grew in both the lymph nodes andlungs, whereas MTLY and BSp73-10AS appeared to prefer the6 See http://www.ncbi.nlm.nih.gov/BLAST/for details.

Table 1. Verification of the in vivometastatic potential of the rat cell lines used

A total of 5 3 105 viable cells in PBS were injected s.c. into the flank of each animal.Three to nine animals were used for each cell line, and the animals were maintained untilthe animals became moribund, or the primary tumor had reached the legal limit as set byGerman animal protection regulations. The animals were then sacrificed, and autopsy wasperformed. Metastases were noted and scored.

Tissue type Cell linePrimarytumor

Animals withlymph nodemetastases

Animals with lungmetastases

Mammary tumor cell linesMTPa 3/3 0/3 0/3MTC 6/6 1/6 2/6MTLN-2 6/6 0/6 2/6MTLN-3 6/6 2/6 2/6MTLY 6/6 6/6 1/6NM081 8/8 0/8 0/8MT450 8/8 8/8 8/8

Pancreatic tumor cell lines1 AS 8/8 0/8 0/810 AS 8/8 7/8 1/8ASML 8/8 7/8 5/8

Prostatic tumor cell linesG subline 8/8 0/8 0/8AT-1 8/8 2/8 6/8AT-3 8/8 1/8 7/8AT-6 9/9 6/9 7/9MatLu 8/8 2/8 7/8

MatLyLu 8 /8 4/8 6/8

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METASTASIS-ASSOCIATED GENE EXPRESSION PROFILES



lymphoid organs. Despite extensive investigation of many organs,metastases were only observed in the lymph nodes or lungs.

Subtractive Cloning of Differentially Expressed Genes.Fromthe cell lines examined and documented in Table 1, we decided tocompare the gene expression profiles of pairs of tumor cells with themost extreme metastatic behavior. Transcripts of the nonmetastaticmammary tumor cell line MTPa were subtracted from those of themetastatic cell line MTLY (this library was denoted ML). The non-metastatic pancreatic cell line BSp73-1AS was subtracted from itsmetastatic counterpart BSp73-ASML (this library was denoted PL).Both subtractive libraries were generated and screened as describedpreviously (10, 13). A total of 1985 and 5000 clones were screenedfrom the mammary and pancreatic tumor libraries, respectively, re-sulting in the isolation of 487 and 494 differentially expressed cDNAclones, which, through sequence analysis, equated to 160 and 119individual genes, respectively7 (Table 2A). Eleven of these geneswere found in both the mammary and pancreatic tumor libraryscreens. Thus, a total of 268 different cDNAs were isolated in the twoscreens. A complete list of all clones obtained will be accessible viaan internet web page.8

As indicated in Table 2, the clones of both carcinoma-specificlibraries were grouped according to their degree of sequence homol-ogy to the described public domain human and rodent genes (18, 19).Sixty-five clones identified from the ML subtraction and 43 clonesidentified from the PL subtraction represent described rat genes (seeTable 2B); an additional 25 ML and 16 PL clones share significanthomologies with known genes from other species, and some of themshow homology to rat genes and could therefore encode new ratfamily members. All sequences with a homology of less than 70% topreviously described genes can be separated into those that demon-strate significant homology to already existing ESTs and those thatshow little or no significant match to any sequence in the publicdatabases. Clones with less than 70% homology to previously de-scribed genes have been classified as novel. On this basis, 70 of 160(ML) and 60 of 119 (PL) clones represent previously unidentifiedsequences.7

By probing Northern blots with randomly selected clones, we

convincingly verified that the SSH method does indeed identifydifferentially expressed genes (Fig. 1). The normalization step of SSHenriches for rare transcripts and thereby allows the identification ofboth high and low abundance transcripts. As indicated in Fig. 1,clonesC andB isolated from the PL and ML, respectively, representgenes expressed at low levels. Conversely, clonesF (PL) andL (ML)represent transcripts of high abundance.

Cancer cells from different tumor types likely require commonproperties during metastasis, and therefore recurrent patterns of me-tastasis-related gene expression should be found. Indeed, within thelist of metastasis-related cDNAs for which sequences are already inthe databases, we find gene products that have been described previ-ously by others as associated with tumor progression (21–51).8 Fur-thermore, several genes were identified as being metastasis associatedin both the PL and ML subtractions (Fig. 2). Moreover, several genesisolated in SAGE screens to identify human colonic and pancreatictumor-specific genes were also found in the ML SSH and PL SSHlibraries (Fig. 2).

Expression of cDNA Sequences in Several Metastatic TumorCell Lines. To determine to what extent metastatic tumor cells ofdifferent origin share a basic set of gene products expressed in themetastatic phenotype, we analyzed the expression of randomly se-lected clones in several tumor progression models (Fig. 3). For thispurpose, two additional tumor systems were used, namely, a pair ofmammary tumor cell lines and a group of related prostatic cancer celllines (15, 17). The metastatic behavior of these tumor cell lines wasverified by in vivo passaging (Table 1). Additionally, two humanMDA-MB cell lines reported to be metastatic in immunocompromisedmice (52, 53) were included in the analysis.

By direct Northern analysis, we found that several of the se-lected clones were differentially expressed in metastasizing celllines from tumor models other than the model from which theclones were originally isolated. cDNA clones whose expressioncorrelates with metastatic potential in more than one tumor modelare the most likely candidates for general relevance in the meta-static process. Genes shown to be differentially expressed in allfour systems tested were CD24, S7, B23, oncomodulin, and HB2.Genes clearly differentially expressed but not correlating withmetastatic potency in all tumor models tested were choline kinase,megakaryocyte potentiating factor, metastasis-associated factor,c-Met, mirf 6, cytokeratin 8, uPAR, HMG-1, and the novel clones

7 It is possible that some of the novel sequences identified originate from the samegene. We have assumed that each novel EST we identified corresponds to one gene. Themost recent database search was performed on November 24, 2000.

8 http://igtmv1.fzk.de/www/itg/sleeman/sleeman.html.

Table 2. Statistical sequence analysis

A.

ML PL

Clonesscreened

Diff. expressedclonesa

% Diff. expressedclones

Diff. expressedgenes

Clonesscreened

Diff. expressedclones

% Diff. expressedclones

% Diff. expressedgenes

1985 487 25% 160 5000 494 10% 119

B.

Database blast search for described human and rodent genes

For the 160 mammary carcinoma metastasis-specific genes For the 119 pancreas carcinoma metastasis-specific genes

.90 % 70–90% .90 % 70–90% ,70 %65 (41%) 25 (16%) 70 (44%) 43 (36%) 16 (13%) 60 (50%)

C.

Database blast search for ESTs

For the 70 sequences,70% For the 60 sequences,70%

.90 % 70–90% 90 % 70–90% 70 %33 (47%) 20 (29%) 17 (24%) 30 (50%) 11 (18%) 19 (32%)

a Diff., differentially.

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23, 25, 56, 62, 63, 82, 154, 162, and 163. The 26 clones used forthis analysis represent only 10% of all of the clones identified andwere randomly selected. The remaining 242 as yet untested genesare expected to show a similar degree of correlation betweenexpression and metastatic potential.

Selected Examples of the Metastatic Gene Expression Patternin Human Primary and Metastatic Carcinomas. The ultimate testfor the validity of our differential isolation approach is the exclusiveor strongly enhanced expression of candidate clones in human cancer,which we tested byin situhybridization analysis. We concentrated ourstudy on two cDNA clones, CD24 and S7, which showed the bestcross-hybridization to human sequences (Fig. 3) and are highly ex-pressed in the metastatic rat cell lines tested.

In situ hybridizations were performed on tumor sections fromprimary tumors as well as from corresponding metastases andnonneoplastic tissue from colorectal and breast carcinomas. In thecolorectal carcinomas, CD24 mRNA expression was weakly de-tectable in normal colonic mucosa but highly abundant in tumorcells and, to a lesser extent, in the cells of the tumor stroma (Fig.4). No marked difference in the expression levels was foundbetween the primary tumors and the corresponding lymph nodemetastases. S7 was very weakly expressed in normal colorectalmucosa and was highly abundant in colorectal tumor cells. Therewere no marked differences in the expression levels of S7 betweenthe primary tumors and the corresponding lymph node metastases.There was, however, a striking difference in the expression be-tween S7 and CD24 in that CD24 was not only overexpressed inthe invasive carcinoma but was also overexpressed in the adjacentnoninvasive areas with mild atypia (compare Fig. 4,j–l). Further-more, the expression of CD24 in the tumor stroma was morepronounced than the expression of S7.

In the breast carcinomas, CD24 expression levels were essentiallycomparable in the nonneoplastic mammary parenchyma and in thecarcinoma cells of the primary tumors as well as the correspondingmetastases (Fig. 5). In general, the hybridization signals obtained forS7 were much weaker than those obtained for CD24. In comparisonwith nonneoplastic mammary gland parenchyma and primary carci-noma, a moderate overexpression of S7 was seen in lymph nodemetastases (Fig. 5).

Thus, genes whose expression we identified as being metastasisassociated in several rodent tumor systems also show a tumor stage-dependent expression behavior in the human cancers investigated,particularly those of colorectal origin, proving the validity of ourapproach.

DISCUSSION

It is clear from many studies that tumor cells that can metasta-size have gained properties not possessed by their nonmetastasiz-ing counterparts. These additional properties must be reflected inchanged gene expression patterns. However, it is completely un-known how many and which genes are differentially expressedbetween metastasizing and nonmetastasizing cells from a giventumor. Furthermore, there is no indication as to what extent thesedifferentially expressed genes are absolutely required for metasta-sis formation by other tumor types or to what extent there isredundancy in the gene expression patterns that lead to metastaticcompetence. Here we present the first reported attempt to describeand compare the complete sets of genes expressed in metastasizingbut not in nonmetastasizing tumor cells.

From our experiments, we conclude that metastasizing tumor cellsfrom different tumor types up-regulate many common genes. Firstly,many of the known genes isolated in the PL and ML screens havepreviously been associated with tumor progression. Secondly, theNorthern blots in Fig. 3 show that many of the differentially expressedclones from the PL and ML screens are also expressed in a metastasis-specific fashion in other model systems. Thirdly, the two genesselected forin situ hybridization analysis are strongly up-regulated inhuman tumor material (Figs. 4 and 5). Fourthly, several genes wereidentified as being metastasis associated by both the PL and MLsubtractions.

We also conclude that metastasizing tumor cells up-regulatelarge pools of genes. Genes identified as being metastasis associ-ated in both the PL and ML subtractions were comparatively fewin number (a total of 11). This could be interpreted to mean thatmammary and pancreatic cancer cells use widely divergent subsetsof genes to metastasize or that there is extensive redundancy in therequirement of genes whose expression is associated with metas-tasis formation, either because the same property can be providedby many different genes or groups of genes or because many genesare up-regulated but not necessary for metastasis formation. How-ever, other data suggest that the yield of clones differentiallyexpressed in our subtractions was not exhaustive. Randomly se-lected clones identified as being expressed in a metastasis-specificmanner in the ML but not PL subtraction were used to screenNorthern blots of the pancreatic tumor cells (Fig. 3). Here, 12 of 16clones proved to be up-regulated in the metastatic BSp73-ASMLbut not in the BSp73-1AS nonmetastatic pancreatic carcinomacells. Similarly, seven of seven clones from the PL screen that werenot isolated in the ML screen were nevertheless more highlyexpressed in the metastasizing MTLY mammary carcinoma cells

Fig. 1. Northern analysis of a number of positively identified clonesfrom the ML and PL screens. To verify that the sequences isolated inthe two SSH screens were indeed differentially expressed between thenonmetastasizing cell line and its metastasizing counterpart, we testedrandomly selected cDNAs by direct Northern analysis. Each lane wasloaded with 2mg of poly(A)1 RNA. The poly(A)1 RNA was isolatedfrom MTPa and MTLY cells (ML) or from BSp73-1AS, BSp73-10AS,and BSp73-ASML cells (PL). The blots were hybridized with the testcDNAs (B–L) or with glyceraldehyde-3-phosphate dehydrogenase (A),which serves as a loading control. For the ML library:B, HB2;C, mirf6; D, novel clone 56;E, B 23;F, IQGAP2;G, novel clone 163;H, novelclone 154;I, novel clone 155;J, novel clone 159;K, cytokeratin 19; andL, novel clone 165. For the PL library:B, novel clone 75;C, novel clone100,D, novel clone 135;E, caveolin-1;F, testin 2;G, uPAR;H, novelclone 72; I, ezrin; J, HMG-1; K, Src SH3-binding protein; andL,calgizzarin.

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than in the nonmetastasizing MTPa cells. Thus, although we iso-lated many genes as being expressed in a metastasis-specificmanner in the PL (119 genes) and ML (160 genes) screens, weanticipate that many more genes are also differentially expressed inmetastatic cells of the rat mammary and pancreatic carcinomamodels. This suggests that metastasizing tumor cells up-regulate alarge pool of genes compared with their nonmetastasizing coun-terparts.

On the basis of our current data, we cannot draw any firmconclusions about the degree of redundancy in gene expressionrequired for metastatic competence. There clearly is redundancy in

the genes that are up-regulated during metastatic progression be-cause several of the clones from the PL and ML screens were notsignificantly up-regulated in a metastasis-specific manner in alltumor models or were even down-regulated (e.g., uPAR, novelgenes 23 and 82; see Fig. 3). This is to be expected: for everyqualitative genetic change that is advantageous for metastaticprogression, there will be many others that are either deleteriousfor the tumor cell or neutral in terms of conferring metastaticproperties on the tumor cell. Furthermore, some genes are likely tobe up-regulated in a cell type-specific manner.

Several laboratories have made attempts to screen for cancer-associated transcripts using different methods [differential display,representational differentational analysis, SAGE, and SSH (10,54 –57)]. Of those techniques capable of efficient expression pro-filing, SAGE has the advantage that even moderate changes intranscription can be detected, but it suffers from the fact that thesequence information it produces is only 13-bp long. Not only isthis problematic for nucleotide sequence comparison algorithms,but it is also not ideal for use in techniques such asin situhybridization and Northern blotting. On the other hand, subtractivecloning techniques such as SSH yield cDNA fragments of 200 –800 bp, which overcomes the problems associated with SAGE.Furthermore, SSH enriches differentially expressed genes withhigh efficiency irrespective of their relative abundance. The com-bination of SSH with high-throughput screening methods, forexample, using microarrays (58), provides a powerful method forthe rapid identification of differentially expressed genes. SSHsuffers from the drawback that it does not readily detect transcrip-tional differences less than 5-fold. SAGE and SSH have never beencompared directly using the same cell system. However, the datapresented in this study verify that SSH is a valuable tool foridentifying differentially expressed genes in tumors and othercellular contexts.

Because the data presented here represent the first extensive de-scription of genes expressed specifically in metastasizing cells, com-parison with previous differential screens is informative but needs tobe interpreted with caution. The only other extensive descriptions ofgenes up-regulated in tumors used differential hybridization to ar-rayed cDNAs, SAGE, or SSH. These studies either compared normaltissue with tumor material or ER-positive cells with ER-negative cells,and no investigation of metastasis-related gene expression has beenmade (11, 58–60) When the differentially expressed sequences inthese analyses are compared with the sequences identified in the MLand PL screens, few of the sequences are identical. This is illustratedby a comparison of human pancreatic tumor-specific genes identifiedby SAGE with metastasis-specific genes expressed in rat pancreatictumor cells identified by SSH (Fig. 2). At first sight, this wouldsuggest that many of the genes identified by SAGE as being up-regulated in the tumor material do not contribute to the metastaticphenotype but rather are concerned with tumorigenesis. However, itshould be borne in mind that the different screens do not constitute anexhaustive list of up-regulated genes. Furthermore, these comparisonsare preliminary because ESTs are not always made accessible in thedatabases, and identities may be overlooked if the ESTs are short.

With respect to molecular functions of the genes we report to beexpressed in a metastasis-specific fashion, several of the knownsequences isolated in the PL and ML screens have previously beenreported to be involved in the process of metastasis. For example,c-Met is the receptor for hepatocyte growth factor/scatter factor. Alarge body of evidence implicates this receptor-ligand pair in theprocesses of tumorigenesis, invasion, migration, and metastasis (re-viewed in Ref. 61). Interestingly, another gene identified as beingup-regulated in metastasizing cells is ezrin, a cytoplasmic substrate for

Fig. 2. Sequence comparison between different screens.A illustrates a comparison ofthe genes isolated in the ML and PL screens presented in this study. Only 10 genes wereisolated in both screens.B demonstrates the comparison between the SAGE screen fortumor-related genes in colon and pancreatic cancer (11) and our SSH analysis, based onthe sequences published on the SAGE web site (http://welchlink.welch.jhu.edu). ESTshave been excluded inB because EST accession numbers have not been made accessiblein the databases.C lists the identity of the genes that were found in more than one of thescreens analyzed inA andB.

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c-Met required for hepatocyte growth factor-mediated morphogenesiswhose expression is associated with the metastatic phenotype (62, 63).We also isolated uPAR in our screens, which binds urokinase-typeplasminogen activator and is believed to be essential for activation ofnumerous proteases involved in invasion (64). As a further example,elevated caveolin protein levels have been associated with humanprostate carcinoma progression (65), and we report here that caveolinis a gene up-regulated in metastatic cells.

The data presented here would suggest that some of the clones wehave identified as being expressed in a tumorigenesis-related fashioncould also prove to serve as new markers for further elucidation of themolecular events in the dysplasia-carcinoma sequence. Others mayserve as targets for therapeutic intervention. An impressive correlationbetween expression and metastatic potential was seen with CD24, S7,and several novel clones. CD24 is expressed on B-cell precursors,neutrophils, neurons, and keratinocytes (66, 67), and its expressionhas not previously been associated directly with metastasis. However,it is expressed on leukemic cells of the B-cell lineage and on varioussolid tumors such as small cell lung carcinoma, neuroblastoma, rhab-domyosarcoma, and renal cell carcinoma (68–71). CD24 was alsoidentified by a SSH screen to identify genes differentially expressedbetween ER-positive and -negative cells lines (58). Significantly,

reduction or absence of CD24 expression on acute lymphoblasticlymphoma cells predicts a better prognosis for patients (72). As asurface protein able to bind to P-selectin, CD24 could promote me-tastasis formation by mediating binding of circulating tumor cells toendothelium (32).

Although several ribosomal components have been reported to beup-regulated in cancer, S7 has not previously been associated withtumor growth and metastasis. It is a component of the translationmachinery, and its RNA binding properties are required for the correctfolding of the 16S rRNA (73). Our observations call for a novelfunction for S7. Enhanced S7 expression in tumor cells is not simplya correlate of enhanced proliferation rate because only very weakexpression is observed in the highly proliferative colonic mucosa. Incolorectal carcinomas, we observed that S7 is homogeneously ex-pressed and up-regulated specifically in invasive carcinomas andmetastases. Moreover, S7 expression is also clearly enhanced inmetastases of the mammary carcinomas we investigated. Our datasuggest that S7 may be useful in certain instances as a marker fortumor staging and indicate that additional experiments are warrantedto investigate whether S7 plays a functional role in invasive tumorgrowth.

In conclusion, this study is the first reported attempt to describe the

Fig. 3. Comparison of expression profiles of novel genes and known tumor-associated molecules in cell lines of differing metastatic potential. The figure is composed ofNorthern blots from four different rat tumor systems and two human breast carcinoma cell lines. Except for the NM081/MT450 pair, all of the tumor systems used are comprisedof clonal cell lines derived from a common primary tumor that differ only in their capacity to metastasize (see Table 1). This metastatic potential is indicated. Each lane onthe Northern blots was loaded with 2mg of poly(A)1 mRNA. The blots were hybridized with various differentially expressed cDNAs isolated in the mammary (A) and pancreatic(B) SHH screens. The hybridization probes used were randomly chosen from the metastasis-associated genes isolated in the ML and PL screens and represent 10% of the isolatedgenes in each case. A rough classification (correlative index) is given to describe the correlation between expression of the gene and the metastatic potential of the cell linesinvestigated. Genes whose expression is up-regulated in all metastatic cell lines and absent or significantly reduced in all nonmetastatic lines were scored111. Genes thatare clearly differentially but not exclusively expressed in metastatic cells were arbitrarily scored11 or 1, depending on the strength of the correlation of expression withmetastatic potential.C, representative example of ethidium bromide-stained RNA gels before Northern blotting to demonstrate equivalence of loading.

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profile of genes expressed specifically in metastasizing tumor cellsusing a direct comparative method and proves that SSH is a usefultool in identifying differentially expressed genes. We demonstrate thata large number of genes are up-regulated during progression tometastatic competence, and we have confirmed the association of theexpression of certain genes with the metastatic phenotype. More

significantly, we have connected the expression of already character-ized genes that have not previously been linked with tumor metastasiswith the metastatic phenotype. Most importantly, we have isolatedtotally novel metastasis-associated genes. These genes represent a richsource of candidates for application in the diagnosis and therapy ofhuman cancer.

Fig. 4. Detection of CD24 and S7 expression inhuman colorectal cancer byin situ hybridization.Sections of formaldehyde-fixed and paraffin-embedded human colorectal carcinomas, corre-sponding nonneoplastic mucosa, and lymph nodemetastases were hybridized with fluorescence-labeled sense and antisense RNA and subsequentlycounterstained with H&E.a–f, CD24 expression ina poorly differentiated adenocarcinoma of the co-lon, staged as pT4b; pN2; pV1 (a–c, antisense;eandf, sense control). Note the marked overexpres-sion of CD24 in the carcinoma (b;CA, carcinoma;M, muscularis propria) as compared with the non-neoplastic mucosa at the resection margin (a).g–i,S7 expression in the same samples shown ina–f.Note the marked overexpression of S7 in the pri-mary cancer as compared with the nonneoplasticmucosa. Furthermore, there is even a slightly stron-ger hybridization signal in the metastasis than inthe primary tumor (compareh andi). j–m, detailedanalysis of CD24 and S7 expression in noninvasiveareas with nuclear atypia (starsin j and k) andinvasive carcinoma (j, S7 antisense;k, CD24 anti-sense) and the corresponding nonneoplastic mu-cosa at the resection margin (l, CD24 antisense;m,CD24 sense). Note that CD24 is already overex-pressed in noninvasive lesions with nuclear atypia(comparek andj), whereas overexpression of S7 isstrictly restricted to the invasive carcinoma (j).Magnification:a–i, 3 60; j–m, 3 120.

Fig. 5. Detection of CD24 and S7 expression inhuman breast cancer byin situ hybridization. Sec-tions of formaldehyde-fixed and paraffin-embed-ded human invasive ductal breast carcinomas (bande), the corresponding nonneoplastic mammaryparenchyma (aandd), and a lymph node metastasis(c andf) were hybridized with fluorescence-labeledsense and antisense RNA and subsequently coun-terstained with H&E.a–c, CD24 antisense probe;d–f, S7 antisense probe. Note that CD24 expressionis clearly detected in nonneoplastic lobules, andthere is no striking difference in the expressionlevel between nonneoplastic parenchyma, primarycarcinoma, and metastasis. S7 expression is notseen in nonneoplastic parenchyma and primary car-cinoma but becomes detectable in the metastasis.a–f, 3120.

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ACKNOWLEDGMENTS

We thank Andrea Fuchsbichler for able assistance with thein situ hybrid-izations and Christian Ahrens for preliminary data analysis.

Note Added in Proof:

While this manuscript was under review, Clarket al. (Nature406: 532–535, 2000)

reported the isolation of genes using microarray screening whose expression correlates

with metastatic potential.

REFERENCES

1. Tlsty, T. D. Cell-adhesion-dependent influences on genomic instability and carcino-genesis. Curr. Opin. Cell Biol.,10: 647–653, 1998.

2. Fidler, I. J. Tumor heterogeneity and the biology of cancer invasion and metastasis.Cancer Res.,38: 2651–2660, 1978.

3. Sleeman, J. P. The lymph node as a bridgehead in the metastatic dissemination oftumors. Recent Results Cancer Res., 157:55–81, 2000.

4. Pencil, S. D., Toh, Y., and Nicolson, G. L. Candidate metastasis-associated genes ofthe rat 13762NF mammary adenocarcinoma. Breast Cancer Res. Treat.,25: 165–174,1993.

5. Daigneault, L., Beaulieu, R., Filion, M., Gaboury, L., Royal, A., and Babai, F.Cloning and identification of genes differentially expressed in metastatic and non-metastatic rat rhabdomyosarcoma cell lines. Clin. Exp. Metastasis,13: 345–356,1995.

6. Salesiotis, A. N., Wang, C. K., Wang, C. D., Burger, A., Li, H., and Seth, A.Identification of novel genes from stomach cancer cell lines by differential display.Cancer Lett.,91: 47–54, 1995.

7. van Groningen, J. J., Bloemers, H. P., and Swart, G. W. Identification of melanomainhibitory activity and other differentially expressed messenger RNAs in humanmelanoma cell lines with different metastatic capacity by messenger RNA differentialdisplay. Cancer Res.,55: 6237–6243, 1995.

8. Ishiguro, T., Nakajima, M., Naito, M., Muto, T., and Tsuruo, T. Identification ofgenes differentially expressed in B16 murine melanoma sublines with differentmetastatic potentials. Cancer Res.,56: 875–879, 1996.

9. Hashimoto, Y., Shindo-Okada, N., Tani, M., Takeuchi, K., Toma, H., and Yokota, J.Identification of genes differentially expressed in association with metastatic potentialof K-1735 murine melanoma by messenger RNA differential display. Cancer Res.,56: 5266–5271, 1996.

10. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B.,Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., and Siebert, P. D.Suppression subtractive hybridization: a method for generating differentially regu-lated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA,93:6025–6030, 1996.

11. Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R. H., Hamilton, S. R.,Vogelstein, B., and Kinzler, K. W. Gene expression profiles in normal and cancercells. Science (Washington DC),276: 1268–1272, 1997.

12. Sgroi, D. C., Teng, S., Robinson, G., LeVangie, R., Hudson, J. R., Jr., and Elkahloun,A. G. In vivo gene expression profile analysis of human breast cancer progression.Cancer Res.,59: 5656–5661, 1999.

13. von Stein, O. D., Thies, W. G., and Hofmann, M. A high throughput screening forrarely transcribed differentially expressed genes. Nucleic Acids Res.,25: 2598–2602,1997.

14. Matzku, S., Komitowski, D., Mildenberger, M., and Zoller, M. Characterization ofBSp73, a spontaneous rat tumor, and itsin vivo selected variants showing differentmetastasizing capacities. Invasion Metastasis,3: 109–123, 1983.

15. Isaacs, J. T., Isaacs, W. B., Feitz, W. F., and Scheres, J. Establishment and charac-terization of seven Dunning rat prostatic cancer cell lines and their use in developingmethods for predicting metastatic abilities of prostatic cancers. Prostate,9: 261–281,1986.

16. Neri, A., and Nicolson, G. L. Phenotypic drift of metastatic and cell-surface propertiesof mammary adenocarcinoma cell clones during growthin vitro. Int. J. Cancer,28:731–738, 1981.

17. Kim, U. Pathogenesis and characteristics of spontaneously metastasizing mammarycarcinomas and the principle of metastasis. J. Surg. Oncol.,33: 151–165, 1986.

18. Tatusova, T. A., and Madden, T. L. BLAST 2 Sequences, a new tool for comparingprotein and nucleotide sequences. FEMS Microbiol. Lett.,174: 247–250, 1999.

19. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., andLipman, D. J. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acids Res.,25: 3389–3402, 1997.

20. Osterreichische Gesellschaft fur Pathologie Histologische Tumorklassifikation, 2nded. Wien, New York: Springer Verlag, 1994.

21. Di Renzo, M. F., Poulsom, R., Olivero, M., Comoglio, P. M., and Lemoine, N. R.Expression of the Met/hepatocyte growth factor receptor in human pancreatic cancer.Cancer Res.,55: 1129–1138, 1995.

22. Ferno, M., Bendahl, P. O., Borg, A., Brundell, J., Hirschberg, L., Olsson, H., andKillander, D. Urokinase plasminogen activator, a strong independent prognosticfactor in breast cancer, analysed in steroid receptor cytosols with a luminometricimmunoassay. Eur. J. Cancer,32A: 793–801, 1996.

23. Sivaparvathi, M., Sawaya, R., Gokaslan, Z. L., Chintala, S. K., Rao, J. S., andChintala, K. S. Expression and the role of cathepsin H in human glioma progressionand invasion. Cancer Lett.,104: 121–126, 1996.

24. Nasu, Y., Timme, T. L., Yang, G., Bangma, C. H., Li, L., Ren, C., Park, S. H.,DeLeon, M., Wang, J., and Thompson, T. C. Suppression of caveolin expressioninduces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancercells. Nat. Med.,4: 1062–1064, 1998.

25. Wauters, C. C., Smedts, F., Gerrits, L. G., Bosman, F. T., and Ramaekers, F. C.Keratins 7 and 20 as diagnostic markers of carcinomas metastatic to the ovary. Hum.Pathol.,26: 852–855, 1995.

26. Yokoyama, N., Shirai, Y., and Hatakeyama, K. Immunohistochemical detection oflymph node micrometastases from gallbladder carcinoma using monoclonal anticy-tokeratin antibody. Cancer (Phila.),85: 1465–1469, 1999.

27. Schoenfeld, A., Luqmani, Y., Sinnett, H. D., Shousha, S., and Coombes, R. C. Keratin19 mRNA measurement to detect micrometastases in lymph nodes in breast cancerpatients. Br. J. Cancer,74: 1639–1642, 1996.

28. Rosel, M., Claas, C., Seiter, S., Herlevsen, M., and Zoller, M. Cloning and functionalcharacterization of a new phosphatidyl-inositol anchored molecule of a metastasizingrat pancreatic tumor. Oncogene,17: 1989–2002, 1998.

29. Bernaert, D., Brewer, L. M., MacManus, J. P., and Galand, P. Immunocytochemicaldetection of the onco-developmental protein oncomodulin in pre-neoplastic andneoplastic hepatocellular lesions during hepatocarcinogenesis in rats. Int. J. Cancer,43: 719–727, 1989.

30. Amundadottir, L. T., Nass, S. J., Berchem, G. J., Johnson, M. D., and Dickson, R. B.Cooperation of TGFa and c-Myc in mouse mammary tumorigenesis: coordinatedstimulation of growth and suppression of apoptosis. Oncogene,13: 757–765, 1996.

31. LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. The inhibitors ofapoptosis (IAPs) and their emerging role in cancer. Oncogene,17: 3247–3259, 1998.

32. Aigner, S., Ramos, C. L., Hafezi-Moghadam, A., Lawrence, M. B., Friederichs, J.,Altevogt, P., and Ley, K. CD24 mediates rolling of breast carcinoma cells onP-selectin. FASEB J.,12: 1241–1251, 1998.

33. Kondo, T., Minamino, N., Nagamura-Inoue, T., Matsumoto, M., Taniguchi, T., andTanaka, N. Identification and characterization of nucleophosmin/B23/numatrin whichbinds the anti-oncogenic transcription factor IRF-1 and manifests oncogenic activity.Oncogene,15: 1275–1281, 1997.

34. Nygaard, S. J., Haugland, H. K., Kristoffersen, E. K., Lund-Johansen, M., Laerum,O. D., and Tysnes, O. B. Expression of annexin II in glioma cell lines and in braintumor biopsies. J. Neurooncol.,38: 11–18, 1998.

35. Gill, Z. P., Perks, C. M., Newcomb, P. V., and Holly, J. M. Insulin-like growthfactor-binding protein (IGFBP-3) predisposes breast cancer cells to programmed celldeath in a non-IGF-dependent manner. J. Biol. Chem.,272: 25602–25607, 1997.

36. Pomerantz, J., Schreiber-Agus, N., Liegeois, N. J., Silverman, A., Alland, L., Chin,L., Potes, J., Chen, K., Orlow, I., Lee, H. W., Cordon-Cardo, C., and DePinho, R. A.The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neu-tralizes MDM2’s inhibition of p53. Cell,92: 713–723, 1998.

37. Tanaka, M., Adzuma, K., Iwami, M., Yoshimoto, K., Monden, Y., and Itakura, M.Human calgizzarin; one colorectal cancer-related gene selected by a large scalerandom cDNA sequencing and Northern blot analysis. Cancer Lett.,89: 195–200,1995.

38. Kobayashi, T., Hirayama, Y., Kobayashi, E., Kubo, Y., and Hino, O. A germlineinsertion in the tuberous sclerosis (Tsc2) gene gives rise to the Eker rat model ofdominantly inherited cancer. Nat. Genet.,9: 70–74, 1995.

39. Xiang, Y. Y., Wang, D. Y., Tanaka, M., Suzuki, M., Kiyokawa, E., Igarashi, H.,Naito, Y., Shen, Q., and Sugimura, H. Expression of high-mobility group-1 mRNA inhuman gastrointestinal adenocarcinoma and corresponding non-cancerous mucosa.Int. J. Cancer,74: 1–6, 1997.

40. Hernandez-Alcoceba, R., Saniger, L., Campos, J., Nunez, M. C., Khaless, F., Gallo,M. A., Espinosa, A., and Lacal, J. C. Choline kinase inhibitors as a novel approachfor antiproliferative drug design. Oncogene,15: 2289–2301, 1997.

41. Bang, Y. J., Pirnia, F., Fang, W. G., Kang, W. K., Sartor, O., Whitesell, L., Ha, M. J.,Tsokos, M., Sheahan, M. D., Nguyen, P.,et al. Terminal neuroendocrine differenti-ation of human prostate carcinoma cells in response to increased intracellular cyclicAMP. Proc. Natl. Acad. Sci. USA,91: 5330–5334, 1994.

42. Hecht, J. T., Hogue, D., Wang, Y., Blanton, S. H., Wagner, M., Strong, L. C.,Raskind, W., Hansen, M. F., and Wells, D. Hereditary multiple exostoses (EXT):mutational studies of familial EXT1 cases and EXT-associated malignancies. Am. J.Hum. Genet.,60: 80–86, 1997.

43. Wu, C. G., Habib, N. A., Mitry, R. R., Reitsma, P. H., van Deventer, S. J., andChamuleau, R. A. Overexpression of hepatic prothymosina, a novel marker forhuman hepatocellular carcinoma. Br. J. Cancer,76: 1199–1204, 1997.

44. Fulchignoni-Lataud, M. C., Olchwang, S., and Serre, J. L. The fragile X CGG repeatshows a marked level of instability in hereditary non-polyposis colorectal cancerpatients. Eur. J. Hum. Genet.,5: 89–93, 1997.

45. Kondoh, G., Yomogida, K., Dohmae, K., Nozawa, M., Koga, M., Nonomura, N.,Miki, T., Okuyama, A., and Nishimune, Y. Coexpression of multiple Sertoli cell andLeydig cell marker genes in the spontaneous testicular tumor of F344 rat: evidence forphenotypical bifurcation of the interstitial cell tumor. Jpn. J. Cancer Res.,88:839–845, 1997.

46. Berta, G. N., Ghezzo, F., D’Avolio, A., Zulian, P., Carbone, V., Racca, S., Vercellino,V., and Di Carlo, F. Enhancement of calcyclin gene RNA expression in squamous cellcarcinoma of the oral mucosa, but not in benign lesions. J. Oral Pathol. Med.,26:206–210, 1997.

47. Kojima, T., Oh-eda, M., Hattori, K., Taniguchi, Y., Tamura, M., Ochi, N., andYamaguchi, N. Molecular cloning and expression of megakaryocyte potentiatingfactor cDNA. J. Biol. Chem.,270: 21984–21990, 1995.

48. Manni, A., Grove, R., Kunselman, S., and Aldaz, M. Involvement of the polyaminepathway in breast cancer progression. Cancer Lett.,92: 49–57, 1995.

1576




49. Lamb, R. F., Ozanne, B. W., Roy, C., McGarry, L., Stipp, C., Mangeat, P., and Jay,D. G. Essential functions of ezrin in maintenance of cell shape and lamellipodialextension in normal and transformed fibroblasts. Curr. Biol.,7: 682–688, 1997.

50. Bleuel, K., Popp, S., Fusenig, N. E., Stanbridge, E. J., and Boukamp, P. Tumorsuppression in human skin carcinoma cells by chromosome 15 transfer or throm-bospondin-1 overexpression through halted tumor vascularization. Proc. Natl. Acad.Sci. USA,96: 2065–2070, 1999.

51. Celis, J. E., Ostergaard, M., Basse, B., Celis, A., Lauridsen, J. B., Ratz, G. P., Andersen,I., Hein, B., Wolf, H., Orntoft, T. F., and Rasmussen, H. H. Loss of adipocyte-type fattyacid binding protein and other protein biomarkers is associated with progression of humanbladder transitional cell. Cancer Res.,56: 4782–4790, 1996.

52. Zhang, R. D., Fidler, I. J., and Price, J. E. Relative malignant potential of humanbreast carcinoma cell lines established from pleural effusions and a brain metastasis.Invasion Metastasis,11: 204–215, 1991.

53. Hendrix, M. J., Seftor, E. A., Seftor, R. E., and Trevor, K. T. Experimental co-expression of vimentin and keratin intermediate filaments in human breast cancercells results in phenotypic interconversion and increased invasive behavior. Am. J.Pathol.,150: 483–495, 1997.

54. Liang, P., and Pardee, A. B. Differential display of eukaryotic messenger RNA bymeans of the polymerase chain reaction. Science (Washington DC),257: 967–971,1992.

55. Sokolov, B. P., and Prockop, D. J. A rapid and simple PCR-based method forisolation of cDNAs from differentially expressed genes. Nucleic Acids Res.,22:4009–4015, 1994.

56. Lisitsyn, N., and Wigler, M. Cloning the differences between two complex genomes.Science (Washington DC),259: 946–951, 1993.

57. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. Serial analysis ofgene expression. Science (Washington DC),270: 484–487, 1995.

58. Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O., and Weigel, R. J. CombiningSSH and cDNA microarrays for rapid identification of differentially expressed genes.Nucleic Acids Res.,27: 1517–1523, 1999.

59. Gress, T. M., Muller-Pillasch, F., Geng, M., Zimmerhackl, F., Zehetner, G., Friess,H., Buchler, M., Adler, G., and Lehrach, H. A pancreatic cancer-specific expressionprofile. Oncogene,13: 1819–1830, 1996.

60. Kang, D. C., LaFrance, R., Su, Z. Z., and Fisher, P. B. Reciprocal subtractiondifferential RNA display: an efficient and rapid procedure for isolating differentiallyexpressed gene sequences. Proc. Natl. Acad. Sci. USA,95: 13788–13793, 1998.

61. Vande Woude, G. F., Jeffers, M., Cortner, J., Alvord, G., Tsarfaty, I., and Resau, J.Met-HGF/SF: tumorigenesis, invasion and metastasis. CIBA Found. Symp.,212:119–130, 1997.

62. Crepaldi, T., Gautreau, A., Comoglio, P. M., Louvard, D., and Arpin, M. Ezrin is aneffector of hepatocyte growth factor-mediated migration and morphogenesis in epi-thelial cells. J. Cell Biol.,138: 423–434, 1997.

63. Akisawa, N., Nishimori, I., Iwamura, T., Onishi, S., and Hollingsworth, M. A. Highlevels of ezrin expressed by human pancreatic adenocarcinoma cell lines with highmetastatic potential. Biochem. Biophys. Res. Commun.,258: 395–400, 1999.

64. Rabbani, S. A., and Xing, R. H. Role of urokinase (uPA) and its receptor (uPAR) ininvasion and metastasis of hormone-dependent malignancies. Int. J. Oncol.,12:911–920, 1998.

65. Yang, G., Truong, L. D., Timme, T. L., Ren, C., Wheeler, T. M., Park, S. H., Nasu,Y., Bangma, C. H., Kattan, M. W., Scardino, P. T., and Thompson, T. C. Elevatedexpression of caveolin is associated with prostate and breast cancer. Clin. CancerRes.,4: 1873–1880, 1998.

66. Shirasawa, T., Akashi, T., Sakamoto, K., Takahashi, H., Maruyama, N., andHirokawa, K. Gene expression of CD24 core peptide molecule in developing brainand developing non-neural tissues. Dev. Dyn.,198: 1–13, 1993.

67. Magnaldo, T., and Barrandon, Y. CD24 (heat stable antigen, nectadrin), a novelkeratinocyte differentiation marker, is preferentially expressed in areas of the hairfollicle containing the colony-forming cells. J. Cell Sci.,109: 3035–3045, 1996.

68. Perri, R. T., Royston, I., LeBien, T. W., and Kay, N. E. Chronic lymphocyticleukemia progenitor cells carry the antigens T65, BA-1, and Ia. Blood,61: 871–875,1983.

69. Droz, D., Zachar, D., Charbit, L., Gogusev, J., Chretein, Y., and Iris, L. Expressionof the human nephron differentiation molecules in renal cell carcinomas. Am. J.Pathol.,137: 895–905, 1990.

70. Jackson, D., Waibel, R., Weber, E., Bell, J., and Stahel, R. A. CD24, a signal-transducing molecule expressed on human B cells, is a major surface antigen on smallcell lung carcinomas. Cancer Res.,52: 5264–5270, 1992.

71. Akashi, T., Shirasawa, T., and Hirokawa, K. Gene expression of CD24 core polypep-tide molecule in normal rat tissues and human tumor cell lines. Virchows Arch.,425:399–406, 1994.

72. Lavabre-Bertrand, T., Duperray, C., Brunet, C., Poncelet, P., Exbrayat, C.,Bourquard, P., Lavabre- Bertrand, C., Brochier, J., Navarro, M., and Janossy, G.Quantification of CD24 and CD45 antigens in parallel allows a precise determinationof B-cell maturation stages: relevance for the study of B-cell neoplasias. Leukemia(Baltimore),8: 402–408, 1994.

73. Wimberly, B. T., White, S. W., and Ramakrishnan, V. The structure of ribosomalprotein S7 at 1.9 A resolution reveals ab-hairpin motif that binds double-strandednucleic acids. Structure,5: 1187–1198, 1997.

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2001;61:1569-1577. Cancer Res Andrea Nestl, Oliver D. Von Stein, Kurt Zatloukal, et al. Phenotype in Rodent and Human TumorsGene Expression Patterns Associated with the Metastatic

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Gene Expression Patterns Associated with the Metastatic ... · tor, ezrin, HMG-1, oncomodulin, cathepsin, and caveolin were thereby isolated. Half of the metastasis-associated clones