[CANCER RESEARCH 58. 3561-3565. August 15. W8|

Advances in Brief

Identification of a Novel Metastasis-suppressor Region on Human Chromosome 121

Hue H. Luu, Gregory P. Zagaja, Zita Dubauskas, Stephen L. Chen, Robert C. Smith, Kounosuke Watabe,Yayoi Ichikawa, Tomohiko Ichikawa, Elizabeth M. Davis, Michelle M. Le Beau, and Carrie W. Rinker-Schaeffer2

Departments of Surgery. Section of Urology ¡H.H. L, G. P. Z., Z. D., S. L C., K. C. S., C. W. R-S.] and Medicine. Section of Heinulology/Oncology ¡E.M. D.. M. M. L/. University

of Chicago, Chicago. Illinois: Department of Medicai Microhiology/ltnmunolítgy. Southern Illinois University School of Medicine, Springfield. Illinois ¡K.W.¡:Department ofUrologv. Teikvo University School of Medicine. Ichihuru Hospital, Ichihara-shi. Chiha 29V-OI, Japan [Y. I., T. /./,' and The Prostate Cancer Program. The Università' of Chicago

Cancer Research Center, Chicago. Illinois 60637 /C. W. R-S.j

Abstract

There is a critical need for markers that can be used to predictaccurately the malignant potential of histológica! prostate cancers (J. T.Isaacs, Am. J. Pathol., 150: 1511-1521, 1997). Metastasis-suppressor genes

are attractive candidates for marker development because, by definition,their loss should be associated with the acquisition of metastatic ability. Inan effort to identify such genes, a single copy of human chromosome 12,tagged with the neomycin resistance gene, was introduced into highlymetastatic Dunning AT6.1 prostate cancer cells by microcell-mediatedchromosomal transfer. Thirty-two AT6.1-12 clonal cell lines were estab

lished and the region(s) of chromosome 12 retained was determined bysequence tagged site-based PCR analysis. Representative AT6.1-12 clones

containing overlapping regions of chromosome 12 were characterizedcytogenetically and were shown to have a normal complement of parentalAT6.1 rat chromosomes. Fluorescence in situ hybridization, performed onrepresentative Aid. I-12 hybrids, demonstrated a single human chromosome 12-specific signal. The metastatic ability of six representative cloneswas tested in iinmunodeficient mice. All of the ATO. I-I2 clones showed thesame in vivo growth rates as the control AT6.1-neo cells. Clonal cell linesthat contained a conserved ~70-cM portion of chromosome 12 (e.g.,

AT6.1-12-8, -8-1, and -8-3), showed a >30-fold suppression in the number

of macroscopic surface lung métastases.Mice that received injections ofthese cells developed a mean number 4 lung métastaseswhereas mice thatreceived injections of other AT6.1-12 hybrids (lacking the ~70-cM region)

or AT6.1-neo control cells, developed a mean number of 140 métastases.

Interestingly, histológica! examination of the lungs of the mice that received injections of AT6.1-12-8 cells showed essentially no microscopicmétastases.These findings suggest that a gene(s) encoded by the ~70-cM

portion of human chromosome 12 suppresses an early step in the metastatic cascade.

Introduction

In 1990, Carter and Coffey (1) described the "increasing dilemma" of prostate cancer in which "an increase in the discovery

of small volume cancers will increase the necessity to distinguishprostate cancer that will remain truly latent from those that willprogress into an aggressive clinical form." It has been suggested

that 80% of the histologically localized lesions diagnosed areclinically insignificant (i.e., not life-altering or life-threatening) (2,3). However, we are presently unable to predict which individuallocalized cancers will be significant. Moreover, of the patients with

Received 4/27/98: accepted 6/25/98.The costs of publication of"this article were defrayed in part by the payment of page

charges. This article must therefore he hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this faci.

1This work was supported by University of Chicago Surgery Research Committee

Grant; Cancer Research Foundation Young Investigator Award and NIH First Award R29CA69487 02 (C. W. R-S.). Howard Hughes Medical Institute (H. H. L., C. W. R-S.),University of Chicago RESCUE Fund <C. W. R-S.). NIH POI CA40046 (M. M. L.), andGrants-in-aid 09470340 and 08265106 from the Ministry of Education. Science. Sports

and Culture. Japan (T. 1.).•¿�To whom requests for reprints should be addressed, at Section of Urology. Depart

ment of Surgery. 5841 South Maryland MC6038. Chicago. IL 60637. Phone: (773)702-3132: Fax: (773) 702-1001; E-mail: crinker@surgery.bsd.uchicago.edu.

clinically significant disease, only one-half will have cancers confined to the prostate, whereas the remaining patients will haveundetectable micrometastatic disease (i.e., incurable cancer) at thetime of initial diagnosis (4). Thus, there is a critical need formarkers that will predict accurately the clinical importance ofmicroscopic lesions. As a result, there has been a concerted effortover the past 5 years to identify genes that regulate prostate cancermetastasis. These studies have used microcell-mediated chromosomal transfer to functionally identify metastasis-suppressor activities/genes on human chromosomes 8, 10. 11, and 17 (5-8).Specifically, KAI1 and CD44 have been identified as prostatecancer metastasis-suppressor genes encoded by human chromosome 11 (7, 9). Furthermore, decreased expression of KAI1 hasbeen correlated with increased malignancy of prostate, non-smallcell lung, and pancreatic cancers (10-12).

As an extension of this effort to identify genes regulating prostatecancer metastasis, human chromosome 12 was tested for a metastasis-suppressor activity. This chromosome is an appropriate candidatebecause of the aberrations of chromosome 12, such as loss of het-erozygosity, associated with prostate, germ cell, ovarian, pancreatic,and gastric cancers (13-17). In addition, prostate cancer tumor suppressor activity has been functionally localized to chromosome bands12pter-ql3 (18). Thus, a single copy of human chromosome 12 wastransferred into highly metastatic Dunning AT6.1 rat prostate cancercells, and the resultant hybrids were tested for their metastatic ability.Our present study identities an ~70-cM portion of human

chromosome 12, consisting of 2 distinct regions of 38.5 cM(D128345^D12S106) and 31.5 cM (D12S321->qter), that encodes anovel metastasis-suppressor activity.

Materials and Methods

Cell Lines and Culture. The Dunning AT6.1 rat prostatic cancer cellline is highly metastatic. anaplastic, and androgen-independent (19). The

A9( 12) fibrosarcoma cell line carries a single copy of human chromosome12 tagged with an integrated neomycin resistance gene (the gift of Dr. J. T.Isaacs. The Johns Hopkins University School of Medicine, Baltimore.MD). A9(12) cells were used to donate human chromosome 12 to AT6.1cells by microcell-mediated chromosomal transfer (8). The AT6.l-neo cell

line was established by transfection of AT6.1 cells with the pSV2Neoplasmid. Resultant AT6.1-12 hybrids and AT6.1-neo transfectants were

selected in standard cell growth media containing 500 /xg/ml G418 andindividually cloned. All of the AT6.1 and AT6.l-derived cell lines weregrown in standard RPMI 1640 with L-glutamine (Cell Grò) containing 8%

PCS (Life Technologies, Inc.). penicillin (100 units/ml)/streptomycin (100jug/ml), and 250 nM dexamethasone (Sigma Chemical). The AT6.1-neo andAT6.1-12 hybrids were also grown in standard media with the addition of

G418 (500 ¿ig/ml) (Life Technologies, Inc.). Incubation conditions werestandard. 37°Cwith 5% CO,.

Genomic DNA Preparation and Molecular Analysis of Cell Lines.Genomic DNA was isolated and quantified from cultured cells as described byChekmareva et al. (8). The regions of human chromosome 12 that wereretained by the AT6.1-12 hybrids were determined by PCR amplification of

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NOVEL METASTASIS SITI'RI-SSOR REGION ON CHROMOSOME 12

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Fig. I. Molecular and cylogenetic analysis of AT6.1-12 microcell hybrids. In A, genomic DNA was isolated and analyzed by PCR for the retention of chromosome 12 STS markersas shown. O. marker lost: •¿�.marker retained. The relative cytogenelic locations and genetic distances of the STS markers (cMI are indicated. B. example of PCR-amplified products(DI2SX7. D12S340). In C. karyolype analysis of AT6.1 and AT6.1-12-8 show that the AT6.I-12-8 hybrid contains a normal complement of rat chromosomes in addition to human

chromosome 12.

STS3 markers.4 The relative genetic distances of the STS markers wereaccording to the National Center for Biotechnology Information Database.5

Total human genomic DNA (Sigma) and AT6.1-neo genomic DNA were used

as positive and negative controls for chromosome 12 amplification, respectively. Amplification with ß-actinprimers was used as a control for DNAintegrity and quality. Amplification conditions using STS and ß-actinprimers

(Research Genetics) were as described by the manufacturer. PCR productswere separated by electrophoresis through 2.5% 3:1 agarose gel (ContinentalLaboratories) in 1 X Tris-borate EDTA buffer and detected by ethidium

' The abbreviations used are: STS, sequence tagged site: FISH, fluorescence IB j/lu

hybridi/ation: SCID. severe combined immunodeficient: hpf. high power field.4 Address: www.resgen.com.5 Address: www.ncbi.nih.gov.

bromide staining. A 123-bp DNA ladder (Life Technologies, Inc.) was used as

a molecular weight marker.

Karyotype Analysis and FISH. The rat chromosomal complement wasanaly/ed using a trypsin-Giemsa banding technique as described previously by

Ichikawa et al. (20). A minimum of 40 Giemsa-banded metaphase cells foreach cell line (AT6.1 parental, AT6.1-12-1, -2, -8. and -9) were examined. In

addition. 10 Giemsa-banded spreads were karyotyped for each cell line. FISH

was performed as described previously (8) with the following modifications.

The probes used included an a satellite probe for the centromere(CEP12â„¢SO. Vysis. red signal) of human chromosome 12 and a whole-

chromosome painting probe for this chromosome (WCP12â„¢SG. Vysis, green

signal). Dual color FISH was performed by cohybridi/ing chromosome 12centromere-specific and painting probes, and 20 metaphase cells from each cell

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NOVEL MLTASTASIS SITI'KHSSOR RECIOS ON CHROMOSOME

Fig. 2. FISH analysis. AT6.1-12 hybrids and AT6.1-neo were analyzed using human chromosome 12 painting (WCPI2â„¢SG) and ccntromeric-specific (CEPI2â„¢SO, Vysis) probes.

A. AT6.l-neo cells contain no material from human chromosome 12. B and C. AT6.1-12-2 cells have a small ring chromosome that contains the centromere (C. arrow) as well aschromosomal material from human chromosome 12 (B. arrow). D, AT6.1-12-9 cells have a small segment of human chromosome 12 inserted into a submetacentric rat chromosome(arrow). E and F. AT6.1-12-8 cells have a rearranged human chromosome 12 resulting from a deletion in the distal long arm del( 12Kq22q24.1). The del( I2q) hybridi/es to both the

centromere (F, arrow) and whole chromosome painting (E, arrow) probes.

line were analyzed. Slides were analyzed with a dual-pass FITC-rhodamine

filter (Chroma Technology. Inc.).In Vivo Studies of Growth and Metastasis. Spontaneous metastasis as

says were performed as described previously (8). Briefly. 4-6 week old maleCB17 SCID mice (Taconic-Lab Animals and Services) received injections s.c.in the flank with 2 X l O5AT6.1 -neo, AT6.1 -12-1, -2, -8, -8-1, -8-3. or -9 cells.

A total of five to eight animals were used per cell line tested. The tumorvolume was determined as an index of tumor growth (8). At 42-45 days after

receiving the injection, the animals were killed, the lungs were excised, and thenumber of macroscopic métastases(>1 mm) were determined.

Histology. The left lung of animals bearing either the suppressed AT6.1-12-8 or unsuppressed AT6.1-12-9 tumors was fixed in 10% buffered formalinand was paraffin embedded. Six random discontinuous 5-/J.M sections were

taken and stained with H&E (University of Chicago Histology Laboratory.Chicago, IL). A total of 425 random hpfs (X 100) were examined per groupand the number of microscopic (<300 cells) and/or macroscopic (>300 cells)

metastatic foci were counted.

Results and Discussion

Highly metastatic AT6.1 cells were used as the recipient for mi-

crocell transfer of human chromosome 12 tagged with a neomycinresistance gene (8). As a control for selection in neomycin, AT6.1cells were also transfected with the pSV2neo plasmid (8). A total of32 AT6.1-12 hybrids were established and the region of chromosome12 retained in each was determined by STS-based PCR analysis. The

summary of STS markers retained in six representative cell lines areshown (Fig. 1/4). Examples of PCR products are shown (Fig. Iß).Allof the 32 AT6.1-12 hybrids retained markers betweenD12S314—»D12S345,suggesting that this may be the location of the

neomycin resistance gene. Four clones retained a larger portion of

chromosome 12. Importantly, clones 8, 8-1 and 8-3 retain a conserved~70-cM region of chromosome 12. consisting of two distinct regionsof 38.5 cM (D12S345-^D12S106) and 31.5 cM (D12S321^qter),

respectively. Karyotype analysis was conducted to assess the numberand integrity of the rat chromosomes in representative clones (AT6.1-12-1, -2, -8, and -9) and parental AT6.1 cells. The karyotype of theparental AT6.1 and the AT6.1-12-8 cells are shown in Fig. 1C. Thesestudies showed that the AT6.1-12-1, -2, -8, and -9 cells had the same

karyotype [44,XY,del(3),+4,+12, del(15)] as the parental AT6.1cells. Moreover, the hybrid AT6.1-12-8 retained a rearranged human

chromosome 12 that seems to have a deletion in the distal long arm,del (12)(q22q24.1), which is in agreement with our STS-based PCR

mapping results (Fig. LA).FISH studies using chromosome 12-specific painting and cen

tromere-specific probes were performed on AT6.1-12-2, -8, and -9

cells to: (a) document the retention of human chromosome 12; (b)determine the number of copies of this chromosome; and (c)identify rearrangements between rat chromosomes and humanchromosome 12. AT6.1-neo cells were used as a negative control.There was no chromosome 12-specific signal in the AT6.1-neocontrol cells (Fig. 2/4). All of the AT6.1-12 hybrids had a singlechromosome 12-specific signal. AT6.1-12-2 cells have a small ring

chromosome comprised entirely of human chromosome 12. Thisrearranged chromosome contains the centromere as well as non-

centromeric material from human chromosome 12 (Fig. 2, B andC). AT6.1-12-9 cells have a small segment of chromosome 12inserted in a submetacentric rat chromosome (Fig. ID). AT6.1-

12-8 cells (Fig. 2, E and F) have a rearranged human chromosome

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Table 1 In vivo growth and metástasis of microcell hybrids and AT6.1-neo

control cells

CelllineAT6.AT6.AT6.AT6.AT6.AT6.AT6.-12-8-12-8-1-12-8-3-12-1-12-2-12-9-neoTumordoubling

time ±SE(days)4.55.15.64.54.15.15.0±0.4±0.5±0.2±0.2±0.3±0.5±0.6Mean

no.of lung métastases/

animal ±SE"4

±19±30133

±17117±22137

±20170±11No.

ofanimals8557778

' P-value < 0.005 as determined by (-lest. Macroscopic métastasesdefined as > 1 mm.

Fig. 3. Spontaneous metastasis assay. 4-6 week old CB17/SCID mice were giveninjections of 2 X IO5 cells s.c.; at the experimental end point (day 42-45). the animals

were sacrificed, the lungs were excised and fixed in 10^ formalin, and the number ofmacroscopic métastases(>l mm) were determined. Representative lungs from animalsbearing AT6.I-12-8. AT6.1-I2-9. and AT6.1-neo cells are shown.

12 resulting from a deletion in the distal long arm,del( 12)(q22q24.1 ). which is in agreement with our STS-based PCR

mapping results and karyotype analysis (Fig. 1, A and C).To determine the metastatic ability of the AT6.1-12 hybrids, six

representative clones (AT6.1-12-1, -2, -8, -8-1, -8-3, and -9) were

injected s.c. into SCID mice. AT6.1 cells transfected with thepSV2neo plasmid (AT6.1-neo) were used as a positive control. All of

the cell lines showed the same in vivo tumor growth rate (Table 1). Nodifference in other aspects of tumorigenicity. such as latency orpercentage of animals developing tumors, was observed (data notshown). At the experimental end point (42-45 days postinjection), the

animals were killed, their lungs excised and fixed, and the number ofmacroscopic lung métastasescounted. Representative lungs excisedfrom AT6.1-12-8, AT6.1-12-9, and AT6.1-neo tumor-bearing animals

are shown in Fig. 3. There was a dramatic suppression in the numberof macroscopic lung métastasesobserved in animals bearing AT6.1-12-8, -8-1, or -8-3 tumors as compared with AT6.1-12-1, -2, -9, orAT6.1-neo control tumor-bearing animals (Table 1). Specifically,

animals that received injections of the suppressed cell lines developeda mean of four visible lung métastases.In contrast, animals thatreceived injections of AT6.1-neo control or AT6.1-12-1, -2, or -9hybrids (lacking the ~70-cM region) developed a mean of 140

macroscopic lung métastases(P <0.005). These results demonstratethat the ~70-cM region of human chromosome 12, present in the

AT6.1-12-8, -8-1, and -8-3 cell lines, encodes a metastasis-suppressoractivity. This suppression is specific for the ~70-cM region becausethe AT6.1-12 hybrids that lack this region (AT6.1 -12-1, -2, and -9) arenot suppressed. It must be stressed that hybrids such as AT6.1-12-1,-2, and -9 provide critical controls for such studies. Because they

retain a portion of chromosome 12 but are not suppressed, theyprovide evidence that the suppression associated with the ~70-cM

region is not an artifact of microcell transfer or the result of clonalheterogeneity.

Although visible lung métastasescould occasionally be observed inthe AT6.1-12-8 tumor-bearing animals at the experimental end point,they were very small (—1mm in diameter). Given these few métas

tases, we were interested to see whether the absence of macroscopicmétastaseswas concomitant with an absence of microscopic métastases. Thus, lungs from six mice, bearing either the suppressed AT6.1-12-8 tumors or the unsuppressed AT6.I-12-9 tumors, were examinedhistologically. Six discontinuous H&E-stained sections from six in

dependent lungs were examined. As shown in Fig. 4, animals thatreceived injections of the suppressed AT6.1-12-8 cells showed essen

tially no microscopic or macroscopic métastases.In contrast, animalsthat received injections of the unsuppressed AT6.1 -12-9 cells showed

numerous microscopic as well as macroscopic métastases.Specifically, examination of 425 random hpfs of lungs from animals giveninjections of suppressed AT6.1-12-8 cells showed that only 1.4% of

AT6.1-12-8 AT6.1-12-9

40 X 100X 40 XI

100X

-•*T•¿�$:'*%

afeFig. 4. Histológica! examination of lungs from tumor-bearing animals. Formalin-fixed lungs from six mice bearing eilher AT6.1-12-8 or AT6.I-12-9 tumors were embedded in

paraffin, and six discontinuous 5-/U.Msections were taken for H&E staining. Representative sections: («)AT6.1-12-8 (X40); (b) AT6.1-12-8 (X100); (c) AT6.1-12-9 X40; and (</>AT6.I-12-9 (XIOO). The arrow in (r) illustrates a macroscopic metastasis.

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NOVEL METASTASIS-SUPPRESSOR REGION ON CHROMOSOME 12

Table 2 Each sel of lung sections was examined for ¡hepresence andJar absence ofmacroscopic or microscopic métastases.Results from the examination of 425 randomhpfat XI00 magnification. A macroscopic metastasis is defined as a focus of greater

than 300 cells

Cell line No métastasesMicroscopicmétastases

Macroscopicmétastases

AT6.1-12-8 94.6% (402 of 425 hpf) 1.4% (6 of 425 hpf) 4% ( 17 of 425 hpf)AT6.1-12-9 6.6% (28 of 425 hpf) 31.5% ( 134 of 425 hpf) 88.9% (378 of 425 hpf)

the hpfs (6 of 425) contained microscopic métastasesand 4% (17 of425) contained macroscopic métastases(Table 2). Importantly, 94.6%of the hpfs had no métastases.In contrast, a similar examination oflungs from animals given injections of the unsuppressed AT6.1-12-9

cells showed that 31.5% of the hpfs (134 of 425) had microscopicmétastasesand 88.9% of the hpfs (378 of 425) had macroscopicmétastases.Micrometastases often coexisted with macroscopic métastases in the same hpf. Only 6.6% of the hpfs (28 of 425) had nomicroscopic or macroscopic métastases.The absence of microscopicand macroscopic métastasesin the suppressed AT6.1-12-8-bearing

animals suggests that individual cells are unable to metastasize to thelungs and/or proliferate to form detectable métastases.

In the present study, we have identified an ~70-cM portion of

human chromosome 12, consisting of two distinct regions of 38.5 cMand 31.5 cM, that suppresses metastasis. The presence of chromosome12 does not affect tumorigenicity, latency, or in vivo growth rate of theAT6.1 rat prostatic cells. This is in contrast to the findings of Beruheet al. (18), which identified a tumor suppressor activity encoded byhuman chromosome bands 12pter-ql3. The recipient cell line used intheir study was the human prostate cancer cell line DU-145.

Furthermore, our present study demonstrates that the mechanism ofchromosome 12-mediated metastasis suppression is distinct from thatof chromosome 17 (8). The metastasis-suppressor activity encoded byan ~70-cM region of human chromosome 17 confers a state of"dormancy," in which micrometastases are present but unable to formmacroscopic métastases.6In sharp contrast, the absence of both mac

roscopic and microscopic métastasesin the suppressed AT6.1-12tumor-bearing animals demonstrates that the metastasis suppressor

activity encoded by chromosome 12 blocks an early step in themetastatic cascade. Potential mechanisms of chromosome 12-medi-ated metastasis suppression include increased cell-cell adhesion, de

creased cell motility, and/or decreased cell invasion. Studies to distinguish between these potential mechanism(s) and to identify thegene(s) mediating this biological activity are presently underway.

Acknowledgments

We thank the contributors of the RESCUE Fund and Dr. Charles B.Brendler for support of this work. A special thanks to Drs. Barbara A. Yoshida

6 M. A. Chekmareva, M. M. Kadkhodaian, C. M. P. Hollowell. H. Kim. B. A. Yoshida.

H. H. Luu, W. M. Stadier, and C. W. Rinker-Schaeffer. submitted for publication.

and Arthur Christiane for critical review of the manuscript. Also thanks to Drs.Michael A. Simon, Terranee D. Peabody. and Parker Gibbs for their assistance.

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