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Advances in Brief |
Jake Gittlen Cancer Research Institute [M. J. S., R. S. S., D. R. W.] and Department of Medicine [M. F. V.], The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-2390
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Metastasis-regulatory genes can be broadly categorized as either metastasis-promoting or metastasis-suppressing. Analogous to the role of oncogenes in tumorigenesis, metastasis promoters drive conversion from nonmetastatic to metastatic. Although similar in other respects, metastasis suppressors are distinguishable from tumor suppressors in that the former block only metastasis when introduced into metastatic tumor cells (i.e., not tumorigenicity). As expected, tumor suppressors suppress both phenotypes because tumorigenicity is a prerequisite to metastasis (1) . To date, only six metastasis suppressor genes (Nm23, KISS1, Kai1, E-cadherin, MKK4, TIMPs) have been shown to functionally suppress metastasis using in vivo models (reviewed in Ref. 2 ).
Two general approaches were used to identify these metastasis-controlling genes. The first involved comparison of gene expression in poorly or nonmetastatic cells with matched metastasis-competent cells. The second took advantage of clinical observations that identified nonrandom chromosomal changes that occur during tumor progression. This information localized the gene(s) from which cloning could commence. In this study, we combined aspects of both strategies to identify a novel, functional breast carcinoma metastasis suppressor gene.
A recent cataloging of differential gene/protein gene expression and chromosomal abnormalities occurring as breast carcinoma acquires metastatic potential (2) revealed that some karyotypic changes commonly occur in early-stage breast cancer (8p, 13q, 16q, 17p, 17q, and 19p), whereas others typically occur later in breast cancer progression (1p, 1q, 3p, 6q, 7q, 11p, and 11q). Among the most common changes in both familial or sporadic breast carcinoma are losses of genetic material on chromosome 11q, which occurs in 40–65% of cases. There are several regions spanning the q-arm of chromosome 11 for which associations have been made with breast cancer progression. Among the most common are amplifications and deletions involving regions near band 11q13. Within this region, there is evidence supporting the existence of a number of critical genes, including tumor-promoting, tumor-suppressing, metastasis-promoting and metastasis-suppressing genes. The genes int-2, hst, bcl-1, glutathione S-transferase, CCND1, and EMS-1, which map to 11q13, are amplified in breast cancer at a frequency between 3 and 20%. There exists a high-frequency involvement of rat chromosome 1 (which is syntenic to human chromosome 11) in the development and progression of rat mammary tumors (3) . Therefore, based on these observations and high-frequency deletions involving 11q13-q14 in late-stage, metastatic breast carcinomas, we tested the hypothesis that chromosome 11q encodes a metastasis suppressor gene. Upon finding that introduction of a normal human chromosome 11 into metastatic MDA-MB-435 (435) human breast carcinoma suppressed metastasis without affecting tumorigenicity (4) , we set out to identify the gene(s) responsible. We report here the isolation and functional characterization of a metastasis suppressor gene from this region.
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MATERIALS AND METHODS |
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BRMS15 transfectants were derived after transfection of full-length BRMS1 cDNA (see below) cloned into the constitutive mammalian expression vector, pcDNA3 (Invitrogen, San Diego, CA). All cell lines were cultured in a 1:1 (v/v) mixture of DMEM and Ham’s F-12 medium supplemented with 5% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 1% nonessential amino acids, and 1.0 mM sodium pyruvate, but no antibiotics or antimycotics. Transfected cells and neo11/435 hybrids also received 500 µg/ml Geneticin (G-418; Life Technologies, Inc., Gaithersburg, MD). BRMS1-transfected 435 cells acquired an unexplained acute sensitivity to trypsin; therefore, cultures were thereafter passaged using 2 mM EDTA solution in calcium- and magnesium-free Dulbecco’s PBS. Hybrid clones and transfectants were used before passage 11 in all cases to minimize the impacts of clonal diversification and phenotypic instability. For all functional and biological assays, cells between 70 and 90% confluence were used with viability >95%. All lines were routinely checked and found to be negative for Mycoplasma spp. contamination using the GenProbe method (Fisher Scientific, Pittsburgh, PA).
Cell line nomenclature was developed to identify the origin and nature of each cell line as unambiguously as possible. Single-cell clones are identified by the parental cell line name preceding a "." followed by a clonal designation. Uncloned populations are identified by a "-" after the parental cell line name. Microcell hybrids are identified by the tagged chromosome number followed by a "/" (e.g., neo11/435.A3 is single cell clone A3 derived after microcell-mediated transfer of chromosome 11 into MDA-MB-435). Where appropriate, numbers in parentheses following the cell line designation indicate the number of subcultures following cloning or establishment of the cell line. Numbers preceded by a "TE" indicate that the cells were passaged in a mixture of 0.125% trypsin-2 mM EDTA in calcium- and magnesium-free Dulbecco’s PBS. Numbers preceded by a "P" indicate the cells were passaged using EDTA alone.
Transfections.
BRMS1 was cloned into the constitutive mammalian expression
vector, pcDNA3 (Invitrogen). To detect BRMS1 protein expression, a
chimeric molecule was also constructed with an NH2-terminal
epitope tag (SV40T epitope 901; Ref. 5
). Epitope-tagged and native
BRMS1 plasmids as well as pcDNA3 vector only were
transfected into 435 and 231 cells by electroporation. After selection
in G-418, single-cell clones were isolated by limiting dilution.
Stable transfectants were assessed for their expression by RNA blot
and, as appropriate, by immunoblot.
DD-RTPCR.
To identify differences in mRNA expression between metastatic and
nonmetastatic neo11/435 hybrid cells, a DD-RTPCR approach was
undertaken. The methods used involved modifications of the method
described by Liang and Pardee (6)
as available in the
Delta Differential Display kit (Clontech Laboratories, Inc., Palo Alto,
CA). To minimize the impact of clonal heterogeneity, a mixture of equal
parts mRNA from three neo11/435 hybrid clones [neo11/435.A3 (TE5),
neo11/435.B1 (TE4), and neo11/435.D1 (TE10)] were used. To reduce the
chances of proceeding with irrelevant cDNAs, we validated findings at
several intermediate steps during the experiment. In short, every step
was replicated using independent samples. Once PCR products were
validated, they were used as probes to examine differential expression
in Northern blots using progressively more extensive series of
cell-line mRNAs. As full-length cDNA clones were obtained, replicate
Northern blots were done with those as well to verify prior results.
Differentially expressed cDNAs and the primers used for the initial DD-RTPCR reactions were as follows: BRMS1 (P9/P9); F5A3 (P9/T5), 8A3 (P3/T4); adenine phosphoribosyltransferase (P1/T9); N-acetyl-galactosamine-6-sulfate sulfatase (P6/P6); hexokinase II (P10/T8):
P1: 5'-ATTAACCCTCACTAAATGCTGGGGA-3'
P3: 5'-ATTAACCCTCACTAAATGCTGGTGG-3'
P6: 5'-ATTAACCCTCACTAAATGCTGGGTG-3'
P9: 5'-ATTAACCCTCACTAAATGTGGCAGG-3'
P10: 5'-ATTAACCCTCACTAAAGCACCGTCC-3'
T4: 5'-CATTATGCTGAGTGATATCTTTTTTTTTCA-3'
T5: 5'-CATTATGCTGAGTGATATCTTTTTTTTTCC-3'
T8: 5'-CATTATGCTGAGTGATATCTTTTTTTTTGC-3'
T9: 5'-CATTATGCTGAGTGATATCTTTTTTTTTGG-3'
F5A3 and 8A3 were provisional nomenclature used during the initial part of these studies.
Chromosomal Localization of BRMS1.
BRMS1 cDNA was used to screen bacterial artificial
chromosome and P1 artificial chromosome libraries at Genome Systems,
Inc. (St. Louis, MO). Bacterial artificial chromosome clones 412(n24)
and 536(h18) harbored BRMS1 as confirmed by direct
sequencing.6
The genomic sequence was determined using the 412(n24 clone). DNA was
isolated and labeled with digoxigenin dUTP by nick translation and
combined with sheared human DNA before hybridization to metaphase
chromosomes derived from phytohemagglutinin-stimulated peripheral blood
leukocytes in a solution containing 50% formamide, 10% dextran
sulfate, and 2x SSC. Specific hybridization signals were detected by
exposing the hybrid cell lines to antidigoxigenin antibodies followed
by counterstaining with DAPI. Specific labeling was seen along the
proximal long arm of a group C chromosome, which was subsequently
confirmed to be chromosome 11 based on cohybridization with genomic
probes known to map to 11p15 and 11cen. Measurements of 71 of 80
specifically labeled chromosomes 11 in metaphase spreads demonstrated
that BRMS1 is located at a position that is 19% of the
distance from the centromere to the telomere of chromosome 11q. This
corresponds to band 11q13.1-q13.2 (data not shown).
Metastasis Assays.
Immediately prior to injection, cells (7–11 passages after
transfection) at 80–90% confluence were detached with a 2
mM EDTA solution. Cells were washed, counted on a
hemacytometer, and resuspended in ice-cold HBSS to a final
concentration of 2.5 x 106
cells/ml for 231
cells and 1 x 107
cells/ml for 435 cells.
MDA-MB-231 cells and derivatives (0.5 x 106
in 0.2 ml) were injected i.v. into the lateral tail vein of 3- to
4-week-old female athymic mice (Harlan Sprague-Dawley, Indianapolis,
IN). Mice were killed 4 weeks post-injection and examined for the
presence of metastases. Lungs were removed, rinsed in water, and fixed
in Bouin’s solution before quantification of surface metastases as
described previously (7)
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Similar procedures were used for the spontaneous metastasis assay using MDA-MB-435 cells, except that 1 x 106 cells (0.1 ml) were injected into exposed axillary mammary fat pads of anesthetized 5- to 6-week-old female athymic mice. When the mean tumor diameter reached 1.5–2.0 cm, tumors were surgically removed under ketamine:xylazine (80–85 mg/kg:14–16 mg/kg) anesthesia, and the wounds were closed with sterile stainless steel clips. Four weeks later, mice were sacrificed, and visible metastases were counted. Lung tissues were handled as above. Metastases were also observed in the ipsilateral and contralateral axillary lymph nodes of control mice. Occasional recurrences developed at the site of tumor removal, but the presence of hematogenous metastases did not necessarily correlate with presence of recurrent tumor.
Animals were maintained under the guidelines of the NIH and the Pennsylvania State University College of Medicine. All protocols were approved by the Institutional Animal Care and Use Committee. Food and water were provided ad libitum.
Statistical Analyses.
The number of lung metastases was compared for BRMS1
transfectants and corresponding parental and vector-only-transfected
MDA-MB-435 and MDA-MB-231 cells. For experimental metastasis assays,
one-way ANOVA followed by Tukey’s Honestly Significant Difference
post hoc test was used. For spontaneous metastasis assays, a
Kruskal-Wallis ANOVA of ranks procedure was used with Dunn’s
post hoc test. Statistical differences in adhesion and
motility assays were done using Student’s t test comparing
BRMS1-transfected to vector-only-transfected cells.
Calculations were performed using SigmaStat statistical analysis
software (Jandel Scientific, San Rafael, CA). Statistical significance
was designed as P 
0.05 using two-tailed
tests.
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RESULTS |
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. Eighteen bands amplified reproducibly in replicate RT-PCR reactions
(data not shown). Of those, six fragments exhibited 
5-fold higher
mRNA expression in neo11/435 hybrid cell clones as detected by Northern
blotting using the PCR product as a probe and quantified by phosphor
image analysis (Fig. 1, b and c
). None of the PCR
products detected mRNAs expressed exclusively in the
metastasis-suppressed cells. The differentially expressed cDNA inserts
were sequenced, and homology to known genes and ESTs was assessed by
comparing with the GenBank/European Molecular Biology
Laboratory/DDBJ/PDB combined database. Three of the cDNAs were
homologous to known human genes
(N-acetylgalactosamine-6-sulphatase, adenine
phosphoribosyltransferase, and hexokinase II). The remaining three cDNA
fragments were novel and became priorities for further study. Here we
report on the isolation and functional characterization of one of these
novel cDNAs, BRMS1.
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TripIEx
library after this tissue was found to express high levels of all three
novel cDNAs. Tissue-specific splice variants were not expected because
only a single band of 
1.5 kb was detected. This pattern was
replicated using full-length BRMS1 cDNA as a probe.
BRMS1 is widely expressed to varying levels in every normal
human tissue examined (Fig. 1d). Nucleotide sequence analysis of BRMS1 cDNA initially revealed no significant homologies to any known genes, ESTs, or proteins deposited in the databases. Regions of BRMS1 cDNA showed numerous homologies to short ESTs isolated from fetal liver and spleen as well as the human tumor cell line HeLa, consistent with the wide tissue expression observed in the multitissue Northern blots. BRMS1 has been detected in Southern blots in multiple species at the DNA level, including yeast, human, mouse, rat, rabbit, and cow (data not shown). A murine cDNA recently has been isolated and is 89.8% homologous at the nucleotide level,7 suggesting that the gene is relatively well conserved. The BRMS1 human cDNA sequence was submitted to GenBank as a novel human gene with an Accession Number of AF159141.
After the BRMS1 sequence was submitted, a deposited partial sequence (Accession Number AL050008; designated "hypothetical human protein sequenced by AGOWA within the cDNA sequencing consortium of the German Genome Project") had 91% homology at the predicted protein level for the region bounded by amino acids 60–244. However, no information regarding function for AL050008 is available. This may indicate that there is a BRMS1 gene family.
Computerized analysis (DNAsis; Hitachi Software) shows that the
BRMS1 cDNA length is 1485 bp with the largest open reading
frame of 741 bp (from nucleotides 122 to 862; Fig. 2
).BRMS1 encodes a novel protein of 246 amino acids
(Mr 
28,500), a result confirmed using
in vitro transcription and translation (data not shown). The
genomic structure of BRMS1 is organized as 10 exons spanning
10 kb.8
Exon 1 is untranslated. Fluorescence in situ hybridization
mapping places the location of BRMS1 gene at human
chromosome 11q13.1-q13.2 (Fig.
3a).
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To assess the effect of BRMS1 on breast carcinoma biological behavior, BRMS1 was transfected into two independently derived metastatic human breast carcinoma cell lines, MDA-MB-435 (435) and MDA-MB-231 (231). The morphologies of 435 and 231 BRMS1-transfected cells were not noticeably or uniformly different from parental cells. Nor were in vitro growth rates or saturation densities different. In the experiment shown, BRMS1-transfected cells exhibited a slight delay following seeding; however, this did not impact routine culture. BRMS1 transfectants also tended to aggregate more readily after detachment. However, these properties were not evident in every clone isolated. Surprisingly, 435 cells, but not 231 cells, transfected with BRMS1 acquired an acute sensitivity to trypsin (data not shown). Whereas parental cells previously were routinely passaged using a mixture of trypsin and EDTA, the BRMS1 transfectants died when exposed to even low concentrations of trypsin. Therefore, subsequent cultures were handled using EDTA to detach the cells from the substrata.
Clones representing low, medium, and high BRMS1 mRNA and protein expression (the latter were evaluated using epitope-tagged BRMS1) were chosen for in vivo functional studies. The transfected cells were then tested for tumorigenicity and metastasis in athymic mice. BRMS1-transfected 231 cells were injected i.v. and assessed for their ability to form macroscopic metastases in lung. Compared with vector-only transfectants, BRMS1 transfectants exhibited a dose-dependent, significant (P < 0.001) decrease in metastatic potential (Fig. 3c). Similar conclusions were drawn using mRNA to assess expression (data not shown). This implies that the epitope tag does not deleteriously influence BRMS1 functionality. As expected, the ability of 231 cells to form progressively growing tumors in the mammary fat pad was not suppressed. Because the parental 231 cell line does not metastasize from an orthotopic site, metastatic suppression using the spontaneous metastasis assay could not be assessed.
Parental and BRMS1-transfected MDA-MB-435 cells grew
progressively after injection into an orthotopic (i.e.,
mammary fat pad) site. Growth of BRMS1-transfected 435
tumors was somewhat delayed compared with the parental and/or vector
controls. In general, once the tumors began to grow, their sizes were
1 week behind the parental, metastatic populations, suggesting that
rate of tumor growth, once established, is the same. Failure to
suppress tumorigenicity indicates that BRMS1 is not a tumor
suppressor gene. The histologic appearance of parental 435 and
BRMS1 transfectants were similar, except that the latter
exhibited fewer fibrous bands in the stromal compartment of the tumors
(data not shown). Whereas tumorigenicity was unaltered, the incidence
and number of metastases to lung and regional lymph nodes were
significantly (P = 0.004) suppressed in the
MDA-MB-435 BRMS1 transfectants (Table 1)
. Parental and vector-only transfectant cells formed axillary lymph node
and lung metastases in 100% of the mice injected, whereas in the
BRMS1 transfectants, the incidence dropped by 50–90%. Of
the metastases that formed, all were noticeably smaller than the
parental lesions at a comparable time following injection. Even if the
metastases were given more time to grow, most did not develop into
grossly visible lesions. BRMS1 expression was still detectable in the
BRMS1-transfected 435 locally growing tumors. Because
parental 435 cells do not form lung metastases following i.v.
injection, i.v. inoculation studies like those with 231 were not done.
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DISCUSSION |
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The mechanism by which BRMS1 suppresses metastasis is still not fully determined. In vitro assays assessing individual steps in the metastatic cascade predict a complex role for this molecule. The step(s) at which BRMS1 functions are downstream of local invasion: invasive cords are observed at the edge of locally growing tumors. This finding is consistent with the lack of gross changes in the ability of the cells to produce and activate matrix metalloproteinase-2 and -9 as detected by zymography.6 Efficient invasion occurs despite a modest but reproducible decrease in motility as measured using in vitro wound migration assays. Adhesion to fibronectin, laminin, and type IV collagen are likewise unaffected by BRMS1 expression in 231 and 435 cells.12
Analysis of the predicted BRMS1 amino acid sequence hints that BRMS1 interacts with other proteins (i.e., phosphorylation sites, coiled-coil periodicity, and leucine zipper). Because these motifs are often found in components of transcriptional machinery, we hypothesized that BRMS1 might suppress metastasis by regulating expression of other metastasis-suppressor genes; however, there appears to be no correlation between the expression of Kai1, Nm23, KiSS1 or E-cadherin with BRMS1.6 Although this hypothesis is not formally disproved, these data argue that it is not the case.
BRMS1 transcript (1.5 kb) was detected in every human tissue examined, albeit at different levels. The uniform size of BRMS1 transcript argues that it is not alternatively spliced in various tissues. Homology to BRMS1 DNA was detected using Southern blotting. This suggests that BRMS1 is relatively well conserved; however, the genomic organization is apparently different (data not shown).
Compared with the many normal tissues examined, BRMS1 mRNA expression was very low in the 435 and 231 cells by both RT-PCR and poly(A)+-enriched mRNA Northern blots. Analysis of protein levels awaits the development of BRMS1-specific antibodies (in progress). An RNA blot composed of a panel of human breast carcinoma cell lines with different malignant properties—MCF10A, MCF7, T47DCO, MDA-MB-435, MDA-MB-231, LCC15, SUM185, SUM1315, and MKL-4 (8, 9, 10, 11, 12) was probed with full-length BRMS1 cDNA to assess expression levels. These cell lines were chosen because all have characteristics that labeled them as "aggressive." In our hands, however, only 435 and 231 are reproducibly metastatic in athymic mouse models. BRMS1 mRNA expression was high in LCC15 and MKL-4, but expression was also observed in MCF10A, T47D, SUM185, and SUM1315 cell lines.6 Sequencing is underway to determine whether BRMS1 is wild-type or mutant in these cell lines.
In summary, we found a new human breast carcinoma metastasis suppressor gene by DD-RTPCR comparison of metastatic 435 cells and metastasis-suppressed neo11/435 cells. The BRMS1 gene maps to a "hot spot" in breast cancer progression, human chromosome 11q13, further supporting the likelihood that BRMS1 is important in human breast cancer progression toward metastasis. In general, low expression of BRMS1 correlates with the metastatic potential in human breast carcinoma cell lines in nude mice. It will be necessary to further analyze the BRMS1 gene in other breast carcinomas during various stages of progression. Presently, it is not possible to state whether defects in human breast carcinoma are due to down-regulation of BRMS1, mutation, or both. Such experiments will require collection of matched samples from primary tumors and metastases. Because the most lethal attribute of breast cancer cells is their ability to spread and colonize distant sites, understanding BRMS1 function may help prevent metastasis and improve breast cancer survival.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by Grant
DAMD-96-1-6152 (to D. R. W.) from the United States Army Medical
Research and Material Command with additional support from the NIH
(Grant CA-62168), the National Foundation for Cancer Research, and the
Jake Gittlen Memorial Golf Tournament. 
2 Contributed equally to this work.
3 Present address: Department of Urology,
University of Virginia, Charlottesville, VA 22908.
4 To whom requests for reprints should be
addressed, at Jake Gittlen Cancer Research Institute, Mailstop H059,
Room C7810, The Pennsylvania State University College of Medicine, 500
University Drive, Hershey, PA 17033-2390.
5 The abbreviations used are: BRMS1,
breast cancer metastasis suppressor-1; DD-RTPCR, differential display;
DAPI, 4',6-diamidino-2-phenylindole; RT-PCR, reverse transcription-PCR;
EST, expressed sequence tags.
6 R. S. Samant, M. T. Debies, and D. R.
Welch, unpublished observations.
7 R. S. Samant, M. T. Debies, and D. R.
Welch, manuscript in preparation.
8 R. S. Samant, M. T. Debies, M. J. Seraj,
and D. R. Welch, manuscript in preparation.
9 http://www.genebio.com/prosite.html.
10 http://www.psort.nibb.ac.jp:8800.
12 T. Sakamaki and D. R. Welch, unpublished
observations.
Received 2/ 4/00. Accepted 4/18/00.
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V. Urquidi, D. Sloan, K. Kawai, D. Agarwal, A. C. Woodman, D. Tarin, and S. Goodison Contrasting Expression of Thrombospondin-1 and Osteopontin Correlates with Absence or Presence of Metastatic Phenotype in an Isogenic Model of Spontaneous Human Breast Cancer Metastasis Clin. Cancer Res., January 1, 2002; 8(1): 61 - 74. [Abstract] [Full Text] [PDF]
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K. W. Hunter, K. W. Broman, T. L. Voyer, L. Lukes, D. Cozma, M. T. Debies, J. Rouse, and D. R. Welch Predisposition to Efficient Mammary Tumor Metastatic Progression Is Linked to the Breast Cancer Metastasis Suppressor Gene Brms1 Cancer Res., December 1, 2001; 61(24): 8866 - 8872. [Abstract] [Full Text] [PDF]
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M. M. Saunders, M. J. Seraj, Z. Li, Z. Zhou, C. R. Winter, D. R. Welch, and H. J. Donahue Breast Cancer Metastatic Potential Correlates with a Breakdown in Homospecific and Heterospecific Gap Junctional Intercellular Communication Cancer Res., March 1, 2001; 61(5): 1765 - 1767. [Abstract] [Full Text]
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B. A. Yoshida, M. M. Sokoloff, D. R. Welch, and C. W. Rinker-Schaeffer Metastasis-Suppressor Genes: a Review and Perspective on an Emerging Field J Natl Cancer Inst, November 1, 2000; 92(21): 1717 - 1730. [Abstract] [Full Text] [PDF]
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