Genomic Analysis of a Spontaneous Model of Breast Cancer Metastasis to Bone Reveals a Role for the Extracellular Matrix

Bedrich L. Eckhardt,1 Belinda S. Parker,1 Ryan K. van Laar,1 Christina M. Restall,1 Anthony L. Natoli,1 Michael D. Tavaria,1 Kym L. Stanley,1 Erica K. Sloan,1 Jane M. Moseley,2 and Robin L. Anderson1

1Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Melbourne, Victoria, Australia and 2Department of Medicine, University of Melbourne, St. Vincent’s Hospital, Fitzroy, Victoria, Australia

Abstract involvement in several tissues, including bone, lung, lymph A clinically relevant model of spontaneous breast cancer node, and liver (1). The selective distribution of metastases is metastasis to multiple sites, including bone, was dictated by several factors, including the pattern of vascular characterized and used to identify genes involved in flow from the primary site, complementary adhesive contacts, metastatic progression. The metastatic potential of and molecular interactions between the tumor cell and the several genetically related tumor lines was assayed stroma at the secondary site (2). using a novel real-time quantitative RT-PCR assay of Breast cancer metastases have a strong avidity for bone tumor burden. Based on this assay, the tumor lines were (3, 4), leading to metastases that cause intractable pain, spinal categorized as nonmetastatic (67NR), weakly metastatic cord compression, bone fractures, and hypercalcemia (5, 6). to lymph node (168FARN) or lung (66cl4), or highly Current therapies, including cytotoxic chemotherapy and the metastatic to lymph node, lung, and bone (4T1.2 and administration of bisphosphonates, are rarely curative but can 4T1.13). In vitro assays that mimic stages of metastasis alleviate symptoms arising from bone metastases (7). An showed that highly metastatic tumors lines were more improved understanding of the biological and genetic regula- adhesive, invasive, and migratory than the less tion of breast cancer metastasis to bone is essential to identify metastatic lines. To identify metastasis-related genes novel and more effective molecular targets for therapy. in this model, each metastatic tumor was array profiled The advent of microarray technology has greatly enhanced against the nonmetastatic 67NR using 15,000 mouse the search for genetic regulators and markers of metastasis. cDNA arrays. A significant proportion of genes relating Indeed, arrays have been employed to identify genetic patterns to the extracellular matrix had elevated expression in that are predictive of metastatic relapse (8-10). They have also highly metastatic tumors. The role of one of these genes, been used in animal models of metastasis to determine POEM, was further investigated in the model. In situ molecular mechanisms that dictate metastatic spread (11-14). hybridization showed that POEM expression was Animal models provide an essential and powerful resource to specific to the tumor epithelium of highly metastatic investigate mechanisms of metastasis. However, breast cancer tumors. Decreased POEM expression in 4T1.2 tumors metastasis research is dominated by the use of xenograft models significantly inhibited spontaneous metastasis to the of experimental metastasis, typically involving the injection of lung, bone, and kidney. Taken together, our data human breast tumor cells directly into the circulatory system of support a role for the extracellular matrix in metastatic immunocompromised mice, resulting in metastases in either progression and describe, for the first time, a role for lung (following tail vein injection) or bone (following POEM in this process. (Mol Cancer Res 2005;3(1):1–13) intracardiac injection; refs. 14-17). These models have been valuable for studying the final stages of metastasis, and when Introduction coupled with microarray analysis, they can identify genes that Metastasis is the main cause of morbidity and mortality in regulate the colonization of specific tissues (13, 14). However, cancer patients. In late-stage breast cancer, patients have tumor these models of experimental metastasis involve human tumors in a mouse host and may lack some of the critical tumor-host interactions. In addition, they do not encompass the initial Received 6/1/04; revised 11/19/04; accepted 11/30/04. stages of primary tumor growth, invasion, and metastasis from Grant support: Department of Defense Breast Cancer Research Program grants an orthotopic site. Thus, array analysis using these experimental DAMD17-98-1-8144 (R.L. Anderson) and DAMD17-01-1-0371 (M.D. Tavaria), metastasis models provides little insight into the biology of Susan G. Komen Breast Cancer Foundation predoctoral fellowship (E.K. Sloan), and NIH/National Cancer Institute grant ROI CA90291 (R.L. Anderson). spontaneous metastasis. The costs of publication of this article were defrayed in part by the payment of A clinically relevant animal model of spontaneous breast page charges. This article must therefore be hereby marked advertisement in cancer metastasis to multiple sites, including bone, is now accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Robin L. Anderson, Trescowthick Research Laboratories, available (18). Several syngeneic tumor lines with a spectrum of Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, metastatic phenotypes have been isolated from a spontaneous Victoria, Australia 8006. Phone: 61-3-9656-1284; Fax: 61-3-9656-1411. E-mail: [email protected] mammary tumor in a BALB/cfC3H mouse (19). When injected Copyright D 2005 American Association for Cancer Research. into the mammary gland of mice, these tumor lines are either

nonmetastatic (67NR; ref. 20), produce spontaneous metastases ALN along with numerous metastatic foci in the lungs (Fig. 1C). to lymph node (168FARN; ref. 21), lung (66cl4; ref. 22), or both When analyzed by q-RT-PCR, high RTB signals were detected in (4T1; ref 20). 4T1.2 and 4T1.13 tumor lines are rare variants the lung (54% tumor tissue), ALN (29%), femur (17%), spine isolated from 4T1 cells that closely mimic the metastatic (10%), kidney (10%), and heart (8%; Fig. 1B). A similar distribution of human breast cancer (18). When grown as a metastatic pattern was observed for the 4T1.13 tumor line. Bone primary tumor in the mammary fat pad of a mouse, these tumor lysis was evident in bone metastases derived from 4T1.2 and lines develop overt metastases in bone, lung, and lymph nodes 4T1.13 tumors (data not shown), consistent with previous reports (18). Furthermore, mice bearing 4T1.2 and 4T1.13 tumors of these tumor lines (18, 24). Tumor signals were also detected in occasionally develop hind limb paralysis and have elevated the brain, liver, and spleen in mice harboring 4T1.2 and 4T1.13 plasma levels of calcium and parathyroid hormone-related tumors albeit to a minor extent (<1%). Thus, 4T1.2 and 4T1.13 protein (PTHrP), two pathologic hallmarks of the human disease can spontaneously metastasize to several organs, but preferential (18, 23). With a diverse range of metastatic capacities, these growth occurs in specific tissues (lung, ALN, and bone). The genetically related tumor lines provide a powerful model to presence of tumor within the ALN of mice bearing 4T1.2 and investigate the molecular events that dictate metastasis of breast 4T1.13 tumors suggests that these lines can also metastasize via cancer. a lymphogenous route. For more accurate analysis of metastasis, a real-time The extent of metastasis from 4T1.2 and 4T1.13 tumors to quantitative reverse transcription-PCR (q-RT-PCR) assay for ALN and lung was f20-fold higher than metastases produced the measurement of tumor burden within a tissue has been by 168FARN and 66cl4 tumors, respectively, indicating that developed. All tumor lines are tagged with a neomycin 4T1.2 and 4T1.13 have a greater metastatic capacity than resistance gene, allowing them to be detected by q-RT-PCR 168FARN and 66cl4. Furthermore, the metastatic distribution of genomic DNA. Using this model of spontaneous breast of 4T1.2 and 4T1.13 tumors is similar to that observed in cancer metastasis, critical metastasis-related genes can be advanced human breast cancer and, to our knowledge, provides identified. Gene expression profiles of metastatic tumors in the only syngeneic mouse model of spontaneous breast cancer this model were compared with the nonmetastatic 67NR using metastasis to bone. cDNA arrays consisting of 15,000 mouse gene elements. Array Representative sections of the kidney, heart, lung, spine, and analysis revealed altered expression of extracellular matrix femur provide further evidence of the difference in extent of (ECM) genes in highly metastatic tumors. One of these genes, metastatic dissemination of the 4T1.2 and 66cl4 tumors POEM, was shown to have a functional role in spontaneous (Fig. 1C). Metastatic lung nodules from 4T1.2 tumors were breast cancer metastasis in the model. larger and more numerous compared with those in the lungs of mice with 66cl4 tumors. Overt metastases in the spine and femur were detected in 4T1.2 and 4T1.13 tumors but not from Results 66cl4 tumors (Fig. 1C; data not shown). These histologic data Characterization of the Metastatic Dissemination of agree with the RTB values determined by q-RT-PCR. Several Syngeneic Mammary Tumor Lines The spontaneous metastatic distribution of each tumor line Increased Adhesion, Migration, and Invasion, but Not was profiled using a sensitive q-RT-PCR assay for tumor Angiogenesis and Proliferation, Correlate with Enhanced burden (Fig. 1A). Tumor cells were inoculated into the fourth Metastatic Capacity inguinal mammary glands of mice. Mice were culled and The processes of angiogenesis, proliferation, cell motility, tissues were excised for genomic DNA extraction when the invasion, and adhesion are critical for the formation of distant average primary tumor weight of each group was f1.5 g or if metastases. Thus, we determined whether highly, weakly, and the mice were displaying signs of distress. The result of a screen nonmetastatic tumor lines differed in these metastasis-related for spontaneous metastasis in mice bearing different tumor lines processes using functional assays. As the recruitment of a is shown in Fig. 1B. Corresponding tissues were also removed blood supply is of vital importance for tumor growth in vivo, from a non-tumor-bearing mouse to measure background the angiogenic capacity of each tumor line was measured. signal. The highest RTB signal detected in any tissue from a After 6 days, Matrigel plugs containing tumor cells were non-tumor-bearing mouse was 31; thus, RTB values below this removed from mice to assess hemoglobin content as a were considered background. As expected, strong RTB scores surrogate marker for the extent of angiogenesis. The use of were evident in all primary tumors. Consistent with previous hemoglobin content as a measure of angiogenesis has been observations (20), no RTB signals were detected in any tissue validated previously (25). Whereas, as expected, all tumor tested from mice bearing nonmetastatic 67NR tumors, con- lines could stimulate angiogenesis (6- to 8-fold higher firming that this tumor line cannot develop spontaneous compared with Matrigel without cells; P < 0.01), there were metastases. no significant differences observed between any tumor lines.3 Mice bearing 168FARN and 66cl4 tumors presented with The doubling time of each line was calculated as the rate of enlarged axillary lymph node (ALN) and metastatic lung log-phase growth between 24 and 96 hours. All metastatic lines nodules, respectively. This was reflected in modest RTB scores of f200 (2% of the tissue consisted of metastatic cells) in both the ALN of 168FARN tumor-bearing mice and the lungs of mice with 66cl4 tumors. These tumors did not metastasize to any other 3 Supplementary data for this are available at Molecular Cancer Research Online tissues (Fig. 1B). 4T1.2 tumor-bearing mice also had enlarged (http://mcr.aacrjournals.org/).

FIGURE 1. Metastatic dissemination of several genetically related tumor subpopulations by q-RT-PCR. A. Tumor burden within a tissue was measured using a novel multiplexed q-RT-PCR assay. This assay quantitates the relative amount of neomycin (tumor tissue) compared with vimentin (endogenous mouse tissue) from the genomic DNA extracted from the tissue of interest. B. Distribution of spontaneous metastases in mice bearing different tumor lines was measured using the q-RT-PCR assay described above. Average RTB scores from an experiment using five mice per tumor line. Tumor lines were grouped into three categories based on metastatic burden: nonmetastatic (67NR), weakly metastatic (168FARN and 66cl4), and highly metastatic (4T1.2 and 4T1.13). C. Presence of metastases (M) identified by q-RT-PCR were confirmed by H&E staining of representative sections of kidney, heart, femur, spine, and lung of mice bearing 66cl4 or 4T1.2 tumors. Visible metastases were confirmed in tissues identified with tumor burden by q-RT-PCR assay. N, normal tissue. Bar, 0.5 mm.

had faster in vitro doubling times than 67NR; however, genes were expressed at higher levels in the weakly and highly doubling time could not distinguish highly from weakly metastatic tumors, respectively. Genes with at least 4-fold metastatic tumor lines.3 The in vivo growth rates of the primary altered expression between the two groups are displayed in tumors were also monitored, but again no correlation was found Table 1. with metastatic potential or in vitro proliferation.3 Adhesion is critical for metastasis related events, such as the initiation of cellular motility and for the extravasation into a tissue (26); hence, the adhesion of the tumor lines to Matrigel (a substrate similar to the ECM components of basement membranes) was measured. After 30 minutes of adhesion, the highly metastatic tumor lines displayed a significant 2-fold increase in adhesion to Matrigel compared with 67NR (P < 0.01; Fig. 2A). There was no difference in adhesion between the weakly metastatic tumor lines and 67NR. At later time points, all cell lines showed a similar extent of adhesion to Matrigel (data not shown). Chemotactic migration of the tumor lines was determined using a modified Boyden chamber assay. Migration was measured as the number of cells that were able to traverse from the apical to basal surface through pores within the membrane of the insert in response to a chemoattractant (10% FCS). 4T1.2 and 4T1.13 clearly showed a 10- to 20-fold increase in migration compared with other tumor lines (P < 0.01; Fig. 2B). The invasive capacity of the tumor lines was examined by their ability to invade through a Matrigel barrier. Highly metastatic 4T1.2 and 4T1.13 cells were 4- to 10-fold more invasive than weakly metastatic tumor lines (P < 0.01; Fig. 2C). Interestingly, although the nonmetastatic 67NR showed negli- gible migration (Fig. 2B), this tumor line was clearly able to invade through a Matrigel barrier. In fact, the level of invasion displayed by 67NR was significantly greater than that of 168FARN and 66cl4 but significantly less than 4T1.2 and 4T1.13 (P < 0.01). The invasion response was not seen in the negative controls (no chemoattractant), indicating a possible interplay between Matrigel and FCS to stimulate an invasive phenotype of 67NR in vitro. Despite this invasive phenotype, 67NR was incapable of metastasizing in vivo (Fig. 1B).

Identification of Genes Associated with Highly and Weakly Metastatic Tumors To identify genes associated with metastasis in our model, pooled RNA from five primary tumors was arrayed using RNA from the nonmetastatic 67NR primary tumor as a common reference. Array experiments were repeated four times for each tumor line, including two replicates with dye reversals to control for dye incorporated bias (as described in Materials and Methods). To identify genes that were altered between the two FIGURE 2. Differences in adhesion, migration, and invasion distin- metastatic phenotypes, we grouped the expression profiles of guish weakly and highly metastatic tumor lines. A. Ability of tumor cells to 168FARN and 66cl4 tumors (weakly metastatic group) and adhere to Matrigel-coated surfaces after 30-minute incubation at 37jC. B. Chemotactic migration of tumor cells toward a 10% FCS chemoattractant compared these with the grouped expression profiles of 4T1.2 using a modified Boyden chamber assay. After 5 hours, the number of and 4T1.13 tumors (highly metastatic group). Gene elements cells traversing the membrane was counted. C. Invasion of tumor cells (n = 216) were identified with significant (P < 0.01 between the through a Matrigel barrier in response to a 10% FCS chemoattractant. 3 After 24 hours, the number of cells invading through the Matrigel and two groups) and z2-fold difference in expression. Of the 216 traversing the membrane was counted. In migration and invasion assays, genes, 125 were known genes, 9 were duplicated genes, and 82 five fields per sample were counted and averaged. Columns, mean of two experiments with triplicate samples; bars, SE (in all assays). *, P < 0.05; were either expressed sequence tags or had weak homology to **, P < 0.01, Student’s t test for metastatic tumor line compared with 3 genes from other species. Of the 125 known genes, 36 and 89 67NR.

A survey of the current literature on the genes presented in Reduced POEM expression was detected by RT-PCR, which Table 1 revealed that 65% have been associated previously has been shown previously to be an accurate measure of gene with human cancer, 33% with human breast cancer, and 21% knockdown (30). A 60% reduction in POEM expression was with metastasis. The close association of the mouse genes with observed in 4T1.2pRS-POEM compared with 4T1.2 and human cancer shows their clinical relevance, especially to 4T1.2pRS-GFP cells (P < 0.01; Fig. 5A). Decreased POEM breast cancer. Further support for the use of this model to expression was stable for at least 8 weeks in culture (data not identify genetic regulators of metastasis comes from the 21% shown). The 4T1.2pRS-GFP and 4T1.2pRS-POEM lines were of the genes that have been associated previously with injected into the mammary glands of BALB/c mice (15 per metastasis. Eight genes identified by microarray analysis were group) and tumor growth was monitored. Reduced POEM selected for verification by q-RT-PCR. Analysis of replicate expression did not alter primary tumor growth rate (Fig. 5B) or samples from 66cl4 and 4T1.2 tumors confirmed a close the final tumor weight (Fig. 5C). Reduced POEM expression at correlation with the level of expression as revealed by the end of the experiment was confirmed in 4T1.2pRS-POEM microarray analysis (Fig. 3). primary tumors as analyzed by RT-PCR of three primary tumors Of the genes listed in Table 1, 18 (41%) were associated from each group (Fig. 5D). Metastatic burden was reduced with adhesion, migration, or invasion, all aspects of the significantly in the lungs, spine, and kidney but not the in heart metastatic process that distinguished the highly metastatic of mice bearing 4T1.2pRS-POEM tumors (P < 0.05; Fig. 5E). phenotype in our model. Interestingly, a significant number of In a repeat experiment, similar results were found, with a genes encoding ECM molecules (9 of 89) were altered in reduction in lung and bone metastases, but no difference in highly metastatic tumors (P = 0.037; Table 2). One of these ALN metastases between groups (data not shown). genes, POEM, was examined further for functional relevance in metastasis. Discussion In this study, we have identified a set of genes linked to spontaneous breast cancer metastasis, and we have reported a POEM Expression in Primary Tumors sensitive q-RT-PCR assay of tumor burden. Several geneti- Although this recently identified ECM protein has been cally related tumor lines were assayed to reveal that 67NR is implicated in cellular adhesion and morphogenesis (27, 28), nonmetastatic, 66cl4 spontaneously metastases to lung and no association with cancer has been reported. POEM was 168FARN metastases to lymph nodes (20, 21). 4T1.2 and expressed 30- to 80-fold higher in highly metastatic compared 4T1.13 are highly metastatic variants, derived from the lung with weakly metastatic tumors (Table 1; Fig. 3). Furthermore, metastasizing 4T1 tumor line, and spread to several organs RT-PCR analysis indicated that the cell lines also express including heart, kidney, lung, lymph node, and bone. Low POEM in vitro and that the level of expression is f30-fold metastatic burden was also noted in the brain, spleen, and higher in 4T1.2 cells compared with 67NR and 66cl4 cells (data liver. The pattern of secondary growth observed in 4T1.2 and not shown). 4T1.13 tumors is similar to that of human metastatic breast To confirm the microarray result, POEM expression was cancer. Moreover, these tumors cause the characteristic in vivo measured by in situ hybridization in primary tumors of varying markers of metastatic bone disease, including pathologic metastatic potential. Using the mouse embryo as a positive fractures, hypercalcemia, and increased plasma PTHrP (18). control, it was found that the POEM antisense riboprobes To our knowledge, this is the only mouse model where overt specifically stained the inner muscle layer of the stomach as bone metastases are observed following spontaneous metas- reported previously (28). This staining was not observed using tasis from the mammary gland. the control sense riboprobes, indicating that any staining The genetic regulation of breast cancer metastasis is an observed was due to the presence of POEM RNA transcripts area of intense research (8, 9, 14, 31, 32). With the advent of (Fig. 4). POEM expression was compared between primary microarray technology, the transcriptional profile of a tumor tumors of the mouse model, revealing a lack of expression in can be obtained and linked to its phenotype. Previous array the nonmetastatic and weakly metastatic primary tumors and studies have been useful in delineating genes involved in the high level POEM expression in the highly metastatic 4T1.2 metastatic process (11, 12, 14, 33); however, the interpreta- primary tumor. The staining was epithelial cell specific (Fig. 4). tion of such data is limited by the restraints of the model The expression of POEM in 4T1.2 primary tumors indicates being used. For example, array analysis on tumor lines that that this gene may have a role in promotion of metastasis. form experimental metastases in bone following intracardiac inoculation is ideal for the identification of genes that Reduced POEM Expression Impairs Tumor Growth and regulate bone colonization but not for genes that are involved Metastasis in the earlier events of metastasis. Here, primary tumors in Using RNA interference we tested the effect of decreased the spontaneous metastasis model were profiled to identify POEM expression on tumor growth and metastasis in the 4T1.2 genes associated with aggressive metastatic disease. tumor model. We engineered the previously described pRe- We identified a panel of 216 genes with altered expression troSuper (pRS) vector (29) to encode short-hairpin RNAs between highly and weakly metastatic tumors. Interestingly, a specific for either POEM or GFP (nonsilencing control) and large number of genes identified using this model have been transfected these constructs into 4T1.2 cells, creating tumor associated previously with human breast cancer or metastatic lines 4T1.2pRS-POEM and 4T1.2pRS-GFP, respectively. progression, thus demonstrating the relevance of this model

Table 1. Genes with Differential Expression between Highly and Weakly Metastatic Tumors and Their Correlation to Cancer and Metastasis-Related Processes

Gene Genbank Symbol Fold difference* Associated withc Metastatic processesb accession no. Human Human Metastasis Adhesion Migration Invasion cancer breast cancer

Cell growth and survival Caspase recruitment BG085048 Card10 15.33 n domain family, member 10 Angiomotin-like 1 AW537935 Amotl1 8.29 nn Keratin complex 1, acidic, BG077966 Krt1-18 7.31 nn n nn gene 18 Junction plakoglobin BG077247 Jup 6.68 nn n Kruppel-like factor 4 (gut) BG069413 Klf4 0.22 nn Semaphorin E BG086959 Sema3c 0.21 nn n n Growth arrest specific 1 BG087671 Gas1 0.09 n Placenta and embryos BG078428 Pem 0.03 oncofetal gene Metabolism Heat shock protein 1 BG070071 Hspb1 7.61 nn ATP citrate lyase AW538652 Acly 5.12 nn S100 calcium binding BG072801 S100a9 4.77 nn nnn protein A9 (calgranulin B) Endothelin-converting enzyme 1 BG083532 LOC230857 4.66 nn ATPase, Na+/K+ transporting, AW544616 Atp1b1 4.45 b1 polypeptide Solute carrier organic anion BG072114 Slco2a1 4.44 transporter family, member 2a1 UDP-N-acetyl-a-D-galactosamine/ BG068045 Galnt3 4.39 nnnn polypeptide N-acetyl-galactosaminyltransferase 3 Peptidylprolyl isomerase C BG065249 Ppic 0.21 n Very low density lipoprotein receptor BG084234 Vldlr 0.10 nn Epoxide hydrolase 1, microsomal BG072453 Ephx1 0.10 n N-acetylated a-linked acidic BG082322 Naalad2 0.03 dipeptidase 2 Proteolysis Serine protease inhibitor 4 BG079624 SPI4 70.77 nnn Serine protease inhibitor, BG079254 Spint1 25.49 nn nnnn Kunitz type 1 Serine protease inhibitor, BG085206 Spint2 7.02 nn nnnn Kunitz type 2 Transglutaminase 2, BG074617 Tgm2 4.23 nn nn C polypeptide Cell communication POEM BG074516 POEM 86.33 n Cell adhesion molecule-related/ BG088280 Cdon 22.52 nn nn down-regulated by oncogenes Tumor necrosis factor BG072211 Tnfrsf19 19.11 receptor superfamily, member 19 Protein tyrosine phosphatase, BG088014 Ptprf 15.98 n receptor type F Anthrax toxin receptor 1 BG069464 Antxr1 9.59 n PTK7 protein tyrosine BG086779 Ptk7 9.04 nn kinase 7 Lectin, galactose-binding, BG074082 Lgals2 7.00 nnnn soluble 2 Matrix c-carboxyglutamate BG074366 Mglap 6.15 n (Gla) protein Transforming growth factor b2 BG067564 Tgfb2 5.58 nn n n F11 receptor BG075763 F11r 4.66 n Gap junction membrane BG063176 Gjb3 4.09 nnn channel protein b3 Myotubularin-related protein 2 BG083487 Mtmr2 4.10 Currently unknown Glucocorticoid-induced gene 1 BG070494 Gig1 28.64 DEAH (Asp-Glu-Ala-His) BG071382 Dhx40 14.22 box polypeptide 40 Plexin B1 BG085089 Plxnb1 8.62 n Tumor protein D52-like 1 BG080670 Tpd52l1 5.47 nn Zinc finger protein 503 BG074838 Zfp503 5.26

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Table 1. Genes with Differential Expression between Highly and Weakly Metastatic Tumors and Their Correlation to Cancer and Metastasis-Related Processes (Cont’d)

c b Gene Genbank Symbol Fold difference* Associated with Metastatic processes accession no. Human Human Metastasis Adhesion Migration Invasion cancer breast cancer

Latexin BG086942 Lxn 0.20 X-linked lymphocyte-regulated 3b BG081054 Xlr3b 0.12 G protein-coupled receptor 124 BG073664 Gpr124 0.10

*Gene expression is presented as a fold difference derived by dividing the expression from highly metastatic tumors by the expression from weakly metastatic tumors. Values indicate fold expression in highly metastatic tumors. cAssociations between individual genes and either human breast cancer, metastasis, or other human cancer was assigned (n) by surveying current literature using a Pubmed (National Center for Biotechnology Information) search for each gene and representative category. bAssociations between individual genes with metastatic processes were assigned (n) by surveying current literature using a Pubmed (National Center for Biotechnology Information) search for each gene and representative category. for the identification of clinically relevant genes. To narrow Notably, a significant proportion of genes that had the spectrum of genes, we adopted a previously described elevated expression in highly metastatic tumors were method of linking known genes to their putative biological those that encode ECM proteins. These include matrix g-carboxyglutamate protein, POEM, laminin a and h functions and then correlating this with the metastatic 5 1 subunits, and several procollagen isoforms. Previous array processes that were altered between tumor phenotypes (12). studies of other metastatic tumors have also revealed altered In other studies, metastatic tumor lines have been shown expression of ECM genes (11, 37). Interaction between the to display a greater capacity to adhere to the ECM, to ECM and the tumor cell can alter physiologic processes proliferate, to attract a blood supply, and to acquire a motile within the tumor microenvironment and thereby influence and invasive phenotype (12, 34–36). Using functional assays metastatic progression (38, 39). ECMs have also been that mimic these processes, we found that adhesion, implicated in epithelial cell differentiation and branching migration, and invasion, but not tumor angiogenesis or morphogenesis within the mammary gland, biological events proliferative capacity, best characterized the differences that reflect the invasion of breast cancer cells (40). observed between highly and weakly metastatic tumors in Depending on the subunit composition, laminins exert varied our model. cellular effects (41). The a5 and h1 subunits (major

FIGURE 3. Differential expression of can- didate genes identified by microarray analysis was confirmed by RT-PCR. Comparative expression levels of a panel of eight genes were assayed by RT-PCR to validate microarray results. Columns, fold difference in gene expression of 4T1.2 tumors compared with 66cl4 tumors. Full names of the eight genes analyzed are shown in Table 1.

Table 2. Genes Encoding ECM That Were Altered in Highly components of laminin-10) have been detected in breast Metastatic Tumors cancer lesions and implicated in the motility and invasion of human colon carcinoma cells via interactions with a3h1 and a6h4 Genbank accession no. Gene Fold difference a h integrins (42). A role for laminin-10 in breast cancer (highly/weakly) 6 4 metastasis remains unresolved. Overexpression of the matrix BG074516 POEM 86.33 Gla protein in human breast carcinoma lines and in human BG074366 Matrix c-carboxyglutamate 6.15 metastatic melanoma has been reported (11, 43). Likewise, (Gla) protein many of the procollagen isoforms have also been associated BG067011 Procollagen, type V, a 1 3.60 with metastatic progression. The amino-terminal propeptide BG075779 Procollagen, type VI, a 1 3.26 AA408762 Laminin, a 5 3.19 of procollagen I has been implicated as a marker for prostate BG075864 Procollagen, type VI, a 2 3.01 cancer metastasis to bone (44), whereas procollagen isoforms BG087985 Laminin B1 subunit 1 2.56 V, VI, and XVIII are involved in remodeling of the tumor BG072504 Procollagen, type XVIII, a 1 2.38 BG087142 Procollagen, type I, a 2 2.02 microenvironment and in promoting cellular adhesion and motility (37, 45-47).

FIGURE 4. In situ hydridization of POEM in 67NR, 66cl4, and 4T1.2 primary tumors. Gut lining of a mouse day 15 embryo was used as a positive control for POEM expression using a mixture of POEM antisense riboprobes, with sense riboprobes as a negative control. Serial sections of primary tumors were stained with H&E and with the sense and antisense probes. 3,3V-Diaminobenzidine staining (brown) reveals positive riboprobe annealing. Sections were counterstained with hematoxylin (blue). Bar, 50 Am.

FIGURE 5. Reduced POEM in the bone metastasizing tumor line 4T1.2 inhibits spontaneous metastasis. A. RT-PCR analysis of POEM expression in cultured cells. 4T1.2pRS-GFP and 4T1.2pRS-POEM lines were produced by stable transfection of vectors encoding short-hairpin RNA specific for either GFP or POEM, respectively. Gene expression is displayed relative to GAPDH. Columns, average of three replicates; bars, SE. B. Tumor cells were injected into the fat pad of mice (15 mice per group) and tumor growth was monitored by caliper measurements (solid line, 4T1.2pRS-GFP; dashed line, 4T1.2pRS- POEM). Mice were culled and tissues were harvested for assay of metastatic burden on day 30. C. Average weights of the primary tumors at the end of the study indicate no difference between the two groups. D. At the end of the experiment, POEM expression in three primary tumors from each group was analyzed by RT-PCR. E. q-RT-PCR analysis of metastatic tumor burden in tissues from mice bearing 4T1.2pRS-GFP (open columns)or 4T1.2pRS-POEM tumors (black columns). Columns, average RTB for each tissue; bars, SE. *, P < 0.05.

POEM is a novel secreted ECM molecule that showed a avh3, avh5, avh6, and a4h7 (27), although the favored receptor striking increase in expression (30- to 80-fold) in the highly pairing is the a8h1 integrin (27, 28). It is thought that adhesive metastatic 4T1.2 and 4T1.13 lines. Whereas POEM expression and survival signals are conveyed through a8h1, which is has not been implicated previously in cancer, we have shown a similarly expressed in 66cl4 and 4T1.2 cells.4 However, in selective reduction in breast cancer metastasis to the lung, tumors with decreased POEM expression (i.e., 66cl4), these kidney, and bone using the 4T1.2 model. As POEM has been signals may not be effectively conveyed and thus result in a reported to be involved in kidney morphogenesis and the weaker metastatic phenotype. The precise mechanism of POEM development of bone (preosteoblastic cells; refs. 27, 28), the function and the involvement of a8h1 integrin in tumor growth expression of POEM by the tumor cells may be critical for and metastasis in this model are the subjects of ongoing studies. establishment in these sites. The function of human POEM is yet to be investigated POEM consists of five epidermal growth factor–like due to the lack of a full-length clone of the human gene. domains, an Arg-Gly-Asp integrin binding motif and a meprin, Recently, using a high-throughput screen for transmembrane A5 protein and receptor protein-tyrosine phosphatase A (MAM) and secreted proteins, a hypothetical human protein, domain (27). The Arg-Gly-Asp and MAM domains have been LOC255743 (National Center for Biotechnology Information linked to adhesion, spreading, and survival of preosteoblastic reference NM_198278), was identified with high similarity cells (25). As these processes are crucial to metastasis, they provide an explanation for the diminished metastatic capacity of 4T1.2 cells with reduced POEM expression. The Arg-Gly-Asp site in POEM binds to several integrin receptors, including 4 B.L. Eckhardt, unpublished results.

(82% identity) to POEM (48). Further analysis of isolated. Tumor burden for each individual tissue was LOC255743 using SAGE map (National Center for Biotech- measured using q-RT-PCR incorporating Taqman chemistry nology Information) identified two SAGE tags5 that were (Applied Biosystems, Foster City, CA). Genomic DNA was detectable in libraries derived from normal kidney, thyroid, subjected to multiplexed q-RT-PCR to detect the cycle brain, and lung as well as libraries from two patients with threshold (Ct) for vimentin (total mouse tissue) and neomycin invasive ductal breast carcinoma, one patient with metastatic (tumor cells only). By comparing the Ct values of vimentin gastric cancer, and one patient with a grade 3 brain and neomycin (DCt), a score for relative tumor burden was astrocytoma (49). These observations implicate POEM in calculated using the following formula: Relative tumor human tumor progression; however, further study is required burden = 10,000 Â 1/2CorrDCt. CorrDCt is a DCt value to establish a role for POEM in the growth and metastasis of that includes correction for the difference in neomycin copy human breast cancer. number in each tumor line as determined by multiplexed q- In summary, we have characterized a unique, clinically RT-PCR. Note that genomic DNA (not RNA) is being relevant model of breast cancer metastasis at both genomic and amplified in this assay; hence, difference in vimentin gene phenotypic levels. By combining our model with array expression between tissues is not a factor. Using this technology, we have identified a set of metastasis-related formula, a tissue without tumor scores zero, whereas a tissue genes, some of which have been implicated in human breast composed entirely of tumor cells scores 10,000. PCR was cancer, but many remain to be characterized. A significantly done using an ABI Prism 7000 thermocycler (Applied high proportion of these genes were ECM molecules, which is Biosystems) using standard cycling methods. All PCR consistent with current views on the importance of cell-ECM reagents were obtained from Applied Biosystems, except interactions in metastasis (39). Finally, we have shown that for neomycin and vimentin forward and reverse primers POEM, a gene identified by array analysis, has a role in (GeneWorks, Adelaide, South Australia, Australia). Sequen- primary breast tumor growth and in spontaneous metastasis. ces for primers and probes used in Taqman assays were designed using Primer Express version 2.0 software (Applied Biosystems).3 Multiplex PCR reactions consisted of the Materials and Methods following primer/probe concentrations: vimentin probe 50 Cell Culture and Reagents nmol/L, vimentin forward and reverse primers 50 nmol/L, The tumor lines 67NR, 168FARN, 66cl4, and 4T1 were neomycin probe 75 nmol/L, and neomycin forward and provided by Dr. Fred Miller (Karmanos Cancer Institute, Detroit, reverse primers 150 nmol/L. MI). These lines are clonal populations of cells derived from a spontaneous mammary carcinoma arising in a BALB/cfC3H mouse (19). The 4T1 line was further cloned to isolate lines Microarray Design, Hybridization, Scanning, and Analysis 4T1.2 (18) and 4T1.13. All lines were transfected with a plasmid The NIA mouse 15K cDNA clone set (50) contains 15,272 containing the neomycin resistance gene to discriminate tumor elements, comprising 13,739 unique UniGene identifiers f cells from endogenous mouse tissue in a q-RT-PCR assay (UniGene Build 127) and 30% novel genes (51). PCR (see below). Cells were maintained in a-MEM medium sup- products from the clone set were purified and arrayed onto plemented with 10% FCS, penicillin, and streptomycin (1:100 glass slides using in-house facilities according to published dilution, Life Technologies, Rockville, MD) and incubated at methods (52). RNA was isolated from tumors as described previously (53). The purity of the RNA was confirmed by 37jC with 5% CO2. Cells were not allowed to reach con- fluence and were passaged for a maximum of 4 weeks. Before spectrophotometry and the quality was checked by electro- experimentation, viability was assessed by trypan blue exclusion. phoresis on a formaldehyde-1% agarose gel. For the cDNA microarray analysis, pools of five primary tumors were used. RNA from the nonmetastatic 67NR tumor was used as a Detection of Tumor Growth and Spontaneous Metastases reference for all the other tumor lines. Microarray analysis for Female BALB/c mice (6-8 weeks old, ARC, Perth, each primary tumor was repeated four times using dye Western Australia, Australia) were anaesthetized and injected reversals for two arrays to account for dye-incorporated bias. with 1 Â 105 viable cells into the fourth mammary fat pad. Total RNA (100 Ag) was labeled with fluorescent Cy3 or Cy5 When palpable, tumor growth was monitored twice weekly dyes, hybridized, and washed as described (53). Arrays were using electronic calipers: tumor volume = (length Â width2)/ scanned using an Agilent confocal laser scanner (Agilent 2. Entire organs, including primary tumor, lung, liver, ALN, Technologies, Palo Alto, CA), and fluorescence intensities spleen, both kidneys, both femurs, spine, brain, and heart, from both Cy3 and Cy5 channels of each scanned image were were excised from mice once the average primary tumor quantified using GenePix Pro version 4.1 (Axon Instruments, weight of a group of mice was 1.5 g or sooner if mice Union City, CA). Spots displaying altered morphology or with displayed signs of distress. All mice bearing one tumor line low fluorescence intensities in both channels were excluded were sacrificed on the same day. Tissues were snap frozen in from the data set. Data were imported into Genespring liquid nitrogen and homogenized and the genomic DNA was version 6 (Silicon Genetics, Redwood City, CA), and each clone was averaged over the four replicates and normalized using the LOWESS regression model (54). Nonparametric ANOVA (Kruskal-Wallis test) with the Benjamini and 5 SAGE tags scrutinized were GTAAAGGTAT and CATTTTTAAT. Hochberg false discovery rate correction for multiple testing

was used to identify genes differentially expressed between almost completely. After 30 minutes, cells were washed and highly and weakly metastatic tumors. All expression data and stained with 2% crystal violet in 50% methanol. Bound dye was microarray experimental details were stored on a local released with 1% SDS, and absorbance was measured at MIAME-complaint relational database system (55). 550 nm.

RT-PCR Analysis Migration and Invasion Assays To validate results obtained from microarray analysis, real- Migration and invasion assays were done according to time RT-PCR of selected genes was done. cDNA was previously established methods (56). The time points used in synthesized from primary tumor RNA, primed with random the migration (5 hours) and invasion (24 hours) assays were hexamers (Promega, San Luis Obispo, CA), and reverse optimized previously to reveal the most significant differences transcribed by SuperScript II transcriptase (Promega) accord- between the tumor lines. ing to manufacturer’s instructions. RT-PCR was done on an ABI Prism 7000 thermocycler using SYBR Green I In situ Hybridization chemistry (Applied Biosystems). Primers used in all PCR A mixture of 450- to 550-bp riboprobes for POEM was analyses were designed using the Primer Express version 2.0 generated by amplification of the POEM DNA region of interest program.3 PCR consisted of 20 ng cDNA, 0.1 Amol/L and insertion into a PCR cloning vector (pGEM-T Easy Vector forward and reverse primers, and 2Â SYBR Green I master System, Promega) containing a T7/SP6 bidirectional promoter mix reagent. Standard cycling procedures were employed. system for generating sense and antisense transcripts. T7 and SP6 Specific amplicon formation with each primer pair was polymerase were used for in vitro transcription, with generation confirmed by dissociation curve analysis and by visualization of sense and antisense probes depending on the orientation of of a single band on a 2% agarose gel. Gene expression was the insert. The in vitro transcription included labeling of measured relative to glyceraldehyde-3-phosphate dehydroge- transcripts with FITC (using FITC-UTP according to manufac- nase (GAPDH) using the following formula: Relative turer’s instruction, Roche Diagnostics, Basel, Switzerland). transcript abundance = 10,000 / 2 (Ct À Ct ). Gene GAPDH Tissues were fixed overnight in 10% buffered formalin at GAPDH was used as a baseline for RT-PCR analysis, 4jC and paraffin embedded. Sections of a 15.5-day embryo and because microarray experiments showed no significant 67NR, 66cl4, and 4T1.2 primary mammary tumors were used change in GAPDH expression between the tumor lines. for in situ hybridization. Individual riboprobes were used initially to determine those that gave the best signal with In vivo Angiogenesis Assay minimal background DNA binding, and these were pooled into The angiogenic capacity of each tumor line (five mice per a cocktail to be used for all subsequent hybridizations group) was measured by the recruitment of vascular endothe- (including sense and antisense cocktails). The protocol was as lium toward tumor cells embedded in a Matrigel plug in a described previously, including dewaxing and fixation of 6 BALB/c mouse. Cells (2 Â 10 ) were resuspended in 200 AL tissues and pretreatment for access to target nucleic acid Matrigel (BD Biosciences, Franklin Lakes, NJ) and injected s.c. sequence, except that FITC labeling replaced DIG (57, 58). into BALB/c mice. Plugs were removed 6 days later and assayed Riboprobe/FITC signal was detected and amplified using the for hemoglobin content (Sigma Diagnostics, St. Louis, MO). GenPoint Fluorescein Tyramide Signal Amplification System (DakoCytomation, Carpenteria, CA). The signal was visualized Sulforhodamine B Proliferation Assay using 3,3V-diaminobenzidine staining followed by nuclear Cells (500) were fluorescence-activated cell sorted based counterstaining with hematoxylin. on propidium iodide exclusion into 96-well plates. At 12-hour time points, cells were fixed in 10% trichloroacetic acid, Preparation of 4T1.2 Cells with Decreased POEM rinsed, and stained in 0.4% sulforhodamine B dissolved in 1% Expression acetic acid. Protein-bound dye was released with 10 mmol/L The mammalian expression vector pRS, a gift from Tris base, and the absorbance was measured by spectropho- Dr. Reuven Agami (Netherlands Cancer Institute, Amsterdam, tometry at 550 nm. Five replicate wells were used per time the Netherlands), was used for the expression of short-hairpin point. In vitro growth rate was calculated from readings over a RNA (29). A synthesized oligonucleotide encoding a short- period of 72 hours. hairpin RNA sequence specific for POEM was ligated into the BglII/HindIII sites of pRS vector (pRS-POEM). The oligonu- Adhesion Assay cleotide encoded a gene-specific 19-bp sequence corresponding Plates (96-well) were coated overnight at 4jC with Matrigel to nucleotides 1,328 to 1,346 downstream of the transcription diluted 1:50 in PBS. Excess Matrigel was aspirated, and the start site of POEM (5V-AGGACGACCCAGGTATTCT-3V), wells were rinsed with PBS, blocked for 1 hour at 37jC with which was separated by a 9-bp noncomplementary spacer (5V- 2% bovine serum albumin, and rinsed again in PBS. Cells (1 Â TTCAAGAGA-3V) from the reverse complement of the same 105) were seeded in serum free a-MEM medium and incubated 19-bp sequence. A nonsilencing vector control was similarly at 37jC with 5% CO2 for 30 minutes. The 30-minute time point fashioned to target GFP (5V-CCACTACCTGAGCACCCAG- was chosen because it showed the best resolution between the 3V; pRS-GFP), a protein not expressed in mammalian cells. tumor lines. At later time points, all tumor lines had adhered Functionality of the pRS-GFP vector was shown when transient

transfection of this vector greatly diminished GFP expression in 20. Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary 293 cells stably expressing GFP (data not shown). pRS-GFP or tumor. Cancer Res 1992;52:1399 – 405. pRS-POEM were transfected into neomycin-tagged 4T1.2 cells 21. Aslakson CJ, Rak JW, Miller BE, Miller FR. Differential influence of organ using LipofectAMINE 2000 reagent according to manufactur- site on three subpopulations of a single mouse mammary tumor at two distinct steps in metastasis. Int J Cancer 1991;47:466 – 72. er’s instructions. Stable cell lines were selected by culturing the 22. Miller BE, McInerney D, Jackson D, Miller FR. Metabolic cooperation cells in a-MEM medium containing 5 Ag/mL puromycin for 2 between mouse mammary tumor subpopulations in three-dimensional collagen weeks. Following single cell cloning of both 4T1.2pRS-GFP gel cultures. 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Mol Cancer Res 2005;3(1). January 2005 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Genomic Analysis of a Spontaneous Model of Breast Cancer Metastasis to Bone Reveals a Role for the Extracellular Matrix1 1 Department of Defense Breast Cancer Research Program grants DAMD17-98-1-8144 (R.L. Anderson) and DAMD17-01-1-0371 (M.D. Tavaria), Susan G. Komen Breast Cancer Foundation predoctoral fellowship (E.K. Sloan), and NIH/National Cancer Institute grant ROI CA90291 (R.L. Anderson).

Bedrich L. Eckhardt, Belinda S. Parker, Ryan K. van Laar, et al.

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