Bcl-2-Mediated Cell Survival Promotes Metastasis of EpH4 hMEKDD Mammary Epithelial Cells

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogenactivated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis. (Mol Cancer Res 2004;2(10):551– 6) Introduction The process of metastasis is highly complex and involves dissemination of cells from the primary tumor through the lymphatic and vascular systems coupled with a tissue-selective ability to colonize distant tissues and organs (1). Mechanisms controlling cellular proliferation, migration, and invasiveness have been identified as key regulators of primary tumor development, but it is unclear whether these are the critical mechanisms that control metastasis. The identification of genes regulating breast cancer metastasis has been the subject of intense investigation (2). A recent study using the MDA-MB231 human breast carcinoma cell xenotransplant model identified a multigene expression program that regulated bone-specific metastasis (3). These data suggest that genes that control metastasis may play a limited role in primary tumor development. Constitutive activation of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK pathway is a frequent event in tumors derived from the breast, colon kidney, and lung (4-7). Hyperactivation of the MEK signal transduction pathway is a frequent event in human breast cancer (4) that correlates strongly with increased lymph node involvement and metastasis (7). However, the molecular targets and outcomes of MEK-MAPK signal transduction are highly cell specific and tumor type specific. Transfection of constitutively active MEK1 induced matrix metalloproteinase expression (8) and mediated transformation of fibroblast (8, 9) and epithelial (10, 11) cell lines, but it is unclear which activities downstream of MEK-MAPK signal transduction are required to promote spontaneous metastasis. We have chosen to use the mammary fat pad transplantation model to dissect mechanisms regulating primary tumor development and spontaneous metastasis in immune-competent mice (12). Previous studies showed that MEK-transformed EpH4 mouse mammary epithelial cells were highly invasive in vitro and tumorigenic but showed limited metastatic ability (11). One of the critical steps in the metastatic cascade is the detachment of cells from the primary tumor and their subsequent dissemination through the vascular system. Detachment of epithelial cells from the extracellular matrix has been shown to induce apoptosis (13), and it has been proposed that suppression of apoptosis could promote primary tumor development and metastasis. Apoptosis from reduced extracellular matrix attachment may be mediated by the resulting disorganization of the cytoskeleton (14). Disruption of the actin cytoskeleton in human mammary epithelial cells by latrunculin-A, a monomeric G-actin binding compound that inhibits actin polymerization, has been shown to potently induce apoptosis, which could be rescued by Bcl-2 (15). In addition to its well-described antiapoptotic role, expression of Bcl-2 in a rat glioma cell line resulted in the induction of matrix metalloproteinase activity (16). Therefore, the effect of Bcl-2 to enhance primary tumor development and metastasis of EpH4 parental and MEK-transformed cells was determined. The results clearly show that Bcl-2 enhanced Received 7/16/04; revised 8/31/04; accepted 9/1/04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jan Pinkas, Genzyme Corporation, 5 Mountain Road, Framingham, MA 01701-9322. Phone: 508-271-3966; Fax: 508-620-1203. E-mail: [email protected] Copyright D 2004 American Association for Cancer Research. Mol Cancer Res 2004;2(10). October 2004 551 Research. on September 15, 2017. © 2004 American Association for Cancer mcr.aacrjournals.org Downloaded from the metastatic capacity of MEK-transformed EpH4 cells without affecting cell motility and invasiveness or primary tumor growth. This indicates that distinct mechanisms regulate primary tumor development and metastasis. Results To address the role of the cell survival protein Bcl-2 in breast tumorigenesis and metastasis, we used the EpH4 mouse mammary epithelial cell line (17) and MEK-transformed EpH4 cell lines that had been shown previously to be highly tumorigenic when transplanted into cleared mammary fat pads (11). Fulllength human Bcl-2 was stably expressed in EpH4 parental and MEK expressing cell lines, and Western blot analysis identified multiple clones expressing high levels of Bcl-2 as compared with parental EpH4 cells (Fig. 1A). Equivalent protein loading was determined by probing for h-actin expression (Fig. 1B). The Bcl-2 expressing clones were morphologically indistinguishable from the parental EpH4 and MEK expressing cell lines, but the Bcl-2 expressing clones did show slightly reduced rates of proliferation in vitro (data not shown). The reduced rate of proliferation is most probably due to the growth inhibitory effect of full-length Bcl-2 that is independent of its antiapoptotic effect (18). Disruption of actin polymerization in MCF10A human mammary epithelial cells using latrunculin-A has been shown previously to induce apoptosis through a poly(ADP-ribose) polymerase (PARP)–dependent mechanism that could be rescued by Bcl-2 (15). Loss of cytoskeletal organization is thought to signal cell death in response to reduced attachment to the extracellular matrix by inducing loss of Akt phosphorylation and reducing Bcl-2 expression. To determine whether disruption of the actin cytoskeleton induced apoptosis in mouse mammary epithelial cells, EpH4 parental and MEK expressing cells were treated with latrunculin-A for 28 hours, and the cleavage of PARP was determined to measure apoptotic commitment (Fig. 1C). EpH4 parental and MEKtransformed cells were equally sensitive to latrunculin-Ainduced cytoskeletal disruption and underwent apoptosis as determined by nearly complete cleavage of PARP. Expression of Bcl-2 efficiently rescued both parental EpH4 and MEKtransformed cells from latrunculin-A-induced apoptosis (Fig. 1C). Matrigel-coated Boyden chambers represent one of the most frequently used in vitro models of invasiveness. This assay mimics the early steps of tumor invasion in vivo by requiring proteolytic degradation of the basement membrane before cells can gain access to the pores in the filter inserts. Studies examining the ability of Bcl-2 to enhance migration and invasion have been mixed depending on the cell line examined (19, 20), suggesting that this represents an indirect effect of Bcl-2 expression. Modified Boyden chamber assays were done to quantify the migratory and invasive capacity of the Bcl-2 expressing EpH4 cell lines. MEK-transformed EpH4 cells have been shown previously to have increased migratory and invasive capacity as compared with parental EpH4 cells (11), and the ability of Bcl-2 to enhance this activity was examined (Fig. 2). These results clearly show that expression of Bcl-2 was not sufficient to enhance the in vitro migration or invasion rates of parental EpH4 or MEK-transformed cells. The effect of Bcl-2 expression in EpH4 cell lines on primary tumor development and metastasis in vivo was determined using the mammary transplantation model (12). EpH4 parental, hPBcl-2, and hMEKDD/Bcl-2 cells were transplanted (5 10 cells/10 AL) into cleared number 4 mammary fat pads of syngeneic BALB/c hosts, and the animals were monitored for tumor development. Mammary fat pads transplanted with EpH4 hPBcl-2 cell lines did not form tumors over the course of the experiment (4-5 months; Table 1), and these results are comparable with previous studies with parental EpH4 cells (11). However, EpH4 hMEKDD/Bcl-2 cells rapidly formed mammary tumors of 1 to 2 cm by 5 to 7 weeks (Table 1). Comparison of EpH4 hMEKDD (11) and hMEKDD/Bcl-2 outgrowths showed that the kinetics of primary tumor development were indistinguishable, and both sets of clones formed poorly differentiated mammary carcinomas characterized by intense stromal reactivity (data not shown). The spectrum and kinetics of metastasis were measured by detailed histopathologic analyses of internal organs and tissues from tumor-bearing animals for macroscopic metastatic lesions. Previous studies with animals bearing EpH4 hMEKDD tumors FIGURE 1. Bcl-2 overexpression inhibits apoptosis induced by disruption of the actin cytoskeleton. Western blot analysis of (A) Bcl-2 and (B) h-actin protein levels in EpH4 parental and hMEKDD cell lines. C. Parental EpH4 cells (CON ) or stable lines expressing empty vector (h2), MEK, Bcl-2, or both MEK and Bcl-2 (as indicated) were treated with vehicle (0.1% ethanol) or latrunculin-A (2.5 mmol/L) for 28 hours. Cell lysates were subjected to SDS-PAGE and Western blotting using a polyclonal anti-PARP antibody, which recognizes both full-length PARP and the caspase-cleaved form (DPARP ). Pinkas et al. Mol Cancer Res 2004;2(10). October 2004 552 Research. on September 15, 2017. © 2004 American Association for Cancer mcr.aacrjournals.org Downloaded from showed that metastatic cells were readily reisolated from several internal organs such as lung and spleen (11), but macroscopic lesions that represented tumor cells, which had extravasated and invaded into the adjoining parenchyma, were difficult to identify (Table 1; Fig. 3A). However, animals bearing hMEKDD/Bcl-2 mammary tumors presented with numerous macroscopic metastatic lesions that had invaded into surrounding lung tissue, and the difference in the metastatic burden between the groups was dramatic (Table 1; Fig. 3). The increase in metastasis in animals bearing EpH4 hMEKDD/Bcl2 mammary tumors seemed to be specific to the lung, because no increase in metastasis was seen in any other tissues (data not shown). These data clearly show that cell survival factors such as Bcl-2, which do not accelerate primary tumor development, can dramatically enhance metastasis. Discussion The presence of metastasis-promoting genes is the subject of intense debate, and several recent studies argue that the metastatic potential of a primary human tumor can be determined by its gene expression signature (21, 22). It is unclear whether these gene expression signatures that are predictive of disease recurrence and metastasis correspond to clonal variants within the primary tumor that have enhanced metastatic ability or to the primary tumor as a whole. Karyotypic and genomic analyses of breast carcinomas showed that metastases frequently shared common features of the primary tumor from the same patient, supporting the clonal progression model (23, 24) However, a recent report challenges this paradigm and presents evidence that tumor cells disseminate early and develop metastatic lesions independent from the primary tumor (25, 26). In either case, these studies support the contention that genomic changes in cells in the primary tumor, which probably result in changes in gene expression, can effect metastatic progression. Several recent studies have showed that modulation of gene expression can alter the metastatic potential of tumor cells. A recent study elucidated a multigenic expression program that regulates bone-specific metastasis of MDA-MB-231 human breast carcinoma cells (3). Interestingly, many of the genes identified code for osteolytic factors and cytokines that are not predicted to promote primary tumor growth but should enhance the ability of disseminated tumor cells to thrive in the bone microenvironment (3). Gene expression analysis in a murine model of osteosarcoma identified the cytoskeletal protein Ezrin as being strongly associated with metastasis (27). Further functional studies showed that Ezrin expression provided an essential survival signal for tumor cells that allowed their establishment of metastases in the lung (28). These studies reveal the importance of survival signals in enhancing metastatic progression of osteosarcoma. Metastatic human breast carcinoma cell lines were shown to be resistant to cell death induced by disruption of the actin cytoskeleton in vitro , and this phenotype could be reproduced in nontumorigenic cell lines by overexpression of Bcl-2 (15). Based on these results, an unbiased cytoskeleton-based functional genetic screen identified the antiapoptotic Bcl-xL protein as an enhancer of metastasis independent of any effects on primary FIGURE 2. Expression of Bcl-2 does not enhance migratory and invasive potential of EpH4 cells expressing constitutively activated MEK1 in modified Boyden chamber assays in vitro . EpH4 parental (CON ), hMEKDD-47, and three independent hBcl-2 and hMEKDD/Bcl-2 cell lines that had (A) migrated through uncoated filter inserts or (B) invaded through Matrigel-coated filter inserts were fixed, stained, and counted. Each experiment was done in triplicate. Columns, average; bars, SD. Table 1. Tumor Development in EpH4 Mammary Fat Pad Transplants Lung Clone Outgrowth* Tumor Metastasisc EpH4 phP-Bcl-2-19 13/16 0/16 0 EpH4 phP-Bcl-2-27 12/14 0/14 0 EpH4 phP-Bcl-2-47 18/22 0/22 0 EpH4 phMEKDD-47 22/22 22/22 0 EpH4 phMEKDD-112 16/16 16/16 0 EpH4 phMEKDD-116 16/16 16/16 0 EpH4 phMEKDD/Bcl-2-1 18/18 18/18 5 F 3 EpH4 phMEKDD/Bcl-2-13 16/16 16/16 7 F 5 EpH4 phMEKDD/Bcl-2-22 20/20 20/20 12 F 6 *Outgrowth measured by whole mount analysis or by H&E; staining of formalinfixed transplanted mammary fat pads. cMetastasis scored by counting macroscopic metastatic lesions using a dissecting microscope. Values represent average F SD. Bcl-2 Promotes Metastasis of EpH4 hMEKDD Cells Mol Cancer Res 2004;2(10). October 2004 553 Research. on September 15, 2017. © 2004 American Association for Cancer mcr.aacrjournals.org Downloaded from tumor development (29). In support of this finding are previous studies demonstrating that overexpression of Bcl-2 and Bcl-xL in metastatic human breast carcinoma cell lines can enhance survival of disseminated tumor cells in the vasculature of immune-deficient animals bearing s.c. xenografts (30, 31). The mechanisms regulating metastasis are complex and still poorly understood, and evidence is increasing that genes involved in metastasis may not be identified in standard transformation assays. The data presented here show that Bcl-2 expression had no effect on in vitro proliferation and invasion assays but that it markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms controlling metastasis can be regulated independently from primary tumor development and suggest an important role for antiapoptotic proteins such as Bcl-2 in enhancing pulmonary metastasis in human patients. Materials and Methods Cell Culture EpH4 mouse mammary epithelial cells derived from BALB/c mice were obtained from Hartmut Beug of the Research Institute of Molecular Pathology (Vienna, Austria). Cells were cultured in monolayer on tissue culture plastic (Corning, Corning, NY) in DMEM supplemented with 10% bovine calf serum, penicillin-streptomycin (100 Ag/mL each), and L-glutamine (2 mmol/L). Latrunculin-A (Biomol, Plymouth Meeting, PA) was reconstituted as a 5 mmol/L solution in ethanol and stored at 20jC and all experiments with latrunculin-A were done in serum-free DMEM. Vectors and Transfections To target high-level expression in EpH4 hMEKDD cells, a new expression construct was created. The pCAGGS vector containing the cytomegalovirus IE enhancer/chicken h-actin promoter (32) was modified by inserting a SV40-Puromycin cassette from pPur (Clontech, Palo Alto, CA) and expanding the multiple cloning site by ligating an oligonucleotide linker containing XhoI, KpnI, HindIII, EcoRI, BamHI, BglII, NotI, and ClaI restriction sites to create phP. The new vector (phP) uses the strong chicken h-actin promoter to drive transgene expression, contains an extended multiple cloning site, and has a puromycin resistance cassette for selection in eukaryotic cells. Full-length human Bcl-2 was subcloned from pCDNA3-Bcl-2 (15) into phP and then transfected into EpH4 cells using Fugene-6 (Roche, Indianapolis, IN). After selection in complete medium supplemented with 2 Ag/mL puromycin (Sigma Chemical Co. St. Louis, MO) for 14 days, individual clones were isolated and expanded. Western Blotting EpH4 cells grown in complete medium or treated in serumfree DMEM containing 0.1% ethanol or latrunculin-A were washed with PBS and then lysed in 150 mmol/L NaCl, 50 mmol/L Tris (pH 7.6), 10 mmol/L sodium pyrophosphate, 10 mmol/L NaF, 2 mmol/L EDTA, 1% NP40, 0.1% SDS, 0.5% deoxycholate, 1 mmol/L sodium orthovanadate, 10 mmol/L h-glycerophosphate, and Complete protease inhibitor tablets (Roche). Protein concentration in the lysates was measured using Bradford reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. Equivalent amounts of protein were separated by SDS-PAGE on 12% polyacrylamide gels and then FIGURE 3. Increased spontaneous lung metastasis in EpH4 hMEKDD/Bcl-2 mammary transplants. H&E-stained; sections of (A and B) EpH4 hMEKDD47 and (C and D) EpH4 hMEK/Bcl-2-22 lung metastases. A and B. Arrowhead, the largest lung metastasis seen in EpH4 hMEKDD mammary transplants. Pinkas et al. Mol Cancer Res 2004;2(10). October 2004 554 Research. on September 15, 2017. © 2004 American Association for Cancer mcr.aacrjournals.org Downloaded from transferred to Immobilon-P membranes (Millipore, Bedford, MA) using a SemiPhor transfer apparatus (Hoeffer, Piscataway, NJ). Western blotting was done according to the manufacturer’s protocols with the following antibodies: polyclonal anti-PARP (H-250, SantaCruzBiotechnology, SantaCruz,CA),monoclonal anti-h-actin (AC-15, Sigma Chemical), and monoclonal antiBcl-2 (Transduction Laboratories, Lexington, KY). Proteins were visualized using a horseradish peroxidase– linked secondary antibody (Jackson ImmmunoResearch, West Grove, PA) and a chemiluminescent detection system (enhanced chemiluminescence, Amersham, Sunnyvale, CA). Boyden Chamber Assays Migration and invasion assays were done using modified Boyden chambers with filter inserts for 24-well dishes containing 8 Am pores (Becton Dickinson, Franklin Lakes, NJ) as described previously (11, 33). Uncoated filters were used for migration assays, and Matrigel-coated filters were used for invasion assays. Cells (1 10) were plated into 300 AL of serum-free mammary epithelial growth medium in the upper chamber, and the lower chamber was filled with 500 AL of mammary epithelial growth medium (Clonetics, San Diego, CA). After 24 hours in culture, cells were fixed in 2.5% glutaraldehyde in PBS for 5 minutes and then stained with 0.5% toluidine blue in 2% Na2CO3 for 5 minutes. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed in PBS. Cells on the underside of the filters were counted by microscopic inspection. Each clone was plated in triplicate per experiment, and each experiment was repeated twice. Mammary Gland Transplantation BALB/c mice were obtained from Charles River Labs (Wilmington, MA) and were maintained according to theInstitutional Animal Care and Use Committee–approvedguidelines. EpH4 cells were transplanted into the clearednumber 4 mammary fat pads of 21to 25-day-old femalesyngeneic BALB/c mice by standard procedures (11, 34).Briefly, EpH4 cells were grown to confluence, harvested, andresuspended in PBS; then, 5 10 cells/10 AL were injectedinto both cleared number 4 mammary fat pads distally to thelymph node using a Hamilton syringe. Starting at 1 week aftertransplantation, the mice were palpated biweekly for mammarytumors. EpH4 hMEKDD and hMEKDD/hPBcl-2 transplantsgave rise to palpable mammary tumors within 7 to 10 days,and the animals were sacrificed 5 to 7 weeks post-transplantwith mammary tumors 1 to 2 cm. EpH4 control and hPBcl-2transplanted mammary fat pads were either isolated at8 weeks post-transplant to assess outgrowth or observed for>6 months to monitor long-term tumor development. Tissueswere fixed in 10% neutral-buffered formalin (Fisher, Pittsburgh,PA) and were processed for routine H&E; staining andhistopathology. References1. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth ofcancer cells in metastatic sites. Nat Rev Cancer 2002;2:563 –72. 2. Welch DR, Steeg PS, Rinker-Schaeffer CW. Molecular biology of breastcancer metastasis. Genetic regulation of human breast carcinoma metastasis.Breast Cancer Res 2000;2:408 –16. 3. Kang Y, Siegel PM, Shu W, et al. A multigenic program mediating breastcancer metastasis to bone. Cancer Cell 2003;3:537 –49. 4. Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression ofmitogen-activated protein kinase in human breast cancer. J Clin Invest 1997;99:1478–83. 5. Salh B, Marotta A, Matthewson C, et al. Investigation of the Mek-MAPkinase-Rsk pathway in human breast cancer. Anticancer Res 1999;19:731–40. 6. Hoshino R, Chatani Y, Yamori T, et al. Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors.Oncogene 1999;18:813–22. 7. Mueller H, Flury N, Eppenberger-Castori S, Kueng W, David F, EppenbergerU. Potential prognostic value of mitogen-activated protein kinase activityfor disease-free survival of primary breast cancer patients. Int J Cancer 2000;89:384–8. 8. Welch DR, Sakamaki T, Pioquinto R, et al. Transfection of constitutivelyactive mitogen-activated protein/extracellular signal-regulated kinase kinase con-fers tumorigenic and metastatic potentials to NIH3T3 cells. Cancer Res 2000;60:1552–6. 9. Cowley S, Paterson H, Kemp P, Marshall CJ. Activation of MAP kinasekinase is necessary and sufficient for PC12 differentiation and for transformationof NIH 3T3 cells. Cell 1994;77:841 –52. 10. Montesano R, Soriano JV, Hosseini G, Pepper MS, Schramek H.Constitutively active mitogen-activated protein kinase kinase MEK1 disruptsmorphogenesis and induces an invasive phenotype in Madin-Darby canine kidneyepithelial cells. Cell Growth Differ 1999;10:317–32. 11. Pinkas J, Leder P. MEK1 signaling mediates transformation and metastasis ofEpH4 mammary epithelial cells independent of an epithelial to mesenchymaltransition. Cancer Res 2002;62:4781 –90. 12. Kittrell FS, Oborn CJ, Medina D. Development of mammary preneoplasiasin vivo from mouse mammary epithelial cell lines in vitro . Cancer Res 1992;52:1924– 32. 13. Frisch SM, Screaton RA. Anoikis mechanisms. Curr Opin Cell Biol 2001;13:555–62. 14. Flusberg DA, Numaguchi Y, Ingber DE. Cooperative control of Aktphosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilamentsand microtubules in capillary endothelial cells. Mol Biol Cell 2001;12:3087 –94. 15. Martin SS, Leder P. Human MCF10A mammary epithelial cells undergoapoptosis following actin depolymerization that is independent of attachment andrescued by Bcl-2. Mol Cell Biol 2001;21:6529 –36. 16. Oliver L, Tremblais K, Guriec N, et al. Influence of bcl-2-related proteins onmatrix metalloproteinase expression in a rat glioma cell line. Biochem BiophysRes Commun 2000;273:411–6. 17. Reichmann E, Schwarz H, Deiner EM, et al. Activation of an induciblec-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion. Cell 1992;71:1103 –16. 18. Coultas L, Strasser A. The role of the Bcl-2 protein family in cancer. SeminCancer Biol 2003;13:115–23. 19. Del Bufalo D, Biroccio A, Leonetti C, Zupi G. Bcl-2 overexpressionenhances the metastatic potential of a human breast cancer line. FASEB J 1997;11:947– 53. 20. Miyake H, Hara I, Yamanaka K, Gohji K, Arakawa S, Kamidono S.Overexpression of Bcl-2 enhances metastatic potential of human bladder cancercells. Br J Cancer 1999;79:1651 –6. 21. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature ofmetastasis in primary solid tumors. Nat Genet 2003;33:49 –54. 22. van de Vijver MJ, He YD, van’t Veer LJ, et al. A gene-expression signa-ture as a predictor of survival in breast cancer. N Engl J Med 2002;347:1999– 2009. 23. Kuukasjarvi T, Karhu R, Tanner M, et al. Genetic heterogeneity and clonalevolution underlying development of asynchronous metastasis in human breastcancer. Cancer Res 1997;57:1597 –604. 24. Pandis N, Teixeira MR, Adeyinka A, et al. Cytogenetic comparison ofprimary tumors and lymph node metastases in breast cancer patients. GenesChromosomes Cancer 1998;22:122– 9. 25. Schmidt-Kittler O, Ragg T, Daskalakis A, et al. From latent disseminatedcells to overt metastasis: genetic analysis of systemic breast cancer progression.Proc Natl Acad Sci U S A 2003;100:7737–42. 26. Gray JW. Evidence emerges for early metastasis and parallel evolution ofprimary and metastatic tumors. Cancer Cell 2003;4:4 – 6.Bcl-2 Promotes Metastasis of EpH4 hMEKDD Cells Mol Cancer Res 2004;2(10). October 2004555 Research.on September 15, 2017. © 2004 American Association for Cancermcr.aacrjournals.orgDownloaded from 27. Khanna C, Khan J, Nguyen P, et al. Metastasis-associated differences in geneexpression in a murine model of osteosarcoma. Cancer Res 2001;61:3750– 9. 28. Khanna C, Wan X, Bose S, et al. The membrane-cytoskeleton linker ezrin isnecessary for osteosarcoma metastasis. Nat Med 2004;10:182–6. 29. Martin SS, Ridgeway AG, Pinkas J, et al. A cytoskeleton-based functionalgenetic screen identifies Bcl-xL as an enhancer of metastasis, but not primarytumor growth. Oncogene 2004;23:4641 –5. 30. Rubio N, Espana L, Fernandez Y, Blanco J, Sierra A. Metastatic behavior ofhuman breast carcinomas overexpressing the Bcl-x(L) gene: a role in dormancyand organospecificity. Lab Invest 2001;81:725–34. 31. Fernandez Y, Gu B, Martinez A, Torregrosa A, Sierra A. Inhibition ofapoptosis in human breast cancer cells: role in tumor progression to the metastaticstate. Int J Cancer 2002;101:317 –26. 32. Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expressiontransfectants with a novel eukaryotic vector. Gene 1991;108:193 –9. 33. Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, Bissell MJ. Matrixmetalloproteinase stromelysin-1 triggers a cascade of molecular alterations thatleads to stable epithelial-to-mesenchymal conversion and a premalignantphenotype in mammary epithelial cells. J Cell Biol 1997;139:1861–72. 34. DeOme KB, Faulkin LJ, Bern HA, Blair PB. Development of mammarytumors from hyperplastic alveolar nodules transplanted into gland-free mammarypads of female C3H mice. Cancer Res 1959;19:515– 9.Pinkas et al. Mol Cancer Res 2004;2(10). October 2004556 Research.on September 15, 2017. © 2004 American Association for Cancermcr.aacrjournals.orgDownloaded from 2004;2:551-556.Mol Cancer ResJan Pinkas, Stuart S. Martin and Philip LederMEKDD Mammary Epithelial CellsβBcl-2-Mediated Cell Survival Promotes Metastasis of EpH4 Updated versionhttp://mcr.aacrjournals.org/content/2/10/551Access the most recent version of this article at: Cited articleshttp://mcr.aacrjournals.org/content/2/10/551.full.html#ref-list-1This article cites 33 articles, 11 of which you can access for free at: Citing articles/content/2/10/551.full.html#related-urlsThis article has been cited by 3 HighWire-hosted articles. Access the articles at: E-mail alertsrelated to this article or journal.Sign up to receive free email-alerts SubscriptionsReprints [email protected] atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications [email protected] atTo request permission to re-use all or part of this article, contact the AACR Publications Research.on September 15, 2017. © 2004 American Association for Cancermcr.aacrjournals.orgDownloaded from