Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.     

Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis

Metastasis is the primary cause of death in patients with breast cancer. Overexpression of c-myc in humans correlates with metastases, but transgenic mice only show low rates of micrometastases. We have generated transgenic mice that overexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF) in the mammary gland, which develop high rates of pulmonary macrometastases. Gene expression profiling revealed a set of deregulated genes in Myc/VEGF tumors compared to Myc tumors associated with the increased metastatic phenotype. Cross-comparisons between this set of genes with a human breast cancer lung metastasis gene signature identified five common targets: tenascin-C(TNC), matrix metalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signaling blockade or knockdown of TNC in MDA-MB-435 cells resulted in a significant impairment of cell migration and anchorage-independent cell proliferation. Mice injected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrated a significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapse after surgical removal of the primary tumor and (3) incidence of lung metastasis. Our results demonstrate that VEGF induces complex alterations in tissue architecture and gene expression. The TNC signaling pathway plays an important role in mammary tumor growth and metastases, suggesting that TNC may be a relevant target for therapy against metastatic breast cancer.     

Overexpression of Bcl-xL in Human Breast Cancer Cells Enhances Organ-Selective Lymph Node Metastasis

Lymph node metastasis are the first prognostic factor in breast cancer diagnosis and an early event in metastatic spread. To assess the role of anti-apoptotic proteins in lymph node metastatic progression of human breast cancer cells we analyzed the metastatic activity of MDA-MB-435 cells transfected with the Bcl-xL gene, after orthotopic inoculation in Nude Balb/c and in SCID mice. The luciferase gene was introduced by permanent transfection in the 435/Bcl-xL and 435/Neo cells and used as a tumor marker to measure the number of tumor cells lodged in lymph nodes. We found that 435/Bcl-xL tumor cells had enhanced organ-specific metastatic activity, preferentially lodging in peripheral lymph nodes, where at 45 days post-implantation we found 7 x 10(6) +/- 6 x 10(6) 435/Bcl-xL.luc and 2 +/- 1.1 435/Neo.luc luciferase tagged tumor cell equivalents (TCEs). Metastases were abrogated in mice in which orthotopic tumors were induced with 435/Bcl-xL-antisense cells. Additionally, in vitro experiments show that in 435 cells Bcl-xL-antisense can override the emergence of resistance to apoptosis induced by TNF- alpha and TGF- beta in cells overexpressing Bcl-xL, increasing also adhesion to extracellular matrix proteins. These results point to the relevance of Bcl-xL overexpression inducing lymph node metastasis of breast cancer cells, and to the value of this gene as a target for therapy in order to prevent metastasis.     

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.     

Induction of apoptosis and inhibition of c-erbB-2 in breast cancer cells by flavopiridol.

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity against a number of human tumor cell lines, both in vitro and when grown as xenografts in mice. It is presently being investigated as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Because breast cancer is the most common cancer and second leading cause of cancer-related deaths in women in the United States, we investigated whether flavopiridol could be an effective agent against a series of isogenic breast- cancer cell lines having different levels of erbB-2 expression and differential invasion and metastatic characteristics. Flavopiridol was found to inhibit the growth of MDA-MB-435 (parental) and 435.eB (stable transfectants) cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. Induction of apoptosis was also observed in these cell lines when treated with flavopiridol, as measured by DNA laddering, PARP, and CPP32 cleavages. We also found modest up-regulation of Bax and down-regulation of Bcl-2, but there was a significant down-regulation of c-erbB-2 in flavopiridol-treated cells. Gelatin zymography showed that flavopiridol inhibits the secretion of matrix metalloproteinase (MMP; MMPs 2 and 9) in the breast cancer cells and that the inhibition of c-erbB-2 and MMPs may be responsible for the inhibition of cell invasion observed in flavopiridol-treated cells. Collectively, these molecular effects of flavopiridol, however, were found to be independent of c-erbB-2 overexpression, suggesting that flavopiridol may be effective in all breast cancer. From these results, we conclude that flavopiridol inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells. These findings suggest that flavopiridol may be an effective chemotherapeutic or preventive agent against breast cancer.     

高转移性人乳腺癌细胞亚克隆的筛选

目的筛选高转移潜能的人乳腺癌细胞亚克隆,为人类乳腺癌转移相关的体内外研究提供实验对象。方法通过有限稀释法分离和培养人乳腺癌细胞MDA-MB-435S的单克隆;采用细胞生物学方法体外评价亚克隆的克隆形成、增殖、运动和侵袭能力;将MDA-MB-435S亚克隆细胞通过乳腺脂肪垫接种于免疫双缺陷SCID鼠,验证体外筛选克隆的体内转移能力。定量资料采用独立样本t检验进行统计学分析。结果筛选得到MDA-MB-435S的高转移亚克隆14-E5;14-E5与亲代细胞呈梭形、伪足较长的细胞形态明显不同,体积比亲代细胞小,呈多边形,触角增多;细胞的体外克隆形成能力、运动能力、侵袭能力及体内自发转移能力均较亲代显著增强(P0.050);细胞周期S期和G2/M期比例比亲代减少,增殖能力较亲代降低(t=7.047,P=0.002);细胞异质黏附能力、体内成瘤率与亲代之间的差异无统计学意义(P0.050)。结论筛选并建立了高转移潜能人乳腺癌细胞亚克隆细胞株14-E5,可以用于乳腺癌转移基因筛选、转移机制研究、抗转移药物的研发和评价抗转移实验性治疗疗效。     

Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13

We previously showed that introduction of a normal, neomycin-tagged human chromosome 11 reduces the metastatic capacity of MDA-MB-435 (435) human breast carcinoma cells by 70-90% without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 11. To identify the gene(s) responsible, differential display comparing chromosome 11-containing (neo11/ 435) and parental, metastatic cells was done. We describe the isolation and functional characterization of a full-length cDNA for one of the novel genes, designated breast-cancer metastasis suppressor 1 (BRMS1), which maps to human chromosome 11q13.1-q13.2. Stably transfected MDA-MB-435 and MDA-MB-231 breast carcinoma cells still form progressively growing, locally invasive tumors when injected into mammary fat pads but are significantly less metastatic to lungs and regional lymph nodes. These data provide compelling functional evidence that breast-cancer metastasis suppressor 1 is a novel mediator of metastasis suppression in human breast carcinoma.     

Regulation of breast cancer cell motility by insulin receptor substrate-2 (IRS-2) in metastatic variants of human breast cancer cell lines.

Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation, protect cells from apoptosis, and enhance metastasis. In this study, we examined the IGF signaling pathway in two breast cancer cell lines selected for metastatic behavior. LCC6 was selected for growth as an ascites tumor in athymic mice from parental MDA-MB-435 cells (435P). The MDA-231BO cell line was derived from osseous metastases that formed after intracardiac injection of the MDA-MB-231 cell line in athymic mice. Compared to the parental cell lines, IGF-I treatment enhanced IRS-2 phosphorylation over IRS-1 in the metastatic variants. IGF-I stimulated cell migration in the variant cells, but not in the parental cells. To determine the role for IRS-2 in IGF-mediated motility, we transfected MDA-231BO cells with an anti-sense IRS-2 construct. Transfected cells had decreased levels of IRS-2 with diminished IGF-mediated motility and anchorage independent growth when compared to control cells. However, adherence to fibronectin was enhanced in the transfected cells compared to MDA-231BO cells. Our data show that breast cancer cells selected for metastatic behavior in vivo have increased IRS-2 activation and signaling. In these cells, IGF-I enhances cell adhesion and motility suggesting that IRS-2 may mediate these aspects of the malignant phenotype.     

Treatment of human breast cancer cells with antisense RNA to the type I insulin-like growth factor receptor inhibits cell growth, suppresses tumorigenesis, alters the metastatic potential, and prolongs survival in vivo

The type I insulin-like growth factor receptor (IGF-IR) plays an important role in the growth and transformation of breast cancer cells. In this study, we investigated the effects of treatment with an antisense IGF-IR construct on cells from the highly metastatic estrogen receptor-negative human breast cancer cell line MDA-MB-435s. The cells carrying the antisense IGF-IR had a markedly reduced expression of IGF-IR, had a significant decrease in cell proliferation, and lost the ability to form colonies in soft agar. There was a delay in tumor formation and a dramatic reduction in tumor size when cells carrying the antisense IGF-IR were injected into either nude or severe combined immunodeficient (scid) beige mice. We have also provided data that show that the scid beige mouse is a more suitable model for studying metastasis of the MDA-MB-435s cells. All of the scid beige mice injected with cells carrying the control construct had metastasis to the lungs, whereas lungs from the nude mice had no apparent metastatic sites after 11 weeks. When cells carrying antisense IGF-IR were injected subcutaneously in scid beige mice, the animals had a significant increase in survival compared with mice injected with cells carrying the control construct. Taken together, these results indicate that the IGF-IR can play a critical role in the progression of breast cancer. Our studies provide a basis for the development of future treatment strategies targeting the IGF-IR in metastatic breast cancer.     

Dietary fat and breast cancer metastasis by human tumor xenografts

Human breast cancer cell lines growing as xenografts in athymic nude mice have been used to examine the effects of dietary fat and fatty acids on tumor progression. The estrogen independent MDA-MB-435 cell line has the advantage that it metastasizes consistently to the lungs and forms quantifiable secondary nodules when injected into the mammary fat pads. With these breast cancer cells, the stimulating effects of polyunsaturated omega-6 fatty acids on both primary tumor growth and metastasis were demonstrated; in contrast, the long-chain omega-3 fatty acids were inhibitory. The model can also be adapted to examine dietary fatty acids, and inhibitors of their metabolism, as experimental adjuvant therapy after surgical excision of the primary tumors. Unfortunately, estrogen dependent human breast cancer cells do not metastasize, or do so rarely, in nude mice; in consequence, it is not possible to use the model to study estrogen-fatty acid interactions on the metastatic process. In addition to metastasis from a primary location, intravenous injection of MDA-MB-435 cells into the nude mouse host, particularly when combined with studies using Matrigel-based in vitro invasion assays, permits further dissection of the steps in the metastatic cascade which are influenced by dietary fatty acids. The results obtained by these several approaches have demonstrated distinct roles for the cyclooxygenase and lipoxygenase-mediated products of omega-6 fatty acid metabolism, and suggest new approaches to experimental breast cancer therapy.     

Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin

Pigment epithelium-derived factor (PEDF) plays an important role in the tumor growth and metastasis inhibition. It has been reported that PEDF expression is significantly reduced in breast cancer, and associated with disease progression and poor patient outcome. However, the exact mechanism of PEDF on breast cancer metastasis including liver and lung metastasis remains unclear. The present study aims to reveal the impact of PEDF on breast cancer. The orthotopic tumor mice model inoculated by MDA-MB-231 cells stably expressing PEDF or control cells was used to assess liver and lung metastasis of breast cancer. In vitro, migration and invasion experiments were used to detect the metastatic abilities of MDA-MB-231 and SKBR3 breast cancer cells with or without overexpression of PEDF. The metastatic-related molecules including EMT makers, fibronectin, and p-AKT and p-ERK were detected by qRT-PCR, Western blot, and Fluorescent immunocytochemistry. PEDF significantly inhibited breast cancer growth and metastasis in vivo and in vitro. Mechanically, PEDF inhibited breast cancer cell migration and invasion by down-regulating fibronectin and subsequent MMP2/MMP9 reduction via p-ERK and p-AKT signaling pathways. However, PEDF had no effect on EMT conversion in the breast cancer cells which was usually involved in cancer metastasis. Furthermore, the study showed that laminin receptor mediated the down-regulation of fibronectin by PEDF. These results reported for the first time that PEDF inhibited breast cancer metastasis by down-regulating fibronectin via laminin receptor/AKT/ERK pathway. Our findings demonstrated PEDF as a dual effector in limiting breast cancer growth and metastasis and highlighted a new avenue to block breast cancer progression.     

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.     

Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer

Introduction  

The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism.     

Relative malignant potential of human breast carcinoma cell lines established from pleural effusions and a brain metastasis.

Three human breast cancer cell lines from pleural effusions (MDA-MB-435, MDA-MB-231, MDA-MB-468) and one from a brain metastasis (MDA-MB-361) were tested for growth potential in vitro (minimum serum requirements, growth in semisolid agarose), for tumorigenicity and metastasis in nude mice following injection into the mammary fatpad (m.f.p.) and intravenously (i.v.), and for formation of experimental brain metastasis following injection into the carotid artery (i.a.). Colony-forming efficiency of the breast cancer cells in dense agarose corresponded with metastatic potential of the m.f.p. tumors. The results consistently ranked the 4 cell lines as follows: MDA-MB-435 greater than MDA-MB-231 greater than MDA-MB-468 greater than MDA-MB-361, from the most to the least aggressive. The exception was for tumor growth in the brain after i.a. injection of cells, where MDA-MB-361 cells were ranked as second highest for tumor take, but with the slowest growth rate. These results indicate that in this series of breast carcinomas, the cells from a brain metastasis (MDA-MB-361) have a lower malignant potential than cells from visceral metastases.     

Metastasis from human breast cancer cell lines

Summary Immunodeficient animals, principally nude mice, when used in appropriately designed studies have been shown to be useful for the experimental analysis of human breast cancer metastasis. As with many other human tumors, the implantation of breast cancer cells into an anatomically appropriate tissue (the mammary fatpad) results in increased tumor take and incidence of metastasis for certain cell lines compared with subcutaneous injection. Testing a number of widely available human breast cancer cell lines identified the MDA-MB-435 cell line as the most metastatic, producing lung and lymph node metastases in a high proportion of nude and severe combined immunodeficient (SCID) mice after injection in the mammary fatpad. Mixing human breast cancer cells with normal fibroblasts or with Matrigel also increases the tumor incidence and growth rates in nude mice. Different routes of injection can be used to assess the ability of human breast cancer cells to form metastatic lesions in the lungs (i.v. injection), the liver (injection in the spleen), the brain (direct or intracarotid artery injection) and the bone marrow and bone (injection into the left ventricle of the heart). These different approaches demonstrate the potential of experimental studies of human breast cancer growth and metastasis using immunodeficient mice; this model is valuable for experiments that test the role of metastasis-associated genes and the efficacy of novel forms of therapy.     

Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo

PURPOSE: Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. EXPERIMENTAL DESIGN: Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. RESULTS: Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms. CONCLUSIONS: Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.     

Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75-1 and ZR75-30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA-MB-231 and MDA-MB-435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies.     

Antimetastatic and antitumor effects of fluoropyrimidines alone and combined with taxanes in a murine model of breast cancer metastatic to the lung

To evaluate the antitumor efficacy against metastatic breast cancer of fluoropyrimidines alone and combined with other chemotherapeutic agents, we developed a murine model of breast cancer metastatic to the lung by orthotopically implanting MDA-MB-435S breast tumors into mice. MDA tumor cells greatly metastasized to lung tissue only after the orthotopically implanted tumors were surgically removed. Measurement of the expression of enzymes involved in 5-FU metabolism showed significantly higher activity of dihydropyrimidine dehydrogenase (DPD) and lower activity of thymidylate synthase (TS) in the MDA metastases than in the orthotopically implanted tumors. Based on the enzymatic properties of metastatic tumors, the minimum toxic doses of UFT (17.5 mg/kg/day) as a DPD-inhibitory fluoropyrimidine (DIF), and of 5'-DFUR (120 mg/kg/day) as a non-DIF, were orally administered to mice with pulmonary metastasis of the breast tumor. The results showed that UFT significantly inhibited the growth of pulmonary metastases of the breast tumors, but 5'-DFUR did not. UFT seemed to inhibit the growth of the pulmonary metastases of the breast tumors in combination with paclitaxel (50 mg/kg) more than in combination with 5'-DFUR, although the antitumor efficacy of neither combination was significantly different from that of paclitaxel alone. These results suggest that combination of DIF with other chemotherapeutic drugs, such as taxanes, is required to attain high antimetastatic and antitumor efficacy against breast tumor metastases, based on the molecular characteristics of the metastatic tumors.