Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor (CD87)

The serine protease urokinase-type plasminogen activator, uPA, when bound to its specific receptor, uPAR (CD87), plays a significant role in tumor cell invasion and metastasis. In breast cancer, enhanced uPA antigen in the primary tumor is correlated with poor prognosis of the patient. In an in vivo nude mouse model, we tested tumor growth and metastasis of human breast carcinoma cells that had been transfected with an expression plasmid encoding a soluble form of uPAR (suPAR). We explored, whether suPAR/uPA interaction reduces the binding of uPA to cell surface-associated uPAR, and, as a consequence, could suppress tumor growth and metastasis of the human breast cancer cell line MDA-MB-231 BAG. Overexpressed, secreted suPAR was shown to bind and thus scavenge the uPA secreted by the transfected lines suPAR3 and suPAR10. In vitro, an overexpression of suPAR did not alter the proliferation rate of the transfected tumor cells, nor did it affect the expression of uPA. Overexpression of suPAR led to a reduction in the plasminogen activation-related proteolytic activity of breast carcinoma cells. Primary tumor growth in the mammary fat pad of nude mice was followed up for 52 days. Overexpression of suPAR correlated with a reduction in tumor growth (from day 21, reaching 30% by day 34) as well as lung colonization (lung metastasis-positive mice in suPAR3: 4 of 17; suPAR10: 3 of 10; parental MDA-MB-231 BAG: 13 of 18). We conclude that suPAR overexpression leading to effective scavenge of uPA impairs proteolysis as well as the tumor growth and metastatic potential of breast carcinoma cells in vivo.     

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.     

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.     

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.     

Synergy of Nab-paclitaxel and Bevacizumab in Eradicating Large Orthotopic Breast Tumors and Preexisting Metastases

Introduction

Patients with metastatic disease are considered incurable. We previously showed that nabpaclitaxel (nanoparticle albumin-embedded paclitaxel) combined with anti-vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, eradicates orthotopic small-sized breast tumors and metastasis. Here, we assessed this therapy in two models of advanced (450–600 mm³) breast tumors and delineated VEGF-A-dependent mechanisms of tumor resistance.

Methods

Mice with luciferase-tagged advanced MDA-MB-231 and MDA-MB-435 tumors were treated with saline, nab-paclitaxel (10 or 30 mg/kg), bevacizumab (4 mg/kg), or combined drugs. Lymphatic and lung metastases were measured by luciferase assay. Proinflammatory and survival pathways were measured by ELISA, Western blot and immunohistochemistry.

Results

Nab-paclitaxel transiently suppressed primary tumors by 70% to 90% but had no effect on metastasis. Coadministration of bevacizumab increased the response rate to 99%, including 71% of complete responses in MDA-MB-231-bearing mice treated concurrently with 30 mg/kg of nab-paclitaxel. This combinatory regimen significantly reduced or eliminated preexisting lymphatic and distant metastases in MDA-MB-231 and MDA-MB-435 models. The mechanism involves paclitaxel-induced NF-κB pathway that upregulates VEGF-A and other tumor prosurvival proteins.

Conclusions

Bevacizumab prevents tumor recurrence and metastasis promoted by nab-paclitaxel activation of NF-κB pathway. Combination therapy with high-dosed nab-paclitaxel demonstrates the potential to eradicate advanced primary tumors and preexisting metastases. These findings strongly support translating this regimen into clinics.     

Combined treatment with verapamil,a calcium channel blocker,and B428, a synthetic uPA inhibitor,impairs the metastatic ability of a murine mammary carcinoma

Urokinase plasminogen activator (uPA) and metalloproteinases (MMP) play key roles in invasion and metastasis, degrading extracellular matrix compounds and modulating tumor cell motility. Their regulation is an attractive therapeutic target for controlling tumor metastasis. Previously we have demonstrated that urokinase overexpression in murine mammary tumor cells is regulated by a Ca2+-dependent pathway and that blockage of Ca2+ channels by verapamil partially inhibited their invasive and metastatic ability. Moreover, the catalytic inhibition of uPA by a synthetic uPA inhibitor B428 reduced local tumor invasiveness but not tumor cell dissemination. We evaluated the effect of a combined treatment with verapamil and B428 on the murine mammary carcinoma F3II behavior in vivo and in vitro. In vivo administration of the combined treatment was not associated to an overt toxicity. Only the daily combined treatment, beginning after tumor take, reduced the incidence and the number of spontaneous lung metastasis, while no differences were found in the subcutaneous growth of the primary tumor. Interestingly, a remarkable reduction in plasma MMP-9 activity was found associated to metastasis impairment. In addition, the number of experimental lung metastases was also significantly diminished, with respect to the control group, only when both compounds were co-administered daily, beginning three days after i.v. tumor cell injection. In vitro, both compounds, either separately or combined, could inhibit secreted uPA activity. F3II cell migration was significantly inhibited by incubation with 50 microM verapamil, 15 microM B428 or the co-treatment with 7.5 microM B428 + 25 microM verapamil. The cell spread was also significantly reduced when F3II cells were exposed to the compounds, with an additive effect when B428 + verapamil combination was used. The combination of two compounds acting through different molecular targets may be useful to improve the control of metastatic dissemination.     

Multi-kinase inhibitor E7080 suppresses lymph node and lung metastases of human mammary breast tumor MDA-MB-231 via inhibition of vascular endothelial growth factor-receptor (VEGF-R) 2 and VEGF-R3 kinase

PURPOSE: Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor. EXPERIMENTAL DESIGN: We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD). RESULTS: In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor. CONCLUSIONS: Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.     

Macrophages mediate inflammation-enhanced metastasis of ovarian tumors in mice

The tumor microenvironment is known to have a profound effect on tumor progression in a highly context-specific manner. We have investigated whether peritoneal inflammation plays a causative role in ovarian tumor metastasis, a poorly understood process. Implantation of human ovarian tumor cells into the ovaries of severe combined immunodeficient mice resulted in peritoneal inflammation that corresponds temporally with tumor cell dissemination from the ovaries. Enhancement of the inflammatory response with thioglycolate accelerated the development of ascites and metastases. Suppression of inflammation with acetyl salicylic acid delayed ascites development and reduced tumor implant formation. A similar prometastatic effect for inflammation was observed when tumor cells were injected directly into the peritoneum of severe combined immunodeficient mice, and in a syngeneic immunocompetent mouse model. Inflammation-modulating treatments did not affect primary tumor development or in vitro tumor cell growth. Depletion of peritoneal macrophages, but not neutrophils or natural killer cells, reduced tumor progression, as assessed by ascites formation and peritoneal metastasis. We conclude that inflammation facilitates ovarian tumor metastasis by a mechanism largely mediated by macrophages, and which may involve stromal vascular endothelial growth factor production. The confirmation of these findings in immunocompetent mice suggests relevance to human disease. Identifying the mechanisms by which macrophages contribute to tumor metastasis may facilitate the development of new therapies specifically targeting immune cell products in the tumor microenvironment.     

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.     

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.     

Development and gene expression profiling of a metastatic variant of the human breast cancer MDA-MB-435 cells

We have developed a model system of late stage metastatic progression by isolating a highly malignant variant of human breast cancer cells from the parental MDA-MB-435 cell line. These cells, isolated from early lung metastasis, displayed increased anchorage independent growth in vitro and when transplanted ortho-topically into nude mice showed accelerated tumor growth rate and early lung spontaneous metastasis when compared to its parental counterpart. These cells, designated as MDA-MB-435-F-L, also showed intense wide spread early skeletal metastasis in vertebrae, mandible, femur, tibia and skull as detected by fluorescence imaging in an experimental bone metastasis model. Gene expression profiles from cDNA microarray showed up or downregulation of the expression of several significant genes regulating angiogenesis, apoptosis, ECM remodeling and metastasis in the MDA-MB-435-F-L cells in comparison to the parental cells. Among the up or downregulated genes, some have also been implicated in the survival of breast cancer patients. As such, the candidate genes selected in this breast cancer progression model system may serve as biomarkers of metastatic progression and also as potential tumor targets for breast cancer therapy.     

Flaxseed and its components reduce metastasis after surgical excision of solid human breast tumor in nude mice

This study determined the effect of 10% flaxseed (FS) and its components, secoisolariciresinol diglycoside (SDG) and flaxseed oil (FO) alone or in combination (SDG+FO), on the metastasis and recurrence of human breast tumor after excision in nude mice. Mice were injected orthotopically with human breast cancer cells (MDA-MB-435) and fed basal diet (BD). When the tumors reached an average size of 110 mm(2) (0.9 g), surgical excisions were performed, and the mice were assigned to one of five diet groups for 7 weeks. The total incidence of metastasis was significantly lower in the FS, SDG, and SDG+FO groups. Reduced lung and lymph node metastases were observed in the FS and SDG+FO groups. In the FS and FO groups, a greater reduction in lung and total metastases was found when excised tumors were 0.9 g. There was no significant difference in tumor recurrence among groups. In conclusion, FS and its components inhibited tumor metastasis but not tumor recurrence after surgical excision of the primary tumor.     

LIM kinase 1 increases tumor metastasis of human breast cancer cells via regulation of the urokinase-type plasminogen activator system

Mammalian LIM kinase 1 (LIMK1) phosphorylates and inactivates the actin-binding and -depolymerizing factor cofilin and induces actin cytoskeletal changes. LIMK1 is reported to play an important role in cell motility, but the mechanism of induction of cell motility and the role of LIMK1 in tumor growth, angiogenesis and invasion are poorly understood. Here we show that expression of LIMK1 in MDA-MB-435 human breast cancer cells enhanced cell proliferation and cell invasiveness and promoted in vitro angiogenesis. Since tumor metastasis requires degradation of the extracellular matrix by the serine protease urokinase type plasminogen activator (uPA), we examined the role of LIMK1 in the regulation of uPA/uPAR system. LIMK1 overexpression in breast cancer cells upregulated the uPA system, increased uPA promoter activity, induced uPA and uPAR mRNA and protein expression and induced uPA secretion. In contrast, cells transfected with the catalytically inactive LIMK mutant D460N-LIMK1 did not exhibit these phenotypic changes. Blocking antibodies against uPA and uPAR suppressed LIMK1-induced cell invasiveness. In addition, LIMK1 overexpression increased tumor growth in female athymic nude mice, promoted tumor angiogenesis and induced metastasis to livers and lungs, possibly by increasing uPA expression in the tumors. Finally, LIMK1 and uPAR were coordinately overexpressed in human breast tumors. These results suggested an important role for LIMK1 signaling in breast cancer tumor growth, angiogenesis and invasion and a regulatory connection between LIMK1 and the uPA system.     

Calcitonin inhibits invasion of breast cancer cells: involvement of urokinase-type plasminogen activator (uPA) and uPA receptor

There is a growing body of evidence indicating that calcitonin (CT) and calcitonin receptor (CTR) are involved in the regulation of cell growth, differentiation, and survival and in tissue development. However, the precise functional role of CT/CTR in breast cancer is still unknown. It is well established that the urokinase plasminogen activator (uPA) system plays an important role in breast cancer invasion and metastasis. The goal of this study was to investigate the effects of CT on regulation of the uPA system and invasive capacity of breast cancer cells. In the highly invasive MDA-MB-231 cell line, 10(-8) M CT decreased both uPA and uPAR mRNA and protein expression which was associated with inhibition of the extracellular signal-regulated kinase (ERK) 1/2 pathway. Furthermore, two weeks of CT administration to nude mice inhibited the expression of uPA mRNA in primary tumors by 25% (P<0.05), as compared to control, untreated animals. CT also inhibited the invasiveness of MDA-MB-231 cells by 37% (10(-8) M CT, P<0.05), as determined by a Matrigel invasion assay. To the best of our knowledge, this is the first report describing a direct effect of CT on breast cancer cell invasion. Our data might suggest a close link between CT signaling, the uPA-mediated pathway, and breast cancer invasion.     

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.     

Antitumor effects of a monoclonal antibody that binds anionic phospholipids on the surface of tumor blood vessels in mice.

PURPOSE: We recently reported that anionic phospholipids, principally phosphatidylserine, become exposed on the external surface of viable vascular endothelial cells in tumors, possibly in response to oxidative stresses present in the tumor microenvironment. In the present study, we tested the hypothesis that a monoclonal antibody directed against anionic phospholipids might exert antitumor effects by causing vascular damage in tumors. EXPERIMENTAL DESIGN: A new mouse immunoglobulin G3 monoclonal antibody, 3G4, was raised that binds anionic phospholipids in the presence of serum or beta2-glycoprotein I. The antibody was tested for its ability to localize to tumor vessels and exert antitumor effects in mice. RESULTS: 3G4 recognized anionic phospholipids on the external membrane of H(2)O(2)-treated endothelial cells and in vitro. It localized specifically to tumor vascular endothelium and to necrotic tumor cells after injection into severe combined immunodeficient mice bearing orthotopic MDA-MB-435 tumors. Treatment with 3G4 retarded the growth of four different tumors in mice. It reduced the growth of established orthotopic MDA-MB-231 and MDA-MB-435 human breast tumors in mice by 75% and 65% respectively, large L540 human Hodgkin's tumors by 50%, and small syngeneic Meth A fibrosarcomas by 90%. Histologic examination revealed vascular damage, a reduction in vascular density, and a reduction in tumor plasma volume. Treatment with 3G4 induced the binding of monocytes to tumor endothelium and infiltration of macrophages into MDA-MB-435 and MDA-MB-231 tumors. No toxicity to the mice was observed. CONCLUSIONS: 3G4 localizes specifically to complexes of anionic phospholipids and serum proteins on the surface of vascular endothelial cells in tumors in mice. This results in damage to tumor vasculature and suppression of tumor growth.     

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.     

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.     

Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.