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Department of Internal Medicine, Division of Hematology and Oncology [Q. P., K. L. v. G., J. I., K. M. B., G. J. B., S. D. M.], Departments of Pathology [C. G. K.] and Human Genetics [D. M. R., R. D. D., G. J. B.], and Comprehensive Cancer Center [Q. P., C. G. K., K. L. v. G., K. M. B., S. D. M.], University of Michigan Medical School, Ann Arbor, Michigan 48109, and Laboratory of Viral Oncogenesis, Division of Hematology and Oncology, Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021 [C. B., M. D. C., E. A. M.]
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; (d) IL-6; and (e) IL-8. In addition, TM inhibits vessel network formation and suppresses nuclear factor (NF)
B levels and transcriptional activity. Our study suggests that a major mechanism of the antiangiogenic effect of copper deficiency induced by TM is suppression of NFB, contributing to a global inhibition of NFB-mediated transcription of proangiogenic factors. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Animal Models of Breast Cancer.
SUM149 cells (1 x 106 cells) were orthotopically injected into the upper left mammary fat pad of 10-week-old female athymic nude mice. Cells were trypsinized, washed, and resuspended in Hank’s buffered saline solution (HBSS) at a density of 1 x 106 cells/200 µl. Mice were anesthetized using 10 mg/ml ketamine, 1 mg/ml xylazine, and 0.01 mg/ml glycopyrrolate, and an incision below the thoracic left mammary fat pad was made. Using a 27-gauge needle, the cell suspension was injected into the exposed mammary fat pad, and the wound was closed with a single wound clip. After a brief recovery period, tumor-implanted mice were randomly assigned and gavaged with water (control; n = 7) or 0.7 mg/day TM (n = 7) daily for 7 weeks. Tumor volume was measured weekly and calculated as (length x width2)/2.
Mouse mammary tumor virus-Her2/neu transgenic mice were purchased from Jackson Laboratory. At 
100 days old, female mouse mammary tumor virus-Her2/neu mice were randomly assigned to two groups and gavaged with water (control; n = 22) or 0.75 mg/day TM (n = 15) for the entire experimental protocol. Mice were monitored weekly for overt palpable tumors, and disease-free survival curve was calculated using Log-rank analysis.
Quantification of Microvessel Density.
Tumors from control or TM-treated mice were resected, immersion fixed in 10% buffered formalin, and paraffin embedded. Intratumoral microvessel density was assessed with CD31 staining (DAKO) using the vascular hotspot technique. Sections were scanned at low power to determine areas of highest vascular density. Within this region, individual microvessels were counted in three separate random fields at high power (x400 magnification). The mean vessel count from the three fields was used. A single countable microvessel was defined as any endothelial cell or group of cells that was clearly separate from other vessels, stroma, or tumor cells without the necessity of a vessel lumen.
Conditioned Media from SUM149 Cells.
SUM149 cells were plated at a density of 2 x 105 cells in 100 mm2 dishes. Cells were treated with vehicle or 0.1 nM TM for 72 h. Conditioned media was collected, centrifuged for 5 min at 2500 rpm, and divided into 1-ml aliquots. Quantikine human VEGF, basic FGF/FGF2, and IL-1 ELISAs (R&D Systems, Inc., Minneapolis, MN) were used to measure protein levels of the 165 amino acid species of VEGF, basic FGF/FGF2, and IL-1. ELISAs for IL-6 and IL-8 were performed by the University of Maryland Cytokine Core Laboratory.6
Rat Aortic Ring Assay.
Aorta was removed from a freshly sacrificed Sprague Dawley rat and rinsed in ice-cold HBSS containing penicillin and streptomycin. Segmental rings, 1 mm in width, were cut from the aorta and embedded in a 50-µl aliquot of 10 mg/ml Matrigel in six-well plates. Rings were incubated overnight at 37°C in serum-free media and then exchanged for conditioned media from control or TM-treated SUM149 cells. Subsequently, rings were incubated for 4 days at 37°C and analyzed by phase-contrast microscopy for microvessel outgrowth.
Transient Transfection and Reporter Gene Assay.
SUM149 or HME cells (1 x 105) were transfected transiently with 1 µg of pNFB (Clontech Laboratories, Inc.) and 0.05 µg pRL-TK (Clontech Laboratories, Inc.) with FuGene6 transfection reagent (Roche Biochemicals). pRL-TK, a Renilla luciferase vector, was cotransfected to normalize for transfection efficiency. After a 24-h recovery period, transfected cells were incubated in fresh medium with or without the addition of 2 nM CuSO4. Cells were treated subsequently with vehicle, TM (1 nM), TNF (2 pM), or TM and TNF for 24, 48, or 72 h. Cells were harvested in passive lysis buffer, and the activities of the firefly luciferase and Renilla luciferase were quantified on a Monolight 2010 luminometer (Analytical Luminescence Laboratory) using the dual luciferase assay system (Promega Corp.).
Electrophoretic Mobility Shift Assay.
Nuclear extracts from SUM149 cells were incubated with 32P-labeled B consensus sequence in a buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT for 30 min at 25°C. Protein-DNA complexes were resolved on a high ionic strength 5% polyacrylamide gel containing 0.5 x Tris-borate EDTA buffer [380 mM glycine, 45 mM Tris base (pH 8.5), 45 nM boric acid, and 2 mM EDTA]. Supershift analysis was performed as described above except nuclear extracts were preincubated with p50, RelA, p52, c-Rel, or RelB antibody (Upstate Biotechnology) for 30 min on ice.
Western Blot Analysis.
Proteins were harvested from SUM149 cells using radioimmunoprecipitation assay buffer (1 x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 0.3 mg/ml aprotinin; Sigma Chemical Co.). Aliquots (20 µg) were mixed with Laemelli buffer, heat denatured for 3 min, separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Nonspecific binding was blocked by overnight incubation with 2% BSA in Tris-buffered saline with 0.05% Tween 20 (Sigma Chemical Co.). Immobilized proteins were probed using antibodies specific for p50 or RelA (Upstate Biotechnology). Protein bands were visualized by enhanced chemiluminescence (Amersham-Pharmacia Biotech).
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. Tumors in the control group were highly vascularized as shown by immunohistochemical staining with a CD31 antibody (Fig. 1B)
. In contrast, the smaller tumors resected from TM-treated mice were only sparsely vascularized (mean vessel count; 16 ± 2 versus 26 ± 3, P < 0.04, n = 3), providing further evidence that copper deficiency induced by TM is antiangiogenic.
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; (d) IL-6; and (e) IL-8 (P < 0.05; Fig. 2A
). Moreover, IL-6 and IL-8 mRNA expression was decreased by TM (data not shown). To test whether the decrease in proangiogenic mediators was sufficient to impair the angiogenic potential of the conditioned media, we used the rat aortic ring assay, an in vitro model of angiogenesis that resembles the outgrowth of primordial vessels from a host vessel under an appropriate stimulus (Fig. 2B)
. Rat aortic rings incubated with conditioned media from TM-treated SUM149 cells showed a marked reduction in vessel outgrowth when compared with rings incubated with conditioned medium from untreated SUM149 cells. In addition, primordial blood vessels failed to grow when conditioned media from untreated SUM149 cells were supplemented with TM before incubation with aortic rings. Because copper may be involved in the regulation of the biological activity of the prototype members of the FGF gene family (11)
, we tested if copper deficiency could suppress the stimulatory effect of exogenous FGF2 on HUVECs. TM blocked FGF2-stimulated HUVEC tubule formation in a dose-dependent manner, without affecting HUVEC proliferation (data not shown). Although previous studies have suggested that copper may be required for endothelial cell proliferation and migration (1
, 2)
, our study indicates, for the first time, that absence of copper is able to block exogenous FGF2-mediated organization of endothelial cells into a primordial vessel network. Our data demonstrate that copper deficiency inhibits tumor angiogenesis by two different mechanisms: (a) reduction in the level of extracellular proangiogenic mediators released by cancer cells; and (b) inhibition of endothelial cell differentiation.
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B Protein Levels and Transcription.B activity to oncogenesis has emerged in recent years. The NFB transcription factor is known to regulate genes important for invasion, angiogenesis, and metastasis. These include proangiogenic factors, such as VEGF, IL-6, and IL-8, matrix metalloproteinases, urokinase plasminogen activator, and cell adhesion molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (12, 13, 14, 15, 16)
. Recently, blocking NFB activity in human ovarian cancer cells was reported to inhibit VEGF and IL-8 expression, resulting in a decrease in tumor angiogenesis (17)
. To investigate the NFB dependence of proangiogenic factors in SUM149 cells, we transfected these cells with the super-repressor IB (S32AS36A). Conditioned media from SUM149 super-repressor IB clones had significantly lower amounts of VEGF, IL-6, and IL-8 in comparison with the empty vector-transfected SUM149 cells; 45 ± 5% inhibition for VEGF, 37 ± 4% inhibition for IL-6, and 58 ± 5% inhibition for IL-8 (P < 0.05, n = 3). Because TM was able to decrease these proangiogenic factors to a similar extent, we sought to determine whether copper deficiency induced by TM is modulating NFB-mediated signaling. SUM149 and nontumorigenic-immortalized HME cells were transiently transfected with pNFB, a vector that contains four tandem copies of the B consensus sequence upstream of the luciferase reporter gene (Fig. 3)
. Endogenous NFB activity was shown to be 2.5-fold higher in SUM149 cells in comparison with HME cells. This is consistent with our observation that p50 protein levels were significantly higher in SUM149 cells (data not shown). Several studies using human breast cancer cells also reported that overexpression of p50 results in constitutive NFB activity (18
, 19)
. After treatment for 24 h, TM inhibited luciferase activity by 62 ± 2% (P < 0.001, n = 6) in SUM149 cells and 34 ± 2% (P < 0.001, n = 3) in HME cells. Moreover, TM completely blocked TNF-stimulated NFB activity in both cell lines. Similar results were observed at 48 and 72 h demonstrating that TM is also able to inhibit NFB activity on a sustained basis without affecting cell survival under these conditions. In Fig. 4A
, we analyzed the binding of nuclear proteins from SUM149 cells to a labeled oligonucleotide spanning the B consensus sequence. Extracts from TM-treated cells showed a decrease in nuclear protein binding to the B consensus sequence. In addition, supershift analysis revealed that the predominant NFB components in SUM149 cells are p50, p52, and RelA. When cells were cultured with added copper (2 nM CuSO4 addition), TM partially lost its ability to regulate B binding and NFB transcriptional activity. In addition, copper repletion partially reversed TM inhibition of IL-6 and IL-8 mRNA expression (data not shown), consistent with restoring NFB’s ability to enhance transcription of these genes. Interestingly, p50 and RelA protein levels were reduced after treatment with TM in SUM149 cells, suggesting that TM may be suppressing NFB activity by decreasing levels of NFB component proteins.
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B signaling is suppressed after TM treatment in vitro. Because VEGF, IL-6, and IL-8 are NFB-regulated genes, we anticipate that the decreased secretion of these proteins may be a direct consequence of TM’s ability to inhibit NFB activity. This leads to the suggestion that TM is limiting tumor neovascularization by repressing the ability of cancer cells to release NFB-dependent inflammatory cytokines and proangiogenic mediators into the extracellular compartment and thus limiting the autocrine and/or paracrine effects of these proteins to stimulate angiogenesis in the tumor microenvironment. TM also retards the release of IL-1 in SUM149 cells. Consistent with our observation, the release of IL-1 is dependent on the oxidative function of intracellular copper and blocked with TM treatment.7
Interestingly, IL-1 was reported to enhance NFB activity resulting in an increase in IL-6 (20)
. IL-1 is able to induce the expression of VEGF and FGF2 in human endothelial and melanoma cells (21
, 22)
. From these observations, we further hypothesize that TM may be limiting tumor angiogenesis by directly blocking the release of IL-1 from tumor cells to prevent additional NFB stimulation in tumor and endothelial cells in close proximity.
TM Prevents the Development of de Novo Clinically Overt Tumors in Her2/neu Transgenic Mice.
Our results suggest that the inhibition of proangiogenic factors within the tumor microenvironment accompanies copper deficiency. This leads to the corollary that malignant clones that arise in a copper-deficient milieu may not be able to stimulate sufficient neovascularization for growth beyond a few millimeters. We designed an experimental protocol to determine the effectiveness of TM in retarding or preventing the growth of mammary tumors in female Her2/neu transgenic mice. Extensive clinical studies have shown that overexpression of Her2/neu in patients with breast cancer correlates overall with poorer prognoses (23
, 24)
. Female Her/neu mice develop focal mammary adenocarcinomas, which eventually metastasize to the lungs (25)
. These mice develop single or multifocal mammary tumors approximately at 205 days of age (25)
. By initiating TM treatment 90–120 days before tumors became clinically evident, we surmised that the TM-treated mice would be copper deficient throughout the key period of tumor development when angiogenesis is required for continued tumor growth. Female Her2/neu transgenic mice (100 days old) were gavaged with water (control) or 0.75 mg/day TM (n = 22 for control group and n = 15 for TM group). Cp levels were maintained at 10–30% of baseline for the entire protocol. Depending on the individual mouse, 2–4 weeks were required to achieve the anticipated end point of copper depletion. We chose this end point to investigate whether the level of copper deficiency that would be tolerable in humans could inhibit the angiogenic switch in Her2/neu mice. TM-treated and control mice did not differ in weight or general health as evidenced by social behavior and level of activity. Fig. 5A
depicts the Kaplan-Meier plot for disease-free survival of TM-treated and control mice. By 218 days, 50% of the control mice and none of the TM-treated mice had overt tumors. Log-rank analysis demonstrated that the TM-treated mice had a statistically significant prolongation of disease-free survival compared with the control group (P < 0.0147). At the conclusion of the experiment, with a median follow-up time of 221 days, palpable tumors were not observed in the TM-treated mice that remained copper deficient with Cp levels 
30% of baseline. However, when TM-treated mice were released from therapy, measurable tumors were observed by 13 ± 5 (n = 5) days postrelease. The restoration of copper in these mice, previously copper deficient for >7 months, appears to be sufficient to enable tumor growth to proceed at the normal rate within 2 weeks. Therefore, it is clear that these TM-treated Her2/neu mice retained the capacity to develop macroscopic mammary tumors, and copper deficiency appears to act as a barrier for their appearance. It is important to note that Her2/neu-positive breast cancer cells have enhanced NFB activity (26
, 27)
. In light of our observations, this implies that in a copper-deficient environment, nascent breast cancer tumors with Her2/neu overexpression have impaired ability to activate the angiogenic switch attributable, in part, to decreased production of proangiogenic factors, perhaps as a consequence of NFB inhibition by TM.
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). These large buds were widespread throughout the mammary glands of TM-treated Her2/neu mice. Histological analyses of these structures clearly showed several discrete microscopic foci of hyperplastic or neoplastic epithelium interspersed among scant normal ducts lined by a single layer of normal cells. These foci contained pleomorphic cells, with increased cytoplasmic clearing and some prominent reddish nucleoli, often filling the duct entirely. These neoplastic foci also were observed in the mammary glands of vehicle-treated and TM-treated/release Her2/neu mice. The rapid development of overt tumors in mice released from TM treatment strongly suggests that the cells of the microtumors in TM-treated mice are neoplastic. Interestingly, the morphology of the cells from the microtumors of TM-treated mice is strikingly similar to the cells from the bulky tumors that developed in vehicle- or TM-treated mice that were released from therapy (Fig. 5C1-3
). This observation further supports our supposition that these microtumors are comprised of completely transformed cells, but failed to acquire neovascularization, and thus have remained noninvasive and relatively contained in a dormant state with minimal or no angiogenesis.
Taken together, these results support our initial clinical observations indicating that copper deficiency induced by TM is a potent approach to inhibit tumor angiogenesis with minimal adverse effects. TM exerts its antiangiogenic action at least in part through restriction of the extracellular appearance of proangiogenic factors and suppression of NFB activity. The observation that TM suppresses NFB activity is potentially exciting from a clinical perspective because constitutive NFB activity is linked to the development of resistance to chemotherapy or radiotherapy (28
, 29) . Inhibition of NFB by TM, in turn, may restore the sensitivity of resistant cancer cells and recapitulate the efficacy of chemotherapy or radiotherapy-induced apoptosis. Moreover, the strong suppressive effect on tumor growth when the angiogenic switch is inhibited before or at the time of neoplastic transformation in mammary epithelium suggests a promising role for TM as a chemopreventative agent for use in carriers of cancer susceptibility genes.
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FOOTNOTES |
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1 Supported in part by NIH Grants R01CA77612 (to S. D. M.), P30CA46592, M01-RR00042, and AI-19192 (to E. A. M.), American Cancer Society RPG-99-207-01-MBC (to E. A. M.), FDA FD-U-000505 (to G. J. B.), and the Tempting Tables Organization, Muskegon, MI. 
2 G. J. B. and S. D. M. are consultants and have a financial interest in Attenuor, LLC, which has licensed TM as an anticancer compound from the University of Michigan.
3 To whom requests for reprints should be addressed, at University of Michigan Medical Center, 7217 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0948. Phone: (734) 764-2248; Fax: (734) 615-2719; E-mail: smerajve{at}umich.edu
4 The abbreviations used are: TM, tetrathiomolybdate; Cp, ceruloplasmin; IL, interleukin; FGF, fibroblast growth factor; HME, human mammary epithelial; HUVEC, human umbilical vein endothelial cell; TNF, tumor necrosis factor; NFB, nuclear factor B; VEGF, vascular endothelial growth factor.
5 J. A. Marrero and B. G. Redman, personal communication.
6 Internet address: http://www.cytokinelab.com.
7 A. Mandinova, S. Bellum, C. Bagala, R. Soldi, I. Micucci, M. Landriscina, F. Tarantini, I. Prudovsky, and T. Maciag. The stress-induced release of the pro-inflammatory cytokine IL1 is Cu2+-dependent. Proc. Natl. Acad. Sci. USA, submitted for publication, 2002.
Received 5/15/02. Accepted 7/10/02.
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