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[Cancer Research 65, 3209-3217, April 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Nuclear Factor-{kappa}B–Dependent Mechanisms in Breast Cancer Cells Regulate Tumor Burden and Osteolysis in Bone

Andrew H. Gordon1,2, Regis J. O'Keefe2, Edward M. Schwarz2, Randy N. Rosier2 and J. Edward Puzas2

1 Biomedical Engineering, University of Rochester and 2 Department of Orthopaedics and Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, Rochester, New York

Requests for reprints: J. Edward Puzas, Department of Orthopaedics and Center for Musculoskeletal Research, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 665, Rochester, NY 14642. Phone: 585-275-3664; Fax: 585-756-4727; E-mail: Edward_Puzas{at}URMC.Rochester.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A central mediator of a wide host of target genes, the nuclear factor-{kappa}B (NF-{kappa}B) family of transcription factors, has emerged as a molecular target in cancer and diseases associated with bone destruction. To evaluate how NF-{kappa}B signaling in tumor cells regulates processes associated with osteolytic bone tumor burden, we stably infected the bone-seeking MDA-MB-231 breast cancer cell line with a dominant-negative mutant I{kappa}B that prevents phosphorylation of I{kappa}B{alpha} and associated nuclear translocation of NF-{kappa}B. Blockade of NF-{kappa}B signaling in MDA-MB-231 cells by the mutant I{kappa}B decreased in vitro cell proliferation, expression of the proinflammatory, bone-resorbing cytokine interleukin-6, and in vitro bone resorption by tumor/osteoclast cocultures while reciprocally up-regulating production of the proapoptotic enzyme caspase-3. Suppression of NF-{kappa}B transcription in these breast cancer cells also reduced incidence of in vivo tumor-mediated osteolysis after intratibial injection of tumor cells in female athymic nude mice. Immunohistochemistry showed that the cancerous lesions formed in bone by MDA-MB-231 cells express both interleukin-6 and the p65 subunit of NF-{kappa}B at the bone-tumor interface. NF-{kappa}B signaling in breast cancer cells therefore promotes bone tumor burden and tumor-mediated osteolysis through combined control of tumor proliferation, cell survival, and bone resorption. These findings imply that NF-{kappa}B and its associated genes may be relevant therapeutic targets in osteolytic tumor burden.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A coordinated balance exists between osteoclastic bone resorption and osteoblastic bone formation in normal bone remodeling (1). When metastatic breast cancer cells invade and seed themselves within the bone microenvironment, this balance becomes disrupted, usually leading to rapid bone loss. Uncontrolled production of an array of osteoclast-activating factors, including cytokines, growth factors, hormones, enzymes, adhesion molecules, and chemotactic factors, underlie progression of this disease state. Various pathologic conditions result, most notably bone fracture, bone pain, and hypercalcemia, which all contribute to increased morbidity among cancer patients with advanced bone tumors and metastases (2).

The clinical hallmark of disrupted bone remodeling in aggressively metastasizing breast cancers is extensive osteoclast-mediated bone destruction (3). Osteoclasts, which seem to be required for progression of osteolytic tumor burden, differentiate and activate in response to stimulation by proinflammatory cytokines, such as tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-1, and IL-6, all known to be secreted by breast cancer cells (4–6). Our data and those of other laboratories indicate that the cytokines produced by the cancer cells not only stimulate bone resorption by host osteoclasts but also induce other cells in the microenvironment to secrete high levels of the same cytokines (5, 7). This sustained release of catabolic cytokines drives a rapid loss of bone. Attempting to individually block the effect of any or all of these cytokines may not be the most practical approach to this clinical problem. Ideally, a distinct therapeutic target governing production of a spectrum of such bone-resorbing factors could be identified and exploited to more effectively reduce or prevent osteolytic bone destruction.

Many of the proinflammatory cytokines involved in stimulating osteoclastic activity, including TNF-{alpha}, IL-1, and IL-6, fall under the control of nuclear factor-{kappa}B (NF-{kappa}B) transcription (8). Targeting the NF-{kappa}B family of transcription factors holds significant promise in the treatment of various cancers and diseases associated with abnormal bone resorption (9–12). NF-{kappa}B controls progression of oncogenesis and associated disease states through suppression of apoptosis and cell differentiation as well as promotion of cell proliferation and migration. Many of the steps in the NF-{kappa}B pathway have been delineated. The dimeric form of the transcription factor resides in the cell cytoplasm bound to an inhibitor (of nuclear factor) of {kappa}B (I{kappa}B) protein. Various molecular stimuli can activate I{kappa}B kinases, which in turn phosphorylate I{kappa}B and target it for degradation in the 26S proteasome. After dissociating from I{kappa}B, the NF-{kappa}B dimer may then translocate to the nucleus and regulate expression of multiple genes potentially involved in cancer cell survival, growth, and osteolysis (9, 10). Blocking this single pathway therefore may inhibit diverse arrays of biological interactions that promote osteolytic tumor progression.

We suppressed NF-{kappa}B transcription and associated gene expression in the tumor cells by using a dominant-negative mutant I{kappa}B{alpha} (mI{kappa}B) superrepressor of NF-{kappa}B signaling (13). This repressor was stably infected into the MDA-MB-231 cell line to determine the role of NF-{kappa}B signaling in cytokine production, apoptosis, proliferation, and bone resorption at a skeletal site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and culture conditions. The MDA-MB-231 breast cancer cell line was a gift from Dr. Theresa Guise (San Antonio, TX) and the 293GP viral packaging cell line was a gift from Dr. Inder Verma (Salk Institute, La Jolla, CA). MDA-MB-231 and 293GP cells were grown in DMEM (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1% penicillin/streptomycin, and nonessential amino acids (Invitrogen). Cells were grown in a humidified 37°C atmosphere of 5% CO2.

Stable expression of lacZ and mI{kappa}B expression vectors. MDA-MB-231 cells stably expressing a dominant-negative inhibitor of NF-{kappa}B, an I{kappa}B{alpha} with serine-to-alanine mutations at positions 32 and 36 (mI{kappa}B), were generated by infecting wild-type MDA-MB-231 cells with a recombinant retrovirus technique described previously and adapted for the MDA-MB-231 cell line (13, 14). Retrovirus was produced using 293 kidney fibroblasts stably expressing the Moloney gag and pol (293GP) under control of the cytomegalovirus promoter-enhancer. 293GP cells were plated in 10 cm culture dishes and grown to 50% confluency at 37°C in 10 mL DMEM/10% FBS/1% penicillin/streptomycin. Fresh serum-free DMEM was added before transfection. Vesicular stomatitis virus envelope (4 µg) downstream of the cytomegalovirus promoter-enhancer, pLXSN retroviral vector (25 µg; consisting of L = long terminal repeat promoter, X = lacZ or mI{kappa}B promoter, S = SV40 promoter, and N = neomycin resistance gene), and pRSV-ß-galactosidase (2 µg) containing the bacterial lacZ gene downstream of the Rous sarcoma virus promoter-enhancer (to aid in assessing transfection efficiency of the mI{kappa}B) were transfected into 293GP cells using LipofectAMINE 2000 (Invitrogen). Transfected 293GP cells were incubated for 24 hours, after which retroviral supernatant was collected and treated with 10 µg/mL polybrene (Sigma, St. Louis, MO). Supernatants were filter sterilized and stored at –80°C until use. Transfection efficiency was evaluated by staining for ß-galactosidase by fixing 293GP cells with 5 mL fixing solution [2% formaldehyde/0.2% glutaraldehyde/PBS (pH 7.4)] per dish for 5 minutes at 4°C. Cells were washed twice with 1x PBS and stained with 3 mL staining solution [5 mmol/L potassium ferricyanide, 5 mmol/L ferrocyanide, 2 mmol/L MgCl, PBS (pH 7.4), X-gal in DMSO (1 mg/mL) at a 1:40 dilution]. Stained cells were incubated for 2 to 4 hours at 37°C. Wild-type MDA-MB-231 cells were then stably infected with lacZ or mI{kappa}B retrovirus supernatant collected from the packaging line. Neomycin-resistant MDA.lacZ and MDA.mI{kappa}B cells from infected cell pools were selected for 2 weeks in 800 µg/mL G418 (Invitrogen) and obtained by limiting dilution.

Luciferase assay. Our transfection reagents were 1.5 µg pNF-{kappa}B-Luc (firefly luciferase driven by a TATA box with five NF-{kappa}B sites in the enhancer element) and 2 ng pRL-SV40 (Renilla luciferase driven by SV40) reporter constructs (Promega, Madison, WI) complexed to 3 µL LipofectAMINE 2000 transfection reagent in 100 µL serum-free culture medium. Cells (n = 300,000) seeded in six-well plates (Falcon, Franklin Lakes, NJ) in 3 mL culture medium were transfected by adding 100 µL LipofectAMINE 2000-plasmid complex. Luciferase activity was assayed after 24 hours using the Dual Luciferase Reporter Assay System (Promega). Reporter assays were conducted on unstimulated cells after a 30-minute stimulation with 40 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) and 2 µmol/L ionomycin A23187 (Calbiochem, San Diego, CA), a common regimen used for NF-{kappa}B activation (15, 16).

Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was used to assess NF-{kappa}B DNA binding as described previously (14). Nuclear protein extract (5 µg) was mixed with 2 µg poly(deoxyinosinic-deoxycytidylic acid) and DNA binding buffer [50 mmol/L NaCl, 5 mmol/L HEPES (pH 7.5), 5 mmol/L EDTA, 5 mmol/L EGTA, 30% glycerol, 1.25 µg bovine serum albumin] in a total volume of 10 µL and incubated on ice for 20 minutes. NF-{kappa}B and Oct-1 consensus oligonucleotides (Santa Cruz Biotechnology, Santa Cruz, CA) were end labeled by the use of T4 polynucleotide kinase and [32P]CTP (New England Nuclear, Boston, MA) and 20,000 counts per minute (cpm) of 32P-labeled oligonucleotide were added to the binding reaction and incubated for 30 minutes at room temperature. Complexes were then separated on 6% polyacrylamide gel under nondenaturing conditions at 125 V for 3 hours. Gels were dried on Whatman No. 3M paper and DNA-protein complexes were visualized by autoradiography. To identify the p65 NF-{kappa}B subunit of the heterodimeric complex, EMSA supershift was done by preincubating nuclear protein extracts with antibody specific for the active NF-{kappa}B p65 subunit (Santa Cruz Biotechnology) for 20 minutes on ice.

Western blot analysis. Caspase-3 and I{kappa}B{alpha} protein expression was analyzed by Western blot as described previously by separating 20 µg whole cell or cytoplasmic protein extract on a 10% SDS-polyacrylamide gel, with subsequent transfer onto Immobilon-P membrane (Millipore, Bedford, MA; ref. 14). Membranes were blocked and probed with polyclonal antibodies to caspase-3 or I{kappa}B{alpha} (Santa Cruz Biotechnology) in 5% nonfat dried milk in a solution of PBS containing 0.1% Tween 20. After two 30-minute washes in PBS-0.1% Tween 20, blots were incubated with horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 hour followed by two 30-minute washes in PBS-0.1% Tween 20. Immunoreactive bands were detected using the enhanced chemiluminescence system (Amersham, Piscataway, NJ) after exposure of the membrane to Hyperfilm enhanced chemiluminescence for 30 to 60 seconds.

ELISA detection and quantitation of cytokines. Cell lines were plated on six-well plates at a cell density of 104/mL and grown in 2 mL DMEM/10% FBS/1% penicillin/streptomycin to near confluence. After incubation at 37°C for 16 hours, conditioned medium samples were collected and stored at –80°C until assayed for cytokine levels by ELISA. Protein standards, primary capture, and secondary biotin antibody pairs were obtained for human TNF-{alpha}, IL-1{alpha}, IL-6 (PharMingen, La Jolla, CA), IL-1ß (Endogen, Woburn, MA), macrophage colony-stimulating factor (M-CSF), IL-11, and IL-17 (R&D Systems, Minneapolis, MN). Cytokine levels for 100 µL of each supernatant sample were evaluated using commercially available ELISA reagents (PharMingen).

Real-time reverse transcription-PCR. Total mRNA from MDA-MB-231 cells was reversed transcribed using the Advantage RT-for-PCR kit (Clontech, Palo Alto, CA) and PCR amplified using Platinum PCR Supermix (Invitrogen). PCR conditions for amplification were initial denaturation of 3 minutes at 94°C and subsequent denaturation for 25 seconds at 94°C, annealing for 25 seconds at 55°C, and extension for 50 seconds at 72°C (36 cycle). Quantitative PCR analysis was done using the Rotor-Gene 2000 Centrifugal Real-time PCR System (Corbett Research, Sydney, New South Wales, Australia). RNA samples were quantified relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal controls in each cell type and stimulatory condition (17). IL-6 fragments (81 bp) were amplified with forward primer 5'-GGTACATCCTCGACGGCATCT-3' and reverse primer 5'-GTGCCTCTTTGCTGCTTTCAC-3' (Life Technologies, St. Paul, MN; ref. 18). Human parathyroid hormone-related protein (PTHrP) mRNA levels were evaluated using primers specific for a 285-bp PTHrP fragment (5'-GCGACGATTCTTCCTTCACC-3' and 5'-AGAGTCTAACCAGGCAGAGC-3, corresponding to the sense and antisense strands of exon 4 of human PTHrP; ref. 19). GAPDH fragments (178 bp) were amplified with forward primer 5'-GACATCAAGAAGGTGGTGAA-3' and reverse primer 5'-TGTCATACCAGGAAATGAGC-3' (20).

In vitro bone marrow/tumor cell cocultures. Transverse slices of femoral diaphyseal bovine cortical bone (4.0 x 4.0 x 0.3 mm) cut with a low-speed diamond saw (Buehler, Evanston, IL) were used as the substrate for generating osteoclast-derived lacunae according to the method of Dempster et al. as modified by our laboratory (21). Bone slices were cleaned by ultrasonication in multiple changes of sterile water and stored at –20°C until used. Long bones (tibias and femurs) were removed from 4- to 6-day-old euthanized rats, dissected free of adherent soft tissue, and minced with a scalpel blade into 2 or 3 mL prewarmed medium 199 with Hank's salts (Sigma; pH 7.0), buffered with 10 mmol/L HEPES containing 100 µg/mL penicillin/streptomycin. The resulting bone fragment and cell suspension was gently triturated 10 times with a transfer pipette. An aliquot (110 µL) of the bone cell suspension was then transferred to individual wells of 96-well plates, each well containing a single bone slice pre-wetted with 100 µL of the above medium. Following a settling period of 30 to 45 minutes at 37°C, medium was replaced with fresh 199 with Earle's salts (Sigma) containing 10% heat-inactivated FBS, parathyroid hormone (10 nmol/L), and 1,25(OH)2-vitamin D3 (10 nmol/L) and adjusted to pH 6.8. Forty-eight hours after plating bone marrow cell suspensions onto bone slices, 10,000 tumor cells from each cell type (MDA-MB-231 and MDA.mI{kappa}B), suspended in 200 µL of the above culture medium, were plated on each cortical bone slice. Tumor cells were cocultured with the bone marrow cell suspensions for 10 days on bone slices, changing medium every 48 hours. After 10 days, slices were removed, washed gently with 1x PBS, scraped free of cell debris, and stained with toluidine blue. Resorption area of each lacunae was quantified with Osteometrics software (Atlanta, GA). Digital image analysis generated a number expressing the percentage of the 16 mm2 bone slice surface that was resorbed.

Intratibial injection model of tumor-mediated bone resorption. Female athymic nu/nu mice (NIH/Charles River, Bethesda, MD) were given 60 mg/kg ketamine and 4 mg/kg xylazine i.p., providing ~15 to 20 minutes of deep anesthesia before tumor cell injection. The injection site was sterilized with isopropyl alcohol, cleaned with Betadine, and allowed to dry. After making a small incision (5 mm) revealing the tibial tuberosity, a small hole was made with a 25 gauge needle. Then, a Hamilton syringe (10 µL capacity, 26S gauge) was inserted in the proximal end of the tibia, and 105 tumor cells suspended in 10 µL PBS were injected into the intramedullary space. Radiographs were taken immediately after injection, while the mice remained anesthetized, to confirm correct insertion of the syringe without further fracture of surrounding bone. Radiographs were taken at 0, 14, and 28 days to track osteolytic lesion progression.

Radiographs and measurements of osteolytic lesion area. Animals were exposed for 6 seconds to X-ray in a prone position against XOMAT-AR film (Kodak, Rochester, NY) using a Faxitron system (Wheeling, IL) set to a tube voltage of 36 kVp. All radiographs were evaluated without knowledge of treatment groups. Radiographs were scanned into digital TIFF format, and quantitation of in vivo osteolytic lesion area was done using image analysis software (NIH Scion Image, Frederick, MD and Adobe Systems Photoshop, San Jose, CA).

Histology/histomorphology. At sacrifice on day 28, tibias were dissected from all mice, fixed in 10% formalin, decalcified in EDTA, and embedded in paraffin. Sections were cut using a standard microtome, placed on poly-L-lysine-coated glass slides, and stained with orange G and H&E. Additional sections were stained for tartrate-resistant acid phosphatase (TRAP), human IL-6, and human p65 (Santa Cruz Biotechnology). Osteoclast number expressed per millimeter of tumor-bone interface was measured from TRAP-stained sections using digital imaging (NIH Scion Image and Adobe Systems Photoshop).

Tumor cell growth. To investigate whether tumor growth was altered in sites other than bone, tumor cell suspensions (5 x 106 cells/0.1 mL PBS) were inoculated s.c. into female athymic nude mice. Mice were sacrificed at day 28 and the tumors were excised and weighed. To confirm cell proliferation rates in vitro, [3H]thymidine incorporation assays were done as described previously (22, 23). Briefly, cell cultures of 20,000 cells in 24-well plates (Becton Dickinson, Franklin Lakes, NJ) were labeled with 10 µCi/mL [3H]thymidine (New England Nuclear) in the presence of 5 mmol/L unlabeled [3H]thymidine (to control for differences in radioactive [3H]thymidine pools) in culture medium. After 4 hours of incubation, the DNA was precipitated and centrifuged and the pellet was redissolved in NaOH. Radioactivity was determined by liquid scintillation spectrometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of MDA.mI{kappa}B and MDA.lacZ cells. To investigate the role of NF-{kappa}B in bone tumor burden and associated tumor-mediated osteolysis, a recombinant retrovirus containing a mI{kappa}B expression cassette and a neomycin drug resistance marker were used to generate cells that stably express the mI{kappa}B. The mI{kappa}B cassette was mutated by replacing Ser32 and Ser36 with alanines. The mI{kappa}B binds NF-{kappa}B, but because the mI{kappa}B cannot be phosphorylated at two key sites NF-{kappa}B signal transduction is suppressed. The mutant therefore behaves as a dominant-negative inhibitor by blocking NF-{kappa}B gene expression. Using this technique, neomycin-resistant MDA.mI{kappa}B cells were isolated after stable infection of wild-type MDA-MB-231 cells. A lacZ retroviral vector was also stably infected into wild-type MDA-MB-231 cells to create neomycin-resistant MDA.lacZ control cells.

To examine stability and functionality of the newly isolated MDA.mI{kappa}B cells, functional activation of NF-{kappa}B transcription was examined by luciferase reporter assay. Suppression of functional NF-{kappa}B activation was exhibited by MDA.mI{kappa}B cells when compared with wild-type MDA-MB-231 and MDA.lacZ controls (Fig. 1A). EMSAs were done on isolated nuclear protein extracts to assess NF-{kappa}B/DNA binding activity. Wild-type MDA-MB-231 cells possessed constitutive NF-{kappa}B/DNA binding activity with or without stimulation by PMA/ionomycin for 30 minutes (Fig. 1B). The active p65 NF-{kappa}B subunit of the p50/p65 heterodimer was identified as a component of this shift, as evidenced by a supershift, after preincubating with antibody to human p65 for 30 minutes on ice (Fig. 1B). NF-{kappa}B/DNA binding associated with the p65 subunit, otherwise seen in nuclear extracts from MDA-MB-231 cells, was lost in MDA.mI{kappa}B cells (Fig. 1B). These experiments therefore show the effectiveness of the dominant-negative mI{kappa}B in reducing NF-{kappa}B activation in MDA.mI{kappa}B cells.



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  		Figure 1. Stability and functionality of MDA.mI{kappa}B cells. A, constitutive and induced NF-{kappa}B transcription is suppressed in MDA.mI{kappa}B cells. NF-{kappa}B luciferase reporter activity is observed to increase significantly after activation of MDA-MB-231 and MDA.lacZ control cells, but not MDA.mI{kappa}B cells, with PMA (40 ng/mL) and ionomycin (2 µmol/L) for 30 minutes *, P < 0.02 versus unstimulated and P < 0.01 versus PMA/ionomycin regimen. B, NF-{kappa}B p65-DNA binding activity is suppressed in MDA.mI{kappa}B cells. As analyzed by EMSA, constitutive NF-{kappa}B/DNA binding is seen in unstimulated (–) wild-type (WT) MDA-MB-231 nuclear extracts and after stimulation (+) with PMA (40 ng/mL) and ionomycin (2 µmol/L) for 30 minutes. To identify the active p65 NF-{kappa}B subunit of the heterodimeric complex, nuclear extracts from wild-type MDA-MB-231 cells were preincubated with antibody to human p65 for 30 minutes on ice. The p65-DNA binding otherwise observed in wild-type MDA-MB-231 extracts is lost in MDA.mI{kappa}B cells. COLD, extracts treated with unlabeled, nonradioactive probe. C, I{kappa}B{alpha} is not degraded in MDA.mI{kappa}B clones. Western analysis shows that after stimulation with PMA (40 ng/mL) and ionomycin (2 µmol/L) I{kappa}B{alpha} levels decrease in MDA-MB-231 and MDA.lacZ control cells, whereas I{kappa}B{alpha} levels in MDA.mI{kappa}B cells remain the same. Endogenous I{kappa}B{alpha} therefore is replaced in MDA.mI{kappa}B cells by the mI{kappa}B, which cannot be degraded. D, an active form of the apoptosis marker caspase-3 (p20) is expressed in MDA.mI{kappa}B cells. Western analysis of whole cell lysates from tumor cells shows that after stimulation with TNF-{alpha} (10 ng/mL) for 0, 12, and 24 hours levels of the caspase-3 precursor CPP32 (~32 kDa) decrease and levels of an active subunit of caspase-3, p20 (~17 kDa), increase in MDA.mI{kappa}B cells but not in MDA-MB-231 and MDA.lacZ control cells.

 
Further demonstration of the efficacy of the dominant-negative mI{kappa}B was shown by immunoblotting. Cytoplasmic extracts from wild-type MDA-MB-231, MDA.lacZ, and MDA.mI{kappa}B clones were probed for I{kappa}B{alpha} levels by Western blot analysis. Wild-type MDA-MB-231, MDA.lacZ, and MDA.mI{kappa}B cells produced similar amounts of I{kappa}B{alpha} before PMA/ionomycin treatment. However, 30 minutes after stimulation with PMA/ionomycin, I{kappa}B{alpha} levels in both wild-type MDA-MB-231 and MDA.lacZ cells decreased substantially, whereas I{kappa}B{alpha} levels in MDA.mI{kappa}B cells remained essentially unchanged. This supports the expectation that the predominant form of I{kappa}B in the MDA.mI{kappa}B cells can no longer be degraded following stimulation (Fig. 1C).

Tumor cell survival is tightly regulated by NF-{kappa}B signaling, which protects against TNF-{alpha}-induced apoptosis (24). To assess TNF-{alpha}-induced apoptosis in MDA.mI{kappa}B cells, we employed caspase-3 as a biological marker. Caspase-3 whose activity is up-regulated in cells with reduced NF-{kappa}B signaling is required for DNA fragmentation and morphologic changes typical of cells undergoing apoptosis (25, 26). On induction of caspase-3 activity during apoptosis, the inactive 32-kDa proenzyme (CPP32) is cleaved at an aspartate residue, yielding the active subunit p20 (~17-21 kDa; ref. 27). Thus, expression of the p20 active form of caspase-3 serves as an indicator of apoptosis. After treatment with TNF-{alpha} for 12 and 24 hours, increased expression of active caspase-3 (p20) was observed in whole cell protein extracts of MDA.mI{kappa}B clones, whereas in wild-type MDA-MB-231 and MDA.lacZ extracts no expression of active caspase-3 was detected (Fig. 1D). These results support the notion that MDA-MB-231 cells are rendered susceptible to TNF-{alpha}-induced apoptosis after inhibition of NF-{kappa}B signaling.

Screening of cytokine production in MDA-MB-231 cells. Breast cancer cells promote in vivo osteolysis by inducing host osteoclast activity (4). To elucidate how tumor-derived NF-{kappa}B signaling increases bone-resorptive activity, MDA-MB-231 cells were screened for secretion of NF-{kappa}B-mediated factors with potential roles in bone resorption. Specifically, we examined levels of IL-1{alpha}, IL-1ß, IL-6, IL-11, IL-17, M-CSF, and TNF-{alpha}, all of which are cytokines implicated in bone-resorptive processes and NF-{kappa}B signaling (6, 8, 28–34). MDA-MB-231 cells showed significantly higher constitutive levels of IL-11 in comparison with TNF-{alpha}, IL-1{alpha}, IL-1ß, IL-6, IL-17, and M-CSF (data not shown). However, after cytokine-induced activation of NF-{kappa}B signaling with IL-1ß or TNF-{alpha} for a prolonged period (16 hours), only IL-6 production increased significantly (data not shown).

MDA-MB-231 cells produce significant amounts of IL-6, a potent stimulator of osteoclast activity and formation whose gene promoter possesses NF-{kappa}B consensus motifs (28–30). These observations suggested that IL-6 secretion could be down-regulated after inhibition of NF-{kappa}B signaling in MDA-MB-231 cells. As measured by ELISA, MDA.mI{kappa}B cells secreted up to 99% less IL-6 than wild-type MDA-MB-231 cells after stimulation (n = 3; P < 0.01; Fig. 2A). This result was verified by real-time reverse transcription-PCR (RT-PCR), which shows suppression of IL-6 mRNA levels in MDA.mIB cells treated with IL-1ß (n = 3; P < 0.01; Fig. 2B). Secretion of IL-11, whose production was not further stimulated by IL-1ß, was observed to be slightly up-regulated in MDA.mI{kappa}B clones (data not shown; n = 3; P < 0.02). MDA-MB-231 cells were also assayed for NF-{kappa}B-mediated PTHrP mRNA expression. PTHrP is a causal factor in tumor-mediated osteolysis whose fragments activate NF-{kappa}B signaling in osteoblasts (35, 36). Real-time RT-PCR did not show a statistically significant difference in PTHrP mRNA expression between wild-type MDA-MB-231 and MDA.mI{kappa}B cells (data not shown). NF-{kappa}B therefore does not seem to control PTHrP mRNA expression in MDA-MB-231 cells.



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  		Figure 2. Control of IL-6 production and bone resorption in vitro by NF-{kappa}B signaling in MDA-MB-231 cells. A, suppression of NF-{kappa}B signaling decreases IL-6 secretion by MDA-MB-231 cells in vitro. IL-6 protein concentrations were assessed by ELISA before and after stimulation with IL-1ß (10 ng/mL) or PMA (40 ng/mL) and ionomycin (2 µmol/L) for 16 hours. Columns, mean; bars, SE. *, P < 0.01 versus wild-type cells stimulated with IL-1ß or PMA/ionomycin. B, suppression of NF-{kappa}B signaling decreases IL-6 mRNA expression in MDA-MB-231 cells in vitro. Real-time RT-PCR shows increased IL-6 mRNA expression (relative to GAPDH control mRNA) in wild-type MDA-MB-231 cells stimulated with IL-1ß (10 ng/mL) for 16 hours compared with unstimulated wild-type MDA-MB-231 and MDA.mI{kappa}B cells before and after stimulation. *, P < 0.01 versus all other cell types and conditions. C, NF-{kappa}B transcription in MDA-MB-231 cells controls their capacity to stimulate bone resorption in vitro. Bone resorption was evaluated by measuring lacunar area of bovine cortical bone wafers (4 x 4 x 1 mm). Bone marrow (BM) cells were cocultured with either wild-type MDA-MB-231 (MDA.WT) with or without anti-human IL-6 antibody (1 µg) or MDA.mI{kappa}B cells for 10 days and compared with two different controls without tumor cells: bone marrow cells cultured alone or treated with parathyroid hormone (BM + PTH). MDA.mI{kappa}B cultures show a substantial decrease in bone resorption compared with wild-type MDA-MB-231 cultures. Wild-type MDA-MB-231 cultures treated with anti-human IL-6 antibody also show a marked decrease in lacunar area compared with untreated wild-type MDA-MB-231 cultures. Columns, mean (n = 3); bars, SE. *, P < 0.05, lesion area statistically significant from BM and BM + PTH controls (n = 3). **, P < 0.05, lesion area statistically significant from BM + MDA.WT group (n = 3).

 
Inhibition of nuclear factor-{kappa}B signaling in MDA-MB-231 cells down-regulates their capacity to stimulate bone resorption in vitro. Next, we examined whether MDA.mI{kappa}B cells were altered in their ability to stimulate bone resorption in vitro. We cocultured wild-type MDA-MB-231 and MDA.mI{kappa}B cells with rat bone marrow cultures and quantified osteoclast lacunae formation in vitro on cortical (bovine) bone wafers using computerized imaging software. In comparison with bone marrow cell culture controls, addition of MDA-MB-231 cells significantly increased bone resorption lacunar area. Substitution of wild-type MDA-MB-231 cells with MDA.mI{kappa}B cells in this coculture, however, showed suppression of this osteolytic activity. Because inhibition of NF-{kappa}B signaling down-regulates IL-6 expression in MDA-MB-231 cells, wild-type MDA-MB-231/bone marrow cocultures were also treated with anti-human IL-6 antibody to ascertain any role tumor-derived IL-6 may have in in vitro bone resorption. A marked decrease in lacuna area was observed in wild-type MDA-MB-231 cocultures pretreated with anti-human IL-6 antibody (Fig. 2C). Thus, inhibition of NF-B signaling, as well as blockade of tumor cell–derived IL-6, down-regulates the ability of breast cancer cells to promote bone resorption.

Effect of the dominant-negative mI{kappa}B on in vivo osteolytic lesion formation by MDA-MB-231-derived tumor cell lines in female athymic nude mice. Our in vitro observations provided sufficient rationale to test whether inhibition of NF-{kappa}B signaling could suppress bone tumor growth and associated tumor-mediated bone resorption in vivo. Osteolytic lesion formation by MDA.mI{kappa}B cells in bone compared with MDA-MB-231 and MDA.lacZ controls was evaluated by intratibial injection in female athymic nude mice. After injecting the tumor cells (n = 10 per treatment group), radiographs were taken at days 0, 14, and 28. Radiography verified a high percentage of osteolytic lesions in MDA-MB-231 and MDA.lacZ treatment groups (Fig. 3A). At day 28, animals were sacrificed for histology.



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  		Figure 3. Establishment of osteolytic tumor lesions by intratibial injection. A, suppression of osteolytic tumor burden in mice implanted with MDA.mI{kappa}B cells. Tumor cells (105) were implanted in the proximal end of tibiae of female athymic nude mice. Arrows, approximate location and direction of tumor cell injection. Mice were X-rayed at days 0, 14, and 28 to track progression of osteolytic tumor lesions formed by wild-type MDA-MB-231, MDA.lacZ, and MDA.mI{kappa}B cells. B, suppression of osteolytic lesions in mice implanted with MDA.mI{kappa}B cells. On sacrifice at day 28, tibiae were harvested, fixed in formalin, and embedded in paraffin. Sections were stained with orange G and H&E. These images show the proximal end of representative tibiae where the tumor cells were injected. Note replacement of bone marrow with tumor cells in wild-type MDA-MB-231 and MDA.lacZ tissue sections. Magnification, x4.

 
Histologic specimens revealed replacement of bone marrow with tumor cells in the intratibial cavity of mice injected with either wild-type MDA-MB-231 or MDA.lacZ cells. In most mice injected with MDA.mI{kappa}B clones, however, the marrow remained intact (Fig. 3B). Tumor lesions formed by intratibial injection of MDA.mI{kappa}B cells were observed to be of significantly smaller magnitude than tumors formed by MDA-MB-231 and MDA.lacZ control cells. Frequency of osteolytic lesions (Table 1), osteolytic lesion area (Fig. 4A), and number of osteoclasts found at the tumor-bone interface (Fig. 4B) were significantly decreased in MDA.mIB-bearing mice when compared with wild-type MDA-MB-231 and MDA.lacZ treatment groups. High numbers of TRAP-positive osteoclasts were seen at the tumor-bone interface (Fig. 4C), consistent with the pivotal role of osteoclasts in tumor-mediated bone destruction (4).


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  		Table 1. In vivo osteolytic tumor frequency and correlating immunohistochemistry

 


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  		Figure 4. Quantification and visualization of in vivo osteolysis. A, in vivo osteolytic lesion area quantified from radiographs of tibias on days 14 and 28. Osteolytic tumor lesion area was measured using computerized imaging analysis. Points, mean measurements from midsections of right tibias of mice injected with wild-type MDA-MB-231, MDA.lacZ, or MDA.mI{kappa}B cells (8-10 mice per group); bars, SE. *, P < 0.01 versus wild-type MDA-MB-231 and MDA.lacZ. B, inhibition of NF-{kappa}B signaling in MDA-MB-231 cells decreases osteoclast number per millimeter of bone in vivo. Histomorphometry of right tibias from female athymic nude mice injected with 105 tumor cells at day 0. Tibias were harvested at day 28 and stained with TRAP to count osteoclasts. Midsections of right mouse tibia. *, P < 0.01 versus MDA-MB-231 and MDA.lacZ. C, in vivo TRAP staining of osteoclasts at tumor-bone interface. After 28 days, tibias were harvested, decalcified, sectioned, and stained for TRAP. Implanted MDA.lacZ-positive control cells stimulating TRAP-positive osteoclasts (OC) at the tumor-bone interface. Similar sections were obtained for all mice implanted with tumor cells that developed osteolytic tumor lesions. Magnification, x20.

 
Immunohistochemistry of all tumors formed showed production of both NF-{kappa}B-p65 and IL-6 protein (Table 1; Fig. 5). The most intense IL-6 staining was found predominantly near areas where tumor cells interfaced with bone, consistent with the notion that IL-6 plays an important role in tumor-mediated bone resorption. These observations suggest that the presence of the NF-{kappa}B-p65 subunit and IL-6 correlates with formation of in vivo osteolytic lesions by MDA-MB-231 cells.



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  		Figure 5. Representative IL-6 and p65 immunohistochemistry from osteolytic tumor lesions. MDA-MB-231 cells characteristically stain positive for IL-6 and p65 in vivo near areas of osteoclastic bone resorption. Brown, positive stains for IL-6 and p65, respectively. Magnification, x20.

 
Effect of inhibition of nuclear factor-{kappa}B signaling in breast cancer cells on tumor growth. To determine if the effect of NF-{kappa}B signaling on bone tumor growth was indeed specific to bone, tumor cells (5 x 106/100 µL PBS) were injected s.c. into female athymic nude mice. S.c. tumor growth for each treatment group (n = 5) was monitored by palpation until sacrifice at day 28. On excision at day 28, tumors were weighed. Tumor mass (n = 5; P < 0.05 versus MDA-MB-231 and P < 0.01 versus MDA.lacZ; Fig. 6) differed significantly between both MDA-MB-231 or MDA.lacZ controls and MDA.mIB cells. In vitro [3H]thymidine incorporation assays confirmed MDA.mIB cells possessed a reduced capacity to proliferate compared with wild-type MDA-MB-231 cells ([3H]thymidine DNA incorporation: wild-type MDA-MB-231 = 441.3 cpm and MDA.mI{kappa}B = 252.7 cpm, n = 3; P < 0.01). Therefore, MDA.mI{kappa}B cells show a decreased capacity to proliferate, independent of bone, as evidenced by their reduced s.c. tumor mass in vivo and decreased [3H]thymidine incorporation in vitro.



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  		Figure 6. Suppression of N F-{kappa}B signaling in MDA-MB-231 cells decreases s.c. tumor proliferation in vivo. Tumor cells (5 x 106) were injected s.c. into female athymic nude mice to assess tumor proliferation in vivo in sites other than bone. Tumors grew for 28 days before excision. Tumor weight was recorded following excision at day 28 (n = 5). *, P < 0.05 versus MDA-MB-231 and MDA.lacZ.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Patients presenting with bone cancers show significantly lower relative survival rates than patients with soft tissue cancers (37). Therefore, validating a therapeutic target, such as NF-{kappa}B, governing production of numerous factors relevant to bone tumor and metastasis progression, remains of great interest. This study distinguished NF-{kappa}B signaling as a key regulator of a tumor cell's capacity to survive in bone and form osteolytic tumors. Here, we show the essential role NF-{kappa}B transcription may play in bone tumor cell survival, proliferation, bone resorption, and overall in vivo bone tumor burden. MDA-MB-231 breast cancer cells stably infected with a mI{kappa}B, which suppressed NF-{kappa}B transcription, displayed decreased in vitro bone resorption, decreased production of the bone-resorbing cytokine IL-6, and increased production of the active form of caspase-3, a known biological marker of apoptosis. Disruption of NF-{kappa}B signaling in MDA-MB-231 cells also reduced in vivo tumor-mediated osteolysis, corresponding with suppressed in vivo production of IL-6 and the active p65 subunit of NF-{kappa}B.

NF-{kappa}B-mediated production of IL-6 by MDA-MB-231 cells may be of critical significance to the capacity of MDA-MB-231 cells to flourish in vivo, particularly in bone. Inhibition of NF-{kappa}B signaling suppressed several biological phenomena (apoptosis, proliferation, cytokine production, and bone resorption) that combine to augment in vivo bone tumor growth and tumor-mediated osteolysis. IL-6 affects all of these processes and therefore warrants closer examination as a therapeutic target (38, 39). Production of IL-6 by breast cancer cells confers increased resistance to chemotherapeutic agents and plays a prominent role in several inflammatory disease states, including cancer-related anorexia and cachexia (40, 41).

Moreover, by increasing the pool of early osteoclast precursors available to mature into active, bone-resorbing osteoclasts, increased amounts of IL-6 secreted by tumor cells with active NF-{kappa}B signaling could profoundly dysregulate normal bone remodeling and cause bone destruction. IL-6 likely works in concert with other bone-resorbing factors produced by breast cancer cells, such as PTHrP (35, 42). Augmented PTHrP expression can increase bone resorption by stimulating osteoblast RANKL mRNA expression, which in turn fosters bone-resorbing activity by the increased number of osteoclasts made available by IL-6 (43). PTHrP fragments also activate NF-{kappa}B transcription in osteoblasts, leading to IL-6 gene induction and suggestive of an autocrine/paracrine role for PTHrP in NF-{kappa}B-mediated osteolytic tumor burden (36, 44).

Other NF-{kappa}B-related mechanisms, not directly dependent on RANKL activation of osteoclasts, exist in bone through which IL-6 may exert a bone-resorptive effect (45). These pathways could include steroid hormone-regulated bone formation. Estrogen, whose loss is shown to result in IL-6 mediated stimulation of osteoclastogenesis, could protect bone via estrogen receptor interference with NF-{kappa}B binding (46, 47). Estrogen also induces production of the proapoptotic caspase-3 protein in association with preosteoclast apoptosis (48). This increase in caspase-3 can be explained by estrogen interference with NF-{kappa}B signaling. Production of caspase-3 and apoptosis of tumor cells also correlates with decreased bone tumor burden in the MDA-MB-231 bone metastasis model (49). Therefore, a strong rationale exists to further investigate the potential reciprocal relationship between estrogen and NF-{kappa}B signaling in cancer cells and how this affects osteolytic tumor burden. Because MDA-MB-231 cells are estrogen receptor negative and unable to suppress NF-{kappa}B signaling in this manner, this could explain why these cells significantly proliferate in bone (38).

Our observations here strengthen the notion that NF-{kappa}B-regulated IL-6 production by tumor cells causes dysregulation of bone remodeling, supportive of bone destructive processes, through multiple signaling pathways. An observed increase in IL-11 production by MDA.mI{kappa}B cells, however, sharply contrasts with previous studies supporting its role as a stimulator of osteoclast formation and bone resorption and also as a predictor or promoter of osteolytic tumor burden or metastases (45, 50). IL-11 possesses anti-inflammatory properties and is reported to inhibit NF-{kappa}B activation by up-regulating production of I{kappa}B (51). In this manner, IL-11 may override its known role as an osteoclast growth factor in favor of tumor suppression. Contrary to previous reports, therefore, IL-11 could function to inhibit as opposed to promote osteolysis in bone tumors with increased NF-{kappa}B activation.

Several therapeutic approaches targeting NF-{kappa}B signaling show promise in treating various cancers and inflammatory diseases and might be similarly applied to treatment of tumor-mediated osteolysis. Consistent with the results of our study, an NF-{kappa}B decoy oligonucleotide significantly stimulated apoptosis, up-regulated caspase-3, and inhibited IL-6 expression in osteoclasts (52). This kind of decoy could decrease the numbers of osteoclasts available to induce osteolytic bone destruction and simultaneously suppress tumorigenic effects dependent on NF-{kappa}B activation (52, 53). Treating with proteasome inhibitors could prevent I{kappa}B degradation, otherwise required for NF-{kappa}B nuclear translocation. Velcade (PS-341), a proteasome inhibitor that suppresses degradation of I{kappa}B and abrogates IL-6 signaling, is currently used in patients with multiple myeloma (9, 54–56). Evaluating this agent in patients with metastatic or other native bone cancers would be a logical next step. Cytokine-induced NF-{kappa}B activation can be suppressed using a cell-permeable NF-{kappa}B essential modifier regulatory protein (NEMO)-binding domain peptide, which blocks association of NEMO with I{kappa}B kinase complexes upstream of NF-{kappa}B (57). Administration of the NEMO-binding domain peptide reduces NF-{kappa}B-mediated bone resorption in vivo and selectively blocks NF-{kappa}B activation induced by proinflammatory cytokines without disrupting basal NF-{kappa}B activity, an important consideration given the dual role of NF-{kappa}B activation in both proper immune system function and pathogenesis of disease (8, 57–59).

Collectively, our data support a potential role for therapeutics like these that could disrupt NF-{kappa}B signaling in cancers causing osteolysis. This investigation presents evidence on how NF-{kappa}B transcription may tightly control bone tumor cell survival and proliferation and associated bone resorption. Although there exist signaling pathways in tumor cells that promote osteolytic tumor burden but do not directly depend on NF-{kappa}B transcription, NF-{kappa}B remains a central target to consider when contemplating potential therapeutics in this regard.


   Acknowledgments
 
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.

We thank Brendan Boyce for critical review of histology and Janet Cushing, Lisa Flick, Loralee Gehan, Jennifer Harvey, Willis Huang, Barbara Stroyer, and Michael Zuscik for excellent technical assistance.

Received 11/10/04. Revised 1/16/05. Accepted 1/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schwarz EM, O'Keefe RJ. Breakthrough in bone: the molecular mechanism of osteoclast/osteoblast coupling revealed. Curr Opin Orthop 2000;11:329–35.
  2. Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2002;2:584–93.[CrossRef][Medline]
  3. Yoneda T. Cellular and molecular mechanisms of breast and prostate cancer metastasis to bone. Eur J Cancer 1998;34:240–5.
  4. Clohisy DR, Ramnaraine ML. Osteoclasts are required for bone tumors to grow and destroy bone. J Orthop Res 1998;16:660–6.[CrossRef][Medline]
  5. Pederson L, Winding B, Foged NT, Spelsberg TC, Oursler MJ. Identification of breast cancer cell line-derived paracrine factors that stimulate osteoclast activity. Cancer Res 1999;59:5849–55.[Abstract/Free Full Text]
  6. Manolagas SC. Role of cytokines in bone resorption. Bone 1995;17:63–7S.[Medline]
  7. O'Keefe RJ, Teot LA, Singh D, Puzas JE, Rosier RN, Hicks DG. Osteoclasts constitutively express regulators of bone resorption: an immunohistochemical and in situ hybridization study. Lab Invest 1997;76:457–65.[Medline]
  8. Barnes PJ, Karin M. Nuclear factor-{kappa}B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997;336:1066–71.[Free Full Text]
  9. Yamamoto Y, Gaynor RB. Therapeutic potential of inhibition of the NF-{kappa}B pathway in the treatment of inflammation and cancer. J Clin Invest 2001;107:135–42.[Medline]
  10. Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-{kappa}B. J Clin Invest 2001;107:241–6.[CrossRef][Medline]
  11. Franzoso G, Carlson L, Xing L, et al. Requirement for NF-{kappa}B in osteoclast and B-cell development. Genes Dev 1997;11:3482–96.[Abstract/Free Full Text]
  12. Iotsova V, Caamano J, Loy J, Yang Y, Lewin A, Bravo R. Osteopetrosis in mice lacking NF-{kappa}B1 and NF-{kappa}B2. Nat Med 1997;3:1285–9.[CrossRef][Medline]
  13. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U. Control of I{kappa}B-{alpha} proteolysis by site-specific, signal-induced phosphorylation. Science 1995;267:1485–8.[Abstract/Free Full Text]
  14. Andela VB, Schwarz EM, Puzas JE, O'Keefe RJ, Rosier RN. Tumor metastasis and the reciprocal regulation of prometastatic and antimetastatic factors by nuclear factor {kappa}B. Cancer Res 2000;60:6557–62.[Abstract/Free Full Text]
  15. Kazmi SM, Plante RK, Visconti V, Taylor GR, Zhou L, Lau CY. Suppression of NF{kappa}B activation and NF{kappa}B-dependent gene expression by tepoxalin, a dual inhibitor of cyclooxygenase and 5-lipoxygenase. J Cell Biochem 1995;57:299–310.[CrossRef][Medline]
  16. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NF-{kappa}B/I{kappa}B family: intimate tales of association and dissociation. Genes Dev 1995;9:2723–35.[Free Full Text]
  17. Ginzinger DG. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 2002;30:503–12.[CrossRef][Medline]
  18. Starkie RL, Arkinstall MJ, Koukoulas I, Hawley JA, Febbraio MA. Carbohydrate ingestion attenuates the increase in plasma interleukin-6, but not skeletal muscle interleukin-6 mRNA, during exercise in humans. J Physiol 2001;533:585–91.[Abstract/Free Full Text]
  19. Bouizar Z, Spyratos F, Deytieux S, de Vernejoul MC, Jullienne A. Polymerase chain reaction analysis of parathyroid hormone-related protein gene expression in breast cancer patients and occurrence of bone metastases. Cancer Res 1993;53:5076–8.[Abstract/Free Full Text]
  20. Jordan NJ, Kolios G, Abbot SE, et al. Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells. J Clin Invest 1999;104:1061–9.[Medline]
  21. Dempster DW, Murrills RJ, Horbert WR, Arnett TR. Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts. J Bone Miner Res 1987;2:443–8.[Medline]
  22. Puzas JE, Brand JS. The effect of bone cell stimulatory factors can be measured with thymidine incorporation only under specific conditions. Calcif Tissue Int 1986;39:104–8.[Medline]
  23. Zuscik MJ, Puzas JE, Rosier RN, Gunter KK, Gunter TE. Cyclic-AMP-dependent protein kinase activity is not required by parathyroid hormone to stimulate phosphoinositide signaling in chondrocytes but is required to transduce the hormone's proliferative effect. Arch Biochem Biophys 1994;315:352–61.[CrossRef][Medline]
  24. Wang CY, Mayo MW, Baldwin AS Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-{kappa}B. Science 1996;274:784–7.[Abstract/Free Full Text]
  25. Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998;273:9357–60.[Abstract/Free Full Text]
  26. Cardoso SM, Oliveira CR. Inhibition of NF-{kappa}B renders cells more vulnerable to apoptosis induced by amyloid ß peptides. Free Radic Res 2003;37:967–73.[CrossRef][Medline]
  27. Kang KH, Lee KH, Kim MY, Choi KH. Caspase-3-mediated cleavage of the NF-{kappa}B subunit p65 at the NH2 terminus potentiates naphthoquinone analog-induced apoptosis. J Biol Chem 2001;276:24638–44.[Abstract/Free Full Text]
  28. Lacroix M, Siwek B, Marie PJ, Body JJ. Production and regulation of interleukin-11 by breast cancer cells. Cancer Lett 1998;127:29–35.[CrossRef][Medline]
  29. Sotiriou C, Lacroix M, Lagneaux L, Berchem G, Body JJ. The aspirin metabolite salicylate inhibits breast cancer cells growth and their synthesis of the osteolytic cytokines interleukins-6 and -11. Anticancer Res 1999;19:2997–3006.[Medline]
  30. Libermann TA, Baltimore D. Activation of interleukin-6 gene expression through the NF-{kappa}B transcription factor. Mol Cell Biol 1990;10:2327–34.[Abstract/Free Full Text]
  31. Van Bezooijen RL, Papapoulos SE, Lowik CW. Effect of interleukin-17 on nitric oxide production and osteoclastic bone resorption: is there dependency on nuclear factor-{kappa}B and receptor activator of nuclear factor {kappa}B (RANK)/RANK ligand signaling? Bone 2001;28:378–86.
  32. Bitko V, Velazquez A, Yang L, Yang YC, Barik S. Transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of NF-{kappa}B and is inhibited by sodium salicylate and aspirin. Virology 1997;232:369–78.[CrossRef][Medline]
  33. Yamada H, Iwase S, Mohri M, Kufe D. Involvement of a nuclear factor-{kappa}B-like protein in induction of the macrophage colony-stimulating factor gene by tumor necrosis factor. Blood 1991;78:1988–95.[Abstract/Free Full Text]
  34. Mancino AT, Klimberg VS, Yamamoto M, Manolagas SC, Abe E. Breast cancer increases osteoclastogenesis by secreting M-CSF and upregulating RANKL in stromal cells. J Surg Res 2001;100:18–24.[CrossRef][Medline]
  35. Guise TA, Yin JJ, Taylor SD, et al. Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 1996;98:1544–9.[Medline]
  36. Guillen C, Martinez P, de Gortazar AR, Martinez ME, Esbrit P. Both N- and C-terminal domains of parathyroid hormone-related protein increase interleukin-6 by nuclear factor-{kappa}B activation in osteoblastic cells. J Biol Chem 2002;277:28109–17.[Abstract/Free Full Text]
  37. Storm HH. Survival of adult patients with cancer of soft tissues or bone in Europe. Eur J Cancer 1998;34:2212–7.
  38. Chiu JJ, Sgagias MK, Cowan KH. Interleukin 6 acts as a paracrine growth factor in human mammary carcinoma cell lines. Clin Cancer Res 1996;2:215–21.[Abstract/Free Full Text]
  39. Papanicolaou DA, Wilder RL, Manolagas SC, Chrousos GP. The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 1998;128:127–37.[Abstract/Free Full Text]
  40. Conze D, Weiss L, Regen PS, et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Res 2001;61:8851–8.[Abstract/Free Full Text]
  41. Trikha M, Corringham R, Klein B, Rossi JF. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence. Clin Cancer Res 2003;9:4653–65.[Abstract/Free Full Text]
  42. De La Mata J, Uy HL, Guise TA, et al. Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo. J Clin Invest 1995;95:2846–52.
  43. Thomas RJ, Guise TA, Yin JJ, et al. Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 1999;140:4451–8.[Abstract/Free Full Text]
  44. Benitez-Verguizas J, Esbrit P. Proliferative effect of parathyroid hormone-related protein on the hypercalcemic Walker 256 carcinoma cell line. Biochem Biophys Res Commun 1994;198:1281–9.[CrossRef][Medline]
  45. Kudo O, Sabokbar A, Pocock A, Itonaga I, Fujikawa Y, Athanasou NA. Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone 2003;32:1–7.[Medline]
  46. Jilka RL, Hangoc G, Girasole G, et al. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science 1992;257:88–91.[Abstract/Free Full Text]
  47. Galien R, Garcia T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-{kappa}B site. Nucleic Acids Res 1997;25:2424–9.[Abstract/Free Full Text]
  48. Zecchi-Orlandini S, Formigli L, Tani A, et al. 17ß-Estradiol induces apoptosis in the preosteoclastic FLG 29.1 cell line. Biochem Biophys Res Commun 1999;255:680–5.[CrossRef][Medline]
  49. Hiraga T, Williams PJ, Mundy GR, Yoneda T. The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases. Cancer Res 2001;61:4418–24.[Abstract/Free Full Text]
  50. Sotiriou C, Lacroix M, Lespagnard L, Larsimont D, Paesmans M, Body JJ. Interleukins-6 and -11 expression in primary breast cancer and subsequent development of bone metastases. Cancer Lett 2001;169:87–95.[CrossRef][Medline]
  51. Trepicchio WL, Wang L, Bozza M, Dorner AJ. IL-11 regulates macrophage effector function through the inhibition of nuclear factor-{kappa}B. J Immunol 1997;159:5661–70.[Abstract]
  52. Penolazzi L, Lambertini E, Borgatti M, et al. Decoy oligodeoxynucleotides targeting NF-{kappa}B transcription factors: induction of apoptosis in human primary osteoclasts. Biochem Pharmacol 2003;66:1189–98.[CrossRef][Medline]
  53. Lin MT, Chang CC, Chen ST, et al. Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-{kappa}B-dependent XIAP up-regulation. J Biol Chem 2004;279:24015–23.[Abstract/Free Full Text]
  54. Hideshima T, Chauhan D, Hayashi T, et al. Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades via caspase-dependent downregulation of gp130 in multiple myeloma. Oncogene 2003;22:8386–93.[CrossRef][Medline]
  55. Hideshima T, Chauhan D, Richardson P, et al. NF-{kappa}B as a therapeutic target in multiple myeloma. J Biol Chem 2002;277:16639–47.[Abstract/Free Full Text]
  56. Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist 2003;8:508–13.[Abstract/Free Full Text]
  57. May MJ, D'Acquisto F, Madge LA, Glockner J, Pober JS, Ghosh S. Selective inhibition of NF-{kappa}B activation by a peptide that blocks the interaction of NEMO with the I{kappa}B kinase complex. Science 2000;289:1550–4.[Abstract/Free Full Text]
  58. Aggarwal BB. Nuclear factor-{kappa}B: the enemy within. Cancer Cell 2004;6:203–8.[CrossRef][Medline]
  59. Jimi E, Aoki K, Saito H, et al. Selective inhibition of NF-{kappa}B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo. Nat Med 2004;10:617–24.[CrossRef][Medline]




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