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Tumor Biology |
B in Breast Cancer1
Departments of Biochemistry [R. R-M., A. M. T., G. E. S.], Pathology and Laboratory Medicine [E. L-B.], and Medicine [D. C. S.] and the Program in Research on Women’s Health [R. R-M., E. L-B., D. C. S., A. M. T., G. E. S.], Boston University School of Medicine, Boston, Massachusetts 02118-2394; and Celgene Signal Research Division, San Diego, California 92121 [F. M.]
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ABSTRACT |
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B (NF-B)/Rel transcription factors regulate genes that control cell proliferation, survival, and transformation. In normal breast epithelial cells, NF-B/Rel proteins are mainly sequestered in the cytoplasm bound to one of the specific inhibitory IB proteins, whereas in breast cancers they are activated aberrantly. Human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples express constitutively high levels of nuclear NF-B/Rel. To begin to understand the mechanism of this aberrant NF-B/Rel expression, in this study we measured the activity of the major kinases implicated in regulation of IB stability, namely IKK
, IKKß, and protein kinase, CK2 (formerly casein kinase II). Hs578T, D3-1, and BP-1 breast cancer cell lines displayed higher levels of IKK, IKKß, and CK2 activity than untransformed MCF-10F mammary epithelial cells. Inhibition of IKK activity upon expression of dominant negative kinases or of CK2 activity by treatment with selective inhibitors decreased NF-B/Rel activity in breast cancer cells. Inactivation of the IB kinase complex in Hs578T cells via expression of a dominant negative IKK
/NF-B essential modulator/IKK-associated protein 1 reduced soft agar colony growth. Thus, the aberrant expression of CK2 or IKK kinases promotes increased nuclear levels of NF-B/Rel and transformation of breast cancer cells. Furthermore, primary human breast cancer specimens that displayed aberrant constitutive expression of NF-B/Rel were found to exhibit increased CK2 and/or IKK kinase activity. These observations suggest these kinases play a similar role in an intracellular signaling pathway that leads to the elevated NF-B/Rel levels seen in primary human mammary tumors and, therefore, represent potential therapeutic targets in the treatment of patients with breast cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B3
/Rel is a family of dimeric transcription factors distinguished by the presence of a 300-amino acid region, termed the Rel homology region, which determines much of its function (1)
. Classical NF-B is a heterodimer composed of a RelA (p65) and p50 subunit. In most cells, other than B lymphocytes, NF-B/Rel proteins are sequestered in the cytoplasm bound to the specific IB inhibitory proteins, of which IB- is the paradigm. Although the v-rel gene, carried by the highly oncogenic avian reticuloendotheliosis virus strain T, is able to cause tumors in birds, the role of NF-B in mammalian cancers was less clear for many years (2)
. Several oncogenic mammalian viruses were shown to activate NF-B. For example, the product of the tax gene of the HTLV-1 virus activates NF-B (3)
, which we showed mediates transactivation of the c-myc promoter (4)
. Recently, we and others have demonstrated a role for NF-B/Rel factors in breast cancer (5
, 6)
. High levels of nuclear NF-B/Rel were found in human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples. In contrast, untransformed breast epithelial cells and normal rat mammary glands contained low basal levels (5
, 6) .
Several laboratories have found that increased NF-B expression in tumor cells correlates with decreased stability of IB proteins. For example, we recently showed that elevated levels of NF-B in the D3-1 and BP-1 cell lines, derived by in vitro transformation of MCF-10F breast epithelial cells by 7,12-dimethylbenz(a)anthracene and benzo[a]pyrene, respectively, correlated with a decrease in the half-life of IB- protein (7)
. Much progress has been made in elucidating the kinases that regulate IB- stability. These appear to function via phosphorylation of NH2-terminal or COOH-terminal sites of IB-. A variety of agents that induce NF-B/Rel have been found to mediate activation of NF-B via phosphorylation of IB on two NH2-terminal serine residues in a large multi-subunit complex (8)
. The IB kinase complex consists of two IB kinases, IKK or IKK-1 and IKKß or IKK-2 (8)
. In addition, there is a Mr 48,000 essential component, alternatively termed NEMO, IKK, or IKKAP1 (9, 10, 11)
. The IKK/NEMO/IKKAP1 protein is essential for function of the IB kinase complex, and mutants can completely inhibit all of the IKK kinase function (9, 10, 11)
. The IKK and IKKß protein serine kinases contain a leucine zipper and a helix-loop-helix motif in the COOH-terminal region and a kinase domain in the NH2-terminal region (8)
. Activation of the IB kinase complex is mediated via phosphorylation of either IKK or IKKß (12
, 13)
. IB- is then recruited into the IB kinase complex, where it is phosphorylated by the functional IKK/IKKß heterodimer at serine residues at positions 32 and 36. The NH2-terminal IKK phosphorylation sites have been recently shown to play a role in the signal-induced degradation of both free and NF-B-bound forms of IB- (14)
. IB- phosphorylation is followed by ubiquitination and rapid degradation, allowing for migration of the released NF-B to the nucleus (15)
.
In addition to the NH2-terminal residues, it has been shown that phosphorylation of serines and/or threonines within the COOH-terminal PEST domain of IB- affects the stability of the protein (16, 17, 18)
. The kinase responsible for this phosphorylation has been identified as the serine/threonine protein kinase CK2 (CK2, formerly casein kinase II). CK2 phosphorylates IB- preferentially at Ser-283, Ser-288, T-291, and Ser-293 within the PEST domain (18
, 19)
. CK2 is a ubiquitously expressed and constitutively active kinase that exists in cells as a heterotetrameric protein containing two catalytic (/, /', or '/') and two regulatory (ß/ß) subunits (20
, 21)
. CK2-mediated phosphorylation of IB- in the PEST domain has been implicated in the basal and signal-dependent turnover of free and NF-B-bound IB- (14
, 16
, 18
, 19)
. The mechanisms of the basal degradation of IB- are not fully understood, although it has been suggested that it involves IB complex phosphorylation, ubiquitination, and degradation by the 26S proteasome (14)
or, alternatively, a calpain-mediated mechanism (22)
. These findings have implicated CK2 in control of intrinsic IB- stability and, thereby, in constitutive activation of NF-B, although this remains to be proven directly.
To begin to evaluate the mechanism of aberrant activation of NF-B in breast cancer, in this study we have assessed the activity of the IKK and IKKß components of the IB kinase complex and of CK2 in human breast cancer cell lines and primary breast cancer specimens. We report that breast cancer cells in culture display elevated IKK and CK2 kinase activity. Inhibition of these activities reduces NF-B activity. Furthermore, multiple primary breast cancer specimens that display aberrant constitutive expression of NF-B exhibit increases in either IKK or CK2 kinase activity. We conclude that aberrant activation of IKK or CK2 leads to elevated nuclear NF-B activity, which in turn can result in enhanced survival and transformed phenotype of breast cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection Conditions.
For transient transfection, cells were incubated for 12 to 16 h at 37°C with a solution of DNA and Fugene reagent according to the manufacturer’s directions (Boehringer Mannheim). To evaluate transcriptional activity, cells were transfected in duplicate with wt (E8-CAT) and mutant (dmE8-CAT) NF-B element-thymidine kinase promoter-CAT reporter vectors, containing two copies of either the wt or mutant NF-B element from upstream of the c-myc promoter (4)
. CAT assays were performed as described previously (5)
. Alternatively, cells were transfected in triplicate with NF-B element-luciferase reporter vector, driven by 3 NF-B elements from upstream of the MHC class I promoter, kindly provided by Dr. A. Chan (Mt. Sinai School of Medicine, New York, NY; Ref. 24
). Luciferase assays were performed as described previously (25)
. The CMV promoter ß-gal reporter vector pON407, in which the five putative NF-B sites within the CMV promoter have been removed (26)
, was used to normalize transfection efficiency as described previously (27)
. SD was obtained using the Student t test. The pRC-ßactin-IKKSS/AA vector, which expresses a phosphorylation-defective mutant IKKSS/AA that functions as a dominant negative version of IKK, and the parental pRC-ßactin vector were as described (13)
. The pRC-ßactin-IKKSS/AA vector insert was subcloned into the pcDNA3 vector yielding pcDNA3-IKKSS/AA. The plasmids pCMV-IKKßSS/AA and pCMV-IKKßSS/EE, allowing expression of a dominant negative mutant flag-tagged IKKß, and a constitutively active flag-tagged IKKß, respectively, have been described previously (13)
. The vector directing expression of dominant negative IKK/NEMO/IKKAP1 (9)
was kindly provided by Drs. D. Rothwarf and M. Karin (University of California San Diego, La Jolla, CA).
Human Breast Cancer Specimen Analysis.
Primary human breast cancer tissue specimens were obtained from patients undergoing surgery for treatment of breast cancer with approval of the Institutional Review Board of Boston Medical Center. Tumors were processed for steroid receptor analysis, and any remaining tissue was considered discarded material and used for subsequent analysis of NF-B, and IKK and CK2 kinases. Tissues were stored frozen at -75°C until samples were processed for nuclear and cytoplasmic protein fractionation. Samples were pulverized on dry ice using a Bessman Tissue Pulverizer (Spectrum). Frozen tissue powder was homogenized (0.5 g/ml) in TEGT/MO buffer [50 mM Tris/HCl, 1 mM EDTA, 10% (v/v) glycerol, 10 mM monothioglycerol, and 10 mM sodium molybdate (pH 7.4) containing 0.02% sodium azide] using a Polytron. After the initial burst, proteolytic inhibitors were added to a final concentration as follows: 0.5 mM PMSF, 1 µg/ml leupeptin, 100 µg/ml aprotinin, 10 µg/ml pepstatin, and 100 µg/ml bacitracin. Homogenates were centrifuged for 10 min at 3000 rpm at 2°C to isolate crude nuclei. The nuclear pellet was washed 3x with buffer, and nuclear proteins were extracted as described previously (5)
. The supernatant was centrifuged at 100,000 x g, and the clear cytosolic extract was removed and stored frozen for analysis.
EMSA.
The sequence of the wt URE NF-B-containing oligonucleotide from the c-myc gene (4)
is as follows: wt, 5'-GATCCAAGTCCGGGTTTTCCCCAACC-3'. The core element is underlined. The mutant URE has two G to C-bp conversions, indicated in bold, which block NF-B/Rel binding: 5'-GATCCAAGTCCGCCTTTTCCCCAACC-3'. The sequence of the Sp1 oligonucleotide is 5'-ATTCGATCGGGGCGGGGCGACC-3'. The sequences of the PU.1- and TCF-1-containing oligonucleotides are as follows: PU.1, 5'-GATCTACTTCTGCTTTTG-3'; and TCF-1, 5'-GGGAGACTGAGAACAAAGCGCTCTCACAC-3' (28)
. Nuclear extracts from breast tissue samples or breast cell lines were prepared, and samples (2.5–5 µg) were subjected to EMSA as described (5)
. For antibody supershift analysis, the binding reaction was first performed in the absence of the probe, the appropriate antibody was added, and the mixture was incubated for 16 h at 4°C. The probe was then added. The reaction was incubated an additional 30 min at 25°C, and the complexes resolved by gel electrophoresis, as above. Antibodies used included: anti-RelA subunit, sc-372; anti-p50 subunit, sc-114; anti-p52 subunit, sc-7386; and anti-c-Rel subunit, sc-71 (all of these were from Santa Cruz Biotechnology, Santa Cruz, CA). Where indicated, either 250 ng of IB--GST fusion protein, GST alone, excess unlabeled wt, or mutant oligonucleotide was added to the binding reaction just before addition of the probe. Data were quantified by densitometry using a Molecular Dynamics densitometer.
Immunoblotting.
Samples were separated by electrophoresis in polyacrylamide-SDS gels, transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA), and subjected to immunoblotting, as described previously (27)
. Antibodies specific for IKK (M-280), IKKß (H-470), and FLAG-pCruz Octa expression vector-encoded fusion proteins (D-8) were purchased from Santa Cruz Biotechnology. Phospho-IB- (Ser-32)-specific antibody was from New England Biolabs (Beverly, MA). The rabbit polyclonal antibody specific for the CK2 subunit of CK2 was from Stressgen (Victoria, British Columbia, Canada). A monoclonal antibody specific for ß-actin (AC-15) was purchased from Sigma Chemical Co.
IKK Kinase Assay.
To prepare WCEs, cells were washed with PBS, resuspended in cold kinase assay lysis buffer [20 mM Tris (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na3VO4, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, and 0.25% NP40], and lysed by sonication. Debris was removed by centrifugation, and extracts precleared with protein A-Sepharose beads (Amersham Pharmacia Biotech AB) for 1 h at 4°C. The IKK complexes were isolated by immunoprecipitation from a 500-µl reaction mixture of PD buffer [40 mM Tris (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na3VO4, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, and 0.1% NP40) containing 150 µg of cytoplasmic proteins and 1 µg of antibody against either IKK (M-280), IKKß (H-470), or Flag-tag (D-8). After washing, one-third of the immunoprecipitate was subjected to a kinase assay at 30°C for 45 min in kinase buffer C [20 mM HEPES (pH 7.7), 2 mM MgCl2, 10 µM ATP, 3 µCi of [-32P]ATP, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM Na3VO4, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM DTT] containing 200 ng of wt GST-IB- fusion protein (GST-wtIB-) as substrate (13)
. Alternatively, a truncated (amino acid 1-54) mutant IB--GST, in which Ser-32 and Ser-36 were replaced by alanines (A32,36mut GST-IB-), was used to assess kinase specificity. (Concentrations of the GST fusion protein preparations were monitored in Coomassie Blue-stained SDS-polyacrylamide gels by comparison to BSA standards.) The kinase reaction was stopped by the addition of 2 x SDS-PAGE sample buffer, subjected to SDS-PAGE analysis, and visualized by autoradiography. The remaining fraction was subjected to immunoblot analysis, as described above. SD was obtained using the Student t test.
CK2 Kinase Assay.
For evaluation of IB-phosphorylation directed by CK2, 10- to 20-µg WCEs prepared using kinase assay lysis buffer were diluted to 10-µl final volume with the same buffer. After the addition of 15 µl of buffer D [100 mM Tris (pH 8.0), 100 mM NaCl, 50 mM KCl, 20 mM MgCl2, 100 µM Na3VO4, and 10 µCi of [-32P]GTP], reactions were incubated at 30°C for 30 min in the presence of 200 ng GST-wtIB- as substrate. Alternatively, GST-
2IB-, with a deletion of amino acids 269–317 in the COOH-terminal PEST domain of IB- or GST-3CIB- with three point mutations at S283A, T291A, and T299A (17)
, kindly provided by Dr. J. Hiscott (Institut Lady Davis de Recherches Medicales, Montreal, Quebec, Canada), were used as substrates. Where indicated, 20–80 µM apigenin, 1–25 µg/ml emodin, or 0.58–1.46 mM CK2-specific peptide substrate RRREEETEEE (Sigma Genosys Inc.) was added to the kinase reaction. Alternatively, recombinant CK2 was used (New England Biolabs). The kinase reaction was stopped, and the products were processed as above.
Focus Formation Assay.
Hs578T cells were transfected in P100 dishes using Fugene, as described above. After 16 h, cells were plated at 1 x 104/ml in top plugs consisting of complete medium and 0.8% SeaPlaque agarose (FMC Bioproducts, Rockland, ME). Plates were subsequently incubated for 18 days in humidified incubator at 37°C. Cells were stained with 0.5 ml of 0.0005% crystal violet, and colonies were counted using a dissecting microscope.
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RESULTS |
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or transfected with plasmids encoding flag-tagged dominant negative mutated IKKßSS/AA or constitutively active mutant IKKßSS/EE. WCEs were prepared from cultures of the four cell lines at 70% confluence, and samples containing equal amounts of proteins were immunoprecipitated with an IKKß kinase specific antibody. Alternatively, with the transfected cell extracts, a flag antibody was used. One-third of the immunoprecipitated material was used in in vitro phosphorylation assays with full length GST-wtIB- as substrate, and protein was labeled with [-32P]ATP (Fig. 1A
, top panel). The remainder was subjected to immunoblotting for IKKß protein or flag epitope, as indicated (Fig. 1A
, bottom panel). Consistent with prior studies (13)
, ectopic expression of the dominant negative IKKß SS/AA resulted in reduced IKKß kinase activity as judged by decreased IB- phosphorylation when compared with expression of IKKßSS/EE (Fig. 1A)
. This decrease occurred despite the higher total levels of IKKß protein (Fig. 1A
, bottom panel), consistent with a dominant negative effect. The IKKß kinase activity in Hs578T was modestly stimulated by treatment with TNF- for 10 min (Fig. 1A)
. IKK activity was specific for Ser-32 and Ser-36 of IB-, because replacement with alanine at both sites in the GST-IB- substrate eliminated phosphorylation (data not shown).
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B- kinase activity directed by IKKß was increased in the tumor cell lines compared with MCF-10F (Fig. 1A)
. When normalized for level of expression of IKKß protein detected in the immunoblot (Fig. 1A
, bottom panel), compared with MCF-10F cells, values obtained in this and a duplicate experiment for the Hs578T, D3-1, and BP-1 tumor cells were 1.6- and 1.4-fold, 2.6- and 2.0-fold, and 1.4- and 1.4-fold, respectively. The increases in all of the cells were statistically significant (P < 0.05). A similar analysis was next performed for IKK. IKK kinase activity was significantly lower in the untransformed MCF-10F cells compared with all of the three breast cancer cell lines (Fig. 1B)
. Immunoprecipitation of IKK brought down approximately equivalent amounts of endogenous protein from the four lines (Fig. 1B)
. Compared with the MCF-10F cells, Hs578T, D3-1, and BP-1 tumor cells displayed higher IKK activities in this and a duplicate experiment of 1.7- and 1.6-fold, 2.0- and 4.2-fold, and 1.9- and 5.0-fold, respectively. Thus, breast tumor cell lines displayed increased IKK and IKKß kinase activity compared with untransformed human mammary epithelial cells.
Kinase Inactive IKK or IKKß Inhibits NF-B Activity in Hs578T Human Breast Cancer Cells.
To assess the role of active IKK kinases in the induction of NF-B seen in the breast tumor cells (5
, 7)
, the effects of inhibition of IKKs on NF-B activity and binding were evaluated. IKK activity was modulated by transfection with plasmids encoding kinase-inactive mutants IKKSS/AA and IKKßSS/AA. The transcriptional activity of NF-B was evaluated by cotransfection with a reporter plasmid driven by wt (E8-CAT) or mutated (negative control, dmE8-CAT) NF-B-binding elements. In Hs578T cells, functional activation of NF-B was reduced 1.9-fold (P < 0.05) upon transfection with IKKSS/AA and 2.2-fold (P < 0.05) by IKKßSS/AA, compared with the cognate parental vectors (pcDNA3 and pCMV-Neo, respectively; Fig. 2
). This observation is consistent with the reduction of IKK kinase activity in transfected cells seen above (Fig. 1A)
. Thus, these findings suggest that IB- turnover is mediated by both active IKK and IKKß.
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B Activity in D3-1 and BP-1 Human Breast Cancer Cells.B transcriptional activity and selected the dominant negative IKKß. D3-1, BP-1, and parental MCF-10F cells were cotransfected with the kinase inactive IKKßSS/AA or parental pCMV-Neo vectors and the wt and dm E8-CAT reporter constructs, as above. In the presence of the parental pCMV-Neo vector, the two transformed lines displayed higher levels of NF-B activity compared with the MCF-10F cells, as expected (Fig. 3)
. The increase in activity was greater with the D3-1 than the BP-1 line. NF-B activity was greatly reduced in D3-1 and BP-1 cells upon transfection with IKKßSS/AA, whereas no significant change was observed in the MCF-10F cells (Fig. 3)
. Thus, inhibition of IKKß activity reduces NF-B activity in both the D3-1- and BP-1-transformed breast cancer cell lines.
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B- in the PEST domain has been implicated in the basal turnover of IB- in immune cells (14
, 18
, 19)
. Thus, to explore the potential role of CK2 on NF-B levels in breast cancer, a CK2 kinase assay was developed using IB- as a specific substrate. GST fusion proteins of either wt IB- (GST-wtIB-) or mutant 269–317 IB- (GST-2 IB-), which has a deletion of amino acids 269–317 in the COOH-terminal PEST domain (17)
, were used as positive and negative controls, respectively. Recombinant CK2 enzyme was assayed in the presence of [-32P]GTP instead of [-32P]ATP, because both ATP and GTP can be used as phosphate donors by CK2 whereas the IKKs can only use ATP. Recombinant CK2 phosphorylated GST-wtIB- to a much greater extent than GST-2 IB- (Fig. 4A)
. Similar results were obtained with an IB- mutant with three point mutations (S283A, T291A, and T299A, GST-3C IB-) that had been shown previously (17)
to reduce phosphoryl group transfer by CK2 in vitro (data not shown).
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B- fusion proteins as substrates in in vitro CK2 phosphorylation assays. Kinase assays demonstrated strong preferential phosphorylation of GST-wtIB- compared with GST-2 IB- or GST-3C IB- (Fig. 4B)
. To verify the involvement of CK2, two selective pharmacological inhibitors of CK2, apigenin or emodin, were used either with cells in culture or with cell extracts (29
, 30)
. Apigenin is a plant flavonoid that has been shown to be more effective in vitro, although it can also inhibit intracellular CK2 activity (31)
. Hs578T cells were incubated for 2 h in the presence of 20, 40, or 80 µM apigenin or a volume of carrier DMSO equivalent to 80 µM (Fig. 4C
, left panel). A decrease of 1.5–2-fold in phosphorylation of GST-wtIB- was observed with the extracts. Alternatively, WCEs were treated with similar concentrations of apigenin (Fig. 4C
, right panel). A dose-dependent inhibition was noted that was more potent than in cells, as seen previously. The natural plant anthraquinone derivative, emodin, has been shown to inhibit CK2 activity by competitively binding to its ATP-binding site (30)
. The addition of emodin similarly caused a dose-dependent decrease in CK2 activity both in Hs578T cells or in extracts with almost complete inhibition at 25 µg/ml (Fig. 4C)
. Thus, apigenin and emodin reduced GST-wtIB- kinase activity when added to extracts or to cells in culture. Lastly, to confirm the role of CK2 in the observed phosphorylation, a CK2-specific peptide substrate RRREEETEEE was added as competitor (Fig. 4D)
. The addition of the peptide substrate RRREEETEEE effectively reduced phosphorylation of the GST-wtIB- substrate. Thus, the assay for CK2 activity using GST-IB- as a substrate appears to be specific. Furthermore, the results indicate that Hs578T cells display CK2 IB- kinase activity.
Next, we compared the relative CK2 activity in the untransformed MCF-10F mammary epithelial parental cells with levels in the carcinogen-transformed lines BP-1 and D3-1 and the Hs578T breast cancer cells. The CK2 kinase activity was clearly higher in all of the tumor cells compared with the MCF-10F line (Fig. 5A)
. Compared with MCF-10F cells, the relative increase in CK2 kinase activity was 2.2-, 2.5-, and 2.2-fold in Hs578T, BP-1, and D3-1 cells, respectively. Increased CK2 activity is most often attributable to increased levels of CK2 protein expression. To assess the relative levels of CK2 protein in the cell lines, immunoblot analysis was performed for the CK2 subunit of CK2 using the WCEs (Fig. 5B)
. The Hs578T, D3-1, and BP-1 cells expressed higher levels of CK2 than MCF-10F cells expressed. Equal loading was confirmed by analysis for ß-actin expression. (A slightly lower ß-actin level was routinely detected in the Hs578T cells; data not shown.) The results from this and a duplicate experiment were quantified. Compared with the MCF-10F cells, an approximate 2.2±0.3-fold, 1.6±0.1-fold, and 1.6±0.3-fold increase in the level of CK2 protein was found in the Hs578T-, BP-1-, and D3-1-transformed lines, respectively. Thus, IB- kinase activity directed by CK2 is increased in these breast tumor cell lines, and this increase can be explained by an increase in levels of CK2 protein.
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B Activity in Hs578T Cells.B in breast cancer cell lines, we chose the CK2-selective inhibitor apigenin. Hs578T cells were incubated for 2.5 h in the presence of 20, 60, or 100 µM apigenin or with the concentration of carrier DMSO equivalent to 100 µM (-apigenin). Nuclear extracts were prepared and analyzed for NF-B-binding activity by EMSA (Fig. 6A)
. Two major complexes were seen in the untreated Hs578T cells. Successful competition with wt but not mutant oligonucleotide and inhibition upon addition of IB- confirmed the specificity of the NF-B binding (Fig. 6B)
. Antibody supershift analysis indicated that complex 2 represents p50/RelA, and complex 1 is a homodimer of p50, consistent with our previous observations with this line (7)
. Apigenin reduced the formation of both bands 1 and 2 in a dose-dependent fashion. Little change was noted at 20 µM, whereas a substantial decline in the upper p50/RelA complex was observed at 60 µM. At a concentration of 100 µM, reduced formation of both complexes was noted. In contrast, little effect was seen on the binding of Sp1 (Fig. 6A)
. When these results were scanned and normalized to the Sp1-binding activity, a 70% decrease in classical NF-B was noted with 60 µM apigenin. At 100 µM apigenin, decreases of 95% and 80% in p50/RelA and p50 homodimers, respectively, were observed. Next, we verified that the NF-B transcriptional activity is decreased by inhibition of CK2 using either apigenin or emodin. After transfection with vectors expressing an NF-B element luciferase reporter plus SV40-ß-gal, for normalization, cells were incubated overnight and then treated with either 60 µM apigenin or 25 µg/ml emodin. The treatments were performed for 6 h to ensure sufficient time for the transcriptional activity to decline and for decay of the luciferase protein. Apigenin treatment resulted in an approximate 43% decline in NF-B activity (39.2 ± 3.8% and 45.6 ± 6.1% in two separate experiments). Emodin treatment caused an approximate 30% drop in NF-B activity (35.9 ± 3.6 and 24.9 ± 1.8% in two separate experiments). Taken together, these results suggest that inhibition of CK2 leads to a reduction of nuclear NF-B-binding activity in Hs578T breast cancer cells.
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B luciferase reporter activity was measured in Hs578T cells cotransfected with increasing doses of the dominant negative IKKß expression vector and treated for 6 h with 60 µM apigenin. A dose-dependent drop in NF-B activity was seen with the dominant negative IKKß expression vector, which reached a plateau at 40% inhibition with a 1.5-µg expression vector DNA (Fig. 6C)
. Furthermore, addition of apigenin increased the extent of inhibition (by 37 to 48%) at every dose of dominant negative IKKß expression vector used; e.g., NF-B activity was inhibited a total of 71% in cells treated with apigenin and 1.5-µg dominant negative IKKß expression vector DNA. These results indicate that the effects of IKK and CK2 on NF-B are additive.
Inhibition of NF-B Reduces Anchorage-independent Growth of Hs578T Human Breast Cancer Cells.
Next, we assessed the role of NF-B in transformed phenotype using anchorage-independent growth of Hs578T breast cancer cells. Because the dominant negative C IKKAP1 construct, encoding a COOH-terminal deletion-mutant of IKK/NEMO/IKKAP1, has been shown to inhibit activities of both IKK and IKKß (8, 9, 10)
, it was selected for analysis here. Using transfection into Hs578T cells, we first confirmed the ability of expression of the C IKKAP1 vector to decrease classical NF-B-binding levels by 70% (data not shown). Cultures of Hs578T cells were then transfected in triplicate with parental pcDNA3EE vector or with 3- or 10-µg C IKKAP1 vector and assessed for growth in soft agar. The colonies/high power field were as follows: for pcDNA3EE, 132 ± 34; 3-µg C IKKAP1, 84 ± 16; and 10-µg C IKKAP1, 57 ± 11. As expected, transfection with a vector expressing full-length IKK/NEMO/IKKAP1 had no effect on NF-B activity or colony formation (data not shown). Thus, a dose-dependent reduction in colony numbers was seen with transfection of the C IKKAP1 vector compared with the parental vector. Taken together, these results indicate that breast cancer cells display a substantial increase in activity of kinases that induce NF-B activity and that this induction can promote the transformed phenotype, as measured by anchorage-independent growth of these cells.
Primary Breast Cancers, Displaying Increased NF-B-Binding Activity, Have Either Increased IKK or CK2 Kinase Activities.
Next, we asked whether these three IB kinases are activated in primary human breast cancer specimens and whether NF-B induction correlates with kinase activation. Two sets of human primary breast tumors were studied. The pathological characteristics and steroid receptor data that were available from these patient cases (Table 1
; and data not shown) show that 9 of 16 were estrogen receptor (ER) positive, 2 of 16 were ER intermediate, and 5 of 16 were ER negative, whereas 14 of 16 were progesterone receptor (PR) positive. Nuclear extracts were prepared from frozen breast tumors and used for NF-B binding by EMSA. Because potential contamination with hematopoietic cells could significantly affect the analysis, our strategy was to also test for such contamination using a binding assay for PU.1 and TCF-1, which are present in B lymphocytes, neutrophils, mast or myeloid cells, and T cells. Fig. 7
and Fig. 8
show data obtained from the analysis of the first group of six patient samples, and Table 1
presents the findings for every PU.1- and TCF-1-negative sample from the two sets analyzed.
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, left panel). These findings were confirmed using immunoblot analysis (data not shown). Compared with Jurkat T cells, only low and comparable levels of binding to TCF-1 was detected in the tumor extracts (data not shown). Two of the samples (6731 and 6712) that tested negative for PU.1 were found to express relatively high levels of NF-B binding (Fig. 7A
, right panel). The gels were subjected to densitometry, and the results of the analysis of all of the samples that tested negative for PU.1 and TCF-1 are presented in Table 1
.
To identify which NF-B subunits are present in the nuclear extracts, immunoblot analysis was performed using antibodies specific for RelA (p65), c-Rel, p50, and p52 (Fig. 7B)
. Both samples 6712 and 6731 displayed nuclear p65 proteins, as did 6679. When c-Rel was assessed, only 6731 displayed detectable levels of expression. The p50 or p52 subunits were detected in both patient samples. (The p50 in sample 6712 had a slightly faster mobility than p50 in sample 6731.) Sample 6731 displayed somewhat more expression of p50 (band 1) than p52, whereas sample 6712 expressed more p52 than p50 (Fig. 7B)
. Thus, sample 6731 contains transactivating subunits RelA and c-Rel, whereas 6712 contains RelA. The data for expression of RelA and c-Rel in the two sets of patient samples are summarized in Table 1
. Essentially, all of the tumors tested positive for the p50 or p52 subunit (data not shown). Of the 10 PU.1/TCF-1-negative breast cancer samples characterized, 3 displayed only low levels of NF-B binding, whereas 1 had a minimally elevated level, and 6 showed substantially elevated levels of NF-B binding.
Cytoplasmic extracts from tumors of these patients were then tested for CK2 IB- kinase activity using GST-wtIB- as substrate (Fig. 8A
; and data not shown). Two of the six primary tumor samples from the first set of patients showed elevated CK2 IB- kinase activity, patients 6731 and 6712 (Fig. 8A)
. As a negative control for the kinase assay, two samples were tested with GST-2 IB- as substrate. The extracts failed to phosphorylate this IB- protein containing a deletion of the PEST domain sequences. Lastly, to confirm the specificity of the assay for CK2, the selective inhibitor apigenin was added to the reaction with wtIB- as substrate. Apigenin dramatically reduced IB- phosphorylation with samples 6680 and 6731, confirming the reactions were mediated by CK2. The results from the two sets of patients were quantified by densitometry; the data for the PU.1/TCF-1-negative samples are presented in Table 1
. Three of the specimens displayed low levels of CK2 activity (between 231 and 415 densitometry units), whereas the remaining specimens had either modestly increased (758 to 1106 densitometry units) or substantially elevated levels (1809 to 6143 densitometry units; Table 1
). The three specimens with low CK2 also displayed low or minimally elevated NF-B binding. Six samples displayed elevated levels of CK2 and NF-B binding, whereas only one specimen (6885) showed high IB- CK2 kinase activity without detectable high NF-B nuclear activity.
Activities of the IKK and IKKß kinases were evaluated after immunoprecipitation with their specific antibodies, and the data are presented in Fig. 8, B and C
, respectively. The results for PU.1/TCF-1-negative tumors are shown in Table 1
. Tumor samples 6731 and 8364 exhibited modestly increased levels of IB- kinase activity directed by IKK and increased NF-B-binding activity. Consistent with data obtained with tumor cell lines, increased IKK activity in primary tumors did not seem to be attributable to increased levels of protein expression (Fig. 8B
; and data not shown). In the analysis of IKKß kinase activity, most of the samples yielded values between approximately 100 and 300 densitometry units; however, cytosolic extracts from three specimens (8361, 8364, and 8385) displayed greatly elevated IKKß activity (1164, 1395, and 980 densitometry units, respectively). All of these three specimens displayed high NF-B-binding activity. No phosphorylation was detected using a mutant of IB- at Ser-32 and Ser-36 as substrate, confirming the specificity of the kinase assay (data not shown). Thus breast cancers display activation of CK2, IKK, IKKß, or various combinations of these kinases, which correlate and are, therefore, likely responsible for the aberrant NF-B activation in these primary tumors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IB kinase activities. Inhibition of any of these activities in the breast cancer cells resulted in reduced functional NF-B/Rel. Reduced NF-B levels, via expression of a dominant negative IKK/NEMO/IKKAP1 to repress the IB kinase complex, resulted in loss of soft agar colony formation ability. Previously, we and others (5
, 6)
demonstrated that primary breast cancer samples from patients or from a carcinogen-induced rodent model, as well as cell lines in culture, are typified by aberrant activation of NF-B. In contrast, only low levels of NF-B/Rel factors are present in the nuclei of normal breast epithelial cells (5
, 7) . These results, which were extended to additional patients here, have been confirmed recently by other laboratories (6
, 32, 33, 34)
. For example, Cogswell et al. (34)
showed that breast tumors compared with adjacent normal tissues display increased mRNA and protein expression of the NF-B subunits p50, p52, c-Rel, as well as Bcl-3, which could enhance p50 activity. As discussed below, the mechanisms mediating activation of NF-B/Rel factors, which regulate expression of genes that control cell proliferation, survival, and transformation, are under investigation in many laboratories. In this study for the first time, aberrant induction of IKK, IKKß, and CK2, key kinases that regulate IB stability and NF-B/Rel activation, is demonstrated in breast cancer.
Aberrant nuclear NF-B activity has been reported in multiple cancers, including the human cutaneous T-cell lymphoma HuT-78, primary adult T-cell leukemias, acute lymphoblastic leukemia, and pancreatic adenocarcinomas (2
, 35, 36, 37, 38)
. In hematopoietic and solid tumors, mutations or modulation in the expression of the IB- protein, as well as amplification, overexpression, or gene rearrangement of the nfkb1, nfkb2, bcl3, c-rel, or relA genes have been noted (2)
. Mutations of IB- have been reported that decrease affinity for NF-B in Hodgkin lymphomas (39)
, whereas other studies (7
, 40
, 41)
have correlated increased NF-B expression in tumor cells of various types with decreased stability of IB proteins. Furthermore, products of several oncogenes have been found to activate NF-B. In breast cancer, overexpression of the HER-2/Neu receptor, which is found in approximately 30% of patients, or of the epidermal growth factor receptor was found to induce p50/RelA complexes specifically (42, 43, 44)
. Consistent with these findings, in our two studies, nuclear RelA was observed in several primary human breast tumor samples. In contrast, only low levels of RelA were seen by Cogswell et al. (34)
, which likely reflect differences in the patient populations studied. Oncogenic Raf and Ras proteins (27
, 45
, 46)
and the HTLV-1 tax protein (4
, 47)
have also been found to induce NF-B activity in multiple cell types. The details of pathways responsible for the increased NF-B in tumor cells are only beginning to be resolved.
IKK kinase has been implicated in the persistent NF-B nuclear activity in HTLV1-infected T lymphocytes (48)
, Hodgkin lymphomas (40)
, and melanoma cells (41)
. Recently, we demonstrated that activation of NF-B by oncogenic Raf is mediated by a Mek to IKKß pathway (49)
. Ras has been found to induce NF-B via phosphatidylinositol 3'-kinase to IKK and also via the Raf pathway (49)
. In this study, all of the breast cancer cell lines studied displayed increased activity of both IKK and IKKß. Furthermore, tumors from three patients were shown to have very high levels of IKKß activity, and tumors from two patients displayed modestly elevated levels of IKK. These latter findings suggest that differential pathways leading to increased activation of these kinases occurred in the various tumor specimens. It should be noted, however, that both IKK and IKKß can form heterodimers or homodimers that can phosphorylate IB- (13)
. Unfortunately, the amino acids comprising the activation loop serines are nearly identical for IKK and IKKß, making it difficult to develop antibodies specific for the active forms of IKK or IKKß kinases. In future studies, the use of two-dimensional gel electrophoresis will be explored to determine whether the increase in activity results from activation of IKK or IKKß or from both kinases.
Elevated CK2 levels have been reported in multiple human tumors, including squamous cell carcinoma, colorectal tumors, and leukemias (50, 51, 52)
. In one study, immunohistochemistry suggested that human breast tumors also had elevated levels of CK2 protein (53)
. In this study, we find that high levels of CK2 activity were found in many of the breast tumor specimens and in all of the cancer cell lines studied. Enforced CK2 expression in T cells within mice was sufficient to induce T lymphomas (54)
. More recently, we have found that transgenic overexpression of CK2 in the mammary gland leads to breast tumors in mice (55)
. Consistent with the work presented in this study, cells derived from these mammary tumors were found to contain functional NF-B (55)
. Interestingly, the CK2 promoter has been found to be regulated by NF-B (56)
. Thus, a positive feedback loop regulation may play a role in the elevated CK2 and NF-B expression seen in breast cancer. To date, there are no reliable dominant negative kinase inactive versions of CK2, which may reflect the complex nature of the tetramer interaction. Thus, to inhibit CK2 we used the selective inhibitors, apigenin and emodin (29, 30, 31)
. Apigenin or emodin treatment reduced constitutive NF-B activity, suggesting CK2-mediated phosphorylation of IB- is important in the aberrant NF-B activation seen in breast cancer cells. These findings are consistent with previous studies (57
, 58) showing the inhibition of NF-B activity induced by lipopolysaccharide or interferon- in macrophages or by TNF- in endothelial cells can be repressed by apigenin or emodin, although no analysis of the CK2 activity was done in these reports. The mechanism by which CK2 modulates IB- stability remains to be determined, and the involvement of proteasome-dependent and -independent pathways have been reported (14
, 17
, 59)
. Overall, more work is required to determine the extent of involvement of these kinases in various cancers and the mechanisms of their activation.
Transfection of Hs578T cells with a dominant negative IKK/NEMO/IKKAP1 reduced NF-B binding, transcriptional activity, and colony formation in soft agar. Transfection of the dominant negative IKKß vector also inhibited colony formation, although more modestly, consistent with the fact that it only partially affects IKK activity (data not shown). Using other methods to inhibit NF-B/Rel factors, either a decrease in proliferation or survival has been seen. For example, Higgins et al. (60)
demonstrated inhibition of growth of diverse tumor cells in vivo using antisense oligonucleotides to the p65 subunit. We demonstrated that microinjection of IB- in Hs578T breast cancer or WEHI 231 B cells in culture led to apoptosis of 20–30% of cells (5
, 61)
. In preliminary experiments, inducible expression of the IKKß dominant negative protein in stable D3-1 and Hs578T cell lines was found to reduce NF-B activity and growth rate (data not shown). NF-B/Rel factors are known to control genes that mediate cell proliferation (e.g., c-myc and cyclin D1; Refs. 4
, 62
), survival (e.g., IAP, Bcl-XL, and Bcl-2; Refs. 61, 62, 63
), and metastasis (e.g., urokinase plasminogen activator and metalloproteinases; Refs. 63
, 64
). Thus, the different responses seen between the various methods of inhibition and cell types likely result from the magnitude, extent, and kinetics of inhibition of NF-B, as well as upon the nature of the subunits induced and coacting factors present that control expression of genes regulated by this family.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Grants from the Association pour la Recherche sur le Cancer (to R. R-M.), the Fondation Bettencourt-Schueller (to R. R-M.), Massachusetts Department of Public Health Breast Cancer Program (to E. L-B. and R. R-M.), Department of Army DAMD 17-98-1 (to G. E. S.), and NIH RO1 CA 82742 (to G. E. S. and D. C. S.). 
2 To whom requests for reprints should be addressed, at Department of Biochemistry, Boston University Medical School, 715 Albany Street, Boston, MA 02118-2394. Phone: (617) 638-5097; Fax: (617) 638-5339; E-mail: gsonensh{at}bu.edu
3 The abbreviations used are: NF-B, nuclear factor-B; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electric mobility shift analysis; GST, glutathione S-transferase; IKKAP1, IKK-associated protein 1; NEMO, NF-B essential modulator; PMSF, phenylmethylsulfonyl fluoride; WCE, whole cell extract; PNPP, p-nitrophenyl phosphate; wt, wild-type; TNF, tumor necrosis factor; ER, estrogen receptor; PR, progesterone receptor; HTLV, T-cell leukemia virus; ß-gal, ß-galactosidase.
Received 11/ 8/00. Accepted 3/ 2/01.
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) or equivalent volume of carrier DMSO (
). Cultures were then harvested, normalized for ß-gal activity, and assayed for luciferase activity. The values for luciferase normalized for ß-gal activity are represented.
