Cancer Research Cancer Epigenetics EMT and Cancer Progression and Treatment
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eddy, S. F.
Right arrow Articles by Sonenshein, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eddy, S. F.
Right arrow Articles by Sonenshein, G. E.
[Cancer Research 65, 11375-11383, December 15, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Inducible I{kappa}B Kinase/I{kappa}B Kinase {varepsilon} Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-{kappa}B Activation in Breast Cancer Cells

Sean F. Eddy1,3, Shangqin Guo1,3, Elizabeth G. Demicco1,3, Raphaëlle Romieu-Mourez1,3, Esther Landesman-Bollag2,3, David C. Seldin2,3 and Gail E. Sonenshein1,3

Departments of 1 Biochemistry and 2 Medicine and 3 the Women's Health Interdisciplinary Research Center, Boston University School of Medicine, Boston, Massachusetts

Requests for reprints: Gail E. Sonenshein, Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. Phone: 617-638-4120; Fax: 617-638-4252; E-mail: gsonensh{at}bu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aberrant activation of nuclear factor-{kappa}B (NF-{kappa}B) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of I{kappa}B kinase {alpha} (IKK{alpha}), IKKß, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKK{varepsilon} has been characterized as a potential NF-{kappa}B activator. Here, we provide evidence that implicates IKK-i/IKK{varepsilon} in the pathogenesis of breast cancer. We show IKK-i/IKK{varepsilon} expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKK{varepsilon} and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKK{varepsilon} expression correlated with CK2{alpha} expression in mammary glands and breast tumors derived from MMTV-CK2{alpha} transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKK{varepsilon} mRNA and protein levels. Inhibition of CK2{alpha} via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKK{varepsilon} levels. Expression of a kinase-inactive IKK-i/IKK{varepsilon} mutant in breast cancer cells reduced NF-{kappa}B activity as judged by transfection assays of reporters driven either by NF-{kappa}B elements or the promoters of two NF-{kappa}B target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKK{varepsilon} mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKK{varepsilon}, which is an important mediator of the activation of NF-{kappa}B that plays a critical role in the pathogenesis of breast cancer. (Cancer Res 2005; 65(24): 11375-83)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nuclear factor-{kappa}B (NF-{kappa}B)/Rel is a family of dimeric transcription factors distinguished by the presence of a 300-amino-acid region, termed the Rel homology domain, which determines much of its function (1). Classic NF-{kappa}B is a heterodimer composed of a RelA (p65) and a p50 subunit. In most cells, NF-{kappa}B/Rel proteins are sequestered in the cytoplasm bound to the specific I{kappa}B inhibitory proteins, of which I{kappa}B-{alpha} is the paradigm. Whereas 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-{kappa}B in mammalian cancers was less clear for many years (2), although several oncogenic mammalian viruses were shown to activate NF-{kappa}B. For example, the product of the tax gene of the human T-cell lymphotrophic virus-1 activates NF-{kappa}B (3), which we showed mediates transactivation of the c-myc promoter (4, 5). Recently, we and others have shown a role for NF-{kappa}B/Rel factors in breast cancer (6, 7). High levels of nuclear NF-{kappa}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 (6, 7).

The increased NF-{kappa}B/Rel activity in tumor cells has been correlated with a decrease in stability of I{kappa}B proteins, in particular of I{kappa}B-{alpha}, which permits the released NF-{kappa}B subunits to translocate into the nucleus (8). To begin to elucidate the mechanism of this increased turnover, we recently characterized the activity of several kinases implicated in I{kappa}B-{alpha} turnover. Breast cancer specimens and tumor cells displayed higher levels of activity of either the I{kappa}B kinase {alpha} (IKK{alpha}) or IKKß proteins or of the serine/threonine protein kinase CK2 (9, 10). Phosphorylation of I{kappa}B-{alpha} at two serine residues (Ser32 and Ser36) by IKK{alpha} or IKKß, which are present in a large IKK complex containing multiple copies of a regulatory subunit NEMO/IKK{gamma} (11, 12), leads to I{kappa}B-{alpha} ubiquination and subsequent proteasome-mediated degradation in the canonical NF-{kappa}B induction pathway. In addition to the NH2-terminal phosphorylation of I{kappa}B-{alpha}, it has been shown that COOH-terminal phosphorylation via CK2 within the COOH-terminal PEST (Ser283, Ser289, Thr291, and Ser293) domain of I{kappa}B-{alpha} also affects its stability (13, 14). CK2 is a ubiquitously expressed tetrameric protein kinase containing two catalytic ({alpha}/{alpha}, {alpha}'/{alpha}, or {alpha}'/{alpha}') and two regulatory (ß/ß) subunits (15, 16). Recent evidence suggests that CK2 activity can be altered by cellular stress, including UV irradiation (17, 18). CK2-mediated phosphorylation of I{kappa}B-{alpha} has been implicated in basal and signal-independent turnover of I{kappa}B-{alpha} (13, 19–21). These findings have implicated CK2 in control of intrinsic I{kappa}B-{alpha} stability and thus activation of NF-{kappa}B. Importantly, we showed that CK2 levels are elevated in primary human breast cancer specimens as well as in established cell lines with elevated NF-{kappa}B activity (9, 10, 22, 23).

A recently discovered inducible IKK protein also leads to I{kappa}B-{alpha} phosphorylation and NF-{kappa}B activation. Initially isolated from mice and termed IKK-i, it is also known as IKK{varepsilon} in humans (24, 25). IKK-i/IKK{varepsilon} is part of an independent complex containing TANK and TRAF (26). TANK-binding kinase-1 (TBK-1), which is highly homologous to IKK-i/IKK{varepsilon}, binds to TANK and TRAF and may form an alternative IKK complex consisting of IKK-i/IKK{varepsilon} and TBK-1 (27). IKK-i/IKK{varepsilon} seems to specifically phosphorylate I{kappa}B-{alpha} at Ser36 (24, 25). Although the significance of only one phosphorylation event at the NH2 terminus of I{kappa}B-{alpha} is not yet entirely clear, it may predispose I{kappa}B-{alpha} towards Ser32 phosphorylation and subsequent degradation (25). Furthermore, IKK-i/IKK{varepsilon} directly phosphorylates transcription factors (26), including NF-{kappa}B/Rel factors (28, 29). Given the aberrant activation of NF-{kappa}B in malignancies, we investigated the role of IKK-i/IKK{varepsilon} in breast cancer. Here, we show for the first time IKK-i/IKK{varepsilon} induction in primary human breast cancers, rodent mammary tumors, and cell lines in culture. Furthermore, we implicate CK2 in the elevated IKK-i/IKK{varepsilon} levels and show a link between IKK-i/IKK{varepsilon} and activation of NF-{kappa}B in breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 and have previously been described (30).

Transgenic mice. Creation of the transgenic MMTV-c-rel and MMTV-CK2 transgenic was previously described (22, 30). Breeding of MMTV-CK2 mice and MMTV-c-rel mice created bitransgenic MMTV-CK2 X MMTV-c-rel mice. Mice were housed in a two-way barrier at the Boston University School of Medicine Transgenic mouse facility in accordance with the regulations of the American Association for the Accreditation of Laboratory Animal Care. Wild-type (WT) mice used in the study were age and pregnancy matched. All tissues were frozen immediately after extraction in liquid nitrogen and stored at –80°C.

Carcinogen treatment of mice. Twenty virgin female FVB/N mice, housed in a two-way barrier, were treated according to a protocol approved by the Boston University Institutional Animal Care and Use Committee. Mice were each given six weekly 1.0-mg doses of 7,12-dimethylbenz(a)anthracene (DMBA) in 0.2 mL of sesame oil by oral gavage, beginning at 5 weeks of age. Mice were then mated continuously to provide an oscillating hormonal environment and followed until either tumors developed or the mice died. By 34 weeks of age (29 weeks after beginning DMBA treatment), all mice had developed tumors. Mice bearing tumors >0.5 cm were euthanized by CO2 inhalation and necropsied. Mammary tumors and grossly normal mammary glands from parous age-matched control FVB mice were excised and frozen on dry ice and stored at –80°C. Whole cell protein extracts were prepared by homogenizing frozen tumors or mammary gland specimens in lysis buffer containing a cocktail of protease inhibitors [50 mmol/L Tris-HCl (pH 8), 1% NP40, 125 mmol/L NaCl, 1 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL aprotinin, 1 µg/mL pepstatin, 1 µg/mL leupeptin, 1 mmol/L Na3VO4, and 10 mmol/L sodium pyrophosphate].

Cell culture and treatment conditions. Hs578T and MDA-MB-231 breast cancer cell lines were grown in standard culture medium, as described by the American Tissue Culture Collection (Manassas, VA). MCF-10F, D3-1, and a DMBA-transformed MCF-10F derivative (31) were cultured as described (9). The MMTV-HER-2/neu NF639 cell line was derived from mammary tumors expressing HER-2/neu and cultured as previously described (32). Human HEK293T endothelial derived kidney cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). NIH 3T3 fibroblast cell lines were cultured as described (10). Where indicated, cells were incubated with 25 or 50 µmol/L apigenin (Sigma, St. Louis, MO) dissolved in DMSO or treated with vehicle DMSO alone.

Plasmids and transfection analyses. The pCDNA3-FLAG-IKK{varepsilon} and pCDNA3-FLAG-IKK{varepsilon} K38A vectors expressing IKK{varepsilon} and kinase-inactive IKK{varepsilon}, respectively, were a kind gift of T. Maniatis (Harvard University, Cambridge MA; ref. 24). The pRC/CMV-HA-CK2{alpha}', pRC/CMV-HA CK2{alpha} K68M, and pRC/CMV-myc-CK2ß vectors were provided by D. Litchfield (University of Western Ontario, Ontario, Canada; ref. 33). To evaluate NF-{kappa}B activity, two-copy WT or mutant NF-{kappa}B element/thymidine kinase promoter/chloramphenical acetyl transferase (CAT) reporter vectors (E8-CAT and mut-E8-CAT, respectively; ref. 5) or a six-copy NF-{kappa}B element–driven luciferase reporter construct, kindly provided by G. Rawadi (Hoechst-Marion-Roussel, Romainville, France; ref. 34), were used. NF-{kappa}B-driven promoter constructs used include (a) cyclin D1 promoter containing WT (–66 wt-Luc) or mutant (–66 mut-Luc) NF-{kappa}B elements (kind gift of R.G. Pestell, Lombardi Comprehensive Cancer Center, Washington, DC; refs. 30, 35); (b) relB promoter containing WT (p1.7 relB) or mutant (p1.7 mut-relB) versions of the two NF-{kappa}B elements, prepared as described previously (36). For transfection into six-well or P100 plates, 4 or 10 µg total DNA, respectively, were transfected per sample. For transient transfection into Hs578T and MDA-MB 231 cells, cells were incubated for 16 to 24 hours with DNA and GenePorter2 (Gene Therapy Systems, San Diego, CA). Transfections into MCF-10F, D3-1, and NF639 cell lines were done by incubating for 16 hours in the presence of DNA and Fugene 6 Transfection Reagent (Roche, Indianapolis, IN). The calcium phosphate method of transfection (37) was used with HEK293T cells. CAT and luciferase assays were done as described in Sovak et al. (6) and Romieu-Mourez et al. (30), respectively. Cotransfection of an SV40-ß-galactosidase (SV40-ß-gal) expression vector was used to normalize for transfection efficiency, as described (9). Where indicated, SD was calculated and significance determined using the Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.005).

Immunoblotting. Cytoplasmic protein extracts were prepared in RSB buffer [10 mmol/L Tris (pH 7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.5% NP40] or TKM buffer [10 mmol/L Tris (pH 7.6), 10 mmol/L KCl, 5 mmol/L MgCl2, 0.2% NP40], where indicated. Nuclear extracts were prepared in DR buffer [20 mmol/L HEPES (pH 7.9), 420 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 20% glycerol] or radioimmunoprecipitation assay (RIPA) buffer [50 mmol/L Tris (pH 7.6), 150 mmol/L NaCl, 1% NP40, 0.1% SDS, 5 mmol/L EDTA, and 1% sodium sarcosyl]. Whole cell extracts were prepared in RIPA buffer or in kinase lysis buffer [20 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl]. Protease and phosphatase inhibitors were added to each protein preparation (10 mmol/L NaF, 25 mmol/L ß-glycerophosphate, p-nitrophenyl phosphate, 1 mmol/L Na3VO4, 1 mmol/L DTT, 0.5 mmol/L PMSF, 5 µg/mL leupeptin). Protein concentration was determined by Lowry assay using the Bio-Rad reagent (Bio-Rad, Hercules, CA). Whole cell extracts for reporter assays were prepared in 1x reporter lysis buffer (Promega, Madison, WI).

Antibodies. IKK-i/IKK{varepsilon} antibodies (sc-9913, sc-5694), I{kappa}B-{alpha} (sc-203), I{kappa}B-ß (sc-945), and hemagglutinin (HA) (sc-805) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against I{kappa}B-{varepsilon} and c-Myc were a gift from N. Rice and M. Ernst (National Cancer Institute, Fredrick, MD) and S. Hann (Vanderbilt University, Nashville, TN), respectively. Competition assays for IKK-i/IKK{varepsilon} were done using 2 µg cognate peptide (sc-9913P). Antibodies against cyclin D1 (06-137) were purchased from Upstate Biotechnology (Lake Placid, NY). FLAG (F-4092) and ß-actin (AC-15) antibodies were purchased from Sigma.

IKK-i/IKK{varepsilon} kinase assay. Protein extracts and immunoprecipitations were done as previously described (9, 10). Briefly, 300 µg (in 500 µL kinase lysis buffer) whole cell extracts were precleared with protein A/G agarose Plus beads (Santa Cruz Biotechnology) and bovine anti-goat horseradish peroxidase secondary antibody (Santa Cruz Biotechnology) for 1 hour at 4°C before the addition of 2 µg of IKK-i/IKK{varepsilon} antibody. Whole cell extracts were split into three aliquots. One third was used for a kinase assay with GST-I{kappa}B-{alpha} as a substrate and one third for a kinase assay with GST-2N-I{kappa}B-{alpha} containing S32A and S36A mutations. All kinase assays were done as described (9). The remaining one third of immunoprecipitated proteins were subject to immunoblot analysis for IKK-i/IKK{varepsilon} using an antibody directed to a different epitope (K-14).

Reverse transcription-PCR. RNA was isolated using Trizol (Invitrogen) reagent according to the manufacturer's protocol and was quantified by measuring A260. The A260/A280 ratios were between 1.8 and 2. RNA samples were prepared as described (30). PCR were done in a Thermal Cycler (MJ Research, Watertown, MA) for 32 cycles under these conditions: 94°C for 60 seconds, 57°C for 45 seconds, and 72°C for 55 seconds. Primer pairs for PCR were as follows: ikk-i/ikk{varepsilon} (forward, nucleic acid position 504) 5'-CGGAAGCTGAACCACCAGAA-3' and (reverse, 976) 5'-CCAGTGGCTGCATGGTACAA-3'; hmgb1 (forward, 725) 5'-AGGAGGATGAAGAGGAATGAG-3' and (reverse, 1025) 5'-GACTGTACCAGGCAAGGTTA-3'; ß-actin (forward, 516) 5'-CACTGGCATCGTGATGGACT-3' and (reverse, 923) 5'-CGGATGTCCACGTCACACTT-3'.

Soft agar transformation assay. Stable Hs578T breast cancer cells were plated at 5 x 103/mL in 0.35% top agarose (SeaPlaque Agarose, FMC Bioproducts, Rockland, ME) with a base agarose of 0.7% supplemented with complete medium and 1 mg/mL G418 (Sigma). Plates were subsequently incubated for 16 days in humidified incubator at 37°C. Cells were stained with 0.5 mL of 0.0005% crystal violet, and colonies were counted visually under x8 magnification.

Matrigel invasion assay. Matrigel (BD Biosciences, San Jose, CA) was diluted to a working concentration of 6.3 mg/mL. For Matrigel assays, 200 µL of Matrigel were added into a 24-well tissue culture plate and incubated at 37°C for 30 minutes. A single-cell suspension of NF639 cells (5 x 105/mL) in serum-free medium (DMEM) was made by passing the cell suspension five times through a 21.5-gauge needle. Ten microliters (5,000 cells) were mixed with 190 µL of Matrigel and plated onto the solidified bottom layer. Complete medium was added, and the plate incubated at 37°C for 3 to 6 days and photographed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IKK-i/IKK{varepsilon} is expressed in primary human breast cancer specimens. To begin to assess the role of IKK-i/IKK{varepsilon} in breast cancer, cytoplasmic extracts from six human breast cancer tissue specimens were analyzed for expression of IKK-i/IKK{varepsilon}. A band of the appropriate molecular weight for IKK-i/IKK{varepsilon} (80 kDa) was seen in four of six samples (Fig. 1A, left). Analysis of ß-actin levels confirmed that loading was essentially equivalent. To verify the specificity, a duplicate blot was subjected to immunoblotting in the presence of 2 µg of cognate peptide (Fig. 1A, right). Detection of the 80-kDa band was completely eliminated with the addition of the peptide. Several lower molecular weight bands were detected and were also eliminated upon addition of the cognate peptide; therefore, they likely represent IKK-i/IKK{varepsilon} degradation products (data not shown). These findings indicate that IKK-i/IKK{varepsilon} is indeed expressed in multiple primary breast cancer specimens.



View larger version (43K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. IKK-i/IKK{varepsilon} is expressed in breast cancer cells. A, cytoplasmic extracts were prepared from the indicated human breast tumor specimens. Samples (50 µg) were used to prepare duplicate blots, which were subjected to immunoblotting with 2 µg of antibody specific for IKK-i/IKK{varepsilon} (Q-15) in the absence or presence of excess (2 µg) cognate IKK-i/IKK{varepsilon} peptide, as specific competitor. One blot was stripped and reprobed with an antibody against ß-actin to confirm loading. B, samples of whole cell extracts (WCE; 50 µg) from DMBA-induced mouse mammary tumors were subjected to immunoblotting for IKK-i/IKK{varepsilon} (SPC, spindle cell carcinoma; SSC, squamous cell carcinoma; MAC, microacinar tumor). All samples from a single gel. Coomassie blue stain (Coom. Blue) was used to control for equal loading. C, left, samples of whole cell extracts (50 µg) prepared from human mammary epithelial cells MCF-10F, the DMBA-transformed D3-1 cell line, and MDA-MB 231 (MB 231) breast cancer cells were subjected to immunoblotting for IKK-i/IKK{varepsilon} expression and ß-actin to control for loading. Right, whole cell extracts (25 µg) from MCF-10F, Hs578T, and MDA-MB 231 breast cancer cell lines were subjected to immunoblotting for IKK-i/IKK{varepsilon} and ß-actin to control for loading. D, whole cell extracts were prepared in kinase assay lysis buffer and precleared with Protein A/G agarose beads and anti-goat secondary IgG horseradish peroxidase–conjugated antibody. IKK-i/IKK{varepsilon} complexes were immunoprecipitated from 300 µg of protein using an NH2-terminal-derived antibody. One third was used for a kinase assay on a GST-WT-I{kappa}B-{alpha} as substrate (KA), one third for a kinase assay on a GST-2N-I{kappa}B-{alpha} as substrate (KA), and one third for Western blotting for IKK-i/IKK{varepsilon} (K-14, a COOH-terminal-derived antibody). Immunoblotting (IB) of whole cell extracts shows endogenous levels of IKK-i/IKK{varepsilon} from MCF10F, D3-1, and Hs578T cells and ß-actin to control for loading (WCE IB).

 
IKK-i/IKK{varepsilon} is expressed in DMBA-induced mammary tumors and human breast cancer cell lines. We next tested extracts isolated from mammary tumors induced in female FVB mice by treatment with DMBA, which were primarily squamous cell carcinomas, with other tumors including a spindle cell carcinoma and a microacinar tumor.4 Whole cell extracts isolated from seven mammary tumors as well as from the normal mammary glands from three age-matched parous FVB female control mice (FVB 4, FVB7, and FVB8) were subjected to immunoblotting for expression of IKK-i/IKK{varepsilon}. Six of seven tumors showed substantially elevated levels of IKK-i/IKK{varepsilon} compared with the mammary glands of nontreated control mice (Fig. 1B). Thus, IKK-i/IKK{varepsilon} expression is increased in many carcinogen-induced mouse mammary tumors.

To further characterize IKK-i/IKK{varepsilon} in breast cancer, we turned to human cell lines in culture. Whole cell extracts were prepared from the untransformed breast epithelial MCF-10F cell line and its DMBA-induced derivative line D3-1 (31) along with the breast cancer cell lines MDA-MB 231 and Hs578T and subjected to immunoblot analysis for IKK-i/IKK{varepsilon} (Fig. 1C). A higher level of IKK-i/IKK{varepsilon} expression was observed in MDA-MB-231, Hs578T, and D3-1 cells compared with MCF-10F cells. MDA-MB 468 and T47D cells also displayed elevated IKK-i/IKK{varepsilon} levels (data not shown). To measure IKK-i/IKK{varepsilon} kinase activity, IKK-i/IKK{varepsilon} was immunoprecipitated from extracts prepared from MCF-10F, D3-1, and Hs578T cells and subjected to a kinase assay using as substrate either GST-WT-I{kappa}B-{alpha} or a mutant GST-2N-I{kappa}B-{alpha} protein (with phosphorylation sites Ser32 and Ser36 mutated to alanines; Fig. 1D). Of the two transformed cell lines, Hs578T exhibited a higher level of IKK-i/IKK{varepsilon} activity than the D3-1 transformed cells, although they seemed to contain almost equal levels of protein as judged by immunoblotting (Fig. 1D). Little or no IKK-i/IKK{varepsilon} activity was detectable in the MCF-10F cells, which also contained a much lower level of IKK-i/IKK{varepsilon} kinase protein (Fig. 1C and D). Thus, breast cancer cell lines contain an elevated level of IKK-i/IKK{varepsilon}.

IKK-i/IKK{varepsilon} expression is elevated in CK2{alpha}-expressing transgenic murine mammary tumors and glands. We next sought to determine whether mammary tumors in transgenic mice display elevated levels of IKK-i/IKK{varepsilon} and selected the following mouse models: MMTV-CK2{alpha}, expressing the catalytic subunit of CK2 and the MMTV-c-rel, expressing the c-Rel NF-{kappa}B subunit. Approximately 30% of female MMTV-CK2{alpha} transgenic mice developed a variety of mammary tumors at a median age of 23 months (22), and 31.6% of female MMTV-c-rel mouse developed one or more tumors at an average age of 19.9 months (30). Cytoplasmic extracts from mammary glands of age-matched and pregnancy-matched WT mice were compared with those from tumors and histologically normal mammary glands of transgenic MMTV-CK2{alpha} (Fig. 2A) and MMTV-c-rel mice (Fig. 2B). Expression of IKK-i/IKK{varepsilon} protein was extremely low in the extracts from mammary glands of WT control animals. The MMTV-CK2{alpha} mouse tumor (7367T) displayed substantially higher levels than the WT-1 and WT-2 mouse mammary gland (Fig. 2A), whereas four of five tumors derived from MMTV-c-rel displayed a higher level of IKK-i/IKK{varepsilon} protein compared with the mammary glands of these two WT mice (Fig. 2B). As control for protein loading, the gel was stained with Coomassie, which indicated essentially equal loading. Interestingly, we noted that the histologically normal mammary glands of the MMTV-CK2{alpha} mice all seemed to contain substantial levels of IKK-i/IKK{varepsilon} compared with the mammary glands of WT animals, whereas low levels were present in the histologically normal glands of the MMTV-c-rel transgenic mouse.



View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. IKK-i/IKK{varepsilon} expression in mouse tumor models. Cytoplasmic extracts were prepared from mammary glands of age-matched WT mice and histologically normal mammary glands from transgenic mice (N) and transgenic mouse tumor (T), as indicated. Samples (50 µg) were subjected to immunoblotting for IKK-i/IKK{varepsilon} expression. Coomassie blue (Coom. Blue) staining of duplicate gels was done to confirm essentially equal loading. A, MMTV-CK2{alpha} transgenic mice. B, MMTV-c-rel transgenic mice. C, MMTV-CK2{alpha} X MMTV-c-rel bitransgenic mice.

 
We have recently prepared a bitransgenic MMTV-CK2 x MMTV-c-rel mice, which exhibited ~50% incidence of breast tumor formation with the average age at onset of 20.8 months.5 Histologically normal mammary glands of bitransgenic MMTV-CK2{alpha} x MMTV-c-rel mice were removed from the three animals that developed tumors and the levels of IKK-i/IKK{varepsilon} protein compared (Fig. 2C). Substantial IKK-i/IKK{varepsilon} expression was detected in cytoplasmic extracts of histologically normal mammary glands from these mice. The tumors from the same animal displayed somewhat higher IKK-i/IKK{varepsilon} levels. These findings are similar to the data obtained with the MMTV-CK2{alpha} mice and suggest a role for CK2 in the induction of IKK-i/IKK{varepsilon} expression.

Ectopic CK2 elevates IKK-i/IKK{varepsilon} levels in NIH 3T3 fibroblasts, MCF-10F breast epithelial cells, and HEK293T cells. To investigate the ability of CK2 to increase IKK-i/IKK{varepsilon} levels, isolated clones of CK2{alpha} stable lines (termed clone 5 and clone 6) in NIH 3T3 cells (10) were analyzed for expression IKK-i/IKK{varepsilon} compared with control NIH 3T3 cells (Fig. 3A, left). Higher levels of IKK-i/IKK{varepsilon} were seen in clone 5 and clone 6 compared with the clones infected with the pBABE control viral vector. To examine the effects of ectopic CK2 expression in breast epithelial cells, MCF-10F cells, which have low levels of CK2 activity (9) and IKK-i/IKK{varepsilon} expression (Fig. 1B), were transiently transfected with vectors expressing HA-CK2{alpha}' and myc-CK2ß, or parental empty expression vectors, as control. IKK-i/IKK{varepsilon} expression was increased compared with control MCF-10F cells transfected with empty vector DNA (Fig. 3A, right).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Ectopic CK2 expression induces IKK-i/IKK{varepsilon} in NIH 3T3 fibroblasts, MCF-10F breast epithelial cells, and HEK293T cells. A, left, clones of NIH 3T3 cells infected with either pBABE-CK2{alpha} retrovirus expressing CK2{alpha} or a pBABE-GFP vector (pBABE) were isolated as described previously (10). Whole cell extracts (50 µg) were subjected to immunoblotting for either IKK-i/IKK{varepsilon} or ß-actin, which confirmed equal loading. Right, MCF-10F breast epithelial cells were grown in P100 plates and transiently transfected with 10 µg pRc/CMV (EV) or 5 µg pRc/CMV-HA-CK2{alpha}' and 5 µg pRc/CMV-myc-CK2ß (CK2{alpha}'/CK2ß). Whole cell extracts were prepared, and samples (100 µg) were immunoblotted for IKK-i/IKK{varepsilon}. Duplicate blots were probed for HA and c-Myc to confirm expression of HA-CK2{alpha}' and Myc-CK2ß. B, cultures of HEK293T cells (in six-well P60 dishes) were transfected with 1 µg of pRC/CMV-HA-CK2{alpha}' or pRC/CMV-myc-CK2ß DNA alone or in combination and enough pCDNA3 (CMV driven) vector to make a total of 2 µg DNA. Protein extracts (50 µg) were subjected to immunoblot analysis for IKK-i/IKK{varepsilon} expression. To confirm expression of CK2{alpha}' and CK2ß, the blot was stripped and reprobed with antibodies specific for HA and c-Myc, respectively. Equal loading was confirmed by probing the blot with a ß-actin antibody. C, HEK293T cells were transfected as above with pRC/CMV-HA-CK2{alpha}' or pRC/CMV-myc-CK2ß DNA alone or in combination, or with 2 µg of pCDNA3-FLAG-IKK{varepsilon} expressing FLAG-tagged IKK-i/IKK{varepsilon}, as a positive control. RNA was prepared using Trizol reagent. Samples (1 µg) were used for first-strand cDNA synthesis, and a 2-µL aliquot (total 50 µL) used to perform RT-PCR analysis for ikk-i/ikk{varepsilon}, hmgb1, and ß-actin. Two independent experiments were performed.

 
To further investigate the ability of CK2{alpha} to increase IKK-i/IKK{varepsilon} levels, cultures of HEK293T cells were transfected with vectors expressing either the catalytic CK2{alpha}' subunit as a HA-tagged protein (pRc/CMV-HA-CK2{alpha}') or the regulatory subunit CK2ß as a c-Myc-epitope tagged protein (pRc/CMV-myc-CK2ß), or a combination of both vectors. Whole cell extracts were subjected to immunoblotting for IKK-i/IKK{varepsilon} and for the expression of the transfected CK2 subunits (Fig. 3B). Expression of either CK2 subunit alone or in combination led to a substantial increase in IKK-i/IKK{varepsilon} levels.

To assess whether CK2 acted at a pretranslational level to increase IKK-i/IKK{varepsilon} expression, semiquantitative reverse transcription-PCR (RT-PCR) was done using total RNA isolated from the HEK293T cells transfected as above (Fig. 3C). As a positive control, cells were transfected with a vector expressing IKK-i/IKK{varepsilon}. Ectopic expression of either CK2 subunit led to a substantial induction of ikk-i/ikk{varepsilon} mRNA levels, whereas analysis of hmgb1, which has been associated with estrogen responsive breast cancer (38–41), and ß-actin showed no differential expression (Fig. 3C). These data strongly suggest a role for CK2 in the induction of IKK-i/IKK{varepsilon}.

Inhibition of CK2 activity decreases IKK-i/IKK{varepsilon} levels in breast cancer cells. As an initial test of the effects of CK2 on IKK-i/IKK{varepsilon} expression, breast cancer cells, which display high CK2 activity (9), were treated with apigenin, a selective inhibitor of CK2. Hs578T cells were incubated for 6 hours in the presence of either 25 or 50 µmol/L apigenin or with an equal volume of carrier DMSO. RNA was isolated and subjected to semiquantitative RT-PCR (Fig. 4A). Compared with control cells treated with DMSO, ikk-i/ikk{varepsilon} mRNA expression decreased by 2.5-fold at 25 µmol/L apigenin and by 2.9-fold with 50 µmol/L apigenin (Fig. 4A) compared with the ß-actin controls. Furthermore, treatment of Hs578T, or D3-1 or MDA-MB 231 breast cancer cells with 50 or 25 µmol/L apigenin, respectively, for 6 hours resulted in substantial decreases in IKK-i/IKK{varepsilon} protein levels (Fig. 4B). Because pharmacologic inhibitors may also affect other kinases, a vector expressing a kinase-inactive CK2{alpha} subunit, which functions as a competitive inhibitor with endogenous CK2{alpha}, was used (10, 33). Hs578T and D3-1 breast cancer cells were transfected with a vector expressing HA-tagged kinase-inactive CK2{alpha} K68M or an empty vector control DNA, and cytoplasmic extracts were analyzed. Hs578T and D3-1 cells expressing the kinase-inactive CK2{alpha} K68M displayed lower levels of IKK-i/IKK{varepsilon} compared with control cells (Fig. 4C). Similar results were obtained using kinase-inactive CK2{alpha}' K69M (data not shown). These findings indicate that CK2 activity induces expression of IKK-i/IKK{varepsilon} in breast cancer cells.



View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Inhibition of CK2 activity in breast cancer cells reduces IKK-i/IKK{varepsilon} expression. A, Hs578T breast cancer cells were grown in six-well plates and treated with 25 or 50 µmol/L apigenin for 6 hours. RNA was harvested using Trizol reagent. An RNA sample (1 µg) was used for first-strand synthesis, and a 2-µL aliquot was used for RT-PCR analysis of ikk-i/ikk{varepsilon}, along with hmgb1 and ß-actin to control for loading. B, Hs578T, D3-1, and MDA-MB-231 breast cancer cells were treated with apigenin for 6 hours at a concentration of 25 or 50 µmol/L (as indicated). Whole cell extracts were immunoblotted for IKK-i/IKK{varepsilon} expression. Blots were reprobed with a ß-actin antibody to confirm equal loading. C, Hs578T and D3-1 cells, grown in six-well plates, were transfected with 2 µg of either a kinase-inactive HA-tagged CK2{alpha} construct (pRc/CMV-HA-CK2{alpha} K68M, labeled CK2{alpha} KI) or empty parental vector. Cells were harvested 16 to 24 hours after transfection and lysed in RIPA buffer, and samples of whole cell extracts (60 µg, Hs578T or 75 µg, D3-1) were subjected to immunoblotting for IKK-i/IKK{varepsilon}, HA (for CK2{alpha} KI), and ß-actin as loading control.

 
IKK-i/IKK{varepsilon} controls I{kappa}B-{alpha} turnover and NF-{kappa}B activity in breast cancer cells. Previous studies showed that IKK-i/IKK{varepsilon} phosphorylates I{kappa}B-{alpha} on Ser36 (24, 25). To determine the in vivo effects of IKK-i/IKK{varepsilon} on steady-state levels of I{kappa}B proteins in HEK293T cells, pCDNA3-FLAG-IKK{varepsilon} expression vector (IKK{varepsilon}) or empty vector pCDNA3 DNA (EV) were transfected into HEK293T cells. Cytoplasmic extracts, prepared 48 hours after transfection, were assessed for I{kappa}B proteins by immunoblotting. Ectopic IKK-i/IKK{varepsilon} expression caused a dramatic reduction in the total amount of cytoplasmic I{kappa}B-{alpha} (Fig. 5A). Only a modest drop in I{kappa}B-ß levels was detected, and no effect was seen on levels of I{kappa}B-{varepsilon}. Furthermore, ectopic IKK-i/IKK{varepsilon} expression in HEK293T cells induced functional NF-{kappa}B activity as judged by NF-{kappa}B element–driven reporter constructs (data not shown).



View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. IKK-i/IKK{varepsilon} controls I{kappa}B-{alpha} turnover and NF-{kappa}B activity. A, HEK293T cells, grown in six-well plates, were transfected with 2 µg of empty vector pCDNA3 (EV) or pCDNA3-FLAG-IKK{varepsilon} (IKK{varepsilon}). Forty-eight hours after transfection, cytoplasmic extracts were prepared, and samples (20 µg) were subjected to immunoblotting for I{kappa}B-{alpha}, I{kappa}B-ß, and I{kappa}B-{varepsilon} expression. Blots were stripped and probed with ß-actin to control for equal loading. A control blot was probed with a monoclonal FLAG antibody detecting FLAG-IKK-i/IKK{varepsilon} fusion protein. B, MMTV-HER-2/neu NF639 cells were cotransfected, in triplicate, with 2 µg of pCDNA3-FLAG-IKK{varepsilon} K38A (IKK{varepsilon} K38A) or pCDNA3 (EV), 0.5 µg E8-CAT or mut-E8-CAT reporter construct and 0.5 µg of SV40-ß-gal for normalization of transfection efficiency. Normalized relative activity of the E8-CAT to mut-E8-CAT. C, Hs578T cells were cotransfected, in triplicate, with 1 µg of either E8-CAT or mut-E8-CAT reporter construct along with 1 µg of SV40-ß-gal, to normalize for transfection efficiency, and increasing concentrations of IKK{varepsilon} K38A (0.5, 1, and 2 µg) while maintaining a 4 µg total DNA concentration with parental pCDNA3 vector. Whole cell extracts were prepared, normalized for transfection efficiency, and subjected to CAT assays. Columns, means; bars, SD. Significance was determined using the Student's t test. D, whole cell extracts (20 µg) from the transfected Hs578T cells in (C) were subjected to immunoblot analysis for the FLAG epitope, to confirm IKK-i/IKK{varepsilon} expression. The blots were stripped and reprobed with antibodies for I{kappa}B-{alpha} and I{kappa}B-ß and subsequently for ß-actin, which confirmed equal loading.

 
To verify a functional role of IKK-i/IKK{varepsilon} in the regulation of NF-{kappa}B in breast cancer, a kinase-inactive IKK-i/IKK{varepsilon} (IKK{varepsilon} K38A) was used, which serves as a dominant negative for IKK-i/IKK{varepsilon} activity. HER-2/neu receptor–driven mouse mammary tumor NF639 cells were used because CK2 activity in these cells had previously been implicated in the induction of NF-{kappa}B (10). NF639 cells were cotransfected with IKK{varepsilon} K38A expression vector or empty parental vector pCDNA3 in the presence of either E8-CAT or mut-E8-CAT, driven by WT or mutant NF-{kappa}B sites, respectively. IKK{varepsilon} K38A caused a nearly 2-fold reduction in NF-{kappa}B activity (Fig. 5B, left). Confirmation of transfection was done by immunoblotting for IKK-i/IKK{varepsilon} (data not shown). We next assessed the effects in Hs578T breast cancer cells of an increasing dose of the IKK{varepsilon} K38A expression on E8-CAT and mut-E8-CAT promoter activity (Fig. 5C). Expression of the kinase-inactive IKK-i/IKK{varepsilon} resulted in a dose-dependent reduction of NF-{kappa}B activity. A nearly 5-fold reduction of E8-CAT activity was seen at 2 µg of IKK{varepsilon} K38A (P < 0.005), reducing the activity of the WT E8-CAT almost to basal reporter activity seen with the mut-E8-CAT. Using a monoclonal FLAG-specific antibody in immunoblot analysis of the remaining extracts confirmed the expression of IKK{varepsilon} K38A. Furthermore, when these extracts were analyzed for levels of I{kappa}B-{alpha} and I{kappa}B-ß, a dramatic increase in the levels of I{kappa}B-{alpha} was observed with increasing doses of IKK{varepsilon} K38A. In contrast, no change was detected in levels of I{kappa}B-ß (Fig. 5D). These results show that IKK-i/IKK{varepsilon} plays a role in the constitutive activation of NF-{kappa}B in breast cancer cells and suggest that I{kappa}B-{alpha} is the preferential I{kappa}B target.

IKK{varepsilon} K38A reduces activity of NF-{kappa}B–driven cyclin D1 and relB promoters in breast cancer cells. To test the effects of the kinase-inactive IKK-i/IKK{varepsilon} on natural promoters that are driven by NF-{kappa}B elements, cotransfection analysis was done using the cyclin D1 and relB promoters, which are each driven by two NF-{kappa}B elements (35, 36). Cotransfection of the kinase-inactive IKK{varepsilon} K38A vector in Hs578T breast cancer cells resulted in a dose-dependent decrease in activity of the WT cyclin D1 luciferase reporter construct (–66 wt-Luc), whereas the mutant version (–66 mut-Luc) was largely unaffected (Fig. 6A). Similarly, cotransfection of IKK{varepsilon} K38A with p1.7 relB-Luc relB promoter reporter construct, containing two WT NF-{kappa}B sites, reduced luciferase activity to that seen with a mutant construct, p1.7 mut-relB-Luc, containing mutations in the two identified NF-{kappa}B elements (Fig. 6B). To ensure the effects were not cell type specific, D3-1 cells and MDA-MB 231 breast cancer cells were similarly cotransfected with either IKK{varepsilon} K38A or pCDNA3 in the presence of the WT p1.7 relB-Luc relB promoter construct (Fig. 6C). The kinase-inactive IKK-i/IKK{varepsilon} caused a decrease in relB promoter activity in these two lines.



View larger version (42K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 6. Inhibition of IKK-i/IKK{varepsilon} kinase in breast cancer cells reduces cyclin D1 and relB expression and transformed phenotype. A, Hs578T cells were cotransfected, in triplicate, with either 1.0 µg of cyclin D1 –66 wt-Luc or –66 mut-Luc reporter construct along with 1.0 µg of SV40-ß-gal and increasing concentrations of IKK{varepsilon} K38A (0.5, 1, and 2.0 µg) while maintaining a 4 µg total DNA concentration with empty pCDNA3 vector. B, Hs578T cells were cotransfected, in duplicate, with 1.0 µg of either p1.7relB or p1.7 mut-relB luciferase reporter construct along with 1.0 µg of SV40-ß-gal and 2.0 µg of either IKK{varepsilon} K38A or pCDNA3. Columns, averages; bars, SE. C, D3-1 (left) cells were cotransfected, in duplicate, with either 1.0 µg p1.7 relB luciferase or p1.7 kB mut-relB luciferase reporter construct along with 1.0 µg of SV40-ß-gal and 2.0 µg of either pCDNA3-FLAG-IKK{varepsilon} K38A or pCDNA3. Luciferase activity was measured and normalized to ß-gal activity. Columns, averages; bars, SE. Alternatively, MDA-MB 231 (right) cells were cotransfected, in triplicate, and processed as above. SD was calculated and significance determined using the Student's t test. D, Hs578T and D3-1 cells in P100 plates were transfected with 10 µg of pCDNA3-FLAG-IKK{varepsilon} K38A (IKK{varepsilon} K38A) or pCDNA3 (EV). Cytoplasmic, nuclear, or whole cell extracts (WCE) were isolated (as indicated), and samples (20 µg) were subjected to immunoblotting for RelB and cyclin D1 expression. E, Hs578T breast cancer cells stably expressing IKK{varepsilon} K38A or pCDNA3 were plated, in triplicate, in soft agar. Following incubation at 37°C for 16 days, the plates were stained with crystal violet to visualize the cells and photographed using a Kodak digital camera. Colonies are shown at x3.2 magnification and counted at x8 magnification in three randomly selected fields of view. F, NF639 cells were plated in Matrigel in 12-well plates (5,000 per well), in triplicate, and assessed for their ability to grow between 3 and 6 days. The plates were photographed using an Orca ER camera at x5 magnification in a Zeiss Axiovert 200M microscope.

 
Next, we assessed whether inhibition of IKK-i/IKK{varepsilon} affects endogenous NF-{kappa}B-driven gene expression. Hs578T cells were transiently transfected with IKK{varepsilon} K38A or parental vector. Nuclear and cytoplasmic extracts were subjected to immunoblot analysis. Inhibition of IKK-i/IKK{varepsilon} led to reduced RelB and cyclin D1 expression (Fig. 6D). Similar results were obtained in D3-1 cells (Fig. 6D). Thus, IKK-i/IKK{varepsilon} plays an important role in control of functional NF-{kappa}B activity and its downstream targets in breast cancer cells.

IKK{varepsilon} K38A reduces breast cancer cell growth in soft agar and colony formation in Matrigel. Because the aberrant expression of NF-{kappa}B cancer cells has been implicated in promoting anchorage-independent growth (10, 42), Hs578T cells stably expressing IKK{varepsilon} K38A or pCDNA3 were used to assess the functional role of IKK-i/IKK-{varepsilon} in this measure of transformed phenotype. Western blotting and reporter assays confirmed the presence of IKK{varepsilon} K38A and the ability of IKK{varepsilon} K38A stably expressing cells to reduce NF-{kappa}B-driven reporters (data not shown). Cells were plated, in triplicate, and assayed for their ability to grow in soft agar. As seen in Fig. 6E, expression of IKK{varepsilon} K38A resulted in a dramatic reduction in colony number, as judged by counting using a dissecting microscope. These data indicate that the induction of IKK-i/IKK{varepsilon} in breast cancer cells promotes a transformed phenotype.

Lastly, we created stably expressing IKK{varepsilon} K38A NF639 cell lines to perform qualitative Matrigel invasion assays. Cells expressing pCDNA3 grow invasively in Matrigel, whereas cells stably expressing IKK{varepsilon} K38A show a much less invasive phenotype (Fig. 6F). Inhibition of IKK-i/IKK{varepsilon} in these mammary cancer cells reduces invasiveness of HER-2/neu-transformed tumor cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we show for the first time the functional role of IKK-i/IKK{varepsilon} in the aberrant activation of NF-{kappa}B in breast cancer and implicate CK2 in the induction of IKK-i/IKK{varepsilon} mRNA and protein levels. IKK-i/IKK{varepsilon} expression was detected in primary human breast tumor specimens and cell lines and in mammary tumors induced by DMBA treatment or that appeared in MMTV-CK2{alpha} and MMTV-c-rel transgenic animals and in MMTV-CK2{alpha} x MMTV-c-rel bitransgenic mice. We noted that histologically normal mammary glands of MMTV-CK2{alpha} and MMTV-CK2{alpha} x MMTV-c-rel bitransgenic mice also displayed high levels of IKK-i/IKK{varepsilon} expression, whereas mammary glands from MMTV-c-rel transgenic mice did not, suggesting a role for CK2 activity in regulation of IKK-i/IKK{varepsilon}. Of interest, elevated CK2{alpha} expression was seen in mammary tumors from DMBA-treated mice compared with the normal mammary glands of parous untreated control animals.4 Ectopic expression of CK2 subunits induced IKK-i/IKK{varepsilon} levels. Conversely, inhibition of CK2 in breast cancer lines reduced endogenous IKK-i/IKK{varepsilon} levels. Previous studies from our lab have shown that a hallmark of breast cancer is the aberrant activation of NF-{kappa}B, which promotes tumor cell survival, growth, and transformed phenotype (6, 10, 43). Consistent with these observations, inhibition of IKK-i/IKK{varepsilon} reduced the ability of breast cancer cells to grow in soft agar and form colonies in Matrigel. Thus, our findings implicate the induction of IKK-i/IKK{varepsilon} by CK2 as a new signaling pathway in the activation of NF-{kappa}B and identify IKK-i/IKK{varepsilon} as a potential new chemotherapeutic target.

IKK-i/IKK{varepsilon} was first identified as a murine IKK-like protein with ~30% amino acid identity to IKK{alpha} and IKKß in the kinase domain and expression inducible with lipopolysaccharide (LPS) treatment (25). Subsequently, the human form of IKK-i/IKK{varepsilon} was also identified, cloned, and shown to display a marked increase in activity, but not expression, upon treatment with the protein kinase C activator phorbol 12-myristate 13-acetate (24). In our studies, a lack of strict correlation was noted in the level of detectable protein compared with activity of IKK-i/IKK{varepsilon} in D3-1 and Hs578T cells, suggesting the possible role of additional activation steps. Because of the sequence similarities between IKK-i/IKK{varepsilon} and IKK{alpha} and IKKß, it was tested for its ability to phosphorylate I{kappa}B-{alpha} and found to specifically phosphorylate Ser36 of I{kappa}B-{alpha} and not Ser32. It was believed that the IKK-i/IKK{varepsilon} kinase makes up part of an alternate IKK complex that lead to NF-{kappa}B/Rel activation. Recent studies have indicated that this complex does not play a role in toll-like receptor-mediated induction of NF-{kappa}B that has been implicated in LPS signaling because LPS treatment activated NF-{kappa}B/Rel in IKK-i–/– MEFs (44). However, these findings do not rule out a role for IKK-i/IKK{varepsilon} in NF-{kappa}B signaling in other systems. Our data shows that IKK-i/IKK{varepsilon} plays a role in aberrant constitutive NF-{kappa}B activation in breast cancer, suggesting that its activation may be organ or signal specific.

Our results confirm the ability of IKK-i/IKK{varepsilon} to phosphorylate I{kappa}B-{alpha} in vitro, and we also showed its ability to regulate I{kappa}B-{alpha} levels and NF-{kappa}B activity in cells in culture. In contrast, the steady-state levels of I{kappa}B-ß (and those of I{kappa}B-{varepsilon}) were much less affected by ectopic expression of IKK{varepsilon} or IKK{varepsilon} K38A in vivo, although we have observed that IKK-i/IKK{varepsilon} can also phosphorylate I{kappa}B-ß in vitro (data not shown). Overall, our findings suggest that I{kappa}B-{alpha} is a preferential in vivo I{kappa}B target of IKK-i/IKK{varepsilon}. Consistent with previous work (24), we observed that the inhibition by IKK{varepsilon} K38A led to a decrease in activities of the NF-{kappa}B element–driven promoters cyclin D1 and relB. IKK-i/IKK{varepsilon} (in addition to other kinases) has recently been reported as a kinase capable of phosphorylating the p65 NF-{kappa}B subunit at Ser536, which may enhance its ability to bind to the interleukin-8 promoter (28). Thus, IKK-i/IKK{varepsilon} may act on multiple levels to regulate NF-{kappa}B activity. Recent studies also showed that IKK-i/IKK{varepsilon} had the ability to phosphorylate c-Jun and affect downstream target genes, mmp-3 and mmp-13 (45), suggesting IKK-i/IKK{varepsilon} involvement in multiple signaling pathways (25, 44–48). Of note, we have recently shown that c-Jun regulates relB expression (36); thus, it is conceivable that IKK-i/IKK{varepsilon} may work on multiple targets (NF-{kappa}B and activator protein) in regulating relB gene expression in breast cancer.

Studies from our lab have shown that transforming growth factor-ß1 (TGF-ß1) treatment reduces NF-{kappa}B activity in breast cancer cells (43). TGF-ß1 treatment of NMuMG normal murine mammary epithelial cells leads to a detectable suppression of basal IKK-i/IKK{varepsilon} mRNA expression by 1 hour and to a nearly complete suppression between 6 and 24 hours (49). Consistent with a role for CK2 in regulating IKK-i/IKK{varepsilon}, hepatocytes treated with TGF-ß1 show reduced CK2 activity and stabilization of I{kappa}B-{alpha} levels (50). Breast cancer cell lines and tumor specimens have been shown to express elevated levels of CK2 (9, 10, 22, 23, 50). Taken together, these results suggest that IKK-i/IKK{varepsilon} may be affected by TGF-ß1 treatment through its effects on CK2 activity, and suppression of IKK-i/IKK{varepsilon} expression may further enhance I{kappa}B-{alpha} stability. Overall, our studies identify a novel role for IKK-i/IKK{varepsilon} in NF-{kappa}B/Rel activation in breast cancer.


   Acknowledgments
 
Grant support: Department of Army grant DAMD 17-01 10158 (S.F. Eddy) and NIH grants P01 ES11624 (G.E. Sonenshein and D.C. Seldin) and RO1 CA71796 (D.C. Seldin).

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 T. Maniatis, D. Litchfield, R. Pestell, R. Rawadi, C. Paya (Mayo Clinic, Rochester, MN), G. Bren (Mayo Clinic), S. Hann, N. Rice, and M. Ernst for generously providing cloned DNAs and antibodies.


   Footnotes
 
4 N. Currier et al. Oncogenic signaling pathways activated in DMBA-induced mouse mammary tumors. Toxicol Pathol (in press). Back

5 S. Guo, unpublished observations. Back

Received 5/ 9/05. Revised 8/27/05. Accepted 9/15/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Siebenlist U, Franzoso G, Brown K. Structure, regulation and function of NF-{kappa}B. Annu Rev Cell Biol 1994;10:405–55.[CrossRef][Medline]
  2. Rayet B, Gelinas C. Aberrant rel/nfkb genes and activity in human cancer. Oncogene 1999;18:6938–47.[CrossRef][Medline]
  3. Smith MR, Greene WC. Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes. Genes Dev 1990;4:1875–85.[Abstract/Free Full Text]
  4. Duyao MP, Kessler DJ, Spicer DB, et al. Transactivation of the c-myc promoter by human T cell leukemia virus type 1 tax is mediated by NF {kappa} B. J Biol Chem 1992;267:16288–91.[Abstract/Free Full Text]
  5. Duyao MP, Buckler AJ, Sonenshein GE. Interaction of an NF-{kappa} B-like factor with a site upstream of the c-myc promoter. Proc Natl Acad Sci U S A 1990;87:4727–31.[Abstract/Free Full Text]
  6. Sovak MA, Bellas RE, Kim DW, et al. Aberrant nuclear factor-{kappa}B/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997;100:2952–60.[Medline]
  7. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ, Jr., Sledge GW, Jr. Constitutive activation of NF-{kappa}B during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17:3629–39.[Abstract]
  8. Kim DW, Sovak MA, Zanieski G, et al. Activation of NF-{kappa}B/Rel occurs early during neoplastic transformation of mammary cells. Carcinogenesis 2000;21:871–9.[Abstract/Free Full Text]
  9. Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Traish AM, Mercurio F, Sonenshein GE. Roles of IKK kinases and protein kinase CK2 in activation of nuclear factor-{kappa}B in breast cancer. Cancer Res 2001;61:3810–8.[Abstract/Free Full Text]
  10. Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Sonenshein GE. Protein kinase CK2 promotes aberrant activation of nuclear factor-{kappa}B, transformed phenotype, and survival of breast cancer cells. Cancer Res 2002;62:6770–8.[Abstract/Free Full Text]
  11. Yamaoka S, Courtois G, Bessia C, et al. Complementation cloning of NEMO, a component of the I{kappa}B kinase complex essential for NF-{kappa}B activation. Cell 1998;93:1231–40.[CrossRef][Medline]
  12. Rothwarf DM, Zandi E, Natoli G, Karin M. IKK-{gamma} is an essential regulatory subunit of the I{kappa}B kinase complex. Nature 1998;395:297–300.[CrossRef][Medline]
  13. Barroga CF, Stevenson JK, Schwarz EM, Verma IM. Constitutive phosphorylation of I {kappa} B {alpha} by casein kinase II. Proc Natl Acad Sci U S A 1995;92:7637–41.[Abstract/Free Full Text]
  14. Lin R, Beauparlant P, Makris C, Meloche S, Hiscott J. Phosphorylation of I{kappa}B{alpha} in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol Cell Biol 1996;16:1401–9.[Abstract]
  15. Guerra B, Issinger OG. Protein kinase CK2 and its role in cellular proliferation, development and pathology. Electrophoresis 1999;20:391–408.[CrossRef][Medline]
  16. Allende JE, Allende CC. Protein kinases. 4. Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation. FASEB J 1995;9:313–23.[Abstract/Free Full Text]
  17. Keller DM, Zeng X, Wang Y, et al. A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol Cell 2001;7:283–92.[CrossRef][Medline]
  18. Kato T, Jr., Delhase M, Hoffmann A, Karin M. CK2 is a C-terminal I{kappa}B kinase responsible for NF-{kappa}B activation during the UV response. Mol Cell 2003;12:829–39.[CrossRef][Medline]
  19. Schwarz EM, Van Antwerp D, Verma IM. Constitutive phosphorylation of I{kappa}B{alpha} by casein kinase II occurs preferentially at serine 293: requirement for degradation of free I{kappa}B{alpha}. Mol Cell Biol 1996;16:3554–9.[Abstract]
  20. Pando MP, Verma IM. Signal-dependent and -independent degradation of free and NF-{kappa} B-bound I{kappa}B{alpha}. J Biol Chem 2000;275:21278–86.[Abstract/Free Full Text]
  21. McElhinny JA, Trushin SA, Bren GD, Chester N, Paya CV. Casein kinase II phosphorylates I {kappa} B {alpha} at S-283, S-289, S-293, and T-291 and is required for its degradation. Mol Cell Biol 1996;16:899–906.[Abstract]
  22. Landesman-Bollag E, Romieu-Mourez R, Song DH, Sonenshein GE, Cardiff RD, Seldin DC. Protein kinase CK2 in mammary gland tumorigenesis. Oncogene 2001;20:3247–57.[CrossRef][Medline]
  23. Landesman-Bollag E, Song DH, Romieu-Mourez R, et al. Protein kinase CK2: signaling and tumorigenesis in the mammary gland. Mol Cell Biochem 2001;227:153–65.[CrossRef][Medline]
  24. Peters RT, Liao SM, Maniatis T. IKK{varepsilon} is part of a novel PMA-inducible I{kappa}B kinase complex. Mol Cell 2000;5:513–22.[CrossRef][Medline]
  25. Shimada T, Kawai T, Takeda K, et al. IKK-i, a novel lipopolysaccharide-inducible kinase that is related to I{kappa}B kinases. Int Immunol 1999;11:1357–62.[Abstract/Free Full Text]
  26. Nomura F, Kawai T, Nakanishi K, Akira S. NF-{kappa}B activation through IKK-i-dependent I-TRAF/TANK phosphorylation. Genes Cells 2000;5:191–202.[Abstract]
  27. Pomerantz JL, Baltimore D. NF-{kappa}B activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. EMBO J 1999;18:6694–704.[CrossRef][Medline]
  28. Buss H, Dorrie A, Schmitz ML, Hoffmann E, Resch K, Kracht M. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{{kappa}}B at serine 536 is mediated by multiple protein kinases including I{{kappa}}B kinase (IKK)-{{alpha}}, IKK{ß}, IKK{{varepsilon}}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. J Biol Chem 2004;279:55633–43.[Abstract/Free Full Text]
  29. Bonnard M, Mirtsos C, Suzuki S, et al. Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-{kappa}B-dependent gene transcription. EMBO J 2000;19:4976–85.[CrossRef][Medline]
  30. Romieu-Mourez R, Kim DW, Shin SM, et al. Mouse mammary tumor virus c-rel transgenic mice develop mammary tumors. Mol Cell Biol 2003;23:5738–54.[Abstract/Free Full Text]
  31. Calaf G, Russo J. Transformation of human breast epithelial cells by chemical carcinogens. Carcinogenesis 1993;14:483–92.[Abstract/Free Full Text]
  32. Elson A, Leder P. Protein-tyrosine phosphatase {varepsilon}. An isoform specifically expressed in mouse mammary tumors initiated by v-Ha-ras OR neu. J Biol Chem 1995;270:26116–22.[Abstract/Free Full Text]
  33. Penner CG, Wang Z, Litchfield DW. Expression and localization of epitope-tagged protein kinase CK2. J Cell Biochem 1997;64:525–37.[CrossRef][Medline]
  34. Rawadi G, Zugaza JL, Lemercier B, et al. Involvement of small GTPases in Mycoplasma fermentans membrane lipoproteins-mediated activation of macrophages. J Biol Chem 1999;274:30794–8.[Abstract/Free Full Text]
  35. Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS, Jr. NF-{kappa}B controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999;19:5785–99.[Abstract/Free Full Text]
  36. Wang X, Sonenshein GE. Induction of the RelB NF-{kappa}B subunit by the cytomegalovirus IE1 protein is mediated via Jun kinase and c-Jun/Fra-2 AP-1 complexes. J Virol 2005;79:95–105.[Abstract/Free Full Text]
  37. Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987;7:2745–52.[Abstract/Free Full Text]
  38. Brezniceanu ML, Volp K, Bosser S, et al. HMGB1 inhibits cell death in yeast and mammalian cells and is abundantly expressed in human breast carcinoma. FASEB J 2003;17:1295–7.[Abstract/Free Full Text]
  39. Flohr AM, Rogalla P, Meiboom M, et al. Variation of HMGB1 expression in breast cancer. Anticancer Res 2001;21:3881–5.[Medline]
  40. He Q, Liang CH, Lippard SJ. Steroid hormones induce HMG1 overexpression and sensitize breast cancer cells to cisplatin and carboplatin. Proc Natl Acad Sci U S A 2000;97:5768–72.[Abstract/Free Full Text]
  41. Zhang CC, Krieg S, Shapiro DJ. HMG-1 stimulates estrogen response element binding by estrogen receptor from stably transfected HeLa cells. Mol Endocrinol 1999;13:632–43.[Abstract/Free Full Text]
  42. Pianetti S, Guo S, Kavanagh KT, Sonenshein GE. Green tea polyphenol epigallocatechin-3 gallate inhibits HER-2/neu signaling, proliferation, and transformed phenotype of breast cancer cells. Cancer Res 2002;62:652–5.[Abstract/Free Full Text]
  43. Sovak MA, Arsura M, Zanieski G, Kavanagh KT, Sonenshein GE. The inhibitory effects of transforming growth factor ß1 on breast cancer cell proliferation are mediated through regulation of aberrant nuclear factor-{kappa}B/Rel expression. Cell Growth Differ 1999;10:537–44.[Abstract/Free Full Text]
  44. Kravchenko VV, Mathison JC, Schwamborn K, Mercurio F, Ulevitch RJ. IKKi/IKK{varepsilon} plays a key role in integrating signals induced by pro-inflammatory stimuli. J Biol Chem 2003;278:26612–9.[Abstract/Free Full Text]
  45. Sweeney SE, Hammaker D, Boyle DL, Firestein GS. Regulation of c-Jun phosphorylation by the I {kappa} B kinase-epsilon complex in fibroblast-like synoviocytes. J Immunol 2005;174:6424–30.[Abstract/Free Full Text]
  46. McWhirter SM, Fitzgerald KA, Rosains J, Rowe DC, Golenbock DT, Maniatis T. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci U S A 2004;101:233–8.[Abstract/Free Full Text]
  47. Peters RT, Maniatis T. A new family of IKK-related kinases may function as I {kappa} B kinase kinases. Biochim Biophys Acta 2001;1471:M57–62.[Medline]
  48. Sharma S, tenOever BR, Grandvaux N, Zhou GP, Lin R, Hiscott J. Triggering the interferon antiviral response through an IKK-related pathway. Science 2003;300:1148–51.[Abstract/Free Full Text]
  49. Xie L, Law BK, Aakre ME, et al. Transforming growth factor ß-regulated gene expression in a mouse mammary gland epithelial cell line. Breast Cancer Res 2003;5:R187–98.[CrossRef][Medline]
  50. Cavin LG, Romieu-Mourez R, Panta GR, et al. Inhibition of CK2 activity by TGF-ß1 promotes I{kappa}B-{alpha} protein stabilization and apoptosis of immortalized hepatocytes. Hepatology 2003;38:1540–51.[Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Zhang, X. Wu, A. J. Lee, W. Jin, M. Chang, A. Wright, T. Imaizumi, and S.-C. Sun
Regulation of I{kappa}B Kinase-related Kinases and Antiviral Responses by Tumor Suppressor CYLD
J. Biol. Chem., July 4, 2008; 283(27): 18621 - 18626.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Belguise, S. Guo, S. Yang, A. E. Rogers, D. C. Seldin, D. H. Sherr, and G. E. Sonenshein
Green Tea Polyphenols Reverse Cooperation between c-Rel and CK2 that Induces the Aryl Hydrocarbon Receptor, Slug, and an Invasive Phenotype
Cancer Res., December 15, 2007; 67(24): 11742 - 11750.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Belguise, S. Guo, and G. E. Sonenshein
Activation of FOXO3a by the Green Tea Polyphenol Epigallocatechin-3-Gallate Induces Estrogen Receptor {alpha} Expression Reversing Invasive Phenotype of Breast Cancer Cells
Cancer Res., June 15, 2007; 67(12): 5763 - 5770.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
V. Giroux, J. Iovanna, and J.-C. Dagorn
Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance
FASEB J, October 1, 2006; 20(12): 1982 - 1991.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Adli and A. S. Baldwin
IKK-i/IKK{epsilon} Controls Constitutive, Cancer Cell-associated NF-{kappa}B Activity via Regulation of Ser-536 p65/RelA Phosphorylation
J. Biol. Chem., September 15, 2006; 281(37): 26976 - 26984.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eddy, S. F.
Right arrow Articles by Sonenshein, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eddy, S. F.
Right arrow Articles by Sonenshein, G. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online