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We previously showed that the signal transcription factor Nuclear Factor-kappaB (NF-κB) is aberrantly activated and that inhibition of NF-κB induces cell death and inhibits tumorigenesis in Head and Neck Squamous Cell Carcinomas (HNSCC). Thus, identification of specific kinases underlying the activation of NF-κB could provide targets for selective therapy. Inhibitor KappaB Kinase (IKK) is known to activate NF-κB by inducing N-terminal phosphorylation and degradation of its endogenous inhibitor, Inhibitor-κB (IκB). Casein Kinase II (CK2) was previously reported to be over expressed in HNSCC cells, and to be a C-terminal IκB kinase, but its relationship to NF-κB activation in HNSCC cells is unknown. In this study, we examined the contribution of IKK and CK2 in the regulation of NF-κB in HNSCC in vitro. NF-κB activation was specifically inhibited by kinase dead mutants of the IKK1 and IKK2 subunits or siRNA targeting the β subunit of CK2. CK2 and IKK kinase activity, as well as NF-κB transcriptional activity, were shown to be serum responsive, indicating that these kinases mediate aberrant activation of NF-κB in response to serum factor(s) in vitro. Recombinant CK2α was shown to phosphorylate rIKK2, as well as to promote immunoprecipitated IKK complex from HNSCC to phosphorylate the N-terminal S32/S36 of IκBα. We conclude that the aberrant NF-κB activity in HNSCC cells in response to serum is partially through a novel mechanism involving CK2 mediated activation of IKK2, making these kinases candidates for selective therapy to target the NF-κB pathway in HNSCC.
The Nuclear Factor-κB (NF-κB/Rel) proteins, a family of nuclear transcription factors and their inhibitors (IκBs), are normally involved in regulating a wide range of intracellular processes including response to oxidative stress, inflammation, growth factors, injury, and programmed cell death (1). Aberrant NF-κB activation has been detected in a variety of cancers, including head and neck squamous cell carcinomas (HNSCC) (1–3). We discovered that NF-κB1/RelA (p50/p65) activation is increased with tumor progression in murine SCC, and in cell lines and tumor specimens from patients with HNSCC (4–8). Increased nuclear staining of the phospho-activated form of p65 has recently been demonstrated in the majority of high grade squamous dysplasias and HNSCC specimens from patients, and correlated with decreased survival, supporting the wider importance of NF-κB in tumor development and clinical outcome of HNSCC (9). We have also demonstrated that NF-κB is an important modulator of the altered pattern of gene expression and malignant phenotype (10), indicating that NF-κB activation is an important target for therapy. Specific inhibition of NF-κB by over-expression of an IκBα with S32/S36 phosphorylation site mutations (IκBαM) restored altered gene expression, and inhibited SCC cell proliferation, survival, migration, angiogenesis, tumorigenesis and radiation resistance in experimental models (7, 10, 11). Bortezomib, an inhibitor of proteasome and IκB degradation, had similar effects in preclinical and a recent phase I clinical study (8, 12). However, the extent of inhibition of the proteasome, NF-κB, and tumor response was limited by the dose of bortezomib achievable without significant side effects (8). Thus, identification of the specific kinases underlying the signal phosphorylation of IκBα and activation of NF-κB could provide more selective targets for therapy in patients with HNSCC.
The eponymous Inhibitor-KappaB Kinase (IKK) plays a critical role in activating NF- κB-mediated events in cell survival and the immune response (2). The classical IKK complex and pathway is comprised of IKK1 (IKKα), IKK2 (IKKβ) and NEMO (IKKγ) subunits. An alternate pathway involving IKK1 has also been described (2). In normal cells, the classical IKK complex plays a critical role in integrating responses to various stimuli, resulting in IKK2- mediated phosphorylation of S32/S36 in the N-terminus of IκBα, and NF-κB activation. Casein Kinase II (CK2) is another stress activated protein kinase that participates in the transduction of signals that promote cell growth and survival (13), and has been implicated in NF-κB activation (14–18). In distinction to IKK2, CK2 has been shown to directly phosphorylate the C-terminal PEST domain of IκBα, and has been recently shown to be a C-terminal IκBα kinase responsible for ultraviolet light (UV) induced NF-κB activation (14–16). CK2 may also promote transactivation by phosphorylation of the RelA subunit of NF-κB (17, 18). CK2 is over-expressed and activated in HNSCC cell lines and tumor specimens, and associated with poor prognosis (19, 20). Furthermore, anti-sense RNA targeting CK2α was found to inhibit proliferation of an HNSCC cell line (21), similar to our findings with inhibition of NF-κB. These observations suggested a potential relationship and a role for CK2 in upstream signaling and activation of NF-κB in HNSCC.
The signals mediating NF-κB activation in HNSCC have not been elucidated. CK2, IKK and NF-κB may be activated in response to a variety of cytokines, growth, and serum factors (13, 17, 18, 22–26). In the present study, we examined the contribution of CK2, IKK and serum to aberrant NF-κB activity in HNSCC.
HNSCC cell lines from the University of Michigan Squamous Cell Carcinoma (UM-SCC) series were obtained from Dr. T.E. Carey, University of Michigan and previously described (27). All UM-SCC cell that are used for this study were maintained in MEM medium (Invitrogen, Carlsbad, CA) with 10% FBS. Supplemented Serum Free Medium (SFM) was the same MEM medium supplemented with Non-Essential Amino Acids, Insulin-Transferrin-Selenium-G, Vitamin Solution, Fibronectin, (Invitrogen, Carlsbad, CA), and trace elements (Biofluid, Camarillo, California). Human keratinocytes (HEKa) were purchased and maintained in supplemented 154 CF Medium with 200 mM CaCl2 (Cascade Biologics Inc. Portland, OR). All cells were maintained at 37°C in 5% CO2 atmosphere.
Anti-IKK2 and CK2β antibodies were purchased from BD Pharmingen (San Jose, CA). Anti IκBα, IKK1 and -NEMO antibodies were purchased from Santa Cruz (Santa Cruz, CA). Wild type and mutant IκBα-GST fusion proteins and pcDNA3-IKK vectors were kind gifts from Dr. Keith Brown, National Institute of Allergy and Infectious Disease, NIH.
10 μg protein of whole cell lysates were used for each assay with standard Immunobloting procedures. Total proteins that were transferred onto the membrane were stained and quantified for equal loading control and for normalizing the intensities of the detected specific protein bands.
Nuclear extracts were obtained by using a Nuclear Extract Kit (Active Motif, Carlsbad, CA). 5μg nuclear extract was used for EMSA with 32P-NF-κB response element oligonucleotide (Sense: 5’-AGT TGA GGG GAC TTT CCC AGG C-3’, antisense: 3’-TCA ACT CCC CTG AAA GGG TCC G-5’, Promega Cat#: E3292, Madison, WI). 32P-OCT-1 was used as a positive control for loading.
Cells were transfected by jetPEI complexed with control or cis-pNF-κB-luc cis-reporter plasmids (PathDetect® in Vivo Signal Transduction Pathway cis-Reporting Systems NF-κB (5×) [TGGGGACTTTCCGC], Cat#: 219077, Stratagene, San Diego, CA, http://www.stratagene.com/manuals/219073.pdf). After 24 hours, luciferase activities were measured with a dual-luciferase system from Promega (San Luis Obispo, CA) using a Vector II spectrophotometer (Perkin Elmer, Boston, MA). pCIS-CK plasmid was employed as a negative control for the pNF-κB-luc cis-reporter plasmid while pFC-MEKK plasmid (both obtained from Stratagene, San Diego, CA) was co-transfected with pNF-κB-luc cis-reporter plasmid to serve as a positive control. The firefly-luciferase activities of triplicate samples were averaged following normalization by corresponding renilla-luciferase activity co-transfected in the same wells. The NF-κB-luciferase activity is presented as the ratio of NF-κB-luc/CK-luc.
Cells were transfected with CK2β-Stealth-RNAi (Sense: 5’-CCAGCAACUUCAAGAGCCCAGUCAA, Anti-Sense 5’-UUGACUGGGCUCUUGAAGUUGACGG), a corresponding Scramble Stealth RNAi control (Sense 5’-CCACAACUUGAACGAACCUGGCCAA, Anti-Sense 5’-UUGGCCAGGUUCGUUCAAGUUGUGG) (both designed by Invitrogen BLOCK-iT™ RNAi Designer https://rnaidesigner.invitrogen.com/rnaiexpress/Invitrogen, Carlsbad, CA), or a universal negative control siRNA (Cat# 4611, Ambion, Austin, TX). and Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 48 hours followed by evaluation assays. Transfection efficiency and cell viability were monitored by FITC labeled RNA oligo, DAPI and Dead Cell Dye respectively (Invitrogen, Carlsbad, CA).
IκBα, NFKB2/p100 and CK2β mRNA levels from the cells were quantified by QuantiGene Assay kit with probe sets designed to target each of these mRNAsspecifically (Genospectra, Fremont, CA). The results were normalized to corresponding 18sRNA levels.
Cells were lysed in 25mM HEPES, 150mM NaCl, 1% Triton X100, 10% glycerol, 5mM EDTA, 2mM DTT, and protease and phosphotase inhibitor cocktails. IKK was immunoprecipitated (IP) from cell lysates by polyclonal anti-NEMO antibody (Santa Cruz, Santa Cruz, CA). Kinase assay was performed in kinase buffer (20mM HEPES, pH7.5, 10mM MgCl2, 1mM DTT, 1mM sodium ortho vanadate, 25mM β-glycerol phosphate) by incubating IKK complex with either N-terminal 72 amino acid of wild-type IκBα (S32/S36)-GST or mutated IκBα (S32G/S36A)-GST fusion proteins as substrate at 30°C for 30 minutes. The products from the kinase reaction were separated on a 4–12% SDS Gradient gel and, then transferred onto a nitrocellulose membrane. IKK kinase activity was quantified by the intensity of the 33P-IκBα-GST band detected by autoradiography and normalized as a ratio to the corresponding quantified protein levels of IκBα and, then, IKK2 detected from immunolblot analysis on the same membrane.
Cell lysates were prepared as described above for IKK Kinase Assay. CK2 kinase activity was measured by using a CK2 Kinase Assay Kit with a CK2 specific peptide substrate (Upstate, Charlottesville, VA; for details refer to http://www.upstate.com/img/coa/17-132-18642.pdf). 20 μg total protein of the lysate from each reaction was incubated along with a PKA inhibitor and [γ33P]-ATP in assay dilution buffer for 10 minutes at 30°C. The phosphorylated substrate is then separated by P81 phosphocellulose paper and quantified by a scintillation counter. Active CK2 enzyme was used as positive control. Spike assay was also employed to ensure accuracy.
We previously showed that NF-κB is activated in several human HNSCC cell lines from the University of Michigan Squamous Cell Carcinoma (UM-SCC) series (6, 7). To select representative cell lines for further analysis, we compared NF-κB DNA binding activity in a panel of 9 UM-SCC cell lines derived from 7 patients, and in non-malignant human keratinocytes (HEKa). Increased NF-κB DNA binding activity was detectable in 8/9 UM-SCC cell lines from 6/7 patients relative to HEKa (Fig. 1A), similar to the frequency of increased NF- κB activation observed in HNSCC tumor relative to normal squamous mucosa specimens (9). To determine if variations in DNA binding are reflected by variations in functional activation, NF-κB luciferase reporter activity was compared in UM-SCC-1, -6, -9 and -11A and B cell lines. Variable NF-κB reporter activities were observed in these cells (Fig. 1B). Thus, UM-SCC cell lines demonstrated aberrant NF-κB activation, with a prevalence and variability similar to that reported for HNSCC tumor specimens (8, 9).
UM-SCC-6 showed significantly higher NF-κB reporter activity when compared to the other cell lines (p<0.01) while UM-SCC-9 showed significantly lower NF-κB reporter activity compared to the other cell lines (p<0.05 between UM-SCC-11B and UM-SCC-9, and p< 0.01 to the other three cell lines). Protein levels of IκBα, a protein both induced by and degraded with activation of NF-κB, were compared between UM-SCC-6 and UM-SCC-9 cells to determine the relative differences in NF-κB activation (Fig 1 C). Cells were treated with 50μg/ml cycloheximide for variable times as indicated. IκBα levels were quantified by the intensity of the band on immunoblot and normalized to total protein levels detected on the membrane. Fig. 1C shows that the endogenous IκBα protein expression level in UM-SCC-6 cells is ~1.8 fold that in UM-SCC-9 cells, consistent with the relatively greater activation of NF-κB detected in UM-SCC-6 cells. The data are representative of 2 independent experiments. UM-SCC-6 cells also showed a ~50% shorter IκBα half-life (T½ ~1.5 vs. 3 hrs, Fig. 1C), and 2-fold difference in IKK kinase activity for phosphorylation of S32/S36 IκBα compared to UM-SCC-9 cells (Fig1 D). These data confirm that UM-SCC-6 cells exhibit greater NF-κB activity, and signal kinase activation by IKK than UM-SCC-9 cells. Based on all of these observations, we chose UM-SCC-6 and 9 for further studies based upon the range of strong and weak NF-κB activation detected in these two cell lines.
The IKK complex has previously been shown to mediate phosphorylation of IκBα and regulate canonical activation of NF-κB by proinflammatory cytokines (13, 28). The role of IKK in aberrant NF-κB activation in HNSCC was examined by co-transfection of amino acid substituted dominant positive (S176E/S180E, IKK1EE and S177E/S181E, IKK2EE), dominant negative (S176A/S180A, IKK1AA and S177A/S181A, IKK2AA), Kinase dead IKK (K44A, IKK1KA and IKK2KA), or wild type IKK1 or IKK2 vectors together with cis-pNF-κB-luc in UM-SCC-6 cells (28). Fig. 2 A and B show that constitutively active IKK1EE and IKK2EE induced over a 20-fold increase in NF-κB reporter activity compared to that of control cells (p<0.001). Co-transfection and expression of additional wild-type IKK1 or IKK2 induced a ~1.5 (p<0.05) and 4-fold (p<0.001) increase of NF-κB activity respectively. Thus, IKKEE and wild-type both increased NF-κB activity, serving as a positive control for the transfection and responsiveness of the cells. In contrast, the IKK2KA partially reduced the NF-κB reporter activity by ~65% (p<0. 001) while IKK1KA reduced activity by ~50% (p<0.001). The partial inhibition of NF-κB reporter activity observed with either kinase dead IKK1KA or IKK2KA in UMSCC-6 cells is consistent with the partial ~50–80% inhibition we have observed in repeated experiments with IKK1KA or IKK2KA, IKK1 or IKK2 siRNA, or IKK2 inhibitor PS-1145 in UMSCC-11A and 11B (L. Nottingham, J. Ricker, unpublished observations). The contribution of both IKK1 and IKK2 to NF-κB activation is also supported by their independent roles in processing of p100 to p52 (29) and degradation of IκBs (28), both of which were observed after cycloheximide treatment in UM-SCC-6 cells (Fig. 1C and data not shown). In addition, co-transfection of the phosphoacceptor mutants IKK2AA did not inhibit NF-κB activity (p>0.10) and that of IKK1 AA increased NF-κB activity (p<0.05) in UM-SCC-6 (Fig. 2 A and B), suggesting that activation of these IKKs and NF-κB may occur via a mechanism other than that previously reported for classical NF-κB activation by TNF and IL-1 (28).
CK2 has been reported to induce NF-κB activation (14–18). Elevated CK2 activity has been demonstrated in HNSCC and linked to cell proliferation and patient survival (19–21). Treatment of UM-SCC cells with apigenin, an agent that inhibits CK2 kinase activity (30), strongly inhibited NF-κB reporter activity in all 4 UM-SCC cell lines (Fig. 3A, UM-SCC-6 and -11A p<0.001, UM-SCC-9 and -11B p<0.05). Depletion of the regulatory subunit CK2β has previously been shown to inhibit CK2 mediated activation of NF-κB by UV light (16). To examine whether CK2 specifically contributes to aberrant NF-κB activity, small interference RNA (siRNA) targeting CK2β was used. Transfection of UM-SCC-6 and UM-SCC-9 cells with human CK2β siRNA resulted in a ~90% decrease of CK2β mRNA in UM-SCC-6 cells and ~75% decrease of that in UM-SCC-9 cells (Fig. 3B for UM-SCC-6 cells p<10−9 and C, for UM- SCC-9 cells p<10−5). The decreased expression of CK2β resulted in a corresponding 82% reduction in NF-κB reporter activity in UM-SCC-6 (Fig. 3D, p<0.05). We also observed an ~60% reduction of NF-κB reporter activity in UM-SCC-9 cells, but this did not reach the level of statistical significance, with the variance observed at the low level of activation detected in this cell line (Fig. 3E, p=0.09). The inhibition of NF-κB reporter activity observed was not due to non-specific inhibition of mRNA expression by siRNA or transfection, as no significant reduction was observed with unrelated or scrambled control siRNAs. Consistent with CK2β siRNA inhibitory effect on NF-κB reporter activity, we also observed a decrease in expression of NF-κB inducible genes IκBα (58%, p<10−6) and NFKB2 (68%, p<10−7) in anti-CK2β siRNA transfected UM-SCC-6 cells, when compared to that of CK2β scramble control siRNA transfected cells (Fig 3 F). Comparable cell viability among different siRNA treatments during the assay was monitored by DAPI staining and propidium iodide staining to exclude the possibility that effects were the result of cell death (data not shown). Together with data using inhibitor apigenin, these siRNA data indicate that CK2 is involved in increased NF-κB activation and expression of its target genes in UM-SCC 6 cells.
Aberrant NF-κB activation in response to growth factor receptors has previously been observed in breast cancer (31, 32). To explore the contribution of serum to the aberrant activation of NF- κB in UM-SCC cells, we examined the effects upon NF-κB reporter activity of replacing FBS with supplemented serum-free media (SFM). A panel of four UM-SCC lines newly changed to SFM (SFM-new) or serially adapted to SFM (SFM-adp) before transfection, each showed a progressive reduction of NF-κB reporter activity when compared to cells grown in 10% FBS that is not heat-inactivated (FBS-NHI) (Fig.4A, p< 0.01 for both SFM-new and SFM-adp comparing to FBS-NHI; p<0.01 between SFM-new and SFM-adp). Accordingly, ~50% decrease of endogenous IκBα mRNA level was also observed in SFM-adp UM-SCC-6 cells comparing to that in FBS-NHI cells (p<10−5, data not shown). Heat-inactivation of FBS (FBS-HI) to 56°C also demonstrated a significant decrease in NF-κB reporter activity (Fig. 4A, p<0.01 when compared to FBS-NHI). The NF-κB reporter activities from the cells that were grown in FBS-HI are also significantly higher than those in SFM-adp (p<0.01), but were not statistically different when compared to those grown in SFM-new (p>0.05). Co-transfection and expression of MEKK, an up-steam activator of NF-κB, increased NF-κB reporter activity under each of the conditions, indicating that NF-κB in UM-SCC cells remained responsive to signal activation (data not shown). The results shown here represent the average of 4 experiments. Two different sources of serum were employed to exclude the possibility of single batch effects of the serum. Together, these results indicate that NF-κB activation is increased in UM-SCC cells in response to serum, and that these serum components are temperature sensitive.
CK2 is known to mediate signal responses to serum (22–26) and we observed that the extent of reduction of NF-κB reporter activity, in the absence of serum, was similar to that observed following inhibition of CK2 by either apigenin or CK2β siRNA. This led to the hypothesis that CK2 may mediate the activation of NF-κB by serum component(s). To examine this hypothesis, we first compared CK2 kinase activities by a CK2 specific peptide substrate from UM-SCC-6 cells that were cultured either in FBS or SFM. CK2 activity from cell lysate that were cultured in SFM was significantly reduced by 50% compared to that in FBS (p<0.001 Fig 4B). Applying apigenin resulted in significant further reduction of CK2 kinase activity in both of the cultures (p<0.05) (Fig 4B) and strongly inhibited a positive control with recombinant CK2α alone. A PKA inhibitor was employed to block non-specific kinase activity in these assays. Spiking cell lysates with a known amount of rCK2α demonstrated a ~70% rate of recovery of CK2 activity by the assay (data not shown). Additionally, we also detected an statistically significant ~30% decrease of CK2β (p<0.01, Fig 4 C) and an ~50% decrease of IκBα (p<10−6, data not shown) mRNA expression level in the cells that were cultured under SFM compared to that under FBS-NHI, suggesting that a serum dependent decrease in CK2 expression contributes to reduced NF-κB activity and expression of target gene IκBα. The differences in CK2 kinase activity between SFM and FBS indicates that CK2 activity is increased in response to serum in UM-SCC-6 cells and suggests that CK2 may play an important role in mediating the increased NF-κB activity in response to serum.
In Fig. 2, we showed that co-transfection of kinase dead IKK2KA inhibited NF-κB activity in UM-SCC-6 cells by ~65% (Fig. 2A) and that SFM also reduces NF-κB activity in these cells (Fig. 4A). We next examined the effect of serum on IKK kinase activity in UM-SCC- 6 cells. IKK kinase activity from whole cell lysate of UM-SCC-6 cells cultured with FBS or SFM was measured as described in methods. A ~50% decrease of IKK kinase activity was observed from cells that were cultured in SFM when compared to that in FBS in two independent experiments (Fig 4D and data not shown). The change in IKK kinase activity is consistent with the magnitude of contribution of IKK2 to NF-κB activation observed in Fig. 2. Specific phosphorylation of S32/S36 IκBα, the phospho-acceptor sites for IKK2, was ensured by comparison with a negative control S32G/S36A IκBα mutant substrate (Fig. 4D). Thus, our data indicates that IKK mediates signal phosphorylation of IκBα, and activation of NF-κB in response to serum by UM-SCC-6 cells.
Since the data above indicate that both CK2 and IKK mediate the response to serum and contribute to NF-κB activity in UM-SCC-6 cells, we wondered if CK2 could modulate IKK2 mediated phosphorylation of IκBα. In this regard, protein sequence analysis revealed 11 putative CK2 phosphorylation consensus motifs in IKK2. Based on prediction algorithms, 6 of these motifs had a score (probability to be phosphorylated by CK2) higher than 0.92 on a 0 to 1 scale (data not shown). This analysis suggested that IKK2 could be a potential substrate of CK2 and, hence, IKK kinase activity could be regulated by CK2. We examined whether IKK2 is a substrate of CK2 in an in vitro CK2 kinase assay by employing rCK2α and rIKK2 (Fig. 5A). In this assay, we used 0.5 and 5 ng of rCK2α with rIKK2 (200ng/reaction). Since CK2 can use both ATP and GTP, to ensure the CK2 specificity, we used GTP rather than ATP as a phospho-donor. Our result showed a low level of auto-phosphorylation by IKK2, which may be caused by the high concentration of rIKK2 used. This was confirmed by comparing GTP to ATP as a phospho-donor, which showed more dramatic auto-phosphorylation by IKK2 itself (data not shown). On top of this IKK2 auto-phosphorylation, we detected an rCK2α dose-dependent increase in phosphoryation of rIKK2. In the presence of 0.5ng and 5ng rCK2α, a ~40% and 2.2 fold increase of normalized phosphorylated IKK2 was observed when compared to control. Apigenin completely suppressed CK2α phosphorylation of IKK2. The result shown here is an average of two parallel measurements. Thus, these data provide direct evidence that IKK2 is a substrate of CK2.
To determine if CK2 could modulate IKK2 kinase activity as part of the IKK complex, anti-NEMO antibody immunoprecipitated IKK complexes were incubated with different amounts of catalytically active rCK2α and GTP under the conditions optimized for CK2 kinase assay, followed by washing out of CK2, prior to the IKK kinase assay. Since the substrate IκBα-GST fusion protein used only contains the N-terminal 72 amino acids, the assay excludes that CK2 phosphorylation is due to the IκBα C-terminal PEST domain. Again S32G/S36A mutant IκBα-GST was used as a negative control to ensure IKK specific phophorylation of the S32/S36 of the N-terminus IκBα. The kinase activities were normalized to protein levels of IκBα and IKK2. Fig. 5B shows the rCK2α dose dependent increase of IKK kinase activity detected as IKK mediated phosphorylation of wild type, but not S32G/S36A mutant IκBα-GST. A 13% and 60% increase of IKK kinase activities from those that were preincubated with 0.5ng and 5ng rCK2α respectyively was observed compared to that of non-treated IP-IKK complexes. When the IP-IKK complex was incubated with 5ng rCK2α in the presence of 40μM apigenin, the CK2α induced IKK kinase activity was completely blocked. A ~70% increase in IKK kinase activity was detected with saturating amounts of rCK2α (10ng and 20ng) in an independent experiment (data not shown). Since IKK2 phosphorylation of IκBα is dependent on complexing with the NEMO subunit, this data provides evidence supporting the hypothesis that CK2 may directly regulate IKK2 activity for phosphorylation of IκBα in UM-SCC-6 cells.
We previously discovered that NF-κB is aberrantly activated and promotes tumorigenesis in human HNSCC and murine SCC, (4–8, 10). Nuclear NF-κB activation has been broadly demonstrated and associated with progression in intraepithelial pre-malignant and malignant squamous neoplasms of the head and neck as well as uterine cervix (9, 33). We have shown that expression of an S32A/S36A IκBα mutant unresponsive to IKK phosphorylation strongly inhibited NF-κB activation in HNSCC and murine SCC (7, 10). Tamatani et al detected increased IKK-mediated phosphorylation of IκBα and NF-κB activation in 3 HNSCC cell lines relative to that observed in 5 primary gingival keratinocyte cultures, indicating that IKK and NF- κB are aberrantly activated together in HNSCC (34). Gapany et al showed that cytosolic CK2 expression and activity are also increased in HNSCC tumor specimens and cell lines (19). In the present study, we directly examined the hypothesis that CK2 promotes IKK mediated aberrant activation of NF-κB in HNSCC. NF-κB activation was specifically inhibited by siRNA targeting the β subunit of CK2, and kinase dead mutants of the IKK1 and IKK2 subunits, implicating both the alternative and classical IKK pathways in NF-κB activation. We found that CK2 contributes to the activation of IKK and NF-κB in response to serum factor(s). rCK2α was shown to phosphorylate rIKK2, as well as to promote immunoprecipitated IKK complex from HNSCC to phosphorylate the N-terminal S32/S36 of IκBα. We conclude that the aberrant NF-κB activity in HNSCC cells in response to serum is partially through a novel mechanism involving CK2 mediated activation of IKK2, making these kinases candidates for selective therapy to target the NF-κB pathway in HNSCC. This is the first study to identify a potential mechanism linking the independent clinical pathologic observations that CK2 is an upstream regulator of IKK and NF- κB activation in HNSCC.
Several observations from this and other studies in our laboratory suggested an alternative mechanism of activation and role of IKK in NF-κB activation in HNSCC. First, we observed that both IKK1 and IKK2 contribute to NF-κB activation. The inhibition of NF-κB reporter activity in different UM-SCC cell lines by specific inhibition of either IKK2 with kinase dead IKK2KA, IKK2 siRNA or IKK2 inhibitor PS-1145, or by that of IKK1 with IKK1KA or IKK1 siRNA along were significant, reproducible, but incomplete. While IKK2 has been shown to mediate activation of NF-κB1/Rel A (p50/p65), IKK1 has been reported to promote processing of p100 to p52 (NF-κB2), an alternative NF-κB activation pathway (29). Consistent with this, we found evidence for higher expression levels of endogenous NF-κB inducible genes IκBα and NF-κB2 (data not shown), increased degradation rate of IκBs, and processing of p100 following cycloheximide treatment in UM-SCC-6 cells data not shown). Furthermore, the IKK2AA mutant did not inhibit NF-κB activation, and the IKK1AA mutant enhanced activation of NF-κB activity. These results suggested that the signaling mediated by these IKK subunits in HNSCC may result from signal(s) and/or mechanism(s) that are distinct from those mediating classical activation of IKK and NF-κB (28).
CK2 is a highly conserved pleiotropic and ubiquitous serine and threonine kinase with a wide range of substrates involved in carcinogenesis and tumor progression. More than 300 CK2 substrates have been identified (13, 35), the majority of which are proteins that are involved in transcription, cell cycle regulation, cell proliferation, cell survival, gene expression and signal transduction. CK2 phosphorylation has been shown to inhibit apoptosis, and favor cell proliferation and oncogenic transformation (13, 36). In this study, we provide evidence that CK2 is a key mediator of overall NF-κB activation, and functions to enhance IKK kinase phosphorylation of IκBα. The CK2 inhibitor apigenin or specific siRNA targeting CK2β were sufficient to inhibit NF-κB activity, indicating that the over-expression of CK2 and increased CK2 activity found in prior studies (19, 20) may be responsible for mediating the aberrant activation of NF-κB in HNSCC.
CK2 has previously been shown to phosphorylate multiple sites in the C- terminal PEST domain of IκBα, including the response involved in ultraviolet (UV) light-induced NF-κB activation (14, 16). CK2 has also been reported to phosphorylate S529 of the RelA/p65 subunit (17, 18). In addition to these targets, we have found that catalytically active recombinant CK2α (rCK2α) can phosphorylate both recombinant IKK2 (rIKK2) or IKK1 (rIKK1, data not shown) in vitro, and that incubation of IKK complex with rCK2α resulted in increased IKK2 kinase activity for the phosphorylation of S32/S36 N-terminus of IκBα, a novel and distinct function from that of CK2 as a known C-terminal PEST domain IκB kinase. This finding is also supported by protein sequence analysis of IKK1 and 2, which reveals multiple CK2 phosphorylation motifs. Additionally, the decreased IKK kinase activity in UM-SCC-9 and SFM cultured UM-SCC-6 cells were associated with lower expression levels of CK2β in these cells, adding another line of evidence that CK2 is an upstream regulator of IKK and NF-κB activation.
CK2 has been linked to aberrant NF-κB activity in cancer cells derived from human breast, hepatic, and colon carcinomas. (30, 37–41). However the relative contribution of CK2 to NF-κB activation appears to vary among different cancers. Compared to CK2 activation of NF-κB in other type of cancer cells (30, 37–41), our CK2β siRNA data indicates a dominant effect of CK2 as a holo-enzyme on NF-κB activation in UM-SCC cells. On the other hand, our data does not rule out the possibility of direct effects of CK2 on activating the NF-κB pathway at other levels, as multiple CK2 phosphorylation motifs have been identified in every member of the NF-κB and IκB families. Thus, the presence of CK2 target sites in IκB and p65 as well as the potential sites in IKK and other NF-κB and IκB family members could explain why we observed nearly complete inhibition of NF-κB activity by blocking CK2 activity. More detailed characterization of the molecular basis of the interaction between CK2 and IKK is needed, and may lead to additional specific targets for cancer treatment.
An important finding of the present study is the demonstration that CK2 and IKK mediate the altered activation of NF-κB in response to serum factor(s). The increase in CK2 and NF-κB activation in the majority of HNSCC specimens relative to normal mucosa (9, 20) indicate that the response of these pathways to serum factor(s) is altered and precedes culture, rather than being a mere consequence of serum induction in culture. Indeed, we have shown that the increased NF-κB activity in murine SCC cells occurs with tumor progression in vivo, and favors tumorigenesis and metastasis in the host environment (5). Consistent with this, the increased nuclear localization of NF-κB observed in human squamous dysplasias has been shown to be associated with higher risk of progression in a recent clinical pathologic study (9).
The factor(s) in serum and/or the host environment that contribute to induction of CK2, IKK and NF-κB remain to be elucidated, and may provide additional targets for therapy. Autocrine factors produced by HNSCC (42, 43) or paracrine factors produced by tumor stromal fibroblasts (44) have been shown to enhance activation of NF-κB in HNSCC. A number of components contained in serum, including cytokines, growth factors, different types of albumins, zinc, copper, and free thiols, have been reported to affect NF-κB activity (13, 24–26,31, 43–49). Since the serum factors contributing to NF-κB activation in UM-SCC were heat sensitive, labile protein factors seem the most likely candidates.
We have previously evaluated the growth factor EGF and cytokine IL-1 as possible candidates (42, 43), since they are factors that are known to activate NF-κB in non-malignant and malignant cells. Although NF-κB was found to be inducible by EGF, C225, the specific antagonist of anti-EGFR antibody, inhibited only inducible, but not aberrant NF-κB activity, making the EGFR receptor an unlikely candidate for the aberrant NF-κB activation in HNSCC (42). We have previously obtained evidence that the IL-1/IL-1Receptor (IL-1R) pathway may be one of the stimuli contributing to the aberrant activation of NF-κB in HNSCC. We found that transient expression of an intracellular form of the IL-1Receptor Antagonist (IL-1RA) could significantly inhibit NF-κB reporter activity and cytokine IL-8 gene expression in UM-SCC-9 and 11B cell lines (43). Recently, interruption of the IL-1 signal pathway by expression of siRNA knocking down expression of IL-1R1, or a dominant negative mutant of the essential Toll receptor linker MyD88, was found to strongly inhibit NF-κB activation in 5/6 UM-SCC lines, including the UM-SCC-6, 9 and 11A and B cell lines (L. Bagain et al, unpublished observations).
Interestingly, a recent study in malignant keratinocyte lines suggests that the IL-1 pathway and intracellular IL-1R Antagonist may play a role in regulating signal degradation of IκBα and other transcription factors by the COP9 (CSN) signalosome that includes CK2 (50). Such an alternative pathway for activation of NF-κB by IL-1 and possibly other factors could explain the enhancement of NF-κB reporter activity by IKK1 AA and lack of inhibitory effect we observed with IKK2AA, both of which are deficient in the phosphoacceptor sites mediating classical activation of NF-κB. It would be interesting if this signalosome containing CK2 interacts with the IKK signalosome and other NF-κB pathway components. Dissection of the role of CK2 or other intermediate kinases as possible oncogenes mediating aberrant activation of IKK and NF-κB in response to exogenous factors in HNSCC may also be important to development of therapy.
In summary, we found that CK2 contributes to the activation of IKK and NF-κB in response to serum factor(s), which suggests that CK2 and IKK2 are key candidates for targeting the NF-κB pathway in HNSCC. This is the first study to identify a potential mechanism linking the independent clinical pathologic observations that CK2 and NF-κB are activated and associated with decreased prognosis in HNSCC. Further clinical and molecular studies are indicated to confirm the role and further elucidate the factor(s) and mechanisms involved in CK2 activation of IKK and NF-κB in HNSCC.
Grant support: NIDCD Intramural Research Project Z01-DC-00016 (C. Van Waes), and Howard Hughes Medical Research Institute-NIH Scholars Program (J Yeh). Critical review and comments of Keith Brown of the National Institute of Allergy and Infectious Disease, and David Gius of the National Cancer Institute are appreciated.