We previously discovered that NF-κB is aberrantly activated and promotes tumorigenesis in human HNSCC and murine SCC, (4
). 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
). 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
). 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
), 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
). 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
) 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
). CK2 has also been reported to phosphorylate S529 of the RelA/p65 subunit (17
). 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
). 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
), 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
) 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
) 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
). 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
), 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.