We show that overexpressing ΔNp63α in primary murine keratinocytes leads to the nuclear accumulation of phosphorylated, transcriptionally active c-Rel, which is required to maintain aberrant proliferation mediated by overexpressed ΔNp63α. In these cells, and in human SCC cells endogenously expressing these proteins, ΔNp63α and phospho-c-Rel physically associate in the nuclei and on the p21WAF1 promoter.
c-Rel was originally identified as the cellular counterpart of the v-Rel oncogene, known to cause lymphomas. c-Rel plays an important role in normal cellular homeostasis21,35,36
, including that of the epidermis21
, and enhanced nuclear c-Rel has been associated with solid and hematopoietic cancers41,42
. In contrast to numerous studies of the NF-κB heterodimer p50:p65, the role of c-Rel in transformation of squamous epithelium remains largely unexplored. However, several studies point to the oncogenic propensity of dysregulated c-Rel expression in other systems. Retroviral overexpression of full length wildtype c-Rel can transform primary spleen cells in vitro25
. Furthermore, forced overexpression of c-Rel in vivo
under control of the MMTV-LTR promoter resulted in mammary tumorigenesis, and correlated with induction of NF-κB target genes including c-myc
and cyclin D124
. Treatment of these c-Rel-transformed mammary tumor cells with dimethylbenzanthracene in vitro
resulted in epithelial to mesenchymal transition43
The transforming ability of c-Rel both in vitro
and in vivo
is dependent on the presence of its transactivation domain44,45
. c-Rel’s transformation capacity can be enhanced by mutations and deletions within the transactivation domain, suggesting that the strength of transactivation activity can determine the potency of c-Rel44,46
. The transactivation domain of c-Rel contains multiple phosphorylation sites and variable levels of phosphorylation have been shown to influence transactivation of distinct sets of target genes47
. In this report we show that, in addition to being phosphorylated, the c-Rel that is modulated by ΔNp63α has transcriptional NF-κB reporter-enhancing and p21 gene-repressing activity. Future studies will aim to identify the impact of sustained ΔNp63α elevation on c-Rel target gene expression.
Regulation of NF-κB is a dynamic process38–40
. In the classical paradigm of NF-κB regulation, cytoplasmic IκB proteins retain NF-κB in an inactive state, with NF-κB nuclear translocation following IκB degradation. Once within the nucleus, NF-κB induces resynthesis of IκBs, and IκBα and IκBε can dissociate NF-κB from DNA and usher it to the cytoplasm via
their nuclear export functions39,40
. IκBβ can function in its phosphorylated form to dissociate NF-κB from DNA, while unphosphorylated IκBβ forms a ternary complex with NF-κB and DNA and can protect it from dissociation by IκBα or IκBε48
. Our data support a model whereby enhanced ΔNp63α expression results in nuclear accumulation of c-Rel without disrupting IkB:c-Rel cytoplasmic interactions or causing degradation of the IκBs (). We have shown that c-Rel physically interacts with ΔNp63α in the cell nucleus and propose that this association inhibits nuclear, but not cytoplasmic, interaction of c-Rel with the IκB proteins by blocking binding. This results in enhanced nuclear accumulation of c-Rel due to the inability of IκBαand IκBε to interact with and remove c-Rel. The c-Rel that accumulates in the nuclei of ΔNp63α overexpressing cells is phosphorylated and transcriptionally active, as determined by reporter gene assay, and can interact in a complex with ΔNp63α on the p21WAF1 promoter to block promoter activity.
The physical association between ΔNp63 and phosphorylated c-Rel requires the α-COOH-terminus of ΔNp63α; like ΔNp63α, TAp63α also physically associates with c-Rel, while ΔNp63p40
does not (). Although less is understood about the role of TAp63 in cancer development, dysregulated TAp63α has been reported to influence the development and progression of chemically-induced skin tumors49
. Whether the downstream effects of TAp63α in this context are mediated by c-Rel remains to be determined.
It was initially proposed that overexpression of ΔNp63 in human cancers blocks the tumor suppressor activity of p5350
. It has recently been shown that the ability of ΔNp63α to repress p73-dependent apoptosis enhances the survival of a subset of squamous cell carcinoma cells17
. The data presented herein support a novel mechanism whereby overexpression of ΔNp63α induces dysregulation of the proto-oncogene c-Rel via
physical association, resulting in loss of normal keratinocyte growth regulation. Enhancement of transcriptionally active c-Rel and activation of downstream effectors could be a means whereby ΔNp63α influences the growth and phenotypic characteristics of human cancers. Consistent with our model, a recent clinical trial targeting constitutively active NF-κB in HNSCC via
a proteasome inhibitor was found to block nuclear localization of Rel-A, but not c-Rel (Allen et al, manuscript submitted). The findings presented here suggest that distinct NF-κB complexes can promote proliferation of keratinocytes, act in concert with other NF-κB dimers to promote an aggressive cancer phenotype, and offer novel targets and useful biomarkers for optimizing therapeutic efficacy in this subset of poorly responsive cancers.