Here we provide evidence for a new mechanism and model of interaction between members of the TP53 and NF-κB families, in co-regulating the transcriptome and phenotype in squamous epithelia of HNSCC and ΔNp63 TG mice (). ΔNp63, the predominant p63 isoform overexpressed in most HNSCC and in the basilar layers of squamous epithelia, demonstrates a versatile ability to coordinately regulate expression of a broad program of genes that overlap the NF-κB/REL and TP53/p63 transcriptomes. Furthermore, ΔNp63 displays novel interactions with NF-κB family members, c-REL and RELA, through co-binding to newly predicted and known p63 and NF-κB promoter regulatory sites of NF-κB/REL target genes. ΔNp63 promoted proliferation, cell cycle, migration, and inflammatory cytokine expression by HNSCC in vitro. These observations are consistent with studies demonstrating an increase in ΔNp63 accompanied by proliferative and integrin adhesion protein markers, as well as inflammatory cell infiltration throughout the tumor microenvironment in vivo. In addition, the overexpression of ΔNp63α in TG mice led to suprabasilar modulation of c-REL and cytokine genes, as well as the development of hyperplasia and inflammation in the epidermis. This model supports the causal function of ΔNp63 as a key transcriptional regulator of proliferation and inflammation in squamous epithelia.
Our results demonstrate a novel bifunctional role of ΔNp63 in coordinating known NF-κB or TP53 downstream genes important in proliferation and cell survival. We demonstrated that ΔNp63 siRNA inhibited gene expression of known NF-κB target genes CCND1
, and BCL-XL
which, upon individual knockdown, were previously shown to inhibit proliferation and survival of UMSCC in vitro
). ΔNp63 reciprocally repressed expression and transactivation activity of TP53 family members TP53, TAp63, and p73, as well as target genes, such as p21CIP1
which mediate growth arrest and apoptosis. ΔNp63 was previously reported as a repressor of TP53 and p73 target genes p21Cip1
in squamous epithelial, HNSCC, and breast cancer cells (14
). Predominant nuclear expression of ΔNp63 and deficiency of TAp63 was also observed in a subset of non-SCC tumors in mice heterozygous for p63 and TP53 alleles (36
), as in human HNSCC with altered p63 and TP53 (14
). Variability in the effects of ΔNp63-modulated gene expression in the UM-SCC line overexpressing mtTP53 compared to UMSCC deficient for wtTP53, suggests that effects of ΔNp63 may potentially be modified by TP53 status (22
). ΔNp63 and TP53 share high homology of the transactivation and DNA binding domains, and differentially bind to ΔNp63/TP53 sites, and may thereby competitively inhibit or alter one another’s transactivation function (8
). Consistent with this, we previously found that the cross-talk between TP53 and NF-κB in differentially modulating BCL-XL and BAX expression is dampened in the subset of UM-SCC cells overexpressing mtTP53 (25
). The relationship between ΔNp63 and Ki67 proliferation marker histoscores in HNSCC and co-staining in suprabasilar layers of few hyperplastic mucosa samples in tissue array, as well as in skin of ΔNp63 TG mice, provide additional evidence for a relationship between increased nuclear ΔNp63 and proliferation in vivo
A novel and unexpected finding from this study is the demonstration that ΔNp63 promotes a broad NF-κB program that includes multiple inflammatory cytokine genes. The inflammatory cytokines modulated by ΔNp63 in HNSCC cells and ΔNp63 TG mice largely overlap with the NF-κB-regulated cytokine repertoire, which we previously identified in HNSCC culture supernatants, patient serum, and tumor specimens (37
). Further, many of these ΔNp63 and NF-κB co-regulated inflammatory factors have been individually shown to promote the aggressive malignant behavior that leads to poor prognosis of HNSCC (4
). Proinflammatory cytokines have been shown to attract infiltrating neutrophils and macrophages that enhance angiogenesis, and greatly promote cancer cell survival and metastasis (41
). Consistent with this, we observed increased nuclear ΔNp63 is linked with infiltrating inflammatory cells in HNSCC, and in the dermis of ΔNp63 TG mice ( and ) The clinical importance of our findings that ΔNp63 promotes inflammation and aggressive malignant phenotypes is further supported by a recent report demonstrating that oral leukoplakias with increased ΔNp63 expression and inflammatory cell infiltration, exhibit a higher rate of cancer development and worse prognosis (42
The role of ΔNp63 in modulation of multiple cell adhesion genes such as integrin α6β4 and laminins, migration by wound healing and Matrigel assays in vitro
, and an association with suprabasilar integrin β4 expression in HNSCC tumors in situ
, suggest it may also function in promoting cell migration during invasion/metastasis in tumor progression. Consistent with this, we previously demonstrated that increased suprabasilar α6β4 expression occurs in more than 70% of HNSCC with higher invasive/metastatic potential in a prospective clinical study, and promotes laminin mediated adhesion and migration (43
). In addition, a causal role for ΔNp63 in modulating a broad adhesion gene program, including β4 integrin, was independently demonstrated in breast epithelial cells (39
). Knockdown of ΔNp63 by shRNA down-modulated adhesion by 24 and 48 hours, and this could be partially restored by expression of α6β4. The finding that ΔNp63 promotes a similar broad adhesion and survival gene program in HNSCC and breast cancer cell lines suggests that such dysregulation may be of broader importance in epithelial cancers.
Remarkably, we found that the promoters of multiple ΔNp63 and NF-κB modulated genes contain nearby or overlapping p63 and NF-κB binding sites (), providing the potential for members of the two families to coordinately or physically interact on nearby or same site. Indeed, ΔNp63, c-REL and RELA family members demonstrated co-binding to the same or nearby promoter sites of several of these genes by ChIP and EMSA supershift analyses (, ). Interestingly, while ΔNp63 co-binding with c-REL was detected on both p63 and REL regulatory sites, RELA binding was not detected on the p63 sites of IL-8, CSF2 and YAP promoters. We have also obtained evidence for binding of ΔNp63 and c-REL, but not RELA, to the p63 site of p21Cip1
promoter in primary murine keratinocytes and HNSCC by ChIP and EMSA (17
). In contrast, ΔNp63 co-binding with RELA was principally detected on REL sites by supershift analysis. Thus far, it appears, ΔNp63 regulates NF-κB target genes through interaction with c-REL on p63 binding sites, and with c-REL and RELA via REL sites.
Supporting a novel functional role of ΔNp63 in co-regulating NF-κB gene activation, knockdown or overexpression of ΔNp63 modulated activation of NF-κB-specific reporter and IL-8 promoter was dependent on NF-κB regulatory sites. ΔNp63α bound the NF-κB/REL site on the proximal promoter was required for ΔNp63α as well as TNF-α induced IL-8 promoter transactivation (, ). Knockdown of RELA exhibited a more potent inhibitory effect than knockdown of c-REL on transactivation of the basal IL-8 reporter activity, while the relative contribution of c-REL increased when ΔNp63 was overexpressed (). These data are consistent with the differential contribution of the three transcription factors to gene regulation under different conditions of activation and binding specificity. As with ΔNp63 in promoting IL-8 transcription, a recent report implicates p63 in IL-6 expression via a NF-κB promoter binding site in tonsillar crypt epithelium in palmoplantar pustulosis (46
The broad role of ΔNp63 in both promoting NF-κB and antagonizing TP53, is consistent with its ancestral co-evolution with NF-κB as a multi-functional transcriptional coordinator (47
). With the mutation or inactivation of TP53, aberrant activation of ΔNp63 together with NF-κB/RELs, appears to mediate a broad primordial gene program and pro-survival response. In that context, it may not be surprising that ΔNp63 serves as a master control to versatilely co-regulate NF-κB, TP53 and other gene programs to promote proliferation, survival, migration, and inflammation in cancer. Based on evidence that ΔNp63-overexpressing HNSCC and other cancers are more sensitive to cisplatin chemotherapy-induced ΔNp63 degradation (29
), ΔNp63 and target gene signatures may warrant further investigation as potential biomarkers for selection of cisplatin and other therapies that target ΔNp63, c-REL and REL activation in cancer.