Here we unveil evidence for a new paradigm (), whereby an inflammatory factor, TNF-α, can promote nuclear interaction of c-REL with ΔNp63α, and TAp73 translocation to the cytoplasm, inhibiting the compensatory ability of TAp73 to activate key genes that mediate growth arrest and apoptosis in HNSCC with mutant TP53. Our findings establish a novel link between TNF-α expression and resistance (6
), nuclear activation of proto-oncogene c-REL (13
), and dysregulation of p63/p73 family transcription factors (4
), which have been independently implicated in cancer progression. TNF-α-induced c-REL/ΔNp63α interactions and TAp73 dissociation were demonstrated at unique p63 DNA regulatory sites within the promoters of important growth arrest and pro-apoptotic genes. The role of TNF-α induced c-REL in these specific interactions with ΔNp63α/TAp73, did not appear to involve RELA, nor c-REL transactivation domain residues involved in binding to κB enhancers shared with other NF-κB/REL family members. These findings suggest that TNF-α-induced c-REL has a distinct function in inactivating TAp73 in p63/p73 promoter-regulated proapoptotic gene expression, complementing that previously demonstrated for RELA in promoting κB-regulated prosurvival genes important in the malignant phenotype (10
). Furthermore, our findings suggest that targeting TNF-α signaling, c-REL, or other regulators of these c-REL-ΔNp63α-TAp73 interactions, could enhance TAp73 function, potentially helping in prevention or treatment of cancers with altered TP53 status.
A novel finding of this study is the demonstration of the role of TNF-α in coordinating interactions between nuclear c-REL/ΔNp63α and reciprocal dissociation of ΔNp63α/TAp73 complexes, linking these previously reported components and interactions into a common dynamic mechanism. Previously, we discovered nuclear interactions between murine c-Rel and overexpressed ΔNp63α in keratinocytes, and human c-REL and ΔNp63α in HNSCC (15
). In murine keratinocytes, c-Rel and ΔNp63α were shown to prevent growth arrest, suggesting that corresponding c-REL/ΔNp63α complexes in HNSCC may also contribute to loss of growth control and promote the malignant phenotype. Independently, overexpressed ΔNp63α was shown to interact with TAp73, and promote survival in HNSCC (29
). However, the relationship between these components, factor(s) modulating them, and basis for TAp73 inactivation were unknown. Here, we found that either c-REL or TAp73 interact with ΔNp63α, but we detected minimal interaction between c-REL and TAp73, suggesting that c-REL or TAp73 alternately complex withΔNp63α. Supporting this model, we further established that TNF-α dynamically promotes nuclear c-REL/ΔNp63α interaction and reciprocal dissociation of ΔNp63α/TAp73. This is accompanied by nuclear-cytoplasmic translocation of TAp73, thus providing a basis for the inactivation of TAp73. TNF-α-induced c-REL was an important component of this mechanism, as c-REL siRNA attenuated dissociation ofΔNp63α/TAp73, while c-REL overexpression had a similar promoting effect on the dissociation and cytoplasmic translocation of TAp73. These effects of TNF-α or c-REL in promoting increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 in a subset of HNSCC lines may help explain the similar distribution we observed in HNSCC tumors, where increased TNF-α expression, as well as amplification and nuclear localization of c-REL have been detected (7
These dynamic nuclear c-REL-ΔNp6α-TAp73 interactions modulated by TNFα or c-REL, were mirrored on established and novel predicted p63 regulatory sites in the promoters of several growth arrest and apoptotic genes. EMSA results indicated TNF-α induces reciprocal modulation between c-REL or TAp73 in binding to an established p63 site sequence of the p21WAF1
). ΔNp63α binding remained relatively unchanged by TNF-α, but ΔNp63α knockdown inhibited ChIP binding of both c-REL and TAp73 to the p21WAF1
promoter, indicating that ΔNp63α bound to this p63 site is a key anchor for c-REL and TAp73 transcription factors. While NOXA and PUMA
were also previously identified as p63/p73 target genes (29
), specific binding sites for p63/p73 were not previously resolved. Using a bioinformatics approach, we predicted p63 binding sites in these genes. We observed similar specificity in binding and modulation by TNF-α of c-REL and TAp73 co-bound with ΔNp63α on p21WAF1, NOXA, and PUMA
promoters by ChIP assay. We have recently demonstrated the capability of TNF-α to modulate c-REL/ΔNp63α interaction on p63 sites of additional genes by ChIP and EMSA (28
), and obtained further evidence for the specificity of c-REL in interacting withΔNp63α at p63 regulatory sites. Preliminary ChIP sequencing results indicate that c-REL, p63 and/or p73 factors bind at many additional loci (H. Lu, unpublished data). Together, these findings suggest that the dynamic modulation of nuclear interactions involving these transcription factors observed in co-IP analysis, are likely related to their specific interactions on p63/p73 sites of multiple gene promoters.
Our results further revealed the important and reversible function of nuclear c-REL in attenuating the compensatory ability of TAp73 to promote expression of these key growth arrest and apoptotic genes. Knockdown of c-REL potentiated the expression of p21WAF1
, further supporting the biologic and potential therapeutic relevance of c-REL-ΔNp63α-TAp73 interactions observed on their promoters. Moreover, the modulation of these growth arrest and apoptotic genes was specifically observed in UMSCC 22A from a subset overexpressing TAp73 andΔNp63α with mtTP53. This effect was not seen in UMSCC 1, from a subset with attenuation of expression and function of both TAp73 and wtTP53, which we have shown can result from other therapeutically reversible mechanisms (17
). Consistent with these findings, c-REL modulation affected proliferation and apoptosis in UMSCC22A but not UMSCC 1. Conversely, overexpressed c-REL inhibited the expression of p21WAF1 and anti-proliferative effects when TAp73 was re-expressed in TAp73-deficient UMSCC 1, supporting an important role for c-REL in inhibiting the compensatory ability of TAp73. Together, these results highlight the functional importance and potentially reversible nature of c-REL-mediated inhibition of TAp73 function in HNSCC overexpressing TAp73, ΔNp63α and mtTP53.
Our findings in UMSCC lines are likely to be of broader relevance in HNSCC and other cancers. Increased nuclear c-REL/ΔNp63α and nuclear and cytoplasmic TAp73 was observed in a majority of HNSCC tumor specimens. Similarly, increased ΔNp63α and TAp73 was previously seen in an independent panel of HNSCC tumors and lines, and linked with inactivation of TAp73 (5
). Breast cancer specimens also show increased ΔNp63 and TAp73 immunostaining, and this most often seen in specimens with mtTP53 status (33
). Subsets exhibiting increased expression of ΔNp63 in HNSCC and breast cancer have also been reported to be more sensitive to the chemotherapy drug cisplatin (33
), underscoring the potential clinical relevance of identifying and selecting agents active in these tumor subsets.
Inflammatory mediator TNF-α is identified as a key modulator of dynamic interactions of c-REL with ΔNp63α, and inactivation of TAp73. This mechanism could therefore contribute to the acquired resistance and promoting effects of TNF-α produced by inflammatory cells during tumorigenesis and metastatic tumor progression of HNSCC and other cancers (6
). Supporting this hypothesis, we show here that combining TNF-α with overexpression of c-REL not only negated the inhibitory effect of TNF-α, but enhanced proliferation over that observed with either alone. Further, human HNSCC tumors and epithelia of K5-ΔNp63α transgenic mice that exhibit increased TNF-α expression, inflammation, and epithelial proliferation (7
), showed greater nuclear c-REL/ΔNp63α, and distribution of TAp73 between the nucleus and the cytoplasm. Previous findings support targeting TNF-α, or the canonical signal pathway which activates c-REL, for prevention or therapy of SCC. Knockout of TNF-α or TNF receptor-1, or TNF-α inhibitors, have been shown to reduce chemical carcinogenesis and malignant progression of SCC of the skin and other epithelial cancers (6
). We previously showed that blocking canonical pathway signaling increased TNF-α cytotoxicity in HNSCC in vitro
, and induced apoptosis and regression of established murine and human SCC in vivo
). However, early phase clinical trials with TNF-α or proteasome inhibitors, which inhibit canonical pathway activation, have demonstrated limited potential to slow disease progression in patients with advanced cancers (6
Consequently, other molecular requirements for coordinated modulation of c-REL, ΔNp63α and TAp73 interactions on the promoters of target genes merit investigation as targets for therapy. Unique structural characteristics of c-REL have previously been implicated in TNF-α-induced, TBK and IKKepsilon phosphorylation, dimerization, DNA binding, or transactivation (35
). One of our laboratories found that the ΔNp63 α-domain, critical for oligomerization (5
), is necessary for interaction with c-Rel (15
). Cisplatin induced IKKα and β activation have been reported to promote degradation ofΔNp63α and stabilization of p73, suggesting DNA damaging agents and these kinases may be important modulators of these important components of the mechanism described herein (37