ΔNp63 Versatilely Regulates a BroadNF-κBGene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Abstract

Head and neck squamous cell carcinoma (HNSCC) and many epithelial malignancies exhibit increased proliferation, invasion and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-κB family members ΔNp63, c-REL and RELA. However, the mechanisms of crosstalk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. Here we demonstrate thatΔNp63 regulates a cohort of genes involved in cell growth, survival, adhesion and inflammation, which substantially overlaps with the NF-κB transcriptome. ΔNp63 with c-REL and/or RELA are recruited to form novel binding complexes on p63 or NF-κB/REL sites of multiple target gene promoters. Overexpressed ΔNp63- or TNF-α-induced NF-κB and inflammatory cytokine IL-8 reporter activation depended upon RELA/c-REL regulatory binding sites. Depletion of RELA or ΔNp63 by siRNA significantly inhibited NF-κB-specific, or TNF-α-induced IL-8 reporter activation. ΔNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with the above, an increase in nuclear ΔNp63 accompanied by increased proliferation (Ki67), and adhesion (β4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared to the basilar pattern of protein expression and minimal inflammation seen in non-malignant mucosa. Further, overexpression of ΔNp63α in squamous epithelia in transgenic mice leads to increased suprabasilar c-REL, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals ΔNp63 as a master transcription factor that in coordination with NF-κB/RELs, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC.

SOX2 and p63 colocalize at genetic loci in squamous cell carcinomas

“…Indeed, loss of function of ΔNp63 enhances terminal maturation in stratified squamous epithelium (41). On one hand, ΔNp63α overexpression has been shown in a rat cell line to accelerate tumor growth in nude mice (42), and transgenic mice overexpressing ΔNp63α develop epidermal hyperplasia (43). Tp63 was recently shown to be required for maintenance of tumors, as inducible genetic deletion of all its isoforms led to blockade of chemically induced SCC formation in mice (44).…”

TP63 (tumor protein p63)

“…Epithelial differentiation: Keratin 1 (KRT1) -KRT1 is a marker for terminal differentiation in the mammalian epidermis (Lessin et al, 1988;Huang et al, 2012). Through protein-protein interactions with other transcription and cofactors, TP63 also contributes to the transcriptional regulation of genes involved in cellular differentiation, proliferation/survival, growth suppression, apoptosis, adhesion, inflammation, and metabolism (Perez and Pietenpol, 2007;Viganò and Mantovani, 2007;Yang et al, 2011). Recent work by our laboratory has identified protein-protein interactions between ∆Np63α, TAp73, and c-REL that function to regulate key genes involved in growth arrest and apoptosis of mutant p53 head and neck squamous cell carcinoma (HNSCC) .…”

“…Apoptosis: p53-Upregulated Modulator of Apoptosis (PUMA) -PUMA binds to BCL2 to induce rapid and profound apoptosis by cytochrome c release (Yu et al, 2001;Flores et al, 2002). Additionally, recent work has shown that a complex of ∆Np63α, with TAp73 or c-REL, can modify expression of key growth arrest and apoptotic genes such as p21WAF1, NOXA, and PUMA Yang et al, 2011). Adhesion: Integrin Alpha 6 (ITGA6) -ITGA6 is a cell surface adhesion molecule that may help regulate migration and layer formation, especially in epithelial cells.…”

“…Mouse models deficient in ITGA6 display severe blistering of the skin and other epithelia and die shortly after birth (Georges-Labouesse et al, 1996;Carroll et al, 2006). Loss of endogenous p63 in mammary epithelial cells has been shown to induce detachment and cell death, presumably because p63 regulates expression of key adhesion molecules such as ITGA6, ITGB1, ITGB4 other ECM components, and cadherinscatenins (Carroll et al, 2006;Yang et al, 2011). Inflammation: ∆Np63 overexpression has been observed in head and neck squamous cell carcinoma (HNSCC), which are associated with inflammation.…”

“…Inflammation: ∆Np63 overexpression has been observed in head and neck squamous cell carcinoma (HNSCC), which are associated with inflammation. TNF-α, a potent pro-inflammatory cytokine, promoted NF-κB, c-REL and RELA complexes with nuclear ∆Np63, to promote a broad-spectrum production of cytokines and chemokines Yang et al, 2011). In addition, in squamous epithelia of ∆Np63α transgenic mice, severe inflammation, skin lesions and erythema were observed after ∆Np63 expression was induced.…”