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The p53 Homologue Np63 Interacts with the Nuclear Factor-B Pathway to Modulate Epithelial Cell Growth

¹ Division of Monoclonal Antibodies, Center for Drug Evaluation and Research, Food and Drug Administration; ² Head and Neck Surgery Branch, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland

Requests for reprints: Wendy C. Weinberg, Laboratory of Immunobiology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, 29B Lincoln Drive, NIH Building 29B, Room 3NN04, HFD-123, Bethesda, MD 20892. Phone: 301-827-0709; Fax: 301-827-0852; E-mail: wendy.weinberg{at}fda.hhs.gov.

	Abstract

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The p53 homologue Np63 is overexpressed and inhibits apoptosis in a subset of human squamous cell carcinomas (SCC). Here, we report that in normal keratinocytes overexpressing Np63 and in human squamous carcinoma cells, Np63 physically associates with phosphorylated, transcriptionally active nuclear c-Rel, a nuclear factor-B family member, resulting in increased c-Rel nuclear accumulation. This accumulation and the associated enhanced proliferation driven by elevated Np63 are attenuated by c-Rel small interfering RNA or overexpression of mutant IBM, indicating that c-Rel–containing complex formation is critical to the ability of elevated Np63 to maintain proliferation in the presence of growth arresting signals. Consistent with a role in growth regulation, Np63-c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human squamous carcinoma cells and normal keratinocytes overexpressing Np63. The relationship between Np63 and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of primary head and neck SCC. This is the first report indicating that high levels of Np63 interact with activated c-Rel in keratinocytes and SCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers. [Cancer Res 2008;68(13):5122–31]

	Introduction

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Considerable debate has focused on the role of the p53 homologue p63 in human cancer pathogenesis (1). Dysregulation of p63 is observed in most squamous cancers, with p63 gene amplification and/or overexpression reported in squamous cell cancers of the head and neck (HNSCC), lung, cervix, and skin (2–4). Functional determination of the consequences of p63 overexpression is complicated by the existence of multiple protein variants of p63, which show overlapping and opposing functions.

The p63 gene is transcribed as two classes: TA and N (5). TAp63 isoforms contain an NH₂-terminal p53-like transactivation domain and are capable of transactivating known p53-responsive genes as well as distinct sequences (5–7). In contrast, N isoforms lack this domain due to alternate promoter usage and can block transactivation by either p53 or TAp63 isoforms while still harboring direct transactivation potential (8–10). p63 overexpression in human cancers has been predominantly associated with Np63 isoforms (1, 2, 4). Additional complexity within each class of isoform is derived from COOH-terminal alternative splicing, giving rise to TAp63, TAp63β, TAp63, Np63, Np63β, and Np63. Unlike p53, the p63 gene is critical for normal development of stratified squamous epithelium (11, 12), and several studies have indicated a requirement for temporal regulation of individual p63 isoforms in both development and maintenance of mature epidermis (13–15). However, the specific contribution of each of the known isoforms remains a subject of active investigation.

Previously, we used primary murine epidermal keratinocytes and adenoviral vectors to mimic Np63 overexpression observed in human SCCs. We showed that overexpressed Np63 maintains keratinocyte proliferation and blocks morphologic and biochemical differentiation despite the presence of signals that induce growth arrest and differentiation (10, 16). Np63 overexpression was subsequently shown by others to promote survival in a subset of HNSCCs by physical association with and blockade of transcription of apoptosis genes by another p53 family member, p73 (17). To gain mechanistic insight into the altered growth regulation of murine keratinocytes associated with elevated Np63 expression, we profiled extracts from keratinocytes overexpressing Np63 or β-galactosidase (β-gal) for differential transcription factor binding, which provided evidence for a novel form of regulation of nuclear factor-B (NF-B) by Np63.

NF-B is widely expressed, with effects that are cell type and context dependent. Dysregulation of NF-B activity is associated with multiple human diseases including cancer (18), and therapeutics targeting constitutive NF-B activity are the subject of clinical trials in oncology (19). The NF-B family consists of five subunits, which function as homodimers and heterodimers. Rel-A, Rel-B, and c-Rel contain a transactivation domain, whereas p50/105 and p52/100 do not. Within the normal epidermis, NF-B plays an important role in regulating homeostasis (20, 21). During the development and progression of SCC, the NF-B₁-Rel-A (p50/p65) heterodimer has been implicated in promotion or repression of the malignant phenotype dependent on the context (22, 23).

Here, we show that murine keratinocytes overexpressing Np63 accumulate transcriptionally active c-Rel in their nuclei and that nuclear c-Rel accumulation is required to maintain Np63-mediated proliferation in the presence of signals that normally induce growth arrest. Accumulation of c-Rel is also seen in the nuclei of tumor specimens and cell lines of human HNSCCs expressing endogenous Np63. Additionally, Np63 and c-Rel physically interact. Their association is observed in vitro in both human and murine cells and has been confirmed in murine cells in vivo on the promoter of the cyclin-dependent kinase (CDK) inhibitor p21WAF1. These findings provide a mechanism whereby c-Rel contributes to the altered growth regulation of Np63-overexpressing keratinocytes. This is the first report showing Np63-mediated regulation of active c-Rel, which is known for its oncogenic propensity (24, 25), and implicates Np63-c-Rel complexes in human HNSCC.

	Materials and Methods

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Cell culture. Primary keratinocytes isolated from C57BL/6NCr mice were cultured in 0.05 mmol/L Ca²⁺-containing medium to maintain proliferation, and induced to differentiate by elevating Ca²⁺ levels to 0.12 mmol/L (10). HNSCC cell lines UM-SCC-11A, UM-SCC-22B, and UM-SCC-38 have been described previously (26). N-ethylmaleimide (NEM), a thiol modifier, was added to the culture medium immediately following adenoviral transduction to block in vivo phosphorylation (27).

Gene transfer. Adenoviruses (Np63, Np63^p40, IBM, and β-gal) and transduction methodology were described previously (2, 10, 16, 28).

Reporter constructs, NF-B (29) or p21WAF1 (30), were transfected using Lipofectamine Reagent System (Life Technologies), or using Lipofectamine 2000 for cotransfections with small interfering RNA (siRNA). Activity relative to protein concentration was determined via the Luciferase Assay System (Promega Corp.).

Reporter assay-only transfections. Keratinocytes were transfected 17 h after adenoviral transduction with the NF-B reporter construct (3 µg) and harvested 24 h after transfection.

c-Rel siRNA and reporter assay transfections. Keratinocytes were transfected with a siRNA pool (c-Rel or nontargeting, 200 pmol; Dharmacon) plus NF-B reporter construct (1.5 µg) 24 h before adenoviral introduction of Np63 or β-gal. Samples were harvested 24 h later.

Transcription factor binding assay. Nuclear extracts from Np63-overexpressing, Np63^p40-overexpressing, or β-gal–overexpressing keratinocytes (31) were used to screen the Panomics DNA Array I.

Western blot analysis. Primary antibodies used were the following: Rel-A (F-6), Rel-B (C-19), c-Rel (C), p100/52 (447), p105/50 (E-10), IB (C21), IBβ (C20), IB (M121), p63 DNA-binding domain (4A4), and p63 -domain (H129; all from Santa Cruz Biotechnology); actin (AC-15; Sigma Immuno Chemicals); and keratin 10 and filaggrin (Babco). Signal was detected using horseradish peroxidase–linked anti-mouse, anti-goat, or anti-rabbit secondary antibodies.

Phosphatase assay. Nuclear extracts were incubated in 1x SAP buffer ± 10 units shrimp alkaline phosphatase (SAP; Promega) for 3 h at 37°C followed by inactivation at 65°C.

Bromodeoxyuridine incorporation analysis. Fluorescence-activated cell sorting (FACS) analysis was performed as described previously (16). Seventeen hours after adenoviral infection (Np63 or β-gal), cells were maintained in fresh 0.05 mmol/L Ca²⁺-containing medium or switched to 0.12 mmol/L for 24 h, with addition of 10 µmol/L bromodeoxyuridine (BrdUrd) for the final 4 h. siRNA experiments were performed as described except that keratinocytes were transfected with the siRNA pools as noted 12 h before adenoviral transduction.

Coimmunoprecipitation analysis. Lysates were precleared with appropriate antibody and beads and then incubated overnight at 4°C with c-Rel (4A4), p63 (H129), Rel-A (F-6), or control antibody. Protein A/G Plus beads were added for the final hour, and samples were washed four times with PBS, resolved by SDS-PAGE, and analyzed. The ExactaCruz F reagent system (Santa Cruz Biotechnology) was used for cases where the same species was used to generate the primary antibody for immunoprecipitation and Western blot analysis.

Reverse transcription-PCR. Primary murine keratinocytes were transfected with siRNA pools (c-Rel or nontargeting, 200 pmol) 8 h before adenoviral transduction (Np63 or β-gal). Seventeen hours later, cultures were exposed to 0.12 mmol/L Ca²⁺ for 15 h or maintained in 0.05 mmol/L Ca²⁺-containing medium. RNA was harvested via the Qiagen RNeasy Plus Mini and reverse transcribed (1 µg) using the AccuScript High Fidelity First-Strand cDNA Synthesis kit (Stratagene) with an oligo(dT) primer. Target sequences were amplified from cDNA pool aliquots in 1x reaction buffer [10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂], 400 µmol/L of each deoxynucleotide triphosphate, 5 units AmpliTaq DNA polymerase (Applied Biosystems), and 250 ng of each primer. Following a 3-min hot start, the reaction profile was the following: denaturation, 94°C, 30 s; annealing, 30 s [p21WAF1: 57°C, 22 cycles, 478 bp product; hypoxanthine phosphoribosyltransferase (HPRT): 51°C, 25 cycles, 526 bp product]; elongation, 72°C, 45 s. The primer sequences were as follows: p21WAF1, 5'-AATCCTGGTGATGTCCGACCTGTT-3' (forward) and 5'-AGACCAATCTGCGCTTGGAGTGAT-3' (reverse); HPRT, 5'-CGTCGTGATTAGCGATGATGA-3' (forward) and 5'-TTCAAATCCAACAAACTCTGGC-3' (reverse). PCR products were quantified using Spot densitometry software on an Alpha Innotech imaging system.

Electrophoretic mobility shift assays. The LightShift Chemiluminescent electrophoretic mobility shift assay (EMSA) kit was used (Pierce) with oligonucleotides from the p21WAF1 promoter p63-binding site #1 (p63BS#1; 32): 5'-TGGCCATCAGGAACATGTCCCAACATGTTGAGCTCTGGCA-3' (forward) and 5'-TGCCAGAGCTCAACATGTTGGGACATGTTCCTGATGGCCA-3' (reverse). Oligonucleotides were end labeled using the 3' Biotin end-labeling kit (Pierce) and incubated with nuclear extract (6 µg/reaction) before resolution (4% acrylamide). For radioactive EMSAs, oligonucleotides were 5' end labeled using T4 polynucleotide kinase (New England Biolabs) and [-³²P]dATP. Nuclear extracts (6 µg/reaction) were incubated at room temperature with 1 µL of labeled probe (20,000 cpm) and resolved by gel electrophoresis.

Chromatin immunoprecipitation. Np63-overexpressing or β-gal–overexpressing keratinocytes were fixed in 1% formaldehyde solution for 10 min. The reaction was stopped by the addition of 1x glycine buffer. Following washing, cells were scraped into PBS. Chromatin was isolated and sheared enzymatically for 10 min (ChIP-IT Express kit, Active Motif). Samples were immunoprecipitated overnight at 4°C with antibodies to c-Rel, p63 H129, or IgG control. The chromatin was eluted and cross-links were reversed before proteinase K digestion. Following a 3-min hot start, the PCR profile was the following: denaturation, 94°C, 30 s; annealing, 58°C, 30 s; elongation, 72°C, 30 s for 35 cycles; 220 bp product. The primer sequences were as follows: p21-binding site #1, 5'-ACTAGCTTTCTGGCCTTCAGGAAC-3' (forward) and 5'-CCTGATACATGTCACAAGATACATACCACC-3' (reverse).

Immunostaining. Patient-matched carcinoma and normal stratified squamous epithelium biopsies were obtained under Institutional Review Board–approved NIH protocol 04-C-0141 in the outpatient clinic. Frozen sections (10 µm) on silanated glass were fixed with 4% paraformaldehyde/PBS at 4°C for 5 min. Nonspecific binding was blocked with 5.5% serum/TBS, and endogenous tissue peroxidase was quenched with 0.6% H₂O₂/TBS before incubation with primary antibodies, c-Rel (C), Np63 (N-16), or isotype control (diluted 1:100 in 3% bovine serum albumin/TBS) overnight at 4°C. Samples were then incubated with biotinylated secondary antibody and then avidin-biotin complex (Vectastain Elite ABC kit, Vector Laboratories) and 3,3'-diaminobenzidine (1–5 min depending on target antigen) to reveal immune complexes. Sections were counterstained with Gill's formula hematoxylin (Vector Laboratories), dehydrated, cleared, and mounted using Permount (Fisher).

	Results

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Np63 overexpression promotes nuclear c-Rel accumulation. Np63 expression is associated with the proliferative compartment of normal stratified squamous epithelium and this protein is overexpressed in SCCs (2, 3, 5). Previously, we showed that elevated exogenous Np63 maintains primary murine keratinocytes in an undifferentiated proliferative state in the presence of signals that normally induce growth arrest and differentiation (10, 16). To identify downstream targets of elevated Np63 that may mediate these effects in squamous epithelium, nuclear extracts prepared from keratinocytes overexpressing Np63 or β-gal were screened for differential transcription factor regulation. Differential binding to a NF-B consensus sequence was observed in samples overexpressing Np63 compared with β-gal controls (data not shown), indicating the potential involvement of NF-B in the altered growth regulation associated with Np63 overexpression.

Five NF-B subunits form heterodimers/homodimers that display differential binding affinity for multiple NF-B consensus sequences (33). To confirm the altered NF-B–binding activity observed with elevated Np63 and to determine which subunits were involved, we profiled nuclear extracts from Np63-overexpressing versus β-gal–overexpressing keratinocytes by Western blotting. c-Rel was enhanced in nuclei of keratinocytes overexpressing Np63 relative to β-gal control, whereas nuclear levels of the other NF-B subunits were unaffected (Fig. 1A ).

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Np63 overexpression results in increased nuclear levels of transcriptionally active phosphorylated c-Rel in murine keratinocytes. A, elevated Np63 expression specifically enhances nuclear levels of the NF-B subunit c-Rel. Western blots of nuclear extracts from primary murine keratinocytes harvested 21 h after adenoviral introduction of human Np63 or β-gal. Cultures were maintained in medium containing 0.05 mmol/L Ca2+ throughout or exposed to 0.12 mmol/L Ca2+ for the final 4 h. c-Rel levels are increased in the nuclei of Np63-overexpressing keratinocytes. B, c-Rel is phosphorylated in response to elevated Np63. Western blot analysis of nuclear extracts from keratinocytes overexpressing Np63 or β-gal cultured under control conditions (top) or in the presence of the thiol modifier NEM at concentrations noted for 21 h following adenoviral introduction to block in vivo phosphorylation (bottom left). The upper species is eliminated in the presence of NEM. Bottom right, phosphorylation was confirmed by Western blot analysis of nuclear extracts derived from keratinocytes overexpressing Np63 incubated in the presence or absence of SAP for 3 h at 37°C. A nonincubated control nuclear extract (NE) is included for visual reference. SAP treatment results in the loss of the upper phosphorylated species. C, Np63 overexpression results in NF-B–mediated transactivation; -domain of Np63 is required. NF-B–responsive reporter gene activity in keratinocytes overexpressing Np63, Np63p40 (Np63 lacking -domain), or β-gal. Samples were harvested 24 h after transfection. Columns, mean of triplicate samples of a representative experiment performed thrice; bars, SD. D, c-Rel is required for NF-B–mediated transactivation following Np63 overexpression. Keratinocytes were cotransfected with NF-B reporter construct and c-Rel–targeting siRNA or control siRNA 24 h before adenoviral infection with Np63 or β-gal. Samples were harvested 24 h after adenoviral introduction. Right, Western blot reveals depletion of c-Rel in whole-cell lysates at time of harvest. NT, nontargeting siRNA. Left, fold increase in NF-B reporter gene activity in Np63-overexpressing cultures compared with β-gal control cultures in the presence or absence of c-Rel siRNA. Triplicate wells were averaged and are presented as fold increase relative to β-gal controls that are normalized to 1.0. Experiment was performed twice with consistent results; representative experiment is shown.

Nuclear c-Rel accumulated in response to elevated Np63 expression in keratinocytes is transcriptionally active. Phosphorylation of c-Rel is known to positively affect its transactivation capacity (34). To assess whether c-Rel enhancement resulting from Np63 elevation affects NF-B transcriptional activity, keratinocytes were transfected with a NF-B–responsive luciferase reporter construct (29) following adenoviral introduction of Np63 or β-gal. This revealed a 6- to 17-fold increase in NF-B reporter activity in Np63-overexpressing keratinocytes relative to β-gal controls (Fig. 1C). Using this assay, we also compared keratinocytes overexpressing Np63^p40, a truncated form of Np63 lacking the entire -COOH terminus that is present in Np63 (Fig. 1C). In contrast to Np63, no reporter gene activity was observed in keratinocytes overexpressing Np63^p40, indicating a requirement for the -tail of p63 in mediating this NF-B transactivation activity. Repetition of the NF-B reporter assay in keratinocytes in which c-Rel had been reduced using siRNA before the adenoviral introduction of Np63 or β-gal revealed a >50% reduction in fold increase of reporter activity relative to samples in which c-Rel was not targeted (Fig. 1D). The siRNA silencing of c-Rel was incomplete (Fig. 1D, Western blot); therefore, this degree of reduction in activity underscores the critical contribution of c-Rel to Np63-induced NF-B–mediated transactivation in keratinocytes.

Enhanced nuclear NF-B levels are required for sustained proliferation mediated by Np63. c-Rel is critical for antigen-dependent B-cell proliferation and T-cell receptor–mediated T-cell proliferation and has been implicated in the maintenance of normal keratinocyte proliferation (21, 35, 36). A substantial block in nuclear accumulation of c-Rel was achieved by hindering NF-B nuclear translocation through the introduction of an adenovirus encoding the IBM superrepressor (Fig. 2A , Western blot; ref. 28). This approach reduced Np63-induced nuclear accumulation of c-Rel to levels approximating those in β-gal control cultures. FACS analysis revealed that β-gal control cultures underwent normal Ca²⁺-induced growth arrest in both the presence and absence of the IBM superrepressor (note decrease in S-phase fraction; Fig. 2A, histogram). Consistent with previous results (10), Np63-overexpressing keratinocytes do not arrest in response to 0.12 mmol/L Ca²⁺ (Fig. 2A, histogram, –IBM). Blocking NF-B subunit translocation with the IBM superrepressor restored responsiveness to Ca²⁺-induced growth arrest in Np63-overexpressing keratinocytes (Fig. 2A, histogram, +IBM).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Enhanced nuclear c-Rel is required for Np63-mediated loss of keratinocyte growth regulation but not differentiation defects. A to C, flow cytometry analysis of BrdUrd incorporation in keratinocytes overexpressing Np63 or β-gal under proliferating (0.05 mmol/L Ca2+) or differentiating (0.12 mmol/L Ca2+) conditions. A, blocking NF-B nuclear translocation with the IBM superrepressor restores normal Ca2+-mediated growth regulation to Np63-overexpressing keratinocytes. Primary murine keratinocytes (1.5 d after plating) were coinfected with adenovirus encoding Np63 or β-gal in combination with IBM superrepressor or empty vector control. Seventeen hours after infection, the medium was changed; cells were maintained for a further 24 h in 0.05 or 0.12 mmol/L Ca2+ and pulsed with BrdUrd (10 µmol/L) for the final 4 h. Columns, mean of triplicate samples from a representative experiment performed thrice; bars, SD. Right, Western blot of corresponding nuclear extracts confirms that coinfection with the IBM superrepressor effectively reduces levels of nuclear c-Rel in Np63-overexpressing keratinocytes. B, c-Rel siRNA knockdown partially restores normal growth arrest in Np63-overexpressing keratinocytes. Keratinocyte cultures were transfected with c-Rel or nontargeting siRNA 12 h before introduction of Np63 or β-gal by adenovirus. Decreasing c-Rel levels by siRNA results in an overall reduction of proliferation in all cultures (right panel versus left panel of histogram) and partially restores Ca2+-mediated growth arrest to Np63-overexpressing keratinocytes (right side and right panel of histogram). Columns, mean of triplicate samples from a representative experiment; bars, SD. Right, Western blot of whole-cell lysates confirms that c-Rel expression is reduced in keratinocytes transfected with c-Rel siRNA. C, Rel-A does not contribute to the aberrant growth arrest response observed in Np63-overexpressing keratinocytes. Keratinocytes were transfected with Rel-A–targeted siRNA to deplete Rel-A levels or with nontargeting siRNA as control. Depleting Rel-A by siRNA has no effect on keratinocyte proliferation under these conditions. Columns, mean of triplicate samples from a representative experiment; bars, SD. Right, Western blot of whole-cell lysates confirms that Rel-A siRNA effectively reduces Rel-A expression in these cultures. D, blocking NF-B nuclear translocation does not restore induction of markers of terminal differentiation in Np63-overexpressing keratinocytes. Western blot of whole-cell lysates from keratinocytes overexpressing Np63 or β-gal ± IBM superrepressor and exposed to 0.12 mmol/L Ca2+ for 24 h. Blocking NF-B nuclear translocation does not restore the Ca2+-mediated induction of the early marker of keratinocyte differentiation, keratin 10, or the late marker, filaggrin.

Rel-A has been implicated in the maintenance of normal keratinocyte proliferation (21); therefore, we also used Rel-A–targeting siRNA to assess the contribution of Rel-A to growth regulation in Np63-overexpressing keratinocytes. In contrast to studies using c-Rel siRNA, depleting Rel-A did not restore normal growth arrest to Np63-overexpressing keratinocytes (Fig. 2C). Based on these findings, and our observation that Np63 overexpression does not alter nuclear levels of Rel-A (Fig. 1A), we conclude that Rel-A does not participate in the aberrant growth arrest response in keratinocytes overexpressing Np63.

In conjunction with loss of Ca²⁺-mediated growth regulation, elevation of Np63 protein expression blocks the onset of squamous morphology as well as the induction of keratinocyte differentiation-specific gene expression (10, 16). Blocking c-Rel translocation with the IBM superrepressor did not restore expression of differentiation markers to Np63-overexpressing keratinocytes, indicating that NF-B subunits do not participate in this aspect of Np63 biological activity (Fig. 2D).

Enhanced nuclear levels of c-Rel in response to elevated Np63 result from altered intracellular localization without altering cytoplasmic IB:c-Rel interactions. Both N and TAp63 isoforms modulate gene transcription (8, 10, 37). To address whether the enhanced nuclear NF-B levels reflect the activity of Np63 as a transcription factor, total levels of each subunit in whole-cell lysates were assessed. No changes in total cellular expression of c-Rel, Rel-A, or Rel-B were observed between Np63-overexpressing and control cultures (Fig. 3A ), indicating that enhanced nuclear c-Rel levels resulted from altered intracellular localization. p50/105 and p52/100 were undetectable under these conditions (data not shown).

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Mechanism of nuclear c-Rel enhancement is not dependent on disruption of cytoplasmic IB-c-Rel interactions. A, Np63 overexpression does not alter total levels of cellular NF-B. Western blots of whole-cell lysates from primary mouse keratinocytes harvested 21 h after adenoviral introduction of human Np63 or β-gal. Cultures were maintained in medium containing 0.05 mmol/L Ca2+ or exposed to 0.12 mmol/L Ca2+ for the final 4 h. B, levels of IB regulatory proteins are not decreased in Np63-overexpressing keratinocytes that accumulate nuclear c-Rel. Western blot of whole-cell lysates of keratinocytes overexpressing β-gal or Np63. C, Np63 overexpression does not inhibit normal cytoplasmic interactions between c-Rel and the IB proteins. Coimmunoprecipitation analysis of whole-cell lysates from keratinocytes overexpressing Np63 or β-gal.

As IB levels are maintained in Np63-overexpressing keratinocytes, we speculated that Np63 might perturb normal cytoplasmic interactions between NF-B and the IBs. We focused on c-Rel, as this is the only subunit altered in Np63-overexpressing keratinocytes. Coimmunoprecipitation analyses of whole-cell lysates revealed that the normal associations between IB, IBβ, or IB and c-Rel remain intact in the presence of overexpressed Np63 (Fig. 3C).

Np63 and c-Rel physically associate in the nuclei of keratinocytes expressing high levels of Np63. In addition to sequestering NF-B in the cytoplasm, IB and IB can disrupt NF-B-DNA interactions in the nucleus and, through nuclear export signals, can actively shuttle NF-B back into the cytoplasm (39, 40). IB/IB shuttling is mediated by CRM1, which when blocked results in enhanced nuclear NF-B accumulation. A failure in shuttling, potentially due to a blocked association between c-Rel and IB/IB, could result in nuclear accumulation of c-Rel in Np63-overexpressing keratinocytes. Unlike our findings in whole-cell lysates (Fig. 3C), we detected no association between c-Rel and IB, IBβ, or IB in nuclear extracts derived from either β-gal–overexpressing or Np63-overexpressing keratinocytes (data not shown). However, coimmunoprecipitation analyses revealed a physical interaction between Np63 and c-Rel in nuclear extracts of Np63-overexpressing keratinocytes (Fig. 4A ). In contrast to c-Rel, interaction was not seen between Np63 and Rel-A in Np63-overexpressing keratinocytes (data not shown).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Np63 and c-Rel physically interact in a phosphorylation, p63 -domain–dependent manner. A, Np63 and c-Rel physically interact in the nuclei of Np63-overexpressing keratinocytes. Coimmunoprecipitation analysis of nuclear extracts from keratinocytes overexpressing Np63 or β-gal. Nuclear extracts were immunoprecipitated with antibody to p63 (left) or c-Rel (right) and probed for c-Rel or p63, as noted. B, the -domain but not the N-domain of p63 is required for the interaction between p63 and c-Rel. Nuclear extracts from keratinocytes overexpressing Np63p40 (a truncated form of Np63 lacking the -COOH terminus), TAp63, or β-gal were immunoprecipitated with antibody to c-Rel and probed for p63. C, Np63 associates with phosphorylated c-Rel. Coimmunoprecipitation analysis of nuclear extracts from keratinocytes overexpressing Np63 or β-gal resolved alongside Np63 or β-gal nuclear extracts (No IP). Comigration and Western blot reveal that the upper, phosphorylated species of c-Rel interacts with Np63. D, protein phosphorylation is required for association between Np63 and c-Rel. Coimmunoprecipitation analysis of nuclear extracts from keratinocytes overexpressing Np63 or β-gal. Culture with NEM for 1 d following adenoviral infection to block in vivo phosphorylation eliminates the interaction between Np63 and c-Rel.

Association between Np63 and phospho-c-Rel requires the p63 -domain and phosphorylation.

To address which c-Rel species interacts with Np63, coimmunoprecipitation reactions were resolved on a gel next to nonimmunoprecipitated nuclear extracts from keratinocytes overexpressing Np63 or β-gal (Fig. 4C). This revealed that the upper, phosphorylated form of c-Rel is the predominant species that interacts with Np63 in the nuclei of keratinocytes overexpressing Np63. To determine if phosphorylation is necessary for this physical interaction, keratinocytes overexpressing Np63 were cultured in the presence of 10 µmol/L NEM and then subjected to coimmunoprecipitation. Blocking protein phosphorylation by NEM treatment abrogated the interaction between Np63 and c-Rel (Fig. 4D).

Overexpressed Np63 physically associates with c-Rel on the p21WAF1 promoter. Previously, we showed (16) that overexpression of Np63 in primary murine keratinocytes blocks induction of the CDK inhibitor p21WAF1 in response to elevated extracellular Ca²⁺. Others have shown that Np63 binds to and acts as a transcriptional repressor for the p21WAF1 promoter (32). Because enhanced levels of c-Rel are critical to the ability of Np63 to maintain proliferation under conditions that normally induce growth arrest (Fig. 2A and B), we asked whether c-Rel also regulates p21WAF1. Cotransfection assays using a luciferase reporter under control of the p21WAF1 promoter confirmed that, like Np63, c-Rel negatively regulates the p21WAF1 promoter (Fig. 5A ). Keratinocytes cotransfected with the p21WAF1 promoter construct in combination with a human c-Rel cDNA exhibited a >50% decrease in luciferase activity relative to the control samples (left), whereas use of siRNA to decrease endogenous c-Rel levels resulted in enhanced p21WAF1 promoter activity compared with control (right).

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Np63 and c-Rel both negatively regulate the CDK inhibitor p21WAF1 and interact in vitro and in vivo at a p63-binding site on the p21WAF1 promoter. A, modulating c-Rel levels alters p21WAF1 reporter gene activity. p21WAF1 reporter gene activity following cotransfection in combination with a human c-Rel cDNA construct, c-Rel–targeted siRNA, or controls. Overexpression of c-Rel represses p21WAF1 reporter gene activity (left), whereas reducing c-Rel expression levels with targeted siRNA enhances expression of a p21WAF1 luciferase reporter construct (right). B, incomplete silencing of c-Rel by targeted siRNA allows slight restoration of induction of endogenous p21WAF1. Semiquantitative reverse transcription-PCR analysis of p21WAF1. Ca2+-mediated induction of p21WAF1 is unaffected by siRNA knockdown of c-Rel in control keratinocytes overexpressing β-gal. Consistent with previous results (16, 32), Ca2+-mediated p21WAF1 induction is blocked by the overexpression of Np63. siRNA knockdown of c-Rel results in a small but reproducible induction of p21WAF1 in Np63-overexpressing keratinocytes in response to 0.12 mmol/L Ca2+ (15% in c-Rel–targeted versus 5% induction in nontargeted siRNA controls), as determined by spot densitometry analysis using an Alpha Innotech imaging system. Experiment was repeated with consistent results. C, Np63 and c-Rel physically associate on the p21WAF1 promoter in vitro. EMSA analysis of nuclear extracts from keratinocytes overexpressing Np63 or β-gal. The p63BS#1 from the p21WAF1 promoter used in the reporter gene assays was biotin labeled and used in the binding reactions. A protein-DNA complex seen only in the presence of overexpressed Np63 is supershifted with a c-Rel antibody and interrupted with a p63-specific antibody. The experiment was performed four times with consistent results; representative experiment is presented. D, Np63 and c-Rel physically associate on the p21WAF1 promoter in vivo. ChIP analysis was performed on samples derived from keratinocytes overexpressing Np63 or β-gal using the antibodies noted. PCR primers were designed to flank the p63BS#1 from the p21WAF1 promoter. Association of the c-Rel-Np63 complex with p63BS#1 is observed. Input DNA: PCR products generated using DNA template from total genomic DNA. Lane labeled "-ve" indicates an absence of DNA in the PCR reaction. M, molecular weight marker. Results shown are representative of two independent experiments.

The p21WAF1 promoter contains two known p53 response elements, which have been previously shown to bind p63 (32). EMSAs performed using "p63BS#1" (32), which corresponds to the p53/p63 consensus site in the reporter constructs used in Fig. 5A, revealed that nuclear extracts derived from Np63-overexpressing keratinocytes produced a DNA-protein complex that could be supershifted with a c-Rel antibody, showing a physical association in vitro between Np63 and c-Rel on the p21WAF1 promoter (Fig. 5C) consistent with a role for c-Rel in regulating p21WAF1. In addition to the p53/p63 consensus binding sites, promoter analysis of the p21WAF1 promoter sequence revealed the presence of one potential c-Rel/p65–binding sequence,³ but EMSAs performed with oligonucleotides to this sequence did not reveal binding (data not shown). The association between Np63 and c-Rel on p63BS#1 of the p21WAF1 promoter was confirmed in vivo by chromatin immunoprecipitation (ChIP) analysis of Np63-overexpressing versus β-gal control keratinocytes using antibodies to either c-Rel or p63. As shown in Fig. 5D, a c-Rel-Np63 complex in Np63-overexpressing keratinocytes occupies this p53/p63 consensus site in vivo.

Np63 and c-Rel are strongly expressed throughout HNSCCs. To determine whether the association between Np63 and c-Rel extends to normal and malignant human squamous epithelia, immunostaining was performed on human squamous mucosa and HNSCC tumor samples. Nuclear expression of both p63 and c-REL is associated with the basilar proliferative compartment of normal human mucosa, as defined by Ki67 immunostaining (Fig. 6A ). Nuclear colocalization shown by strong nuclear staining of both proteins is diffusely seen throughout SCC tissue samples (Fig. 6A). Increased, diffuse nuclear costaining of Np63 and c-REL was observed in the malignant squamous epithelia of 13 of 16 (81%) of HNSCC specimens examined, indicating that such nuclear colocalization is common in HNSCC.

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Endogenous Np63 and c-Rel expression are correlated and expanded in primary human cancers and associate in the nuclei of human SCC cells. A, nuclear expression patterns of Np63 and c-Rel are expanded and associated in primary human SCCs. Immunostaining of normal mucosa and SCC tissue sections with p63 and c-Rel. The proliferative compartment of normal mucosa is identified by Ki67 immunoreactivity. B, endogenous Np63 and c-Rel are present in nuclei of human HNSCC lines. Western blots of nuclear extracts prepared from SCC lines. Mouse keratinocytes overexpressing Np63 or β-gal are included as controls. C, endogenous nuclear Np63 and c-Rel physically interact in SCC cells. Coimmunoprecipitation analysis of UM-SCC-38 nuclear extracts. Nuclear extracts were immunoprecipitated with antibody to c-Rel (left) or p63 (right) and probed for c-Rel or p63, as noted. D, endogenous nuclear Np63 and c-Rel derived from squamous carcinoma cell lines are associated on the p21WAF1 promoter in vitro. EMSA analyses of nuclear extracts derived from the HNSCC cell line UM-SCC-46. A 32P-labeled probe using the p63BS#1 from the p21WAF1 promoter was used in these reactions. A protein-DNA complex is seen and can be partially supershifted with a c-Rel antibody. Use of the smaller 32P tag in the HNSCC experiments allowed for a supershift band to be seen with the p63 antibody as well.

Endogenous Np63 and c-Rel physically associate in nuclei of HNSCC cell lines.

	Discussion

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We show that overexpressing Np63 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 Np63. In these cells, and in human SCC cells endogenously expressing these proteins, Np63 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 homeostasis (21, 35, 36), including that of the epidermis (21), and enhanced nuclear c-Rel has been associated with solid and hematopoietic cancers (41, 42). In contrast to numerous studies of the NF-B 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 wild-type c-Rel can transform primary spleen cells in vitro (25). Furthermore, forced overexpression of c-Rel in vivo under control of the mouse mammary tumor virus long terminal repeat promoter resulted in mammary tumorigenesis and correlated with induction of NF-B target genes, including c-myc and cyclin D1 (24). Treatment of these c-Rel–transformed mammary tumor cells with dimethylbenzanthracene in vitro resulted in epithelial to mesenchymal transition (43).

The transforming ability of c-Rel both in vitro and in vivo is dependent on the presence of its transactivation domain (44, 45). The transformation capacity of c-Rel can be enhanced by mutations and deletions within the transactivation domain, suggesting that the strength of transactivation activity can determine the potency of c-Rel (44, 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 genes (47). In this report, we show that, in addition to being phosphorylated, the c-Rel that is modulated by Np63 has transcriptional NF-B reporter-enhancing and p21 gene-repressing activity. Future studies will aim to identify the effect of sustained Np63 elevation on c-Rel target gene expression.

Regulation of NF-B is a dynamic process (38–40). In the classic paradigm of NF-B regulation, cytoplasmic IB proteins retain NF-B in an inactive state, with NF-B nuclear translocation following IB degradation. Once within the nucleus, NF-B induces resynthesis of IBs, and IB and IB can dissociate NF-B from DNA and usher it to the cytoplasm via their nuclear export functions (39, 40). IBβ can function in its phosphorylated form to dissociate NF-B from DNA, whereas unphosphorylated IBβ forms a ternary complex with NF-B and DNA and can protect it from dissociation by IB or IB (48). Our data support a model whereby enhanced Np63 expression results in nuclear accumulation of c-Rel without disrupting IB-c-Rel cytoplasmic interactions or causing degradation of the IBs (Fig. 3). We have shown that c-Rel physically interacts with Np63 in the cell nucleus and propose that this association inhibits nuclear, but not cytoplasmic, interaction of c-Rel with the IB proteins by blocking binding. This results in enhanced nuclear accumulation of c-Rel due to the inability of IB and IB to interact with and remove c-Rel. The c-Rel that accumulates in the nuclei of Np63-overexpressing cells is phosphorylated and transcriptionally active, as determined by reporter gene assay, and can interact in a complex with Np63 on the p21WAF1 promoter to block promoter activity.

The physical association between Np63 and phosphorylated c-Rel requires the -COOH terminus of Np63; like Np63, TAp63 also physically associates with c-Rel, whereas Np63^p40 does not (Fig. 4B). Although less is understood about the role of TAp63 in cancer development, dysregulated TAp63 has been reported to influence the development and progression of chemically induced skin tumors (49). Whether the downstream effects of TAp63 in this context are mediated by c-Rel remains to be determined.

It was initially proposed that overexpression of Np63 in human cancers blocks the tumor suppressor activity of p53 (50). It has recently been shown that the ability of Np63 to repress p73-dependent apoptosis enhances the survival of a subset of SCC cells (17). The data presented herein support a novel mechanism whereby overexpression of Np63 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 Np63 influences the growth and phenotypic characteristics of human cancers. Consistent with our model, a recent clinical trial targeting constitutively active NF-B in HNSCC via a proteasome inhibitor was found to block nuclear localization of Rel-A but not c-Rel.⁵ The findings presented here suggest that distinct NF-B complexes can promote proliferation of keratinocytes, act in concert with other NF-B 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.

	Disclosure of Potential Conflicts of Interest

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No potential conflicts of interest were disclosed.

	Acknowledgments

 
Grant support: Intramural projects Z01 BO 04006-06 LIMB (Center for Drug Evaluation and Research, Food and Drug Administration) and Z01-DC-00016 (National Institute on Deafness and Other Communication Disorders, NIH).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. D. Guttridge and D. Sidransky for providing IBM superrepressor and Np63^p40 adenoviruses, Dr. T. Gilmore for providing c-Rel cDNA, Dr. Bin Yan for p21WAF1 promoter analysis, Drs. M. Stacey Ricci and David Gius for critical reading, and Drs. Christophe Cataisson and Stuart Yuspa for helpful discussions.

	Footnotes

 
³ B. Yan et al., unpublished data.

⁴ H. Lu, unpublished observations.

⁵ C. Allen, K. Saigal, L. Nottingham, P. Arun, Z. Chen, C. VanWaes. Bortezomin-induced apoptosis with limited clinical response is accompanied by inhibition of canonical but not alternative NF-B pathway subunits in based and neck cancer. Clin Cancer Res. In press 2008.

Received 11/ 5/07. Revised 3/31/08. Accepted 4/21/08.

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