Dnp63 Versatilely Regulates a Broad NF-Kb Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Cancer Tumor and Stem Cell Biology Research

DNp63 Versatilely Regulates a Broad NF-kB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Xinping Yang1, Hai Lu1, Bin Yan1, Rose-Anne Romano2, Yansong Bian1, Jay Friedman1, Praveen Duggal1, Clint Allen1,3, Ryan Chuang1, Reza Ehsanian1,4,5, Han Si1, Satrajit Sinha2, Carter Van Waes1, and Zhong Chen1

Abstract Head and neck squamous cell carcinoma (HNSCC) and many other epithelial malignancies exhibit increased proliferation, invasion, and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-kB family members DNp63, cRel, and RelA. However, the mechanisms of cross-talk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. In this study, we showed that DNp63 regulates a cohort of genes involved in cell growth, survival, adhesion, and inflammation, which substantially overlaps with the NF-kB transcriptome. DNp63 with cRel and/or RelA are recruited to form novel binding complexes on p63 or NF-kB/Rel sites of multitarget gene promoters. Overexpressed DNp63- or TNF-a–induced NF-kB and inflammatory cytokine interleukin-8 (IL-8) reporter activation depended on RelA/cRel regulatory binding sites. Depletion of RelA or DNp63 by small interfering RNA (siRNA) significantly inhibited NF-kB–specific, or TNF-a–induced IL-8 reporter activation. DNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with these data, an increase in nuclear DNp63, accompanied by increased proliferation (Ki-67) and adhesion (b4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared with the basilar pattern of protein expression and minimal inflammation seen in nonmalignant mucosa. Furthermore, overexpression of DNp63a in squamous epithelial cells in transgenic mice leads to increased suprabasilar cRel, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals DNp63 as a master transcription factor that, in coordination with NF-kB/Rels, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC. Cancer Res; 71(10); 3688–700. 2011 AACR.

Introduction as well as frequent abnormalities of the TP53 and NF-kB transcription factor families (1–5). HNSCC and epidermal Head and neck squamous cell carcinomas (HNSCC) and malignancies are known to carry a high mutation rate for other epithelial cancers display coordinated changes in cell TP53,butTP53 mutation alone is insufficient to account for proliferation, migration, and inflammatory cell infiltration, cancer development and differences in prognosis. This is attributable, in part, to the diversity of mutations or other Authors’ Affiliations: 1Tumor Biology Section, Head and Neck Surgery mechanisms of inactivation that affect TP53 functions (6, 7). Branch, National Institute on Deafness and Other Communication Dis- In addition, more recent studies have pointed to redundant orders, NIH, Bethesda, Maryland; 2Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and distinct functions of other TP53 family members, such and Life Sciences, Buffalo, New York; 3Clinical Research Training Program as p63, in the modulation of gene expression and the supported jointly by NIH and Pfizer Inc.; 4Howard Hughes Medical Insti- – tute–NIH Research Scholars Program; and 5Graduate Partnership Pro- malignant phenotype (8 10). p63 consists of 6 isoforms gram of NIH and Oxford University, United Kingdom differing in the N- or C-terminal region as a result of Note: Supplementary data for this article are available at Cancer Research alternative promoter usage or splicing (11). TAp63 isoforms Online (http://cancerres.aacrjournals.org/). contain full-length N-terminal transactivating domains, C. Van Waes and Z. Chen contributed equally as senior authors. whereas DNp63 has a truncated N-terminus. The p63 iso- Corresponding Author: Zhong Chen, Head and Neck Surgery Branch, forms share high sequence homology with the TP53 DNA- NIDCD/NIH, 10/5D55, MSC-1419, Bethesda, MD 20892. Phone: 301-435- binding domain (12) and, thus, enable TA and DNp63 iso- 2073; Fax: 301-594-4643; E-mail: [email protected] or Carter Van Waes, Head and Neck Surgery Branch, NIDCD/NIH, 10/CRC, Rm 4- forms to transactivate or inhibit TP53 target genes, respec- 2732, Bethesda, MD 20892. Phone: 301-402-4216; Fax: 301-402-1140. tively. DNp63 is the most abundant isoform detected in the E-mail: [email protected]. basal layer of mucosa, skin, other epithelial tissues (8, 13), doi: 10.1158/0008-5472.CAN-10-3445 and in HNSCC (14). Furthermore, p63 isoforms regulate a 2011 American Association for Cancer Research. broad range of target genes (13, 15, 16), although the

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different partners and mechanisms by which they coordi- embedded HNSCC and mucosal tissue arrays were obtained nate such gene activation remain incompletely defined. from Cybrdi. Images were acquired using an Aperio Scan- Recently, we have shown that DNp63 can form a novel scope, and the staining intensity was quantified using the complex with the transcriptionally functional NF-kB family Aperio Cell Quantification Software (Aperio). member cRel to bind a p63 regulatory site and repress expression of p21Cip1, thus enhancing proliferation of murine Short interfering RNA keratinocytes (17). The NF-kB/Rel family includes 5 function- The short interfering RNA (siRNA) targeting the unique ally and structurally related proteins, including RelA and cRel, exon in the N-terminal of DNp63 was made by Integrated DNA which are often classified together as components of the Technologies (Supplementary Table S1), based on the pre- canonical NF-kB pathway (18). Furthermore, aberrant activa- vious publication (24). siRNAs targeting TAp63, total p63,or tion of RelA is observed in the majority of HNSCCs and many other genes were purchased from Dharmacon or Qiagen. other malignancies and has been shown to regulate a broad gene program to promote cancer progression, inflammation, Real-time reverse transcriptase PCR metastasis, and resistance to therapies (1, 4, 19). However, The RNA isolated was subjected to reverse transcription RelA, cRel, and other members and transcription factors of the and PCR as described (25). PCR primers for DNp63 and TAp63 NF-kB family were found to be variably activated and sensitive were synthesized (Supplementary Table S1), and other pri- to proteasome inhibition in different subsets of HNSCC lines mers were purchased (Applied Biosystems). The gene expres- and specimens obtained from patients resistant to therapy sion was normalized to 18S rRNA as an internal control. (20, 21). These findings suggested that cRel, RelA, and other transcription factors might contribute to coregulation of Reporter gene assays NF-kB target gene expression and the malignant phenotype Reporter gene activities were assayed using the Dual-Light in HNSCC and epidermal tumorigenesis. System Kit (Applied Biosystems) and measured by Wallac The finding that DNp63 and cRel can bind and down- VICTOR2 1420 Multilabel Counter (Perkin Elmer). Differences regulate a classical TP53 target gene suggested a new para- in reporter activity due to nonspecific transfection or digm by which these 2 transcription factors can interact and gene-related antiproliferative or cytotoxic effects were nor- coregulate target gene(s). In the present study, we explored malized using either b-Gal or WST-1 Cell Proliferation the hypothesis that DNp63, RelA, and cRel members of the NF- Reagent (Roche Diagnostics) according to the manufacturer's kB family can interact together to more broadly regulate instructions. transcription of TP53/p63 and NF-kB/Rel target genes that are known to be important in pathologic alterations in HNSCC Prediction of transcription binding sites and squamous epithelial cells. The binding sites in the promoter regions of NF-kB, p63, or TP53 target genes were identified using the published binding Materials and Methods and experimental information after querying National Center for Biotechnology Information and other databases. The novel Cell lines p63 and NF-kB binding sites were predicted using Genomatix The UM-SCC cell lines used were originally obtained from and position-weighted matrices (PWM) built on published Dr. Thomas E. Carey, University of Michigan, in 2000. TP53 information (see Supplementary Methods). sequence analysis for exons 4 to 9 was done in our laboratory as previously reported (22). DNA was sent for sequence Chromatin immunoprecipitation assay genotyping in 2008 and Fall 2010 to compare and verify their Protein–DNA complexes were precipitated using the anti- unique origin from original stocks, as recently described (23). bodies indicated, and PCR was carried out using specific The 9 loci analyzed included D3S1358, D5S818, D7S820, primers as described in Supplementary Methods. D8S1179, D13S317, D18S51, D21S11, FGA, vWA, and amelo- genin. Primary human epidermal keratinocytes (HEKA) were Images of cultured cell morphology, wound healing, purchased and cultured in accordance with the man- and cell migration through Matrigel ufacturer's protocol (Invitrogen) and used within 5 passages. Images of cultured cells and wound-healing experiments HEKA and human oral mucosal keratinocytes (HOK) exhibit were taken under an Olympus IX70 inverted microscope at similar global gene expression patterns for DNp63 and other room temperature using the SPOT RT Color Digital Camera genes presented in this study (data not shown). (Diagnostic Instrument, Inc.) and the SPOT Advanced version 4.0.1 software program. Images of cells migrating through Immunohistochemical analysis Matrigel were acquired using an Aperio Scanscope, and Frozen HNSCC tissues were obtained from the Cooperative quantification was done using the Aperio Cell Quantification Human Tissue Network. Detailed immunohistochemistry Software program (Aperio). (IHC) protocol is presented in the Supplementary Informa- tion. The staining patterns and morphology were observed DNp63a transgenic mouse model and under an Olympus BX51 microscope, and images were cap- immunofluorescence tured by using an Olympus DP70 camera and DP70 Image A Tet-responsive DNp63a transgenic (TG) mouse model Manager software. De-identified formalin-fixed and paraffin- has been developed by crossing HA-DNp63a with Tet-OFF TG

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animals under bovine K5 promoter (K5-tTA; ref. 26). Expres- unpublished data; Supplementary Methods). RelA and cRel sion of DNp63a in TG mouse was induced after birth, and sites were highly represented in the proinflammatory cytokine immunofluorescence staining was done as described in Sup- and NF-kB gene clusters, whereas NF-kB1 sites predominated plementary Methods. Immunofluorescence images were in the adhesion gene cluster (Fig. 1B). Literature review and observed under a Nikon FXA fluorescence microscope, cap- annotated Ingenuity Pathway Analysis (IPA) linked many of tured using a Nikon digital camera, and analyzed using the modulated genes via related physical, signal, or transcrip- ImageJ, Adobe Photoshop, and Adobe Illustrator software tional interactions to DNp63 and NF-kB (Fig. 1C) or other programs. pathways (Supplementary Table S3). These findings supported the hypothesis that DNp63 regulates a broad gene program Statistical analysis that partially overlaps with the NF-kB transcriptome. Data are presented as mean SD, and the statistical analysis relative to the control is calculated (t-test, *P < 0.05). DNp63, cRel, and RelA binding to predicted sites of target gene promoters detected by chromatin Results immunoprecipitation We searched for putative p63 and/or NF-kB–specific reg- DNp63 regulates a broad gene program that overlaps ulatory sequences in the promoter regions of p63 target genes with NF-kB targets identified in this study using Genomatix (B. Yan, unpublished Nuclear DNp63 staining of intermediate and strong inten- data; Supplementary Methods). Many of the DNp63 target sity was observed throughout malignant epithelia in 18 of 21 genes contained predicted p63 and/or NF-kB binding sites (85%) frozen HNSCC specimens when compared with the (Fig. 2A; Supplementary Table S4), consistent with coregula- basilar, localized, staining pattern seen in squamous mucosa tion and a potential functional interaction between the 2 (Supplementary Fig. S1A). Proliferating human keratinocytes factors (Fig. 1A and C; Supplementary Table S3). To test this (HEKA) and HNSCC (UM-SCC) lines predominantly expressed hypothesis, chromatin immunoprecipitation (ChIP) assays DNp63 mRNA and protein, with most HNSCC lines possessing were conducted in cell lines that express DNp63 at relatively mutant (mt) TP53 expressing higher levels than those with high (UM-SCC46, Fig. 2B) or lower levels (UM-SCC1, not wild-type (wt) TP53 (Supplementary Fig. S1B). To investigate shown). We detected significant basal p63 binding activity the broad role of DNp63 in regulating gene expression, we on 13 sites of 8 promoters, including a known p63 binding selected UM-SCC1 and UM-SCC6 lines with wtTP53 and site in the p21Cip1 gene, which served as a positive control intermediate DNp63 expression, as well as UM-SCC22B and (Supplementary Table S4; refs. 17, 27). IL-8, CSF2, PDGFA, and UM-SCC38 lines with mtTP53 and higher DNp63 expression YAP1 promoters were additionally selected for ChIP analyses level (Supplementary Fig. S1B). p63 was knocked down by with multiple antibodies, without or with TNF-a treatment specific siRNA targeting DNp63, total p63 (Supplementary (Fig. 2B). Antibodies included for ChIP assay were selected on Fig. S2A; Supplementary Table S1), or TAp63 (not shown), the basis of previous findings that, in HNSCC, DNp63 can form a confirming distinctive specificity of DNp63 knockdown. A protein complex with cRel (17), canonical RelA/p65 is often broad gene panel, including NF-kB and TP53 family targets, activated (18, 28), and IkB kinase alpha (IKKa) may bind to was tested (Fig. 1A; Supplementary Fig. S2B; Supplementary DNp63 (13, 29). ChIP results revealed that p63 and cRel Table S2). In UM-SCC1 cells, DNp63 knockdown led to sig- exhibited higher basal binding activities at most predicted nificant modulation of genes critical in inflammation, includ- binding sites, and TNF-a potently induced cRel binding to all ing members of the interleukin-1 (IL-1) family, CSF2, IL-6, and sites. In addition, as expected, TNF-a greatly induced RelA/p65 IL-8; in growth regulation and apoptosis, such as PDGFA, binding activity in the previously defined proximal p65/cRel TGF-a, TP53, p73, MDM2, Bcl-2, and Bcl-xL, and in adhesion, site in the IL-8 promoter (30). Minimal basal and TNF- such as ITGA6, ITGB4, and ICAM1 (Fig. 1A). Similar results a–induced IKKa-binding activities were detected (Fig. 2B). were obtained for most genes in UM-SCC6, UM-SCC22B, and UM-SCC38 cells, although some differed between wtTP53 and Coordinate binding of p63, cRel, and/or RelA protein mtTP53 lines (Supplementary Fig. S2B; Supplementary complexes to p63 and NF-kB regulatory elements Table S2). Together, these results suggested that DNp63 could To investigate whether DNp63, cRel, and RelA bind to the coregulate a broad gene program, including several known same cognate elements present in these promoters, electro- NFkB and TP53 target genes. phoretic mobility shift assays (EMSA) were carried out with nuclear extracts from UM-SCC1 (Fig. 3) and UM-SCC46 (not Systems biology analyses predict transcription binding shown). Multiple protein complexes were bound to the oli- motifs and network between DNp63- and NF-kB– gonucleotide containing the sequence of RelA/cRel site of the regulated genes IL-8 promoter [main bands (MB); Fig. 3A, lanes 2 and 4]. Next, we compared 80 genes modulated by DNp63 or RelA Remarkably, supershift with an anti-RelA antibody (lane 6), or knockdown, or treatment with NF-kB inducer TNF-a, and an antibody against the p63 C-terminus (H129; lane 7), atte- found a broad overlap of 26 genes (Fig. 1B). We then predicted nuated most MBs. Furthermore, lesser supershift of MB NF-kB binding motifs on the proximal promoters (500 activity was detectable using 2 different cRel C-terminal þ100 bp) of these genes using the Genomatix software antibodies (lanes 10 and 11). A protein complex was bound program and PWMs based on published data (B. Yan, to the oligonucleotide containing the sequence of the p63 site

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IL-1A IL-1B IL-1R1 IL-1R2 IL-1RN CSF2 A 0.0 0.0 0.0 2.5 * 2.0 0 −10 −3.0 −4.0 2.0 * 1.5 * − −0.5 20 * 1.5 −30 * − − 1.0 6.0 8.0 * * −40 − 1.0 * * * 1.0 * −50 −9.0 −12.0 0.5 * * 0.5 −60 * −1.5 − −12.0 * −16.0 * * 0.0 0.0 70 * IL-6 IL-8 PDGFA TGF-α p53 0.5 p73 * 0 0.0 0.0 1.5 3.0 0.0 − * * − −4 0.5 −1.0 * 0.5 * * 1.0 2.0 − −1.0 * 1.0 * −8 −2.0 * * −1.5 −1.5 0.5 1.0 − −12 * −3.0 * 2.0 −2.0 * −2.5 0.0 0.0 Relative gene expression Relative −16 * − Figure 1. Knockdown of DNp63 * * −2.5 4.0 * ICAM1 regulates a broad gene expression MDM2 Bcl-2 Bcl-xL ITGA6 ITGB4 0.0 0.0 0.0 0.0 0.0 0.0 program that overlaps with the −0.5 −0.5 NF-kB transcriptome and −1.0 −1 −1.0 −0.5 −0.5 network. A, relative fold-change in −1.5 −1.0 −2 * − D −2.0 * * −2.0 1.0 mRNA expression after Np63 * * * −1.5 −2.5 * −1.0 * * siRNA transfection of UM-SCC1 −3 − −1.5 −3.0 * 3.0 * −2.0 * * for 24, 48, or 72 hours (white, gray, * −3.5 * −4 * −1.5 − or black bars); *, P < 0.05 versus −4.0 * −2.5 * 2.0 control siRNA. B, heat map for up- (red) or downregulated (green) BC D mRNA expression after Np63, κ B1 RelA siRNA, or TNF-a treatment. Δ Np63 KO RELA KO TNF Tx RELA NF- cRel IL-1B IL-1R2 Dots indicate predicted RelA, NF- BIRC2 k ICAM1 B1, and cRel binding sites in the CDKN1A IL-1RN IL-8 proximal promoter regions ( 500 CSF2 IL-1A bp þ100 bp of transcription IL-8 PDGFA IL-1R1 start site). Gene categories are as IL-1B Rel TGFER1 follows: blue, growth and IL-1A RELA apoptosis; pink, cytokines; black, IL-6 TGFB1 adhesion; brown, NF-kB. C, IPA ICAM1 NF-KB1 TGFA illustrates important functional LAMA3 Bcl-xL TGFB2 networks between DNp63- LAMB3 CSF2 IL-6 modulated genes and RelA, cRel, ITGA5 ITGB4 CCND1 or NF-kb1. ITGA2 IGFBP3 Bcl-2 ITGA6 TP53 ITGA5 ITGA6 ITGB4 TP63 CDKN1A CCND1 LAMB3 BBC3 EGFR ERBB3 BIRC2 YAP1 ERBB4 LAMC2LAMA3 Rela TP73 MDM2 NF-KB1 AREG NRG2 cRel IGFBP3 EGR1 Transcription regulator IKBKE Cytokine/growth factor IL1-RN Transporter IL1-R2 Transmembrane receptor TNFAIP2 Kinase 0 +− Others

of IL-8 promoter, and was attenuated by both p63-specific (Fig. 3C, lanes 20–22). For the predicted CSF2 promoter Rel antibodies (Fig. 3B, lanes 15 and 16) and shifted with anti-p63 site, RelA and p63 were both detected by supershift (lanes 24– H137 antibody (lane 16). Although the RelA antibody had no 26), and, furthermore, the anti-cRel antibody significantly significant effect (lane 14), the cRel antibody attenuated MB decreased the MBs (lanes 27 and 28). We obtained similar binding activity, indicative of cRel cobinding to the predicted results for the YAP promoter p63 site (Fig. 3D). Both anti-p63 p63 binding site (lane 18). A predicted CSF2 promoter p63 site antibodies attenuated MBs (lane 31 and 32), with H137 detect- oligonucleotide-bound complex was shifted or attenuated ing 2 supershifted bands (lane 32). Although no supershift was with anti-p63 antibodies but not with RelA antibody detected with anti-RelA (lane 30), anti-cRel attenuated MB

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IL-8 A TSS CSF2 p63(−1,425) c-Rel/p65(−83) p63(+845)

p63(−1,415) cRel(+702)

YAP1 PDGFA p63(−1,485) p63(−2,324) cRel/p65/p50 (−2,218)

p63(−1,486) p50(−1,609) p63(−2,314) p50(−2,261)

p63 cRel/p65/p50 cRel/p65 cRel p50

Figure 2. Distinct DNp63, cRel, B and RelA binding activities on p63- or NF-kB–responsive 20 15 IL-8 promoter IL-8 promoter elements. A, p63 and NF-kB/Rel − Ctrl − p63 site ( 1,424 bp) # p65/c-Rel site ( 83 bp) binding sites [with base pairs from 15 TNF # # transcription start site (TSS)] on * 10 * * gene promoters were predicted by 10 # Genomatix by including user- # * 5 # defined p63 PWM as described in 5 * * * # * * * * Supplementary Methods. B, ChIP assay conducted using anti-p63, 0 0 cRel, IKKa, RelA, and isotype IgG p63a cRel IKKα p65 IgG p63a cRel IKKα p65 antibodies without [control (Ctrl)] or with TNF-a treatment, followed CSF2 promoter PDGFA promoter by real-time PCR. Mean relative 20 + 10 p63 site ( 845 bp) # p63 site (−1,495 bp) binding activity SD for # P < * 8 triplicates; 0.05 compared with 15 # * isotype (*) or between control and * 6 TNF-a treatment (#). 10 * # 4 * # * * * 5 # 2 # Relative binding activity Relative 0 0 IgG p63a cRel IKKα p65 IgG p63a cRel IKKα p65 20 15 YAP promoter YAP promoter # − # p63 site ( 2,324 bp) cRel site (−2,218 bp) 15 * * 10 10 * # * 5 # # # # 5 * * * * * * * * * 0 0 IgG p63a cRel IKKα p65 IgG p63a cRel IKKα p65

binding activity (lane 34). These data provide evidence for NF-kb or RelA/cRel site–dependent IL-8 reporter gene activ- coordinate and differential binding of distinct p63, cRel, and/or ities (construct: 133 bp; ref. 30); however, the effects were RelA complexes to p63 and NF-kB/Rel regulatory elements of less potent than with RelA knockdown (Fig. 4A, top row). multiple genes. Conversely, overexpression of DNp63 increased NF-kB and IL-8 reporter gene activities more potently than TAp63 p63 and TNF-a regulate NF-kB, TP53, p21Cip1,orIL-8 (Fig. 4A, middle row), at a level similar to the classical transcription activity NF-kBinducerTNF-a (Supplementary Fig. S3, left column). Thereafter, we investigated how p63 modulated NF-kBor In contrast, overexpressed DNp63 weakly stimulated TP53 TP53-mediated transcriptional activity (Fig. 4; Supplemen- while repressing TP53 target p21Cip1 reporter activity tary Fig. S3). DNp63 knockdown markedly decreased specific (Fig. 4A, bottom row).

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AB Probe alone Probe + extract Cold probe (wt) Cold probe (mt) Probe + extract Anti-ReIA Anti-p63 α (H129) Isotype Probe + extract Anti-cRel (S) Anti-cRel (M) Isotype Isotype Anti-RelA Anti-p63 α (H129) Anti-p63 (H137) Isotype Anti-cRel (M)

ReIA p63 p63 cRel

MB MB MB MB

1234 5678 9 10 11 12 13 14 15 16 17 18 IL-8 ReIA/c-Rel IL-8 p63

CD α (H129) α (H129) α (H129) Probe + extract Anti-RelA Anti-p63 Probe + extract Anti-RelA Anti-RelA Anti-p63 (H137) Anti-p63 Anti-p63 Anti-p63 (H137) Isotype Anti-cRel (M) Isotype Anti-p63 (H137) Isotype Anti-cRel (M)

RelA p63 p63 p63

MB MB MB MB MB

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 CSF2 p63 CFS2 cRel YAP p63

Figure 3. Coordinate binding of p63, cRel, and/or RelA protein complexes to p63 and NF-kB regulatory elements. EMSA using nuclear extract from UM-SCC1 treated with TNF-a, incubated with 32P-labeled oligonucleotide probes for known IL-8 promoter RelA/cRel (83 bp, lanes 1–12; A) and predicted p63 (1,425 bp, lanes 13–18; B) sites; CSF2 promoter p63 (þ845 bp, lanes 19–22; C) or cRel (þ702 bp, lanes 23–28) sites; and a YAP promoter p63 site (2,324 bp, lanes 29–34; D). Experimental conditions include labeled probe alone; labeled probes plus nuclear extracts; labeled probe and nuclear extract plus 50-fold excess unlabeled wild-type (wt) or mutant (mt) competitor DNA; anti-RelA, anti-p63 (H129 or H137), anti-cRel (S, Santa Cruz; M, Millipore), or isotype control antibodies.

Transcriptional regulation of the IL-8 gene by DNp63 was observed using the minimal –133-bp promoter construct further examined in UM-SCC1 cells using luciferase reporter containing the RelA/cRel binding site, for which we estab- constructs with serial deletions and point mutations (Fig. 4B; lished p63, cRel, and RelA cobinding activities in the preceding ref. 30). Consistent with observations discussed in the pre- paragraphs (Fig. 3A, lanes 5–12). Furthermore, deletion or ceding sections, overexpression of DNp63 exhibited a stimu- point mutation of this RelA/cRel site (83) or nearby AP-1 site latory pattern for most of the promoter constructs that was (128 bp) had a profound impact on both TNF-a- and DNp63- similar to that of the NF-kB–inducing cytokine TNF-a. Delet- induced reporter activity (Fig. 4B, bottom panel), indicating ing the distal predicted p63-binding site (1,424 bp) had a that DNp63- and TNF-a–induced IL-8 activation are both minimal effect, whereas increased reporter activity was dependent on these sites.

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NF-κB 1.5 IL-8 − A 1.5 1.0 B IL-8 reporter activity (×10 3) 1.0 0.7 Figure 4. DNp63 and TNF-a 1.0 1.0 0 100 200 300 400 500 600 modulate NF-kB, IL-8, TP53,and * * p21 0.4 reporter activities. A, relative * 0.4 * 1,481 0.5 0.2 0.5 * NF-kB–specific, NF-kB– dependent IL-8 (133 bp), TP53- 0.0 0.0 * 272 specific, and TP53-dependent siRNA Ctrl RelA ΔNp63 Ctrl RelA ΔNp63 * p21Cip1 luciferase reporter * 133 NF-κB * activities 48 hours after 15 10 IL-8 transfection of UM-SCC1, with p63 * * * pLpc D 12 9.6 8 6.5 * AP-1 98 Ctrl, RelA, or Np63 siRNAs, or pLpc+TNF NF-IL6 D a 9 * 6 overexpression of Ctrl, Np63 , 6.1 ΔNp63 Exp NF-κB * 50 or TAp63a vectors, as indicated. 6 4 b P < 1.0 1.1 Normalized with -Gal. *, 0.05 3 1.0 2 IL-8 0 100 200 300 400 500 600 versus Ctrl. B, relative 0 0 luciferase reporter activity for Vector Ctrl ΔNTA Ctrl ΔNTA * serially deleted or binding site- Relative luciferase activity luciferase Relative 133 * specific point mutant promoter 150 TP53 4.0 p21 * constructs, which include p63, * 2.9 NF-kB Mut 106.9 * NF-kB, AP-1, and NF-IL6 binding 3.0 * 100 sites. UM-SCC1 cells were 2.0 * AP-1 Mut transfected with indicated reporter 1.0 * 50 * * constructs plus Ctrl (pLpc), TNF-a 6.7 1.0 0.4 D a 1.0 * NF-IL6 Mut (20 ng/mL), or Np63 expression 0 0.0 * vectors. Mean SD of reporter Δ Δ Vector Ctrl N TA Ctrl N TA activities of 6 replicates, adjusted for cell density by WST-1 48 hours after transfection. *, P < 0.05 versus baseline. C, IL-8 promoter C (133 bp) reporter activity. Left, 6.0 60 a 3.0 Basal 40 TNF-α Ctrl Vector ΔNp63 basal (without) or with TNF- for 24 hours preceded by Ctrl or * 30 DNp63 siRNA for 46 hours. Right, 4.0 40 2.0 * * cotransfected with Ctrl or DNp63 20 expression vector, and Ctrl, RelA, 1.0 2.0 20 * or cRels siRNAs for 70 hours. 10 * * Mean SD for 4 replicates, after

IL-8 reporter activity normalization for cell density by 0 0 0 0 P < siRNA Ctrl ΔNp63 Ctrl ΔNp63 Ctrl RelA cRel Ctrl RelA cRel WST-1. * 0.05 versus Ctrl.

To further test the dependency of IL-8 promoter activity on phases in UM-SCC1 and UM-SCC6 cell lines (Fig. 5A and B; DNp63, RelA, or cRel transcriptional components, IL-8 repor- Supplementary Fig. S4A and B). Furthermore, DNp63 deple- ter activity was measured under combinatory knockdown or tion induced development of larger and flatter cells of differ- inducing conditions (Fig. 4C). DNp63 depletion partially inhib- entiated cell morphology (Fig. 5C), reduced wound-healing ited basal IL-8 reporter activity and almost completely sup- motility in scratch assay (Fig. 5D; Supplementary Fig. S4C), pressed TNF-a–induced IL-8 reporter activation (2 of the left and cell migration in classical Matrigel assay by up to 35% (P < panels). Conversely, basal level (control vector) IL-8 reporter 0.05; Supplementary Fig. S4D). Thus, the effect of modulation activity was more strongly suppressed by depletion of RelA of DNp63 upon multiple vital biological functions is consistent than cRel, whereas DNp63 overexpression enhanced the rela- with its role in the regulation of a broad gene program. tive contribution of cRel (2 of the right panels). These results are consistent with the relatively greater binding of RelA and Increase in DNp63, Ki-67, and b4 integrin DNp63 relative to cRel to the IL-8 RelA/cRel binding site as immunostaining and inflammation in HNSCC shown by EMSA (Fig. 3A), and previous evidence indicating compared with mucosa RelA is the major NF-kB family subunit responsible for bind- We further examined the pattern of expression of DNp63, ing and transactivation of the IL-8 promoter in HNSCC (28). markers of proliferation (Ki-67) and adhesion (b4 integrin), and cellular inflammation in human HNSCC and mucosa DNp63 modulates proliferation, differentiation, cell specimens. An increase in nuclear, localized, DNp63 staining cycle, survival, and migration in HNSCC cell lines was observed throughout the malignant epithelia in most Knockdown of DNp63, but not TAp63, markedly inhibited HNSCC samples from independent series of 21 frozen (e.g. cell proliferation, increased sub-G0 DNA fragmentation and Fig. 6A; Supplementary Fig. S1) and 20 fixed tumor tissues (e.g., G0–G1 blockade, and decreased S-phase and G2–M cell-cycle Fig. 6B). In contrast, nuclear DNp63 was predominantly

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AB15 90 Sub-G0 G0/G1 80 No treatment 10 2.5 70 Lipofectamine 60 2.0 Control siRNA 5 50 ΔNp63 siRNA D 1.5 0 40 Figure 5. Np63 regulates TAp63 siRNA 14 S 14 G /M HNSCC proliferation, cell cycle, 2 1.0 12 12 differentiation, and migration. UM- 10 10 0.5 8 SCC1 cells were transfected with ∗ 8 Proliferation (MTT) Proliferation ∗ ∗ % of cells (serum free) 6 6 Lipofectamine (L), control (C), ∗ 4 4 D 0.0 Np63, or TAp63 siRNA as 24 48 72 96 2 2 0 0 indicated. A, cell proliferation L CdNTA L C dNTA measured by MTT assay. *, Time (h) siRNA siRNA P < 0.05 versus control. B, DNA – – cell-cycle distribution (G0/G1 S CD G2/M) at 48 hours and DNA Time (h) 0 24 47 fragmentation (sub-G0) measured at 72 hours by flow cytometry. C, UM-SCC6 UM-SCC1 photomicrographs of cell None morphology after 96 hours (100; scale bar, 100 mm). D, wound assay, UM-SCC1 monolayers Control were scratched 24 hours after siRNA

indicated transfections, and Control siRNA wound closure was photographed and measured at indicated time DeltaNp63 points (40; scale bar, 1,000 mm). siRNA Δ Np63 siRNA TAp63 siRNA

localized in the basal layers of 2 patient-matched mucosa DNp63 TG mice exhibit epithelial hyperplasia and available from the series of frozen tissues (Fig. 6A; Supple- inflammation mentary Fig. S1), and 6 mucosal specimens from the series of To determine the effects of DNp63 overexpression in squa- fixed tissues (Fig. 6B). Interestingly, we observed similar mous epithelia in vivo, we examined a recently established TG patterns of staining for the Ki-67 proliferative marker and mouse model in which DNp63a was conditionally induced in b4 integrin adhesion marker throughout HNSCC tumor speci- skin under the control of a keratin-5 promoter (26, 31). DNp63 mens, and a basilar pattern of expression for these markers in TG mice began to develop skin lesions and erythema within mucosa (Fig. 6A). We found a positive and statistically sig- 1 month of induction (Fig. 7A) and showed hyperplastic and nificant correlation between DNp63 and Ki-67 IHC histoscores hyperproliferative epidermis with diffuse infiltration of inflam- for 19 of 20 (95%) HNSCC and 6 mucosal tissue specimens matory cells in subadjacent stroma (Fig. 7B). In TG mice, from the tissue array (Fig. 6B). One outlier among the HNSCC nuclear DNp63 expression extended to suprabasilar layers of samples exhibited higher Ki-67 staining but low DNp63 IHC the hyperplastic epidermis, whereas the endogenous DNp63 staining, possibly related to a different mechanism of Ki-67 expression was restricted in the innermost basal layer of the protein expression. Among 3 mucosa samples with relatively epidermis and hair follicles in wild-type mice (Fig. 7C, upper higher DNp63 and intermediate Ki-67 staining, epithelial left). Furthermore, cRel expression was increased and colo- hyperplasia with expansion of basilar nuclear layers was calized together with DNp63 in the epidermis, and induced in observed (e.g., Fig. 6A, top vs. bottom panel of mucosa). stromal infiltrating cells (Fig. 7C, top right). As in hyperplastic Additionally, increased nuclear DNp63 in HNSCC was accom- human mucosal specimens (Fig. 6A and B), DNp63 and pro- panied by adjacent focal or diffuse infiltration of inflammatory liferation marker Ki-67 were increased in suprabasilar layers of cells in 9 of the 20 specimens (45%; P ¼ 0.04 through Fisher the thickened TG epidermis exhibiting cytokeratin-K14 exact test, not shown). Thus, the ability of DNp63 to broadly (CK) staining (Fig. 7C, bottom panels). The altered gene- regulate gene expression and critical biological processes in expression profile expressed in the skin of TG mice includes tumor cells in vitro was supported by an increase in nuclear inflammatory cytokines, adhesion molecules, and other genes DNp63, proliferation and adhesion markers, and inflammatory expressed in UM-SCC cell lines (Fig. 7D). Together, these cell infiltration in HNSCC specimens when compared with results indicate that overexpression of DNp63 in 2 indepen- mucosa in vivo. dent biological systems, HNSCC and TG mice, promotes

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Δ β A H&E Np63 Ki67 Integrin 4 pan CK B Mucosa (+) HNSCC (++) HNSCC (+) Mucosa Δ Np63 Ki-67 Tonsillar SCC Tonsillar Mucosa 120100806040200 Ki-67 Tongue SCC Tongue

C Tongue Sinus 0 20406080100 120 140 ΔNp63 H&E

HNSCC Mucosa Coef 0.550 0.827 P value 0.015 0.042 Δ Np63

Figure 6. Diffuse DNp63, Ki-67, and b4 integrin staining with inflammation in HNSCC compared with basilar staining pattern and lack of inflammation in mucosa. A, frozen sections of tonsillar and tongue SCC with matched mucosa were stained with hematoxylin and eosin (H&E), and DNp63, Ki-67, b4 integrin, and pan-CK antibodies by immunoperoxidase (400; scale bar, 200 mm). B, tissue array of HNSCC was immunostained and quantified using histoscore system (200; scale bar, 200 mm). Linear regression and Pearson correlation for DNp63 and Ki-67. Coefficient of the slope and statistical significance are shown. C, DNp63 staining in a tongue and sinus HNSCC, with adjacent infiltrating inflammatory cells. Left, 400; scale bar, 200 mm. The black rectangle frames highlight the areas shown at 1,000 in right panels; scale bar, 80 mm.

epithelial hyperproliferation, NF-kB/Rel–regulated cytokine of DNp63a in TG mice led to suprabasilar modulation of cRel gene expression, and inflammation. protein and cytokine genes as well as the development of hyperplasia and inflammation in the epidermis. This model Discussion supports the causal function of DNp63 as a key transcriptional regulator of proliferation and inflammation in squa- In this article, we provide evidence for a new mechanism mous epithelia. and model of interaction between members of the TP53 and Our results show a novel bifunctional role of DNp63 in NF-kB families in coregulating the transcriptome and phe- coordinating known NF-kB or TP53 downstream genes notype in squamous epithelia of HNSCC and DNp63 TG mice important in proliferation and cell survival. We showed (Fig. 7E). DNp63, the predominant p63 isoform overex- that DNp63 siRNA inhibited gene expression of known NF- pressedinmostHNSCCandinthebasilarlayersofsqua- kB target genes CCND1, Bcl-2,andBcl-xL that, upon indi- mous epithelia, shows a versatile ability to coordinately vidual knockdown, were previously shown to inhibit pro- regulate expression of a broad program of genes that overlap liferation and survival of UM-SCC in vitro (25, 32–35). the NF-kB/Rel and TP53/p63 transcriptomes. Furthermore, DNp63 reciprocally repressed expression and transactiva- DNp63 displays novel interactions with NF-kBfamilymem- tion activity of TP53 family members TP53, TAp63, and p73, bers, cRel and RelA, through cobinding to newly predicted as well as target genes such as p21CIP1 and PUMA,which and known p63 and NF-kB promoter regulatory sites of mediate growth arrest and apoptosis. DNp63 was previously NF-kB/Rel target genes. DNp63 promoted proliferation, cell reported as a repressor of TP53 and p73 target genes cycle, migration, and inflammatory cytokine expression by p21Cip1, NOXA,andPUMA in squamous epithelial and HNSCC in vitro. These observations are consistent with HNSCC (14, 17, 27). Furthermore, predominant nuclear studies showing an increase in DNp63 accompanied by expression of DNp63 and deficiency of TAp63 were observed proliferative and integrin adhesion protein markers, as well in a subset of non-SCC tumors in mice heterozygous for p63 as inflammatory cell infiltration throughout the tumor and TP53 alleles (36), similar to observations in human microenvironment in vivo. In addition, the overexpression HNSCC with altered p63 and TP53 (14). Variability in the

3696 Cancer Res; 71(10) May 15, 2011 Cancer Research

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effects of DNp63-modulated gene expression in the UM-SCC be partially restored by expression of a6b4. The finding that cell line overexpressing mtTP53, compared with UM-SCC DNp63 promotes a similar broad adhesion and survival gene deficient for wtTP53, suggests that effects of DNp63 may program in HNSCC and breast cancer cell lines suggests potentially be modified by TP53 status (22, 25, 32). DNp63 that such dysregulation may be of broader importance in and TP53 share high homology of the transactivation and epithelial cancers. DNA-binding domains and differentially bind to DNp63/ Remarkably, we found that the promoters of multiple TP53 sites; furthermore, these may thereby competitively DNp63- and NF-kB–modulated genes contain nearby or over- inhibit or alter one another's transactivation function (8, 9). lapping p63 and NF-kB binding sites (Fig. 2A), providing the Consistent with this finding, we previously found that the potential for members of the 2 families to coordinately or cross-talk between TP53 and NF-kB in differentially mod- physically interact on the same or nearby sites. Indeed, family ulating Bcl-xL and Bax expression is dampened in the members DNp63, cRel, and RelA showed cobinding to the subset of UM-SCC cells overexpressing mtTP53 (25, 32). same or nearby promoter sites of several of these genes by The relationship between DNp63 and Ki-67 proliferation- ChIP and EMSA supershift analyses (Figs. 2 and 3). Interest- marker histoscores in HNSCC and costaining in supraba- ingly, although DNp63 cobinding with cRel was detected on silar layers of a few hyperplastic mucosal samples in tissue both p63 and Rel regulatory sites, RelA binding was not array, as well as in skin samples from DNp63 TG mice, detected on the p63 sites of IL-8, CSF2, and YAP promoters. provides additional evidence for a relationship between Furthermore, we obtained evidence for binding of DNp63 and increased nuclear DNp63 and proliferation in vivo. cRel, but not RelA, to the p63 site of p21Cip1 promoter in A novel and unexpected finding from this study is the primary murine keratinocytes and HNSCC by ChIP and EMSA demonstration that DNp63 promotes a broad NF-kBpro- (17, 45). In contrast, DNp63 cobinding with RelA was princi- gram that includes multiple inflammatory cytokine genes. pally detected on Rel sites by supershift analysis. Thus far, it The inflammatory cytokines modulated by DNp63 in HNSCC appears that DNp63 regulates NF-kB target genes through cells and DNp63 TG mice largely overlap with the NF-kB– interaction with cRel, on p63 binding sites, and cRel and RelA regulated cytokine repertoire, which we previously identified via Rel sites. in HNSCC culture supernatants, patient serum, and tumor Supporting a novel functional role of DNp63 in coregu- specimens (37, 38). Furthermore, many of these DNp63 and lating NF-kB gene activation, knockdownoroverexpression NF-kB coregulated inflammatory factors have been indivi- of DNp63-modulated activation of NF-kB–specific reporter dually shown to promote the aggressive malignant behavior and IL-8 promoter was dependent on NF-kBregulatory that leads to poor prognosis of HNSCC (4, 19, 22, 25, 37–42). sites. DNp63a bound to the NF-kB/Rel site on the proximal Proinflammatory cytokines have been shown to attract promoter was required for DNp63a-andTNF-a–induced infiltrating neutrophils and macrophages that enhance IL-8 promoter transactivation (Figs. 3 and 4). Knockdown of angiogenesis, and they greatly promote cancer cell survival RelA exhibited a more potent inhibitory effect than knock- and metastasis (41). Consistent with this, we observed that down of cRel on transactivation of the basal IL-8 reporter increased nuclear DNp63 level is linked with infiltrating activity, whereas the relative contribution of cRel increased inflammatory cells in HNSCC, and in the dermis of DNp63 when DNp63 was overexpressed (Fig. 4C). These data are TG mice (Figs. 6C and 7B). The clinical importance of our consistent with the differential contribution of the 3 tran- findings that DNp63 promotes inflammation and aggressive scription factors to gene regulation under different condi- malignant phenotypes is further supported by a recent tions of activation and binding specificity. As with DNp63 in report showing that oral leukoplakias with increased promoting IL-8 transcription, a recent report implicates p63 DNp63 expression and inflammatory cell infiltration exhibit in IL-6 expression through a NF-kB promoter binding site a higher rate of cancer development and worse prognosis in tonsillar crypt epithelium in palmoplantar pustulosis (42). (46). TheroleofDNp63 in modulation of multiple cell adhe- The broad role of DNp63, both in promoting NF-kB and sion genes, such as a6b4integrinand laminins,migrationby antagonizing TP53, is consistent with its ancestral coevolution wound healing, and Matrigel assays in vitro, and an associa- with NF-kB as a multifunctional transcriptional coordinator tion with suprabasilar b4 integrin expression in HNSCC (47–49). With the mutation or inactivation of TP53, aberrant tumors in situ suggest it may also function in promoting cell activation of DNp63 together with NF-kB/Rels appears to migration during invasion and metastasis in tumor progres- mediate a broad primordial gene program and prosurvival sion. Consistent with this, we have previously shown that response. In that context, it may not be surprising that DNp63 increased suprabasilar a6b4expressionoccursinmore serves as a master control to versatilely coregulate NF-kB, than 70% of HNSCC with higher invasive–metastatic poten- TP53 and other gene programs to promote proliferation, tial in a prospective clinical study, and that this integrin survival, migration, and inflammation in cancer. Based on promotes laminin-mediated adhesion and migration (43, evidence that DNp63-overexpressing HNSCC and other can- 44). In addition, a causal role for DNp63 in modulating a cers are more sensitive to cisplatin-chemotherapy–induced broad adhesion gene program, including b4integrin,was DNp63 degradation (29, 50), DNp63 and target gene signatures independently shown in breast cancer epithelial cells (39). may warrant further investigation as potential biomarkers for Knockdown of DNp63 by short hairpin RNA (shRNA) down- selection of cisplatin and other therapies that target DNp63, regulated adhesion by 24 and 48 hours, and adhesion could cRel, and Rel activation in cancer.

3698 Cancer Res; 71(10) May 15, 2011 Cancer Research

Disclosure of Potential Conflicts of Interest providing DNp63 and TAp63 expression vectors. We also express appreciation to Ms. Cindy Clark (NIH library) for editing of the manuscript.

No potential conflicts of interest were disclosed. Grant Support Acknowledgments This project is supported by NIDCD Intramural projects ZIA-DC-00016, ZIA- DC-00073, and ZIA-DC-00074 and a grant from NIH R01AR049238 (S. Sinha). The authors thank Drs. Liesl Nottingham, Mathew Brown, Ning T. Yeh The costs of publication of this article were defrayed in part by the payment (NIDCD/NIH), and Chris Silvin (NHGRI/NIH) for their technical assistance; Drs. of page charges. This article must therefore be hereby marked advertisement in Paul Meltzer (NCI/NIH), Maranke Koster (University of Colorado, Denver), and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Yong-Jun Liu (The University of Texas MD Anderson Cancer Center) for critique of the manuscript; Drs. Kathryn E. King and Wendy C. Weinberg (FDA) for providing lysates of cells overexpressing DNp63 and their scientific recommen- Received September 21, 2010; revised February 21, 2011; accepted March 17, dations for the project; and Drs. James W. Rocco and Leif W. Ellisen for 2011; published online May 16, 2011.

References 1. Karin M. Nuclear factor-kappaB in cancer development and progres- 20. Allen C, Saigal K, Nottingham L, Arun P, Chen Z, Van Waes C. sion. Nature 2006;441:431–6. Bortezomib-induced apoptosis with limited clinical response is 2. Stiewe T. The p53 family in differentiation and tumorigenesis. Nat Rev accompanied by inhibition of canonical but not alternative nuclear Cancer 2007;7:165–8. factor-{kappa}B subunits in head and neck cancer. Clin Cancer Res 3. Dey A, Tergaonkar V, Lane DP. Double-edged swords as cancer 2008;14:4175–85. therapeutics: simultaneously targeting p53 and NF-kappaB path- 21. Chen Z, Ricker JL, Malhotra PS, Nottingham L, Bagain L, Lee TL, et al. ways. Nat Rev Drug Discov 2008;7:1031–40. Differential bortezomib sensitivity in head and neck cancer lines 4. Van Waes C. Nuclear factor-kappaB in development, prevention, and corresponds to proteasome, nuclear factor-kappaB and activator therapy of cancer. Clin Cancer Res 2007;13:1076–82. protein-1 related mechanisms. Mol Cancer Ther 2008;7:1949–60. 5. Tergaonkar V, Pando M, Vafa O, Wahl G, Verma I. p53 stabilization is 22. Yan B, Yang X, Lee TL, Friedman J, Dong G, Yeh NT, et al. Genome- decreased upon NFkappaB activation: a role for NFkappaB in acqui- wide identification of novel expression signatures reveal distinct sition of resistance to chemotherapy. Cancer Cell 2002;1:493–503. patterns and prevalence of binding motifs for p53, nuclear factor- 6. Belyi VA, Levine AJ. One billion years of p53/p63/p73 evolution. Proc kappaB and other signal transcription factors in head and neck Natl Acad Sci U S A 2009;106:17609–10. squamous cell carcinoma. Genome Biol 2007;8:R78. 7. Strano S, Dell’Orso S, Di Agostino S, Fontemaggi G, Sacchi A, 23. Brenner JC, Graham MP, Kumar B, Saunders LM, Kupfer R, Lyons Blandino G. Mutant p53: an oncogenic transcription factor. Oncogene RH, et al. Genotyping of 73 UM-SCC head and neck squamous cell 2007;26:2212–9. carcinoma cell lines. Head Neck 2010;32:417–26. 8. Deyoung MP, Ellisen LW. p63 and p73 in human cancer: defining the 24. Thurfjell N, Coates PJ, Vojtesek B, Benham-Motlagh P, Eisold M, network. Oncogene 2007;26:5169–83. Nylander K. Endogenous p63 acts as a survival factor for tumour cells 9. Harmes DC, Bresnick E, Lubin EA, Watson JK, Heim KE, Curtin JC, of SCCHN origin. Int J Mol Med 2005;16:1065–70. et al. Positive and negative regulation of deltaN-p63 promoter activity 25. Lee TL, Yang XP, Yan B, Friedman J, Duggal P, Bagain L, et al. A novel by p53 and deltaN-p63-alpha contributes to differential regulation of nuclear factor-{kappa}B gene signature is differentially expressed in p53 target genes. Oncogene 2003;22:7607–16. head and neck squamous cell carcinomas in association with TP53 10. Lanza M, Marinari B, Papoutsaki M, Giustizieri ML, D’Alessandra Y, status. Clin Cancer Res 2007;13:5680–91. Chimenti S, et al. Cross-talks in the p53 family: deltaNp63 is an anti- 26. Romano RA, Ortt K, Birkaya B, Smalley K, Sinha S. An active role of apoptotic target for deltaNp73alpha and p53 gain-of-function the DeltaN isoform of p63 in regulating basal keratin genes K5 and mutants. Cell Cycle 2006;5:1996–2004. K14 and directing epidermal cell fate. PLoS One 2009;4:e5623. 11. Candi E, Dinsdale D, Rufini A, Salomoni P, Knight RA, Mueller M, et al. 27. Westfall MD, Mays DJ, Sniezek JC, Pietenpol JA. The Delta Np63 TAp63 and DeltaNp63 in cancer and epidermal development. Cell alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in Cycle 2007;6:274–85. vivo and has transcriptional repressor activity that is reduced by Hay– 12. Perez CA, Ott J, Mays DJ, Pietenpol JA. p63 consensus DNA-binding Wells syndrome-derived mutations. Mol Cell Biol 2003;23:2264–76. site: identification, analysis and application into a p63MH algorithm. 28. Ondrey FG, Dong G, Sunwoo J, Chen Z, Wolf JS, Crowl-Bancroft CV, Oncogene 2007;26:7363–70. et al. Constitutive activation of transcription factors NF-(kappa)B, AP- 13. Koster MI, Dai D, Marinari B, Sano Y, Costanzo A, Karin M, et al. p63 1, and NF-IL6 in human head and neck squamous cell carcinoma cell induces key target genes required for epidermal morphogenesis. Proc lines that express pro-inflammatory and pro-angiogenic cytokines. Natl Acad Sci U S A 2007;104:3255–60. Mol Carcinog 1999;26:119–29. 14. Rocco JW, Leong CO, Kuperwasser N, DeYoung MP, Ellisen LW. p63 29. Chatterjee A, Chang X, Sen T, Ravi R, Bedi A, Sidransky D. Regulation mediates survival in squamous cell carcinoma by suppression of p73- of p53 family member isoform DeltaNp63alpha by the nuclear factor- dependent apoptosis. Cancer Cell 2006;9:45–56. kappaB targeting kinase IkappaB kinase beta. Cancer Res 2010; 15. Testoni B, Borrelli S, Tenedini E, Alotto D, Castagnoli C, Piccolo S, 70:1419–29. et al. Identification of new p63 targets in human keratinocytes. Cell 30. Mori N, Mukaida N, Ballard DW, Matsushima K, Yamamoto N. Human Cycle 2006;5:2805–11. T-cell leukemia virus type I Tax transactivates human interleukin 8 16. Pozzi S, Zambelli F, Merico D, Pavesi G, Robert A, Maltere P, et al. gene through acting concurrently on AP-1 and nuclear factor-kap- Transcriptional network of p63 in human keratinocytes. PLoS One paB-like sites. Cancer Res 1998;58:3993–4000. 2009;4:e5008. 31. Romano RA, Smalley K, Liu S, Sinha S. Abnormal hair follicle devel- 17. King KE, Ponnamperuma RM, Allen C, Lu H, Duggal P, Chen Z, et al. opment and altered cell fate of follicular keratinocytes in transgenic The p53 homologue DeltaNp63alpha interacts with the nuclear factor- mice expressing DeltaNp63alpha. Development 2010;137:1431–9. kappaB pathway to modulate epithelial cell growth. Cancer Res 32. Lee TL, Yeh J, Friedman J, Yan B, Yang X, Yeh NT, et al. A signal 2008;68:5122–31. network involving coactivated NF-kappaB and STAT3 and altered p53 18. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell modulates BAX/BCL-XL expression and promotes cell survival of 2008;132:344–62. head and neck squamous cell carcinomas. Int J Cancer 2008; 19. Allen CT, Ricker JL, Chen Z, Van Waes C. Role of activated nuclear 122:1987–98. factor-kappaB in the pathogenesis and therapy of squamous cell 33. BrownMS,DialloOT,HuM,EhsanianR,YangX,ArunP,etal.CK2 carcinoma of the head and neck. Head Neck 2007;29:959–71. modulation of NF-kappaB, TP53, and the malignant phenotype in

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head and neck cancer by anti-CK2 oligonucleotides in vitro or in with other biomarkers, predicts the development of oral cancer in vivo via sub-50-nm nanocapsules. Clin Cancer Res 2010;16:2295– patients with leukoplakia. Clin Cancer Res 2009;15:6284–91. 307. 43. Van Waes C, Kozarsky KF, Warren AB, Kidd L, Paugh D, Liebert M, 34. Pernas FG, Allen CT, Winters ME, Yan B, Friedman J, Dabir B, et al. et al. The A9 antigen associated with aggressive human squamous Proteomic signatures of epidermal growth factor receptor and survival carcinoma is structurally and functionally similar to the newly defined signal pathways correspond to gefitinib sensitivity in head and neck integrin alpha 6 beta 4. Cancer Res1991;51:2395–402. cancer. Clin Cancer Res 2009;15:2361–72. 44. Van Waes C, Surh DM, Chen Z, Kirby M, Rhim JS, Brager R, et al. 35. Worden B, Yang XP, Lee TL, Bagain L, Yeh NT, Cohen JG, et al. Increase in suprabasilar integrin adhesion molecule expression in Hepatocyte growth factor/scatter factor differentially regulates human epidermal neoplasms accompanies increased proliferation expression of proangiogenic factors through Egr-1 in head and neck occurring with immortalization and tumor progression. Cancer Res squamous cell carcinoma. Cancer Res 2005;65:7071–80. 1995;55:5434–44. 36. Flores ER, Sengupta S, Miller JB, Newman JJ, Bronson R, Crowley D, 45. Lu H, Duggal P, Allen C, Yan B, Yang X, Cohen J, et al. Nuclear c-REL et al. Tumor predisposition in mice mutant for p63 and p73: evidence displaces p73 in partnering with deltaNp63 to suppress p21 expres- for broader tumor suppressor functions for the p53 family. Cancer Cell sion and promote proliferation in head and neck cancers [abstract]. In: 2005;7:363–73. Proceedings of the 101st Annual Meeting of the American Association 37. Chen Z, Malhotra PS, Thomas GR, Ondrey FG, Duffey DC, Smith for Cancer Research;2010 Apr 17–21; Washington, DC. Philadelphia CW, et al. Expression of proinflammatory and proangiogenic cyto- (PA): AACR; 2010. Abstract nr 3900. kines in patients with head and neck cancer. Clin Cancer Res 1999; 46. Koshiba S, Ichimiya S, Nagashima T, Tonooka A, Kubo T, Kikuchi T, 5:1369–79. et al. Tonsillar crypt epithelium of palmoplantar pustulosis secretes 38. Allen C, Duffy S, Teknos T, Islam M, Chen Z, Albert PS, et al. Nuclear interleukin-6 to support B-cell development via p63/p73 transcription factor-kappaB-related serum factors as longitudinal biomarkers of factors. J Pathol 2008;214:75–84. response and survival in advanced oropharyngeal carcinoma. Clin 47. Yang A, Zhu Z, Kapranov P, McKeon F, Church GM, Gingeras TR, Cancer Res 2007;13:3182–90. et al. Relationships between p63 binding, DNA sequence, transcrip- 39. Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, et al. tion activity, and biological function in human cells. Mol Cell p63 regulates an adhesion programme and cell survival in epithelial 2006;24:593–602. cells. Nat Cell Biol 2006;8:551–61. 48. Vigano MA, Mantovani R. Hitting the numbers: the emerging network 40. Chen Z, Ehsanian R, Van Waes C. Nuclear transcription factors and of p63 targets. Cell Cycle 2007;6:233–9. signal pathways. In: Myers J, editor. Oral cancer metastasis. New 49. Sullivan JC, Kalaitzidis D, Gilmore TD, Finnerty JR. REL homology York: Springer Science and Human Press; 2008, p. 197–229. domain-containing transcription factors in the cnidarian Nematostella 41. Richmond A. NF-kappa B, chemokine gene transcription and tumour vectensis. Dev Genes Evol 2007;217:63–72. growth. Nat Rev Immunol 2002;2:664–74. 50. Leong CO, Vidnovic N, DeYoung MP, Sgroi D, Ellisen LW. The p63/p73 42. Saintigny P, El-Naggar AK, Papadimitrakopoulou V, Ren H, Fan YH, network mediates chemosensitivity to cisplatin in a biologically defined Feng L, et al. DeltaNp63 overexpression, alone and in combination subset of primary breast cancers. J Clin Invest 2007;117:1370–80.

3700 Cancer Res; 71(10) May 15, 2011 Cancer Research

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Cancer Correction Research Correction: DNp63 Versatilely Regulates a Broad NF-jB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inﬂammation

In this article (Cancer Res 2011;71:3688–700), which was published in the May 15, 2011, issue of Cancer Research (1), the graph in Fig. 7D was mislabeled because of a production error. The corrected ﬁgure appears below.

A C

ΔNp63 cREL ΔNp63 Np63 TG Δ

B Wild type

Ki67 CK Np63 TG Δ Wild type

DE Carcinogenesis TNF-α 10,000 d 16 19041094 1,000 107 43 30 100 27 12 10 3 ΔNp63 cRel NF-κB -0.7-0.5 1

0.1 Nucleus 1 mo ΔNp63/cRel ΔNp63/cRel cRel NF-κB 1,000 229 174 64 p63 NF-κB NF-κB 100 40 Gene expression 6 7 10 2.5 3 -0.6-0.2 1 Proliferation Δ Apoptosis κ 0.1 Np63 Inflammation NF- B 2

β β Migration

II-1 II-6 Cxcl 1 Cxcl 2 Cxcl 5 CSF Icam 1 Itgb 2 Tgf Igfbp6 Transcriptomes

Figure 7.

Reference 1. Yang X, Lu H, Yan B, Romano R, Bian Y, Friedman J, et al. DNp63 versatilely regulates a broad NF-kB gene program and promotes squamous epithelial proliferation, migration, and inﬂam- mation. Cancer Res 2011;71:3688–700.

Published OnlineFirst November 17, 2011. doi: 10.1158/0008-5472.CAN-11-3437 2011 American Association for Cancer Research.

www.aacrjournals.org 7323 ∆Np63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Xinping Yang, Hai Lu, Bin Yan, et al.

Cancer Res 2011;71:3688-3700.

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