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[Cancer Research 65, 2314-2320, March 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

IGFBP-3 Is a Direct Target of Transcriptional Regulation by {Delta}Np63{alpha} in Squamous Epithelium

Christopher E. Barbieri1,4, Carmen A. Perez1,4, Kimberly N. Johnson1,4, Kim A. Ely2, Dean Billheimer3 and Jennifer A. Pietenpol1,4

Departments of 1 Biochemistry, 2 Pathology, and 3 Biostatistics and 4 Center in Molecular Toxicology, the Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee

Requests for reprints: Jennifer A. Pietenpol, 652 Preston Research Building, Vanderbilt University Medical Center, Nashville, TN 37232. Phone: 615-936-1512; Fax: 615-936-1790; E-mail: j.pietenpol{at}vanderbilt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
{Delta}Np63{alpha} is a nuclear transcription factor that maintains epithelial progenitor cell populations, is overexpressed in several epithelial cancers, and can negatively regulate apoptosis. However, the mechanisms by which {Delta}Np63{alpha} promotes cell survival are unclear. {Delta}Np63{alpha} has been reported to act as a transcriptional repressor, but specific target genes directly repressed by {Delta}Np63{alpha} remain unidentified. Here, we present evidence that {Delta}Np63{alpha} functions to negatively regulate the proapoptotic protein IGFBP-3. Disruption of p63 expression in squamous epithelial cells increases IGFBP-3 expression, whereas ectopic expression of {Delta}Np63{alpha} down-regulates IGFBP-3. {Delta}Np63{alpha} binds to sites in the IGFBP-3 gene in vivo and can modulate transcription through these sites. Furthermore, {Delta}Np63{alpha} and IGFBP-3 expression patterns are inversely correlated in normal squamous epithelium and squamous cell carcinomas. These data suggest that IGFBP-3 is a target of transcriptional repression by {Delta}Np63{alpha} and that this repression represents a mechanism by which tumors that overexpress p63 may be protected from apoptosis.

Key Words: p53 • squamous • carcinoma • repression


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
p63 is a homologue of the tumor suppressor p53 (1–4). At least six different p63 transcripts can be expressed due to alternate promoter usage and alternative splicing (1, 5); all of these encode a DNA-binding domain with significant homology (~60% identity) to that of p53. Consistent with this sequence conservation, p63 proteins can bind to p53-consensus DNA sequences in vitro and in vivo (6, 7). Isoforms of p63 have different NH2 termini with either transactivating (TA) or dominant negative ({Delta}N) activities; however, in epithelial cells, {Delta}Np63{alpha} is the predominant form expressed (6, 8–11). The {Delta}Np63{alpha} protein has been reported to act as a transcriptional repressor in vitro and in animal models and can strongly oppose p53- or TAp63-mediated transactivation (1, 6, 12, 13). {Delta}Np63{alpha} is expressed primarily in the proliferative, basal compartment of epithelia, including epidermis, oral mucosa, cervix, vaginal epithelium, urothelium, prostate, and breast (1, 8, 9). Expression of {Delta}Np63{alpha} decreases in differentiating cells in vitro and in vivo, and studies suggest that {Delta}Np63{alpha} is specifically expressed in epidermal stem cells possessing the highest proliferative capacity (6, 14, 15).

Further insight to {Delta}Np63{alpha} function is provided by animal models in which p63 expression is disrupted. Unlike p53 –/– mice, which are developmentally normal but rapidly develop tumors (16), p63 –/– mice display gross developmental abnormalities. The most striking of these is a complete lack of all stratified squamous epithelia and their derivatives, including epidermis, mammary glands, prostate, and other tissues (17, 18). This phenotype is recapitulated in zebrafish, in which disruption of {Delta}Np63 results in lack of epidermal morphogenesis (12, 13). Taken together, these data suggest that p63 is critical for the survival or proliferative capacity of epithelial stem cells.

{Delta}Np63{alpha} is overexpressed in several epithelial cancers, often as a result of gene amplification (2, 19–22). Overexpression of a {Delta}Np63 isoform in Rat-1A cells increases colony growth in soft agar and xenograft tumor formation in nude mice, supporting the view that p63 acts as an oncogene (19). In addition, {Delta}Np63{alpha} must be down-regulated in order for UVB-induced apoptosis to occur (23). It is hypothesized that {Delta}Np63{alpha} promotes the survival and maintenance of proliferative capacity of both epithelial stem cells and cancer cells. However, the mechanisms by which {Delta}Np63{alpha} promotes cell survival, in particular which target genes are regulated by {Delta}Np63{alpha}, remain to be elucidated.

Here, we present evidence that {Delta}Np63{alpha} functions to negatively regulate insulin-like growth factor binding protein 3 (IGFBP-3) at the transcriptional level. IGFBP-3 induces apoptosis in a number of cell types, and its expression inhibits the growth of xenograft tumors (24–28). IGFBP-3 was identified as a target gene of the tumor suppressor p53 almost 10 years ago (29); however, the role of IGFBP-3 in p53-mediated apoptosis remains unclear. In contrast, subsequently identified proapoptotic p53 target genes such as Noxa and PUMA have been shown to play unequivocal roles in p53-mediated apoptosis (30–32). Regulation of IGFBP-3 expression by p63 may provide a stronger link between IGFBP-3–mediated apoptosis and the p53 family of transcription factors. The data presented herein suggest that IGFBP-3 is a target of transcriptional repression by {Delta}Np63{alpha} and that this repression represents a mechanism by which tumors that overexpress p63 may be protected from apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture. The human keratinocyte cell line HaCaT was generously provided by P. Boukamp [German Cancer Research Center (DKFZ), Heidelberg, Germany]; the squamous carcinoma cell lines SCC-1 and SCC-6 were gifts from T. Carey (University of Michigan, Ann Arbor, MI); the squamous carcinoma cell line SCC-012 was kindly provided by D. Sidransky (Johns Hopkins University, Baltimore, MD). HaCaT, SCC-1, SCC-6, and H1299 (ATCC) human large cell lung carcinoma cells were cultured in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. SCC-012 cells were cultured in RPMI 1640 supplemented with 10% FCS and 1% penicillin-streptomycin. The A-549 human lung adenocarcinoma cell line (American Type Culture Collection, Manassas, VA) was cultured in DMEM supplemented with 10% FCS, 10 µg/mL insulin, and 1% penicillin-streptomycin. All cells were cultured at 37°C with 5% CO2.

Cell Transfection and Small Interfering RNA. The following targeting sequence was used for small-interfering siRNA–mediated knockdown of p63: (5'-AACAGCCATGCCCAGTATGTA-3'). Targeting oligonucleotides for p63 were designed as previously described (33). pCEP-H1 {phi} and pCEP-H1 p63 expression vectors were generated as previously described (34). {Delta}Np63{alpha} was ectopically expressed in A-549 cells using pCEP4-{Delta}Np63{alpha} as previously described (6). HaCaT, SCC-1, SCC-6, SCC-012, and A-549 cells were transfected using Fugene 6 (Roche, Indianapolis, IN). Cells were selected with hygromycin B 48 hours after transfection and harvested for Western or Northern analysis as described below.

Flow Cytometry. Cells were trypsinized, and 1 x 106 cells were analyzed by flow cytometry. Cells were incubated with 50 µg/mL propidium iodide (Sigma, St. Louis, MO), and DNA content was measured using a FACSCalibur (Becton Dickinson, Palo Alto, CA). Data were analyzed using Cell Quest software (Becton Dickinson); 15,000 events were analyzed for each sample.

Western Analysis. Cells were harvested and lysates prepared as previously described (35). Western analysis was done as previously described (35) with the following primary antibodies: {alpha}-p63 monoclonal antibody Ab-1 (Oncogene Research Products, Calbiochem, San Diego, CA), {alpha}-ß-actin polyclonal antibody I-19 (Santa Cruz Biotechnology, Santa Cruz, CA), {alpha}-poly(ADP-ribose) polymerase polyclonal antibody (Cell Signaling, Beverly, MA), and {alpha}-IGFBP-3 goat polyclonal antibody (Diagnostic Systems Laboratories, Webster, TX). A Fluor-S Max MultiImager (Bio-Rad, Hercules, CA) was used to quantify Western signals.

Luciferase Assays. Upper and lower strand oligonucleotides representing IGFBP-3 Box A and Box B binding sites, as well as analogous sites with key mutations (Mut-Box A and Mut-Box B) as previously described (29), were used for construction of luciferase reporter plasmids. Oligonucleotide sequences are available upon request. Complementary oligonucleotides were annealed, and four copies of each binding site were concatamerized and cloned into the SmaI site of pGL3promoter. H1299 cells were transiently transfected with the pGL3promoter-Box A, -Box B, -MutBox A, and -MutBox B reporter plasmids and expression vectors encoding p53, {Delta}Np63{alpha}, and TAp63{gamma}. The TAp63{gamma} cDNA was kindly provided by Dr. Chikashi Ishioka (Department of Clinical Oncology, Tokohu University, Japan). All transfections were done using Fugene 6, and cells were harvested 30 hours after transfection. Luciferase activity measurements were done using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI).

Chromatin Immunoprecipitation Assay. Cross-linking and harvesting of HaCaT and SCC-1 cells was done as previously described (6). The lysates were divided into aliquots, and 1 mg of protein extract was immunoprecipitated with {alpha}-p63 rabbit polyclonal antibody (H129; Santa Cruz) or {alpha}-Bax rabbit polyclonal antibody (N20; Santa Cruz) as previously described (6). IGFBP-3 Box A, Box B, and Exon 1, and p21 site 2 PCR amplifications were done and PCR DNA products were resolved using 6% polyacrylamide gels (acrylamide-bisacrylamide [19:1]) in 1x Tris acetate-EDTA buffer. Primer sequences and PCR conditions are available upon request. Gels were stained with ethidium bromide.

Tissue Collection and Immunohistochemistry. Tissue samples were taken from individuals treated at Vanderbilt University Medical Center with institutional review board approval. Paraffin-embedded samples used for analysis were all evaluated by the study pathologist, Dr. Ely. Sections of 5-µm-thick paraffin-embedded tissue microarrays were deparaffinized and rehydrated with xylene and ethanol. For p63 immunostaining, antigen retrieval was done by microwaving slides in 0.1 mol/L citrate buffer for 10 minutes. Slides were incubated 10 minutes in 3% hydrogen peroxide in methanol to exhaust endogenous peroxidase activity then incubated with a 1:50 dilution of p63 Ab-1 for 1 hour at 25°C. The Dako (Carpinteria, CA) LSAB2 kit was used to develop the slides. For IGFBP-3 staining, slides were incubated 10 minutes in 3% hydrogen peroxide then blocked for 20 minutes with 2.5% goat serum. Slides were incubated with IGFBP-3 rabbit polyclonal antibody (Diagnostic Systems Laboratories) overnight in a 1:100 dilution at 4°C. Biotin-labeled anti-rabbit secondary antibody was applied for 30 minutes, then slides were incubated for 30 minutes with VECTASTAIN Elite ABC reagent. The DAB substrate kit for peroxidase (Vector Laboratories, Burlingame, CA) was used to develop slides. The slides were counterstained with hematoxylin. Protein expression was determined and scored by the study pathologist. Statistical significance of the relationship between p63 and IGFBP-3 expression was determined using Fisher's exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Disruption of p63 Expression Sensitizes Cells to Apoptosis. It has been previously reported that down-regulation of {Delta}Np63{alpha} expression is required for UV radiation–induced apoptosis in mouse epidermis (23). To determine if loss of {Delta}Np63{alpha} expression would sensitize squamous cell lines to apoptosis in vitro, we disrupted p63 expression in SCC-1 cells using siRNA, and examined sensitivity to apoptosis-inducing agents. siRNA-mediated knockdown of p63 led to an approximate 2-fold increase in apoptosis as measured by poly(ADP-ribose) polymerase cleavage and subdiploid cells (Fig. 1A and B). This effect was p63 dependent, because H1299 cells, which do not express endogenous {Delta}Np63{alpha}, did not show a significant increase in apoptosis when transfected with p63-specific siRNA (Fig. 1A and B). These data confirm previous observations that {Delta}Np63{alpha} plays a role in regulation of apoptosis in squamous epithelial cells.



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  		Figure 1. Knockdown of p63 results in increased sensitivity to apoptosis. A, SCC-1 and H1299 cells were transfected with control (con) or p63 siRNA vectors. After selection with hygromycin, cells were treated with 20 µg/mL cisplatin (CDDP), 100 J/m2 UVC (UV), 100 nmol/L paclitaxel (Tax), or untreated ({phi}). Untreated, cisplatin- and UV-treated cells were harvested 24 hours after treatment; paclitaxel-treated cells were harvested 48 hours after treatment and p63, ß-actin, and PARP protein levels were analyzed by Western blot; PARP cleavage was quantified using the Fluor-S Max MultiImager. B, SCC-1 and H1299 cells were treated as indicated for A and were processed for flow cytometric analysis; DNA content was analyzed and the percentage of sub-2N cells quantified for three independent experiments; bars, SD.

 
Disruption of p63 Expression Leads to Up-regulation of IGFBP-3. To identify target genes regulated by {Delta}Np63{alpha} that could affect apoptosis, we disrupted p63 expression in SCC-1 cells using siRNA and evaluated global gene expression using the Affymetrix (Santa Clara, CA) U133A GeneChip (data not shown). IGFBP-3 expression was increased ~2.5-fold following siRNA-mediated knockdown of p63 expression. With respect to p53 target genes, this effect was specific for IGFBP-3, as we did not observe changes in the expression of other target genes such as p21, Mdm2, Bax, Noxa, and PUMA. To validate these microarray results, we examined IGFBP-3 protein expression in a panel of transformed (SCC-1, SCC-012, SCC-6) and nontransformed (HaCaT) squamous cell lines in which p63 expression had been disrupted. {Delta}Np63{alpha} is the predominant, if not only, p63 protein expressed in these cell lines (ref. 36 and data not shown). siRNA-mediated knockdown of p63 expression led to an increase in IGFBP-3 protein levels in all cell lines examined (Fig. 2A). The IGFBP-3 protein was up-regulated ~2- to 3-fold by disruption of p63 expression (Fig. 2B), consistent with increases in IGFBP-3 mRNA seen in microarray analyses. IGFBP-3 protein levels were also increased in the conditioned media of HaCaT and SCC-1 cells in which p63 expression had been disrupted, indicating an increase in secreted IGFBP-3 protein as well (Fig. 2C). To confirm that this up-regulation of IGFBP-3 was due specifically to siRNA-mediated disruption of p63, we transfected A-549 cells, which do not express detectable p63, with p63-targeting siRNAs. We did not observe any change in IGFBP-3 levels (Fig. 2D), suggesting that the up-regulation of IGFBP-3 is not due to an off-target effect of the siRNA. Furthermore, transfection of HaCaT cells with a control siRNA targeting GFP did not affect IGFBP-3 protein levels (Fig. 2E). Northern analyses showed changes in IGFBP-3 mRNA levels consistent with protein levels in all cell lines (data not shown). We conclude from these data that disruption of endogenous p63 expression leads to up-regulation of IGFBP-3 at both the mRNA and protein level.



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  		Figure 2. Disruption of p63 expression leads to up-regulation of IGFBP-3. A, HaCaT, SCC-1, SCC-6, and SCC-012 cells were transfected with p63 siRNA and selected with hygromycin. p63, IGFBP-3, and ß-actin protein levels were analyzed by Western blot. B, IGFBP-3 protein levels were quantified using the Fluor-S Max MultiImager. Relative IGFBP-3 protein level, normalized ratio of IGFBP-3 signal in at least three independent experiments; bars, SD. C, 10 µL of conditioned media from HaCaT and SCC-1 cells transfected with p63 siRNA as described above were analyzed by Western blot for IGFBP-3 protein levels. D, A-549 cells were transfected with p63 siRNA and selected as described above. IGFBP-3 and ß-actin protein levels were analyzed by Western blot and quantified using the Fluor-S Max MultiImager. Relative IGFBP-3 protein level, normalized ratio of IGFBP-3 signal in at least two independent experiments; bars, SE. E, HaCaT cells were transfected with control or GFP siRNA vectors and selected as described above. IGFBP-3 and ß-actin protein levels were analyzed by Western blot and quantified using the Fluor-S Max MultiImager. Relative IGFBP-3 protein level, normalized ratio of IGFBP-3 signal in at least three independent experiments; bars, SD. F, ectopic expression of {Delta}Np63{alpha} decreases expression of IGFBP-3. A-549 cells were transfected with pCEP4 alone (vec) or pCEP4-{Delta}Np63{alpha} ({Delta}Np63{alpha}), then selected with hygromycin. p63, IGFBP-3, and ß-actin protein levels were analyzed by Western blot. Representative of at least three independent experiments.

 
To determine if ectopic expression of {Delta}Np63{alpha} in cells that do not express endogenous p63 could negatively regulate IGFBP-3 expression, we transfected A-549 cells with {Delta}Np63{alpha}. Ectopic expression of {Delta}Np63{alpha} decreased expression of IGFBP-3 (Fig. 2F). These results show that {Delta}Np63{alpha} negatively regulates IGFBP-3 expression in both transformed and nontransformed cell lines. Furthermore, all cell lines used lack functional p53; therefore, the observed {Delta}Np63{alpha}-mediated effects on IGFBP-3 expression are p53 independent.

p63 Is Bound to Specific Sequences in the IGFBP-3 Gene In vivo. The DNA-binding domain of {Delta}Np63{alpha} has significant homology to that of p53, and numerous studies have reported that p63 proteins can bind to p53 consensus DNA sequences (6, 7, 37, 38). To further investigate the mechanism by which {Delta}Np63{alpha} can negatively regulate expression of IGFBP-3, we examined sequences in the IGFBP-3 gene that have previously been identified as p53 binding sites (29). These sites, named Box A and Box B, are located in the second and third intron of the IGFBP-3 gene, respectively (Fig. 3A). The sequences of Box A and Box B and the p53 consensus are shown in Fig. 3B. To determine if p63 protein is bound to Box A and Box B sites in vivo, we used chromatin immunoprecipitation assays (39). In HaCaT and SCC-1 cells, IGFBP-3 Box A and Box B DNA were bound to immunopurified p63 protein at levels significantly higher than nonspecific background (Fig. 3C). p63 has been previously reported to bind to both p53-consensus sites in the p21 promoter (6, 7); thus, p21 site 2 was used as a positive control in this assay. There was little, if any, binding of p63 to exon 1 of IGFBP-3, demonstrating that p63 binding is specific to Box A and Box B in this genomic region. Collectively, these data show that {Delta}Np63{alpha} is bound to the Box A and Box B sites in the IGFBP-3 gene in transformed and nontransformed squamous cell lines.



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  		Figure 3. p63 is bound to specific sequences in the IGFBP-3 gene in vivo. A, schematic representation of the IGFBP-3 gene, showing locations of Box A and Box B. Base pair numbers are relative to the translational start site. B, sequences of the Box A and Box B sites and the p53 consensus site. Underlined residues in Box A and Box B, deviation from the perfect consensus. C, rapidly growing HaCaT and SCC-1 cells were formaldehyde cross-linked, and DNA fragments immunoprecipitated with p63-specific antibodies were PCR amplified by using primers flanking the Box A and Box B binding sites, as well as exon 1 of IGFBP-3 and p53 binding site 2 in the p21 promoter. Genomic, PCR products that were generated using DNA template derived from total genomic DNA harvested from rapidly growing cells. To control for nonspecific binding, non–cross-linked lysates were immunoprecipitated with antibodies against p63, and cross-linked lysates were immunoprecipitated with antibodies against a nonspecific protein, Bax. Each ethidium bromide–stained gel shows one representative result of at least three independent experiments.

 
p63 Proteins Can Modulate Transcription through the Box A and Box B Sites. To determine if {Delta}Np63{alpha} could affect transcription through its binding to the IGFBP-3 Box A and Box B sites, we used luciferase reporter plasmids containing the wild-type Box A and Box B sites as well as the mutant sites Mut-Box A and Mut-Box B. For these assays we used TAp63{gamma}, as it has an identical DNA-binding domain to that of {Delta}Np63{alpha}, but strongly activates transcription, providing a facile readout of p63 regulation of transcription through the Box A and Box B sites. Consistent with our in vivo DNA-binding data, p53 and TAp63{gamma} were able to activate transcription through the Box A and Box B sites, but not the Mut-Box A and Mut-Box B sites (Fig. 4A). TAp63{gamma} activated transcription from both sites ~4- to 5-fold, whereas p53-mediated transactivation was comparable, activating 5- to 6-fold (Fig. 4A). H1299 do not express endogenous IGFBP-3, therefore we were unable to determine if TAp63{gamma} expression was able to induce endogenous IGFBP-3 in these cells (data not shown). {Delta}Np63{alpha} effectively inhibited both p53- and TAp63{gamma}-mediated transactivation through both Box A and Box B sites, reducing luciferase activity encoded by the reporter constructs by 89% to 100% (Fig. 4B). These data are consistent with the observed {Delta}Np63{alpha}-mediated regulation of IGFBP-3 expression in Fig. 2.



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  		Figure 4. p63 proteins can modulate transcription through the Box A and Box B sites. A, p53 and TAp63{gamma} can transactivate a heterologous promoter through wild-type, but not mutant, Box A and Box B sites. H1299 cells were cotransfected with luciferase reporter plasmids containing the indicated DNA sequence and either p53 or TAp63{gamma}. Cells were harvested 30 hours after transfection and assayed for luciferase activity. B, {Delta}Np63{alpha} can inhibit p53 or TAp63{gamma}-mediated transactivation through Box A and Box B sites. H1299 cells were cotransfected with luciferase reporter plasmids containing the indicated DNA sequence and either p53 or TAp63{gamma} with and without {Delta}Np63{alpha}. Cells were harvested 30 hours after transfection and assayed for luciferase activity. Representative of at least three independent experiments; bars, SD.

 
{Delta}Np63{alpha} and IGFBP-3 Protein Expression Patterns Are Inversely Correlated in Normal Epithelium and Squamous Cell Carcinomas In vivo. To determine if the regulation of IGFBP-3 expression by {Delta}Np63{alpha} observed in squamous epithelial cell lines was seen in vivo, we examined p63 and IGFBP-3 protein expression in tissue sections of normal oral epithelium and squamous cell carcinomas of the head and neck. As previously reported, p63 protein expression is restricted to the basal region of normal epithelium (Fig. 5A; refs. 1, 8, 9 ). In contrast, IGFBP-3 protein was expressed primarily in the differentiating suprabasal cells of the epithelium (Fig. 5A). We further examined this phenomenon in human tumors using a tissue microarray consisting of tissue from 49 patients with squamous cell carcinomas of the head and neck. Consistent with previous reports, we found that 67% (33 of 49) of squamous cell carcinomas of the head and neck overexpressed p63 (19, 40; representative staining patterns are seen in Fig. 5B, top two panels). Of these p63-overexpressing tumors, only 12% (4 of 33) were positive for IGFBP-3 expression (Fig. 5B, top, left). Conversely, 50% of tumors that do not overexpress p63 express IGFBP-3 (Fig. 5B, bottom, left). Of note, in select tumors positive for both p63 overexpression and IGFBP-3 expression, it was difficult to determine if IGFBP-3 expression was localized to the stroma or tumor epithelium (Fig. 5B, top, left); however, this did not impact the scoring. Fisher's exact test indicates a significant negative relationship between p63 and IGFBP-3 expression (P = 0.01). We conclude from these data that p63 and IGFBP-3 expression patterns are inversely correlated in normal and transformed squamous epithelium. This is consistent with our model that {Delta}Np63{alpha} negatively regulates the expression of IGFBP-3.



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  		Figure 5. {Delta}Np63{alpha} and IGFBP-3 protein expression patterns are inversely correlated in normal human epithelium and squamous cell carcinomas in patients. A, normal human oral epithelium was immunostained with antibodies against p63 and IGFBP-3 as described in Materials and Methods. Note positive staining for p63 in basal epithelium and IGFBP-3 staining in upper layers of epithelium. B, A tissue microarray consisting of 49 human head and neck squamous cell carcinomas was immunostained with antibodies against p63 and IGFBP-3 as described in Materials and Methods. p63 and IGFBP-3 staining were evaluated by the study pathologist and scored for intensity of staining (range, 0-3) and percentage of cells staining. For p63, tumors with greater than 50% of cells staining and an intensity score of at least 2 were considered positive for p63 overexpression. Tumors were grouped into four classes based on p63 and IGFBP-3 expression (+/+, +/–, –/+, and –/–). p63 (left) and IGFBP-3 (right) staining of representative tumors from each class with the number of tumors in each class reported below. All photomicrographs, magnification x200. Fisher's exact test determined that a significant negative relationship existed between p63 overexpression and IGFBP-3 expression; P = 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{Delta}Np63{alpha} is the predominant isotype of p63 expressed in normal and transformed squamous epithelium. The {Delta}Np63{alpha} protein is overexpressed in several epithelial cancers, often as a result of genomic amplification, and numerous studies have highlighted the oncogenic potential of this protein (19, 21, 22, 40–43). Furthermore, animal models indicate that {Delta}Np63 is critical for the maintenance of progenitor cells in stratified epithelium (12, 13, 17, 18), and {Delta}Np63{alpha} has been observed to negatively regulate apoptosis in vivo (23). However, the mechanisms by which {Delta}Np63{alpha} executes these functions, particularly which target genes are directly regulated by p63, are poorly defined. A microarray screen has identified a large number of potentially regulated genes, including IGFBP-3, using ectopically overexpressed p63 proteins in a nonepithelial cell line (44). Here, we present evidence that IGFBP-3 is a direct transcriptional target of negative regulation by endogenous {Delta}Np63{alpha} in squamous epithelial cells and tissues. siRNA-mediated knockdown of p63 expression in human keratinocytes and squamous carcinoma cells resulted in an up-regulation of IGFBP-3 at the mRNA and protein levels; conversely, introduction of {Delta}Np63{alpha} into a cell line lacking any p63 expression reduced IGFBP-3 expression. {Delta}Np63{alpha} was observed to bind to sites in the IGFBP-3 gene in vivo, and could modulate transcription through these sites. Finally, observations about the relationship between {Delta}Np63{alpha} and IGFBP-3 in cell culture models were extended to normal and transformed human tissues, in which expression patterns of p63 and IGFBP-3 showed significant negative correlation.

IGFBP-3 is a growth-inhibitory, proapoptotic protein in both cell culture and animal models. Two mechanisms mediate its proapoptotic effects: an IGF-dependent mechanism and an IGF-independent mechanism (reviewed in ref. 45). The apoptotic effects of IGFBP-3 in cell culture and animal models are clinically significant as well; decreased IGFBP-3 expression is correlated with unfavorable prognosis in human cancers (46, 47). Additional studies are necessary to determine whether negative regulation of IGFBP-3 by {Delta}Np63{alpha} has prognostic significance in head and neck squamous cell carcinomas and other p63-overexpressing cancers.

{Delta}Np63{alpha} can bind to p53 consensus DNA binding sites and act as a transcriptional repressor in vitro and in vivo (6, 7, 13). Numerous studies have reported that {Delta}Np63{alpha} acts as a dominant negative with respect to p53 function (1, 6, 12, 48). Despite these observations, there is a remarkable paucity of data demonstrating the ability of {Delta}Np63{alpha} to negatively regulate specific p53 target genes in intact cells or animal models (38, 44). In fact, paradoxical transactivation of p53 target genes by ectopically expressed {Delta}Np63{alpha} has been reported (44, 49). Significantly, several studies reporting the repressive effects of {Delta}Np63{alpha} have used the approach of disrupting endogenous p63 expression rather than relying solely on ectopic overexpression of p63 proteins (12, 13, 38). This implies that ectopic {Delta}Np63{alpha} may not be able to function in the same manner as endogenous protein. This idea is supported by inconsistent data concerning the activity of {Delta}Np63{alpha} in luciferase reporter assays (50), and a recent report suggests that investigators should use caution in interpreting data from such assays due to the complexity of the p53 family of transcription factors (51). Continued research into the biological and biochemical functions of {Delta}Np63{alpha} will be necessary to resolve these questions.

It is clear from mouse and zebrafish models that the major biological role of p63 is the maintenance of stratified epithelia (12, 13, 17, 18) . Several lines of evidence suggest that {Delta}Np63{alpha} is a nuclear transcription factor that executes this function by acting as a transcriptional repressor (6, 13). It stands to reason that the identification of the specific target genes repressed by {Delta}Np63{alpha} will greatly enhance our understanding of the mechanisms by which p63 promotes the viability and proliferative capacity of epithelial cells. In this study, we present evidence that the proapoptotic IGFBP-3 is one such target. Without question, the continued identification of target genes regulated by p63 will further elucidate signaling pathways involved in maintenance of stratified epithelium, and provide insight to the interaction of the p53 family of transcription factors.


   Acknowledgments
 
Grant support: NIH grants CA70856 and CA105436 (J.A. Pietenpol), training grants GM073407 and CA009385 (C.E. Barbieri), and ES00267 and CA68485 (Core services).

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. Anna Spagnoli (Vanderbilt University Medical Center) for kindly providing IGFBP-3 cDNA, David Sidransky (Johns Hopkins), Petra Boukamp (German Cancer Research Center-DKFZ, Heidelberg, Germany), and Tom Carey (University of Michigan) for providing cell lines. We thank members of the Pietenpol and Cortez laboratories for critical reading of the manuscript and helpful discussions.

Received 9/26/04. Revised 1/ 5/05. Accepted 1/12/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998;2:305–16.[CrossRef][Medline]
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