Purpose and Experimental Design: It has been proved recently that ΔNp63 may play an oncogenic role in the tumorigenic pathway of squamous cell cancers. To gain additional insight into this pathway, we examined global patterns of gene expression in cancer cells after ΔNp63 gene introduction using the oligonucleotide microarray approach.

Results: We found that S100A2 might be a target of the ΔNp63 pathway. To confirm the data obtained from oligonucleotide microarray, we then examined the interaction of ΔNp63 to S100A2. S100A2 induction was strictly dependent on ΔNp63 expression by ΔNp63 transgene and Northern analysis. ΔNp63 transactivated the S100A2 promoter, and significantly more fold changes were seen in ΔNp63-introduced cells than in p53-introduced cells, suggesting that ΔNp63 may be a novel stimulator of the S100A2 promoter.

Conclusion: Taken together, this evidence would seem to suggest that S100A2 is a novel downstream mediator of ΔNp63.

p53 is the most commonly inactivated gene in human cancer, and the loss of critical p53 pathways is central to tumorigenesis (1, 2, 3). A new human p53 homologue, p63, was isolated recently using a degenerate PCR approach, which showed that p63 was located on chromosome 3q28 (4, 5, 6). The DNA binding domain and the oligomerization domain of p63 display a strong conservation of amino acid residues with p53, raising the possibility that human p63 may also bind key p53 DNA binding sites in the human genome and/or interact with p53. A transcript that lacked the NH2-terminal TA2 domain of p53 (ΔNp63) was found to act in a dominant-negative fashion and to be able to suppress p53 TA.3 We additionally examined ΔNp63 status and observed ΔNp63 overexpression in head and neck cancer cell lines and primary lung cancers associated with a low-level increase in chromosomal copy number (7). Moreover, we found that increased expression of ΔNp63 in mouse fibroblast cells led to a transformed phenotype. Conversely, no evidence of a tumor-suppressive function of ΔNp63 in these cancers was found. In addition, proliferating human keratinocytes and various epithelial neoplastic cells and lesions predominantly express the ΔNp63 isotypes (8, 9, 10). We found recently that high ΔNp63 gene expression was significantly associated with lymph node metastasis in esophageal squamous cell cancer (11). These data suggested that ΔNp63 might play an oncogenic role in squamous cell cancers. Subsequently, we found that the ΔNp63 isotype forms complexes with p53, resulting in the caspase-dependent degradation of ΔNp63 protein (12). Therefore, the ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or induce epithelial proliferation. However, we do not yet know how ΔNp63 would advance the tumorigenic pathway in these cancers.

In an effort to gain additional insight into this pathway, we examined global patterns of gene expression in cancer cells after ΔNp63 gene introduction using oligonucleotide microarray approach. Here we identified S100A2, a Ca2+ binding protein, as a novel downstream mediator of ΔNp63.

ΔNp63 Adenovirus.

A full-length ΔNp63γ (p40) cDNA was cloned from a human prostate cDNA library, and ΔNp63 adenovirus was constructed as described previously (4, 7). Briefly, ΔNp63γ cDNA was subcloned into the shuttle vector, pAdTrack-CMV. The resultant plasmid was linearized by digesting with the restriction endonuclease PmeI and subsequently cotransformed into Escherichia coli BJ5183 cells with an adenoviral backbone plasmid, pAdEasy-1. Recombinants were selected for kanamycin resistance, and recombination was confirmed by restriction digest analysis. The linearized recombinant plasmid was then transfected into the adenovirus packaging cell line, HEK-293.

Oligonucleotide Microarray.

A human osteosarcoma cell line, Saos2, was infected by 10 multiplicities of infection of a ΔNp63 adenovirus or an empty adenovirus. In 24 h, cells were lysed, and total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The extracted total RNA was purified using an RNeasy column (Qiagen, Austin, TX).

Affymetrix (Santa Clara, CA) microarray analysis was done according to the manufacturer’s instructions. Briefly, total RNA (5 μg) was reverse transcribed to cDNA using T7-(dT)24 primer. Biotin-labeled cRNA was synthesized from cDNA using the MEGAscript In Vitro Transcript Kit (Ambion, Austin, TX). cRNA was fragmented to an average size of 50–100 nucleotides by incubating at 95°C for 35 min in 40 mm Tris-acetate (pH 8.1) containing 100 mm potassium acetate and 30 mm magnesium acetate, and then hybridized to human GeneChip (Human Genome Arrays U95A; Affymetrix) containing ∼12,000 human genes. The hybridized oligonucleotide microarrays were scanned using a confocal scanner (Affymetrix). The scanned data obtained from each microarray were normalized to correct for small differences in the amounts of each cRNA probe applied to the microarrays and were processed for average difference values using Affymetrix software. Fold changes of gene expression induced by the infection of ΔNp63 adenovirus were calculated by comparing the fluorescent intensity of each array with respect to the control. Seven genes were chosen for candidates of downstream mediator of ΔNp63, according to several criteria set in advance and validated by the following analyses.

Northern Analysis.

Saos2 sarcoma cell line was purchased from the American Type Culture Collection. Cultured cell lines were lysed in guanidine buffer, and total RNA was isolated using the CsCl gradient method. Northern blot hybridization using the cDNA probe was performed as described previously (13). cDNA included the 3′ part of the S100A2 gene or the whole open reading frame of the ΔNp63 gene. The human β-actin gene was used as an internal control to standardize the relative amount of RNA in each lane.

Luciferase Assay.

The luciferase reporter construct including the S100A2 promoter was made as described previously in another paper (14). Briefly, a 2269-bp DNA fragment of the S100A2 promoter was generated by PCR amplification from normal esophageal tissue DNA. The primers used were: PA25-Xho (sense), 5′-CTGCTCGAGTTTGTACAGGACAGAACAGGTAGA and PA23-Hind (antisense), 5′-CTGAAGCTTGGCAGAGACAGACCCAGGAAG. The PCR fragments were subcloned into pGL-Basic-3 luciferase reporter plasmid (Promega, Madison, WI) at the restriction sites of XhoI and HindIII.

Saos2 cells were plated in triplicate in six-well plates at a concentration of 106 cells/well. The Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) was used for transfection according to the manufacturer’s instructions. The luciferase reporter construct was cotransfected with a ΔNp63 adenovirus, a p53 adenovirus, or an empty adenovirus. p53 adenovirus was kindly provided by Dr. Bert Vogelstein of Johns Hopkins University (Baltimore, MD). Cells were washed twice with 2 ml of PBS and then lysed in 200 μl (per well) of 1× lysis buffer (Promega). After a brief spin to pellet large debris, 20 μl of supernatant was added to 100 μl of reconstituted luciferase assay reagent (Promega). Light emission was detected using a luminometer. The results are presented as the fold change over the empty adenovirus after normalizing the β-galactosidase activity.

To evaluate the transcriptional effects of ΔNp63, we studied a human osteosarcoma cell line (Saos2) infected by a ΔNp63 adenovirus or an empty adenovirus (control). The Saos2 cell line does not express p53 or ΔNp63 as described previously (7), and ΔNp63 might not be degraded by p53. Therefore, changes in gene expression were evaluated using an oligonucleotide microarray. Using Affymetrix software, we calculated the fold changes of gene expression induced by the infection of ΔNp63 adenovirus by comparing the fluorescent intensity of each array with respect to the control. This analysis revealed that 6 genes were overexpressed and 1 gene was repressed in ΔNp63-expressed Saos2 cells (Table 1). Interestingly, 2 of the overexpressed genes (M87068 and AI539439) were derived from the same gene, S100A2. The S100A2 protein is a member of the S100 family of Ca2+-binding proteins, which are involved in signal transduction processes and consequently in the regulation of proliferation and differentiation (15). It has been reported that basal cell and SCCs showed strong S100A2 immunoreactivity in neoplastic cells corresponding to basal cells, but were nonreactive or faintly reactive for other S100 proteins (16). This indicated that S100A2 exhibited the same distribution to ΔNp63 in human tissues and suggested that S100A2 might be a target of the ΔNp63 pathway. The other genes except S100A2 have not been examined.

To additionally confirm the data obtained from the oligonucleotide microarray, we performed Northern analysis using ΔNp63 and a S100A2 cDNA probe (Fig. 1). S100A2 mRNA was induced in 24 h after infection, whereas ΔNp63 began to be expressed at 4 h and was maximally expressed at 24 h. S100A2 induction was strictly dependent on ΔNp63 expression.

As described previously (14), a putative p53 binding site, 5‘-GGGCATGTGTGGGCACGTTC (italics indicate two mismatches to the consensus sequence), located at −3811 bp upstream from the translation initiation site, has been identified. ΔNp63 lacks the NH2-terminal TA domain but contains DNA binding and the hetero-oligomerization domain that are common to p53. Therefore, ΔNp63 may bind the S100A2 promoter and activate its transcription. To examine whether ΔNp63 is able to bind to this putative binding site, we generated a luciferase reporter construct driven by a 2269 bp promoter fragment containing the p53 binding sites and used it in a TA/luciferase assay. The results showed that p53 and ΔNp63 transactivated the S100A2 promoter, and significantly more fold changes were seen in ΔNp63-introduced cells than in p53-introduced cells, suggesting that ΔNp63 may be a novel stimulator of the S100A2 promoter (Fig. 2).

Expression of ΔNp63 in SCC was first detected in the head and neck, lung, and esophagus in a previous study. Moreover, we found that increased expression of ΔNp63 in mouse fibroblast cells led to a transformed phenotype. To gain additional insight into this pathway, we examined global patterns of gene expression in cancer cells after ΔNp63 gene introduction using oligonucleotide microarray approach, and identified S100A2 as a novel downstream mediator of ΔNp63. S100A2 had been originally suggested to function as a tumor suppressor gene in breast cancer (17). Other reports also suggested that S100A2 expression was suppressed in the course of the tumorigenic pathway (18). On the other hand, Xia et al.(19) reported that S100A2 was strongly expressed in bulk specimens of basal and SCCs of the skin and oral cavity. Moreover, Villaret et al.(20) found, using subtractive and microarray technology, that the S100A2 gene was significantly overexpressed in head and neck SCC compared with normal tissue. These results indicated the possibility that S100A2 would have an oncogenic function, especially in SCC.

The present emerging model supports the presence of transcriptional cross-talk among p53, ΔNp63, and S100A2. This critical interaction may balance the oncogenic and growth-stimulating activity of ΔNp63 in tumorigenesis with its ability to induce epithelial proliferation during development.

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.

2

The abbreviations used are: TA, transactivation; SCC, squamous cell carcinoma.

Fig. 1.

Time course of S100A2 expression induced by ΔNp63 at the indicated time in Saos2 cells.

Fig. 1.

Time course of S100A2 expression induced by ΔNp63 at the indicated time in Saos2 cells.

Close modal
Fig. 2.

Activation of S100A2 promoter by p53 or ΔNp63. Luciferase reporter construct including the S100A2 promoter was cotransfected with a ΔNp63 adenovirus, a p53 adenovirus, or an empty adenovirus. The results are presented as fold change; bars, ± SD.

Fig. 2.

Activation of S100A2 promoter by p53 or ΔNp63. Luciferase reporter construct including the S100A2 promoter was cotransfected with a ΔNp63 adenovirus, a p53 adenovirus, or an empty adenovirus. The results are presented as fold change; bars, ± SD.

Close modal
Table 1

Overexpressed or repressed genes in Saos2 cells following Δ Np63 gene introduction

Genbank no.DescriptionExpression levelFold change
contaΔ Np63b
Overexpressed genes     
 M64347 Human novel growth factor receptor mRNA, 3 cds 432.4 1389.2 4.1 
 X80907 H. sapiens mRNA for p85 β subunit of phosphatidyl-inositol-3-kinase 74.5 350.6 3.5 
 AB018302 H. sapiens mRNA for KIAA0759 protein 219.2 441.9 2.8 
 M87068 Definition = HUMCAN H. sapiens CaN19 mRNA 397.3 1312.3 2.7 
 AL049341 H. sapiens mRNA; cDNA DKFZp566A163 (from clone DKFZp566A163) 82.3 276.3 2.7 
 AI539439 te51e07.x1 Homo sapiens cDNA 401.4 906.3 2.3 
Repressed gene     
 AB014520 Homo sapiens mRNA for KIAA0620 protein 364.5 52.1 −3.8 
Genbank no.DescriptionExpression levelFold change
contaΔ Np63b
Overexpressed genes     
 M64347 Human novel growth factor receptor mRNA, 3 cds 432.4 1389.2 4.1 
 X80907 H. sapiens mRNA for p85 β subunit of phosphatidyl-inositol-3-kinase 74.5 350.6 3.5 
 AB018302 H. sapiens mRNA for KIAA0759 protein 219.2 441.9 2.8 
 M87068 Definition = HUMCAN H. sapiens CaN19 mRNA 397.3 1312.3 2.7 
 AL049341 H. sapiens mRNA; cDNA DKFZp566A163 (from clone DKFZp566A163) 82.3 276.3 2.7 
 AI539439 te51e07.x1 Homo sapiens cDNA 401.4 906.3 2.3 
Repressed gene     
 AB014520 Homo sapiens mRNA for KIAA0620 protein 364.5 52.1 −3.8 
a

Absolute expression level in empty adenovirus-infected cells.

b

Absolute expression level in Δ Np63 adenovirus-infected cells.

We thank Drs. David Sidransky and Bert Vogelstein for various plasmids. We also thank Masumi Taguchi for technical assistance.

1
Hollstein M., Metcalf R. A., Welsh J., Montesano R., Harris C. C. Frequent mutation of the p53 gene in human esophageal cancer.
Proc. Natl. Acad. Sci. USA
,
87
:
9958
-9961,  
1990
.
2
Baker S. J., Fearon E. R., Nigro J. M., Hamilton S. R., Preisinger A. C., Jessup J. M., Tuinen P., Ledbetter D. H., Barker D. F., Nakamura Y., White R., Vogelstein B. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas.
Science (Wash. DC)
,
244
:
217
-221,  
1989
.
3
Takahashi T., Nau M. M., Chiba I., Birrer M. J., Rosenberg R. K., Vinocour M., Levitt M., Pass H., Gazdar A. F., Minna J. D. p53: a frequent target for genetic abnormalities in lung cancer.
Science (Wash. DC)
,
246
:
491
-494,  
1989
.
4
Trink B., Okami K., Wu L., Sriuranpong V., Jen J., Sidransky D. A new human p53 homologue.
Nat. Med.
,
4
:
747
-748,  
1998
.
5
Osada M., Ohba M., Kawahara C., Ishioka C., Kanamaru R., Katoh I., Ikawa Y., Nimura Y., Nakagawara A., Obinata M., Ikawa S. Cloning and functional analysis of human p51, which structurally and functionally resembles p53.
Nat. Med.
,
4
:
839
-843,  
1998
.
6
Yang A., Kaghad M., Wang Y., Gillett E., Fleming M. D., Dt̂sch V., Andrews N. C., Caput D., McKeon F. p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.
Mol. Cell
,
2
:
305
-316,  
1998
.
7
Hibi K., Trink B., Patturajan M., Westra W. H., Caballero O. L., Hill D. E., Ratovitski E. A., Jen J., Sidransky D. AIS is an oncogene amplified in squamous cell carcinoma.
Proc. Natl. Acad. Sci. USA
,
97
:
5462
-5467,  
2000
.
8
Parks B. J., Lee S. J., Kim J. I., Lee S. J., Lee C. H., Chang S. G., Park J. H., Chi S. G. Frequent alteration of p63 expression in human primary bladder carcinomas.
Cancer Res.
,
60
:
3370
-3374,  
2000
.
9
Parsa R., Yang A., McKeon F., Green H. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes.
J. Investig. Dermatol.
,
113
:
1099
-1105,  
1999
.
10
Nylander K., Coates P. J., Hall P. A. Characterization of the expression pattern of p63α and ΔNp63α in benign and malignant oral epithelial lesions.
Int. J. Cancer
,
87
:
368
-372,  
2000
.
11
Hibi K., Nakayama H., Taguchi M., Kasai Y., Ito K., Akiyama S., Nakao A. AIS overexpression in advanced esophageal cancer.
Clin. Cancer Res.
,
7
:
469
-472,  
2001
.
12
Ratovitski E. A., Patturajan M., Hibi K., Trink B., Yamaguchi K., Sidransky D. p53 associates with and targets ΔNp63 into a protein degradation pathway.
Proc. Natl. Acad. Sci. USA
,
98
:
1817
-1822,  
2001
.
13
Hibi K., Nakamura H., Hirai A., Fujikake Y., Kasai Y., Akiyama S., Ito K., Takagi H. Loss of H19 imprinting in esophageal cancer.
Cancer Res.
,
56
:
480
-482,  
1996
.
14
Tan M., Heizmann C. W., Guan K., Schafer B. W., Sun Y. Transcriptional activation of the human S100A2 promoter by wild-type p53.
FEBS Lett.
,
445
:
265
-268,  
1999
.
15
Schafer B. W., Heizmann C. W. The S100 family of EF-hand calcium binding proteins.
Trends Biochem. Sci.
,
21
:
134
-140,  
1996
.
16
Shrestha P., Muramatsu Y., Kudeken W., Mori M., Takai Y., Ilg E. C., Schafer B. W., Heizmann C. W. Localization of Ca2+-binding S100 proteins in epithelial tumours of the skin.
Virchows Arch.
,
432
:
53
-59,  
1998
.
17
Lee S. W., Tomasetto C., Swisshelm K., Keyomarsi K., Sager R. Down-regulation of a member of the S100 gene family in mammary carcinoma cells and reexpression by azadeoxycytidine treatment.
Proc. Natl. Acad. Sci. USA
,
89
:
2504
-2508,  
1992
.
18
Feng G., Xu X., Youssef E. M., Lotan R. Diminished expression of S100A2, a putative tumor suppressor, at early stage of human lung carcinogenesis.
Cancer Res.
,
61
:
7999
-8004,  
2001
.
19
Xia L., Stoll S. W., Liebert M., Ethier S. P., Carey T., Esclamado R., Carroll W., Johnson T. M., Elder J. T. CaN19 expression in benign and malignant hyperplasias of the skin and oral mucosa: evidence for a role in regenerative differentiation.
Cancer Res.
,
57
:
3055
-3062,  
1997
.
20
Villaret D. B., Wang T., Dillon D., Xu J., Sivam D., Cheever M. A., Reed S. G. Identification of genes overexpressed in head and neck squamous cell carcinoma using a combination of complementary DNA subtraction and microarray analysis.
Laryngoscope
,
110
:
374
-381,  
2000
.