Identification of S100A2 As a Target of the Np63 Oncogenic Pathway

4282 Vol. 9, 4282–4285, September 15, 2003 Clinical Cancer Research Identification of S100A2 as a Target of the ⌬Np63 Oncogenic Pathway 1 2 Kenji Hibi, Shin-ichi Fujitake, p53. A transcript that lacked the NH2-terminal TA domain of Tsunenobu Takase, Yasuhiro Kodera, p53 (⌬Np63) was found to act in a dominant-negative fashion 3 Katsuki Ito, Seiji Akiyama, Masatoshi Shirane, and to be able to suppress p53 TA. We additionally examined ⌬Np63 status and observed ⌬Np63 overexpression in head and and Akimasa Nakao neck cancer cell lines and primary lung cancers associated with Gastroenterological Surgery, Nagoya University Graduate School of a low-level increase in chromosomal copy number (7). More- Medicine, Nagoya 466-8560 [K. H., S. F., T. T., Y. K., K. I., S. A., over, we found that increased expression of ⌬Np63 in mouse A. N.], and Department of Product Research, Nippon Roche Research Center, Kanagawa 247-8530 [M. S.], Japan fibroblast cells led to a transformed phenotype. Conversely, no evidence of a tumor-suppressive function of ⌬Np63 in these cancers was found. In addition, proliferating human keratino- ABSTRACT cytes and various epithelial neoplastic cells and lesions predom- Purpose and Experimental Design: It has been proved inantly express the ⌬Np63 isotypes (8–10). We found recently recently that ⌬Np63 may play an oncogenic role in the that high ⌬Np63 gene expression was significantly associated tumorigenic pathway of squamous cell cancers. To gain with lymph node metastasis in esophageal squamous cell cancer additional insight into this pathway, we examined global (11). These data suggested that ⌬Np63 might play an oncogenic patterns of gene expression in cancer cells after ⌬Np63 role in squamous cell cancers. Subsequently, we found that the gene introduction using the oligonucleotide microarray ⌬Np63 isotype forms complexes with p53, resulting in the approach. caspase-dependent degradation of ⌬Np63 protein (12). There- Results: We found that S100A2 might be a target of the fore, the ability of p53 to mediate ⌬Np63 degradation may ⌬Np63 pathway. To confirm the data obtained from oligo- balance the capacity of ⌬Np63 to accelerate tumorigenesis or nucleotide microarray, we then examined the interaction of induce epithelial proliferation. However, we do not yet know ⌬Np63 to S100A2. S100A2 induction was strictly dependent how ⌬Np63 would advance the tumorigenic pathway in these on ⌬Np63 expression by ⌬Np63 transgene and Northern cancers. analysis. ⌬Np63 transactivated the S100A2 promoter, and In an effort to gain additional insight into this pathway, we significantly more fold changes were seen in ⌬Np63-intro- examined global patterns of gene expression in cancer cells after duced cells than in p53-introduced cells, suggesting that ⌬Np63 gene introduction using oligonucleotide microarray ap- ⌬Np63 may be a novel stimulator of the S100A2 promoter. proach. Here we identified S100A2, a Ca2ϩ binding protein, as Conclusion: Taken together, this evidence would seem a novel downstream mediator of ⌬Np63. to suggest that S100A2 is a novel downstream mediator of ⌬ Np63. MATERIALS AND METHODS ⌬Np63 Adenovirus. A full-length ⌬Np63␥ (p40) cDNA INTRODUCTION was cloned from a human prostate cDNA library, and ⌬Np63 p53 is the most commonly inactivated gene in human adenovirus was constructed as described previously (4, 7). ⌬ ␥ cancer, and the loss of critical p53 pathways is central to Briefly, Np63 cDNA was subcloned into the shuttle vector, tumorigenesis (1–3). A new human p53 homologue, p63, was pAdTrack-CMV. The resultant plasmid was linearized by di- isolated recently using a degenerate PCR approach, which gesting with the restriction endonuclease PmeI and subsequently showed that p63 was located on chromosome 3q28 (4–6). The cotransformed into Escherichia coli BJ5183 cells with an ad- DNA binding domain and the oligomerization domain of p63 enoviral backbone plasmid, pAdEasy-1. Recombinants were display a strong conservation of amino acid residues with p53, selected for kanamycin resistance, and recombination was con- raising the possibility that human p63 may also bind key p53 firmed by restriction digest analysis. The linearized recombinant DNA binding sites in the human genome and/or interact with 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 ⌬Np63 adenovirus or an empty adenovirus. In 24 h, cells were Received 2/3/03; revised 5/28/03; accepted 5/28/03. lysed, and total RNA was isolated using the TRIzol reagent The costs of publication of this article were defrayed in part by the (Invitrogen, Carlsbad, CA) according to the manufacturer’s in- 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. 1 To whom requests for reprints should be addressed, at Gastroenter- ological Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan. Phone: 81-52-744- 2 The abbreviations used are: TA, transactivation; SCC, squamous cell 2245; Fax: 81-52-744-2255; E-mail: [email protected]. carcinoma. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 4283 Table 1 Overexpressed or repressed genes in Saos2 cells following ⌬ Np63 gene introduction Expression level Genbank no. Description conta ⌬ Np63b Fold change Overexpressed genes M64347 Human novel growth factor receptor mRNA, 3 cds 432.4 1389.2 4.1 X80907 H. sapiens mRNA for p85 ␤ subunit of 74.5 350.6 3.5 phosphatidyl-inositol-3-kinase AB018302 H. sapiens mRNA for KIAA0759 protein 219.2 441.9 2.8 M87068 Definition ϭ HUMCAN H. sapiens CaN19 mRNA 397.3 1312.3 2.7 AL049341 H. sapiens mRNA; cDNA DKFZp566A163 (from 82.3 276.3 2.7 clone DKFZp566A163) 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 Ϫ3.8 a Absolute expression level in empty adenovirus-infected cells. b Absolute expression level in ⌬ Np63 adenovirus-infected cells. structions. The extracted total RNA was purified using an subcloned into pGL-Basic-3 luciferase reporter plasmid (Pro- RNeasy column (Qiagen, Austin, TX). mega, Madison, WI) at the restriction sites of XhoI and HindIII. Affymetrix (Santa Clara, CA) microarray analysis was Saos2 cells were plated in triplicate in six-well plates at a done according to the manufacturer’s instructions. Briefly, total concentration of 106 cells/well. The Lipofectamine (Life Tech- RNA (5 ␮g) was reverse transcribed to cDNA using T7-(dT)24 nologies, Inc., Gaithersburg, MD) was used for transfection primer. Biotin-labeled cRNA was synthesized from cDNA using according to the manufacturer’s instructions. The luciferase the MEGAscript In Vitro Transcript Kit (Ambion, Austin, TX). reporter construct was cotransfected with a ⌬Np63 adenovirus, cRNA was fragmented to an average size of 50–100 nucleotides a p53 adenovirus, or an empty adenovirus. p53 adenovirus was by incubating at 95°C for 35 min in 40 mM Tris-acetate (pH 8.1) kindly provided by Dr. Bert Vogelstein of Johns Hopkins Uni- containing 100 mM potassium acetate and 30 mM magnesium versity (Baltimore, MD). Cells were washed twice with 2 ml of acetate, and then hybridized to human GeneChip (Human Ge- PBS and then lysed in 200 ␮l (per well) of 1ϫ lysis buffer nome Arrays U95A; Affymetrix) containing ϳ12,000 human (Promega). After a brief spin to pellet large debris, 20 ␮lof genes. The hybridized oligonucleotide microarrays were supernatant was added to 100 ␮l of reconstituted luciferase scanned using a confocal scanner (Affymetrix). The scanned assay reagent (Promega). Light emission was detected using a data obtained from each microarray were normalized to correct luminometer. The results are presented as the fold change over for small differences in the amounts of each cRNA probe the empty adenovirus after normalizing the ␤-galactosidase applied to the microarrays and were processed for average activity. difference values using Affymetrix software. Fold changes of gene expression induced by the infection of ⌬Np63 adenovirus were calculated by comparing the fluorescent intensity of each RESULTS array with respect to the control. Seven genes were chosen for To evaluate the transcriptional effects of ⌬Np63, we stud- candidates of downstream mediator of ⌬Np63, according to ied a human osteosarcoma cell line (Saos2) infected by a ⌬Np63 several criteria set in advance and validated by the following adenovirus or an empty adenovirus (control). The Saos2 cell line analyses. does not express p53 or ⌬Np63 as described previously (7), and Northern Analysis. Saos2 sarcoma cell line was pur- ⌬Np63 might not be degraded by p53. Therefore, changes in chased from the American Type Culture Collection. Cultured gene expression were evaluated using an oligonucleotide mi- cell lines were lysed in guanidine buffer, and total RNA was croarray. Using Affymetrix software, we calculated the fold isolated using the CsCl gradient method. Northern blot hybrid- changes of gene expression induced by the infection of ⌬Np63 ization using the cDNA probe was performed as described adenovirus by comparing the fluorescent intensity of each array previously (13). cDNA included the 3Ј part of the S100A2 gene with respect to the control. This analysis revealed that 6 genes or the whole open reading frame of the ⌬Np63 gene. The human were overexpressed and 1 gene was repressed in ⌬Np63-ex- ␤-actin gene was used as an internal control to standardize the pressed Saos2 cells (Table 1). Interestingly, 2 of the overex- relative amount of RNA in each lane. pressed genes (M87068 and AI539439) were derived from the Luciferase Assay. The luciferase reporter construct in- same gene, S100A2. The S100A2 protein is a member of the cluding the S100A2 promoter was made as described previously S100 family of Ca2ϩ-binding proteins, which are involved in in another paper (14).

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