BRCA 15083 del 19 Mutant Allele Selectively UpRegulates Periostin Expression In vitro and In vivo

Purpose: The aim of this study was to explore the gene expression pattern produced by the cancer-associatedBRCA15083del19 foundermutationbyusingamicroarrayanalysis. Suchamutation, identified ina subsetof familialbreast cancer patients, involves adeletionat the 3¶ endof the BRCA1messenger leading, in themature protein, to the ablationof theBRCT tandemdomain. Experimental Design:We generated HeLa cells stably expressing both exogenous wild-type (HeLa/BRCA1), used as a control, and 5083del19 BRCA1 (HeLa/BRCA1) alleles; gene chips were then used to investigate any changes in the transcription profile induced by the 5083del19 BRCA1mutant compared with controls. Results: Among the genes showing perturbation of their expression, periostin was found to be up-regulated in HeLa/BRCA1cells to an extent of 72-fold versus HeLa/ and 76-fold versus HeLa/BRCA1cells.This finding was validated both in vitro in breast cancer cell lines harboring mutations of BRCA1and in vivo by immunohistochemistry of breast cancer specimens bearing the 5083del19 BRCA1mutation as well as byWestern blot analysis of sera obtained from patients and healthy carriers of the same mutation. Conclusions:Our results suggest that periostin overexpression, whose product is released from cells in the extracellular fluids, might be a potential marker for early cancer detection in a specific subset of hereditary breast carcinomas triggered by cancer-associated BRCA1mutations that affect the BRCT tandem domain. Hereditary breast/ovarian cancer is characterized by a high degree of clinical and genetic heterogeneity. Approximately 10% to 15% of ovarian carcinomas occur secondary to an inherited mutation in BRCA1 or BRCA2 (1, 2). Inherited mutations in BRCA1 confer lifetime risks of breast cancer of 60% to 80% and of ovarian cancer of >25% to 50% (3, 4). A better understanding of the molecular events of breast/ovarian transformation would facilitate improved surveillance, prevention, and therapy. On the other hand, the development of human cancers is a multistep complex process by which cancer cells acquire the ability to overcome the restraints imposed by the surrounding normal tissue internal environment (5). This process is believed to be driven by the intrinsic genomic instability of cancer cells to express genes that confer selective advantages under the adverse growth conditions associated with a rapidly expanding tumor mass. Recently, gene expression pattern differences between various types of human cancers and their corresponding normal tissues have been determined by using the serial analysis of gene expression and gene array analyses (6–10). In particular, in breast cancer, such investigations have led to the application of gene array analysis in the surveillance, prevention, prognosis, and therapy of patients according to the molecular signatures of the individual tumors (9, 11–13). BRCA1 is a tumor suppressor gene whose germ-line mutations predisposes to breast and ovarian cancer (1, 14) and regulates multiple nuclear processes including DNA repair and recombination, checkpoint control of the cell cycle, transcription, chromatin remodeling, and ubiquitination (15–19). BRCA1 encodes a nuclear phosphoprotein of f220 Kd, functionally organized in domains located at the NH2 and COOH termini of the molecule (20). In particular, the COOHterminal BRCT domain is an evolutionarily conserved region characterized by hydrophobic clusters of amino acids that are thought to stabilize the three-dimensional structure of the protein (21). Along with the role in the stability of protein conformation, the BRCT domain is involved in protein-protein interactions with many different molecules (22). The majority of Human Cancer Biology Authors’Affiliations: Department of Experimental and Clinical Medicine,‘‘Magna Græcia’’ University of Catanzaro; Azienda Ospedaliera ‘‘Pugliese-Ciaccio’’ of Catanzaro, Catanzaro, Italy, and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio Received12/19/07; revised 5/12/08; accepted 6/25/08. Grant support: Ministero Universita' e Ricerca Scientifica eTecnologica COFIN 2006, Regione Calabria (POR 2000-2006), and Associazione Italiana Ricerca sul Cancro. 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 with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Francesco Costanzo, Department of Experimental and Clinical Medicine, ‘‘Magna Græcia’’ University of Catanzaro, ‘‘Salvatore Venuta’’ Campus,Viale Europa, 88100 Catanzaro, Italy. Phone: 39-961-3694097; Fax: 39961-3694112; E-mail: fsc@unicz.it. F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-5208 www.aacrjournals.org Clin Cancer Res 2008;14(21) November1, 2008 6797 Research. on July 9, 2017. © 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from cancer-associated BRCA1 mutations affect the BRCT tandem, resulting in truncated products lacking one or two BRCT domains (23). These findings together with the observation that deletion of the BRCA1 BRCT domains is responsible for tumor development in mice (24) show that BRCT domains play a central role in the BRCA1 tumor suppressor function. Our group recently identified a BRCA1 mutation consisting of a heterozygous frame shift mutation causing a large 19-bp deletion in exon 16, leading to a stop codon at position 1,670 that was indicated as BRCA1 founder mutation (5083del19; ref. 25). The aim of this study was to explore the gene expression pattern of this cancer-associated BRCA1 5083del19 founder mutation by using a microarray analysis. Here, we show that periostin, whose product is released from cells in the extracellular fluids (26), was significantly up-regulated both in HeLa/BRCA1 cells compared with HeLa/ cells and HeLa/BRCA1 cells and in mammary tumor specimens and sera from individuals harboring the BRCA1 5083del19 mutation. Materials andMethods Cell culture and DNA transfections. HeLa, human cervix adenocarcinoma cells (parental and stable clones), and MCF-7 and MDA-MB231, human sporadic breast cancer cells, were grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin. HCC1937, human hereditary breast cancer cells, were grown in RPMI 1640 (Life Technologies) containing 15% fetal bovine serum and 1% streptomycin/penicillin. All cells were grown at 37jC in a 5% CO2 atmosphere. HeLa cells were stably transfected with pcDNA3.1/BRCA1 (HeLa/BRCA1) using the calcium phosphate precipitation method and subsequently selected in 400 Ag/mL G418 (Invitrogen Life Technologies). Moreover, HeLa stable clones (HeLa/, HeLa/BRCA1) and HCC1937 stable clones (HCC1937, HCC1937/BRCA1) were generated as described previously (27, 28). Plasmids. The entire open reading frame of BRCA1, cloned into pcDNA3.1 vector (Invitrogen Life Technologies), was already available in our laboratory. Site-directed mutagenesis of the BRCA1 insert was obtained using the QuickChange Kit (Stratagene). The BRCA1 mutant (pcDNA3.1/BRCA1) was generated with the following primers: forward 5¶-gtccatggtggtgtctttatgctcgtgtacaag-3¶ and reverse 5¶-cttgtacacgagcataaagacaccaccatggac-3¶. The construct was verified by DNA