RESEARCH ARTICLE Proteomic analysis of BRCA1-depleted cell line reveals a putative role for replication protein A2 up-regulation in BRCA1 breast tumor development Julien Bouley 1,2 , Ce ´dric Pionneau 2 , Justine Varinot 3 , Denis Biard 4 , Catherine Genestie 3 , Martine Antoine 5 , Florence Coulet 1,6 , Marc-Henri Stern 7 , Dominique Stoppa-Lyonnet 8 and Florent Soubrier 1,6 1 UMRS 956, Universite ´ Pierre et Marie Curie (UPMC), and INSERM, Paris, France 2 Plateforme Post-ge ´ nomique P3S, UPMC, Paris, France 3 Ho ˆ pital Pitie ´ -Salpe ˆ trie ´ re, Assistance Publique Ho ˆ pitaux de Paris (APHP), De ´ partement d’anatomie Pathologique, Paris, France 4 CEA-DSV-IRCM/INSERM U935, Institut A. Lwoff-CNRS, Villejuif, France 5 Ho ˆ pital Tenon, APHP, De ´ partement de Pathologie, Paris, France 6 Ho ˆ pital Pitie ´ -Salpe ˆ trie ` re, APHP, De ´ partement de Ge ´ ne ´ tique, Paris, France 7 Institut Curie, Centre de Recherche & INSERM U830, Paris, France 8 Institut Curie, De ´ partement de Biologie Tumorale and Universite ´ Paris Rene ´ Descartes, Paris, France Received: May 29, 2009 Revised: November 16, 2009 Accepted: November 17, 2009 Purpose: Germline mutations in BRCA1 result in a strong predisposition to breast cancer, with frequent loss of heterozygosity of the remaining wild-type allele. The development of BRCA1 tumors is likely to depend on additional genetic alterations and gene expression changes which follow growth and DNA repair defects associated with BRCA1 deficiency. The identification of these modifications offers an opportunity to find surrogate markers of BRCA1 tumors. Here, we sought to identify differentially expressed proteins related to BRCA1 depletion. Experimental design: We used isogenic HeLa cells either stably knocked-down or not for BRCA1 (BRCA1 KD ) and compared protein profiles of these cells by DIGE. Results: We detected increased levels of Replication protein A2 (RPA2) in BRCA1 KD cells as compared to control cells. RPA2 is an essential protein required for DNA replication and repair. We further demonstrated that depletion of RPA2 subunit delays growth of BRCA1 KD respect to isogenic control cells. Strikingly, elevated levels of RPA2 were more frequently observed in BRCA1 tumors when triple-negative tumors from BRCA1 mutation carriers (n 5 13) and non-carriers (n 5 36) were stained in situ for RPA2. Conclusions and clinical relevance: RPA2 up-regulation may thus be involved in the growth and/or survival of BRCA1 tumor cells and useful in immunohistochemical discrimination of triple-negative BRCA1 tumors. Keywords: BRCA1 / Inherited breast tumor / Replication protein A / Surrogate markers 1 Introduction Breast cancer is the most prevalent cancer in women and about 5% of breast cancer cases are inheritable, caused by mutations of tumor suppressor genes, such as breast-cancer- associated gene-1 and -2 (BRCA1 and BRCA2/FANCD1) [1]. BRCA1 is the most frequently mutated tumor suppressor gene found in familial breast cancers and BRCA1 mutation Correspondence: Professor Florent Soubrier, INSERM U956 Faculte ´ de Me ´ decine Pitie ´ -Salpe ˆ trie ` re, 6e `me e ´ tage, 91 Bd de l’Ho ˆ pital, 75634 Paris, Cedex 13, France E-mail: florent.soubrier@upmc.fr Fax: 33-1-40-77-97-28 Abbreviations: BrdU, bromodeoxyuridine; dpt, days post-trans- fection; DSBs, double-strand breaks; ER, estrogen receptor; HR, homologous recombination; IHC, immunohistochemical; NDKA, nucleoside diphosphate kinase A; PR, progesterone receptor; RPA, replication protein A; TNP, triple negative phenotype & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com Proteomics Clin. Appl. 2010, 4, 489–498 489 DOI 10.1002/prca.200900107